Anthony W Ashton¹, Thi Yen Loan Le², Mahidi Mardini³, Celso E. Gomez-Sanchez⁴ and Anastasia Susie Mihailidou^*⁵
¹The Kolling, Royal North Shore Hospital & University of Sydney, Sydney, Australia, ²The Kolling, Royal North Shore Hospital & University of Sydney, St Leonards NSW, Australia, ³The Kolling, Royal North Shore Hospital & University of Sydney, Australia, ⁴University of Mississippi Medical Center, Jackson, MS, ⁵The Kolling, Royal North Shore Hospital & University of Sydney, Sydney NSW, Australia

Increased levels of aldosterone (Aldo) lead to cardiac damage by pro-oxidant and pro-apoptotic action during myocardial infarction (MI). Free radical scavenger Tempol, prevents Aldo-induced cardiac damage during experimental MI to the same extent as mineralocorticoid receptor (MR) antagonists, suggesting a role for redox balance. Further, low dose MR antagonists alone prevented reperfusion injury by preventing apoptosis. Aim: Since activation of oxidative stress triggers apoptosis, we examined the role of redox balance in the cardioprotective action of low dose MR antagonists. Methods:We used our ex-vivo rat heart model of ischemia-reperfusion (I-R) with spironolactone (SPIRO, 10 nM), Tempol (100 μM) or Aldo (10 nM) perfused 15 mins. prior to ischemia (30 min) and throughout reperfusion (2.5hr) and apoptosis measured using in-situ nick end-labeling (TUNEL) assay. A membrane impermeable Aldo analogue, aldosterone-3-carboxymethoxylamine-TFP ester reacted with 8-branched PEG amine (Aldo-PEG) was used to determine contribution of non-genomic pathways. Redox balance was determined by measuring superoxide levels (lucigenin-enhanced chemiluminescence) and estimation of reduced glutathione (GSH)-to-oxidised glutathione (GSSG) ratio. In separate studies, cultured rat cardiomyocytes (H9c2) were treated with buthionine sulfoximine (BSO), a glutathione synthesis inhibitor, to simulate oxidative stress (±10 nM SPIRO) and Sgk-1 and PAI-1 transcripts used as a measure of MR activation. Results: I-R significantly increased superoxide levels [166 ± 10% (I-R, N=8) vs 100 ± 7% (sham I-R, N=8), P<0.05], while decreasing GSH:GSSG ratio [2 ± 0.3 (I-R, N=5) vs 4 ± 0.3 (sham I-R, N=5), P<0.05], indicating loss of antioxidant defence. This correlated with activation of apoptosis [8 ± 0.6% (I-R, N=8) vs 2 ± 0.5%, (sham I-R, N=5), P<0.05]. SPIRO alone restored redox balance (3 ± 0.1, N=8) and prevented I-R-induced apoptosis, whereas Tempol alone did not prevent I-R induced apoptosis, despite restoring redox balance. In contrast, Aldo potentiated I-R induced decrease in GSH levels (2 ± 0.4, N=7) and aggravated apoptosis (17 ± 1.2%, N=6). Activity of Aldo-PEG was similar to Aldo to increase superoxide production in H9c2 cells, whereas only Aldo aggravated cardiac damage. In separate studies, redox state was manipulated in H9c2 cells by BSO (20 μM) normalising the effect of SPIRO, confirmed by decreased GSH levels. BSO-induced oxidant stress increased both Sgk-1 and PAI-1 transcription (1.6 ± 0.18 and 1.2 ± 0.04 -fold change) indicating MR activation; SPIRO blocked these increases despite absence of anti-oxidant action. Conclusion: Our findings suggest that the cardioprotective actions of MR antagonists involve both maintaining redox balance as well as preventing MR activation.

Nothing to Disclose: AWA, TYLL, MM, CEG, ASM

PP06-1 12739 1.0000 SAT-0839 A Role of Genomic and Non-Genomic Pathways in the Cardioprotective Action of Mineralocorticoid Receptor Antagonists 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Cardiovascular Endocrinology Saturday, June 21st 11:30:00 AM PP06 4892 11:15:00 AM Renin-Angiotensin-Aldosterone System/Endocrine Hypertension Poster Preview

Carmela Maniero^*¹, Junhua Zhou¹, Elena AB Azizan¹, Ian McFarlane², Sudeshna G Neogi², Cheryl A. Brighton¹, Luis Galietta³, Paolo Scudieri³ and Morris J Brown¹
¹University of Cambridge, Cambridge, United Kingdom, ²Genomics CoreLab, Cambridge National Institute for Health Research (NIHR) Biomedical Research Centre (BRC), Cambridge, United Kingdom, ³Istituto Giannina Gaslini, Genova, Italy

Somatic mutations in genes encoding Ca_v1.3 and Na⁺/K⁺-ATPase delineate a zona glomerulosa (ZG) subtype of aldosterone producing adenomas (APA).¹ In order to identify molecules which are markers of a ZG-phenotype and may be influenced by gain-of-function mutations in an adrenal-selective Ca²⁺ channel, we compared the transcriptome of adjacent ZG and zona fasciculata (ZF), and investigated whether genes upregulated in ZG may influence aldosterone production.

1) RNA was isolated by laser capture microdissection from ZF, ZG and tumour (T) of 14 APA and 7 phaeochromocytoma patients. An Affymetrix microarray analysis was performed comparing ZG, ZF and T. The expression of upregulated genes was validated by qPCR. 2) Putative Ca²⁺-sensitive genes were analysed for protein expression by immunohistochemistry ± western blotting. 3) Subcellular localisation of a putative Ca²⁺-activated chloride channel was determined by immunofluorescence microscopy of transfected HEK293 cells. 4) The channel’s role in regulating aldosterone production was studied by transfection of H295R cells. 5) We looked for mutations in these genes from whole exome sequencing of ten ZG-like APA.

Results are summerized as follow: 1) 28 genes were at least 5-fold over-expressed in ZG vs ZF. VSNL1 was 23.5-fold (p=3.6x10^-23), and ANO4 19.9-fold higher (p=6.6x10^-24) in ZG than ZF; although both were also increased in APAs, expression was 2- and 10-fold lower, respectively, than in ZG. qPCR confirmed 168- and 54-fold upregulation, respectively, of VSNL1 and ANO4 in ZG vs. ZF. 2) IHC showed selective staining of both VSNL1and ANO4 in ZG adjacent to either phaeocromocytoma or APA. Western blot was consistent with IHC. 3) Aldosterone secretion from H295R cells was reduced from 27.3 to 11.8 pg/ml after transection with ANO4, and CYP11B2 mRNA expression fell 24-fold. 4) HA-tagged ANO4 was localized in the membrane.5) We found rare germline coding variant in VSNL1 (K153T), predicted to cause loss of function (SIFT=0) in one APA, but this not found on a further 56 patients during the replication sequencing.

In conclusion, two of the most ZG-selective genes are VSNL1, a Ca²⁺-sensor previously reported in APAs, whose transfection protects H295R cells from apoptosis²; and ANO4, a member of the anoctamin family including at least two Ca²⁺-activated chloride channels. Both VSNL1 and ANO4 may function to protect normal ZG cells from Ca²⁺ activation, and contribute to negative feedback of aldosterone from APAs. Relatively lower expression in APAs may augment the consequence of Ca²⁺ activation – by Ca_v1.3 and other mutations.

Nothing to Disclose: CM, JZ, EAA, IM, SGN, CAB, LG, PS, MJB

PP06-2 16199 2.0000 SAT-0840 A Putative Calcium-Sensitive Genes, VSNL1 and ANO4, ARE Upregulated in Human Zona Glomerulosa CELLS and MAY PLAY a Role in Regulation of Aldosterone Production 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Cardiovascular Endocrinology Saturday, June 21st 11:30:00 AM PP06 4892 11:15:00 AM Renin-Angiotensin-Aldosterone System/Endocrine Hypertension Poster Preview

Andrew S Powlson^*¹, Olympia Koulouri¹, Elena AB Azizan¹, Carmela Maniero¹, Kevin Taylor², Franklin Aigbirhio¹, Brendan Koo², Heok K Cheow², John Buscombe², Mark Gurnell¹ and Morris J Brown¹
¹University of Cambridge, Cambridge, United Kingdom, ²Addenbrooke's Hospital, Cambridge, United Kingdom

Adrenal vein sampling (AVS) is the current gold-standard for distinguishing unilateral and bilateral disease in primary aldosteronism (PA). However it is technically demanding and invasive, and in many centres is frequently unsuccessful or ambiguous. Metomidate (MTO), a potent ligand of CYP11B1 and CYP11B2, can be C¹¹H₃-labelled as a PET tracer (¹¹C-MTO), offering a rapid non-invasive alternative to AVS for localising unilateral aldosterone-producing adenomas (APAs)¹.

Here, we report a retrospective analysis of the 61 sequential patients with PA referred for ¹¹C-MTO PET-CT in the three years since our formal comparison with AVS. All patients had a definite or possible adenoma on previous CT or MRI, or an AVS suggestive of lateralisation. ¹¹C-MTO PET-CT was unequivocally positive, by our previously validated criteria, and led to a recommendation for surgery in 26 out of the 61 patients (42.6%). In all 12 cases operated to date following local surgical referral for unilateral adrenalectomy adrenocortical histology (including immunohistochemistry for CYP11B2) was positive, and there was biochemical cure of PA (normalisation of the aldosterone-to-renin ratio (ARR)); six local patients await adrenalectomy. In a further 8 ‘tertiary’ referrals from abroad, or another centre in the UK, lateralisation by ¹¹C-MTO PET-CT led to a recommendation for adrenalectomy but further clinical outcome is currently unknown.

The 26 patients in whom unilateral disease was successfully confirmed fell into one of five indications for ¹¹C-MTO PET-CT: [i] AVS technically unsuccessful with failure to cannulate one of the adrenal veins; [ii] technically adequate AVS, but without unequivocal lateralisation: [iii] AVS not possible (unable to safely withdraw spironolactone or epleronone); [iv] no clear cut abnormality on cross-sectional imaging; [v] patient choice not to undergo AVS. ¹¹C-MTO PET-CT was also valuable in providing strong evidence against lateralisation, especially in patients with a definite unilateral adenoma on CT or MRI but no resolution by AVS.

Finally, where multiple nodules co-exist within a single or both glands, ¹¹C-MTO PET-CT has accurately identified the causative tumour (confirmed by cell-culture, gene-expression and genotyping). We speculate that this may facilitate non-surgical targeted nodule-specific ablation or selective surgical adenomectomy.

In conclusion, analysis of ¹¹C-MTO PET-CT in 61 sequential patients supports its use as an adjunct to AVS in diagnostically challenging cases and, in addition, provides a non-invasive alternative.

Disclosure: MG: Principal Investigator, Ipsen, Advisory Group Member, Novartis Pharmaceuticals. Nothing to Disclose: ASP, OK, EAA, CM, KT, FA, BK, HKC, JB, MJB

PP06-3 16188 3.0000 SAT-0838 A 11C-Metomidate PET-CT Facilitates Definitive Diagnosis in Patients with Primary Aldosteronism and Ambiguous Previous Imaging: A Sequential Analysis of 61 Cases 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Cardiovascular Endocrinology Saturday, June 21st 11:30:00 AM PP06 4892 11:15:00 AM Renin-Angiotensin-Aldosterone System/Endocrine Hypertension Poster Preview

Amalia Sertedaki^*¹, Athina Markou², Gregory A Kaltsas³, Ioannis Androulakis⁴, Theodora Pappa², Aggeliki Gouli², Labrini Papanastasiou², Achilles Zacharoulis², Apostolos Karavidas², Despoina Ragkou², Dimitrios Vlachakis⁵, Sophia Kossida⁵, Emilie Campanac⁶, Dax A Hoffman⁶, Maria De La Luz⁷, Paraskevi Xekouki⁸, Constantine A Stratakis⁹, Evangelia Charmandari¹, George P. Chrousos¹⁰ and Georgios P Piaditis²
¹Athens University, School of Medicine, Athens, Greece, ²“G. Gennimatas” General Hospital, Athens, Greece, ³University of Athens School of Medicine, ⁴“G. Gennimatas” General Hospital, Athens, Greece, Greece, ⁵Biomedical Research Foundation of the Academy of Athens, Greece, ⁶National Institutes of Health, ⁷National Institute of Health, Bethesda, ⁸National Institute of Health, Bethesda, MD, ⁹National Institutes of Health (NIH), Bethesda, MD, ¹⁰University of Athens, School of Medicine, Athens, Greece

Background: Aldosterone secretion by the adrenal zona glomerulosa is regulated by angiotensin II, K⁺and ACTH. Somatic and germline mutations of the KCNJ5 gene expressed in the zona glomerulosa and encoding the Kir3.4 inward rectifying K⁺ channel have been identified in patients with Familial Hyperaldosteronism (FH) type III. FH type I, on the other hand, is due to a chimeric gene originating from the unequal crossing over between the CYP11B1 and CYP11B2 genes.

Objective and Hypotheses: The aim of this study was to investigate the presence of KCNJ5 gene mutations or the CYP11B1/CYP11B2 chimeric gene in patients with hypertension and an increased aldosterone response to ACTH stimulation, but no evidence of primary aldosteronism.

Patients and Methods: We studied 23 hypertensive patients aged 35-68 years with normal findings on adrenal computed tomography scan and increased aldosterone response to ACTH (0.03 μg, iv). Using the 97.5% of CI of aldosterone concentrations and the aldosterone/renin ratio (ARR) of a population of 61 healthy controls, we defined increased aldosterone response the presence of both aldosterone >1300 pmol/L and ARR > 77 pmol/mIU. Genomic DNA was isolated from peripheral blood leucocytes in all subjects. The coding region of KCNJ5 gene was PCR-amplified and sequenced. The chimeric gene CYP11B1/CYP11B2 was PCR amplified as previously described. Electrophysiological and structural biology studies were performed to determine the effect of the mutations identified.

Results: The chimeric gene CYP11B1/CYP11B2 was not detected in any of our patients. Two novel, heterozygous KCNJ5 mutations were detected: the p.V259M c.775G>A in exon 2 and the p.Y348N, c.1042T>A in exon 3. The in silico analysis showed that the mutations were deleterious and that the amino acids V259 and Υ348 are highly conserved among species. Electrophysiological studies revealed that the Y348N showed greater rectification and a significantly less negative reversal potential of -1.3 ± 11.8 mV than the wild-type. The V259M did not appear more rectified than the wild-type, given that the reversal potential was not significantly different (-18.7 ± 10.5 mV). Structural biology studies revealed that the amino acid positions 259 and 348 are very important to the overall 3D arrangement of the KCNJ5 protein.

Conclusions: Two novel mutations of the KCNJ5 gene were detected in two hypertensive patients with an increased aldosterone response to ACTH stimulation. These findings indicate that the K⁺channel might be involved in the pathophysiology of idiopathic hypertension.

Nothing to Disclose: AS, AM, GAK, IA, TP, AG, LP, AZ, AK, DR, DV, SK, EC, DAH, MD, PX, CAS, EC, GPC, GPP

PP06-4 14614 4.0000 SAT-0837 A Mutations of the KCNJ5 Gene in Patients with Hypertension and Increased Aldosterone Response to ACTH 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Cardiovascular Endocrinology Saturday, June 21st 11:30:00 AM PP06 4892 11:15:00 AM Renin-Angiotensin-Aldosterone System/Endocrine Hypertension Poster Preview

Sapna Sanjay Shah^*, Claudia E Ramirez, Alvin C Powers, Cyndya Shibao and James M Luther
Vanderbilt University Medical Center, Nashville, TN

Impaired insulin secretion is an early indicator of type 2 diabetes risk but has not been completely investigated in association with the metabolic syndrome, another risk factor for type 2 diabetes. We hypothesized the severity of the metabolic syndrome is associated with impaired insulin secretory response and insulin sensitivity. A total of 109 hyperglycemic clamps in 96 individuals (mean age 45.3±1.2 years, 56% female, 22% African American) were performed at Vanderbilt University Medical Center. Standardized glucose priming dose followed by a variable infusion rate was adjusted to maintain plasma glucose at 200mg/dL for 120 minutes. The metabolic syndrome defined by ATP III criteria was present in 78% of the cohort: increased waist circumference (80.7%), elevated blood pressure (79%), decreased HDL (70%), elevated fasting glucose (50.5%), and elevated triglycerides (20%). Outcomes included acute glucose stimulated insulin response (AIR = area under insulin curve from 0-10 minutes corrected for baseline insulin secretion), late phase insulin response (LIR = average of insulin values from 90-120 minutes), insulin sensitivity index (ISI = average glucose infusion rate/LIR), and disposition index (DI = AIR x ISI). Outcome variables and metabolic syndrome components were analyzed using fixed effects models adjusted for age, gender, and race. ISI declined nonlinearly when stratified by number of metabolic syndrome components. ISI negatively correlated with age (-0.17 [95% CI -0.01 to -0.32] per year, p=0.040), waist circumference (-0.29 [95% CI -0.17 to -0.42] per cm, p<0.001), and male gender (-3.7 [95% CI -0.19 to -7.3] versus females, p=0.039) and positively correlated with HDL (0.18 [95% CI 0.04 to 0.32] per mg/dL, p=0.01). ISI did not correlate with race, systolic blood pressure, triglycerides, or fasting glucose. DI was assessed to adjust the insulin secretory response for insulin sensitivity. DI correlated inversely with number of metabolic syndrome components (-511.1 [95% CI -215.3 to -806.8, p<0.001), age (-67.6 [95% CI -31.6 to -103.6] per year, p<0.001) and in Caucasians (-1391.9 [95% CI -498.4 to -2285.5] versus African Americans, p=0.003). When individual metabolic syndrome components were analyzed as continuous variables, DI correlated inversely with waist circumference (-48.6 [95% CI -19.1 to -78.1] per cm, p=0.002) and fasting glucose (-41.6 [95% CI -12.5 to -70.7per mg/dL, p=0.06) and correlated positively with HDL (33.1 [95% CI 0.11 to 66.1] per mg/dL, p=0.049). DI did not correlate with systolic blood pressure, triglycerides, or gender. These data demonstrate a negative association between beta cell function and number of metabolic syndrome components adjusted using multivariate analysis. Therefore, an insulin secretion defect is evident in individuals with the metabolic syndrome prior to progression to overt type 2 diabetes.

Disclosure: ACP: Ad Hoc Consultant, Boehringer Ingelheim Pharma GmbH & Co. KG, Ad Hoc Consultant, GNF/Novartis. Nothing to Disclose: SSS, CER, CS, JML

PP01-2 14932 2.0000 SAT-1052 A Beta Cell Function Is Negatively Associated with Metabolic Syndrome Severity 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Diabetes & Glucose Metabolism Saturday, June 21st 11:30:00 AM PP01 4893 11:15:00 AM Basic Mechanisms of Glucose Homeostasis and Diabetic Complications Poster Preview

Xiaoyu Li^*¹, Mark A Clements¹, K Kover¹, Dara J Watkins¹, Mengwei Zang², Dan Heruth¹, Wayne V Moore¹ and Yun Yan¹
¹Children's Mercy Hospital and University of Missouri-Kansas City, Kansas City, MO, ²Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA

Background: Chronic hyperglycemia in diabetes causes endothelial cell dysfunction and, eventually, macro- and microvascular diseases. Thioredoxin-interacting protein (TXNIP) has been recently described as a regulator of the cellular redox state. TXNIP over-expression induced by high glucose concentration decreases thioredoxin reductase activity and renders cells more susceptible to oxidative stress and apoptosis. Metformin is a first line therapy for type 2 diabetes and an antioxidant effect has been reported. The effect and the mechanism of modification of TXNIP expression by metformin in endothelial cells are unknown.

Objective: To determine the impact of metformin on TXNIP over-expression induced by high glucose concentration in endothelial cells.

Materials and Methods: The cells (a mouse aortic endothelial cell line) were maintained at 37 ⁰C with 5% CO₂. Metformin and Compound C, an AMP-activated protein kinase (AMPK) inhibitor, were purchased from Sigma and Millipore, respectively. The treated cells were harvested for qRT-PCR and western blot analyses. To study metformin effects on TXNIP promoter activity, we transfected cells with a construct containing a TXNIP promoter-driven luciferase or the pGL3 basic vector prior to the following treatments. Relative firefly luciferase activity was determined using the Dual Luciferase Assay Kit (Promega) after 24h incubation.

Results: The cells were divided into 5 groups: 1) normal glucose (5.5 mM), 2) high glucose (25 mM), 3) normal glucose with metformin (2 mM), 4) high glucose with metformin (2 mM), 5) 25 mM mannitol (osmotic control) and treated for 24 hours . TXNIP expression was significantly increased by high glucose after 24 h compared to normal glucose at both the mRNA level (10 fold, p<0.01) and the protein level (4 fold, p<0.05). The expression at the protein level was suppressed by metformin (2 fold, p<0.05). Metformin also suppressed TXNIP promoter activity (3 fold, p<0.05). The effect of metformin on suppressing TXNIP promoter activity is partially attenuated by pretreatment with Compound C (10 µM) under high glucose conditions (1.5 fold, p<0.05).

Conclusion: High glucose concentration induces the pro-oxidant gene TXNIP. Metformin suppresses high glucose concentration induced TXNIP over-expression through activation of AMPK activity. Further delineation of the mechanisms of metformin action on TXNIP over-expression induced by high glucose concentration will provide evidence for using metformin as a potential adjunctive treatment for patients with type 1 diabetes.

Disclosure: MAC: Consultant, Medtronic Minimed. WVM: Investigator, Versartis, Inc. Nothing to Disclose: XL, KK, DJW, MZ, DH, YY

PP01-3 15585 3.0000 SAT-1058 A Metformin Suppresses Thioredoxin-Interacting Protein (TXNIP) over-Expression in Endothelial Cells: The Involvement of AMP-Activated Protein Kinase 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Diabetes & Glucose Metabolism Saturday, June 21st 11:30:00 AM PP01 4893 11:15:00 AM Basic Mechanisms of Glucose Homeostasis and Diabetic Complications Poster Preview

Sabyasachi Sen^*¹ and Nagendra Yadava²
¹The George Washington University, Washington, DC, ²Pioneer Valley Life Science Institute

MSCs are multipotent cells that differentiate into fat, muscle, bone and cartilage cells. High Glucose (HG) exposure of bone marrow derived MSCs leads to: increased reactive superoxide (ROS) accumulation both in cytosol and mitochondria ( analysed by FACS using Mitosox Red dye) and increased intra-cellular lipid droplet accumulation. We hypothesized that intra-cellular antioxidant up-regulation specifically mitochondrial (super-oxide dismutase-2), may help reduce superoxide accumulation in mitochondria which will subsequently help reduce fat droplet accumulation and associated cellular inflammation.

We exposed hMSCs to HG (25mM) and normal glucose (NG, 5.5 mM) and interrogated mitochondrial superoxide accumulation and cellular oxygen consumption rate (OCR using Seahorse) and interrogated response to mitochondrial complex I-IV 4 substrate addition. We used AdSOD2 to up-regulate SOD2 prior to HG exposure. HG exposure increased TNF α (Tumor Necrosis Factor, 4 fold) and IL6 (interleukin6, 6-fold) mRNA and increased lipid accumulation (2.5 fold) and led to impaired OCR on SeaHorse and impaired Mitochondrial Complex 1.

With prior SOD2 upregulation in MSCs, TNF α and IL6 mRNA expression was reduced in HG and improved Complex-1 function.

Next we delivered the SOD2 upregulated MSC intra-peritoneally to obese diabetic (db/db) mice. We confirmed homing-in of eGFP labeled MSC, delivered IP, to different fat pockets. The mice that received MSC-SOD2 (experimental) showed reduced adipocyte and systemic inflammation (IL6 and TNF alpha levels) compared the mice that received MSC-Null (control). Interestingly the experimental group showed consistent reduction in fat mass (Echo-MRI), local adipocyte inflammation, systemic inflammation in serum and marked improvement in glucose tolerance tests (GTT).

We conclude that local delivery of mitochondrial superoxide dismutase using stem cells as a gene delivery vehicle reduces inflammation and glucose tolerance tests in vivo. We plan to carry out further glucose clamp studies to consolidate our findings. In conclusion local rather than systemic up-regulation of a mitochondrial anti-oxidant helps in reducing local and systemic inflammation, total fat mass and improves glucose mediated insulin response in obese diabetic mouse models.

Nothing to Disclose: SS, NY

PP01-4 15951 4.0000 SAT-1063 A Genetically Modified Mesenchymal Stem Cells (MSCs) Help Reduce Inflammation and Glucose Mediated Insulin Response in Obese Diabetic Mouse Models 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Diabetes & Glucose Metabolism Saturday, June 21st 11:30:00 AM PP01 4893 11:15:00 AM Basic Mechanisms of Glucose Homeostasis and Diabetic Complications Poster Preview

Le Min^*¹, Min Nie², Junping Wen², Sekoni D. Noel³, Rona S. Carroll³ and Ursula B Kaiser⁴
¹Brigham and Women's Hospital, Harvard Medical School, Boston, MA, ²Brigham and Women's Hospital, Boston, MA, ³Brigham and Women's Hospital/Harvard Med School, Boston, MA, ⁴Brigham and Women's Hospital and Harvard Medical School, Division of Endocrinology/Diabetes, Boston, MA

The important roles of G protein-coupled receptors (GPCRs) in the central regulation of reproductive function have been well documented. These GPCRs include GnRHR, KISS1R, NK3R, and PROKR2. Many mutations in these GPCRs have been identified in patients with GnRH deficiency. GPCRs are integral membrane proteins that possess seven transmembrane domains connected by three extracellular and three intracellular loops. Alignment of GPCRs has identified many conserved amino acids across GPCRs. Mutations occurring in conserved residues are usually thought to cause loss of function, whereas mutations in non-conserved residues are regarded as more likely to be benign. We performed receptor alignment by CLUSTAL 2.0.12 Multiple Sequence Alignments and then ran BoxShade to identify non-conserved, conserved and highly conserved amino acids - defined as having no conservation at that position, 50% or more similar residues (i.e., sharing physicochemical properties), or 50% or more identical residues, respectively - in human GnRHR, KISS1R, NK3R and PROKR2. Notably, all of these receptors are primarily Gq-coupled. To specify conserved amino acids across GPCRs, we also included AT1a, a Gq-coupled receptor, and b2AR, a Gs-coupled receptor, in the alignment. Through detailed literature searches, we found a total of 51 reported naturally occurring missense mutations resulting in amino acid substitutions in GnRHR, KISS1R, NK3R, and PROKR2, identified in patients with GnRH deficiency. Among these, 26 mutations were in non-conserved amino acids, 11 in conserved amino acids, and 14 in highly conserved residues. Seventeen of the 26 mutations in non-conserved residues were reported to have undergone experimental testing for functional effects; 14/17 caused loss of function, whereas 3/17 displayed normal function. Nine of 11 mutations in conserved residues were tested experimentally; all caused loss of function. All 14 mutations in highly conserved residues were demonstrated to cause loss of function. In conclusion, as expected, all mutations tested that occurred in conserved and highly conserved amino acid residues are loss-of-function mutations. Surprisingly, most mutations in non-conserved amino acid residues are also loss-of-function mutations. It is, therefore, important to perform functional testing for mutations occurring in non-conserved amino acids to determine whether they are associated with loss of function or normal function.

Nothing to Disclose: LM, MN, JW, SDN, RSC, UBK

PP08-1 16461 1.0000 SAT-0346 A Predicting the Functional Effects of Mutations in GPCRs Involved in the Neuroendocrine Regulation of Reproduction By Sequence Alignment and Conservation Analysis 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Growth Factor/Tyrosine Kinase/G Protein-Coupled Receptor/Intracelluar Signaling & Biology Saturday, June 21st 11:30:00 AM PP08 4903 11:15:00 AM New Insights in GPCR Structure and Function Poster Preview

Shilpi Mahajan^*¹, Juan A Oses-Prieto², Dimitris Grammatopoulos³, Alma L Burlingame² and Aditi Bhargava¹
¹UCSF, San Francisco, CA, ²University of California San Francisco, ³Univ of Warwick Biomed Res Inst, Coventry, United Kingdom

The corticotropin-releasing factor (CRF) family mediates the classic “fight or flee” stress response by activating the HPA axis. The effectors and responders consist of the neuropeptides CRF, urocortins (Ucn1-3), and two GPCRs: CRF₁ and CRF₂. Two protein-coding variants of CRF₂ have been reported (CRF_2a and CRF_2b). CRF₁ and CRF_2a-bcontain putative N-terminal signaling peptide (NTSP) that is cleaved-off after mediating internalization and/or endoplasmic reticulum (ER) targeting. Usually the transmembrane domain 1 of the mature GPCRs mediates ER targeting and insertion, and the role of putative N-terminal signal peptide in ligand-mediated trafficking and signaling remains unclear. We hypothesized that the NTSP of CRF_2b is key in determining its subcellular localization because it interacts with specific proteins and acts as a rheostat to respond differentially to its four distinct ligands in a context-dependent manner. We generated HEK cells stably expressing full-length CRF_2b (FL-CRF_2b) or deleted NTSP (ΔSP-CRF_2b). FL-CRF_2b HEK cells showed robust intracellular Ca²⁺ peak [Ca²⁺]_i responses after stimulation with 10-100nM doses of Ucn3, but only at high (100nM) dose of Ucn1 or Ucn2. Surprisingly, and unlike CRF_2a, FL-CRF_2b also showed measurable [Ca²⁺]_i after challenge with high doses (75-100nM) of CRF only. Unexpectedly, HEK cells expressing ΔSP-CRF_2b displayed a left-shift in dose-response for kinetics of Ca²⁺ stimulation as activation of [Ca²⁺]_i responses was seen even at 10-50nM of Ucn1-2 and showed a 50% increase in peak [Ca²⁺]_i responses to 30-100nM CRF stimulation as compared with FL-CRF_2b, suggesting increased receptor sensitivity. Next, we determined if this differential signaling was due to differential trafficking of the ΔSP-CRF_2b and CRF₁ to distinct subcellular organelles. Cell-surface labeling of epitope-tagged receptor showed that while CRF₁ trafficked to early endosomes where it co-localized with EEA1 after stimulation with either CRF or Ucn1, CRF_2b did not co-localize with EEA1 after stimulation with either CRF or Ucn1-3. Because deletion of the SP resulted is distinct receptor signaling and trafficking, we determined if the SP determines subcellular localization after ligand stimulation via interaction with other regulatory proteins. By co-immunoprecipitation and mass spectrometry analysis, we identified calnexin, a lectin and ER-targeting chaperone protein that is part of the CRF receptor complex, only after stimulation with CRF and/or Ucn2. We previously showed that CRF₂^-/- acinar cells have dramatic ER ultrastructure damage and activate ubiquitination of unfolded proteins in response to chemical stress that is more pronounced in males than females. Thus, we propose a novel mechanism by which the CRF₂ receptor forms a complex with calnexin to regulate subcellular targeting of proteins and signaling at baseline and during stress.

Nothing to Disclose: SM, JAO, DG, ALB, AB

PP08-2 16590 2.0000 SAT-0347 A N-Terminal Signaling Peptide of CRF Receptor 2b Is a Key Mediator of Ligand-Dependent Trafficking and Signaling 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Growth Factor/Tyrosine Kinase/G Protein-Coupled Receptor/Intracelluar Signaling & Biology Saturday, June 21st 11:30:00 AM PP08 4903 11:15:00 AM New Insights in GPCR Structure and Function Poster Preview

Juliane Dinter^*¹, Jessica Mühlhaus¹, Carolin Leonie Piechowski¹, Anne Müller¹, Daniela Nürnberg², Annette Grueters², Josef Köhrle¹, Chun-Xia Yi³, Matthias H. Tschöp³, Heiko Krude¹, Gunnar Kleinau¹ and Heike Biebermann¹
¹Charité - Universitätsmedizin Berlin Campus Virchow, Berlin, Germany, ²Charité - Universitätsmedizin Berlin Campus Virchow, Berlin, ³Helmholtz Center Munich, Garching, Germany

The thyroid hormone derivative 3-iodothyronamine (T1AM) is known to activate the GPCR trace amine-associated receptor 1 (TAAR1) via Gs/adenylate cyclase pathway. T1AM is more and more gaining medical interest as therapeutic target. Recent studies in rodents have shown that T1AM has cardio- and neuroprotective effects with treatment options in humans for intensive care patients such as infarct or stroke patients. Moreover, significantly higher T1AM concentrations were observed in diabetic patients. Prior to taking treatment options for humans into account all effects and functions of T1AM as well as species transferability need to be elucidated.

We speculated that T1AM affects other receptors than TAAR1 because T1AM injection produced similar effects in WT and Taar1 ko mice. A possible candidate could be Taar5 for T1AM as potential target for mediating T1AM effects which is from the same receptor class and moreover highly conserved across species.

In situ hybridization for Taar1 and Taar5 in mouse brains revealed overlapping expression profiles in the amygdala and ventromedial hypothalamus. Consequently, we functionally characterized the signaling properties of human and mouse TAAR5. We confirmed recent data that the volatile amine dimethylethylamine is an agonist for Gs activation at mTaar5, however, not at hTAAR5. Further investigations revealed elevated basal activity for ERK activation and inositoltriphosphate (IP3) accumulation at hTAAR5 and mTaar5 and additional basal Gs activity for mTaar5. In response to 3-T1AM, no signaling via Gs, Gq, Gi, G12/13 and ERK was observed at hTAAR5 and mTaar5, while T1AM acted as inverse agonist at hTAAR5 by decreasing its basal IP3 and ERK activity. In order to elucidate the particular determinants involved in the ascertained signaling differences between hTAAR5 and mTaar5 chimeric receptors were created, with the objective of transferring murine signaling properties to hTAAR5. We identified 6 amino acids in the ligand binding and G protein-coupling region to be involved.

Our data demonstrated that transferring signaling properties of Taar5 from mouse to human situation ought to be treated warily. The in vitro data suggest that hTAAR5 functions as a target for T1AM and exhibits inhibitory T1AM effects on Gq- and ERK signaling. In consequence, T1AM acts on distinct molecular components which their interaction with T1AM and each other need to be understood in order to use T1AM as pharmacological agent.

Nothing to Disclose: JD, JM, CLP, AM, DN, AG, JK, CXY, MHT, HK, GK, HB

PP08-3 13734 3.0000 SAT-0348 A Identification of a New Target for 3-Iodotyonamine (T1AM) 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Growth Factor/Tyrosine Kinase/G Protein-Coupled Receptor/Intracelluar Signaling & Biology Saturday, June 21st 11:30:00 AM PP08 4903 11:15:00 AM New Insights in GPCR Structure and Function Poster Preview

Anne Müller^*¹, Franziska Meyer², Gunnar Kleinau¹, Heiko Krude¹, Annette Grüters-Kieslich¹ and Heike Biebermann¹
¹Charité - Universitätsmedizin Berlin Campus Virchow, Berlin, Germany, ²Charité Universitätsmedizin Berlin, Berlin, Germany

G-protein coupled receptors (GPCRs) play an important role in hypothalamic body weight regulation. Promoting orexigenic signals the ghrelin receptor (GHSR) and the melanocortin 3 receptor (MC3R), both expressed in the arcuate nucleus on Neuropeptide Y/Agouti-related peptide-neurons are key players in appetite control. Recently it was shown that both receptors function in a signaling network with other GPCRs. To elucidate deeper insights into the complex network of GPCR function in hypothalamic weight regulation we established a hypothalamic murine screening system based on bimolecular fluorescence complementation to detect further interactions of these GPCRs. With that screening procedure we identified a variety of different new interaction partners.

In the present study the impact of the screening hit Ring Finger Protein 11 (RNF11) was investigated. RNF11 is an ubiquitin ligase, part of the A20-complex and required to downregulate inflammatory NFκB-signaling, communicated by the Tumor Necrosis Factor α (TNFα) receptor for instance. In a first step we confirmed the RNF11/MC3R and the RNF11/GHSR-interaction. Next we demonstrated that RNF11 downregulates MC3R-signaling in response to α- and γ-Melanocyte-Stimulating Hormone. In contrast GHSR-signaling, basal and with ghrelin-stimulation was not affected by RNF11. In addition and in concordance with these findings we showed that the MC3R potentiates the cellular NFκB-signaling in response to TNFα, while the GHSR did not. RNF11 in coexpression with the MC3R decreased the TNFα-mediated NFκB-signaling, as expected. These data indicate a hint for a potential MC3R/TNFR-interaction in which RNF11 downregulates simultaneously TNFR and MC3R.

Recently it was shown that obesity is associated with low-grade inflammation of the hypothalamus. This is the first time that a molecular link between GPCRs, expressed in the arcuate nucleus of the hypothalamus and hypothalamic inflammation could be demonstrated indicating a huge spectrum of complexity in energy metabolism.

Nothing to Disclose: AM, FM, GK, HK, AG, HB

PP08-4 14616 4.0000 SAT-0345 A RNF11: A Potential Link Between Chronic Inflammation in Obesity, Hypothalamic GPCR-Signaling and Control of Energy Homeostasis 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Growth Factor/Tyrosine Kinase/G Protein-Coupled Receptor/Intracelluar Signaling & Biology Saturday, June 21st 11:30:00 AM PP08 4903 11:15:00 AM New Insights in GPCR Structure and Function Poster Preview

Laura E. Dichtel^*¹, Kevin C.J. Yuen², Miriam A. Bredella¹, Anu V. Gerweck³, Brian M. Russell³, Ariana D. Riccio³, Michelle H. Gurel³, Stacy E. Legg², Patrick M. Sluss³, Beverly MK Biller¹ and Karen K. Miller¹
¹Massachusetts General Hospital/Harvard Medical School, Boston, MA, ²Oregon Health and Science University, Portland, OR, ³Massachusetts General Hospital, Boston, MA

Context: Obesity is associated with diminished GH secretion, which may result in the overdiagnosis of GHD in overweight/obese patients with pituitary disorders. Although BMI-appropriate peak GH cut-offs for the diagnosis of GHD have been established for the GHRH-arginine test, there are no BMI-specific peak GH cut-offs for the GST, which is currently the favored dynamic test for assessing GHD in the U.S.

Objective: To determine a peak GST GH cut-off value for the diagnosis of GHD in overweight/obese individuals.

Subjects and Methods: Controls (n=47) were otherwise healthy young men with BMI ≥25 kg/m² on no medications. Total pituitary deficiency (TPD) cases (n=20) were defined as BMI of ≥25 kg/m² and ≥3 non-GH pituitary deficiencies. Diagnoses included pituitary adenoma (n=9), craniopharyngioma (n=5), and other pituitary disorders (n=6), while treatments included TSS (n=12) and cranial irradiation (n=6). A partial pituitary deficiency (PPD) group (n=41) was defined as men with BMI ≥25 kg/m²and 1-2 non-GH pituitary hormone deficits. IM glucagon 1 mg (<90 kg) or 1.5 mg (≥90 kg) was administered and serum GH (Immulite 2000, Siemens Medical) was measured every 30 min for 4 h. TPD cases were used as the gold standard for defining GHD in ROC analysis. Abdominal fat was measured by cross-sectional CT at L4 in controls.

Results: Mean BMI did not differ between TPD cases and controls (32±7 vs 34±3 kg/m², p=0.1), while age of controls was lower than TPD cases, as expected (33±7 vs 45±14 y, p=0.004). Mean age (45±13 y) and BMI (33±7 kg/m²) of PPDs was not significantly different than TPD cases (p=0.8 and p=0.7, respectively). Median peak GH was 0.4 ng/ml (95% CI 0.4-4.1) in TPD cases and 3.0 ng/ml (95% CI 0.7-16.1) in controls. Using the standard peak GH cut-off of 3 ng/ml, 95% (19/20) of TPD cases and 45% (21/47) of controls were classified as GHD. In ROC curve analysis of controls vs TPD cases, a value of 0.94 ng/ml provided the greatest sensitivity (94%) and specificity (90%) for diagnosing GHD in overweight/obese men. Using a cut-off of 1 ng/ml, 6% (3/47) of controls and 90% (18/20) of TPD cases would be classified as GHD. Additionally, 88% (18/21) of controls and 5% (1/19) of TPD cases who failed using the standard cut-off would be reclassified as GH sufficient. When the cut-off of 1 ng/ml was applied to the PPD group, 59% (24/41) were classified as GHD, as compared to 80% (33/41) using the standard cut-off. BMI (R= -0.35, p=0.02), visceral adipose tissue (R= -0.32, p=0.03) and waist circumference (R= -0.31, p=0.04) negatively correlated with peak GH levels in controls.

Conclusion: Nearly 50% of young, healthy men with a BMI of ≥25 kg/m² failed a GST using the standard cut-off of 3 ng/ml. This suggests that overweight/obese men with pituitary disease are at significant risk of being misclassified as GHD by GST using this cut-off. A GH cut-off of 1 ng/ml by GST may reduce the rate of overdiagnosis of GHD in these patients.

Disclosure: KCJY: Principal Investigator, Pfizer, Inc., Advisory Group Member, Pfizer, Inc., Investigator, Novo Nordisk, Advisory Group Member, Novo Nordisk. BMB: Consultant, Pfizer, Inc., Consultant, Novo Nordisk. Nothing to Disclose: LED, MAB, AVG, BMR, ADR, MHG, SEL, PMS, KKM

PP03-1 13147 1.0000 SAT-0676 A Overweight/Obese Adults with Pituitary Disorders Require Lower Peak Growth Hormone (GH) Cut-off Values on Glucagon Stimulation Testing (GST) to Avoid Overdiagnosis of Growth Hormone Deficiency (GHD) 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Neuroendocrinology and Pituitary Saturday, June 21st 11:30:00 AM PP03 4906 11:15:00 AM GHRH, GH & IGF Biology Poster Preview

Yujun Gan^*, Andrew J. Paterson, Yue Zhang, Ashiya Buckels, Jing Jiang and Stuart J Frank
University of Alabama at Birmingham, Birmingham, AL

Growth hormone (GH) is a powerful somatogenic and metabolic regulatory hormone that signals via the GH receptor (GHR) and the GHR-associated JAK2 tyrosine kinase. GH activates the STAT5 transcription factor and expression of the insulin-like growth factor-1 (IGF-1) gene and others. We previously demonstrated that GHR interacts physically and functionally with the IGF-1 receptor (IGF-1R), such that, even in the absence of IGF-1, IGF-1R’s presence augments GH-induced STAT5 phosphorylation and IGF-1 gene expression. Using IGF-1R-floxed mouse osteoblasts, our recent studies (1) showed that adenovirus (Ad)-mediated expression of IGF-1R, but not insulin receptor (IR), rescued the diminished GH-induced STAT5 phosphorylation observed in Ad-Cre-infected (IGF-1R-deficient) osteoblasts. Further, reexpression of IGF-1R/IR chimeras with swaps of the related alpha chain extracellular domain L1, CR, and L2 regions indicated that the IGF-1R alpha chain CR-L2 region was required for functional collaboration with GHR signaling. We now report the effects of treatment of GH-responsive cells with conditioned medium (CM) from cells programmed to express soluble truncated fragments of either IGF-1R or IR alpha chain extracellular domains that include the L1-CR-L2 regions of each receptor. We first verified the presence of immunodetectable soluble (sol) IGF-1R and sol IR in the CM of HEK-293 cells infected with Ad-sol IGF-1R or Ad-sol IR, respectively. Pretreatment of each of three GH-responsive cells (mouse osteoblasts, mouse 3T3-F442A preadipocytes, and human LNCaP prostate cancer cells) with sol-IGF-1R CM, but not sol IR CM, resulted in substantial reduction of GH-induced acute STAT5 phosphorylation. Further, sol IGF-1R CM specifically inhibited GH-induced expression of IGF-1 mRNA, as assessed by RT-PCR, in osteoblasts. By immunoblotting, we observed a GH-dependent association of sol IGF-1R, but not sol IR with cells, suggesting that the inhibitory effect of the sol IGF-1R is related to interaction with GHR or a GHR-associated cell surface protein. Studies of the mechanism(s) of this inhibitory effect of sol IGF-1R on GH signaling are underway and may have therapeutic implications.

Nothing to Disclose: YG, AJP, YZ, AB, JJ, SJF

PP03-2 13299 2.0000 SAT-0673 A Effect of a Soluble IGF-1 Receptor Extracellular Domain Fragment on GH Signaling 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Neuroendocrinology and Pituitary Saturday, June 21st 11:30:00 AM PP03 4906 11:15:00 AM GHRH, GH & IGF Biology Poster Preview

Jose Cordoba-Chacon^*¹, Neena Majumdar², Edward O List³, John J Kopchick³ and Rhonda D. Kineman⁴
¹University of Córdoba; Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC); Hospital Universitario Reina Sofia, and CIBER de la Fisiopatología de la Obesidad y Nutrición (CIBERobn) / University of Illinois at Chicago, Córdoba, Spain, ²Jesse Brown VA Medical Center/University of Illinois at Chicago, Chicago, IL, ³Ohio University, Athens, OH, ⁴University of Illinois at Chicago/Jesse Brown VA Medical Center, Chicago, IL

The liver partitions nutrients for appropriate utilization/storage by other tissues. Defects in hepatic nutrient processing can lead to inappropriate fat accumulation (hepatosteatosis), insulin resistance, hyperglycemia, hyperlipidemia and associated pathologies. The liver is a major target of growth hormone (GH), where developmental-, gender- and disease-related changes in GH secretion/action are associated with alterations in hepatic nutrient processing. However, since GH receptors (GHR) are ubiquitously expressed and are required for normal structural growth, it has been difficult to tease apart the direct vs. indirect effects of experimental or disease-related changes in circulating GH levels on adult hepatic metabolism. In order to better define the primary actions of GH on hepatic metabolism in adults, we have knocked down the GH receptor (GHR) in the liver of mice (aLivGHRkd), by treating 10wk-old GHR^fl/fl mice (1) with adeno-associated vectors expressing liver-specific, Cre recombinase. Specifically, 7d post-virus infection, hepatic GHR mRNA expression was reduced (<1.5% of controls) and hepatic GHR protein was undetectable by western-blot in aLivGHRkd mice. Hepatic glycogen and triglyceride (TG) content was dramatically increased in male, but not female mice. The increase in hepatic nutrient storage could not be attributed to changes in systemic or hepatic insulin sensitivity or GH-mediated increases in lipolysis. Interestingly, under both basal and fasted conditions, hepatic PPARγ mRNA and protein levels were increased and this was associated with consistent changes in expression of PPARγ target genes. The lack of an effect of aLivGHRkd in female mice may be in part mediated by estrogen, because livers of ovariectomized (OVX) aLivGHRkd mice accumulated TG and increased expression of lipogenic genes, which was blocked by estrogen replacement. Interestingly, an increase in lipogenesis was not evident in OVX-females with intact hepatic GHR, as compared to SHAM-operated controls. These results clearly demonstrate that the hepatic GHR, or estrogen alone, are sufficient to suppress lipogenesis. It is possible that estrogen may modify GHR through regulation of pStat5 (2). However, estrogen may also bypass the effects of hepatic GHR resistance and act directly to suppress the activity of lipogenic transcription factors. Studies are ongoing to differentiate between these possibilities.

Nothing to Disclose: JC, NM, EOL, JJK, RDK

PP03-3 14851 3.0000 SAT-0674 A Sexual Dimorphic Impact of Adult-Onset Hepatic GH Resistance on Glucose and Lipid Metabolism 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Neuroendocrinology and Pituitary Saturday, June 21st 11:30:00 AM PP03 4906 11:15:00 AM GHRH, GH & IGF Biology Poster Preview

Andrew S Powlson^*¹, Anand K Annamalai¹, Alison J Webb¹, Samantha Moir², John M Shneerson² and Mark Gurnell¹
¹University of Cambridge, Cambridge, United Kingdom, ²Papworth Hospital, Cambridge, United Kingdom

Context:Sleep disordered breathing (SDB), including obstructive sleep apnoea (OSA), leads to excessive daytime somnolence, impaired quality of life, and predisposes to premature cardiovascular and metabolic disease. It is a common complication of acromegaly. In many centres, screening for SDB involves an initial questionnaire [e.g. Epworth Sleepiness Scale (ESS)], which then triggers overnight oximetry to measure desaturation index (DI). However, polysomnography (PSG) remains the gold-standard for confirming and defining the true extent of SDB. Since 1980, forty studies have reported sleep data in subjects with acromegaly, but PSG was performed in fewer than half, and many cohorts have included a predominance of previously treated patients, thus confounding attempts to dissect primary disease and secondary treatment effects.

Study aims:

(i) To determine the prevalence of SDB in the largest cohort of patients with newly-diagnosed, treatment naïve, acromegaly studied to date.

(ii)To investigate whether screening tools such as ESS and DI accurately capture the true extent of SDB in acromegaly.

(iii)To determine the extent to which sleep architecture is disrupted in acromegaly.

Study design:ESS, DI, and PSG were performed in 40 consecutive newly diagnosed patients (22 males, 18 females, mean age 55, range 23-78 yr) referred to our tertiary centre between 2004 and 2013.

Results: OSA, defined on PSG by the apnoea-hypopnoea index (AHI), was a common finding (78%) (mild OSA n=12; moderate OSA n=5; severe OSA n=14). However, in contrast DI markedly underestimated the extent of SDB (mild OSA n=11; moderate OSA n=7; severe OSA n=3). ESS also failed to predict the presence of SDB in many patients (ESS>11, n=12). Consistent with the finding of a high rate of OSA, patients exhibited an increased arousal index, with consequent marked disruption of the normal sleep cycle, despite the majority (n=34) exhibiting normal sleep latency and sleep period time. Twenty-seven patients spent longer than predicted in stage 1 sleep, while the deeper sleep stages were dramatically attenuated in many patients (reduced stage 2, n=26; reduced slow wave sleep, n=26; reduced REM sleep, n=31).

Discussion: Our findings confirm that the majority of patients with acromegaly have SDB. Importantly, we have shown that in acromegaly this is associated with disrupted sleep architecture – specifically attenuation of the deeper sleep stages resulting from regular arousal. Moreover, we have found that ESS and DI are poor screening tools for SDB in acromegaly and, if used alone, may fail to diagnose this important complication.

Disclosure: MG: Principal Investigator, Ipsen, Advisory Group Member, Novartis Pharmaceuticals. Nothing to Disclose: ASP, AKA, AJW, SM, JMS

PP03-4 15767 4.0000 SAT-0675 A Sleep Disordered Breathing in Acromegaly Revisited: Novel Insights from the Largest Study of Polysomnography in De Novo Acromegaly 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Neuroendocrinology and Pituitary Saturday, June 21st 11:30:00 AM PP03 4906 11:15:00 AM GHRH, GH & IGF Biology Poster Preview

Hidenori Fukuoka^*¹, Genzo Iguchi¹, Ryusaku Matsumoto², Hironori Bando², Kentaro Suda², Hitoshi Nishizawa¹, Michiko Takahashi¹, Naoko Inoshita³, Song-Guang Ren⁴, Shozo Yamada⁵ and Yutaka Takahashi²
¹Kobe University Hospital, Kobe, Japan, ²Kobe University Graduate School of Medicine, Kobe, Japan, ³Toranomon Hospital, Tokyo, Japan, ⁴Cedars-Sinai Med Ctr/UCLA, West Hollywood, CA, ⁵Toranomon Hosp, Tokyo, Japan

Objectives: ACTH-producing tumors express ErbB receptors, and EGFR (ErbB1) has been shown to be a therapeutic target for these adenomas (1). To extend these observations, we now assessed other ErbB receptor family (ErbB2-4) molecules as potential anti-tumor therapeutic targets using the pan-ErbB receptor tyrosine kinase inhibitor (TKI), canertinib.

Methods: We treated primary cultured cells derived from resected human ACTH-producing pituitary adenomas (n = 14) with canertinib or with gefitinib, an EGFR mono-TKI, and analyzed effects on POMC expression. We also determined whether expression levels (ranging from - to ++) of tumor ErbB receptor family members were determinants of TKI-responsiveness.

Results:In three EGFR (+) tumors, POMC expression levels were suppressed by gefitinib treatment (42 ± 29 %, p < 0.01). In two EGFR (-) tumors POMC levels were not altered by gefitinib, but were suppressed by canertinib treatment (34 ± 9 %, p = 0.04). However, in normal human pituitary cells, POMC levels were not decreased by canertinib suggesting tumor specificity of the drug. In two ErbB3 (-) tumors, POMC levels were significantly suppressed by canertinib more effectively than in 5 ErbB3 (++), or 5 ErbB3 (+) tumors (p < 0.01, p < 0.01). ErbB4 was detected in all 14 tumors, and 7 ErbB4 (++) tumors showed the most profound reduction in POMC levels (mean reduction of 63 %) achieved by canertinib than 6 ErbB4 (+) tumors (p < 0.01), suggesting that canertinib specifically suppresses POMC expression via ErbB4.

Conclusion:Canertinib suppresses POMC expression in human ACTH-producing pituitary adenomas. This effect was associated with high adenoma cell expression levels of ErbB4 and low expression of ErbB3. The pan-ErbB TKI canertinib could be a targeted drug for drug-naïve as well as for gefitinib-resistant ACTH-secreting tumors.

Nothing to Disclose: HF, GI, RM, HB, KS, HN, MT, NI, SGR, SY, YT

PP09-1 15873 1.0000 SAT-0639 A ErbB Mediate ACTH Suppression By Canertinib in Human Pituitary Corticotroph Adenoma Cells 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Neuroendocrinology and Pituitary Saturday, June 21st 11:30:00 AM PP09 4908 11:15:00 AM Pituitary Tumors Poster Preview

Daniel Bengtsson^*¹, Marianne Andersen², Dominique Maiter³, Katarina Berinder⁴, Ulla Feldt-Rasmussen⁵, Aase Krogh Rasmussen⁵, Gudmundur Johannsson⁶, Charlotte Hoybye⁴, Aart Jan van der Lely⁷, Maria Petersson⁸, Oskar Ragnarsson⁹, Henrik Daa Schroeder¹⁰ and Pia Burman¹
¹University Hospital, Malmö, Sweden, ²Odense Univ Hosp, Denmark, ³Univ of Louvain, Brussels, Belgium, ⁴Karolinska Univ Hospital, Stockholm, Sweden, ⁵National Univ Hosp, Copenhagen, Denmark, ⁶Sahlgrenska Univ Hosp, Gothenburg, Sweden, ⁷Erasmus MC, Rotterdam, Netherlands, ⁸Department of Endocrinology, Metabolism and Diabetology, Karolinska University Hospital, Stockholm, Sweden, ⁹Department of Endocrinology, Sahlgrenska University Hospital, Gothenburg, Sweden, ¹⁰Univ Hosp, Odense, Denmark

Atypical pituitary adenomas and pituitary carcinomas constitute a clinical challenge. The tumors usually progress in spite of radiotherapy, and respond poorly to conventional medical therapy and cytotoxic drugs. About 66% of carcinoma patients die within 12 months after diagnosis. Temozolomide (TMZ) is an oral alkylating drug with good tolerability, approved for medical treatment of malignant gliomas. The experience of its use in aggressive pituitary tumors is limited.

Here we report on 20 patients with aggressively growing pituitary tumors treated with TMZ for a median of 6 months (range 2-23) at 7 specialist centers in Europe. Hormone producing tumors were confirmed in 16 (80%) patients (ACTH n=3, GH 4, GH/PRL 2, PRL 7). Ki-67 levels ranged from 2-23% in atypical adenomas, and 5-90% in carcinomas. Time to metastases was shortest in ACTH producing carcinomas. Immunohistochemistry of tumor O6-methylguanine DNA methyltransferase (MGMT) was performed at a single laboratory.

Outcome: Of 7 pituitary carcinomas, complete regression of metastases was observed in 2 (which persists after 3 and 7 yrs off TMZ), regression >50% followed by stable disease in 1, transient regression > 50% in 1, and progressive growth in 3. Four patients were alive at 1.2 -7 yrs after start of TMZ. In 13 patients with atypical pituitary tumors there was a clinically relevant decrease in size (by 25-80%) in 7, no change in 3, and progressive growth in 3. In 3 of the 20 patients where TMZ was paused after an initial response, a second treatment period was less effective. One patient developed liver metastases after cessation of TMZ. The MGMT staining tended to correlate with the clinical response.

In summary, TMZ improved outcome in about half of the patients with atypical pituitary adenomas and pituitary carcinomas. TMZ can thus be of benefit and a valuable treatment option in patients with uncontrolled pituitary tumors.

Nothing to Disclose: DB, MA, DM, KB, UF, AKR, GJ, CH, AJV, MP, OR, HD, PB

PP09-2 13403 2.0000 SAT-0638 A Treatment with Temozolomide in 20 Patients with Atypical Pituitary Tumors and Pituitary Carcinomas - the Experience of the North-European Neuroendocrine Network 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Neuroendocrinology and Pituitary Saturday, June 21st 11:30:00 AM PP09 4908 11:15:00 AM Pituitary Tumors Poster Preview

Kyohei Hayashi^*¹, Akira Takeshita¹, Hisanori Suzuki¹, Noriaki Fukuhara¹, Naoko Inoshita², Hiroshi Nishioka², Shozo Yamada¹ and Yasuhiro Takeuchi¹
¹Toranomon Hosp, Tokyo, Japan, ²Toranomon Hospital, Tokyo, Japan

Background: Crooke’s cell adenoma (CCA) is a rare histologic subtype of Cushing’s disease (CD), characterized by massive Crooke’s hyaline change in corticotroph adenoma. CCA is generally aggressive presenting as invasive macroadenomas, and resistant to both surgery and radiotherapy. Therefore, the patients with CCA often require additional postoperative medical therapies. Although the effectiveness of dopamine agonist cabergoline, somatostatin analog pasireotide, as well as alkylating agent temozolomide have been reported in some proportions of CD, whether such drugs would be valuable therapeutic options for the management of CCA is unknown. The aim of this study is to predict drug susceptibilities of CCA by comparison with those of other histological types of CD.

Methods: Tumor samples from 59 CD patients were collected during surgery at Toranomon Hospital between 2004-2013. Forty-three samples were non-CCAs and 16 samples including 3 boarder line cases were CCAs. Clinical features were compared between the two groups with respect to tumor size, cavernous sinus invasion, and radical resection rate. After extracting total RNA from the samples, the mRNA expressions of the proopiomelanocortin (POMC), dopamine receptor type 2 (D2R), somatostatin receptor subtype-2 (SSTR2), SSTR5, and O⁶-methylguanine-DNA methyltransferase (MGMT) were determined with quantitative reverse-transcription-PCR analysis (qRT-PCR) using SYBR green. Results were normalized with GAPDH and expressed relative to the mean expressions of 9 normal pituitary samples.

Results: In clinical features, rates of macroadenomas, cavernous sinus invasion and curative resection were 93.8% vs 30.2%, 87.5% vs 53.5% and 43.8% vs 78.3% in CCAs and non-CCAs, respectively, confirming aggressive clinical behaviors of CCAs compared with non-CCAs. In qRT-PCR analysis, the expressions of POMC, D2R, SSTR2, SSTR5, and MGMT were significantly (P<0.05) lower in CCAs as compared with non-CCAs (mean±SEM=131.7±122.2% vs 355.8%±363.9%, 2.9±3.5% vs 21.2±31.4%, 5.9±5.3% vs 10.8±12.8%, 8.4±13.4% vs 25.1±34.1%, 24.6±29.3% vs 65.6±57.9%, respectively).

Conclusion: Our study suggests that the patients with CCAs are resistant to cabergoline and pasireotide compared with those with non-CCAs because of lower expressions of their cognate receptors. On the other hand, lower MGMT expression in CCAs may predict a better response to temozolomide because MGMT proteins repair DNA damage induced by alkylating agents such as temozolomide.

Nothing to Disclose: KH, AT, HS, NF, NI, HN, SY, YT

PP09-3 13189 3.0000 SAT-0637 A Predicting Drug Susceptibilities of Crooke's Cell Adenoma, an Aggressive Variant of Cushing's Disease 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Neuroendocrinology and Pituitary Saturday, June 21st 11:30:00 AM PP09 4908 11:15:00 AM Pituitary Tumors Poster Preview

Marie Helene Schernthaner-Reiter^*¹, Giampaolo Trivellin¹, Maria V Nesterova¹, Laura C. Hernandez Ramirez², Elena Daniela Aflorei³, Maria De La Luz Sierra¹, Constantine A Stratakis¹ and Márta Korbonits³
¹National Institutes of Health (NIH), Bethesda, MD, ²Barts and The London School of Medicine, Queen Mary University of London, London, United Kingdom, ³William Harvey Research Institute, Barts and The London School of Medicine, Queen Mary University of London, London, United Kingdom

M.H.S.-R. and G.T. share the first authorship; C.A.S. and M.K. share the senior authorship of this abstract.

Background: Germline mutations in the Aryl hydrocarbon receptor-interacting protein (AIP) gene cause mostly somatotropinomas (SM) in a subset of familial isolated pituitary adenomas (FIPA). AIP has been shown to interact with two phosphodiesterases (PDE), thereby suggesting a link to the cAMP-PKA pathway, which is involved in sporadic SMs that carry GNAS1 mutations and SMs in Carney complex that are due to PRKAR1A(R1a) mutations. R1a binds the PKA catalytic subunit Ca.

Aim of the study: To study possible interactions between AIP and the PKA pathway in pituitary tumors.

Methods: The SM (GH3) and HEK293 cell lines were used for experiments. AIP, R1a, and Ca overexpression and knockdown were performed by transient transfection with AIP-MYC, R1a-HA, and Ca-HA expression vectors or Aip siRNA, respectively. Both wild-type (wt) and p.R304* AIP, a mutation with a shorter half-life (1), were employed. cAMP levels and PDE activity were measured. Intracellular localization and levels of AIP and R1a were assessed by immunofluorescence (IF). Co-immunoprecipitation (Co-IP) was performed to assess whether AIP interacts with R1a or Ca.

Results: Aip knockdown in GH3 cells led to significantly increased cAMP levels, as shown previously (2), and increased PDE activity. Co-IP suggested direct interaction of AIP with R1a, but not with Ca. IF confirmed that AIP levels were lower in HEK293 overexpressing p.R304* compared to wt AIP. In addition, when p.R304* was co-transfected with R1a, R1a levels were lower than during co-transfection with wt AIP. In GH3 cells, endogenous R1a expression was reduced during AIP knockdown.

Conclusions: We show that AIP knockdown simultaneously leads to increased cellular cAMP levels and PDE activity, indicating that an overactive PKA pathway could be compensated by PDE-mediated cAMP degradation. Furthermore, these preliminary data provide evidence for a functional overlap of AIP with the PKA pathway, possibly through stabilization of R1a by direct interaction with AIP, suggesting a potential mechanism for the contribution of AIP to pituitary tumorigenesis.

Disclosure: MK: Researcher, Pfizer, Inc., Researcher, Novartis Pharmaceuticals, Researcher, Syntaxin, Consultant, Pfizer, Inc., Consultant, Chiasma, Consultant, Sanofi, Consultant, Syntaxin. Nothing to Disclose: MHS, GT, MVN, LCH, EDA, MDLLS, CAS

PP09-4 16714 4.0000 SAT-0640 A Interaction of AIP with the cAMP-Dependent Protein Kinase (PKA) Pathway and Its Role in Pituitary Tumor Formation 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Neuroendocrinology and Pituitary Saturday, June 21st 11:30:00 AM PP09 4908 11:15:00 AM Pituitary Tumors Poster Preview

Zhixiao Wang^*¹ and Shaloo Gupta²
¹Eisai, Inc., Woodcliff Lake, NJ, ²Kantar Health, Princeton, NJ

Background: Obesity has been associated with significant burden and can negatively affect patients’ quality of life. Different weight loss (WL) methods are available and may lead to different results. Treatment satisfaction is not only associated with the effectiveness of the WL method, but can be a driver for long-term compliance and commitment, which is essential in weight management. The objective of this study was to explore treatment satisfaction associated with different WL methods.

Methods: Data were obtained from the 2012 National Health and Wellness Survey (NHWS). The NHWS is an annual Internet-based survey, using stratified random sampling to ensure demographic representativeness of the adult U.S. population (N=71,157). Patients were categorized as had a WL procedure (e.g., gastric bypass, LAP-BAND@) or using a prescription medication for WL (Sur/Rx), vs. using self-modification WL techniques (e.g., diet, exercise, over-the-counter medication, weight management programs, and WL supplements). Sur/Rx patients were matched on demographics, comorbidities, insurance status, smoking and alcohol use, obesity class, and had non-WL surgery in the past 12 months to self-modification patients, via propensity scores (1:2). Overall satisfaction with current WL methods (1 [extremely dissatisfied] to 7 [extremely satisfied]) was assessed. Chi-square tests and ANOVAs were used to determine significant differences after matching.

Results: Of the 22,927 obese (BMI≥30) patients, 58.4% took no current action to lose weight, 2.3% were in the Sur/Rx group and 39.3% were in the self-modification group. The average age was 50.6 (SD=15.3), 50.0% were female. The Sur/Rx group reported being extremely/very satisfied more frequently than the self-modification group (39.3% vs. 20.2%, p<0.001). There was no difference in treatment satisfaction between those using Rx and those whom had a surgical procedure (p>0.05). Similar results were found in overweight and obese patients (BMI≥27) with ≥1 weight related comorbidity (type 2 diabetes, hypertension, or dyslipidemia). Satisfaction was higher for the Sur/Rx group vs. the self-modification group (44.4% vs. 19.7%, p<0.001).

Conclusion: Almost 60% of the obese U.S. population did not take steps to lose weight, suggesting the need for more education on obesity awareness/prevention. Among those who took measures to lose weight, satisfaction with WL methods was greater for the Sur/Rx vs. the self-modification group.

Disclosure: ZW: Employee, Eisai. SG: Consultant, Eisai.

PP05-1 12522 1.0000 SAT-0926 A Satisfaction with Different Weight Loss Methods Among Obese Patients: An Analysis of the 2012 U.S. National Health and Wellness Survey 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Obesity, Adipocyte Biology and Appetite Saturday, June 21st 11:30:00 AM PP05 4912 11:15:00 AM Peripheral Regulation of Energy Homeostasis Poster Preview

Rachel K Crowley^*¹, Conor Woods², Beverly Hughes³, Joanna Gray⁴, Theresa McCarthy⁴, Susan Hughes⁵, Cedric H Shackleton⁵, Nicola Crabtree⁵, Peter Nightingale⁴, Paul M Stewart⁵ and Jeremy W Tomlinson⁵
¹Centre for Endocrinology, Diabetes and Metabolism, University of Birmingham, Birmingham, United Kingdom, ²St Vincent's University Hospital, Dublin, Ireland, ³Centre for Endocrinology, Diabetes and Metabolism, University of Birmingham, UK, ⁴Queen Elizabeth Hospital, Birmingham, United Kingdom, ⁵University of Birmingham, Birmingham, United Kingdom

Obesity is a major risk factor for hypertension and is associated with adverse cardiovascular outcomes. Excess mineralocorticoid production is associated with hypertension in Conn’s syndrome. The role of mineralocorticoid metabolism in hypertension associated with obesity remains unclear.

In order to investigate the role of corticosteroids in hypertension and obesity, we conducted a longitudinal observational study of 57 obese or overweight individuals over 5 years. The participants underwent yearly dual energy X-ray absorptiometry (DXA) scans, 24-hour urine collections for steroid metabolites, subcutaneous fat biopsy and measurement of blood pressure. Total body water (litres) was calculated by multiplication of free fat mass (grams) on DXA by the constant 0.732. Urinary steroids were measured by gas chromatography / mass spectrometry. Expression of the NR3C2 gene for the mineralocorticoid receptor was measured by real time PCR. Generalised estimating equations were used to analyse the change in variables over time. Linear regression was used to assess prediction of final visit systolic blood pressure. Statistical significance was accepted at p < 0.05.

Systolic blood pressure increased during the study (p = 0.01) at a rate of approximately 1mmHg / year. Serum sodium increased from 140 mmol/L to 142 mmol/L (p < 0.001) but the increase in calculated total body water did not reach significance (p = 0.06). The urinary mineralocorticoid metabolite tetrahydroaldosterone increased during the study (p < 0.001) as did urinary cortisol:cortisone (F/E) ratio (p = 0.003) which indicated the presence of increased circulating aldosterone, and reduced 11β- hydroxysteroid dehydrogenase type 2 activity in the kidney. Expression of NR3C2 in subcutaneous adipose tissue at baseline visit was predictive of final visit systolic blood pressure 4 or 5 years later (p = 0.03).

The increase in systolic blood pressure and in serum sodium during the study suggests increased mineralocorticoid activity. The MR could have been activated by either aldosterone or by cortisol, since inactivation of cortisol to cortisone by 11β-HSD2 was falling over time. These findings suggest a role for MR antagonists in this patient group. It is unclear what role the MR plays in adipose tissue and why MR gene expression in adipose tissue would be predictive of later systolic blood pressure. It is possible that an adipokine is secreted in response to MR activation that contributes to development of hypertension in obese and overweight patients.

Nothing to Disclose: RKC, CW, BH, JG, TM, SH, CHS, NC, PN, PMS, JWT

PP05-2 16975 2.0000 SAT-0927 A Systolic Blood Pressure Increases with Time in Obese Patients and Is Predicted By Adipose Expression of the Mineralocorticoid Receptor Gene 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Obesity, Adipocyte Biology and Appetite Saturday, June 21st 11:30:00 AM PP05 4912 11:15:00 AM Peripheral Regulation of Energy Homeostasis Poster Preview

Suman Srinivasa^*¹, Kathleen V Fitch¹, Kimberly Wong¹, Eva Petrow² and Steven K. Grinspoon³
¹Massachusetts General Hospital/Harvard Medical School, Boston, MA, ²Massachusetts General Hospital, Boston, MA, ³Mass General Hosp, Boston, MA

Metabolic abnormalities and body composition changes are common in HIV-infected patients on chronic antiretroviral therapy. We previously demonstrated that intervention with lifestyle modification (LSM) or metformin significantly improves metabolic indices in HIV-infected patients, and that HIV-infected patients have unique brown adipose tissue-like fat depots associated with improved resting energy expenditure (REE). In this study, we investigated whether irisin and FGF21, both known to play a role in “beiging” of white adipose tissue, increase after intervention with LSM or metformin, providing a potential mechanism by which metabolic indices improve in HIV-infected patients with fat redistribution. This is the first study to our knowledge investigating irisin in any HIV cohort and to assess changes in irisin and FGF21 in an HIV cohort in response to LSM or metformin. 50 HIV-infected patients with the metabolic syndrome were previously randomized to receive LSM and/or metformin over 12 months. Baseline comparisons were made to 50 healthy controls well-matched on age, gender, race, and waist circumference. Circulating levels of FGF21 and irisin were assessed at baseline and after 12 months of intervention in association with metabolic parameters. At baseline, HIV-infected patients demonstrated increased log FGF21 (2.13±0.06 vs. 1.98±0.05 pg/mL, P=0.05) and log irisin (0.33±0.02 vs. 0.17±0.04 mg/mL, P=0.003) levels compared to healthy controls. In a subset of HIV-infected patients with increased waist circumference (WC) per National Cholesterol Education Panel guidelines (n=32), FGF21 was positively associated with fasting insulin (ρ=0.42, P=0.02) and HOMA-IR (ρ=0.45, P=0.01), while irisin was negatively associated with fasting insulin (ρ=-0.37, P=0.04) and HOMA-IR (ρ=-0.38, P=0.03). There was no association to these glucose homeostasis parameters in controls with increased WC. After 12 months, HIV-infected patients randomized to LSM had a significant decrease in FGF21 compared to those not randomized to LSM (-10[-35, 22]) vs. 40[0, 94] % change, P =0.01), and the change in FGF21 was strongly associated with improved REE (ρ=-0.34, P=0.05), maximal oxygen consumption (ρ=-0.38, P=0.02), and respiratory quotient (ρ=0.40, P=0.02). Irisin levels were unaffected by LSM or metformin. These data may suggest that HIV-infected patients with the metabolic syndrome present with compensatory increases in FGF21 and/or an FGF21 resistant state. Our data suggest that LSM may help to overcome FGF21 resistance, reducing FGF21 levels and improving energy homeostasis. Irisin levels are elevated in HIV-infected patients, but not influenced by LSM or metformin.

Nothing to Disclose: SS, KVF, KW, EP, SKG

PP05-3 12178 3.0000 SAT-0924 A Effects of Lifestyle Modification and Metformin on Irisin and FGF21 Among HIV-Infected Patients with the Metabolic Syndrome 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Obesity, Adipocyte Biology and Appetite Saturday, June 21st 11:30:00 AM PP05 4912 11:15:00 AM Peripheral Regulation of Energy Homeostasis Poster Preview

F Xavier Pi-Sunyer^*¹, Arne Astrup², Ken Fujioka³, Frank Lyons Greenway III⁴, Alfredo Halpern⁵, Michel Krempf⁶, David C Lau⁷, Carel W Le Roux⁸, Rafael Violante Ortiz⁹, Christine Bjørn Jensen¹⁰ and John Wilding¹¹
¹St Luke's Roosevelt Hosp, New York, NY, ²University of Copenhagen, Frederiksberg C, Denmark, ³Scripps Clinic, La Jolla, CA, ⁴Pennington Biomed Res Ctr, Baton Rouge, LA, ⁵Universidade Sao Paulo, Sao Paulo, Brazil, ⁶Université de Nantes, Nantes, France, ⁷University of Calgary, Calgary, AB, Canada, ⁸University College Dublin, Dublin, Ireland, ⁹Instituto Mexicano del Seguro Social, Cuidad Madero, Tamaulipas, Mexico, ¹⁰Novo Nordisk A/S, Søborg, Denmark, ¹¹University of Liverpool, Liverpool, United Kingdom

Obesity is associated with prediabetes, a significant risk factor for development of type 2 diabetes mellitus (T2DM) and its accompanying complications. This trial investigated the effects of liraglutide 3.0 mg, as adjunct to diet and exercise, on weight loss (primary endpoint), prediabetes prevalence and onset of T2DM (ADA 2010 criteria) over 56 weeks. The effects of liraglutide 3.0 mg cessation were investigated in a subsequent 12‑week re-randomized period.

Adults (BMI ≥27 kg/m² with ≥1 comorbidity or ≥30 kg/m²) were advised on a 500 kcal/day deficit diet and exercise program and randomized 2:1 to once-daily s.c. liraglutide 3.0 mg or placebo. Randomization was stratified by prediabetes status (ADA 2010 criteria). At week 56, individuals without prediabetes and on liraglutide 3.0 mg were re-randomized 1:1 to liraglutide 3.0 mg or placebo (diet and exercise continued). The trial has an ongoing 2-year extension for individuals with prediabetes. Clinicaltrials.gov ID: NCT01272219.

Of 3731 randomized individuals (age 45.1 years, 78.5% female, body weight 106.2 kg, BMI 38.3 kg/m², 61.2% with prediabetes), 71.9% on liraglutide 3.0 mg and 64.4% on placebo completed 56 weeks.

At week 56, individuals on liraglutide 3.0 mg (n=2432) had lost 8.0% (8.4 kg) of body weight compared to 2.6% (2.8 kg) on placebo (n=1220) (estimated treatment difference [ETD] 5.4% [5.6 kg], p<0.0001, LSmeans, full analysis set with LOCF, ANCOVA).

Liraglutide 3.0 mg improved fasting and post-load glycemia compared to placebo (ETD FPG ‑6.9 mg/dL, PG [OGTT, area under the curve] ‑36.4 h*mg/dL, HbA_1c ‑0.23 %-points; p<0.0001 for all). Accordingly, of those with prediabetes at screening, more individuals had reverted to normoglycemia on liraglutide 3.0 mg (69.7%) than on placebo (32.1%) at week 56 (estimated odds ratio [OR] 4.85, p<0.0001, LSmeans, logistic regression). Likewise, of those with normoglycemia at screening, more individuals had progressed to prediabetes on placebo (19.9%) than on liraglutide 3.0 mg (6.9%) at week 56 (OR 3.3, p<0.0001). Few individuals developed T2DM during treatment, but more did so on placebo (14 individuals, 1.3 events/100 patient years of exposure [PYE]) than on liraglutide 3.0 mg (4 individuals, 0.2 events/100 PYE) (OR 0.12, p=0.0003).

From week 56 to 68, individuals re-randomized from liraglutide 3.0 mg to placebo regained more weight (2.9%) than individuals staying on liraglutide 3.0 mg (0.7%) (ETD 2.2%, p<0.0001), and more individuals progressed to prediabetes on placebo (from 8.0% to 22.4%, observed means) than on liraglutide 3.0 mg (from 9.1% to 8.6%) (p<0.0001). No individuals developed T2DM.

In conclusion, consistent with the combined effects on body weight and glycemia, liraglutide 3.0 mg, as adjunct to diet and exercise, was superior to placebo in reducing the prevalence of prediabetes and T2DM after 56 weeks of treatment. Continued treatment was required to sustain these effects.

Disclosure: FXP: Medical Advisory Board Member, Novo Nordisk, Medical Advisory Board Member, Weight Watchers, Medical Advisory Board Member, Johnson &Johnson, Medical Advisory Board Member, Vivus USA, Medical Advisory Board Member, Zafgen, Medical Advisory Board Member, Eisai. AA: Advisory Group Member, BioCare, Advisory Group Member, Pathway Genomics Corp, Advisory Group Member, Vivus USA, Board Member, Dentacom Aps, Shareholder, Dentacom Aps, Consultant, Arena Pharmaceuticals Inc, Consultant, Basic Research , Consultant, Boehringer Ingelheim Pharma GmbH & Co KG, Consultant, Gelesis, Consultant, Gerson Lehrman Group, Consultant, Novo Nordisk, Consultant, S-Biotek, Consultant, Twinlab. KF: Consultant, Eisai, Speaker Bureau Member, Eisai, Consultant, NPS, Speaker Bureau Member, NPS, Consultant, Vivus USA, Speaker Bureau Member, Vivus USA, Consultant, Isis, Consultant, NaZura, Consultant, Novo Nordisk, Consultant, Zafgen, Consultant, Orexigen, Speaker Bureau Member, Abbott Laboratories, Speaker Bureau Member, Takeda, Researcher, Eisai, Researcher, Novo Nordisk, Researcher, NPS, Researcher, Orexigen, Researcher, Enteromedics, Researcher, Shire, Speaker Bureau Member, WeightWatchers. FLG III: Patent holder, Patent holdings, Consultant, Align2Action, Consultant, BARONova, Consultant, Basic Research, Consultant, Citius Pharmaceutical, Editorial Board member, Diabetic Living, Consultant, Eisai, Consultant, General Nutrition Corp, Consultant, Medacorp, Consultant, Guidepoint Global, Consultant, Japan Tobacco Pharmaceutical Division, Medical Advisory Board Member, Jenny Craig, Consultant, Lithera Inc, Patents licencee and stockholder, NeuroQuest, Scientific Board Member, Novo Nordisk, Stockholder, NuMe Health, Scientific Board Member, Orexigen, Stockholder, Origin Biomed, Advisory Group Member, Pam Labs, Stockholder, PlenSat, Consultant, Thetis Pharmaceuticals, Consultant, Unigene Labs, Advisory Group Member, Zafgen, Researcher, Novo Nordisk, Researcher, Hanmi Pharmaceuticals. AH: Advisory Group Member, Novo Nordisk, Speaker Bureau Member, Novo Nordisk. MK: Medical Advisory Board Member, Astra Zeneca, Medical Advisory Board Member, Bristol-Myers Squibb, Medical Advisory Board Member, Novo Nordisk. DCL: Researcher, Astra Zeneca, Researcher, Boehringer Ingelheim, Researcher, Bristol-Myers Squibb, Researcher, Eli Lilly & Company, Researcher, Novo Nordisk, Medical Advisory Board Member, Amgen, Medical Advisory Board Member, Astra Zeneca, Medical Advisory Board Member, Boehringer Ingelheim, Medical Advisory Board Member, Bristol-Myers Squibb, Medical Advisory Board Member, Jansen Pharmaceuticals, Medical Advisory Board Member, Eli Lilly & Company, Medical Advisory Board Member, Novo Nordisk, Medical Advisory Board Member, Roche Pharmaceuticals, Medical Advisory Board Member, Valeant Pharmaceuticals, Speaker Bureau Member, Amgen, Speaker Bureau Member, Astra Zeneca, Speaker Bureau Member, Boehringer Ingelheim, Speaker Bureau Member, Bristol-Myers Squibb, Speaker Bureau Member, Eli Lilly & Company, Speaker Bureau Member, Merck & Co., Speaker Bureau Member, Novo Nordisk, Speaker Bureau Member, Valeant Pharmaceuticals. CWL: Medical Advisory Board Member, Novo Nordisk. RV: Medical Advisory Board Member, Merck Sharp & Dohme, Medical Advisory Board Member, Boehringer Ingelheim, Medical Advisory Board Member, Novo Nordisk, Medical Advisory Board Member, Bristol-Myers Squibb, Medical Advisory Board Member, Astra Zeneca, Medical Advisory Board Member, Eli Lilly & Company, Researcher, Eli Lilly & Company, Researcher, Novo Nordisk, Speaker Bureau Member, Merck Sharp & Dohme, Speaker Bureau Member, Boehringer Ingelheim, Speaker Bureau Member, Novo Nordisk, Speaker Bureau Member, Bristol-Myers Squibb, Speaker Bureau Member, Astra Zeneca. CBJ: Employee, Novo Nordisk, Employee, Novo Nordisk. JW: Speaker Bureau Member, Novo Nordisk, Medical Advisory Board Member, Novo Nordisk, Speaker Bureau Member, Eli Lilly & Company, Speaker Bureau Member, Jansen Pharmaceuticals, Medical Advisory Board Member, Jansen Pharmaceuticals, Speaker Bureau Member, Bristol-Myers Squibb, Researcher, Bristol-Myers Squibb, Medical Advisory Board Member, Bristol-Myers Squibb, Speaker Bureau Member, Boehringer Ingelheim, Speaker Bureau Member, Astra Zeneca, Medical Advisory Board Member, Astra Zeneca, Medical Advisory Board Member, Astellas, Researcher, Novo Nordisk, Speaker Bureau Member, Merck & Co..

PP05-4 12489 4.0000 SAT-0925 A Liraglutide 3.0 Mg Reduces the Prevalence of Prediabetes and Delays Onset of Type 2 Diabetes in Overweight and Obese Adults: Results from Scale Obesity and Prediabetes, a Randomized, Double-Blind and Placebo-Controlled 56-Week Trial 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Obesity, Adipocyte Biology and Appetite Saturday, June 21st 11:30:00 AM PP05 4912 11:15:00 AM Peripheral Regulation of Energy Homeostasis Poster Preview

Miyuki Shibata^*¹, Ryoichi Banno¹, Takeshi Onoue¹, Taku Tsunekawa¹, Koichi Adachi¹, Yoshihiro Ito¹, Motomitsu Goto¹, Hiroshi Arima¹ and Yutaka Oiso²
¹Nagoya University Graduate School of Medicine, Japan, ²Nagoya Univ Grad Schl of Med, Nagoya, Japan

Peripheral signals reflecting energy balance are integrated in the hypothalamic arcuate nucleus, and alter feeding behavior as well as energy expenditure. Two potent orexigenic neuropeptides, neuropeptide Y (NPY) and agouti-related protein (AgRP), are co-expressed in the same neurons in the arcuate nucleus. The central administration of these peptides was shown to increase food intake consumption, whereas the acute ablation of AgRP neurons reportedly caused severe anorexia and weight loss. In addition, it was reported that pharmacological activation of AgRP neurons markedly reduced energy expenditure.

Glucocorticoids are key hormones that regulate energy balance by affecting many peripheral neuroendocrine signals and metabolites. In rodents, genetic or diet-induced obesity is reportedly prevented by adrenalectomy, and restored by glucocorticoid replacement. An excess of glucocorticoids causes obesity, whereas their depletion leads to marked anorexia in humans. Our previous studies demonstrated that (1) glucocorticoids increased the expression of NPY and AgRP mRNA, (2) insulin inhibited NPY mRNA expression only in the presence of glucocorticoids, and (3) ghrelin increased NPY mRNA only in the presence of glucocorticoids. These data suggest that glucocorticoids are not only orexigenic but also play a permissive role in the energy balance.

In this study, we examined the effects of AgRP neuron-specific deletion of glucocorticoid receptors (GRs) (KO) on energy balance in mice. Female and male KO mice on high fat diet (HFD), which was started at the age of 3 weeks, showed mild decreases in body weight (BW) at the age of 6 weeks compared to control (WT) mice, and the differences in BW between KO and WT mice remained significant until 16 weeks old. On a chow diet, the female KO mice showed slightly but significantly attenuated weight gain compared to WT mice after 11 weeks, but there were no significant difference in BW in males between genotypes. The analysis of female mice on HFD showed that visceral fat pad weight and serum leptin levels were decreased in KO mice compared to WT mice, whereas no differences were detected in brown adipose tissue (BAT) weight. In addition, female KO mice on HFD showed increases in uncoupling protein-1 mRNA expression in the BAT as measured by RT-PCR.

Thus, it is demonstrated that the absence of GRs signaling in AgRP neurons resulted in decreased body weight and adiposity under HFD conditions, suggesting that GRs signaling in AgRP neurons is required for the homeostatic regulation of energy expenditure.

Nothing to Disclose: MS, RB, TO, TT, KA, YI, MG, HA, YO

PP07-1 13690 1.0000 SAT-0875 A Agrp Neuron-Specific Deletion of Glucocorticoid Receptor Leads to Decreased Body Weight and Adiposity in Mice 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Obesity, Adipocyte Biology and Appetite Saturday, June 21st 11:30:00 AM PP07 4913 11:15:00 AM Central Regulation of Energy Homeostasis Poster Preview

Elizabeth P. Bless^*, Tejaswini Reddy, Kalpana D Acharya and Marc J Tetel
Wellesley College, Wellesley, MA

Perimenopausal women tend to gain fat weight, increasing the likelihood of related health concerns including heart disease, cancers and type 2 diabetes. While the drop in estradiol associated with menopause is a known factor in weight gain, the mechanism by which this occurs is not entirely understood, but is likely due to cross-talk between estrogens and leptin. Leptin and estradiol have overlapping functions in energy homeostasis and fertility, and receptors for these hormones are localized in the same regions in the hypothalamus which is considered the energy homeostasis center of the brain. Mammalian adult neurogenesis, the birth of new neurons, is well identified in the olfactory bulb and the hippocampus. More recently, new neurons have been discovered in the adult male rodent hypothalamus. Furthermore, some of these new neurons are leptin-sensitive and influenced by diet. Here, we tested the hypothesis that diet and hormonal status modulate hypothalamic neurogenesis in the adult female mouse. Adult mice were ovariectomized and implanted with capsules containing estradiol (E2) or oil (Veh). Within each group, mice were fed a high fat diet (HFD) or maintained on standard chow (STND). All animals were administered BrdU into the lateral ventricle of the brain for 9 days and sacrificed 34 days later after an injection of leptin to induce phosphorylation of signal transducer of activation and transcription 3 (pSTAT3). Areas of the hypothalamus involved in energy homeostasis: the arcuate, ventromedial hypothalamus and dorsomedial hypothalamus were immunohistochemically labeled for BrdU (newly born cells), Hu (neuronal marker) and pSTAT3 (leptin sensitive). As predicted, while mice on a HFD became obese, estradiol protected against obesity. Interestingly, in all three brain regions, HFD increased the number of new cells while E2 inhibited this effect. Moreover, the number of new leptin-sensitive neurons (BrdU-Hu-pSTAT3) in the arcuate was increased by HFD, while E2 attenuated this induction. These results suggest that adult hypothalamic neurogenesis in the adult female mouse is modulated by diet and hormonal status and is related to energy homeostasis. We are currently exploring the possibility that a subpopulation of these new neurons is also estrogen-sensitive.

Nothing to Disclose: EPB, TR, KDA, MJT

PP07-2 14809 2.0000 SAT-0877 A Estradiol and Diet Modulate Energy Homeostasis and Hypothalamic Neurogenesis in the Adult Female Mouse 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Obesity, Adipocyte Biology and Appetite Saturday, June 21st 11:30:00 AM PP07 4913 11:15:00 AM Central Regulation of Energy Homeostasis Poster Preview

Shi-Bing Yang^*¹, Yuh Nung Jan² and Lily Yeh Jan²
¹University of California San Francisco, san francisco, CA, ²University of California San Francisco/HHMI, San Francisco, CA

Role of Hypothalamic K_ATP Channel in Developing Age-Dependent Diabetes in mice

Aging is important risk factor for diabetes, however, little is known about the mechanism for aging to increase diabetes risk. ATP-sensitive Potassium (K_ATP) channel in the hypothalamus has been proposed as a key molecule in regulating energy homeostasis, and interestingly, mice lacking Kir6.2, the pore-forming subunit of the K_ATP channel, develop diabetes in older age. As recent evidence reveals that the K_ATP channel activity in the hypothalamus is regulated in an age-dependent manner for midlife obesity, we hypothesize that this age-dependent regulation of K_ATP channel activity in the hypothalamus may also contribute to the development of diabetes in older mice. We found that pharmacological manipulation of the K_ATP channel in the brain is sufficient to recapitulate the age-dependent diabetes phenotype in the Kir6.2 knockout mice, as blocking central neuronal K_ATP channel by glibenclamide induces diabetes while opening K_ATP channel in the brain ameliorates diabetes in older mice but not in young adult mice. Furthermore, re-expressing Kir6.2 in the leptin receptor (lepR) expressing neurons in the hypothalamus reverses the age-dependent diabetes in older Kir6.2 knockout mice. Lastly, genetic activation of K_ATP channel in the pro-opiomelanocortin (POMC) neurons improves glucose tolerance and this glucose lowering effect is abolished in the Kir6.2 knockout background. Our study reveals that down-regulation of K_ATP channel in hypothalamic neurons contributes to age-dependent diabetes.

Nothing to Disclose: SBY, YNJ, LYJ

PP07-3 17037 3.0000 SAT-0878 A Role of Hypothalamic KATP Channel in Developing Age-Dependent Diabetes in Mice 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Obesity, Adipocyte Biology and Appetite Saturday, June 21st 11:30:00 AM PP07 4913 11:15:00 AM Central Regulation of Energy Homeostasis Poster Preview

Hillary Lauren Woodworth^*, Hannah Marie Batchelor, Janaan Meyers, Raluca Bugescu, Juliette Anne Brown and Gina Marie Leinninger
Michigan State University, East Lansing, MI

The lateral hypothalamic area (LHA) acts in concert with midbrain dopamine (DA) neurons to modulate the motivation to move, drink or feed, and these adaptive behaviors regulate energy balance. We hypothesized that LHA neurons expressing the neuropeptide neurotensin (Nts) regulate distinct DA-mediated circuits and behaviors to contribute to energy balance. We previously demonstrated that LHA Nts neurons project into the midbrain and regulate DA neurons that co-express neurotensin receptor 1 (NtsR1). We therefore examined the projections of midbrain NtsR1 neurons by injecting a cre-mediated anterograde tract tracer into the midbrain of transgenic NtsR1^Cre mice. We found that midbrain NtsR1 neurons synapse within the ventral striatum, including the nucleus accumbens and olfactory tubercle, which are important sites for modulating motivated intake and locomotor activity. To determine if the LHA Nts neuronal circuit regulates DA-mediated behaviors we utilized the D3q Designer Receptor Activated Exclusively by Designer Drugs (DREADD) system to selectively activate LHA Nts neurons. We injected an AAV that mediates the cre-dependent expression of the D3q-DREADD into the LHA of Nts^cre mice, permitting the selective activation of LHA Nts neurons in response to the DREADD ligand, clozapine-N-oxide (CNO). Acute activation of LHA Nts neurons with CNO increased water intake and locomotor activity, but not chow intake in sated animals. Further, action via the LHA Nts neuronal circuit increased the expression of cFos (a marker of neuronal depolarization) in the ventral striatum. Collectively, these data suggest that LHA Nts neurons modulate mesolimbic DA circuits to promote locomotor activity and water intake, and thus regulate adaptive behaviors that impact energy balance.

Nothing to Disclose: HLW, HMB, JM, RB, JAB, GML

PP07-4 13985 4.0000 SAT-0876 A Lateral Hypothalamic Neurotensin Neurons Regulate Locomotor Activity and Water Intake Via the Mesolimbic Dopamine System 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Obesity, Adipocyte Biology and Appetite Saturday, June 21st 11:30:00 AM PP07 4913 11:15:00 AM Central Regulation of Energy Homeostasis Poster Preview

Lucia Gagliardi^*¹, Marni A Nenke¹, Tilenka Rosemary Jenni Thynne¹, Jenny von der Borch¹, Wayne A Rankin², David Ernest Henley³, Jane Sorbello⁴, Warrick J Inder⁴ and David J Torpy¹
¹Royal Adelaide Hospital, Adelaide, Australia, ²SA Pathology, Adelaide, Australia, ³Sir Charles Gairdner Hospital, Guildford, WA, Australia, ⁴Princess Alexandra Hospital, Woolloongabba QLD, Australia

Background: Cortisol is a hormone essential for life; plasma levels reflect secretion by the adrenal zona fasciculata. Key regulators of cortisol production include the light-entrained circadian rhythm, ultradian pulsatility and stress. Patients with Addison’s disease (AD) report impaired well-being [1, 2]. It has been postulated that this is due to an inability of standard release oral glucocorticoid to mimic the circadian variation in cortisol. In contrast, continuous subcutaneous hydrocortisone infusion (CSHI) is able to produce circadian variation in cortisol levels and is feasible and safe [3]. Anecdotal data suggest CSHI therapy improves health status in patients with AD but no placebo-controlled trial has been performed [3].

Hypothesis: CSHI therapy improves health status relative to standard oral glucocorticoid therapy.

Methods: We undertook a multicentre, randomised, double-blind, placebo-controlled clinical trial (CTN080310). CSHI was administered using a proprietary insulin infusion pump. Participants were randomly assigned to each of two 4 week treatment periods: (1) thrice daily oral hydrocortisone (HCT) with placebo subcutaneous infusion and (2) oral placebo with CSHI. Crossover followed a 2 week washout period. Participants took a prescribed bolus dose on waking, mimicking the cortisol-awakening response, and a bolus with lunch, to mimic the cortisol response to food. In addition, they were asked to take a “stress” bolus on experience of a predefined psychic stress. CSHI rates were as previously described [3]. Health status questionnaires (Addison’s quality of life questionnaire [AddiQoL], Short Form-36 [SF-36], Chalder Fatigue Scale [CFS] and General Health Questionnaire-28 [GHQ-28]) were performed at the beginning and end of each treatment period. Between treatment differences were validated by salivary cortisol day profiles and 24hr urine cortisol, performed at baseline and twice during each treatment period. Treatment preference was recorded at the end of the study.

Results: Ten participants (2M, 8F) completed the study, mean age 49y (33-70). Baseline health status scores were consistent with mild impairment. Few stress boluses were administered. Questionnaire scores (mean±SD) with CSHI vs oral HCT were: AddiQoL 100.3 ±9.6 vs 94.5±12.4, SF-36 physical component summary 51.4±6.7 vs 52.1±8.2 and mental component summary 52.5±8.3 vs 52.3±10.1, CFS 11.9±1.7 vs 12.9±4.6, GHQ-28 14.7±3.1 vs 13.2±3.1. No statistically or biologically significant differences were observed between the treatments. Five participants preferred CSHI, four preferred oral HCT and one was uncertain.

Conclusion: CSHI therapy did not improve health status in ten AD participants with mild deficits in well-being at baseline. Treatment preferences suggested non-superiority of CSHI therapy over standard oral glucocorticoid replacement.

Nothing to Disclose: LG, MAN, TRJT, JV, WAR, DEH, JS, WJI, DJT

PP02-1 14151 1.0000 SAT-0821 A Continuous Subcutaneous Hydrocortisone Infusion Therapy in Addison's Disease; Effects on Health Status in a Randomised Placebo-Controlled Clinical Trial 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 11:30:00 AM PP02 4917 11:15:00 AM Hypothalamic-Pituitary-Adrenal Axis Poster Preview

Ribal Al Aridi^*¹, Katia El Sibai², Laure Sayyed Kassem¹, Nadine El Asmar³ and Baha M Arafah⁴
¹University Hospitals,Case Medical Center, Cleveland, OH, ²University Hospitals Case Medical Center, Cleveland, OH, ³UH Case Medical Center/ Case Western Reserve University, Cleveland, ⁴Case Western Reserve Univ, Cleveland, OH

Background: Earlier studies from our institution demonstrated that HPA function is activated during critical illness as evident by the rise in serum free cortisol levels (1,2). In critically ill (CI) patients with low albumin, serum total cortisol was lower than that seen in others with near normal (near-NL) albumin even though the two groups had similar free cortisol levels. The impact of activation of HPA function during critical illness on secretion of other ACTH-dependent steroids (DHEA and DHEA-S) is not well studied. Some studies showed serum DHEA-S levels in CI patients to be low. In the current investigation, we studied alterations in serum DHEA, DHEA-S along with total and free cortisol levels in CI patients before and after cosyntropin stimulation. We calculated the molar ratio of cortisol to DHEA to investigate comparative alterations in secretion of the two steroids. We postulated that critical illness is associated with enhanced secretion of glucocorticoids more so than adrenal androgens, such that the molar ratio of total or free cortisol to DHEA would be higher than normal. In light of its binding to albumin in the circulation, we also postulated that serum DHEA-S would be normal or elevated in CI patients with near-NL albumin but low in those with low albumin.

Methods: Baseline and cosyntropin stimulated levels of cortisol, free cortisol, DHEA, and DHEA-S were measured in 51 CI subjects and 32 healthy subjects of similar ages and gender distribution. While 29/51 of the CI subjects had near-NL albumin (mean 3.2g/dL), the remaining 22/51 had albumin <2.5 (mean 1.9) g/dL.

Results: Although both groups of CI patients had higher than normal baseline serum total cortisol, the respective levels were lower in patients with low albumin (13.2±6.6mcg/dL) than those with near-NL albumin (20.9±11.4mcg/dL) and both groups had equally elevated serum free cortisol levels. Similar pattern was noted in the two CI groups after cosyntropin stimulation. Baseline and post-cosyntropin serum DHEA levels in both groups of CI were similar and not different from those of healthy subjects. In contrast, serum DHEA-S in patients with low albumin (51±28mcg/dL) were lower (p<0.005) than those observed in patients with Near-NL albumin (101±84 mcg/dL) and those of healthy subjects (105±70mcg/dL). The molar ratios of baseline free cortisol to DHEA in the two groups of CI patients (20.5±16.2 Vs 31±42) were higher (p <0.0001) than those of normal subjects (3.3±3.5) and these ratios did not change with cosyntropin stimulation.

Conclusion: Critical illness is associated with higher than normal ratio of cortisol to DHEA secretion that continues at equimolar concentration with cosyntropin stimulation. The persistence of normal DHEA secretion in this setting is yet another evidence against impaired adrenal function during critical illness. The low serum DHEA-S levels observed in some CI patients are due to decreased albumin.

Nothing to Disclose: RA, KE, LS, NE, BMA

PP02-2 14245 2.0000 SAT-0822 A Discordance Between Glucocorticoids and Adrenal Androgen Secretion during Critical Illness: The Impact of Alteration in Binding Proteins 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 11:30:00 AM PP02 4917 11:15:00 AM Hypothalamic-Pituitary-Adrenal Axis Poster Preview

Carmen A Carrasco^*¹, Alejandro Martínez-Aguayo¹, Fidel Allende¹, Sandra Solari¹, Carmen Campino¹, Carolina Mendoza², Carolina A Loureiro², Francisca Grob¹, Rodrigo Bancalari¹, Carolina Valdivia¹, Cristobal A Fuentes¹, Andrea Vecchiola¹, Alejandra Tapia¹, Hernan Garcia Bruce² and Carlos E Fardella¹
¹Pontificia Universidad Catolica de Chile, Santiago, Chile, ²Pontificia Universidad Católica de Chile, Santiago, Chile

Introduction: Midnight salivary cortisol (MSC) is a reliable way to screen hypothalamic pituitary adrenal axis. Few data are available concerning normal value in children and adolescent and all of them used radioimmunoassay. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has the advantage to eliminate interference from drugs and cortisol metabolites and recent publications demonstrate utility in adults.

Aim: To evaluate MSC measured by LC-MS/MS in children and compare to adult values.

Methods: We prospectively included 29 children (56% female, median of age 13 years [range 10-17] and 26% obese) and 50 adult volunteers (62% female, median of age 46 years [range 19-63] and 25% obese). Criteria for obesity was body mass index (BMI) > 95^thpercentile for age and gender in children and BMI > 30 in adults. Interview and physical examination excluded symptoms of Cushing disease, drugs or pathologies known to interfere with cortisol axis. They collected two consecutive MSC (MSC1, MSC2) and 24 hours urinary free cortisol (UFC) at home; morning plasma cortisol (F) was measured at 9:00 am. F and UFC were measured by ECLIA (normal value UFC 13.6-106.7 µg/g creatinine). MSC was collected using polyester Salivette and measured by LC-MS/MS. The range of cortisol assay was 0.02-1 µg/dL, the detection limit was 0.02 µg/dL and the inter assay coefficient of variation was <5.6%, with minimum volume requirement of 500 µL. Normal values in adults < 0.1µg/dL were previously established by Mayo Medical Laboratories.

Results: Median of MSC1 and MSC2 in children were 0.02 µg/dL (range 0.02-0.08) and 0.02 µg/dL (range 0.02-0.06) respectively. No differences were found between children and adults in MSC1 (median 0.02 µg/dL vs.0.02 µg/dL, p 0.1), MSC2 (median 0.02 µg/dL vs. 0.02 µg/dL, p 0.1), UFC (median 45.1 µg/g creatinine vs. 42.4 µg/g creatinine, p 0.5) or F (median 12.2 µg/dL vs. 13.4 µg/dL, p 0.3). Regression analyses revealed no correlation between gender or BMI in MSC, UFC or F. MSC values below the assay lower limit of quantification were found in 87% of children and 76% of adult samples. Six children and ten adults were excluded of the analyses because of inadequate saliva volume.

Conclusion: Our results suggest that MSC measured by LC-MS/MS in children above 10 years had similar values than adults. Inadequate volume of saliva sample is one limitation. To the best of our knowledge, this is the first report of normal values of MSC measured by LC-MS/MS in children.

Nothing to Disclose: CAC, AM, FA, SS, CC, CM, CAL, FG, RB, CV, CAF, AV, AT, HG, CEF

PP02-3 12103 3.0000 SAT-0818 A Assessment of Midnight Salivary Cortisol in Children and Adolescents Measured By Liquid Chromatography-Tandem Mass Spectrometry 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 11:30:00 AM PP02 4917 11:15:00 AM Hypothalamic-Pituitary-Adrenal Axis Poster Preview

Katarzyna G Zarnecki^*¹, Nicole Kelly², William Peppard³, David Herrmann⁴ and James W. Findling⁵
¹Medical College of Wisconsin, Milwaukee, WI, ²University of Colorado Hospital, ³Froedert Memorial Lutheran Hospital, ⁴Froedtert Memorial Lutheran Hospital, ⁵Medical College of Wisconsin, Menomonee Falls, WI

Background

Cushing’s syndrome (CS) is a disorder of diverse etiologies leading to cortisol excess. Etomidate (ET) is an anesthetic induction agent that suppresses adrenal steroidogenesis by inhibiting 11 beta hydroxylase with subhypnotic doses. In patients where rapid control of prodigious hypercortisolism is required and oral therapy is problematic, a continuous intravenous therapy of etomidate has been shown to be an effective intervention.

Patients

We describe 6 patients with ACTH dependent CS in whom we employed a continuous intravenous etomidate infusion (iET) for control of severe hypercortisolism prior to more definitive medical or surgical therapy from January 2008 to January 2014. Five cases involved an ectopic ACTH producing tumor and one patient had an ACTH pituitary tumor and acute psychosis. The mean basal pre-treatment ACTH and cortisol were 419 pg/mL (range 93-1002) and 138 ug/dL (range 32-245).

Results

ET was initiated for preoperative management of hypercortisolism, except in one patient where ET was initiated to control hypercortisolemic metabolic derangements while awaiting metyrapone availability. Goal cortisol level was 10-20 ug/dL. Goal was achieved in all but one patient in whom iET was suspended due to the initiation of palliative care due to extensive tumor progression. The average time to goal was 76 hrs (range 29-134 hrs). All patients received a 5 mg ET bolus prior to a starting infusion rate of 0.02 mg/kg/hr. The rates were increased by 0.01-0.02 mg/kg/hr every 4-6 hrs. The mean rate of cortisol change was 1.93 ug/dL/hr. The average infusion rate at goal was 0.07 mg/kg/hr with a maximum infusion rate of 0.1 mg/kg/hr. Sedation was monitored by the Richmond Agitation Sedation Scale and the maximum infusion rate was below sedative doses in all patients.

Discussion

ET is commonly used as an induction anaesthetic agent, but its adrenostatic effects limit its infusion for prolonged maintenance of sedation. However, subhypnotic doses by means of a continuous infusion may have desirable adrenal suppressive effects. Studies have shown that normalization of cortisol at a dose of 0.1 mg/kg/hr or lower may be an effective strategy to control hypercortisolism. Based on our experience, we have developed a standard iET protocol in our institution: an ET 5 mg bolus is followed by an infusion at 0.02 mg/kg/hr with dose titration in increments of 0.01-0.02 mg/kg/hr every 4 to 6 hrs based on serum cortisol changes. A maximum dose of 0.3 mg/kg/hr is recommended based on previous studies showing a sedative effect at these rates.

Conclusion

From our cumulative experience, we have now developed a standardized titrated iET protocol which should provide clinicians with a simple, safe and effective means to lower serum cortisol in patients with severe clinical, metabolic, and neuropsychiatric consequences of prodigious hypercortisolism as a bridge to more definitive medical or surgical therapy.

Nothing to Disclose: KGZ, NK, WP, DH, JWF

PP02-4 14311 4.0000 SAT-0819 A Continuous Etomidate Infusion for the Management of Severe Hypercortisolism in ACTH-Dependent Cushing's Syndrome: A Case Series 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 11:30:00 AM PP02 4917 11:15:00 AM Hypothalamic-Pituitary-Adrenal Axis Poster Preview

Amina M AL-Awadi^*¹, Layal Saleh², Entissar S. Al-Zaman³, Naeema A. Mahmoud³, Fabiola Lisa Saldanha¹ and Wassim Y Almawi⁴
¹Arabian Gulf University, Manama, Bahrain, ²Pennsylvania State University, State College, PA, ³Salmaniya Medical Complex, Manama, Bahrain, ⁴Arabian Gulf Univ, Manama, Bahrain

Context: Adiponectin is an adipocyte-secreted insulin-sensitizing adipokine, with anti-diabetic and anti-atherosclerotic properties. Adiponectin exists in plasma as three oligomeric isoforms, of which the high molecular weight (HMW) is reported as the most biologically active form of adiponectin in regulating glucose homeostasis.

Objective: We investigated whether decreased total and HMW-adiponectin, and altered HMW/total adiponectin ratio are independent predictors of polycystic ovary syndrome (PCOS).

Subjects and Methods: Study subjects included 122 PCOS women and 89 control women. PCOS was evaluated according to 2003 Rotterdam criteria. Plasma total and HMW adiponectin were measured by ELISA.

Results: PCOS women had significantly reduced plasma total adiponectin (P = 0.031), HMW adiponectin (P = 0.026), and total/HMW adiponectin ratio (P = 0.002) compared to control women. Logistic regression analysis revealed that HMW adiponectin levels and HMW/total adiponectin ratio, more so than total adiponectin, were independently and negatively associated with PCOS, after adjusting for age, body mass index, waist-to-hip ratio, and HOMA-IR. The receiver operator characteristic (ROC) area under the curve (AUC) for predicting PCOS for HMW adiponectin (0.679 ± 0.037), and HMW adiponectin/total adiponectin ratio (0.653 ± 0.039), were larger than that for total adiponectin (0.537 ± 0.041). Categorizing levels of total adiponectin, HMW adiponectin, and HMW/total adiponectin ratio in quartiles demonstrated that PCOS was positively associated with low quartiles in HMW adiponectin (P <0.001) and HMW/total adiponectin (P <0.001), but not total adiponectin (P = 0.178). Logistic regression analysis confirmed the association of low HMW adiponectin [OR(95% CI) = 5.42(2.31 – 12.72)] and HMW/total adiponectin ratio [OR(95% CI) = 7.11(2.66 – 18.97)] with PCOS. Serum HMW adiponectin and HMW/total adiponectin ratio were inversely correlated with age, BMI, hirsutism, fasting insulin, HOMA-IR, and positively correlated with serum LDL-cholesterol. On the other hand, total adiponectin was negatively correlated with waist-hip ratio and serum LH levels.

Conclusions: Reduction in adiponectin secretion is an independent risk factor for PCOS. Our results demonstrate that changes in HMW adiponectin serum levels and HMW/total adiponectin ratio are better better predictors for the presence of PCOS when compared with total adiponectin serum levels.

Nothing to Disclose: AMA, LS, ESA, NAM, FLS, WYA

PP04-2 16699 2.0000 SAT-0019 A High-Molecular Weight (HMW) Adiponectin and HMW/Total Adiponectin Ratio Are Better Predictors of Polycystic Ovary Syndrome (PCOS) Than Total Adiponectin 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Reproductive Endocrinology Saturday, June 21st 11:30:00 AM PP04 4919 11:15:00 AM Female Reproductive Endocrinology Poster Preview

Pedro E Martinez^*¹, Peter John Schmidt¹, Karla D Thompson¹, Lynnette K. Nieman², A Leslie Morrow³, Dahima Cintron⁴ and David R Rubinow⁵
¹National Institute of Mental Health, Bethesda, MD, ²NIH, Bethesda, MD, ³UNC Schl of Med, Chapel Hill, NC, ⁴University of Puerto Rico-Medical Sciences Campus, ⁵Univ of North Carolina at Chaple, Chapel Hill, NC

Background: Evidence suggests that premenstrual dysphoric disorder (PMDD) represents an abnormal affective response to normal levels of progesterone (PROG). In rodents, the exposure to or withdrawal from a PROG metabolite, allopregnanolone (ALLO), modulates GABA-A receptor subunit conformation, brain excitability and anxiety-like behavior. Despite no clear evidence of abnormal plasma levels of ALLO in PMDD, the onset of PMDD symptoms could reflect sensitivity to changing levels of ALLO during the luteal phase. We examined the effects on PMDD of preventing the luteal phase increase in ALLO by administering dutasteride (DUT), a blocker of 5 alpha reductase (type I&2) critical for the conversion of PROG to ALLO.

Methods: Women with PMDD (n=16) and asymptomatic controls (ACs) (n=16) (mean [SD] ages = 41.8 [6.1], 41.5 [4.8] years, respectively) participated in a double-blind, placebo-controlled, cross-over trial of DUT, conducted over two menstrual cycles. Subject samples were further subdivided to receive one of two doses of DUT: a low dose (LD) 0.5mg/day and a high dose (HD) 2.5mg/day. Outcome measures included daily symptom self-ratings. In the HD group, plasma levels of sex steroids and the corresponding 5 alpha- and 5 beta-reduced metabolites were assayed by GC/MS-MS. Results were analyzed with ANOVA-R and Bonferroni t-tests.

Results: In women with PMDD on LD DUT (n=8), we observed no significant effects of DUT on luteal phase symptom appearance or severity compared with either baseline or PBO conditions – all women continued to meet criteria for PMDD. In contrast, HD DUT in women with PMDD (n=8) significantly decreased luteal phase symptom scores (i.e., irritability, anxiety, sadness, breast pain, bloating, and food cravings) compared with both baseline and PBO conditions (F_6,36[range]= 4.8-8.7; p[range]= .001-.03). Seven of these eight women no longer met criteria for PMDD. Luteal phase plasma PROG levels consistent with ovulation were observed and did not differ in women with PMDD on HD DUT compared with PBO. In contrast, significant luteal phase increases in plasma ALLO levels were observed in all women on PBO but not on HD DUT (F_1,13=10.1, p<.01). Both doses of DUT were well-tolerated in both PMDD and AC groups.

Discussion: This pilot study suggests that DUT (2.5 mg per day) effectively treats both affective and physical symptoms in PMDD (without the induction of dysphoric symptoms in ACs) by preventing exposure to increased ALLO levels in the luteal phase. These preliminary therapeutic results require replication in a larger RCT. Nonetheless, PMDD symptoms were eliminated in the context of “normal” ovulation and reduced luteal phase ALLO secretion despite the absence of differences in plasma PROG secretion between DUT and placebo. These data suggest that the mechanism underlying DUT’s effects in PMDD involves the prevention of a luteal phase increase in ring A-reduced metabolites of PROG.

Nothing to Disclose: PEM, PJS, KDT, LKN, ALM, DC, DRR

PP04-3 12177 3.0000 SAT-0020 A Effects of Dutasteride, a 5 Alpha-Reductase Type I Inhibitor, on Pmdd Symptoms: Results of a Pilot Study 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Reproductive Endocrinology Saturday, June 21st 11:30:00 AM PP04 4919 11:15:00 AM Female Reproductive Endocrinology Poster Preview

Clare A Flannery^*¹, Anne M Rowzee², Farrah Saleh¹, Gina Choe¹, Pinar Kodaman¹, Teresa L Wood³ and Hugh S Taylor¹
¹Yale University School of Medicine, New Haven, CT, ²New Jersey Medical School/Rutgers University, Newark, NJ, ³New Jersey Med Sch, Rutgers Biomedical & Health Sciences, Newark, NJ

Women with obesity, PCOS, or Type 2 Diabetes Mellitus are at risk for endometrial hyperplasia and adenocarcinoma. The expression of insulin-like growth factor 1 receptor (IGF-1R) is well-characterized in the endometrium during decidualization and is known to promote cancer growth. The insulin receptor has two splice variants, IR-A and IR-B, which have mitogenic and metabolic roles, respectively; IR-A and IR-B expression are not well characterized in the endometrium. We hypothesized that IR-A and IR-B mRNA expression in endometrial tissue vary differently across the menstrual cycle, suggesting distinct roles in the physiology of normal endometrium. In addition, we hypothesized that the mitogenic receptors IR-A and IGF-1R have higher expression in endometrial adenocarcinoma.

We developed a highly specific quantitative PCR assay to quantify and compare IR-A, IR-B, and IGF-1R expression. We determined receptor expression in endometrial tissue from cycling women using no hormonal medication (n=45, mean age 36+/-1, range 20-48 years) and women with endometrial adenocarcinoma, type 1 (n=10, mean age 58+/-4). Gene expression was normalized to actin, and analyzed for each phase of the menstrual cycle: early proliferative (EP, D1-7), late proliferative (LP, D8-14), early secretory (ES, D15-21), and late secretory (LS, D22-28).

IR-A increased dramatically during the early proliferative phase, with a mean mRNA expression 20 fold-higher than either IR-B or IGF-1R (p=0.002). IGF-1R was at its lowest expression during EP (p<0.03). During LP, IR-B and IGF-1R rose slowly, reaching their individual maximum expressions during ES (p<0.01). In LP and ES phases, the relative ratios of IR-A: IR-B: IGF-1R were 1:1:1, but then altered to 3:2:1 in LS phase due to an increase in IR-A and decrease in IGF1-R expression. In endometrial adenocarcinoma tissue, the mean relative expressions of IR-A: IR-B: IGF-1R were similar (1.5: 1.6: 1; p=NS), and most resembled the levels seen during ES phase. Receptor expression did not vary with either age or body mass index in normal tissue or histological grade in malignant tissue.

The dramatic rise of IR-A corresponds to the rapid increase in endometrial thickness seen during the early proliferative phase, indicating IR-A is likely the predominant isomer responsible for glandular proliferation in normal endometrial physiology. In contrast, IR-B and IGF-1R reached their maximum expressions during the early secretory phase, indicating a role in decidualization involving the differentiation of stromal cells and accumulation of glycogen in epithelial cells. Surprisingly, IR-B was equal in expression to the mitogenic receptors IR-A and IGF-1R in endometrial adenocarcinoma. This is distinct from that reported in prostate, breast, lung, and colon cancer and supports a role for metabolic IR signaling in endometrial adenocarcinoma.

Nothing to Disclose: CAF, AMR, FS, GC, PK, TLW, HST

PP04-4 15170 4.0000 SAT-0017 A Differential Expression of IR-a, IR-B and IGF-1R in Endometrium Reflects Physiology during the Menstrual Cycle and Demonstrates a Distinct Expression Signature in Endometrial Adenocarcinoma 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Reproductive Endocrinology Saturday, June 21st 11:30:00 AM PP04 4919 11:15:00 AM Female Reproductive Endocrinology Poster Preview

Juliette Abeillon - du Payrat^*¹, Karim Chikh², Patricia Bretones³, Pascal Gaucherand⁴, Olivier Claris⁴, Anne Charrié⁵, Veronique Raverot⁶, Jacques Orgiazzi⁷, Francoise Borson-Chazot⁸ and Claire Bournaud⁹
¹Hospices Civils de Lyon, Groupement hospitalier est, Bron, France, ²Hospices Civils de Lyon , Centre hospitalier Lyon Sud, Pierre-Bénite, France, ³Hospices Civils de Lyon, Groupement Hospitalier est, Bron, ⁴Hospices Civils de Lyon, Groupement Hospitalier Est, Bron, ⁵Hospices Civils de Lyon, Centre Hospitalier lyon Sud, Pierre - Bénite, ⁶Hospices Civils de Lyon, Lyon, France, ⁷Université Lyon 1, Pierre-Bénite, ⁸Hospices Civils de Lyon, Groupement Hospitalier est, Bron Cedex, France, ⁹Hospices Civils de Lyon, Lyon Cedex 03, France

Context: Hyperthyroidism occurs in 1% of neonates born of mothers with active or past Graves’ disease (GD). Recent guidelines for management of GD during pregnancy were edicted from studies conducted with first-generation Thyroid Binding Inhibitory Immunoglobulin (TBII) assays.

Objective: This retrospective study was conducted in order to specify second-generation TBII threshold predictive of fetal and neonatal hyperthyroidism, and to identify other factors that may be helpful in predicting neonatal hyperthyroidism.

Methods: We included 47 neonates born in the Lyon area from 45 mothers harboring measurable TBII during pregnancy. TBII measurements were performed in all mothers; bioassays in 18, and fetal thyroid ultrasonographies (US) in 21.

Results: Three neonates were born with hypothyroidism and 9 with hyperthyroidism, including 5 with severe hyperthyroidism requiring treatment. All hyperthyroid neonates were born of mothers with TBII > 5 IU/L in second trimester and TSH-receptor Stimulating Antibodies (TSAb, measured by bioassay)> 400%. These associated criteria identified at risk pregnancies with 100% sensitivity and 85% specificity. Fetal thyroid US was normal in 4 out of 5 hyperthyroid neonates. None of the mothers of hyperthyroid neonates that required antithyroid drug during pregnancy could stop treatment before delivery. Analysis of TBII evolution showed six unexpected cases of increasing TBII values during pregnancy.

Conclusion: TBII values above 5 IU/L require performing a bioassay. In such cases, TSAb values above 400% are strong predictors of neonatal hyperthyroidism. Fetal thyroid US, if not repeated, is not as sensitive as previously described

Nothing to Disclose: JA, KC, PB, PG, OC, AC, VR, JO, FB, CB

PP12-3 12961 2.0000 SAT-0528 A Predictive VALUE of Maternal Second Generation Tbii ASSAY for Neonatal Autoimmune Hyperthyroidism 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Thyroid/HPT Axis Saturday, June 21st 11:30:00 AM PP12 4923 11:15:00 AM Thyroid Autoimmunity Poster Preview

jin-An Zhang^*¹, Qiu Qin², Ronghua Song², Ling Xiao² and Wenjuan Jiang²
¹Jinshan Hospital of Fudan University, Shanghai, China, ²Jinshan Hospital of Fudan University

Abnormal microRNAs (miRNAs) expression is found in many diseases including autoimmune diseases. However, little is known about the role of miRNA regulation in Graves’ disease (GD). Here, we simultaneously detected different expressions of miRNAs and mRNAs in thyroid tissues via a high-throughput genomics approach, known as microarray. Totally 7 specimens of thyroid tissue from 4 GD patients and 3 controls were obtained by surgery for microarray analysis. Then, 30 thyroid specimens (18 GD and 12 controls) were also collected for further validation by qRT-PCR. Statistical analysis showed that the expressions of 5 specific miRNAs were increased significantly while those of other 18 miRNAs were decreased in thyroid tissue of GD patients (FC≥1.3 or≤0.77 and p<0.05). In addition, the transcription of 1271 mRNAs was up-regulated, while the expression of 777 mRNAs transcripts was down-regulated (FC≥2.0 or≤0.5 and p<0.05). Furthermore, integrated analysis of differentially expressed miRNAs and their target mRNAs demonstrated that 2 miRNAs (miR-22 and miR-183) were increased while their target mRNAs were decreased. 3 miRNAs (miR-101, miR-197 and miR-660) were decreased while their target mRNAs were increased. The above findings from microarray screening were confirmed by quantitative real-time PCR (qRT-PCR) in more samples. Thus, our study highlights the possibility that miRNA-target gene network may be involved in the pathogenesis of GD and could provide new insights into understanding the pathophysiological mechanisms of GD.

Nothing to Disclose: JAZ, QQ, RS, LX, WJ

PP12-4 16530 3.0000 SAT-0529 A Aberrant Expression of Micrornas and Target Genes in Lesioned Tissues of Graves' Disease 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Thyroid/HPT Axis Saturday, June 21st 11:30:00 AM PP12 4923 11:15:00 AM Thyroid Autoimmunity Poster Preview

Susmeeta T. Sharma^*¹, Jack A Yanovski², Smita Baid Abraham¹ and Lynnette K. Nieman³
¹Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, ²NIH, Bethesda, MD, ³National Institutes of Health, Bethesda, MD

Background: Dexamethasone (dex) suppression tests (DST) are used for screening and differential diagnosis of Cushing’s syndrome (CS). The 1mg overnight (LD) DST is used to diagnose CS, the dex-suppressed CRH stimulation (Dex-CRH) test to differentiate CS from pseudocushings (PCS) while the 8mg overnight (HD) DST is used to differentiate Cushing’s disease (CD) from ectopic ACTH syndrome (EAS). We assessed the utility of dex levels in improving the diagnostic accuracy of these tests.

Methods: Retrospective study of patients (pts) with CS, PCS and normal volunteers (NV) who had a dex level measured as part of LDDST, HDDST or Dex-CRH test (1-2). A post-dex cortisol (F) level ≥1.8mcg/dl in the LDDST and a 15min post-CRH F level ≥1.4 mcg/dl in the Dex-CRH test suggested CS (2-3). A ≥69% suppression of F levels in HDDST indicated CD (4). Dex levels <140 ng/dl for LDDST and <1600ng/dl for HDDST were considered low.

Results: LDDST (N=77): Post-dex F was abnormal in 44 pts, 37 of these did not have CS on follow-up. Proportion of pts with low dex levels was similar in those with incorrect or correct LDDST results (P=0.7). Three of five pts with an abnormal result and low dex levels (44-117ng/dl) had suppressed post-dex F levels after a 2mg overnight DST.

HDDST (N=56): Results were not consistent with the final diagnosis (CD or EAS) in 13 (23%) pts. Of these, five had low dex levels (400-1220 ng/dl). Proportion of pts with low dex levels was similar between those with correct and incorrect HDDST results (P=0.5). HDDST in one pt with ACTH-dependent CS suggested EAS (28% suppression) with low dex level. IPSS indicated CD. After a doubled dex dose (16mg), F levels suppressed by 76%, changing the HDDST result to CD.

Dex-CRH (N=139): Results were consistent with the final diagnosis in 133 pts (74 CS, 20 NV, 39 PCS). Six pts with an abnormal result had dex levels <500 (247-493) ng/dl. Of these, repeat testing with doubled dex dose (1mg every 6 hours) in two pts led to higher dex levels (610, 757 ng/dl) and normal F level in one. Two pts with abnormal result were on OCPs, one with a known high cortisol binding globulin (CBG) level. None had CS on follow-up.

There was no correlation between dex and post-dex F levels in LDDST, 15min post-CRH F levels in Dex-CRH test and % suppression of F post-dex in HDDST (P=NS).

Conclusion: Low dex and high CBG levels can account for false positive (FP) DST and Dex-CRH test results. Use of a higher dex dose in pts with low dex levels can help decrease FP results.

Nothing to Disclose: STS, JAY, SBA, LKN

OR02-1 16622 1.0000 A Utility of Measurement of Dexamethasone Levels in the Diagnostic Testing for Cushing's Syndrome 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 1:00:00 PM OR02 4719 11:30:00 AM Diagnosing and Treating Cortisol Excess and Deficiency Oral

Ty Brian Carroll^*, Bradley R Javorsky and James W. Findling
Medical College of Wisconsin, Milwaukee, WI

Background:

Transsphenoidal surgery (TSS) is first-line treatment for most patients with Cushing’s disease (CD); however, post-surgical recurrence is an ongoing concern that may arise years after initial remission. While there are currently no standardized guidelines for post-TSS follow-up in patients with CD, conventional tools for assessing recurrence include urinary free cortisol (UFC) and 1 mg overnight dexamethasone suppression testing (DST). More recently, late-night salivary cortisol (LNSC) has also gained recognition as a diagnostic tool for both de novo and recurrent CD. However, timely recognition and treatment may be delayed when biochemical results are discordant.

Methods:

To study this diagnostic difficulty more closely we retrospectively reviewed clinic records from 2/2006 to 7/2013 at our institution to identify patients with proven post-TSS CD recurrence and normal UFC.

Results:

We identified 10 female patients with recurrent CD who had normal UFC at time of recurrence. All patients underwent primary TSS; mean time to CD recurrence was 3.5 years (range 1–9) after initial clinical and biochemical remission. Five patients had evidence of tumor on MRI at time of recurrence. All patients had normal renal function and underwent testing with LNSC, DST, and UFC. All 10 patients had normal UFC; however, DST was abnormal in 8, and all 10 had ≥1 elevated LNSC measurement. All patients with available follow-up data (9/10) demonstrated significant improvement upon treatment. Two patients underwent repeat TSS; in both cases, pathology confirmed ACTH-staining pituitary adenoma and the patient achieved clinical and biochemical remission. Six patients received pharmacological therapy (5 with mifepristone, 1 with cabergoline) and all have shown clinical and/or biochemical improvement. One patient, who had normal UFC and DST with abnormal LNSC, underwent bilateral adrenalectomy and, among other clinical improvements, experienced a 55-kg weight loss and resolution of hypertension.

Conclusion:

While UFC is generally considered an accurate diagnostic tool for CD, our case series indicates that UFC is not a reliable marker of recurrence and supports the hypothesis that LNSC may be more sensitive than UFC or DST for detection of recurrent CD. Prompt intervention when LNSC is elevated, despite normal UFC, may yield significant clinical benefit for many patients with CD.

Disclosure: TBC: Consultant, Corcept. JWF: Consultant, Corcept, Consultant, Novartis Pharmaceuticals. Nothing to Disclose: BRJ

OR02-2 12799 2.0000 A Late-Night Salivary Cortisol for the Diagnosis of Recurrent Cushing's Disease: Evidence of Clinical Benefit from Early Detection 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 1:00:00 PM OR02 4719 11:30:00 AM Diagnosing and Treating Cortisol Excess and Deficiency Oral

Eleni Daniel^*¹, Simon J B Aylwin², Stephen G Ball³, Kristien Boelaert⁴, Daniel Cuthbertson⁵, Christina Daousi⁵, Julian Richard Davis⁶, William Drake⁷, Ashley B. Grossman⁸, Mark Gurnell⁹, Niki Karavitaki¹⁰, Tara Kearney¹¹, Karim Meeran¹², Aled Rees¹³, P J Trainer¹⁴ and John Newell-Price¹⁵
¹University of Sheffield, Sheffield, United Kingdom, ²King's College Hospital, London, United Kingdom, ³Newcastle University, Newcastle Upon Tyne, United Kingdom, ⁴University of Birmingham, Birmingham, ⁵University of Liverpool, Liverpool, United Kingdom, ⁶University of Manchester, Manchester, United Kingdom, ⁷St Bartholomew's Hospital, London, United Kingdom, ⁸University of Oxford, Oxford, United Kingdom, ⁹University of Cambridge & Addenbrooke's Hospital, Cambridge, United Kingdom, ¹⁰Oxford Center for Diabetes, Oxford, United Kingdom, ¹¹Salfrod Royal NHS Foundation Trust, Salford, United Kingdom, ¹²Imperial College NHS Healthcare Trust, London, United Kingdom, ¹³Cardiff University, Cardiff, United Kingdom, ¹⁴Department of Endocrinology, Manchester, United Kingdom, ¹⁵University of Sheffield and Department of Endocrinology, Sheffield Teaching Hospitals, Sheffield, United Kingdom

Background: Metyrapone is widely used in the UK for the control of cortisol excess in Cushing’s syndrome, but its use is not standardised. The few published reports on metyrapone use pertain to limited patient numbers.

Method:A retrospective survey was conducted across 13 tertiary centres in England and Wales. Using a standardised proforma, extensive data including monitoring and safety information were collected for patients with Cushing’s syndrome on metyrapone therapy between 1997 and 2013. Eucortisolemia was defined according to the monitoring test used as a mean cortisol ‘day curve’ value ≤300nmol/l, a urinary free cortisol bellow the upper limit of normal (ULN) or a 9am serum cortisol <ULN (but <600nmol/l).

Results: 195 patients received metyrapone (160 as monotherapy). Average age was 49.6 +/-15.7 years: 87.2% had metyrapone in conjunction with other interventions (surgery, radiotherapy or chemotherapy) while 12.8% had cortisol-lowering treatment alone. Dose-titration was used in 81% of patients, whereas 19% had a block-and-replace regimen.

A total of 138 patients received metyrapone monotherapy for a mean duration of 162 days before any other intervention took place. The etiology of Cushing’s syndrome in this subgroup was: pituitary-dependent disease [CD, 59% (macroadenoma 32% of CD)], ectopic ACTH syndrome (EAS, 17%), adrenocortical carcinoma (ACC, 4%), adrenal adenoma (AA, 17%) and other benign adrenal disease (3%). Hypokalemia was actively managed with potassium levels increasing during metyrapone therapy (3.90mmol/L Vs 3.68mmol/L, p=0.0026). In this subgroup, 74% achieved eucortisolemia on varying doses: CD 1370mg, EAS 2080mg, AA 1170mg, ACC 750mg daily in divided doses.

The preferred monitoring method was by cortisol ‘day-curves’, followed by 9am cortisol and urinary free cortisol. Overall, 25.3% of patients developed side effects, most commonly gastrointestinal upset and hypoadrenalism. 88% of adverse events were managed as outpatients; 36% of patients treated for more than one month had ≤2 monitoring assessments and insufficient dose titration.

Conclusion: This is the largest report of metyrapone use. Metyrapone was effective in achieving eucortisolemia in over 70% of patients without any other cortisol-lowering intervention, with a satisfactory safety profile. A variety of monitoring regimens were used, but greater standardisation of practice and more active dose titration is needed.

Disclosure: ED: Investigator, HRA Pharma. SJBA: Principal Investigator, HRA Pharma. SGB: Principal Investigator, HRA Pharma. KB: Principal Investigator, HRA Pharma. DC: Principal Investigator, HRA Pharma. CD: Principal Investigator, HRA Pharma. JRD: Principal Investigator, HRA Pharma. WD: Principal Investigator, HRA Pharma. ABG: Principal Investigator, HRA Pharma. MG: Principal Investigator, HRA Pharma. NK: Principal Investigator, HRA Pharma. TK: Principal Investigator, HRA Pharma. KM: Principal Investigator, HRA Pharma. AR: Principal Investigator, HRA Pharma. PJT: Principal Investigator, HRA Pharma. JN: Principal Investigator, HRA Pharma.

OR02-3 13753 3.0000 A Clinical Effectiveness of Metyrapone Monotherapy in 195 Patients with Cushing's Syndrome 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 1:00:00 PM OR02 4719 11:30:00 AM Diagnosing and Treating Cortisol Excess and Deficiency Oral

Elena Valassi^*¹, Alicia Santos², Romana T. Netea-Maier³, Richard A. Feelders⁴, Thierry Brue⁵, John A. Wass⁶, Philippe Chanson⁷, Maria Yaneva⁸, Stylianos Tsagarakis⁹, Kathrin Zopf¹⁰, Olivier Chabre¹¹, Irina V Komerdus¹², Miklos Toth¹³, Holger Franz¹⁴, C J Strasburger¹⁵, Steven W.J. Lamberts⁴, P J Trainer¹⁶ and Susan M Webb¹⁷
¹IIB-Sant Pau and Department of Endocrinology/Medicine, Hospital Sant Pau, Universitat Autònoma de Barcelona (UAB), ISCIII; Barcelona, Spain, ²IIB-Sant Pau and Department of Endocrinology/Medicine, Hospital Sant Pau, Universitat Autònoma de Barcelona (UAB), 08193, Bellaterra, Cerdanyola del Vallès, and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBER-ER, Unidad 747), ISCIII, Barcelona, Spain, ³Radboud University Nijmegen Medical Center, Nijmegen, Netherlands, ⁴Erasmus Medical Center, Rotterdam, Netherlands, ⁵Centre de Recherche en Neurobiologie et Neurophysiologie de Marseille, France, ⁶University of Oxford, Churchill Hospital, Oxford, United Kingdom, ⁷Univ Paris-Sud, UMR-S693, Le Kremlin Bicêtre, France, Assistance Publique-Hôpitaux de Paris, Hôpital de Bicêtre, Service d’Endocrinologie et des Maladies de la Reproduction, Le Kremlin Bicêtre, Paris, France, Le Kremlin-Bicêtre, France, ⁸Medical University of Sofia, ⁹Evangelisnos Hospital, Athens, Greece, ¹⁰Division of Clinical Endocrinology, Department of Medicine CCM, Charité- Universitätsmedizin, Berlin, Germany, ¹¹Service d’Endocrinologie-Diabétologie-Nutrition, Grenoble Cedex, France, Grenoble, France, ¹²Moscow Regional Research Clinical Institute, Moscow, Russia, ¹³Semmelweis University, Budapest, Hungary, ¹⁴Lohmann and Birkner Health Care Consulting GmbH, Berlin, Germany, ¹⁵Division of Clinical Endocrinology, Department of Medicine CCM, Charité- Universitätsmedizin, Berlin, Germany, Berlin, Germany, ¹⁶Department of Endocrinology, Christie Hospital, Manchester, UK, ¹⁷IIB-Sant Pau and Department of Endocrinology/Medicine, Hospital Sant Pau, Universitat Autònoma de Barcelona (UAB) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBER-ER, Unidad 747), ISCIII; 08025 Barcelona, Spain

Background: Surgery is the treatment of choice for Cushing’s syndrome (CS) but preoperative medical therapy (PMT) may be used to lower circulating cortisol and improve patient’s condition. The most commonly used medications for CS include ketoconazole (KTZ), metyrapone (MTP) and cabergoline (CAB); mifepristone, mitotane, etoposide and aminogluthetimide may also be employed depending on etiology of CS and availability of drugs.

Objective: 1) To study the prevalence of PMT in CS patients throughout Europe; and 2) To evaluate any differences in surgical outcome between patients who received PMT vs. those who did not.

Methods/Design: In October 2013 ERCUSYN included 1023 patients (821 F, 202 M; mean age (+SD) 44.7±13.5 years) from 57 centers in 28 countries. It comprises 669 (65%) pituitary-dependent CS (PIT-CS), 249 (24%) adrenal-dependent CS (ADR-CS), and 105 (11%) from other etiologies, including ectopic (ECT-CS).

Results: Two hundred and twenty-eight CS patients of 930 (25%) with therapy data available took PMT vs. 634 who did not (68%). A subset of 65/930 (7%) was excluded from analysis because only medical treatment was given, but no subsequent surgery. More than half of CS patients who were medically treated before surgery were from the Netherlands, France and Spain. Patients with PIT-CS received PMT more frequently than the other etiologic groups (82% PIT-CS vs. 7% ADR-CS and 10% ECT-CS; p<0.01 for both comparisons). KTZ was the most commonly used medication, given to 171 of 228 patients (75%; alone or in combination) vs. MTP administered to 53 (23%), CAB to 18 (8%), and mifepristone to 10 (4%) patients. Mitotane and aminogluthetimide were taken by 2 and 1 patients, with PIT-CS. Median duration of PMT was 103 days (range:1-1155 days). Median cumulative dose of KTZ and MTP was 66 gr (range:1,6-1314gr) and 120gr, (range: 6-1838.5gr), respectively. Median cumulative dose of CAB was 0.06 gr (range: 0.015-8.6 gr). Of 471 PIT-CS patients in remission in the immediate postoperative period (within 2 weeks since surgery), 326 (69%) had not received any PMT vs. 144 (31%) who had been medically treated (p<0.01). Early hypoadrenalism was reported in significantly more patients without PMT (70%), as compared with those who had taken PMT (30%) (p<0.01). After a median follow-up of 975 days (range:181-8153 days), 319 PIT-CS were in remission, of whom 196 (61%) had not taken PMT vs. 123 (38%) who had been medically treated before surgery (p<0.01).

Conclusions:

- PMT may be associated with lower prevalence of postoperative hypoadrenalism in PT-CS patients; this may be due to decreased negative feedback, leading to recovery of pituitary-adrenal axis function.

- PMT appears to be associated with lower prevalence of long-term remission in PIT-CS patients, although this may be determined by a more severe initial clinical presentation.

Disclosure: TB: Clinical Researcher, Pfizer, Inc., Clinical Researcher, Novo Nordisk, Clinical Researcher, Novartis Pharmaceuticals, Clinical Researcher, Ipsen, Clinical Researcher, Serono, Clinical Researcher, Sandoz. CJS: Advisory Group Member, Chiasma. PJT: Principal Investigator, HRA Pharma. Nothing to Disclose: EV, AS, RTN, RAF, JAW, PC, MY, ST, KZ, OC, IVK, MT, HF, SWJL, SMW

OR02-4 16669 4.0000 A Presurgical Medical Treatment in Patients with Cushing's Syndrome. Results from the European Registry on Cushing's Syndrome (ERCUSYN) 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 1:00:00 PM OR02 4719 11:30:00 AM Diagnosing and Treating Cortisol Excess and Deficiency Oral

Ashwini Mallappa^*¹, Lori-Ann Daley², Ninet Sinaii², Carol Van Ryzin², Hiep Huatan³, Dena Digweed³, David Eckland³, Martin J Whitaker³, Lynnette K. Nieman⁴, Richard J Ross⁵ and Deborah P. Merke²
¹National Institutes of Health Clinical Center, Bethesda, MD, ²National Institutes of Health, Bethesda, MD, ³Diurnal Limited, ⁴NIH, Bethesda, MD, ⁵Univ of Sheffield, Sheffield, United Kingdom

Background: Classic congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is characterized by cortisol and aldosterone deficiency and androgen excess. Current conventional glucocorticoid therapy is suboptimal as it cannot replace the normal cortisol circadian rhythm and inadequate or inappropriate suppression of adrenal androgens are common. Here we present preliminary results of a phase 2 study of Chronocort®, a modified release hydrocortisone capsule formulation.

Methods: The study objectives were: (A) characterize pharmacokinetics and (B) examine disease control following 6 months dose titration. Serial profiling was obtained at baseline (conventional glucocorticoid) and every 2 months. Twice daily Chronocort® was initiated: 20mg at 2300h, 10mg at 0700h. Dose titration was based on clinical status and optimal hormonal ranges (17OHP 300-1200 ng/dL, normal androstenedione (males: 40-150, females: 30-200 ng/dL), with androstenedione prioritized. Chronocort® cortisol pharmacokinetic profile was the primary endpoint. Secondary endpoints included biomarkers of disease control.

Results: 16 adults (8 females; age 29 ±13 years) with classic CAH (12 salt-wasting, 4 simple virilizing) participated. Conventional therapy varied (5 dexamethasone, 7 prednisone, 4 hydrocortisone). Chronocort® cortisol pharmacokinetic profile approximated physiological cortisol secretion. 10 patients required Chronocort® dose adjustments (decrease in 8, increase in 2; mean hydrocortisone equivalent dose conventional vs. 6 months: 16.1 ± 6.4 vs. 14.7 ± 6.4mg/m²).

Serial androstenedione levels were in the normal range in 8 (50%) of patients on conventional therapy compared to 12 (75%) on Chronocort® at 6 months The majority of patients on Chronocort® achieved 17OHP levels within the normal range, rather than within the mildly elevated range currently used for management.

At 6 months, Chronocort® resulted in lower 24-hr (p=0.02), morning (0700-1500; p=0.008), and afternoon (1500-2300; p=0.03) area-under-the-curve androstenedione compared to conventional therapy.

No serious adverse events occurred. Common adverse events were headache, fatigue, early awakening, and anemia. 3 patients had unexpected carpal tunnel syndrome which resolved with wrist splints.

Conclusions: Chronocort®, a novel modified release hydrocortisone capsule formulation, approximates physiological cortisol secretion, and improves biochemical control of CAH. Further analyses are underway.

Disclosure: HH: Employee, Diurnal. DD: Employee, Diurnal. DE: Employee, Diurnal. MJW: Management Position, Diurnal. RJR: Other activities, please specify:, Asterion Ltd, Director, Diurnal Ltd. DPM: Investigator, Diurnal. Nothing to Disclose: AM, LAD, NS, CV, LKN

OR02-5 14644 5.0000 A A Phase 2 Study of Chronocort®, a Modified Release Formulation of Hydrocortisone, in the Treatment of Adults with Classic Congenital Adrenal Hyperplasia 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 1:00:00 PM OR02 4719 11:30:00 AM Diagnosing and Treating Cortisol Excess and Deficiency Oral

Chiara Simeoli^*¹, Teresa Mannarino¹, Maria Cristina De Martino¹, Alessia Cozzolino¹, Davide Iacuaniello¹, Monica De Leo¹, Renata Simona Auriemma¹, Carolina Di Somma¹, Annamaria Colao¹ and Rosario Pivonello²
¹Università Federico II, Naples, Italy, ²Federico II University, Naples, Italy

Life-long glucocorticoid (GC) treatment is needed in patients with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency in order to replace cortisol deficiency and to control ACTH and consequently androgen levels. Therefore, patients with CAH tend to have an increased risk of metabolic syndrome (MS), probably due to cortisol overexposure, caused by multiple daily doses of conventional GCs, unable to mimic cortisol circadian rhythm. The current study aimed at investigating the impact of the switch from twice or thrice daily conventional GCs to once daily dual release hydrocortisone formulation (DR-HC) on metabolic and hormonal profile in a cohort of patients with CAH. Twenty-three patients (15 F, 8 M, 19-29 years) with CAH, chronically treated with hydrocortisone (15-40 mg/day) or prednisone (6.25-12.5 mg/day) and switched to DR-HC (10-40 mg/day) entered the study. Metabolic and hormonal parameters were evaluated before and after short (3 months) and long-term (6 months) DR-HC treatment in the entire group of 23 and in a subgroup of 15 patients, respectively. The insulin resistance was evaluated by calculating the homeostasis model assessment of the insulin resistance index (HOMA-IR) whereas the MS was estimated in line with NCEP ATP III definition. At 3-month-follow-up, fasting plasma glucose (p=0.004) and HDL-cholesterol (p=0.027) levels significantly improved. At 6 month-follow up, fasting plasma glucose (p=0.004) significantly improved and a trend to a significant improvement was registered for fasting serum insulin (p=0.074). Moreover, HOMA-IR also significantly improved (p=0.041); a clear diagnosis of MS was performed in one patient at the baseline, but this was not confirmed after 6 months of DR-HC treatment. No significant change in morning plasma ACTH, 17-OH progesterone and androgens levels and no clinical worsening of symptoms and signs related to hyperandrogenism were observed, but a significant increase in morning serum cortisol levels was registered both after short and long-term follow-up. In conclusion, the switch from conventional GCs to DR-HC significantly improves metabolic parameters and insulin resistance, maintaining an optimal hormone control in patients with CAH due to 21-hydroxylase deficiency.

Disclosure: CS: Consultant, Viropharma. AC: Speaker, Novartis Pharmaceuticals. MD: Consultant, Viropharma. AC: Speaker, Novartis Pharmaceuticals, Consultant, Italfarmaco, Consultant, Pfizer, Inc., Consultant, Ipsen, Consultant, Novartis Pharmaceuticals, Study Investigator, Novo Nordisk, Study Investigator, Merck & Co., Study Investigator, Ferring Pharmaceuticals, Principal Investigator, Lilly USA, LLC, Principal Investigator, Pfizer, Inc., Principal Investigator, Ipsen, Principal Investigator, Novartis Pharmaceuticals, Speaker, Ipsen, Speaker, Pfizer, Inc.. RP: Coinvestigator, Viropharma, Coinvestigator, IBSA, Consultant, Novartis Pharmaceuticals, Consultant, Ipsen, Consultant, Pfizer, Inc., Consultant, Viropharma, Consultant, Ferring Pharmaceuticals, Consultant, Italfarmaco, Speaker, Novartis Pharmaceuticals, Speaker, Novartis Pharmaceuticals, Coinvestigator, Novartis Pharmaceuticals, Principal Investigator, Novartis Pharmaceuticals. Nothing to Disclose: TM, MCD, DI, RSA, CD

OR02-6 15038 6.0000 A The Treatment with Glucocorticoids in Congenital Adrenal Hyperplasia: Short and Long-Term Effects of the Switch from Conventional Glucocorticoids to "Dual Release" Hydrocortisone on Metabolic and Hormonal Profile 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 1:00:00 PM OR02 4719 11:30:00 AM Diagnosing and Treating Cortisol Excess and Deficiency Oral

Stephanie Sisley^*¹, Randy Seeley² and Darleen Sandoval³
¹Baylor College of Medicine, Houston, TX, ²Univ of Cincinnati, Cincinnati, OH, ³University of Cincinnati, Cincinnati, OH

Low vitamin D levels have been correlated to both obesity and the development of type 2 diabetes (T2DM) (1, 2), although no causative mechanisms have been established. Vitamin D receptors are present in the hypothalamus (3), a region important in both weight and glucose regulation. The role of these receptors, though, is unknown. We tested the hypothesis that vitamin D can act in the brain to improve glucose tolerance and weight gain in diet-induced obese animals. 1,25-dihydroxyvitamin D₃, the active form of vitamin D, improved glucose tolerance in DIO rats when given into the third cerebral ventricle (i3vt) 1 hour prior to an intraperitoneal glucose tolerance test. During a hyperinsulinemic euglycemic clamp, i3vt 1,25-dihydroxyvitamin D₃ acutely improved whole body insulin sensitivity, as demonstrated by an 8-fold higher glucose infusion rate in vitamin D treated animals compared to vehicle treated animals (13.19±0.96 vs. 1.55±0.62 mg/kg/min; P < 0.0001; n≥4 per group). I3vt vitamin D3 suppressed hepatic glucose production to 50% of vehicle treated animals (7.36±1.98 vs. 16.40±2.82 mg/kg/min; P = 0.03). This correlated with a 9-fold decrease in the expression of phosphoenolpyruvate carboxykinase (PEPCK), a key enzyme in hepatic gluconeogenesis, in vitamin D treated animals (12.67±6.86 vs. 118.3±66.61 ratio of PEPCK:L32; P = 0.009). No changes occurred between groups in glucose clearance (13.59±0.97 vs. 9.96±1.67 mL/kg/min; P = 0.08). Although i3vt 1,25-dihydroxyvitamin D₃ did not change food intake when given acutely, chronic administration dramatically reduced food intake (179.71±11.27 vs. 484.29±28.84 g/28 days; P < 0.0001; n≥3 per group) and body weight (509.87±9.43 vs. 670.21±38.05 g; P = 0.014) of DIO animals on a high fat diet without changes in energy expenditure. These results demonstrate the ability of vitamin D to act within the brain to dramatically alter glucose homeostasis and weight maintenance in DIO rats and suggest that vitamin D may play a large role in the onset of both obesity and T2DM.

Results expressed as mean±SEM.

Nothing to Disclose: SS, RS, DS

OR13-1 16486 1.0000 A CNS Vitamin D Improves Glucose Tolerance, Hepatic Insulin Sensitivity, and Reverses Diet-Induced Obesity 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 21st 1:00:00 PM OR13 4721 11:30:00 AM Vitamin D Metabolism & Action Oral

Ken Batai^*¹, Adam B Murphy², Ebony Shah³ and Rick A Kittles¹
¹University of Illinois at Chicago, Chicago, IL, ²Northwestern University, ³University of Illinois at Chicago

Vitamin D deficiency is more common among African Americans (AAs) than European Americans (EAs). Epidemiologic evidence links to vitamin D status to many health outcomes, and differences in serum vitamin D [25(OH)D] concentration among racial/ethnic groups are suspected to be one of the sources of health disparities. Two genome wide association studies (GWAS) meta-analyses in European populations identified vitamin D pathway gene Single Nucleotide Polymorphisms (SNPs) associated with serum vitamin D 25(OH)D levels, but a few of these SNPs have been replicated in AAs. Here, we investigated the associations of 39 SNPs in vitamin D pathway genes, including 19 GWAS identified SNPs, with serum 25(OH)D concentrations in 652 AAs and 405 EAs. Linear and logistic regression analyses were performed adjusting for environmental and biological factors which confound associations. The pattern of SNP associations in AAs contrasted from EAs. In AAs, six GWAS identified SNPs in GC, CYP2R1, and DHCR7/NADSYN1 were replicated, while nine GWAS SNPs in GC and CYP2R1 were replicated in EAs. The CYP2R1 SNP, rs12794714, revealed the strongest signal of association in AAs (P=0.01). In EAs, however, another CYP2R1 SNP, rs1993116, was the most strongly associated (P=0.006). Additionally, although not significant after correcting for multiple testing, another GC SNPs, rs115316390, which is unlined to GWAS identified SNPs, was also associated in AAs (P=0.03), but not in EAs. Our models, which take into account genetic variants and environmental variables, account for 20% and 28% of the variance in serum vitamin D levels in AAs and EAs, respectively. Increased attention is focused on the role of vitamin D in many health conditions, and many of them are more common in AAs than other racial groups. Our findings provide insights on the biological and environmental modifiers of serum vitamin D3 and will help guide future studies on the role of vitamin D in health disparities.

Nothing to Disclose: KB, ABM, ES, RAK

OR13-2 16451 2.0000 A Common Vitamin D Pathway Gene Variants Reveal Contrasting Effects on Serum Vitamin D Levels in African Americans and European Americans 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 21st 1:00:00 PM OR13 4721 11:30:00 AM Vitamin D Metabolism & Action Oral

Julie Støy^*¹, Ulla Kampmann¹, Lars Rejnmark², Jørgen Rungby³, Ivan Brandslund⁴, Cramer Christensen⁴, Torben Hansen⁵, Oluf Pedersen⁵ and Niels Møller¹
¹Aarhus University Hospital, Aarhus C, Denmark, ²Aarhus University Hospital, Aarhus C, ³Rigshospitalet, Copenhagen, Denmark, ⁴Vejle Hospital, Vejle, Denmark, ⁵University of Copenhagen, Copenhagen, Denmark

The genetics of osteoporosis have been scrutinized in large-scale genome-wide association studies (GWAS) with a large number of common genetic variants identified, each of which exerts subtle effects on disease susceptibility. In a recent whole exome sequencing project of 2000 Danish individuals, a rare amino acid polymorphism in CD300LG was found to associate with low fasting HDL-cholesterol levels and high triglyceride levels. The rs72836561 CT polymorphism in CD300LG has not been detected in GWAS of osteoporosis, most likely due to its relatively low frequency, but a negative correlation between BMD and HDL-cholesterol has been suggested. Mice studies have shown changes in bone structure and quality in CD300LG knockout mice. We therefore performed a bone-related phenotype characterization of human carriers of the CD300LG risk-allele including an evaluation of BMD and a biochemical profile relevant to bone metabolism.

Methods: 20 healthy males with the CD300LG rs72836561 CT genotype were matched on age and BMI with 20 healthy males with the CC genotype. The 40 study subjects were examined by DEXA scan of the hip, lumbar spine, distal forearm, and whole body. The biochemical profile included markers of bone formation, bone reabsorption, vitamin D turn-over, and calcium homeostasis.

Results: CT-carriers had a tendency to higher whole body BMD (1.19 g/cm² (95 % confidence interval (CI), 1.15-1.24) versus 1.16 (1.11-1.20); p=0.24) and a higher BMD in the hip (0.99 g/cm² (95 % CI, 0.93-1.05) versus 0.95 (0.91-0.99); p=0.21) compared to the CC-carriers. The differences in BMD were statistically significant or borderline significant after adjustment for vitamin D level (p=0.064 and p=0.045, respectively). The same trend was observed for BMD in the lumbar spine and in the forearm (adjusted p-values of 0.22 and 0.06, respectively). Vitamin D levels were higher in the CT-carriers 59.4 nmol/l (95 % CI, 51.7-67.1) compared to the CC-carriers (47.5 nmol/l (39.0-56.1)); p=0.037. Exclusion of two study subjects with daily intake of a vitamin D and calcium supplement, preserved a significant difference in BMD of the hip (p=0.023). A trend towards a higher level of the bone formation markers, osteocalcin and bone-specific alkaline phosphatase, among CC-carriers was observed (p=0.09 and p=0.07, respectively).

Conclusion: The CD300LG rs72836561 CT-genotype may affect vitamin D and bone metabolism in healthy male carriers in terms of higher levels of Vitamin D and higher hip BMD. This points to CD300LG as a potential novel gene in the regulation of bone and vitamin D metabolism. The underlying mechanisms are currently unknown and future studies are warranted to test the possible association in a larger study cohort.

Nothing to Disclose: JS, UK, LR, JR, IB, CC, TH, OP, NM

OR13-3 13627 3.0000 A Hip Bone Mineral Density and Vitamin D Levels Are Increased in Healthy Male Carriers of Arg82Cys in CD300LG and Points to CD300LG As a Novel Gene in Bone and Vitamin D Metabolism 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 21st 1:00:00 PM OR13 4721 11:30:00 AM Vitamin D Metabolism & Action Oral

Klaus Badenhoop^*¹, Dimitra Bogdanou², tje Lin Chung³, Firouzeh Shoghi⁴, Maria Sandler⁴, Claudia Brehm⁴, Sabine Huenecke⁴, Eva Herrmann³, Ulrike Koehl⁵ and Marissa Penna-Martinez²
¹Goethe-University Hospital, Frankfurt, Germany, ²University Hospital, Goethe-University Frankfurt am Main, Germany, ³Goethe University Frankfurt am Main, Germany, ⁴University Hospital Frankfurt am Main, Germany, ⁵Institute for Molecular and Therapeutics (MHH), Hannover, Germany

Introduction: Autoimmune Addison’s disease (AAD) is mediated by adrenal infiltrating T-lymphocytes. The initiation and propagation of AAD is determined by both genetic and environmental factors. One candidate environmental risk factor is vitamin D deficiency. Based on the immunemodulatory effects of vitamin D on immune cells, the aim of this study was to investigate the influence of three months vitamin D therapy on T-cell subpopulations in patients with AAD.Methods: Thirteen patients (7 females, 6 males) with AAD were included in this randomised, placebo controlled, double blind cross-over trial (registered under EudraCT 201002267734). The patients received three months 4000 IU/d Vigantol oil followed by three months placebo oil (AB arm) or placebo/verum (BA arm). The 25(OH)D₃ concentration and T-cell subpopulations were measured at baseline (V1), after three months (V3) and six months (V5). Hereby, EDTA-blood samples were stained by fluorochrome conjugated antibodies and flow cytometric analysis was performed to determine the following T cell phenotypes: T helper cells (=Th;CD3⁺CD4⁺,CD4⁺HLA-DR⁺,CD4⁺HLA-DR⁺[late active]_,CD4⁺CD25⁺CD127^dim/neg) and T cytotoxic cells (=Tc; CD3⁺CD8⁺, CD8⁺HLA-DR⁺,CD8⁺HLA-DR⁺[late active]). In addition 25(OH)D₃ plasma concentration were measured by radioimmunoassay. The influence of the vitamin D therapy was evaluated on the basis on within-subject differences between verum and placebo with regard to the investigated outcome variables. Median differences are given with the Hodges-Lehmann estimate and 95 % confidence interval (CI).Results: Preliminary testing did not detect significant differences in carryover effects between the AB/BA arms. After three months of therapy with Vigantol, the 25(OH)D₃ concentration was significantly elevated (median 20 ng/mL, CI 13-24; p < 10^-3). An absolute percentage reduction of peripheral blood derived CD8⁺HLA-DR⁺ (median 0.75 %, CI 0.1-1.45) and CD8+HLA-DR⁺[late active] (median 4.05 %, CI 0.4-7.2) as well as CD4⁺HLA-DR⁺[late active] (median 1.6 %, CI 0.3-2.6) p ≤ 0.03, respectively was observed. In contrast, changes in the percentages of CD3⁺CD4⁺, CD4⁺HLA-DR⁺ and CD4⁺CD25⁺CD127^dim/neg cell populations were not significant.Conclusion: Vitamin D therapy with 4000 IU/d Vigantol in AAD patients led to an elevation of 25(OH)D₃ concentration. This was accompanied by a reduced autoimmune activity probably due to the inhibition of particular T cell subpopulations. It is important to note that the vitamin D therapy only had an influence on Tc cells and Th cells that expressed the activation marker HLA-DR on their surface but not on regulatory T cells (CD4⁺CD25⁺CD127^dim/neg). A follow-up study with larger numbers is needed to confirm these findings and to extend functional analyses.

Nothing to Disclose: KB, DB, TLC, FS, MS, CB, SH, EH, UK, MP

OR13-4 14771 4.0000 A The Effect of Vitamin D Therapy on Peripheral HLA-DR Expressing T Cell Subpopulations in Patients with Autoimmune Addison's Disease: A Randomized Controlled Trial 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 21st 1:00:00 PM OR13 4721 11:30:00 AM Vitamin D Metabolism & Action Oral

Deep Dutta^*¹, Samim Ali Mondal², Indira Maisnam², Satinath Mukhopadhyay³ and Subhankar Chowdhury²
¹Institute of Post Graduate Medical Education & Research (IPGMER) and Seth Sukhlal Karnani Memorial (SSKM) Hospital, Calcutta, West Bengal, India, ²Institute of Post Graduate Medical Education & Research (IPGMER) and Seth Sukhlal Karnani Memorial (SSKM) Hospital, Calcutta, India, ³Institute of Postgraduate Medical Education & Research (IPGMER) and Seth Sukhlal Karnani Memorial (SSKM) Hospital, Calcutta, India

Objective:

Indian individuals with prediabetes (IPD) have one of the highest rates of progression (≈18% per year) to type2 diabetes (T2DM). Since vitamin-D deficiency has been linked to prediabetes, we aimed to evaluate role of vitamin-D supplementation on progression to T2DM and/ or reversal to normoglycemia in IPD.

Methods:

IPD with persistent impaired fasting glucose (IFG) or impaired glucose tolerance (IGT) over 2 oral glucose tolerance test (OGTT), without any severe co-morbid state or drug intake, having serum 25-OH-vitamin-D (25OHD) ≤30ng/ml were randomized into Group-A [received vitamin-D (cholecalciferol 60,000 U once weekly for 8 weeks then monthly) and calcium (1250mg of calcium carbonate/day equivalent to elemental calcium 500mg) supplementation] and Group-B (received calcium only). IPD with serum 25OHD>30ng/ml were also followed with calcium supplementation (Group-C). All received therapeutic lifestyle modification. OGTT, insulin, 25OHD, lipids, interleukin-6 (IL6), tumor necrosis factor-α (TNF-α) and hsCRP were done at baseline and annually. Data from IPD with at least 1-year follow up were analyzed. The trial is registered with clinical trial registry of India at ctri.nic.in (CTRI/2011/091/000192).

Results:

1946 individuals were initially screened, of which 498 underwent OGTT-1 and 301 underwent OGTT-2. 125 out of 170 finally included IPD (73.52%) had 25OHD ≤30ng/ml. Mean follow-up in Group-A (n=55), B (n=49) and C (n=32) was 28.2±8.83, 29.15±7.69 and 27.51±7.8 months respectively. 25OHD had significant correlation with HOMA2-IR (r=-0.42; P=0.004), TNFα (r=-0.31; P=0.03) and hsCRP (r=-0.31; P=0.03), after adjusting for BMI.

At the end of study, Group-A IPD had significantly higher serum 25OHD (p<0.001), lower FBG (p=0.023), 2hPGBG (p<0.001), TNFα (p=0.002) and IL-6 (p=0.0005) as compared to Group-B and C. Group-A IPD as compared to Group-B had significantly lower progression to diabetes (6/55 vs. 13/49; P=0.04), and higher reversal to normoglycemia (23/55 vs. 10/49; P=0.02). Cox regression revealed baseline 25OHD [Exp(B)=0.921; P=0.049] and 2hPGBG [Exp(B)=1.033; P=0.014] independently predicted progression to diabetes. Hypertension [Exp(B)=0.416; P=0.043] and baseline 25OHD [Exp(B)=1.054; P=0.046] predicted reversal to normoglycemia.

Conclusion:

Vitamin-D supplementation in IPD decreased the rate of progression to diabetes and increased reversal to normoglycemia, presumably by an improvement in IR and systemic inflammation.

Nothing to Disclose: DD, SAM, IM, SM, SC

OR13-5 11046 5.0000 A Vitamin-D Supplementation in Prediabetes Reduced Progression to type2 Diabetes through Decreased Insulin Resistance and Systemic Inflammation: An Open Label Randomized Prospective Study from Eastern India 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 21st 1:00:00 PM OR13 4721 11:30:00 AM Vitamin D Metabolism & Action Oral

Francesco Tona¹, Elena Osto¹, Giulia Famoso¹, Sara Tellatin¹, Sabino Iliceto¹, Nadia Sorgato¹, Mariarosa Pelizzo¹, Andrea Rebellato² and Francesco Fallo^*¹
¹Univ of Padova, Italy, ²Padova University Hospital, Padova, Italy

Primary hyperparathyroidism (PHPT) is associated with high rate of cardiovascular events. We previously showed (Circulation, 2012) that high PTH levels correlate independently with coronary microvascular dysfunction as assessed by coronary flow reserve (CFR) measurement, suggesting a crucial role of PTH in the increased cardiovascular risk of PHPT. Hypovitaminosis D is considered an independent predictor of cardiovascular morbidity and mortality in the general population, and is commonly seen in PHPT. The aim of our study was to dissect in PHPT patients the potential impact of vitamin D deficiency, as determinant of coronary disease, from that of PTH. One-hundred eighteen consecutive patients with PHPT due to solitary parathyroid adenoma (98 female, aged 59±11 years) and without clinical evidence of ischemic heart disease, were studied. PHPT patients were subdivided into two groups accordingto the absence (n=77) or the presence (n=41) of vitamin D deficiency, as defined by serum 25(OH)D levels <20 ng/ml (50 nmol/L). Coronary flow velocity in the left anterior descending coronary artery was detected by transthoracic Doppler echocardiography at rest and during adenosine infusion. CFR was obtained as the ratio of hyperemic diastolic flow velocity (DFV) to resting DFV. The two PHPT groups had comparable age, sex distribution, prevalence of risk factors (including hypertension, diabetes and dyslipidemia) and time from diagnosis. No differences between the two groups were found in serum PTH, calcium as well in CFR values. PTH was inversely related to CFR both in all PHPT patients and in each PHPT group separately (P=0.03). A direct linear correlation between vitamin D and CFR was found in the vitamin D-sufficient PHPT patients (R=0.293, P=0.01), but not in the vitamin D–deficient PHPT group. At multivariable linear regression analysis, vitamin D was an independent determinant of CFR in the vitamin D-sufficient PHPT group (Beta=0.254, P=0.01), regardless PTH levels. Conclusions: In the vitamin D-sufficient PHPT patients, relative lowering of vitamin D concentration may have a negative impact on coronary microvascular function, representing an additional cardiovascular risk factor. The potential impact of vitamin D on CFR is lost in the vitamin D-deficient PHPT patients, due to the distribution within a very narrowed low-level range, and the deleterious effect of high PTH becomes predominant.

Nothing to Disclose: FT, EO, GF, ST, SI, NS, MP, AR, FF

OR13-6 12007 6.0000 A Vitamin D Deficiency and Coronary Microvascular Function in Primary Hyperparathyroidism 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 21st 1:00:00 PM OR13 4721 11:30:00 AM Vitamin D Metabolism & Action Oral

Mitsuhide Naruse^*¹, Mika Tsuiki¹, Kanako Nakao¹, Hironobu Umakoshi¹, Yuichi Fujii², Kohei Kamemura³, Tatsuya Kai⁴, Ryuichi Sakamoto⁵, Tetsuya Tagami⁶, Atsushi Ogo⁷, Yusuke Hirokawa⁸, Yuichi Matsuda⁹ and Tomikazu Fukuoka¹⁰
¹National Hospital Organization Kyoto Medical Center, Kyoto, Japan, ²Hiroshima General Hospital of West Japan Railway Company, Hiroshima, Japan, ³Akashi Medical Center, Akashi, Japan, ⁴Saiseikai Tondabayashi Hospital, Japan, ⁵National Hospital Organization Kyushu Medical Center, Fukuoka, Japan, ⁶NHO Kyoto Medical Center, Kyoto, Japan, ⁷Kyushu Medical Center, Fukuoka, Japan, ⁸Kyoto Medical Center, Kyoto, Japan, ⁹Sanda City Hospital, Sanda, Japan, ¹⁰Matsuyama Red Cross Hospital, Matsuyama, Japan

Adrenal venous sampling (AVS) is recommended as a gold-standard for subtype testing in PA, if adrenal surgery is considered. Besides that AVS requires expertise staffs in the specialized centers, unstandardized method and criteria for subtype classification are another drawback. Aim of the study was to investigate the effects of ACTH loading and different criteria on the subtype classification by AVS. The study was conducted as the multi-center collaborative study in west Japan (WAVES-J). Methods: Seven hospitals participated into the study. AVS data from 288 patients were analyzed. ACTH loading was performed in 270 patients. Catheterization was judged to be successful if selectivity index was > 2 before and > 5 after ACTH loading. Four different criteria were used for subtype classification: 1) Lateralized ratio (LR) >2, 2) LR>2.6, 3) LR>4, and 4) Contralateral ratio (CR) <1.0 before ACTH loading and 1) LR>2.6, 2) LR>4, 3) PAC >14000pg/ml, and 4) CR <1.0 after ACTH loading. Success rate before and after ACTH loading was 60.0% and 85.2%, resulting in the overall success rate of 85.4%. Percentage of unilateral subtype before ACTH loading was 66.5% with LR>2 followed by 60.7% with LR>2.6, 45.1% with LR>4 and 42.8% with CR<1, respectively. Percentage of unilateral subtype after ACTH loading was 41.3% with PAC>14000pg/ml followed by 37.8% with LR>2.6, 30.7% with CR<1, and 29.6% with LR>4, respectively. LR before ACTH loading was increased in 12.7% and unchanged in 34.4%, but decreased in 52.9% after ACTH loading. Subtype classification before ACTH loading (LR>2.0) was unchanged in 55.4% even after ACTH loading (LR>4). By contrast, subtype classification was changed in 44.6% after ACTH loading: unilateral to bilateral in 41.4%, bilateral to unilateral in 1.9%, and unilateral to contra-unilateral in 1.3%. The present study clearly demonstrated that both ACTH loading and type of criteria have profound effects on the subtype classification by AVS. Since AVS is critical in determining treatment of choice, standardization of the methods and decision criteria of AVS is mandatory.

Nothing to Disclose: MN, MT, KN, HU, YF, KK, TK, RS, TT, AO, YH, YM, TF

OR06-1 14353 1.0000 A Effects of ACTH Loading and Different Criteria on the Subtype Classification By Adrenal Venous Sampling in Primary Aldosteronism 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Cardiovascular Endocrinology Saturday, June 21st 1:00:00 PM OR06 4725 11:30:00 AM Hyperaldosteronism and Salt-Sensitive Hypertension Oral

Celso E. Gomez-Sanchez^*¹, Xin Qi², Elise P Gomez-Sanchez³ and Max V Wisgerhof II⁴
¹Univ of Mississippi Med Ctr, Madison, MS, ²University of Mississippi Medical Center, Jackson, MS, ³University of Missippi Medical Cener, Jackson, MS, ⁴Henry Ford Hosp, Detroit, MI

Immunohistochemical and immunofluorescence of CYP11B2, CYP11B1, KCNJ5 and 17Alpha-hydroxylase of the Adrenals in Familial Hyperaldosteronism type 3

Geller et al described family with a syndrome of severe hypertension, hypokalemia, low plasma renin activity and very high levels of aldosterone and 18-oxocortisol which he classified as familial hyperaldosteronism type 3 (FHA3)(1). The hypertension and hypokalemia was resistant to high doses of spironolactone and bilateral adrenalectomy was required for control of the hyperaldosteronism, hypokalemia and hypertension. Genetic analysis determined that this family had a germline mutation in the selectivity filter of the inward-rectifying potassium channel KCNJ5 (T158A).

We performed immunohistochemical and immunofluorescent analysis of the adrenals from 2 of the original FHA3 patients using specific monoclonal antibodies against the CYP11B1 and CYP11B2 enzymes and polyclonal antibodies against the KCNJ5 and 17a-hydroxylase. H & E staining demonstrated marked hyperplasia with distortion of the normal architecture of the adrenal glands with areas of cells resembling zona glomerulosa (ZG) embedded in areas with cells with zona fasciculata (ZF) characteristics. Normally CYP11B2 and KCNJ5 are found in ZG cells, CYP11B1 and 17a-hydroxylase only in ZF. Strongly CYP11B2-immunoreactive cells were found throughout the ZG and ZF. Some of the cells were compact as expected of normal ZG cells, others looked more like lipid-ladened ZF cells.

Triple immunofluorescence confirmed wide distribution of CYP11B2-immunoreactive cells throughout the adrenal gland intercalated with CYP11B1 expressing cells. Some cells co-expressed both the CYP11B2 and CYP11B1 enzymes. CYP11B1 expressing cells co-expressed 17a-hydroxylase as in normal adrenals, but there were also cells that co-expressed CYP11B2 and 17a-hydroxylase, a distinctly abnormal situation. KCNJ5 immunoreactivity was co-expressed with the CYP11B2 enzyme throughout the adrenal, with a few cells co-expressing CYP11B1 and KCNJ5.

In conclusion, adrenals from patients with a T158A germline mutation of the KCNJ5 gene exhibit gross hyperplasia with distorted architecture of the cortex. Normal segregation of enzyme expression is also lost and some cells co-expressed enzymes of ZF cells, CYP11B2 and 17a-hydroxylase, with CYP11B2 normally found only in ZG cells. The extremely high excretion rates of 18-oxocortisol in FHA3 occurs both by synthesis in cells co-expressing CYP11B2 and the 17a-hydroxylase and by commingling of normal ZF cells that co-express CYP11B1 and 17a-hydroxylase and provide substrate to ZG-type cells that express CYP11B2. It is unclear how a mutation of the KCNJ5 gene would alter not only its pattern of expression, but that of the steroidogenic enzymes.

Nothing to Disclose: CEG, XQ, EPG, MVW II

OR06-2 15427 2.0000 A Immunohistochemical and Immunofluorescence of CYP11B2, CYP11B1, KCNJ5 and 17alpha-Hydroxylase of the Adrenals in Familial Hyperaldosteronism Type 3 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Cardiovascular Endocrinology Saturday, June 21st 1:00:00 PM OR06 4725 11:30:00 AM Hyperaldosteronism and Salt-Sensitive Hypertension Oral

Rene Baudrand^*¹, Nidhi Gupta², Amanda Elizabeth Garza², Anand Vaidya³, Jonathan S Williams⁴, Gail K. Adler⁵, Gordon H Williams⁵ and Luminita H Pojoga⁵
¹Pontificia Universidad Catolica de Chile, Santiago, Chile, ²Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, ³Brigham and Women’s Hospital/Harvard Medical School, Boston, MA, ⁴Brigham & Women's Hospital, Harvard Medical School, Boston, MA, ⁵Brigham and Women's Hospital, Harvard Medical School, Boston, MA

Background/Aim: Excess activation of the aldosterone/mineralocorticoid receptor (MR) pathway is associated with impaired glucose metabolism, insulin resistance (IR) and dyslipidemia. Caveolin-1 (cav-1) is a plasma membrane protein involved in glucose and lipid homeostasis and has been proposed as a modulator of MR signaling. Herein - in a translational approach - we conducted 2 studies to investigate the interplay between cav-1 and aldosterone signaling in modulating IR and dyslipidemia. In study 1 we evaluated metabolic dysfunction and MR-mediated pathways in cav-1 null mice. Next in study 2 we assessed the relationship between a prevalent cav-1 variant and aldosterone levels in modulating cardiometabolic outcomes.

Methods/Results: Cav-1 KO mice exhibited higher HOMA, cholesterol and resistin, and lower HDL/LDL ratio (all p <0.001 vs WT). Moreover, cav-1 KO mice displayed hypertriglyceridemia, as well as increased mRNA expression of resistin, retinol-binding protein 4 (RBP4), NADPH oxidase 4 (NOX4) and aldose reductase (AldoR) in liver and/or fat tissues. Further, MR blockade by eplerenone in the cav-1 KO significantly decreased glycemia (68.6±2.1 vs. 98.9±7.1 mg/dL, p <0.01), total cholesterol (p <0.05), plasma resistin (p <0.05) and transcript levels for RBP4, NOX4 and AldoR, but had no effect on insulin or triglyceride levels. Next, available cav-1 gene eQTL data revealed that the minor allele of rs926198 was associated with lower cav-1 expression. Then, we analyzed 556 Caucasians for the association between the cav-1 polymorphism and cardiometabolic outcomes using a dominant model. Fifty-eight percent of genotyped subjects were minor allele carriers of rs926198 and displayed higher odds for IR (HOMA-IR > 2.5, OR 2.26 [1.40 – 3.64]) and low HDL status (<35 mg/dL in men, <39 mg/dL in women, OR 1.54 [1.01 – 3.37]) despite similar baseline characteristics. Interestingly, aldosterone levels positively correlated with HOMA-IR, total cholesterol and resistin and negatively with HDL-C only in minor allele carriers, thus resembling the cav-1 null phenotype.

Conclusion: Our findings in mice and humans suggest that cav-1 may modulate the effect of aldosterone on IR, dyslipidemia and circulating resistin. Further, we show that blocking MR signaling improved glucose, cholesterol and resistin dysregulation in cav-1 deficient states, consistent with a role for MR overactivation in these metabolic pathways. In contrast, hyperinsulinemia and hypertriglyceridemia in cav-1 null mice were not corrected by eplerenone, suggesting that these abnormalities are likely mediated by MR-independent mechanisms.

Nothing to Disclose: RB, NG, AEG, AV, JSW, GKA, GHW, LHP

OR06-3 16030 3.0000 A Caveolin-1 Modulates Aldosterone-Mediated Pathways of Glucose Homeostasis and Lipid Profile 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Cardiovascular Endocrinology Saturday, June 21st 1:00:00 PM OR06 4725 11:30:00 AM Hyperaldosteronism and Salt-Sensitive Hypertension Oral

Amanda Elizabeth Garza^*¹, Chevon M Rariy², Jonathan S Williams³, Rene Baudrand⁴, Jose R Romero¹, Gail K. Adler¹, Luminita H Pojoga¹ and Gordon H Williams¹
¹Brigham and Women's Hospital, Harvard Medical School, Boston, MA, ²Brigham and Women's Hospital, Boston, MA, ³Brigham & Women's Hospital, Harvard Medical School, Boston, MA, ⁴Pontificia Universidad Catolica de Chile, Santiago, Chile

Background: Numerous studies have demonstrated an association between dietary sodium intake and increases in blood pressure (BP). The underlying mechanism(s) resulting in salt sensitivity (SS) of BP in humans remain largely unknown. Aldosterone (ALDO) through mineralocorticoid receptor (MR) activation is an important regulator of sodium and water homeostasis. We recently reported that dietary sodium restriction in mice was associated with increased striatin levels in aortas and kidneys. Furthermore, we demonstrated MR and striatin co-immunoprecipitate in cardiovascular tissues. Striatin is a highly conserved member of the WD-repeat family of proteins that functions as both a scaffolding protein and signal transducer. The aim of this study was to test the hypothesis that striatin modulates SS of BP.

Methods/Results: We examined the relationship between striatin gene polymorphisms and SS BP in 366 Caucasian, hypertensive subjects. Haplotype constructs from 19 SNPs were analyzed using a multivariate logistic model to predict BP response to a dietary salt intervention (High Sodium [HS] minus Low Sodium [LS] diet). Block 1 was significantly associated with salt sensitive blood pressure (P=0.04), driven by two SNPs (rs2540923: OR, 6.25; 95% CI 1.7-20; P=0.01 adjusted) and (rs888083: OR 1.98; 95% CI 1.22-3.22;P=0.005 adjusted).

To further explore the relationship between SS of BP and striatin, we generated a striatin heterozygous knockout (Strn^+/-) mouse model. Sixteen-week old male Strn^+/- and aged-matched littermate wild-type (WT) mice were randomized in a crossover intervention to HS (1.6% NaCl) and LS (0.03% NaCl) diets for 7 days each. BP was measured by tail cuff plethysmography. RT-PCR and western blot data demonstrate significantly decreased striatin mRNA and protein expression in heart, aorta, adrenal, and kidney tissue of Strn^+/-as compared to WT mice. Strn^+/-mice had significantly higher BP on HS as compared with LS diet (LS 105 ± 3 vs HS 114 ± 3 mmHg, P=0.02), whereas WT mice had no change (LS 104 ± 2 vs HS 109 ± 3 mmHg, p=NS). BP response to salt intake was independent of ALDO or renin activity in both the human cohort and the mouse model. MR genomic (ENaC and SGK1) targets were significantly increased, while non-genomic targets (pAkt/Akt) were significantly decreased kidney and heart tissue of Strn^+/- mice compared to WT mice.

Conclusion: Striatin is associated with salt sensitivity of BP in human gene association analysis and in a genetically altered mouse model. Observed reduced striatin expression with salt sensitivity of BP in the absence of RAAS modulation suggests impaired MR signaling. This may implicate striatin in the pathogenesis of salt sensitive blood pressure.

Nothing to Disclose: AEG, CMR, JSW, RB, JRR, GKA, LHP, GHW

OR06-4 16056 4.0000 A Variation in Striatin Gene Is Associated with Salt Sensitive Blood Pressure 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Cardiovascular Endocrinology Saturday, June 21st 1:00:00 PM OR06 4725 11:30:00 AM Hyperaldosteronism and Salt-Sensitive Hypertension Oral

Masao Omura^*¹, Kohzoh Makita², Seishi Matsui³, Maki Nagata³, Yoko Matsuzawa¹, Jun Saito¹ and Tetsuo Nishikawa¹
¹Yokohama Rosai Hosp, Yokohama, Japan, ²Hikarigaoka Hospital, Tokyo, ³Yokohama Rosai Hospital, Yokohama, Japan

Introduction:Unilateral total adrenalectomy (T-Adx) is worldwide performed for unilateral hyperaldosteronism, while normal adrenal tissue attaching to APA is removed even though it dose not cause primary aldosteronism (PA). Since 2010, we have treated CT-detectable APA by partially removing unilateral adrenal lesions containing CT-visible APA (P-Adx) to preserve normal adrenal tissue attaching to the APA based on the diagnosis of APA by selective segmental AVS (SSAVS). Then, we tried to analyze outcomes of clinical and laboratory findings after surgical treatment with P-Adx.

Methods: We performed SSAVS obtaining adrenal effluents at central and more than 2 tributary veins in each adrenal gland after ACTH stimulation to detect the lesions causing hyperaldosteronism in 300 cases with PA. Comparative study on subtype diagnosis of PA by regular AVS with that by SSAVS was performed, and we tried P-Adx according to the detailed localization of APA diagnosed by SSAVS.

Results: The regular AVS could diagnose 114 cases with unilateral PA, who were treated by unilateral T-Adx, and also 186 cases with bilateral PA, who were under medications. On the other hand, SSAVS diagnosed 98 unilateral APA and 39 with bilateral APAs, that could be treated by P-Adx when the adrenal lesions localized by SSAVS were detected as the same portion as APA detected by CT images. SSAVS also diagnosed 36 cases with CT-undetectable small APA and 12 with unilateral hyperplasia. We performed P-Adx in 76 cases with unilateral APA and 32 with bilateral APAs, resulting in complete and partial improvement in hyperaldosteronemia of unilateral APA and of bilateral APAs, respectively.

Conclusion: We can exactly localize the detailed portion of APA by SSAVS, and also differentiate bilateral APAs from IHA. CT-detectable APA is possible to be completely cured by minimally invasive P-Adx.

Nothing to Disclose: MO, KM, SM, MN, YM, JS, TN

OR06-5 14399 5.0000 A Minimally Invasive Surgery of Aldosterone-Producing Adenoma (APA) Detected By a New Adrenal Venous Sampling (AVS) Method 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Cardiovascular Endocrinology Saturday, June 21st 1:00:00 PM OR06 4725 11:30:00 AM Hyperaldosteronism and Salt-Sensitive Hypertension Oral

Hidekazu Nagano^*¹, Takashi Kouno¹, Masanori Fujimoto¹, Eri Komai¹, Akina Shiga¹, Akitoshi Nakayama¹, Tomoko Takiguchi¹, Seiichirou Higuchi¹, Ikki Sakuma¹, Sawako Suzuki¹, Naoko Hashimoto¹, Hisashi Koide¹, Tomohiko Yoshida¹, Ichiro Tatsuno², Koutaro Yokote¹ and Tomoaki Tanaka¹
¹Chiba University Graduate School of Medicine, Chiba, Japan, ²Toho University Sakura Medical C, Sakura-City, Japan

Primary aldosteronism (PA), characterized by the autonomous overproduction of aldosterone, is one of the most common form of secondary hypertension, with a prevalence of approximately 8 to 10% of hypertensive patients. While the two major PA subtypes are bilateral idiopathic hyperaldosteronism and aldosterone-producing adenomas (APA), APA can be cured by surgical resection. Importantly, recent exome sequencing of human APAs identified somatic mutations in K⁺ channel KCNJ5, P-type ATPase gene family, ATP1A1 and ATP2B3, additionally, CACNA1D encoding a voltage-gated calcium channel. However, a little is known about the frequency of these mutations as well as endocrinological pathogenesis in Japanese APA patients. Here, we have performed direct sequencing, RNA-sequence and RT-qPCR analysis using adrenal tumor tissues and examined the relationship between types of mutation, clinical features and molecular biological characteristics including gene expression in steroid hormone synthetic pathways. Gene mutation analysis of 71 APA cDNA samples revealed that KCNJ5 mutations were present in 48 cases (67.5%), ATP1A1 in two cases (2.8%), ATP2B3 in one case (1.4%), CACNA1D in one case (1.4%) and WT in 19 cases (26.7%). Consistent with previous reports, KCNJ5 mutations were more prevalent in female than male (female: 56.0% of KCNJ5mt). Aldosterone renin ratio (ARR), lateralization index of AVS, serum aldosterone levels after adrenocorticotropic hormone (ACTH)- or saline-infusion test were significantly elevated in APA group with mutations, while there was a variation in the presence or absence of mutations. Gene expression analysis using tumor-derived mRNA showed that CYP11B2 expression was correlated to total urine aldosterone and peak levels of serum aldosterone after ACTH stimulation in vivo whereas CYP17/HSD3B2 displayed inverse correlation to them. Furthermore, the expression of CYP11B2 and PCP4, a Ca²⁺ signal transducer, was upregulated in mutant groups with variation. Taken together, our results suggest that the reasonable expression profile of steroidogenic enzymes and Ca²⁺ signaling pathway for autonomous aldosterone production including CYP11B2 in APA represents aldosterone-secreting ability in vivo, with remarkable changes in tumors with somatic mutations of KCNJ5, ATP1A1, ATP2B3 and CACNA1D, concerning mutation frequency and pathogenesis of Japanese APA patients.

Nothing to Disclose: HN, TK, MF, EK, AS, AN, TT, SH, IS, SS, NH, HK, TY, IT, KY, TT

OR06-6 16373 6.0000 A Somatic KCNJ5, ATP1A1, ATP2B3 and CACNA1D Mutations, Its Clinical Features and Gene Expression Profile By RNA-Sequence in Japanese Aldosterone Producing Adenoma 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Cardiovascular Endocrinology Saturday, June 21st 1:00:00 PM OR06 4725 11:30:00 AM Hyperaldosteronism and Salt-Sensitive Hypertension Oral

Kavaljit H Chhabra^*¹, Jessica M. Adams¹, Daniel D Lam¹, Marcelo Rubinstein² and Malcolm James Low³
¹University of Michigan, Ann Arbor, MI, ²INGEBI, CONICET, Buenos Aires, Argentina, ³University of Michigan Medical School, Ann Arbor, MI

Hypothalamic-derived POMC peptides regulate body weight (BW) by modulating food intake and energy expenditure. Consequently, arcuate nucleus-specific (Arc) POMC-deficient mice develop morbid obesity and insulin resistance (1). However, it is not clear if the insulin resistance is purely a secondary effect of obesity or a direct result of POMC-deficiency. Therefore, we determined the role of central POMC in maintaining glucose homeostasis independently of changes in BW by utilizing ArcPOMC-deficient mice that were weight-matched to wildtype (WT) controls by food restriction starting immediately after weaning. The daily allotment of standard low fat chow was provided once a day before lights out to singly housed mice. 8-10 wk old mice were fasted for 6 h on two occasions and blood glucose and plasma insulin were measured before performing insulin- or oral glucose-tolerance tests (ITT 0.5 units/kg BW i.p.; OGTT 2 g/kg BW by gavage). The mice were also challenged with 10% glucose in drinking water while housed in metabolic cages for collection of 24 hr urine samples. Weight-matched ArcPOMC-deficient mice (n = 6 or 7) exhibited hyperinsulinemia (Male: 1.6 ± 0.1 vs. 1.0 ± 0.2; Female: 1.3 ± 0.1 vs. 0.5 ± 0.2 ng/ml) and reduced insulin sensitivity (glucose AUC, Male: 13,610 ± 1,676 vs. 9,150 ± 311; Female: 14,920 ± 940 vs. 9,840 ± 414 mg/dl*min) compared to WT groups (unpaired, two-tailed t-tests, all P < 0.05). Paradoxically, the weight matched ArcPOMC-deficient mice showed improved glucose tolerance (glucose AUC, Male: 20,823 ± 1,774 vs. 25,500 ± 1,607, P < 0.05; Female: 17,266 ± 242 vs. 23,076 ± 2,485 mg/dl*min, P < 0.01) despite insulin resistance (HOMA-IR, Male: 13.1 ± 2.4 vs. 7.4 ± 1.8; Female: 11.9 ± 1.2 vs. 6.4 ± 1.3 mM*mU/L) (P < 0.01). We speculated that this unusual phenotype could be a consequence of reduced glucose reabsorption and thus increased excretion of glucose in urine. Hence, we measured urine glucose levels using Bayer Diastix test strips during OGTT. Indeed, ArcPOMC-deficient mice excreted more glucose than WT (urine glucose: 251-500 vs. 0 mg/dl at the 60 min time point) suggesting reduced renal glucose reabsorption in the mutant mice. The mice were then challenged with 10% glucose in their drinking water. In agreement with the screening results during OGTT, ArcPOMC-deficient mice exhibited profound glycosuria compared to WT mice (urine glucose, Male: 1,030 ± 548 vs. 18.0 ± 7.6; Female: 1,018 ± 818 vs. 15.5 ± 4.2 mg/dl) indicating the suppression of glucose reabsorption in mutant mice. By analogy to the relative protection against hypertension exhibited by obese melanocortin receptor 4-deficient rodents due to decreased sympathetic tone, we speculate that the glycosuria exhibited by weight-restricted ArcPOMC-deficient mice may be the result of decreased renal sympathetic nerve activity (2) leading to down-regulation of one or both of the renal glucose transporters, GLUT2 and SGLT2.

Nothing to Disclose: KHC, JMA, DDL, MR, MJL

OR01-1 11608 1.0000 A Elevated Glycosuria Improves Glucose Tolerance in Arcuate Nucleus-Specific Pomc Deficient Mice Despite the Presence of Insulin Resistance 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Diabetes & Glucose Metabolism Saturday, June 21st 1:00:00 PM OR01 4726 11:30:00 AM Basic Mechanisms of Glucose Homeostasis and Diabetic Complications Oral

Jennifer M. Rojas^*¹, Miles E. Matsen¹, Gregory J. Morton¹, Darko Stefanovski², Richard N Bergman³ and Michael W. Schwartz¹
¹University of Washington, Seattle, WA, ²Cedars-Sinai Medical Center, West Hollywood, CA, ³Cedars-Sinai Medical Center, CA

Following a glucose challenge, glucose disposal occurs via both insulin-dependent and -independent mechanisms. The latter, referred to as glucose effectiveness (GE), is crucial for glucose homeostasis and, like insulin action, is impaired in obesity and type 2 diabetes mellitus. We recently reported that low-dose intracerebroventricular (icv) administration of the gut-derived hormone fibroblast growth factor-19 (FGF19) exerts anti-diabetic effects in genetically obese, leptin-deficient ob/ob mice by potently, rapidly and selectively increasing GE. This effect involves an action at central FGF receptors (FGFR), since glucose lowering (whether FGF19 is given centrally or peripherally) is strongly inhibited by icv pre-treatment with a non-selective FGFR inhibitor (FGFRi; PD173074). As the mechanism underlying increased GE in response to central FGF19 appears to involve increased metabolism of glucose to lactate, we sought to determine whether endogenous neuronal FGFR signaling plays a physiological role in glucose and/or lactate homeostasis and if so, whether the effect involves regulation of GE. To address these questions, we performed icv infusions of either PD173074 (150µg) or vehicle (Veh) in lean, overnight fasted Long-Evans rats 1 week following cannulation of the third ventricle. Thirty min after icv injection, glucose homeostasis was assessed with a frequently sampled intravenous glucose tolerance test (FSIGT) followed by Minimal Model analysis to quantify GE, insulin secretion (AIRg) and insulin sensitivity (Si). As predicted, treatment with icv FGFRi worsened glucose tolerance during the FSIGT (by ~28%; p<0.001) relative to Veh, despite a surprising 3-fold increase of Si (p<0.01). In contrast, GE was reduced (albeit non-significantly; p=0.174) and AIRg did not change. Treatment with icv FGFRi also markedly raised lactate levels at baseline (20 min post icv injection, 10 min before the FSIGT) relative to Veh (p<0.01), and this effect was partially ameliorated following iv glucose injection, suggesting a marked disruption of basal lactate homeostasis. Interestingly, basal glucose values were strongly positively correlated with GE in the Veh-treated controls (r=0.9; p=0.005), and this relationship was lost following icv injection of the FGFRi (r=-0.01; p=ns). A similar correlation was observed between basal lactate levels and GE in control rats, although it did not achieve statistical significance. In conclusion, endogenous central FGFR signaling appears to be required for normal glucose homeostasis, potentially via opposing effects on GE and Si. In addition, basal glucose is a strong predictor of GE measured during the FSIGT in normal rats. Since this relationship is blocked by central FGFR inhibition, we hypothesize that the link between basal glucose levels and GE is centrally mediated.

Nothing to Disclose: JMR, MEM, GJM, DS, RNB, MWS

OR01-2 13259 2.0000 A Central FGF Receptor Signaling Is Required for Normal Glucose Homeostasis 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Diabetes & Glucose Metabolism Saturday, June 21st 1:00:00 PM OR01 4726 11:30:00 AM Basic Mechanisms of Glucose Homeostasis and Diabetic Complications Oral

Mengxi Jiang^*, Meishu Xu and Wen Xie
University of Pittsburgh, Pittsburgh, PA

The steroid sulfatase (STS)-mediated de-sulfation is a critical metabolic mechanism that regulates the chemical and functional homeostasis of endogenous and exogenous molecules. In this report, we first showed that the liver expression of STS was induced in both the high-fat diet (HFD) and ob/ob models of obesity and type 2 diabetes and during the fed to fasting transition. In defining the functional relevance of STS induction in metabolic disease, we showed that over-expression of STS in the liver of transgenic mice alleviated HFD and ob/ob models of obesity and type 2 diabetes, including reduced body weight, improved insulin sensitivity, and decreased hepatic steatosis and inflammation. Interestingly, STS exerted its metabolic benefit through sex-specific mechanisms. In female mice, STS may have increased hepatic estrogen activity by converting biologically inactive estrogen sulfates to active estrogens and consequently improved the metabolic functions, whereas ovariectomy abolished this protective effect. In contrast, the metabolic benefit of STS in males may have been accounted for by the male-specific decreased inflammation in white adipose tissue and skeletal muscle, as well as a pattern of skeletal muscle gene expression that favors energy expenditure. The metabolic benefit in male STS transgenic mice was retained after castration. Treatment with the STS substrate estrone sulfate also improved metabolic functions in both the HFD and ob/ob models. Our results have uncovered a novel function of STS in energy metabolism and type 2 diabetes. Liver-specific STS induction or estrogen/estrogen sulfate delivery may represent novel management of metabolic syndrome.

Nothing to Disclose: MJ, MX, WX

OR01-3 11210 3.0000 A Hepatic over-Expression of Steroid Sulfatase Ameliorates Mouse Models of Obesity and Type 2 Diabetes through Sex-Specific Mechanisms 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Diabetes & Glucose Metabolism Saturday, June 21st 1:00:00 PM OR01 4726 11:30:00 AM Basic Mechanisms of Glucose Homeostasis and Diabetic Complications Oral

Laura Ann Maile^*¹, William Flowers², Kara Stewart², Nikhol Dysart², Stefani Garbacik², Timothy Nichols³, Dwight Bellinger³, David R Clemmons⁴, Katherine Gollahon⁵, Christine Wai⁶, Robin Raymer³, Elizabeth Merricks³ and Kent Passingham³
¹Vasular Pharmaceuticals, Chapel Hill, NC, ²North Carolina State University, ³University of North Carolina at Chapel Hill, ⁴Univ of N Carolina Schl of Med, Chapel Hill, NC, ⁵Vascular Pharmaceuticals, ⁶University of North Carolina at Chapel Hill, Chapel Hill, NC

The aim of this study was to characterize the development of renal impairment in a pig model of streptozotocin (STZ) induced diabetes combined with high fat diet and to examine the effect of a novel anti αVβ3 antibody on the progression of the disease. The anti-C-loop β3 antibody (anti-C-loop) binds to a non-RGD binding region site on the β3 subunit of αVβ3 and is the mouse monoclonal precursor of VPI-2690B, which is currently in Phase 1 clinical trials.

Twenty pigs were made hyperglycemic by the administration of STZ then placed on a high fat diet. 2 weeks later 10 pigs were treated with control F(ab)₂and 10 pigs were treated with anti-C-loop F(ab)₂ every 72 hours for 20 weeks.

After 20 weeks of diabetes, the animals developed proteinuria and histological changes in the kidney. Urinary protein was significantly lower in diabetic pigs that received anti-C-loop antibody compared to those that received control (218 ± 57 vs 115 ± 50 mg protein / gm creatinine, p<0.05). The kidneys of untreated diabetic pigs were characterized by significant histopathology, including expansion of the mesangial matrix and glomerular basement membrane (GBM) and by an increase in podocyte foot width compared to nondiabetic pigs. In the diabetic animals treated with anti C-loop F(ab)₂, the area of the mesangial matrix as a percentage of total glomerular area was reduced (35 ± 5.2% vs 27.4 ± 3.8%; p<0.02),GMB thickness decreased (169 ± 3.1 vs 159.9 ± 7.3 nm, p<0.01) and podocyte foot width decreased (503±15 nm vs. 359 ± 21.2, p<0.05) compared to diabetic animals treated with control F(ab)₂. β3 phosphorylation, a marker of αVβ3 activation, was elevated in the diabetic animals compared to nondiabetic controls. As expected, treatment with anti-C-loop significantly reduced β3 phosphorylation in these animals (13,934 ± 6437 vs 6,730 ± 1524 scanning units; p<0.01).

The results of this study show that, in this pig model of Type 2 diabetes, animals develop the early changes in loss of barrier function and histopathologic changes that are consistent with diabetic nephropathy. The changes are accompanied by activation of the αVβ3 integrin as assessed by phosphorylation of the b3 subunit. Blocking αVβ3 integrin with anti-C-loop antibody leads to prevention of albuminuria and of the early histologic changes of diabetic nephropathy. The results suggest that this antibody may have efficacy in preventing the progression of diabetic nephropathy in patients.

Disclosure: LAM: Employee, Vascular Pharmaceuticals. DRC: Chief Scientific Officer, Vascular Pharmaceuticals. KG: Employee, Vascular Pharmaceuticals. Nothing to Disclose: WF, KS, ND, SG, TN, DB, CW, RR, EM, KP

OR01-4 11805 4.0000 A Targeting a Novel Site on the aphaVbeta3 Integrin Reduces Hyperglycemia-Induced Changes in the Kidney of Diabetic Pigs 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Diabetes & Glucose Metabolism Saturday, June 21st 1:00:00 PM OR01 4726 11:30:00 AM Basic Mechanisms of Glucose Homeostasis and Diabetic Complications Oral

Weiwei Zhao, Wei Gong, Bin Lu, Zhaoyun Zhang, Yiming Li, Yehong Yang^* and Renming Hu
Huashan hospital, Shanghai, China

Although significant advances in the diagnosis and treatment of diabetic nephropathy (DN) have been achieved over the last two decades, the molecular pathogenesis is still unclear. In order to further understand the mechanisms of DN, patients with DN and non-DN were tested by SELDI-TOF-MS with the CM10 protein chip to get the protein spectra maps. Combined with RayBiotech high-throughput antibody microarray, we finally identified the elevated protein resistin-like molecule β (RELMβ), which may involved in the development of DN. High expression of the RELMβ protein was validated in both glomerular mesangium of Sprague Dawley rats with DN and cytoplasm of human mesangial cells (HMCs). High levels of circulating RELMβ were correlated with the development of diabetic albuminuria in community population. Glucose stimulated the production of RELMβ protein derived from HMCs. The overexpression of RELMβ in HMCs promoted the proliferation of mesangial cells and accelerated cell cycle progression, whereas the knockdown of RELMβ decreased cell proliferation and delayed cell cycle progression. However, RELMβ did not work on apoptosis of HMCs. Furthermore, RELMβ triggered HMC proliferation by phosphorylating MAPKs (JNK and p38 MAPK), while the activity of ERK1/2 MAPK was not altered. The proliferation could be reversed by a p38 MAPK inhibitor (SB 202190) in Lenti-RELMβ HMCs. In addition, PCR array analysis demonstrated that 8 signal transduction pathways involved in cell proliferation were modified upon RELMβ modulation. WISP1 gene, which is associated with proliferation, extracellular matrix production, as well as fibrosis (1), is significantly downregulated as 33.27 fold in Lenti-sh-RELMβ HMCs. These findings suggest that RELMβ accelerates the proliferative function of HMCs by targeting the MAPK pathway and cell cycle. RELMβ might be a candidate predictor for DN.

Nothing to Disclose: WZ, WG, BL, ZZ, YL, YY, RH

OR01-5 14757 5.0000 A Resistin-like Molecule β Promotes Renal Injury By Modulating Mesangial Cell Proliferation in the Development of Diabetic Nephropathy 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Diabetes & Glucose Metabolism Saturday, June 21st 1:00:00 PM OR01 4726 11:30:00 AM Basic Mechanisms of Glucose Homeostasis and Diabetic Complications Oral

I George Fantus^*¹, Anu Shah², Elodie AY Masson², Rohan John³ and Mansoor Husain³
¹Mount Sinai Hospital, Toronto, ON, Canada, ²Mount Sinai Hospital, ³University Health Network

Thioredoxin-interacting protein (TxNIP), a member of the α-arrestin domain family of proteins, binds and inhibits the thiol oxidoreductase, thioredoxin. We previously reported that high glucose (HG) upregulated TxNIP in cultured mesangial cells (MC) which in turn, promoted oxidative stress, MAPK/p38 signaling and collagen synthesis. However, the role of TxNIP in diabetic nephropathy (DN) in vivo is unknown. Thus, TxNIP-WT (WT), TxNIP^-/- (KO), TxNIP^+/- (HET), C3H (Control) and Hcb-19 (Spontanous mutation lacking TxNIP) mice were rendered equally diabetic with low dose streptozotocin (STZ). After 24 weeks of diabetes and equivalent hyperglycemia and BP, in contrast to WT, KO mice did not manifest albuminuria, proteinuria or elevation of serum cystatin C or creatinine. EM studies showed thickened glomerular basement membranes and effacement of podocyte foot processes in diabetic WT mice, but not in KO. Similarly, a loss of podocytes (WT-1 staining) and increased urinary nephrin was observed in diabetic WT but not in KO mice. Mesangial expansion (PAS staining), collagen IV and TGFβ (immunohistochemistry, iHC), and renal interstitial fibrosis (Masson’s trichrome) revealed significant increases only in diabetic WT. Consistent with a role of TxNIP in generating oxidative stress and inflammation, while WT diabetic mice showed significantly increased 24 h urinary 8-hydroxy-2-deoxyguanosine, glomerular nitrotyrosine and F4/80 iHC and augmented renal cortex Il-1β mRNA, these parameters were not altered in diabetic KO mice. Furthermore, consistent with our results in MC, renal cortex Nox4 mRNA and glomerular Nox4 staining were increased in diabetic WT but not KO. Diabetic Hcb-19 TxNIP-deficient mice also displayed protection from renal markers of DN in comparison to diabetic C3H controls, similar to KO versus WT. In the presence of diabetes the TxNIP^+/-HETS had a lesser but significantly increased TxNIP mRNA (~70% of WT) and showed only partial protection from DN. Taken together, these data indicate that TxNIP plays a critical role in the development and progression of DN and may represent a promising therapeutic target.

Nothing to Disclose: IGF, AS, EAM, RJ, MH

OR01-6 12187 6.0000 A Thioredoxin-Interacting Protein (TxNIP) Deficiency Protects Against Diabetes-Induced Renal Oxidative Stress, Inflammation and Pathologic Characteristics of Diabetic Nephropathy 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Diabetes & Glucose Metabolism Saturday, June 21st 1:00:00 PM OR01 4726 11:30:00 AM Basic Mechanisms of Glucose Homeostasis and Diabetic Complications Oral

Nora S Kayton^*¹, Greg Poffenberger¹, Chunhua Dai¹, Pedro L Herrera², Dale L Greiner³, Lenny D Shultz⁴ and Alvin C Powers⁵
¹Vanderbilt University Medical Center, Nashville, TN, ²Univ of Geneva Medical Sch, Geneva, Switzerland, ³University of Massachusetts Medical School, Worcester, MA, ⁴The Jackson Laboratory, Bar Harbor, ME, ⁵Vanderbilt Univ Med Ctr, Nashville, TN

Chronic hyperglycemia and resulting “glucotoxicity” are suspected to compromise beta cell function. To examine the effect of hyperglycemia on human beta cells in vivo, we transplanted human islets into NOD-scid Il2rgamma^null (NSG)–Rat Insulin Promoter-Diphtheria Toxin Receptor (RIP-DTR) mice. In this new model, human islets engraft under normoglycemia, and subsequent injection of diphtheria toxin (DT) specifically ablates the pancreatic (mouse) beta cells. In characterizing this model, following transplantation of 500 human islets under the kidney capsule of NSG or NSG-RIP-DTR mice, we found that injection of 5.0 ng DT in NSG-RIP-DTR mice reduced mouse pancreatic insulin content from 47,244 ± 14,905 μg/g of pancreas weight to below detectable levels but did not alter fasting (control 2.07 ± 0.26 vs. DT treated 2.10 ± 0.32 ng/mL, n=6, p>0.05) or glucose/arginine-stimulated human C-peptide (control 3.17 ± 0.37 vs. DT treated 3.75 ± 0.46 ng/mL, n=6, p>0.05) or graft human insulin content (control 187.6 ± 30.31 vs. DT 156.0 ± 42.66 ng; n=5, p>0.05). Engraftment of four thousand human islet equivalents (IEQ) maintained euglycemia after DT injection (mean fed blood glucose of 106.2 ± 3.0 vs. control of 123.0 ± 3.4 mg/dL; n=3, p>0.05), but 2000 human IEQ resulted in hyperglycemia (mean fed blood glucose of 353.2 ± 43.5 vs. control of 123.0 ± 3.4 mg/dL; n=3, p<0.01). The divergence in blood glucose values began 3 days after DT injection and continued for 4 weeks, until the termination of the experiment. We have discovered different consequences for human islets under euglycemic and hyperglycemic conditions. At 2 weeks after DT injection, human insulin release in response to injection of a glucose-arginine bolus did not increase in 2000 IEQ + DT mice (1.13 ± 0.42 ng/mL) but did increase in 4000 IEQ + DT mice (6.19 ± 0.93 ng/mL), compared to controls, which had 2000 human IEQ but received no DT (2.63 ± 0.59ng/mL). This resulted in a greater increase in blood glucose of 2000 IEQ + DT mice compared to the other groups (544.8 ± 23.6 vs. 265.3 ± 24.3 and 264.7 ± 48.1 mg/dL). At 4 weeks after DT injection, human insulin content of grafts from 4000 IEQ + DT mice was significantly higher compared to controls (11409 ± 2244 ng vs. 1351 ± 253 ng, n=6, p<0.01), but 2000 IEQ + DT grafts were unchanged (973.7 ± 300.9, n=5 vs. 1351 ± 253 ng, n=5, p>0.05). Mouse pancreatic insulin content in 4000 and 2000 IEQ DT-treated animals was reduced to 2.7% and 3.4%, respectively, of the content of controls (23.88 ± 5.9, n=5 and 18.90 ± 7.0, n=6 vs. 712.1 ± 60.22 ng, n=4, p<0.0001). Beta cell proliferation and apoptosis are extremely low and not significantly different among the groups. The NSG-RIP-DTR model enabled us to observe that glucose-stimulated insulin release and increases in insulin content are impaired in human beta cells exposed to 4 weeks of hyperglycemia in vivo, suggesting glucotoxic consequences, but does not induce beta cell proliferation or death.

Disclosure: PLH: , Unknown (author has not provided this information. Information will be provided as soon as possible).. DLG: Speaker, Genzyme Corporation, Speaker, Pfizer, Inc., Speaker, Boehringer Ingelheim, Ad Hoc Consultant, Viacord, Ad Hoc Consultant, The Jackson Laboratory. LDS: Speaker, Boehringer Ingleheim, Speaker, Bristol-Myers Squibb, Speaker, Pfizer, Inc., Ad Hoc Consultant, Viacord. ACP: Ad Hoc Consultant, Boehringer Ingelheim Pharma GmbH & Co. KG, Ad Hoc Consultant, Novartis Pharmaceuticals. Nothing to Disclose: NSK, GP, CD

OR10-1 15030 1.0000 A Assessment of Glucotoxic Consequences for Human Beta Cells in Vivo Via the NOD-SCID Il2rgamma^null –RIP-Dtr Mouse 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Diabetes & Glucose Metabolism Saturday, June 21st 1:00:00 PM OR10 4732 11:30:00 AM Nutrient-Insulin Interactions in the Pancreas and Periphery Oral

Ramya Embar Srinivasan^*, Poonam R Sharma and Craig S Nunemaker
University of Virginia, Charlottesville, VA

Background: Type 2 diabetes (T2D) is characterized by insulin resistance and beta-cell failure. Several mechanisms underlying the causes of pancreatic beta-cell failure, which we term “stressors”, have been reported including glucotoxicity, lipotoxicity, endoplasmic reticulum (ER) stress, oxidative stress, and inflammation. We identified several iron-regulating genes in a gene microarray of beta-cell stress, of which Six-transmembrane epithelial antigen of the prostate 4 (STEAP4) was the most upregulated. STEAP-proteins allow cellular uptake of iron by reducing iron to its lower oxidative state. Epidemiological studies have reported a positive association between high body iron stores, increased dietary iron intake and the risk of T2D. We wished to examine whether STEAP4 and other iron-regulating genes participate in islet stress responses.

Objective: Determine how iron-regulating genes are regulated by 1) beta-cell stressors and 2) STEAP4 overexpression in islets.

Methods: Pancreatic islets of 8-12-week-old, male CD1 mice were isolated and cultured. Islets were exposed for 48-hrs to one of the following stressors representing models of beta-cell failure: 20nM rotenone (oxidative stress), 100nM thapsigargin (ER stress), 10pg/ml IL-1B + 20pg/ml IL-6 (low-grade inflammation), 28mM glucose (glucotoxicity), or 50pg/ml palmitate + 100pg/ml oleate + 50pg/ml linolate (lipotoxicity). Islets incubated in standard RPMI 1640 media were used as controls; n=4/condition. The expression of iron-regulating genes STEAP4, ferritin, ferroportin, and lipocalin-2 (LCN2) were measured in islets by RT-PCR. In a second study, islets were transduced with lentivirus for 24 hrs for overexpression of STEAP4. RNA was extracted from transduced islets 72 hrs post transduction to measure expression of various genes by RT-PCR.

Results: When treated with cytokines, thapsigargin and free fatty acids, respectively, fold increases in islet STEAP4 expression were 90.6+/-7.6-fold SEM (p<0.001), 4.1+/-1.0 (p<0.05), 3.4+/-0.9 (p<0.05) and LCN2 were 35.6+/-6.2 (p<0.01), 7.1+/-1.7 (p<0.05), 2.1+/-0.5 (p=0.11). Thapsigargin increased islet ferritin expression by 2.0+/-0.2 (p<0.01); no significant changes in ferroportin were observed. No iron genes were downregulated. Overexpression of STEAP4 (~15-fold) upregulated the iron-sequestering protein LCN2 by 7.9+/-1.7-fold, p<0.05, n=3. Ferritin and ferroportin were not significantly changed.

Conclusion: STEAP4 and LCN2 are linked in their expression and both exceptionally cytokine-sensitive, suggesting these iron-regulating proteins may play a central role in the low-grade chronic inflammation accompanying T2D.

Nothing to Disclose: RES, PRS, CSN

OR10-2 13940 2.0000 A Association of Iron-Regulating Genes and Islet Stress 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Diabetes & Glucose Metabolism Saturday, June 21st 1:00:00 PM OR10 4732 11:30:00 AM Nutrient-Insulin Interactions in the Pancreas and Periphery Oral

Riccarda Granata^*¹, Fabio Settanni¹, Letizia Trovato¹, Davide Gallo¹, Iacopo Gesmundo¹, Rita Nano², Maria Pia Gallo¹, Marina Taliano¹, Loredana Bergandi¹, Marco Volante¹, Giuseppe Alloatti¹, Lorenzo Piemonti², Mauro Papotti¹, Jérôme Leprince³, Hubert Vaudry³, Huy Ong⁴ and Ezio Ghigo¹
¹University of Turin, Turin, Italy, ²San Raffaele Scientific Institute, Milan, Italy, ³University of Rouen, Rouen, France, ⁴University of Montreal, Montreal, QC, Canada

The RFamide neuropeptides 43RFa and 26RFa were found to promote food intake and to exert a variety of peripheral actions through binding to the orphan receptor GPR103. In addition, 26RFa has been recently shown to inhibit pancreatic insulin secretion, whereas 43RFa effect on β-cell function is still unknown, as well as the role of both peptides on β-cell survival. In the present study we sought to investigate 43RFa and 26RFa effects on survival and apoptosis of pancreatic β-cells and human pancreatic islets. Furthermore, we determined the influence of these peptides on insulin secretion and the underlying signaling mechanisms. Our results show that in INS-1E β-cells and human pancreatic islets, both 43RFa and 26RFa prevented cell death and apoptosis induced by serum starvation, cytokine synergism and glucolipotoxicity, via PI3K/Akt- and ERK1/2-induced signaling. Moreover, 43RFa promoted, whereas 26RFa inhibited, glucose- and exendin-4-induced insulin secretion, through Gα_s and Gα_i/o proteins, respectively. The insulinotropic effect of 43RFa, but not the insulinostatic action of 26RFa, was blocked in β-cells knocked-down for GPR103. Finally, 43RFa, but not 26RFa, induced cAMP increase and glucose uptake. In conclusion, because of their survival effects along with the effects on insulin secretion, these findings suggest potential for 43RFa and 26RFa as therapeutic targets in diabetes and metabolic dysfunctions.

Nothing to Disclose: RG, FS, LT, DG, IG, RN, MPG, MT, LB, MV, GA, LP, MP, JL, HV, HO, EG

OR10-3 13684 3.0000 A Rfamide Peptides 43RFa and 26RFa Both Promote Survival of Pancreatic Β-Cells and Human Pancreatic Islets but Exert Opposite Effects on Insulin Secretion 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Diabetes & Glucose Metabolism Saturday, June 21st 1:00:00 PM OR10 4732 11:30:00 AM Nutrient-Insulin Interactions in the Pancreas and Periphery Oral

Rodolfo Guardado-Mendoza^*¹, Carla Perego², Lilia Marisela Jiménez-Ceja³, Giovanna Finzi⁴, Carlo Capella⁴, Edward J. Dick Jr.⁵, Ma. de Lourdes Reyes-Escogido³, Stefano La Rosa⁴, Fausto Sessa⁴, Ralph DeFronzo⁶, Amalia Gastaldelli⁷, Alberto M Davalli⁸ and Franco Folli⁶
¹University of Guanajuato, Department of Medicine and Nutrition, and Hospital Regional de Alta Especialidad del Bajío, Departamento de Investigación, León, Guanajuato, Mexico, ²Universitá degli Studi di Milano, ³University of Guanajuato, ⁴University of Insubria, ⁵Southwest National Primate Research Center, Texas Biomedical Research Institute, ⁶University of Texas Health Science Center, San Antonio, TX, ⁷CNR, Inst of Clinical Physiology, Pisa, Italy, ⁸San Raffaele Scient Inst, Milano, Italy

Background and Aims: The unique composition and architecture of the islet of Langerhans is essential to ensure its normal function (1-3). Morphological and physiological abnormalities are both present in islets of subjects with type 2 diabetes but the relationship between islet cell composition with fasting plasma glucose (FPG) and other metabolic and biochemical parameters is presently unknown. Therefore, we explored the correlation between islet cell abnormalities and FPG and other metabolic markers.

Methods:We measured the relative α-, β- and δ-cell volumes and amyloid deposition with stereology techniques in the pancreas of 40 baboons stratified according to FPG in 4 groups (G1: FPG<80mg/dl n=10; G2: FPG=80-<95mg/dl n=9, G3: FPG=95-125mg/dl n=9, G4: FPG≥125mg/dl n=12) and examined the correlations between islet composition and various functional and metabolic parameters. We performed double and triple immunofluorescence staining to identify apoptotic islet cells as well as electron microscopy to identify the ultra-structural composition of different islet cells.

Results: As compared to G1, G2 showed a significant increase in islet amyloid deposition which increased linearly up to G4. Amyloidosis preceded the decrease in b-cell volume statistically significant only in G4. α-cell volume increased of ~50% in G3 and G4 (p <0.05), while δ-cell volume decreased in these groups of 39 and 31%, respectively (both p<0.05). In G4, glucagon, cholesterol and free fatty acid levels were higher, while insulin and HOMA-B were lower, than in the other groups. Staining of G4 pancreatic sections with the apoptosis marker CCL3, insulin and somatostatin showed ongoing apoptosis of both b- and d-cells which was confirmed by immuno-electron-microscopy. Ultra-structural analysis confirmed a normal structure in α-cells while β- and δ-cells showed multiple signs of cell suffering in baboons with the higher glucose levels.

Conclusions/interpretations: In baboons, the changes in islet composition that matches the increase in FPG concentration are in sequence: i. increased amyloid deposition; ii. increased a-cell- and decreased d-cell volumes; iii. decreased b-cell volume. Timing and nature of these changes suggests a so far unrecognized role of d-cells in diabetes pathogenesis.

Nothing to Disclose: RG, CP, LMJ, GF, CC, EJD, MDLR, SL, FS, RD, AG, AMD, FF

OR10-4 15533 4.0000 A Islet Cell Paracrinopathy and the Relevance of Delta-, Alpha- and Beta-Cell Dysfunction in the Development of Hyperglycemia in Non-Human Primates 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Diabetes & Glucose Metabolism Saturday, June 21st 1:00:00 PM OR10 4732 11:30:00 AM Nutrient-Insulin Interactions in the Pancreas and Periphery Oral

Wenwei Zhang^*¹, So Young Bu², Mara T Mashek², Olga Ilkayeva³, Christopher B. Newgard³, Douglas Mashek² and Terry G Unterman¹
¹University of Illinois at Chicago & Jesse Brown VAMC, Chicago, IL, ²University of Minnesota, St Paul, MN, ³Duke University Medical Center, Durham, NC

FoxO proteins are major targets of insulin action and regulate gluconeogenic, glycolytic and lipogenic gene expression in the liver (JBC 281:10105, 2006). Adipose triacylglycerol lipase (ATGL) mediates the first step in triacylglycerol (TAG) hydrolysis and FoxO1 stimulates ATGL expression in adipose tissue. Based on gene array data, we asked whether FoxO1 also regulates hepatic expression of ATGL and its inhibitor, the G0/G1 switch gene protein 2 (G0S2) and examined their role in mediating effects of FoxO1 on gene expression and metabolism in the liver. Studies in liver-specific transgenic and knockout mice show that FoxO proteins stimulate ATGL and suppress G0S2 expression, and studies in isolated hepatocytes with adenoviral vectors confirm that FoxO1 regulates ATGL and G0S2 expression in liver cells. Metabolic labeling studies demonstrate that FoxO1 stimulates TAG turnover and fatty acid oxidation in isolated hepatocytes, and knocking down ATGL expression or expressing its inhibitor, G0S2, with adenoviral vectors disrupts the effect of FoxO1 on TAG catabolism. FoxO1 also stimulates liver TAG turnover and fatty acid oxidation in vivo, based on serum beta hydroxybutyrate and intrahepatic fatty acylcarnitine levels, and knocking down ATGL in the liver with an adenoviral vector expressing ATGL shRNA disrupts these effects of FoxO1. In addition, post-prandial serum TAG levels are decreased in transgenic mice expressing constitutively active FoxO1 in the liver, reflecting reduced lipogenic and glycolytic gene expression, consistent with previous studies (ibid), and knocking down ATGL reverses these effects and increases glycolytic and lipogenic gene expression and hepatic TAG production and secretion. Similarly, FoxO1 transgenic mice have impaired glucose tolerance reflecting increased gluconeogenic gene expression (ibid), and suppressing ATGL in the liver restores normal glucose and pyruvate tolerance in transgenic mice. Studies in isolated hepatocytes confirm that ATGL is required for the ability of FoxO1 to stimulate glucose production in liver cells. Together, these studies demonstrate that ATGL plays a critical role in mediating effects of FoxO1 on TAG turnover and fatty acid oxidation in the liver, and contributes to the ability of FoxO1 to suppress glycolytic and lipogenic gene expression and stimulate hepatic glucose production. These results indicate that ATGL-mediated TAG catabolism plays a critical role in the integrated regulation of both lipid and carbohydrate metabolism by FoxO proteins in the liver.

Nothing to Disclose: WZ, SYB, MTM, OI, CBN, DM, TGU

OR10-5 16294 5.0000 A Regulation of Hepatic Lipid and Glucose Metabolism By Fox0 Proteins Requires Atgl-Mediated Triacylglycerol Catabolism 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Diabetes & Glucose Metabolism Saturday, June 21st 1:00:00 PM OR10 4732 11:30:00 AM Nutrient-Insulin Interactions in the Pancreas and Periphery Oral

Lucia Russo^*¹, Sumona Ghosh Lester², Saja S Khuder¹, Terry D Hinds², Simon L Abdallah², Simona S Ghanem², Scott L Friedman³ and Sonia M Najjar²
¹University of Toledo College of Medicine and Life Sciences, Toledo, OH, ²University of Toledo, College of Medicine, Toledo, OH, ³Icahn School of Medicine at Mount Sinai, New York, NY

Nonalcoholic fatty liver disease (NAFLD) and its progressive form, nonalcoholic steatohepatitis (NASH), are a global health concern. Whether insulin resistance, commonly associated with obesity, is an obligate metabolic manifestation of the disease is still debated. We have demonstrated that mice with transgenic liver-specific inactivation and with global null deletion of Ceacam1 (Cc1) gene are characterized by impaired insulin clearance in liver, which causes hyperinsulinemia followed by insulin resistance, liver steatosis and visceral obesity. These mice have spontaneous liver fibrosis with a NASH-characteristic chicken-wire collagen deposition pattern. Moreover, high-fat feeding provoked progression to the full spectrum of NASH in both Cc1 mutants. This prompted us to test whether liver-specific loss of CEACAM1 causes insulin resistance and NASH, both being driven by hyperinsulinemia. Clinically, this is bolstered by findings that hepatic CEACAM1 levels are markedly decreased in patients with NAFLD/NASH. Thus, we aimed to determine whether specific deletion of Ceacam1 gene (Cc1) in liver causes insulin resistance and the pathological abnormalities of NAFLD/NASH in L-Cc1^fl/flmice. Conversely, we tested whether exclusive rescuing of CEACAM1 in liver protects Cc1^–/^–xliver+ mice against NAFLD/NASH. L-Cc1^fl/fl null mice exhibited a higher body weight beginning at 2 months of age that was associated with an increase in total fat mass and lower lean mass as compared to their littermate controls starting at 4 months of age. These mice also developed hyperinsulinemia and insulin resistance, as demonstrated by insulin intolerance testing. Conversely, liver-rescued Cc1^–/^–xliver+mice (2 months old) exhibited lower body weight and total fat mass, a higher total lean mass and lower levels in almost all the metabolic parameters compared to Cc1^–/^–littermates. They also exhibited restored insulin sensitivity. We are currently feeding mice with a high fat diet to examine whether null mice are predisposed while rescued mice are protected against NASH-characteristic pathological abnormalities. Furthermore, lentiviral-mediated ShRNA loss of CEACAM1 in immortalized human LX2 stellate cells decreased cellular fat content with a parallel increase in its mobilization into the medium, associated with increased cell proliferation and expression of smooth muscle actin, all consistent with increased stellate cell activation. These data support a key role for CEACAM1 in attenuating hepatic steatosis and stellate cell activation. These findings uncover an important novel role for CEACAM1-dependent pathways in regulating key features of NASH.

Nothing to Disclose: LR, SG, SSK, TDH, SLA, SSG, SLF, SMN

OR10-6 16019 6.0000 A Loss of Liver Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1 (CEACAM1) Causes Insulin Resistance and Nash Pathogenesis in Mice 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Diabetes & Glucose Metabolism Saturday, June 21st 1:00:00 PM OR10 4732 11:30:00 AM Nutrient-Insulin Interactions in the Pancreas and Periphery Oral

Han-Chuan Dai^* and Ya-Xiong Tao
Auburn University, Auburn University, AL

The melanocortin-4 (MC4R) is a G protein-coupled receptor critically involved in regulating energy homeostasis, including both food intake and energy expenditure. More than 170 mutations in the MC4R gene have been identified in different populations of patients, causing early-onset severe obesity. In addition to the activation of the classical Gs-cAMP pathway, the MC4R also activates mitogen-activated protein kinases, especially extracellular signal-regulated kinases 1 and 2 (ERK1/2). We recently showed that in wild-type (WT) and six naturally occurring constitutively active (CAM) MC4Rs, including H76R, S127L, D146N, P230L, L250Q and F280L, the endogenous antagonist Agouti-related protein and three small molecule inverse agonists decrease basal signaling in cAMP pathway but are agonists in the ERK1/2 pathway. In the present study, we performed detailed pharmacological studies on two peptide antagonists (SHU9119 and Compound 10) derived from the endogenous agonist alpha-melanocyte stimulating hormone on these seven MC4Rs. In ligand binding experiments using iodinated NDP-MSH as the tracer, we showed that SHU9119 bound to the seven MC4Rs with high affinity, with IC₅₀ranging from 1.12 to 6.68 nM. However, Compound 10 could not displace iodinated NDP-MSH in any of the seven MC4Rs studied. In signaling experiments measuring cAMP generation, we showed that SHU9119 was a very weak agonist, increasing intracellular cAMP levels at very high concentrations whereas Compound 10 had negligible agonist activity. We then examined potential activation of MC4R in the ERK1/2 pathway by these ligands. We found that upon treatment with 1 nM SHU9119 (which did not increase cAMP levels), all seven MC4Rs had significantly increased pERK1/2 levels, ranging from 2.3 to 6.2-fold of vehicle-treated controls. When treated with 1 nM Compound 10, WT and four mutants (H76R, D146N, P230L and F280L) had significantly increased pERK1/2 levels, ranging from 2.5 to 5.2-fold of vehicle-treated controls. In summary, our study showed SHU9119 and Compound 10, MC4R antagonists at the Gs-cAMP pathway, could serve as agonists in the MAPK pathway, suggesting that these ligands are biased ligands at the MC4R.

Nothing to Disclose: HCD, YXT

OR08-1 16932 1.0000 A SHU9119 and Compound 10 Are Biased Ligands at the Melanocortin-4 Receptor 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Growth Factor/Tyrosine Kinase/G Protein-Coupled Receptor/Intracelluar Signaling & Biology Saturday, June 21st 1:00:00 PM OR08 4737 11:30:00 AM New Insights in GPCR Structure and Function Oral

William F Schwindinger^*¹ and Janet D Robishaw²
¹Bloomsburg University of Pennsylvania, Bloomsburg, PA, ²Geisinger Health System, Danville, PA

Heterotrimeric G-proteins couple cell surface receptors for hundreds of different extracellular signals to a smaller number of intracellular effectors. Heterotrimeric G-proteins are assembled from the protein products of 16 genes encoding α-subunits, 5 genes encoding β-subunits and 12 genes encoding γ-subunits. Both the α-subunit and the βγ-dimer of a heterotrimeric G-protein may participate in signal transduction, sometimes regulating distinct intracellular effectors. Much of what is known about G-protein coupled signal transduction details which specific α-subunit couples a given receptor to a particular intracellular effector. For example Gα_s couples receptors for a number of hormones (e.g. PTH, TSH, MSH, LH) to the stimulation of adenylyl cyclase. This knowledge has permitted a greater understanding of endocrine disorders caused by mutations in the gene encoding Gα_s: pseudohypoparathyroidism and McCune-Albright syndrome. Much less is known about the specific roles of individual γ-subunits in signal transduction. We studied mice with targeted disruption of several γ-subunit genes: Gng3, Gng5, Gng7 and Gng11. Knockout of Gng7 disrupted stimulation of adenylyl cyclase by dopamine and adenosine in the striatum, but did not produce a locomotor phenotype. Knockout of Gng3 disrupted stimulation of potassium currents by a GABA_B agonist, and produced phenotypes of seizure susceptibility and resistance to diet induced obesity. Knockout of Gng5 produced an embryonic lethal phenotype associated with abnormalities in heart and brain development. Knockout of Gng11 resulted in accelerated development of an inflammatory dermatitis. These results indicate that individual γ-subunits have unique roles in signal transduction. Elucidation of the signal transduction pathways for each of these γ-subunits may grant insights into the molecular basis of human diseases such as obesity, epilepsy, Parkinson’s disease, congenital defects and inflammatory conditions.

Nothing to Disclose: WFS, JDR

OR08-2 11190 2.0000 A Further Characterization of G-Protein Gamma-Subunit Knockout Mice 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Growth Factor/Tyrosine Kinase/G Protein-Coupled Receptor/Intracelluar Signaling & Biology Saturday, June 21st 1:00:00 PM OR08 4737 11:30:00 AM New Insights in GPCR Structure and Function Oral

Mi Hye Lee^*¹, Kathryn M. Appleton², Thomas A. Morinelli³, Josh Kwon⁴, Yuri K. Peterson² and Louis M. Luttrell³
¹College of Medicine, Medical University of South Carolina, Charleston, SC, ²College of Pharmacy, Medical University of South Carolina, Charleston, SC, ³Medical University of South Carolina, Charleston, SC, ⁴University of Pittsburgh, Pittsburgh, PA

The arrestins are a small family of G protein-coupled receptor (GPCR) binding proteins that interact with the vast majority of GPCRs. Agonist-induced changes in GPCR conformation promote receptor phosphorylation by GPCR kinases (GRKs), leading to redistribution of arrestins from the cytosol to agonist-occupied receptors on the plasma membrane. Arrestin-binding sterically uncouples GPCRs from their cognate heterotrimeric G proteins, producing receptor desensitization, and links receptors to the clathrin-dependent endocytic machinery to promote their sequestration. Arrestins also function as signaling scaffolds, supporting the assembly of multi-protein signalsome complexes that confer novel G protein-independent signaling functions. In this study, we created a series of arrestin3 intramolecular fluorescent arsenical hairpin (FlAsH) bioluminescence resonance energy transfer (BRET) reporters that monitor changes in arrestin conformation in live cells based on the BRET between an N-terminal RLuc donor and a FlAsH acceptor fluorophore located at each of seven different positions within the protein. We then determined the arrestin3 ‘conformational signature’ generated upon activation of six GPCRs that differ in their G protein-selectivity, trafficking pattern of GPCR-arrestin complexes, and capacity of the receptor to promote arrestin-dependent activation of ERK1/2. We found that GPCRs that bind arrestin3 stably and traffic with it into early endosomes, i.e. angiotensin AT₁, vasopressin V₂, and parathyroid hormone PTH₁, produce similar FlAsH1-7 signatures, whereas the signatures produced by GPCRs that form transient GPCR-arrestin complexes confined to the plasma membrane, i.e. β₂adrenergic, α_1B adrenergic, and sphingosine 1-phosphate S1P₁, were different and more heterogenous. Receptors capable of arrestin-dependent ERK1/2 activation, i.e. AT₁, V₂, PTH₁ and α_1B, produced a characteristic decrease in the BRET signal detected by FlAsH acceptors in the C-terminal domain. Comparing the FlAsH signatures of wild type V₂ and β₂ receptors with those of chimeric V₂-β₂ct and β_2-V₂ct receptors that exhibit reversal of the arrestin3-binding and trafficking patterns, demonstrated that stable arrestin binding and arrestin-dependent ERK1/2 activation correlate with characteristic changes in arrestin conformation. These data suggest that upon GPCR binding, arrestin3 undergoes characteristic conformational rearrangements that relate to its downstream trafficking functions, and that a ‘conformational signature’ monitored by FlAsH BRET may be valuable for receptor and ligand classification.

Nothing to Disclose: MHL, KMA, TAM, JK, YKP, LML

OR08-3 16127 3.0000 A Arrestin3 Adopts GPCR-Specific Conformations That Correlate with Its Trafficking and Signaling Functions 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Growth Factor/Tyrosine Kinase/G Protein-Coupled Receptor/Intracelluar Signaling & Biology Saturday, June 21st 1:00:00 PM OR08 4737 11:30:00 AM New Insights in GPCR Structure and Function Oral

Rory Sleno^*, Dominic Devost, Darlaine Pétrin, Marc-André Bourassa and Terry Hébert
McGill University, Montreal, QC, Canada

Recently, a large number of G protein-coupled receptor (GPCR) structures have provided a wealth of information about different inactive and active receptor conformations. Comparison of active- and inactive-state structures has suggested specific conformational rearrangements which occur during receptor activation. However, these static images only provide a glimpse of the complexity of receptor conformational dynamics and are limited in their utility for drug discovery due to technical challenges of protein crystallization and the loss of the physiological milieu associated with their purification. Here, we propose a system to capture conformational rearrangements induced by ligand binding to GPCRs in live cells and how these might be modulated by partner receptors.

We have designed two panels of conformation-sensitive biosensors to capture structural rearrangements in the receptor for PGF2α (FP) or angiotensin II (AT1R). Our system incorporates intramolecular bioluminescence resonance energy transfer (BRET) between a C-terminally fused Renilla luciferase moiety and a targeted small fluorescent molecule (FlAsH) with binding sites engineered or “walked” into different positions in the third intracellular loop. These biosensors were validated for cell surface localization and signalling competency in that they were capable of activating ERK1/2 mitogen-activated protein kinase when treated with PGF2α or angiotensin II, respectively. Our sensors generate distinct FlAsH tag position-dependent BRET signals in the absence of ligand reflecting their ability to report conformation from different vantage points. We also demonstrate varying degrees of position-dependent BRET following stimulation with either PGF2α or angiotensin II.

Using these conformation-sensitive biosensors, we have investigated conformational rearrangements which occur in the context of a recently identified FP/AT1R heterodimer. FP- or AT1R-based biosensors were co-expressed with WT AT1R or FP respectively. Interestingly, we identified conformational changes in the FP biosensor in the presence of agonist for untagged AT1R which could be blocked by an antagonist for the AT1R but not FP. Inhibition of Gα_q by a small molecule inhibitor abolished the conformational change of the WT partner receptor. We have now begun to interrogate the possibility of feedback from downstream signalling as well as the role of specific G proteins on this transmitted conformational change. To our knowledge this is the first account of relayed conformational rearrangements between protomers of a heterodimer in intact cells.

Nothing to Disclose: RS, DD, DP, MAB, TH

OR08-4 13062 4.0000 A Monitoring Conformational Rearrangements of F Prostanoid-Angiotensin II Type 1 Receptor Heterodimers Using Bret-Based Biosensors 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Growth Factor/Tyrosine Kinase/G Protein-Coupled Receptor/Intracelluar Signaling & Biology Saturday, June 21st 1:00:00 PM OR08 4737 11:30:00 AM New Insights in GPCR Structure and Function Oral

Ashutosh Trehan^*¹, Emmi Rotgers¹, Eleanor Coffey², Jorma Toppari³, Ilpo T. Huhtaniemi⁴ and Adolfo Rivero-Müller¹
¹Institute of Biomedicine, University of Turku, Turku, Finland, ²Biocity, Åbo Akademi University, Turku, Finland, ³University of Turku, Turku, Finland, ⁴Imperial College London, London, United Kingdom

G protein-coupled receptors (GPCRs) constitute a family of cell surface receptors that have been highly targeted by the current generation of drugs, mainly because of their ability to transduce signals from a wide variety of extracellular stimuli such as odor, light, neurotransmitters and hormones, among others. Many GPCRs upon ligand activation, couple to Gαs leading to cyclic AMP (cAMP) production through adenylyl cyclase. Therefore, the development of assays to monitor cAMP production is crucial in studying signaling of GPCRs that couple via Gαs. Moreover, the use of primary cell cultures over established cell lines represent a better system to study the signaling properties of GPCRs, since they are physiologically more similar to their parent tissue. However, currently available methods for detecting cAMP, mainly from primary cell cultures, suffers from two major drawbacks (1) lack of kinetic measurements for cAMP production as the commonly used methods based on competitive ELISA require cell lysis and hence can only measure a single time point, and (2) high variability in measuring successive time points because of the use of different population of primary cells for different time points. Even though the use of fluorescent and luminescent cAMP sensors is becoming more common, their utility in primary cell cultures is limited due to poor transfection efficiency, requirement for extensive normalization controls and photobleaching. We have developed a cell-based bioassay to monitor the kinetics of cAMP production in primary cell cultures. The assay is based on co-culture of primary cells (donor cells) with a separate sensor cell line, which stably expresses a luminescent cAMP sensor (Glosensor 22F, Promega). The activation of GPCRs in primary cells causes cAMP production that is detected by the co-cultured sensor cells thereby causing a luminescent read-out of cAMP production. This co-culture setup leads to kinetic monitoring of cAMP production, reduces variability and also eliminates the need to transfect primary cells. The activation of GPCRs in primary cultures of rat cortical neurons and mouse granulosa cells was tested by this method. The optimization of assay for best signal-to-noise ratio was done by varying the number of sensor and primary cells, using phosphodiesterase inhibitors and by testing the necessity for cell-cell contact. The assay is robust, best performed at room temperature and does not require sophisticated humidified gas chambers for kinetic measurements up to 2 hours. The main utility of the assay lies in monitoring the kinetics of cAMP production in primary cell cultures and in comparing the kinetics of cAMP production from different treatment of the same cells as well as in testing cAMP production from cells of unknown origin.

Nothing to Disclose: AT, ER, EC, JT, ITH, AR

OR08-5 16819 5.0000 A Bioassay to Monitor cAMP Production in Primary Cell Cultures 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Growth Factor/Tyrosine Kinase/G Protein-Coupled Receptor/Intracelluar Signaling & Biology Saturday, June 21st 1:00:00 PM OR08 4737 11:30:00 AM New Insights in GPCR Structure and Function Oral

Kim Carol Jonas^*¹, Francesca Fanelli², Ilpo T. Huhtaniemi³ and Aylin Hanyaloglu¹
¹Imperial College London, London, United Kingdom, ²Univ of Modena & Reggio Emilia, Modena, MO, Italy, ³Imperial Coll of London, London, United Kingdom

G protein-coupled receptors (GPCRs) are the largest family of mammalian receptors, modulating most aspects of endocrine homeostasis. In vitro studies have demonstrated that GPCRs can associate to form dimers and higher order oligomers, acting to diversify receptor functionality. Using the luteinizing hormone receptor (LHR), we have demonstrated the first in vivo relevance of Class A GPCR dimerization by transactivation; targeted co-expression of ligand-binding deficient (LHR^B-), and signalling-defective (LHR^S-) LHR restored the gonadal function and fertility of male LHR knockout mice, showing that dimerisation is a physiologically relevant form of GPCR-mediated signalling. As transactivation is a complex and multifaceted form of signaling, the aim of this study was to mechanistically resolve transactivation at the single molecule level. Using our previously described transactivating LHR mutants, we have developed a multi-color super-resolution imaging approach to detect individual GPCR molecules using photoactivatable dyes and localization microscopy, we term PD-PALM. PD-PALM resolved the molecular composition of wild type (WT) LHR, and transactivating heteromeric LHR^B-/S- complexes to ~8nm. Both WT LHR and heteromeric LHR^B-/S- formed dimers and oligomers, however, heteromeric LHR^B-/S- favored association as oligomeric complexes. These hetero-oligomeric complexes were functionally affected by the ratiometric composition of LHR^B-: LHR^S-. Furthermore, these transactivating heteromers differentially signaled to Gαs and Gαq in a ligand-specific manner. Spatial analysis of individual LHR^B-/S- oligomeric forms by PD-PALM revealed specific spatial arrangements of protomers within trimers and tetramers that interestingly favored excess LHR^B-over LHR^S-. Structural modeling of LHR^B-/S- di/tri/tetramers strongly aligned with the spatial analysis by PD-PALM and identified TM interfaces within these specific arrangements. Combining both super-resolution and structural modeling, with functional analysis, provides an unprecedented molecular insight into how GPCR protomers communicate within individual di/oligomers to modulate their functionality, suggesting mechanisms of how one receptor subtype can fulfill multiple physiological requirements.

Nothing to Disclose: KCJ, FF, ITH, AH

OR08-6 16643 6.0000 A Single Molecule Analysis of GPCR Transactivation Via PD-PALM Reveals Oligomeric Complexes That Regulate Signal Sensitivity 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Growth Factor/Tyrosine Kinase/G Protein-Coupled Receptor/Intracelluar Signaling & Biology Saturday, June 21st 1:00:00 PM OR08 4737 11:30:00 AM New Insights in GPCR Structure and Function Oral

Hana Vakili^*, Yan Jin and Peter A Cattini
University of Manitoba, Winnipeg, MB, Canada

Growth hormone (GH) is a major metabolic homeostatic factor, and its levels are decreased in obese individuals. Exercise is a potent stimulus for GH secretion in healthy individuals and an effective physiological intervention in obesity to rescue the GH deficiency status. However GH levels decrease early in response to both excess caloric (food) intake and insulin release (hyperinsulinemia) before obesity occurs. The underlying mechanism responsible for this suppression of GH is not known. We used a ‘humanized’ transgenic mouse containing the intact human (h) GH gene locus (hGH/CS-TG) to examine whether a high caloric intake results in hyperinsulinemia and remodeling and down-regulation of the hGH gene promoter and synthesis. In addition, we assessed whether promotion of energy usage through physical activity interferes with the negative effect of excess caloric intake on hGH production. Four-week old male hGH/CS-TG mice were assigned to non-exercise and exercise (50 minutes swimming daily) groups and were fed either a high-fat (60 kcal%) or a low-fat (10 kcal%) diet for 3 days. Serum glucose, insulin, C-peptide and hGH levels were assessed by ELISA, and hGH RNA levels were assessed by quantitative polymerase chain reaction. Relative levels of histones H3/H4 hyperacetylation and recruitment of RNA polymerase II at the hGH promoter, were assessed as an indication of promoter accessibility/transcriptional status in pituitary cells by chromatin immunoprecipitation assay. The presence of a long-range interaction between the hGH promoter and a distal upstream enhancer required for efficient expression via ‘looping’ was assessed by Chromosome Conformation Capture (3C) assay. High fat diet for three days created a state of excess insulin but not hyperglycemia, without weight gain. Metabolic changes during excess caloric intake were associated with a decrease in both hGH synthesis and secretion. This was associated with decreases in histone H3/H4 hyperacetylation, RNA polymerase II occupancy and disruption of the chromatin ‘looping’, consistent with reduced hGH promoter function. ‘Exercise’ significantly muted the effect of excess caloric intake on insulin and C-peptide levels. There was also a corresponding blunting of the decrease in hGH promoter hyperacetylation, and gene expression/RNA levels seen in the absence of prescribed physical activity through preservation of ‘looping’. These observations provide the first evidence that metabolism and energy homeostasis target hGH synthesis through changes in the three-dimensional chromatin structure of the GH gene locus. A significant concept that may be derived from our study relates to the dynamic and flexible repression and reactivation of the hGH gene in response to the altered metabolic states, which may provide insight into the changes in GH availability as an early event in pathogenesis of obesity.

Nothing to Disclose: HV, YJ, PAC

OR03-1 11776 1.0000 A Dynamic Chromosomal Reconfiguration of the Human Growth Hormone Gene Locus in Response to Excess Caloric Intake and Physical Activity 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Neuroendocrinology and Pituitary Saturday, June 21st 1:00:00 PM OR03 4740 11:30:00 AM Factors Regulating HPA Axis Function Oral

Rocío Leal-Campanario^*¹, Juan Francisco Martin-Rodriguez², Ainara Madrazo-Atutxa², Victor D Ramos-Herrero¹, Eva Venegas-Moreno³, Agnès Gruart¹, Jose Maria Delgado-Garcia¹, David A Cano⁴ and Alfonso Leal-Cerro⁵
¹Division of Neurosciences, Pablo de Olavide University, Seville, Spain, ²Instituto de Biomedicina de Sevilla (IBiS), Consejo Superior de Investigaciones Científicas, Endocrinology Unit of Virgen del Rocío University Hospital, University of Seville, Sevilla, Spain, ³Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla & Endocrinology and Nutrition Unit, Virgen del Rocio, Seville, Spain, ⁴Endocrinology and Nutrition Unit, Institute of Biomedicine of Sevilla (IBiS), Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, Seville, Spain, ⁵Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío Seville, Sevilla, Spain

Growth hormone (GH) and IGF-I play a significant role in the structure and function of the developmental and mature brain. Moreover, neuroprotective and regenerative effects have been attributed to GH, and particularly IGF-I, in vitro and in vivo experimental models. Interestingly, chronic GH/IGF-I hypersecretion has been suggested to underlie cognitive deficits in acromegaly patients. In this research we aimed to test the hypothesis that chronic GH/IGF-I hypersecretion can result in cognitive deficits by studying cognitive functions in an animal model of chronic GH/IGF-I hypersecretion. Wistar Furth rats were s.c. injected with GH-producing GC cells to induce a GH-secreting tumor. Control rats were injected with PBS. In a group of tumor-implanted rats, tumor was surgically extirpated either 3 or 8 weeks after cell injection. GH and IGF-I levels returned to normal levels upon removal of the tumor. Learning and memory functions were assessed with associative learning tasks 12 weeks after cells implantation. The tests included appetitive operant conditioning and fear learning with the passive avoidance task. Tumor-bearing rats displayed better learning performance than both tumor-resected and control rats. Interestingly, rats in which the tumor was resected at 8 weeks showed better learning and memory performance than both control rats as well as rats in which tumor was resected at 3 weeks. To determine whether increased neurogenesis could underlie improved learning and memory performance, cell proliferation was analyzed by injecting rats with a daily dose of 5-bromo-2'-deoxyuridine (BrdU) (150 mg/kg) for 3 consecutive days and their brains dissected the day after the last BrdU injection for immunohistochemical analysis. Increased neurogenesis (higher number of BrdU-positive cells) in the dentate gyrus of the hippocampus was observed in both tumor-bearing rats and rats in which tumor was resected at 8 weeks compared with control rats. These results were confirmed by quantification of cells positive for doublecortin in the subgranular zone of the dentate gyrus. Altogether, our results suggest that chronic exposure to GH/IGF-I hypersecretion enhances cognitive function and that this effect remains even after GH/IGF-I levels are restored to normal values. Increased adult hippocampal neurogenesis could mediate the cognitive effects of GH/IGF-I hypersecretion.

Nothing to Disclose: RL, JFM, AM, VDR, EV, AG, JMD, DAC, AL

OR03-2 14382 2.0000 A Chronic Growth Hormone/IGF-I Hypersecretion Enhances Learning and Memory and Promotes Neurogenesis in Adult Rats 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Neuroendocrinology and Pituitary Saturday, June 21st 1:00:00 PM OR03 4740 11:30:00 AM Factors Regulating HPA Axis Function Oral

Joseph R Kurian^*¹, Ei Terasawa² and Jon E Levine¹
¹University of Wisconsin-Madison, Madison, WI, ²Univ of Wisconsin, Madison, WI

Puberty and reproductive function depend on the elevation of gonadotropin releasing hormone (GnRH) neuron activity. We previously found that increased activity of GnRH neurons is accompanied by epigenetic changes (1). Specifically, during neuronal maturation, GnRH mRNA levels rise while cytosine residues within the GnRH gene promoter are actively demethylated. Whether active DNA demethylation itself has an impact on neuron development and consequently reproductive function remains unknown. In these studies we aimed to determine whether Tet enzymes, which initiate the process of active DNA demethylation, influence neuronal function and reproduction. First, examining two GnRH neuronal cell lines with immature (GN11) and mature (GT1-7) neuronal characteristics, we found that expression of Tet1 and Tet2 is drastically lower in GN11 compared to the GT1-7 cells. Overexpression of Tet2 in GN11 cells increased GnRH mRNA levels and elevated mean GnRH peptide release in dynamic cultures. While glutamate alone had no impact, exposure of GN11 cells overexpressing Tet2 to glutamate increased GnRH mRNA to levels consistent with mature GT1-7 cells. Second, to evaluate the impact of Tet2 in vivo, we generated two separate sets of mice with selective disruption of Tet2 activity in GnRH (gTKO mice) and kisspeptin (kTKO) neurons. We found minimal impact on the timing of puberty in gTKO mice, perhaps due to developmental activation of the GnRH gene prior to Tet2 disruption. Notably, plasma lutenizing hormone (LH) levels were significantly lower in both male and female adult gTKO compared to WT animals, suggesting Tet2 might be necessary for the maintenance of GnRH neuron function in adulthood. In contrast, disruption of Tet2 in kisspeptin neurons had a more distinguishable influence on pubertal timing in male and female mice. Vaginal opening and regular estrus cycling was significantly delayed in kTKO females and the pubertal rise in plasma LH lagged for both male and female mice compared to WT littermates. Abnormal fecundity was not observed in female kTKO mice; however, for male kTKO animals the interval between pairing with a fertile female and litter birth was significantly longer than for WT males. Together, these studies implicate Tet2 as a mediator of epigenetic control over neuronal maturation and reproductive function. They further suggest that Tet2 activity is necessary for the maintenance of neuroendocrine control of gonadotropin release.

Nothing to Disclose: JRK, ET, JEL

OR03-3 13869 3.0000 A The Methylcytosine Dioxygenase Ten-Eleven Translocase-2 (Tet2) Enables Elevated GnRH Gene Expression and the Typical Development and Maintenance of Reproductive Function 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Neuroendocrinology and Pituitary Saturday, June 21st 1:00:00 PM OR03 4740 11:30:00 AM Factors Regulating HPA Axis Function Oral

Louise Cheryl Gregory^*¹, Simon James Rhodes², Khadija Nuzhat Humayun³, Miles Jonathan Levy⁴, James Greening⁵ and Mehul Tulsidas Dattani⁶
¹UCL Institute of Child Health, London, United Kingdom, ²Indiana Univ.-Purdue Univ. Indianapolis, Indianapolis, IN, ³Aga Khan University, Karachi, Pakistan, ⁴Leicester Royal Infirmary, Leicestershire, United Kingdom, ⁵Leicester Royal Infirmary, Leicester, United Kingdom, ⁶UCL GOS Institute of Child Health, London, United Kingdom

Introduction: The LHX4 gene encodes the LIM/homeobox protein (LHX4), which is a member of the LIM-homeodomain transcription factor protein family. These genes possess two specialized zinc-fingers termed the LIM domains; unique cysteine-rich zinc-binding domains. LHX4 is found in the nucleus and acts as a transcriptional regulator where it is involved in controlling differentiation and development of the pituitary gland and nervous system. To date, only incompletely penetrant or de novo, heterozygous mutations in LHX4 have been described in patients with variable combined pituitary hormone deficiencies (CPHD). Mice homozygous for LHX4 die shortly after birth from immature lungs that fail to inflate.

Objective/hypothesis: To investigate a cohort of patients with congenital hypopituitarism for mutations in LHX4.

Method: We screened 150 patients with CPHD (the vast majority having an ectopic posterior pituitary (EPP)) using PCR and direct sequencing analysis. Upon identification of any variants, control databases (1000 genomes, dbSNP, Exome Variant Server) were consulted for the change.

Results: We identified a novel homozygous missense mutation (c.377C>T, p.T126M) in two deceased male patients of Pakistani origin, born to non-consanguineous parents, with panhypopituitarism. Both sons had low set crumpled ears, small upturned nose with depressed nasal bridge, mid-facial hypoplasia, micropenis and undescended testes. The parents also had a daughter with a depressed nasal bridge. In spite of rapid commencement of hydrocortisone and thyroxine, all three children died within the first week of life. DNA analysis confirmed the presence of a homozygous p.T126M mutation, located in the LIM zinc-finger binding domain 2, predicted to alter protein-protein interaction. Additionally, we identified a novel heterozygous missense mutation (c.1009A>C, p.N337H) in a Caucasian female patient with isolated GHD (peak GH: 1.47ug/L) learning difficulties, obesity and an EPP. The mutation is in the C-terminal domain, possibly affecting protein folding. Both mutations are absent from control databases and are located at highly conserved residues.

Functional studies are currently underway in the form of reporter assays.

Conclusion: We report for the first time to our knowledge a novel homozygous mutation in LHX4 associated with a lethal phenotype; recessive mutations in LHX4 may be incompatible with life.

Nothing to Disclose: LCG, SJR, KNH, MJL, JG, MTD

OR03-4 12164 4.0000 A Novel Lethal Form of Hypopituitarism Associated with the First Recessive LHX4 Mutation 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Neuroendocrinology and Pituitary Saturday, June 21st 1:00:00 PM OR03 4740 11:30:00 AM Factors Regulating HPA Axis Function Oral

Jennifer Park¹, Zhiqiang Cheng¹, Jong Youl Kim², Chia-Ling Tu¹ and Wenhan Chang^*¹
¹University of California San Francisco, San Francisco, CA, ²University of California San Francisco

Adequate balances between mineral, skeletal, and energy metabolism are required to maintain a normal body growth. The hypothalamus (Hyp) regulates growth, skeletal development, and energy homeostasis by controlling autonomic activities and secretion of hormones in the pituitary gland (Pit). We detected robust expression of the extracellular Ca²⁺-sensing receptor (CaSR) in Hyp neurons. To test whether the CaSR, a putative receptor sensing changes in Ca²⁺ homeostasis, modulates neuroendocrine signals to control growth, skeletal, and energy metabolism, we generated ^NeuronCaSR^-/- mice with CaSR knockout (KO) targeted to neurons by breeding floxed-CaSR mice with transgenic Nestin-Cre mice. Genomic DNA, immunoblotting, and qPCR analyses confirmed ablation of CaSR gene, protein, and RNA, respectively, in Hyp, but not in Pit, liver, kidney, and bone. The KO mice were born in normal Mendelian ratios, but had smaller body sizes (by ≈25% in weights) and small undermineralized skeleton compared to floxed-CaSR control (Cont) mice at 2 weeks and 3 months of age. qPCR analyses of RNA from the bones of the KO mice showed reduced expression of IGF-1 (by 30%) and osteocalcin, a marker of mature osteoblast, and increased expression of early osteoblast marker, type I collagen, indicating a delay in skeletal development. In the KO mice, serum growth hormone (GH) and IGF-1 levels were reduced to <10% of Cont mice along with reduced expression of GH and GH-releasing hormone RNA by >60% in the Pit and Hyp, respectively, suggesting that a dysregulated Hyp-Pit-GH/IGF1 axis might have contributed to stunned growth and skeletal phenotypes of the KO mice. Despite their smaller body sizes, the KO mice have (1) increased visceral fats by ≈60%, as determined by DEXA and weights; (2) increased serum leptin levels by 2 folds; and (3) profound glucose intolerance, indicating dysregulated energy metabolism. Despite the elevated serum leptin levels, the expression of agouti-related protein (AGRP) and neuropeptide Y, which enhance feeding and decrease energy expenditure, were increased by 70 and 25%, respectively, while pro-opiomelanocortin (POMC), which suppresses feeding and increases energy expenditure, was decreased by ≈30% in the KO Hyp, indicating reduced sensitivity of POMC and AGRP neurons to leptin. Immunohistochemistry confirmed the expression of CaSR in the POMC and AGRP neurons. Additionally, in the KO mice, RNA levels for thyrotropin-releasing hormone and gonadotropin-releasing hormone were reduced by 35-60% in the Hyp and the expression of thyrotropin, follicle-stimulating hormone, and luteinizing hormone, was reduced by 40-60% in the Pit, indicating dysregulated Hyp-Pit-thyroid/adrenal and Hyp-Pit-Gonadal axes. Together, our data support a central role for neuronal CaSRs as a critical integrator of mineral, skeletal, reproductive, and energy metabolism by controlling hypothalamic functions.

Nothing to Disclose: JP, ZC, JYK, CLT, WC

OR03-5 16592 5.0000 A Neuronal Ca²⁺-Sensing Receptors (CaSRs) Modulate Growth, Energy Metabolism, and Skeletal Development By Controlling Hypothalamic Functions 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Neuroendocrinology and Pituitary Saturday, June 21st 1:00:00 PM OR03 4740 11:30:00 AM Factors Regulating HPA Axis Function Oral

Nastaran Foyouzi^*¹, Sally A Camper¹, Michele Moreira², Marcela M Franca², Aline P Otto², Fernanda de Azevedo Correa², Ivo J P Arnhold², Berenice B Mendonca², Qianyi Ma¹, Jun Z. Li¹, Qing Fang¹ and Luciani R S Carvalho³
¹University of Michigan, Ann Arbor, MI, ²Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo FMUSP, São Paulo, Brazil, ³University of São Paulo, Hospital das Clinicas, São Paulo, Brazil

Mutations in the homeodomain transcription factor OTX2 cause variable and incompletely penetrant effects on craniofacial development that can include the eyes, hypothalamus, and pituitary gland [1, 2]. Mouse studies demonstrate that genetic variation in multiple loci can suppress or enhance the features associated with OTX2 dysfunction, but the genes underlying these loci are unknown [3]. Multiple pieces of evidence support the idea that hypopituitarism results primarily from the critical role of OTX2 in development of the neural ectoderm that gives rise to the hypothalamus, pituitary stalk, and posterior lobe (neurohypophysis) [4]. A proband with hypopituitarism ((GH, TSH, LH/FSH and ACTH deficiency), polydactyly, and normal eyes was ascertained in a large Brazilian pedigree with 4 unaffected siblings, and Sanger sequencing of candidate genes revealed a novel heterozygous variant in OTX2 (p.H230L). This histidine is conserved across all vertebrate species and some in silico analyses predict that the leucine substitution is deleterious. To test this prediction we transfected heterologous (293T) and homologous GnRH neuronal cell lines (GT1-7) with expression vectors for OTX2 and/or the p.H230L variant and assessed transactivation of reporter genes with either a consensus OTX2 binding site or the GnRHR promoter. In both cell lines normal OTX2 produced robust activation of each reporter gene, while mutant OTX2 had no activity. Mixing experiments suggest that the OTX2 variant exerts a dominant negative effect on normal OTX2 function. This is consistent with the idea that the OTX2 variant (p.H230L) causes CPHD in this patient. The proband’s mother is a p.H230L carrier with short stature (-2.4 SD), and 2 unaffected siblings are carriers with normal stature (sister -1.7SD, brother -0.4 SD). All of these individuals have normal basal pituitary hormone levels. The presence of polydactyly in the father and the proband suggests the possibility of digenic disease. No other patients with OTX2 mutations are reported to have digital abnormalities. We carried out exome sequencing on several family members to identify variants in other genes that could contribute to the proband’s phenotype. No deleterious variants were detected in obvious candidate genes for hypopituitarism. Ongoing analysis may reveal candidate genes for polydactyly segregating in this family. Exome sequencing of other large families with incompletely penetrant effects of OTX2 mutations could identify modifier loci that influence the effects of OTX2 variation on normal craniofacial development and function. This study underlines the difficulty of predicting the clinical consequences of deleterious genetic variation.

Nothing to Disclose: NF, SAC, MM, MMF, APO, FDAC, IJPA, BBM, QM, JZL, QF, LRSC

OR03-6 16490 6.0000 A A Novel OTX2 Mutation, p.H230L, Causes Hypopituitarism with Incomplete Penetrance: Exome Sequencing to Identify Modifier Genes 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Neuroendocrinology and Pituitary Saturday, June 21st 1:00:00 PM OR03 4740 11:30:00 AM Factors Regulating HPA Axis Function Oral

Sarah J. Larkin^*¹, Veronica Angela Preda², Niki Karavitaki³, Ashley B Grossman⁴ and Olaf Ansorge¹
¹University of Oxford, Oxford, United Kingdom, ²Royal North Shore Hospital, Sydney, Australia, ³Oxford Center for Diabetes, Oxford, United Kingdom, ⁴Oxford Centre for Diabetes, Endocrinology & Metabolism

Mutations in BRAF and CTNNB1 genetically distinguish papillary and adamantinomatous craniopharyngiomas

Craniopharyngiomas are epithelial, sellar tumours comprising two subtypes: adamantinomatous (aCP) and papillary (pCP). The adamantinomatous form occurs predominantly during childhood whereas the papillary form is more common in adults. aCPs often contain mutations in CTNNB1, encoding β-catenin: a component of the adherens junction and mediator of Wnt signaling. The tumorigenic mechanism underlying pCPs is not well understood. In a large series of craniopharyngiomas (61 aCP, 25 pCP) we show that the adamantinomatous subtype harbours mutations in CTNNB1 in 44% of cases sequenced, while the papillary subtype has BRAF V600E mutations in 91% of cases sequenced, indicating that these lesions are genetically distinct with differing tumorigenic mechanisms, namely Wnt pathway (aCP) and MAPK pathway activation (pCP). An antibody to the BRAF V600E mutation (clone VE1) proved less reliable than sequencing for detection of BRAF V600E mutation, but for specimens with little epithelium, immunohistochemistry may prove useful. While CTNNB1 mutation - when present - is found throughout aCPs, translocation of β-catenin from membrane to cytosol and nucleus is restricted to small cell clusters near the invading tumour front. We observed translocated β-catenin in 100% of aCPs, occurring not only in cell clusters but also in individual cells scattered throughout the tumour. There was no disruption of the adherens junction in these tumours indicating that loss of junction integrity is not associated with β-catenin translocation or mutation. Translocation of β-catenin from membrane to cytosol and nucleus in aCPs and mutation in V600E in pCPs offers a potential marker for differential diagnosis. Furthermore the availability and clinical efficacy of mutant BRAF inhibitors presents an opportunity for directed therapy in patients with pCPs. While mutations in CTNNB1 and BRAF underlie tumorigenesis in the majority of craniopharyngiomas, the tumorigenic event in the remaining population is unknown.

Disclosure: NK: Principal Investigator, HRA Pharma. Nothing to Disclose: SJL, VAP, ABG, OA

OR09-1 16970 1.0000 A Mutations in BRAF and CTNNB1 Genetically Distinguish Papillary and Adamantinomatous Craniopharyngiomas 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Neuroendocrinology and Pituitary Saturday, June 21st 1:00:00 PM OR09 4742 11:30:00 AM Non-Functioning Pituitary Tumors and Craniopharyngioma: Pathology and Survival Oral

Hiroshi Nishioka^*¹, Ozgur Mete², Sylvia L. Asa³, Naoko Inoshita¹, Noriaki Fukuhara⁴, Kentaro Horiguchi⁵ and Shozo Yamada⁴
¹Toranomon Hospital, Tokyo, Japan, ²University Health Network, Toronto, ON, Canada, ³Univ Health Network, Toronto, ON, Canada, ⁴Toranomon Hosp, Tokyo, Japan, ⁵Chiba University Medical School, Chiba, Japan

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Objective

Clinically nonfunctioning pituitary adenomas (NFA) are histologically classified into null cell adenomas (NCA) and silent adenomas. NCA are considered to represent undifferentiated adenomas with no adenohypophysial hormone immunoreactivity and no specific adenohypophysial differentiation. In 2008, Asa et al proposed to add lack of transcription factors for each adenohypophysial cell lineage to the definition of NCA. The aim of the present study is to classify NFA using the new classification system and to detect clinicopathological features of each subtype.

Materials

Based on hormone immunostainings, 516 consecutive cases of NFA, operated on between 2008 and 2011, were initially classified as: 119 NCA, 300 silent gonadotroph adenomas (SGA), 51 silent corticotroph adenomas (SCA), and 46 other silent adenomas. In 119 cases of NCA, immunohistochemistry for transcription factors including SF-1, estrogen receptors (ER), Pit-1, and Tpit was performed.

Results

119 NCA showed lineage-specific differentiation as gonadotrophs (SF-1- and ER-positive), corticotrophs (Tpit-positive), or somatomammothyrotrophs (Pit-1-positive) in 78 cases, 36 cases, and 2 cases, respectively. Only three cases (0.6%) were true NCA, i.e. conventional NCA with no lineage-specific differentiation at the transcription factor level. The 36 ACTH-negative, Tpit-positive adenomas (7.0%) showed female preponderance (P<0.0001), were more frequently giant (P=0.0028) and were associated with cavernous sinus invasion (P<0.0001) compared with 378 SGA; these features were identical to those of the previously classified 51 cases SCA in this series.

Conclusions

Our results show that the use of pituitary transcription factors (SF-1, ER, Tpit, and Pit-1) prevents hormone-immunonegative adenomas from being mistakenly classified as NCA. Our data suggest that 30% of hormone-negative pituitary adenomas are SCA and share distinct clinicopathological features of ACTH-expressing SCA. This histological classification of NFA more accurately characterizes biological behavior and can predict postoperative prognosis.

Nothing to Disclose: HN, OM, SLA, NI, NF, KH, SY

OR09-2 11322 2.0000 A The Crucial Role of Pituitary Transcription Factors in the Accurate Classification of Hormone-Negative Nonfunctioning Pituitary Adenomas 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Neuroendocrinology and Pituitary Saturday, June 21st 1:00:00 PM OR09 4742 11:30:00 AM Non-Functioning Pituitary Tumors and Craniopharyngioma: Pathology and Survival Oral

Laura C. Hernandez Ramirez^*¹, Plamena Gabrovska², Judit Dénes¹, Giampaolo Trivellin³, Serban Radian⁴, Daniel Tilley¹, Francesco Ferraù², Scott Alexander Akker⁵, Ashley B. Grossman⁶, Mônica Gadelha⁷, Márta Korbonits¹ and The International FIPA Consortium²
¹Barts and The London School of Medicine, Queen Mary University of London, London, United Kingdom, ²Barts and The London School of Medicine, London, United Kingdom, ³National Institutes of Health (NIH), Bethesda, MD, ⁴William Harvey Research Institute, Barts and The London School of Medicine, Queen Mary University of London, London, United Kingdom, ⁵St Bartholomew's Hospital, London, United Kingdom, ⁶University of Oxford, Oxford, United Kingdom, ⁷Clementino Fraga Filho University Hospital, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil

Introduction: The variable penetrance displayed by germline AIP mutations (AIPmut) could theoretically be explained by mutation-specific phenotypes or by other disease-modifying genes. Somatic GNAS1 mutations are frequent in sporadic somatotropinomas, and are associated with a relatively benign phenotype. In contrast, an apparent correlation between the germline FGFR4 G388R variant and somatotroph adenoma size and GH levels has been described. We evaluated the influence of the type of AIP mutations and these two disease-modifying genes on the clinical features of pituitary adenoma (PA) patients in a large cohort of familial isolated pituitary adenoma (FIPA) and simplex AIPmut positive patients.

Methods: We studied 37 AIPmut positive FIPA families, including 113 patients and their unaffected relatives, and 34 apparently sporadic young-onset (≤30 years) PA patients with AIPmut. DNA from 22 AIPmut positive paraffin-embedded somatotropinomas was screened for GNAS1 mutations. Additionally, 98 AIPmut positive patients and 108 unaffected AIPmut carriers were screened for FGFR4 G388R.

Results: Of the 31 different AIPmut detected (10 not previously published), 70.9% were predicted to produce a truncated protein. Patients with truncating mutations had a younger age at onset (median 16 [IQR15-25] vs. median 22 [IQR17.3-27.8], P<0.01), and at diagnosis (median 21 [IQR 16-30] vs. median 27 [IQR 20.8-37], P<0.01) than those with non-truncating mutations, concluding that AIPmut truncating mutations cause an earlier disease onset in FIPA. GNAS1 mutations were absent in all the tumors studied. The absence of GNAS1 mutations in AIPmut positive adenomas is concordant with the relative resistance to somatostatin analog therapy described in this setting. Ninety-four (45.6%) of the screened individuals were hetero- or homozygous for FGFR4 G388R; the genotype distribution was similar between patients and carriers. Age at onset/diagnosis, tumor size and extrasellar invasion were not significantly different between GG and GR/RR individuals. The FGFR4 G388R SNP does not seem to influence the disease penetrance or to confer additional features of aggressiveness in AIPmut positive FIPA.

Conclusions: Younger age of onset is characteristic in truncating AIPmut patients compared to those with non-truncating mutations. No somatic GNAS1 mutations are present in AIPmut somatotroph adenomas. The FGFR4 G388R variant does not influence the penetrance or clinical features of AIP-related pituitary adenomas.

Disclosure: ABG: Advisory Group Member, Novartis Pharmaceuticals, Advisory Group Member, Ipsen, Advisory Group Member, HRA Pharma, Advisory Group Member, Viropharma. MG: Collaborator, Novartis Pharmaceuticals, Medical Advisory Board Member, Novartis Pharmaceuticals, Collaborator, Ipsen, Collaborator, Pfizer, Inc., Principal Investigator, Novartis Pharmaceuticals, Researcher, Novartis Pharmaceuticals, Researcher, Pfizer, Inc.. MK: Researcher, Pfizer, Inc., Researcher, Novartis Pharmaceuticals, Researcher, Syntaxin, Consultant, Pfizer, Inc., Consultant, Chiasma, Consultant, Sanofi, Consultant, Syntaxin. Nothing to Disclose: LCH, PG, JD, GT, SR, DT, FF, SAA, TIF

OR09-3 13830 3.0000 A Pituitary Adenomas Harboring AIP Mutations Exhibit Phenotype-Genotype Correlation, but No Association with the Germline FGFR4 G388R Variant or Somatic GNAS1 Mutations 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Neuroendocrinology and Pituitary Saturday, June 21st 1:00:00 PM OR09 4742 11:30:00 AM Non-Functioning Pituitary Tumors and Craniopharyngioma: Pathology and Survival Oral

Mark J McCabe^*¹, Mark J Cowley¹, Jiang Tao¹, Kerith-Rae Dias¹, Tanya Thompson¹, Marcel E Dinger¹ and Ann I McCormack²
¹Garvan Institute of Medical Research, Sydney, Australia, ²St. Vincent's Hospital, Sydney, Australia

Background: In the past few years, new genes involved with familial predisposition to pituitary tumour development have been recognised, including AIP and SDHx. In routine clinical care, these genes are tested individually, guided by clinical presentation with significant cost and efficiency implications. These factors are likely to underestimate the occurrence of familial pituitary tumour predisposition, commonly thought to account for 5% of all pituitary tumours. Furthermore, the clinical management of aggressive pituitary tumours is challenging, particularly when tumours exhibit resistance to standard hormonal agents and temozolomide.

Methods: We have developed a custom next generation sequencing (NGS) panel (Roche/Nimblegen) which contains the 8 known familial pituitary tumour genes (MEN1, AIP, PRKAR1A, p27, SDHA-D), plus 25 genes implicated in embryonic pituitary development and a further 267 genes that have been implicated in various cancers and cancer-related pathways (~0.9 Mb target sequence). Subjects recruited for testing include those under the age of 40 with a sporadic pituitary tumour, or patients with a family history of pituitary tumours or other endocrine neoplasia. DNA extracted from blood is interrogated using our 300-gene panel (Illumina HiSeq 2500 sequencing). Raw sequencing data was analysed by a custom bioinformatic pipeline, with mutations being functionally assessed in silico. In addition, whole exome and transcriptome sequencing was applied to DNA and RNA extracted from blood and tumour of a patient with a highly aggressive ACTH-secreting pituitary tumour, who had failed all standard treatments including temozolomide.

Results: In this ongoing study, sequence analysis using our gene panel detected three patients from 12 tested, with germline mutations in AIP (p.F269F, p.A299V, p.R106C) and verified by Sanger sequencing. In addition, RNA from the aggressive pituitary tumour was found to have high expression of the VEGF signalling pathway which may indicate suitability for trial of Bevacizumab, an inhibitor of VEGF-A.

Conclusions: We have demonstrated the clinical utility of NGS in patients with pituitary tumours. Use of a comprehensive gene panel allows concurrent testing of multiple genes, and potentially exploration of new predisposition genes and otherwise unrecognised clinically significant parallel genomic variations. Whole exomic and RNA expression analyses may allow new treatment targets to be identified.

Nothing to Disclose: MJM, MJC, JT, KRD, TT, MED, AIM

OR09-4 15576 4.0000 A Next Generation Sequencing: Towards a New Clinical Frontier in the Diagnosis and Management of Pituitary Tumours 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Neuroendocrinology and Pituitary Saturday, June 21st 1:00:00 PM OR09 4742 11:30:00 AM Non-Functioning Pituitary Tumors and Craniopharyngioma: Pathology and Survival Oral

Daniel S Olsson^*¹, Eva Andersson², Ing-Liss Bryngelsson³, Anna G Nilsson¹ and Gudmundur Johannsson¹
¹Department of Endocrinology, Sahlgrenska University Hospital, Gothenburg, Sweden, ²Sahlgrenska University Hospital, Gothenburg, Sweden, ³Örebro University Hospital, Örebro, Sweden

Excess mortality has been described in adult patients with craniopharyngioma (CP) in small retrospective studies at tertiary care centers. Our aim was to investigate mortality, morbidity and incidence rate in CP patients and to examine the influence of age at diagnosis and hypopituitarism on the outcome in a nationwide register-based population study.

CP patients were identified and followed in national health registers in Sweden. Search criteria for CP were tested and validated in 28% of the total population where detailed patient records were accessible. Standardized mortality ratios (SMRs), standardized incidence ratios (SIRs) and annual incidence rates were calculated.

Included in the analysis were 307 CP patients (151 men, 156 women) diagnosed between 1987 and 2011. Mean follow-up time was 9 years (range 0-25). Patients with childhood-onset CP (COCP) and adult-onset CP (AOCP) were 106 and 201, respectively. Hypopituitarism was reported in 250 patients, 110 patients had diabetes insipidus (DI) and 54 patients had neither.

Mean annual incidence was 1.7/million inhabitants. The observed number of deaths during the study was 54 compared to the expected number of 14, resulting in an SMR of 3.8, 95% CI 2.9-5.0 (men: 3.2, CI 2.2-4.7: women: 4.9, CI 3.2-7.2). SMR for COCP of 17 (CI 6.3-37) was significantly higher than for AOCP, 3.5 (CI 2.6-4.6). SMR in patients with hypopituitarism was 4.3 (CI 3.1-5.8), with DI 6.1 (CI 3.5-9.7) as compared to 2.7 (CI 1.4-4.6) in patients without hypopituitarism and DI. SMR due to cerebrovascular diseases was increased, 5.1 (CI 1.7-12). The mortality was also increased in patients with prior radiation therapy (SMR 5.9, CI 2.9-11). Average years of life lost was 14.6 for all, 55 for COCP patients and 9.6 for AOCP patients. SIRs for all patients were increased for type 2 diabetes mellitus (6.3, CI 3.8-8.0), cerebral infarctions (7.1, CI 5.0-9.9), fractures (2.1, CI 1.4-3.0) and infections leading to hospitalization (5.9, CI 3.4-9.4). SIR for malignant tumors was not increased, 1.3 (CI 0.8-2.1).

This first population-based study of CP patients demonstrated an excess mortality ratio in CP patients that was markedly higher in COCP than in AOCP. Prior radiotherapy and anterior and posterior pituitary hormone deficiency had a negative impact on the prognosis. CP patients suffered from an increased disease burden of type 2 diabetes mellitus, cerebral infarctions, fractures and severe infections. These findings demand a substantial improvement in the treatment and follow-up of CP patients with the aim to prevent early death.

Disclosure: DSO: Speaker, Pfizer, Inc.. AGN: Consultant, Viropharma. GJ: Speaker, Pfizer, Inc., Speaker, Novo Nordisk, Speaker, Otsuka, Consultant, Viropharma, Consultant, Astra Zeneca. Nothing to Disclose: EA, ILB

OR09-5 13467 5.0000 A Mortality and Morbidity in 307 Patients with Craniopharyngioma – a Population Based Study 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Neuroendocrinology and Pituitary Saturday, June 21st 1:00:00 PM OR09 4742 11:30:00 AM Non-Functioning Pituitary Tumors and Craniopharyngioma: Pathology and Survival Oral

Daniel S Olsson^*¹, Gudmundur Johannsson², Ing-Liss Bryngelsson³, Penelope Trimpou², Anna G Nilsson¹ and Eva Andersson²
¹Department of Endocrinology, Sahlgrenska University Hospital, Gothenburg, Sweden, ²Sahlgrenska University Hospital, Gothenburg, Sweden, ³Örebro University Hospital, Örebro, Sweden

Adult hypopituitarism due to various causes, including non-functioning pituitary adenomas (NFPAs), has been associated with an excess risk of death. The aim was to investigate mortality in NFPA patients and to examine whether gender, hormonal deficiencies and other clinical characteristics influenced the outcome in a nationwide register-based population study.

NFPA patients were identified and followed in national health registers in Sweden. Search criteria for NFPA were tested and validated in 17% of the total population where detailed patient records were accessible. Standardized mortality ratios (SMRs) with 95% confidence intervals (CI) and annual incidence rates with means and 95% CIs were calculated.

Included in the analysis were 2795 patients (1502 men, 1293 women), diagnosed with NFPA between 1987 and 2011. Mean (±SD) age at diagnosis was 58±17 years (men 60±15; women 56±18; p-value <0.001). Hypopituitarism was reported in 1500 patients and diabetes insipidus (DI) in 145 patients. Mean follow-up time was 6 years (range 0-25) with 20 139 patient-years included in the analysis.

Mean annual incidence of NFPA in Sweden was 20.3 (CI 18.8-21.9) cases per million inhabitants. The incidence was significantly higher in men compared to women (21.8, CI 19.7-23.9 vs. 18.9, CI 17.8-19.9; p-value=0.03). The observed number of deaths during the study was 473 compared to the expected number of 431, resulting in a SMR of 1.10, CI 1.00-1.20 (men: 1.00, CI 0.88-1.12; women: 1.29, CI 1.11-1.48). SMR for patients with onset of NFPA ≤40 years of age was 2.68 (CI 1.23-5.09). SMR for patients with hypopituitarism was 1.06, CI 0.94-1.19 (men: 0.94, CI 0.81-1.08; women 1.38, CI 1.12-1.66) and for patients with DI 1.71, CI 1.07-2.58 (men: 1.26, CI 0.60-2.31; women 2.43, CI 1.26-4.25), respectively. SMRs due to cerebrovascular and infectious diseases were increased to 1.73 (CI 1.34-2.19) and 2.08 (CI 1.17-3.44), respectively, whereas death caused by ischemic heart disease was not increased (SMR 1.09, CI 0.88-1.34). SMR for malignant tumors was decreased to 0.76 (CI 0.61-0.94). Mortality in patients with one pituitary surgical treatment was normal (SMR 1.01, CI 0.84-1.20), but increased in patients treated with radiation therapy (2.67, CI 1.79-3.84).

This nationwide study of patients with NFPA demonstrated an excess mortality ratio, mainly due to increased deaths from cerebrovascular and infectious diseases. The worst prognosis was seen in women, patients with early onset of the disease, patients with DI and those who had received radiation therapy.

Disclosure: DSO: Speaker, Pfizer, Inc.. GJ: Consultant, Astra Zeneca, Consultant, Viropharma, Speaker, Otsuka, Speaker, Novo Nordisk, Speaker, Pfizer, Inc.. AGN: Consultant, Viropharma. Nothing to Disclose: ILB, PT, EA

OR09-6 13474 6.0000 A Women and Young Patients with Non-Functioning Pituitary Adenoma Have an Excess Mortality – a Nationwide Study Based on More Than 20 000 Patient-Years 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Neuroendocrinology and Pituitary Saturday, June 21st 1:00:00 PM OR09 4742 11:30:00 AM Non-Functioning Pituitary Tumors and Craniopharyngioma: Pathology and Survival Oral

Leon S Farhy^*, Ralf Manfred Nass, Jianhua Liu, Suzan S Pezzoli, Bruce D Gaylinn and Michael O Thorner
University of Virginia Health System, Charlottesville, VA

Background.Ghrelin is a gut peptide that regulates GH, appetite and metabolism. Its primary source, the stomach X/A-cells, co-secrete two main forms: acyl-ghrelin (AG) and desacyl-ghrelin (DG). Both increase before meals and at midnight on a typical fed day, AG decreases with long-term fasting [1], and AG and DG are inhibited by insulin [2]. AG regulation may depend also on the time elapsed from the last meal, since ghrelin acylation decreases with fasting [3]. Here, we test whether under low insulin conditions, total ghrelin (TG=AG+DG) is still controlled by insulin and also by a separate process of progressive secretion decline.

Methods. Seven men, age (mean±SD) 21±2.9yr; BMI 22±2.9kg/m² were studied twice on the Clinical Research Center. At 8 AM after an overnight fast, blood was sampled every 10 min for 26 hr for AG and DG using an in-house two-site sandwich assay. On one (fed) admission, 3 meals were served at 8 AM, 1 PM and 6 PM. No meals were served on the other (fasting) admission. Changes in circulating ghrelin were assessed during 6-hr periods: P1 (8 AM-2 PM); P2 (2 PM-8 PM); P3 (8 PM-2 AM); P4 (2 AM-8AM). To minimize the confounding effect of meals, periods P1 and P2 on the fed admission were not studied. Ghrelin regulation was analyzed by testing the ability of 3 mathematical models to describe the changes in TG. Model M_I includes suppression by insulin and model M_D assumes a secretion decline as time progresses. A combined model, M_ID, assumes suppression by insulin and separate gradual decline with time. Two information criteria (IC) were used to compare model performance.

Results. In the fed admission AG declined overnight (P3 vs. P4: 32.6±9.8 vs. 23.5±6.28 pg/mL; p=0.035). DG and TG decreased, but not significantly (DG: 70.0±16.70 vs. 67.2±14.74 pg/mL, p=0.62; TG: 102.4±24.02 vs. 90.2±18.42 pg/mL, p=0.18). During the fasting admission, AG declined 3 fold: 28.2±9.67 (P1), 28.3±10.47 (P2), 18.9±6.98 (P3), 9.5±3.81 pg/mL (P4), p=0.002; DG declined 1.3 fold: 83.7±23.70 (P1), 80.3±22.76 (P2), 79.0±22.60 (P3), 65.2±17.56 pg/mL (P4), p=0.02; and TG decreased 1.5 fold: 112.4±27.58 (P1), 106.6±26.70 (P2), 96.41±25.55 (P3), 75.5±19.07 pg/mL (P4), p=0.0003. Model M_I was the worst performer with higher IC values than M_D and M_ID (p<0.05). M_D was outperformed by M_ID during the fasting admission (p=0.02) and had one of the two IC values lower during the fed admission (p=0.02). Overall, model M_IDwas the best performer and explained 64.4% of the variance of TG.

Conclusions. Our findings support the hypothesis that the changes in circulating total ghrelin depend jointly on inhibition by insulin (even under low-insulin conditions) and on a separate process of progressive secretion decline. This process may depend on the time elapsed since the last meal and may be also influenced by the circadian clock.

Nothing to Disclose: LSF, RMN, JL, SSP, BDG, MOT

OR05-1 14874 1.0000 A Evidence for Joint Regulation of Circulating Total Ghrelin By Insulin Suppression and a Separate Process of Secretion Decline in Healthy Young Men 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Obesity, Adipocyte Biology and Appetite Saturday, June 21st 1:00:00 PM OR05 4749 11:30:00 AM Ghrelin and GHRH in Human Obesity Oral

Richard C Brown¹, Anna L Hopkins¹, Charlotte L Lewis¹, Irina A Guschina¹, Helen C Christian², Jeffrey S Davies³ and Timothy Wells^*¹
¹Cardiff University, Cardiff, United Kingdom, ²University of Oxford, Oxford, United Kingdom, ³Swansea University, Swansea, Wales, United Kingdom

Although unacylated ghrelin (UAG) does not activate the growth hormone secretagogue receptor (GHS-R) (1), it does exert a distinct spectrum of biological actions. For example, unlike acylated ghrelin (AG), which promotes adipogenesis in most depots, UAG stimulates adipogenesis in bone marrow (2), but not in intra-abdominal white adipose tissue (WAT) (3). Since a receptor mediating these actions of UAG has not been identified, we investigated the interaction of UAG with GHS-R in bone marrow in greater detail.

As previously shown in rats (2), intra-bone marrow (ibm) infusion of AG and UAG induced adipogenesis in wild-type (WT) mice, increasing the number of tibial marrow adipocytes by 90 and 68% respectively (p<0.001 & 0.05). Surprisingly, these effects of AG and UAG were abolished in loxTB-GHS-R (GHS-R-null) littermates (4), AG- and UAG-treated mice having 100 and 88% of marrow adipocytes in vehicle-treated animals. Since this suggested that UAG may be acylated in bone marrow, we used capillary gas chromatography with flame ionization detection to reveal that isolated tibial marrow adipocytes contain observable amounts of octanoic acid, the substrate used for activation. In addition, fluorescence immunohistochemistry demonstrated that, unlike stomach (which expressed the activating enzyme, ghrelin O-acyl transferase (GOAT), but not the adipocyte-specific marker, PPARγ) and intra-abdominal WAT (which expressed PPARγ, but not GOAT), tibial marrow adipocytes show prominent co-expression of both PPARγ and GOAT. Immunogold electron microscopy revealed that GOAT immunoreactivity was located in the membrane of the lipid trafficking vesicles and the plasma membrane of tibial marrow adipocytes. Finally, although a tibial ibm infusion of UAG increased adipocyte number by 120% in WT littermates (p<0.05), this effect of UAG was completely abolished in GOAT-null mice (5).

Thus, our data demonstrate that the adipogenic effect of UAG in tibial marrow is dependent upon acylation by GOAT and subsequent activation of GHS-R. This suggests not only that GOAT may acylate extracellular UAG, but implies that the local expression of the elements of this hormone activation system, together with the relative physico-chemical properties of AG and UAG, may determine the different activity spectra of these two hormones.

Nothing to Disclose: RCB, ALH, CLL, IAG, HCC, JSD, TW

OR05-2 14853 2.0000 A The Adipogenic Action of Unacylated Ghrelin in Murine Bone Marrow Is Mediated By Ghrelin O-Acyl Transferase and the Growth Hormone Secretagogue Receptor 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Obesity, Adipocyte Biology and Appetite Saturday, June 21st 1:00:00 PM OR05 4749 11:30:00 AM Ghrelin and GHRH in Human Obesity Oral

Philippe Zizzari, Yacine Chebani, Khadidja Chettab, Marie Pastor, Marie Korostelev, Jacques P Epelbaum, Virginie Tolle and Jacques Pantel^*
INSERM UMR-S 894, Université Paris Descartes, Paris, France

The deciphering of the physiological importance of the GH secretagogue receptor (Ghsr), a G protein-coupled receptor (GPCR) depicted as the sole receptor of the pleiotropic hormone ghrelin, was initially compromised by the modest phenotype observed in Ghsr^-/- animals. This lack of a robust response to total loss of Ghsr may result from developmental compensatory signals. Still, the description of rare mutations in the GHSR partially affecting receptor function in patients with short stature or partial GH-deficiency allowed us to ascertain its role in somatic growth (1-2). Thus, we hypothesized that a recently generated line of rats carrying a premature termination codon in the Ghsr gene (Q343X) might be an ideal model to explore Ghsr physiology. Prior to exploring the phenotype of these animals, we deciphered the mechanism of action of this specific mutation using HEK293 cells transfected with HA-tagged and/or eYFP- or rLuc-chimeras of the wild-type (WT) or mutant rat Ghsr. The Q343X mutation does not impair cell surface expression of Ghsr. The functional analysis of this GPCR in calcium flux experiments revealed that, compared to the WT isoform, the mutant isoform has an increased potency as well as a major increase of the maximal response in the presence of increasing concentrations of ghrelin (EC₅₀= 1.5±0.5 nM and 0.5±0.2 nM; E_max= 25±0.6% and 37±0.8 %, respectively, Mean± sem, n=2). Similar patterns of response were also found in reporter gene assays depicting the serum-response elements (SRE) or cAMP-response elements (CRE) pathways. Of note, when subjected to a maximal dose of ghrelin, internalization of the mutated receptor is blunted compared to the WT receptor. Finally, bioluminescence resonance energy transfer (BRET) experiments suggest that this results from a severe impairment of the ligand-induced β-arrestin₂ recruitment. Overall, these in vitro observations are supportive of a gain-of function associated to the Ghsr Q343X mutant allele. Interestingly, our very first in vivo data are consistent with an increased body weight and chow intake in adult rats carrying the mutated allele. To our knowledge, these mutant rats represent the first description in mammals of a functionally significant gain-of-function mutation in the Ghsr. This model therefore deserves an extensive phenotypic evaluation.

Nothing to Disclose: PZ, YC, KC, MP, MK, JPE, VT, JP

OR05-3 14364 3.0000 A Putative Gain-of-Function in Rats Carrying the Ghsr Q343X Mutation 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Obesity, Adipocyte Biology and Appetite Saturday, June 21st 1:00:00 PM OR05 4749 11:30:00 AM Ghrelin and GHRH in Human Obesity Oral

Ralf Manfred Nass^*¹, Leon S Farhy¹, Jianhua Liu¹, James Patrie², Suzan S Pezzoli¹, Bruce D Gaylinn¹ and Michael O Thorner¹
¹University of Virginia Health System, Charlottesville, VA, ²University of Virginia, School of Medicine, Charlottesville, VA

Background: Acyl-ghrelin is a 28-amino acid peptide released from the stomach. It has both orexigenic effects and stimulates growth hormone (GH) release. In-vitro, an increase in beta-1 adrenergic activity stimulates acyl-ghrelin release from cultured ghrelin cells (1). Similar studies in humans have not been reported. We tested the hypothesis that beta-1 adrenergic blockade suppresses circulating acyl-ghrelin concentrations.

Methods: Five healthy young men age (mean ± SD) 25.4 ± 5.1 yr; BMI 24.9 ± 2.6 kg/m² were studied in a single-blind, placebo-controlled study during 4 outpatient admissions on the Clinical Research Unit. The volunteers received each of the following interventions on separate admissions in random order at 0900h: a) atenolol (100 mg) tablet and s.c. glucagon (1 mg); b) placebo tablet and s.c. glucagon; c) atenolol and s.c. saline; d) placebo tablet and s.c. saline. Subjects also took atenolol or placebo at 0900h for 2 days prior to admission. During the admission blood was drawn every 10 min for 7 hr (0800h-1500h) for measurement of acyl-ghrelin, insulin and blood glucose (BG) concentrations. A moving average was computed for each intervention and the fold change from baseline and nadir was analyzed. A Wilcoxon matched pair test was used for statistical analysis; p<0.05 was considered statistically significant.

Results:

There was no significant difference between the mean baseline acyl-ghrelin (pg/ml) concentrations between placebo and atenolol treatments.
In response to glucagon, BG and insulin peaked and then declined. Acyl-ghrelin levels tended to decrease in parallel with the glucagon-stimulated increase in BG and insulin (p=0.125).
Atenolol had no impact on the decrease in acyl-ghrelin (fold change from baseline).
Acyl-ghrelin concentrations rebounded on the decline of BG and insulin; the rebound (fold change from nadir) showed a trend to higher values with glucagon/atenolol when compared to glucagon/placebo (p=0.0625).

Conclusions: Blocking the beta-1 adrenergic receptor did not change baseline acyl-ghrelin levels. Administration of glucagon resulted (indirectly) in an initial decrease followed by a rebound-like increase in acyl-ghrelin levels. There was a trend for higher rebound acyl-ghrelin levels during beta-1 adrenergic receptor blockade.

Nothing to Disclose: RMN, LSF, JL, JP, SSP, BDG, MOT

OR05-4 13312 4.0000 A Effect of Beta-1 Adrenergic Blockade on Acyl-Ghrelin Release in Healthy Young Adults 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Obesity, Adipocyte Biology and Appetite Saturday, June 21st 1:00:00 PM OR05 4749 11:30:00 AM Ghrelin and GHRH in Human Obesity Oral

JI-an Chen¹, Bobby O Guillory² and Jose Manuel Garcia^*³
¹Third Military Medical University, ²Baylor College of Med, Houston, TX, ³MEDVAMC/ Baylor Coll of Med, Houston, TX

Cachexia and muscle atrophy are common and often lethal consequences of cancer and, paradoxically, of chemotherapy administration. The novel hormone ghrelin has been proposed as a treatment for cachexia. However its mechanisms of action are not well-understood. We characterized the molecular pathways involved in muscle atrophy induced by tumor implantation and by cisplatin administration. We also determined the effects of ghrelin in these settings and the mechanisms mediating these effects in muscle.

We show here how multiple pathways interact and are involved in the development of cachexia; either induced by a tumor or, paradoxically, by the chemotherapeutic agent cisplatin. Ghrelin prevented the anorexia, fat loss, muscle loss and weakness induced by cisplatin or tumor. Activation of p38/C/EBPβ, myostatin, and inflammatory cytokines (in tumor-bearing animals) and a decrease in akt and Myogenin/myoD ultimately leads to increased proteolysis, and decreased muscle mass and strength. Ghrelin prevents muscle atrophy by decreasing inflammation, and by downregulating the p38/C/EBPβ/myostatin pathway and increasing akt phosphorylation and activating myogenin and myoD. These changes appear, at least in part, to target muscle cells directly and to be GHSR1a-independent.

Experiments in GHSR-1a KO animals suggest that although this receptor is necessary for ghrelin’s effect on food intake and adipose tissue, it only partially mediates ghrelin’s effect on muscle. Ghrelin also decrease mortality in tumor bearing animals, highlighting the clinical relevance of these findings.

In summary, we characterize the effect of ghrelin in different models of cancer-related cachexia and show how multiple pathways are modulated leading to the prevention of muscle atrophy, weakness and ultimately death.

Disclosure: JMG: Consultant, Helsinn Therapeutics, Investigator, Aeterna Zentaris, Investigator, Helsinn Therapeutics. Nothing to Disclose: JAC, BOG

OR05-5 16096 5.0000 A Ghrelin Prevents Cancer-Related Muscle Wasting through a Combination of Ghsr-Dependent and Independent Mechanisms 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Obesity, Adipocyte Biology and Appetite Saturday, June 21st 1:00:00 PM OR05 4749 11:30:00 AM Ghrelin and GHRH in Human Obesity Oral

Takara L. Stanley^*¹, Meghan N. Feldpausch¹, Jinhee Oh², Karen L Branch³, Hang Lee², Martin Torriani⁴ and Steven K. Grinspoon⁵
¹Massachusetts General Hospital and Harvard Medical School, Boston, MA, ²Massachusetts General Hospital, Boston, MA, ³Massachusetts General Hospital, ⁴Massachusetts General Hospital/ Harvard Medical School, Boston, MA, ⁵Massachusetts General Hospital/Harvard Medical School, Boston, MA

Background: Non-alcoholic fatty liver disease (NAFLD) is common in HIV-infected patients and is strongly associated with increased visceral adiposity. A growth hormone (GH) releasing hormone analogue, tesamorelin, increases endogenous pulsatile GH secretion and reduces visceral adiposity in HIV-infection, but its effects on liver fat are not known.

Objective: To determine the effects of tesamorelin on liver fat in HIV-infected individuals with increased visceral adiposity, with the hypothesis that tesamorelin will significantly reduce liver fat.

Methods: 50 HIV-infected, antiretroviral-treated men and women with abdominal fat accumulation were randomized in a 1:1 ratio to receive tesamorelin 2mg subcutaneously daily or identical placebo for 6 months in a double-blind trial. Primary endpoints were changes in visceral fat and in liver fat as measured by ¹H-magnetic resonance spectroscopy. Secondary endpoints included glucose and insulin sensitivity. Euglycemic hyperinsulinemic clamp was performed in 50% of randomly selected patients.

Results: At baseline, visceral fat and liver fat were strongly positively associated (ρ = 0.42, P = 0.003), and both showed strong associations with measures of glucose homeostasis and lipids. Both VAT (ρ = -0.43, P = 0.003) and liver fat (ρ = -0.44, P = 0.003) were negatively associated with baseline overnight mean GH; neither was associated with baseline IGF-1. Tesamorelin treatment increased endogenous GH secretion, as evidenced by significant increases in IGF-1 (Δ IGF-I Z score +1.1±0.3 vs. -0.1±0.2, tesamorelin (T) vs. placebo (P), p=0.004) and mean overnight GH (Δ +0.35 [0.15, 0.57] vs. -0.01 [-0.07, 0.06] ng/mL, T vs. P, p=0.0007). Tesamorelin significantly reduced VAT (Δ -34±9 vs. +8±11 cm², T vs. P, p=0.005) and liver fat (Δ -2.0 [-6.4, 0.1] vs. +0.9 [-0.6, 3.7] lipid-to-water %, T vs. P, p=0.004). Change in liver fat was associated with change in VAT (ρ = 0.31, P = 0.047) and HOMA-IR (ρ = 0.50, P = 0.001).

Fasting glucose increased in the tesamorelin group between baseline and two weeks (Δ 9±2 vs 2±3mg/dL; T vs. P, p=0.03) but was not different from baseline at 3 months (p=0.20) or 6 months (p=0.56). Mixed effects modeling showed no significant effects of tesamorelin on fasting glucose (p=0.94), fasting insulin (p=0.76), or HOMA-IR (p=0.48) after 6 months. There was a modest but statistically significant increase in HbA1c from baseline to 6 months (Δ 0.20±0.08 vs. 0.02±0.04%; p=0.04, T vs. P). In the clamp subgroup, insulin stimulated glucose uptake (M) significantly changed over the first three months in the tesamorelin group (ΔM -0.4±0.6 vs. +1.3±0.3 mg/kg/min, T vs. P, p=0.03), whereas the overall change from baseline was not significant at 6 months (0.5±0.8 vs. 0.7±0.6, T vs. P, p=0.78).

Conclusion: In HIV-infected individuals with visceral obesity, tesamorelin significantly reduces liver fat in conjunction with decreases in VAT.

Nothing to Disclose: TLS, MNF, JO, KLB, HL, MT, SKG

OR05-6 12198 6.0000 A Growth Hormone Releasing Hormone Analogue Reduces Liver Fat in HIV-Infected Patients 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Obesity, Adipocyte Biology and Appetite Saturday, June 21st 1:00:00 PM OR05 4749 11:30:00 AM Ghrelin and GHRH in Human Obesity Oral

Nasreen Alfaris^*¹, Jesse Chittams², Lisa Diewald², Marion Vetter³ and Thomas Wadden¹
¹University of Pennsylvania, philadelphia, PA, ²University of Pennsylvania, Philadelphia, PA, ³Univ of Pennsylvania, Philadelphia, PA

Little is known about the effects of intentional weight loss on sleep quality or sleep duration in obese individuals not selected for obstructive sleep apnea. This study examined the issue in 390 subjects (M age=51.5±11.5 yr, BMI=38.5±4.7 kg/m², 75.4% female) who participated in a 2-yr randomized trial of behavioral weight loss in primary care (POWER-UP study). As reported previously¹, subjects were randomized to: 1) Usual Care; 2) Brief Lifestyle Counseling (LC); or 3) Enhanced Brief LC, all of which were delivered by physicians and medical assistants in the primary care practices. The three interventions provided subjects different amounts of support to achieve the prescribed diet (1200-1800 kcal/d) and activity (180 min/wk) goals. This study reports changes at months 6 and 24 in weight, sleep quality and duration (assessed by the Pittsburgh Sleep Quality Index questionnaire [PSQI]), and mood, measured by the Patient Health Questionnaire-8 (PHQ-8). Data were analyzed by mixed effects general linear models. Changes in sleep and mood also were compared in subjects who lost ≥5% vs <5% of initial weight, regardless of original group assignment. At month 6, subjects in the three groups lost a mean (SEM) of 2.0±0.5, 3.5±0.5, and 6.6±0.5 kg, respectively; all three groups differed significantly from each other (p<0.05). There were no significant differences between groups in sleep duration, PSQI scores, or mood, although the latter two values declined (favorably) over time (p<0.05). At month 24, subjects in Enhanced Brief LC lost more weight than Usual Care (-4.6±0.7 vs -2.0±0.5 kg, p<0.05) and reported more favorable changes in sleep duration (+13.8±9.0 vs -9.0±9.0 min; p<0.05). There were no differences between groups on PSQI or mood scores, but both values declined (favorably) over time (p<0.05). When examining participants who lost ≥5% vs <5% of initial weight, regardless of original group assignment, at month 6 sleep increased by 21.6±7.2 min in the former group, compared with 1.2±6.0 min for those losing <5% (p<0.05). Similarly favorable changes, with greater weight loss, were observed on the PSQI (-1.6±0.2 vs -0.4±0.2, p < 0.001) and PHQ (-2.5±0.4 vs -0.1±0.3, p<0.0001). At month 24, however, only the differences between the two groups on mood (PHQ) remained significant (p<0.05), and at no time were significant differences observed between the two weight loss categories on sleep duration. The present findings indicate that losing ≥5% of initial weight is associated with significant short-term (6 month) self-reported improvements in sleep quality and mood. Further study is needed of the possible effects of weight regain, as observed in this study, in mitigating short-term improvements in sleep quality.

Nothing to Disclose: NA, JC, LD, MV, TW

OR07-1 13475 1.0000 A Effect of Behavioral Weight Loss on Sleep and Mood: Results from the POWER-up Trial 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Obesity, Adipocyte Biology and Appetite Saturday, June 21st 1:00:00 PM OR07 4750 11:30:00 AM Neuroscience of Human Obesity Oral

Kristen Eckstrand¹, Ronald Cowan¹, Brian Welch¹, Joel Kullberg², Robert Kessler³, Heidi Silver¹, Kevin Dean Niswender¹ and Malcolm Avison^*¹
¹Vanderbilt University Medical Center, Nashville, TN, ²Uppsala University Hospital, Uppsala, Sweden, ³University of Alabama Medical Center, Birmingham, AL

Increased BMI is associated with decreased availability of striatal dopamine (DA) D2 receptors (D2R). This has led to the suggestion that obesity, like classical drug addiction, may be driven, at least in part, by impaired behavioral self-regulation, arising from disruptions in striatal DA tone. The molecular and neural mechanisms underlying such a disruption, and their impacts on behavior remain poorly understood in the context of obesity, however.

We therefore used whole body fat-water (FW-MRI), functional MRI (fMRI), and F18-fallypride PET imaging to measure adipose tissue (AT) distribution, activation of neural circuitry subserving impulse control during a stop signal task (SST), and brain D2R availability respectively, in a cohort of right-handed obese (BMI=37.1 ± 0.7, range 30-39; n=48) volunteers (age 47.1 ± 1, range 30-39 yrs) with T2DM (HOMA-IR=7.8±0.8) who were otherwise healthy, and had no history of insulin therapy.

We hypothesized that increased adiposity, would be associated with decreased availability of striatal D2R, and/or altered activation of neural elements of impulse control in a stop signal task (SST) measured as the BOLD fMRI contrast between stop success and stop error (CON(SS>SE)), and that one or both of these contribute to increased impulsivity in obesity.

Increased visceral (VAT), but not total or subcutaneous, AT burden was associated with decreased striatal D2R availability (R2 = 0.50, p = 0.001), and decreased striatal activation during stopping (CON(SS>SE): R2 = 0.2, p = 0.07). Both striatal D2R and CON(SS>SE) were significant predictors of more impulsive responding (i.e. shorter median Go Response Time - mGRT): mGRT vs D2R, R2 = 0.43, p = 0.036; mGRT vs CON(SS>SE), R2 = 0.42, p = <0.001. After accounting for an independent impact of insulin resistance (Eckstrand et al., abstract submitted), there was a significant association of increased VAT burden with decreased mGRT (increased impulsivity) that was mediated by the VAT-associated reductions in striatal D2R and activation. Thus VAT, independent of total adiposity or insulin resistance, was a predictor of reduced striatal D2R availability and blunted striatal activation during response inhibition, associations that appeared to mediate a VAT-associated blunting of behavioral impulse control.

We believe these findings are the first to demonstrate a link between VAT burden and molecular and functional correlates of impulsivity in the human brain. Furthermore the nature of the association suggests that increased visceral adiposity, independent of BMI, is associated with poorer impulse control.

Disclosure: RC: Investigator, Novo Nordisk. HS: Investigator, Novo Nordisk. KDN: Investigator, Novo Nordisk. MA: Investigator, Novo Nordisk. Nothing to Disclose: KE, BW, JK, RK

OR07-2 16983 2.0000 A Molecular and Neural Bases for Heightened Impulsivity with Visceral Adiposity in Obesity Associated T2DM 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Obesity, Adipocyte Biology and Appetite Saturday, June 21st 1:00:00 PM OR07 4750 11:30:00 AM Neuroscience of Human Obesity Oral

Erin C Hanlon^*¹, Rachel Leproult², Esra Tasali³, Harriet de Wit², Kara Stuhr⁴, Cecilia J Hillard⁴ and Eve Van Cauter²
¹Univ of Chicago, Chicago, IL, ²University of Chicago, Chicago, IL, ³The University of Chicago, Chicago, IL, ⁴Medical College of Wisconsin, Milwaukee, WI

The endocannabinoid (EC) system, a pharmacotherapeutic target for obesity treatment, mediates the hedonic control of feeding, and modulates reward mechanisms. Over activation of the EC system could be involved in excessive hedonically driven eating and the risk of overeating associated with obesity. We examined the 24-h profile of (2-AG), and of its structural analog 2-oleoylglycerol (2-OG), which does not bind EC receptors, in healthy lean and obese subjects.

Sixteen healthy lean subjects, (5 women; age: 23 ±1yrs; BMI: 23.6±1.3kg/m²) and 11 obese but otherwise healthy subjects (7 women; age: 27 ±1yrs; BMI: 39.0±1.7kg/m²), were studied in the laboratory under controlled conditions of energy expenditure and caloric consumption. Blood sampling was performed for 24-h following two nights of normal sleep (2300-0730). Samples taken at 60-min intervals were assayed for levels of 2-AG and 2-OG by liquid chromatography-electrospray ionization-mass spectrometry (LC-ES-MS).

Both lean and obese individuals displayed circadian rhythms for 2-AG and 2-OG, with similar nadir and acrophase concentrations. However, the timing of the nadir was significantly delayed from mid-sleep in lean subjects to early morning in obese individuals (lean 0353 ± 32min vs. obese 0621 ± 39min, p = 0.0065). Moreover, the acrophase was also significantly delayed from early to late afternoon (lean 1223 ± 30min vs. obese 1805 ± 37min, p < 0.001). There was a significant correlation between BMI and the timing of both the nadir and the acrophase, such that the higher the BMI the later the timing of the nadir and acrophase (n=27; p = 0.0006 and p = 0.0035, respectively). When expressed as % of the 24-h mean, over the sleep period from 2300-0700 2-AG and 2-OG levels were higher in obese individuals than in the lean subjects (2-AG: obese 86.9 ± 5.2% vs lean 65.3 ± 4.3%, p = 0.004; 2-OG obese 95.8 ± 3.9% vs. lean 84.4 ± 3.3%, p = 0.035).

This study provides the first demonstration of a circadian rhythm of human plasma EC levels in obese individuals. Moreover, the delay in timings of both 2-AG and 2-OG nadir and acrophase in obese compared to lean individuals reveals that obesity is associated with a circadian misalignment that may contribute to overeating in the later part of the day and adverse metabolic consequences known to be associated with obesity.

Nothing to Disclose: ECH, RL, ET, HD, KS, CJH, EV

OR07-3 17049 3.0000 A Alterations in the Circadian Rhythm of Circulating Levels of the Endocannabinoid (EC) Receptor Ligand, 2-Arachidonoylglycerol (2-AG), in Obese Individuals 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Obesity, Adipocyte Biology and Appetite Saturday, June 21st 1:00:00 PM OR07 4750 11:30:00 AM Neuroscience of Human Obesity Oral

Peter Kühnen^*¹, Daniela Handke¹, Joachim Spranger², Antje Fischer-Rosinsky², Annette Grüters-Kieslich¹ and Heiko Krude¹
¹Charité - Universitätsmedizin Berlin Campus Virchow, Berlin, Germany, ²Charité - Universitätsmedizin Berlin Campus Mitte, Berlin, Germany

Background: POMC plays a major role in central body weight regulation. Recently we have shown in a candidate gene approach, that a POMC hypermethylation variant at the border of the intron2- 3´CpG island in obese children is significantly associated with obesity in children and adolescents. We could also demonstrate that this variant was present before the onset of obesity, is linked to an Alu element that most likely triggers the hypermethylation and that the hypermethylation seems to interfere with an enhancer that binds p300 ¹. Here we extended our epigenetic studies in the POMC locus and analysed the POMC DNA methylation in a second childhood cohort now with another independent method (pyrosequencing) and tested the hypothesis that also adult obese individuals harbour the hypermethylation variant more often compared to normal weight controls.

Methods: We tested the POMC DNA methylation in obese adults (n=92) and in normal weight individuals (n=101). The DNA methylation was analysed in DNA extracted from peripheral blood cells with a sodium-bisulfite based protocol and pyrosequencing.

Results: We could show that - based on pyrosequencing - we could reproduce our initial findings and observed a highly significant enrichment of the POMC hypermethylation variant in obese children compared to normal weight individuals. Moreover we observed now for the first time also a significant higher rate POMC hypermethylation in obese adults compared to age-and sex matched normal weight adult individuals (30,3% variant in obese versus 15,3% variant in normal weight).

Conclusion: Our finding of a highly significant enrichment of the POMC hypermethylation variant in obese children and also in obese adults represent the strongest factor that explain the individual risk for obesity since all so far described genetic variants have shown to have a much lesser impact on the individual BMI compared to the here described POMC locus hypermethylation.

Nothing to Disclose: PK, DH, JS, AF, AG, HK

OR07-4 15908 4.0000 A Pomc DNA Hypermethylation Variant in Highly Associated with Obesity in Children and Adults 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Obesity, Adipocyte Biology and Appetite Saturday, June 21st 1:00:00 PM OR07 4750 11:30:00 AM Neuroscience of Human Obesity Oral

Belinda S Lennerz^*¹, Elke Fröhlich-Reiterer², Ulrich Paetow³, Klaus-Michael Debatin⁴, Georgia Lahr⁵, Franziska Degenhardt⁶, Pamela Fischer-Posovszky⁷, Gudrun Weinhandel², Hans-Josef Boehles³ and Martin Wabitsch⁷
¹Ulm University, Ulm, Germany, ²University Hospital Graz, ³Johann Wolfgang von Goethe University, ⁴University Medical Center Ulm, ⁵University Hospital Ulm, ⁶University Bonn, ⁷University of Ulm, Ulm, Germany

Background: The prevalence of homozygous mutations in the leptin receptor (LEPR) gene was 3% in a cohort of patients with early onset extreme obesity1. 6 of the 8 affected children had consanguineous parents. Despite this report, LEPR mutations are not routinely tested for, and only 11 affected families are published in the literature.

Objective:Our aim is to raise awareness of this monogenetic form of obesity.

Methods:Over the past 12 months, three patients with early onset extreme obesity and mutations in the LEPR gene were presented to our pediatric obesity center and were gathered in a case series.

Results:

34 months old boy, BMI: 40 kg/m2 (+5.4 SDS). A new homozygous deletion with loss of function was identified: exon 3-20.
33 months old boy, BMI 30 kg/m2 (+4.4 SDS). Two published2,3 homozygous missense point mutations were identified: exon 8, c.946C>A and exon 14, c.1938G>T.
32 months old girl, BMI 45 kg/m2 (+5.5 SDS). A published1 homozygous mutation resulting in a deleterious frame shift was identified: Exon 5, c.461dupA (p.N154Kfs*3).

All three patients had in common:

extreme hyperphagia

dramatic weight gain in the first 6 months of life, surpassing the 97th weight percentile between 1 and 6 months of age

extreme obesity at the time of presentation (BMI > +4SDS)

normal-weight, consanguineous parents of Turkish ancestry

Conclusion: Together with the high reported prevalence rate1, this case series suggests that mutations in the LEPR gene are a common cause of early onset extreme obesity. If no other causes are identified, genetic testing of these patients should include sequencing of the LEPR Gene, especially when consanguinity is present.

This research was funded by the Federal Ministry for Education and Research (BMBF, 01GI1120A) and is integrated in the Competence Network Obesity (CNO).

Nothing to Disclose: BSL, EF, UP, KMD, GL, FD, PF, GW, HJB, MW

OR07-5 17059 5.0000 A Mutations in the Leptin Receptor (LEPR) Gene in Patients with Early Onset Extreme Obesity - a Case Series 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Obesity, Adipocyte Biology and Appetite Saturday, June 21st 1:00:00 PM OR07 4750 11:30:00 AM Neuroscience of Human Obesity Oral

Shan He^* and Ya-Xiong Tao
Auburn University, Auburn University, AL

The melanocortin-4 receptor (MC4R) is a Family A G protein-coupled receptor that plays an essential role in regulating energy homeostasis, including both energy intake and expenditure. Defects in MC4R are the most common monogenic form of obesity, with more than 170 distinct mutations identified in humans. In addition to the conventional Gs-stimulated adenylyl cyclase pathway, it has been recently demonstrated that MC4R also activates mitogen-activated protein kinases, especially extracellular signal-regulated kinases 1 and 2 (ERK1/2). We also showed there is biased signaling in the two signaling pathways, with inverse agonists (negative antagonists) in the Gs-cAMP pathway acting as agonists for the ERK1/2 pathway. Herein, we investigated the constitutive and ligand-stimulated activation (pERK1/2) in wild type and 64 naturally occurring MC4R mutations, including Class IV (mutants that have normal cell surface expression and ligand binding but are defective in cAMP signaling) and V (mutants that have normal cell surface expression, binding and cAMP signaling) mutants. We showed that four mutants (P48S, G55V, I125K and M208V) had significantly decreased basal pERK1/2 level, and fifteen mutants (F51L, T112M, N240S, N274S and S295P in class V, N62S, P78L, D90N, L106P, S136F, T162I, R165W, R165Q, R165G and Y302F in class IV) had impaired ligand-stimulated pERK1/2 activation. Defective signaling in Class V mutants might be the cause of obesity observed in patients harboring these mutations. In summary, our studies demonstrated for the first time that the decreased basal or ligand-stimulated ERK1/2 signaling might contribute to obesity pathogenesis caused by mutations in the MC4R gene. We also observed biased signaling in these naturally occurring mutations in the Gs-cAMP-protein kinase A and ERK1/2 pathway.

Nothing to Disclose: SH, YXT

OR07-6 17016 6.0000 A Defect in MAPK Signaling As a Cause for Monogenic Obesity Caused By Inactivating Mutations in the Melanocortin-4 Receptor Gene 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Obesity, Adipocyte Biology and Appetite Saturday, June 21st 1:00:00 PM OR07 4750 11:30:00 AM Neuroscience of Human Obesity Oral

Angela Delaney^*¹, Francois M. Lalonde¹, Jonathan D. Blumenthal¹, Liv S. Clasen¹, Rebecca L. Hicks¹, Alexander H. Denker¹, Jay N. Giedd¹, William F Crowley Jr.² and Ravikumar Balasubramanian²
¹National Institutes of Health, Bethesda, MD, ²Massachusetts General Hospital, Boston, MA

BACKGROUND: Men with idiopathic hypogonadotropic hypogonadism (IHH) have impaired spatial ability compared to healthy controls or men whose hypogonadism was acquired after normal puberty¹. This observation suggests that androgen exposure during puberty plays an important role in neurocognitive development. However, it is not known whether similar neurocognitive defects occur in females with a deficiency of pubertal sex steroids. In addition, the effect of sex-steroid deficiency on structural differences in the developing brain is yet to be directly quantified. Therefore, we hypothesized that the delayed exposure to sex hormones during puberty in both male and female patients with IHH results in neurocognitive deficits as well as structural differences in the developing brain.

METHODS: Neurocognitive testing, structural brain MRI and functional MRI (fMRI) were performed in subjects with IHH (n=16, mean age 24.2 y), including 11 with anosmia (Kallmann syndrome; 5M, 6F) and 5 normosmic subjects (all male), as well as 10 healthy controls (5M, 5F; mean age 30.3 y) matched for age, sex, socioeconomic status, and handedness. MRI scans were acquired using a GE 3 Tesla LX MR750 scanner. Cortical measurements such as thickness, area, and volume were analyzed using FreeSurfer² with sex and mean-centered age at the time of the scan included as covariates.

RESULTS: Complete sexual development did not occur in IHH patients unless treated with sex steroids (mean age at pubertal onset: 16.3 y, M; 14.8 y, F). Puberty onset was normal in controls (12.5 y, M; 9.8 y, F). Accounting for gender, the cortical area was negatively correlated with age in the IHH group and positively or uncorrelated with age in controls in the superior temporal (p < 0.01) and rostral middle frontal (p < 0.01) cortices. fMRI results available from a subset of subjects (11 IHH; 7 controls) showed that the magnitude and extent of blood oxygenation level dependent activation during a face recognition task was reduced bilaterally in occipital regions in the IHH subjects (P < 0.001). Neurocognitive testing in the same subset showed preserved verbal and overall IQ, but significant deficits in several neurocognitive domains in IHH patients (performance IQ (WASI, p=0.009); spatial vs. verbal short term working memory (WMS-III; P=0.047); cognitive flexibility/shift (Trail Making Test, P=0.004); processing speed (WAIS Symbol Search, P=0.022); verbal memory (WRAML2 Verbal Learning, P=0.05) and semantic verbal fluency (P=0.027)).

CONCLUSIONS: 1. In both sexes, pubertal sex steroid deficiency contributes to persistent structural and functional brain differences as well as to neurocognitive deficits primarily involving spatial ability and recognition memory.

2. These findings provide further direct evidence in humans about the critical spatiotemporal role played by appropriately timed pubertal sex steroids during normal brain development.

Nothing to Disclose: AD, FML, JDB, LSC, RLH, AHD, JNG, WFC Jr., RB

OR11-1 16936 1.0000 A Delayed Sex Hormone Exposure during Puberty Is Associated with Differences in Brain Cortical Thickness, Spatial Ability, and Recognition Memory 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Pediatric Endocrinology Saturday, June 21st 1:00:00 PM OR11 4754 11:30:00 AM Puberty: From Bench to Bedside Oral

Ana Paula Abreu^*¹, Delanie B. Macedo², Ana Claudia Silva Reis³, Luciana Ribeiro Montenegro⁴, Andrew Dauber⁵, Diane Beneduzzi², Priscilla Cukier², Leticia Gontijo Silveira⁶, Milena Gurgel Teles⁷, Rona S. Carroll⁸, Gil Guerra Jr.⁹, Guilherme Guaragna Filho¹⁰, Zoran Spasico Gucev¹¹, Ivo J P Arnhold², Margaret Castro¹², Ayrton C. Moreira¹³, Carlos Eduardo Martinelli Jr.¹³, Joel N Hirschhorn¹⁴, Berenice Bilharinho Mendonça², Vinicius N. Brito², Sonir Roberto Antonini¹⁵, Ursula B Kaiser⁸ and Ana Claudia Latronico⁴
¹Brigham and Women's Hospital/Harvard Medical School, Boston, MA, ²Disciplina de Endocrinologia e Metabologia, Laboratório de Hormônios e Genética Molecular/ LIM42, Hospital das Clinicas da Faculdade de Medicina da Universidade de São Paulo, Sao Paulo, Brazil, ³Universidade de Sao Paulo, Ribeirao Preto, Brazil, ⁴Hospital das Clinicas da Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil, ⁵Division of Endocrinology, Boston Children's Hospital and Program in Medical and Population Genetics, Broad Institute, Boston, MA, ⁶Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil, ⁷Hospital das Clinicas da Faculdade de Medicina da Universidade de São Paulo, Sao Paulo, Brazil, ⁸Brigham and Women's Hospital and Harvard Medical School, Division of Endocrinology/Diabetes, Boston, MA, ⁹Medical School, University of Campinas (FCM-UNICAMP), Brazil., São Paulo, Brazil, ¹⁰Medical School, University of Campinas (FCM-UNICAMP), Brazil., ¹¹Univ Kiril & Metodij/Med Sch, Skopje Fyr, Macedonia, ¹²R, ¹³Ribeirao Preto Medical School - University of Sao Paulo, Ribeirao Preto, Brazil, ¹⁴Division of Endocrinology, Boston Children's Hospital and Program in Medical and Population Genetics, Broad Institute, Harvard Medical School, Boston, MA, ¹⁵Ribeirão Preto Medical School - University of São Paulo, Ribeirão Preto, Brazil

Background: We recently identified loss-of-function mutations in the imprinted MKRN3 gene, encoding makorin ring finger protein 3, in patients with GnRH-dependent central precocious puberty (CPP) using whole exome sequencing. MKRN3 is a maternally imprinted gene located on chromosome 15q11.2, in the Prader Willi syndrome critical region. We showed that Mkrn3 is expressed at high levels in the hypothalamus of mice prepubertally and decreased immediately prior to puberty onset, suggesting that MKRN3 acts as a brake on GnRH secretion.

Objectives: To investigate potential MKRN3 sequence variations in patients with apparently sporadic CPP.

Methods: We studied 215 unrelated children (207 girls and 8 boys) with a diagnosis of CPP. All but two of these patients (213 cases) reported no family history of premature sexual development. First-degree relatives of patients with identified MKRN3 variants were included for genetic analysis. All 215 CPP patients were screened for MKRN3 mutations by automatic sequencing.

Results: We identified five novel heterozygous mutations in MKRN3 in eight unrelated girls with CPP. Four were frameshift mutations predicted to encode truncated proteins and one was a missense mutation, which was suggested to be deleterious by in silico analysis. All patients with MKRN3 mutations had classical features of CPP with a median age of onset at 6 years. No major signs of Prader Willi syndrome were detected in the patients with CPP and MKRN3 mutations. Segregation analysis was possible in 5 of the 8 girls with MKRN3 mutations; in all cases, the mutation was inherited on the paternal allele.

Conclusions: We have identified novel inherited MKRN3 defects in children with apparently sporadic CPP, supporting a role of MKRN3 in the control of puberty initiation. Because of the imprinting pattern of MKRN3, the CPP phenotype can be inherited from an apparently asymptomatic father, highlighting the importance of obtaining a detailed family history, especially from the paternal side, in children with apparently idiopathic CPP. MKRN3 gene analysis may provide an additional tool for the diagnosis of CPP, allowing early diagnosis and treatment of children with CPP.

Nothing to Disclose: APA, DBM, ACSR, LRM, AD, DB, PC, LGS, MGT, RSC, GG Jr., GG, ZSG, IJPA, MC, ACM, CEM Jr., JNH, BBM, VNB, SRA, UBK, ACL

OR11-2 16858 2.0000 A Apparently Sporadic Central Precocious Puberty Is Caused By Paternally Inherited Mutations in the Imprinted MKRN3 Gene 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Pediatric Endocrinology Saturday, June 21st 1:00:00 PM OR11 4754 11:30:00 AM Puberty: From Bench to Bedside Oral

Maria Bygdell, Liesbeth Vandenput, Claes Ohlsson and Jenny M Kindblom^*
University of Gothenburg, Gothenburg, Sweden

A secular trend for the timing of menarche has been described in women, but for men, studies of pubertal timing are scarce. Both negative and positive associations between childhood obesity and pubertal timing in men have been reported.

In Sweden, Child Health Care (CHC) centers follow all children regarding growth and general health. We have collected detailed CHC growth data (height and weight) from centrally archived records for all children born 1946 or later in Gothenburg and established a unique population-based cohort, the BMI Epidemiology STudy (BEST; n≈400 000). The overall aim of the well-powered BEST cohort is to determine the role of childhood obesity and pubertal timing for a variety of diseases later in life.

The aim with the present BEST sub-study was to investigate if there is a secular trend for male pubertal timing and if childhood BMI influences pubertal timing. Men born every five years from 1946 to 1991 were evaluated (n=200 for each birth year, n=2000 for the entire sub-cohort). The height measurements were curve-fitted according to the Infancy – Childhood – Puberty (ICP) model and age at Peak Height Velocity (PHV) was calculated. PHV is the maximum growth velocity and represents an established method to objectively determine age at pubertal timing (PHV ≈ 2 years after pubertal onset).

The mean age at PHV was 14.0± 1.1 years. Linear regression analyses revealed that age at PHV was 1.3 months earlier for every 10 year increase in birth year between 1946 and 1991 (p=5.3*10^-12). As expected, a secular trend of increased childhood BMI at eight years of age was observed (p=1.2*10^-12). We next evaluated the impact of childhood BMI on age at PHV and found that age at PHV was 2.1 months earlier for every quintile increase in BMI at eight years of age (p=1.1*10^-21). To determine the independent role of birth year and childhood BMI for age at PHV, both parameters were included in the same model, demonstrating that both birth year and childhood BMI were independently associated with age at PHV (standardized β; birth year β = -0.13, p=8.2*10^-9; childhood BMI β =-0.21, p= 9.8*10^-21).

In conclusion, we provide compelling statistical evidence of a secular trend for pubertal timing in Swedish men. The secular trend of earlier pubertal timing is partly explained by increased childhood BMI but also other factors, to be identified, contribute.

Nothing to Disclose: MB, LV, CO, JMK

OR11-3 14868 3.0000 A A Secular Trend for Pubertal Timing in Swedish Men Born 1946-1991 – the Best Cohort 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Pediatric Endocrinology Saturday, June 21st 1:00:00 PM OR11 4754 11:30:00 AM Puberty: From Bench to Bedside Oral

Casper P. Hagen^*¹, Kaspar Sørensen², Lise Aksglaede¹, Annette Mouritsen¹, Mikkel Grunnet Mieritz², Jeanette Tinggaard¹, Christine Wohlfart-Veje¹, Jørgen Holm Petersen¹, Katharina M Main³, Ewa Rajpert-De Meyts¹, Kristian Almstrup⁴ and Anders Juul¹
¹University of Copenhagen, Faculty of Health and Medical Sciences, Copenhagen, Denmark, ²University of Copenhagen, Faculty of Health and Medical Sciences, Rigshospitalet, Department of Growth and Reproduction, Denmark, Copenhagen, Denmark, ³Rigshospitalet, Copenhagen, Denmark, ⁴University of Copenhagen, Faculty of Health and Medical Sciences, Rigshospitalet, Copenhagen, Denmark

Context: Follicle-stimulating hormone (FSH) stimulates ovarian follicle maturation and estradiol synthesis which is responsible for breast development. Age at pubertal onset varies substantially among healthy girls. Although more than half of the variation is heritable, only a small part has been attributed to specific genetic polymorphisms identified so far.

Objective:We assessed the effect on pubertal onset of three genetic polymorphisms affecting FSH action.

Design and Participants: Combined cross-sectional and longitudinal cohort study of 964 healthy girls, age 8-13 years.

Setting: General community.

Main Outcome Measures: Puberty was defined as Tanner breast stage ≥ 2 by palpation. DNA was isolated from blood and FSHB -211 G>T, FSHR -29 G>A, and FSHR 2039 A>G were genotyped by competitive allele-specific polymerase chain reaction.

Results: Girls homozygous for FSHR -29 AA (reduced FSH receptor expression) entered puberty 7.4 (2.5-12.4) months later than carriers of the common variants FSHR -29 GG+GA, p = 0.003. In a combined model, puberty occurred on average 8.0 months later in carriers of either FSHR -29 AA or FSHB -211 TT (reduced FSH production) compared with girls with at least one wild-type allele (10.63 vs. 9.96 years, p = 0.001). The number of minor alleles (FSHR -29 A and FSHB -211 T) was positively associated with age at pubertal onset (beta 1.9 months, p = 0.025).

Conclusions: For the first time we demonstrate that age at breast development is highly influenced by genetic variation in promoters affecting FSH action. To our knowledge, this is the strongest genetic effect on age at pubertal onset in girls published to date.

Nothing to Disclose: CPH, KS, LA, AM, MGM, JT, CW, JHP, KMM, ER, KA, AJ

OR11-4 15111 4.0000 A Pubertal Onset in Girls Is Strongly Influenced By Genetic Variation in Promoters Affecting FSH Action 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Pediatric Endocrinology Saturday, June 21st 1:00:00 PM OR11 4754 11:30:00 AM Puberty: From Bench to Bedside Oral

Felix Schreiner^*¹, Bettina Gohlke¹, Michaela Hamm¹, Eckhard Korsch² and Joachim Woelfle¹
¹Children's Hospital, University of Bonn, Bonn, Germany, ²Children's Hospital Amsterdamer Straße, Cologne, Germany

Background: Loss-of-function-mutations in the gene encoding the makorin RING finger protein 3 (MKRN3) have recently been reported to underlie familial cases of central precocious puberty (CPP). The imprinted MKRN3 gene is expressed only from the paternal allele, and mutations inherited from the father affect boys and girls equally, which is in contrast to the known female preponderance in idiopathic CPP.

Patients and Methods: In order to select a series of individuals suspective of having monogenetically caused CPP, we searched our single-center database (in total, 47 new non-organic CPP cases between 2005 and 2013) for either idiopathic CPP with evidence of familial clustering (14 cases in six families) or idiopathic CPP in males (two sporadic cases, one of them lost for follow-up). The coding region of the intronless MKRN3 gene was analyzed by genomic sequencing.

Results: By sequencing MKRN3 in six families and one male patient with idiopathic CPP, we identified two further families carrying loss-of-function-mutations. In addition to the previously reported variant c.475_476insC (p.Ala162Glyfs*14), which was detected in two girls who entered puberty at ages 6.8 and 7.5 yrs, we identified a novel mutation, c.331G>T (p.Glu111*), detected in one girl (thelarche at age 6.1 yrs) and his brother (pubarche at age 9.0 yrs and unusually rapid testicular growth to >8.0 ml at age 10.0 yrs). In both families, the MKRN3 mutations were inherited from the fathers, both of them were of normal height and reported to have entered puberty at an “early-to-normal” age. Grandparental DNA was not available in these two families.

Conclusions: We conclude that MKRN3 mutations appear to be a frequent cause of familial CPP. Considering the imprinted mode of inheritance allowing the phenotype to skip one or more generations, MKRN3 mutations may also account for a certain proportion of isolated CPP cases. Remarkably, four out of five MKRN3 mutations described so far encode either a stop codon or a frameshift followed by a premature stop codon, suggesting that the clinical spectrum associated with MKRN3 mutations known to date might represent only the lowest tip of a broader phenotypical range related to functional variants of the MKRN3 gene. Mutation screening in larger cohorts is necessary in order to estimate the prevalence of MKRN3 mutations in idiopathic CPP.

Nothing to Disclose: FS, BG, MH, EK, JW

OR11-5 14772 5.0000 A MKRN3 Mutations in Familial Central Precocious Puberty 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Pediatric Endocrinology Saturday, June 21st 1:00:00 PM OR11 4754 11:30:00 AM Puberty: From Bench to Bedside Oral

Gerhard Binder^*¹, Roland Schweizer¹, Gunnar Blumenstock² and Regina Braun¹
¹University Children's Hospital, Tuebingen, Germany, ²University of Tuebingen, Tuebingen, Germany

Background

The clinical distinction between constitutional delay of growth and puberty (CDGP) and hypogonadotropic hypogonadism (HH) in girls with delayed puberty is sometimes difficult. Accurate endocrine tests would be helpful for counselling and treatment in time. As delayed puberty is rare in females, there is a big lack of studies.

Aim

We assessed the accuracy of inhibin B (INHB) and the GnRH agonist (GnRHa) test for diagnosing HH by retrospective analysis of our data collected during the last 5 years using a strict clinical protocol.

Patients

All prepubertal non-underweight girls (n=20) aged 13.1 to 17.5 years characterized by Tanner breast stage B1 or B2 and low E2 serum levels were tested. Re-examinations were performed every six months. CDGP was defined by spontaneous menarche, HH by no spontaneous progress of puberty during 30 months of follow-up. INHB was measured by ELISA (Beckman Coulter, Inc, U.S.A.), FSH and LH by CLIA (Siemens Health Care Systems, Germany). We assessed the test validity of INHB as well as of stimulated FSH and LH at 4h after a Triptorelin 0.1 mg challenge.

Results

The cohort comprised 12 girls with CDGP and 8 girls with HH. Causes of HH were septo-optic dysplasia, panhypopituitarism, Prader-Willi syndrome, chromosomal aberration, mental retardation syndrome with ataxia and idiopathic (n=3).

Each, basal INHB < 18 pg/mL or stimulated FSH(4h) < 11 IU/L had a sensitivity and a specificity for the detection of HH of 100%. Stimulated LH(4h) < 9 IU/L had a sensitivity of 100%, but a low specificity of 83%. The area under the curve of ROC plot analysis for diagnosing IHH was greatest for INHB < 18 pg/mL (100%) and for stimulated FSH(4h) < 11 IU/L (100%). Performance of stimulated LH(4h) < 9 IU/L was lower (97.4%; 95%-CI: 91.6%-100%).

Conclusions

Inhibin seems to be the ideal parameter to detect HH in girls. GnRHa test is the alternative, but more laborious. In contrast to males, in females with delayed puberty GnRHa stimulated FSH seems to be more specific than stimulated LH to distinguish HH from CDGP.

Nothing to Disclose: GB, RS, GB, RB

OR11-6 14866 6.0000 A Inhibin B and the GnRH Agonist Test for Distinguishing Constitutional Delay of Growth and Puberty from Hypogonadotropic Hypogonadism in Girls 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Pediatric Endocrinology Saturday, June 21st 1:00:00 PM OR11 4754 11:30:00 AM Puberty: From Bench to Bedside Oral

Alexander N Comninos^*¹, Channa N Jayasena², Evi Stefanopoulou³, Adam Buckley⁴, Julianne Mogford², Chioma Izzi-Engbeaya², Risheka Ratnasabapathy², Shakunthala Narayanaswamy², Mohammad A Ghatei², Stephen R Bloom⁴, Myra Hunter³ and Waljit S Dhillo¹
¹Imperial College NHS Healthcare Trust, London, United Kingdom, ²Imperial College London, London, United Kingdom, ³King's College London, United Kingdom, ⁴Imperial College London, United Kingdom

Hot flushes affect millions of women worldwide, yet their precise aetiology remains elusive. Furthermore, existing therapies such as HRT and clonidine are limited by side-effects and variable effectiveness.

Hypothalamic neurokinin B (NKB) expression is markedly increased in post-menopausal women. In addition, the NKB receptor is strongly expressed in hypothalamic thermoregulatory centres. In rodents, ovariectomy (as a model of menopause) results in thermoregulatory tail skin vasodilatation, while ablation of hypothalamic NKB receptor-expressing neurones blocks this effect. NKB is therefore a potential mediator of menopausal hot flushes; however animal studies are limited by their inability to detect symptoms. We investigated for the first time whether NKB could induce symptomatic hot flushes in healthy pre-menopausal women.

We performed a randomised, placebo-controlled, double-blinded, 2-way cross-over study in a temperature-controlled and humidity-controlled clinical research facility. Nine healthy pre-menopausal women (age 34±1.4y, BMI 22.9±1.3kg/m²), in the follicular phase of their menstrual cycle, received two 30min intravenous infusions during the same study visit separated by a 90min washout interval. Infusions were randomised to either NKB or vehicle. Symptoms, skin temperature (measure of cutaneous vasodilatation, determined by skin temperature probe and thermal imaging camera), skin conductance (measure of sweating), and heart rate were recorded minutely peri-infusion and every 10min at other times. BP and reproductive hormone levels were measured every 10min throughout the study visit.

Flushing was reported in 7/9 women during NKB administration, and 0/9 during vehicle administration (p<0.01 vs. vehicle). In total there were 12 episodes of flushing during NKB administration with a mean duration of 3.7±0.6min. Flushing symptoms were localised to the face and upper torso in all women who experienced flushing (7/7). During hot flush episodes, elevations in mean heart rate (+6.1±2.1bpm, p<0.01 vs. mean pre-symptoms), mean skin temperature (+0.2±0.08⁰C, p<0.05 vs. mean pre-symptoms), and alterations in skin conductance suggestive of menopausal flushing were observed. There were no significant changes in BP or reproductive hormone levels (LH, FSH, estradiol) during NKB or vehicle administration.

We demonstrate for the first time that NKB administration to healthy pre-menopausal women results in symptoms and physiological changes analogous to those observed during menopausal hot flushes. These data have important implications for the better understanding of the elusive aetiology of menopausal flushing and for the future clinical development of improved targeted therapies for hot flushes.

Nothing to Disclose: ANC, CNJ, ES, AB, JM, CI, RR, SN, MAG, SRB, MH, WSD

OR04-1 14487 1.0000 A Neurokinin B Administration Induces Menopausal-like Hot Flushes in Healthy Young Women 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Reproductive Endocrinology Saturday, June 21st 1:00:00 PM OR04 4757 11:30:00 AM Female Reproductive Endocrinology Oral

Ali Abbara^*¹, Channa N Jayasena², Chioma Izzi-Engbeaya², Alexander N Comninos³, Richard A Harvey⁴, Juan Gonzalez Maffe⁵, Subair Sarang⁶, Zainab Ganiyu-Dada⁷, Ana Padilha⁸, Mandish Dhanjal⁸, Catherine Williamson⁹, Lesley Regan⁸, Mohammad A Ghatei², Stephen R Bloom¹ and Waljit S Dhillo³
¹Imperial College London, United Kingdom, ²Imperial College London, London, United Kingdom, ³Imperial College NHS Healthcare Trust, London, United Kingdom, ⁴Imperial College NHS Trust, London, ⁵Imperial College London, London, ⁶Imperial College London, ⁷Imperial College London, london, United Kingdom, ⁸Imperial College NHS Trust, ⁹King’s College London, London, United Kingdom

BACKGROUND: The kisspeptins are a group of peptides encoded by the KISS1 gene, which is highly expressed in the placenta. Circulating levels of kisspeptin rise dramatically during normal human pregnancy, reaching levels several thousand-fold higher when compared with levels found outside of pregnancy. Plasma kisspeptin levels measured during early pregnancy may therefore represent a novel predictive marker for assessing the risk of subsequent pregnancy complications. Miscarriage (pregnancy loss prior to 24 weeks of gestation) is the most common complication of pregnancy, affecting 1 in 5 pregnancies.

OBJECTIVE: To determine whether a single measurement of plasma kisspeptin in asymptomatic women attending their antenatal booking visit predicts miscarriage risk.

STUDY DESIGN: Prospective cohort study in single tertiary obstetric centre in London.

PARTICIPANTS: We recruited 993 asymptomatic pregnant women attending their routine antenatal booking visit (mean gestation of 11.2 weeks (range 5.9-29.0) between 2010 and 2012.

MAIN OUTCOME MEASURES: Plasma kisspeptin and serum hCG were measured during the routine booking antenatal visit and pregnancy outcome was prospectively recorded.

RESULTS: Plasma kisspeptin levels were highly correlated with the gestational week of pregnancy (r²=0.57; P<0.0001). Gestational age-corrected (multiples of median; MoM) plasma kisspeptin levels were 60.4% lower (P<0.001), and MoM hCG was 36.1% lower (P<0.001) in women who were later diagnosed with miscarriage when compared with women who did not miscarry. Plasma kisspeptin >1306 pmol/L was strongly associated with a reduced risk of miscarriage, even after adjusting for age, body mass index, gestational age, smoking and blood pressure (Odds Ratio 0.13 [CI 0.08-0.22], P=0.0001). Plasma kisspeptin had a higher diagnostic performance for miscarriage when compared with hCG (ROC area under curve: 0.899±0.025, plasma kisspeptin; 0.775±0.040, serum hCG, P<0.01 vs. plasma kisspeptin).

CONCLUSION: This is the first study demonstrating that a single plasma kisspeptin measurement taken during early pregnancy provides a highly predictive marker for identifying asymptomatic pregnant women with subsequent miscarriage.

Nothing to Disclose: AA, CNJ, CI, ANC, RAH, JG, SS, ZG, AP, MD, CW, LR, MAG, SRB, WSD

OR04-2 14583 2.0000 A Plasma Kisspeptin Measurement during Early Pregnancy Is a Highly Predictive Marker of Subsequent Miscarriage 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Reproductive Endocrinology Saturday, June 21st 1:00:00 PM OR04 4757 11:30:00 AM Female Reproductive Endocrinology Oral

Ruchi Bhabhra^*, Amy Denise Anderson, Christine M. Burt Solorzano, John C. Marshall and Christopher R. McCartney
University of Virginia, Charlottesville, VA

Background: Pulsatile LH (by inference GnRH) secretion is primarily nocturnal (sleep related) in early puberty; but by late puberty, daytime LH pulse frequency (awake) is higher than nighttime pulse frequency (sleep). Mechanisms regulating these changes are unknown. We reported differential sensitivity of the GnRH pulse generator to inhibition by progesterone (P) in early pubertal girls - supraphysiological P levels abolished daytime (awake) but did not suppress nighttime (sleep) pulse frequency. We have shown that the GnRH pulse generator is less sensitive to feedback inhibition by P in women/adolescent girls with hyperandrogenemia (HA). In normal puberty, mean free T gradually increases from 2.8 (Tanner I) to 12.7 pmol/L (Tanner V). We aimed to test the hypothesis that the ability of P to inhibit daytime GnRH secretion is impaired as T levels increase across normal puberty, allowing a gradual increase in daytime LH frequency.

Methods: We studied 49 normal weight girls (Tanner I-V) via q 10 min sampling from 1900 to 0700 h. Subjects were divided into groups based on AM free T (pmol/L): <5 (“low T”), 5-10 (“mid T”), and >10 (“high T”). 39 subjects (14 low T, 8 mid T, 17 high T) did not receive P, while 10 girls (5 low T, 3 mid T, 2 high T) received exogenous P in 2-3 divided doses (mean P = 5.33 ng/mL). LH pulses frequency was determined in two time blocks: 1900-2300 h (daytime/wake) and 2300-0700 h (nighttime/sleep).

Results: Girls with T < 5 had few daytime LH pulses (0.14 ± 0.06 pulses/h [mean ± SEM]), but LH frequency was higher in girls with higher T: 0.56 ± 0.11 (mid T) and 0.74 ± 0.05 (high T) (p = 0.005 for both vs. low T). Nighttime (sleep) LH frequency was similar in all 3 groups: 0.43 ± 0.06 (low T), 0.56 ± 0.07 (mid T), and 0.51 ± 0.04 (high T). In girls receiving exogenous P, daytime LH frequency was 0.10 ± 0.10 (low T), 0.08 ± 0.08 (mid T), and 0.63 ± 0.13 (high T). In each group receiving P, LH frequency during sleep was similar to that in corresponding groups not receiving P: 0.38 ± 0.07 (low T), 0.46 ± 0.08 (mid T), 0.69 ± 0.06 (high T).

Conclusion: These results indicate that P tends to suppress LH pulses during day time (awake) with no effect during the night (sleep). This effect is more prominent when T levels are lower. These data suggest that the progression of GnRH pulse secretion during pubertal maturation is regulated by the gradual increase of T. The physiological overnight increase in P may be adequate to suppress day time GnRH pulses in early pubertal (lower T) girls. However as T rises during pubertal maturation, this process is impaired, allowing a selective increase of daytime pulse frequency. This mechanism may regulate the gradual increase in GnRH pulse secretion during puberty which drives pubertal development.

Nothing to Disclose: RB, ADA, CMB, JCM, CRM

OR04-3 16713 3.0000 A Testosterone Regulates the Evolution of Daytime GnRH Pulse Secretion during Pubertal Maturation in Girls? 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Reproductive Endocrinology Saturday, June 21st 1:00:00 PM OR04 4757 11:30:00 AM Female Reproductive Endocrinology Oral

Marcelle Cedars^*¹, Sunni Mumford², Anne Z. Steiner³, Peter Raymond Casson⁴, Christos Coutifaris⁵, Michael P Diamond⁶, Gregory M Christman⁷, William D Schlaff⁸, Ruben Alvero⁹, Hao Huang¹⁰, Nanette Santoro¹¹, Esther Eisenberg¹², Richard S Legro¹³, Heping Zhang¹⁴ and The Reproductive Medicine Network¹⁵
¹Univ of California San Francisco, San Francisco, CA, ²Eunice Kennedy Shirver National Institute of Child Health and Human Development, Rockville, MD, ³University of North Carolina, ⁴University of Vermont, Burlington, VT, ⁵Hosp of Univ of PA, Philadelphia, PA, ⁶Wayne State Univ, Grosse Pointe Shores, MI, ⁷Univ of Michigan, Ann Arbor, MI, ⁸Jefferson University, Philadelphia, PA, ⁹University of Colorado-Denver, Aurora, CO, ¹⁰Yale University School of Medicine, New Haven, CT, ¹¹University of Colorado Anschutz Medical Campus, Aurora, CO, ¹²Vanderbilt Univ Med Ctr, Nashville, TN, ¹³Penn State University, Hershey, PA, ¹⁴Yale University, New Haven, CT, ¹⁵NICHD

AMH reduces sensitivity of the follicle to FSH stimulation and reduces aromatase activity suggesting a potential for resistance to ovulation even in response to ovulation induction.

Objective: To determine the association between AMH levels and ovulation among the Pregnancy in Polycystic Ovary Syndrome II (PPCOS II) participants.

Methods: This is a secondary analysis of a randomized clinical trial. 750 women with polycystic ovary syndrome (PCOS) were randomized to ovulation induction with either letrozole or clomiphene citrate (mean BMI 35.1 kg/m²). PCOS was defined by Rotterdam Criteria. Females were 18-40y, had at least 1 patent fallopian tube and normal uterine cavity and a male partner with sperm concentration of at least 14 million/mL who consented to regular intercourse. Couples were followed up to 5 treatment cycles to determine ovulation and pregnancy. 748 subjects had AMH measured at baseline. Geometric mean AMH levels were compared between women who ovulated during the trial and women who did not, after adjustment for age, BMI, testosterone, insulin and Ferriman-Gallwey score. Logistic regression was also used to evaluate the association between AMH and ovulation, after adjusting for the same factors.

Results: 619 women ovulated in response to treatment during the study. A lower mean AMH was associated with ovulation (geometric means 5.59 vs. 7.07, p=0.004; odds ratio 0.56, 95% confidence interval 0.42, 0.75). Significance remained when the comparison was adjusted for age, BMI, testosterone, insulin levels, and Ferriman-Gallwey scores (p=0.0001). Adjustment for antral follicle count, smoking, and treatment did not impact the findings. Both age and BMI were negatively associated with AMH levels, but absolute AMH levels still were relatively high (geometric mean 5.81). AMH was also positively associated with testosterone and negatively associated with insulin levels. The fully adjusted ratio of AMH/AFC was also lower (0.14 vs. 0.17, p=0.04) in women who ovulated compared to those who did not ovulate.

Conclusions: In this large cohort of obese women with PCOS, AMH levels and AMH per follicle count were significantly lower among women who ovulated in response to treatment compared to women who never achieved an ovulatory cycle. There were no interactions between AMH levels and treatment (letrozole vs. clomiphene citrate). Lastly, among women with an ovulatory cycle, the AMH levels at baseline were lowest among women taking the smallest dose at their last ovulatory cycle. These results suggest that high AMH is associated with a decreased response to ovulation induction and women with higher AMH levels may require higher doses to achieve ovulation.

Disclosure: MC: Investigator, Ferring Pharmaceuticals. AZS: Consultant, Roche Diagnostics. CC: Board Member, NOVA Therapeutics . NS: Advisory Group Member, Menogenix, Principal Investigator, Bayer, Inc.. RSL: Speaker, Ferring Pharmaceuticals. HZ: Ad Hoc Consultant, Sun Yat-Sen University, Heilongjiang University of Chinese Medicine, Tsinghua University, and Shangdong University, Ad Hoc Consultant, Sun Yat-Sen University, Heilongjiang University of Chinese Medicine, Tsinghua University, and Shangdong University. Nothing to Disclose: SM, PRC, MPD, GMC, WDS, RA, HH, EE, TR

OR04-4 15532 4.0000 A Impact of AMH Level on Chances for Ovulation in Pregnancy in Polycystic Ovary Syndrome II, a Multi-Center Randomized Clinical Trial 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Reproductive Endocrinology Saturday, June 21st 1:00:00 PM OR04 4757 11:30:00 AM Female Reproductive Endocrinology Oral

Mojca Jensterle^*¹, Tomaz Kocjan¹ and Andrej Janez²
¹Univ Med Ctr Ljubljana, Ljubljana, Slovenia, ²University Medical Center Ljubljana, Ljubljana, Slovenia

Context: Phosphodiesterase (PDE) enzymes, including members of PDE4, have been investigated in regulation of endocrine and reproductive functions of ovaries. In addition, selective inhibition of PDE4 enzyme has recently been implicated in the regulation of metabolism with positive effects on glucose homoeostasis and weight reduction.

Objective: The aim of this study was to evaluate whether the PDE4 inhibitor roflumilast affects body weight and hormonal and metabolic status in obese women with polycystic ovary syndrome (PCOS).

Design/Participants/Main Outcome Measure: A 12-week prospective randomized open-label study was conducted with 36 obese women with PCOS diagnosed by the National Institute of Child Health and Human Development criteria who had been pre-treated with metformin (MET). They were randomized to MET 1000 mg BID or combined treatment (COMBI) with MET 1000 mg BID and roflumilast 500 mcg QD. The primary outcome was change in anthropometric measures of obesity. Secondary outcomes included hormonal and metabolic changes.

Results: 31 patients (aged 33.8 ± 7.4 years, BMI 36.4 ± 5.1 kg/m², mean ± SD) completed the study: 16 on MET and 15 on COMBI. Subjects treated with COMBI lost on average 4.2 ± 2.8 kg compared with a 0.9 ± 2.5 kg weight gain in MET group (p<0.001). BMI decreased for 1.6 ± 1.1 kg/m²in COMBI arm compared to increase for 0.9 ± 2.4 kg/m²in MET arm (p= 0.001). Visceral adipose tissue area as assessed by DXA decreased from 136.7 ± 37.8 to 121.2 ± 36.2 cm² in COMBI arm compared to increase from 155.3 ± 61.9 to 166.7 ± 67.2 cm²in MET arm (p=0.02). From baseline to study end both treatment interventions resulted in a significant reduction of androstenedione (p=0.013), free testosterone (p=0.002) and HOMA-IR score (p=0.027) and a significant increase in SHBG (p=0.024), although the between-treatment differences of the changes have not been statistically significant yet.

Conclusion: Roflumilast added to metformin reduced body weight in obese women with PCOS, primarily due to a loss of fat mass. These improvements were associated with beneficial effects on hormonal and metabolic status.

Nothing to Disclose: MJ, TK, AJ

OR04-5 12947 5.0000 A Phosphodiesterase 4 Inhibition As a Potential New Therapeutic Target in Obese Women with Polycystic Ovary Syndrome 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Reproductive Endocrinology Saturday, June 21st 1:00:00 PM OR04 4757 11:30:00 AM Female Reproductive Endocrinology Oral

Natalie D Shaw^*¹, Serene Srouji², Corrine K. Welt³, Kimberly H Cox⁴, Janis H Fox⁵, Judith M. Adams⁶, Patrick M. Sluss⁴ and Janet E. Hall⁷
¹Boston Children's Hospital, Boston, MA, ²Brigham and Wmn's Hosp, Boston, MA, ³The University of Utah, Salt Lake City, UT, ⁴Massachusetts General Hospital, Boston, MA, ⁵Brigham & Women's Hosp, Boston, MA, ⁶MA Gen Hosp, Boston, MA, ⁷Massachusetts General, Boston, MA

Context: Estradiol (E2) levels are maintained within or exceed the normal range in older reproductive-aged women with regular menstrual cycles, whereas levels of inhibin B and anti-mullerian hormone (AMH) begin to decline and FSH levels begin to rise and fertility is decreased. The mechanisms underlying preserved E2 synthesis in older reproductive–aged women in the face of other evidence of declining ovarian function are unknown. The current study was designed to determine if sustained E2 secretion with aging is due to increased granulosa cell aromatase mRNA expression and activity.

Methods: Healthy, regularly cycling older (n=13; 36-45 yrs) and younger (n=14; 22-34 yrs) women were studied. Reproductive hormone and peptide levels were measured in peripheral blood samples throughout the follicular phase. Follicular fluid (FF) and granulosa cells were aspirated from dominant follicles in naturally occurring cycles. FF was assayed for androstenedione (AD), testosterone (T), estrone (E1) and E2 as indices of aromatase activity within the follicle. Inhibin A, inhibin B, and AMH were also measured in FF. Aromatase mRNA expression was determined in granulosa cells using qRT-PCR.

Results: Older women had higher FSH levels (O vs Y: 13.5±0.9 vs. 11.3±0.5 IU/L, p=0.04) than younger women during the early follicular phase in the setting of similar E2 (54.6±5.0 vs. 52.0±2.9 pg/ml, p=0.7) but lower inhibin B (74.0±9.3 vs. 94.3±5.3 pg/ml, p=0.04) and AMH (1.0±0.3 vs. 3.1±0.5 ng/ml, p=0.006) levels. Late follicular phase serum E2 levels were similar in the two groups but older women demonstrated an earlier pre-ovulatory rise in E2 (cycle day 7.4±0.5 vs. 9.9±0.8; p=0.02). Aromatase mRNA expression was 3-fold higher in older compared with younger women (p< 0.001). Although aromatase expression per granulosa cell was higher in older women, there were no age-related differences in FF E2 (O vs. Y: 1169.8±183 vs. 1166.8±215 ng/ml, p=1) or E1 (40.0±7.7 vs. 54.8±14.2 ng/ml, p=0.3) or in aromatase substrate to product ratios (T to E2: 0.03±0.005 vs. 0.02±0.005, p=0.8; AD to E1: 15.8±4.1 vs. 10.6±3.2, p=0.3) after controlling for dominant follicle size.

Conclusions: These studies demonstrate an increase in ovarian aromatase mRNA in older compared with younger reproductive-aged women. Similar FF E2 levels in older and younger women are consistent with previous studies finding fewer granulosa cells in the dominant follicle in women with decreased ovarian reserve marked by increased FSH levels (1). These data suggest that estrogen synthesis is likely to be increased on a per cell basis in conjunction with ovarian aging in women.

Disclosure: CKW: Ad Hoc Consultant, Up To Date. Nothing to Disclose: NDS, SS, KHC, JHF, JMA, PMS, JEH

OR04-6 17075 6.0000 A Evidence That Ovarian Aromatase Action Is Preserved with Aging in Regularly Cycling Women 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Reproductive Endocrinology Saturday, June 21st 1:00:00 PM OR04 4757 11:30:00 AM Female Reproductive Endocrinology Oral

Sin-Gi Park¹, Jong Bhak¹, Jong-Soo Kim¹, Tae-Hyung Kim¹, Jee Hyun An², Sang Youl Rhee³, Hee Kyung Kim⁴, Min Joo Kim⁵, Hyun Jeong Jeon⁶, Taekeun Oh⁶ and Hyung Jin Choi^*⁷
¹Theragen BiO Institute, TheragenEtex, Suwon, Korea, Republic of (South), ²College of Medicine, Korea University, Seoul, Korea, Republic of (South), ³Kyung Hee University School of Medicine, Seoul, Korea, Republic of (South), ⁴Chonnam National University College of Medicine, Gwangju, Korea, Republic of (South), ⁵Korea Cancer Center Hospital, Seoul, Korea, Republic of (South), ⁶Chungbuk National University College of Medicine, Cheongju, Korea, Republic of (South), ⁷Seoul National University College of Medicine, Seoul, Korea, Republic of (South)

Agranulocytosis is a rare but life-threatening side effect of antithyroid drugs. Several reports, including familial cases, suggested that susceptibility to antithyroid drug induced agranulocytosis has a genetic bases. These genetic components could be used to predict the risk of antithyroid drug induced agranulocytosis. However, no tests are currently available to predict the risk of antithyroid drug induced agranulocytosis. In this study, we performed whole-exome sequencing to comprehensively identify the genetic variations responsible for antithyroid drug induced agranulocytosis. For discovery stage 1, whole-exome sequencing was performed for seven antithyroid drug induced agranulocytosis cases. Variant based approach and gene based approach were performed to discover candidate variants or genes for antithyroid drug induced agranulocytosis. For HLA loci, HLA-sequence-based typing was performed. For validation stage 2, Sanger sequencing and HLA-sequence-based typing were performed for eight additional independent antithyroid drug induced agranulocytosis cases. Korean local healthy controls and public HLA database were used as control group. Discovery stage genome-wide association study results revealed strong signals in chromosome 6 (P=2.7×10^-8) and chromosome 19 (P=1.7×10^-14). HLA typing of seven discovery cases and eight validation cases revealed several HLA types, which showed strong association with antithyroid drug induced agranulocytosis (odds ratio=4.6-6.3, P=3×10^-3-5×10^-5). Gene based approach indicated several candidate genes for antithyroid drug induced agranulocytosis. In these genes, several loss of function variants were found, which were common among antithyroid drug induced agranulocytosis cases, but these variants were rare among controls (P=2.0×10^-3-1.8×10^-4). In conclusion, several strong genetic predictors of antithyroid drug induced agranulocytosis were discovered. To avoid the risk of life-threatening agranulocytosis side effect, these pharmacogenomics markers could be tested in clinic before initiation of antithyroid drugs.

Nothing to Disclose: SGP, JB, JSK, THK, JHA, SYR, HKK, MJK, HJJ, TO, HJC

OR12-1 14618 1.0000 A Whole-Exome Sequencing Reveals Strong Genetic Predictors of Antithyroid Drugs Induced Agranulocytosis 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Thyroid/HPT Axis Saturday, June 21st 1:00:00 PM OR12 4764 11:30:00 AM Thyroid Autoimmunity Oral

Jing Li^*¹, Juan Qin¹, Hongmei Zhang¹, Zhongyan Shan² and Weiping Teng³
¹the First Affiliated Hospital of China Medical University, Shenyang, China, ²Institute of Endocrinology, China, ³The Endocrine Institute, Shenyang, China

Alpha-Enolase (ENO1) is a key glycolytic enzyme in the cytoplasm, and also expressed on the surface of immune cells, neurons, glial and endothelial cells (1). Anti-ENO1 autoantibody (ENO1Ab) was reported in several autoimmune disorders, such as autoimmune retinopathy. Aside from enzymatic function, the pathogenic roles of ENO1 have been implicated for its immunogenic capacity, DNA-binding ability and plasmin(ogen) receptor function. ENO1Ab has been found in the sera of patients with steroid responsive encephalopathy associated with autoimmune thyroiditis (2). However, it is not clear whether ENO1Ab develops directly from thyroid autoimmunity or due to other concurrent autoimmune diseases. In this study, we directly examined whether ENO1 was an autoantigen in the pathogenesis of autoimmune thyroiditis based on a classical experimental autoimmune thyroiditis (EAT) animal model induced by thyroglobulin (Tg) immunization. Seven-week-old female CBA/J mice were randomly assigned to two groups and kept in specific pathogen free facilities. One group was first immunized with murine Tg in complete Freund's adjuvant, and then challenged with Tg in incomplete Freund's adjuvant two weeks later. They were sacrificed 4 weeks after the second Tg challenge. The other group was injected only with Freund's adjuvant as control. Sera were collected for determination of TgAb and ENO1Ab by ELISA. Serum ENO1Ab were further identified by western blot using recombinant ENO1 protein and HRP-conjugated anti-mouse IgG. ENO1 expression in the thyroid from non-immunized intact CBA/J mice was detected through both western blot and immunohistochemical staining using rabbit anti-ENO1 IgG. After Tg immunization, the occurrence of EAT was confirmed by significantly elevated serum TgAb and intrathyroidal mononuclear cell infiltration. Serum anti-ENO1 total IgG level was significantly higher in EAT group induced by Tg immunization (OD₄₅₀=0.4509±0.2643; n=17) as compared with that of control group injected only with Freund's adjuvant (OD₄₅₀=0.2416±0.0875; P=0.024; n=10). In western blot experiments, the pooled sera from EAT mice (n=3) was found containing autoantibody against 47 kDa ENO1 protein, and there was a protein band migrating around 47 kDa on SDS-PAGE gel from pooled thyroid extract of non-immunized mice recognized by rabbit anti-ENO1 IgG (n=3). The above western blot experiments had been repeated three times with consistent findings. Immunohistochemical staining using rabbit anti-ENO1 IgG and UltraSensitive™ SP system confirmed ENO1 expression in thyroid follicular cells, which was mainly distributed in the cytoplasm (n=3). The above findings suggest that ENO1 expressed in thyroid follicular cells may act as one of autoantigens in thyroid autoimmunity. Reactions to this multifunctional protein might elicit immune attacks to some extrathyroidal tissues, such as brain, at the same time.

Nothing to Disclose: JL, JQ, HZ, ZS, WT

OR12-2 13218 2.0000 A Direct Identification of Alpha-Enolase As an Autoantigen in the Pathogenesis of Autoimmune Thyroiditis 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Thyroid/HPT Axis Saturday, June 21st 1:00:00 PM OR12 4764 11:30:00 AM Thyroid Autoimmunity Oral

Angela Lombardi^*¹, William B Inabnet III¹, Randall Owen¹ and Yaron Tomer²
¹Icahn School of Medicine at Mount Sinai, New York, NY, ²Icahn School of Medicine at Mount Sinai and James J. Peters VA Medical Center, New York, NY

Background: Amiodarone (AMIO) is one of the most effective antiarrhythmic drugs available. However, its use is often limited by serious side-effects including thyroid dysfunction. Management of AMIO-induced thyrotoxicosis (AIT) remains a very challenging problem especially since the half life of AMIO is several weeks and merely stopping therapy does not result in resolution of thyrotoxicosis. Moreover, the molecular mechanisms underlying AIT are still unclear and therefore, specific therapy cannot be developed. Subtoxic doses of AMIO were recently reported to interfere with protein folding in the endoplasmic reticulum (ER) causing ER stress. Here we sought to evaluate the potential role of ER stress in the induction of AMIO-induced thyroiditis.

Results: To investigate the functional role of AMIO in ER stress, human thyroid cell line ML-1 was treated with increasing concentrations of AMIO (5, 10 or 20 µM); the classic ER stress inducer Thapsigargin (0.5 µM) was used as positive control. AMIO increased mRNA and protein levels of BiP, a typical marker of ER stress, already at a concentration of 5 µM. Time course analysis with AMIO (5 µM) showed that BiP mRNA was significantly increased as early as 16 hours after treatment and remained elevated for up to 72 hours. To explore the contribution of iodine released from AMIO in BiP upregulation, we treated ML-1 cells with different concentrations of iodine (10, 100 or 1000 µM). Intriguingly, none of the concentrations used caused upregulation of BiP, tested both at the mRNA and protein levels. These data demonstrated that AMIO did not induce BiP via iodine-mediated mechanisms. Confirming the BiP data, incubation of ML-1 with 5 µM AMIO for 24 hours significantly increased expression of other ER stress markers including phospho-eIF2α, Chop and spliced-XBP1. Human primary thyrocytes cultured in the same experimental settings, displayed a significant increase of BiP mRNA and protein, mirroring the results obtained in ML-1 cells. Remarkably, in both experimental systems AMIO downreguated thyroglobulin (Tg) protein levels but had no significant effect on Tg mRNA levels suggesting a mechanism involving Tg protein degradation. To test this hypothesis, we pretreated overnight both ML-1 cells and primary thyrocytes with 0.8 µM MG132, a proteasome-specific inhibitor, reversing the AMIO-induced downreguation of Tg protein. Moreover, pretreatment of both ML-1 cells and primary thyrocytes with the chemical chaperone 4-phenylbutyric acid (7.5 mM) for 24 hours completely prevented the effects of AMIO on ER stress induction and Tg downregulation, suggesting an ER stress-dependent proteasome activation.

Conclusions: We demonstrated for the first time that ER stress represents one of the molecular links between AMIO and AIT providing a new therapeutic target in the prevention and treatment of AMIO-induced thyroid dysfunction.

Nothing to Disclose: AL, WBI III, RO, YT

OR12-3 15305 3.0000 A A New Mechanism for Iodine Independent Amiodarone-Induced Thyroiditis with Translational Implications 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Thyroid/HPT Axis Saturday, June 21st 1:00:00 PM OR12 4764 11:30:00 AM Thyroid Autoimmunity Oral

Liping Wu^*¹, Yuan Zhao¹, Bingyin Shi², Chuqi Gao¹, Yue Wang¹, Li Xu¹, Hongjun Lv², Jing Shi¹, Peng Hou² and Meiju Ji²
¹The First Affiliated Hospital of Xi’an Jiaotong University Health Science Center, Xi'an, China, ²The First Affiliated Hospital of Xi’an Jiaotong University School of Medicine, Xi'an, China

Graves’ disease (GD) is more prevalent in females than males. This female preponderance for abnormal autoimmune function has largely gone unexplained. Androgen may be responsible for the decreased susceptibility of males to GD. The current study was designed to explore gender difference and response to androgen treatment in the mice model of GD induced by injection of adenovirus expressing thyrotropin receptor (TSHR) A-subunit. The incidence and the severity of GD were observed in male and female BALB/c mice, castrated males or androgen treated females. The total thyroxine (TT4) levels of males were decreased compared to females (98.71+19.08 nmol/l vs. 167.75+26.44 nmol/l, p=0.048; n=10 per group). However, the free thyroxine (FT4) levels were slightly, but not significantly, lower in males than females (p=0.368). Castration had no apparent influence on the induction of GD vs. sham-operation (TT4: p=0.59; FT4: p=0.81; n≥10 per group). As expected, both testosterone(T) and dihydrotestosterone(DHT) reduced the levels of TT4(T: 88.20+7.0 nmol/l vs. 160.24+24.15 nmol/l，p=0.047; DHT: 84.35+11.98 nmol/l vs.160.24+24.15 nmol/l , p=0.042; n≥10 per group), FT4(T: 13.07+1.46 pmol/l vs. 39.75+9.04 pmol/l, p=0.047; DHT: 13.11+2.57 pmol/l vs.39.75+9.04 pmol/l, p=0.0 47) and the degree of thyroid hyperplasia vs. controls. In addition, the incidence of Graves’ hyperthyroidism was decreased respectively 22% or 30% in androgen(T or DHT) treated females compared to controls. To help understand the androgen influence on the immune system, the immune response of Th1/Th2/Th17/Treg was analysis by measuring the percentage of CD4+INF-γ+, CD4+IL-4+, CD4+IL-17A+, CD4+CD25+, CD4+CD25+Foxp3+ in CD4+ T cells from spleen. The analysis of T cells flow cytometry showed that the expression of CD4+INF-γ+ among CD4+ T cells in males was decreased when compared to females(10.24±0.79 % vs. 12.45±0.68 %, p=0.048), and androgen depletion mildly decreased the percentage of CD4+CD25+ (6.79±0.28 % vs. 7.69±0.33 %, p=0.046) or CD4+CD25+Foxp3 (5.70±0.28 % vs. 6.60±0.33 %, p=0.047)in CD4+ T cells vs. sham-operation. Androgen treatment not only significantly inhibited the expression of CD4+INF-γ+(T: 9.85±0.59 % vs.12.67±0.85 %, p=0.07; DHT: 9.97±0.60 % vs.12.67±0.85 %, p=0.01), but also promoted the expansion of CD4+CD25+ (T: 8.67±0.43 % vs.7.23±0.35 %, p=0.011; DHT: 8.91±0.32 % vs 7.23±0.35 % ，p=0.003) and CD4+CD25+Foxp3(T: 7.74±0.41 % vs.6.17±0.32 %, p=0.005; DHT: 8.14±0.33 % vs.6.17±0.32 %，p=0.001) population within CD4+ T cells compared to controls. Our data suggested that endogenous androgen hasn’t evidently inhibited effect against GD in BALB/c mice. Supplemental exogenous androgen provided protection against Graves’ hyperthyroidism. Supressed Th1 immune response and extended Tregs population may be one mechanism responsible for protective role of androgen.

Nothing to Disclose: LW, YZ, BS, CG, YW, LX, HL, JS, PH, MJ

OR12-4 12999 4.0000 A Exogenous Androgen Is Protective in Experimental Graves' Hyperthyroidism in BALB/c Mice 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Thyroid/HPT Axis Saturday, June 21st 1:00:00 PM OR12 4764 11:30:00 AM Thyroid Autoimmunity Oral

Veitla S Rao^*¹, Allison Lea Galloway², Hongliang LI¹, Alexandria Benbrook¹, Xichun Yu³ and David C Kem³
¹OUHSC, Oklahoma City, OK, ²Freeman Heath System, Joplin, MO, ³Univ of Oklahoma and VAMC, Oklahoma City, OK

Objective: We examined the prevalence of activating autoantibodies (AAb) to beta (B) adrenergic and muscarinic (M) receptors in subjects with Graves’ hyperthyroidism (GH) and atrial tachyarrhythmias including atrial fibrillation (AF).

Methods: Sera from 81 patients including 31 with GH and AF, 36 with GH without AF, 9 with toxic multinodular goiter (TMNG), 5 subacute thyroiditis and 10 healthy control subjects were examined for autoantibodies to B1/2-adrenergic receptors (B1/2AR) and M2 muscarinic receptor (M2R) by ELISA. Some sera were examined for B1/2AR-AAb and M2R-AAb bioactivity using transfected cell-based bioassays.

Results: 45% of patients with GH and AF were ELISA positive for autoantibodies to B1AR and 65% of the patients were positive for autoantibodies to M2R. Seventy eight (78) % were positive for B2AR but these were equally distributed between those with and without AF. Twelve subjects harbored both B1AR and M2R autoantibodies. Using a sensitive receptor-specific cell-based cAMP assay in 20 subjects, 10 with AF and 10 without AF, mean B1AR-AAb activity was elevated in both groups but higher in those with AF than those without AF (P=0.01). Both suppressed toward baseline with the BAR blocker propranolol. Β2AR-AAb activity for both was elevated (P<0.0001) from baseline but equally (P=0.2); and suppressed (P<0.001) to baseline with the B2AR blocker ICI188531. M2R-AAb suppressed forskolin-induced cAMP production in both groups, but with significantly greater suppression (P<0.001) in AF subjects. This activity returned to nearly baseline with the muscarinic blocker atropine. All but one with AF were >45 years of age. Patients with TMNG or subacute thyroiditis had a low prevalence of autoantibodies.

Conclusions: B1AR, B2AR and M2R AAb are elevated in patients with GH and concurrent AF and to a lesser extent in those without AF. Orthosteric activation of these receptors with their normal ligands is known to facilitate AF especially in older subjects. Patients with TMNG have a relatively low prevalence of AAb compatible with the non-autoimmune pathogenesis of their hyperthyroidism. We conclude B1AR-AAb and M2R-AAb, often in combination, serve as allosteric agonists in the presence of excessive thyroid hormone to facilitate AF and tachycardia in older subjects with GH. The role for B2AR-AAb is less clear, but may add to the risk for AF when present with other AAb. Β2AR-AAb also may contribute to the sinus tachycardia so commonly observed with GH.

Nothing to Disclose: VSR, ALG, HL, AB, XY, DCK

OR12-5 15165 5.0000 A Activating Autoantibodies to Beta Adrenergic and Muscarinic Receptors Associate with Atrial Tachyarrhythmias in Patients with Graves' Hyperthyroidism 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Thyroid/HPT Axis Saturday, June 21st 1:00:00 PM OR12 4764 11:30:00 AM Thyroid Autoimmunity Oral

Tania Pilli^*, Silvia Cantara, Valeria Cenci, Sandro Cardinale, Fausta Sestini, Carla Fioravanti and Furio Pacini
University of Siena, Siena, Italy

Introduction: Over the past 10 years, several clinical studies have suggested that selenium supplementation may influence the natural history of AIT. Recently, IFNγ-inducible chemokines (CXCL9, CXCL10 and CXCL11) were shown to be elevated in the AIT patients. The aim of this prospective, randomized, controlled study was to evaluate the effect of two different doses of selenomethionine (80 or 160 µg) versus placebo in euthyroid women with AIT, in terms of reduction of anti-thyroid antibodies and improvement of thyroid hypoechogenicity, during 12 months. Serum levels of selenium, CXCL9, CXCL10 and CXCL11 and their regulators, TNF-α and INF-γ, thyroid functin and volume and the quality of life of AIT patients were also evaluated. Patients and Methods: 60 patients (age 21-65 years) were equally randomized into 3 groups according to the treatment modality: 80 µg of Semet (80-Semet), 160 µg of Semet (160-Semet) or placebo. The abovementioned parameters were measured at baseline and every 3 months. Results: Data available up to 9 months. Patients were moderately in selenium deficient at the baseline (m±SD: 81.78±14.55 µg/l). AbTg levels were significantly reduced in the 160-semet group, from a median of 181.5 to a median of 102.5 U/ml, starting at 3 months (p=0.0125) while they did not show any change in the placebo and 80-semet group; serum levels of CXCL9, CXCL10 and CXCL11, assessed at baseline and at 6 months, were significantly reduced in both 80-Semet (p = 0.008, 0.007 and 0.016 respectively) and 160-Semet (p = 0.001, 0.006 and 0.001 respectively) group, while they remained unchanged or increased in the placebo group. In parallel we observed a significant decrease of TNF-α and IFN-γ in both Semet groups (IFN-γ, p=0.015 in the 80-Semet group and p=0.072 in the 160-Semet group; TNF-α, p=0.013 in the 80-Semet and p=0.006 in the 160-Semet group). Semet supplementation had no effect on AbTPO levels, thyroid hypoechogenicity, function and volume and the quality of life of AIT patients. No side effects were reported. Conclusions: Both doses of Semet (80 and 160 µg) were able to decrease significantly circulating CXCL9, CXCL10 and CXCL11, maybe through TNF-α and IFN-γ reduction, suggesting a positive immune-modulatory effect in AIT patients , as confirmed by the decreased levels of AbTg in the 160-Semet group. However, we hypothesize that a longer Semet supplementation may be needed to achieve a more evident clinical effect.

Nothing to Disclose: TP, SC, VC, SC, FS, CF, FP

OR12-6 15147 6.0000 A IFNγ-Inducible Chemokines Are Down-Modulated By Selenomethionine (Semet) Supplementation in Women with Euthyroid Chronic Autoimmune Thyroiditis (AIT): Comparison Between 2 Doses of Semet (80 μg or 160 μg) Versus Placebo 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Thyroid/HPT Axis Saturday, June 21st 1:00:00 PM OR12 4764 11:30:00 AM Thyroid Autoimmunity Oral

Dimitrios T Papadimitriou^*¹ and Anastasios Papadimitriou²
¹3rd Department of Pediatrics, Attikon University Hospital, Athens Greece, Athens, Greece, ²Attikon University Hospital, Athens, Greece

Background: Hormonal replacement in boys with congenital HH remains a challenge in pediatric endocrinology. Micropenis has been traditionally successfully treated, usually with 3 monthly injections of 25/50 mg of testosterone enanthate in the post-neonatal period or in early infancy, but in cases with bilateral cryptorchidism surgical intervention is required. Even after a successful surgery, the hypoplastic testes with the deficient proliferation of immature Sertoli cells before and during puberty, due mainly to the absence of the male neonatal surge in pulsatile gonadotropin secretion, are responsible for azoospermia and infertility later in life.

Objective: To investigate whether early postnatal daily injections of the commercially available recombinant LH plus FSH preparation (Pergoveris®) could mimic the physiological male mini puberty and successfully resolve bilateral cryptorchidism, result in penis enlargement and restore the responses of the Leydig and Sertoli cells to normal.

Design and Methods: Three neonates with bilateral cryptorchidism in intra-abdominal position and micropenis with absence of neonatal male mini-puberty were treated for 3 months with daily subcutaneous injections of Pergoveris^® (recombinant LH 75 IU and FSH 150 IU), followed monthly. Parents were trained to perform the injections. Case 1 (born 36⁺⁶, AGA) had CHARGE syndrome had a penile length of 2.3 cm at 4 months of age. Case 2 (born 36⁺³, SGA) had Kallmann syndrome and a penile length of 2 cm at 1 month of age. Case 3 (born 37⁺¹, AGA) had septo-optic dysplasia and panhypopituitarism diagnosed soon after borth beacause of symptomatic hypoglycemia and cholestatic jaundice with a penile length of 1.5 cm at 3 months of age.

Results: Mean LH reached high normal C1: 7.22, C2: 4.7, C3: 6.5 IU/L) and mean FSH supranormal levels C1: 132, C2: 88, C3: 77.1 IU/L. Mean Inhibin b and AMH, from subnormal before treatment, reached high normal levels: 241 pg/ml and 1034 pmol/L respectively. Mean Testosterone increased from undetectable levels to C1: 2.22, C2: 3.33 and C3: 0.55 ng/ml. In all cases testes descended in scrotal position by the end of the 1^st in C1, 2^nd in C2 and 3^rdmonth in C3 with a volume of 1.5, 2.5 ml and 2 ml respectively. In cases 1 and 2 who have now reached 3.5 and 4.5 years of age testes have regressed to 0.5 and 1 ml respectively but are still in scrotal position. Case 3 just completed therapy. Penile length increased to 4.5, 3.8 and 4 cm respectively. None presented any adverse events or reactions.

Conclusions: The present regimen mimics neonatal male mini puberty repairing micropenis and cryptorchidism and inducing high-normal activation of Leydig and Sertoli cells.

Nothing to Disclose: DTP, AP

LB-OR01-1 17971 1.0000 A Successful Treatment of Neonatal Micropenis and Bilateral Cryptorchidism Due to Hypogonadotropic Hypogonadism (HH) with 3-Month Daily Subcutaneous Injections of the Recombinant LH Plus FSH Preparation (Pergoveris®) 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Reproductive Endocrinology Saturday, June 21st 1:00:00 PM LB-OR01 5225 11:30:00 AM Origins and Treatment of Reproductive Dysfunction Oral

Anu Bashamboo^*¹, Marie France Portnoi², Marie-Charlotte Dumargne¹, Joelle Bignon-Topalovic¹, Hassan Rouba³, Sandra Rojo¹, Celia Ravel⁴, Luca Persani⁵, John C Achermann⁶, Sophie Christin-Maitre⁷, Jean-Pierre Siffroi² and Ken McElreavey¹
¹Institut Pasteur, Paris, France, ²Trousseau Hospital, Paris, France, ³Institut Pasteur of Morocco, Casablanca, France, ⁴Centre Hospitalier Universitaire de Rennes, Rennes, France, ⁵Istituto Auxologico Italiano, milan, Italy, ⁶UCL Inst of Child Health, London, United Kingdom, ⁷Hôpital Saint-Antoine, Assistance Publique Hôpitaux de Paris, Paris, France

Sox8, a member of the SoxE gene family, is expressed in Sertoli cells during early testis development. The combined action of both Sox9 and Sox8 in murine Sertoli cells is essential for proper testis differentiation. Sox8 null mutants show normal embryonic and early postnatal testis development but develop infertility due to progressive spermatogenic failure. We identified a patient with 46,XY complete gonadal dysgenesis (CGD), who carried a paracentric inversion of the short arm of chromosome 16. The distal breakpoint mapped to 69 kb upstream of the SOX8 gene. Sequencing the SOX8 open reading frame in other cases of 46,XY gonadal dysgenesis revealed a heterozygote missense mutation in the HMG-box. Extending this analyses to unexplained cases of infertility revealed 7 heterozygous missense mutations associated with either azoo- or oligozoospermia (n=274, 2.5%) and 7 heterozygous missense or frameshift mutations associated with ovarian insufficiency (n=153, 4.75%). Sequencing of the SOX8 gene in ancestry-matched control samples (n=840) identified 10 rare or novel missense mutations (1.19%). However, when a subgroup of ancestry-matched control samples of known fertility status and/or with normal semen parameters were analysed, only 2 of 420 (0.047%) carried rare or novel SOX8 missense mutations. These two mutations were predicted to be benign by SIFT and PolyPhen analysis. All of the mutations associated with infertility were located outside the HMG-box of the protein. In-vitro assays, showed that the mutations have a variable effect on the biological function of SOX8. Overall the data demonstrate that, similar to SF-1/NR5A1, heterozygous mutations involving the SOX8 gene contribute to a wide range of reproductive anomalies in both the male and the female.

Nothing to Disclose: AB, MFP, MCD, JB, HR, SR, CR, LP, JCA, SC, JPS, KM

LB-OR01-2 18002 2.0000 A Mutations Involving SOX8 Are Associated with a Spectrum of Human Reproductive Disorders 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Reproductive Endocrinology Saturday, June 21st 1:00:00 PM LB-OR01 5225 11:30:00 AM Origins and Treatment of Reproductive Dysfunction Oral

Andy Videsh Babwah^*¹, Michele Dawn Calder¹, Yee-Ming Chan², Renju Raj³, Adrienne Elbert⁴, Michelle Noonan⁴, Claudia Caligioni⁵, Nathalie Berube⁴, Moshmi Bhattacharya⁴ and Stephanie Beth Seminara⁵
¹University of Western Ontario, London, ON, Canada, ²Massachusetts General Hospital, MA, ³University of Vermont College of Medicine, ⁴University of Western Ontario, ⁵Massachusetts General Hospital, Boston, MA

The hypothalamic kisspeptin signaling system is a major positive regulator of the reproductive neuroendocrine axis, and loss of Kiss1 in the mouse results in infertility; a condition generally attributed to its hypogonadotropic hypogonadism. We demonstrate that in Kiss1^-/- female mice, acute replacement of gonadotropins and estradiol restores ovulation, mating and fertilization; however, these mice are still unable to achieve pregnancy because embryos fail to implant. Progesterone treatment did not overcome this defect. Kiss1^+/- embryos transferred to a wild-type female mouse can successfully implant, demonstrating the defect is due to maternal factors. Kisspeptin and its receptor are expressed in the mouse uterus, and we suggest that it is the absence of uterine kisspeptin signaling that underlies implantation failure. This absence, however, does not prevent the closure of the uterine implantation chamber, proper alignment of the embryo and the ability of the uterus to undergo decidualization. Instead, the loss of Kiss1 expression specifically disrupts embryo attachment to the uterus. We observed that on the day of implantation, Lif, a cytokine that is absolutely required for implantation in mice, is weakly expressed in Kiss1^-/- uterine glands and that the administration of exogenous Lif to hormone-primed Kiss1^-/- female mice is sufficient to partially rescue implantation. Taken together, our study reveals that uterine kisspeptin signaling regulates glandular Lif levels, thereby identifying a novel and critical role for kisspeptin in regulating embryo implantation in the mouse. This study provides compelling reasons to explore this role in other species, particularly livestock and humans.

Nothing to Disclose: AVB, MDC, YMC, RR, AE, MN, CC, NB, MB, SBS

LB-OR01-3 18027 3.0000 A Implantation Failure in Female Kiss1-/- Mice Is Independent of Their Hypogonadic State and Can be Partially Rescued By Leukemia Inhibitory Factor 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Reproductive Endocrinology Saturday, June 21st 1:00:00 PM LB-OR01 5225 11:30:00 AM Origins and Treatment of Reproductive Dysfunction Oral

Jean-Pierre J Bourguignon^*¹, Delphine Franssen¹, Arlette Gerard¹, Benoit Hennuy² and Anne-Simone M Parent¹
¹Developmental Neuroendocrinology Unit, GIGA Neurosciences, University of Liège, Liège, Belgium, ²transcriptomic platform, GIGA, University of Liège, Belgium

We recently reported that neonatal exposure of female rats to 1 or 10 µg/kg.day of diethylstilbestrol could respectively cause late or early puberty and consistent changes in maturation of pulsatile GnRH secretion (1). Endocrine disrupting effects of low bisphenol A (BPA) doses in the µg range are a matter of controversy. We studied the effects of neonatal exposure to a very low dose of 25 ng/kg.day in comparison with 5mg/kg.day. Newborn female rats were exposed to vehicle (corn oil) or BPA injected subcutaneously from postnatal day 1 (PND 1) to 5 or from PND 1 to 15. The rats were followed for vaginal opening (VO) and estrous cyclicity. The GnRH interpulse interval that was known to decrease between PND 10 and 25 was studied ex vivo using hypothalamic explants obtained at PND 15, 20 or 25. Gene expression in the retrochiasmatic hypothalamus was assessed by whole exome RNA-sequencing on PND 20 (3 samples per condition). After neonatal exposure to 25 ng/kg.day of BPA for 15 days, the age at VO was delayed (35.3 ± 0.7 days vs 33.5 ± 0.5 days in controls) while advancement (32.1 ± 0.6 days) was observed using 5 mg/kg.day. The difference in pubertal timing between the two doses was significant. The late VO after exposure to 25 ng/kg.day of BPA was preceded by a significant increase in GnRH interpulse interval (52.5 ± 0.8 min vs 44.6 ± 0.7 min in controls) at PND 20. By contrast, early VO after exposure to 5 mg/kg/d was preceded by a significant decrease in GnRH interpulse interval (40.3 ± 0.1 min vs 42.8 ± 0.4 min). Similar dose-related changes in GnRH secretion were observed after BPA exposure from PND 1 to 5. At PND 20, after exposure from PND 1 to 15, RNA expression of 10 genes showed significant opposing changes in the high vs low BPA dose groups. Fourteen genes displayed an expression that was only affected by 25 ng/kg of BPA. The dose of 5 mg/kg resulted in modified expression level of 472 genes versus controls. A significant difference in level of RNA expression was observed for 1407 genes when comparing the two BPA dose conditions. In conclusion, neonatal exposure to a very low dose of BPA was followed by a delay in pubertal timing with consistent changes in pulsatile GnRH secretion. Changed hypothalamic RNA expression confirmed the effects of the two BPA doses with opposing changes of similar genes in relation to BPA dose and alteration of distinct genes by each of the two doses.

Nothing to Disclose: JPJB, DF, AG, BH, ASMP

LB-OR01-4 17984 4.0000 A Delayed Puberty, Slowed Down GnRH Secretion and Changed Hypothalamic RNA Expression after Neonatal Exposure to a Very Low Environmentally Relevant Dose of Bisphenol a 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Reproductive Endocrinology Saturday, June 21st 1:00:00 PM LB-OR01 5225 11:30:00 AM Origins and Treatment of Reproductive Dysfunction Oral

Almudena Veiga-Lopez^*¹, Sindhu Halubai¹, Lixia Zeng², Charles F Burant², Kurunthachalam Kannan³, Subramaniam Pennathur² and Vasantha Padmanabhan²
¹Univ of Michigan Med Schl, Ann Arbor, MI, ²University of Michigan, Ann Arbor, MI, ³Wadsworth Center, Albany, NY

Human exposure to bisphenol A is a contentious public health issue. Developmental exposure to BPA leads to intrauterine growth restriction and adult metabolic diseases, including insulin resistance. Free fatty acid imbalance and oxidative stress are common traits of disorders manifesting insulin resistance and are thus potential targets for insult from BPA. We hypothesized that mothers exposed to higher levels of BPA will have metabolic imbalance reflected as systemic inflammation via the oxidative stress pathway. Pregnant women were recruited in the first trimester (8 to 14 weeks) and blood samples collected avoiding plastic contact. Unconjugated (uBPA) and glucuronidated BPA (gBPA) concentrations in plasma were quantified using HPLC and MS/MS by one of the laboratory validated through the Round Robin study [1]. Two subsets (n=12 each), one that had low uBPA (Blow:0.08±0.01 ng/ml, mean±SEM:) and another with high uBPA (Bhigh: 27.8±8.4 ng/ml) concentrations were selected to assess 1) the concentration of products of tyrosine oxidation (chlorotyrosine (CT), dityrosine (DT), and nitrotyrosine (NT)), markers for oxidative stress and 2) free fatty acids (FFA). Products of tyrosine oxidation and FFAs were measured using MS/MS and GC. Independent T-test revealed that pregnant mothers with high BPA levels had higher levels of NT (Bhigh: 0.38±0.06 vs. Blow: 0.09±0.008 mM/mol; P=0.001), but not CT or DT compared to those with low BPA. A significant Pearson correlation was found between gBPA and NT levels (r=0.440, P<0.05) and marginally significant between uBPA and NT (r=0.398, P=0.054). This is of relevance, as NT has been implicated in the pathogenesis of chronic diseases including diabetes-induced vascular dysfunction. Levels of 14:0, 14:1, 18:3 (n-3), 20:0, 20:5, 22:0, 22:1, 22:5, and 22:6 FFA were undetectable. FFA 18:3 (n-6), 20:1, and 22:4 levels were detected in £ 3 subjects and not analyzed. FFA 16:0 was higher in the Bhigh group (81.1±9.6nmol/ml) compared to the Blow group (52.7±5.3 nmol/ml; P=0.01). No differences were found in FFAs 16:1, 18:0, 18:1(n-7), 18:1(n-9), 18:2, 20:2, 20:3, and 20:4, between groups. A positive correlation was found between uBPA and 16:0 (palmitic acid; r=0.779, P<0.001) and between uBPA and total FFA (r=0.645, P<0.001), but not gBPA. The correlation between 16:0 and NT tended to be significant (r=0.396, P=0.062). The correlation between increased maternal BPA exposure and increased maternal NT suggests that BPA may play a role in the development of systemic inflammation via the nitrosative stress pathway. Higher palmitic acid and its correlation with increased NT are supportive of palmitate-induced oxidative stress, a mediator of cellular insulin resistance. Further studies are required to determine if palmitic acid and NT may ultimately serve as non-invasive biomarkers for assessing the degree of BPA toxicity and whether they impact offspring well being.

Nothing to Disclose: AV, SH, LZ, CFB, KK, SP, VP

LB-OR01-5 17927 5.0000 A First Trimester Maternal BPA Levels Correlate Positively with Systemic Oxidative Stress in Humans 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Reproductive Endocrinology Saturday, June 21st 1:00:00 PM LB-OR01 5225 11:30:00 AM Origins and Treatment of Reproductive Dysfunction Oral

Peter Laurberg^* and Stine Linding Andersen
Aalborg University Hospital, Aalborg, Denmark

Objective: Birth defects might seriously hamper the life of the child and the parents. Increasing evidence suggests that Methimazole (MMI)/Carbimazole (CMZ) treatment of maternal hyperthyroidism in early pregnancy might lead to severe birth defects (e.g. aplasia cutis, choanal and esophageal atresia). On the other hand, it is less clear if treatment with Propylthiouracil (PTU) is teratogenic.

Methods: Cohort study of all children live-born in Denmark, 1996-2008 (n=817,093). Information on maternal redeemed prescription of antithyroid drug (ATD) and diagnosis of birth defect in the child were obtained from nationwide registers. Logistic regression was used to estimated crude and adjusted odds ratio (OR) with 95% confidence interval (CI) for a diagnosis of birth defects before the age of 2 years (overall and within specific subgroups of birth defects) in children exposed to ATD (MMI/CMZ n=1,097, PTU n=564) in early pregnancy versus non-exposed children (no maternal ATD treatment (n=811,730)). To evaluate the PTU associated birth defects in detail, we subsequently focused on the two subgroups of birth defects which revealed a significant association with PTU (face and neck region and urinary system). As these birth defects might be less severe, the time of diagnosis could be later. Thus, we extended the follow-up period to a median age of 8 years and reviewed individual cases in detail.

Results: In line with previous studies, MMI/CMZ was associated with an increased risk of birth defects and a particular high risk of the previously described embryopathi with severe birth defects. Overall, additional 3.4% children had birth defects after MMI/CMZ exposure in early pregnancy. As a new finding, PTU was also associated with an increased risk of birth defects with additional 2.3% cases, and the risk was confined to two subgroups: the face and neck region and the urinary system. When follow-up was performed to the age of 8 years, a total of 14 cases (face and neck n=7 and urinary system n=7) were identified after PTU exposure and an increased risk was again observed (face and neck; adjusted OR 4.92 (95% CI 2.04-11.86), urinary system; 2.73 (1.22-6.07)) versus non-exposed children. In the face and neck region, the birth defects were dominated by preauricular and branchial sinus/fistula/cyst, and 6 of 7 cases had undergone surgery. In the urinary system, the birth defects were dominated by congenital hydronephrosis and renal cysts, and 3 of 7 cases had undergone surgery.

Conclusions: Both MMI/CMZ and PTU treatment in early pregnancy are associated with an increased risk of birth defects, but the spectrum of malformations is different. The MMI/CMZ associated birth defects are often severe. The PTU associated birth defects seemed less severe, but they did, however, often require surgery. The use of ATD should be restricted as much as possible in early pregnancy. If treatment is considered necessary, PTU should be used.

Nothing to Disclose: PL, SLA

LB-OR01-6 17798 6.0000 A Birth Defects after the Use of the Antithyroid Drugs Methimazole and Propylthiouracil in Early Pregnancy Are Common and May be Serious. a Danish Nationwide Study 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Reproductive Endocrinology Saturday, June 21st 1:00:00 PM LB-OR01 5225 11:30:00 AM Origins and Treatment of Reproductive Dysfunction Oral

Gabriela R V Sousa^*¹, Tamaya C Ribeiro¹, Andre M Faria¹, Beatriz M P Mariani², Antonio M Lerario³, Maria Claudia N Zerbini², Ibere C Soares⁴, Alda Wakamatsu², Venancio A F Alves², Berenice B Mendonca⁵, Maria Candida B V Fragoso⁶, Ana Claudia Latronico¹ and Madson Q. Almeida⁷
¹Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil, ²Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil, ³ICESP/University of Sao Paulo, São Paulo, Brazil, ⁴Faculdade de Ciências da Saúde de Barretos Dr. Paulo Prata, Barretos, Brazil, ⁵Unidade de Endocrinologia do Desenvolvimento, Laboratório de Hormônios e Genética Molecular LIM/42, Disciplina de Endocrinologia, Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, ⁶Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo, ⁷Hospital das Clínicas & Instituto do Câncer do Estado de São Paulo (ICESP), Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil

Introduction: A microRNA family, miR-103/107, attenuates miRNA biosynthesis by targeting Dicer, a key component of the miRNA processing machinery. In human breast cancer, high levels of miR-103 and miR-107 are associated with metastasis and poor outcome. Recently, DICER1 mutations in the RNase IIIb domain were found in 29% of nonepithelial ovarian tumors, predominantly in Sertoli–Leydig cell and embryonic tumors. These mutations were restricted to codons encoding metal-binding sites within the RNase IIIb catalytic center. Aim: Since adrenocortical tumors (ACTs) are also sterodoigenic lesions such as Leydig cell tumors and up to 12% of adrenocortical tumors are diagnosed before 1 yr, which suggests an embryonic origin, we investigated DICER1 mutations in metal biding sites located at the RNase IIIb domain, and studied DICER1 and miR-103 expression in pediatric and adult ACTs. Patients and methods: Four metal-binding sites (codons 1709 and 1705 in exon 24; codons 1810 and 1813 in exon 25) within the RNase IIIb catalytic center of DICER1 were sequenced in 86 ACTs (25 children and 61 adults). DICER1 expression was assessed in 80 ACTs from 23 children (17 clinically benign and 6 clinically malignant) and 57 adults (28 adenomas and 29 carcinomas) by quantitative real-time PCR. Expression of miR-103 was analyzed in a subgroup of this cohort (49 ACTs). Results: No variant was identified in the four metal binding sites within the RNase IIIb catalytic center of DICER1 in pediatric and adult ACTs. DICER1 mRNA levels in ACTs were 5.0 ± 0.8 (range, from 0 to 43.6) in comparison with the normal adrenal gland. DICER1 expression was not associated with overall and disease-free survival in both children and adults. DICER1 and miR-103 expression did not correlate in both children and adults with ACTs. Interestingly, miR-103 overexpression was associated with a reduced overall survival in pediatric ACTs (p= 0.02), but not with disease-free survival. Additionally, miR-103 expression levels were higher in clinically malignant than in clinically benign pediatric ACTs [median (range), 63.5 (30.8-188.6) vs. 31.4 (12.7-72.5), p= 0.08). miR-103 expression did not predict outcome in adult ACTs. Conclusion: DICER1 deregulation was not associated with pediatric and adult adrenocortical tumorigenesis, but miR-103 overexpression was a predictor of reduced overall survival in children with ACTs.

Nothing to Disclose: GRVS, TCR, AMF, BMPM, AML, MCNZ, ICS, AW, VAFA, BBM, MCBVF, ACL, MQA

14496 1.0000 SAT-0781 A Mutation and Expression Analysis of DICER1 and Mir-103 in Pediatric and Adult Adrenocortical Tumors 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0781-0797 4771 1:00:00 PM Adrenal Tumors: ACC & Adrenal Incidentaloma Poster

Kaori Sugawara¹, Ken Matsuda², Naotaka Kogure¹, Akira Uruno³, Ikuko Sato¹, Kyoko Shimizu¹, Rehana Parvin¹, Takeo Yoshikawa³, Masataka Kudo², Akiko Saito-Hakoda¹, Ryo Ito¹, Atsushi Yokoyama¹, Sadayoshi Ito² and Akira Sugawara^*⁴
¹Tohoku Univ Grad School of Med, Sendai, Japan, ²Tohoku University Hospital, Sendai, Japan, ³Tohoku Univ Grad School of Med, ⁴Tohoku Univ Grad School of Med, Sendai Miyagi-Ken, Japan

11β-hydroxylase gene (CYP11B1) encodes the final step enzyme of adrenal cortisol production. In order to examine its transcriptional regulation, we generated a stable human adrenal H295R cell line expressing -1102/-2 of CYP11B1 promoter region fused upstream of liciferase cDNA. The cell line responded 3.0-fold to dbcAMP (1 mM), 2.8-fold to angiotensin II (100 nM), 1.5-fold to ACTH (10 μM), and 2.4-fold to KCl (10.4 mM), which was almost equivalent to the transient transfection study. We next examined the effect of high-glucose on CYP11B1 expression. The stable cell line and H295R cells were incubated with several concentrations of D-glucose. The cellular osmolarity was adjusted by L-glucose. mRNA expression of CYP11B1 was determined by real-time PCR. Interestingly, D-glucose dose- and time-dependently stimulated CYP11B1 transcription as well as CYP11B1 mRNA expression. The high-glucose stimulated CYP11B1 transcription was not affected by treatment with LY333531, a protein kinase (PK) C-β inhibitor, but was partially abrogated by treatment with H89, a PKA inhibitor. These data indicate that high-glucose mediated CYP11B1 transcription activation may at least in part be mediated via the cAMP-PKA pathway independent of PKC-β. It is therefore speculated that high-glucose-induced CYP11B1 expression may induce cortisol secretion, which may result in cortisol-induced hyperphagia and further increase of blood glucose.

Nothing to Disclose: KS, KM, NK, AU, IS, KS, RP, TY, MK, AS, RI, AY, SI, AS

16701 2.0000 SAT-0782 A Generation of a Stable H295R Cell Line Expressing 11beta-Hydroxylase Gene Promoter and the Effect of High-Glucose on Its Expression 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0781-0797 4771 1:00:00 PM Adrenal Tumors: ACC & Adrenal Incidentaloma Poster

Guillaume Assie^*¹, Eric Letouze², Anne Jouinot³, Windy Luscap³, Olivia Barreau⁴, Hanin Omeiri⁵, Stéphanie Rodriguez⁵, Karine Perlemoine¹, Martin Fassnacht⁶, Fernande rené-Corail⁷, Nabila Elarouci⁸, Silviu Sbiera⁹, Matthias Kroiss¹⁰, Bruno Allolio¹¹, Jens Waldmann¹², Marcus Quinkler¹³, Massimo Mannelli¹⁴, Franco Mantero¹⁵, Thomas Papathomas¹⁶, Ronald R de Krijger¹⁷, Antoine Tabarin¹⁸, Veronique Kerlan¹⁹, Eric Baudin²⁰, Frederique Tissier⁵, Bertrand Dousset²¹, Lionel Groussin²², Laurence Amar²³, Eric Laurent Clauser²⁴, Xavier Bertagna¹, Bruno Ragazzon¹, Felix Beuschlein²⁵, Rossella Libe¹, Aurélien de Reynies²⁶ and Jerome Yves Bertherat²⁷
¹INSERM U 1016, CNRS 8104, Institut Cochin, Paris Descartes University, Paris, France, ²Ligue Nationale Contre Le Cancer, Paris, France, ³INSERM U1016, CNRS UMR 8104, Institut Cochin Université Paris Descartes, Paris, ⁴Inserm U1016, CNRS 8104, Institut Cochin, Paris Descartes University, Paris, ⁵INSERM U1016, CNRS UMR 8104, Institut Cochin Université Paris Descartes, ⁶Medizinische Klinik und Poliklinik IV, Klinikum der Universität München, University of Munich, Munich, Germany, ⁷INSERM U 1016, CNRS 8104, Institut Cochin, Paris Descartes University, Paris, Paris, ⁸Ligue Nationale Contre Le Cancer, Paris, ⁹Medizinische Klinik und Poliklinik IV, Ludwig-Maximilians-Universität München, ¹⁰University Hospital Wuerzburg, ¹¹University of Wuerzburg, Wuerzburg, Germany, ¹²Visceral-, Thoracic and Vascular Surgery, University Hospital Giessen and Marburg, Marburg, Germany, ¹³Charité University Medicine Berlin, Campus Mitte, Berlin, Germany, ¹⁴Univ of Florence, Florence, Italy, ¹⁵University of Padova, Padova, Italy, ¹⁶Department of Pathology, Josephine Nefkens Institute, Erasmus MC-University Medical Center, Rotterdam, Netherlands, ¹⁷Erasmus MC, Rotterdam, Netherlands, ¹⁸Universite de Bordeaux II/Hopital Haut Leveque, Pessac, France, ¹⁹CHUBrest - Hopital De La Cavale Blanche, Brest, France, ²⁰Institut Gustave-Roussy, Paris, France, ²¹Department of Digestive and Endocrine Surgery, Assistance Publique Hôpitaux de Paris, Hôpital Cochin, Paris, France, ²²INSERM U 1016, CNRS 8104, Institut Cochin, Paris Descartes University, Paris, ²³Hopital Europeen Georges Pompido, Paris, France, ²⁴Assistance Publique Hôpitaux de Paris, Hôpital Cochin, Paris, France, Paris, France, ²⁵University of Munich, Munich, Germany, ²⁶Ligue Contre Le Cancer, Paris, ²⁷INSERM U 1016, Cochin Institute, Paris Descartes University, Paris, France

Despite the overall poor prognosis, adrenocortical carcinoma (ACC) outcome is heterogeneous. The pathophysiological mechanisms are not well identified. The aim was to perform an integrated genomic analysis of these tumors, to identify the molecular alterations and a molecular classification with potential clinical impact.

130 ACC were included, with a discovery set of 53 ACC from French centers (COMETE network) and an independent validation set of 77 ACC from European centers (ENSAT network). ACCs from the discovery cohort were characterized by exome sequencing, transcriptome, mirnome, methylome and single nucleotide polymorphism (SNP) arrays.

Exome sequencing and SNP array analysis in the discovery cohort revealed recurrent alterations in known drivers (CTNNB1, TP53, CDKN2A, RB1, MEN1) and in genes not previously reported to be altered in ACC (ZNRF3, DAXX, TERT and MED12). These alterations were confirmed in the validation cohort.

MiRna expression identified three clusters of ACC: the Mi1 cluster was characterized by the up-regulation of 11 miRNA from the 506-514 miRNA cluster in Xq27.3, and the down-regulation of 38 miRNAs from the DLK1-MEG3 miRNA cluster in 14q32.2; the Mi2 cluster was characterized by the up-regulation of the 506-514 miRNA cluster; the third cluster (Mi3) presented none of these alterations.

Integration of the different unsupervised omics classifications defined molecular groups, associated with different outcomes. The C1A group of poor outcome was characterized by a specific transcriptome signature, by the accumulation of somatic mutations and by specific DNA methylation alterations. The C1B group of better outcome displayed a distinct transcriptome signature, and the specific deregulation of the two miRNA clusters Mi1 and Mi2.

In conclusion, aggressive and indolent ACC correspond to two distinct molecular entities, driven by different oncogenic alterations.

Disclosure: BA: Consultant, Boehringer-Ingelheim, Coinvestigator, HRA-Pharma, Committee Member, Ipsen. EB: Research Funding, HRA Pharma. Nothing to Disclose: GA, EL, AJ, WL, OB, HO, SR, KP, MF, FR, NE, SS, MK, JW, MQ, MM, FM, TP, RRD, AT, VK, FT, BD, LG, LA, ELC, XB, BR, FB, RL, AD, JYB

15185 3.0000 SAT-0783 A Integrated Genomic Characterization of Adrenocortical Carcinoma 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0781-0797 4771 1:00:00 PM Adrenal Tumors: ACC & Adrenal Incidentaloma Poster

Akiko Saito-Hakoda¹, Akira Uruno², Kyoko Shimizu¹, Naotaka Kogure¹, Rehana Parvin¹, Ikuko Sato¹, Ikuma Fujiwara³, Hiroyuki Kagechika⁴, Yasumasa Iwasaki⁵, Atsushi Yokoyama¹, Sadayoshi Ito⁶ and Akira Sugawara^*¹
¹Tohoku Univ Grad School of Med, Sendai, Japan, ²Tohoku Univ Grad School of Med, ³Tohoku Univ Hosp, Sendai, Japan, ⁴Tokyo Med Dent Univ, ⁵Health Service Center, Kochi, Japan, ⁶Tohoku University Hospital, Sendai, Japan

Retinoids are possible candidates for the novel therapeutics against adrenocorticotropic hormone (ACTH)-dependent Cushing's syndrome. In ENDO 2011 and 2012, we demonstrated that RXR agonist HX630 inhibited ACTH secretion, proopiomelanocortin (Pomc) gene expression, and cell proliferation in AtT20 cells. In the present study, we examined the molecular mechanisms of HX630-mediated Pomc transcription suppression and the effects of HX630 on in vivo tumor formation. Using Pomc promoter deletion mutants, HX630 was demonstrated to suppress Pomc transcription via NurRE, to which Nurr1/Nur77 heterodimer binds. HX630 also decreased Nur77 and Nurr1 mRNA expression as well as Nur77/Nurr1 heterodimer binding to the region determined by ChIP assay. Overexpression of Nurr1 and Nur77 attenuated the HX630-mediated suppression. On the other hand, overexpression of RXR augmented the HX630-mediated suppression, while RXR siRNA attenuated the suppression. It is therefore indicated that HX630-mediated RXR activation may induce the decrease of Nur77 and Nurr1 mRNA expression, resulting in the suppression of Pomc transcription. Furthermore, HX630 inhibited tumor growth, and decreased Pomc mRNA expression in corticotroph tumor cells in female nude mice in vivo. These results therefore indicate that RXR agonist HX630 may be a novel therapeutic candidate for ACTH-dependent Cushing's syndrome.

Nothing to Disclose: AS, AU, KS, NK, RP, IS, IF, HK, YI, AY, SI, AS

16676 4.0000 SAT-0784 A Effects of RXR Agonist HX630 on Pomc Promoter Activity and in Vivo Tumor Formation 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0781-0797 4771 1:00:00 PM Adrenal Tumors: ACC & Adrenal Incidentaloma Poster

Segolene Hescot^*¹, Angelo Paci², Atmane Seck², Abdelhamid Slama³, Say Viengchareun⁴, Severine Trabado³, Sylvie Brailly³, Jacques Young⁵, Eric Baudin⁶ and Marc Lombes⁴
¹INSERM U693, Le Kremlin Bicetre, France, ²Gustave Roussy Institute, Villejuif, France, ³APHP CHU Bicêtre, Le Kremlin Bicetre, France, ⁴Inserm U1185, Le Kremlin-Bicêtre, France, ⁵University Paris Sud, Department of Endocrinology, Bicetre Hospital, INSERM U693, Le Kremlin-Bicêtre, Fance, Le Kremlin Bicêtre, France, ⁶Institut Gustave-Roussy, Paris, France

Mitotane (o,p’DDD) is the most effective treatment of advanced adrenocortical carcinoma (ACC) but its molecular mechanism of action remains poorly understood. It has been suggested that o,p’DDD requires a metabolic transformation into o,p’DDA, carried out by an unknown cytochrome P450 enzyme in adrenal cells. To determine whether o,p’DDA was responsible for pharmacological effects of mitotane, we quantified o,p’DDD and o,p’DDA contents by UV-HPLC analysis with a threshold detection limits of 0.5 and 2.5 mg/l respectively, in human adrenocortical tissues and in subcellular fractions of H295R and SW13 adrenal cells. In both a ACC and a normal adrenal gland of mitotane-treated patients, o,p’DDA was undetectable unlike o’p’DDD, suggesting a lack of cellular uptake and/or in situ production. Furthermore, o,p’DDA was not detected in whole cell homogenates nor in the supernatant of cultured cells after 48 h exposure to 50 µM of o,p’DDD, consistent with the absence of o,p’DDA production in these models. While o,p’DDD concentration was significantly reduced by 40% in the supernatant of H295R cells after 48h exposure, indicating a marked cellular uptake, o,p’DDA levels were not altered upon cell exposure suggesting that it was not efficiently transported into H295R cells. Next, we investigated functional consequences of o,p’DDA compared to o,p’DDD exposure on proliferation, steroidogenesis and mitochondrial activity. Dose-dependent concentration curves (from 10 to 300 µM) showed that o,p’DDA did not inhibit proliferation of H295R and SW13 cells. At 50 µM, o,p’DDA was also unable to inhibit 17-hydroxyprogesterone secretion as opposed to the 70% reduction induced by o,p’DDD. Mitotane significantly decreased expression of genes encoding proteins involved in steroidogenesis whereas o,p’DDA had little or no effect on HSD3B2, CYP21, CYP11B, StAR and CYP11A1 expression. Moreover, o,p’DDA did not alter respiratory chain complex IV (cytochrome c oxidase) activity and gene expression nor induce mitochondrial biogenesis as previously demonstrated with o,p’DDD (1).

Collectively, our results indicate a lack of adrenal cell metabolism and uptake of o,p’DDA and provide in vitro evidence that o,p’DDA is not an effective metabolite of mitotane to control tumor proliferation, steroidogenesis and to impact mitochondrial activity. Better understanding of o,p’DDD pharmacology will enable identification of predictive factors of efficacy for ACC management.

Disclosure: SH: Researcher, HRA Pharma. EB: Research Funding, HRA Pharma. Nothing to Disclose: AP, AS, AS, SV, ST, SB, JY, ML

12996 5.0000 SAT-0785 A Lack of Adrenal Cell Metabolism and Uptake of o,P'dda Excludes Its Prominent Role As an Active Metabolite of Mitotane for Adrenocortical Carcinoma Treatment 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0781-0797 4771 1:00:00 PM Adrenal Tumors: ACC & Adrenal Incidentaloma Poster

Mabrouka Doghman^*¹, Silviu Sbiera², Veronica Granatiero³, Rosario Rizzuto³, Martin Fassnacht⁴ and Enzo Lalli¹
¹IPMC CNRS UMR 7275, Valbonne, France, ²Medizinische Klinik und Poliklinik IV, Ludwig-Maximilians-Universität München, ³Padua University, Padua, Italy, ⁴Medizinische Klinik und Poliklinik IV, Klinikum der Universität München, University of Munich, Munich, Germany

Steroidogenic Factor-1 dosage has a critical role in regulating the proliferation of human adrenocortical cells and to trigger adrenocortical tumorigenesis in mice. We identified FATE1 (Fetal and Adult Testis Expressed) as a new target gene for SF-1 (1).

The aim of this study was to elucidate the localization and the role of FATE1 in adrenocortical cells and also in normal and human adrenocortical tumoral tissue by using different biochemical and immunochemistry approaches.

We showed that FATE1 is an outer mitochondrial membrane protein. By performing immunoprecipitation experiments followed by MS/MS analysis, we identified FATE1-interacting proteins. We found that FATE1 is enriched in MAM (Mitochondrial Associated Membranes), specialized subdomains that play an important role in metabolism, cell survival and cell death as well as in intracellular calcium signaling. By using overexpression and knockdown approaches, we showed that FATE1 overexpression induces a change in mitochondrial morphology and calcium uptake and modulates steroidogenesis, prompting us to explore the impact of FATE1 in the response to chemotherapeutic drugs.

Furthermore, high FATE1 expression is associated with poor clinical outcome in adrenocortical cancer (ACC). A strong negative correlation exists between SF-1 and FATE1 expression with recurrence-free and overall survival in ACC patients.

Taken together, our findings reveal that FATE1 is a potential marker for poor prognosis in human adrenocortical tumors and highlight the novel role of FATE1 on mitochondrial and ER dynamics in adrenocortical cancer cells.

Nothing to Disclose: MD, SS, VG, RR, MF, EL

15704 6.0000 SAT-0786 A Role of FATE1, a Novel Steroidogenic Factor-1 Target, in the Regulation of Mitochondrial Morphology and Function in Human Adrenocortical Cells 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0781-0797 4771 1:00:00 PM Adrenal Tumors: ACC & Adrenal Incidentaloma Poster

Joakim Crona^*¹, Per Hellman² and Peyman Björklund¹
¹Uppsala University, Uppsala, Sweden, ²Uppsala University, Sweden

Background: Pheochromocytomas are characterized by pronounced genetic heterogeneity with more than 14 disease causing genes described to date. Tumours are divided into 3 molecular clusters: 1a (SDHx), 1b (VHL) and cluster 2 (NF1, RET, TMEM127 and MAX). Previous studies suggested common genomic aberrations with sporadic tumours harbouring loss of chromosome 1p and 3q whereas VHL related tumours showed loss of 3p and 11p arms.

Methods: DNA from 103 tumours were investigated by targeted Next generation sequencing (NGS) and high resolution SNP array. Copy number events and allelic imbalances were determined. Relative clonal fractions were calculated from NGS allele frequencies as wells as SNP array B-allele frequencies and log ratios. Phylogenetic trees were determined among matched tumour samples.

Results: Novel and previously described mutation specific copy number alterations were discovered. Copy number neutral loss of heterozygosity were observed in 15/103 tumours, while 37/103 had regions with hemizygous copy number loss that showed variable allelic frequencies. Branched and linear modes of evolution were observed in paired tumours from patients with metastatic disease.

Conclusion: Pheochromocytoma tumours may exhibit inter and intra tumoural heterogeneity. Presence of multiple tumour cell clones should be further investigated for potential impact on patient management and adds additional levels of complexity to the understanding of the genomic landscape of these tumours.

Nothing to Disclose: JC, PH, PB

15050 7.0000 SAT-0787 A Integrative Genomics Reveal Branched Evolution and Clonal Heterogeneity in Pheochromocytoma Tumours 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0781-0797 4771 1:00:00 PM Adrenal Tumors: ACC & Adrenal Incidentaloma Poster

Hannah Yi^*¹, Rita Keil¹, Robert J Handa² and Tao-Yiao J Wu¹
¹Uniformed Services University, Bethesda, MD, ²University of Arizona College of Medicine, Phoenix, AZ

The mPER2 gene is a `clock gene’ important for the regulation of circadian rhythms. Its deletion in mice results in impair diurnal rhythms. The goal of this study is to examine the effect of PER2 gene deletion on depressive and anxiety behaviors in male mice by using the Porsolt’s forced swim tests (FST) and marble burying test (MBT). For MBT, 19 WT and 16 PER2-/- mice were placed in cages with 4 cm of sawdust bedding and 8 black marbles for 30 minutes. The MBT was scored by marking the time each marble was buried for the first time to examine the rate of burying. FST was performed with 12 WT and 12 PER2-/- mice (clear cylinder with 15 cm of water for 6 minutes). Time spent floating rather than swimming was measured. Corticosterone (CORT) levels were enhanced in PER2-/- mice that were restrained. There was no difference (p>0.10) in MCR2 mRNA and 11β-hydroxysteroid dehydrogenase mRNA levels in the adrenals. There was no difference between the rate of burying for WT and PER2-/- mice in the MBT. However, PER2-/- mice spent more time floating in the FST test, suggesting that they exhibited more (p<0.05) depression-like behavior than WT mice. The results of the present study suggest that disruption of the circadian system via mPER2 gene knockout dysregulated the stress axis and is correlated with a depressive behavior. (HY and RK have equal contributions to the work)

Nothing to Disclose: HY, RK, RJH, TYJW

15099 8.0000 SAT-0788 A mPER2 Mutant Mice Show Depressive Behavior 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0781-0797 4771 1:00:00 PM Adrenal Tumors: ACC & Adrenal Incidentaloma Poster

Sofia Pereira^*¹, Valdemar Máximo², Ricardo Coelho², Paula Soares², Manuel Sobrinho Simões², Mariana Pereira Monteiro³ and Duarte Pignatelli⁴
¹Instituto Ciências Biomédicas Abel Salazar - UP, Porto, Portugal, ²IPATIMUP, Porto, Portugal, ³Instituto Ciências Biomédicas Abel Salazar - UP, Matosinhos, Portugal, ⁴Hospital S João, Porto, Portugal

INTRODUCTION: Normal human somatic cells cannot divide indefinitely due to progressive telomeric DNA loss mainly attributed to the lack of TERT (telomerase reverse transcriptase) expression. Activating TERT mutations and expression is able to immortalize some cell types. In adrenocortical tumors, the state of TERT remains unknown.

AIM: The aim of this work was to search for TERT promoter mutations in adrenocortical tumors and hyperplasias and to evaluate the TERT expression in adrenocortical tumors and normal adrenal glands.

METHODS: The expression of TERT was evaluated by immunohistochemistry in adrenocortical carcinomas (n=12), adrenocortical hyperplasias (n=3), adenomas with Cushing syndrome (n=7), non-functioning adenomas (n=11) and adjacent normal adrenal gland (n=8). For the search of promoter TERT mutations in the non-adrenocortical normal tissues, the two hotspots positions located -124 and -146 bp upstream from the ATG start was identified by PCR followed by Sanger sequencing.

RESULTS: TERT promoter mutations were not identified in any of the adrenocortical tumors and hyperplasias. TERT was found to be expressed in the nucleus of 33% of adrenocortical carcinomas and 45% of non-functiong adenomas, while no nuclear TERT expression was observed in adrenal hyperplasias and adenomas with Cushing syndrome. In the normal adrenal gland, all of the cases presented expression of TERT in the nuclei of zona glomerulosa and zona reticularis cells and approx. 25% of the cases presented positive staining in the nuclei of zona fasciculata.

CONCLUSIONS: These results suggest that there are no TERT promoter mutations in adrenocortical tumors and adrenocortical hyperplasias. As to the TERT protein it seems to be expressed in only a few adrenocortical carcinomas and adenomas, while an intense nuclear expression occurs in the normal adrenal gland. This warrants further research in order to understand its role in adrenal physiology.

Nothing to Disclose: SP, VM, RC, PS, MS, MPM, DP

16770 9.0000 SAT-0789 A TERT Promoter Mutations and TERT Expression in Normal and Tumoral Adrenocortical Tissues 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0781-0797 4771 1:00:00 PM Adrenal Tumors: ACC & Adrenal Incidentaloma Poster

Ping Li^*, Xinjue Dai, Yan Hao, Shanmei Shen and Dalong Zhu
Drum Tower Hospital Affiliated to Nanjing University Medical School, Nanjing, P. R. China, China

Although primary aldosteronism (PA) is the most common cause of endocrine hypertension, histopathological methods to differentiate the aldosterone-producing adenoma (APA) with cortisol-producing adenoma (CPA) and non-functioning adenoma (NFA), have not been established. Disabled-2 (Dab2) is a mitogen-regulated phosphoprotein. Previous study demonstrated Dab2 is a specific marker of the zona glomerulosa (ZG) not only in rodents but also in humans. Whether Dab2 can be a powerful tool for histopathological diagnosis of APA remains to be investigated. The objective of this study was to determine the significance of dab2 in APA by investigating the expression of Dab2 in tissue of different adrenocortical adenomas. Real-time PCR and immunohistochemistry were used to detect Dab2 expression in 12 APA samples, 10 CPA samples, 8 NFA samples and 6 normal adrenal samples. In normal adrenal glands, Dab2 protein is expressed strongly in the zona glomerulosa, weakly in zona fasciculata-reticularis, and with no expression in medulla. In APAs, several clusters of cells with positive dab2 staining were detected in 4 out of 12 samples, others (8 out of 12 APA samples) showed extensively weak dab2 staining. In all the CPA and NFA tumors, weak dab2 staining was detected. According to the results of real-time PCR, Dab2 mRNA expression increased significantly in APAs compared with normal adrenal glands. There is no significant difference between normal adrenal glands, CPAs and NFAs in Dab2 mRNA expression. Compared to nontumor portions, APAs also showed higher Dab2 mRNA expression in the tumor. No significant difference was detected between nontumor portions and tumor in Dab2 mRNA expression in CPAs and NFAs. In conclusion, Dab2 was predominantly localized in zona glomerulosa in normal adrenal gland. Increased Dab2 mRNA expression was detected in APAs compared to CPAs, NFAs and normal adrenal glands. Whereas, just sporadically cluster of ZG cells expressing Dab2 were demonstrated in some of the APAs. Dab2 was not an ideal marker in differential diagnosis of APAs with other adrenocortical adenomas in histopathology.

Nothing to Disclose: PL, XD, YH, SS, DZ

11423 10.0000 SAT-0790 A The Expression of Dab2 in Adrenocortical Adenomas 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0781-0797 4771 1:00:00 PM Adrenal Tumors: ACC & Adrenal Incidentaloma Poster

Guilherme Asmar Alencar^*¹, Antonio M Lerario², Mirian Y Nishi¹, Beatriz M P Mariani³, Madson Q. Almeida⁴, Johanne Tremblay⁵, Pavel Hamet⁵, Isabelle Bourdeau⁶, Maria Claudia N Zerbini³, Maria Adelaide Albergaria Pereira¹, Gilberto Carlos Gomes¹, Manoel Souza Rocha¹, Jose Luiz Chambo¹, Andre Lacroix⁷, Berenice B Mendonca⁸ and Maria Candida Barisson Villares Fragoso¹
¹Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil, ²ICESP/University of Sao Paulo, São Paulo, Brazil, ³Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil, ⁴Hospital das Clínicas & Instituto do Câncer do Estado de São Paulo (ICESP), Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil, ⁵Centre hospitalier de l’Université de Montréal (CHUM), Montréal, Canada, ⁶Centre hospitalier de l’Université de Montréal (CRCHUM), Montreal, QC, Canada, ⁷Centre hospitalier de lUniversité de Montréal (CHUM), Montréal, QC, Canada, ⁸Unidade de Endocrinologia do Desenvolvimento, Laboratório de Hormônios e Genética Molecular LIM/42, Disciplina de Endocrinologia, Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo

Background: Primary macronodular adrenal hyperplasia (PMAH) is a rare cause of Cushing's syndrome (CS). Familial clustering of PMAH has been described, suggesting an inherited genetic cause for this condition. Objectives: Identify the gene responsible for PMAH and characterize the clinical aspects of the disease. Methods: Forty-seven individuals of a Brazilian family with PMAH underwent a clinical, laboratory and radiologic evaluation. A SNP-based genome-wide linkage analysis followed by whole-exome sequencing were then performed in selected family members. Additionally, 29 other patients with PMAH and 125 healthy individuals were studied to validate the genetic findings. Moreover, PMAH tissue was also analyzed. Results: Three candidate genomic regions (16p11.2, 16q13 and 16q21) were initially identified by linkage analysis. A heterozygous germline mutation (p.Leu365Pro) was then found by whole-exome sequencing in the armadillo repeat containing 5 (ARMC5) gene (16p11.2). The mutation was confirmed by conventional sequencing in all 16 affected family members and was predicted to be damaging by in silico methods. Pedigree analysis showed that PMAH was inherited in an autosomal dominant fashion, with incomplete penetrance and variable expressivity. Seven additional ARMC5 germline mutations were subsequently identified in five out of 21 patients with apparently sporadic PMAH and in two other families with the disease. Further molecular analysis also identified a somatic mutational event in the adrenal tissue. The 1 mg dexamethasone suppression test (DST) proved to be the best laboratory test for the diagnosis of PMAH (sensibility: 90%; specificity: 87%; NR ≤ 50 nmol/liter). In nearly 38% (6/16) of the patients with familial PMAH, radiological abnormalities were found in only one of the adrenal glands. Distinct responses among family members were observed throughout the in vivo studies for aberrant hormone receptors, favoring the hypothesis that these receptors might be a secondary phenomenon in the pathophysiology of the disease in this family. Conclusions: We demonstrate that germline ARMC5 mutations are a frequent cause of familial PMAH, which is in agreement with data recently published by Assié et al. (1). Biallelic inactivation of ARMC5 is consistent with its role as a potential tumor suppressor gene. Subclinical CS is the most common clinical presentation of familial PMAH and DST demonstrated the highest sensitivity for the detection of this condition. Asymmetry between adrenal glands is a fairly frequent finding in familial PMAH.

Nothing to Disclose: GAA, AML, MYN, BMPM, MQA, JT, PH, IB, MCNZ, MAAP, GCG, MSR, JLC, AL, BBM, MCBVF

11315 11.0000 SAT-0791 A Inherited Autosomal Dominant Mutations in ARMC5 Gene a Frequent Cause of Primary Macronodular Adrenal Hyperplasia 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0781-0797 4771 1:00:00 PM Adrenal Tumors: ACC & Adrenal Incidentaloma Poster

Christopher Ryan Lapensee^*¹, Gary D Hammer², William E. Rainey¹ and Stephen W Hunt III³
¹University of Michigan, Ann Arbor, MI, ²Univ of Michigan, Ann Arbor, MI, ³Atterocor, Inc., Ann Arbor, MI

Adrenocortical Carcinoma (ACC) is a rare cancer of the adrenal cortex (~0.5-2 cases/million), accounting for 0.2% of cancer deaths annually. ACCs are highly aggressive with many patients presenting with metastases upon diagnosis due to difficulty of detection. While mitotane and cytotoxic chemotherapy are often used to treat metastatic ACC, the efficacy of most regimens is extremely limited, toxic, and poorly tolerated, leaving an unmet need for new treatments for ACC. ATR-101 is a novel, oral drug candidate currently in development for the treatment of ACC. ATR-101 is an inhibitor of acyl-coenzyme A:cholesterol O-acyltransferase (ACAT) that catalyzes intracellular esterification of free cholesterol. The adrenal cortex is particularly reliant on the proper maintenance of cholesterol homeostasis, as cholesterol is the precursor of all steroid hormones. In a variety of preclinical studies, ATR-101 has been shown to have selective inhibitory effects on cells derived from the adrenal cortex from a number of species. We are investigating mechanisms by which ATR-101 specifically targets and induces death in cells of the adrenal cortex. In H295R human adrenocortical carcinoma cells, ATR-101 treatment decreases viability and increases cellular toxicity in a time- and dose-dependent manner. Caspase-3 levels are markedly increased by ATR-101 after 4 hours of treatment, indicating activation of apoptosis. ATR-101 does not act as mitochondrial toxin in H295R cells, or alter oxygen consumption in isolated mitochondria. Together, these studies indicate that ATR-101 causes H295R cell death by activating the apoptotic pathway. The inability of ATR-101 to alter oxygen consumption in vitro suggests that ATR-101 induced cell death either does not involve disruption of mitochondrial activity, or an in vivo metabolite of ATR-101 present in the intact adrenocortical cell does indeed result in mitochondrial toxicity. Transcriptional profiling is being conducted to gain insight into genes and pathways altered by ATR-101 in H295R cells.

Disclosure: This work was supported in part by a sponsored research grant funded by Atterocor, Inc. SWH is an employee of Atterocor. WER is a consultant for Atterocor. GDH is a co-founder of Atterocor.

Disclosure: GDH: Founder, Atterocor, Inc.. WER: Consultant, Atterocor Inc. SWH III: Chief Scientific Officer, Atterocor, Inc., Chief Scientific Officer, Atterocor. Nothing to Disclose: CRL

16835 12.0000 SAT-0792 A ATR-101, a Selective and Potent Inhibitor of ACAT1, Causes Apoptosis in H295R ACC Cells 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0781-0797 4771 1:00:00 PM Adrenal Tumors: ACC & Adrenal Incidentaloma Poster

Tamaya C Ribeiro^*¹, Alexander Augusto Lima Jorge², Madson Q. Almeida³, Beatriz M P Mariani⁴, Mirian Y Nishi⁵, Berenice B Mendonca¹, Maria Candida Barisson Villares Fragoso⁶ and Ana Claudia Latronico⁷
¹Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil, ²Faculdade de Medicina da Universidade de Sao Paulo, Sao Paulo, Brazil, ³Hospital das Clínicas & Instituto do Câncer do Estado de São Paulo (ICESP), Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil, ⁴Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil, ⁵University of São Paulo, Hospital das Clinicas, Sao Paulo, Brazil, ⁶Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo, ⁷Hospital das Clinicas da Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil

Context: Insulin-like growth factor 1 receptor (IGF1R) overexpression is a prognostic biomarker of metastatic pediatric adrenocortical tumors. However, the molecular mechanisms that are implicated in its upregulation remain unknown.

Aim: To investigate the potential mechanisms involved in IGF1R overexpression in adrenocortical tumors.

Patients and methods: We studied 64 adrenocortical tumors that were diagnosed in 26 children and 38 adult patients. IGF1R expression was previously studied in 45 of these tumors, and overexpression was detected in 65% and 13% of pediatric and adult tumors, respectively. IGF1R copy number variation was determined using multiplex ligation-dependent probe amplification and confirmed using real time PCR. Automatic sequencing was used to identify IGF1R allelic variants. The expression of microRNAs involved in IGF1R regulation was determined using real time PCR.

Results: IGF1R amplification was detected in an adrenocortical carcinoma that was diagnosed in a 46-year-old woman with Cushing´s syndrome and virilization. IGF1R overexpression (5-times higher than the normal adrenal gland pool) was demonstrated in this case. In addition, gene amplification of other loci was identified in this adrenocortical malignant tumor, but no IGF1R copy number variation was evidenced in the remaining cases. Automatic sequencing revealed three known polymorphisms in the adrenocortical tumors, but they did not correlate with its expression. Expression of miR-100, 145, 375 and 126 did correlate with IGF1R expression.

Conclusion: We demonstrated the amplification and overexpression of the IGF1R gene in only one adrenocortical carcinoma, suggesting that these combined events are uncommon. In addition, IGF1R polymorphisms and abnormal microRNA expression did not correlate with IGF1R upregulation in adrenocortical tumors.

Nothing to Disclose: TCR, AALJ, MQA, BMPM, MYN, BBM, MCBVF, ACL

11331 13.0000 SAT-0793 A Amplification of the Insulin-like Growth Factor 1 Receptor Gene in an Adrenocortical Adenocarcinoma: Searching for Potential Mechanisms of Overexpression 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0781-0797 4771 1:00:00 PM Adrenal Tumors: ACC & Adrenal Incidentaloma Poster

Beatrice Rubin^*¹, Raffaele Pezzani¹, Maria Verena Cicala¹, Maurizio Iacobone², Carla Maria Scaroni¹, Marco Boscaro¹ and Franco Mantero¹
¹University of Padua, Padova, Italy, ²University of Padova, Padova, Italy

Introduction: Adrenocortical tumors (ACT) are common diseases mostly benign, but among them, adrenocortical carcinomas (ACC) appear highly aggressive with metastatic potential. Wnt/β-catenin pathway is frequently switched on in ACT, with β-catenin greatly dephosphorylated and consequently activated. While IWR1 induces an increase in Axin2 protein levels, XAV939 inhibits tankyrase 1 and tankyrase 2 (thus stabilizing Axin), both stimulate β-catenin phosphorylation and degradation.

Aim: To investigate the role of IWR1 and XAV939 alone or in combination with Mitotane in adrenocortical cell lines and in primary adrenocortical tumor cells.

Methods: MTT test was performed on SW13 and H295R cells and 2 ACC and 2 cortisol producing adenomas (CPA) primary cell cultures. Western blot and Immunofluorescence analyzed the expression of IWR1 and XAV939 targets (Tankyrase1, Tankyrase2, Axin1 and Axin2) and β-catenin.

Results: MTT test at 72h revealed a concrete cell viability decrease using XAV939 (10 µM) of 52% for SW13 and 37% for H295R cells. Furthermore IWR1 at 72h (10 µM) induced a moderate cell viability reduction for SW13 cells of 57% and for H295R cells of 30%. In SW13 cells at 72h, combination of Mitotane (10 µM) and XAV939 (10 µM) resulted in cell viability decrement of 15% if compared to XAV939 alone. No appreciably alteration was found in combination regimen for H295R cells. Furthermore XAV939 alone at 24h was effective in 1 ACC primary cell culture, while IWR1 at 24h showed a weak cell viability decrease in 1 ACC and 1 CPA primary cell culture.

Discussion and conclusions: Our preliminary results demonstrated by Western blot the expressions of different IWR1 and XAV939 targets in ACT cells. Moreover IWR1 and XAV939 seem to effectively act on Wnt/β-catenin pathway, providing evidence for their potential role on ACT treatment. Further biomolecular analysis are in progress to substantiate these data.

Nothing to Disclose: BR, RP, MVC, MI, CMS, MB, FM

13775 14.0000 SAT-0794 A New Drugs Switching off Wnt/Beta-Catenin Signaling in Adrenocortical Tumor Cells 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0781-0797 4771 1:00:00 PM Adrenal Tumors: ACC & Adrenal Incidentaloma Poster

Ivan Casaburi^*, Paola Avena, Adele Chimento, Arianna De Luca, Carmela Campana, Francesco Domanico, Emilia Martire, Rosa Sirianni and Vincenzo Pezzi
University of Calabria, Rende, Italy

ACC is a highly aggressive tumor extremely heterogeneous with limited therapeutic options and its pathogenesis involves integration of multiple signals. The study of ACC-associated syndromes has suggested that the IGF-II signaling pathway, p53, or Wnt/β-catenin signaling are currently the most attractive targets to fight ACC (1). Recently, we demonstrated the existence of a functional cross-talk between IGF-II signaling pathway and Estrogen Receptor α (ESR1), a gene overexpressed in ACC that drives estrogen-dependent proliferative effects (2,3). A useful strategy to develop an effective therapy for ACC is to identify a common downstream target of these multiple pathways. A good candidate could be ERRα, an orphan member of the superfamily of hormone nuclear receptors involved in the control of energy metabolism, mitochondrial biogenesis, and cancer progression. Several studies suggested that peroxisome proliferator-activated receptor γ coactivator-1 α and β (PGC-1α or PGC-1β) expression level and/or activity could regulate the transcriptional activity of ERRα and the ERRα/PGC-1 complex is a downstream target of multiple signaling pathways in hormone-dependent cancers (4). ERRα seems to be more expressed in ACC respect to adenoma and normal adrenal where it regulates steroidogenesis (5,6). In this study we investigated the role of ERRα in ACC by using the ERRα inverse agonist, XCT-790 and H295R cell line as experimental model for ACC. We revealed that XCT-790 cell treatment (1-10 µM) determined a dose dependent reduction of ERRα protein content concomitantly with a reduction of adrenocortical cancer H295R cells growth in vitro and in H295R xenograft model in vivo. Flow cytometric analysis indicated that XCT-790 increases cell population within the G0/G1 phase of the cell cycle, without any detectable sub-G1 cells accumulation. Accordingly, XCT-790 treatment decreases cyclin D1, cyclin-dependent kinase-4 (cdk4) protein expression and the phosphorylation status of pRb. Furthermore, XCT-790 treatment increases autophagic vesicles, concomitant with a reduction of PGC1-α protein expression, which is key player in mitochondrial biogenesis. Indeed, the evaluation of c-cic carrier expression, here used as an index of mitochondrial function and mass, suggests that mithophagy could be involved in such inhibitory effect. In conclusion these results, suggest that ERRα depletion could be a new strategy to control ACC growth.

Nothing to Disclose: IC, PA, AC, AD, CC, FD, EM, RS, VP

13884 15.0000 SAT-0795 A Estrogen Related Receptorα (ERRα) As Potential Target for a New Adrenocortical Carcinoma (ACC) Therapy 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0781-0797 4771 1:00:00 PM Adrenal Tumors: ACC & Adrenal Incidentaloma Poster

Mary Anne Pandy Calimon^*¹, Maria Katrina Malabanan Mallonga², Oliver Allan Castillo Dampil³, Pauline Rizelle Toledo¹ and Michael L Villa⁴
¹St. Luke's Medical Center Quezon City, Philippines, ²St. Luke's Medical Center, Quezon City, Philippines, ³St. Luke,s Medical Center, Quezon City, Philippines, ⁴St Lukes Medical Center, San Juan, Philippines

INTRODUCTION: Most adrenal masses are lipid-rich cortical adenomas with low unenhanced attenuation value on CT. Size is one of the criteria used to determine if it should be resected. However, all subjects with an incidentally discovered adrenal mass should still be screened for both catecholamine and cortisol.

METHODOLOGY: This is a retrospective cohort study done at a private tertiary hospital from January 2009 to August 2012. This included 23 admitted patients with a total of 27 adrenal masses detected on CT with corresponding biochemical evaluation. Outcomes of the biochemical tests and the unenhanced attenuation value (Hounsfield units) on CT were correlated using ANOVA with a significant p value < 0.05.

RESULTS: The total number of detected adrenal masses among the 23 patients was 27. Seven of the adrenal masses (25.9%) were functional, 9 (33.3%) were operated on. Urinary metanephrine were elevated in patients with an adrenal mass characterized by a high unenhanced attenuation value (HU 20.29 + 11.53) but this did not differ significantly compared to patients with normal urinary metanephrine (p value < 0.701). No significant difference in size of the tumor as well as the Hounsfield unit in patients with normal or elevated cortisol, renin and aldosterone (p-value 0.664, 0.804 and 0.701, respectively).

CONCLUSION: The Hounsfield unit measurements of adrenal masses were not statistically different among those with normal and elevated biochemical tests. Biochemical screening may still be necessary to determine the functionality of the tumor that will help determine if the adrenal mass should be resected or can be closely monitored.

Nothing to Disclose: MAPC, MKMM, OACD, PRT, MLV

14536 16.0000 SAT-0796 A Correlation of the Biochemical Markers of Adrenal Masses and Their Unenhanced Attenuation Values on Computed Tomography: A Retrospective Cohort Study 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0781-0797 4771 1:00:00 PM Adrenal Tumors: ACC & Adrenal Incidentaloma Poster

Yutaka Watanuki^*¹, Shinobu Takayasu², Kazunori Kageyama³ and Makoto Daimon³
¹Hirosaki University School of Medicine & Hospital, ²Hirosaki Univ Schl of Med, Hirosaki-Shi, Japan, ³Hirosaki University Graduate School of Medicine, Hirosaki Aomori, Japan

Ultraviolet radiation B (UVB) stimulates a-melanocyte-stimulating hormone (a-MSH) production in human keratinocytes and melanocytes. The melanosomal enzyme tyrosinase-related protein 1 (TRP1) is involved in modulation of pigment production in response to the stressor. Recent molecular and biochemical analyses have revealed the presence of corticotropin-releasing factor (CRF) and urocortins (Ucns), together with their corresponding receptors in mammalian skin. Although CRF and Ucns are thought to have potent effects on the skin system, their possible roles and regulation have yet to be fully determined. Our previous findings suggest that in human melanoma HMV-II cells both CRF and Ucn1 regulate TRP1 gene expression via Nurr-1/Nur77, transcription factors that constitute the nuclear receptor 4a subgroup of orphan nuclear receptors, production, independent of pro-opiomelanocortin or a-MSH stimulation.

This study aimed to explore the effects of UVB on Ucn1 gene expression and TRP1 protein levels using human melanoma HMV-II cells. HMV-II cells were found to express mainly Ucn1 mRNA, as well as CRF and Ucn2 mRNA at lower levels; no Ucn3 mRNA was evident. CRF receptor type 1, but not type 2, mRNA was also expressed. UVB stimulated Ucn1 mRNA expression levels, and increased both Nurr-1 and Nur77 mRNA expression levels. UVB also increased TRP1 protein levels. These data suggest that UVB-induced Ucn1 contributes to TRP1 production via the transcriptions, both Nurr-1 and Nur77. Ucn1 would be produced in melanocytes, and act on melanocytes themselves in an autocrine manner.

Nothing to Disclose: YW, ST, KK, MD

13565 17.0000 SAT-0797 A Ultraviolet Radiation B Stimulates Urocortin 1 Gene Expression and Tyrosinase-Related Protein 1 Protein Levels in Human Melanoma Hmv-II Cells 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0781-0797 4771 1:00:00 PM Adrenal Tumors: ACC & Adrenal Incidentaloma Poster

Thomas Kerkhofs^*¹, Luc Derijks², Hester Ettaieb², Jan Den Hartigh³, Kees Neef⁴, Hans Gelderblom³, Henk-Jan Guchelaar³ and Harm Haak⁵
¹Máxima Medical Center, Eindhoven, Netherlands, ²Máxima Medical Center, Eindhoven/Veldhoven, the Netherlands, ³Leiden University Medical Center, Leiden, the Netherlands, ⁴, Maastricht University Medical Center, Maastricht, the Netherlands, ⁵Maxima Medisch Centrum, Eindhoven, Netherlands

Context: Mitotane is the drug of choice in medical treatment of adrenocortical carcinoma. The anti-neoplastic effect appears to be correlated with a minimum plasma level of 14mg/L, but plasma concentration build-up is in general slow due to the long elimination half-life. Consequently, the therapeutic effect sets in after weeks or even months.

Objective: To develop a pharmacokinetic model that enables clinicians to adjust dosing based upon a target drug exposure, which facilitates personalized therapy.

Design and setting: Retrospective multicenter study.

Patients/Interventions: Data on dosing and plasma level measurements performed throughout mitotane therapy were collected in a population of 29 patients from two hospitals. A population pharmacokinetic model was constructed based on data from 20 patients using iterative two-stage Bayesian fitting (ITSB, MWPharm). The model was validated in an independent sample of 9 patients.

Main Outcome Measure: Goodness of fit was evaluated using the root mean squared error.

Results: The concentration-time data were best described by a three-compartment model. The model estimated mitotane clearance at 0.94±0.37L/h and volume of distribution in steady state at 161±68L/kg∙LBM. Mean prediction error was 2.2±0.5mg/L.

Conclusions: A pharmacokinetic model was developed which characterized mitotane by slow clearance and large volume of distribution. The model appears to be able to predict mitotane levels in individual patients with an error margin of 2.2mg/L. The model enables to adapt dosing based on individual plasma level measurements in prospective setting, which improves the prediction’s accuracy. We expect that individualization of mitotane dosing leads to anticipated and more rapid attainment of the therapeutic levels and potentially to improved clinical management of mitotane treatment.

Nothing to Disclose: TK, LD, HE, JD, KN, HG, HJG, HH

11458 1.0000 SAT-0798 A Development of a Pharmacokinetic Model of Mitotane: Towards Personalized Dosing in Adrenocortical Carcinoma 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0798-0817 4775 1:00:00 PM Adrenal Tumors: ACC & Incidentaloma Poster

Olga Martínez Colete^*¹ and Ivette Pereira Venereo²
¹Hospital Clínico-Quirúrgico "Hermanos Ameijeiras", La Habana, Cuba, ²Hospital Clínico-Quirúrgico, La Habana, Cuba

Adrenal incidentalomas (AI) are mostly benign and non-functioning lesions, but it is important to detect the few cases with malignant or hyperfunctioning tumors for immediate resolution. OBJECTIVES: To analyze clinical and imaging characteristics, functional status, surgical criteria, pathological findings and etiological diagnosis in patients diagnosed for AI. METHODS: We performed a retrospective descriptive study in 65 patients diagnosed for AI that were referred to the Endocrinology Department of tertiary referral Hermanos Ameijeiras Hospital, Havana, Cuba, between July 2006 and March 2013. We reviewed data about patient history, clinical findings, hormonal tests results and Computed Tomography (CT) findings. Surgical criteria and pathological findings were analyzed. Kappa Index was calculated to establish the concordance between presumptive diagnosis and pathological findings in patients underwent surgery. We compared clinical and imaging characteristics by dividing patients according to the type of tumor in 5 groups: benign adenoma, (BA), adrenal carcinoma (AC), Mielolipoma/Cyst (M/C), pheocromocitoma (P), and miscellaneous (MISC). Statistical significance was set at p<0,05. RESULTS: 65 patients, mean age: 50,4 yr; female: 48 (73,8%). 12 patients (18,5%) showed hormonal adrenal hyperfunction . 34 patients (52,2%) were referred to the follow-up protocol. 31 patients (47,7%) underwent surgery (7 due to tumor size over 6cm, 12 due to presumptive malignancy in CT scans, 2 due to functioning status and 10 due to mixed criteria). Kappa Index was 0.85 (CI 95%: 0,65-1), meaning an almost perfect concordance between presumptive diagnosis and pathological results. The main diagnosis were: BA : 30 (49,2%, 2 of them cortisol-secreting adenomas); mielolipoma: 10 (15,5%), AC: 9 (13,8%, 5 of them functioning AC); P: 7 (10,8%, 5 of them with normal cathecolamines levels); cysts: 3 (4,7%); and hemangioma, nodular cortical hyperplasia, pseudocyst and ganglioneuroma: 1 (1,5%) each. Patients on AC group showed significant higher levels of DHEA-S (mean 433,1 µg/dl, p=0,001,normal range: 62-492µg/dl). Mean size tumor (cm) was: BA: 3,7; AC: 9,8; P: 8,8; M/C: 4,7; MISC: 6,6 (p<0,001). Mean attenuating values (UH) were: BA: 13,3; AC: 35,6; P: 40,9; M/Q: -7,6; MISC: 17,3 (p<0,001) . CONCLUSIONS: Benign and non-functioning lesions were the main causes for AI. This study showed that functional and malignant tumors can be detected in early stages by a correct interpretation of clinical, hormonal and imaging findings.

Nothing to Disclose: OM, IP

11569 2.0000 SAT-0799 A Adrenal Incidentaloma: A Study of 65 Patients 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0798-0817 4775 1:00:00 PM Adrenal Tumors: ACC & Incidentaloma Poster

Anh Truong^*, Diane Mary Biskobing and Amelia Grover
Virginia Commonwealth University, Richmond, VA

Background: Incidental adrenal lesions are discovered in between 4-10% of patients undergoing imaging scans. The American Association of Clinical Endocrinologists (AACE) and the American Association of Endocrine Surgeons advise that all patients with an adrenal incidentaloma be referred to an endocrinologist or endocrine surgeon for appropriate evaluation¹. AACE guidelines recommend biochemical evaluation to screen for a hormone secreting adenoma and a repeat CT scan within 6 to 12 months to evaluate for growth. This study was conducted to determine whether patients at an academic medical center with adrenal incidentalomas receive the suggested evaluation.

Methods: A list of subjects was generated by the radiology department. Reports between January 2012 and January 2013 were searched for the phrase ‘adrenal nodule’, but excluding the phrase ‘no adrenal nodule’. The initial list identified 885patients; however, 437 were excluded because of a history of cancer, multiple repeat scans outside of the time frame, or no adrenal nodule. A total of 448 remaining patients were reviewed for demographical information, nodule size, labs, and follow up CT scans.

Results: An analysis of the physicians ordering the CT/MRI scans found that the majority of scans were ordered by Emergency Medicine (EM) physicians (23.6%). Other groups ordering large numbers of scans included Internal Medicine and General Surgery. Upon review of the patients’ history, 304 patients had hypertension. Of the hypertensive patients, 71.7% had adrenal masses larger than 1 cm, but only 8.2% had at least one of the recommended labs drawn and only 2.96% of patients had a follow up scan done within 6-12 months of the initial scan. In the two patients with masses larger than 4 cm, neither had labs drawn nor scans ordered. The masses were also examined in the context of patients’ age, lab work, and follow up scans. Of the 448 masses reviewed, 311 had been identified in patients 65 y/o or younger. From this group of patients, 65.6% had masses larger than 1 cm. Only 2.2% of patients in this group had a full lab work up done and only 2.25% had scans ordered. The remaining 137 masses had been seen in patients older than 65 y/o, with 75.9% of these masses found to be larger than 1 cm. From this group of patients, only 2.2% had a full lab work done and 2.2% had follow up scans ordered within 6-12 months.

Conclusion: The majority of patients with incidentalomas did not have any biochemical evaluation or a follow up scan within the 6-12 month timeframe. The largest number of these masses were discovered on scans ordered by the EM department. While guidelines are in place for the management of these patients, it is unclear as to why these guidelines are not followed. Further determination into the cause can be made by evaluating contributing factors such as discontinuity of care, oversight of secondary findings on imaging studies, or lack of awareness of these guidelines.

Nothing to Disclose: AT, DMB, AG

11677 3.0000 SAT-0800 A Evaluation of Adrenal Incidentaloma 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0798-0817 4775 1:00:00 PM Adrenal Tumors: ACC & Incidentaloma Poster

Anand Vaidya^*¹, Rene Baudrand², Linda Wiinberg³, Daniel T Ruan³, Paul Shyn⁴, Robert G Dluhy³, Rana McKay⁵, Toni Choueiri⁶ and Aymen Elfiky⁷
¹Brigham and Women’s Hospital/Harvard Medical School, Boston, MA, ²Pontificia Universidad Catolica de Chile, Santiago, Chile, ³Brigham and Women's Hospital, Harvard Medical School, Boston, MA, ⁴Brigham and Women's Hospital, Harvard Medical School, MA, ⁵Dana-Farber Cancer Institute, ⁶Dana-Farber Cancer Institute, Boston, MA, ⁷Brigham and Women's Hospital, Dana-Farber Cancer Institute, Harvard Medical School

Context:Advanced adrenal cortical carcinoma (ACC) is a rapidly progressive, often hormonally active, malignancy with no effective medical therapies.

Hypothesis/Aims:We performed a retrospective review of our patients with stage IV ACC, hypothesizing that an aggressive approach to repeatedly reduce overall tumor volume, employing surgical and minimally invasive procedures, could improve outcomes and patient well-being when combined with medical therapy.

Methods:Patients with stage IV ACC presenting to our Center for Adrenal Disorders after 2012, when repeated minimally invasive interventions were routinely considered for all ACC patients, were treated when possible with repeated cytoreductions after initial debulking surgery with metastatectomies and thermal ablations to reduce overall tumor volume, in addition to medical therapy (REP-MED) (n=6). Age and gender matched patients in the prior 5 years who only had an initial extensive surgical debulking, followed by medical therapy, were categorized as “INI-MED” (n=8). Patients were included in INI-MED even if they underwent 1-2 metastatectomies if indicated for pain palliation only. Matched patients who received only maximal cytotoxic medical therapy because their disease was too severe to permit safe surgical debulking were categorized as “MED” (n=5). Patients completed validated quality of life (QoL) instruments every 3 months, including CDC-HRQoL, CARES, SF12, and PHQ9. ANOVA and P-trend regressions were used to evaluate the influence of treatment category on outcomes (*P<0.05; **P<0.01; ***P<0.001).

Results:Mean follow-up time was 22 vs 29 vs 6 months for REP-MED vs INI-MED vs MED. During this time, the REP-MED group had the most cytoreductive procedures (4.2 vs 1.2 vs 0*), but there were no differences with respect to the types or number of cytotoxic medical therapies administered, anti-hormonal medical drugs used, or number of ACC specialists involved per patient. During the course of their disease, the REP-MED group had fewer sites of metastases (1.8 vs 2.5 vs 2.4 organs) and fewer overactive adrenal hormonal lines, when compared to the MED group (1 vs 1 vs 2.8 adrenal hormone lines**). While all 3 groups described poor overall mental and physical status, REP-MED had the best QoL scores (CARES: 85 vs 98 vs 122**; SF12: 31 vs 27 vs 25*). During the follow-up time, the REP-MED group had the least progression of disease (50% vs 75% vs 100%*), the longest progression-free time after initial intervention (6 vs 2.6 vs 2.2 months***) and the lowest mortality (0% vs 25% vs 80%*).

Conclusion: This short and small observational study suggests that repeated interventions to reduce tumor volume, in combination with medical therapy, may delay the progression of advanced ACC and improve QoL. Larger and longer studies are needed to validate our findings and assess the impact of this approach on overall survival and well-being.

Nothing to Disclose: AV, RB, LW, DTR, PS, RGD, RM, TC, AE

13091 4.0000 SAT-0801 A The Impact of Repeated Cytoreduction in Combination with Maximal Medical Therapy on Clinical Outcomes and Quality of Life in Advanced Adrenal Cortical Carcinoma 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0798-0817 4775 1:00:00 PM Adrenal Tumors: ACC & Incidentaloma Poster

Yuksel Altuntas^*, Suna Avci, Sayid Shafi Zuhur and Feyza Yener Ozturk
Sisli Etfal Training and Research Hospital, Istanbul, Turkey

Introduction:Adrenal incidentalomas are frequently encountered in clinical practice on radiologic imaging done for unrelated reasons.

Aim:We aimed to evaluate the functional status and the growing rates of adrenal masses followed-up at our department.

Method:Our study included 137 patients (97 female, 40 male, age range: 27-80 years) who were evaluated for adrenal incidentaloma over 1 cm in diameter at Sisli Etfal Training and Research Hospital, Department of Endocrinology between years 2008 and 2013. The archive records of the patients were analyzed retrospectively. All the masses diagnosed with CT or USG had been reevaluated with MR imaging. Hormonal assessment for pheochromocytoma, subclinical Cushing’s syndrome, primary hyperaldosteronism (in hypertensive patients) and non-classic congenital adrenal hyperplasia were performed in all patients. Mean follow-up period was 20,2±19,86 months. The patients were reevaluated after six months and then yearly with MR imaging and biochemically.

Results:According to the hormonal assessment, the patients were classified as 99 (72,3%) non-functional adenoma(NFA), 21 (15%) subclinical Cushing’s syndrome , 9 (6%) pheochromocytoma, 6 (4%) Conn’s syndrome, 2 (1%) congenital adrenal hyperplasia and 6 (4%) malignancy. The latter one consisted 2 patients (1%) with adrenocortical carsinoma and 4 patients (3%) with metastasis (2 lung cancer, 1 renal cell cancer, 1 Non Hodgkin Lenfoma). Patients with functioning adrenal adenomas were significantly younger than the patients with non-functioning tumours (p<0,05). The groups didn’t differ in regards to tumour size. During follow up, none of the patients with non-functional adrenal incidentaloma developed hormonal hyperfunction. Tumour enlargement was determined in 24 patients (17%). Growth rate of adenomas for males was significantly higher than that of females (5 male (12%) vs. 19 female (%19)) (p=0,031). During follow-up period, the enlargement was detected in 20/99 of NFAs (20%; 10,8±8,92 mm) and 4/38 of functional adenomas (10.5%; 10,75±4,35mm). However, the growing rates did not show significant difference between two groups .

Conclusion:Tumor enlargement may occur in a substantial number of adrenal incidentalomas during follow-up. Since the mass enlargement may be a sign of malignancy, long-term follow-up of these patients with imaging techniques is essential.

Nothing to Disclose: YA, SA, SSZ, FY

13374 5.0000 SAT-0802 A The Functional Assessment and Growth Rates of Adrenal Incidentalomas: A Single Center Experience 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0798-0817 4775 1:00:00 PM Adrenal Tumors: ACC & Incidentaloma Poster

Serkan Yener^*¹, Hamiyet Yilmaz², Abdurrahman Comlekci¹ and Tevfik Demir¹
¹Dokuz Eylul University Medical School, Izmir, Turkey, ²Tepecik Hospital, Izmir, Turkey

Subclinical Cushing's Syndrome (sCS) is defined as the mild hypercortisolemic state in subjects with an adrenal incidentaloma. The prevalence of sCS in individuals with an adrenal incidentaloma may vary due to the heterogenicity in diagnostic approach. The overnight dexamethasone suppression test (DST) with 1 mg dexamethasone is considered the most valuable test to screen for sCS. However, becasue of the limitations of any single test of the HPA axis, several additional tests like urinary free cortsiol, midnight serum cortsiol, CRH test or high dose DST have been recommended in previous studies.

As the concept of sCS is the ACTH independent cortsiol secretion of an adrenal adenoma, we aimed to demonstrate DHEAS levels in four different conditions; non functioning adrenal adenoma (NFA), sCS, overt Cushing Syndrome becasue of an adrenal adenoma (CS) and benign non-adenomatous tumors (cysts or myelolipomas) (NAT). In this study, data of consecutive 84 subjects with radiologically benign adrenal tumors with different functional status has been evalauted. All subjects referred to our divison becasue of an incidentally discovered adrenal mass were evalauted with radiological and laboratuary interventions as previously described by several associations. Hormonal evalaution was performed in accordance with the recent guidelines. Breifly, subjects with post-DST cortsiol levels < 1.8 mcg/dl were defined as NFA group while subjects with post DST cortisol levels > 5 mcg/dl were defined as sCS group. Individuals with post DST cortsiol between 1.8-5 mcg/dl were defined as sCS if ACTH level was < 10 pg/ml.

Study group included 18 subjects with sCS, 49 subjects with NFA, 5 subjects with CS and 12 subjects with NAT. Mean age was comparable in NFA, sCS and CS groups (53, 54, 46 years respectively, p>0.05, One way ANOVA, Bonferoni correction). Mean age was lower in NAT group (38 yr. p<0.001 when compared to sCS and NFA groups. Mean DHEAS level was 17.0, 26.9, 98.8 and 170.4 mcg/dl in subjects with CS, sCS, NFA and NAT, respectively (p<0.001, One way ANOVA). DHEAS level was significantly reduced in subjects with CS and sCS when compared to subjects with NFA (p<0.001 for both comparisons, One way ANOVA, Bonferoni correction). Subjects with NAT had the highest DHEAS levels. DHEAS level was significantly and positively correlated with ACTH (r=0.370, p<0.005) and negatively correlated with age (r=-0.357, p<0.005) and post DST cortisol level (r=-0.374, p<0.001).

These results indicate that hypercortisolism related with adrenal adenomas was associated with lower DHEAS levels. The magnitude of DHEAS suppression was significantly associated with the extent of hypercortisolism. This was also clear and independent from the effect of age variations between NFA and sCS groups. Therefore, we suggest routine DHEAS testing in adrenal incidentaloma workup.

Nothing to Disclose: SY, HY, AC, TD

13599 6.0000 SAT-0803 A Pros and Cons of Dheas Testing for Adrenal Incidentaloma Workup 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0798-0817 4775 1:00:00 PM Adrenal Tumors: ACC & Incidentaloma Poster

Joana Menezes Nunes^*¹, Elisabete Rodrigues², Elisabete Rios³, Catarina Eloy⁴, Isabel Carvalho⁵ and Davide Carvalho⁶
¹Endocrinology, Diabetes and Metabolism Department, Centro Hospitalar São João. Faculty of Medicine, University of Porto. Portugal, Porto, Portugal, ²Endocrinology, Diabetes and Metabolism Department, Centro Hospitalar São João. Faculty of Medicine, University of Porto, Porto, Portugal, ³Pathology Department Centro Hospitalar S. João, Porto. Faculty of Medicine, Porto University. Institute of Molecular Pathology and Immunology at the University of Porto. Portugal, Porto, Portugal, ⁴Institute of Molecular Pathology and Immunology at the University of Porto. Portugal, Porto, ⁵Oncologic Registry, Centro Hospitalar S. João, Porto. Portugal, Porto, Portugal, ⁶Endocrinology, Diabetes and Metabolism, Centro Hospitalar São João. Faculty of Medicine, Porto University. Portugal, Porto, Portugal

Introduction: Adrenocortical carcinoma (ACC) is an uncommon and aggressive malignancy, 30-85% presenting distant metastasis at the time of diagnosis. We aimed to investigate the clinical and pathological findings of ACC diagnosed / treated in our hospital from Jan/88 until Jan/13. Methods: Search of adult patients with ICD10-C74 diagnosis and selection of those with ACC for data collection: clinical presentation, imagiologic characteristics, histopathology review, staging according to ENSAT and survival. Results: We identified 31 ACC patients, mean age at diagnosis 54.6±12.6years, 51.6% women. Sixteen died, seven are alive and there was no information about the remaining patients. At diagnosis, 11.8% were asymptomatic, 23.5% presented back pain, 17.6% general malaise, 29.4% hypercortisolism and 17.6% hirsutism / virilization. The average size of the tumor was 11.7±6.6cm, 53.3% localized on the right side, 72.7% with local invasion, 25% inferior vena cava invasion and 63.7% distant metastasis. Two patients were submitted to exploratory laparotomy and ten to adrenalectomy. One patient refused surgery based on religious beliefs. Of the 10 histologies reviewed, 80% presented atypical mitosis, 20% diffuse architecture, 60% ≤25% clear cells, 90% necrosis, 70% venous invasion, 90% sinusoidal invasion, 60% capsule invasion, mean mitotic index 41.2/50HPF (±36.8). One patient was treated with adjuvant radiotherapy and four with mitotane therapy (two stopped because of side effects: rash and liver dysfunction). The remaining patients didn’t start adrenolytic treatment due to death and/or difficulties in acquiring the drug. Two patients, on stage II ENSAT, maintain follow up without mitotane. Three patients were submitted to cytotoxic therapy (cisplatin, etoposide and doxorubicin). Mean overall survival was 59.2±52.4 months for stages 1+2 and 6.2±6.8 months stages 3+4 (ENSAT) (p=0.004). Conclusion: Our series reinforces the importance of early diagnosis by demonstrating a worse prognosis of cases in more advanced stages and the need for a multidisciplinary approach in such a rare malignancy.

Nothing to Disclose: JMN, ER, ER, CE, IC, DC

13837 7.0000 SAT-0804 A Adrenocortical Carcinoma: What's Our Experience in the Past 25 Years? 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0798-0817 4775 1:00:00 PM Adrenal Tumors: ACC & Incidentaloma Poster

Joana Menezes Nunes^*¹, Elisabete Rodrigues², César Esteves³, Rui Cunha Guimarães⁴, Davide Carvalho³ and Isabel Ramos⁵
¹Endocrinology, Diabetes and Metabolism Department, Centro Hospitalar São João. Faculty of Medicine, University of Porto. Portugal, Porto, Portugal, ²Endocrinology, Diabetes and Metabolism Department, Centro Hospitalar São João. Faculty of Medicine, University of Porto, Porto, Portugal, ³Centro Hospitalar São João, Faculty of Medicine, University of Porto, Portugal, ⁴Radiology Department, Centro Hospitalar São João. Faculty of Medicine, Porto University, Portugal, Porto, Portugal, ⁵Radiology Department, Centro Hospitalar São João, Faculty of Medicine, Porto University. Portugal, Porto, Portugal

Introduction: According to actual guidelines, adrenal incidentalomas should have imagiologic evaluation 3-6 months after diagnosis and annually for 2 years and hormonal evaluation annually for 5 years. However, the optimal frequency and duration of follow up are still uncertain issues. Methods: We retrospectively analyzed all patients with at least two imagiologic exams (CT/MR) performed in our hospital, from Jan/04 to Sep/13, whose reports referred adrenal nodule and selected those with incidental diagnosis. Results: Of 251 selected patients, 183 fitted our criteria: 58.6% were women, mean age at diagnosis 65±10.7years, 85 on left adrenal gland with average size 20±7.7mm, 60 on the right one with mean size of 23±10.4mm and 38 bilateral. Six patients didn’t have hormonal evaluation, two are waiting for results, two presented hypercortisolism, two 21-hydroxylase deficiency and two hormonal alterations compatible with pheocromocytoma. After the initial evaluation, adrenalectomy was undertaken in 6 patients: four for hyperfunction and two for size >40mm (<10HU, regular and homogeneous lesions, one of them with imaging features suggesting a myelolipoma). Histologies revealed four cortical adenomas, one oncocytoma and one pheocromocytoma. Of the remaining 177, 37 (20.9%) had imagiologic re-evaluations at 4±2 months (1^st), 96 (54.2%) at 12±3 months (2^nd), 48 (27.1%) at 24±3 months (3^rd), 48 (21.7%) at 36±3 months (4^th), 28 (15.8%) at 48±3 months (5^th), 17 (9.6%) at 60±3 months (6^th), 4 (2.6%) at 72±3 months (8^th) and 8 (4.5%) at 84±3 months. The growth rate ≥5mm observed at the follow-up evaluations was 1^st-10.8%, 2^nd-9.4%, 3^rd-10.4%, 4^th-14.6%, 5^th-0%, 6^th-5.9%, 7^th-0%, 8^th-12.5%. Overall, the mean time of follow up was 43.5±23.4 months and the observed growth was 0.8±1.4 cm (main left incidentaloma) and 0.7±1.5 cm (main right incidentaloma). Seven patients were submitted to adrenalectomy because of growth, which occurred, in four patients, 2y after diagnosis (histologies: adenomas). None of the incidentalomas became hyperfunctioning. Conclusion: As we observed growth of adrenal incidentalomas after the two initial years and in four cases with surgical indication, we propose that imagiologic follow up should be individualized according to the rate of growth and to the adrenal lesion size.

Nothing to Disclose: JMN, ER, CE, RCG, DC, IR

13889 8.0000 SAT-0805 A Adrenal Incidentalomas: From Diagnosis to Follow up 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0798-0817 4775 1:00:00 PM Adrenal Tumors: ACC & Incidentaloma Poster

Anna Kasperlik-Załuska^*¹, Andrzej Cichocki², Maciej Otto³, Katarzyna Roszkowska², Rafal Z Slapa³, Jadwiga Slowinska-Srzednicka⁴, Elzbieta Roslonowska⁴, Wojciech Jeske⁴, Piotr Zdunowski⁵ and Wojciech Zgliczynski⁶
¹Medical Centre of Postgraduate Education, Warsaw, Poland, ²M. Sklodowskiej-Curie Memorial Center of Oncology, Warsaw, Poland, Warsaw, Poland, ³Medical University of Warsaw, Warsaw, Poland, ⁴The Medical Centre of Postgraduate Education, Warsaw, Poland, ⁵Bielanski Hospital, Warsaw, Poland, ⁶Medical Center of Postgraduate, Warsaw, Poland

Most of adrenal incidentalomas (AI) are benign tumors. Malignancy in this group is frequently believed to be characteristic for elderly. We would like to revise this opinion basing on observation of our group of patients with AI.

Material: 2666 patients, 1953 women and 713 men, aged 11 - 87 years (only 4 patients below 18 years old).

Methods: clinical examination, imaging techniques, hormonal assays, histopathological and immunohistochemical studies in patients treated by surgery. Basing on the results of these studies we divided our material into 4 groups: 1/ probably benign tumors - 2377; 2/ adrenal carcinoma (ACC) - 194; 3/ metastatic lesions - 62; 4/ other malignant tumors - 17 (pheochromocytomas, lymphomas, sarcomas, ganglioneuroblastoma) - not evaluated, because of too small number for statistics.Three age compartments were distinguished: up to 30 years old, 31 - 60, over 60 years.

Results In the all group there were 109 patients (4%) up to 30 years old: in the group 1 - 74 (3%), group 2 - 34 (18%), group 3 - 0. Subset ranging 31 - 60 years: group 1 -1281 (54%); group 2 - 114 (59%); group 3 - 28 (45%). Subset over 60 years - group 1 - 1021 (43%); group 2 - 48 (23%); group 3 - 34 (55%).

Conclusions:

1. A domination of patients middle aged , 31 - 60 years, was found in the ACC group.

2. Similar trend was noted in the group with probably benign tumors.

3. A majority of patients over 60 years old was stated only in the group of patients with metastatic infiltrations in the adrenals.

4. Interestingly, the greatest percentage of young patients (18%) was found in the ACC group.

5. In the total group of 256 patients with malignant tumors (ACC and metastases) a majority (55%) was middle aged

The study was supported by 501-1-13/101 and 301-1-08-11/13 CMKP grants.

Nothing to Disclose: AK, AC, MO, KR, RZS, JS, ER, WJ, PZ, WZ

14366 9.0000 SAT-0806 A Malignant Adrenal Incidentaloma - Is It a Tumor of Old People? a Clinical Analysis of a Group of 2666 Patients Observed at a Single Endocrinological Unit 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0798-0817 4775 1:00:00 PM Adrenal Tumors: ACC & Incidentaloma Poster

Mehmet Calan¹, Mine Sencan Eren¹, Serkan Yener¹, Ozhan Ozdogan², Dilek Cimrin², Ali Riza Sisman², Mustafa Secil¹, Hatice Durak³ and Abdurrahman Comlekci^*¹
¹Dokuz Eylul University Medical School, Izmir, Turkey, ²Dokuz Eylul University, ³Dokuz Eylul University Medical School

Introduction: Incidentally-identified adrenal masses, are increasingly detected, due to the widespread clinical use of abdominal imaging. For differentiating between benign and malignant masses, positron emission tomography (PET/CT) imaging is useful with its qualitative analysis. The aim of the present study was to find out the optimum SUVmax cut-off for accurate characterization of adrenal tumors.

Design, methods:The research was designed as a cross-sectional study. Data was collected from subjects who underwent abdominal MRI. Hormonal evaluation included measurements of plasma ACTH and cortisol before and after 1-mg dst sup. test, midnight cortisol, and 24-h urinary-free cortisol; 24-h urinary catecholamine metabolites; and finally, plasma renin activity, and aldosterone. All subjects underwent PET-CT imaging for the assessment of SUVmax of the adrenal mass. 29 subjects who had high-risk lesions underwent surgical excision. Pathological examination of the resected specimens revealed malignant adrenal masses in 14 patients and benign adrenal adenoma in 15 subjects.

Results: A total of 145 subjects (91 women and 54 men, mean age 58.11 ± 10.24) attended the study. The study population consisted of 116 subjects with non-functional and benign adrenal adenoma, 14 with subclinical Cushing’s syndrome, and 15 with malignant adrenal mass. 14.5% of subjects had bilateral adrenal masses. Median adrenal mass size was 26 (10-65) mm in subjects with benign adrenal adenoma, 27 (10-40) mm in subjects with subclinical Cushing’s syndrome, and 4.9 (2.9-6.3) mm in subjects with malignant adrenal mass. Adrenal mass size and SUVmax was significantly higher in subjects with malignant adrenal mass compared to those with non-functional incidentaloma and with subclinical Cushing’s sydrome (p<0.001). SUVmax was found to be 2.3 (1-4.9) in subjects with benign adrenal adenoma, 2.8 (1.6-5.1) in subjects with subclinical Cushing’s syndrome, and 4.9 (2.9-6.3) in subjects with malignant adrenal adenoma. In Pearson’s correlation analysis, SUVmax was found out to be positively correlated with adrenal mass size (r=0.26, p=0.001) and with BMI (r=0.18, p=0.025). Multiple linear regression analysis showed that age, adrenal mass size, and BMI had no direct impact on SUVmax. Upon ROC curve analysis, a SUVmax value of 3.5 (sensitivity 87%; specificity 89%) was found to be the cut-off value for distinguishing between benign and malignant adrenal masses.

Conclusions: PET imaging may be of use if a lesion remains indeterminate in appropriate clinical setting. In the present study, SUV index was observed to be significantly higher in subjects with malignant adrenal mass compared to both subjects with non-functional incidentaloma and with subclinical Cushing’s syndrome and a SUV index of 3.5 was found to be optimal for differentiating between benign and malignant masses of the adrenal.

Nothing to Disclose: MC, MSE, SY, OO, DC, ARS, MS, HD, AC

15082 10.0000 SAT-0807 A Maximum Standardized Uptake Value: To Distinguish Between Benign and Malignant Adrenal Mass 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0798-0817 4775 1:00:00 PM Adrenal Tumors: ACC & Incidentaloma Poster

Joana Simoes-Pereira¹, Daniel Macedo^*², Margarida Silva-Vieira³, Helena Vilar⁴, Maria Conceição Pereira⁴, Valeriano Leite¹ and Maria João Bugalho¹
¹Portuguese Institute of Oncology, Lisbon, Portugal, ²Portuguese Institute of Oncology, Lisbon, Lisbon, Portugal, ³Portuguese Institute of Oncology of Lisbon, Lisbon, Portugal, ⁴Portuguese Cancer Centre of Lisbon, Lisbon, Portugal

Introduction

Adrenocortical Carcinoma (ACC) is a rare tumor, being the 2^nd most aggressive endocrine malignancy (after anaplastic thyroid carcinoma). Herein, we describe the clinical features and outcomes in 27 cases.

Material & Methods

Retrospective analysis of clinical files of patients with histological features of ACC (Weiss criteria ≥ 3 or local invasion/distant metastases) diagnosed and/or followed at our institution between 1997-2013. Data were analyzed using SPSS 20.0.

Results
Mean age at diagnosis: 46 ± 19 years; 70% women; median follow-up: 35 months (mo). Hormonal profile: 33% produced glucocorticoids 15% androgens, 11% glucocorticoids + androgens; 22% were non-functioning; unknown in 19%. ACC was an adrenal incidentaloma in 22%. Hypertension was documented in 37% and secondary diabetes in 3%; pheochromocytoma was excluded in only 44%. Of those who underwent surgery (25), 60% were submitted to adrenalectomy, 24% adrenalectomy + nephrectomy, 12% en bloc resection (4% unknown procedure). Lymph node dissection was only performed in 12%. Resection status: R0 40%, R1 12%, 8% R2, Rx 40%. Type of resection had no impact in our sample’s survival (p=0.119). Average tumor’s size: 12 ± 4 cm. At diagnosis, 56% had adrenal confined disease, 19% infiltration of surrounding tissue, 6% invasion of adjacent organs and 18% distant metastasis. During follow-up, 88% were reclassified as stage IV (ENSAT); their median survival was 60 mo; 5-y survival was 48%. Compared with other imaging scans, FDG-PET: anticipated relapse in 4/10 patients; confirmed images obtained by conventional methods in 5/10; and turned out to be false-negative in 1/10. Mitotane was used in 74% patients; 35% of them reached therapeutic levels (median cumulative dose: 770 g). From these, after a mean time of 30 mo, 51% showed progression of disease. Main adverse event was adrenal insufficiency. Mitotane had no impact on survival (p=0.920); patients who achieved therapeutic levels didn’t show better progression free survival (p=0.687). Chemotherapy (QT) was performed in 33% (most used schemes: Streptozocin and /or Doxorubicin + Etoposide + Cisplatin). Radiotherapy (RT) in 41% (36% palliative, 64% adjuvant). Combined therapy (mitotane + QT) in 18.5%. Currently, 22% are alive with no disease, 15% with distant metastasis; 15% were lost to follow-up; 48% died from ACC. Most living patients were submitted to surgery + mitotane or surgery + mitotane + QT/RT. Median survival: 106 months; 5-y survival: 64%; median disease free survival: 28 mo.

Conclusion
Treatment options beyond surgery are limited making preoperative staging and definition of surgical protocols crucial in the management of these patients. Mitotane monotherapy might be indicated in patients with low tumor burden but requires close monitoring. Combined treatments are likely to improve survival.

Nothing to Disclose: JS, DM, MS, HV, MCP, VL, MJB

15188 11.0000 SAT-0808 A Adrenocortical Carcinoma: Retrospective Analysis of a Portuguese Cancer Centre 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0798-0817 4775 1:00:00 PM Adrenal Tumors: ACC & Incidentaloma Poster

Paolo Dalino Ciaramella^*¹, Maurizio Vertemati², Duccio Petrella¹, Erika Grossrubatscher¹, Benedetta Zampetti¹, Marco Boniardi¹, Raffaele Pugliese¹ and Paola Loli¹
¹Niguarda Ca Granda Hospital, Milan, Italy, ²University of Milan, Milan, Italy

High mitotic index and high nuclear grade are part of representative hallmarks of adrenocortical cancer (ACC), but the analysis of these parameters is known to be operator-dependent. A characteristic neutrophil/T-lymphocytes infiltrate ratio has been often implicated in carcinogenesis, progression and clinical outcome of several cancer types. However, its role in adrenal cortical tumors is unclear. Aim: to assess by computerized morphometry morphological features, vascular and inflammatory pattern in adrenocortical adenomas (ACAs) and carcinomas. Methods: A single Institution series of 11 ACAs and 18 ACCs samples was analyzed using a Kontron-Zeiss KS400 image analyzer. Four consecutive sections 4 µm thick were obtained with a total of 250–300 HPF examined for each case. Immunohistochemistry for Ki67 and CD8/CD15 was obtained to assess proliferation index and inflammatory infiltrate respectively. To minimize subjectivity, particularly relevant when quantitative results are expected, we generated a morphometric model based on analysis of volume fractions occupied by Ki67 positive and negative cells (nuclei, cytoplasm) and inflammatory compartments (CD15+ granulocytes, CD8+ lymphocytes). Lastly, the assessment of Ki-67 by computerized morphometry was compared with pathologist’s evaluation. Results: volume fraction of Ki-67+ cells was higher in ACCs (ACC .11951, ACA .06637; p<0.001); nuclei volume fraction resulted higher in ACCs, in both Ki-67- (ACC .11951, ACA .06637; p<0.001) and Ki-67+ cells (ACC .01293, ACA .00104; p<0.001). Nuclear/cytoplasmic ratio was higher in ACCs, both Ki-67- (ACC .20535, ACA .09260; p<0.001) and Ki-67+ cells (ACC .66141, ACA .27281; p<0.001). Volume fractions of CD15+ (ACC .00312, ACA .00098; p<0.001) and CD8+ cells (ACC .00731, ACA .00356; p<0.05) were also significantly higher in ACCs. Moreover, when comparing morphometric analysis of Ki67+ cells to pathologist’s scores, the data of the point grid analysis revealed significantly lower values compared to conventional histopathology. Conclusions: Our computerized morphometric model is simple, repeatable (lacking observer bias) and flexible, as it can be upgraded to include newly described histological or immunohistochemical features. This method could be integrated into a classification tool to complement conventional histological analysis to achieve quantification of morphological characteristics and histological biomarkers of adrenocortical tumors. In our experience, nuclear/cytoplasmic ratio differs mostly between ACCs and ACAs, both in Ki-67+ and Ki-67- cells. We speculate that neutrophils may play a role in ACC milieu and that the quantitative assessment of inflammatory infiltrate may find a place in the diagnostic algorithm of adrenal benign and malignant tumors.

Nothing to Disclose: PD, MV, DP, EG, BZ, MB, RP, PL

15214 12.0000 SAT-0809 A Quantitative Assessment By Computerized Morphometry of Ki-67 and Intratumoral Inflammatory Infiltrate in Benign and Malignant Adrenocortical Tumors 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0798-0817 4775 1:00:00 PM Adrenal Tumors: ACC & Incidentaloma Poster

Thiago Machado Nogueira^*¹, Roy Lirov², Antonio M Lerario³, Gary D Hammer⁴, Maria Candida B V Fragoso⁵, Elaine M Caoili² and Tobias Else⁶
¹Faculdade de Medicina da Universidade de São Paulo, Sao Paulo, Brazil, ²University of Michigan Health System, Ann Arbor, MI, ³ICESP/University of Sao Paulo, São Paulo, Brazil, ⁴University of Michigan, Ann Arbor, MI, ⁵Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil, ⁶Univ of Michigan, Ann Arbor, MI

Benign adrenal tumors are common (5-10% of adult population) and often discovered incidentally. Adrenocortical carcinoma (ACC) in contrast is a markedly rare neoplasm (incidence 1/million/year). Concern for ACC is a leading cause for imaging follow-up of incidentally discovered adrenal tumors. However, taken the rarity of this malignancy, the benefits of serial imaging have not been established.

The goal of the current study is to define the imaging and clinical characteristics of adrenal lesions in patients later diagnosed with an ACC.

We identified a total of 11 patients (7F, 4M) within the Michigan Endocrine Oncology Repository (MEOR, total 410 patients) with an adrenal lesion <6cm diagnosed ≥5 months prior to ACC diagnoses.

The reason for initial imaging was unrelated to endocrine symptoms in all cases. Mean age at initial diagnosis was 42.4 ± 15.0 years and ACC was diagnosed at 46.8 ±14.5 years

The mean lead time to diagnosis of ACC was 54.2 ± 41.9 months (range: 5 to 126) with 4 ACCs being diagnosed within a 24 month period. The mean size of the initial lesion was 3cm (median 3cm, range 1.5 – 5.9) with 9 tumors < 4cm. In retrospective image review, signs not clearly suggestive of a benign lesion (heterogeneity, attenuation > 10HU, irregular margins, absence of signal drop in MRI, high signal in T2) were evident in all but one patient. Mean increase in the largest diameter was 1.6 ± 1.3cm (range 0.2 – 6.0) per year. However, in several cases a latent phase of stability or slow growth was followed by accelerated growth. 5 of 11 of the lesions had a growth rate (incidentaloma to ACC) lower than 1cm/year.

6 of 11 patients were initially followed with at least one additional imaging. 4 of these lesions were stable over 11, 11, 25 and 103 months and 2 ACCs were diagnosed at 5 and 8 months, with growth of 2.6 and 1.5 cm/year.

In summary, our data show that the time between an initial adrenal lesion and ACC diagnosis varies greatly. Imaging features of only one lesion were suggestive of a benign character; only 4 ACCs were diagnosed within a time frame of 24 months after initial incidentaloma diagnosis and 4 patients developed an ACC despite stability on initial follow-up imaging. These findings raise concerns on the usual recommendations for follow-up imaging. Taken the rarity of ACC, general follow-up imaging, particularly for benign appearing lesions, may not be justified considering patient radiation exposure and overall health care costs. Individualized risk stratification is needed.

Nothing to Disclose: TMN, RL, AML, GDH, MCBVF, EMC, TE

16215 13.0000 SAT-0810 A Radiographic Characteristics of Incidentalomas in Patients Later Diagnosed with Adrenocortical Cancer 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0798-0817 4775 1:00:00 PM Adrenal Tumors: ACC & Incidentaloma Poster

Aung Naing^*¹, Mouhammed Amir Habra², Rashmi Chugh³, Marian Ijzerman⁴, Martin D Phillips⁵ and David C Smith⁶
¹MDAnderson Cancer Center, Houston, TX, ²The University of Texas MD Anderson Cancer Center, Houston, TX, ³University Of Michigan, Ann Arbor, MI, ⁴Atterocor, Inc, Ann Arbor, MI, ⁵Atterocor, Inc., Ann Arbor, MI, ⁶University of Michigan, Ann Arbor, MI

ATR-101 (Atterocor, Inc., Ann Arbor, MI, USA) is a selective inhibitor of ACAT1 (acyl coenzyme A:cholesterol acyltransferase) in clinical development for the treatment of adrenocortical carcinoma (ACC). ACAT1 catalyzes cholesterol ester formation from cholesterol and long-chain fatty acyl-CoA and, in the adrenals, is particularly important in creating a reservoir of substrate for steroid biosynthesis. ATR-101 selectively distributes to the adrenals, inhibits steroidogenesis and causes apoptosis of cells of the adrenal cortex, as well as in H295R ACC cells.

ACC is an ultra-rare malignancy, occurring in about 2 per million population annually. ACC is frequently discovered in Stage 4 and the overall disease survival is approximately 17 months. Tumors often overproduce steroids normally produced in the adrenal cortex and cause therapy-resistant Cushing’s syndrome. Current therapies are toxic, difficult to administer, and poorly effective.

ATR-101-001 is a phase 1 study being conducted at two centers in the United States. It is a “3+3” design: 3 subjects with advanced ACC who have failed or declined existing therapies are enrolled at each dose level for 28 days. If no Dose Limiting Toxicity is observed, three additional subjects are enrolled at a higher dose. Subjects who appear to be deriving benefit may stay on ATR-101 indefinitely. The primary objective is to determine the safety and tolerability of ATR-101 in subjects with advanced ACC. Secondary objectives include efficacy by RECIST, measurement of production of steroid hormones and intermediates, determination of pharmacokinetics and the Maximum Tolerated Dose. The study is open to patients age 18 and over with advanced ACC. Patient’s mitotane level must be 5 ng/ml or less; the QTcF 470 ms or less; and if present, CNS metastases must be treated and inactive.

Full study information is available on ClinicalTrials.gov, Identifier NCT01898715.

Disclosure: MAH: Investigator, Eisai. RC: Study Investigator, Atterocor, Inc. MI: Employee, Atterocor, Inc. MDP: Employee, Atterocor, Inc, Employee, Atterocor, Inc. Nothing to Disclose: AN, DCS

16240 14.0000 SAT-0811 A ATR-101 Phase 1 Clinical Study for Adrenocortical Carcinoma 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0798-0817 4775 1:00:00 PM Adrenal Tumors: ACC & Incidentaloma Poster

Lucyna Papierska^*¹, Jaroslaw B Cwikla², Michal Rabijewski³, Piotr Glinicki⁴, Maciej Otto⁵ and Anna A Kasperlik-Zaluska¹
¹The Medical Centre of Postgraduate Education, Warsaw, Poland, ²University of Warmia and Mazury, The Faculty of Medical Sciences, Olsztyn, Poland, ³Medical University of Warsaw, Warsaw, Poland, Warsaw, Poland, ⁴The Medical Centre of Postgraduate Education,, Warsaw, Poland, ⁵Medical University of Warsaw, Warsaw, Poland

Objective

According to some authors a higher incidence of subclinical hypercortisolaemia is found among patients with bilateral adrenal tumors with benign phenotype than with unilateral ones. The question is whether all patients with bilateral adrenal tumors and subclinical hypercortisolemia should undergo surgery and, if yes, which of the tumors should be removed first.

Patients and methods

The group consisted of 50 patients with benign bilateral adrenal tumors: 25 with subclinical hypercortisolemia and 25 without hormonal activity. 24 patients with subclinical hypercortisolaemia were operated. The adrenal gland for removing was typed basing on scintigraphy or on tumor diameter. Patients with tumors without hormonal hyperfunction were only controlled every 6 months. Measurements of cortisol concentration at 08⁰⁰, 22⁰⁰ and the following morning after dexamethasone suppression were done. Blood morning levels of ACTH, DHEAS, 17OHPG, concentration of glycated hemoglobin and lipid fractions were determined. The measurements were repeated every 6 months. In 15 patients with subclinical hypercortisolaemia adrenal scintigraphy with iodometyl-19norcholesterol was conducted.

Results

In all operated patients the biochemical signs of hypercortisolaemia ceased after surgery. However only in 14 (58%) operated patients the clinical improvement was evident. A clinically significant decrease in blood pressure, glycemia or glycated hemoglobin and BMI was not observed in 10 operated patients with good-controlled arterial hypertension or diabetes despite of evident hypercortisolemia before surgery. Nobody in observed group without hypercortisolemia developed a hormonal hyperfunction or health status deterioration during long-term observation.

Conclusions

Even though unilateral adrenalectomy brought about a regression of subclinical hypercortisolemia in all operated patients with bilateral adrenal tumors, only persons with deteriorated control of diabetes, hypertension or a quick increment of body mass before surgery have experienced benefits from surgical treatment.

Nothing to Disclose: LP, JBC, MR, PG, MO, AAK

16276 15.0000 SAT-0812 A Subclinical Hypercortisolemia in Bilateral Adrenal Incidentalomas - Who Should be Operated? 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0798-0817 4775 1:00:00 PM Adrenal Tumors: ACC & Incidentaloma Poster

Eun Ky Kim^*, Ji Hyun Lee, Eun Roh, Jae Hyun Bae, Jung Hee Kim, Ji Won Yoon, Seong Yeon Kim and Sang Wan Kim
Seoul National University College of Medicine, Seoul, Korea, Republic of (South)

Diagnosis for adrenal metastasis in patients with adrenal incidentaloma is less than 1%. But, among the patients with cancers, upto 50-75% of adrenal mass are reported to be metastasis (1, 2). Hounsfield units (HU) and the size of tumor in CT may help to distinguish between benign and malignant lesions. We investigated radiological features of adrenal incidentaloma in patients with cancer. Among 1029 subjects with adrenal mass detected during 2000-2012, 181 patients had cancer (17.6%). Mean age was 58.6 ± 10.3 years, and 61.9% were male. Renal cell carcinoma is the most common in cancer patients with adrenal mass (17.1%), followed by gastric (13.3%), colorectal (12.7%), lung (11.0%) and breast (11.0%) cancer. Initial size of mass was 21.0 ± 13.6 mm, and 59.7% of tumors were in the left side. 6.6% of patients had bilateral adrenal tumors. Biochemically functioning tumors were in 9.4% of patients. Adrenalectomies were performed to rule out metastasis in 21 patients, which revealed metastasis in 11 patients (52.4%). We compared the cases of adrenal metastasis (M) to the cases of benign adrenal lesions (B). Tumor size was 31.6 ± 14.2 mm (M) versus 26.5 ± 8.0 mm (B) (p=0.338). HU was 37.8 ± 10.9 U (M) versus 16.7 ± 2.1 U (B) (p=0.011). Although statistically not significant, there were more metastatic adrenal tumors in patients with other distant metastasis than in patients without distant metastasis (7 of 10 patients versus 4 of 11 patients). The primary cancer were lung cancer (n=4), renal cell carcinoma (n=2), lymphoma (n=2), hepatocellular carcinoma (n=2), breast cancer (n=1) and others. The cases which had features of benign adrenal tumors in CT (no more than 40 mm mass size and 20 HU) with at least 6 months of follow-up period were also analyzed. Initial size of mass was 16.1 ± 5.8 mm and their change in size was 0.5 ± 1.7 mm. Mean HU was 14.5 ± 24.0 U. Using receiver operating characteristic (ROC) curve, we may suggest that tumor size of 16.5 mm (sensitivity 91%, specificity 57%) and HU of 26 U (sensitivity 99%, specificity 77%) can be used for reference to exclude metastasis in cancer patients with adrenal mass.

Nothing to Disclose: EKK, JHL, ER, JHB, JHK, JWY, SYK, SWK

16783 16.0000 SAT-0813 A Clinical Features of Incidentally Detected Adrenal Mass in Cancer Patients 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0798-0817 4775 1:00:00 PM Adrenal Tumors: ACC & Incidentaloma Poster

Narin Nasiroglu Imga^*¹, Ozgul Ucar Elalmis², Mazhar Muslum Tuna¹, Bercem Aycicek Dogan¹, Deniz Sahin¹, Dilek Berker¹ and Serdar Guler³
¹Ankara Numune Education and Research Hospital, Ankara, Turkey, ²Ankara Numune Education and Research Hospital, Ankara, Turkey, ³Hitit University, Faculty of Medicine, Corum, Turkey

INTRODUCTION: Emerging evidences indicate that patients diagnosed with adrenal incidentaloma may present with cardiovascular complications. Epicardial fat is known to play a role in left ventricle changes. Whether epicardial fat can be associated with increased left ventricle mass (LVM), which is a strong predictor of adverse cardiac events, in patients with adrenal incidentaloma is unknown. We aimed to test the relationship between echocardiographic epicardial fat thickness and LVM in a group of subjects with adrenal incidentaloma.

METHODS: 51 consecutive patients (age 52.6 ± 10.2 years) with adrenal incidentaloma and 35 healthy controls (age 52.1 ± 7.75 years) underwent transthoracic echocardiogram for epicardial fat thickness and LVM measurement. Standard parasternal and apical echocardiographic views were obtained in the left lateral decubitis position. Epicardial fat thickness was measured in parasternal long axis at end-systole. LVM was calculated using the Penn equation. LVM values were divided to body surface area to calculate the LVM index. Non-functional incidentaloma was confirmed by endocrine tests in all subjects.

RESULTS: Epicardial fat thickness was significantly higher in patients with incidentaloma when compared to controls (0.89 ± 0.32 vs 0.74±0.26 mm, p= 0.023). LVM index was also higher in subjects with adrenal incidentaloma than in controls (103.9 ± 29.1 vs 87.2 ± 22.4 g/m²; p=0.006). A multivariate regression analysis which included age, body mass index, epicardial fat tissue thickness, HbA1c, neutrophil/lymphocyte ratio and the Homeostasis Model Assessment insulin resistance (HOMA-IR) showed that epicardial fat thickness was the best correlate ( R²=0.146, beta=0.242, 95% CI=1.505-40.695, p =0.035) of LVM index in overall study patients.

CONCLUSION: In conclusion, we showed that epicardial fat thickness and LVM index are higher in subjects with adrenal incidentaloma. Therefore these patients should be closely followed for increased cardiovascular risk. In addition, epicardial fat thickness independently correlates with LVM index and epicardial fat may be related with earlier cardiac abnormalities in patients with adrenal incidentaloma.

Nothing to Disclose: NNI, OUE, MMT, BAD, DS, DB, SG

16828 17.0000 SAT-0814 A Epicardial Fat Thickness and Left Ventricular Mass in Subjects with Adrenal Incidentaloma 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0798-0817 4775 1:00:00 PM Adrenal Tumors: ACC & Incidentaloma Poster

Maria Antonenko^*¹, Natalya Volkova², Serguey Dimitriadi³, Ilia Davidenko⁴, Igor Reshetnikov⁵ and Irina Dzherieva⁶
¹Rostov State Med Univ, Rostov-on-don, Russia, ²Rostov state medical university, Rostov on Don, ³Rostov Scientific Oncology Institute, Rostov on Don, Russia, ⁴Rostov State Medical University, Rostov-on-Don, Russia, ⁵Rostov State Medical University, Rostov-on-Don, Russia, ⁶Rostov State Medical University, Rostov on Don, Russia

Urologists are the specialists who often deal with adrenal pathology, in other words who operate adrenals frequently. Since there is increased prevalence of adrenal incidentalomas (AI) as well as about 20% of them are presented as conditions requiring an operation, the adherence to the diagnostic protocol for AI by urologists is absolutely urgent. The aim of the study was to inquire about adherence to diagnostic protocol for AI by urologists. There was performed a retrospective analysis of 25 case reports of the patients who had been presented with the diagnosis of AI at urology department. The following parameters were analyzed: carrying-out screen tests for pheochromocytoma, primary aldosteronism (PA) and subclinical Cushing syndrome (CS); primary endocrinologist consultation, interpretation the CT density in Hounsfield units (HU), indication for contrast CT if it was necessary, calculation of washout percentage, indications for adrenalectomy. According to the conducted analysis, there were received next results. General screening for pheochromocytoma was performed to 18 patients among 23. However, it was correctly done only at 4 patients. Screening for PA was indicated to 16 of 23 patients as they had arterial hypertension. Nevertheless, it was executed correctly only in 3 patients. Screening for subclinical CS was carried out to 5 of 23 patients. However, it was done correctly only in 2 patients. It should be also referred that endocrinologist consultation did not preceded hormonal assessment in most cases. 17 patients among 23 had CT density lesion more than 10 HU, therefore, contrast CT should have been performed. However, it was done at 10 patients without calculating washout percentage. In all analysed case reports there were no interpretation of CT density in connection with indications for adrenalectomy. The most predominant indication for operation was the lesion size. The second was the presence of hormonal activity. In accordance with results received, it may be concluded that there is a dual adherence to diagnostic protocol for AI by urologists. On the one hand, hormonal and imaging assessment was preformed. However, on the other hand, it was not completely correct. In our opinion, it might indicate that AI is not being considered as urologic pathology. Since urologists operate many patients with AI, it might be useful to create combined clinical guidelines by endocrine and urologic associations in order to underline that AI are as endocrine as urologic pathology.

Nothing to Disclose: MA, NV, SD, ID, IR, ID

16833 18.0000 SAT-0815 A Adherence to the Diagnostic Protocol for Adrenal Incidentalomas By Urologists 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0798-0817 4775 1:00:00 PM Adrenal Tumors: ACC & Incidentaloma Poster

Christiane Gruetzmacher¹, Marilena Nakaguma^*², Mirela Costa de Miranda³, Livia Mara Mermejo⁴, Margaret Castro⁵, Gabriela Resende⁶, Antonio M Lerario⁷, Berenice B Mendonca⁸, Ana Claudia Latronico⁹, Sonir Roberto Antonini¹⁰, Madson Q. Almeida¹¹ and Maria Candida B V Fragoso¹²
¹Hospital das Clínicas, University of Sao Paulo Medical School, Sao Paulo, São Paulo, Brazil, ²Hospital das Clinicas, University of São Paulo Medical School, São Paulo, SaoPaulo, ³Hospital das Clinicas, University of São Paulo Medical School, São Paulo, Sao Paulo, Brazil, ⁴Centre hospitalier de l'Universite de Montreal, Montral, QC, Canada, ⁵R, ⁶Hospital das Clínicas, University of Sao Paulo Medical School, ⁷ICESP/University of Sao Paulo, São Paulo, Brazil, ⁸Unidade de Endocrinologia do Desenvolvimento, Laboratório de Hormônios e Genética Molecular LIM/42, Disciplina de Endocrinologia, Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, ⁹Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil, ¹⁰Ribeirão Preto Medical School - University of São Paulo, Ribeirão Preto, Brazil, ¹¹Hospital das Clínicas & Instituto do Câncer do Estado de São Paulo (ICESP), Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil, ¹²University of Sao Paulo, Sao Paulo-SP, Brazil

The specific p.R337H TP53 germline mutation in adults with adrenocortical carcinoma

Christiane Gruetzmacher¹ MD, Marilena Nakaguma¹MD, Mirela C de Miranda¹ MD, Livia Mermejo²MD PhD, Margaret de Castro²MD PhD, Gabriela Resende¹MD PhD, Antonio M Lerario¹MD PhD, Berenice B Mendonca¹MD PhD, Ana Claudia Latronico¹MD PhD, Sonir R R Antonini²MD PhD, Madson Q Almeida¹MD PhD, Maria Candida B V Fragoso¹MD PhD

Adrenal Unit, Division of Endocrinology and Metabolism, Hospital das Clínicas, University of Sao Paulo Medical School^1,2, Sao Paulo¹, Ribeirao Preto², Brazil

Background: The incidence of pediatric adrenocortical tumors (ACTs) is notable in Southern Brazil, due to a high frequency of a specific germline mutation (p.R337H) of the TP53 gene. This specific mutation was identified in 78% of children with pediatric ACT. Aim: To evaluate the prevalence of p.R337H germline mutation in an adult cohort of ACC patients from southeastern Brazil, describing the clinical features and outcomes. Methods: A retrospective chart review of 51 ACC patients seen at São Paulo University in São Paulo and Ribeirão Preto from 1990-2013 was performed. We specifically reviewed information regarding personal and family history of cancer, age of diagnosis, tumor size, Weiss score, ENSAT stage and outcomes. Germline p.R337H TP53 mutations were studied in all these patients either by RFLP or by sequencing analysis. Results: Nine out of 51 patients (17.6%) harbored the p.R337H germline mutation. The median age at diagnosis was 26.7 years. The ENSAT stage was II (2/9); III (3/9); IV (4/9), and Weiss score 3 (1/9), 6 (1/9), 7 (2/9), 8 (2/9) and 9 (3/9). Tumor sizes ranged from 6.5-20 cm (mean 12.93 cm). The majority of patients presented Cushing’s syndrome isolated or associated to virilization except for one non-functioning tumor. All except one patient had no personal or family history of other cancers. This patient had a strong family history for breast cancer, consistent with Li Fraumeni-like syndrome. All patients who underwent a complete surgical resection presented a recurrence after 6 to 12 months. Seven patients died from progressive disease after a median time of 16.2 months. Conclusion: We identified a low frequency (17.6%) of p.R337H germline mutation in adult patients with ACC from Southeastern Brazil. The p.R337H mutation in this group was associated with a dismal prognosis.

Nothing to Disclose: CG, MN, MCDM, LMM, MC, GR, AML, BBM, ACL, SRA, MQA, MCBVF

16839 19.0000 SAT-0816 A The Specific p.R337H TP53 Germline Mutation in Adults with Adrenocortical Carcinoma 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0798-0817 4775 1:00:00 PM Adrenal Tumors: ACC & Incidentaloma Poster

Antonio M Lerario^*¹, Francis P Worden¹, Carole Andrea Ramm¹, Elizabeth A Hasseltine¹, Walter M Stadler², Manisha H Shah³, Edem Agamah⁴, Krishna Rao⁵ and Gary D Hammer¹
¹University of Michigan, Ann Arbor, MI, ²University of Chicago, ³The Ohio State University Comprehensive Cancer Center, Columbus, OH, ⁴Central Illinois Hematology Oncology Center, Springfield, IL, ⁵Southern Illinois University School of Medicine, Springfield, IL

Background: Adrenocortical carcinoma (ACC) is an aggressive malignancy, which lacks an effective systemic treatment. In recent years, molecular-targeted therapies have been proposed as therapeutic options for different types of cancer. In ACC, the activation of the IGF system is the most frequent molecular abnormality, suggesting that insulin-like growth factor receptor 1 (IGF1R) would be an interesting therapeutic target. Preclinical data have shown that pharmacological inhibition of IGF1R signaling in ACC cells significantly reduces proliferation and enhances apoptosis and also potentiates mitotane cytotoxic effects. A previous phase I trial with an IGF1R inhibitor has demonstrated biological activity against ACC. Aims: The aim of this study was to assess efficacy of the combination of IMC-A12, a monoclonal antibody that targets the IGF1R, in association with mitotane as a first-line treatment for advanced ACC. Methods. This study was designed as a randomized double-arm phase II trial and consisted of an initial single-arm phase for safety evaluation with IMC-A12 + mitotane, followed by the randomization phase, which included two treatment groups: IMC-A12 + mitotane and mitotane alone. IMC-A12 was dosed at 10mg/kg intravenously as a single one-hour infusion every two weeks. The starting dose for mitotane was 2 grams orally on a daily basis, either in single or divided doses. Dose adjustments were based on symptoms and serum levels (to attain a serum concentration of 14 to 20 mg/dL). The primary end-point was progression-free survival (PFS) according to Response Evaluation Criteria in Solid Tumors (RECIST) criteria. Results. This study was terminated before the randomization phase due to slow accrual and limited efficacy. Therapeutic effects were observed in 8/20 patients, including one partial response and 7 stable diseases (SD). The median for PFS was 6 weeks (range 6 – 48). The median duration of SD was 12 weeks (range 12 – 48). Toxic events included two grade 4 (hyperglycemia and hyponatremia) and one grade 5 (multiorgan failure). Conclusion. Although the regimen demonstrated activity in some patients, the relatively low therapeutic efficacy precluded further studies with this combination of drugs.

Nothing to Disclose: AML, FPW, CAR, EAH, WMS, MHS, EA, KR, GDH

16888 20.0000 SAT-0817 A The Combination of Insulin-like Growth-Factor Receptor 1 (IGF1R) Antibody Cixutumumab and Mitotane As a First-Line Therapy for Patients with Recurrent/Metastatic Adrenocortical Carcinoma: A Multi-Institutional NCI-Sponsored Phase II Study 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0798-0817 4775 1:00:00 PM Adrenal Tumors: ACC & Incidentaloma Poster

Perie Adorable-Wagan^*
The Medical City, Pasig City, Philippines

Bilateral small adrenal glands in a patient with pulmonary tuberculosis

Perie Adorable-Wagan, MD, FPCP¹, Mary Queen Villegas-Florencio, MD, FPSEM², Hilario Tamondong, MD,FPCCP³

Fellow in training¹, Consultant Endocrinologist², Section of Endocrinology and Metabolism, Pulmonologist, Section of Adult Pulmonology³, The Medical City Hospital, Ortigas Avenue, Pasig City, Philippines

Background

Usual CT scan findings of patients with acute PTB have enlarge adrenals with or without calcification, while chronic PTB have smaller adrenal glands, mostly unilateral and rarely bilateral. We report a not so common finding of PTB patient with primary adrenal insufficiency having bilateral small adrenal glands on CT scan.

Clinical case

A 47 year old male with Type 2 DM, with a 5 year history of Pulmonary tuberculosis, presented with generalized body weakness, vomiting and progressive weight loss over 1 month. Laboratory tests showed hyponatremia at Na 118 mmol/L and K 3.60 mmol/L. Thyroid function test were normal. His serum baseline cortisol level was low at 0.7ug/dL, and elevated ACTH of 86 pg/ml. (0-46 pg/ml). ACTH stimulation using 250 mcg IV test showed inadequate cortisol response of 2.10 ug/dL and 2.20 ug/dL at 30 and 60 mins post ACTH respectively. A Chest xray showed PTB with cavitary lesion. He was given Hydrocortisone 30mg/day and Fludrocortisone 0.1mg/day.

Since test for adrenal autoantibodies are not available in the Philippines, information obtained by CT scan is important in the etiological diagnosis of Addison’s disease. A CT scan of the abdomen with IV contrast showed the adrenals are small with the right measuring approximately 2cm in length and the left measuring 3 cm in length. No focal lesions, no abnormal enhancement or calcification seen. Studies show that patients with active pulmonary tuberculosis did not show evidence of adrenal cortical insufficiency (York, 1992, Barnes,1989). Kelestimur evaluated radiological findings of PTB patients and the size of adrenal glands on CT scan. He observed that adrenal glands are enlarged on CT scan while small glands generally indicate either idiopathic atrophy or long-standing tuberculosis (Kelestimur, 1994). Standard TB therapy includes Rifampicin a potent hepatic enzyme inducer that contribute to adrenal insufficiency by accelerating the catabolism of cortisol. (Venter, 2006).

Conclusion: While some of the studies showed normalization of the adrenal function following treatment of anti-TB drugs others have contradicted it. In this case, both adrenal glands are small. It is prudent to re-evaluate this patient if there is recovery of adrenal function upon completion of anti-TB treatment. Close follow up is required to prevent an adrenal crisis.

(1.)Kelestimur, F., Unlu, Yalcin. A hormonal and radiological evaluation of adrenal gland in patients with acute or chronic pulmonary tuberculosis. Clinical Endocrinology (1994) 41, 53-56.

Nothing to Disclose: PA

13260 1.0000 SAT-0747 A Bilateral Small Adrenal Glands in a Patient with Pulmonary Tuberculosis 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0747-0780 4776 1:00:00 PM Adrenal Case Reports 1 - CAH and Adrenal Insufficiency Poster

Mohammed Ahmed^*
King Faisal Spec Hosp/Res Ctr, Riyadh, Saudi Arabia

Introduction

Adreanleukodystrophy(ALD) is X-linked disorder of peroxisomal fatty acid ß-oxidation resulting in abnormal accumulation of very long chain fatty acids (VLCFA). It is caused by mutations in ABCD1 gene, located at Xq28, that encodes ABC transporter. The transporter helps transloate VLCFA into the peroxisome. ABCD1 mutations prevent normal transport of VLCFA into peroxisomes, thereby preventing ß-oxidation. ALD is a heterogamous disorde.The most severely affected tissues are adrenal cortex , testicular Leydig cells & myelin in CNS. ACTH levels are often increased during early life. VLCFA levels are elevated in 99.9% males w/ ALD of all ages . The parameters analyzed are: concentration of C26:0,C 24:0, ratio of C26:0/C22:0 & C24:0/C22:0.

Objective:I present an asymptomatic familial case diagnosed early in life w/ adrenal insufficiency & early brain abnormalities detected on MRI. Definitive treatment using bone marrow transplant (BMT) was instituted. Long-term FU revealed arrest of disease process associated w/ substantial decline in levels of VLCFA , regression of abnormal brain MRI findings, & preserved testicular function.

Case Report: A 2-yr. old asymptomatic male had lost 2 brothers from ALD. Pt. was 10th percentile for height & weight, had no history of seizure disorder, had normal neurological, ophthamological, & audiolgy findings . Lab findings: Normal EEG, serum AM cortisol 239 nmol/l, (RR: 170-356), elevated serum AM ACTH 144 ng/l (RR: 5-60 ng/l), & abnormal synacthen test. GC/MS analysis repeated x2 revealed abnormally increased VLCFA profile (values expressed in ug/ml) C26:0 1.406 (RR: 0.256+/- 0.08), C24:0: 12.45:(RR:0.12+/- 0.01), an abnormal ratio for VLCFA/LCFA profile: C26/C22: 0.248 (RR: 0.019+/- 0.007), C24/C22: 2.196. MRI Brain: minimal increased signal intensity involving bilateral peritrigonal regions consistent w/ early changes of ALD but findings had progressed a year later.

Clinical Course: Treatment consisted of Hormonal replacement using hydrocortisone & florinef, followed later using allogeneic BMT. Serum VLCFA improved w/ C26 declining to 0.544. MRI brain done 2 years post BMT showed substantial improvement & no additional lesions seen compared to previous year. At age 15 years pt. developed normal puberty w/ serum testosterone of 23.67 nmol/l (RR: 9.9-27.8), normal LH & FSH. However, pt. has developed Nelson’s syndrome, serum ACTH increased to 248, & MRI showed 7x 4 mm centrally located lesion. A progressive increase in ACTH level to 2594 warranted increase in hyrdocortisone dose to 5 mg tid w/ a decline in ACTH to 29.

Conclusions: A combination of classical clinical features coupled w/ abnormal plasma levels of VLCFA is sufficient to establish the Dx of X-ALD. ACTH levels are often increased during early life, & should alert to early Dx. Definitive treatment using bone marrow transplant offers a promising approach towards disease arrest.

Nothing to Disclose: MA

13711 2.0000 SAT-0748 A Inherited Neuroendocrine Peroxisomal Disorder Involving Abnormal Very Long Chain Fatty Acids Metabolism: Long-Term Follow up after Allogeneic Bone Marrow Transplant 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0747-0780 4776 1:00:00 PM Adrenal Case Reports 1 - CAH and Adrenal Insufficiency Poster

Alicia Algeciras-Schimnich^*, Hemamalini Ketha, J Paul Theobald and Alice Y Chang
Mayo Clinic, Rochester, MN

Background: Interference in immunoassays is a rare event but a potentially clinically significant problem when it leads to additional diagnostic tests and inappropriate treatments. Previous reports identified falsely elevated ACTH concentrations due to heterophile antibodies and misdiagnosis of Cushing’s disease. We describe a case of misdiagnosed primary adrenal insufficiency that required evaluation beyond commercially available heterophile antibody blocking reagents.

Case: An 18 year old male, presented to the Endocrinology department for evaluation of primary adrenal insufficiency. Prior clinical history included growth hormone deficiency and delayed puberty in the context of secondary hypogonadism. During routine annual evaluation with his local endocrinologist, he was diagnosed with primary adrenal insufficiency based on a noon ACTH concentration of 156 pg/mL and a cortisol concentration of 3.9 μg/dL. The patient was treated with hydrocortisone (20 mg am, 10 mg pm). Patient presented for a second opinion because he was asymptomatic at the time of diagnosis. He denied any weight loss, nausea, fatigue and had no evidence of hyperpigmentation on exam. Baseline 8am cortisol (more than 24 hours from the last hydrocortisone dose) was 8.6 μg/dL (reference interval 7-25 μg/dL) and ACTH was 89 pg/mL (reference interval 10-60 pg/mL). Cortisol concentrations after cosyntropin stimulation were 19 and 22 μg/dL at 30 and 60 minutes, respectively. To investigate possible interference in the Siemens Immulite ACTH immunoassay, ACTH measurements were performed after serial dilutions. ACTH concentration was 83 pg/mL (non-diluted), 200 pg/mL (1:5), 280 pg/mL (1:10) and 420 pg/mL (1:20). This nonlinear response indicated an analytical interference affecting the Siemens Immulite ACTH immunoassay. The analytical interference could not be resolved using heterophile antibodies blocking reagents (Scantibodies Laboratories, Inc.). ACTH concentration measured using the Roche Elecsys immunoassay was 8.9 pg/mL. With the clinical assessment otherwise arguing against adrenal insufficiency and evidence for a falsely-elevated ACTH, the patient was tapered off the hydrocortisone successfully without any difficulty.

Conclusion: In cases where the clinical presentation is inconsistent with the diagnosis of primary adrenal insufficiency, an analytical interference in the ACTH immunoassay should be considered and may need to be evaluated beyond standard heterophile antibody assessment.

Nothing to Disclose: AA, HK, JPT, AYC

15486 3.0000 SAT-0749 A Interference in the Siemens Immulite ACTH Immunoassay Leading to Misdiagnosis of Primary Adrenal Insufficiency 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0747-0780 4776 1:00:00 PM Adrenal Case Reports 1 - CAH and Adrenal Insufficiency Poster

Fatima Al Kaabi^*¹, Nagi Mohammed² and Muhammad Houri³
¹AL Ain Hospital, Al Ain, United Arab Emirates, ²ICLDC, AlAin, ³Al Ain Hospital, Al Ain, United Arab Emirates

Introduction

The autoimmune polyglandular syndromes (APS) include a wide spectrum of autoimmune disorders involving multiple organs. Clinicians should suspect this diagnosis in patients who have multiple autoimmune disorders especially the rare ones like hypoparathyroidism. The time lag between different manifestations could be years. So follow up is crucial.

Case: 25-year-old female student had ( APS-1) diagnosed in adulthood. Her initial presentation was at age 7 with symptomatic hypocalcemia related to primary hypoparathyroidism, followed by alopecia and recurrent mucocutaneous candidiasis. Few years later, she developed primary hypothyroidism. Up to this point the diagnosis of APS was still not made.

She presented to our hospital in January 2012, with fatigue, nausea, vomiting hyperglycemia and dehydration. Physical exam was remarkable for signs of dehydration and chronic nail changes. She had no hyperpigmentation

Diabetic ketoacidosis was diagnosed and treatment initiated.

Because of past medical Hx, Adrenal insufficiency (AI) was suspected and confirmed by short Synachten test. Basal cortisol 10 nmol/l (0.36 mcg/dl) after synacthen 250 mcg IV, cortisol was 256 nmol/L and 189 nmol/L at 30 and 60 minutes respectively. Baseline ACTH <0.03 ( normal range<= 14.0 Pmol/L) Pituitary MRI was unremarkable.

She was discharged in stable condition on hyrodcotisone and insulin.

Few weeks later, a pulmonologist advised her-by error- to taper hydrocortisone down to 5 mg daily. Within days she presented with symptoms and signs of impending adrenal crisis, potassium was 5.3 mmol/l .Alsosterone 0.05 ( normal range 0.08-0.44 nmol/L) Renin > 230.40 milli IU/L ( normal range 7.00-42.60 ) and ACTH was elevated at 25 pmol/l consistent with a diagnosis of primary AI.

Currently she is on Prednisolone,Fludrocortisone, Levothyroxine and Aspart via insulin pump with HbA1c of 6.6 % .

Discussion:

Our patient presented with DKA and Addison’s disease to complete the spectrum of APS type I. The initially low ACTH, was likely a result of mishandled specimen . Even though she had four different manifestation of APS in childhood, APS was not suspected.

(APS1), also known as the autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) syndrome, is a rare autosomal recessive disorder and it is appears to be due to mutations in (AIRE) gene. Diagnosis of APS involves serological measurement of organ-specific autoantibodies and functional testing. Treatment is based on supplementation of the various deficiencies. Clinicians should have high level of suspicion of this disease when patient present with more than one autoimmune manifestation espicially rare ones. For Instance patients who have autoimmune hypoparathyroidism should be monitored for and counseled about the possibility of other endocrinopathies.

Nothing to Disclose: FA, NM, MH

17035 4.0000 SAT-0750 A Autoimmune Polyglandular Syndrome Diagnosed 18 Years after the Initial Presentation of Hypoparathyroidism 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0747-0780 4776 1:00:00 PM Adrenal Case Reports 1 - CAH and Adrenal Insufficiency Poster

Jamil bawerjan Alkhaddo^*¹, J. Bruce Redmon², Ameer Khowaja³ and Asad Saeed⁴
¹University of Minnesota, Minneapolis, MN, ²Univ of MN Med Sch, Minneapolis, MN, ³Hennepin County Medical Center, Minneapolis, MN, ⁴Univ of Minnesota, Minneapolis, MN

78 years old Male diagnosed with 46 XX Disorder of Sex Development (DSD) due to Congenital Adrenal Hyperplasia (CAH), presented with Adenocarcinoma of Cervix

Introduction

46 XX Disorder of Sex Development (DSD) due to congenital adrenal hyperplasia (CAH) is a rare condition with wide spectrum of phenotypic manifestation from ambiguous genitalia to complete virilization. It is usually diagnosed during childhood. We present a case of 46 XX DSD due to CAH diagnosed at age of 78 years in a phenotypic male.

Clinical Case

78 year old male presented with symptoms of hematuria, increase frequency and hesitancy. He was diagnosed with UTI and sepsis. He required mechanical ventilation and pressors for hemodynamic support. A transurethral urinary catheter placement was attempted but failed, a bed side cystoscopy showed obliterated bulbar urethra.

He had been married but did not father any children. He subsequently underwent a CT and alter a MRI of abdomen and pelvis, that showed rudimentary vagina and a Uterus was visualized too, alongwith 11.3x17x13 cm pelvic mass causing mass effect. A 4.2 cm heterogeneous cystic structure was found on left lateral aspect, consistent with cystic ovary. A bilateral adrenal masses seen too, measuring 8.2 cm on right and 14.5 cm on left.

Karyotype analysis was done using Fluorescent In Situ Hybridization (FISH) method, which showed 97 % cells with 46 XX Karyotype. SRY gene was not detected.

After his recovery, his adrenal hormonal profile showed ACTH: 189 pg/mL (10 – 47 pg/mL), 17 OH Progesterone: more than 9000 ng/dL (20 – 190 ng/dL), morning cortisol: 8.2 µg/dL (4 – 22 µg/dL), DHEA – sulfate: 583 µg/dL (80 – 560 µg/dL) and estradiol: 92 pg/mL (6 – 50 pg/mL).

He was diagnosed with 46 XX DSD due to CAH, 21 hydroxlase deficiency. He subsequently underwent resection of pelvic mass. Surgical pathology showed moderately differentiated cervical adenocarcinoma originating from endocervix. Bilateral ovaries and fallopian tubes were also resected. Intraoperative core needle biopsy of right adrenal gland did not show evidence of adrenal malignancy.

Currently he is undergoing radiation treatment with adjuvant chemotherapy with cisplatin followed by 5-fluorouracil.

He wished to continue to live as a male.

Conclusion

This is a rare case 46 XX DSD due to CAH diagnosed at late age of 78 years. He had a diagnosis of cervical adenocarcinoma, non- HPV related. It is of paramount importance that individuals with 46 XX DSD due to CAH receive endocrine (for appropriate hormone replacement) and surgical cares (resection of internal genitalia considering the risk of malignancy). Psychological and social support is also crucial in order to maintain quality of life. If diagnosed early in life then issues related to gender assignment need to be addressed as well.

Nothing to Disclose: JBA, JBR, AK, AS

11928 5.0000 SAT-0751 A 78 Years Old Male Diagnosed with 46 XX Disorder of Sex Development (DSD) Due to Congenital Adrenal Hyperplasia (CAH), Presented with Adenocarcinoma of Cervix 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0747-0780 4776 1:00:00 PM Adrenal Case Reports 1 - CAH and Adrenal Insufficiency Poster

Mohammed Almehthel¹, Ebtesam Qasem² and Ali Saeed Alzahrani^*²
¹King Faisal Specialist Hosp & Research Centre, Riyadh, Saudi Arabia, ²King Faisal Specialist Hospital & Research Centre, Riyadh, Saudi Arabia

Background: Congenital adrenal hyperplasia (CAH) due to 17-alpha-hydroylase (CYP17A1) deficiency is a rare autosomal recessive disorder. Several CYP17A1 gene mutations have been reported (1, 2). We report here a case of a young “female” who has this condition.

Case: An 19-year-old patient, raised as a female, presented with delayed puberty. She was a product of full term, spontaneous vaginal delivery without any complications; particularly there was no history of neonatal crisis. Her childhood and adolescence were uneventful. She presented with primary amenorrhea and lack of secondary sexual characteristics. She has never been told to have hypertension of hypokalemia. Parents are first-degree relatives and she has 5 sisters who all achieved puberity by age 13 years. Physical examination was remarkable for BP of 136/99 mmHg and Tanner I breasts. She has scant axillary and pubic hair. Genital exam showed female external genitalia with small blind vaginal pouch. No clitromegaly and no palpable gonads in the pubic or inguinal areas. She has no alopecia or acne. Laboratory investigations are remarkable for the following(normal values are presented between the brackets): K 2.3 mmol/L (3.5-5.0), CO2 33 mmol/L (22-30), ACTH 124 ng/L (5-60), AM cortisol 154 nmol/L (low normal), peak cortisol after ACTH stimulation was 225 nmol/L (expected >550 nmol/l), renin <0.5 mU/L, Aldosterone 384 pmol/L (normal), deoxycorticosterone 829 ng/dL (3.5-11.5) , 17-hydroxyprogesterone <0.4 nmol/L, DHEAS 0.13 µmol/L (1.8-8.3), testosterone < 0.08 nmol/L, DHT <50 pg/mL, 11-deoxycortisol <5 ng/dL, Estradio 25 pmol/L and progesterone 33 nmol/L. Karyotyping 46XY. MRI of pelvis showed no uterus, cervix or ovaries with distal vagina and bilateral inguinal oval-shaped structure which could represent bilateral undescended testes.

Molecular studies:DNA was extracted from peripheral leucocytes using Puregene DNA extraction kit. Primers and PCR conditions to amplify the 8 exons and the exon-intron boundaries of the CYP17A1 gene were previously described (3). The PCR products were purified and directly sequenced.

Results: A biallelic homozygous mutation changing guanine to adenine in exon 8 (c.G1247A) and changing the codon 416 from CGT to CAT resulting in a substitution of arginine with histidine (R416H). This mutation was previously described in a patient with CYP17A1 deficiency in a heterozygous form as part of a compound heterozygous mutation with another heterozygous mutation in exon 2 (R125Q) but has never been described as an isolated homozygous mutation. It was previously functionally characterized and was shown to abolish the activity of the CYP17A1.

Conclusion: We present a case of CAH with clinical and laboratory investigations consistent with 17-alpha-hydroxylase deficiency which was confirmed by genetic testing showing a mutation in CYP17A1 gene (R416H) that has never been described in a homozygous form.

Nothing to Disclose: MA, EQ, ASA

14913 6.0000 SAT-0752 A Congenital Adrenal Hyperplasia Due to a Previously Undescribed Homozygous Mutation in 17-Alpha-Hydroylase (CYP17A1) Gene 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0747-0780 4776 1:00:00 PM Adrenal Case Reports 1 - CAH and Adrenal Insufficiency Poster

Maria Sonia Baquedano^*¹, Sabrina Madjinca¹, Paula Aliberti¹, Nora Isabel Saraco², Diana M. Warman³, Marco A. Rivarola¹ and Alicia Belgorosky¹
¹Garrahan Pediatric Hospital, Buenos Aires, Argentina, ²Hospital de Pediatria Garrhan, Buenos Aires, Argentina, ³Hospital de Pediatria Garrahan, Buenos Aires, Argentina

17-Hydroxyprogesterone (17OHP) can be converted to DHT via the backdoor pathway without the intermediacy of testosterone. This is a major route to DHT in pathological states in which 17OHP accumulates, including 21-hydroxylase (21OH) and POR deficiencies. However, the relevance of the backdoor pathway to human adrenal physiology is unknown.

Adrenal tissue from a 7 year-old 21OH-deficient patient resistant to hydrocortisone replacement therapy (21OHD-Res), but responsive to high doses of dexamethasone, and normal human adrenal tissues (HAT) were collected from 3 postnatal age groups (Grs): Gr1: <3 months, n=9, fetal zone involution; Gr2: 3 months to 6yr, n=9, pre-adrenarche; and Gr3: >6 to 20yr, n=8, post-adrenarche period.

By quantitative real-time RT-PCR, Gr3 mRNA levels (mean±SD, arbitrary units) of AKR1C1 (1.86±0.64) and AKR1C2 (1.89±0.50) were similar to GR2 but higher (p<0.05) than in GR1 (1.14±0.39 and 1.13±0.32, respectively). AKR1C3 mRNA in Gr3 (3.06±0.78) was higher than in Gr1 (2.00±0.67) and Gr2 (1.24±0.54), p<0.05. SRDA1 and RoDH mRNAs were readily amplified from HAT without difference among age Grs. SRDA2 and AKR1C4 mRNA were undetectable in the three age Grs. In all cases, mRNA expression from 21OHD-Res (AKR1C1, 1.74±0.09; AKR1C2, 1.78±0.09; AKR1C3, 2.99±0.12; SRDA1, 2.47±0.16 and RoDH, 1.8±0.08) was within the ±2SD range of Gr3 HAT. Laser capture microdissection of zona reticularis (ZR) and zona fasciculata (ZF) from Gr3 HAT showed no differences in adrenal zone-specific backdoor enzyme expression, except for a higher ZR expression of AKR1C1 and AKR1C3.

AR mRNA was expressed in micro-dissected ZR (1.22±0.11) and ZF (1.11±0.26) without differences among the 3 age Grs (Gr1, 1.70±0.32; Gr2, 2.12±0.22; Gr3, 2.02±0.21). AR protein expression was confirmed by immunofluorescence. Impaired 21OHD-Res AR mRNA expression (1.22±0.04), below -2SD of HAT Gr3 expression was observed.

These results indicate that postnatal HAT would express all enzymes needed to complete the steps in the backdoor pathway to DHT. The reported activation of the backdoor pathway in 21OHD seems not to be due to a transcriptional activation of the pathway enzymes, suggesting that it might only be related to an excess of the 17OHP substrate. These data support a physiological role of the backdoor pathway in the postnatal human adrenal cortex. Finally, our data might suggest that proper activation of AR signaling is required to control adrenal cortex sensitivity to ACTH in 21OHD.

Nothing to Disclose: MSB, SM, PA, NIS, DMW, MAR, AB

15076 7.0000 SAT-0753 A Postnatal Expression of Androgen Receptor (AR) and the Alternative “Backdoor” Pathway to Dihydrotestosterone (DHT) in Normal and 21-Hydroxylase Deficient Human Adrenal Cortex 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0747-0780 4776 1:00:00 PM Adrenal Case Reports 1 - CAH and Adrenal Insufficiency Poster

Boonchai Boonyawat^*¹, Nawaporn Numbenjapon¹, Piriya Chantrathammachart¹ and Voraluck Phatarakijnirund²
¹Phramongkutklao Hospital, Bangkok, Thailand, ²Phramongkutklao Hospital and College of Medicine, Bangkok, Thailand

Background: Adrenal hypoplasia congenita (AHC) is a rare inherited disorder of adrenal development resulting in hypoplasia of adrenal gland and inability to produce glucocorticoids, mineralocorticoids and sex steroids.. The most prevalence form is an X-linked AHC caused by mutation of the DAX1 gene on chromosome Xp21. Most of X-linked AHC patients present with skin hyperpigmentation and salt losing crisis. To date, almost 200 mutations in the DAX1 have been identified but no data was available in Thailand. Here we report a case of 4-year-old Thai boy with X-linked AHC, the first case of genetically confirmed novel DAX1 mutation in Thailand.

Clinical case: A 4-year-old Thai boy presented with fever and vomiting for 3 days. He was the first child of unrelated parent. Birth history revealed an uneventful pregnancy and delivery. Birth weight was 3,200 gm. The past medical history was unremarkable except his skin progressively more pigmented since early infancy. Physical examination revealed normal vital signs, no dysmorphic feature with normal genital development and addisonian hyperpigmentation which prominent at skin crease, gum and knuckles. Laboratory investigations showed hyponatremia (Na 128 mmol/L), hyperkalemia (K 5.2 mmol/L), and hypochloremia (Cl 94 mmol/L). Hormonal evaluation revealed low baseline cortisol level (0.89 mg/dL) which is not rising after 250 mg ACTH stimulation test. ACTH level was very high (13,270 pg/mL) as well as plasma renin activity (470 ng/dl/hr). Aldosterone and 17-hydroxyprogesterone levels were normal. The karyotype was 46,XY. A diagnosis of X-linked AHC was established. Glucocorticoid and mineralocorticoid replacement therapy were initiated.

Mutation analysis by direct DNA sequencing of all 2 coding exons and exon-intron junction of the DAX1 gene revealed a novel hemizygous GG deletion (c.1148_1149delGG; codon 383). This frameshift mutation resulted in a premature termination codon at the position 387 and was predicted to encode a truncated DAX1 protein missing a portion of ligand binding domain. Molecular analysis from all other family members revealed only a heterozygous for the same mutation in the mother, indicating that the mutation found in the index case was inherited from his carrier mother.

Conclusion: We report a case of X-linked adrenal hypoplasia congenita with established a novel hemizygous frameshift mutation in the DAX1 gene. Mutation analysis is important not only for diagnostic confirmation in the index case but also for carrier detection in his mother which provided a proper management and appropriate genetic counseling for this family.

Nothing to Disclose: BB, NN, PC, VP

15803 8.0000 SAT-0754 A Novel Dax-1 Mutation in a Thai Boy with X-Linked Adrenal Hypoplasia Congenita (AHC): A First Report 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0747-0780 4776 1:00:00 PM Adrenal Case Reports 1 - CAH and Adrenal Insufficiency Poster

Karla Cristina Borromeo Detoya^*¹, Daniela Ciltea², Dylan Timberlake³ and Nairmeen A Haller⁴
¹Akron General Medical Center, Cleveland Clinic Affiliate, Akron, OH, ²Akron Gen Prtnrs Physician Grp, Akron, OH, ³Northeast Ohio Medical University, ⁴Akron General Med Ctr, Akron, OH

Introduction: Autoimmune Polyglandular Syndrome (APS) is a rare condition involving multiple endocrinopathies and associated autoimmune disorders. It is preceded by adrenal failure in almost 50% of cases. Takotsubo Cardiomyopathy (TCM) is a disorder with a poorly understood pathogenesis, leading to transient dysfunction of the middle segments/apex of the left ventricle and subsequent apical systolic dilatation. We report a case of APS type II in a 65-year-old female with recent history of TCM.

Clinical Case: A 65-year-old female diagnosed with TCM based on cardiac catheterization consulted seven months later for progressive and chronic fatigue, hair loss, nail problems, insomnia, eczema and multiple hypopigmented patches. She was being maintained on hormone replacement therapy for hypothyroidism (TSH at 33.3 uIU/mL, n 0.358- 3.74 uIU/mL; free T4 at 0.37 ng/dL, n 0.82-1.77 ng/dL). Workup revealed thyroid peroxidase antibodies (51 IU/mL, n <35 IU/mL) present and was unremarkable for other autoimmune antibodies (anti-thyroglobulin, anti-tissue transglutaminase, anti-cardiolipin, anti-phosphatidylserine, anti-glutamic acid decarboxylase, anti-islet cell, and anti-21 alpha hydroxylase antibodies). ACTH (17 pg/mL, n 6-50 pg/mL) and DHEAS (75 ug/dL, n <146 ug/dL) were both normal. Despite the sensitivity of anti-21 alpha hydroxylase antibody testing, negative results have been documented in diagnosed cases of Addison’s disease. Thus, confirmatory cosyntropin stimulation test was performed and result suggested primary adrenal insufficiency (baseline cortisol at 1.7 ug/dL, 30 minute cortisol at 11.0 ug/dL, 60 minute cortisol at 13.6 ug/dL; n baseline cortisol at least 5 ug/dL, increase in 30 minute cortisol at least 7 ug/dL, 60 minute cortisol at least 18 ug/dL). Autoimmune Polyglandular Syndrome was suspected based on symptomatology, laboratory findings of hypothyroidism, primary adrenal insufficiency and recent clinical findings consistent with dermatomycosis. The patient was then started on hydrocortisone, with good response to treatment.

Conclusion: This is a case of TCM with hypothyroidism,presenting with clinical and laboratory findings consistent with APS II. Three other cases of APS with concomitant TCM have been reported. While TCM is currently of unclear etiology, if related to APS it may be an early sign of the latter disease, as seen in this case. One plausible theory argues that TCM may be triggered by potentiated catecholamine release in APS II due to ACTH stimulation. Other authors cited possible hormonal preconditioning combined with the patient being in distress as factors contributing to the coexistence of these two diseases. This case report supports the possibility that APS II and TCM may share common pathogenetic pathways which may include autoimmunity, hormonal factors, and genetic factors. Further studies are recommended to establish correlation.

Nothing to Disclose: KCBD, DC, DT, NAH

16103 9.0000 SAT-0755 A Autoimmune Polyglandular Syndrome II in a Patient with Takotsubo's Cardiomyopathy 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0747-0780 4776 1:00:00 PM Adrenal Case Reports 1 - CAH and Adrenal Insufficiency Poster

Mohamad Eid^*¹, Swathi Sridhar Sangli², Erwyn Chua Ong³ and Adrienne M. Fleckman⁴
¹Beth Israel Medical Center, ²Beth Israel Medical Center, New York, NY, ³Mount Sinai Beth Israel, New York, NY, ⁴Beth Israel Med Center, a Member of the Mount Sinai Health System, New York, NY

Introduction

Inhaled glucocorticoid (GC) is the mainstay of treatment for asthma and chronic obstructive pulmonary disease (COPD). Although the development of iatrogenic Cushing’s syndrome is well known with the use of systemic (oral) GC, little is known with the use of inhaled GC, with the few case reports restricted only to fluticasone and budesonide. Here we report a first adult case of iatrogenic Cushing’s syndrome due to adverse drug-drug interaction from inhaled mometasone and a concomitant cytochrome P-450 inhibitor.

Case

A 54-year old woman presented with acute kidney injury secondary to multiple medications and dehydration. She was a long-time smoker with COPD on mometasone/formoterol inhaler (Dulera®), and had HIV on tenofovir, raltegravir and darunavir/ritonavir for >10 years. She had developed multiple vertebral fractures, hypertension, impaired glucose tolerance, hyperlipidemia and a recent episode of herpes zoster. Examination was significant for BMI 32 with typical Cushingoid features of facial plethora, “moon facies”, central obesity, abdominal striae, thin arms and legs, bipedal edema with thin blistered skin and multiple bruises on both upper limbs. Laboratory values included a low morning cortisol level (2.9 mg/dL) with undetectable ACTH (< 5 pg/mL). During a prolonged ACTH stimulation test (250 mg Cosyntropin over 12 hours), cortisol rose from baseline 2.55 mg/dL to 28 mg/dL at 7 hours (27.6 mg/dL at 10 hours) consistent with adequate adrenal response. The patient was started on physiologic doses of the short-acting GC hydrocortisone in preparation for adjusting her medications to prevent further iatrogenic Cushing’s syndrome and permitting recovery of the pituitary-adrenal axis.

Discussion

Our patient developed severe unrecognized exogenous Cushing’s syndrome from the concomitant administration of the potent inhaled GC mometasone in conjunction with the Cyp3A4 inhibitor ritonavir. This case demonstrates that it is critical for clinicians (1) to recognize that exogenous glucocorticoids from any route of administration may cause Cushing’s syndrome, with possible adrenal insufficiency on sudden withdrawal; (2) to take detailed drug histories, evaluate the potential for drug-drug interactions when prescribing new medications, and to be aware of interactions with related compounds; (3) to monitor patients on exogenous GC for signs/symptoms of Cushing’s syndrome, and to be aware of the necessity to evaluate patients with COPD and other chronic illnesses who develop rapid/excessive weight gain; and (4) to inform families and take a multidisciplinary approach to prevent complications of systemic side effects.

Nothing to Disclose: ME, SSS, ECO, AMF

12119 10.0000 SAT-0756 A Severe Iatrogenic Cushing's Syndrome from Combined Mometasone and Ritonavir: First Adult Report 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0747-0780 4776 1:00:00 PM Adrenal Case Reports 1 - CAH and Adrenal Insufficiency Poster

Lauren Brian Hawkins^*¹ and Cynthia Anne Burns²
¹Wake Forest University School of, Winston Salem, NC, ²Wake Forest Univ Hlth Sci, Winston Salem, NC

Background: Disseminated Cryptococcosis typically affects immunocompromised patients, most often affecting the CNS and lungs. Very rarely cryptococcus causes adrenal insufficiency. Adrenal disease without evidence of CNS and lung disease is rare.

Clinical Case:We present an atypical case of primary AI secondary to cryptococcal infection without evidence of CNS involvement.

The patient was admitted to the hospital with hypotension, weight loss, and N/V. A 250mcg cosyntropin stimulation test confirmed primary AI: baseline cortisol 1.4 mcg/dL (N 5.5-20.0 mcg/dL), cortisol at 30 minutes 1.8 mcg/dL, cortisol at 60 minutes 1.7 mcg/dL; ACTH 726 pg/mL (N 6.0-50.0 pg/mL). Treatment with hydrocortisone and fludrocortisone was begun. Antibodies for 21-hydroxylase and adrenal tissue were negative.

Abdominal CT revealed symmetric, diffusely enlarged and thickened adrenal glands. He was referred to ID for workup of adrenal infection. Serum cryptococcal Ag titer was positive at 1:64. Testing for HIV was negative. LP showed normal CSF protein (69 mg/dL, N 15-60 mg/dL), WBC (1/mm³, N 0-5/mm³), and glucose (44 mg/dL, N 50-75 mg/dL). CSF cryptococcal Ag was negative. Blood and CSF fungal cultures were negative. Chest CT showed a focus of lobular nodularity with surrounding groundglass attenuation in the RLL and numerous sub-4mm nodules in the right lung. Induction therapy with liposomal amphotericin and flucytosine was given. He is currently taking fluconazole 200mg daily for maintenance therapy.

A low CD4 count at 150 (N 310-3,110) was found. Immunoglobulins were also found to be low, likely secondary to poor T-cell stimulation: IgG 523 mg/dL (N 635-1741 mg/dL), IgA 30 mg/dL (N 70-350 mg/dL), IgM 26 mg/dL (N 70-210 mg/dL). He was referred to Immunology for further workup. He was prescribed weekly SC IG. No cause for his immunodeficiency has been found.

A follow up contrasted abdominal CT seven months after diagnosis showed unchanged adrenal enlargement. There was no contrast enhancement, suggesting no residual functioning tissue. Serum cryptococcal titer most recently is stable at 1:32 after eleven months of treatment

He is followed by Endocrinology, Infectious Disease, and Immunology. He continues hydrocortisone, fludrocortisone, fluconazole, weekly SC immunoglobulin, and PCP and HSV prophylaxis. The fluconazole will be continued indefinitely given his immune deficiency.

Conclusion: This patient’s case is atypical in that Cryptococcus rarely causes primary adrenal insufficiency, and when it does, it is typically associated with cryptococcal meningitis. We could only find three prior case reports of patients with adrenal insufficiency secondary to cryptococcosis without meningoencephalitis. This patient’s case is unique in that his diagnosis of cryptococcosis led to the finding of idiopathic T-cell deficiency and hypogammaglobulinemia, which up to this point had gone undiagnosed.

Nothing to Disclose: LBH, CAB

12590 11.0000 SAT-0757 A Disseminated Cryptococcosis Causing Primary Adrenal Insufficiency in a Patient with Idiopathic T-Cell Lymphopenia 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0747-0780 4776 1:00:00 PM Adrenal Case Reports 1 - CAH and Adrenal Insufficiency Poster

Aqueel Usman¹, Thanh Duc Hoang^*², Vinh Quang Mai¹, Patrick W Clyde¹ and K M Shakir¹
¹Walter Reed National Military Medical Center, Bethesda, MD, ²Naval Medical Center San Diego, San Diego, CA

Background: Bilateral adrenal nodules usually require a thorough investigation to rule out underlying disorders that can have significant adverse clinical implications. Herein we report an elderly male presenting with calcified bilateral macronodules due to non-classic (late onset) congenital adrenal hyperplasia (CAH).

Clinical case: An 80-year-old male was referred for evaluation of adrenal nodules. Patient had no signs or symptoms of Cushing’s syndrome, pheochromocytoma, or any malignancy. Review of systems was essentially negative other than unsteadiness and vertigo. Family history did not reveal any endocrine disorders. Physical examination showed HR 58 bpm, BP 151/71 mmHg, BMI 20.8, no stigmata for Cushing’s syndrome and normal testicular examination. Laboratory tests: serum testosterone 398 ng/dL, androstenedione 58 ng/dL, DHEA 37 mcg/dL, DHEAS 79 mcg/dL, aldosterone/renin ratio 1.4, 17-OHP 187 ng/dL. Abdominal CT scan showed bilateral adrenal nodules with rim calcifications (right 5.5cm x 4.0cm with HU 20.5, left 2.7 cm x 3.5 cm HU 30). A previous CT scan in 2005 showed bilateral adrenal nodules, right 5.3cm x 4.0cm HU 26, left 5.4cm x 3.6 cm HU 24. Stimulation with 250 mcg of ACTH revealed elevated 17-hydroxyprogesterone 1495 ng/dL at 30 minutes and 1464 ng/dL at 60 mintues, confirming a diagnosis of CAH due to 21-hydroxylase deficiency. 11-deoxycortisol, 17-hydroxypregnenolone and cortisol responses were normal. Peripheral blood gene analysis confirmed heterozygous CYP21A2 mutation (Val281Leu).

Discussion: The differential diagnosis of bilateral adrenal nodules involves granulomatous disease, bilateral pheochromocytoma, bilateral primary aldosteronism, adrenal myelolipoma, adrenal metastases from malignancy elsewhere (1, 2). Detailed evaluation did not confirm these disorders; and this patient was diagnosed with late onset heterozygous CAH. It has been suggested that elevated CRH and ACTH may have contributed to adrenal hyperplasia and subsequent formation of adrenal nodules. Adrenal nodules are more common in homozygous than heterozygous patients with CAH although there are some overlaps in 17-OHP responses (2).

Conclusions: Patients with bilateral adrenal nodules should be evaluated for CAH after excluding other underlying disorders. Additionally gene testing may be useful to confirm the diagnosis in patients with borderline response to ACTH stimulation test.

Nothing to Disclose: AU, TDH, VQM, PWC, KMS

11436 12.0000 SAT-0758 A Bilateral Calcified Macronodules in the Adrenal Glands As an Initial Presentation of Congenital Adrenal Hyperplasia 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0747-0780 4776 1:00:00 PM Adrenal Case Reports 1 - CAH and Adrenal Insufficiency Poster

Hebatullah M Ismail^*¹, Fernanda Delgado², Catherine Pihoker¹ and Anita E Beck¹
¹Department of Pediatrics, University of Washington, Seattle, WA, ²University of Washington School of Medicine, Seattle, WA

Background: Congenital isolated adrenocorticotrophic hormone deficiency (IAD) is a rare disorder that can be difficult to diagnose, and missed diagnosis may result in infant mortality. IAD is caused by homozygous or compound heterozygous TBX19 gene mutations. TBX19 mutations can be uncovered by whole exome sequencing (WES), a relatively new diagnostic tool, even if the diagnosis is not suspected in advance.
Clinical Case: We present a 24-month-old Hispanic child with a lifelong history of multiple endocrine abnormalities without a unifying diagnosis. He was a full term infant with a history of feeding difficulties and hyperbilirubinemia, referred to endocrinology for congenital hypothyroidism identified on the newborn screen. He had been started on levothyroxine at 1 week of age, and was seen at 4 weeks. Physical exam was notable for icteric sclera, and he was found to have conjugated hyperbilirubinemia. Subsequent evaluation revealed low cortisol, and he was started on hydrocortisone. During an acute illness he was hypoglycemic, at which time stress doses of hydrocortisone were administered. He also had low serum growth hormone and elevated insulin levels, therefore growth hormone and diazoxide were prescribed. A comprehensive clinical evaluation was insufficient to explain his clinical findings. However, a single nucleotide polymorphism (SNP) microarray demonstrated long stretches of homozygosity representing about 23% of his genome and raised concern for possible consanguinity with recessive disorders. Subsequent WES revealed a homozygous TBX19 mutation that partially explains his presentation and conditions. The hypothyroidism appears to be transient, and his levothyroxine has been since stopped; growth hormone therapy and diazoxide are gradually being discontinued.
Conclusions: We report on a perplexing case of congenital IAD and highlight the utility of WES to establish a molecular diagnosis in endocrine practice. The use of WES in endocrinology has led to other major advances and discoveries: germline mutations in patients with pheochromocytomas, a de novo heterozygous nonsense mutation in a gene encoding thyroid hormone receptor alpha, an inactivating mutation in KISS1, as well as novel mutations in the imprinted gene MKRN3 that causes central precocious puberty in humans. With more widespread availability and advances in technology, WES will continue to serve as a valuable tool in endocrine research and clinical practice.

Nothing to Disclose: HMI, FD, CP, AEB

12511 13.0000 SAT-0759 A An Argument for Application of Whole Exome Sequencing in Puzzling Endocrine Cases 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0747-0780 4776 1:00:00 PM Adrenal Case Reports 1 - CAH and Adrenal Insufficiency Poster

Inderpreet K Dardi and Serge Jabbour^*
Thomas Jefferson University, Philadelphia, PA

DAX-1 (NROB1) Mutation with Adrenal Hypoplasia Congenita & Hypogonadotropic Hypogonadism

Inderpreet K Dardi, MD & Serge A Jabbour, MD; Division of Endocrinology, Department of Medicine; Jefferson Medical College, Thomas Jefferson University , Philadelphia, Pennsylvania

Background: X-linked Adrenal Hypoplasia Congenita is a rare disorder in which the adrenal cortex doesn’t develop normally and manifests as primary adrenal failure in early infancy. It is caused by mutations or deletions in DAX-1 (NROB1) dosage sensitive sex reversal, adrenal hypoplasia gene located on short arm of Chromosome X that is Xp21.Tissues of DAX-1 expression are adrenals, gonads, hypothalamus and pituitary gland and is responsible for controlling development and function of these tissues. More than 100 DAX-1 mutations have been described occurring throughout the coding region. Heterozygous females with this DAX-1 mutation are asymptomatic.

Clinical Case: Mr.KM presented at age 3 weeks when he was noticed to have poor feeding. His sodium was 115mmol/L (135-146 mmol/L) and his potassium was 8.9 mmol/L(3.5-5.3 mmol/L). Subsequent testing revealed an ACTH of 606 pg/ml (9-46 pg/ml), renin activity of 8.56 ng/ml/h (0.25-5.82 ng/ml/h) and undetectable serum cortisol and aldosterone levels. In addition, his 11-deoxycorticosterone and 17-OH-progesterone levels were undetectable. Ultrasound of the abdomen showed no evidence of adrenal tissue. He was started on replacement hydrocortisone and fludrocortisone.

At age 14, he was noted to have delayed puberty. Testing showed FSH of 0.99 mIU/ml (1.5-12.4mIU/ml), LH 0.15 mIU/ml (1.7-8.6 mIU/ml) and testosterone of 22ng/dl (348-1197 ng/dl) with normal prolactin and pituitary MRI, all consistent with hypogonadotropic hypogonadism. DAX-1 mutation was hypothesized and genetic testing was done showing a maternally inherited large deletion of Xp21.2 diagnostic of X-linked Adrenal Hypoplasia Congenita. The patient received androgen replacement with normal attainment of secondary sex characteristics.

Review of his family history indicated the death of his mother’s half-brother with adrenal crisis at age 13 days and death of his maternal grandmother’s brother in early infancy of uncertain etiology. Mr.KM has only one female sibling who tested negative for DAX-1 mutation.

Conclusion: DAX-1 mutation can cause congenital adrenal hypoplasia and usually manifest very early with primary adrenal insufficiency. Most individuals develop hypogonadotropic hypogonadism usually recognized at the time of puberty. Abnormal spermatogenesis with sertoli cell defects is also noted in some patients. Secretion of other pituitary hormones is not impaired.

Nothing to Disclose: IKD, SJ

11835 14.0000 SAT-0760 A Dax-1 (NROB1) Mutation with Adrenal Hypoplasia Congenita & Hypogonadotropic Hypogonadism 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0747-0780 4776 1:00:00 PM Adrenal Case Reports 1 - CAH and Adrenal Insufficiency Poster

Tripti Joshi^* and Shamasunder H Acharya
John Hunter Hospital, New Lambton, Australia

TITLE: A Giant Retroperitoneal Teratoma and Post-Operative Adrenal Insufficiency.

Background: Large retroperitoneal teratomas involving adrenal gland are exceedingly rare.

Clinical case: A 29 year old male with a background of schizophrenia was found to have a large right renal /adrenal mass on CT scan whilst being worked up for hypertension in 2004. He was lost to follow up and represented in 2012 with abdominal discomfort. A repeat CT scan, showed this large and complex mass arising from the right kidney measuring 23 × 23 × 26 cm, encasing the dual renal arteries, causing right urinary tract obstruction. An elective right nephrectomy and removal of the mass was performed through laparotomy and median sternotomy, after consulting multi-disciplinary team.

The histology was suggestive of a mature teratoma with mucinous borderline proliferation with no secretory component arising from retroperitoneum. There was extensive perirenal fibrosis and necrosis. The right adrenal gland was compressed and trapped in the mass.

He had a complicated post-operative course and was hypotensive requiring vasopressors. Unexpectedly, he was found to have a low cortisol level of 276nmol/L at 0500 hrs. and 302nmol/L at 0800hrs on two separate occasions in the intensive care, which appears to be low for his critical illness.

He received hydrocortisone replacement from 3^rdpost –operative day with improvement in his blood pressure.

Repeat cortisol was 205nmol/L with an ACTH level of 19.8pmol/L (RR 0-10). An outpatient short-synacthen test on two occasions found him to be glucocorticoid deficient. The 8 am cortisol was 102 nmol/L with an ACTH level of 5.6pmol/L (RR 0-10) at 0 min followed by a stimulated 60 min level of 206nmol/L. The adrenal antibodies were negative. The renin was 1.3ng/ml/hr. (1.2-2.8), Aldosterone 66 pmol/L (80-1040) with an aldosterone renin ratio of 1.8 (0-30). The 17-hydroxy progesterone was 10.2 nmol/L(<10.3) following synacthen stimulation making classic and non-classic congenital adrenal hyperplasia unlikely.

The tumor markers were negative: AFP 1IU/L (RR<8), HCG <2IU/L (RR <5).

Conclusion: We present a case of large retroperitoneal teratoma involving adrenal gland with post- operative persistent hypocortisolism. Teratomas are not known to be secretory to cause adrenal suppression.

Teratomas are a type of non-seminomatous germ cell neoplasms, arising from pluripotent cells that can differentiate into ectoderm, mesoderm, and endoderm (1). Retroperitoneal teratomas are the least common of the extra-gonadal tumors and involvement of adrenal gland is exceedingly rare. Majority of retroperitoneal teratomas involving adrenal gland are asymptomatic, benign and present as an incidental finding and 6% of retroperitoneal teratomas are malignant. Surgical excision is recommended for symptomatic relief and malignant potential (2).

Nothing to Disclose: TJ, SHA

11525 15.0000 SAT-0761 A A Giant Retroperitoneal Teratoma Post-Operative Adrenal Insufficiency 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0747-0780 4776 1:00:00 PM Adrenal Case Reports 1 - CAH and Adrenal Insufficiency Poster

Melvin Kok Seng Lee^* and Cherng Jye Seow
Tan Tock Seng Hospital, Singapore, Singapore

Introduction:

Licorice is a known cause of hypokalemia. There are reports of life-threatening cardiac arrhythmias secondary to hypokalemia arising from excessive licorice intake. Not many are aware that licorice is an ingredient in preserved plums, a popular local snack. We report a patient who presented with cardiovascular collapse secondary to ventricular fibrillation from severe hypokalemia. She was successfully resuscitated, and subsequent history- taking and detailed biochemical investigation point towards chronic licorice ingestion as the most likely aetiology.

Case report:

A 66 year-old Chinese lady with no significant medical history of note presented with poor oral intake and worsening muscle cramps over a few days. She subsequently had a cardiac arrest. Cardiopulmonary resuscitation and defibrillation was initiated rapidly when ventricular fibrillation was noted. She had no history of hypertension. On examination, she did not look Cushingnoid. Biochemical results: Sodium 135 mmol/L (RI: 134-145), Potassium 1.6 mmol/L (RI: 3.5-5.0), Magnesium 0.6 mmol/L (RI:0.7-1.0) fT4 19 pmol/L (RI:8-21), TSH 2.55 mIU/L (RI: 0.34-5.60), HCO3 29 mmol/L (RI: 19-31). Her spot urinary potassium was 20.9 mmol/L suggesting renal loss. Both aldosterone and renin were low at 217 pmol/L and 0.37 ng/ml/hr respectively. 8am cortisol was suppressed at 45 nmol/L after an overnight 1mg dexamethasone suppression test. The hypokalemia was aggressively corrected and she underwent urgent cardiac catheterisation. This revealed sub-total occlusion (TIMI 1 Flow) of the left anterior descending artery. The cardiologist deemed the occlusion as an unlikely root cause of the cardiovascular collapse. A detailed history was taken from the patient and it surfaced that she had been taking a large amount of preserved plums for the last few years. Her poor oral intake prior to admission had likely exacerbated the hypokalemia resulting in ventricular fibrillation. She was advised to stop intake of preserved plums. Serum potassium rapidly normalised in the ward.

A follow up serum potassium done 2 months after discharge was normal at 4.7mmol/L.

Discussion:

Licorice contains glycyrrhizic acid which inhibits 11-beta hydroxysteroid dehydrogenase Type 2 (an enzyme that inhibits cortisol conversion to cortisone). Excess cortisol accumulates stimulating mineralocorticoid receptors and inducing a state of pseudohyperaldosteronism. Enhanced renal excretion results in clinically significant hypokalemia.

People are unaware of the significant content of licorice in preserved fruit and its side effects. The most commonly reported complications are secondary hypertension and hypokalemic myopathy. Documented fatalities were linked to its arrthymogenic effects from hypokalemia.

We report this case to increase awareness of this often-overlooked cause of hypokalemia and its significant complications.

Nothing to Disclose: MKSL, CJS

13348 16.0000 SAT-0762 A Case Report of a Patient Who Experienced Cardiovascular Collapse Secondary to Severe Hypokalemia from Chronic Consumption of Licorice-Containing Preserved Plums 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0747-0780 4776 1:00:00 PM Adrenal Case Reports 1 - CAH and Adrenal Insufficiency Poster

Milay Luis Lam^*¹, Alan Scott Sacerdote² and Gul Bahtiyar³
¹woodhull Medical Center, Brooklyn, NY, ²Woodhull Med & Mental Hlth Ctr, Brooklyn, NY, ³Woodhull Medical & Mental Health Center, Brooklyn, NY

Background: We have previously reported that vitamin D replacement can ameliorate both classical and non-classical adrenal hyperplasia (NCAH) due to 11-hydroxylase deficiency much as it does with both polycystic ovarian syndrome, possibly due to a reduction in insulin resistance. In this case report we show the biochemical benefit that a patient received from vitamin D replacement and GLP-1 agonist treatment in terms of his 11-deoxycortisol levels.

Clinical Case: Our patient is a 60 year old male being followed in Endocrinology clinic since March 2012, after being hospitalized for bowel obstruction and noticing that he was hyperglycemic. His only past medical history was positive for seizure disorder, for which he was taking oxcarbazepine 300mg and phenobarbital 30mg both three times a day. The latter is known to cause Vitamin D deficiency/insufficiency via increased clearance.

At the time of his diabetes diagnosis he was not taking any insulin sensitizers. His BMI was 36 kg/m² and has been stable in that range. His initial HbA1c by immunoturbidimetry on 3/20/2012 was 11.4% (normal range 0-6.99%) so he was started on an insulin regimen with bedtime glargine and lispro before meals. On 3/20/2012 his 25-OH-vitamin D by liquid chromatography tandem mass spectrometry [LC MS/MS] was 10ng/ml (normal range 30-100 ng/ml). He was started on replacement with ergocalciferol 50 000 IU weekly. The initial measurement of 11- deoxycortisol could not be done since the laboratory lost the corresponding sample.

On 5/22/2012 he had a follow-up visit in Endocrinology clinic where he was started on liraglutide,1.2mg SC, since his HbA1c from 5/3/2012 was still above target at 8.8%, 11 deoxycortisol by LC/MS/MS was found to be 79ng/dl (< 42ng/dl) with a 25-OH-Vitamin D level of 41ng/ml. On 8/6/2012 his HbA1c was 5.8%, 11 Deoxycortisol level was 70ng/dl, 25–OH vitamin D level was 61ng/dl, so on his 8/15/2012 Endocrinology visit, liraglutide was increased to 1.8mg daily.

By 11/8/2012 his 11-deoxycortisol level was 49ng/dl, HbA1c 5.5% and on 2/13/13 11-deoxycortisol level was normal at 34 ng/dl, with a 25-OH-Vitamin D level of 62 ng/ml.

Conclusion: Our findings suggest that vitamin D replacement in combination with a GLP-1 agonist can help in the treatment of NCAH caused by 11- Hydroxylase deficiency by reducing insulin resistance through reductions in inappropriate glucagon secretion and glucose toxicity in addition to weight loss (when it occurs) as well as by a direct effect of Vitamin D, through binding to its adrenocortical receptors and increasing the synthesis of mRNA for 11-hydroxylase.

Nothing to Disclose: ML, ASS, GB

11634 17.0000 SAT-0763 A Normalization of Serum 11- Deoxycortisol in a Patient with Non-Classic Adrenal Hyperplasia Due to 11-Hydroxylase Deficiency Treated with Vitamin D and a Glucacon-like Peptide(GLP)-1 Agonist 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0747-0780 4776 1:00:00 PM Adrenal Case Reports 1 - CAH and Adrenal Insufficiency Poster

Muhammad A Mahmood^*¹ and Christine Irene Oakley²
¹Joan C. Edwards School of Medicine, Marshall University, Huntington, WV, ²Joan C. Edwards School of Medicine Marshall university, Huntington, WV

Objective: To describe an unusual case of adrenal insufficiency (AI) in pregnancy.

Clinical Case: A 26 year-old G1P0 female with microprolactinoma, currently off medical therapy, presented at 32 weeks gestation with sudden onset sharp right upper quadrant (RUQ) pain associated with malaise and vomiting. She was afebrile and normotensive, with mild RUQ tenderness. Laboratory evaluation was unremarkable except leukocytosis. Abdominal ultrasonography was normal. CT scan of abdomen showed a 4.2 x 3.1 cm right adrenal mass likely representing hemorrhage and normal left adrenal. Endocrinology was consulted. Patient recalled that her father underwent adrenalectomy for unknown reason. Plasma metanephrines and normetanephrines were normal. MRI two days later demonstrated stability of adrenal mass and changes consistent with hemorrhage. Her hematocrit was stable. She was discharged, but readmitted four days later with recurrence of abdominal pain more so on left, associated with nausea and fatigue. Vitals were stable and laboratory evaluation unremarkable. Repeat MRI showed stability of right adrenal mass. A morning serum cortisol was 7.2ug/dL (>21ug/dL normal in 3rd trimester). Cosyntropin (250ug) stimulation showed baseline cortisol of 7.5ug/dL, 8.2ug/dL at 30 minutes, and 7.7ug/dL at 60 minutes indicative of AI. Serum ACTH was 286.4pg/mL (normal:7.2-63.3pg/mL). Twenty-one hydroxylase antibodies were ordered, but specimen lost. The patient started hydrocortisone 20mg in the morning and 10mg in the afternoon leading to resolution of her symptoms. She delivered a healthy baby at 37 weeks gestation via elective Cesarean section. She was lost to endocrine follow-up.

Discussion/Conclusion: With a reported incidence of 1/3,000 (1), the diagnosis of AI in pregnancy should be entertained in those with significant malaise and emesis beyond the 1^st trimester, or in those with symptoms of weight loss, salt craving, orthostatic hypotension, hypoglycemia, or electrolyte abnormalities. Primary AI manifests when more than 90% of each adrenal gland is destroyed (2) making primary AI from adrenal hemorrhage unlikely in our case, and autoimmune disease more likely. As cortisol binding globulin levels rise in pregnancy, levels of serum cortisol should be higher, leading to higher diagnostic cutoffs (3). Recognition of AI is vital to prevent maternal adrenal crisis, IUGR and fetal demise (3).

Nothing to Disclose: MAM, CIO

11295 18.0000 SAT-0764 A “an Atypical Presentation of Adrenal Insufficiency in Pregnancy As Recurrent Abdominal Pain” 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0747-0780 4776 1:00:00 PM Adrenal Case Reports 1 - CAH and Adrenal Insufficiency Poster

Lauren Michelle Maiorini^*¹ and Lawrence E Shapiro²
¹Winthrop University Hospital, Mineola, NY, ²Winthrop Univ Hosp, Garden City, NY

Introduction/background:

Adrenocortical insufficiency (AI) is a rare but potentially lethal disease. Compared to the prevalence of adrenal metastases in patients with malignancy, AI in these patients is very uncommon. A salient feature of AI due to adrenal metastases is normal levels of basal serum cortisol.

Clinical Case:

This is a 68 year old female referred for evaluation of hypotension and new onset hyperkalemia. Associated symptoms included severe bilateral flank pain, fatigue and dizziness. She has a history of ovarian carcinoma s/p TAH-BSO. During a chemotherapy treatment session, she became hypotensive and recent labs revealed a potassium level of 6.0 mEq/L. She was known on PET-CT imaging to have bilateral adrenal masses, suspicious for metastatic disease. Given her adrenal masses, hyperkalemia, and hypotension, she was referred to our office for suspected AI. A 250mcg cosyntropin stimulation test was performed. Her baseline serum cortisol level was normal, 15.4 mcg/dL, but the stimulated value was only 16.2 mcg/dL. Despite normal basal cortisol, other values were consistent with adrenal insufficiency: baseline ACTH level of 400 pg/mL, an aldosterone level of <1 ng/dL, and a renin level of 21.55 ng/mL/hour. She was started on Hydrocortisone and Fludrocortisone. Her symptoms of fatigue and orthostasis improved. She died a few months later from her progressive disease.

Discussion:

The adrenal glands are the 4^th most common site of distant metastasis. However, adrenal metastases from gynecological neoplasms are relatively rare. AI is typically found in patients with 4cm or larger bilateral adrenal metastases. The features of AI which are treatable may be masked by the symptoms of end stage metastatic disease so the diagnosis can be missed. A literature review revealed that advanced cancer patients have increased levels of basal cortisol but can have biochemical and clinical features of AI. This elevation in cortisol may be due to an activated hypothalamic-pituitary-adrenal axis from increased physiologic stress & interactions between the tumor, the immune system, and the endocrine system. Thus, “normal” basal cortisol concentrations exist in patients with AI due to adrenal metastases. Hormonal replacement therapy may be life-saving and improve quality of life in patients suffering symptoms of adrenal insufficiency.

Conclusion:

This is a rare case of ovarian cancer metastasizing to the adrenal glands & causing symptoms and biochemical abnormalities consistent with AI. Evaluation of patients with bilateral adrenal metastases & suspected AI must account for the fact that the HPA axis is activated in patients with malignancy to avoid erroneous exclusion of AI by the presence of normal serum cortisol.

Nothing to Disclose: LMM, LES

12214 19.0000 SAT-0765 A Adrenal Insufficiency with Normal Morning Cortisol in a Patient with Metastatic Carcinoma 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0747-0780 4776 1:00:00 PM Adrenal Case Reports 1 - CAH and Adrenal Insufficiency Poster

Jamie M Olejarski^*, Lavanya N Sendos, Kelly L Wirfel and Jeena Mary Varghese
The University of Texas Health Science Center at Houston

Introduction: Acute adrenal crisis is a potentially life threatening condition requiring rapid diagnosis and treatment. We present a case of heparin induced thrombocytopenia associated bilateral adrenal hemorrhage resulting in primary adrenal insufficiency and acute adrenal crisis.

Clinical Case: A 64 year old woman was transferred to our hospital for surgical intervention after sustaining a traumatic T12 burst fracture. She underwent an uncomplicated surgical repair. Four days postoperatively, platelet counts were noted to have fallen from an initial value of 188 to 42 K/cmm. Heparin administered for DVT prophylaxis was discontinued. Later that evening, the patient developed acute neurological changes culminating in complete obtundation and respiratory failure requiring intubation. CT scan of the head revealed acute bilateral posterior cerebral artery infarcts with a large intracranial hematoma with mass effect. Heparin induced thrombocytopenia (HIT) was confirmed by HIT antibody testing. Therapeutic plasma exchange was initiated. Sodium values during this time were also noted to fall acutely from an initial level of 136 to 123 mEq/L with normal potassium levels. A baseline cortisol value was obtained with a level of 1.2 µg/dl. Results of a cosyntropin stimulation test confirmed the diagnosis of acute adrenal insufficiency. CT abdomen revealed acute bilateral adrenal hemorrhage. Steroid therapy and fluid resuscitation were immediately initiated with the eventual addition of mineralocorticoid replacement. Sodium values quickly trended to normal limits. Platelet count slowly improved after discontinuation of heparin and treatment with fondaparinux. The patient gradually made a full recovery and was discharged home on oral steroid replacement.

Conclusion: Although isolated HIT is well documented, bilateral adrenal hemorrhage resulting in primary adrenal insufficiency secondary to heparin induced thrombocytopenia is rare. The rapid clinical deterioration and potential for complete hemodynamic collapse from acute adrenal crisis requires a high degree of clinical suspicion regardless of confounding factors or the complexity of the case.

Nothing to Disclose: JMO, LNS, KLW, JMV

15287 20.0000 SAT-0767 A Bilateral Adrenal Hemorrhage: A Rare Complication of Heparin Induced Thrombocytopenia 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0747-0780 4776 1:00:00 PM Adrenal Case Reports 1 - CAH and Adrenal Insufficiency Poster

Lara Paraskos^*¹ and Atil Yilmaz Kargi²
¹University of Miami, Coral Gables, FL, ²University of Miami Miller School of Medicine, Miami, FL

INTRODUCTION: Congenital Adrenal Hyperplasia (CAH) is a known a cause of nonhyperfunctioning adrenal adenomas. Non-classical CAH presents in late childhood or early adulthood and manifests itself in females as hyperandrogenism, anovulatory infertility and amenorrhea, in males excess androgen does not cause clinical signs and so diagnosis is usually not made. Non-Classical CAH should be ruled out in patients suspected of PCOS as the phenotypes overlap significantly. Elevated serum 17-hydroxyprogesterone (17OHP) has been observed in patients with adrenal adenomas both with and without CAH, suggesting that abnormal tumoral steroidogenesis, rather than CAH, may be the cause in some instances.

CASE PRESENTATION: We present a case of a 48-year-old man referred for evaluation of bilateral adrenal masses.. He initially presented with a 1.5 cm left adrenal adenoma with benign imaging characteristics found incidentally on CT scan of abdomen 10 years prior for evaluation of musculoskeletal pain . Functional workup was negative and the nodule was followed annually for several years with minimal growth. 10 years after the initial diagnosis, MRI of the abdomen showed interval increase of left sided adenoma to 2.3cm and development of a right-sided 2.7cm adrenal adenoma. Dedicated CT abdomen with adrenal mass protocol revealed bilateral adenomas with precontrast density <10 HU. To elucidate an etiology of these adenomas and prevent further testing and imaging, a baseline17OHP level was measured and found to be elevated at 657 ng/dL (33-195), and after ACTH stimulation increased to 3535 ng/dL (33-195). This led to the diagnosis of non-classical CAH.

This diagnosis lead to questions regarding his children, and it was discovered that his 21 year old daughter was previously diagnosed with PCOS. We performed baseline 17-hydroxyprogesterone level testing on the daughter and the levels were significantly elevated at 1127 ng/dL (33-195), confirming a diagnosis of non-classical CAH rather than PCOS.

In this case, elucidating the etiology of the bilateral adenomas as non-classical CAH does not affect the treatment course of the father, but has led to the diagnosis of non-classical CAH in the patient’s daughter who had been previously diagnosed with PCOS. Both father and daughter have been referred to genetics for testing, as it will be useful to have pre-conception genetic counseling in the daughter. Additionally, a diagnosis of non-classical CAH in the father will provide reassurance to treating physicians and eliminate the need for repeat adrenal imaging.

CONCLUSION: We conclude that a diagnosis of non-classical CAH should be considered even in males with adrenal adenomas as this may have implications for family members.

Nothing to Disclose: LP, AYK

14647 21.0000 SAT-0768 A Adrenal Incidentaloma in Father Leads to Diagnosis of Congeital Adrenal Hyperplasia in Daughter 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0747-0780 4776 1:00:00 PM Adrenal Case Reports 1 - CAH and Adrenal Insufficiency Poster

Ursina A. Probst-Scheidegger^*¹, Christa E Flueck², Dagmar l'Allemand³ and Nuria Camats⁴
¹Children's Hospital of Eastern Switzerland, St. Gallen, Switzerland, ²Pediatric Endocrinology and Diabetology, Bern, Switzerland, ³Children's Hospital of Eastern Switzerland, St.Gallen, Switzerland, ⁴University Children's Hospital Bern, Bern, Switzerland

Background: 3β-Hydroxysteroiddehydrogenase (3b-HSD) is a key enzyme in steroidogenesis, responsible for the conversion of Δ5- to Δ4-Steroids. Deficiency in 3b-HSD results in congenital adrenal hyperplasia (CAH) including glucocorticoid deficiency and a various degree of mineralocorticoid and sex steroid deficiency. The molecular etiology of 3b-HSD deficiency lies in a defect in HSD3B2gene, coding for the isoform II of the enzyme, present in adrenals and gonads.

Clinical Case: A healthy full-term female baby, born to a non-consanguineous Swiss couple, was admitted on day of life (DOL) 8 due to increased 17-OH-progesterone (17OHP) in newborn screening. She was in no physical distress, feeding well, and gaining weight appropriately. External genitalia were female without virilisation nor palpable gonads.

Ultrasound revealed female internal genitalia with slightly stimulated uterus and normal ovaries with few follicles. Biochemical analysis revealed increased ACTH (549 ng/l) with low cortisol (92 nmol/l), increased 17OHP (124 nmol/l), normal DHEA and 11-desoxycortisol, and increased renin (116 pg/ml).

The patient was immediately started on hydrocortisone (HC). The following day, she developed salt loss (Na of 129 mmol/l, K of 6.1 mmol/l) and was therefore started on fludrocortisone (FC) and NaCl. On continuous HC and FC replacement psychomotor and physical development were normal.

At 7 months of age, the patient was further evaluated for steroidogenic function. Serum steroids were measured 36h after last dose of HC and FC. Results confirmed glucocorticoid and mineralocorticoid deficiency with large increase in ACTH and renin, low cortisol and aldosterone. There was no increase, whatsoever, in 17OHP, DHEA, androstendione, 11-desoxycortisol and testosterone.

Molecular analysis discovered that the patient was compound heterozygote for 2 mutations in HSD3B2: a previously described c.512G>A (W171X) mutation in the paternal allele, and a novel frameshift mutation c.503delC (A168Vfs*6) in the maternal allele. Both mutations cause two shorter and aberrant gene products.

In retrospect, we interpreted the neonatally increased 17OHP as a product of the placental acitivity of 3β-HSD Type I activity. The absent increase in DHEA at 7 months is probably due to the immature zona reticularis at this age.

Conclusion: Our patient is compound heterozygote for two mutations in HSD3B2 causing two shorter and aberrant peptides. This clinically causes a severe loss of 3β-HSD II activity, leading to a nearly complete deficiency in glucocorticoids, mineralocorticoids and sex steroids in our phenotypically non-virilized female CAH patient. One of the mutations is novel: c.503delC (A168Vfs*6).

Nothing to Disclose: UAP, CEF, DL, NC

12083 22.0000 SAT-0769 A Non-Virilizing Congenital Adrenal Hyperplasia in a Female Patient - Report of a Novel HSD3B2 Mutation 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0747-0780 4776 1:00:00 PM Adrenal Case Reports 1 - CAH and Adrenal Insufficiency Poster

Maria Patricia Gamboa Puno^*, Elizabeth P Pacheco, Adriano Dela Paz, Stephanie Mae O. Ang, Gregorio B. Cortez III and Elizabeth Ann S. Alcazaren
The Medical City, Pasig City, Philippines

Hemorrhage to the adrenal glands is a rare finding occurring more frequently in children than in adults. It is classically associated with meningococcal septicemia (Waterhouse–Friderichsen syndrome). In adults, it is mainly caused by trauma, surgical stress, anticoagulation therapy, or a tumor; however, spontaneous or idiopathic adrenal hemorrhage is extremely rare.

We report a case of a 72 year old male, hypertensive, diabetic, presenting with an a palpable soft, non-tender right lumbar mass. He had no history of trauma nor intake of anti-coagulants and was otherwise well with good functional capacity. A CT scan was done and revealed a large heterogeneous mass lesion with a central hypodense component (likely necrosis) centered in the right retroperitoneal region, with non-contrast Hounsfield units ranging from 15-40 HU. It measured 14.2 x 12.5 x 14.7 cm, with neovascularities and small calcific components. A possible adrenal neoplasm was considered. A hormonal work-up was subsequently done revealing a non-functioning adrenal mass. The results: Plasma Aldosterone-RIA 23.77 ng/dl (NV: 3.0-35.5 ng/dl), Plasma Renin Activity 5.65 ng/ml/hr (0.5-1.9 ng/ml/hr supine; 1.9-6.0 ng/ml/hr upright), ARR: 4.207, Serum Cortisol (8am): 15.7 ug/dl (nv 4.2 – 38.4), 24h urine metanephrine: 1.21 mg/24hr (nv up to 1 mg metanephrine/24 hr ); DHEA-S: 69.5 ug/dl (nv 33.6-249); Progesterone: 0.26 ng/ml (nv 0.2-1.31); Testosterone: 3.31 ng/ml (nv 1.95 – 11.38); Estradiol: 29 pg/ml (nv 20-77). A biopsy of the adrenal mass was then done however results were inconclusive. Exploratory laparotomy and resection of the retroperitoneal mass was done with a histopathologic finding of a diffuse adrenal hemorrhage on the right.

Adrenal hemorrhage is an uncommon condition and is difficult to diagnose because of its nonspecific presentation that the diagnosis is often made at autopsy. The value of imaging modalities with the use of CT scan or MRI allows one to determine certain characteristics of an adrenal mass that can point to a benign or malignant tumor. The size and characteristics of the adrenal mass of this patient on CT scan indicated a possibility of malignancy. Further hormonal work-up was warranted and only on post surgical histopathology was the diagnosis of adrenal hemorrhage made. This patient, with no known predisposing factors for bleeding, with no history of intake of anti-coagulant nor history of trauma suffered from an idiopathic adrenal hemorrhage without any hormonal disturbances.

Nothing to Disclose: MPGP, EPP, AD, SMOA, GBC III, EASA

13354 23.0000 SAT-0770 A Idiopathic Unilateral Adrenal Hemorrhage 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0747-0780 4776 1:00:00 PM Adrenal Case Reports 1 - CAH and Adrenal Insufficiency Poster

Anis Rehman^*¹, Michael Dominic Morocco² and Vidhya Sabapathy³
¹Akron General Medical Center, Cleveland Clinic Affiliate, Akron, OH, ²Endocrine Associates, Akron, OH, ³NorthEast Ohio Medical Univesity, Akron, OH

Introduction:

Anti-Phospholipid Syndrome (APLS) is a frequent accompaniment with Systemic Lupus Erythematosus (SLE). The condition predisposes to thrombosis, however adrenal hemorrhage in such population is exceptionally rare.

Case Report:

A 57-year-old Caucasian female, presented to the emergency room with altered mental status, shortness of breath and decreased urine output for 48 hours. Vital signs revealed hypotension (blood pressure 53/30), tachycardia (pulse 110/minute), and a temperature of 37.0 (°C). Laboratory investigations were significant for decreased sodium 125 mEq/L potassium 3.1 mEq/L, blood glucose (serum sugar 46 mg/dL) and elevated creatinine (Cr 5.23 mg/dL). Chest x-ray showed diffuse bilateral lower lobe infiltrates and VBGS were: pH 7.34, pCO2 43.2 mmHg, pO2 32.7 mmHg, HCO3- 22.9mEq/L, O2% Sat Venous 52.9%.

Thus a preliminary diagnosis of sepsis with acute respiratory failure secondary to pneumonia and acute kidney injury was made and the patient was intubated and shifted to the ICU. CT scan of abdomen and pelvis (without contrast) performed in ICU showed a 2.4 x 4cm right and 3 x 4 cm left adrenal gland abnormality that was consistent with adrenal hemorrhage. Patient had been on warfarin 5 mg daily for APLS with an INR being subtheraputic 1.33, she was switched to heparin drip at 20 units/kg/hour in the ICU. An endocrinology consultation was obtained and the dose of hydrocortisone 50 mg q24h was continued.

The patient initially received one time dose of Piperacillin/tazobactam 3.375 g and vancomycin 1g q12h, however after Infectious Disease consultation, the antibiotics were switched to ciprofloxacin 200 mg q12h IV and meropenum 0.5 gm q24h for highly resistant gram negative rod coverage.

Patient gradually responded over a period of 2-3 days. The patient was extubated on day 5 and was discharged on physiologic cortisol replacement, with hydrocortisone 20 mg qAM and 10 mg qPM.

Discussion:

Adrenal hemorrhage is a relatively uncommon condition with a non-specific presentation (typically flank, back or abdominal pain), but can also present through cardiovascular sequel. Without prompt management, the condition it could lead to adrenal crisis, shock and death with an overall mortality rate of 15%.

Our case illustrates several risk factors for adrenal insufficiency such as sepsis, APLS and use of anti-coagulant therapy. Adrenal hemorrhage is a life threatening condition warranting prompt diagnosis and therapy. Clinicians should maintain a high index of suspicion while managing cases of hypotension in the setting of risk factors. A timely CT scan of abdomen and steroid administration could be life saving in such cases.

Nothing to Disclose: AR, MDM, VS

12393 24.0000 SAT-0771 A Adrenal Insufficiency Due to Bilateral Adrenal Hemorrhage in Anti-Phospholipid Syndrome (APLS) and Systemic Lupus Erythematosus (SLE) 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0747-0780 4776 1:00:00 PM Adrenal Case Reports 1 - CAH and Adrenal Insufficiency Poster

Melissa Sawyer^*¹, Gauri Dhir² and Marc J Laufgraben³
¹Aria Health, Philadelphia, PA, ²Cooper University Hospital, Bala Cynwyd, PA, ³Cooper University Hospital, Camden, NJ

First report of large uterine myelolipoma in a woman with 11-β hydroxylase deficiency

Sawyer ML, Laufgraben MJ, Dhir G.

Department of Endocrinology, Diabetes and Metabolism, Cooper Medical School of Rowan University, Camden, NJ

Department of Medicine, Aria Health, Langhorne, PA

Background: CAH patients are at increased risk of developing adrenal myelolipomas and testicular adrenal rest tumors, but uterine leiomyoma has only been reported in a single case of a woman with 21-hydroxylase deficiency . We provide the first report of a large uterine leiomyoma in a woman with 11-β hydroxylase deficiency.

Case presentation: A 24 yo African American female with 11-β hydroxylase deficiency presented with increasing abdominal girth associated with abdominal pain, nausea and vomiting. She had been diagnosed with CAH due to 11-β hydroxylase deficiency shortly after being born with ambiguous genitalia. She had been treated with hydrocortisone and had corrective vaginal surgery at 7 months. At age 10, she developed HTN requiring treatment with multiple antihypertensive agents. She was nonadherent with her treatment and was lost to follow-up for many years prior to presenting with abdominal signs and symptoms. Abdominal exam revealed a large, soft and nontender pelvic mass extending to xiphisternum suggestive of a uterus at 36 weeks gestation. However, a pregnancy test was negative. CT revealed bilaterally enlarged adrenal glands and a large tumor originating from the uterus measuring 19.5 x 17.0 x 20.5 cm. She underwent total abdominal hysterectomy with bilateral salpingectomy. The resected mass measured 17 x 15 x 17 cm and weighed 3620 gm. Pathology demonstrated features of a typical benign leiomyoma.

Conclusion: We believe this is the first case report of a large uterine leiomyoma presenting in a woman with 11-β hydroxylase deficiency. Leiomyoma in women with CAH is exceedingly rare, with only one prior report of multiple uterine leiomyomas in a woman with 21-hydroxylase deficiency. The growth of uterine leiomyoma is generally associated with stimulation by estrogen and progesterone, and would be expected to be inhibited in a high androgen environment as seen in 21-hydroxylase deficiency and 11-β hydroxylase deficiency. In vitro analysis of hormone receptors in the leiomyoma tissue from such unusual patients would be most illuminating.

Nothing to Disclose: MS, GD, MJL

15411 25.0000 SAT-0772 A First Report of Large Uterine Myelolipoma in a Woman with 11-Beta Hydroxylase Deficiency 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0747-0780 4776 1:00:00 PM Adrenal Case Reports 1 - CAH and Adrenal Insufficiency Poster

YunYing Shi^* and Dorothy Santos Martinez
UCLA, Los Angeles, CA

Introduction: Addison’s disease is often difficult to diagnose due to non-specific presenting symptoms. Hypovolemic hypotension is a commonly associated cardiovascular complication. Rarely, cardiogenic shock can also occur during an Addisonian crisis. We report here a postpartum young woman with undiagnosed Addison’s disease who presented with acute biventricular systolic failure as the primary marker for a fulminate adrenal crisis.

Case: A 32-year-old Caucasian female was admitted from an outside hospital (OSH) for heart transplant evaluation with presumed postpartum cardiomyopathy. Patient’s pregnancy was complicated by hyperemesis gravidarum started 2 months into her pregnancy. She did not have any history of hypotension while pregnant and had an uneventful vaginal delivery of a healthy, full-term boy. Two weeks postpartum, her emesis worsened, accompanied by 30lb weight loss, dizziness, and low blood pressure (BP) requiring IV hydration. Six months postpartum, she presented at OSH due to hemodynamic collapse with left ventricular ejection fraction (LVEF) <10%, requiring intubation and pressor support. Hospital course was complicated by acute renal failure, sepsis, anemia, right lower extremity arterial thrombus, stroke and seizure. On admission, her TSH was 106 mcIU/mL (n: 0.3-4.7 mcIU/mL). Levothyroxine IV 50mcg daily and hydrocortisone 100mg IV q8h were started by OSH. Repeat TFT after 5 days of therapy at our hospital showed elevated TSH (16 mcIU/mL, n: 0.3-4.7 mcIU/mL), borderline-low Free T4 (0.6 ng/dL, n: 0.6-1.7 ng/dL), low Free T3 (122 pg/dL, n: 222-383 pg/dL), and positive thyroid peroxidase antibody and thyroglobulin antibody. Liothyronine 5mcg twice daily was added. Despite rapid full recovery of ventricular function (LVEF 60-65%) in two weeks, she continues to have intermittent low BP on hydrocortisone IV 25mg daily. Further evaluation showed elevated ACTH (2150 pg/mL, n: 4-48 pg/mL), elevated prolactin (152 ng/mL, n: 3.0-23.1 ng/mL), elevated rennin (6.3 ng/mL/hr, n: 0.6-1.6 ng/mL/hr), and normal aldosterone (n<1.0 ng/dL). A diagnosis of Addison’s disease was made. Her 21-hydroxylase antibody was later confirmed to be positive. Patient’s BP improved with the addition of mineralocorticoid. She was eventually discharged in stable condition on oral levothyroxine, hydrocortisone, and fludrocortisone.

Conclusion: New onset Addison’s disease is rare in pregnancy. It has been suggested that during the pregnancy, free cortisol produced by the fetal adrenal cortex can offer some maternal protection. However, the withdrawal of the cortisol source after delivery can result in acute adrenal insufficiency, in our case, leading to the rare presentation of acute ventricular dysfunction. Therefore, a high index of suspicion for Addison’s disease is required in postpartum women presenting with hemodynamic instability with a history of hyperemesis gravidarum.

Nothing to Disclose: YS, DSM

15508 26.0000 SAT-0773 A Addisonian Crisis Presenting As Acute Ventricular Failure in the Postpartum 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0747-0780 4776 1:00:00 PM Adrenal Case Reports 1 - CAH and Adrenal Insufficiency Poster

Vishnu Sundaresh^*¹, Kartik Anand², Tania Rowland³, Michael Constantinescu³ and Sherry Lynn Ryan³
¹Louisiana State University Health Sciences Center, Shreveport, LA, ²Louisiana State University health Sciences Center, Shreveport, LA, ³Overton Brooks VA Med Ctr, Shreveport, LA

Amyloidosis refers to the extracellular deposition of insoluble proteinaceous material affecting multiple organs in its systemic form. Endocrine involvement is infrequent with endocrine dysfunction being extremely rare. We report a case of Systemic Amyloidosis (SA) presenting with primary adrenal insufficiency (PAI) with poly-endocrine gland involvement.

A 63-year-old white male presented with a 3-month history of recurrent episodes of dizziness and syncope. He had cold sensations in the extremities, decreased libido, erectile dysfunction, dysphagia, early satiety, and unintentional 90-pound weight loss pounds in two years. Medical history included papillary thyroid cancer treated with total thyroidectomy and I-131 ablation, autoimmune and post-surgical hypothyroidism, focal gastric amyloidosis secondary to h. Pylori gastritis, and stage 3 kidney disease of unknown etiology. Exam revealed supine hypertension, orthostatic hypotension with inappropriate heart rate response and peripheral sensory neuropathy. Sodium was 133 mmol/L (136–145), potassium 3.8 mmol/L (3.3-5.1), and creatinine 1.8 mg/dL (0.6-1.3). EKG had low voltage complexes with fixed R-R interval. 2D ECHO showed normal systolic function with impaired diastolic relaxation. Gastric emptying study showed severe gastroparesis with T 1/2 855 minutes (normal <110). No obstruction was seen on EGD. A 250-µg cosyntropin stimulation test revealed baseline, 30- and 60-minute cortisol levels of 9.7, 20, and 21 µg/dL (AM 6.7-22.6 ug/dL), respectively. ACTH was 42 pg/mL (7.2-63.3), DHEA-S 38.9 µg/dL (48.9-344.2), aldosterone <1 ng/dL (0.0-30), and renin < 0.16 ng/ml/hr. (1.31-3.95 upright). LH was 2.1 mIU/ml (1.2-8.6), FSH-7.7mIU/ml (1.3-19.3) and total testosterone was 1.1 (1.8-7.8). Repeat testing showed baseline cortisol of 15.9 which stimulated to 17.5 with a baseline ACTH of 14.7. Non-contrast CT of the adrenals and pituitary were normal. Free Lambda light chains were abnormally detected in both serum and urine along with nephrotic range proteinuria. Serum immunoelectrophoresis was normal with an absent M spike. Bone marrow biopsy revealed 4.6% plasma cells. 18-F PET scan ruled out a plasmacytoma. On careful re-examination of biopsies of the stomach, duodenum, bone marrow and previously excised thyroid tissue, all exhibited Congo red staining and apple green birefringence within small vessel walls, confirming SA. Mayo classification was stage II, AL type. Treatment with hydrocortisone and fludrocortisone for presumed PAI due to amyloid infiltration of the adrenals resulted in significant clinical improvement. He is currently receiving chemotherapy in preparation for autologous stem cell transplantation.

Endocrine dysfunction secondary to SA is rare, with subtle presentation. Investigating glandular function may avoid potentially life-threatening consequences of glandular failure.

Nothing to Disclose: VS, KA, TR, MC, SLR

16479 27.0000 SAT-0774 A Amyloid Here, Amyloid There, Amyloid Everywhere! Primary Adrenal Insufficiency Due to Systemic Amyloidosis 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0747-0780 4776 1:00:00 PM Adrenal Case Reports 1 - CAH and Adrenal Insufficiency Poster

Sri Harsha Tella^*¹ and J Christopher Gallagher²
¹Creighton University, Omaha, NE, ²Creighton University Medical Center, Omaha, NE

Background: Treatment with the benzodiazepines atypical antipsychotics is frequently associated with development of obesity, insulin resistance and drowsiness. Treatment-induced weight gain has been suggested to be the main contributing factor of diminished insulin sensitivity. Here we describe a case of benzodiazepine and antipsychotic medication induced hypocortisolism.

Case:We describe a case of 32 year old homeless Caucasian male with past medical history of anxiety, depression, bipolar disorder, obsessive compulsive disorder and opioid abuse (methadone) was brought into ER by squad as he overdosed himself with 15 pills of clonazepam 2 mg. Initial vital signs on presentation are Temp: 99.7 F, Pulse: 97/min, BP: 60-70/40-50 mm Hg, RR: 12/min. Chest X ray and CT head were unremarkable. He was transferred to the ICU for management of hypotension. He was given 8 liters of 0.9% normal saline over a period of 6 hours (including 3 liters of bolus) and blood pressure came up to 80-90/50-60 mm Hg. His laboratory evaluation showed normal electrolytes, creatinine and white count. Serum cortisol levels were tested which showed 1.7 mcg/dl at 04 50 AM and 0.6 mcg/dl at 06 00 AM. 250 mcg ACTH stimulation test was done which showed serum cortisol of 2.8 mcg/dl, 14.4 mcg/dl and 16.7 mcg/dl at 0, 30 and 60 minutes respectively. ACTH, testosterone, LH and TSH at baseline were 3.8 pg/ml, 979 ng/dl, 2.2 IU/l and 0.75 microIU/ml respectively. On further review of records, patient had normal blood pressures and serum cortisol in the past before the initiation of these medications. We discontinued clonazepam from his medication list and added prednisone 40 mg that has increased the blood pressures to normal range in our patient. He was discharged home on after fast taper with follow up in clinic which showed normal blood pressure in 1 month.

Discussion:Benzodiazepines and Opioids can cause endocraniopathy by acting on GABA receptors there by inhibiting the secretion of corticotrophin releasing hormone (CRH). In this patient, the acute drop in serum cortisol might be due to overdose of clonazepam that can inhibit CRH. This is interesting since CRF may have neurotropic effects related to the behavioral responses to stress, and the inhibitory effect thus may represent a mechanism of action for the anxiolytic effect of benzodiazepines. Surprisingly, this effect is dose dependent and can be seen as soon as 2 hours. So, in our patient this acute drop in blood pressures and cortisol levels are mostly likely related to more potent and efficacious benzodiazepine Clonazepam.

Conclusion: Benzodiazepines can cause in acute lowering of serum cortisol levels and one should recognize this effect and treat accordingly to prevent the morbidity or mortality from acute drop in cortisol levels. Moreover, chronic use of opioids, antipsychotics and benzodiazepines can cause GABA induced suppression of CRH release leading to low cortisol levels.

Nothing to Disclose: SHT, JCG

12950 28.0000 SAT-0775 A Relationship of Benzodiazepines with Serum Cortisol Levels: A Hidden Fact 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0747-0780 4776 1:00:00 PM Adrenal Case Reports 1 - CAH and Adrenal Insufficiency Poster

Cesar Abuchaibe^*¹ and Omar Naeem Akhtar²
¹The Jewish Hospital of Cincinnati - Mercy Health, Cincinnati, OH, ²Mercy Health, Mason, OH

Background

To present a case of exogenous Cushing’s syndrome after use of a OTC joint supplement from Cuba.

This case illustrates how difficult it can be demonstrate the presence of corticosteroids in OTC medications especially foreign medications.

In this case the patient was not aware that the Herbal medication that she brought from Cuba was the one responsible for her clinical syndrome. Therefore physicians should pay close attention on the history for asking new medications, even herbs or foreign medications.

As we can illustrate in this case the label telling the composition of the medication never showed or mentioned the inclusion of steroids

Presentation

67 y/o Hispanic female who presented to the endocrine clinic for the management of Diabetes mellitus type 2, Hypertension and severe osteoporosis. During the initial encounter the patient appeared clinically Cushingoid. Her physical examination exhibited obesity, round facies, supraclavicular fat, thin skin and lower extremity edema.

She had been evaluated for Cushing’s Syndrome by previous endocrinologist. Work up in the past for endogenous Cushing’s was negative on multiple occasions.

She had also been tested for exogenous Cushing’s; having multiple positive screens for synthetic steroids. She had not had any steroids injections in the past and she denied the use of steroids orally. She did admit that she had been taking an OTC joint supplement from Cuba called ATRIN^® for the past 3 to 4 years.

Having the suspicion that this foreign OTC joint medication could have been causing the exogenous Cushing’s’ resulting in adrenal suppression; she was advised in several visits to stop taking this medication. The patient tried to stop taking the OTC medication but she had fatigue and joint pain after stopping it.

On our initial visit, we had the same suspicion of Exogenous Cushing’s, source being the OTC supplement.

A sample of the medication was sent to the Mayo Clinic to be analyzed

Composition was as follows:

4 mg of prednisolone

100 ug of hydrocortisone

She was taking 5-6 pills a day.

Am cortisol was low (1.6mcg/dl) after holding the supplement, indicating secondary adrenal insufficiency. It was stopped and she was started on steroid replacement which was gradually tapered.

After stopping it, she lost 30lbs, insulin and oral hypoglycemic were stopped and osteoporosis improved.

Her Synthetic glucocorticoid screening was also negative for the first time

Conclusion

Even though herbal medications appear to be harmless, caution should be taken in those from other countries. Patient with Exogenous Cushing’s should be started on steroid replacement before stopping the supplement due to secondary adrenal insufficiency.

Nothing to Disclose: CA, ONA

14155 29.0000 SAT-0776 A Exogenous Cushing's Syndrome after Use of OTC Joint Supplement 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0747-0780 4776 1:00:00 PM Adrenal Case Reports 1 - CAH and Adrenal Insufficiency Poster

Vrinda Agrawal^*¹ and Ramaswami Nalini²
¹Baylor College of Medicine, Houston, TX, ²Baylor Colg of Med, Houston, TX

Introduction:

Long term glucocorticoid therapy may result in suppression of hypothalamic-pituitary-adrenal function leading to significant morbidity. We report a case of a Cushingoid appearing male with adrenal insufficiency secondary to the use of ‘Rumoquin’ – an over the counter (OTC) pain medication from Mexico.

Clinical Case:

A 57 year old Hispanic male with coronary artery disease and uncontrolled hypertension was referred to endocrinology clinic for evaluation of suspected adrenal insufficiency after he was found to have undetectable morning cortisol levels on two separate occasions. Patient’s physical examination revealed a Cushingoid appearing male, rounded facies with central adiposity and marked skin bruising. This prompted the primary care physician to obtain a morning cortisol level. He was asymptomatic otherwise. Morning cortisol was <0.2 ug/dL (normal 3.09-22.4 ug/dL) along with an ACTH level of <1.1 pg/mL (normal 7.2-63.3 pg/mL). He failed a 1 mcg ACTH stimulation test with a baseline cortisol of 1.3 ug/dL, ACTH of 53.3 pg/mL (normal 7.2-63.3 pg/mL) and a peak cortisol of 2.0 ug/dL. With a suspicion for exogenous steroid use, detailed questioning revealed that the patient had been taking ‘Rumoquin’, an OTC Mexican pain medication for more than 5 years for arthritis related pain. Further review of this medication divulged that this contained 0.75mg of dexamethasone. Patient also reported symptoms of lightheadedness and increased lethargy upon missing a single pill of this medication. A diagnosis of adrenal insufficiency was made secondary to chronic exogenous corticosteroid use. He was advised to stop Rumoquin and was started on replacement doses of hydrocortisone with an aim to taper it off gradually.

Conclusion:

This case illustrates the importance of a detailed history taking including a complete medication history encompassing OTC medications, creams or supplements as quite often patients are unaware that these contain steroids. Physicians should be increasingly aware of such practice to recognize the potential effects of long term corticosteroid use in any form, and to educate the patients, thereby preventing any resulting morbidity and mortality.

Nothing to Disclose: VA, RN

16535 30.0000 SAT-0777 A Over the Counter Pain Medications from Mexico and Adrenal Suppression: Beware! 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0747-0780 4776 1:00:00 PM Adrenal Case Reports 1 - CAH and Adrenal Insufficiency Poster

Ravi Kant^*¹, Hillary Barnes Loper², Kashif M. Munir² and Rana Malek²
¹AnMed Health and Medical University of South Carolina, ²University of Maryland School of Medicine, Baltimore, MD

Although fluticasone (FC) is a potent glucocorticoid, it has minimal systemic effects at recommended dosages due to rapid metabolism by cytochrome P450 3A4 (CYP3A4). We report the first case to our knowledge of iatrogenic Cushing's syndrome (CS) that was presumably caused by the inhibition of CYP3A4 metabolism of FC by duloxetine (DL).

A 52 year old man presented with a 10 week history of lower extremity weakness, extreme fatigue, night sweats, easy bruising and 45 lbs weight gain. Past medical history included allergic rhinitis and ulcerative colitis (UC) managed with FC (50 mcg/act) nasal spray 2 puffs/nostril for 5 years and intermittent mesalamine, respectively. DL had been started for depression several weeks prior to the onset of symptoms. Physical examination revealed severe facial plethora, central obesity and trace pedal edema. Laboratory investigations for CS were discordant with mildly elevated 24 hr urine cortisol [24 UFC (52.2-71.8; normal <50 mcg/24 hr)] but normal 1 mg dexamethasone suppression test [DST (0.5-0.6 mcg/dl)] and mid-night salivary cortisol levels (0.3-1.4; normal 0.3-4.3 mcg/dl). Additional evaluation to support the diagnosis of CS, including ACTH 50 (6-50 pg/ml), 24 hr urine 17-OH corticosteroids 1.7 (3.0-10.0 mg/24 hr), MRI-pituitary, and CT-chest, abdomen and pelvis, was unremarkable. Interestingly, urine FC 17-B carboxylic acid level (U-FCA) was elevated at 266 (normal <10 pg/mL), suggesting iatrogenic CS. Discontinuation of FC led to resolution of hypercortisolism within 4 weeks (1mg DST 0.7 and 24 UFC 32.2). Loss of steroid suppression from FC resulted in a UC flare, 12 lbs weight loss, and continued fatigue over the next 3 months. EMG and gastrocnemius muscle biopsy were performed for myopathy evaluation. Muscle biopsy showed many non-specific findings suggestive of myotonic dystrophy.

Clinically significant drug interactions in patients receiving concomitant FC and CYP3A4 inhibitors, such as ritonavir, are well known.¹ DL has been shown to cause time dependent reversible inhibition of CYP3A4, which can potentially alter FC metabolism and lead to systemic corticosteroid effect although no such case has been reported.² Our patient tolerated chronic FC therapy for 5 years, but developed clinical features consistent with iatrogenic CS shortly after initiating DL. This led us to suspect a significant drug interaction between DL and FC as a potential etiology for his symptoms. In addition to elevated U-FCA levels, resolution of hypercortisolism symptoms and development of UC exacerbation after discontinuation of FC further support our hypothesis. The patient’s myopathy is probably explained by another etiology as suggested by EMG and muscle biopsy, however, glucocorticoid-induced myopathy cannot be entirely excluded. FC should be cautiously administered to patients who are receiving DL and providers should be wary of clinical clues that may indicate CS.

Nothing to Disclose: RK, HBL, KMM, RM

14884 31.0000 SAT-0778 A Iatrogenic Cushing's Syndrome in a Patient Treated with Duloxetine and Fluticasone 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0747-0780 4776 1:00:00 PM Adrenal Case Reports 1 - CAH and Adrenal Insufficiency Poster

César Esteves¹, Celestino Neves^*², Maria João Matos¹ and Davide Carvalho¹
¹Centro Hospitalar São João, Faculty of Medicine, University of Porto, Portugal, ²São João Hospital, Faculty of Medicine, University of Porto, Porto, Portugal

Introduction: Cases of iatrogenic Cushing syndrome or adrenal failure are usually the result of the administration of oral glucocorticoids for a long period. Inhaled or topic corticoids aren't usually considered to be responsible for significant abnormalities in the glucocorticoid axis. However, there are exceptions. Aims: To report a case of adrenal failure in a patient treated with itraconazole and inhaled budesonide therapy. Clinical case: Female, 63 years old, under follow-up in a Pulmonology consultation for allergic broncopulmonary aspergillosis. She was treated with itraconazole 400 mg od for 7 months until July 2011 and kept under therapy with budesonide/formoterol 160/4,5 µg / inhalation bid, tiotropium bromide 18 µg/inhalation od and mometasone furoate 50 µg/pulverization od. She was referred to the Endocrinology appointment due to skin hyperpigmentation and weight loss after initiation of therapy with itraconazole, and a morning serum cortisol of 0.05 µg/dL. At the time of the first Endocrinology appointment she complained of subsequent weight gain with centripetal fat deposition and moon face. She had an ACTH below the limit of detection, normal potassium and low aldosterone (0.9 ng/dL). After itraconazole therapy suspension, ACTH increased to 64.1 ng/L, cortisol 9.7 and 18.8 µg/dL before and after the administration of Synacthen, respectively, and aldosterone to 10.3 ng/dL. Due to worsening of aspergillosis, she initiated treatment with itraconazole, with reduction of cortisol levels to 4.0 and 9.3 µg/dL before and after the administration of Synacthen, respectively. Discussion: The present case is representative of itraconazole effects on the pituitary-adrenal axis, in individuals treated with budesonide, which is an inhaled glucocorticoid metabolized by the cytochrome P450 3A4. Mometasone furoate is also a glucocorticoid for nasal pulverization metabolized by the same cytochrome but its systemic absorption is minimal and its contribution in the present case is undetermined. Itraconazole can also induce abnormalities of mineralocorticoid metabolism. Conclusion: Itraconazole is an antifungal agent, considered safe in terms of its effects on the glucocorticoid axis. The occurrence of pituitary-adrenal axis suppression was previously described for the association of itraconazol and budesonide. There are no described cases associated with mometasone furoate. However, it might be considered as it shares the same metabolic pathways as budesonide.

Nothing to Disclose: CE, CN, MJM, DC

15978 32.0000 SAT-0779 A Hypothalamic-Pituitary-Adrenal Axis Suppression Related to Itraconazole and Budesonide Association Therapy 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0747-0780 4776 1:00:00 PM Adrenal Case Reports 1 - CAH and Adrenal Insufficiency Poster

Ashwini Mallappa¹, Lori-Ann Daley², Carol Van Ryzin² and Deborah P. Merke^*²
¹National Institutes of Health Clinical Center, Bethesda, MD, ²National Institutes of Health, Bethesda, MD

Background: Heterogeneous quality-of-life (QoL) has been reported in patients with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency. Patients with classic CAH require lifelong glucocorticoid therapy. Disease and treatment-related co-morbidities such as obesity, hyperandrogenism, metabolic syndrome, and the use of long-acting glucocorticoid therapy have been suggested to contribute to poor QoL outcomes.

Methods: Health related quality of life (QoL) was assessed by administering the Short Form Health Survey (SF-36) to adult patients with classic CAH enrolled in a Natural History Study at the National Institutes of Health Clinical Center in Bethesda, MD. This 36-item survey measures subjective physical and mental health within the past four weeks across eight domains: physical functioning, role - physical, bodily pain, general health, vitality, social functioning, role- emotional and mental health. Scores in each domain range from 0 - 100 with higher scores indicating better health; a score of 50 represents the population mean with a standard deviation of plus or minus 10.

Results: 48 patients (25 female; 29 ± 13 years) with classic CAH (33 salt-wasting, 15 simple-virilizing) participated in this study. Overall, patients reported an above average QoL (mean total SF36 score: females 65.1 ± 25.9; males 84.3 ± 14.6). QoL was significantly reduced in females compared to males in the physical functioning (p=.033), role-physical (p=.009), bodily pain (p=.014), general health (p=.003), vitality (p=.003), social functioning (p=.004), mental health (p=.003), physical health summary (p=.002), mental health summary (p=.005), and total SF-36 score (p=.003). Salt-wasting CAH females reported the lowest scores. Younger age and lower BMI significantly contributed to higher QoL, but only for females. Total QoL and subgroup scores were not associated with glucocorticoid dose or type, marital/relationship status, or socioeconomic status.

Conclusions: In a population of adult patients with classic CAH reporting overall good quality of life, female patients consistently report lower health status than males. Further studies are necessary to elucidate the factors contributing to impaired QoL in classic CAH females.

Disclosure: DPM: Investigator, Diurnal. Nothing to Disclose: AM, LAD, CV

14709 3.0000 SAT-0820 A Quality of Life in Adults with Classic Congenital Adrenal Hyperplasia: Females Have Reduced Quality of Life Compared to Males 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0818-0831 4780 1:00:00 PM Hypothalamic-Pituitary-Adrenal Axis 1 - CAH, AI & Cushing's Poster

Ribal Al Aridi^*¹, Katia El Sibai², Nadine El Asmar³, Warren Selman⁴ and Baha M Arafah⁵
¹University Hospitals,Case Medical Center, Cleveland, OH, ²University Hospitals Case Medical Center, Cleveland, OH, ³UH Case Medical Center/ Case Western Reserve University, Cleveland, ⁴UH Case Medical Center, Cleveland, OH, ⁵Case Western Reserve Univ, Cleveland, OH

Background: PRL has been proposed as a modulating factor in DHEA-S secretion. Yet, studies in vivo and in vitro have yielded variable conclusions. The aim of this study is to assess the acute and chronic influence of changes in PRL levels on DHEA and DHEA-S secretion.

Materials: We examined changes in serum DHEA, DHEA-S and PRL levels during the first 48hrs after transphenoidal surgery (TSS) in 16 women with prolactinoma (group 1; mean age 26.9 ± 9.9yrs) and in a control group of 10 women who had resection of non-secreting pituitary adenomas (group 2; mean age 32.4 ± 6.8 yrs). Blood samples for measurements of PRL, ACTH, cortisol, DHEA and DHEA-S were taken at baseline and at 2-4, 6-8, 12-18, 24, 36 and 48hrs postop in both groups. In a separate investigation, we examined the effects of medical treatment of hyperprolactinemia (using dopamine agonists) on serum DHEA-S levels in 9 patients (5F, 4M) with prolactinoma (mean age 45.5 ± 9.2yrs). In the latter group, PRL and DHEA-S levels were measured before initiation of treatment and at 2-4 months (mo) intervals for the first year of therapy. All studied patients had NL-HPA function and were not on medications that affect HPA axis.

Results: In the surgically treated prolactinoma group, PRL levels decreased acutely during the first few postop hours reaching normal levels 6-8 hrs following TSS. In both groups of patients who underwent TSS, plasma ACTH levels increased similarly reaching a peak during the first 2-4 postop hrs after which they decreased gradually to normal by 12-18hrs. Similar increases in serum cortisol levels were noted in both groups. Patients with prolactinoma had higher baseline DHEA levels than those with non functioning tumors (8 ±6 ng/ml Vs 3.8±2.5 ng/ml; p<0.05) even though the 2 groups were of similar age and gender. With the acute rise in ACTH at 2-4hrs, serum DHEA levels increased in both groups but were consistently higher in patients with prolactinoma in the first 24hrs. Serum DHEA-S levels in patients with prolactinoma were similar during the first 24hrs to those of non functioning tumors. However, DHEA-S levels in patients with prolactinoma decreased from their baseline values at 48hrs (171 to 131mcg/dL; P 0.009). In medically treated prolactinoma, PRL and DHEA-S decreased in a parallel manner at 2-4 mo post treatment and reached a plateau at 4-6 mo of continued therapy.

Conclusion: Patients with prolactinoma had higher serum DHEA and DHEA-S levels that decreased with surgical or medical treatment of the disease. In surgically treated prolactinomas, the effect of PRL was eliminated at 6-8 hrs postop once PRL levels normalized. The higher levels of DHEA at baseline and during the early postop period in patients with prolactinoma suggest potentiation of ACTH influence on DHEA secretion by hyperprolactinemia. In the absence of acute ACTH stimulation as observed in medically treated prolactinoma, PRL was noted to be a modest modulator of DHEA-S secretion.

Nothing to Disclose: RA, KE, NE, WS, BMA

14248 6.0000 SAT-0823 A Prolactin (PRL) Is a Modulator of DHEA and DHEA-S Secretion 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0818-0831 4780 1:00:00 PM Hypothalamic-Pituitary-Adrenal Axis 1 - CAH, AI & Cushing's Poster

Hideki Katakami^*¹, Seiichi Hashida² and Isolated ACTH Deficiency Study Group³
¹Teikyo Univ Chiba Medical Center, Ichihara, Japan, ²Tokushima Bunri University, Tokushima, Japan, ³IAD consortium, Japan

Isolated ACTH deficiency (IAD) is a rare disease. Our previous epidemiological study of IAD in a Japanese cohort, population size: 1.15-1.17 million-inhabitants, has shown that its prevalence and incidence were 19.1 cases/million and 0.9 cases/year/million, respectively, whereas 85.9 case/million and 5.3 cases/year/million, respectively, for acromegaly in the same cohort. There were both IAD of young onset (JIAD, 11-21yr, 4M+1F, n=5) and IAD of mature onset (MIAD, 36-96yr, 16M+4F, n=20) of acquired form and idiopathic etiology. Noted initial symptoms in JIAD and MIAD were hypoglycemia and weight loss, respectively. Thyroidal autoantibodies were positive in 1/4 of JIAD and 4/11 of MIAD. Pituitary atrophy on MRI or CT was found in 3/4 of JIAD and 4/18 of MIAD.

On the other hand, selectively impaired ACTH secretion causes secondary adrenal insufficiency. Conventional measurements for plasma ACTH levels (2.0/mL for ECLIA or 5.0pg/ml for IRMA) are not sensitive enough to characterize ACTH secretory dynamics in Px with IAD, or to make differential diagnosis among conditions or diseases which cause ACTH insufficiency. We have developed a novel ultrasensive EIA (immune complex transfer-EIA, ICT-EIA) for ACTH, LDV 0.1pg/mL (0.2attomole/assay). To characterize corticotroph function among acquired ACTH insufficiency, including acquired form of IAD (n=18), autoimmune hypophysitis (AH, n=6), CNS germ cell tumors (GT, n=16), post irradiation hypopituitarism (RH, n=6), pituitary tumors (PTm, n=24) and normal controls (CNT, n=28), we measured plasma ACTH levels before and after provocative tests. Despite low plasma ACTH levels in Px with GT or PTm, ACTH responded significantly to stimulatory (CRH or GHRP-2) tests. However, Px with IAD or AH showed no ACTH responses to stimulatory tests. Pharmacological doses of synthetic steroids acutely suppressed plasma ACTH levels to 1.5-4.0pg/ml in Px without pituitary diseases.

The present results show that the prevalence and incidence of IAD are about 1/5 of those of acromegaly, and there are two forms of IAD, i.e., JIAD and MIAD. The corticotroph function was most severely impaired in Px with IAD or AH (0.6-1.2pg/ml), followed by Cushing’ syndrome (1.2-3.5pg/ml), PTm (3.8-8.6pg/ml) and/or irradiation (3.8-8.6pg/ml) vs. CNT (7.8-43.0pg/ml). These results suggest that the novel ultrasensive ICT-EIA for ACTH is useful to establish the diagnosis of IAD or AH.

Sources of Research Support: Health and Labor Sciences Research Grant, Japan

Nothing to Disclose: HK, SH, IADS

16438 7.0000 SAT-0824 A Isolated ACTH Deficiency: Epidemiology, Clinical Features and Secretory Dynamics of Plasma ACTH By a Novel Ultrasensitive EIA 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0818-0831 4780 1:00:00 PM Hypothalamic-Pituitary-Adrenal Axis 1 - CAH, AI & Cushing's Poster

Yasin Simsek^*¹, Zuleyha Karaca², Halit Diri¹, Sulbiye Aribas¹, Fatih Tanriverdi², Kursad Unluhizarci² and Fahrettin Kelestimur²
¹Faculty of Medicine, Erciyes University, Kayseri, Turkey, ²Erciyes University Medical School, Kayseri, Turkey

Aim

Insulin tolerance test (ITT) is accepted as the gold standard test for the assessment of growth hormone (GH)–insulin like growth factor (IGF-1) axis and hypothalamic-pituitary-adrenal (HPA) axis. The goal is to achieve a blood glucose level ≤40 mg/dl during ITT for an effective test hence correct assessment of HPA and GH-IGF-1 axes. In this study, we aimed to compare GH and cortisol responses of patients who achieved biochemical hypoglycemia or not during ITT.

Material-Method

Eighty-six patients with pituitary disorders were included in the study. All patients had clear symptoms of hypoglycemia during ITT and blood glucose levels were obtained during these symptoms at the beginning of ITT. Since the patients were clearly symptomatic The patients were divided into two groups according to their plasma glucose level was ≤ 40 mg/dl or >40 mg/dl during ITT.

Results

The mean age of the patients between 2 groups were found to be similar. The patients had similar body mass index and waist circumference in both groups. The mean blood glucose level was significantly lower in the hypoglycemic group (19.3±0.9) than in non-hypoglycemic group (52.0±2.3 mg/dl). Basal cortisol levels of the patients were similar in both groups. When two groups were compared in terms of peak cortisol and GH responses to insulin-induced hypoglycemia, no statistically significant differences were found.

Conclusion

Present data suggest that clinically symptomatic hypoglycemia is more important than biochemically confirmed glycemic level ≤40 mg/dl during ITT.

Nothing to Disclose: YS, ZK, HD, SA, FT, KU, FK

12788 8.0000 SAT-0825 A Is Biochemical Hypoglycemia Necessary during Insulin Tolerance Test? 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0818-0831 4780 1:00:00 PM Hypothalamic-Pituitary-Adrenal Axis 1 - CAH, AI & Cushing's Poster

Peter Kamenický^*¹, Alban Redheuil², Charles Roux³, Sylvie Salenave⁴, Jacques Young⁵, Elie Mousseaux⁶ and Philippe Chanson⁷
¹Univ Paris-Sud, UMR-S693, Assistance Publique-Hôpitaux de Paris, Le Kremlin Bicêtre, France, ²Université Pierre et Marie Curie, Département d’Imagerie Cardiovasculaire, Paris, France, ³AP-HP, Hôpital Européen George Pompidou, Service de Radiologie Cardiovasculaire, Paris, France, ⁴AP-HP, Hôpital de Bicêtre, Service d'Endocrinologie et des Maladies de la Reproduction, Le Kremlin Bicêtre, France, ⁵Université Paris-Sud, Faculté de Médecine Paris-Sud & AP-HP, Hôpital de Bicêtre, Service d’Endocrinologie et des Maladies de la Reproduction, Le Kremlin-Bicêtre, France, ⁶Université Paris Descartes, Faculté de Médecine & AP-HP, Hôpital Européen George Pompidou, Service de Radiologie Cardiovasculaire, Paris, France, ⁷Univ Paris-Sud, UMR-S693, Le Kremlin Bicêtre, France, Assistance Publique-Hôpitaux de Paris, Hôpital de Bicêtre, Service d’Endocrinologie et des Maladies de la Reproduction, Le Kremlin Bicêtre, Paris, France, Le Kremlin-Bicêtre, France

Background Cardiac structure and function in Cushing’s syndrome has been evaluated in few echocardiographic studies that reported left ventricular (LV) hypertrophy with LV dysfunction. Echo-based measurements require assumptions about LV geometry, which may limit the accuracy of mass and volume measurements especially in obese patients. Little is known about right ventricular (RV) and left atrial (LA) size and function in this setting. The aim of this study was to evaluate LV, RV and LA structure and function by cardiac magnetic resonance (CMR), currently the reference modality in such assessment.

Methods and Results Eighteen patients with active Cushing’s syndrome and 18 normotensive volunteers matched for age, sex and body mass index were studied by CMR. In patients, imaging was repeated after normalization of cortisol secretion. Patients compared to controls had lower LV, RV and LA ejection fractions (P<0.001 for all) despite comparable end-diastolic volumes, ruling out pre-load differences. Patients had markedly increased end-diastolic LV segmental thickness in the basal (P<0.001), mid-LV (P<0.001) and apical (P<0.001) short axis planes associated with a trend towards increased LV mass. However, only one patient had LV hypertrophy according to the conventional CMR threshold. Treatment of hypercortisolism improved ventricular and atrial systolic performance, as reflected by a 15% increase in LV ejection fraction (P=0.029) and 45% increase in LA ejection fraction (P<0.001) with a trend for an 11% increase in RV ejection fraction. The end-diastolic segmental thickness decreased by 37% in the basal (P<0.001), 34% in the mid-LV (P<0.001) and 35% in the apical (P<0.001) short axis planes. LV mass index and end-diastolic LV mass/volume ratio decreased by 17% (P<0.001) and 10% (P=0.002) after treatment, pointing to more excentric LV geometry. The treatment-related decrease in LV mass was independently associated with changes in glucose metabolism (r²=0.82, P<0.001) and BMI (r²=0.61, P=0.017). Late-gadolinium enhancement was absent in all patients.

Conclusion Cushing’s syndrome is associated with subclinical biventricular and LA systolic dysfunction that are reversible after treatment in the absence of dense myocardial fibrosis. Despite skeletal muscle atrophy, Cushing’s syndrome patients have increased LV wall thickness and a tend to have increased LV mass, reversible upon normalization of cortisol excess.

Nothing to Disclose: PK, AR, CR, SS, JY, EM, PC

14742 9.0000 SAT-0826 A Cardiac Structure and Function in Cushing's Syndrome Assessed By Cardiac Magnetic Resonance Imaging 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0818-0831 4780 1:00:00 PM Hypothalamic-Pituitary-Adrenal Axis 1 - CAH, AI & Cushing's Poster

A Ram Hong^*¹, Jung Hee Kim¹, Yoon Ji Kim¹, Kyeong Seon Park¹, Eun Ky Kim¹, Sang Wan Kim², Chan Soo Shin¹ and Seong Yeon Kim¹
¹Seoul National University College of Medicine, Seoul, Korea, Republic of (South), ²Seoul Metropolitan Government Boramae Medical Center, Seoul, Korea, Republic of (South)

Salivary cortisol has been used as a diagnostic test for Cushing’s syndrome. However, the diagnostic performance of morning salivary cortisol for adrenal insufficiency was less established. Furthermore, ACTH-stimulated salivary cortisol has rarely been evaluated in identifying adrenal insufficiency. We aimed to investigate the diagnostic utility of morning basal and ACTH-stimulated salivary cortisol in assessing adrenal insufficiency in Korean adults.

We prospectively included 120 subjects (female, n=70) in Seoul National University Hospital, who was subjected to the short Synacthen test (SST) to identify adrenal insufficiency. A peak serum cortisol level below 18 μg/dl during the SST was used to diagnose adrenal insufficiency. Saliva was collected by chewing a cotton swab and saliva-collecting device. Salivary cortisol levels were measured using an enzyme immunoassay kit. Serum and saliva sampling were taking from 0800 h to 0900 h in the morning.

Thirty-five patients were diagnosed with hypocortisolism according to the SST results. Age, gender, BMI, serum albumin, and serum creatinine were not different in between normal and adrenal insufficiency group. Basal and peak salivary cortisol levels were positively correlated with basal (r=0.538) and peak serum cortisol levels (r=0.750), respectively (all P <0.001). Basal salivary cortisol level in adrenal insufficiency group was significantly lower than that in normal group (0.130 ± 0.288 vs. 0.260 ± 0.195 μg/dl, P <0.001). Receiver-operating characteristic (ROC) analysis allowed that the cutoff levels with at least 95% sensitivity and specificity were 0.378 and 0.068 μg/dl, respectively (area under the curve (AUC), 0.815). The optimal cutoff value of peak salivary cortisol was 0.477 μg/dl (sensitivity, 91.8%; specificity, 94.3%; AUC, 0.960). Subjects with peak salivary cortisol level above 0.477 μg/dl but peak serum cortisol level below18 μg/dl (n=2) had lower serum albumin levels compared with those with concordant response. In addition, subjects with peak salivary cortisol level below 0.477 μg/dl but peak serum cortisol level above 18 μg/dl (n=7) had higher serum creatinine levels than those with concordant response.

Morning basal salivary cortisol measurement can be used as a noninvasive screening tool in assessing adrenal insufficiency. The diagnostic performance of peak salivary cortisol measurement after the SST was comparable with serum cortisol measurement.

Nothing to Disclose: ARH, JHK, YJK, KSP, EKK, SWK, CSS, SYK

16589 10.0000 SAT-0827 A Diagnostic Utility of Morning Basal and Stimulated Salivary Cortisol in Assessing Adrenal Insufficiency in Korean Adults 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0818-0831 4780 1:00:00 PM Hypothalamic-Pituitary-Adrenal Axis 1 - CAH, AI & Cushing's Poster

Luciane Carneiro de Carvalho^*¹, Regina Martin Matsunaga¹, Aline Machado Zamboni¹, Elaine M F Costa¹, Sorahia Domenice¹, Rosana Barbosa Silva¹, Margaret de Castro², Livia M Mermejo², Fernanda B. Coeli-Lacchini³, Rosana Quezado⁴, Virginia Ribeiro Teixeira⁴, Fabricia Torres Gonçalves⁵, Alexandre Jose Faria Carrilho⁶, Kenny Yelena Del Toro Camargo⁷, Gabriela Paula Finkielstain⁸, Ignacio Bergada⁹, Giselle Fernandes Taboada¹⁰ and Berenice Bilharinho Mendonça¹
¹University of São Paulo, Hospital das Clinicas, São Paulo, Brazil, ²University of São Paulo, Faculdade de Medicina de Ribeirão Preto, Ribeirão Preto, Brazil, ³Ribeirao Preto Medical School - University of Sao Paulo, Ribeirao Preto, Brazil, ⁴Federal University of Ceara, Fortaleza, Brazil, ⁵Federal University of Uberlândia, Uberlândia, Brazil, ⁶State University of Londrina, Londrina, Brazil, ⁷Unidad Médica Villa Country, Barranquilla, Colombia, ⁸Centro de Investigaciones Endocrinológicas (CEDIE); Hospital de Niños Dr. Ricardo Gutiérrez, Buenos Aires, Argentina, ⁹Centro de Investigaciones Endocrinológicas Dr. César Bergadá, Buenos Aires, Argentina, ¹⁰Federal University Fluminense, Niteroi, Brazil

Congenital adrenal hyperplasia due to P450c17 deficiency is a rarely reported in 46,XX patients. Report the clinical, laboratory, genetic and ovarian imaging in 46,XX patients. We evaluated 18 Brazilian patients belonging to 12 families, and reviewed 10 cohorts with deficiency of P450c17 activity, 7 due to CYP17 defects and 3 due to POR defects, 46 patients were 46,XX, and 13 patients had complete clinical, laboratory, genetic and ovarian evaluation. From our cases, most patients had primary amenorrhea (83%) and 17% had secondary amenorrhea; 89% of the patients had blood hypertension at diagnosis. The ultrasound showed an increase of at least one of the ovaries in 75% of the patients before treatment and ovarian macrocists in 56%; three of them had previous surgery for twisting or ovarian rupture. The patients were treated with dexamethasone, estrogen and progesterone with ovarian volume reduction. Around 80% of patients in the literature also showed ovarian enlargement. We observed a high incidence of emotional disorders such as depression and anxiety (13/18) in our cohort but no reports were found in the literature. All patients showed basal elevated basal LH and progesterone levels, and decreased androgen levels. In our cases, the molecular study showed that 17 patients have inactivating mutation in the CYP17 gene and 1 in POR gene. Two novel mutations were identified in CYP17 gene, the p.R362H in exon 6 and p.G478S in exon 8. The most prevalent mutation in CYP17 was p.W406R, followed by p.P428L. In cases of literature, the clinical and laboratory data are similar to those of our patients, although psychological disorders were not reported. In literature review the patients came from the 5 continents and there was a predominance of Chinese and Brazilian with defects in CYP17 whereas defects in POR were most reported in European and American subjects. Mutations in the CYP17 found in Brazilian patients differ from those found in most patients already reported, but the mutation found in POR gene in one of our patient (p.A287P) is the most prevalent in the literature. In this review on thirty-one 46,XX patients with deficiency of P450c17 activity, we emphasize the importance of basal progesterone measurement for this diagnosis and the high prevalence of ovarian macrocists with risk of twisting and of psychiatric disorders.

Nothing to Disclose: LCD, RMM, AMZ, EMFC, SD, RBS, MD, LMM, FBC, RQ, VRT, FTG, AJFC, KYDTC, GPF, IB, GFT, BBM

15896 11.0000 SAT-0828 A Phenotype and Genetic Aspects of 46,XX Patients with Congenital Dysfunction of the P450c17 Activity 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0818-0831 4780 1:00:00 PM Hypothalamic-Pituitary-Adrenal Axis 1 - CAH, AI & Cushing's Poster

Mary Ellen Vajravelu^*¹, Jared Tobolski², Evanette Burrows³, Marianne Chilutti³, Vaneeta Bamba⁴, Steven Matthew Willi¹ and Shana E. McCormack⁴
¹Children's Hospital of Philadelphia, Philadelphia, PA, ²Drexel University College of Medicine, Philadelphia, PA, ³Children's Hospital of Philadelphia, ⁴The Children's Hospital of Philadelphia, Philadelphia, PA

Background: Corticotropin-releasing Hormone (CRH) stimulation testing is used to evaluate suspected adrenocorticotropic hormone (ACTH) deficiency in at-risk children, but the clinical factors that determine maximal cortisol response are not known. Previous studies have not systematically evaluated the effect of age and body surface area (BSA), particularly in young children (age < 6 years).

Objective: To determine the effect of age and BSA on peak cortisol response to CRH in children referred for clinical testing.

Subjects/Setting: 288 children, ages 30 days – 18 years, undergoing their first outpatient CRH stimulation testing at a tertiary referral center, 2007-2013

Design/Methods: Retrospective observational study. All subjects received 1mcg/kg corticorelin per testing protocol. Bioinformatics-based extraction of clinical and laboratory values, as well as manual record review, were performed to abstract variables of interest. Univariate and multivariate regression analyses were performed.

Results: Subjects were 70% male, with mean age (+/- SD) 8.8 +/- 4.6 years (154 days-17.8 years), 76% pre-pubertal. Peak cortisol was 21.2 mcg/dl +/- 8.0; 24% were <18 mcg/dL. Indications for testing included: short stature with concern for GH deficiency, divided into those found to be GH sufficient >10 ng/mL (n=39, group 1) or insufficient (n=38, group 2), anatomic and/or syndromic risk for pituitary insufficiency (n=62, group 3), neoplasm with treatments increasing risk for cortisol deficiency (n=56, group 4), previous steroid exposure (n=58, group 5), known hypopituitarism (n=23, group 6), and other (n=12, group 7). Subjects with incomplete anthropometric data (documentation within 3 months of testing) were excluded from subsequent analyses (n=31). Among all subjects with available data, peak cortisol was inversely correlated with age (r=-0.15, p=0.01) and BSA (r=-0.21, p=0.0009). The association between peak cortisol and BSA held in each of groups 1 and 3, with similar trends in groups 2 and 4 (r=-0.3,p=0.06; r=-0.22, p=0.11). In groups 1-4, peak cortisol was more likely to be >=18 mcg/dL (88%) than in groups 5 and 6 (43%; p<0.001). Multivariate regression analysis was performed to determine the independence of these physiologic effects, excluding groups 5 and 6 due to high failure rates (66% and 37%, respectively). When both age and BSA were included in the model, BSA remained negatively correlated with peak cortisol (p<0.001); after accounting for BSA, age was positively associated with peak cortisol (p=0.004). These results held even after sex, pubertal status, and indication for testing were included in the model.

Conclusions: Despite the use of a weight-based dosing protocol, peak cortisol response to CRH is independently negatively associated with BSA in children referred for clinical testing. The clinical implications of this finding will be the focus of additional study.

Nothing to Disclose: MEV, JT, EB, MC, VB, SMW, SEM

12144 12.0000 SAT-0829 A Peak Cortisol Response to Corticotropin-Releasing Hormone Is Related to Age and Size in Children Referred for Clinical Testing 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0818-0831 4780 1:00:00 PM Hypothalamic-Pituitary-Adrenal Axis 1 - CAH, AI & Cushing's Poster

Maya Beth Lodish^*¹, Yasaman Ardeshirpour², Ali Afshari², Evgenia Gourgari³, Margaret Farmar Keil⁴, Elena Belyavskaya⁴, Charalampos Lyssikatos⁵, Victor Chernomordik⁶, Amir Gandjbakhche⁶ and Constantine A Stratakis⁷
¹National Institutes of Health, Bethesda, MD, ²NIH, ³Georgetown University Hospital, Washington, DC, ⁴Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH)., Bethesda, MD, ⁵Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH), Bethesda, MD, ⁶NICHD, Bethesda, MD, ⁷National Institutes of Health (NIH), Bethesda, MD

In this study, we used non-invasive multi-spectral imaging (MSI) before and after surgery (transphenoidal surgery (TSS), bilateral adrenalectomy, or surgical resection of pulmonary carcinoid) to quantify changes of facial plethora in patients with Cushing’s syndrome (CS) as an early assessment of their cure. As a follow-up to our previous pilot study in 5 patients, we have expanded the use of this technology to a larger cohort. Twenty patients with CS (14 F, mean age 19.8 ± 12.3) were studied. Among these patients, 17 patients had ACTH secreting pituitary tumors (Cushing Disease, CD), 2 had ACTH-independent adrenocortical tumors and one had an ectopic ACTH-secreting pulmonary carcinoid. In the single case that required two surgical procedures to achieve cure of CD, MSI data after the first non-curative TSS considered “pre surgery” and the MSI after the re-operation was used to assess correlation with cure. Two patients with CD were excluded from the study, one secondary to severe facial acne and the second due to fever on the day of imaging. MSI was performed on the right cheek of the patients prior to TSS and a mean of 5.6 ± 3.9 days after surgery using 4 wavelengths (700,750, 800 and 850nm) with a Near Infrared CCD based multispectral imaging system. Spectral analysis, using the two-layer skin model along with a curvature correction algorithm, was applied to quantify the fraction of blood volume of the tissue (2). Data are presented as mean ± SD. A two-tailed Fisher’s exact test was used to examine the association between cure of CS and change in MSI data. Fourteen of the 18 patients were surgically cured of disease with post-operative measurements of plasma cortisol < 2 μg/dl as previously described (1). Pre-op AM cortisol values of patients were 23.9 ± 18.8 μg/dL. The post-op AM cortisol in the 14 cured patients reduced to 1.33 ± 0.62 μg/dL, whereas the AM cortisol level in the non-cured patients is decreased only to11.9 ± 2.9 μg/dL. In all patients, who entered remission for their CS, the mean value of tissue blood content (volume percentage), observed at the patient right cheek, is significantly reduced, after surgery, relative to one, measured before treatment, i.e., 17 ± 2.6 versus 14.7 ± 3 %. To the contrary, in all five patients, who were not surgically cured, blood volume percentage at their right cheeks, increased from pre- to post-operative measurements, %(18.6 ± 3.9 to 21.6 ± 4.3), p-value ≤0.0001. Clinical data, obtained from 18 patients, indicate that a decrease in facial plethora after surgery, evidenced by blood volume decrease, is well correlated with cure of CS. Thus, discussed novel technology is a promising early marker of cure of patients with Cushing’s syndrome after surgery, along with biochemical data

Nothing to Disclose: MBL, YA, AA, EG, MFK, EB, CL, VC, AG, CAS

12888 13.0000 SAT-0830 A Non-Invasive Multi-Spectral Imaging for Pre and Post-Operative Assessment of Patients with Cushing's Syndrome 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0818-0831 4780 1:00:00 PM Hypothalamic-Pituitary-Adrenal Axis 1 - CAH, AI & Cushing's Poster

Oskar Ragnarsson^*¹, Anders F. Mattsson², John Patrick Monson³, Helena Filipsson Nyström⁴, Gudmundur Johannsson¹, Ann-Charlotte Akerblad⁵ and Maria Koltowska Häggström⁶
¹Sahlgrenska University Hospital, Gothenburg, Sweden, ²Pfizer Health AB, Sollentuna, Sweden, ³London Clinic Ctr for Endo, London, United Kingdom, ⁴Sahlgrenska University Hospital, Gothenburg, ⁵Endocrine Care, Pfizer Health AB, Sollentuna, Sweden, ⁶Uppsala University, Uppsala, Sweden

Introduction

Adult hypopituitary patients have impaired quality of life (QoL). This study analyzed: a) the impact of glucocorticoid (GC) replacement on QoL, b) the impact of GC doses on QoL, and c) the influence of ACTH insufficiency on the effect of growth hormone (GH) replacement on QoL. The main hypothesis was that ACTH insufficient patients experience a dose dependent deterioration in QoL.

Patients and study design

This was a retrospective analysis of data from 2,737 hypopituitary patients obtained from KIMS (Pfizer International Metabolic Database). All patients had GH deficiency. Data at baseline and after one year of GH replacement were analyzed.

Methods

Data on background characteristics were obtained from KIMS as reported by participating centres. QoL was assessed by the QoL-assessment of GH deficient adults (QoL-AGHDA). Higher scores (up to 25) denote poorer QoL. Total score and 5 dimensions (memory, tiredness, tenseness, social isolation and self-confidence) were analyzed with linear regression. P-values <0.05 were considered as statistically significant.

Results

Nine hundred eighty seven patients (36%) were ACTH sufficient and 1,750 (64%) were ACTH insufficient. The mean ± SD hydrocortisone (HC) dose was 22.3 ± 8.7 mg (median 20.0). After adjustment for age, gender, weight, etiology, KIMS entry year, country, onset of pituitary disease (childhood versus adult), TSH deficiency and serum IGF-I SDS, no difference in mean QoL score was observed between the ACTH sufficient [11.1 (95% CI 10.6-11.7)] and ACTH insufficient patients [11.0 (95% CI 10.5 – 11.5); P =0.81]. There was an association with increasing HC dose and worse QoL. Patients on HC ≤10 mg had the best and patients with doses ≥25 mg the poorest QoL. There were also statistically significant associations between increasing HC dose and problems with tiredness, tenseness and social isolation. At one year on GH replacement, QoL improved at a similar rate in ACTH sufficient and insufficient patients. No association was observed between HC dose and the improvement in QoL.

Conclusion

Hypopituitary patients with untreated GH deficiency on GC replacement have similar QoL as ACTH sufficient patients, however it should be noted that QoL-AGHDA is a GHD-sensitive measure. In patients with ACTH insufficiency, an adverse dose-dependent association with QoL was observed that may be explained by supraphysiological GC exposure or an empirical increase in GC dose in patients with negatively affected QoL.

Disclosure: OR: Speaker, Pfizer, Inc.. AFM: Employee, Pfizer, Inc.. GJ: Speaker, Pfizer, Inc., Speaker, Novo Nordisk, Speaker, Otsuka, Consultant, Viropharma, Consultant, Astra Zeneca. ACA: Employee, Pfizer, Inc.. MK: Employee, Pfizer, Inc.. Nothing to Disclose: JPM, HF

12975 14.0000 SAT-0831 A Glucocorticoid Replacement Therapy and Quality of Life: A Study in 2,737 Hypopituitary Patients 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Adrenal HPA Axis Saturday, June 21st 3:00:00 PM SAT 0818-0831 4780 1:00:00 PM Hypothalamic-Pituitary-Adrenal Axis 1 - CAH, AI & Cushing's Poster

Karin Amrein^*¹, Christian Schnedl², Alexander Holl³, Regina Riedl⁴, Kenneth B. Christopher⁵, Tadeja Urbanic-Purkart⁴, Andrea Berghold⁴, Thomas R Pieber⁶, Christoph Pachler⁴, Andreas Waltensdorfer⁴, Andreas Münch⁴, Tatjana Stojakovic⁴, Egbert Bisping⁴, Wolfgang Toller⁴ and Harald Dobnig⁷
¹Medical University of Graz, Graz, Austria, ²Medical University Graz, Graz, Austria, ³Department of Neurology, ⁴Medical University of Graz, ⁵Renal Division, Brigham and Women's Hospital, ⁶Medical University of Graz, Austria, ⁷Schilddruesen|Endokrinologie|Institut, Graz, Austria

Introduction

Observational studies have shown an association between low vitamin D status and poor outcomes including increased mortality in critically ill patients. Whether this relationship is causal remains unclear to date.

Methods

In this randomized, double-blind, placebo-controlled single-center trial, five intensive care units assigned a mixed medical and surgical population of 480 adult patients with vitamin D deficiency (≤20 ng/ml) to either vitamin D or placebo. Vitamin D3 was given orally or via nasogastric tube once at a dose of 540,000 IU followed by 5 monthly maintenance doses of 90,000 IU.

Results

Vitamin D3 administration led to an immediate and sustained rise of mean 25-hydroxyvitamin D levels in the interventional vs. the placebo group: 13.0±4.0 vs.13.1±4.3 (baseline); 35.5±20.6 vs. 14.5±5.1 ng/ml (day 7; P<0.001). The primary endpoint, length of hospital stay, was comparable between groups (median 20.1 vs. 19.3 days, P=0.98). Hospital- and 6-month mortality rates in contrast, tended to be lower in the vitamin D group [HR 0.81 (95%CI 0.58-1.11), P=0.18 and HR 0.78 (0.58-1.04), P=0.09, respectively]. In the predefined subgroup analysis of patients with severe vitamin D deficiency (≤12 ng/ml, n=200), hospital- and 6-month mortality rates were significantly reduced [HR 0.56 (95%CI 0.35-0.90), P=0.01 and HR 0.60 (0.39-0.93), P=0.02, respectively]. At the 6-month follow up, patients with severe vitamin D deficiency at baseline did not show any significant changes in investigated parameters compared to the placebo group whereas patients with less severe vitamin D deficiency (baseline 25(OH)D level >12 and ≤20 ng/ml) showed significantly improved grip strength of the right hand as well as a higher physical component summary score of the SF-12 questionnaire. Adverse event rates including hypercalcemia, falls and fractures were similar in both groups.

Conclusions

High-dose vitamin D3 in critically ill patients did not change the length of hospital stay but tended to decrease hospital- and 6-month mortality. In patients with severe vitamin D deficiency, vitamin D3 reduced all cause mortality, as compared with placebo. (ClinicalTrials.gov number: NCT01130181)

Nothing to Disclose: KA, CS, AH, RR, KBC, TU, AB, TRP, CP, AW, AM, TS, EB, WT, HD

12989 1.0000 SAT-0229 A Correction of Vitamin D Deficiency in Critically Ill Patients: A Randomized Placebo-Controlled Trial 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 21st 3:00:00 PM SAT 0229-0266 4784 1:00:00 PM Vitamin D Metabolism & Action Poster

Shariq Rashid Masoodi^*¹, Khalid J Farooqui², Abdul Hamid Zargar³, Arshad Iqbal Wani⁴, Mir Iftikhar Bashir⁴, Syed Mudassar⁴ and Mushtaq Siddiqi⁵
¹Sher-i-Kashmir Institute of Medical Sciences, Srinagar, Jammu and Kashmir, India, ²Sher-i-Kashmir Institute of Medical Sciences, ³Center for Diabetes & Endocrine Care, Srinagar, India, ⁴Sher-i-Kashmir Institute of Medical Sciences, Srinagar, India, ⁵Sher 1 Kashmir Institute of Medi, Srinagar, J&K, India

BACKGROUND: There is a worldwide epidemic of Vitamin D deficiency with Kashmir Valley (Northern India) being no exception. About 83% of apparently healthy adults in the Kashmir valley are reported to be vitamin D deficient despite adequate exposure to sunlight and adequate consumption of dairy products. A positive correlation between vitamin D deficiency, glucose intolerance and impaired insulin secretion has been demonstrated in subjects with PCOS. Serum levels of 25-hydroxyvitamin D [25(OH) D] are inversely correlated with insulin resistance (IR) in subjects with PCOS. Apart from vitamin D deficiency, PCOS is also very common in Kashmir Valley.

OBJECTIVE: To determine the association between vitamin D status and components of Metabolic Syndrome (Met-S) in women with PCOS and the effects of vitamin D supplementation on IR parameters.

STUDY DESIGN: In this randomized double-blind, placebo-controlled trial, 123 eligible enrolled subjects were randomly divided into three groups of 41 each: Group I: Active Vitamin D3 Tablets (1000U/tab), twice a day plus placebo sachets; Group II: Placebo Tablets plus Active Vitamin D3 sachets (60,000U/sachet); and Group III: Tablets: Placebo; sachets: Placebo. Effects of Vitamin D supplementation, 2,000 IU per day (Group I) or 60,000 IU per month (Group II) were compared with those of placebo (Group III) on measures of IR at 3 months.

METHODS: The fasting blood glucose, insulin, 25(OH) D, HOMA-IR and QUICKIE were measured at baseline and after treatment.

RESULTS:Hypovitaminosis D was present in 115 out of 123 PCOS women (93.4%). Of the 115 subjects with hypovitaminosis D, 98 (79.7%) had vitamin D deficiency (< 20 ng/ml). Using the IDF criteria Met-S was identified in 30 subjects (24.4%). Baseline 25(OH) D did not show any correlation with components of Met-S. The mean 25(OH) D levels increased from 13.76±10.60 ng/dl to 25.71±15.04 ng/dl after intervention (86.84% change).Post Vitamin D supplementation, 48(39%) subjects achieved vitamin D sufficiency as compared to 8(6.5%) at baseline. Similarly the number of subjects with severe, moderate and mild vitamin deficiency before intervention i.e. 55(44.7%), 43(35%) and 17(13.8%) respectively improved to 25(20.3%), 19(15.4%) and 31(25.2%) after intervention. The mean HOMAIR did not show any significant change overall (2.30±4.13 vs. 3.96±6.82), but in 53 subjects in whom Vitamin D levels increased by two-fold with a post-supplementation 25(OH)D level of ≥20 ng/ml, a statistically significant improvement in HOMA-IR was observed (P= 0.025).

CONCLUSIONS: Vitamin D3 supplementation equivalent to 2000 IU daily for 3 months normalises vitamin D status in around half of the predominantly Vitamin D deficient subjects with PCOS, but improves insulin resistance in only those with significant increase in vitamin D levels (two-fold increase with a post-supplementation 25(OH)D level of 20 ng/ml or more)

Nothing to Disclose: SRM, KJF, AHZ, AIW, MIB, SM, MS

16938 2.0000 SAT-0230 A Polycystic Ovary Syndrome and Vitamin D Status - Impact of Vitamin D Supplementation on Insulin Resistance 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 21st 3:00:00 PM SAT 0229-0266 4784 1:00:00 PM Vitamin D Metabolism & Action Poster

Kyu-jin Kim^*¹, Bo-Yeon Kim¹, Chan-Hee Jung¹, Chul-Hee Kim², Sung-koo Kang² and Jioh Mok¹
¹Soonchunhyang University School of Medicine, Bucheon hospital, Bucheon, Korea, Republic of (South), ²Soonchunhyang University Bucheon Hospital, Soonchunhyang University College of Medicine, Bucheon city, Gyeonggi-do, Korea, Republic of (South)

Existing evidence suggests that vitamin D deficiency may contribute to the development of atherosclerosis. The aim of our cross-sectional study was to assess the association of circulating vitamin D metabolite concentrations with insulin resistance and subclinical atherosclerosis in patients with T2DM.

Two hundred thirty seven T2DM patients who were measured serum 25-hydroxyvitamin D(25(OH)D) at diabetes clinic, from January 2009 to September 2013 (89 males and 148 females, mean age 60 years, mean duration of DM 8.5 years) were enrolled. Viatamin D status was categorized into three groups, deficiency [25(OH)D < 10ng/mL], insufficiency [10ng/mL ≤ 25(OH)D < 20ng/mL] and sufficiency [20ng/mL ≤ 25(OH)D]. Then, basic characteristics, insulin resistance, subclinical atherosclerosis marker and diabetic microangio pathies were analyzed by intergroup differences according to vitamin D status. Carotid atherosclerosis was assessed by B-mode ultrasonography. In addition, the patients were evaluated diabetic microangiopathies and baPWV and ABI to evaluate atherosclerotic burden.

The prevalence of deficiency (<10 ng/ml) and sufficiency (≥20 ng/ml) of vitamin D in T2DM patients was 25.3% and 24.1%, respectively. In intergroup analysis of variance, vitamin D deficiency had positive correlation with hsCRP level (p=0.025), fasting insulin level (p=0.045), c-peptide level (p=0.004) and HOMA-IR level (p=0.021). And, there was significant difference in cIMT mean values by degrees(0.66±0.17 vs. 0.59±0.15 vs. 0.540± 0.16 mm; p=0.044) of subclinical atherosclerosis markers between groups. But, there was no significant difference in prevalence of retinopathy and nephropathy. In relation analysis of vitamin D status with insulin resistance, subclinical atherosclerosis and microangiopathies, vitamine D deficiency was related to fasting c-peptide level (r=-0.174, p=0.011) and HOMA-IR level(r=-0.144, p=0.047).

In this present study, vitamin D deficiency were associated with insulin resistance, inflammation and atherosclerosis. But, there was not a correlation between vitamin D deficiency and diabetic microangiopathies

Nothing to Disclose: KJK, BYK, CHJ, CHK, SKK, JM

13190 3.0000 SAT-0231 A Association Vitamin D Status with Subclinical Atherosclerosis in Patients with Type 2 Diabetes Mellitus 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 21st 3:00:00 PM SAT 0229-0266 4784 1:00:00 PM Vitamin D Metabolism & Action Poster

Asma Javed^*¹, Adrian Vella¹, Prabhakara P Balagopal², Philip Fischer¹, Amy Weaver¹, Francesca Piccinini³, Chiara Dalla Man³, Claudio Cobelli³, Paula Giesler¹, Jeanette Laugen¹ and Seema Kumar¹
¹Mayo Clinic, Rochester, MN, ²Res Div, Jacksonville, FL, ³University of Padova, Padova, Italy

Effect of Vitamin D supplementation on Insulin Sensitivity and Beta Cell Function in Obese adolescents

Background: Despite the strong relationship between Vitamin D deficiency and obesity, ambiguity exists on its potential relationship with insulin sensitivity and β-cell function in obese adolescents.

Objective: To determine if Vitamin D supplementation has any impact on insulin sensitivity (S_i)and pancreatic β-cell function as assessed by oral glucose tolerance test (OGTT) in obese adolescents.

Study Design and Methods: The study design was a 12 week double blinded, randomized, comparison of the effect of vitamin D supplementation on S_i and β-cell function (Disposition Index or DI) in obese, Caucasian adolescents (BMI > 95^th percentile) aged 12-18 years. The subjects were randomized to receive either 400 IU/ day of Vitamin D3 supplementation (n=23) or 2000 IU/day (n=24) for 12 weeks. Each subject underwent a 7-sample 75 g oral glucose tolerance test (OGTT) with glucose, insulin and C peptide used to calculate S_iand DI using the oral minimal model before and after Vitamin D supplementation.

Results: A total of 47 subjects (age: 15.0±1.9 years) with mean BMI percentile 98.1±1.2 were randomized to 2000 IU/day (n=24) or to 400 IU/day (n=23) of Vitamin D supplementation. Mean 25(OH)D concentration was 24.0±8.1 ng/mL with 37 (78.7%) subjects having 25(OH)D level less than 30 ng/mL. There was no correlation between plasma 25(OH)D levels and insulin sensitivity or DI in the subjects at baseline. There was a modest but statistically significant increase in the 25(OH)D level in the 2000 IU/day arm (3.1 ± 6.5 ng/mL, p=0.04) but not in the 400 IU/day group (0.8 ± 4.2 ng/mL, p=0.40). There was no significant change in the S_i in either group, (-4.97 ± 15.94, p-value= 0.28) in the 2000 IU arm and (0.42 ± 4.29, p = 0.81) in the 400 IU group. The change in S_idid not differ between groups (p-value 0.12).

There was no significant change in DI in either group (2000 IU: 1865.2 ± 6833.7, p-value 0.22 and 400 IU: 274 ± 824.9, p-value 0.44). The change in DI also did not differ between groups (p-value 0.2).

Conclusions: Our study explores the relationship between Vitamin D status and dynamic measures of insulin sensitivity and β-cell function in obese Caucasian adolescents. Vitamin D supplementation at 2000 IU/day and 400 IU/day for 12 weeks in obese adolescents did not impact dynamic measures of insulin sensitivity and β-cell function. Given the modest increase in 25 (OH) D concentrations with these supplementation doses, further studies with higher doses of vitamin D and longer duration are warranted.

Disclosure: AV: Advisory Group Member, Sanofi. Nothing to Disclose: AJ, PPB, PF, AW, FP, CD, CC, PG, JL, SK

13291 4.0000 SAT-0232 A Effects of Vitamin D Supplementation on Insulin Sensitivity and Beta Cell Function in Obese Adolescents 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 21st 3:00:00 PM SAT 0229-0266 4784 1:00:00 PM Vitamin D Metabolism & Action Poster

Angela Mojica^*¹, Kathleen Bethin² and Lucy D Mastrandrea¹
¹University at Buffalo, Buffalo, NY, ²University at Buffalo School of Medicine and Biomedical Sciences, Buffalo, NY

Higher rates of vitamin D deficiency have been found among obese children and adolescents. Several studies have shown an inverse relationship between 25(OH) D levels and fasting glucose levels as well as insulin resistance (1) (2). The outcome of treating vitamin D deficiency on glucose homeostasis in children hasn’t been examined in large randomized prospective controlled studies. The aims of this study are to examine the relationship between 25-OH D level and insulin resistance in obese/overweight children and the effect of vitamin D supplementation on insulin sensitivity in subjects with low vitamin D by measuring changes in Homeostasis Model of Assessment - Insulin Resistance (HOMA-IR). We also sought to compare the magnitude of this effect in relation to race and baseline vitamin D level (deficient vs. insufficient). Using a prospective clinical trial design, we recorded BMI, vitamin D intake, acanthosis nigricans, fasting glucose and insulin, and 25-OH D levels in obese/overweight children (age 10 – 18yr) seen at a subspecialty endocrine clinic and at a General Pediatric practice from January 2013 to January 2014. These assessments were done at baseline and 6 months after vitamin D supplementation per the Endocrine Society Guidelines in subjects with low vitamin D levels. A total of 52 subjects (31 female) were recruited for the study. 92% of the subjects had 25-OH D levels below 30 ng/mL (52% had 25-OH D below 20 ng/mL and 40% 21-29 ng/mL). African American subjects were more likely to be diagnosed with Vit D deficiency (25OHD < 20 ng/ml; p = 0.01). Although there were no significant differences in HOMA-IR among the vitamin D deficient and insufficient groups, 51% and 43% of the subjects had HOMA-IR levels above 2.5 and 3.16 respectively. Vitamin D levels trended toward a negative correlation with insulin levels (r= -0.26; p=0.06), but this correlation was not significant for fasting glucose (r=-0.08; p=0.58) or HOMA-IR (r=-0.22; p=0.11). Odds ratio of having insulin resistance based on HOMA-IR >2.5 and >3.16 was 0.78 (p=0.0025) and 0.84 (p=0.0094) respectively. Analysis of 6 month follow-up data for 12 subjects demonstrated that BMI is a stronger predictor of HOMA-IR change than vitamin D level. Subjects with 25-OH D below 30 ng/mL tend to have elevated fasting insulin; however it is unclear whether vitamin D supplementation alone improves insulin resistance.

Nothing to Disclose: AM, KB, LDM

11123 5.0000 SAT-0233 A Effect of Vitamin D Deficiency/Insufficiency Treatment on Insulin Resistance in Obese and Overweight Children 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 21st 3:00:00 PM SAT 0229-0266 4784 1:00:00 PM Vitamin D Metabolism & Action Poster

Mansi Mehta^*¹, Barbara Tommasulo¹, Renee Pekmezaris², Andrzej Kozikowski², Meredith Akerman¹, Nooshi Karim¹, Judy Beizer², Sidhu Jasdeep¹, Stuart A Weinerman² and Gisele Wolf-klein¹
¹North Shore LIJ Health System, Manhasset, NY, ²North Shore LIJ Health System

Background: Older adults and particularly nursing home residents are known to be at risk for Vitamin D deficiency. Vitamin D deficiency has been implicated as a contributing factor in a multiplicity of diseases including Type 2 diabetes mellitus (DM). We proposed to assess the association of vitamin D deficiency and insufficiency and HbA1C (Haemoglobin A1C) levels in elderly patients with type 2 DM.

Methods: A retrospective chart review study was conducted in a nursing home, using electronic medical records of all patients over the age of 55 from 1/1/07 to 12/31/12. All diabetic residents met American Diabetes Association criteria for diagnosis of type 2 DM. Vitamin D levels, alkaline phosphatase levels, phosphorus, parathyroid hormone levels, calcium and albumin levels were recorded as well as demographics, co-morbidities, mobility, medications and supplements. Vitamin D deficiency was defined as <20 ng/dl, insufficiency as 20-29 ng/dl, and sufficiency >= 30 ng/dl. The Kruskal-Wallis test was used to compare the three groups for continuous variables. The chi-square test or Fisher’s exact test, as deemed appropriate, was used to compare the three groups for categorical variables.

Results: In the 120 patients analyzed, average age was 80 years (range: 56-97), with 71% female and 92% Caucasian. Average body weight was 73 kg (range: 40-149 kg) with BMI of 26 (range: 17-46). In this nursing home population, 37% of subjects were vitamin D deficient, 35% were insufficient and only 28% were sufficient. As expected, there was a significant difference among the three Vitamin D groups for alkaline phosphatase levels (p<0.002), with those subjects that were vitamin D deficient having the highest median levels. However, none of the other variables collected, namely subject age, weight, BMI, serum albumin, calcium levels, phosphorus, HbA1C levels and daily calcium and Vitamin D supplements, were significantly associated with Vitamin D serum levels.

Conclusions: Our study does not support the association between the severity of vitamin D deficiency or insufficiency and elevated HbA1C in elderly Type 2 diabetic patients residing in long term care institutions.

Nothing to Disclose: MM, BT, RP, AK, MA, NK, JB, SJ, SAW, GW

11261 6.0000 SAT-0234 A Association Between Vitamin D Deficiency and HbA1C Levels in Elderly Patients with Type 2 Diabetes in Long Term Care Institutions 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 21st 3:00:00 PM SAT 0229-0266 4784 1:00:00 PM Vitamin D Metabolism & Action Poster

Hwa Young Kim^*¹, Young Ah Lee¹, Jung Hae Woon Jung¹, Lee Gyung Min Lee¹, Chung Seung Joon Chung¹, Jieun Lee², Yoon Ju Young Yoon³, Choong Ho Shin⁴ and Sei Won Yang¹
¹Seoul Natl Univ College of Med, Seoul, Korea, Republic of (South), ²Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea, Republic of (South), ³National Cancer Center, Goyang, Korea, Republic of (South), ⁴Seoul National University Children's Hospital, Seoul, Korea, Republic of (South)

Many studies suggest that vitamin D deficiency is more common in individuals with type 1 diabetes (T1DM) compared to controls, but mechanisms contributing to the vitamin D deficiency in T1DM remains unclear. With increased urinary loss of vitamin D binding protein (VDBP), a major transport protein for vitamin D metabolites in plasma, the risk of vitamin D deficiency may also be increased as the recirculation of 25-hydroxyvitaminD (25-OHD) is impaired. In this study, we examined whether there was an increased urinary loss of VDBP in pediatric T1DM patients without microalbuminuria and examined for a correlation between VDBP and circulating 25-OHD levels. We also aimed to identify risk factors influencing low vitamin D levels in pediatric T1DM.

Subjects with T1DM without microalbuminuria (n=45) and age-matched healthy control subjects (n=29), aged 9–14 yr, residing in Seoul and the Gyeonggi-Do in Korea (37°N) were studied during January and March 2013. Height, weight and pubertal stage were evaluated. The percentage of body fat was measured by bioelectrical impedance analysis (inbody). A questionnaire was used to assess the amount of daylight outdoor activity and vitamin D intake. Serum levels of calcium, phosphorus, intact parathyroid hormone, 25-OHD, 1,25 dihydroxyvitamin D and VDBP, as well as urinary levels of VDBP, microalbumin and creatinine (Cr) were measured. 25-OHD deficiency was defined as ≤20 ng/mL.

T1DM subjects were older and showed lower physical activity compared with the control group. However, the percentage of body fat and amount of vitamin D intake were not different between the two groups. There was no difference in the frequency of vitamin D deficiency between the T1DM (55.6%) and control group (44.8%) during the winter season. Serum 25-OHD and VDBP levels were not different. Whereas the urinary microalbumin to Cr ratio (mACR) was not different, the urinary VDBP to Cr ratio (VDBPCR) in T1DM patients was higher than in the control group (P=0.016). VDBPCR was positively correlated with mACR in both T1DM subjects and control groups (P=<0.001). In the univariate analysis, serum 25-OHD levels did not correlate with serum VDBP nor urinary VDBPCR. Age (P=0.050) and daylight outdoor hours (P=0.035) positively correlated with 25-OHD levels. Multivariate regression analysis including known risk factors for vitamin D deficiency such as age, gender, percentage of body fat, vitamin D intake, daylight outdoor hours and urinary VDBPCR showed that the factors affecting 25-OHD level in T1DM patients were daylight outdoor hours (β = 2.881, P = 0.009) and vitamin D intake (β = 2.342, P = 0.050).

In pediatric T1DM patients without microalbuminuria, urinary loss of VDBP was increased in proportion to urinary mACR. However, urinary VDBPCR was not correlated with serum 25-OHD levels. The factors associated with 25-OHD level during winter period were daylight outdoor hours and vitamin D intake.

Nothing to Disclose: HYK, YAL, JHWJ, LGML, CSJC, JL, YJYY, CHS, SWY

11293 7.0000 SAT-0235 A A Lack of Association Between Vitamin D-Binding Protein and 25-Hydroxyvitamin D Concentrations in Pediatric Type 1 Diabetes without Microalbuminuria 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 21st 3:00:00 PM SAT 0229-0266 4784 1:00:00 PM Vitamin D Metabolism & Action Poster

Asma Javed^*, Ravinder J. Singh, Aida N Lteif and Seema Kumar
Mayo Clinic, Rochester, MN

BACKGROUND:Vitamin D deficiency is prevalent worldwide, particularly in the obese. In this patient population, higher doses of vitamin D are needed to treat vitamin D deficiency. There is insufficient data on the safety or efficacy of high dose once monthly vitamin D supplementation in obese adolescents. Poor compliance is often a concern in adolescence which makes once monthly dosing regimens a suitable option. We conducted a prospective study to determine the short term safety and efficacy of once monthly 100,000 IU vitamin D3 in obese adolescents.

METHODS: 19 obese adolescents (BMI > 95^thpercentile) aged 13-18 years with vitamin D deficiency or insufficiency (serum 25 (OH)D concentration less than 30 ng/ml) received Vitamin D3 100,000 IU orally once a month for three months. Each subject completed a short calcium questionnaire (SCQ) before and after vitamin D supplementation to assess daily calcium intake. Serum calcium, urine calcium and 25(OH)D levels were measured at baseline and 1, 2 and 3 months after starting vitamin D supplementation. Parathyroid hormone (PTH) levels were obtained at baseline and 3 months. Changes in 25 (OH)D levels and other biochemical variables were assessed using paired t-tests. Linear regression analyses were used to assess associations between change in 25 (OH) D and various biochemical variables.

RESULTS:Mean age was 15.8 ± 1.7 years, mean BMI was 36.1 ± 6.04 kg/m2 and BMI z score was 2.35 ± 0.56. Mean 25(OH)D concentrations increased from 22.4 ± 4.9 to 34.8 ± 6.7 (ng/ml) (P < 0.01). 25(OH)D levels increased to above 30 ng/mL in 15/19 (79%) of subjects by 3 months. Serum calcium increased by a mean of 0.23 ± 0.01 mg/dL (p < 0.01) but none of the patients were noted to have serum calcium above the reference range. Serum phosphorus concentrations did not change. There was no significant change in urine calcium to creatinine ratio following vitamin D supplementation (P = 0.32). There was a significant decline in PTH (pg/ml) (-7.4 ± 0.6 P=0.01) but none of the subjects had a suppressed PTH. There was no change in calcium intake as assessed by SCQ (P = 0.12).

CONCLUSION: In obese adolescents with inadequate levels of 25(OH)D, vitamin D (3) at a dose of 100,000 IU orally once a month for 3 months appears to be effective in achieving 25(OH)D levels above 30 ng/mL. Hypercalcemia, hypercalciuria and PTH suppression were not found in this short term study. Long term studies are warranted to study the safety and efficacy of this regimen in obese adolescents.

Nothing to Disclose: AJ, RJS, ANL, SK

13535 8.0000 SAT-0236 A Short Term Safety and Efficacy of High Dose Vitamin D Supplementation in Obese Adolescents with Vitamin D Deficiency 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 21st 3:00:00 PM SAT 0229-0266 4784 1:00:00 PM Vitamin D Metabolism & Action Poster

Michael Yuri Torchinsky^* and Xu Zhang
University of Mississippi Medical Center, Jackson, MS

Background: Low serum 25-OH vitamin D levels are more common in obese children; however, the clinical significance of these findings has been unclear (1-3).

Objectives: The objective was to determine the prevalence of secondary hyperparathyroidism in obese children with serum 25-OH vitamin D <20 ng/ml.

Methods: We reviewed medical records from the Pediatric Endocrine Clinic over a period of one year and identified forty children aged 3 to 18 years with BMI >95 percentile, who had serum 25-OH vitamin D, intact parathyroid hormone (PTH), serum calcium, phosphorus, albumin, alkaline phosphatase, blood urea nitrogen, creatinine, and urine calcium-to-creatinine ratio measured. Spearman rank correlation coefficient was evaluated to measure association between continuous variables. Fisher’s exact test was used to assess association between categorical variables.

Results: Vitamin D deficiency, defined as serum 25-OH vitamin D level <20 ng/ml, was found in twenty eight out of forty obese children, including six out of eleven children aged 3 to 10 years (54%), and twenty two out of twenty nine children aged 11 to 18 years (76%). The mean 25-OH vitamin D level decreased with age (r=-0.52, p<0.001). Only fifteen out of twenty eight obese children with vitamin D deficiency had secondary hyperparathyroidism, defined as serum PTH >65 pg/ml, including four out of six children aged 3 to 10 years (66%), and eleven out of twenty two children aged 11 to 18 years (50%). Secondary hyperparathyroidism was seen in six out of seven patients with serum 25-OH vitamin D level <15 ng/ml (86%), and only in nine out of twenty one patients with 25-OH vitamin D between 15 and 20 ng/ml (43%). All twelve obese patients with serum 25-OH vitamin D >20 ng/ml had serum PTH within the normal range 10 to 65 ng/ml. There was significant negative association between serum 25-OH vitamin D concentration and incidence of secondary hyperparathyroidism (p<0.001). Urine calcium-to-creatinine ratio inversely correlated with serum PTH levels (r=-0.44, p=0.004).

Conclusions: Obese children with serum 25-OH vitamin D levels <20 ng/ml include patients who have vitamin D deficiency that adversely affects bone metabolism through secondary hyperparathyroidism and those who are vitamin D sufficient based on normal serum PTH and have low 25-OH vitamin D levels presumably due to high volume of distribution of vitamin D in excessive body fat. Measuring serum PTH in conjunction with 25-OH vitamin D may be useful for identifying those obese children who can benefit most from vitamin D replacement. Increase in severity of vitamin D deficiency with age underscores the need for early screening.

Nothing to Disclose: MYT, XZ

11104 9.0000 SAT-0237 A Decreased Serum 25-OH Vitamin D Levels Are Not Always Associated with Secondary Hyperparathyroidism in Obese Children 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 21st 3:00:00 PM SAT 0229-0266 4784 1:00:00 PM Vitamin D Metabolism & Action Poster

Preneet Cheema Brar^*¹, Bonita H Franklin² and Nipapat Visavachaipan³
¹New York University School of Medicine, New York, NY, ²NYU School of Medicine, NY, ³Bumrungrad International Hospital, Thailand

Background: The childhood obesity epidemic had led to an exponential rise in the prevalence of pre diabetes and /or Type 2 diabetes. Vitamin D deficiency results in decreased insulin sensitivity (ability of insulin to promote peripheral glucose uptake) and fasting hyperglycemia has been correlated with Vitamin D deficiency in obese adolescents and children.^1,2

Aims: To study insulin sensitivity and secretory indices derived from an oral glucose tolerance test (OGTT) in obese adolescents with vitamin D deficiency before and immediately after normalization of serum 25(OH) vitamin D levels.

Methods: In a single blinded randomized placebo controlled study (cross over design with planned sample size= 20) obese adolescents with vitamin D deficiency; 25 (OH) vitamin D ≤ 20 ng/dl (50 nmol/L) were recruited. Adolescents were assigned to receive 50,000 IU of ergocalciferol (group A) once weekly or placebo (group B) for 6 weeks. At week 7, subjects were reassigned to receive vitamin D if they were in group B or placebo if they were in Group A. Study subjects had an OGTT and screening labs (25 (OH) Vitamin D, PTH, CMP, calcium, phosphorous and urine calcium/ creatinine ratio) at baseline, week 7 and then again at week 12 on the completion of study.

Indices derived from OGTT were:

1) Whole body sensitivity index (WBISI):10,000/√(fasting glucose(mg/dL)x fasting insulin(uIU/mL)x(mean glucose (mg/dL) x mean insulin (uIU/mL)

2) Insulinogenic index: 30 min insulin- fasting insulin (uIU/mL) ⁄ 30 min glucose- fasting glucose (mg/dL)

Results: To date four subjects have completed the study: Hispanic females (mean ± SD) age 16.1± 1years, BMI 34.5± 4.5. Baseline labs were: HbA1c 5.3± 0.2, fasting glucose 81± 5.1, and fasting insulin 16.5± 5.3uIU/ml. PTH 48.5± 13.8 ng/dl (15- 75), calcium 9.1± 0.1 mg/dl (8.3- 10.3), phosphorous 4.3± 0.2mg/dl ( 2.7- 4.5), alkaline phosphatase 95± 19 mg/dl (39-390) and urine calcium/creatinine were normal at baseline and on completion as we monitored for hypercalcemia.

Mean 25 (OH) Vit D was 19.4± 1.4 ng/dl before treatment. In 3 of 4 subjects vit D did not normalize to ≥ 30 ng/dl (23± 6.7 mg/dl) on completion of the study, while it did in one patient (30.2 ng/dl). The mean insulin during OGTT (total of six levels during OGTT) decreased by 57% from 88± 36 to 51± 10 IU/L from start to completion of the study, while mean glucose values and HbA1c remained unchanged. WBISI improved significantly from 2.2± 0.3 to 4.3± 0.6, a 51% increase in this sensitivity index.

Conclusions: These results, though preliminary, suggest that even marginal increases in the Vitamin D levels in deficient insulin resistant obese adolescents appears to improve their hyperinsulinemia. More intriguing is that insulin sensitivity index WBISI improved after 6 weeks of vitamin D treatment, which if confirmed at study completion could have important therapeutic implications for insulin resistance.

Nothing to Disclose: PCB, BHF, NV

13978 10.0000 SAT-0238 A Efficacy of Vitamin D on Glucose Homeostasis in Obese Adolescents 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 21st 3:00:00 PM SAT 0229-0266 4784 1:00:00 PM Vitamin D Metabolism & Action Poster

Gonca Tamer^*¹, Ilkay Kartal¹, Ismet Tamer², Ayse Kubat Uzum³, Banu Mesci¹, Gül Sagun¹, Damla Coksert Kilic¹, Safiye Arik¹ and Meral Mert⁴
¹Medeniyet University, Göztepe Training and Research Hospital, Istanbul, Turkey, ²Kartal Training and Research Hospital, Istanbul, Turkey, ³Istanbul University, Istanbul Medical Faculty, Istanbul, Turkey, ⁴Bakirköy Training and Research Hospital, Istanbul, Turkey

Objective: Vitamin D deficiency is suggested to be a risk factor for atherosclerosis. Since high lipid levels have important roles in the pathogenesis of atherosclerosis, the aim of our study is to compare fasting lipid concentrations of vitamin D sufficient subjects with those of vitamin D insufficient subjects.

Materials and Methods: Three hundred and fifteen non-smoking premenopausal female volunteers without diabetes mellitus were included in the study. According to their serum 25(OH)D vitamin levels, subjects were divided into 2 subgroups as 195 subjects (61.90%) with vitamin D insufficiency (VDIS Group) and 120 vitamin D sufficient subjects (38.10%) (VDS Group). Total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglyceride, and non-HDL-C levels of two groups were compared.

Results: Serum mean HDL-C level of VDIS Group was significantly lower (p=0.002) and means of LDL-C, triglyceride and non-HDL-C levels of VDIS Group were significantly higher (p=0.01, p=0.02, p=0.004, respectively) than those of VDS group. There were no significant differences between serum parathormone, calcium and phosphorus levels of VDIS and VDS groups (p=0.78, p=0.11, p=0.76, respectively). No significant difference was found between obese and nonobese subjects with vitamin D insufficiency in serum TC, LDL-C, TG, HDL-C and non-HDL-C levels (p=0.399, p=0.471, p=0.198, p=0.271,p=0.215, respectively).

Conclusion: Dyslipidemia may be more common in vitamin D insufficiency independent of diabetes mellitus and hyperparathyroidism. Further studies are needed to determine whether vitamin D insufficiency is a casual factor for dyslipidemia.

Nothing to Disclose: GT, IK, IT, AKU, BM, GS, DCK, SA, MM

11610 11.0000 SAT-0239 A Vitamin D Insufficiency May be a Casual Factor for Dyslipidemia 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 21st 3:00:00 PM SAT 0229-0266 4784 1:00:00 PM Vitamin D Metabolism & Action Poster

Anthony Chinedu Anyanwu^*¹, Olufemi Adetola Fasanmade², Herbert Babatunde Coker³ and Augustine E Ohwovoriole⁴
¹Division of EDM, Dept of Medicine, Lagos University Teaching Hospital, Lagos, Nigeria, ²Lagos University Teaching Hospital, Lagos, Nigeria, ³Faculty of Pharmaceutical Sciences, University of Lagos, Lagos, Nigeria, ⁴University of Lagos, Yaba Lagos, Nigeria

BACKGROUND

Diabetes Mellitus (DM) is a progressive disease associated with multiple complications. Improvement of glycaemic control reduces the risk of complications. Reports suggest that vitamin D supplementation improves glycaemia. However, there are no data on the vitamin D status or its relationship to glycaemic control in diabetic Nigerians.

RESEARCH QUESTION AND OBJECTIVE

Does vitamin D supplementation improve glycaemic control in Type 2 diabetes mellitus (T2DM) Nigerians with vitamin D deficiency?

The objective of this study was to determine the effect of vitamin D supplementation on glycaemic control in type 2 DM participants with vitamin D deficiency.

METHODOLOGY

A prospective randomized placebo controlled trial, involving type 2 DM participants attending the Diabetes Clinic of the Lagos University Teaching Hospital, Lagos, Nigeria. We studied 42 T2DM participants with poor glycaemic control and vitamin D deficiency. Participants were randomized into one of two treatment groups i.e. treatment and placebo arms. Levels of serum vitamin D, fasting plasma glucose (FPG), HbA1c, calcium, phosphate and albumin were determined. Daily doses of 3000 IU vitamin D₃ were given to the participants in the treatment arm while placebo was given to the placebo arm. Glycaemic status was determined at baseline and after 12 weeks of treatment. Results were expressed as Mean±SD and percentages. Comparisons between treatment groups were made using Wilcoxon, Chi square and Z-tests. P values < 0.05 were considered statistically significant.

RESULTS

The mean age of the participants was 52.5 ± 2.2 years in the treatment group and 51.1 ± 1.9 years in the placebo group (p > 0.05). There were 10 (58.8%) females and 7 (41.2%) males in the treatment group and 9 (56.3%) females and 7 (43.7%) males in the placebo group (χ² = 0.02, p = 0.88). The Mean±SD baseline HbA1c was 8.1±0.5% in the treatment arm and 7.7±0.5% in the placebo arm (p > 0.05). The mean (95%CI) baseline FPG was 156(130-181) mg/dl in the treatment arm and 150(121-180) mg/dl in placebo arm (p>0.05). Vitamin D₃ supplementation resulted in a significant increase in serum vitamin D level, serum calcium and decrease in FPG in the treatment arm compared to placebo. There was a 0.7% reduction in the mean HbA1c level in the treatment group, while the mean HbA1c increased by 0.4% in the placebo arm after 12 weeks of treatment. The number of participants with good glycaemic control increased by 33% in the treatment arm but reduced by 9% in the placebo arm (p< 0.05)

CONCLUSION

Vitamin D₃supplementation in persons with T2DM and vitamin Ddeficiency results in a significant improvement in glycaemic control. Diabetic patients with vitamin D deficiency will benefit from vitamin Dsupplementation. However, optimal dose of vitamin D, duration of supplementation and the underlying mechanism for glycaemic control need to be determined.

Nothing to Disclose: ACA, OAF, HBC, AEO

14693 12.0000 SAT-0240 A Vitamin D Supplementation Improves Glycaemia in Vitamin D Deficient Nigerians with Diabetes Mellitus 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 21st 3:00:00 PM SAT 0229-0266 4784 1:00:00 PM Vitamin D Metabolism & Action Poster

Jinny Cai^*¹, Morri Markowitz² and Ping Zhou³
¹University of Southern California, ²Albert Einstein College of Medicine, Bronx, NY, ³Children's Hospital at Montefiore, Albert Einstein College of Medicine, Bronx, NY

Background

There is evidence that thyroid hormone levels may be regulated by vitamin D (1-3). For example, low vitamin D and slightly elevated TSH levels have been described together in obese populations, a state now considered pro-inflammatory. As an immune modulator, vitamin D may affect thyroid functioning in patients with autoimmune thyroiditis. However, population based studies supporting this relationship are lacking.

The purpose of this study is to examine the relationship between serum 25-hydroxyvitamin D levels (25(OH)D) and thyroid related measures in people with and without evidence of autoimmune thyroid activity.

Data, Design, Method, and Analysis

This is a retrospective cross-sectional study using NHANES data collected between 2001-2002. Data on anthropomorphic measures, 25(OH) D, TSH, FT4, anti-thyroglobulin and anti-thyroid peroxidase antibodies levels are abstracted.

Pearson correlations are calculated. To examine the role of autoimmunity, we divided the data into two groups based on thyroid antibody positivity. Means of 25(OH) D levels of the two groups were calculated and compared.

We further analyzed the data in the available pediatric subset (ages 12-20 years). For these analyses we divided this group into four subgroups: lean, normal, overweight, and obese (<4.9%, 5-84.9%, 85-94.9%, and =>95% respectively) based on BMI percentile for age and sex. Group means of 25(OH) D, TSH, and FT4 were compared.

Results

In the original data set (N=2006), 25(OH) D levels show a strong correlation with BMI (-0.18, p<0.0001, weight (-0.12, p< 0.0001) and height (0.08, p<0.0001). TSH shows a strong positive correlation with both anti-thyroglobulin antibody (0.097 p<0.0001) and anti-thyroid peroxidase antibody (0.31, p<0.0001). It also shows a negative correlation with FT4 (-0.16, p<0.0001). However, 25(OH) D levels were not significantly correlated with any of the thyroid related measures.

Similarly, the difference between mean 25(OH) D levels in the groups stratified by antibody status was not statistically significant (22.2 Vs. 21.4).

In the thyroid antibody negative group (N=611), 25(OH) D shows no correlation with either TSH or FT4, but has a strong negative correlation with BMI (-0.18, p<0.0001).

In the pediatric analyses, compared to the BMI normal group, the mean 25(OH) D levels of all three other groups are significantly lower. The mean of the FT4 is slightly lower in the overweight group; however, differences between groups were small and may not have biological meaning. For TSH, the mean value in the obese group is statistically significantly higher than the normal BMI group, but the mean values for both groups are in the normal range.

Conclusion

No relationship was found between thyroid function (TSH, and Free T4 levels) and 25(OH) D levels regardless of thyroid antibody or weight status.

Nothing to Disclose: JC, MM, PZ

11516 13.0000 SAT-0241 A Relationship Between Thyroid Hormones and Vitamin D 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 21st 3:00:00 PM SAT 0229-0266 4784 1:00:00 PM Vitamin D Metabolism & Action Poster

Amra Osmancevic^*¹, Taye Demeke², Anne Lene Krogstad³, Håkan Sinclair⁴, Eva Angesjö⁵, Gamal El-Gawad⁶ and Kerstin Landin-Wilhelmsen⁷
¹Department of Dermatology, Gothenburg, Sweden, ²Lärjedalen Primary Health Care, Gothenburg, Sweden, ³Section for Climate Therapy, Oslo, Norway, ⁴Department of Geriatric Medicine, Borås, Sweden, ⁵Brämhult Primary Health Care, Alingsås, Sweden, ⁶Gamlestadens Primary Health care, Gothenburg, Sweden, ⁷Section for Endocrinology, Institution of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden

Background: Sun exposure is the strongest factor affecting vitamin D status in man. Vitamin D deficiency is a common health problem among immigrants with dark skin and with reduced sun exposure. There is limited information about how common and how serious vitamin D deficiency is in this population.

Aim: The aim was to compare the effect of two doses of oral vitamin D intake/placebo on serum 25(OH)D levels in healthy Somalian women (latitude 0-10°N) in Sweden (latitude 57° North).

Subjects: A total of 102 women from Somalia (mean age 34.2 years, 0.95CI: 32.3 - 36.0) living in Sweden >2 years participated.

Methods: A randomized, double-blind, placebo-controlled study was performed with two doses of vitamin D. Oral drops containing 800 IU, 1600 IU cholecalciferol and similar amounts of placebo were given during 12 weeks. Blood tests including 25-hydroxyvitamin D (25(OH)D) were monitored before and every 6^thweek throughout 6 months, i.e. 3 months follow-up after the treatment.

Results: The majority of women (n=71; 70%) were vitamin D deficient, 25(OH)D < 10 ng/ml (25 nmol/l) at the start of the study.

After six weeks of treatment only 50 subjects (49%) entered the first study visit. The mean increase in serum 25(OH)D after six weeks in subjects treated with 800 IU (n=14) was 6.0 ng/ml (14.9 nmol/l) (0.95CI: 9.0 - 20.8.) and the mean increase for subjects treated with 1600 IU (n=17) was 8.7 ng/ml (21.8 nmol/l) (0.95CI: 11.3 - 32.2.) The subjects in the placebo group (n=19) had no increase in 25(OH)D. There was a dose dependent increase in serum 25(OH)D levels (P=0.024).

After twelve weeks only 35 patients (34%) remained in the study. The mean increase in serum 25(OH)D after twelve weeks in patients treated with 800 IU (n=13) was 7.1 ng/ml (17.8 nmol/l) (0.95CI: 6.2 - 29.3.) and the mean increase for patients treated with 1600 IU (n=12) was 12.0 ng/ml (29.9 nmol/l) (0.95CI: 17.5 - 42.4.) There was no significant difference between serum 25(OH)D increase in the groups treated with 800 IU and 1600 IU, probably due to the low number of remaining participants.

During the follow-up period, after the per-oral vitamin D treatment was terminated, the serum 25(OH)D levels decreased but were still above baseline levels in the treatment groups. The placebo group remained unchanged in 25(OH)D levels throughout the study.

Conclusions: Vitamin D deficiency is a very common problem in immigrants living at higher latitudes. Treatment with Vitamin D in Somalian women living in Sweden increased the serum 25(OH)D levels dose dependently compared with placebo during 3 months. The effect was maintained for another 3 months. One third of the subjects dropped out from the study for unknown reasons indicating that this study group was hard to reach and, by that, difficult to treat.

Nothing to Disclose: AO, TD, ALK, HS, EA, GE, KL

16090 14.0000 SAT-0242 A Vitamin D Treatment in Somalian Women Living in Sweden - a Randomized, Double-Blind, Placebo-Controlled Study 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 21st 3:00:00 PM SAT 0229-0266 4784 1:00:00 PM Vitamin D Metabolism & Action Poster

Nasser Mohmmed Al-Daghri^*¹, Majed S Alokail¹, Antigoni Manousopoulou², Omar Al-Attas³, Khalid Alkharfy⁴, Yousef Al-Saleh⁵, Sobhy Yakout³, Shaun Sabico³, Harvey Johnston⁶, Theodoros I Roumeliotis⁶, Akul Singhania⁶, Christopher Woelk⁶, Paul Townsend⁷, George P. Chrousos⁸ and Spiro D Garbis⁶
¹King Saud University, Riyadh, Saudi Arabia, ²University of Southampton, United Kingdom, ³King Saud University, Riyadh, ⁴King Saud University, ⁵King Saud University for Applied Health Sciences, ⁶Southampton University, ⁷University of Manchester, Manchester, United Kingdom, ⁸First Department of Pediatrics, Athens, Greece

Low vitamin D status or hypovitaminosis D has been associated with a plethora of adverse extra-skeletal health consequences, including obesity, metabolic syndrome, cardiovascular disease and cancer. However, a causal molecular link at the serological protein level remains to be established. This proof-of-principle study compared the proteomic profiles of age-matched non-diabetic, overweight and obese females (n=22) and males (n=20) that attained a Vitamin D sufficient status after a 12-month intervention compared to those in women (n=17) and men (n=20) that participated in the same intervention but did not achieve vitamin D sufficiency. The intervention protocol was based on the increased consumption of vitamin D-rich foods and sun exposure. Non-targeted, depletion-free quantitative proteomics with bioinformatics in silico interpretations were used to analyze the whole serum specimens. This novel method profiles more than double the number of proteins otherwise captured by other proteomic methods to date. Over 2500 proteins were profiled of which ~14% changed positively or negatively with vitamin D status correction (p<0.05). The change ranged from negative 2.2 to positive 3.6 on a log₂ ratio, included novel and known proteins, and showed marked sexual dimorphism. These data, thus, provide a sexually dimorphic signature of vitamin D repletion. The known proteins identified are involved in the canonical pathways of intermediary metabolism, blood coagulation, tumorigenesis and apoptosis. These multiple, mildly modulated proteins and their potentially epistatic functions may on the one hand explain the beneficial effects of vitamin D and on the other the elusive and frequently controversial mainstream clinico-pathological indicators. The identified previously unknown proteins could serve as novel vitamin-D status correction molecular signatures and help create testable hypotheses on its potential metabolic, cardioprotective and anti-cancer effects in men and women.

Nothing to Disclose: NMA, MSA, AM, OA, KA, YA, SY, SS, HJ, TIR, AS, CW, PT, GPC, SDG

11291 15.0000 SAT-0243 A The Effect of Vitamin D Status Correction on the Human Milieu Intérieur Proteome: A Year-Long Prospective Interventional Study 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 21st 3:00:00 PM SAT 0229-0266 4784 1:00:00 PM Vitamin D Metabolism & Action Poster

Jo Eun Kim^*¹, Yong-ho Lee², Yun Ho Roh³, Yumie Rhee¹, Dae Ryong Kang³ and Sung-Kil Lim¹
¹Yonsei University College of Medicine, Seoul, Korea, Republic of (South), ²Department of Internal Medicine, Severance Hospital, Endocrine Research Institute, Yonsei University College of Medicine, Seoul, Korea, Republic of (South), ³Yonsei University College of Medicine

Although mild-to-moderate chronic kidney disease (CKD) and vitamin D insufficiency are prevalent in the elderly population worldwide and are closely associated with sarcopenia, their influence on bone mineral density (BMD) has not been determined. We aimed to assess the effects of vitamin D insufficiency and CKD on BMDs in the elderly population and their relationships with sarcopenia and parathyroid hormone levels. We designed a cross-sectional study with nationally representative samples of 6949 subjects aged≥55 years from the Korea National Health and Nutrition Examination Surveys (KNHANES) conducted between 2008 and 2011. Appendicular skeletal muscle mass and BMD were examined by dual energy X-ray absorptiometry; serum 25-hydroxyvitamin D [25(OH)D] was determined, and glomerular filtration rate was estimated using the CKD-EPI equation. The study population was divided into four groups according to vitamin D and CKD status. BMDs in the total hip and femoral neck as well as femoral bone geometry were markedly deteriorated in stage 3 and 4 CKD subjects with vitamin D insufficiency [25(OH)D <20 ng/ml] compared to other groups, regardless of gender. The prevalence of osteopenia, osteoporosis and sarcopenia were significantly higher in CKD subjects with vitamin D insufficiency. Multivariable logistic regression analyses demonstrated that CKD subjects with vitamin D insufficiency showed a significantly increased risk of osteoporosis or osteopenia, which was mainly mediated by elevated levels of parathyroid hormone and sarcopenia in these groups. In conclusion, the combination of mild-to-moderate CKD and vitamin D insufficiency was more closely associated with deteriorated BMDs in a geriatric population, linked with hyperparathyroidism and sarcopenia.

Nothing to Disclose: JEK, YHL, YHR, YR, DRK, SKL

12460 16.0000 SAT-0244 A The Combination of Vitamin D Insufficiency and Mild-to-Moderate Chronic Kidney Disease Is Significantly Associated with Deterioration of Bone Mineral Density and Bone Geometry Via Hyperparathyroidism and Sarcopenia-the Korea National Health and Nutrition Examination Surveys 2008-2011 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 21st 3:00:00 PM SAT 0229-0266 4784 1:00:00 PM Vitamin D Metabolism & Action Poster

Yanzhi Cai¹, Chandrama Shrestha¹, Shangli Ji¹, Lingyun Liang¹, Xiangbing Wang² and Zhongjian Xie^*¹
¹The Second Xiangya Hospital, Central South University, Changsha, China, ²Rutgers University-RWJMS, New Brunswick, NJ

Graves’ disease (GD) is one of the commonly encountered endocrine diseases in China. Studies over the past few years have suggested the association of vitamin D deficiency in myriad autoimmune diseases, including GD. However, it is unclear whether the vitamin D binding protein (DBP) plays a role in altering vitamin D levels in patients with GD. To address this issue, forty female patients of GD without remission after antithyroid drugs (ATD) therapy [23 patients on methimazole (MMI), 6 patients on propylthiouracil (PTU) and 9 on MMI and PTU alternately] and 40 age- and body mass index (BMI)-matched female control subjects were included in the study. Levels of total calcium, albumin, triiodothyronine (FT₃), free thyroxine (FT₄), thyroid-stimulating hormone (TSH), thyroid stimulating hormone receptor antibody (TRAb), total 25OHD, intact parathyroid hormone (iPTH), and DBP were measured. The results showed that both total and free 25OHD levels in GD patients were higher than that in controls (p<0.05). Levels of iPTH were increased in GD patients compared with controls (P<0.05). There was, however, no significant difference in DBP levels between the two groups. A positive correlation of DBP levels with total 25OHD levels (r=0.423, p<0.01) and a negative correlation of DBP levels with free 25OHD levels (r=-0.587, p<0.01) or bioavailable 25OHD levels (r=-0.573, p<0.01) were observed. There was no correlation of DBP levels with levels of FT_3,FT_4,TSH, iPTH, calcium, or albumin, age, or BMI. The level of iPTH was negatively correlated (r=-0.466; p<0.01) with the level of calcium. No correlation of 25OHD levels with FT_3,FT_4,TSH and TRAb levels was observed. The level of total 25OHD in patients on MMI was higher than those on PTU (p<0.05). There was no significant difference in levels of free 25OHD, bioavailable 25OHD, iPTH, DBP, and calcium between patients on MMI and patients on PTU. Therefore, we conclude that although DBP levels are not changed in GD patients, DBP levels are negatively associated with free 25OHD. Therefore, DBP may play a role in altering bioavailable 25OHD levels in patients with GD. Measuring DBP and calculating the bioavailable 25OHD in patients with GD might be useful for assessing true vitamin D status in such patients.

Nothing to Disclose: YC, CS, SJ, LL, XW, ZX

12580 17.0000 SAT-0245 A The Vitamin D-Binding Protein Level Is Associated with Vitamin D Status in Graves' Disease 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 21st 3:00:00 PM SAT 0229-0266 4784 1:00:00 PM Vitamin D Metabolism & Action Poster

William Wilkison^*¹, Benjamin M Buehrer², Bentley Cheatham³, Susan Walker³, Shigeru Nakano⁴, Kenji Katsuno⁴ and Masayuki Isaji⁴
¹Islet Sciences, RALEIGH, NC, ²Zen-Bio, Inc., RTP, NC, ³BHV Pharma, RTP, NC, ⁴Kissei Pharmaceuticals, Matsumoto City, Japan

The worldwide increase in obesity has fostered a corresponding increase in the incidence of non-alcoholic fatty liver disease (NAFLD). NAFLD is a key risk factor in the development of non-alcoholic steatohepatitis (NASH), a serious condition that can lead to hepatic failure. The etiology of NASH is not certain but the combination of insulin resistance and oxidative stress has been shown to be important. Remogliflozin etabonate (RE), the prodrug of remogliflozin, is an inhibitor of the sodium glucose-dependent renal transporter 2 (SGLT2). RE has been shown to reduce HbA1C and also improve insulin sensitivity. In the current study, we examined the effects of a 4-week RE treatment in a diet-induced mouse model of hepatic steatosis. Compared to obese untreated animals, RE caused reductions of liver triglycerides (40% decrease) and liver weight (50% decrease). In addition, reductions in ALT (80% decrease) and AST (35% decrease) were observed. Finally, thiobarbituric acid (TBARS), a surrogate marker of the oxidative stress levels, was significantly decreased (30%) in plasma and liver homogenates from RE treated animals. The TBAR results suggested that RE may have intrinsic antioxidant activity. We directly measured the oxygen radical antioxidant capacity (ORAC) in three different SGLT2 inhibitors: RE, canagliflozin and dapagliflozin. Interestingly, only remogliflozin, the active species derived from RE, had any significant ORAC activity. In summary, Remo suppressed the increase in both AST and ALT, reduced hepatic triglyceride content and weight, and reduced markers of oxidative stress in both plasma and liver. Based on these observations, we propose that RE may be an ideal compound for the treatment of NASH and NAFLD due to its insulin-sensitizing and anti-oxidant properties.

Disclosure: WW: Management Position, Islet Sciences, Inc., Founder, BHV Pharma. BC: Management Position, BHV Pharma. SW: Collaborator, BHV Pharma. Nothing to Disclose: BMB, SN, KK, MI

13979 18.0000 SAT-0246 A Remogliflozin Etabonate Improves Non-Alcoholic Steatohepatitis 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 21st 3:00:00 PM SAT 0229-0266 4784 1:00:00 PM Vitamin D Metabolism & Action Poster

Manoj Kumar^*¹, Deep Dutta², Samim Ali Mondal³, Shivaprasad K S⁴, Abu hena hesanoor Reza⁴ and Satinath Mukhopadhyay⁵
¹Institute of Post Graduate Medical Education And Research and SSKM Hospital, Kolkata, India, ²Institute of Post Graduate Medical Education & Research (IPGMER) and Seth Sukhlal Karnani Memorial (SSKM) Hospital, Calcutta, West Bengal, India, ³Institute of Post Graduate Medical Education & Research (IPGMER) and Seth Sukhlal Karnani Memorial (SSKM) Hospital, Calcutta, India, ⁴Institute of Post Graduate Medical Education And Research and SSKM Hospital, ⁵Institute of Postgraduate Medical Education & Research (IPGMER) and Seth Sukhlal Karnani Memorial (SSKM) Hospital, Calcutta, India

Abstract

Background and aims:Vitamin-D is believed to modulate glucose homeostasis and cytokines. Fetuin-A (FetA) secreted predominantly from liver is believed to modulate insulin resistance (IR) and inflammation, by being the principle ligand for free fatty acid induced activation of toll like receptor-4 (TLR-4) and nuclear factor‑κB (NF‑κB). Tumor necrosis factor-α (TNF-α), interleukin-1beta (IL1β) and interleukin-6 (IL6) are the principle cytokines implicated in IR. Interleukin-1 receptor antagonist (IL1ra) is the predominantly anti-inflammatory cytokine. This study aimed to evaluate the relation between Vitamin-D, FetA, cytokines and IR in normal individuals (NI), individuals with prediabetes (IPD) and newly diagnosed treatment naïve type-2 diabetes (T2DM) patients.

Material and methods:30 NI (Group-I), 57 IPD (Group-II) and 37 T2DM (Group-III) who had persistent normoglycemia, IFG and/or IGT and blood glucose in diabetic range respectively, over 2 OGTTs over a week, underwent estimation of insulin 25-hydroxyvitamin-d (25OHD) (Architect, Abbott, USA), TNF-α, IL6, IL1β, IL1ra and FetA (ELISA, Ray Biotech, USA).

Results: 75% and 41.1% individuals had 25OHD ≤30 and ≤20 ng/ml respectively. Mean age of NI, IPD and T2DM was 39.7±7.7, 42.4±8.8 and 42.2±6.7 years (P-value 0.18) respectively. Significantly increased FetA (489.2±103.7 vs 443.8±116.6 ng/ml; p=0.02), IL1β (7.9±7.7 vs 6.1±2.9 pg/ml; p=0.03), fasting glucose (112.4±22.4 vs 105.5±22.8 mg/dl; p=0.04) and IR (HOMA2-IR 1.5±0.99 vs 1.1±0.8; p=0.01) were observed in individuals with metabolic syndrome (MetS; IDF Criteria; n=46) compared to those without MetS (n=78).

In NI HOMA2-IR had positive correlation with BMI (r=0.57; p=0.02), IL1β (r=0.52; p=0.02), and FetA (r=0.49; p=0.04). Serum 25OHD had inverse correlation with IL-6 (r= -0.47; p=0.006), IL1β(r= -0.56; p=0.01) and positive correlation with IL1ra (r=0.42; p=0.02). BMI positively correlated with IL1ra (r=0.45; p=0.01).

Among IPD HOMA2-IR had positive correlation with BMI (r=0.39; p<0.001) and TNFα (r=0.23; p=0.03). Serum 25OHD had inverse correlation with HOMA2IR (r= -0.31; p=0.001), TNFα (r= -0.28; p=0.003), IL6 (r= -0.26; p=0.006), Ilβ (r= -0.27; p=0.02), FetA (r=0.31; p=0.01) and positive correlation with IL1ra (r=0.33; p=0.006). IL1ra was inversely correlated with TNFα (r= -0.3; p=0.01) and IL6 (r= -0.28; p=0.03).

In T2DM, 25OHD had positive correlation with IL1ra (r=0.36; p=0.02) and negative correlation with FetA (r=0.35; p=0.03).

Conclusion: The inverse correlation of vitamin-D with inflammatory cytokines along with positive correlation with IL1ra supports anti-inflammatory role of vitamin-D. It may be hypothesized that vitamin-D may exert its beneficial effects on IR through decreased inflammation and modulation of FetA, as evidenced by significant inverse correlation of 25OHD with FetA in IPD and T2DM.

Nothing to Disclose: MK, DD, SAM, SK, AHHR, SM

12286 19.0000 SAT-0247 A Serum Vitamin-D May Modulate Fetuin-a, Cytokines and Insulin Resistance Across the Spectrum of Glycemia 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 21st 3:00:00 PM SAT 0229-0266 4784 1:00:00 PM Vitamin D Metabolism & Action Poster

Anne E Sumner¹, Caroline K Thoreson¹, Michelle Y O' Connor², James C Reynolds³, Madia Ricks² and Stephanie T Chung^*¹
¹NIDDK, NIH, Bethesda, MD, ²NIDDK/NIH, Bethesda, MD, ³National Institutes of Health, Bethesda, MD

The Institute of Medicine (IOM) defines vitamin D insufficiency as 25(OH)D <20 ng/mL, while the Endocrine Society uses the threshold <30 ng/mL. By either standard, African descent populations have a high prevalence of low 25(OH)D. The significance and etiology of low 25(OH)D levels in African descent populations are unknown. Therefore, in 82 Africans living in America (74% male; age 39±1y (mean±SE), range 22-63y; BMI 28±0.5 kg/m², range 20-41 kg/m²), we measured 25(OH)D, parathyroid hormone (PTH), ionized calcium, phosphorous, and whole body bone mineral density (BMD) by dual-energy X-ray absorptiometry scan. Africans with 25(OH)D <20 ng/mL were compared to Africans with 25(OH)D ≥20 ng/mL. Then Africans with 25(OH)D <30 ng/mL were compared to Africans with 25(OH)D ≥30 ng/mL. In the entire cohort, 25(OH)D levels were 21±1 ng/mL, range 10-36 ng/mL. Most importantly, 50% had 25(OH)D <20 ng/mL and 83% had 25(OH)D <30 ng/mL. Ionized calcium levels were 1.2±0.01 mmol/L, range 1.1-1.3 mmol/L. Phosphorous levels were 3.3±0.1 mg/dL, range 2.5-4.4 mg/dL. Nine percent had T-scores defined as osteopenic (-1.0 to -2.5) and no one had T-scores defined as osteoporotic (<-2.5). PTH levels were 44±2 pg/mL, range 20-93 pg/mL, but 10% had PTH levels above the upper limit of normal. Adjusting for age and sex, and using the 25(OH)D cutoff of above and below 20 ng/mL, there was no difference by group in PTH, ionized calcium, phosphorous or whole body BMD. Using the threshold of 30 ng/mL, the results were similar. Overall, 50% of Africans are vitamin D insufficient by IOM thresholds, and >80% of Africans are vitamin D insufficient by Endocrine Society thresholds. Yet, less than 10% of Africans have any evidence of vitamin D insufficiency such as secondary hyperparathyroidism or decreased bone density. Therefore, the level of vitamin D which indicates insufficiency in Africans needs fresh evaluation.

Nothing to Disclose: AES, CKT, MYO, JCR, MR, STC

12862 20.0000 SAT-0248 A Vitamin D Levels Defined As Insufficient By the Institute of Medicine and the Endocrine Society May be Adequate in Africans 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 21st 3:00:00 PM SAT 0229-0266 4784 1:00:00 PM Vitamin D Metabolism & Action Poster

Mohammad Nabil Soliman Atta, Samir Naim Assaad^*, Aliaa Ali El-Aghoury, Hassan El-Prince, Mohammad Ibrahim Sayed Ahmed and Manal Ahmad Aboulfadl Aboulela
University of Alexandria, Alexandria, Egypt

Recent studies have linked low vitamin D to atherosclerosis and cardiovascular events. Hypovitaminosis D and hypoadiponectinemia have been reported in metabolic syndrome, a collection of risk factors that increases chance of developing heart disease and stroke. Sunny areas as middle eastern countries are not immune against vitamin D deficiency. The aim of this study was to determine the relation of vitamin D status and adiponectin to serum endothelin (an index of endothelial dysfunction) and carotid intima media thickness (CIMT)(a surrogate marker for atherosclerosis) in subjects with metabolic syndrome.

SUBJECTS & METHODS: The study included forty male patients with metabolic syndrome (Met S), aged between 25-35 years, and selected according to the new IDF definition (2006). Twenty healthy men of matched age served as a control group. BMI, waist circumference (WC), and skin fold thickness (triceps, subscapular) were measured. Fasting plasma glucose (FPG) was determined. Serum was used for the estimation of calcium, phosphorus, LDL-cholesterol, HDL-cholesterol, triglycerides, 25 OH cholecalciferol (25 OH Vit D3) (by Elisa), adiponectin (by Elisa), and endothelin-1 (by Elisa). Carotid intima media thickness (CIMT) was assessed using duplex ultrasonography (7.5-10 MHz transducer).

RESULTS: The mean serum 25 OH Vit D3 was 27.6 ± 19.6 ng/ml in Met S patients versus 33.7 ± 17.9 ng/ml in controls. The difference was not statistically significant (P=0.111). However, 77.5% of Met S patients were vitamin D insufficient (serum 25 OH Vit D3 <30 ng/mL) versus 55% in controls. A negative correlation was observed between serum 25 OH Vit D3 and both BMI and WC in Met S (p=0.038 and p=0.006, respectively). There was no correlation between serum 25 OH Vit D3 and serum LDL-cholesterol, HDL-cholesterol, triglycerides, FPG, or blood pressure. Mean serum endothelin-1 was higher while mean serum adiponectin was lower in Met S subjects versus healthy controls (62.7 ± 32.9 pg/ml vs 17.6 ± 5.7 pg/ml, 0.50 ± 0.19 ng/ml vs 1.56 ±0.56 ng/ml, respectively, p<0.001). CIMT was greater in Met S patients versus controls (0.67 ± 0.14 mm and 0.54 ± 0.05 mm, p<0.001). In Met S subjects, serum 25 OH Vit D3 correlated negatively with serum endothelin-1 and CIMT (p=0.01 and p=0.03, respectively); and directly with serum adiponectin (p=0.034). An inverse relation was observed between serum adiponectin and endothelin-1 (p<0.001).

CONCLUSIONS: In patients with Met S, serum vitamin D is inversely correlated with measures of whole body (BMI) and central (WC) adiposity. In these subjects, low vitamin D status is associated with hypoadiponectinemia. The increased serum endothelin-1 and CIMT observed in Met S is related to vitamin D insufficiency.

Nothing to Disclose: MNSA, SNA, AAE, HE, MISA, MAAA

13442 21.0000 SAT-0249 A Hypovitaminosis D and Hypoadiponectinemia in Metabolic Syndrome: Relation to Endothelin and Carotid Intima-Media Thickness 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 21st 3:00:00 PM SAT 0229-0266 4784 1:00:00 PM Vitamin D Metabolism & Action Poster

Anuradha Khadilkar^*¹, Vivek Patwardhan², Shashi Chiplonkar², Zulf M Mughal³ and Vaman Khadilkar²
¹Hirabai Cowasji Jehangir Medical Research Institute, Pune, Maharashtra, India, ²Hirabai Cowasji Jehangir Medical Research Institute, Jehangir Hospital, Pune, India, ³Royal Manchester Children’s Hospital, Manchester, United Kingdom

Vitamin D status as assessed by serum 25(OH)D concentrations is influenced by sunlight exposure and dietary intake. In tropical countries such as India, abundance of sunlight along with dietary inadequacy of vitamin D coupled with higher prevalence of metabolic syndrome may have varying impact on Vitamin D status and lipid concentrations. In the skin, Cholecalciferol (Vitamin D₃) and cholesterol are synthesized from the common substrate dehydrocholesterol where sunlight and DHCR compete for the same substrate. Those with high serum DHCR concentrations may have less efficient Vitamin D synthesis for given exposure of sunlight.

To explore these associations, a cross sectional study was performed in apparently healthy adult men. A random sample of 200 men (40-60 yrs) were selected from people undergoing preventive health checks. Exclusion criteria were i) fasting blood sugar level (FBSL) >125 mg/dl, ii) abnormal glutamic pyruvic transaminase (SGPT >65 IU/ L ), iii) abnormal creatinine ( 1.2 mg/dl), iv) on treatment with statins or v) on Vitamin D supplements. Eligible 172 subjects (mean age 48 ± 6 yrs ) were included in the study with a written informed consent. Sunlight exposure was assessed using validated sunlight exposure questionnaire. Lipid profile, serum 25(OH)D, blood sugar and serum DHCR were estimated on a fasting blood sample using standardize procedures.

Vitamin D deficiency (25(OH)D < 20ng/ml) was found in 56% of cases. Sunlight exposure per day was less than 60 minutes in 28%, between 60-120 minutes in 52% and more than 120 minutes in 20% cases. 25(OH)D was positively associated with sunlight exposure (r=0.47, p=0.0001) and negatively with DHCR (r= -0.30, p=0.013). At lower sunlight exposure (< 60 minutes/day) 25(OH)D was positively associated with High Density Lipoprotein cholesterol (r=0.36, p=0.011) and HDL-C:LDL-C ratio (r=0.216, p=0.145). With increasing sunlight exposure ( > 120 min/day) relationship turned to a negative association with HDL-C ( r= -0.386, p=0.022) and HDL-C:LDL-C ratio (r = -0.338, p=0.047). Generalized linear model analysis adjusted for DHCR showed that HDL-C is influenced positively by serum 25(OH)D (beta=0.143, p=0001) and triglyceride is negatively influenced by serum 25(OH)D (beta=0. 814, p=0.0001).

Interrelationship of vitamin D, HDL-C and DHCR appear to vary for a given level of DHCR or sunlight exposure.

Nothing to Disclose: AK, VP, SC, ZMM, VK

14440 22.0000 SAT-0250 A Duration of Sunlight Exposure and Levels of Dehydrocholesterol Reductase (DHCR) Affect Relationship Between Vitamin D and Lipids 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 21st 3:00:00 PM SAT 0229-0266 4784 1:00:00 PM Vitamin D Metabolism & Action Poster

Nigel J. Clarke^*¹, Mildred Goldman¹, Patrick William Mason², Khan Viec¹, Michael Phillip Caulfield³, Michael John McPhaul⁴ and Richard E Reitz⁵
¹Quest Diagnostics Nichols Inst, San Juan Capistrano, CA, ²Quest Diagnostics, Chantilly, VA, ³Quest Diagnostics Nichols Institute, San Juan Capistrano, CA, ⁴Quest Diagnostic, San Juan Capistrano, CA, ⁵Quest Diagnostic Inc, San Juan Capistrano, CA

Background

Vitamin D is derived from dietary sources or from the action of ultraviolet light on 7-dehydrocholesterol and undergoes a number of enzymatic modifications that lead to the synthesis of active Vitamin D metabolites or metabolites with reduced biological activity. Among these, epimerization at the 3-hydroxyl group leads to the synthesis of 3-epi 25-hydroxy vitamin D (3EVD). Described first in biological systems experiments employing in vitro incubation of vitamin D in cell culture, this molecule has been reported as having distinct activities when compared to 25-hydroxy vitamin D (25OHVD). Measurements of vitamin D have been conducted using a variety of methodologies and have led to conflicting assessments of the quantities of 3EVD D₃ that are measured.

Method

The present manuscript describes the development and use of a simple, CLIA validated, LC/MS/MS method to quantitate 3EVD₃ in 3528 subjects, including 309 children (162 less than 2 years of age) and 232 pregnant women.

Results

Our findings demonstrate that although 3EVD₃ constitutes a significant proportion of measureable 25OHVD₃ in subjects less than 1 year of age, 3EVD₃levels are negligible in most subjects older than one year of age.

Conclusions

It is important to choose the correct 25-OH Vitamin D assay dependent on the age of the patient. Patients under 1 year of age should be run on an LC-MS/MS assay proven to not have potential contributions from any 3-Epi 25-OH Vitamin D present in the sample.

Disclosure: NJC: Employee, Quest Diagnostics. MG: Employee, Quest Diagnostics. PWM: Employee, Quest Diagnostics. KV: Employee, Quest Diagnostics. MPC: Employee, Quest Diagnostics. MJM: Employee, Quest Diagnostics. RER: Employee, Quest Diagnostics.

11603 23.0000 SAT-0251 A The Measurement of 3-Epi-25-Hydroxyvitamin D By Mass Spectrometry in Clinical Specimens Detects Inconsequential Levels in Adult Subjects 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 21st 3:00:00 PM SAT 0229-0266 4784 1:00:00 PM Vitamin D Metabolism & Action Poster

Gülay Karagüzel^*¹, Nil Palanci¹, Sevgi Bahadir¹ and Selçuk Yaman²
¹Karadeniz Technical University, School of Medicine, Trabzon, Turkey, ²Karadeniz Technical University, School Of Medicine, Trabzon, Turkey

Background and Objectives: Alopesia areata (AA) is an inflammatory hair loss and there is a role of the immune system in the pathogenesis of the disease. It has been reported that vitamin D plays an important role in cutaneous immune modulation as well as calcium regulation. We investigated serum 25-hydroxyvitamin D [25(OH)D] levels in children with AA and evaluated the efficacy of oral vitamin D treatment in addition to topical treatment on the hair regrowth of AA patients.

Methods: Thirty (10 boys, 20 gils) newly-diagnosed AA (mean age 10.5±2.9 years) patients without systemic autoimmune diseases and 30 sex- and age-matched healthy children as controls were included in the study. Lesion sizes and levels of 25(OH)D, PTH, calcium, phosphorus, alkaline phosphatase were measured at baseline and sixth month. Vitamin D deficiency was defined as 25(OH)D level <20 ng/ml. Both patients and controls who diagnosed vitamin D deficiency were treated with oral vitamin D for six months. All the patients were also treated with topical ointment.

Results: Serum 25(OH)D levels of the patients with AA and controls were 25.3±19.4 ng/ml and 21.3±12.5 ng/ml, respectively (p >0.05). The frequency of vitamin D deficiency was similar in patients and controls (p >0.05). Serum levels of 25(OH)D and calcium were increased and PTH levels were decreased significantly after six months of the treatment in children with vitamin D deficieny (p <0.05). In patients with AA who received oral vitamin D treatment in addition to topical ointment (n= 15), lesion size decreased from 59.9±58.3 cm² to 14.0±20.1 cm² after 6 months of treatment (p <0.005). In patients who received only topical ointment (n= 15), lesion size increased from 29.5±33.6 cm² to 38.9±41.7 cm² (p >0.05).

Conclusions: This is the first prospective study in childhood and our results showed that there was no statistically significant difference in 25(OH)D levels between the patients with AA and controls. However, we found a marked reduction in lesion size in AA patients who received vitamin D treatment in addition to topical ointment. Therefore, we suggest that vitamin D treatment besides topical ointments could be successful on the hair regrowth of AA patients.

*This study was supported Karadeniz Technical University Research Project Unit under protocol No: 2010.114.003.08.

Nothing to Disclose: GK, NP, SB, SY

13693 24.0000 SAT-0252 A Vitamin D Status and the Effects of Vitamin D Treatment in Children with Alopesia Areata 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 21st 3:00:00 PM SAT 0229-0266 4784 1:00:00 PM Vitamin D Metabolism & Action Poster

Goknur Yorulmaz^*¹, Aysen Akalin², Narguler Tomus³, Murat Senol³, Pınar Yildirim⁴ and Muzaffer Bilgin⁴
¹Eskisehir State Hospital, Eskisehir, Turkey, ²OGUTF, Eskisehir, Turkey, ³Eskisehir State Hospital, ⁴Eskisehir Osmangazi University

Introduction: Gestational transient thyrotoxicosis (GTT), nonautoimmune hyperthyroidism results from the direct stimulatory effects of hCG on the thyroid and is most often present as subclinical hyperthyroidism observed transiently in the first half of gestation. Although hyperthyroidism arising from primary thyroid disease is rare in pregnancy, transient gestational hyperthyroidism is not uncommon. The prevalence of low vitamin D levels may be increasing globally.

Objectives: Our aim in this study was to evaluate the vitamin D levels in pregnants with gestational thyrotoxicosis.

Methods: Sixteen pregnants with thyrotoxicosis and 9 age matched non-pregnant healthy women were included in study. The ages, thyroid function tests, gestational weeks, β- Hcg levels, thyroid ultrasound, 25 hydroxyvitamin D and 1,25 hydroxyvitamin D levels were evaluated. Vitamin D levels were evaluated in pregnants and healthy women who referred in november and december.

Results: None of the individuals in both groups had thyroid disease in the past. Anti TPO, antithyroglobulin, TSH receptor antibodies were negative. Three of the pregnant women had nodules in ultrasound, there was no nodule and thyroid sizes were normal in the remaining patients. All pregnants had hyperemesis.

There was a positive correlation between Hcg levels and free T4 levels. There was no relation between gestational week and free T4 levels.

The vitamin D levels of both patients and control group were low and the difference was not considered significant statistically. 1,25 hydroxyvitamin D levels were higher in pregnants compared with control group and the difference was significant.

Results: The low levels of vitamin D in both groups were considered as a seasonal effect. The high levels of 1,25 dihydroxyvitamin D in pregnants may be due to increased maternal renal 1α-hydroxylase activity and the synthesis and secretion of calcitriol by the placenta.

Nothing to Disclose: GY, AA, NT, MS, PY, MB

13420 25.0000 SAT-0253 A Vitamin D Levels in Pregnants with Gestational Thyrotoxicosis 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 21st 3:00:00 PM SAT 0229-0266 4784 1:00:00 PM Vitamin D Metabolism & Action Poster

Preneet Cheema Brar^*¹, Sara K Kim², Nipapat Visavachaipan³, Maria F Contreras¹ and Brenda Kohn¹
¹New York University School of Medicine, New York, NY, ²New York University School of Medicine, ³Bumrungrad International Hospital, Thailand

Background: Obese adolescents are at high risk for vitamin D deficiency (VDD) because Vitamin D is sequestrated in adipose tissue. Vitamin D deficiency is defined as 25 (OH) D levels < 20 ng/ml; 50 nmol/L. Observational studies in adolescents support an association between VDD, Type 2 diabetes and metabolic syndrome.

Objective: a) To determine the prevalence of VDD in an inner city multi ethnic cohort of obese adolescents and b) to study the efficacy of 50,000 IU of weekly PO vitamin D2 (Ergocholecalciferol) X 6 weeks in normalizing 25 (OH) D levels (based on published Endocrine Society guidelines, 2011)

Methods: In a retrospective design from July 2011- 2013, we reviewed metabolic profile, pre and post treatment 25(OH) D levels of obese adolescents with VDD (> 95% weight for age). Though different regimens for vitamin D supplementation were used only obese adolescents who received 50,000 IU once weekly for either 4- 6 weeks were studied.

Results: Of the 53 obese adolescents identified (56% were female, age: 11.7± 4.4 years; BMI: 32.3± 7.7; mean± SD) only 3 adolescents had 25 (OH) D levels which were sufficient (>30 ng/ml; 75 nmol/L). Average 25 (OH) D levels in this obese cohort was 21± 8 ng/dl. 40 subjects had a record of being treated and the pretreatment 25 (OH) D level was 15.9± 4.6 ng/dl. While 83% received treatment for 6 weeks, a subset were treated for 4 weeks. Of those adolescents, post treatment 25 (OH) D levels were available in only 34%. 8 (44%) normalized their 25 (OH) D levels and this level went above 30 ng/ml (34.8± 4.4 ng/dl) while 10 adolescents (56%) failed to optimize their level to above > 30 ng/ml.

Conclusions: These results illustrate the following points:

Obese adolescents have invariably VDD and correction of their deficiency should be considered an effective addition to the management of their obesity to maximize non skeletal benefits of Vitamin D such as its role in glucose homeostasis.
Obese adolescents may require 2-3 fold higher doses (as suggested by the Endocrine society for adults) to optimize levels of 25(OH) D levels (>30 ng/dl) and this treatment course may need to be repeated 2-3 times/year to maintain optimal levels.
We suggest that adipose tissue is dynamically altered in obese adolescents which affects local metabolism of Vitamin D, it’s reentry into the circulation and metabolic clearance rates, an area that deserves investigation.

Nothing to Disclose: PCB, SKK, NV, MFC, BK

13929 26.0000 SAT-0254 A Efficacy of Treatment of Obese Vitamin D Deficient Adolescents Based on Endocrine Society Guidelines 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 21st 3:00:00 PM SAT 0229-0266 4784 1:00:00 PM Vitamin D Metabolism & Action Poster

Marija Pfeifer^*¹, Tinkara Duhovnik², Marija Troskot², Ajda Bicek² and Bogdan Lorber²
¹University Medical Center Ljubljana, Ljubljana, Slovenia, ²University Medical Center, Ljubljana, Slovenia

Background:Carbamazepine (C) and oxcarbazepine (O) are antiepileptic drugs that induce P450 24A1 (24-hydroxylase), the enzyme involved in vitamin D inactivation. Longtime treatment with antiepileptic drugs and its effect on vitamin D metabolism may cause vitamin D insufficiency.

Aim:To assess the relation between long-term treatment with C and O and vitamin D serum levels and to evaluate the difference in vitamin D levels, bone turnover markers and BMD in the group of epileptic patients on antiepileptic treatment and age matched healthy controls. In the intervention part of the study the effects of vitamin D supplementation on these parameters were assessed in patients only.

Methods:Prospective case-control study was performed in 32 patients (aged 28 to 59 years) treated with C or O for at least one year, mean duration of treatment 10.3 ± 7.5 (SD) years; twenty-two healthy hospital staff aged 29 to 60 years formed the control group at baseline. Serum concentrations of vitamin D, calcium and bone turnover markers [type 1 procollagen amino-terminal-propeptide (PINP), skeletal alkaline phosphatase (S-ALP), carboxy-terminal telopeptide (CTX)] were assessed in patients before and after one year of vitamin D replacement. BMD was measured using dual-energy X-ray absorptiometry. The control group hasn’t been treated with vitamin D.

Results:There was no difference in serum vitamin D concentrations between patients and controls at baseline. Both groups were vitamin D insufficient, hospital staff controls having vitamin D deficiency (levels below 50 nmol/L) in 59.1% as compared to only 46.9% of patients. No significant difference was observed in Z scores of BMD. With one year vitamin D supplementation in epileptic patients treated with antiepileptic drugs, increase in vitamin D concentrations, in BMD at the distal third of radius (p<0,0001) and of the bone formation marker S-ALP was observed as well as the borderline significant increase in BMD of the lumbar spine (p = 0,051).

Conclusions: There was no difference in serum vitamin D concentrations and BMD in patients treated with C or O as compared to healthy hospital staff controls. Vitamin D insufficiency was found in both groups while the proportion of vitamin D deficiency was greater in the control group. One-year vitamin D supplementation in epileptic patients resulted in improvement of BMD at radius and of borderline improvement at lumbar spine. Bone formation marker S-ALP increased. We recommend replacement of vitamin D, and measurement of BMD in patients on antiepileptic drugs. Hospital staff represents a risk group for vitamin D insufficiency and deserves further evaluation.

Nothing to disclose: PM, DT, TM, BA, LB.

Nothing to Disclose: MP, TD, MT, AB, BL

14783 27.0000 SAT-0255 A Effects of Vitamin D Replacement on BMD and Bone Turn-over Markers in Epileptic Patients Treated with Carbamazepine (C) or Oxcarbazepine (O) 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 21st 3:00:00 PM SAT 0229-0266 4784 1:00:00 PM Vitamin D Metabolism & Action Poster

Joely A. Straseski^*¹ and Sara P Wyness²
¹Univ of Utah School of Medicine, Salt Lake City, UT, ²ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT

Objective:Analyze the performance of 6 automated total 25-hydroxyvitamin D assays using 25(OH)D2/D3 and 25(OH)D3 only samples.

Methods: Access 2 and DxI 800 (Beckman Coulter*), ARCHITECT i2000_SR(Abbott), ADVIA Centaur XP (Siemens), Liaison XL (DiaSorin) and Modular E170 (Roche Diagnostics) assays were evaluated for imprecision, method comparison and concordance. Imprecision used control material tested in duplicate twice daily for 5 days. Method comparisons used residual serum samples with endogenous D2 and D3 (n=50) or D3 only (n=86). Comparisons with all 136 samples were intended to simulate real-world laboratory testing. Results were compared to in-house LC-MS/MS (traceable to NIST SRM 972) using Passing-Bablok regression and Bland-Altman bias plots. Acceptability criteria were CV <10% and bias <15.8%.

Results:Imprecision was acceptable for all assays except E170 and Centaur with imprecisions of 11%. Regression analysis of all samples compared to LC-MS/MS showed under-recovery of ARCHITECT, DxI, E170 and Liaison while Access and Centaur over-recovered. Compared to D2/D3 samples, E170 and Centaur showed the greatest improvement in slope without D2 while Liaison was unaffected. Also, E170 under-recovered with D2/D3 and over-recovered in the absence of D2. Access, Centaur and DxI assays exhibited the opposite effect. Constant bias improved without D2 present for all assays except ARCHITECT and E170. Testing all samples, Centaur had the lowest overall bias; E170 and Liaison did not meet acceptable limits. Testing D2/D3 samples, DxI and Access had the lowest bias; ARCHITECT, E170 and Liaison did not meet acceptable limits. Without D2, the Liaison still exceeded this limit; ARCHITECT had the lowest bias. Concordance with LC-MS/MS at 20 ng/mL ranged 77% (Centaur) to 89% (DxI). ARCHITECT, E170 and Liaison concordance improved without D2.

Overall, Access and DxI had slopes close to 1 and acceptable bias for all sample groups. Liaison had the lowest slopes and was not affected by D2. While ARCHITECT slope and intercept were not greatly affected by D2, bias and concordance improved without D2 present. E170 and Centaur assays were most affected by D2, based on improvements in slope, intercept or bias when D2 was absent.

Conclusions:It is important to consider the effects of D2 and D3 on individual assay performance. Assessing performance using total vitamin D may mask possible interferences in supplemented populations.

*Assays pending US FDA approval

Nothing to Disclose: JAS, SPW

14865 28.0000 SAT-0256 A Performance Characteristics of Six Automated 25-Hydroxyvitamin D Assays: Mind Your 3's and 2's 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 21st 3:00:00 PM SAT 0229-0266 4784 1:00:00 PM Vitamin D Metabolism & Action Poster

Cecilia Fenili^*, Ana Maria Sequera, Gabriela Ruibal, Mirta Gurfinkiel and Andrea Kozak
Argentine Society for Endocrinology and Metabolism, CABA, Argentina

At the present, the Vitamin D (VIT D) status is frequently measured in risk populations, in big cities and mainly in winter. Also it is accepted that the VIT D deficiency is a negative prognostic factor associated to different pathologies, and this has increased its measurement. There have been developed various automatic systems for 25-OH VIT D, that for being faster and less laborious are replacing the manual RIAs. Aim: To compare and establish by ROC plot analysis the consensus cutoff values for deficiency of VIT D (accepted for RIA) and the cutoff limits with greater sensitivity (S) and specificity (Sp) in the other automatic systems evaluated. Materials and Methods: 254 serum samples (collection in winter time) from healthy adults (18 - 45 years) not supplemented with VIT D, were included. Samples were aliquoted and stored at -20ºC during the study. Total 25 OH VIT D was tested in all samples by four methods: radioimmunoassay (RIA, DIASORIN); chemiluminiscence (CLIA1, ADVIA Centaur,Siemens Diagnostics), electrochemiluminiscence (ECLIA, Cobas e411, Roche Diagnostics) and chemiluminiscence (CLIA2, Architect, Abbott Laboratories). Cutoff levels with best S and Sp were calculated by ROC plot analysis. Samples with VIT D values ≤ 20.0 ng/ml by RIA(consensus cut-off) were interpreted as true positives. Results: VIT D values (ng/ml) (median and range) by RIA were 24.0 (7.0-94.0), with 39 % sample results below 20 ng/ml; by CLIA1 were 18.7 (7.2-49.8) with 57 % sample results < 20 ; by ECLIA were 23.7 (3.0-60.4) with 33 % sample results < 20 and by CLIA2 were 17.8 (4.1-57.3) with 58 % sample results < 20. Significant differences were found between RIA and CLIA1, CLIA1 vs ECLIA and CLIA2 vs (RIA, vs ECLIA, vs CLIA1) (p< 0.001, Friedman, Dunn test). No significant differences were found between RIA vs ECLIA (p= NS).ROC plot analysis showed that samples with VIT D ≤ 20.0 ng/ml by RIA would have by CLIA1 a cutoff of 19.62 ng/ml (S: 83.3 %, Sp: 56.5, AUC: 0.727), by ECLIA a cutoff of 19.60 ng/ml (S: 70.8 %, Sp: 81.7, AUC: 0.818) and by CLIA2 a cutoff of 13.1 ng/ml (S: 63.9 %, Sp: 88.9, AUC: 0.823). Conclusions: the cut-off obtained with greater sensitivity (S) and specificity (Sp) in the automatic systems evaluated are not all equivalents to the RIA. We consider necessary the unique cut-off used to be revised to define as insufficiency as sufficiency of VIT D, such as performance of different methods in the follow up of patients under supplementation treatment.

Nothing to Disclose: CF, AMS, GR, MG, AK

14902 29.0000 SAT-0257 A Is It Correct to Use the Same Vitamin D Insufficient or Sufficient Cut-off Value If We Measure with Different Immunoassays? 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 21st 3:00:00 PM SAT 0229-0266 4784 1:00:00 PM Vitamin D Metabolism & Action Poster

Tatiane Vilaca^*¹, Marília Brasilio Rodrigues Camargo², Olguita Ferreira³ and Marise Lazaretti-Castro⁴
¹Universidade Federal de Sao Paulo UNIFESP, Sao Paulo, Brazil, ²Universidade Federal de Sao Paulo UNIFESP, ³CETEC Centro Tecnológico de Minas Gerais, ⁴Universidade Federal de São Paulo UNIFESP, Sao Paulo SP, Brazil

Background

Strontium (Sr) is an alcaline Earth metal, like calcium (Ca). Both are absorbed by the same pathways. Previous studies had shown that Sr could be used as a surrogate of Ca absorption. Vitamin D has a main role in this function. The aim of this study was to investigate whether vitamin D supplementation in deficient women with low bone mass could enhance Ca absorption.

Patients and methods

Twenty-five vitamin D deficient women (25OHD<50 nmol/L, according to the Endocrino Society guidelines) were submitted to a four-hour strontium overload test. After an overnight fast, blood was drawn for baseline determinations (creatinine, total Ca, PTH, 1,25(OH)₂D). All the patients received 1 gr of Sr ranelate dissolved in deionized water. Sr determinations were made at 0, 30, 60, 120 and 240 min. The women were treated until reaching sufficient vitamin D levels (25OHD>75 nmol/L) and the test was repeated. The fraction of Sr absorbed dose (FAD) was used to evaluate Ca absorption, calculated by the formula: FAD_t = [(Sr_t – Sr₀) x 15% body weight]/ Sr administered dose .

Results

Changing vitamin D status from deficient to sufficient resulted in a significant increase in 1,25(OH)2D (24.97 ± 4.64 x 34.62 ± 9.14 pg/mL p< 0.001) and a reduction in PTH (73.87 ± 37.50 x 58.24 ± 20.13 pg/mL p=0.006), but no change was detected in calcium absorption. The FAD did not differ in any of the moments evaluated (30, 60, 120 or 240 min) before and after treatment.

Discussion

The relation between vitamin D levels and Ca absorption is controversial. Heaney et all proposed 25OHD concentrations 80-90 nmol/L as the threshold for maximum Ca absorption. On the other hand, Need and colleagues described its impairment only with 25OHD< 10 nmol/L, suggesting that severe vitamin D deficiency is needed to compromise this function. Recently, evaluating postmenopausal women, Gallagher et all found a correlation between Ca absorption and 1,25 (OH)₂D but not 25OHD. They hypothesized that at very low levels of 25OHD the lack of substrate causes a decrease in 1,25(OH)₂D production and harms mineral absorption. At not so low levels, secondary hyperparathyroidism is effective to enhance 1,25 (OH)₂D enough to maintain adequate Ca absorption. This theory is in agreement with our findings, once there were not severely vitamin D deficient women in our sample.

Conclusion

In patients with mild vitamin D deficiency, cholecalciferol supplement does not enhance intestinal calcium absorption.

Nothing to Disclose: TV, MBRC, OF, ML

15298 30.0000 SAT-0258 A No Change in Calcium Absorption in Postmenopausal Women before and after Vitamin D Oral Administration Using Strontium As a Surrogate 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 21st 3:00:00 PM SAT 0229-0266 4784 1:00:00 PM Vitamin D Metabolism & Action Poster

Riyad Sulimani^*¹, Ashry Gad², Suliman Alshehri³, Abdulaziz Alothman² and Assim A Alfadda⁴
¹King Saud University, RIYADH, Saudi Arabia, ²King Saud University, ³Ministry of health , Saudi Arabia, ⁴College of Medicine, King Saud University, Riyadh, Saudi Arabia

Aim : To study the prevalence of vitamin D deficiency among Saudi female students and its seasonal variation

Methods : This is a cross sectional study investigating the prevalence of vitamin D deficiency among Saudi female students - in intermediate and secondary schools- in the Riyadh region , Saudi Arabia . Demographic data of the girls including their exposure to sunlight and life style were obtained. Vitamin D deficiency was defined as level below 50 nmol/l while insufficiency was defined as levels between 50-75 nmol/l.

Results : A total of 2167 students were recruited . Their mean age was 16.13± 1.77 years Fasting morning samples for vitamin D estimation were taken during the summer months of May and June and during the winter months of December , January and February. A total number of students recruited during summer was 2167 while repeat samples for winter were obtained from 1048 students . The mean winter vitamin D level was 39.5 ± 28 nmol/l while it was 26.17 ± 18 nmol/l in summer (p=0.000). The majority of the girls were minimally exposed to sun with 35 % exposed for 1-10 minutes during winter months; but with practically no exposure during the hot summer months. Their consumption of dairy products was inadequate with mean calcium intake of 283±212.3 /day.

Discussion: It is observed that vitamin D deficiency occurs with high frequency among Saudi female students. The deficiency appears to be more profound during summer months – temperatures reaching up to 107 f -in contradiction to reports from other countries with more temperate climates where vitamin D deficiency is observed more frequently during winter months. In summer, direct sun exposure is tradionally completely avoided in Saudi Arabia which could explain the difference in vitamin D levels between winter and summer months.

Conclusion : It is concluded that vitamin D deficiency is common among Saudi adolescent girls. Attention to vitamin D supplementation is more needed especially during summer months to prevent severe vitamin D deficiency. Our observation also suggest that even some casual sun exposure for short periods may significantly raise vitamin D levels.

Nothing to Disclose: RS, AG, SA, AA, AAA

15025 31.0000 SAT-0259 A Vitamin D Deficiency Is More Prevalent in Summer Among Saudi Female Students 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 21st 3:00:00 PM SAT 0229-0266 4784 1:00:00 PM Vitamin D Metabolism & Action Poster

Kerstin Landin-Wilhelmsen^*¹, Taye Demeke², Amra Osmancevic³, Penelope Trimpou⁴, Göran Oleröd⁵, Anders Harald Lindahl⁵ and Lars Wilhelmsen⁶
¹Section for Endocrinology, Institution of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden, ²Lärjedalen Primary Health Care, Gothenburg, Sweden, ³Department of Dermatology, Gothenburg, Sweden, ⁴Sahlgrenska University Hospital, Gothenburg, Sweden, ⁵Department of Clinical Chemistry, Gothenburg, Sweden, ⁶Institution of Medicine, Gothenburg, Sweden

Background: Reference or recommended levels of serum vitamin D, 25(OH)D have been discussed. There are still uncertainties if serum 25(OH)D levels <10 ng/ml (<25 nmol/l) should be denoted deficiency and >10<20 ng/ml (>25<50 nmol/l) should be considered as insufficiency.

Aim: The aim was to study the prevalence of serum 25(OH)D according to the recommended concentrations, in relation to season, body composition, life style factors, bone measures and fractures in the population during 13 years follow-up.

Methods: A random population sample of men and women from the World Health Organization (WHO, MONItoring of trends and determinants for CArdiovascular disease), project, Gothenburg, Sweden, (latitude 57°North) was studied in 1995, n=608, 25-64 years and re-examined in 2008, n=412 aged 38-78, participation rate 68%. Serum 25(OH)D was measured with a RIA method (DiaSorin, Stillwater, MN, USA). Body composition and bone mass were measured, physical activity, coffee consumption and smoking data were asked for via standardized questionnaires. X-ray verified fractures were retrieved from the hospital registers until 2012.

Results: In 2008, serum 25(OH)D was similar in men, mean 24.0+10.4 ng/ml (60.0+26.1 nmol/l) range 6-60 ng/ml (15-150 nmol/l) and women 24.8+9.1 ng/ml (62.0+22.7 nmol/l) range 7-65 ng/ml (17-162 nmol/l), and increased with increasing age.

Four (1%) had vitamin D deficiency; 25(OH)D levels <10 ng/ml (<25 nmol/l) and 259/412 (63%) had vitamin D insufficiency; levels >10<20 ng/ml (>25<50 nmol/l). The figures were 5% versus 49% in 1995.

There was no seasonal variation in 25(OH)D in 2008 as seen 13 years ago with high levels during the summer months. Serum 25(OH)D did not correlate with degree of physical activity, which was a plausible explanation for being outdoors, as it did 13 years ago. Serum 25(OH)D correlated inversely and independently with body fat and serum parathyroid hormone.

The fracture incidence increased with age. Women aged 45-64 years who had vitamin D insufficiency in 1995 suffered from more fractures (28%) during 17 years follow-up than women of the same age with sufficient vitamin D concentrations (16%), (p=0.02; OR 1.11-3.48). Calcium/vitamin D supplementation was rare in 1995 and was used by 9% in 2008 and their 25(OH)D levels were higher than those without these agents at similar age.

Conclusions: Serum 25(OH)D increased with increasing age. In contrast to the findings 13 years ago, no seasonal variation and no correlation between 25(OH)D and degree of physical activity were seen. Serum 25(OH)D correlated inversely with body fat and parathyroid hormone in men and women. The majority of this Swedish random population sample had Vitamin D levels considered as insufficient.

Nothing to Disclose: KL, TD, AO, PT, GO, AHL, LW

15959 32.0000 SAT-0260 A The Majority of Men and Women from a Random Population Sample in Sweden Are Vitamin D Insufficient, the WHO Monica Project, Gothenburg 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 21st 3:00:00 PM SAT 0229-0266 4784 1:00:00 PM Vitamin D Metabolism & Action Poster

Selin Dincer¹, Sebila Dokmetas^*² and Fatih Kilicli³
¹Cumhuriyet University, ²Medipol University, Istanbul, Turkey, ³Cumhuriyet University, Sivas, Turkey

Background: There is controversy about the diagnostic features, dose, duration and criteria concerning vitamin D deficiency. In this study, we investigated the relation of vitamin D levels with symptoms, demographic, clinical and laboratory parameters and the changes induced by treatment in patients with vitamin D deficiency living in a Central Anatolia city of Turkey.

Methods: Five hundred and thirty-three patients having 25-OHD levels <30 ng / mL and age > 18 years living in a city with a continental climate were examined. Patients' age, symptoms, clothing style, health and satisfaction in their own health state, treatment duration were recorded. Patients on treatment were followed-up for 1-6 months.

Results: Mean serum 25(OH)D at the time of diagnosis was 7.59±4.41 ng/ml. 74%of the patients had serious deficiency, 24.2% had deficiency, 1.3% had insufficiency. Mean serum 25(OH)D after treatment was 24.42±14.77 ng/ml. By treatment, 57.6% of the cases reached the target values, 25.4%had insufficiency, 14.7% had deficiency and 2.3% had near the border serious deficiency of vitamin D. 74.4% of the women had protective way of clothing and 25.6% weared daily clothes leaving the head exposed to sunlight. We have found that response to treatment decreased as the body mass index increased. Abundant bone and muscle pain were present at diagnosis in 61.4% of the patients, 79% were not satisfied with their health status. Ca, PTH and ALP levels were normal at the time of diagnosis. 25(OH) D levels at diagnosis was 7.85±4.06 at the age of 30-49, 8.44±5.95 at the age of 50-69 and 6.34±3.84 at the age of 15-29. There was significant increase by age.

Conclusion: We found high percentage of vitamin D deficiency in people living in a city of Middle Anatolia especially in women at reproductive age. Treatment should be continued until a sufficient level of 25(OH)D is reached.

Nothing to Disclose: SD, SD, FK

15052 33.0000 SAT-0261 A Evaluation of Response to Vitamin D Treatment in Patients with Osmeomalacia 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 21st 3:00:00 PM SAT 0229-0266 4784 1:00:00 PM Vitamin D Metabolism & Action Poster

Helenius Jan Kloosterboer^*¹, Peter Schot² and Marjanne Prins²
¹KC2, Oss, Netherlands, ²OrgaNext Research

NDD is a novel fixed dose combination of nandrolone decanoate (25 mg) and vitamin D3 (28.000IU) /0.5 ml sesame oil in development for mitigation of disuse atrophy and loss of physical function in elderly women recovering after hip fracture surgery at risk of vitamin D deficiency.

To determine the safety, tolerability and multiple dose pharmacokinetics of subcutaneously injected NDD, 16 healthy women (65-78 years of age) received two 0.5 ml injections at day 1 and subsequently one 0.5 ml injection at day 8, 15 and 22. Blood samples were taken day at screening, day 1 (pre-dose), 2, 3, 4, 8, 11, 15, 16, 18, 22, 23, 24, 25, 35, 50 and 112. To correct for seasonal changes in vitamin D3 (D3) and 25-(OH) vitamin D3 (25-(OH) D3) levels, 14 women were included in a control group. In the control group blood samples were collected at screening, day 25 and 112. During the study safety and tolerability were monitored in both groups. In serum levels of nandrolone, D3 and 25-(OH) D3 were measured using validated LCMS/MS assays.

Nandrolone shows first order kinetics: After dose 5 AUC_0-72 is 143.6 h*ng/ml, C_max, T max , T _½ and K_elare 2.89 ng/ml, 46.3h, 478.5h and 1.67/h*10^-3,respectively. For D3 and 25-(OH) D3 C_max, T max, T _½ and K_elcould not be calculated because mean serum levels did not significantly change after the first and last injections. Baseline-corrected AUC_0-72 after dosing at day 1 was -23.6 and -21.0 h*ng/ml and after dosing at day 22 160.8 and -25.1 h*ng/ml for D3 and 25-(OH) D3, respectively. At day 25 D3 and 25-(OH) D3 levels in the NDD group were not significantly different from those in the control group. Between day 25 and 112 serum levels of 25-(OH) D3 declined in the control group but not in the NDD group. The injections were well tolerated and no clinically relevant (serious) adverse events occurred. In the NDD group one subject discontinued the study because of Lyme disease.

In a post-hoc analysis the results of the 25-(OH) D3 measurements in the NDD group were analyzed depending on the screening level (> or < 30 ng/ml). Baseline corrected levels at day 25 and 112 in the group < 30 ng/ml were + 4.7 and +6.4 ng/ml, in the group > 30 ng/ml the baseline corrected levels were -3 and - 8 ng/ml.

In conclusion: multiple injections with NDD are safe and well tolerated, both ND and D3 are released from the oil depots and effects on the 25-(OH) D3 level are dependent on the screening level, suggesting that 25-(OH) D3 levels are regulated.

Nothing to Disclose: HJK, PS, MP

16640 34.0000 SAT-0262 A Safety, Tolerability and Multiple Dose Pharmacokinetics of a Fixed Dose Combination of Nandrolone Decanoate + Vitamin D3 in Healthy Elderly Females (65 years of age or older) 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 21st 3:00:00 PM SAT 0229-0266 4784 1:00:00 PM Vitamin D Metabolism & Action Poster

Ye An Kim^*¹, Kyoung Min Kim¹, Sung Hee Choi², Soo Lim³, Jae Hoon Moon⁴, Sang Wan Kim⁵, Eu Jeong Ku¹, Kyong Yeun Jung¹, Chan Soo Shin⁶ and Hak Chul Jang⁷
¹Seoul National University College of Medicine, Seoul, Korea, Republic of (South), ²Seoul Nat Univ Bundang Hosp, Seongnam-Si, Korea, Republic of (South), ³Seoul National University Bundang Hospital, Seongnam, Korea, Republic of (South), ⁴Seoul National University College of Medicine, Korea, Republic of (South), ⁵Seoul Metropolitan Government Boramae Medical Center, Seoul, Korea, Republic of (South), ⁶Seoul Nat Univ College of Med, Seoul, Korea, Republic of (South), ⁷Seoul National University Bundang, Seongnam-Si, Korea, Republic of (South)

Background

Dietary vitamin C might have a protective role in bone loss in postmenopausal women. Few studies evaluated the relative contribution of vitamin C on bone mineral density (BMD) in adjunction with vitamin D, which is an important regulator of bone metabolism.

Objective

We investigated the association between dietary vitamin C intake and bone mineral density (BMD) in postmenopausal women using the data from the Third Korean National Health and Nutrition Examination Survey (KNHANES IV, 2009).

Design

Dietary information was assessed with a 24-hour dietary recall questionnaire. BMD was measured at lumbar spine and hip using dual x-ray absorptiometry. Subjects with premenopausal state, renal insufficiency (Cr≥1.4mg/dl), histories of osteoporosis treatment, estrogen/progesterone users, and supplementary vitamin and mineral users were excluded.

Results

A total of 5690 women were enrolled and 2073 subjects aged 50 years and older were included. BMDs for all sites showed significant positive correlations with the dietary vitamin C tertiles with more strong relationships for hip than that of lumbar spine ( r=0.148 for lumbar spine, r=0.221 for femur neck, r=0.214 for total hip, p<0.001, respectively). Moreover, subjects with osteoporosis at lumbar spine or hip showed significant lower values of dietary vitamin C intake than subjects without osteoporosis of identical site (74.4 ± 66.2 to 94.1 ± 78.6 for lumbar spine, 65.5 ± 56.6 to 94.3 ± 79.2 for femur neck, 46.3 ± 38.2 to 89.2 ± 76.0 for total hip, p <0.001, respectively). These positive associations between dietary vitamin C and BMD were not consistently observed in premenopausal women. Interestingly, the associations with dietary vitamin C intake and BMD at each site were stronger in subjects with low serum vitamin D less than 50 nmol/L. Lower dietary vitamin C intakes (<100mg/d) showed higher prevalence of osteoporosis (OR = 1.505, 95% CI 1.086-1.948, p = 0.009) after adjusting dietary calcium and vitamin D. In age 50s and 70s or older groups, lower dietary vitamin C was associated with osteoporosis while there was no association between dietary vitamin C and osteoporosis in 60s.

Conclusions

Dietary vitamin C intake showed positive relationships with BMDs at all skeletal sites in postmenopausal women. These results could suggest that dietary vitamin C have protective effects on bone loss of postmenopausal women and could be encouraged.

Nothing to Disclose: YAK, KMK, SHC, SL, JHM, SWK, EJK, KYJ, CSS, HCJ

15444 35.0000 SAT-0263 A Favorable Effect of Dietary Vitamin Con Bone Mineral Density in Postmenopausal Women (KNHANES IV 09') 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 21st 3:00:00 PM SAT 0229-0266 4784 1:00:00 PM Vitamin D Metabolism & Action Poster

Nasser M. Rizk^*¹, Esma Ahmedou Moud Moud², Salwa Saeed², Amina Saleh Fadel¹, Samah ebrahim Ahmed², Fadheela Dadbakhsh Arzemohmmed² and Ayman El-Menyar³
¹Qatar University, Doha, Qatar, ²Qatar University, Qatar, ³Hammad medical coroprtaion, HMC, Doha, Qatar

Background: Several studies indicate an association between vitamin-D deficiency and coronary artery disease ^1,2.

Objectives: The aim of this study was to evaluate the relation between different grades of vitamin-D deficiency and coronary artery disease, and to determine if there was a relation between vitamin-D deficiency and the severity of the coronary artery lesion among CAD patients.

Methods: A prospective-retrospective case–control association study included 110 healthy volunteers and 129 CAD patients. They were enrolled from the cardiology department-Heart Hospital, Hammed Medical Corporation (HMC)-Doha, Qatar, from November 2011 to November 2012. Based on the severity of coronary arteries stenosis, CAD patients were divided into: group 1 (severe stenosis with > 50% luminal lesion) and group 2 (minimal stenosis with luminal lesion ≤ 50%). 25 hydroxy-vitamin D (Vit D) and vitamin-D binding protein (Vit D –BP) were performed by Eliza in Biomedical labs – Qatar University. Data are expressed as mean with 95% CI of the mean unless mentioned otherwise.

Results: The mean ± SD age in years of study subjects was (56.00±9.1) for CAD patients and (44.77±16.3) for healthy controls. Males were (76.7%) among CAD and (72.15%) among healthy controls. CAD had significantly lower mean values of Vitamin D (16.30, (10.10-30.39) than the healthy controls subjects (21.21, (13.2- 27.5) ng/ml, p =0.011 but had significantly higher (Vit D –BP) (3720, 3660-3750) than for healthy subjects (3650, 3610-3736) pg/ml, p=0.003. CAD patients with severe stenosis had significantly lower mean values of Vit D (12.7, 9.33-26.9) than CAD with minimal or no stenosis (19.8, 11.7-32.8) , with p value =0.036, but no significant difference for Vit D-BP between both groups with p=0.419. For CAD patients with severe stenosis, the groups with deficient (<10 ng/ml) and insufficient levels (10-30 ng/ml) of vitamin D had the highest percentage (58.8%) of patients with the numbers of blood vessels affected (2 or more) than patients with optimal Vit D (>30 ng/ml) level (52,32%), with p=0.737. CAD patients with optimal levels of vitamin-D had the highest percentages (60.70%) with ejection fraction (EF) (> 50%), while CAD with insufficient/deficiency vit-D group had the highest percentage (51.10%) of severe EF (< 50%). Spearman’s correlation revealed that Vitamin-D levels, has significant correlation with Vit D-BP (r=0.322, p=0.022); and troponin (r=-0.289, p=0.019); but insignificant with the numbers of blood vessels affected (r=-0.063, p=0.620); and the ejection fraction (r=-0.069, p=0.637).

Conclusion:The results of the present study suggest that Vit D could have an impact on the severity of the coronary artery lesion independent on the numbers of blood vessels affected. Further investigations are needed to explore the possible mechanisms of Vit D deficiency on CAD.

Nothing to Disclose: NMR, EAMM, SS, ASF, SEA, FDA, AE

16885 36.0000 SAT-0264 A Association Between Vitamin D and Coronary Artery Disease in Qatar 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 21st 3:00:00 PM SAT 0229-0266 4784 1:00:00 PM Vitamin D Metabolism & Action Poster

Julianne Cook Botelho^*, Ashley Ribera, Hans C Cooper and Hubert W Vesper
Centers for Disease Control and Prevention, Atlanta, GA

Laboratory measurements are used to make patient care and public health decisions. However, the accuracy and reliability of these tests prevent appropriate detection, treatment and prevention of diseases. The aim of the CDC Standardization Programs for testosterone (T), estradiol (E2), and vitamin D [25(OH)D] are to standardize clinical measurements such that accurate and comparable measurements are obtained regardless of the measurement procedure, location, and time. To support the clinical and research community the CDC has established Reference Services, Standardization-Certification Programs, and Quality Assurance Monitoring Services.

As part of the Reference Laboratory Services the CDC has established higher order reference measurement procedures for total testosterone, total estradiol, and 25-hydroxyvitamin D2 and D3 in serum using LC-MS/MS. These measurement procedures are traceable to primary reference materials and to JCTLM certified reference measurement procedures. The CDC collaborates with the clinical, research, metrological and proficiency testing (PT) communities to assign target values to materials. These materials are used for method comparisons, calibration, and accuracy controls. CDC Standardization-Certification Programs are operating for T and E2 with the Hormone Standardization (HoSt) Program and 25(OH)D with the Vitamin D Standardization-Certification Program (VDSCP). In both of these programs, 4 blinded challenges are sent over the period of a year. Bias and imprecision assessments using established protocols and final assessment are made using criteria derived from biological variability. At present, 22 participants are enrolled in the HoSt-T Program and 28 in VDSCP. Participants include clinical, academic, and pharmaceutical laboratories as well as immunoassay manufacturers. Approximately 80% of participants have meet the established criteria and successful laboratories are published on the CDC website (http://www.cdc.gov/labstandards/hs.html). The CDC is also working with external quality assessment and PT providers on accuracy-based surveys to allow the performance of clinical laboratories to be monitored and the impact of standardization programs on patient care to be assessed. In addition the CDC provides quality assurance monitoring services to help assure accuracy over time for research studies and clinical trials by providing accuracy based blinded quality control materials.

CDC Standardization Programs collaborate closely with stakeholders such as the Partnership for Accuracy in Hormone Testing (PATH) and The Endocrine Society to assure clinical and public health needs are met. One example of this collaboration is through the development of common reference ranges for T. Through these efforts and collaborations the CDC is working towards standardizing testosterone, estradiol and vitamin D measurements.

Nothing to Disclose: JCB, AR, HCC, HWV

17065 37.0000 SAT-0265 A CDC Standardization Programs – Testosterone, Estradiol, and Vitamin D 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 21st 3:00:00 PM SAT 0229-0266 4784 1:00:00 PM Vitamin D Metabolism & Action Poster

Pratibha Shahajirao Pawal^*
T. N. Medical College & B.Y.L. Nair hospital, Mumbai, Mumbai

Title: Study Of Various Vitamin D3 Supplementation Protocols That Are Used In Community Practice For Treatment Of Hypovitaminosis D

Authors: Pawal PS,Joshi AS,Lathia T,Mittal S,Sharma BR,Dalwadi PP,Bhagwat NM, Chadha MD, Varthakavi PK.

Acknowledgement: Dhoyle JP, Rahate S,Halankar S.

ABSTRACT

Introduction: Hypovitaminosis D is widely prevalent in India (70-90%). Different dosing protocols have been used for Vitamin D(VITD) supplementation; however there is no universally accepted regimen.
- Objectives: Comparison of serial VITD concentrations attained with various VITD supplementation protocols in VITD deficient subjects.
- Methods: 242 subjects were screened.105 consenting subjects( age 20-60 years) with Hypovitaminosis D(VITD <16ng/ml) participated in the study and were randomized to receive 4 different supplemental regimens i.e. single dose 6lacIU orally, single dose 6lacIU intramuscular, 60000IU once weekly for 8 weeks and 2000IU daily for 8 weeks.
- Outcome measures: Primary outcome measure was the serial serum VITD concentration attained. VITD, Parathyroid Hormone(PTH), calcium, phosphorus, alkaline phosphatase were monitored at baseline, 1,3,6,8 & 12 weeks of therapy.

Results: 66.5% subjects were found to be vitamin D deficient (<20ng/ml). Baseline VITD concentrations for Oral, IM, Weekly and Daily groups were 10.6±3.7; 9.7±4.5; 8.9±4.4 and 10.2±4.3 ng /ml respectively. VITD concentrations increased significantly in all groups at 3 weeks(P <0.001).After 8 weeks, VITD concentrations for Oral, IM, Weekly and Daily groups were 34.6±9.4; 40.6±15.1 ; 40.2±13.9 and 24.7±7.7ng/ml respectively. VITD concentration decreased from 8 weeks to 12 weeks in all groups except in IM group which maintained steady increasing trend from 8wks to 12 wks.VITD level >100ng/ml was observed in 40% of subjects in oral group while none of other groups showed VITD >100ng/ml at all time points. All groups were compared with mean percentage increment in VITD concentration at different time points. Serum intact PTH was increased in only in 40% of subjects with VITD <16 ng/ml.

Conclusions: Only IM mega dose group showed sustained increment of VITD concentration at 12 weeks without VITD toxicity or hypercalcaemia. Only oral group had VITD levels >100 ng/ml(40%) without clinical manifestations of hypervitaminosisD .Oral loading dose is adequate for maintaining VITD sufficiency only for duration of 3 months & need for resupplementation at 3 monthly interval. Weekly group also showed decline in VITD sufficiency at 12 weeks indicates need for retesting and supplementation.2000IU daily is not adequate to achieve sufficiency in VITD deficient subjects.

Nothing to Disclose: PSP

15555 38.0000 SAT-0266 A Study of Various Treatment Protocols for Hypovitaminosis D 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 21st 3:00:00 PM SAT 0229-0266 4784 1:00:00 PM Vitamin D Metabolism & Action Poster

Elizabeth Parra Garnica^*, Pritisheel Banga and Soe Naing
University of California, San Francisco, Fresno Medical Education Program, Fresno, CA

Background: Primary Hyperparathyroidism (PHPT) is rarely diagnosed during pregnancy. Acute pancreatitis is a rare complication of PHPT and is associated with significant maternal and fetal morbidity during pregnancy. However, there is a paucity of information and recommendations in the literature on the appropriate management of this serious complication.

Clinical Case: A 28-year-old pregnant woman, who conceived twins with IVF treatment, was admitted to the hospital with a one day history of severe epigastric pain and vomiting at 17.5-weeks’ gestation. She did not have any significant past medical history and she was on prenatal vitamins only. Her blood tests revealed elevated lipase of 3356 (NR 12-53 U/L) and amylase of 2967 (NR 20-130 U/L). Abdominal ultrasound showed an enlarged pancreas and large volume ascites. There was hypercalcemia with albumin-corrected serum calcium of 13.5 mg/dL, ionized calcium of 7.11 (NR 4.48-5.28 mg/dL) and PTH of 177.9 (NR 14.0-72.0 pg/mL). She was therefore diagnosed with acute pancreatitis due to PHPT and a multidisciplinary team consisting of surgeons, obstetricians, an endocrinologist, gastroenterologists and hospitalists were immediately involved in her care. She was given aggressive IV fluid hydration, IV furosemide, and subcutaneous calcitonin. Her calcium level came down to 11.5 mg/dL 24 hours later. There was also drastic improvement in clinical and biochemical parameters of acute pancreatitis within 3 days. On her 5^th day of hospitalization, she underwent parathyroidectomy in which right superior parathyroid adenoma was removed. Serum calcium and PTH levels were normalized post-operatively and she was discharged home on 7^thday of hospitalization. Her serum calcium and PTH remained within normal range when she was last reviewed at 28 weeks of gestation.

Conclusion: Acute pancreatitis due to PHPT during pregnancy is very rare and is associated with significant maternal and fetal morbidity. Aggressive intravenous hydration, loop diuretics and subcutaneous calcitonin appeared to be effective and safe initial medical therapies. Parathyroidectomy should be considered as soon as the patient is stabilized. This case illustrated that early recognition and prompt management by a multidisciplinary team is vital for the successful outcome.

Nothing to Disclose: EP, PB, SN

14407 1.0000 SAT-0193 A Acute Pancreatitis Due to Primary Hyperparathyroidism during Pregnancy: Multidisciplinary Team Approach for the Successful Outcome 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 21st 3:00:00 PM SAT 0193-0213 4787 1:00:00 PM Hyperparathyroidism and Metabolic Bone Disease Poster

Kaniksha Desai^*¹ and Shon Edward Meek²
¹Mayo Clinic Florida, Jacksonville, FL, ²Mayo Clinic-Jacksonville, Jacksonville, FL

Background: Osteogenic osteomalacia is a rare tumor which results in hypophosphatemia. This is often secondary to mesenchymal tumors. Very rarely it has been reported in patients with neurofibromatosis. Definitive treatment is localization of the tumor and surgical removal. Here we present a case where a woman presented with progressive weakness, inability to walk, and multiple fractures and was found to have a nerve sheath tumor in her left calf and was diagnosed with neurofibromatosis.

Case: 51 year old woman presented to the clinic with 4-5 years of progressive weakness with a decreased ability to walk and was also noted to have bilateral feet fractures and a hip fracture. She was previously worked up for muscle weakness but no secondary etiology was found. During her examination she was noted to have multiple skin lesions including café-au-lait macules and axillary freckling which were biopsied and consistent with neurofibromatosis. She was also noted to have significant proximal muscle weakness, especially in her lower extremities. During her workup here she was noted to have low phosphorus level of 1.8 mg/dL and a low Vitamin D level of 12.7 ng/ml and a normal PTH level. Further laboratory studies showed that she had an elevated fibroblast growth factor 232 H RU/mL (normal <180 RU/mL) and urinary phosphorus level of 466 which was consistent with hypophosphatemic osteomalacia. She was treated with Neutra-Phos and cholecalciferol and was noted to have a slight improvement in her weakness and an increase in her phosphorus levels to 2.1 mg/dL. She had an octreotide scan which did not show any focal lesion and then proceeded to have a PET scan which showed increased uptake in the calf muscle. She had an MRI completed which showed a 2.7 cm mass in the left gastrocnemius muscle. She subsequently was taken to surgery and had the tumor removed which showed pathology consistent with a nerve sheath tumor.

In conclusion, a detailed physical exam and measurements of basic labs including calcium and phosphorus measurements are important in evaluating patients with weakness and fractures.

Nothing to Disclose: KD, SEM

14822 2.0000 SAT-0194 A Oncogenic Osteomalacia Secondary to a Schwannoma in a Patient with Neurofibromatosis That Presented with Weakness and Multiple Fractures 2014-06-24 Chicago 2014-06-17 16th International Congress of Endocrinology & the Endocrine Society’s 96th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 21st 3:00:00 PM SAT 0193-0213 4787 1:00:00 PM Hyperparathyroidism and Metabolic Bone Disease Poster

Rodolfo J Galindo^*¹, Ageliki S Valsamis², Isabela J Romao² and Yael Tobi Harris²
¹Hofstra University - School of Medicine, Great Neck, NY, ²Hofstra North Shore LIJ School of Medicine, Great Neck, NY

Introduction: Etiologies of hypercalcemia associated with cancer include humoral hypercalcemia of malignancy (i.e. tumoral production of parathyroid-related peptide [PTHrP]), local osteolytic changes, calcitriol-mediated and rarely ectopic hyperparathyroidism.

Clinical case: A 58-year-old man presented with syncope and falls during orthostatic postural changes. He also reported malaise, weight loss, polyuria and diffuse abdominal pain. He had been diagnosed with anal squamous cell carcinoma one year prior, and had responded well to chemotherapy. No systemic disease had been found on initial staging. His medical history also included bipolar disorder (never treated with lithium). His medications included gabapentin and docusate. On presentation, he was bradycardic to 48 bpm and had orthostatic hypotension. Physic