Thang S Han1, Nils P. Krone2, Debbie S Willis3, Gerard S Conway4, Stephanie Hahner5, Aled Rees6, Roland H Stimson7, Brian R Walker7, Wiebke Arlt8 and Richard J Ross*9
1Royal Holloway University of London (ICR2UL) and Ashford and St Peter's NHS Foundation Trust, Surrey, United Kingdom, 2University of Sheffile, Birmingham, United Kingdom, 3British Endocrine Society, Bristol, United Kingdom, 4University College London, London, United Kingdom, 5Univ Hosp of Wuerzburg, Wurzburg, Germany, 6Cardiff University, Cardiff, United Kingdom, 7University of Edinburgh, Edinburgh, United Kingdom, 8University of Birmingham, Birmingham, United Kingdom, 9University of Sheffield, United Kingdom

 

Context: Quality of life (QoL) has been variously reported as normal or impaired in adults with congenital adrenal hyperplasia (CAH). To explore the reasons for this discrepancy we investigated the relationship between QoL, glucocorticoid treatment and other health outcomes in CAH adults.

Methods: Cross-sectional analysis of 151 adults with 21-hydroxylase deficiency (50M: 47 with classic and 3 with non-classic CAH; 101F: 75 with classic and 26 with non-classic CAH) aged 18-69 years in whom QoL (SF-36), glucocorticoid regimen, anthropometric and metabolic measures were recorded. Relationships were examined between QoL, type of glucocorticoid (hydrocortisone, prednisolone and dexamethasone), and dose of glucocorticoid expressed as prednisolone dose equivalent (PreDEq). QoL was expressed as z-scores calculated from matched controls (14,430 subjects from UK population). Principal components analysis (PCA) was undertaken to identify clusters of associated clinical and biochemical features and the principal component (PC) scores used in regression analysis as predictor of QoL.

Results: QoL scores were associated with type of glucocorticoid treatment for vitality (P=0.002) and mental health (P=0.011), with higher z-scores indicating better QoL in patients on hydrocortisone than in patients receiving prednisolone or dexamethasone (P<0.05). QoL did not relate to PreDEq or mutation severity. PCA identified three PCs (PC1, disease control; PC2, adiposity and insulin resistance; PC3, blood pressure and mutations) that explained 61% of the variance in observed variables. Stepwise multiple regression analysis demonstrated that PC2 (comprising waist circumference, serum triglycerides, HOMA-IR and HDL-cholesterol) was associated with QoL scores, specifically impaired physical functioning, bodily pain, general health, Physical Component Summary Score (P<0.001) and vitality (P=0.002).

Conclusions: Increased adiposity, insulin resistance and use of prednisolone or dexamethasone are associated with impaired QoL in adults with CAH independently of mutation severity. Intervention trials are required to establish whether choice of glucocorticoid treatment and/or weight loss can improve QoL in CAH adults.

 

Disclosure: SH: Advisory Group Member, Viropharma. RJR: Founder, Diurnal. Nothing to Disclose: TSH, NPK, DSW, GSC, AR, RHS, BRW, WA

FP03-1 5428 1.0000 SAT-1 A Quality of Life in Adults With Congenital Adrenal Hyperplasia Relates to Glucocorticoid Treatment, Adiposity and Insulin Resistance: United Kingdom Congenital Adrenal Hyperplasia Adult Study Executive (CaHASE) 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 11:30:00 AM FP03 2203 11:00:00 AM Glucocorticoids & Glucocorticoid Actions Poster Preview


Margreet A.E.M. Wagenmakers*1, Sean H.P.P. Roerink1, Linda Gil1, Theo S. Plantinga1, Johannes W.A. Smit1, Romana T. Netea-Maier1 and Ad RMM Hermus2
1Radboud University Nijmegen Medical Center, Nijmegen, Netherlands, 2Radboud University Medical Center, Nijmegen

 

Context: Centripetal obesity is associated with systemic low grade inflammation and an increased cardiovascular risk profile. Patients in long-term remission of Cushing’s syndrome (CS) report persisting abdominal fat accumulation despite overall weight loss. However, this has not been adequately objectified because previously published studies on this topic have major methodological limitations and show conflicting results[1-3].

Objective: The aim of this study was to investigate body composition and adipose tissue distribution of patients in long-tem remission of CS, in combination with risk factors for cardiovascular disease.

Patients and methods: Fifty-eight patients that had been in remission of CS for at least 5 years (69% pituitary CS and 31% adrenal CS, 79 % women, age 51 ± 12 years, BMI 26.5 ± 4 kg/m2) were included and compared to 58 age-, gender- and BMI-matched healthy control subjects. In all patients presence of hormonal deficiencies had been objectified according to international consensus criteria and all deficiencies had been adequately treated for at least 5 years. The subjects underwent a clinical evaluation and a Dual Energy X-ray Absorptiometry scan to assess body composition. Furthermore laboratory parameters, including adipokine and cytokine profiles were measured.

Results: Compared to the matched control subjects, the patients in long-term remission of CS had a greater waist circumference (p<0.01), a smaller thigh circumference (p<0.01), a higher waist-to-hip ratio (p<0.01) and a higher hip-to-thigh-ratio (p<0.01). Systolic and diastolic blood pressure did not differ between the two groups. As measured with DEXA scanning, patients had a higher percentage of truncal fat mass (p=0.01) while the percentage of total fat mass and lean body mass did not differ. The truncal fat mass to leg fat mass ratio was higher in the patients (p<0.01).

Patients had lower adiponectin levels (p<0.01), higher leptin levels (p>0.01) and higher resistin levels (p=0.04) than control subjects. Furthermore they had a more adverse plasma lipid profile with lower HDL levels (p=0.03) and higher fasting triglyceride levels (p<0.01) although total cholesterol and LDL levels did not differ.  IL-6, IL-8, TNF-α and MCP-1 levels were not significantly different between the two groups. Nor were creatinine, glucose, Hba1c and insulin levels. The centripetal adipose tissue distribution was present in both men and women and all significant differences remained significant if the 22 patients who used glucocorticoids were excluded from the analyses.

Conclusion: Even after long-term remission, patients that suffered from CS in the past continue to have a centripetal adipose tissue distribution and an adverse adipokine and plasma lipid profile. This probably contributes to the remaining decreased quality of life and persistent increased cardiovascular risk profile in these patients.

 

Nothing to Disclose: MAEMW, SHPPR, LG, TSP, JWAS, RTN, ARH

FP03-2 6962 2.0000 SAT-2 A Persistent Centripetal Fat Distribution and Metabolic Abnormalities in Patients in Long-Term Remission of Cushing's Syndrome 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 11:30:00 AM FP03 2203 11:00:00 AM Glucocorticoids & Glucocorticoid Actions Poster Preview


Matthew D Taves*1, Adam W Plumb2, Chunqi Ma2, Benjamin A Sandkam3, David A Close4, Ninan Abraham2 and Kiran K Soma2
1University of British Columbia, Vancouver, BC, Canada, 2University of British Columbia, 3Simon Fraser University, Burnaby, BC, 4University of British Columbia, Vancouver, BC

 

Glucocorticoids (GCs) are secreted by the adrenal glands and circulate through the blood to coordinate organismal physiology. GCs play a paradoxical role in development, as they are critical for maturation of organs such as the liver and heart but simultaneously impair growth and neural development. The stress hyporesponsive period (SHRP), when circulating GCs are minimal soon after birth, avoids the harmful effects of GCs but conversely deprives organs of GCs where they are needed. Thus, temporal changes in systemic GC levels cannot meet the conflicting GC requirements of different organs during development. Interestingly, while the adrenals are traditionally considered the sole source of GCs, other organs also express GC-synthetic enzymes, suggesting that developing organs can independently regulate their local GC levels to correspond with organ-specific requirements. We quantified GCs in developing and adult mice and found that during the SHRP, GC levels in multiple organs were higher than in whole blood, consistent with constitutive local GC synthesis. Cortisol and its precursor deoxycortisol, widely considered to be absent in mice, were elevated in primary lymphoid organs (thymus and bone marrow), with cortisol concentrations in marrow up to 1000-fold higher than in circulating blood. Corticosterone and its precursors were elevated in lymphoid organs and liver, while deoxycorticosterone (both a GC and mineralocorticoid) was elevated in brain and heart. In lymphoid organs and heart, local GC elevation was most dramatic in the embryo and neonate, then decreased with age, showing that GC levels in different organs can be regulated independently of GC levels in blood. Thus, especially during early development, quantification of circulating steroids can be very misleading with regard to steroid levels in specific organs or tissues. Within the developing organism, there is clearly a “mosaic” of different steroids and different steroid concentrations, to match organ-specific developmental requirements. GC effects in different tissues should therefore be re-evaluated in the context of local (not just systemic) GC patterns.

 

Nothing to Disclose: MDT, AWP, CM, BAS, DAC, NA, KKS

FP03-5 3118 5.0000 SAT-3 A Widespread compartmentalization of glucocorticoid physiology during mouse development 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 11:30:00 AM FP03 2203 11:00:00 AM Glucocorticoids & Glucocorticoid Actions Poster Preview


Caroline Costa Mesquita*1, Danilo Silva Ferreira1, Ana Paula Lima Barbosa1, Joseane Morari1, Licio Augusto Velloso2 and Gabriel Forato Anhe3
1State University of Campinas, 2University of Campinas, Campinas, Brazil, 3State Univeristy of Campinas, Campinas, SP, Brazil

 

Rodents produce corticosterone in a circadian fashion with peaking values approximately 2 hours before lights off. Among the myriad of functions attributed to corticosterone, this hormone is known to stimulate food intake by reducing hypothalamic Corticotrophin-Releasing Hormone (CRH) expression. Apart from this, the precise mechanism leading to downregulation of CRH still remains to be described. Recent publications show that the Unfolded Protein Response (UPR) established in the hypothalamus is able to stimulate food intake and body weight gain. Dexamethasone (DEX), a synthetic glucocorticoid, was described to specifically activate the ATF6 branch of the UPR in different cell types. The aim of this study was to investigate if glucocorticoids regulate CRH expression and food intake through a mechanism dependent on ATF6. Male Wistar rats were kept under a light/dark cycle (12h/12h) with standard chow ad libitum. Rats were sacrificed in different moments of the light dark cycle (ZT0, ZT4, ZT8, ZT12, ZT16 or ZT20). Different set of animals were adrenalectomized (ADX) or subjected to sham surgery (SHAM). Intraperitoneal injections with DEX were performed at ZT1 (2, 20 or 200 mg/kg). Hypothalami removed from the rats were used for western blot detection of Activating transcription factor 6 (ATF6), cAMP response element-binding protein 1 (CREB1) and CREB co-activator (CRTC2). Tissues were also processed for quantification of CRH mRNA by Real Time PCR. CRTC2 association with CREB1 or ATF6 was determined by co-immunoprecipitation. ATF6 expression was maximal at ZT12 and minimal at ZT0. This was paralleled by increased ATF6/CRTC2 association at ZT16 (higher than ZT4). In contrast, CRTC2/CREB1 association was higher in ZT4 than in ZT16. Hypothalamic CRH expression peaked at ZT16. ADX rats have reduced ATF6 content and association with CRTC2. Moreover, ADX rats displayed increased CRTC2/CREB1 association, higher CRH expression at the end of the dark phase and reduced food intake. Acute exposition to DEX in vivo increased food intake, ATF6 expression and association with CRTC2 and decreased CRTC2/CREB1 association. Our data suggest that physiological levels of glucocorticoids might repress CRH expression and stimulate food intake through an ATF6-dependent pathway. We propose that ATF6 may associate with CRTC2 thus decreasing the formation of the CRTC2/CREB1 complex. These events, by extension, would decrease CREB1 transcriptional activity over CRH.­

 

Nothing to Disclose: CCM, DSF, APLB, JM, LAV, GFA

FP03-6 6148 6.0000 SAT-6 A ATF6 Association With CRTC2 As A Possible Mechanism Through Which Glucocorticoids Controls Hypothalamic Crh Expression and FOOD Intake 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 11:30:00 AM FP03 2203 11:00:00 AM Glucocorticoids & Glucocorticoid Actions Poster Preview


Aarthi Arasu*1, Peggy M Cawthon2, Li-Yung Lui2, Jane A Cauley3, Kristine E Ensrud4 and Steven R Cummings5
1UCLA, Torrance, CA, 2California Pacific Medical Center, San Francisco, CA, 3University of Pittsburgh, Pittsburgh, PA, 4University of Minnesota, Minneapolis, MN, 5CA Pacific Med Ctr, San Francisco, CA

 

Context: Dickopff-1 (DKK-1), an osteocyte-secreted inhibitor of the Wnt pathway, decreases bone formation. We have shown that sclerostin, another Wnt antagonist, increased risk of hip fracture in older women independent of bone mineral density.

Objective: To test the hypothesis that elevated serum DKK-1 levels are associated with increased risk of hip and vertebral fractures in older women.

Design, Setting, and Participants: SOF prospectively studied a community based cohort of 9704 women aged ≥65 years.  DKK-1 levels were measured by the Biomedica ELISA assay in serum collected in 1989-90 in 228 women with subsequent hip fractures, 109 women with new vertebral fractures, and 227 women randomly selected from the cohort. Average follow-up time was approximately 9.8 years for hip fractures and 3.5 years for vertebral fractures.  

Results: The risk of hip fracture increased across quartiles of serum DKK-1 (test for trend, p<0.01).  Women in the highest quartile of DKK-1 (43.2-174.9 pmol/L) had about twice the risk of hip fracture (HR 2.2, 95% CI 1.2-3.8) compared to women in the lowest quartile (4.8 – 21.5 pmol/L).  The risk of vertebral fracture increased by 39% for every 1 standard deviation (22.6 pmol/L) increase in DKK-1 levels (OR 1.39, 95% CI 1.09-1.78).  Results were similar after adjusting for age, body mass index, estrogen use, history of fracture since age 50, and BMD for both hip and vertebral fractures.  Serum DKK-1 levels did not significantly correlate with total hip bone mineral density

Conclusions: We conclude that higher serum DKK1 levels are associated with a greater risk of hip and vertebral fractures in older women. 

 

Nothing to Disclose: AA, PMC, LYL, JAC, KEE, SRC

FP10-1 3945 1.0000 SAT-224 A Elevated Serum Dickopff-1 Levels Increase Risk of Hip and Vertebral Fracture in Older Caucasian Women 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 15th 11:30:00 AM FP10 2225 11:00:00 AM Osteoporosis & Other Metabolic Bone Diseases Poster Preview


Claire Theret1, Laure Esterle2, Pierre-Francois Souchon3, Gwennaelle Roussey4, Emma Allain-Launay4, Anya Rothenbuhler5, Georges Deschenes6, Catherine Chaussain7, Dominique Prie8, Peter Kamenicky9, Caroline Silve10 and Agnes Linglart*11
1Hôpital de Lons le saunier, 2APHP Hôpital Bicêtre, Le Kremlin Bicetre, France, 3Reims hospital, Reims, France, 4Nantes hospital, Nantes, France, 5Bicetre Hospital, France, 6Robert Debre hospital, Paris, France, 7Paris descartes University, Paris, France, 8Necker hospital, Paris, France, 9Fac Med Paris-Sud, Le Kremlin Bicetre, France, 10Faculte de Med Xavier Bichat and INSERM U986, Paris, France, 11CHU de Bicetre, LE KREMLIN BICETRE, France

 

X-linked hypophosphatemic rickets (XLHR), due to loss of function mutations in the endopeptidase PHEX, is the most frequent form of HR with elevated FGF23. PHEX is expressed by osteoblasts, osteocytes and odontoblasts; its precise function in controlling circulating FGF23 level is still unclear. FGF23, secreted by osteoblasts and osteocytes regulates phosphate handling and vitamin D metabolism mainly through its action on kidney. Extra renal effects of FGF23, including in bone, have been very recently suspected from FGF23 overexpression or underexpression in mouse models. Specific missense mutations of FGF23 prevent FGF23 cleavage and inactivation, and thus result in the rare autosomal dominant form of HR (ADHR) with elevated circulating FGF23. Although XLHR and ADHR manifest as HR and elevated FGF23, only patients with XLHR present a specific loss in PHEX function within PHEX expressing tissues (bone and teeth).

Aim

Examine the role of PHEX on bone and mineral metabolism by comparing the phenotype of patients with high FGF23 and HR due to PHEX or FGF23 mutations.

Patients

6 patients with FGF23 mutation and ADHR (4 children and 2 untreated adults); 23 patients with PHEX mutation and XLHR (18 children and 5 untreated adults); XLHR and ADHR patients were matched for age at start of treatment

Results

Children with FGF23 mutations were diagnosed earlier (1.5±0.0 yrs) than children with PHEX mutations (2.3±0.2 yrs, p=0.03), with similar leg bowing (intercondylardistance 7.9±2.3 and 5.0±0 .7, respectively, p>0.05). At diagnosis, ADHR patients presented with bone demineralization (semi-quantitative assessment on X-rays) and fractures in one patient, whereas none of the 18 XLHR patients had bone demineralization or fractures. In addition, ADHR patients had significantly higher alkaline phosphatases than XLHR patients (2037±439 and 649±103, p=0.01, respectively). Phosphate, PTH and urinary calcium excretion were similar in both groups. Patients follow up revealed that, in opposition to XLHR, vitamin D analogs and phosphate supplements easily restored serum phosphate levels in ADHR; final height of untreated ADHR adults appears higher (-1.2 and -1 SD) than that of untreated XLHR (-3,2±1.3 SD).

Conclusion

Despite the limited number of patients, we pinpointed differences in the phenotypes of ADHR and XLHR. This suggests that the phenotype associated with PHEX deficiency does not uniquely result from FGF23 excess, yet advocates for a direct role of PHEX on bone mineralization and growth.


 

Nothing to Disclose: CT, LE, PFS, GR, EA, AR, GD, CC, DP, PK, CS, AL

FP10-2 5965 2.0000 SAT-228 A Patients With Mutations In Phex Or FGF23 Share FGF23 Excess But Present Distinct BONE and Mineral Metabolism Features 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 15th 11:30:00 AM FP10 2225 11:00:00 AM Osteoporosis & Other Metabolic Bone Diseases Poster Preview


Alexander Terence Faje*1, Pouneh K. Fazeli2, Karen K. Miller1, Erinne Neubecker Meenaghan1, Madhusmita Misra1 and Anne Klibanski1
1Massachusetts General Hospital/Harvard Medical School, Boston, MA, 2Neuroendocrine Unit, Massachusetts General Hospital/Harvard Medical School, Boston, MA

 

Adolescent girls and adult women with anorexia nervosa (AN) have low areal bone mineral density (aBMD) compared to normal-weight controls.  Studies have demonstrated an increased risk of fracture in adults with AN.  It is unknown whether the risk of childhood fracture is increased in adolescents with AN.  Measurement of aBMD has a limited ability to predict fractures in healthy children.  The relationship between aBMD and fracture prevalence has not been examined in adolescents with AN.

Objective: To (i) determine whether the prevalence of childhood fracture is increased in adolescent girls with AN compared to normal-weight controls and (ii) examine whether reductions in aBMD are associated with fracture risk in girls with AN.

Design and Methods: 418 females (310 with active AN and 108 normal-weight controls) 12-22 years of age were studied.  Subjects were recruited consecutively through print and electronic advertisements and by referral from regional practitioners in the northeastern United States.  Body composition and aBMD measurements of the whole body, whole body less head, lumbar spine, hip, and radius were assessed by dual-energy x-ray absorptiometry (DXA).

Results: AN subjects and normal-weight controls did not differ for chronological age, sexual maturity, or height.  The prevalence of prior fracture was 59.8% higher in those with AN compared to controls (31.0% versus 19.4%, p=0.02).  The distribution of fracture sites did not differ in the two groups.  aBMD was significantly lower at all sites in the AN group compared to controls (p values 0.007 to <0.0001).  Lower aBMD and lumbar bone mineral apparent density were not associated with a higher prevalence of fracture in the AN or control group on univariate or multivariate analyses.  Compared to controls, fracture prevalence was significantly higher in the subgroup of girls with AN who had modest reductions of aBMD (Z-scores > -1 or -1.5).

Conclusions: For the first time, we show that the risk of childhood fracture is significantly higher in AN than in a normal-weight population.  Increased fracture prevalence is observed in this cohort of subjects with AN even without significant reductions of aBMD.  Serial measurement of aBMD, the presence of microarchitectural deterioration, or additional unspecified risk factors may have greater value for the prediction of childhood fracture in AN.  Prospective studies are needed to determine effective predictors of site-specific fractures in this high risk population.

 

Nothing to Disclose: ATF, PKF, KKM, ENM, MM, AK

FP10-5 7225 5.0000 SAT-229 A Fracture Risk and Areal Bone Mineral Density in Adolescent Girls With Anorexia Nervosa 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 15th 11:30:00 AM FP10 2225 11:00:00 AM Osteoporosis & Other Metabolic Bone Diseases Poster Preview


Andre B. Araujo*1, Robert Chang2, Nicholas Dagincourt2, Shan Chen2, Gretchen Chiu1, Elizabeth Suarez2, Rachael Gerber2, Julia Akeroyd2 and Carrie G. Wager2
1New England Research Institutes, Watertown, MA, 2New England Research Institutes, Inc., Watertown, MA

 

The relative influence of fat vs. lean tissue on bone strength has been the subject of numerous epidemiologic and clinical studies, and data are mixed. Cross-sectional data from our Boston Area Community Health/Bone (BACH/Bone) Survey indicate a strong association of lean mass (LM) vs. fat mass (FM) with bone strength among men. Unfortunately, relatively few population-based longitudinal studies of men have examined this issue. The objective of this study was to examine the relation of baseline LM and FM with bone loss at the femoral neck. We used data from the BACH/Bone Survey, a population-based cohort study of 1219 racially diverse men who were examined at baseline and follow-up. Femoral neck bone mineral density (fnBMD) as well as total body LM and FM were assessed by dual x-ray absorptiometry (DXA) using the same scanner at baseline and follow-up. Multivariable linear regression was used to examine the independent associations of LM and FM (quartiles (Q) and continuous) assessed at baseline with percent (%) change in fnBMD, controlled for baseline fnBMD, age, race/ethnicity, income, smoking, type 2 diabetes mellitus, and self-rated health. A total of 692 men (70% of eligible men) with mean±SD baseline age 51±12y completed follow-up examinations 7.0±0.6y later. Estimated (relative standard error; RSE) % decline in fnBMD was 5.0(0.2)% during follow-up (annualized decline, 0.71%/y), with steeper declines in men 70+ y (6.8(0.9)%), and among black (6.0(1.1)%) and Hispanic (6.1(1.1)%) vs. white (4.8(1.1)%) men. Estimated (RSE) % declines in fnBMD were 4.4(0.5)% in Q1 of LM compared with 5.2(0.5)% among men in Q4, whereas in Q1 for FM, fnBMD declined by 5.5(0.5)% vs. 4.7(0.5)% among men in FM Q4. In multivariable models, FM (Q, p=0.05, continuous, p=0.02) but not LM (Q, p=0.12, continuous, p=0.11) was significantly associated with change in fnBMD. For FM quartiles, least-square means were as follows: Q1, -6.1%; Q2, -5.7%; Q3, -4.7%, and Q4, -4.2%. Similar results, although not significant (p=0.16), were observed for BMI considering normal, overweight, and obese groups. For each 10kg decrease in LM, the decline in fnBMD was 0.8±0.3% greater. Variation in the influence of FM or LM on fnBMD decline according to age decade or race/ethnicity was not present. In conclusion, declines in fnBMD in this relatively young and racially diverse population-based cohort of men are consistent with previous studies. In addition, baseline levels of FM are strongly predictive of declines in fnBMD over a 7y follow-up period, independent of LM and other potential confounders. Although recent opinion suggests a potentially deleterious influence of FM on skeletal outcomes, these data show that elevated FM is protective against bone loss in middle-aged men, and as such, are consistent with other studies showing the negative effect of low weight on bone loss and fractures.

 

Disclosure: ABA: Principal Investigator, Abbott Laboratories, Principal Investigator, GlaxoSmithKline, Consultant, Lilly USA, LLC. Nothing to Disclose: RC, ND, SC, GC, ES, RG, JA, CGW

FP10-6 8594 6.0000 SAT-225 A Body Composition in Relation to Bone Loss At the Femoral Neck At the Hip Among Racially/Ethnically Diverse Middle-Aged Men 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 15th 11:30:00 AM FP10 2225 11:00:00 AM Osteoporosis & Other Metabolic Bone Diseases Poster Preview


Maria Moisidou*, Sevasti Karaliota, Styliani Ourailidou, Elisavet Kodela, Panagiotis Tsakanikas and Katia/Catherine P Karalis
Biomedical Research Foundation of the Academy of Athens, Athens, Greece

 

Food deprivation, to the extreme state of starvation, has serious impact upon several physiologic functions including immunity and metabolism. Emerging evidence supports the critical role of immune cells in the physiological changes associated with reduced food intake, with the main focus on amino acid deprivation and its downstream implications.  Although profound changes in lipid storage, with liver steatosis remaining the hallmark finding, are associated with starvation, data on the specific role of immune cells on lipid metabolism remains limited. The aim of this study was to elucidate the role of lymphocytes in starvation in mice, with an emphasis in the development of non-alcoholic fatty liver disease, NAFLD. For this purpose, we compared the responses of the lymphocyte-deficient rag1-/- and wild-type mice to starvation–induced NAFLD. To our surprise, macroscopically the livers of rag1-/- starved mice had normal appearance and minimal signs of lipid deposition were revealed by histology, in contrast to the abnormal appearance and the profound signs of steatosis in the wt mice. Genomic analysis of liver tissue from both groups identified significantly activated lipid oxidation pathway in the rag1-/- compared to the wt tissue. Interestingly, the adipocytes’ size of rag1-/- mice was also significantly smaller from that of the wt mice, indicating more efficient lipolysis in fat stores. In support of the above, indirect calorimetry analysis under normal feeding and refeeding states, showed significant decrease in the respiratory exchange ratio of the rag1-/- mice, as expected in association with more efficient lipid versus carbohydrate utilization. Similar experiments in the model organism Zebrafish, confirmed the differences in the lipid deposition in rag1-/- mutants in association with altered caloric intake. Our results provide evidence for the critical role of lymphocytes in starvation-induced hepatic lipid metabolism in experimental animal models. On-going studies aim to characterize the exact metabolic pathways and identify the particular factors involved in the lymphocyte-driven effects on lipid metabolism.

 

Nothing to Disclose: MM, SK, SO, EK, PT, KPK

FP05-1 6752 1.0000 SAT-727 A Contribution of the Immune System On Lipid Metabolism 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Cardiovascular Endocrinology Saturday, June 15th 11:30:00 AM FP05 2232 11:00:00 AM Lipids: Regulation & Mechanism of Disease Poster Preview


Andrzej Jan Milewicz*1, Lukasz Laczmanski2, Justyna Kuliczkowska2, Agnieszka Lenarcik2, Katarzyna Kolackov2, Maurycy Pawlak2, Felicja Lwow3 and Marek Bolanowski4
1Medical University Wroclaw, Wroclaw, Poland, 2Wroclaw Medical University, Wroclaw, Poland, 3University School of Physical Education, Wroclaw, Poland, 4Wroclaw Univ Med School, Wroclaw, Poland

 

Nonalcocholic fatty liver disease (NFLD) may be evident in woman with PCOS, both conditioning associating with metabolic disorders. Endocannabinoids modulate eating behavior; hence, endocannabinoid genes may contribute to the biological vulnerability to eating disorders, obesity, fat cumulation and others. The aim of our study were to develop the hypothetical role of the CNR1 polymorphisms in etiology of nonalcocholic fatty liver disease in woman with PCOS (diagnosis on the ESHRE/ASRM criteria) in comparison to homogenous healthy control.

208 woman with PCOS and 120 woman as control were study. NFLD were diagnose on the basis of USG and biochemical markers. Genomic DNA were isolated using standard method. To polymorphisms identification PCR and minisequencing were used. Reaction products were separated on Genetic Analyser ABI 3100. For statistic analysis Hardy-Weinberg Equilibrium was used.

62% woman with PCOS presented NFLD in comparison to 48% in control. Our results showed significantly higher frequency of the A/A genotype of the rs806381 (p<0. 001258) and G/G genotype of the rs10485170 (p<0.000027) in woman with PCOS and NFLD versus PCOS woman without NFLD. These specific correlation was not observed in control group. We observed also significantly higher (p<0.001718) frequency of the A/A genotype of rs1049353 CNR1 polymorphism vs. controls. As well as G/G genotype of the rs806381 CNR1 polymorphism (p<0.000086) vs. controls. The relation between CNR1 polymorphisms to hormonal and metabolic profile will discuss.

Our preliminary results suggest potencial role of the CNR1 polymorphisms in etiology of the NFLD specially in PCOS woman what need further study.

 

Nothing to Disclose: AJM, LL, JK, AL, KK, MP, FL, MB

FP05-2 5530 2.0000 SAT-724 A Connection of the CNR1 Polymorphisms (rs806368, rs12720071, rs1049353, 806381, rs10485170, rs6454674, rs2023239) With Nonalcocholic Fatty Liver Disease in PCOS and Healthy Controls 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Cardiovascular Endocrinology Saturday, June 15th 11:30:00 AM FP05 2232 11:00:00 AM Lipids: Regulation & Mechanism of Disease Poster Preview


Ho Cheol Hong*1, Hae Yoon Choi1, Jae Hee Ahn1, Nam Hoon Kim1, Yoon Jung Kim1, Hye-Jin Yoo2, Hee Young Kim3, Ji A Seo4, Sin Gon Kim5, Nan Hee Kim6, Kyoung-Mook Choi7, Sei Hyun Baik1 and Dong Seop Choi8
1College of Medicine, Korea University, Seoul, Korea, 2Korea University Guro Hospital, Seoul, Korea, Republic of (South), 3Korea Univ ANAM Hospital, Seoul, Korea, Republic of (South), 4Korea Univ Ansan Hosp, Ansan City, Korea, Republic of (South), 5College of Medicine, Korea University, Seoul, Korea, Republic of (South), 6Korea Univ Ansan Hosp, Ansan, Korea, Republic of (South), 7Korea Guro Univ Hosp, Seoul, Korea, Republic of (South), 8Korea UNIV HOSP, Seoul, Korea, Republic of (South)

 

Objective: Progranulin and C1q/TNF-related protein-3 (CTRP3) were recently discovered as novel adipokines which may link obesity with altered regulation of glucose metabolism, chronic inflammation and insulin resistance.
Research Design and Methods: We examined circulating progranulin and CTRP3 concentrations in 127 subjects with (n = 44) or without metabolic syndrome (n = 83). Furthermore, we evaluated the relationship of progranulin and CTRP3 levels with inflammatory markers and cardiometabolic risk factors, including high-sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6), estimated glomerular filtration rate (eGFR), and adiponectin serum concentrations, as well as carotid intima-media thickness (CIMT).
Results: Circulating progranulin levels are significantly related with inflammatory markers, hsCRP (r = 0.30, P = 0.001) and IL-6 (r = 0.30, P = 0.001), whereas CTRP3 concentrations exhibit a significant association with cardiometabolic risk factors, including waist circumference (r = -0.21), diastolic blood pressure (r = -0.21), fasting glucose (r = -0.20), triglyceride (r = -0.34), total cholesterol (r = -0.25), eGFR (r = 0.39) and adiponectin (r = 0.26) levels. Serum progranulin concentrations were higher in patients with metabolic syndrome than those of the control group (199.55 [179.33, 215.53] vs. 185.10 [160.30, 204.90], P = 0.051) and the number of metabolic syndrome components had a significant positive correlation with progranulin levels (r = 0.227, P =0.010). In multiple regression analysis, IL-6 and triglyceride levels were significant predictors of serum progranulin levels (R2 = 0.251). Furthermore, serum progranulin level was an independent predictor for increased CIMT in subjects without metabolic syndrome after adjusting for other cardiovascular risk factors (R2 = 0.365).
Conclusions: Serum progranulin levels are significantly associated with systemic inflammatory markers and were an independent predictor for atherosclerosis in subjects without metabolic syndrome.

 

Nothing to Disclose: HCH, HYC, JHA, NHK, YJK, HJY, HYK, JAS, SGK, NHK, KMC, SHB, DSC

FP05-3 5312 3.0000 SAT-736 A Implication of Progranulin and C1q/TNF-related Protein-3 (CTRP3) on Inflammation and Atherosclerosis in Subjects With or Without Metabolic Syndrome 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Cardiovascular Endocrinology Saturday, June 15th 11:30:00 AM FP05 2232 11:00:00 AM Lipids: Regulation & Mechanism of Disease Poster Preview


Mark A Sperling*1, Yong Fan2, Xing Fang2, Asako Tajima2 and Massimo Trucco2
1Children's Hosp - Pittsburgh, Pittsburgh, PA, 2Division of Immunogenetics, Pittsburgh, PA

 

Progression of hepatic steatosis to fibrosis and adenoma formation in liver specific growth hormone receptor knockout (GHRLD) mice

Yong Fan, Xing Fang, Asako Tajima, Massimo Trucco, Mark A Sperling.

Department of Pediatrics, Children’s Hospital, University of Pittsburgh

Introduction:Non-alcoholic fatty liver disease (NAFLD) is one of the most common forms of chronic liver disorders closely related to obesity and insulin resistance. Non-alcoholic steatohepatitis (NASH) develops from NAFLD, and is characterized by lipid accumulation with evidence of hepatocellular damage, inflammation and progressive fibrosis, ultimately resulting in cirrhosis and adeno-carcinoma formation. We and others have shown that liver-specific deletion of GHR or JAK2/STAT5 signaling in mice leads to NAFLD (1-3). We investigated the progression of NAFLD over the lifespan in mice with GHRLD.

Methods: We performed sequential histology and histochemical analysis of liver samples from mice with GHRLD at 44 to 72 weeks of age on normal chow, followed by quantitative PCR for markers of inflammation (TNFalpha, CCL3, IL1a, IL1b), fibrosis (Col1A2, Col3A1, MMP13, TGFb), and liver regeneration (Egr-1). Results are reported from at least 5 mice in each group at 40-72 weeks comparing GHRLD to controls.

Results:In GHRLD, distinct nodular structure was first histologically observed at ~ 40 weeks. Microscopy (H&E) revealed the presence of areas with diminished steatosis, replaced by fibrosis and adenoma formation. Trichrome-Masson staining revealed marked increase of collagen deposition throughout the GHRLD sections. An increased mitotic index was apparent in the adenomas, in conjunction with increased markers of apoptosis. Real-time PCR analysis of key markers of inflammation revealed a 3-5 fold increase in TNFa, IL1a, IL1b, CCL3 and MMP13, confirming the severity of inflammation. The expression of fibrotic markers, Col1A2 and Col3A1, were 15-20 fold increased, together with a 70% increase in TGFb transcripts. The marker of liver regeneration, Egr-1, was 25-35 fold increased in the GHRLD liver.    

Conclusion: Abrogation of GHR signaling in the liver results in hepatic steatosis due to increased synthesis and decreased export of triglyceride, which can be rescued by adenovirus-mediated GHR, but not by IGFI (1). In time, hepatic steatosis progresses to NASH, with increased markers of inflammation and fibrosis, ultimately leading to adenoma formation. Since obesity, a common precursor of NAFLD, is a state of deficient GH secretion and action (4), the GHRLD model could be used to dissect the molecular mechanisms and to identify potential targets for therapy in these pathological processes.

References:

  1. Fan et al. (2009), J Biol Chem. 284 (30): 19937-44.
  2. Sos et al. (2011), J Clin Invest. 121 (4): 1412-23.
  3. Barclay et al. (2011), Endocrinology 152 (1): 181-192.
  4. Williams et al. (1984), N Eng J Med. 311: 1403-7.

 

Nothing to Disclose: MAS, YF, XF, AT, MT

FP05-4 8875 4.0000 SAT-728 A Progression of Hepatic Steatosis to Fibrosis and Adenoma Formation in Liver Specific Growth Hormone Receptor Knockout (GHRLD) Mice 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Cardiovascular Endocrinology Saturday, June 15th 11:30:00 AM FP05 2232 11:00:00 AM Lipids: Regulation & Mechanism of Disease Poster Preview


Thiago Matos Ferreira de Araujo*1, Juliana de Almeida Faria2, Andrezza Pinheiro Kinote2, Licio Augusto Velloso3 and Gabriel Forato Anhe4
1University of Campinas - UNICAMP, Campinas, Brazil, 2University of Campinas - UNICAMP, 3University of Campinas, Campinas, Brazil, 4State Univeristy of Campinas, Campinas, SP, Brazil

 

Background and aims: The “lipotoxicity” caused by excess of fatty acids are known to contribute to nonalcoholic steatohepatitis (NASH) and cell death in hepatocytes due to activation of ER stress-related pathways. AKT is a serine/threonine protein kinase that is essential for regulating cell growth, proliferation, survival and the interaction with environmental stimuli. Classically, AKT is fully activated by phosphoinositide-dependent kinase 1 (PDK1) and mammalian Target of Rapamycin (mTOR) complex 2, leading to inhibition of apoptosis. Alternatively, transitory AKT activation was demonstrated to cause cell death through yet not described mechanisms. ER stress triggers the Unfolded Protein Response (UPR) that is comprised by three independent branches; the PKR-like ER kinase (PERK), the activating transcription factor (ATF)-6 and serine/threonine-protein kinase/endoribonuclease Ire1. The aim of this study is to demonstrate if short-term AKT activation by palmitate is linked to ER stress and apoptosis in HepG2 cells.

Materials and methods: HepG2 cells were treated with palmitate (0,5 mmol/l) pre-conjugated with albumin, rapamycin (500 nmol/l) and/or AKT Inhibitor (100 nmol/l). Cells were also transfected with siRNA targeted to Raptor or a siRNA targeted to Rictor. A scrambled siRNA was used as control. mTORC2/AKT pathway and UPR were assessed by Western blotting. Cell death was assessed by DNA fragmentation.

Results: Palmitate-induced apoptosis was maximal after 12h of treatment. CHOP, a marker of ER-stress-dependent apoptosis was also modulated by palmitate and peaked 12h after exposition to palmitate. Processed ATF6 was upregulated 3h after palmitate exposition and these levels were sustained up to 12h. Palmitate also activated PERK and increased ATF4 expression (respectively after 6 and 12h of treatment). The events were preceded by short-term and transitory AKT activation by palmitate. By inhibiting mTORC1/2 with rapamycin we observed a suppression of palmitate-induced UPR activation and apoptosis. Similarly inhibition of apoptosis and ER stress was also observed with the use of a specific inhibitor for AKT (“AKT inhibitor”) and Rictor knockdown with siRNA.

Conclusion: Our results demonstrated that palmitate induces a short-term and transitory AKT activation that leads to long term activation of UPR and apoptosis. This response is likely to be triggered by mTORC2 activation. All together, those results could help to the understanding of NASH physiopathology.

 

Nothing to Disclose: TMFD, JDAF, APK, LAV, GFA

FP05-5 6387 5.0000 SAT-726 A Temporary AKT Activation Contributes to Endoplasmic Reticulum Stress and Apoptosis in Hepatocytes Induced By Palmitate 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Cardiovascular Endocrinology Saturday, June 15th 11:30:00 AM FP05 2232 11:00:00 AM Lipids: Regulation & Mechanism of Disease Poster Preview


Rasheed Ivey*1, Indrani Sinha-Hikim1, Merra Deasi1, Chang-Sung Shin1, Theodore C Friedman1 and Amiya P Sinha-Hikim2
1Charles R. Drew University, Los Angeles, CA, 2Charles R Drew University of Medicine and Science, Los Angeles, CA

 

Background and Objective: Smoking is a major risk factor for diabetes and cardiovascular disease and may contribute to non-alcoholic fatty liver disease (NAFLD). The health risk associated with smoking is exaggerated by obesity and is the leading causes of morbidity and mortality worldwide. We recently demonstrated that combined treatment with nicotine and a high-fat diet (HFD) triggers greater oxidative stress, activates hepatocellular apoptosis, and exacerbates HFD-induced hepatic steatosis (Endocrinology 153: 5809-5820, 2012). Given that hepatocellular apoptosis plays a pivotal role in the pathogenesis of NAFLD, using this model of exacerbated hepatic steatosis, we elucidated the signal transduction pathways involved in HFD plus nicotine-induced liver cell death.

Experimental Design: Adult C57BL6 male mice were fed a normal chow diet or HFD with 60% of calories derived from fat and received twice daily IP injections of 0.75 mg/kg BW of nicotine or saline for 10 weeks.

Results: Light and electron microscopy revealed markedly higher lipid accumulation with lower content of endoplasmic reticulum and glycogen in hepatocytes from mice on HFD plus nicotine, compared to mice on HFD alone. Addition of nicotine to HFD further resulted in an increase in the incidence of hepatocellular apoptosis and was associated with activation of caspase 2 (an initiator caspase working upstream of kinase activation and mitochondria-dependent apoptotic pathway), p38 mitogen-activated protein kinase (MAPK), induction of inducible nitric oxide synthase (iNOS), and perturbation of the BAX/BCL-2 ratio.

Conclusions: Together, our data indicate the involvement of caspase 2, p38 MAPK, and iNOS that though activation of intrinsic pathway signaling promotes hepatocellular apoptosis and further worsen HFD-induced hepatic steatosis in obese mice. Targeting the caspase 2-mediated death pathway may have a protective role in development and progression of NAFLD.

 

Nothing to Disclose: RI, IS, MD, CSS, TCF, APS

FP05-6 4561 6.0000 SAT-723 A Additive Effects of Nicotine and High-Fat Diet On Hepatic Steatosis and Hepatocellular Apoptosis in Mice: Involvement of Caspase 2 and Inducible Nitric Oxide Synthase-Mediated Intrinsic Pathway Signaling 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Cardiovascular Endocrinology Saturday, June 15th 11:30:00 AM FP05 2232 11:00:00 AM Lipids: Regulation & Mechanism of Disease Poster Preview


Nagesha Guthalu Kondegowda*1, Anais Lynda Mozar2, Adolfo Garcia-Ocana3 and Rupangi C Vasavada3
1University of Pittsburgh, Pittsburgh, PA, 2Vasavada Lab, New York, NY, 3Mount Sinai School of Medicine, New York, NY

 

Finding ways to expand endogenous functional β-cell mass is an important goal for the treatment of diabetes.  Osteoprotegerin (OPG), a member of the Tumor Necrosis Factor (TNF) receptor superfamily, is upregulated in four distinct models of rodent β-cell expansion, leading us to hypothesize that OPG may directly enhance β-cell proliferation.

To examine this, C57BL6 male mice were injected for 7 days with mouse recombinant OPG (mOPG) (2–2000ng/g body weight) or saline as control (n=3-8 mice/group). Treatment with mOPG increased β-cell proliferation, analyzed by bromodeoxyuridine (BrdU) and insulin co-staining, by 2-3-fold at the lower doses (2-250ng) and significantly by 4-8-fold at the higher doses (500-2000ng) versus control. Longer (30 days) treatment with mOPG (10-500ng/g body weight) also resulted in a significant increase in β-cell proliferation at the higher dose. 

To determine if OPG has a similar effect on human β-cells, human islet cell cultures (n=3-5) were treated with 25–200ng/ml of human recombinant OPG (hOPG) and β-cell proliferation was assessed using Ki-67 co-staining with insulin. There was a significant 3-4-fold increase in human β-cell proliferation with the higher hOPG doses. This confirmed our earlier observation of the pro-proliferative effect of hOPG on human β-cell proliferation by BrdU staining. We have previously shown that hOPG also improves survival of human β-cells, against glucolipotoxicity (GLT) and cytokine-induced cell death. 

OPG actions are mediated through inhibition of the receptor activator of nuclear factor kappa-B ligand (RANKL) or the TNF-related apoptosis-inducing ligand (TRAIL) pathways in other tissues. To examine the mechanism of OPG action in human β-cells, we treated human islet cell cultures with denosumab (1-200ng/ml), a human monoclonal antibody against RANKL, which acts as a partial functional equivalent of OPG, affecting only the RANKL but not the TRAIL pathway. Denosumab significantly increased not only human β-cell proliferation by 2-3-fold as observed by both BrdU and Ki-67 staining, but also significantly ameliorated (~60%) GLT-induced human β-cell death (n=3-4), implying that the pro-survival and proliferative effects of OPG are mediated through inhibition of the RANKL pathway. 

Overall, our studies reveal that OPG can enhance rodent and human β-cell proliferation as well as improve human β-cell survival, likely through the RANKL pathway.

 

Nothing to Disclose: NG, ALM, AG, RCV

FP13-1 5692 1.0000 SAT-837 A Osteoprotegerin Increases Rodent Beta Cell Proliferation in Vivo, and Enhances Human Beta Cell Proliferation and Survival Through Inhibition of RANKL 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Diabetes & Glucose Metabolism Saturday, June 15th 11:30:00 AM FP13 2249 11:00:00 AM GI Peptides, Beta Cells & Glycemia Poster Preview


Lane Jaeckle Santos*1 and Rebecca Anne Simmons2
1University of Pennsylvania, Philadelphia, PA, 2Univ Penn Med Ctr-BRB II/III, Philadelphia, PA

 

Intrauterine growth retardation (IUGR) is linked to the later development of type 2 diabetes (T2D). We have developed an animal model of IUGR, which leads to the development of T2D in adulthood. Inflammation is associated with T2D and is a critical component of metabolic syndrome in both human and experimental models, but it is unknown whether inflammation is causal or secondary to the abnormal metabolic state. We hypothesized that IUGR induces fetal inflammation in the pancreatic islet, which in turn leads to decreased islet vascularity and impaired insulin secretion.

Microarray analysis of fetal and PD14 IUGR islets showed marked changes in expression of genes regulating immune mediated inflammation, macrophage activation and angiogenesis. Histological examination of e19 and PD14 IUGR islets show decreased capillary density, and invasion by T-lymphocytes and macrophages. Levels of IL-2, IL-4 and IL-10 were significantly elevated in fetal islet lysates, consistent with Th2 immune response. By PD14, systemic levels of insulin, leptin and the pro-inflammatory cytokines mcp1 and RANTES, which recruit macrophages to sites of inflammation and amplify their effects, were significantly elevated, demonstrating systemic inflammation and the beginning of metabolic syndrome. To determine whether Th2 inflammation is responsible for the abnormal ß-cell phenotype, animals received neutralizing IL-4 antibody treatment or vehicle at PD days 1-5. Neutralizing IL-4 treatment restored islet capillary density by PD14, reduced circulating insulin levels, and ameliorated systemic inflammation. At 15 weeks of age, IUGR animals treated with neutralizing IL-4 antibody showed completely normal insulin secretion in isolated perifused islets at 15 weeks.

Our results demonstrate that adult-onset diabetes secondary to IUGR is both preceded by and caused by fetal islet inflammation, resulting in immune cell invasion, inflammatory cytokine release, decreased islet vascularity and increased insulin resistance. Administration of neutralizing IL-4 antibodies at the neonatal stage suppresses inflammatory cytokine levels, normalizes islet vascularity, and permanently restores insulin sensitivity, demonstrating a novel role for Th2 immune responses in the induction and progression of T2D and metabolic syndrome. At the neonatal stage, inflammation and vascular changes are reversible, and may define an important developmental window for therapeutic intervention to prevent adult onset diabetes.

 

Nothing to Disclose: LJ, RAS

FP13-2 6517 2.0000 SAT-835 A Neutralizing IL-4 Rescues Inflammation in Neonatal Islets and Prevents β-Cell Failure in Adult IUGR Rats 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Diabetes & Glucose Metabolism Saturday, June 15th 11:30:00 AM FP13 2249 11:00:00 AM GI Peptides, Beta Cells & Glycemia Poster Preview


Mieke Baan*, Muhammad K Ahmed, Carly R Kibbe, Jeremy A Lavine and Dawn Belt Davis
University of Wisconsin, Madison, WI

 

Diabetes mellitus (DM) is caused by a reduced number of functional β-cells in the pancreas. Identifying factors that can protect β-cells from apoptotic stressors is important in developing effective diabetes therapies. The hormone cholecystokinin (CCK) is expressed in pancreatic islets of obese mice and is necessary for maintenance of β-cell mass in obesity (1). CCK treatment ex vivo prevents islet apoptosis secondary to thapsigargin-induced ER-stress (1). To test whether overexpression of CCK could protect β-cells from apoptosis in vivo, we generated transgenic mice with Cck expression driven by the mouse insulin promoter (MIP-CCK). As a model of in vivo β-cell apoptosis we used Akita mice, as they carry a missense mutation in the insulin gene causing misfolding of the protein, resulting in ER overload and β-cell apoptosis (2). Heterozygotes develop a diabetic phenotype around one month of age. MIP-CCK/Akita+/- males were compared with Akita+/- males. At 4 weeks of age, random fed glucose concentrations were elevated in both groups, but not significantly different (246 +/- 124 mg/dL vs 224 +/- 83 mg/dL, respectively, p=0.70, n=7). However, at 14 weeks MIP-CCK/Akita+/- males have less hyperglycemia than controls (429 +/- 53 mg/dL vs 289 +/- 102 mg/dL, n=7-9, p=0.0057). We examined pancreatic morphology with insulin and TUNEL staining in 14-16 week old animals.  By this stage, there is massive β-cell loss and ongoing apoptosis is no longer detectable.  However, the CCK overexpressors had more residual fractional β-cell area than controls (0.44% vs. 0.07%, n=2, p=0.015). In addition, mean islet size was larger in MIP-CCK/Akita+/- (4550 unit area vs. 1041 unit area, p=0.047). In summary, localized β-cell expression of CCK protects from ER-stress mediated β-cell loss in vivo, and CCK directed therapies may provide benefit in maintaining β-cell mass in diabetes.

 

Nothing to Disclose: MB, MKA, CRK, JAL, DBD

FP13-3 7391 3.0000 SAT-836 A β-Cell Expression of Cholecystokinin Ameliorates the Diabetic Phenotype in Akita Mice 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Diabetes & Glucose Metabolism Saturday, June 15th 11:30:00 AM FP13 2249 11:00:00 AM GI Peptides, Beta Cells & Glycemia Poster Preview


Kathryn Lisa Hackman*1, Gregory Snell2 and Leon Bach3
1Monash University, Melbourne, Australia, 2The Alfred Hospital, Melbourne, Australia, 3The Alfred, Melbourne, Australia

 

Diabetes (DM) is a known risk factor for mortality following lung transplantation (LTx) but few studies have systematically determined DM status both before and after LTx and assessed its effect on survival.

All 386 patients who underwent LTx at our institution between 1/1/2001 – 31/7/2010 were retrospectively assessed for DM using patient files and all available pathology test results. We categorised all patients’ DM status prior to and following LTx. Patients whose DM status was unknown (n= 9) or who had transient DM that then resolved (n= 10) were excluded from analyses. Kaplan-Meier and multivariate Cox regression analyses were used to assess median survival of the remaining 367 patients according to diabetes category and to determine whether DM independently affected survival after allowing for other risk factors.

There was a significant difference in survival between groups (p < 0.001). Sixty-seven of the 171 patients without DM died (39%). Estimated median survival in this group was 3677 days. Patients with new onset DM post LTx and those with pre- and post-LTx DM respectively had increased mortality rates of 59% and 49%, and significantly shorter median survivals of 1583 (CI 222-1148) and 1834 (CI 1198 – 2470) days.

On multivariate analysis, time-dependent DM status was the strongest predictor of mortality, conferring a 5.6-fold increased risk of mortality (95% CI 4.0-7.8, p <0.001). Other predictors were use of cyclosporin rather than tacrolimus (HR 1.8, 95% CI 1.2-2.7, p= 0.005) and baseline triglycerides (HR 1.6, CI 1.2-2.1, p<0.001). Protective factors were negative donor/recipient CMV status (HR 0.6, CI 0.4–0.9, p = 0.018) and underlying cystic fibrosis or bronchiectasis (HR 0.5, CI 0.4-0.8, p<0.001). In contrast, duration on LTx waitlist, smoking history, age, sex and BMI did not predict survival. Cause of death did not differ significantly in relation to DM status, and bronchiolitis obliterans (BOS) was the main cause, accounting for half of all deaths occurring after the first 3 months.

Diabetes is an important and potentially modifiable risk factor for mortality following lung transplantation. The minimal difference in survival between patients with pre-transplant vs new-onset DM post-LTx suggests that post transplant hyperglycaemia may have relatively rapid effects. It is possible that hyperglycaemia hastens the development of BOS. Further studies are warranted to investigate the effect of glycaemic control on lung transplant outcome.

 

Nothing to Disclose: KLH, GS, LB

FP13-6 5216 6.0000 SAT-839 A Diabetes Is the Major Risk Factor for Mortality in Lung Transplant Recipients 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Diabetes & Glucose Metabolism Saturday, June 15th 11:30:00 AM FP13 2249 11:00:00 AM GI Peptides, Beta Cells & Glycemia Poster Preview


Cynthia L Bethea*1, Oleg Varlamov2, Paul Kievit2 and Charles T Roberts Jr.3
1OR Regional Primate Res Ctr, Beaverton, OR, 2Oregon National Primate Research Center, Beaverton, OR, 3Oregon National Primate Research Center, Oregon Health & Science University, Beaverton, OR

 

Clinical and animal data suggest that atypical antipsychotics such as (OLZ) induce significant metabolic changes that are serious side effects of their primary use. Since controlled human studies are problematic and rodent data may be poorly translatable, we have started to develop a macaque model of OLZ-induced metabolic disease. A female Japanese macaque was administered OLZ (5 mg/kg/day) for 6 months, with dietary changes instituted at 2-month intervals; i.e., OLZ+restricted chow, OLZ+unrestricted chow, OLZ+western-style diet (WSD), and placebo+WSD. Weight was accessed weekly, with glucose tolerance tests and Dexa scans performed at baseline and every 2 months. On OLZ+restricted chow, weight increased 14% over baseline; on OLZ+unrestricted chow, weight increased further and stabilized at 26% over baseline; on OLZ+WSD, the animal found the diet unpalatable and weight returned to 14% over baseline, and remained similar on placebo+WSD. Body fat increased from 14% at baseline to 22%, 31%, 28% and 19% at 2, 4, 6 and 8 mo, respectively, indicating that body fat was elevated on OLZ regardless of diet and declined upon OLZ removal. OLZ alone induced partial glucose intolerance, while subsequent access to unlimited chow resulted in increased post-prandial hyperglycemia and loss of first-phase and decreased second-phase insulin secretion, which was exacerbated upon addition of WSD. After removal of OLZ but continued WSD, a more normal insulin secretory pattern was seen and hyperglycemia was reduced, although this was achieved through hyperinsulinemia, suggesting continued insulin resistance. There was no evidence for elevated fasting glucose. To evaluate adipose-specific effects, visceral (V) and subcutaneous (SC) adipose tissue biopsies were obtained at baseline and after OLZ+unrestricted chow and OLZ+WSD to evaluate adipocyte size, and lipolysis and insulin-stimulated fatty acid uptake. Adipocyte diameter was increased in SQ and, to a lesser degree, in V fat by OLZ alone. Basal lipolysis was unaffected by OLZ or diet, but isoproterenol-stimulated lipolysis was increased by OLZ in V and SQ fat, and subsequently reduced by addition of WSD. Insulin-stimulated fatty acid uptake was inhibited in both depots by OLZ alone and not further influenced by diet. We conclude that OLZ can exert metabolic effects that are independent of diet but that can be exacerbated by dietary excess, and that these changes involve alteration of both islet and adipose function.

 

Nothing to Disclose: CLB, OV, PK, CTR Jr.

FP02-4 6045 4.0000 SAT-788 A Olanzapine Modulation of Metabolic Status in a Non-Human Primate 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Diabetes & Glucose Metabolism Saturday, June 15th 11:30:00 AM FP02 2261 11:00:00 AM Obesity and Diabetes: Drugs & Interventions Poster Preview


Nora Alghothani*, Kathleen M Dungan and Prathima Pangulur
The Ohio State University, Columbus, OH

 

Background:  Two earlier cases have documented a discrepancy between A1C levels and glycemic control in patients with diabetes and HS. This however has not been reported for mild HS with normal hemoglobin despite a high incidence of diabetes in this hemolytic disorder.

Clinical Case:  A 22 year old Caucasian woman with type 1 diabetes diagnosed at age 11 was referred to Endocrinology for persistent hyperglycemia. Her regimen consisted of Lantus 16 units twice daily and Humalog 8 units with meals. Her initial A1C was 5.1% (n , 4.3 - 6.1 %) and routine chemistry and CBC revealed normal creatinine (0.67 mg/dL, n 0.60 - 1.10 mg/dL), hemoglobin (12.7 g/dL, n 11.1-15.5g/dL), hematocrit (35.7%, n 35-45%), MCV (85.5 fL, n 81-100 fL), MCHC (35.5 g/dL, n 32-36 g/dL), and RBC distribution (14.7, n 11.6-14.8). Exam was normal without hepatosplenomegaly. Given self-reported hyperglycemia, fructosamine, which measures the glycosylation of serum proteins rather than hemoglobin, was checked and was elevated (358 umol/L, n 200-285 umol/L). A peripheral smear was then obtained to assess for the presence of schistocytes or other RBC abnormalities that may falsely lower A1C, and found to be normal. Patient was eventually started on subcutaneous continuous insulin infusion. She had professional retrospective continuous glucose monitoring over a 6 day period that demonstrated marked hyperglycemic excursions, particularly in the evenings, with mean sensor glucose of 228 mg/dL +/- 102, and corresponding A1C of 5.4% and fructosamine of 365 umol/L. Throughout her 5 years of follow up, A1C ranged 4.0-5.6% and fructosamine 289-435 umol/L. She otherwise remained in overall good health with improved glycemic control. About 4 years after initial presentation, she reported a new diagnosis of spherocytosis in her mother, which prompted a work up for HS. A reticulocyte count was elevated at 5.4% (n 0.5 - 1.5 %) and an osmotic fragility test was supportive of HS with increased erythrocyte osmotic fragility due to spherocytosis. She was started on folic acid and recently became pregnant.

Conclusion:  This is the first case demonstrating a marked distortion of A1C in HS with normal hemoglobin. Given the relatively high frequency of mild HS, early testing should be considered in patients with an apparent discrepancy in A1C and meter readings, even in the absence of anemia. Alternate glycemic markers such as fructosamine are necessary to monitor chronic diabetic control in affected patients.

 

Nothing to Disclose: NA, KMD, PP

FP02-5 5419 5.0000 SAT-758 A A1C An Inaccurate Measure of Glycemic Control in a Woman With Hereditary Spherocytosis (HS) Despite Normal Hemoglobin Levels 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Diabetes & Glucose Metabolism Saturday, June 15th 11:30:00 AM FP02 2261 11:00:00 AM Obesity and Diabetes: Drugs & Interventions Poster Preview


Katherine Quinn Philla*1, Andrew Jacob Bauer1 and Siri Atma W Greeley2
1Walter Reed National Military Medical Center, Bethesda, MD, 2University of Chicago, Chicago, IL

 

Background: Although monogenic neonatal diabetes may be caused by mutations in over 20 different genes, the most common are activating heterozygous mutations in KCNJ11, encoding the Kir6.2 subunit of the ATP-sensitive potassium (KATP) channel, highly expressed in pancreatic beta-cells and brain. Mutated KATPchannels typically have decreased sensitivity to ATP inhibition, hampering insulin secretion even during hyperglycemia. Oral sulfonylureas (SU) have been demonstrated as an effective treatment in the majority of cases, where the likelihood of success is largely predicted by the particular mutation. The few previous reports of the P333L mutation suggested insensitivity to SU therapy.

Clinical Case: A 13-month old (mo) Caucasian male was diagnosed at 3 mo with insulin-requiring diabetes complicated by diabetic ketoacidosis. He was otherwise healthy and had normal growth and development. His insulin dose was 0.3 units/kg/day delivered via an insulin pump and most recent HbA1c was 9.5% (eAG 226 mg/dL). Given his young age of diabetes onset, genetic testing was ordered and revealed the dominant heterozygous KCNJ11mutation P333L.  Previously reported cases with this mutation failed an attempt to transition to SU. However, inpatient transition from insulin to SU therapy was attempted for this patient. Glyburide (glibenclamide) titrated up to a 0.3mg/kg/day was successful with complete discontinuation of insulin after 6 days. Pre-SU fasting c-peptide was <0.1 ng/mL (normal fasting 0.80-3.10 ng/mL) but post-SU fasting c-peptide increased to 0.47 ng/mL after 3 days of therapy and was normalized further to 1.86 ng/mL by 3-month outpatient follow up. HbA1c decreased to 6.7% (eAG 146mg/dl) at the 7 month follow-up. There were no adverse events observed. Complete blood count and complete metabolic profile results as well as neurodevelopment remained normal throughout treatment. Excellent diabetes control on oral SU monotherapy has been sustained throughout his 12-month follow up and the patient’s family regularly voices satisfaction on the decreased intensity of care required on the new oral regimen.

Conclusion: This case demonstrates the critical importance of molecular genetic diagnosis leading to the first successful transition from insulin to SU therapy in a patient with the P333L mutation in KCNJ11 mutation after few previously reported cases were unable to transition off of insulin and exhibited neurodevelopmental disability. Further study of these important rare cases will help to clarify the factors that influence the likelihood of successful SU treatment and neurodevelopmental outcome. Furthermore, this case highlights the variability of predicted response based exclusively on genotype and reemphasizes the importance, and potential life-altering impact, of genetic screening for patients suspected of having monogenic forms of diabetes.

 

Nothing to Disclose: KQP, AJB, SAWG

FP02-6 5158 6.0000 SAT-759 A First Report of Successful Transition From Insulin to Oral Sulfonylurea Therapy in a Patient With Monogenic Neonatal Diabetes Due to the P333L Mutation in KCNJ11 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Diabetes & Glucose Metabolism Saturday, June 15th 11:30:00 AM FP02 2261 11:00:00 AM Obesity and Diabetes: Drugs & Interventions Poster Preview


Ying Liu*1, Philip Alton Berry1, Yue Zhang1, Jing Jiang1, Kurt R. Zinn2 and Stuart J Frank2
1University of Alabama at Birmingham, Birmingham, AL, 2Univ of Alabama at Birmingham, Birmingham, AL

 

Growth hormone receptor (GHR) and prolactin receptor (PRLR) are single membrane-spanning proteins in the cytokine receptor superfamily. In humans, GH binds and activates both GHR and PRLR, while PRL binds and activates only PRLR. Structural and biochemical studies, as well as FRET and BRET data, suggest GHR and PRLR each form homodimers that may be modified by ligand binding. We previously demonstrated GH-dependent disulfide linkage of GHR homodimers (1), as well as association between GHR and PRLR that is enhanced by GH (2) in human cells that endogenously express these receptors. With the goal of better understanding the mechanism(s) and significance of GHR-PRLR association, we are developing a split luciferase complementation assay. Herein, we report our initial results. For studies of protein-protein association, the luciferase complementation assay tests the ability of expression of one protein molecularly fused to an N-terminal fragment of luciferase to be close enough to the second protein fused to a C-terminal fragment of luciferase to allow reconstitution of intact luciferase activity and measurement of bioluminescence as a report of the closeness of the two proteins. The assay features great sensitivity and specificity with a very low background signal. GHR-GHR homodimerization was initially examined with this system. We created chimeras that included either full-length human GHR (GHR) or human GHR truncated after residue 322 in the proximal cytoplasmic domain (TGHR). Each was fused with either Nluc (residues 1-398 of firefly luciferase) or Cluc (luciferase residues 394-550) with a flexible peptide linker between the GHR and luc portions. Plasmids encoding each fusion or a control with GHR fused to full-length luciferase were transfected alone or in combination into the JAK2-expressing, GHR-deficient human fibrosarcoma cell line, gamma2A-JAK2. Expression of each protein was verified by immunoblotting and the response to GH with JAK2 and/or STAT5 phosphorylation validated that each chimera achieved cell surface presentation. To date, we have confirmed specific luciferase complementation with coexpression of GHR-Nluc/GHR-Cluc and TGHR-Nluc/TGHR-Cluc. As a negative control, coexpression of GHR-Nluc with an estrogen receptor-Cluc fusion yielded no signal. Stronger complementation was observed with TGHR-Nluc/TGHR-Cluc than with GHR-Nluc/GHR-Cluc coexpression, in agreement with published FRET/BRET data. The effect of GH on these complementation signals is currently being evaluated and analogous PRLR fusions are being constructed. These studies should allow fine mapping of GHR-PRLR association determinants.

 

Nothing to Disclose: YL, PAB, YZ, JJ, KRZ, SJF

FP01-1 5406 1.0000 SAT-88 A Development of a Luciferase Complementation Assay to Investigate GH Receptor Dimerization 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Neuroendocrinology and Pituitary Saturday, June 15th 11:30:00 AM FP01 2278 11:00:00 AM Cell Specific GH & IGF-1 Signaling Poster Preview


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

 

Growth hormone (GH) regulates growth and metabolism by signaling through the GH receptor (GHR), a cytokine receptor linked to the JAK2 tyrosine kinase. GH activates the STAT5 transcription factor to promote expression of genes including insulin-like growth factor-1 (IGF-1). IGF-1 binds the IGF-1 receptor (IGF-1R), a heterotetrameric (alpha2-beta2) tyrosine kinase growth factor receptor structurally related to the insulin receptor (IR). In addition to the GH -> GHR -> IGF-1 -> IGF-1R pathway, we previously demonstrated that GH induces a protein complex including GHR, JAK2, and IGF-1R (1). Further, deletion of the floxed IGF-R in murine osteoblasts by treatment with an adenovirus directing the expression of the Cre recombinase (Ad-Cre) renders the cells less sensitive to acute GH-induced STAT5 activation and downstream IGF-1 mRNA accumulation. Diminished GH-induced STAT5 phosphorylation in Ad-Cre-treated osteoblasts was at least partially rescued by coinfection with adenoviruses encoding either full length IGF-1R (Ad-IGF-1R) or IGF-1R lacking the beta chain intracellular domain (Ad-IGF-1R-trunc) (2). Reasoning that the IGF-1R extracellular portion (alpha chain or extracellular part of the beta chain) may mediate this functional interaction with GH signaling, we pursued reconstitution studies in the osteoblast model. Although structurally related to IGF-1R, the adenovirally-expressed IR (Ad-IR) failed to rescue GH-induced STAT5 phosphorylation in Ad-Cre-treated cells. We thus created chimeras, swapping homologous extracellular regions of IR into the IGF-1R backbone. Both IR and IGF-1R possess the so-called L1, cysteine-rich (CR), and L2 domains t the N-terminal regions of the alpha chain. We created Ad-IR-L1/IGF-1R and Ad-IR-L1-CR-L2/IGF-1R, in which either the L1 domain alone or the L1, CR, and L2 domain region of IR replaces the corresponding region of the IGF-1R, respectively. Each chimera was detected by immunoblotting and migrated at the expected position in SDS-PAGE. In functional tests, we found that Ad-IR-L1/IGF-1R, but not Ad-IR-L1-CR-L2, rescued GH-induced STAT5 phosphorylation in Ad-Cre-treated osteoblasts. These data suggest that a determinant(s) of the IGF-1R alpha chain extracellular domain, likely in the CR-L2 region, specifically allows IGF-1R to collaborate with GHR and JAK2 to allow robust GH-induced acute STAT5 phosphorylation. Further mapping and mechanistic studies are underway.

 

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

FP01-6 5447 6.0000 SAT-90 A Functional Collaboration of IGF-1R, But Not IR, With Acute GH Signaling in Osteoblasts 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Neuroendocrinology and Pituitary Saturday, June 15th 11:30:00 AM FP01 2278 11:00:00 AM Cell Specific GH & IGF-1 Signaling Poster Preview


Smita Salian-Mehta*1, Mei Xu2, Connor Nash3, Mathew Scott Stratton4, Timothy A McKinsey4, Donald R Menick5, Stuart Allen Tobet6 and Margaret E Wierman7
1University of Colorado Denver, Aurora, CO, 2University of Colorado School of Medicine, Aurora, CO, 3Colorado state university, Fort Collins, CO, 4University of Colorado Denver, 5Medical University of South Carolina, 6CO State Univ, Fort Collins, CO, 7University of Colorado School of Medicine and Research Service VAMC, Aurora, CO

 

Abnormal sexual maturation and infertility in mice and humans result from disruption of Gonadotropin releasing hormone (GnRH) neuron development or function. Microarray analysis of GT1-7 (differentiated) as compared to NLT (undifferentiated) mouse GnRH neuronal cell lines revealed an upregulation of most HDACs and particularly class-IIa HDAC9. This class shuttles from cytoplasm to nucleus to classically act as transcriptional repressors although recent cytoplasmic action has been suggested during differentiation and development. Increases in HDAC9 by microarray (6-fold), RT-PCR (2.9-fold), Immunoblot (10-fold) and specific deacetylase activity in GT1-7 cells suggested that HDAC9 might play a role in GnRH neuronal development. Over-expression of hHDAC9 in NLT GnRH neuronal cells (low endogenous HDAC9) protected cells from serum withdrawal induced apoptosis as assessed by cleaved caspase 3 (0.6 vs.1.2 fold, compared to controls) and by Hoechst staining of condensed nuclei (0.5 fold), confirming a pro-survival role for HDAC9. Silencing of HDAC9 (by 80%) in GT1-7 cells, however, did not alter   caspase-3 cleavage, suggesting there may be redundancy with other Class II HDACs (4,5 and/or 7).The N-terminus of class-IIa HDACs is critical for protein-protein interactions (binding to transcription factors like MEF2) and C-terminus encodes the HDAC domain. To ask the role of the domains and nuclear versus cytoplasmic location to mediate HDAC9’s pro-survival effects, WT,  N- and C- terminal HDAC9 mutants were tested. Immunocytochemistry demonstrated WT hHDAC9 protein was expressed preferentially in the nucleus (N>C), whereas HDAC-N was only nuclear and HDAC9-C (deacetylase domain) was exclusively cytoplasmic. In response to growth factor withdrawal induced apoptosis,  both WT and C-terminal, but not N-terminal HDAC9 mutants showed decreased caspase 3/7 luminescence as compared to vector control (0.8-fold, p=0.05), suggesting that nuclear localization may not be critical for this cell specific effect. To ask if loss of HDAC9 had adverse effects on GnRH neuron development, GnRH neuron numbers were counted in brains of WT and HDAC9 null mice (E11-E16 based on crown-rump length). A 37% decrease in GnRH neurons was observed in HDAC9 null (210±82, n=5) compared to WT (335±87, n=5) embryonic brains. Thus our data supports the role of HDAC9 to promote neuron survival across GnRH neuronal development via a unique cytoplasmic rather than nuclear site of action.

 

Nothing to Disclose: SS, MX, CN, MSS, TAM, DRM, SAT, MEW

FP04-1 8720 1.0000 SAT-136 A Understanding Histone Deacetylase 9 (HDAC9) Function in GnRH Neuron Biology 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Neuroendocrinology and Pituitary Saturday, June 15th 11:30:00 AM FP04 2290 11:00:00 AM GnRH & Gonadotroph Biology & Signaling Poster Preview


Yewei Xing*1, Mohamad Zubair1, Holly A Ingraham2 and Gary D Hammer1
1University of Michigan, Ann Arbor, MI, 2University of California San Francisco, San Francisco, CA

 

Sf1 (Nr5A1) is a critical nuclear receptor involved in adrenocortical development and homeostasis. Sf1 knockout mice are born with adrenal aplasia while Sf1 haplo-insufficient mice exhibit defects in homeostasis of the adult gland. In addition to gene dosage, post-translational modifications including reciprocal phosphorylation and SUMOylation also play important roles in regulating Sf1 function.  Specifically, our prior studies reported that SUMOylation of Sf1 inhibits CDK7-mediated phosphorylation of Sf1.  Despite the reported decreased expression of a subset of SUMO-sensitive genes in the presence of SUMOylated Sf1, the physiological role of SUMOylation of Sf1 in adrenocortical development and homeostasis remains unclear. In this study, the SUMO-deficient Sf1 knock-in mouse (Sf-1 that is unable to be SUMOylated) was crossed with the FAdE-Ad4bp-LacZ transgenic reporter line.  The Fetal Adrenal Enhancer of Sf1 activates transcription in a spatially and temporally restricted manner – only in the fetal zone of the adrenal cortex and only during embryonic life.

We found that the expression of LacZ was abnormally maintained in an abnormally retained fetal zone through P30 in the mutant male mouse. A similar retention of the fetal zone has been observed in mice and in patients with loss-of-function mutations in Dax1 (Nr0B1). This result is in contrast to the normal regression of the fetal zone at puberty in wildtype male mice. Because we have shown previously that FAdE-mediated expression of Sf1 is maintained by auto-activation of FAdE by Sf1 itself, we hypothesize that SUMOylation of Sf1 is important for full extinction of FAdE enhancer activity and ultimate regression of the fetal zone.  Further in-vitro studies using FAdE-luciferase constructs confirm that SUMO-tagged Sf1 is markedly deficient in activating FAdE activity in 293T cells compared to SUMO-deficient Sf1.  Future efforts will focus on examining the molecular mechanism by which SUMOylation of Sf1 together with Dax1 regulates FAdE activity during development.

 

Disclosure: GDH: Consultant, orphagen, Founder, Atterocor, Consultant, Embera, Advisory Group Member, Embera, Consultant, HRA Pharma, Consultant, Corcept, Consultant, Isis, Advisory Group Member, orphagen, Consultant, OSI-Astella. Nothing to Disclose: YX, MZ, HAI

FP06-1 4660 1.0000 SAT-385 A Sumoylation Of SF1 Regulates MICE Adrenal Development 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Nuclear Receptors and Steroid Hormone Action Saturday, June 15th 11:30:00 AM FP06 2305 11:00:00 AM Steroid Hormone Actions, Biosynthesis & Metabolism Poster Preview


Samantha Rose Lewis*1, William A Ricke2 and Joan Susan Jorgensen3
1University of Wisconsin-Madison, Madison, WI, 2University of Wisconsin, Madison, WI, 3Univ of Wisconsin, Madison, WI

 

The dependence of prostate cancer on androgens provides a targeted means of treating advanced disease. Unfortunately, androgen deprivation therapies that block gonadal steroid synthesis ultimately become ineffective, leading to the deadly form of prostate cancer. While there are likely many ways this transition to treatment refractive cancer can occur, one important factor is the ability of prostate adenocarcinoma cells to acquire machinery for de novo steroidogenesis and therefore, fuel their own growth. The mechanisms by which prostate cancer cells initiate and maintain steroidogenesis are unknown. We hypothesize that Steroidogenic Factor 1 (NR5A1, ADBP4, SF1), a key regulator of steroidogenesis in normal endocrine tissues, is expressed in castration resistant prostate cancer where it stimulates aberrant steroidogenesis and fuels malignant growth. Notably, SF1 is not expressed in normal prostate tissue. Our results indicated that SF1 was absent in benign prostate cell lines as expected, but present in aggressive prostate cancer cell lines. When ectopic SF1 expression was induced in benign prostate epithelial cells (BPH1), increased steroidogenic enzyme expression, steroid synthesis, and cell growth was observed. Converse experiments using shRNA-mediated knockdown of SF1 in an aggressive prostate cancer cell line (WR3) diminished steroidogenic activity and inhibited cell growth. SF1 depleted cells also exhibited signs of decreased cell cycle progression and defects in cell division. Xenograft studies were performed to evaluate the role of SF1 in tumor growth. Results comparing control versus SF1 deficient prostate cancer cells showed that knockdown of SF1 substantially impaired tumor growth under the kidney capsule in both castrated and intact nude mouse hosts. Based on these data, we conclude that aberrant SF1 expression in aggressive prostate cancers stimulates steroidogenesis and promotes aggressive tumor growth. These findings present a new potential mechanism and therapeutic target for deadly castration resistant prostate cancer.

 

Nothing to Disclose: SRL, WAR, JSJ

FP06-3 8649 3.0000 SAT-384 A Steroidogenic Factor 1 Drives Aggressive Prostate Cancer Cell Proliferation and Plays a Critical Role in Tumor Growth 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Nuclear Receptors and Steroid Hormone Action Saturday, June 15th 11:30:00 AM FP06 2305 11:00:00 AM Steroid Hormone Actions, Biosynthesis & Metabolism Poster Preview


Maria Magdalena Szwarc*, Ramakrishna Kommagani, San-Pin Wu, Sophia Y Tsai, Ming-Jer Tsai, Francesco J. DeMayo and John P Lydon
Baylor College of Medicine, Houston, TX

 

Steroid receptor coactivator-2 (SRC-2) is a member of the p160/SRC family of coregulators, which also includes SRC-1 and SRC-3. Members of this coregulator class exert a wide-spectrum of physiological processes, ranging from mammary morphogenesis to metabolic homeostasis. Importantly, deregulation of SRC expression levels is a causal factor for many tissue pathologies in both human and mouse. In the case of the endometrium, clinical studies reveal that SRC-2 and SRC-3 levels are elevated in endometrial biopsies from patients diagnosed with polycystic ovary syndrome (PCOS). Significantly, the endometrium of PCOS patients displays severe defects in functionality, including increased endometrial cancer susceptibility and miscarriage rate. Elevated expression of both SRC-2 and SRC-3 has also been found in the hyperplastic and neoplastic endometrium. Collectively, these descriptive findings suggest a causal link between elevated expression of one or both coregulators and the emergence of these endometrial disorders.  To address this proposal further, we engineered a SRC-2 overexpressor (SRC-2: OE) mouse in which high levels of human SRC-2 expression are specifically targeted to cells that express the progesterone receptor. Long term-breeding studies, a decidual response assay, gonadotropin-induced superovulation, and measurement of serum hormone levels were conducted on SRC-2:OE mice to evaluate fertility status. Although ovulation and serum hormone levels are normal, six month breeding studies show that elevated levels of endometrial SRC-2 result in a severe subfertility defect. Importantly, an artificial decidual response assay revealed that the SRC-2:OE endometrium exhibits an impaired ability to undergo decidualization, an essential cellular process that enables embryo implantation to occur. The inability of the SRC-2:OE endometrium to decidualize is also reflected at the molecular level by a marked decrease in the induction of the decidual biomarkers, Follistatin, Wingless-related MMTV integration site 4, Bone morphogenetic protein 2 and Heart and neural crest derivatives expressed transcript 2. Furthermore, short- and long-term estradiol treatment reveals that perturbation of SRC-2 levels markedly potentiates estradiol-induced uterine epithelial hyperplasia, providing strong support for SRC-2 in the promotion of unopposed estrogen-action. We conclude that tight control of SRC-2—independent of changes in SRC-3 levels—is mandatory not only for normal endometrial functionality but also to prevent unscheduled endometrial hyperplasia which can lead to cancer.

 

Nothing to Disclose: MMS, RK, SPW, SYT, MJT, FJD, JPL

FP06-4 5984 4.0000 SAT-381 A Perturbation of Steroid Receptor Coactivator-2 Expression Levels Leads to Abnormal Steroid Hormone Responsiveness in the Murine Endometrium That Underpins a Severe Sub-Fertility Phenotype 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Nuclear Receptors and Steroid Hormone Action Saturday, June 15th 11:30:00 AM FP06 2305 11:00:00 AM Steroid Hormone Actions, Biosynthesis & Metabolism Poster Preview


Say Viengchareun*1, Ingrid Lema2, Vixra Keo3, Larbi Amazit4, Jerome Nevoux3, Laetitia Martinerie5, Nadia Cherradi6 and Marc Lombes7
1Inserm U1185, Le Kremlin-Bicêtre, France, 2INSERM U693, Le Kremlin Bicêtre, France, 3Inserm U693, Le Kremlin-Bicetre, France, 4inserm u693, le kremlin bicetre, France, 5INSERM U693, Le Kremlin Bicetre, France, 6INSERM U1036, Grenoble, France, 7Fac de Medicine Paris-SUD, Le Kremlin-Bicetre, France

 

The Mineralocorticoid Receptor (MR, NR3C2) mediates sodium-retaining action of aldosterone. MR is highly expressed in the distal nephron where the renal corticopapillary gradient generates strong variations in extracellular fluid tonicity. This osmotic gradient, pivotal for the regulation of ion and water transport, is impaired during renal development and in various kidney diseases. These pathophysiological conditions are accompanied by variations of renal MR expression; however, the mechanisms regulating MR expression remain sparse.

We showed previously that post-transcriptional events control renal MR abundance (Viengchareun Mol Endo 2009). Here, using cortical collecting duct KC3AC1 cells, we show that the RNA Binding Protein Tis11b (a member of tristetraprolin/ZFP36 family) induces a dramatic reduction of MR expression under hypertonicity. Consistent with a Tis11b-mediated MR downregulation mechanism, hypertonicity induces a 2-4-fold increase of Tis11b mRNA and protein levels, concomitant with the decreased MR expression. In contrast, RNAi-dependent reduction of Tis11b expression induces the increase of MR expression level. We suggest that Tis11b modulates MR mRNA stability/degradation through binding to Adenine/Uridine Rich Elements (AURE), located in the 3’-UnTranslated Region (3’-UTR) of MR mRNA. Eight highly conserved AURE were indeed identified in the 3’-UTR of MR mRNA.

To examine whether MR transcript was a direct target of Tis11b, the entire 3’-UTR of MR mRNA was subcloned downstream of a luciferase reporter gene and truncated mutants, carrying or lacking AURE, were generated. Cotransfection assays in HEK 293 cells showed that Tis11b induces a 2-fold reduction in luciferase activity of AURE-containing constructs. RNA-ChiP assays demonstrated that endogenously expressed Tis11b physically interacts with MR mRNA AURE in KC3AC1 cells. Finally, to demonstrate the physiological relevance of our findings, we challenged the renal osmotic gradient and demonstrated that mice present with a modification in renal MR expression under water deprivation or diuretic treatment (furosemide, indapamide).

Our data are important to explain the partial aldosterone resistance associated with cyclic variations of renal MR expression during the perinatal period. Whether such regulatory post-transcriptional mechanisms occur in other MR target tissues (cardiovascular and central nervous system) or are altered in kidney diseases and hypertension remains to be established.

 

Nothing to Disclose: SV, IL, VK, LA, JN, LM, NC, ML

FP06-5 8325 5.0000 SAT-383 A Renal Mineralocorticoid Receptor Expression Is Regulated By Tis11b-Mediated Post-Transcriptional Control: Pathophysiological Implications 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Nuclear Receptors and Steroid Hormone Action Saturday, June 15th 11:30:00 AM FP06 2305 11:00:00 AM Steroid Hormone Actions, Biosynthesis & Metabolism Poster Preview


Elizabeth Louise Kilby*1 and Thomas Hugh Jones2
1University of Sheffield, Sheffield, United Kingdom, 2Barnsley District Gen Hosp, Barnsley S Yorkshire, United Kingdom

 

Evidence suggests that testosterone is atheroprotective and has been shown to reduce salient cardiovascular risk factors which include lowering cholesterol.  Macrophage liver x receptor α (LXRα) stimulates cholesterol efflux and this ability means LXRα agonists are a potential therapy for atherosclerosis.  It was therefore proposed that testosterone acts to reduce the features of atherosclerosis by acting through LXRα.  THP-1 macrophages were used, as they express the androgen receptor (AR) and are therefore responsive to testosterone.  Cells were treated with 10-8 M testosterone (24, 48 and 72 hr) either alone or in combination with Flutamide (AR inhibitor) or LXRα antagonist and gene expression and protein levels between control and treated cells were assessed by qPCR and western blotting, respectively.  Changes in protein localisation were observed by immunofluorescence.  Using the fluorescent cholesterol analogue dehydroergosterol (DHE) and time-lapse microscopy we have been able to directly observe the effect of testosterone on cholesterol efflux.  Testosterone significantly increased LXRα expression in macrophages.  In addition, testosterone increased the expression of genes downstream of LXRα which encode proteins involved in cholesterol efflux and metabolism, including ABCA1 (ATP-binding cassette transporter A1), APOE (apolipoprotein E), FAS (fatty acid synthase) and SREBP1c (sterol regulatory element-binding protein 1c).  Corresponding protein levels were also increased in response to testosterone.  Blocking LXRα activity inhibited the effect of testosterone, demonstrating testosterone increases ABCA1, APOE, FAS and SREBP1c expression by activating LXRα.  Testosterone was shown to act via its AR to activate LXRα, as blocking the AR inhibited the effect of testosterone on LXRα and its downstream targets.  Testosterone increased the rate of cholesterol efflux from macrophages and an increase in ABCA1 protein at the cell membrane suggests this may result from an increase in intracellular trafficking of cholesterol towards the cell membrane.  We provide evidence that testosterone activates LXRα and acts through this nuclear receptor to control the expression of LXR-target genes to stimulate cholesterol efflux.  We therefore hypothesize that testosterone exerts its anti-atherogenic effects in part through the activation of LXRα and LXR-target genes. 

 

Disclosure: THJ: Consultant, Bayer Healthcare, Researcher, Bayer Healthcare, Speaker, Bayer Healthcare, Consultant, Eli Lilly & Company, Consultant, Pro Strakan, Researcher, Pro Strakan, Speaker, Pro Strakan, Consultant, Merck, Consultant, Clarus. Nothing to Disclose: ELK

FP06-6 9259 6.0000 SAT-386 A Testosterone Stimulates Cholesterol Efflux From Human Macrophages: A Potential Therapy for Atherosclerosis 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Nuclear Receptors and Steroid Hormone Action Saturday, June 15th 11:30:00 AM FP06 2305 11:00:00 AM Steroid Hormone Actions, Biosynthesis & Metabolism Poster Preview


Chimaroke Edeoga*1, Sotonte Ebenibo2 and Samuel Dagogo-Jack1
1University of Tennessee Health Science Center, Memphis, TN, 2University of Tennessee, Memphis, TN

 

Purpose: Leptin regulates body weight and fasting (basal) plasma leptin levels are closely correlated with body fat mass, but dynamic leptin responses to secretagogues have not been well-studied.  Fasting hypoleptinemia has been suggested as a risk factor for future weight gain in Pima Indians, but data are lacking for other populations, and the relationship between dynamic leptin secretion and future weigh change is unknown.  We tested the hypothesis that stimulated leptin levels better predict future weight change than basal leptin levels.

Subjects and Methods: We studied 254 (127 white, 127 African American {AA}) nondiabetic subjects (mean{SD} age 44.2 + 10.6 y) enrolled in our ongoing Pathobiology of Prediabetes in a Bi-racial Cohort (POP-ABC) study1.  At baseline and annually, POP-ABC participants underwent physical examination, OGTT, and measurements of body fat (DEXA) and fasting leptin levels, among other assessments. Dynamic leptin secretion was assessed at baseline, using the method of Dagogo-Jack et al 2(with a reduced dose, 2 mg, of dexamethasone {dex}). The interactions among basal and post-dex plasma leptin levels and changes in weight, BMI, and total fat mass during the ensuing 1 year were analyzed using ANOVA and linear regression.

Results: There were no gender or ethnic differences in the correlation between fasting leptin and total fat mass (r= ~ 0.81). The peak plasma leptin response to dex (% baseline) showed marked individual variability (-9% to +760%). However, the mean change was similar (~100% above baseline) in men and women and in AA and Caucasians. Fasting leptin levels were inversely correlated with peak dex-induced leptin (r= -0.26, P=0.037). Fasting leptin levels had a marginal relationship with 1-yr changes in weight (P=0.0496) and no significant relationship with total fat mass (P=0.3394). In contrast, the changes in leptin following dex significantly predicted 1-yr trajectories in weight (P=0.0016) and total fat mass (P=0.0023). These findings were similar in men and women and in AA and Caucasians. When the data were analyzed by percentiles of leptin response to dex, subjects in the <25th, 25-50th, and 51-75th percentiles maintained stable (or decreasing body fat), whereas those in the >75-90th and >90thpercentiles of dynamic leptin secretion gained fat mass during the ensuing 1 year.

Conclusion: Our data indicate that basal leptin levels convey uncertain information regarding the risk of future adiposity in African Americans and Caucasians, whereas stimulated leptin levels reliably predict 1-yr trajectories in weight and fat mass. The Dexamethasone-Leptin Test thus might be of prognostic value in predicting future changes in adiposity.

 

Nothing to Disclose: CE, SE, SD

FP09-5 5240 5.0000 SAT-697 A Basal Versus Stimulated Leptin Levels for Predicting Trajectories of Future Weight and Fat Mass 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Obesity, Adipocyte Biology and Appetite Saturday, June 15th 11:30:00 AM FP09 2314 11:00:00 AM Obesity: Physiologic Responses to Energy Balance Disruption Poster Preview


Suhyun Lee*1, Claudio Alberto Mastronardi2, Rachel Li3, Gilberto J. Paz-Filho4, Paul Smith5, Julio Licinio6 and Ma-Li Wong7
1John Curtin School of Medical Research, Australian National University, Canberra, Australia, 2Australian National University, Forrest, Australia, 3The John Curtin School of Medical Research, The Australian National University, Canberra, Australia, 4Australian National University, Acton, Australia, 5Canberra hospital, Canberra, Australia, 6The Australian National Univ, Canberra, Australia, 7The Australian National Univ, Canberra, ACT, Australia

 

Introduction Currently, antidepressants are among the most frequently prescribed classes of drugs. Over 164 million antidepressant prescriptions were issued in the USA in 2009, and 12.3 million antidepressant scripts were written for more than 1.6 million Australians in 2008. In the last decade, there has been a rise in antidepressant use and a concomitant rise in the rates of overweight and obesity. Significant weight gain (an increase of 7% or more over baseline weight) is associated with the use of most antidepressants. However, the pathophysiological mechanisms of this association are still poorly understood.

Methods Male Sprague-Dawley rats subjected to a novel paradigm, consisting of short-term exposure to recurrent restraint stress and antidepressants for 2 weeks, followed by long-term high-fat diet intake, were studied for 295 days. Body and organs weights, and behaviour were characterised.     

Results: During the post-stress recovery period, obesity prone rats treated with fluoxetine had increased body weight (R-FX) in comparison to the control group treated with saline (R-C) and non-restraint control group (NRCF) (R-FX vs R-C: 610.6 ±15.52vs503.4±10.75g, P <0.0001; R-FX vs NRCF: 610.6 ±15.52vs566.1±14.55g, P<0.05). Fluoxetine-treated animals also had heavier bones in comparison to control groups (R-C vs R-FX: 1.995±0.06vs2.39±0.07g, P<0.0001; NRCF vs R-FX: 1.996±0.03vs2.39±0.06g, P<0.0001). Furthermore, spleen weight in saline treated animals was significantly decreased, while antidepressant treated group did not differ when compared to non-restraint group (R-C vs NRCF: 0.640±0.03vs0.745±0.02, P<0.05). At the behavioural level, open field studies showed that antidepressant treated animals were significantly less anxious than saline treated animals during post-restraint period (R-C vs R-FX: 0.092 ± 0.005vs0.1139 ± 0.004 CD/TD ratio, P <0.01).

Conclusion Our study suggests that short-term exposure to stress and antidepressants leads to long-term body weight gain accompanied with increased bone and spleen weights. These findings may implicate different pathophysiological mechanisms in stress and antidepressant related obesity when compared to obesity that is solely diet-induced.

 

Nothing to Disclose: SL, CAM, RL, GJP, PS, JL, MLW

FP09-6 6857 6.0000 SAT-677 A Understanding the Long Term Effect of Weight Gain Induced By Short Term Exposure of Antidepressants 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Obesity, Adipocyte Biology and Appetite Saturday, June 15th 11:30:00 AM FP09 2314 11:00:00 AM Obesity: Physiologic Responses to Energy Balance Disruption Poster Preview


Anne Müller*1, Timo D. Müller2, Kirk Habegger3, Carolin Leonie Piechowski1, Chun-Xia Yi4, Richard DiMarchi5, Heiko Krude6, Paul T. Pfluger2, Gunnar Kleinau1, Matthias H. Tschöp4 and Heike Biebermann6
1Charité - Universitätsmedizin Berlin Campus Virchow, Berlin, Germany, 2Helmholtz Center Munich, Munich, Germany, 3University of Cincinnati, Cincinnati, 4Helmholtz Center Munich, Garching, Germany, 5Indiana University, Bloomington, 6Charité - Universitätsmedizin Berlin, Berlin, Germany

 

Signaling of the ghrelin receptor (GHSR1a) is of upmost importance for energy metabolism. Recently it was shown that the GHSR1a can interact with a variety of other G protein coupled receptors (GPCRs) including the melanocortin 3 receptor, dopamin 1 receptor and serotonin 2c receptor. Here we investigated the role of the orphan G protein coupled receptor 83 (GPR83) in modulating the function of GHSR1a. The GPR83 is an orphan GPCR that is expressed widely in the brain, including in hypothalamic nuclei governing energy balance. We show that hypothalamic expression of GPR83 is decreased in diet-induced obese (DIO) mice compared to lean mice. Furthermore, hypothalamic expression is reduced following fasting and increased upon re-feeding. GPR83 is co-localized with GHSR1a in the arcuate nucleus (ARC), and in vitro studies indicate that GPR83 forms heterodimers with GHSR1a, leading to reduced activation of GHSR1a by its ligand ghrelin. Studies in GPR83-knockout mice show that loss of GPR83 potentiates the effect of central and peripheral ghrelin treatment on food intake and adiposity. GPR83-knockout mice have normal body weight and glucose tolerance when fed a regular chow diet. However, glucose intolerance and obesity were not observed in response to high-fat diet despite relative hyperphagia. Taken together, these data suggest that the orphan GPR83 acts as a modulator of ghrelin action in response to nutrient availability and might act also through other pathways to regulate systemic metabolism in response to high-fat diet. Our data support the notion that GHSR1a is part of a hypothalamic GPCR interactome which have to be unravelled when GHSR1a is used as target to combat obesity.

 

Nothing to Disclose: AM, TDM, KH, CLP, CXY, RD, HK, PTP, GK, MHT, HB

FP08-2 5568 2.0000 SAT-649 A The Orphan G Protein Coupled Receptor 83 (GPR83) Modulates Ghrelin Receptor Signaling in Vitro and in Vivo 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Obesity, Adipocyte Biology and Appetite Saturday, June 15th 11:30:00 AM FP08 2315 11:00:00 AM Obesity: Novel Mechanisms of Body Weight Regulation Poster Preview


Joon S Kim*1, Janette H Quennell1, Mohammed Z Rizwan1 and Greg M Anderson2
1University of Otago School of Medical Sciences, Dunedin, New Zealand, 2University of Otago, Dunedin, New Zealand

 

The adipose-derived hormone leptin and the sex steroid 17b-estradiol (E2) are critical regulators of body weight. While it is well accepted that leptin acts predominantly via its long-form receptor (LepRb) to induce the phosphorylation of signal transducer and activator of transcription 3 (STAT3), the mechanism underlying E2’s anorexigenic effects are largely unknown. It is thought that the main receptors of these hormones, LepRb and estrogen receptor-alpha (ERα), share an intracellular cross-talk. Consistent with this, E2 is unable to exert its anti-obesity effects in a neural STAT3 knockout mouse model, suggesting that STAT3 plays the critical common pathway via which these two hormones exert their effects (1). A series of experiments were designed to explore the possible interaction between leptin and E2 on STAT3. We first characterized the colocalization of LepRb with ERα in the mouse hypothalamus to determine the extent to which a direct relationship between LepRb-STAT3 and ERα signaling was possible. By crossing LepRb-Cre and Tau-GFP reporter mice, GFP expression was able to be colocalized with ERα immunoreactivity. A relatively low percentage of GFP-positive cells coexpressing ERα were observed in the arcuate nucleus (24.7%), ventromedial hypothalamus (25.7%), caudal dorsomedial hypothalamus (26.0%), and ventral premamillary nucleus (34.8%). However, a higher coexpression was recorded in the preoptic area (88.3%), rostral dorsomedial (63.4%) and lateral hypothalamus (62.3%). To explore the effects of E2 on leptin-induced STAT3 phosphorylation, ovarectomized mice with low, medium, or high levels of chronic E2 replacement received an acute leptin challenge (0.02 mg/kg sc). We observed no increase in the degree of leptin-induced phosphorylated STAT3-positive cell numbers in the arcuate nucleus with higher estrogenic states. Finally, mice with deletion of STAT3 specifically in LepRb-expressing cells were ovarectomised and given either chronic E2 or vehicle implants to test whether E2's weight reducing effects were dependent on LepRb-STAT3 signaling. After 8 days there was no difference in body weight between E2 treatment and controls; data collection still in progress. Here we report quantified analysis of LepRb and ERα colocalization in the murine hypothalamus while also revealing previously undescribed coexpressing populations. Furthermore, we suggest that E2 is likely to exert anorexigenic effects independently of LepRb-STAT3 signaling.

 

Nothing to Disclose: JSK, JHQ, MZR, GMA

FP08-3 7707 3.0000 SAT-709 A Anorexigenic Estradiol Effects Do Not Involve Leptin-STAT3 Signaling 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Obesity, Adipocyte Biology and Appetite Saturday, June 15th 11:30:00 AM FP08 2315 11:00:00 AM Obesity: Novel Mechanisms of Body Weight Regulation Poster Preview


Gang Xi*1, Melissa Solum1, Christine Wai1, Clifford J Rosen2 and David R Clemmons1
1University of North Carolina at Chapel Hill, Chapel Hill, NC, 2Maine Medical Cntr Research Inst, Scarborough, ME

 

IGFBP2 contains two heparin binding domains (HBD), localized in the linker (HBD1) and C-terminal regions (HBD2). HBD2 shares sequence similarity with HBDs in other IGFBPs. We reported that an HBD1 peptide stimulated osteoblast growth but HBD2 had no effect. IGFBP2 prevents the development of obesity but the roles of the HBD domains have not been determined. Therefore we investigated if peptides containing the HBD1 or 2 sequences could replicate the effect of the whole molecule on fat development. The results showed that addition of wild type IGFBP-2 markedly inhibited the differentiation of primary preadipocytes isolated from IGFBP-2 -/- mice. Both HBD1 and HBD2 inhibited fat cell differentiation but HBD2 was more effective. Substitution of key charged residues in the HBD1 or HBD2 regions resulted in attenuation of their ability to inhibit differentiation. The HBD2 mutant form of IGFBP2 which contained an intact HBD1 sequence was less effective, suggesting that the HBD2 domain might play more important role in inhibiting adipogenesis. To determine the effect of the HBD1 and HBD2 peptides on fat development in vivo, these two peptides were pegylated and administered to IGFBP-2-/- mice (50ug S.C., 3 times/week) for 12 weeks. Interestingly, both peptides significantly suppressed body weight gain after 12 weeks, compared to a control peptide. MRI scanning showed that both peptides reduced the gain of fat mass. However, HBD2 was more effective than HBD1 (e.g. 65 ± 15% reduction vs. 30 ± 3% reduction). In addition, although administration of both peptides significantly reduced the accumulation of both inguinal and visceral fat mass, HBD2 was to be more effective. For example, inguinal fat mass and visceral fat mass were reduced 35 ± 8% (p<0.05) of 28 ± 3% (p<0.05), respectively, in HBD2 treated mice, compared to control peptide treated mice. In contrast, they were reduced 21± 4% (p<0.05) and 6 ± 3% (p, NS) in HBD-1 treated mice, respectively. In addition, administration of both peptides reduced the triglyceride content in the inguinal fat pad and plasma adiponectin but only HBD2 significantly increased the plasma leptin levels compared to control peptide.This study clearly demonstrates that the HBD2 domain of IGFBP2 is the primary region that accounts for its ability to inhibit adipogenesis in vitro and fat development in vivo. Since the the HBD1 domain is more effective in osteoblasts, this suggests that the roles of the HBD domains may be cell type specific.

 

Nothing to Disclose: GX, MS, CW, CJR, DRC

FP08-4 8511 4.0000 SAT-650 A Specific Heparin Binding Domains Within IGFBP2 Mediate Its Ability to Inhibit Preadipocyte Differentiation 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Obesity, Adipocyte Biology and Appetite Saturday, June 15th 11:30:00 AM FP08 2315 11:00:00 AM Obesity: Novel Mechanisms of Body Weight Regulation Poster Preview


Jose Manuel Garcia*1, Bobby O Guillory2 and Tripti Halder3
1MEDVAMC/ Baylor Coll of Med, Houston, TX, 2Baylor College of Med, Houston, TX, 3MEDVAMC/ Baylor Coll MEd, Houston, TX

 

The prevalence of obesity increases dramatically with age and is associated with an increase risk of diabetes, cardiovascular disease, cancer and overall mortality. Aging is also associated with a decline in muscle function, leading to poor quality of life and increased mortality. Nevertheless, the mechanisms contributing to the development of obesity and muscle dysfunction in aged individuals have not been fully characterized. Ghrelin is an appetite-stimulating hormone and GH secretagogue that is known to decrease energy expenditure and to increase food intake, adiposity and body weight. The purpose of this study was to characterize the role of ghrelin during aging.

Body weight, body composition, food intake, locomotor activity, energy expenditure (EE), muscle strength and endurance were compared between young adult (6 month-old) and old (19 month old) ghrelin wild type (WT) and knock-out (KO) c57bl/6 male mice. Older animals had higher body weight and fat mass measured by NMR than younger animals in both genotypes. Although there was no difference in these parameters between young WT and KO animals, old KO animals had significantly lower body weight and fat mass than WT. Daily food intake and respiratory quotient (RQ) did not differ between groups. However, EE adjusted by LBM was significantly decreased by age in WT but not in KO animals. Spontaneous 24-h locomotor activity tended to decrease with aging in both genotypes although this difference did not reach significance. Treadmill performance and grip strength declined during aging and were improved with deletion of ghrelin.

In summary, in our model aging is associated with an increase in body weight and adiposity, and decreased EE, endurance and muscle strength. The increase in body weight and fat mass was ameliorated by deletion of the ghrelin gene only in aged animals. In spite of the known orexigenic effects of ghrelin at pharmacologic doses, the deletion of ghrelin did not cause changes in food intake. However, it prevented the decrease in EE seen with aging in WT animals. These data suggest that the differences in body weight and body composition induced by deletion of ghrelin in aged animals are due to differences in EE. Muscle performance was improved by deletion of ghrelin in spite of its known anabolic properties.

 

Disclosure: JMG: Principal Investigator, Aeterna Zentaris, Principal Investigator, Helsinn Therapeutics. Nothing to Disclose: BOG, TH

FP08-5 8647 5.0000 SAT-710 A Ghrelin Deletion Prevents Aging Associated Obesity and Muscle Dysfunction 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Obesity, Adipocyte Biology and Appetite Saturday, June 15th 11:30:00 AM FP08 2315 11:00:00 AM Obesity: Novel Mechanisms of Body Weight Regulation Poster Preview


Poonamjot Deol*1, Jane R Evans2, Soo Han1, Karthikeyani Chellappa1 and Frances M. Sladek3
1University of California Riverside, CA, 2University of California, Riverside, Riverside, CA, 3University of California, Riverside, CA

 

Metabolic Effects of Dietary Linoleic Acid and Fructose

Poonamjot Deol, Jane Evans, Soo Han, Karthikeyani Chellappa, Frances M. Sladek

Department of Cell Biology and Neuroscience, University of California, Riverside, CA 92521

The incidence of obesity in the U.S. has increased from 15% to 35% in the last 40 years and is expected to rise to 42% by 2030. Paralleling this increase in obesity are a number of dietary changes, most pronounced of which is a >1000 fold increase in consumption of soybean oil from 0.01 to11.6 kg/yr/capita from 1909-1999: soybean oil consists of 50-60% linoleic acid (LA), so the energy intake from LA has increased from 2% to >7%/day (1). LA is an essential fatty acid and a precursor to arachidonic acid, which is linked to inflammation, a key player in obesity, diabetes, cancer, etc. Another component of the American diet that has increased substantially in the last four decades is fructose, primarily in the form of high fructose corn syrup in processed foods and sodas (2). The roles of both LA and fructose in the current obesity epidemic are under intense scrutiny but are not well understood and seldom compared side-by-side (3).

To investigate effects of LA and fructose on obesity and diabetes, we designed a series of specialized isocaloric diets to mimic the American diet; the diets are high in saturated fats (HFD, 40%kcal total fat) and supplemented, or not, with soybean oil to achieve 10%kcal LA (LA-HFD) and 25.9%kcal fructose. C57/BL6 mice on LA-HFD showed increased weight gain, adiposity and diabetes, as well as impaired glucose tolerance and insulin insensitivity compared to the low-LA HFD. They also had fatty livers with significant ballooning injury and fibrosis, exhibited changes in crypt length in the proximal colon, shrunken cecums and spleen hypoplasia. Though the high fructose diets did not cause as much obesity or diabetes as the high LA diets, they did cause a very fatty liver and rectal prolapse. Transgenerational effects of LA were examined by mating female mice on LA-HFD to vivarium chow (VC) fed males: pups born to LA-HFD dams had a higher wean weight and gained weight faster on both VC and LA-HFD than pups born to VC-fed dams. These results suggest that dietary LA in a high fat background causes obesity and diabetes and that both LA and fructose contribute to fatty liver and have negative effects on gut health. Finally, results from RNAseq of livers from mice on HFD and LA-HFD will provide insights into mechanisms responsible for effects on the liver, and possibly obesity and diabetes.

 

Nothing to Disclose: PD, JRE, SH, KC, FMS

FP08-6 6690 6.0000 SAT-708 A Metabolic Effects Of Dietary Linoleic ACID and Fructose 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Obesity, Adipocyte Biology and Appetite Saturday, June 15th 11:30:00 AM FP08 2315 11:00:00 AM Obesity: Novel Mechanisms of Body Weight Regulation Poster Preview


Jan Idkowiak*1, Angela E Taylor1, Donna M O'Neil1, Sandra Subtil1, Raymon Vijzelaar2, Renuka Dias1, Zaki K Hassan-Smith1, Rakesh Amin3, Timothy Barrett1, Paul M Stewart1, Jeremy Kirk4, Celia Moss4, Cedric H Shackleton1 and Wiebke Arlt1
1University of Birmingham, Birmingham, United Kingdom, 2MRC Holland, Amsterdam, Netherlands, 3Great Ormond Street Hospital for Children, London, United Kingdom, 4Birmingham Children's Hospital, Birmingham, United Kingdom

 

The enzyme Steroid Sulfatase (STS) cleaves sulfate groups from steroid hormones, including the adrenal androgen metabolite DHEAS (1). STS deficiency (STSD) due to inactivating deletions or mutations in the STS gene causes X-linked ichthyosis (OMIM 308100), a skin condition characterized by dry scales due to the epidermal accumulation of cholesterol sulfate. A defect in DHEA sulfation - the opposite enzymatic reaction of STS - results in androgen excess due to increased conversion of DHEA to active androgens (2). The aim of this study was to investigate androgen metabolism in patients with STSD in order to explore whether this defect would have the opposite effect on circulating androgens.

We recruited 30 male patients with STSD (age 6-30 years) and 45 age-matched healthy controls. We genetically confirmed the diagnosis of STSD in all subjects identifying either complete (n=27) or partial deletions (n=1) of the STS gene; two patients harbored a hemizygous missense mutation (p.R454C). The KAL1 locus was intact in all patients. There were no apparent abnormalities in the physical development of the STSD patients. Urinary steroid metabolomics (gas-chromatography/mass-spectrometry) revealed decreased excretion of active androgen precursor metabolites (Androsterone, An and Etiocholoanolone, Et) over androgen precursor metabolites (DHEA, 16hydroxy-DHEA, pregnenediol and 5-pregnenetriol) as compared to controls (p<0.001). 5α-reductase activity assessed as the ratio of 5α-reduced tetrahydrocortisol (THF) over THF (5αTHF/THF) was significantly increased in STSD (p<0.001). Serum steroid measurements (liquid chromatography/ tandem mass spectrometry) revealed decreased DHEA levels in all STSD age-groups (p<0.001) but testosterone was only lower in the adult subgroup (p=0.009). Cholesterol-sulfate was grossly elevated in all STSD subjects but DHEAS levels did not differ significantly. However, the ratio of DHEA/DHEAS was lower in STSD patients (p<0.001).

Our study demonstrates that though physical/pubertal development does not seem to be impaired in STSD, the steroid metabolome of these patients indicate a mild androgen deficiency with elevation of androgen precursors. We hypothesize that this reflects a mechanism of compensation, with further evidence of up regulation of peripheral androgen activation due to increased 5α-reductase activity. This illustrates for the first time in vivo that STS contributes to androgen metabolism in the context of DHEA sulfation.

 

Nothing to Disclose: JI, AET, DMO, SS, RV, RD, ZKH, RA, TB, PMS, JK, CM, CHS, WA

FP11-2 6841 2.0000 SAT-598 A Compensated Mild Androgen Deficiency in Boys With Steroid Sulfatase Deficiency: Evidence From Steroid Metabolomics 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Pediatric Endocrinology Saturday, June 15th 11:30:00 AM FP11 2335 11:00:00 AM Pediatric Endocrinology Poster Preview


Felix Schreiner*1, Christine Poralla1, Christoph Haertel2, Matthias Heckmann3, Joachim Woelfle1, Peter Bartmann1, Egbert Herting2, Wolfgang Goepel2 and (GNN) German Neonatal Network4
1Children's Hospital, University of Bonn, Bonn, Germany, 2Children's Hospital, University of Luebeck, Luebeck, Germany, 3Children's Hospital, University of Greifswald, Greifswald, Germany, 4GNN, Luebeck, Germany

 

Background: Apart from its beneficial effects on lung maturation, prenatal betamethasone administration has been shown to reduce the incidence of necrotizing enterocolitis, sepsis, and intraventricular hemorrhage in preterm infants. However, little is known about the role of endogenous differences in corticoid sensitivity arising from polymorphisms in the glucocorticoid receptor (GR) gene.

Study design: We analyzed GR polymorphisms BclI, N363S and R23K with respect to recorded neonatal outcome parameters in a German Neonatal Network (GNN) multicenter cohort comprising 2.211 very low birth weight preterm (VLBW)  infants.

Results:  Birth parameters were not different between genotype groups. Variants BclI and R23K were associated with a significantly higher risk of culture-positive neonatal sepsis (Bcll homozygosity: OR 1.85, p=0.002; R23K-carrier: OR 2.08, p=0.016; adj. for gestational age). In addition, infants carrying BclI alleles were more likely to develop bronchopulmonary dysplasia (OR 1.41 per allele [95% CI 1.14-1.75], p < 0.01 adj. for gestational age and mechanical ventilation). A similar relative risk was seen in the subcohort of infants with antenatal betamethasone treatment (OR 1.44, p < 0.01), whereas no such effect was detectable in the small subgroup without steroid treatment (n=185, OR 1.03, n.s.). N363S-carrier tended to have lower mean arterial blood pressure at the first day of life (total cohort: beta=-0.050, p=0.010; BclI-/R23K-wildtype subcohort: beta -0.055, p=0.074) and were more likely to require catecholamine treatment (p<0.01). However, none of the analyzed GR variants significantly influenced incidence rates of intraventricular hemorrhage (IVH), necrotizing enterocolitis (NEC), or retinopathy (ROP).

Conclusion: This study supports the hypothesis that endogenous differences in glucocorticoid sensitivity mediated by GR gene polymorphisms affect the neonatal outcome of VLBW preterm infants. In order to confirm the observed associations, analysis of a replication cohort is ongoing.

 

Nothing to Disclose: FS, CP, CH, MH, JW, PB, EH, WG, G

FP11-3 8448 3.0000 SAT-600 A Glucocorticoid Receptor Gene Polymorphisms and Neonatal Outcome of VLBW Preterm Infants: Preliminary Results from a German Multicenter Study 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Pediatric Endocrinology Saturday, June 15th 11:30:00 AM FP11 2335 11:00:00 AM Pediatric Endocrinology Poster Preview


Rajiv B. Kumar*1, Run Zhang Shi2, Nicole K. Schleifer1 and Tandy Aye1
1Stanford University Medical Center, Stanford, CA, 2Stanford University, Palo Alto, CA

 

Background:  Current methods to measure cortisol levels, either by venipuncture or 24 hour urine collection, are impractical for the pediatric population. Salivary cortisol measurement using an antibody based assay is available; however there is cross-reactivity, and limited pediatric normative data.  We report preliminary data from an ongoing study using mass spectrometry to measure salivary cortisol in healthy children.                                                                                                    

Design:  Children age 3-17y without chronic medical illness including pituitary or adrenal pathologies and/or recent use of systemic glucocorticoids participated in the study.  After viewing detailed instructions on salivary sample collection, the subjects/caregivers collected samples on two different dates at their usual bedtime, midnight, and upon waking the following morning. 

Results: 183 viable samples have been submitted from 37 children to date: 18 subjects were <8y, 10 were 8-12y, and 9 were 13-17y.  Salivary cortisol values (ng/dL) are reported as mean ± SD (range): Bedtime All = 45.3 ± 81.7 (<20-557), <8y = 66.1 ± 117.0 (<20-557), ≥8y = 28.6 ± 25.3 (<20-135); Midnight All = 44.5 ± 84.9 (<20-583), <8y = 73.9 ± 140.3 (<20-583), ≥8y = 30.2 ± 30.3 (<20-135); Morning All = 244.7 ± 261.9 (<20-1790), <8y = 274.0 ± 323.7 (<20-1790), ≥8y = 219.1 ± 193.7 (<20-1100).  Caregivers for children <8y noted difficulty in sample collection including emotional distress.  For this group, we noted wide cortisol variability and frequently insufficient quantity of saliva (especially at midnight).  The bedtime and midnight cortisol levels for all groups were comparable (p-values >0.80).  The morning levels were significantly higher than the bedtime and midnight values (p-values <0.01).  

Conclusion:  These results suggest that adult norms may not apply to the pediatric population.  Additionally, bedtime is comparable to midnight measurement, and morning measurement does demonstrate diurnal variation in cortisol secretion. Salivary cortisol measurement by mass spectrometry may be an attractive alternative to the current techniques, though this testing may not be optimal for children <8y.

 

Nothing to Disclose: RBK, RZS, NKS, TA

FP11-4 4584 4.0000 SAT-597 A Assessment of Normal Salivary Cortisol Values in Children Using Mass Spectrometry 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Pediatric Endocrinology Saturday, June 15th 11:30:00 AM FP11 2335 11:00:00 AM Pediatric Endocrinology Poster Preview


Rebecca Ann Hicks*1, Jennifer Kuang Wei Yee1, Catherine S Mao1, Martin Kharrazi2, Steve Graham2, Fred Lorey2 and Wai-Nang Paul Lee1
1Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA, 2California Department of Public Health, Richmond, CA

 

Introduction: In newborn screening for congenital adrenal hyperplasia (CAH), samples with elevated 17-α-hydroxyprogesterone (17-OHP) above the birth weight-adjusted intermediate cutoff values are subject to steroid profiling (second-tier testing). Samples with elevated 17-OHP and a ratio of (17-OHP+androstenedione)/cortisol > 1 are considered to be presumptive positive for CAH. Under steady state conditions, the 17-OHP/11-deoxycortisol ratio in steroid synthesis is a reflection of the changes in 21-α-hydroxylase enzyme activity. The purpose of this study is to explore the use of direct precursor–to-product ratios as additional criteria for the evaluation of these second-tier samples.

Methods: Deidentified dried blood spot samples from confirmed CAH cases identified by newborn screen (N=8) and second-tier samples (N=197) were obtained from the California State Newborn Screening Program with the approval of the Institutional Review Board. Samples (~6.25 mm circular spots) were extracted and processed using methanol:water in the ratio of (9:1).  Deuterated steroids were added to each sample before extraction as isotope internal standards. 17-OHP, 11-deoxycortisol (11-DOC), androstenedione (A4) and cortisol in the blood spots were quantified using liquid chromatography-tandem mass spectrometry. The 17-OHP/11-DOC and 17-OHP/A4 ratios were calculated. 

Results: 17-OHP/11-DOC >1.6 (Pd) and 17-OHP/A4 >2 (Pa) in CAH-positive samples reflect diminished 21-α-hydroxylase activity and elevated androgen production. Using these ratios as criteria for decreased cortisol and increased adrenal androgen production, the second-tier samples were classified into four groups according to Pd, Pa, Nd (17-OHP/11-DOC <1.6) and Na (17-OHP/A4 <2). The proportions of adrenal profiles differed by birth weight (BW) greater or less than 1500 grams, with χ2(3, N = 197) = 83.38, p <0.001.

Conclusion: Steroid profiles of second-tier samples are clearly dependent on birth weight categories. Infants with BW <1500 had an androgen (A4)-producing phenotype associated with elevated 17-OHP, contributing to many false positives by the second-tier criteria. For infants with BW >1500, additional criteria based on 17-OHP/11-DOC and 17-OHP/A4 ratios may be helpful to further reduce the false positive rate in newborn screening of CAH.

 

Nothing to Disclose: RAH, JKWY, CSM, MK, SG, FL, WNPL

FP11-5 4328 5.0000 SAT-601 A Precursor-to-Product Ratios of Second-tier Samples in Newborn Screening Of Congenital Adrenal Hyperplasia 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Pediatric Endocrinology Saturday, June 15th 11:30:00 AM FP11 2335 11:00:00 AM Pediatric Endocrinology Poster Preview


Kristy A Brown*1, Seungmin Ham2, Sarah Jayne Meachem3, Catherine S. Choong4, Adrian K Charles5, Gareth S Baynam6, Timothy W Jones7, Nirukshi U Samarajeewa8 and Evan R Simpson9
1Prince Henry's Institute, Clayton VIC, Australia, 2Prince Henry's Institute, Clayton Victoria, Australia, 3Prince Henry's Inst Med Rsrch, Clayton VIC, Australia, 4Princess Margaret Hospital for Children, Subiaco, Australia, 5Genetic Services of Western Australia, Australia, 6Princess Margaret Hospital for Children, Australia, 7Princess Margaret Hosp, Western Australia, Australia, 8Prince Henry's Institute, Australia, 9Prince Henry's Inst of Med Res, Clayton VIC, Australia

 

Peutz Jeghers syndrome (PJS) is an autosomal dominant disorder characterized by the association of gastrointestinal harmatomatous polyps and mucocutaneous pigmentation which is due to mutations in the STK11 gene that encodes the LKB1 protein. PJS males may have estrogen excess manifesting as gynecomastia and an advanced bone age. We and the group of Santen have previously described an increase in testicular aromatase expression in PJS patients. However, the underlying mechanism has not yet been explored. The aim of this study was to characterize the role of LKB1 in regulating the expression of aromatase in the testes of two boys with PJS via signaling pathways involving pAMPK and CRTC1, CRTC2, and CRTC3. We studied testicular biopsies from two boys with STK11 mutations; a 13-year-old boy and an unrelated 4-year-old boy with prepubertal gynaecomastia and advanced bone age. Loss of heterozygosity of STK11 in Sertoli cells of abnormal cords of PJS testis samples, measured by the absence of LKB1 immunofluorescence staining, was associated with loss of p21 expression and decreased phosphorylation of AMPK, known downstream targets of LKB1, as well as the increased expression of aromatase.  The nuclear localization of the potent stimulator of aromatase CRTC3, which arises as a consequence of decreased activity of AMPK, was increased in cells where aromatase was detected. In conclusion, loss of heterozygosity of the STK11 gene leads to an increase in aromatase expression associated with increased CRTC3 nuclear localization, thereby providing a mechanism whereby PJS is associated with estrogen excess.

 

Nothing to Disclose: KAB, SH, SJM, CSC, AKC, GSB, TWJ, NUS, ERS

FP11-6 8007 6.0000 SAT-599 A Estrogen Excess Phenotype Associated With Loss of Heterozygosity of the STK11 Gene in the Testis of Two Boys With Peutz Jeghers Syndrome 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Pediatric Endocrinology Saturday, June 15th 11:30:00 AM FP11 2335 11:00:00 AM Pediatric Endocrinology Poster Preview


Tassiane Alvarenga Oliveira*1, Marina Cunha Silva1, Mirian Y Nishi2, Andrea Maciel Guerra3, Gil Guerra Jr.3, Elaine Maria Frade Costa4, Berenice B Mendonca5 and Sorahia Domenice4
1Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HC-FMUSP), 2University of São Paulo, Hospital das Clinicas, Sao Paulo, Brazil, 3Grupo Interdisciplinar de Estudos da Determinação e Diferenciação do Sexo, Faculdade de Ciências Médicas, Universidade Estadual de Campinas (FCM-UNICAMP), Campinas, SP, Brazil., 4Disciplina de Endocrinologia e Metabologia - Laboratório de Hormônios e Genética Molecular/ LIM42, Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil, 5Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo FMUSP, São Paulo, Brazil

 

Introduction Persistent Müllerian Duct Syndrome (PMDS) is defined as the lack of Müllerian derivatives regression in an otherwise normally virilized XY male. This rare disorder is due to mutations in the anti-Müllerian hormone (AMH) gene or in the type II AMH receptor (AMHR- II) gene. A variety of germ cell tumors have been reported in patients with PMDS. The presence of a longstanding cryptorchidism has been associated with the presence of gonadal tumors in these patients. However, in adult male Amh or Amhr-II  transgenic mice the presence of Leydig cells hyperplasia and testes tumor have been described. Aim To describe the presence of testicular tumors in patients with PMDS. Patients 11 patients with PMDS were evaluated.  The patients presented with age from 0.3 to 37 yrs. 8 of them were at postpubertal age (15 to 38 yrs). The main complaints were the presence of inguinal hernia and/or cryptorchidism. 3 patients were referred by tumor mass. All subjects had male external genitalia and uni (1 patient) or bilateral cryptorchidism (10 patients). All of them had 46,XY karyotype and present Müllerian derivatives demonstrated by histological analysis. Bilateral gonadectomy was done in 6 patients and orchidopexy in 5 patients (2 unilateral and 3 bilateral). 2 patients who underwent bilateral orchidopexy lost the follow up and in 1 patient it was done gonadectomy 8 yrs later. AMH and AMHR2 mutations were identified in 6 and 2 patients respectively. Results Testicular tumors were diagnosed in 3 patients (2 abdominal and 1 scrotal mass). At the time of diagnosis, the age of patients was 38, 33 and 23 yrs. The histological study showed an embryonal carcinoma (EC), a seminoma (S), and a bilateral S in previously undescended testes placed in the scrotum (8 yrs after orchidopexy) , respectively. AMH mutations were identified in patients with EC and S; and AMHR2 mutations in the patient with bilateral S. Discussion In PMDS, the testes seem to be histologically normal but the incidence of testicular tumor is 5 - 18%. This incidence is similar to the cryptorchidic testes. A higher frequency of testicular cancer was observed in our group of postpubertal patients (37.5%). Conclusions The frequency of testicular tumors in PMDS patients may be higher than that regularly described. Later orchidopexy not offers protection against future testicular malignancy. AMH and AMHR-II have been considered tumor suppressor genes and the role of AMH in the tumor testis development could not be ruled out.

 

Nothing to Disclose: TAO, MCS, MYN, AM, GG Jr., EMFC, BBM, SD

FP12-3 6958 3.0000 SAT-558 A Testicular Tumors in Patients With AMH and Amhr-II Genes Mutations 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Reproductive Endocrinology Saturday, June 15th 11:30:00 AM FP12 2353 11:00:00 AM Reproductive Hormones: Vital Effects Evidenced In Human Cohorts & Experimental Models Poster Preview


Kim Mileski1, Luiz Guilherme Grossi Porto*2 and Adriana Lofrano Alves Porto3
1Faculty of Health Sciences - University of Brasilia, Brasilia, Brazil, 2University of Brasilia, Brasilia, Brazil, 3University of Brasilia, Brasilia DF, Brazil

 

INTRODUCTION: Congenital male hypogonadism (CH) is a rare clinical disorder characterized by delayed or absent pubertal development and insufficient testosterone (T) production. T deficiency is known to result in significant physical impairment. CH patients commonly report weakness and fatigue, even during Testosterone Replacement Therapy (TRT). OBJECTIVE: To compare the isokinetic quadriceps muscle performance between men with and without CH. METHODS: Men with CH under TRT were recruited from the Gonadal and Adrenal Diseases Clinics of the University Hospital of Brasilia for a physical test evaluation (CH group; n=9). Control group was recruited among healthy community men with no clinical hypogonadism criterion (CG; n=16)1. CH and control volunteers had similar age (17 - 46 yrs), BMI, waist circumference and physical activity level (IPAQ questionnaire) (p>0.05). Muscle performance (strength and endurance) was measured by knee extension on isokinetic dynamometer equipment Biodex System 3®, as proposed by the American Society of Exercise Physiology2. We analyzed the highest Peak Torque (PT) by a strength protocol (2 sets of 4 maximum repetitions at 60º/s with 1 min rest between sets); the Total Work (TW) by a endurance protocol (1 set of 30 maximum repetitions at 180º/s) and the Fatigue Index (FI) by the percentage decrease between the last 5 and the first 5 repetitions of the endurance protocol. Variables were measured on day one (D1 - 7 days after T administration) and on day two (D2 - 14 days after T administration). CG was randomly assigned for D1 and D2. Unpaired t-test was used with p<0.05, after Shapiro-Wilk test. RESULTS: On D1, CH and CG values were similar for PT (251.3±53.3 vs 228.5±32.0 N.m; p=0.19), TW (3914.0±853.4 vs 3661.0±464.2 J; p=0.34) and FI (40.4±6.5 vs 40.0±12.3%; p=0.93). A similar pattern was observed on D2., with PT = 243.2±55.4  vs 235.4±29.8 N.m (p=0.65); TW = 3929.0±921.5 vs 3720.0±471.7 J (p=0.46) and FI = 42.9±7.3 vs 41.8±12.2% (p=0.80), respectively. Testosterone on CH was higher on D1 (616.2±335.0 ng/dL) compared to D2 (231.9±84.0 ng/dL; p=0.007). T for CG were similar on both protocol days (346.1±149.6 vs 330.1±127.6 ng/dL; p=0.46) CONCLUSION: Quadriceps muscle performance in males with CH under TRT were similar to paired healthy men, independently of T values. Our findings suggest that TRT was effective in maintaining quadriceps muscle performance in congenital hypogonadic patients, similarly to the controls.

 

Nothing to Disclose: KM, LGGP, ALAP

FP12-4 8568 4.0000 SAT-556 A COMPARISON OF THE ISOKINETIC QUADRICEPS MUSCLE PERFORMANCE BETWEEN MEN WITH AND WITHOUT CONGENITAL HYPOGONADISM 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Reproductive Endocrinology Saturday, June 15th 11:30:00 AM FP12 2353 11:00:00 AM Reproductive Hormones: Vital Effects Evidenced In Human Cohorts & Experimental Models Poster Preview


Shannon Danielle Sullivan*1, Hong Wang2, Marc R. Blackman3 and Suzanne Groah4
1Medstar Washington Hospital Center, Washington, DC, 2Medstar Health Research Institute, Hyattesville, MD, 3Washington DC VAMC, Bethesda, MD, 4Medstar National Rehabilitation Hospital, Washington, DC

 

Background: 300,000 individuals with spinal cord injury (SCI) currently reside in the U.S., with injuries most commonly occuring in men between the ages of 15 and 24. Men with SCI undergo an accelerated aging process post-injury, characterized by development of sarcopenic obesity and metabolic syndrome, which together heighten overall cardiovascular disease (CVD) risk. Indeed, CVD is a leading cause of death among men with SCI who achieve long-term post-injury survival. Hypogonadism is also a frequent complication of SCI in men, which may contribute to development of sarcopenic obesity and metabolic syndrome post-injury.  To date, however, few studies have investigated the prevalence or cardiometabolic consequences of T deficiency in men with SCI. 

Methods: In this pilot study, we investigated the prevalence of T deficiency in 36 young (age 18-50 years) men with chronic (≥ 1 yr) SCI and the relationships between total T (TT) and free T (fT) and cardiometabolic risk components, including body composition [BMI, waist-to-hip ratio (WHR), central body fat %], insulin sensitivity [insulin sensitivity index (ISI), fasting and 2hr blood glucose on oral glucose tolerance test (OGTT), HbA1C%, SHBG], lipid profiles [fasting total cholesterol (TC), HDL, LDL, VLDL, and triglycerides (TG)], and systemic inflammation (hsCRP, IL-6) in a subset (n=14) of these men.  Due to small sample size for correlation analyses, the non-parametric Spearman Correlation coefficient was used to calculate correlations between TT/fT and cardiometabolic risk factors.  Correlation coefficients [r] ≥ 0.3 were considered fair to strong correlations; P<0.05 was considered significant.

Results: The prevalence of low T, defined as serum TT<300 ng/dL and/or serum fT< 9 ng/dL, was 33% (n=12/36).  14 of these 36 men with SCI have undergone the cardiometabolic risk assessments listed above, 36% (n=5/14) of whom had TT<300 ng/dL [(n=2)(mean±s.d. TT, 458±163; range 120-686 ng/dL)] or fT<9 ng/dL [(n=3) (mean±s.d. fT, 11.0±3.7; range 2.6-16.0 ng/dL)].  Among these 14 men, TT was significantly related to the calculated fT (r = 0.81, P<0.01).  Low TT was associated with less favorable BMI (r= -0.37), WHR (r= -0.74), TG (r= -0.33), HDL (r= 0.83), VLDL (r= -0.42), FBG (r= -0.60), 2hr OGTT glc (r= -0.36), ISI (r= 0.49), SHBG (r= 0.67), and % central fat (r= -0.41). Low fT was associated with less favorable WHR (r= -0.74), HDL (r= 0.66), FBG (r= -.30), 2hr OGTT glc (r= -0.34), and % central fat (r= -0.34). Associations of TT with CRP, IL-6, and HbA1C%, and of fT with BMI, TG, VLDL, CRP, IL-6, ISI, and HbA1C were poor (r<0.3).

Conclusion: T deficiency is common in young men with chronic SCI and is associated with increased cardiometabolic risk. Thus, screening for hypogonadism is warranted in this population, and the effects of physiologic T replacement therapy on cardiometabolic risk reduction and prevention of CVD should be investigated.

 

Nothing to Disclose: SDS, HW, MRB, SG

FP12-5 6641 5.0000 SAT-557 A Hypogonadism Is Associated With Increased Cardiometabolic Risk in Young Men With Chronic Spinal Cord Injury 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Reproductive Endocrinology Saturday, June 15th 11:30:00 AM FP12 2353 11:00:00 AM Reproductive Hormones: Vital Effects Evidenced In Human Cohorts & Experimental Models Poster Preview


Lori Ann Cooper*1, Stephanie T. Page1, Thomas J. Walsh1, John K. Amory2, Bradley D. Anawalt1 and Alvin M. Matsumoto3
1University of Washington, Seattle, WA, 2Univ of WA Med Ctr, Seattle, WA, 3VA Puget Sound Hlth Care Sys, Seattle, WA

 

Background: Sex hormone binding globulin (SHBG) is affected by many conditions, most notably obesity and aging.  Circulating testosterone (T) is mostly bound to SHBG and albumin; only 1-2% is unbound or “free”.  Alterations in SHBG affect total T (TT), but not free T levels.  Increasing age is associated with higher SHBG levels, whereas obesity is associated with lower SHBG levels.  It is not clear whether older, obese men have higher, lower, or similar SHBG levels compared to non-obese, younger men. 

Hypothesis: We hypothesized that increasing age is associated with a relatively greater effect on SHBG and TT levels than obesity.  We also tested whether TT is an insensitive test of biochemical hypogonadism in obese and non-obese older men.

Methods: Using a cohort of 3671 men evaluated for hypogonadism at the VA Puget Sound from 1997-2007, we compared TT, calculated free T, and SHBG among subgroups of younger and older (age <65 or ≥65 years old) and non-obese and obese (body mass index, BMI <30 or ≥30 kg/m2).  We also calculated the sensitivity of various TT thresholds for these groups to exclude biochemical hypogonadism (calculated free T <34 pg/mL).

Results: SHBG levels were higher in older men than in younger men (49 ±24 [mean±SD] vs 42 ±27 nmol/L, p<0.001).  SHBG levels were lower in obese men than in non-obese men (36 ±22 vs 50 ±27 nmol/L, p<0.001).  Differences in TT were similar to those for SHBG.  The differences in SHBG and TT associated with obesity were proportionately greater than those associated with older age.  In addition, in obese men ≥65, a TT value ≥280 ng/dL had a sensitivity of 100% to exclude biochemical hypogonadism (i.e. all had normal calculated free T levels).  A sensitivity ≥98% was not seen in the younger (non-obese and obese) and older non-obese subgroups until TT ≥400 ng/dL.  In all groups, the ability to rule in biochemical hypogonadism was poor, and a specificity of 98% was not reached until TT<150 ng/dL.

Summary: 1) Obesity has a significantly greater effect on SHBG and TT levels than aging.  2) A TT ≥280 ng/dL is highly sensitive to exclude biochemical hypogonadism in older obese men, but is insensitive in younger (non-obese and obese) and older non-obese men.

Conclusions: In older obese patients, a normal TT level (≥280 ng/dL) reliably excludes biochemical hypogonadism, but TT is a poor screening test for biochemical hypogonadism in younger (non-obese and obese) men and for non-obese older men.

 

Disclosure: AMM: Investigator, Abbott Laboratories, Investigator, GlaxoSmithKline, Consultant, Lilly USA, LLC, Consultant, GTx, Editor, Up To Date, Grant Review Panel, Partnership for Clean Competition. Nothing to Disclose: LAC, STP, TJW, JKA, BDA

FP12-6 5383 6.0000 SAT-554 A Obesity Has a Greater Influence Than Aging On Serum SHBG and Total Testosterone (TT) in Men, and a Normal TT Is Sufficient to Exclude Biochemical Hypogonadism in Older, Obese Men 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Reproductive Endocrinology Saturday, June 15th 11:30:00 AM FP12 2353 11:00:00 AM Reproductive Hormones: Vital Effects Evidenced In Human Cohorts & Experimental Models Poster Preview


Hernan E Gonzalez*1, Rodrigo Martinez1, Lorena M Mosso2, Sergio Vargas1, Soledad Urra1 and Alexis Kalergis1
1Pontificia Universidad Católica de Chile, Santiago, Chile, 2P Univ Catolica, Santiago, Chile

 

INTRODUCTION: Fine needle aspiration (FNA) biopsy is the standard approach of choice to determine the nature of thyroid nodules. However, in approximately 15-20 % of cases the cytology is reported as indeterminate, including atypia of undetermined significance and follicular neoplasm (Bethesda III and IV). In these cases, surgery is frequently recommended although only 15 to 25% of theses cases prove to be cancer. Recent molecular testing assays have emerged with improved diagnostic accuracy; however, they require a second FNA and analysis is performed in a centralized service setting. We present a novel gene signature that uses 10 genes, is highly predictive and suitable for point of care molecular diagnosis.

METHODS: Systematic literature search for potential biomarkers related papillary thyroid cancer (PTC) was performed. Eighteen genes were selected.  Fresh samples from PTC and benign thyroid nodules were obtained in the operating room from 215 patients (108 cancer – 107 benign). Gene expression was analyzed by quantitative real-time PCR. Patients were randomly divided into two sets, a training set (68 PTC and 42 benign) and a testing set (38 PTC and 67 benign). Two independent algorithms were trained; the first one identified and classified patients with outlier gene profiles  (Outlier Classification System - OCS) (algorithm 1) and the second used linear discriminant analysis (LDA) (algorithm 2). The training set was analyzed sequentially in two steps: first, outliers were identified and classified by the OCS, and the remaining non-classified data followed to LDA. Analytical accuracy was determined by receiving operating characteristics curves (ROC) analysis.

RESULTS: The new classifier used ten genes, and showed high analytical accuracy; area under the curve (AUC) 0.98, sensitivity 98 %, specificity 93%, positive predictive value (PPV) 95% and negative predictive value (NPV) 96%. Analytical accuracy of the classifier remained remarkably high in the independent testing set; AUC 0.95, sensitivity 95 %, specificity 90%, PPV 83% and NPV 98%. In a second testing set performed in 65 fresh FNA samples (49 benign and 16 PTC), the assay correctly classified 62 samples, with sensitivity 88%, specificity 96%, PPV 88% and NPV 96%.

CONCLUSION: We have developed a new highly predictive gene signature that accurately classifies thyroid nodules. It effectively classifies benign nodules, making it potentially useful to identify patients that do not require surgery. This signature uses only ten genes making it suitable for the development of kit for point of care molecular diagnosis. Final validation set of patients with indeterminate nodules is underway to prove clinical usefulness of this new assay.

 

Nothing to Disclose: HEG, RM, LMM, SV, SU, AK

FP14-1 3559 1.0000 SAT-414 A New Highly Predictive Gene Signature for Indeterminate Thyroid Nodules for Point of Care Molecular Diagnosis 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Thyroid/HPT Axis Saturday, June 15th 11:30:00 AM FP14 2373 11:00:00 AM Thyroid Cancer: Insights into Diagnosis & Treatment Poster Preview


Becky Thai Muldoon*1, Michele Gage2, Merica Shrestha3, Alexander Stojadinovic1 and Henry B Burch1
1Walter Reed National Military Medical Center, Bethesda, MD, 2Walter Reed National Military Medical Center, 3Eisenhower Army Medical Center, Evans, GA

 

Background:  After an initially benign FNA, thyroid nodules are still monitored for growth or development of suspicious clinical or ultrasound features, owing to a false-negative rate from 0.4-13%.  Such nodules frequently undergo a second FNA procedure in order to further reduce the FNA false-negative rate.  We sought to determine the outcome of repeat thyroid FNA following an initially benign result.

Methods:  All thyroid FNAs performed at the Walter Reed Army Medical Center during a 10-year period from September 2001-August 2011 were retrospectively reviewed.  All patients who had a repeat FNA after an initially benign result were considered for inclusion.  A strict correlation between the biopsy site, location and size of nodule on ultrasound and ultimate pathology report for those undergoing surgery was ensured. FNA results were classified as insufficient, benign, malignant, or indeterminate (includes atypical lesions, follicular neoplasm/suspicious for follicular neoplasm, suspicious for malignancy), and the pathology result was categorized as either benign or malignant.  The outcome of repeat FNA was then categorized for each nodule.

Results:  Of 3013 patients that had FNA, 439 patients had more than one FNA.  In those with more than one FNA, 104 patients had repeat biopsy of a nodule with a previously benign FNA and did not have surgery and 71 patients had a repeat of a benign FNA and ultimately did go to surgery.  In those patients that did not go to surgery, there were a total of 237 nodules with benign FNA that were repeated, in which the repeat FNA was benign in 228 nodules (96%), indeterminate in 4 nodules (1.7%, all were atypical), and inadequate in 5 nodules (2.1%).  Of the 71 patients that had surgery, 45 patients (63%) had repeatedly benign FNA result after the initial benign FNA and all but one patient had benign surgical pathology except for 1 who had malignant surgical pathology corresponding to the nodule. Three patients had concurrent incidental microcarcinomas.  Two patients went to surgery after repeat FNA was insufficient (both had benign surgical pathology),  and 22 patients had repeat FNA that was indeterminate, leading to surgery, with no malignancy found;  3 patients had incidental microcarcinoma in areas not previously sampled.  In 2 patients, repeat biopsy yielded a malignant FNA result, but only 1 of those had malignant surgical pathology; the second patient had an incidental microcarcinoma elsewhere in the gland.

Conclusion:   Our results show that repeat of a benign FNA generally leads to another benign result or one that is indeterminate, resulting ultimately in unnecessary surgery.  For those that did go to surgery, the majority of those nodules with initial benign FNA ultimately had benign surgical pathology of those nodules.  Stricter criteria need to be applied in the selection of thyroid nodules for a repeat FNA following a previously benign result.

 

Disclosure: HBB: Consultant, Up To Date. Nothing to Disclose: BTM, MG, MS, AS

FP14-2 7541 2.0000 SAT-417 A An Assessment Of The Outcome Of Repeat Thyroid FINE-Needle Aspiration In Nodules Previously Found To Have A Benign Cytology 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Thyroid/HPT Axis Saturday, June 15th 11:30:00 AM FP14 2373 11:00:00 AM Thyroid Cancer: Insights into Diagnosis & Treatment Poster Preview


Amit Bhargava*1, Sameera Tallapureddy1, Sarah Varghese2 and Beatriz Tendler1
1University of Connecticut, Farmington, CT, 2University of Connecticut, Hartford, CT

 

Background: Rheumatoid nodules occur in 30% of patients with active rheumatoid arthritis (RA), and commonly involve sites like the extensor surface of the forearm. The authors present a unique case of the development of rheumatoid nodules in the thyroid bed of a patient, post thyroidectomy.

Clinical Case: A 46-year-old Caucasian lady, with active RA and Hashimoto’s thyroiditis, on hydroxychloroquine, prednisone and levothyroxine, presented with a goiter. She complained of dysphagia and a sensation of airway compression. The thyroid was enlarged. On ultrasound, the right lobe measured 7.9x3.4x3.3cm, the left lobe 8.3x3.3x3.1cm, and isthmus 2.1cm. TSH was 4.22 uU/ml (0.34-5.60). Due to a concern of worsening tracheal compression and growth of a nodule on levothyroxine, a total thyroidectomy was done (thyroid gland- 79g). Repeat ultrasound showed no remaining tissue. Pathology revealed several small neoplasms ranging from a well-encapsulated adenoma to highly atypical follicular and papillary Hurthle cell lesions, in the setting of Hashimoto’s thyroiditis. Due to this, low dose radioactive iodine (RAI) 33.4 mCi was given. 4 months later, the patient complained of neck fullness. A large solid nodule of mixed echogenicity (5.6x3.3x2.3cm) was seen in the right level VI of the neck, and solid tissue of mixed echogenicity (2.9x2.3x1.7cm) on the left. Repeat surgery yielded a 11g aggregate of soft, tan irregular tissue from the right, and 1g from the left.  Pathology from the right showed Hashimoto's thyroiditis. The left tissue specimen had areas of granuloma formation with fibrinoid necrosis and palisading histiocytes, consistent with the histology of a rheumatoid nodule. No malignant foci were seen. No further RAI was given and the patient remains disease free 4 years later.

Conclusion:  Rheumatoid nodules have not been reported in the thyroid bed. Their pathogenesis is not clear. Post-operative release of TNF- α and local vascular damage may have triggered nodule formation in this case. Rheumatoid nodules must be kept in the differential diagnosis of an enlarging thyroid, in the setting of active RA. This is especially relevant if there is a triggering factor such as small vessel trauma. Fine needle aspiration biopsy may show granuloma formation and be the most cost-effective initial step. Early identification of these nodules will help decrease morbidity from unnecessary interventions and result in treatment that is both timely and appropriate.

 

Nothing to Disclose: AB, ST, SV, BT

FP14-4 7665 4.0000 SAT-419 A A Thyroid Nodule with a Twist 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Thyroid/HPT Axis Saturday, June 15th 11:30:00 AM FP14 2373 11:00:00 AM Thyroid Cancer: Insights into Diagnosis & Treatment Poster Preview


Sapna Sanjay Shah*1, Annis M. Marney2, Cindy Vnencak-Jones3, Shubhada M Jagasia4 and Leon Lucien Parks III5
1Vanderbilt Univ, Nashville, TN, 2University of Vermont, South Burlington, VT, 3Vanderbilt University, 4Vanderbilt Eskind Diab Clinic, Nashville, TN, 5Vanderbilt Eskind Diabetes Cli, Nashville, TN

 

Background:    Malignant struma ovarii, a rare germ cell tumor, presents diagnostic and treatment controversies. We present a case of simultaneous malignant struma ovarii and intrathyroidal papillary thyroid carcinoma.   

Clinical Case: A 49 year old woman presented with a 3 month history of increasing abdominal fullness and distension. Laboratory workup demonstrated a normal complete blood count, basic metabolic profile, thyroid stimulating hormone and free thyroxine, and minimally elevated CA-125 of 22.5 μ/ml (ref 0.0-19.0 μ/ml).  She was evaluated with exploratory laparotomy for large, mobile, right-sided pelvic mass and was found to have malignant struma ovarii.  Pathology was consistent with papillary thyroid carcinoma.  She was then found to have multinodular goiter. Surgical pathology after total thyroidectomy revealed follicular variant papillary thyroid microcarcinoma. BRAF mutation analysis showed distinct mutations with BRAF mutation V600E in the primary intrathyroidal tumors and BRAF mutation K601E in the malignant struma ovarii.  The patient has been followed for the last five years and has been recurrence free with thyroid stimulating hormone in the low normal range and undetectable unstimulated thyroglobulin levels and no concerning lymph nodes on neck ultrasound.

Conclusion: Two previous cases report coincident malignant struma ovarii and intrathyroidal carcinoma. To our knowledge, this is the first case of concurrent malignant struma ovarii and papillary thyroid carcinoma utilizing molecular analysis of pathology specimens.  BRAF mutation K601E in the strumal focus compared to the more common BRAF mutation V600E in the thyroidal foci convincingly shows that the focus of malignant struma is an independent process. The application of molecular analysis of thyroid tumors expands our understanding of thyroid tumor biology. This analysis demonstrated that tumor pathogenesis in this setting involves similar molecular mechanisms to those implicated in tumors developing within the thyroid gland. Molecular testing may be a useful adjunct to cytological-pathological diagnosis of thyroid malignancy in struma ovarii and improve tumor prognostication.

 

Disclosure: AMM: Coinvestigator, Merck & Co.. Nothing to Disclose: SSS, CV, SMJ, LLP III

FP14-6 7169 6.0000 SAT-415 A Utility of Thyroid Molecular Mutation Analysis in Malignant Struma Ovarii 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Thyroid/HPT Axis Saturday, June 15th 11:30:00 AM FP14 2373 11:00:00 AM Thyroid Cancer: Insights into Diagnosis & Treatment Poster Preview


John P Wiebe*1, Roxana Schillaci2, Patricia Virginia Elizalde3 and Martin Alfredo Rivas4
1Univ of Western Ontario, London, ON, Canada, 2Instituto de Biología y Medicina Experimental, CONICET, Buenos Aires, Argentina, 3Instituto de Biología y Medicina Experimental (IBYME-CONICET), Buenos Aires, Argentina, 4Vall d'Hebron Institute of Oncology, Barcelona, Spain

 

Previous studies have shown that  progesterone (P) is converted to 5alpha-dihydroprogesterone (5aP) in breast tissue and human breast cell lines by the action of 5alpha-reductase (5a-red) [1-3], and that 5aP stimulates proliferation of human mammary cells in vitro regardless of their estrogen responsiveness, estrogen (ER)/progesterone (PR) receptor levels, and whether they are nontumorigenic or tumorigenic [1,4]. Studies with human breast cancer cells in which the conversion of P to 5aP was blocked by a 5a-red inhibitor [5] provided in vitro proof-of-principle that the cancer promoting actions are due to 5aP and not to P. The objective of the studies reported here was to determine in a P-sensitive in vivo mouse model if the presumptive P-induced mammary cell tumorigenesis is due to the 5aP metabolite. METHODS. BALBc mice were challenged with C4HD (ER+/PR+) murine mammary cells, which have been shown to form tumors when treated with the synthetic progestin MPA [6,7] or with P [8]. Cells and mice were treated with various doses and combinations of P, 5aP and/or the 5a-red inhibitor, finasteride (FIN), and the effects on cell proliferation (in vitro) and induction and growth of tumors (in vivo) were monitored. Hormone levels in serum and tumors were measured by specific RIA and ELISA tests. RESULTS. In vitro, C4HD cell proliferation was stimulated equally by treatment with P and 5aP; the increases with P were blocked by FIN and were reinstated with 5aP. In vivo, either 5aP or P induced tumorigenesis; the P-induced tumorigenesis was blocked by FIN and reinstated by concomitant treatment with 5aP. Hormone measurements showed significantly higher levels of 5aP in serum from mice with tumors (8.7 ±1.9 ng/ml) than without tumors (2.1±0.8 ng/ml) and significantly higher levels in tumors (32.7±6.0 ng/g) than in respective sera. CONCLUSIONS. The results indicate that the stimulation of C4HD tumor growth in BALBc mice treated with P is due to the P metabolite 5aP, formed at elevated levels in mammary tumors as a result of the 5a-red action on P. These are the first in vivo studies indicating that the P metabolite 5aP, rather than P itself, is the cancer-promoting hormone that stimulates tumor induction and growth in ER+/PR+ breast cells exposed to progesterone.

 

Nothing to Disclose: JPW, RS, PVE, MAR

FP07-1 8580 1.0000 SAT-295 A Progesterone-induced stimulation of PR-positive murine mammary tumorigenesis is due to the progesterone metabolite, 5alpha-dihydroprogesterone (5aP) 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Cancer/Neoplasia Saturday, June 15th 11:30:00 AM FP07 2378 11:00:00 AM New Players in Hormonal Control of Breast & Prostate Cancer Poster Preview


Hong Zhao*1, Robert T Chatterton Jr.1, John S Coon V1, David C Brooks1, Dolores Huberts1, Francesco J. DeMayo2 and Serdar Ekrem Bulun3
1Northwestern University, Chicago, IL, 2Baylor College of Medicine, Houston, TX, 3Northwestern Univ, Chicago, IL

 

Aromatase is a key enzyme for estrogen biosynthesis. Long-term cumulative exposure to estrogen increases the breast cancer risk in women.  Recently it has been recognized that metabolic reprogramming is tumor hallmark, and an important regulator of cellular energy homeostasis that is often altered is AMP-activated protein kinase (AMPK).  Metformin, a front-line antidiabetic drug, lowers serum glucose levels via activating AMPK pathway in the liver. In vitro studies show that activated AMPK inhibits adipose tissue aromatase expression via suppression of binding of cAMP-responsive element binding protein (CREB) to aromatase promoters II/I.3, leading to decreased estrogen formation. However, there is no mouse model for study of the in vivo role of metformin on aromatase expression and carcinogenesis in mammary tissue via the AMPK pathway because mice lack aromatase expression in their mammary glands.   To address role of alteration of AMPK metabolic pathway in the most common breast cancer risk, i.e estrogen, we generated transgenic humanized aromatase (Aromhum) mice to mimic the human aromatase distribution pattern in the mouse, and crossed this mouse with an ERBB2 transgenic mouse to generate ERBB2 and Aromhum/ERBB2 (AE) mice. Fifty six percent of ERBB2 (only) mice developed mammary tumors within 6 months, as expected.  AE mice had a significantly higher (86%, p<0.05) tumor incidence at this time.  Thus, the human aromatase transgene increased the rate of mammary tumor growth in ERBB2 mice. Metformin treatment (125 mg/kg /day for 21 days) significantly decreased blood glucose levels by 35 % in AE mice over this time as compared to vehicle treatment. Immunihistochemistry demonstrated that AMPKα was expressed in both stromal and mammary epithelial cells. Phosphorylation of AMPKα was increased in breast tissues and mammary tumors of AE mice after metformin treatment, and phosphorylation of CREB, a downstream effector of the AMPK pathway, which mediates aromatase expression, was significantly decreased.  In conclusion, metformin upregulated the AMPK pathway in normal breast tissues and breast tumors of AE mice and may lead to decreased mammary aromatase expression and estrogen production, and reduced tumorigenesis.  This study provides a fundamental mechanism for testing strategies for prevention of breast cancer using the AMPK activator metformin in women with type 2 diabetes.

 

Nothing to Disclose: HZ, RTC Jr., JSC, DCB, DH, FJD, SEB

FP07-5 9329 5.0000 SAT-293 A Metformin Upregulates AMPK Pathway in Humanized Aromatase Expressing ERBB2 Mice 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Cancer/Neoplasia Saturday, June 15th 11:30:00 AM FP07 2378 11:00:00 AM New Players in Hormonal Control of Breast & Prostate Cancer Poster Preview


Monika E Schmidmayr*1, Johanna Lohmaier2, Loreen Richter2, Peter Luppa3, Almut Artmann4, Tibor Schuster5, Marion Kiechle6 and Vanadin Regina Seifert-Klauss7
1Tech Univ Muenchen, Munich, Germany, 2Technische Universitaet Muenchen, 3Technische Universitaet Muenchen, Munich, 4Praxis fuer Brustgesundheit, Munich, 5Technische Universitaet, Munich, 6Technische Universitaet Muenchen, Munich, Germany, 7Frauenklinik der TU Muenchen, Munich, Germany

 

Introduction

Vitamin D deficiency has been linked to higher grade breast tumors, thereby possibly affecting prognosis in breast cancer. Mammographic density is an established risk factor for breast cancer, while high density breast tissue may also delay diagnosis. The association of serum vitamin D and mammographic density has not been studied prospectively in a sufficiently large sample to date.

Methods

In this cross sectional study women with a clinical indication for mammography were recruited for a standardized interview on reproductive history, risk factors for breast cancer, diet, activity and chronic diseases. Serum 25-OH-vitamin D (sVD), calcium, phosphate and creatinine were measured and breast density by ACR classification documented. Patients with >= BIRADS IV received a breast biopsy.

Results

A significant difference in sVD was found between women with ACR 3 vs. ACR4 (p = 0.04) after multivariate adjustment for other risk factors, but not when comparing ACR 1 or 2 with ACR 4. Out of 1090 recruited women, 111 (10%) were biopsied, 53 (4.9%) were diagnosed with DCIS or invasive carcinoma. In a 1:2 matching analysis, matching criteria were age (+/- 5years), BMI (+/- 2kg/m²), menopausal status, family history, vitamin D or hormone intake. Patients with carcinoma or DCIS had mean sVD values of 16.1ng/ml vs. 16.7ng/ml in the control group (n=106). 96% of all women were vitamin D deficient. Regression analysis showed a non-significant 13% risk reduction for malignancy per 10ng/ml increase of sVD (OR 0.87, 95% CI 0.51-1.47, p=0.602).

Conclusion

Low serum vitamin D may independently influence very high breast density, thereby reducing mammographic sensitivity and increasing the risk of higher grade tumors.

 

Nothing to Disclose: MES, JL, LR, PL, AA, TS, MK, VRS

FP07-6 7136 6.0000 SAT-292 A Serum 25-Hydroxyvitamin D, Mammographic Density and Breast Biopsy Results 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Cancer/Neoplasia Saturday, June 15th 11:30:00 AM FP07 2378 11:00:00 AM New Players in Hormonal Control of Breast & Prostate Cancer Poster Preview


Nadia Cherradi*1, Rossella Libe2, Guillaume Assie3, Olivia Barreau4, Jerome Yves Bertherat5, Xavier Bertagna5, Jean-Jacques Feige1 and Olivier Chabre6
1INSERM U1036, Grenoble, France, 2INSERM Unité 1016, Centre National de la Recherche Scientifique Unité Mixte de Recherche 8104, Institut Cochin, Paris, France, 3INSERM U 1016, CNRS 8104, Institut Cochin, Paris Descartes University, Paris, France, 4Inserm U1016, CNRS 8104, Institut Cochin, Paris Descartes University, Paris, 5Hopital Cochin, Paris, France, 6University Hospital Grenoble, Grenoble, France

 

Adrenocortical carcinoma (ACC) is a rare cancer with poor prognosis. Local and distant recurrences occur in a subset of tumors classified as “aggressive” ACC (aACC), as opposed to “non-aggressive” ACC (naACC). In this study, we investigated whether tissue and serum microRNAs (miRNAs) are predictive of ACC aggressiveness. MiRNA expression profiles were determined using microarrays in a test series of adrenocortical adenomas (ACA), naACC and aACC. Eight miRNAs were selected for further validation by quantitative RT-PCR (validation cohort: 10 ACA, 9 naACC, 9 aACC, and 3 normal adrenocortical tissues). Quantification of selected miRNAs was performed in serum samples from 56 subjects (19 healthy controls, 14 ACA, 9 naACC and 14 aACC patients). MiR-195 levels were significantly decreased in both tumor and serum samples of ACC patients relative to ACA patients and healthy controls. MiR-139-5p and miR-376a levels were significantly increased in aACC compared to naACC patients in tumor samples only. Very interestingly, miR-483-5p, a miRNA which is located within the second intron of insulin-like growth factor Igf2 gene, was dramatically increased in 80 % of ACC compared to ACA tumor samples. In addition, serum miR-483-5p was detected only in aACC patients, suggesting that its release in the bloodstream is related to advanced disease. Receiver operating characteristic (ROC) analyses indicated that serum miR-195 and miR-483-5p had a high diagnostic value for ACC and aACC patients, respectively. Kaplan-Meier analyses revealed that low circulating levels of miR-195 and high circulating levels of miR-483-5p were associated with poor prognosis (p=0.0314 and p=0.0003, respectively) and short recurrence-free survival of ACC patients (p=0.0097 and p=0.0004, respectively). In conclusion, the present study reports for the first time that circulating miR-195 and miR-483-5p are promising non-invasive biomarkers with a highly specific prognostic value for the clinical outcome of ACC patients.

 

Nothing to Disclose: NC, RL, GA, OB, JYB, XB, JJF, OC

LB-FP-1 9674 1.0000 SAT-LB-01 A Serum miR-195 and miR-483-5p are predictive of recurrence risk in adrenocortical cancer patients 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Miscellaneous/Other Saturday, June 15th 11:30:00 AM LB-FP 2670 11:00:00 AM Late-Breaking Featured Poster Presentation Poster Preview


Ping Yi*1, Zhao Wang2, Qin Feng1, Charles Edward Foulds1, Rainer B. Lanz1, Grigore D. Pintilie2, Steven J. Ludtke2, Michael F. Schmid2, Wah Chiu2 and Bert W O'Malley1
1Baylor College of Medicine, Houston, TX, 2Baylor College of Medicine

 

Estrogen receptor (ER) is a transcription factor critical for development, reproduction, metabolism and cancer. ER function hinges on its ability to recruit primary and secondary coactivators. Portions of ER bound to small peptides of coactivator proteins have been studied structurally, but it is unclear how intact ER and coactivators are assembled into a transcriptionally active complex on DNA. Here we use cryo-EM to determine the quaternary structure of an active complex of DNA-bound ER alpha, steroid receptor coactivator 3 (SRC-3) and a secondary coactivator (p300). Combined with our cryo-EM maps of p300 and other structural information, we identify the spatial relationships in the complex. Each of the two ligand-occupied ER alpha monomers independently recruits one SRC-3 molecule via one of its transactivation function domains; the two SRC-3s in turn bind one molecule of p300 through multiple contacts. The arrangement of these components can explain how p300 is accessible to nearby histones without steric hindrance from the other proteins in the complex. Our structure resolves the stoichiometry of this multi-component assembly of a transcriptionally active nuclear receptor complex.

 

Nothing to Disclose: PY, ZW, QF, CEF, RBL, GDP, SJL, MFS, WC, BWO

LB-FP-2 9643 2.0000 SAT-LB-03 A Structure of a DNA-Bound Estrogen Receptor and Coactivator Complex 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Miscellaneous/Other Saturday, June 15th 11:30:00 AM LB-FP 2670 11:00:00 AM Late-Breaking Featured Poster Presentation Poster Preview


Elena AB Azizan1, Hanne Poulsen2, Junhua Zhou1, Michael Clausen2, Carmela Maniero1, Elena Bochukova3, Wanfeng Zhao4, Lalarukh Haris Shaikh3, Cheryl A. Brighton1, Tanja Dekkers5, Bas Tops6, Anthony P Davenport1, Benno Küsters6, Jiri Ceral7, Giles Yeo3, Ian McFarlane8, Miroslav Solar7, Jaap Deinum9, I Sadaf Farooqi10, Poul Nissen2 and Morris Jonathan Brown*1
1University of Cambridge, Cambridge, United Kingdom, 2Aarhus University, Denmark, 3University of Cambridge, United Kingdom, 4Addenbrookes Hospital, Cambridge, United Kingdom, 5Radboud University Nijmegen Medical Center, Nijmegen, Netherlands, 6Radboud University Nijmegen Medical Centre, Netherlands, 7Charles University - Faculty of Medicine in Hradec Kralove, Czech Republic, 8Genomics CoreLab, Cambridge National Institute for Health Research (NIHR) Biomedical Research Centre (BRC), Cambridge, United Kingdom, 9Radboud university medical center, Nijmegen, Netherlands, 10Wellcome Trust-MRC Institute of Metabolic Science, Addenbrooke’s Hospital, Cambridge, United Kingdom

 

Phenotypic differences between APAs with or without mutations of KCNJ5 prompted us to look for novel somatic mutations in a subgroup of ‘wild-type’ APAs. Ten were selected with histological features and gene expression profiles resembling normal adrenal zona glomerulosa (ZG) (1). APA and paired germline DNA were sent to BGI (Shenzen, China) for whole exome sequencing. Replication of novel mutations was tested in each of: 89 APAs of unknown genotype from two independent cohorts; 32 APAs of zona fasciculata (ZF)-like phenotype ± KCNJ5 mutations (n=20); and ten cortisol-secreting or non-secretory adenomas. APAs with novel mutations were compared by histology and immunohistochemistry (IHC) with KCNJ5-mutant APAs; and transcriptomes of the genotypically different APAs were compared by microarray both with each other and with adjacent ZG and ZF from which RNA had been separately collected by laser capture microdissection. The function of expressed mutants was studied in Xenopus oocytes and human adrenocortical (H295R) cells.

Exome sequencing showed nine of the 10 APAs to have a novel somatic mutation in one or other of two genes which encode either a Na+,K+-ATPase (n=4) or voltage-dependent Ca2+ channel (Cav) subunit (n=5). Two of the mutations occurred twice in the 10 samples, including the L104R mutation of ATP1A1 discovered independently, and replicated in 7/199 unselected APAs (2). This, and a 100-104del mutation spanning the same L104 residue, not only blocked Na+,K+ transport but caused a large inward leak of Na+ and H+ in oocytes; ATP1A1 L104R caused a 2-3 fold increase in aldosterone secretion and CYP11B2 expression in human adrenocortical cells. 5/39 APAs in a Czech cohort diagnosed in some cases by adrenal vein sampling (AVS) alone (i.e. with normal adrenal CT), had the L104R (n=4) or Cav mutations, compared to only 2/50 patients in a less selected Dutch cohort, and 0/42 of the controls. The function of the Cav mutations is not yet certain; but their clustering, conserved positions and recurrence in further APAs support a causal role. All APAs with the novel Na+,K+-ATPase or Cav mutations were <2 cms in diameter (eight were < 1cm), and had >40% compact cells. Unsupervised cluster analysis of the microarray data separated KCNJ5 mutant APAs from those with the new mutations; qPCR confirmed a cluster of genes which were more highly expressed both in normal ZG than ZF, and in APAs with the new mutations than the KCNJ5 mutants. IHC showed the Na+,K+-ATPase and Cavproteins themselves to be more abundant in ZG than ZF, and in the APAs of ZG-like phenotype.

The Na+,K+-ATPase and Cav mutations appear to define a subtype of APA, probably arising in ZG. The small size of the APAs (due partly to the more compact cells) and their higher prevalence in cohorts where absence of adenoma on CT or MRI did not preclude further investigation, suggest that ZG-like APAs are an easily overlooked, potentially curable cause of hypertension.

 

Nothing to Disclose: EAA, HP, JZ, MC, CM, EB, WZ, LH, CAB, TD, BT, APD, BK, JC, GY, IM, MS, JD, ISF, PN, MJB

LB-FP-3 9713 3.0000 SAT-LB-06 A EXOME SEQUENCING REVEALS FREQUENT SOMATIC MUTATIONS OF NA+/K+-ATPASE AND VOLTAGE-DEPENDENT CA2+ CHANNEL SUBUNITS IN A ZONA GLOMERULOSA-LIKE SUBTYPE OF ALDOSTERONE-PRODUCING ADENOMAS (APA) 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Miscellaneous/Other Saturday, June 15th 11:30:00 AM LB-FP 2670 11:00:00 AM Late-Breaking Featured Poster Presentation Poster Preview


Raymond D Blind*1, Holly A Ingraham1, Robert Fletterick2, Elena P Sablin3 and Debanu Das4
1University of California San Francisco, San Francisco, CA, 2Univ of CA - San Francisco, San Francisco, CA, 3University of California San Francisco, 4Joint Center for Structural Genomics, Stanford Synchrotron Radiation Lightsource

 

Steroidogenic Factor-1 (SF-1, NR5A1) is a nuclear receptor with essential endocrine functions in steroidogenic tissues in adults and throughout development. Prior work by our group has shown this transcription factor interacts with both phosphatidylinositol (4,5) bisphosphate (PIP2) & (3,4,5) triphosphate (PIP3), but atomic resolution details of how these potent signaling lipids bind SF-1 remain unclear. Here, we report two crystal structures of the human SF-1 ligand binding domain complexed to PIP2 and PIP3 at 3.0Å resolution, both co-crystallized with a peptide corresponding to the coactivator PGC-1α. Initial examination of electron-density maps corresponding to the PIP2 and PIP3-bound structures reveals novel electron densities that define the ligand positions, demonstrating how the head groups of PIP2 and PIP3 are coordinated by SF-1. Previous studies suggested PIP3 might be registered by SF-1 residues in regions that correspond with human SF-1 mutations associated with 46 X,Y sex reversal in patients. We are currently refining the models to define any unique coordination features of these ligands by the receptor. These data, coupled with follow-up functional studies, should reveal how these signaling phospholipids bind and regulate SF-1.

 

Nothing to Disclose: RDB, HAI, RF, EPS, DD

LB-FP-4 9742 4.0000 SAT-LB-05 A Dual crystal structures of Steroidogenic Factor-1 complexed with PIP2 and PIP3 reveal independent modes of inositol headgroup coordination 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Miscellaneous/Other Saturday, June 15th 11:30:00 AM LB-FP 2670 11:00:00 AM Late-Breaking Featured Poster Presentation Poster Preview


Christine G Lee*1, Aaron Baraff2, Richard Smith3, Erin Baker3, Vladislav Petyuk3, Peggy M Cawthon4, Douglas C Bauer5, David Gibbs1, Arie Baratt1, Shannon McWeeney1, Jodi Lapidus1 and Eric S Orwoll1
1Oregon Health & Science University, 2University of Washington, 3Pacific Northwest National Laboratory, 4California Pacific Medical Center, San Francisco, CA, 5UCSF, San Francisco, CA

 

Introduction: Despite concerns about the public health burden of obesity, there are few targets for preventing or treating obesity. The discovery of markers and mediators of obesity is needed for elucidating mechanisms of obesity and its complications. Given the high prevalence of obesity in older adults, we developed a new population-based proteomic approach to identify peptides and proteins associated with obesity in older men.

Methods: We performed a cross-sectional analysis of 2473 ambulatory, community-dwelling men ages ≥65 years enrolled in the Osteoporotic Fractures in Men Study from 6 sites in the U.S. The categorization of obese (BMI≥30), overweight (BMI 25.0-29.9) and normal weight (BMI 18.5-24.9) was based on WHO criteria. High-throughput quantitative proteomic analysis was performed on serum samples using a multi-dimensional approach coupling liquid chromatography, ion-mobility separation, and mass spectrometry (LC-IMS-MS). Peptides that were differentially abundant in obese versus normal weight men were identified using analysis of covariance adjusted for multiple comparisons using the Storey method with a false discovery rate (FDR) q-value < 0.05. Models also included adjustments for age, site, comorbidities, lifestyle factors, and medication use.  Meta-analytic methods accounting for correlated metrics were used to generate protein-obesity association rankings from averaged differential abundance and combined p-values for peptides of each protein.

Results: Among the older men, 536 (21.7%) were obese and 650 (26.3%) were of normal weight. Of 18485 identified serum peptides, 1237 were associated with obesity in fully adjusted models with a q-value<0.05, and these peptides mapped to 169 proteins with a q-value < 0.05. Among these proteins were well-known markers of obesity: obese men had a higher abundance of C-reactive protein (q-value=0.003) and a lower abundance of adiponectin (q-value=0.005). The 5 top-ranking proteins associated with obesity were zinc-alpha-2 glycoprotein (ZAG2), glutathione peroxidase 3 (GPX3), vitamin D-binding protein (DBP), putative zinc-alpha-2-glycoprotein-like 1 (ZAGL1), and afamin. Obese men had 20-26% lower abundance of ZAG2, GPX3, DBP, and ZAGL1, and 34% higher abundance of afamin compared to normal weight men, q-values<10-10.

Conclusion: We have demonstrated a rapid and broad assessment of peptides and proteins associated with obesity using a novel, population-based proteomic approach. Traditional obesity markers like C-reactive protein were identified, and our findings support prior reports of decreased ZAG2 and GPX3 expression in adipose tissue of obese subjects and gene polymorphisms in DBP associated with high adiposity. Among the top ranking 5 proteins were 2 not previously reported with obesity, afamin and ZAGL1. Further research to understand the biologic roles of these proteins in obesity is needed.

 

Nothing to Disclose: CGL, AB, RS, EB, VP, PMC, DCB, DG, AB, SM, JL, ESO

LB-FP-5 9757 5.0000 SAT-LB-04 A Novel Markers of Obesity: a Population-Based Proteomic Approach 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Miscellaneous/Other Saturday, June 15th 11:30:00 AM LB-FP 2670 11:00:00 AM Late-Breaking Featured Poster Presentation Poster Preview


Shehzad Basaria*1, Thomas G Travison2, Maithili Davda3, Philip E Knapp3, Eric Scott Bachman4, Alexander Walley5, Daniel Alford5, Shalender Bhasin6 and Robert Edwards7
1Brigham and Women's Hospital - Harvard Medical School, Boston, MA, 2Brigham and Women's Hospital, Boston, MA, 3Boston University Medical Center, Boston, MA, 4Novartis Inst. for Biomedical Rs, Swampscott, MA, 5Boston University, 6Boston Univ Schl of Med, Weston, MA, 7Brigham and Women's Hospital

 

Background:Opioid analgesics suppress the hypothalamic-pituitary-gonadal axis; consequently, androgen deficiency is common in men treated with opioid analgesics. In animal studies, testosterone displays anti-nociceptive properties and improves pain perception. However, the effects of testosterone replacement on pain perception and quality of life (QOL) in opioid-treated men have not been studied in a randomized-controlled trial.

Objective: To determine whether testosterone replacement improves pain perception and tolerance, and QOL in men with opioid-induced androgen deficiency.

Methods: 84 men (age 18-64 yrs) with opioid-induced androgen deficiency were randomized to 5 gm of transdermal testosterone gel or placebo for 14 weeks. Two weeks post-randomization, dose titration was performed to achieve serum testosterone concentrations between 500 and 1000 ng/dl. The primary outcome was clinical pain (subjective pain perception) that was measured by the Brief Pain Inventory (BPI) questionnaire. Secondary outcomes included Quantitative Sensory Testing (QST) of pain threshold and tolerance; and measures of QOL using the SF-36 questionnaire. All outcomes were measured at baseline and at week 12. Total testosterone was measured by liquid chromatography tandem mass spectrometry (LC-MS/MS). Free testosterone was calculated. Statistical comparisons were by Student’s t-tests.

Results: 65 men [testosterone (36), placebo (29)] completed the trial. The two groups were well-matched at study entry. At baseline, mean (SD) age was 49 (±8) yrs, BMI 33 (±7) kg/m², total testosterone 228 (±91) ng/dl and free testosterone 44 (±21) pg/ml. Both total and free serum testosterone levels significantly increased in the testosterone arm. At 12-weeks, men randomized to testosterone showed nominal mean improvements on the pain interference subscale of BPI, but neither this nor overall changes in composite BPI scores were significantly different between the two groups.  The men randomized to testosterone experienced significant improvements in pressure pain threshold (p<0.031), mechanical pain intensity (p=0.049) and cold presser pain after-sensations (p=0.08). The testosterone-treated group also showed a trend toward improvement in aspects of role limitation due to emotional problems as measured by MOS-SF-36.

Conclusion: Testosterone administration in men with opioid-induced androgen deficiency for 12-weeks was associated with a greater reduction in several measures of pain sensitivity during QST. As improvements in laboratory-based indices of central sensitization often precede improvement in clinical pain, trials of longer duration might be necessary to definitively address the anti-nociceptive role of testosterone.

 

Nothing to Disclose: SB, TGT, MD, PEK, ESB, AW, DA, SB, RE

LB-FP-6 9734 6.0000 SAT-LB-02 A Effects of Testosterone Replacement on Pain Perception, Pain Tolerance and Quality of Life in Men with Opioid-Induced Androgen Deficiency: A Randomized Controlled Trial 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Miscellaneous/Other Saturday, June 15th 11:30:00 AM LB-FP 2670 11:00:00 AM Late-Breaking Featured Poster Presentation Poster Preview


Irit Hochberg*1, Dave Bridges2, Alan R Saltiel3, Ariel L Barkan4 and William F Chandler2
1Rambam Health Care Campus, Haifa, Israel, 2University of Michigan, Ann Arbor, MI, 3University of California, San Diego, La Jolla, CA, 4Univ of Michigan, Ann Arbor, MI

 

Context: Growth hormone has clinically important effects on adipose tissue, including stimulation of lipolysis and lipid oxidation; enhancing of lipoprotein lipase (LPL); inhibition of conversion of cortisone to cortisol and decrease in insulin sensitivity. Patients with acromegaly have impaired insulin sensitivity and increased lipolysis.
Aim: The objective of this study was to determine the effect of chronic excess growth hormone in acromegaly on gene expression in adipose tissue in humans.
Experimental design: We compared global gene expression in subcutaneous fat biopsies from 9 acromegaly patients undergoing transsphenoidal pituitary adenomectomy with that of 11 controls undergoing a similar surgery for non-functioning pituitary adenoma. The patients underwent pre-operative clinical and metabolic profiling including assessment of HOMA-IR. Explants of adipose tissue from patients were assayed ex-vivo for lipolysis. mRNA was analysed by next-generation sequencing and bioinformatic analysis of transcript expression was performed.
Results: We observed enhanced ex vivo lipolysis in adipose tissue explants from acromegaly patients, which could be potentially explained by over a 6 fold induction in beta adrenergic receptor-3 expression and LPL expression that was 2 fold higher compared to controls. Interestingly, TSH-R expression was induced 6 fold, possibly contributing to induction of lipolysis. Expression of TCF7L2, a diabetes susceptibility gene whose expression in adipose tissue has been correlated with diabetes, was significatnly higher in acromegaly patients, and could be a factor in the growth hormone-induced insulin resistance. 11β-hydroxysteroid dehydrogenase type 1 expression was 4 fold lower in acromegaly patients. As expected, adipose tissue IGF-1 and IGF-BP3 expression was higher (3.7 fold and 2.4 fold respectively)  in acromegaly patients. Bioinformatic analysis identified over a hundred additional differentially expressed genes and transcripts in adipose tissue of acromegaly subjects compared to controls, including gene clusters for growth, lipid metabolism, energy homeostasis and apoptosis.
Conclusions: We have identified the acromegaly gene expression signature in human adipose tissue. The significance of altered expression of TCF7L2, beta adrenergic receptor-3, TSH-R and other significantly modified genes to the insulin resistance and enhanced lipolysis in acromegaly will enhance our understanding of the metabolic changes in fat tissue which are associated with acromegaly.

 

Nothing to Disclose: IH, DB, ARS, ALB, WFC

OR01-1 6827 1.0000 A The acromegaly gene expression signature in human adipose tissue reveals possible new mechanisms for enhanced lipolysis and insulin resistance 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Neuroendocrinology and Pituitary Saturday, June 15th 1:00:00 PM OR01 2195 11:30:00 AM Cell Specific GH & IGF-1 Signaling Oral


Xingbo Liu*, Adam Jara, Edward O List, Darlene E Berryman and John J Kopchick
Ohio University, Athens, OH

 

Heart failure occurs when the heart cannot pump enough blood and oxygen to support other organs. It is the primary cause of more than 55,000 deaths each year in US, and the five year survival rate is less than 50%. There is accumulating evidence correlating GH deficiency with heart failure. GH has been shown to play roles in cardiac growth, myocardial contractility and vascular function. To better understand the mechanism by which GH affects cardiac function, we explored cardiac calcium channel gene expression in tamoxifen-inducible, cardiac-specific GHR disrupted (iC-GHRKO) mice. iC-GHRKO mice and controls (n=5) were injected with tamoxifen (80mg/kg) at four months of age. Two weeks after injection, hearts were dissected and total RNA was isolated. Using quantitative PCR, we evaluated RNA transcript levels of four key calcium channels involved in normal cardiac contraction: ATPase calcium transporting, slow twitch 2 (ATP2A2); cardiac specific sodium/calcium exchange (NCX1); cardiac ryanodine receptor 2 (RYR2); and voltage-dependent, L type calcium channel (CACNA1C). Compared to controls, levels of RYR2 were significantly reduced by 14%, ATP2A2 levels were significantly reduced by 35%, and NCX1 levels were significantly increased by 17% in iC-GHRKO mice. Levels of CACNA1C in iC-GHRKO mice were similar to controls. These changes are similar to what is observed in the failing heart and suggest that GH may be an important factor in cardiac calcium homeostasis. Future studies will focus on confirming these results at the protein level.

 

Nothing to Disclose: XL, AJ, EOL, DEB, JJK

OR01-2 7615 2.0000 A Cardiac calcium channel expression in heart-specific GH receptor gene disrupted mice 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Neuroendocrinology and Pituitary Saturday, June 15th 1:00:00 PM OR01 2195 11:30:00 AM Cell Specific GH & IGF-1 Signaling Oral


Takahiro Sawada*1, Daiki Arai1, Xuefeng Jing1, Masayasu Miyajima2, Qingfa Chen1, Kazuki Kawakami1, Kenryo Furushima1 and Kazushige Sakaguchi1
1Institute of Advanced Medicine, Wakayama Medical University, Wakayama, Japan, 2Wakayama Medical University, Wakayama, Japan

 

The GH-IGF1 axis plays an important role in postnatal body growth. We reported a novel signaling pathway that regulates postnatal body growth through EphA4, a member of the Eph family of receptor tyrosine kinases and a mediator of the cell-cell contact-mediated signaling (1). EphA4 forms a complex with GH receptor (GHR), Janus kinase 2 (JAK2) and signal transducers and activators of transcription 5B (STAT5B) and enhances Igf1 expression predominantly via the JAK2-dependent pathway with some direct effect on STAT5B. We also found that EphA4 regulates IGF1 signal transduction through PI-3 kinase, but not the signal through MAP kinase which is generally the main player of cell proliferation. Epha4 knockout mice show a gene dose-dependent short stature and low levels of Igf1 mRNA expression in liver and many other tissues despite normal plasma GH levels. In this study, we examined molecular interaction mechanisms that regulate STAT5B activation and IGF1 synthesis via the GHR/EphA4/JAK2 complex. We constructed several deletion and chimeric mutants of GHR and EphA4, and studied their interactions. EphA4 binds to GHR at both extracellular and intracellular domains. We have also determined that STAT5B binds to the amino-terminal kinase domain of EphA4. To study the importance of GHR for the JAK2-independent and EphA-mediated activation of STAT5B, we suppressed GHR expression in Jak2-/- mouse fibroblasts using lentivirus-mediated expression of Ghr shRNA. Upon stimulation with ephrin-A1, reduction of GHR expression decreased STAT5B phosphorylation, suggesting a supporting function of GHR in this signaling pathway. On the other hand, amplification mechanisms of the JAK2-dependent signal by EphA4 are now being studied using mutants of GHR and EphA4. They appear to be related with the complex formation of GHR/EphA4/JAK2/STAT5B, in which STAT5B binds to both GHR and EphA4 to be a substrate of JAK2. Thus, we have clarified two molecular mechanisms how EphA4 interacts with other molecules in enhancing IGF1 production; direct binding and activation of STAT5B, and enhancement of the GHR/JAK2-mediated STAT5B activation through complex formation with GHR, JAK2 and STAT5B.

 

Nothing to Disclose: TS, DA, XJ, MM, QC, KK, KF, KS

OR01-3 6725 3.0000 A Molecular mechanisms of EphA4-mediated signal modulation of the growth hormone (GH)-Insulin-like growth factor 1 (IGF1) axis 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Neuroendocrinology and Pituitary Saturday, June 15th 1:00:00 PM OR01 2195 11:30:00 AM Cell Specific GH & IGF-1 Signaling Oral


Hitoshi Nishizawa*1, Genzo Iguchi2, Hidenori Fukuoka2, Michiko Takahashi1, Masaaki Yamamoto1, Kentaro Suda1, Hironori Bando1 and Yutaka Takahashi1
1Kobe University Graduate School of Medicine, Kobe, Japan, 2Kobe University Hospital, Kobe, Japan

 

Background

Nonalcoholic steatohepatitis (NASH) is a serious liver disease because it progresses to cirrhosis and the prevention of fibrosis is crucial for the treatment. An aberrant activation of hepatic stellate cells (HSCs) plays a key role in the progression of fibrosis and it has been reported that an induction of cellular senescence inactivates HSCs and suppresses fibrosis (Cell 2008, 134, 657). Recently we have reported that nonalcoholic fatty liver disease (NAFLD)/NASH are frequently associated with adult GH deficiency (AGHD) and GH replacement therapy ameliorates these conditions (EJE 2012, 167, 67, Gastroenterology 2007, 132, 938). In addition, GH deficient rat exhibited NASH, GH or IGF-I restores these changes (GH & IGF-I Res 2012, 22, 64). These results indicate that GH/IGF-I play important roles in liver and may have a potential therapeutic application for NASH. The aim of this study was to apply GH or IGF-I for a treatment of general NASH and to clarify the underlying molecular mechanisms.

Methods

Methionine-choline-deficient diet-fed db/db (MCD db/db) mouse is one of established animal models for general NASH. We examined metabolic and histological effects of GH or IGF-I administration on MCD db/db mice and analyzed oxidative stress, mitochondrial function, and activation status of HSCs. We further investigated the involvement of cellular senescence in the effect of IGF-I using p53-null (p53KO) mice.

Results

IGF-I reduced visceral adiposity and improved insulin sensitivity in MCD db/db mice. Histologically, IGF-I, rather than GH, significantly ameliorated steatosis and fibrosis in the liver. In a mechanistic insight, IGF-I treatment restored mitochondrial function in hepatocytes concomitant with a reduced oxidative stress. In addition, we found that IGF-I receptor was strongly expressed in HSCs, and IGF-I induced cellular senescence and inhibited the activation of HSCs in vitro and vivo, suggesting that IGF-I prevents fibrosis via modulating HSCs function by inducing the cellular senescence. Convincingly, in MCD-fed p53KO mice, in which cellular senescence was impaired, IGF-I did not show any effect on the prevention of fibrosis.

Conclusion

The present data demonstrate that IGF-I plays a central role in the liver and prevents the progression of NASH by multiple underlying mechanisms. In particular, IGF-I decreases oxidative stress and improves mitochondrial function. Further, IGF-I directly regulates HSCs function via inducing the cellular senescence in a p53-dependent manner. These results reveal a novel mechanism of IGF-I action and suggest a potential therapeutic application for the treatment of general NASH.

 

Nothing to Disclose: HN, GI, HF, MT, MY, KS, HB, YT

OR01-4 6696 4.0000 A IGF-I prevents the progression of hepatic fibrosis in nonalcoholic steatohepatitis by inactivating hepatic stellate cells in a p53 dependent manner 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Neuroendocrinology and Pituitary Saturday, June 15th 1:00:00 PM OR01 2195 11:30:00 AM Cell Specific GH & IGF-1 Signaling Oral


TAO WANG*1, Muriel Babey1, Yongmei Wang2, Candice Tahimic1, Frankie Fong1, Alicia Menendez1, Zhiqiang Cheng3 and Daniel D Bikle4
1Veterans Affairs Medical Center and University of California, san francisco, CA, 2UCSF, San Francisco, CA, 3University of California San Francisco, San Francisco, CA, 4VA Med Ctr 111N-UCSF, San Francisco, CA

 

Insulin-like Growth Factor I (IGF-I) signaling plays an important role in osteoblastic differentiation, cell proliferation and the synthesis of collagen and other matrix  proteins . Mice with osteoblast-specific loss of the IGF-1 receptor (IGF-IR) have decreased osteoblast proliferation, increased apoptosis and reduced bone both trabecular and cortical. We tested the hypothesis that loss of IGF-1R results in decreased bone formation and impaired healing following fracture.  To determine the role of IGF-IR in fracture healing, a closed tibial fracture was induced in female IGF-I flox/flox / Collagen 2.3kb driven Cre (IGF-IR KO) and IGF-I flox/flox (CON) mice aged 12 weeks. The tibial fracture callus was evaluated by micro CT, bone histomorphometry  and gene expression 10, 21, and 28d after fracture. Bone formation was assessed with two flurochrome labels prior to euthanasia. The results show that osteoblast-specific deletion of IGF-IR results in decreased bone formation, bone remodeling, and mineralizing process, as well as delayed cartilage turnover in IGF-IR KO mice. These studies identify an important role of IGF-I signaling during fracture healing, suggesting that loss of IGF-IR in osteoblasts results in delayed chondrocyte maturation and conversion into bone resulting in delayed union. Therefore, enhancing IGF-I signaling may provide a means to enhance endochondral bone formation resulting in improved  fracture healing.

 

Nothing to Disclose: TW, MB, YW, CT, FF, AM, ZC, DDB

OR01-5 8057 5.0000 A Osteoblast-specific Knockout of the Insulin-like Growth Factor I Receptor (IGF-IR) Gene Impairs Endochondral Bone Formation in Unstable Fracture Healing 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Neuroendocrinology and Pituitary Saturday, June 15th 1:00:00 PM OR01 2195 11:30:00 AM Cell Specific GH & IGF-1 Signaling Oral


Ashiya Buckels*1, Yue Zhang1, Yujun Gan1, Yao Huang2, Jing Jiang1 and Stuart J Frank1
1University of Alabama at Birmingham, Birmingham, AL, 2St. Joseph's Hospital and Medical Center, Phoenix, AZ

 

Growth hormone (GH) triggers intracellular signaling by binding GH receptor (GHR) and activating the GHR-associated cytoplasmic tyrosine kinase, JAK2. We previously found (1) that GH-induced GHR tyrosine phosphorylation by JAK2 is required for downstream activation of the STAT5 transcription factor, but not for activation of JAK2 itself; however, a GHR rendered resistant to tyrosine phosphorylation by mutation of its intracellular tyrosine residues was also resistant to GH-induced downregulation. These and other data (2) suggest that the pace and degree of GH-induced GHR downregulation are correlated with the degree of GH-induced JAK2 activation and GHR tyrosine phosphorylation. We have also observed that the ability of GH to induce acute downstream signaling (activation of STAT5) is reduced by Cre-lox-mediated silencing of IGF-1 receptor (IGF-1R) expression in murine osteoblasts (3). We now investigate the relationship between IGF-1R levels, acute GH signaling, and GH-induced GHR downregulation in a separate system – human LNCaP prostate cancer cells. We compared LNCaP cells that express an shRNA targeting the IGF-1R (shIGF-1R cells) to control LNCaP cells in which an empty vector is expressed (vector cells). We verified substantial IGF-1R protein reduction in shIGF-1R cell extracts and found that acute GH-induced GHR, JAK2, and STAT5 tyrosine phosphorylation were reduced in the shIGF-1R cells over a 2-30 min exposure at various GH concentrations. Our previous conclusions about the relationship between GH-induced GHR tyrosine phosphorylation and GH-induced GHR downregulation predicted that the pace and degree of GHR loss in GH-treated shIGF-1R cells would be less than in vector cells. In fact, we observed the opposite result. GH induced more rapid and complete loss of GHR protein, as assessed by specific immunoblotting, in the cells with reduced IGF-1R. These data suggest that the presence of IGF-1R (even unliganded) contributes very proximally to GH signaling by augmenting GH-induced JAK2 activation and GHR phosphorylation, but may also stabilize the activated GHR, perhaps prolonging its signaling. The mechanism of this novel impact of IGF-1R on the fate of the GH-activated GHR is yet unknown, but could relate to effects on GHR endocytosis or post-endocytic GHR trafficking.

 

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

OR01-6 5491 6.0000 A IGF-1 Receptor Contributes to GH Signaling and Influences GH-induced GHR Downregulation in LNCaP Human Prostate Cancer Cells 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Neuroendocrinology and Pituitary Saturday, June 15th 1:00:00 PM OR01 2195 11:30:00 AM Cell Specific GH & IGF-1 Signaling Oral


Matthew Hill*1, Nicole P. Bowles2, Ilia Nicholas Karatsoreos3, Cecilia J Hillard4 and Bruce S McEwen5
1Univ of Calgary, Calgary, AB, Canada, 2The Rockefeller University, New York, NY, 3Washington State University, Pullman, WA, 4Medical College of Wisconsin, Milwaukee, WI, 5Rockefeller Univ, New York, NY

 

Glucocorticoids are known to promote the development of metabolic syndrome through a multitude of pathways including the regulation of both feeding pathways and metabolic processes. Recent evidence has demonstrated that glucocorticoids possess the ability to increase the production and release of endocannabinoid molecules. Endocannabinoids are potent regulators of appetite, energy balance and metabolic processes through both central and peripheral regulation of feeding and metabolism. The aim of the current study was to determine the role of the endocannabinoid signaling in glucocorticoid-mediated obesity and metabolic syndrome. In our model, male mice are given free access to 100 µg/ml corticosterone, or vehicle solution, for 4 weeks, at the end of which markers of obesity and metabolic markers were examined. In wild-type or vehicle treated mice, glucocorticoid administration resulted in an increase in total body weight, the weight of the abdominal/gonadal fat pads, fatty liver (as indicated by elevated Oil Red O staining, elevated ALT and hepatic triglyceride content), adipocyte expansion and dramatic elevations in the circulating levels of triglycerides, insulin, leptin, and glucose. Glucocorticoid administration also increased feeding behaviour, nearly tripling food intake over vehicle exposed animals; however, food restriction did not attenuate the effects of glucocorticoid exposure on obesity, indicating that while feeding behaviour is increased the ability of glucocorticoids to promote obesity is through a more direct effect on metabolic processes. With respect to the endocannabinoid system, corticosterone treatment surprisingly resulted in a reduction in the tissue levels of the endocannabinoid 2-AG within the hypothalamus, with no effects on the other endocannabinoid anandamide. Conversely, within the liver and the circulation, anandamide levels were dramatically increased by glucocorticoids, while 2-AG levels were found to decrease. Interestingly, we found that CB1 receptor deficient mice were protected from the effects of glucocorticoids on obesity and metabolic processes. Similarly, global pharmacological blockade of the CB1 receptor throughout the corticosterone treatment regimen also attenuated the effects on obesity. More so, administration of a peripherally restricted CB1 receptor antagonist also blocked glucocorticoid-induced obesity and metabolic changes. Taken together, these data indicate that glucocorticoids increase hepatic production of anandamide, which in turn contributes to the development of obesity and metabolic syndrome. In support of this hypothesis, recent work has highlighted the hepatic endocannabinoid system as a necessary component of diet-induced obesity, demonstrating that this system may be a critical player in the development of obesity, through both diet and hormonal pathways.

 

Nothing to Disclose: MH, NPB, INK, CJH, BSM

OR03-1 7523 1.0000 A Glucocorticoids Promote Obesity and Metabolic Syndrome Through a Hepatic Endocannabinoid Mechanism 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 1:00:00 PM OR03 2197 11:30:00 AM Glucocorticoids & Glucocorticoid Actions Oral


Stuart Andrew Morgan*1, Iwona Bujalska2, Laura Louise Gathercole2, Zaki K Hassan-Smith2, Paul Michael Stewart2, Jeremy W Tomlinson2 and Gareth Geoffrey Lavery2
1University of Birmingham, United Kingdom, 2University of Birmingham, Birmingham, United Kingdom

 

Glucocorticoids (GC), such as prednisolone, are widely prescribed for their anti-inflammatory and immunosuppressive properties. However, they have significant side-effects including insulin resistance and hepatic steatosis. 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) converts 11-dehydrocorticosterone (11DHC) to active corticosterone (CORT) and thus amplifies local GC action. We hypothesise that enhanced local GC regeneration of exogenously administered GCs by 11β-HSD1 may contribute to the adverse side-effect profile. To test this hypothesis, 6-week old male 11β-HSD1-/- and wildtype (WT) mice were treated with CORT(100μg/mL), 11DHC(100μg/mL) or vehicle via the drinking water. After 5 weeks, animals underwent glucose tolerance testing and were sacrificed for assessment of metabolic parameters. As anticipated, 11DHC treated 11β-HSD1-/- mice were indistinguishable from vehicle treated mice. CORT and 11DHC treated WT mice displayed impaired glucose tolerance, hepatic steatosis and increased hepatic expression of the fatty acid transporter CD36. However, 11β-HSD1-/- CORT treated mice were protected from glucose intolerance, hepatic steatosis and had lower hepatic CD36 expression. In CORT treated WT adipose tissue, 11β-HSD1 and hormone sensitive lipase (HSL) expression were elevated, associated with increased circulating free fatty acid (FFA) levels. Conversely, CORT treated 11β-HSD1-/- mice were protected from elevated HSL expression and increased circulating FFAs. Finally, intramyocellular diacylglyceride (DAG) content was elevated in CORT and 11DHC treated WT mice, whereas CORT treated 11β-HSD1-/- mice were protected from increased DAG levels, a possible factor in improved glucose tolerance. Importantly, both WT and 11β-HSD1-/- CORT treated mice had a similar increased circulating CORT (500nmol/mL) compared to vehicle controls (50nmol/mL). These data demonstrate that 11β-HSD1-/- mice are protected from the adverse effects of exogenous GC excess, and suggest that local GC regeneration may contribute significantly to the adverse effect profile of therapeutic GC use. This raises the possibility of using selective 11β-HSD1 inhibitors as an adjunctive therapy to limit the side-effects of GC treatment.

 

Nothing to Disclose: SAM, IB, LLG, ZKH, PMS, JWT, GGL

OR03-2 8166 2.0000 A 11ß-HSD1 Knockout Mice are Protected from the Adverse Metabolic Effects of Exogenous Glucocorticoid Excess 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 1:00:00 PM OR03 2197 11:30:00 AM Glucocorticoids & Glucocorticoid Actions Oral


Ash I Taylor*1, Lynne E Ramage1, Donald R Dunbar1, Mark Nixon2, Thomas M Stulnig3, Maximilian Zeyda3, Ruth Andrew1 and Brian R Walker1
1University of Edinburgh, Edinburgh, United Kingdom, 2University of Texas Health Science Centre, Houston, TX, 3Medical University of Vienna, Vienna, Austria

 

Human plasma contains both cortisol and corticosterone, the major glucocorticoid in rodents, at a ratio of ~10:1. Export of cortisol, but not corticosterone, from CNS by ABCB1 (MDR1, p-glycoprotein) explains lower cortisol:corticosterone ratios in cerebrospinal fluid than plasma and may allow corticosterone to exert disproportionate feedback on the HPA axis. Conversely, in cell models, corticosterone but not cortisol is transported by another ABC transporter, ABCC1 (MRP1). We hypothesised that tissue-specific distribution of ABCB1 and ABCC1 may render some tissues differentially sensitive to corticosterone or cortisol.

Using microarray, RT-PCR and qPCR in a library of human tissue cDNA samples, we showed similar expression of ABCB1 and ABCC1 in kidney, liver and thymus; however, ABCC1 was more highly expressed than ABCB1 in peripheral blood mononuclear cells, skeletal muscle and adipose tissue. In differentiated SGBS cells, a human adipocyte cell line, mRNA and protein for ABCC1 were present, and ABCB1 was not expressed. This contrasts with murine subcutaneous and visceral adipose tissue and differentiated 3T3-L1 cells in which both ABCB1 and ABCC1 are expressed. Influx of 3H-corticosterone and 3H-cortisol into the intracellular fraction in SGBS cells was similar over 24h, however, efflux of corticosterone from cells was faster than that of cortisol at 4h (2.3-fold; p<0.05), 8h (2.8-fold; p<0.01) and 24h (3.2-fold; p<0.01). Incubation with the ABCC1 inhibitor MK-571 for 24h increased intracellular accumulation of 3H-corticosterone (3.5-fold; p<0.001) to a greater extent than 3H-cortisol (1.2-fold; p<0.001). In human adipose biopsies, ABCC1 transcript levels were higher in subcutaneous (n=6) than visceral (n=8) adipose tissue (1.6-fold; p<0.01) in lean individuals, and higher in subcutaneous (1.5-fold; p<0.01) but not visceral adipose in obese (n=8) compared with lean subjects.

Thus, expression of ABCC1 may render human adipose tissue more responsive to variations in cortisol than corticosterone, in contrast with CNS where expression of ABCB1 confers differential sensitivity to corticosterone. This mechanism may have evolved in humans to exploit the presence of two endogenous glucocorticoids and increase the plasticity of tissue-specific glucocorticoid action. In obesity, up-regulation of ABCC1 may protect subcutaneous, but not visceral, adipose tissue from corticosterone and thereby contribute to glucocorticoid-dependent visceral fat accumulation.

 

Nothing to Disclose: AIT, LER, DRD, MN, TMS, MZ, RA, BRW

OR03-3 7292 3.0000 A The ABC Transporter ABCC1 Exports Corticosterone but Not Cortisol from Human Adipocytes and is Upregulated in Subcutaneous but Not Visceral Adipose Tissue in Obesity 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 1:00:00 PM OR03 2197 11:30:00 AM Glucocorticoids & Glucocorticoid Actions Oral


Katherine Elizabeth Wynne-Edwards*, Heather E Edwards and Trina M Hancock
University of Calgary, Calgary, AB, Canada

 

Context. Fetal stress is relevant to newborn outcomes, and should be signalled from fetus to mother. Corticosterone is rarely quantified in human clinical endocrinology and is found at much lower concentrations than cortisol. However, fetal corticosterone is a candidate hormone as a fetal stress signal. Objective. Test the hypothesis that preferential fetal corticosterone synthesis occurs in response to fetal intra-partum stress. Design. Cross-sectional comparison of paired serum corticosteroid concentrations in umbilical artery and vein from 300 women providing consent at admission to a General Hospital Labor and Delivery unit. Pre-term and multiple births were excluded, leaving 265 healthy deliveries. Main Outcome Measures. Corticosterone and cortisol concentrations determined by LC-MS/MS for umbilical cord venous (V) and arterial (A) samples and used to calculate fetal synthesis (A-V) and proportional fetal synthesis ([A-V]/V). Chart-derived criteria stratified samples by type of delivery, maternal regional analgesia, augmentation of contractions, and clinical rationale for emergent Caesarian delivery. Results. Cortisol concentrations were higher than corticosterone concentrations, however, the fetus preferentially secretes corticosterone (148% vs 49% proportional increase for cortisol) and differentially secretes corticosterone as fetal stress increases. Fetal corticosterone synthesis is elevated after passage through the birth canal relative to Caesarian deliveries. For vaginal deliveries, augmentation of contractions does not affect corticosteroid concentrations whereas maternal regional analgesia decreases venous (maternal) concentrations and increases fetal synthesis. Fetal corticosterone synthesis is also elevated after C-section indicated by cephalopelvic disproportion after labor whereas cortisol is not. Conclusions. The full-term fetus preferentially secretes corticosterone in response to fetal stress during delivery. Fetal corticosterone could serve as a biomarker of fetal stress.

 

Nothing to Disclose: KEW, HEE, TMH

OR03-4 9082 4.0000 A THE HUMAN FETUS PREFERENTIALLY SECRETES CORTICOSTERONE, RATHER THAN CORTISOL, IN RESPONSE TO INTRA-PARTUM STRESSORS 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 1:00:00 PM OR03 2197 11:30:00 AM Glucocorticoids & Glucocorticoid Actions Oral


Annalisa Gastaldello*, Mark Nixon, Chenjing Yang, Philippa T Saunders, Brian R Walker, Karen Elizabeth Chapman and Ruth Andrew
University of Edinburgh, Edinburgh, United Kingdom

 

Glucocorticoids (GCs) are highly effective anti-inflammatory drugs, however their chronic use is limited by serious metabolic side effects. The 5α-reduced metabolite of corticosterone (B), 5α-tetrahydrocorticosterone (5αTHB), binds the GC receptor (GR) and suppresses inflammation in vitro and in vivo. Here the underlying molecular mechanisms of the anti-inflammatory action of 5αTHB were explored in cell models of ligand-induced GR phosphorylation, nuclear localisation and gene transcription. Data are mean±SEM (of 4 experiments, each in triplicates; * p<0.05).

Phosphorylation of Ser211GR, known to influence GR nuclear localisation and gene transcription, was assessed by Western blot in A549 cells treated (1h) with vehicle, corticosterone (B) (1μM) or 5αTHB (1-30μM). Phosphorylation was not induced by 5αTHB alone, in contrast to B (fold induction over vehicle: 1.4±0.4 (5αTHB, 1μM), 6.6±1.6* (B)). Co-incubation of B and 5μTHB did not significantly alter GR phosphorylation compared to B alone.

To determine mobility of ligand-bound GR, localisation of green fluorescent protein tagged-rat GR (GR-GFP) transfected in HEK293 cells was monitored by fluorescence microscopy. Nuclear translocation of GR-GFP by 5αTHB (1μM) was incomplete (82.7±1.5% reaching the nucleus within 5h). Translocation with B (1μM) was complete within 30mins. Compared to a sub maximal dose of B alone (3nM, 45mins), co-incubation with 5αTHB (1μM) resulted in a greater proportion (3x) of GR-GFP nuclear localisation. Nuclear export after steroid washout (24h) was observed only with 5αTHB (32.1±6.1% remaining). The rate of recovery from nuclear photobleaching suggested that 5αTHB-bound GR-GFP was more mobile in the nucleus than B-bound GR-GFP (half-life: 5αTHB 3.12±0.38* vs B 4.40±0.42s).

Ligand ability to induce transcription of GR dimer- and multimer-dependent reporter genes (MMTV and PNMT respectively) was tested in A549 cells. In contrast to B, 5αTHB (0.1-3μM; 24h) was unable to activate either reporter plasmid (fold induction over vehicle MMTV-Luc: 5αTHB 1.2±0.04; B 16.9±1.1*; PNMT-Luc: 5αTHB 1.6±0.6; B 3.1±0.5*).

Effects of B and 5α-THB on mRNA levels of metabolic genes were investigated in BWTG3 mouse hepatoma cells naturally expressing GR. While B (1μM, 16h) significantly increased the levels of mRNA encoding the GC-induced genes tyrosine aminotransferase (Tat) and gamma-glutamyltransferase 1 (Ggt1) (fold over vehicle: x23±3, x1.4±0.2 respectively), 5α-THB (1-30μM) did not. However, 5α-THB (1-3μM) suppressed the induction of Tat (by 67±4%) and Ggt1(by 33±5%) by B alone.

In conclusion, 5αTHB exhibits the properties of a partial agonist. In its presence, GR translocates slowly, does not become phosphorylated at Ser211 and fails to increase the abundance of metabolic genes. However, 5αTHB restrains the ability of B to activate metabolic genes, protecting tissues from excess glucocorticoid action.

 

Nothing to Disclose: AG, MN, CY, PTS, BRW, KEC, RA

OR03-5 5598 5.0000 A Investigation into the Molecular Mechanisms Underlying the Anti-inflammatory Properties of 5α-Tetrahydrocorticosterone 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 1:00:00 PM OR03 2197 11:30:00 AM Glucocorticoids & Glucocorticoid Actions Oral


Diana Cruz Topete*1, Robert H Oakley1, Rongqin Ren2, Monte S Willis3 and John A Cidlowski4
1National Institute of Environmental Health Sciences, Research Triangle Park, NC, 2National Institutes of Environmental Health Sciences, Research Triangle Park, NC, 3McAllister Heart Institute, University of North Carolina, Chapel Hill, NC, 4NIEHS/NIH, Research Triangle Pk, NC

 

Glucocorticoids are the primary regulators of the stress response, and numerous observations have suggested that glucocorticoids exert direct effects on cardiomyocytes. However, the role of glucocorticoids in cardiac physiology remains elusive.To investigate the in vivo role of glucocorticoid signaling in the heart, the Cre/LoxP system was used to generate mice with a cardiomyocyte-specific deletion of GR (cardioGRKO). The CardioGRKO mice die prematurely due to the development of cardiovascular disease. By three months of age, cardioGRKO mice display left ventricular function abnormalities, characterized by decreases in ejection fraction and fractional shortening. Supporting these findings, histological evaluation showed an increase in the incidence of cardiomyocyte hypertrophy in cardioGRKO mice. Interestingly, adrenalectomy did not have any effects on the severity of the disease. Analysis of the gene expression profiles showed that 302, 1189, and 925 genes were dysregulated in cardioGRKO hearts as compared to controls at 1, 2 and 3 months of age, respectively. The Ingenuity Pathway Analysis library was used to identify the most significantly altered signaling pathways in the hearts of cardioGRKO mice. Cardiovascular Disease was among the highest-ranked pathways, and further analysis highlighted several novel glucocorticoid-regulated genes involved in cardiac hypertrophy. These findings revealed an unprecedented role for GR in the maintenance of normal cardiac homeostasis, and suggest that the pharmacological modulation of GR signaling in cardiomyocyte is a promising new target to treat heart disease.

 

Nothing to Disclose: DC, RHO, RR, MSW, JAC

OR03-6 6244 6.0000 A Knockout of the Glucocorticoid Receptor in Cardiomyocytes Leads to Spontaneous Cardiovascular Disease and Death 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 1:00:00 PM OR03 2197 11:30:00 AM Glucocorticoids & Glucocorticoid Actions Oral


Gherardo Mazziotti*1, Antonio Bianchi2, Teresa Porcelli3, Marilda Mormando4, Filippo Maffezzoni3, Alessandra Cristiano3, Antonella Giampietro2, Laura De Marinis2 and Andrea Giustina3
1Azienda Ospedaliera Carlo Poma of Mantua, Mantua, Italy, 2Catholic University, School of Medicine, Rome, Italy, 3University of Brescia, Brescia, Italy, 4Catholic University of the Sacred Heart, Rome, Italy

 

Context. Cross-sectional studies showed an elevated prevalence of vertebral fractures in acromegaly. However, no data are available on incident vertebral fractures in this clinical setting.

Objective. To investigate the incidence and risk factors of vertebral fractures in patients with acromegaly.

Design. Three-year prospective study.

Setting. Referral centers.

Subjects. 88 patients with acromegaly  (33 females, 55 males; mean age 50, range 21-88) and 106 control subjects, matched for sex and age (43 females and 63 males, mean age 55 years, range: 33-79), attending out-patient Bone clinics.

Main Measures. Patients and control subjects were evaluated for incidence of vertebral fractures using a quantitative morphometric approach on spine X-ray, which was performed at baseline and after 3 years of follow-up. At the same time-points, patients with acromegaly were also evaluated for bone mineral density (BMD) with DXA at lumbar spine and femoral neck.

Results. After 3-year follow-up, 37 patients with acromegaly (42.0%) and 4 control subjects (3.8%) experienced incident vertebral fractures (p<0.001). The incident of vertebral fractures was significantly higher in patients with active disease as compared to those who were with controlled/cured acromegaly at the study entry (62.5% vs. 25.0%; p<0.001). Risk of incident vertebral fractures was significantly associated with hypogonadism (OR: 6.6 C.I.95% 1.1-43.3; p=0.047), change in femoral neck BMD (OR: 0.80, C.I.95% 0.67-0.96; p=0.02) and prevalent vertebral fractures at the study entry (OR: 6.9, C.I.95% 1.1-43.6; p=0.039) only in patients with controlled/cured acromegaly, whereas in patients with active disease the fracture risk was not influenced by the above clinical factors but it was significantly associated with duration of active acromegaly (OR: 1.6, C.I.95% 1.1-2.3; p=0.01).

Conclusions. This prospective study demonstrates high rate of incident vertebral fractures both in patients with active and controlled acromegaly.

 

Disclosure: GM: Ad Hoc Consultant, Novartis Pharmaceuticals, Ad Hoc Consultant, Ipsen. AG: Consultant, Ipsen, Consultant, Novartis Pharmaceuticals, Clinical Researcher, Amgen. Nothing to Disclose: AB, TP, MM, FM, AC, AG, LD

OR10-1 8288 1.0000 A VERTEBRAL FRACTURES IN PATIENTS WITH ACROMEGALY: A 3-YEAR PROSPECTIVE STUDY 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 15th 1:00:00 PM OR10 2222 11:30:00 AM Osteoporosis Oral


Amy H Warriner*1, Wilson Smith2, Jeffrey R Curtis2, Kenneth G Saag1 and Elizabeth Delzell2
1Univ of Alabama at Birmingham, Birmingham, AL, 2University of Alabama at Birmingham

 

Background: Bone mineral density is lower in people with HIV (HIV+) compared to HIV-negative (HIV-) persons. However, it is still unclear if these changes convey an increased fracture risk among HIV+ persons. Use of a large administrative database, such as Medicare, allows for assessment of a large, diverse population.

Methods: Retrospective cohort study using Medicare beneficiaries and their medical claims from 1999-2010. From the national random 5% sample of Medicare beneficiaries, we selected subjects of any age; continuously enrolled for ≥12 months in Medicare parts A and B and not in a Medicare Advantage plan. All patients had a 12-month baseline period, after which follow-up began and continued until the earliest of fracture, loss of Medicare coverage, death, or 12/31/2010. We classified beneficiaries as HIV+ or HIV- based on claims during the baseline and follow-up periods using a previously developed HIV case-finding algorithm. We identified fracture outcomes during follow-up using claims data on diagnoses and procedures and included all fracture types (hip, spine, femur, pelvis, tibia/fibula, ankle, clavicle, humerus, radius/ulna, carpal bones, foot, rib). We used multivariable Poisson regression to estimate the rate ratio (RR) for the association between HIV status and fracture, adjusting for demographic factors and comorbidities assessed during baseline. Fracture occurrences at individual fracture sites were determined for HIV+ and HIV- groups.

Results:  The overall study population included 13,221 HIV+ and 2,500,442 HIV- beneficiaries.  The HIV+ population was more likely to be African-American, male, and younger, compared to the HIV- population. Crude fracture rates were 13.8 per 1000 person years in HIV+ patients vs. 20.5 per 1000 person years in HIV-patients. However, the RR of fracture was 1.67 (95% Confidence Interval, CI, 1.55-1.80) for HIV+ compared to HIV- subjects after adjustment for age, gender, year, geographic region, race, Medicare entry reason, Medicare/Medicaid dual-eligibility, hepatitic C and Charlson score.  The elevated association between HIV and fracture risk was present in both younger beneficiaries (<65 years old, RR 1.32, 95% CI 1.21 – 1.45) and older beneficiaries (≥65 years old, RR 1.52 (1.34 – 1.73) after adjustment. The most common fractures in younger HIV+ patients were hip, vertebral, ankle, and wrist.

Conclusions:  The risk of fracture is approximately 50% higher in HIV+ Medicare patients when compared to a HIV- group of Medicare patients after adjusting for demographics and other fracture risk factors.  The risk is present for both older and younger HIV+ persons but common “osteoporotic” fractures may occur at a younger age in HIV+ patients.

 

Nothing to Disclose: AHW, WS, JRC, KGS, ED

OR10-2 6274 2.0000 A Fracture Among Older and Younger HIV+ Medicare Beneficiaries 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 15th 1:00:00 PM OR10 2222 11:30:00 AM Osteoporosis Oral


Sowmya Srinivasan*1, Malini Chandra2, Mary Patton1, Amer A Budayr1 and Joan C. Lo2
1Kaiser Permanente Oakland Medical Center, Oakland, CA, 2Kaiser Permanente Northern California, Oakland, CA

 

Introduction: Hip fractures are associated with significant morbidity and mortality in postmenopausal women. This study examines contemporary rates of re-hospitalization and mortality following proximal hip fracture in older women within an integrated healthcare delivery system.

 Methods: Hospitalization records were used to identify women age ≥65 years in Kaiser Permanente Northern California who experienced a proximal hip fracture during 2000-2008.  Demographic and clinical characteristics were obtained from health plan databases.  Outcomes included hospital discharge disposition, re-hospitalization within 90 days after discharge, and all-cause mortality at 6 and 12 months after hip fracture.

 Results:  There were 10,650 women with an index hip fracture during 2000-2008. The majority (84.7%) were age ≥75 years and of white race (82.8% white, 2.8% black, 5.4% Hispanic, 3.7% Asian, 5.3% of other/unknown race). During the index hospitalization, 262 (2.5%) died prior to discharge, 8167 (76.7%) were discharged to a skilled nursing facility, and 1913 (18.0%) were discharged to home, with or without home health services. There were 2056 (19.3%) women who were re-hospitalized within 90 days of discharge. Excluding orthopedic indications, pneumonia and cardiovascular disease were the most common reasons for readmission. At 6 months and 1 year, the overall mortality rate was 17.2% and 23.2%, respectively, and increased substantially with age.  The 1 year mortality was 14.1% for women age 65-74, 17.8% for age 75-84 and 32.6% for age ≥85 years old.  One year mortality also varied by race/ethnicity with rates higher among whites compared to Hispanics and Asians, but not different compared to blacks. For age 65-74, Hispanics had a lower mortality than whites and for age ≥85, both Hispanics and Asians had a lower mortality compared to whites. In multivariable logistic regression, older age, white race and higher comorbidity index were associated with greater odds of death within 1 year following hip fracture. 

 Conclusions: Hip fracture mortality rates are high among older postmenopausal women, particularly those of white race, with a 2-fold difference in mortality between those age ≥85 years and age 65-74 years. Women with hip fracture were also at high risk for re-hospitalization within 90 days of discharge, especially for cardiac and pulmonary diagnoses.  Future studies should examine demographic and clinical predictors of increased morbidity following hip fracture.

 

Nothing to Disclose: SS, MC, MP, AAB, JCL

OR10-3 7648 3.0000 A Morbidity and Mortality following Proximal Hip Fracture in Older Women 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 15th 1:00:00 PM OR10 2222 11:30:00 AM Osteoporosis Oral


Pouneh K. Fazeli*1, Alexander Terence Faje2, Stephen T Russell3, Clifford J Rosen4, Mary Larsen Bouxsein5 and Anne Klibanski2
1Neuroendocrine Unit, Massachusetts General Hospital/Harvard Medical School, Boston, MA, 2Massachusetts General Hospital/Harvard Medical School, Boston, MA, 3Massachusetts General Hospital/Neuroendocrine Unit, Boston, MA, 4Maine Medical Center Research Institute, Scarborough, ME, 5Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA

 

Anorexia nervosa (AN) is a psychiatric disorder characterized by extreme, self-induced starvation affecting 0.5-1% of college-aged women in the US. The disorder is associated with multiple medical comorbidities, including significant bone loss and a seven-fold increased risk of fracture compared to controls. Microarchitectural methods of evaluating bone structure allow for the evaluation of both cortical and trabecular parameters and finite element analysis (FEA) enables assessment of bone strength, a predictor of fracture risk independent of BMD. Women with AN have decreased trabecular parameters, decreased cortical thickness and decreased bone strength compared to healthy controls as assessed by FEA. Hormonal predictors of measures of bone strength in women with AN have not been previously investigated. We hypothesized that two major nutritionally dependent hormones affecting bone, IGF-1 and leptin, would be predictors of decreased bone strength in this population. We studied 40 women [20 with AN, mean age +/- SD: 28.1 +/- 5.1 yrs and 20 healthy controls (HC): 27.5 +/- 3.2 yrs; p=0.66]. We measured stiffness and failure load of the distal radius and distal tibia by FEA of high resolution peripheral quantitative CT images and serum IGF-1 and leptin levels. By design, AN had a significantly lower % ideal body weight compared to HC (AN: 78.3 +/- 8.1% vs HC: 101.7 +/- 5.9%; p<0.0001). AN had significantly lower stiffness and failure load as compared to HC in both the radius (stiffness: AN: 67.1 +/- 17.3 KN/mm vs HC: 77.2 +/- 13.1 kN/mm, p=0.04; failure load: AN: 3.4 +/- 0.9 kN vs HC: 3.9 +/- 0.6 kN, p=0.03) and tibia (stiffness: AN: 183.3 +/- 44.8 kN/mm vs HC: 219.9 +/- 28.7 kN/mm, p<0.01; failure load: AN: 9.2 +/- 2.2 kN vs HC: 11.0 +/- 1.4 kN; p<0.01). Mean serum leptin and IGF-1 levels were significantly lower in AN compared to HC (Leptin: AN: 2.4 +/- 2.7 ng/mL vs HC: 11.1 +/- 7.0 ng/mL, p<0.0001; IGF-1: AN: 176.8 +/- 66 ng/mL vs HC: 245.6 +/- 61.9, p=0.001). IGF-1 was significantly associated with tibial stiffness (R=0.58; p<0.01) and failure load (R=0.61; p<0.01) and radial stiffness (R=0.57; p<0.01) and failure load (R=0.57; p<0.01) in AN but not in HC. There were no significant associations between leptin and stiffness or failure load in either group. Our data support the hypothesis that IGF-1 is a predictor of bone strength in AN.  Further studies are needed to delineate other potential hormonal predictors of bone strength in AN.

 

Nothing to Disclose: PKF, ATF, STR, CJR, MLB, AK

OR10-4 7223 4.0000 A IGF-1 is Associated with Bone Strength in Anorexia Nervosa 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 15th 1:00:00 PM OR10 2222 11:30:00 AM Osteoporosis Oral


Andre B. Araujo*1, Nicholas Dagincourt2, Shan Chen2, Gretchen Chiu1, Elizabeth Suarez2, Robert Chang2, Rachael Gerber2, Julia Akeroyd2, Carrie G. Wager2, Benedetta Bartali2 and Mary Larsen Bouxsein3
1New England Research Institutes, Watertown, MA, 2New England Research Institutes, Inc., Watertown, MA, 3Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA

 

Although recent studies and opinion suggest that obesity in men is associated with increased fracture risk, few studies have examined the relationship between body composition and bone microarchitecture. We obtained cross-sectional data from 692 men aged 38-86y  enrolled in the Boston Area Community Health/Bone (BACH/Bone) Survey, a population-based cohort study of racially diverse men. Microarchitecture at the distal radius (N=443) and tibia (N=473) was measured by high-resolution peripheral quantitative computed tomography (HR-pQCT, XtremeCT, Scanco), with CVs <5%. Total body lean mass (LM) and fat mass (FM) were assessed by dual-energy x-ray absorptiometry (DXA). Multivariable linear regression was used to examine the independent associations of LM and FM with cortical and trabecular parameters, controlled for age, race/ethnicity, physical activity, body mass index (BMI), self-rated health, smoking, and cancer. Mean (±SD) age was 58.0±11.6y, with black, Hispanic, and white men representing 30.9%, 29.5%, and 39.6% of the sample, respectively. BMI was 29.0±5.3kg/m2 while FM and LM were 21.85±8.73kg and 54.9±8.1kg, respectively. Bivariate results showed that higher LM was consistently associated with both cortical and trabecular parameters in both the radius and tibia. In contrast, FM was significantly and positively associated only with trabecular parameters in the radius, and exhibited comparatively greater associations with both trabecular and cortical parameters in the distal tibia. These results were largely preserved in multivariable models. For instance, a 10kg increase in LM was associated with a significant increase of 0.07 and 0.22 in trabecular number (per 1/mm) in the distal radius and tibia, respectively, whereas FM was not associated with any parameter in the radius in multivariable models. LM was also significantly associated with higher trabecular density and total area, but lower cortical density and trabecular thickness in the distal tibia. FM was positively associated with trabecular number and negatively associated with cortical density and thickness in the distal tibia. In conclusion, LM and FM appear to play strong, if complex, roles in determining bone microarchitectural properties in middle-aged and older men. Overall, trabecular and cortical parameters in the weight-bearing tibia were more strongly related to body composition than those in the radius, consistent with the important role of mechanical loading influencing the determinants of  bone strength.

 

Disclosure: ABA: Principal Investigator, Abbott Laboratories, Principal Investigator, GlaxoSmithKline, Consultant, Lilly USA, LLC. Nothing to Disclose: ND, SC, GC, ES, RC, RG, JA, CGW, BB, MLB

OR10-5 8444 5.0000 A Body composition in relation to bone microarchitecture among racially/ethnically diverse middle-aged and older men 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 15th 1:00:00 PM OR10 2222 11:30:00 AM Osteoporosis Oral


Michael R McClung*1, E Michael Lewiecki2, Pei-Ran Ho3, Michael Anthony Bolognese4, Beiying Ding3, Michelle Geller3, Cynthia O'Malley3, Rachel B Wagman3 and Paul D Miller5
1Oregon Osteoporosis Center, Portland, OR, 2New Mexico Clinical Research & Osteoporosis Center, Albuquerque, NM, 3Amgen Inc., Thousand Oaks, CA, 4Bethesda Health Research Center, Bethesda, MD, 5Colorado Center for Bone Research, Lakewood, CO

 

Background: Osteoporosis is a chronic disease that requires long-term treatment with pharmacologic therapy to ensure sustained anti-fracture benefit. Denosumab (DMAb) reduced the risk for vertebral, non-vertebral, and hip fractures (1); and treatment for up to 8 years was associated with continued gains in bone mineral density (BMD) and persistent reduction in markers of bone turnover (2). To understand osteoporosis management strategies after long-term DMAb treatment, we report findings after 1 year of observational follow-up from a phase 2 study extension.

Methods: Subjects who completed the phase 2 extension study were eligible to enter an observational phase designed to understand real-world osteoporosis management strategies. During the observational year, participants received osteoporosis management at the discretion of their treating physician and returned to the clinic after 1 year to undergo BMD assessment and complete an osteoporosis management questionnaire. Incidence of serious adverse events and fractures was collected. All analyses were descriptive.

Results: Of 138 eligible subjects, 82 enrolled in the observational phase and completed the final visit. The majority of subjects (65 [79%]) did not receive any osteoporosis prescription medication (excluding calcium and Vitamin D), with “my doctor felt I no longer needed a medication” given as the most common reason (23 [35%]). Osteoporosis medications taken by the remaining 17 subjects (21%) included alendronate (7 [41%]), DMAb (5 [29%]), risedronate (4 [24%]), ibandronate (2 [12%]) and teriparatide (2 [12%]). In subjects treated with DMAb for 8 years during the double-blind and extension studies (n=52), overall BMD at the end of the 1-year observational phase showed a mean decrease of 6.7% at the lumbar spine and 6.6% at the total hip compared to the end of treatment. In the subset of these 52 subjects who took osteoporosis medication during the observation period (n=10), BMD showed a smaller decline (‑3.7% at lumbar spine and ‑4.1% at total hip). No new safety concerns were identified; 8 subjects (9.8%) experienced 17 fractures with all subjects having at least 1 predisposing risk factor for fracture.

Conclusion: Findings from this 1-year observational phase showed that the majority of subjects were not prescribed further treatment for osteoporosis. Consistent with the mechanism of action of DMAb, treatment cessation was associated with a reversibility of the effect. For subjects who transitioned to another osteoporosis therapy, there was evidence of attenuation of bone loss. A limitation of this study is inherent selection bias with retention of a small number of subjects over 9 years. In conclusion, for patients at high risk for fracture, continuing osteoporosis therapy after DMAb discontinuation seems appropriate.

 

Disclosure: MRM: Consultant, Amgen, Investigator, Amgen, Consultant, Merck & Co., Consultant, Eli Lilly & Company, Consultant, Novartis Pharmaceuticals, Investigator, Merck & Co., Speaker Bureau Member, Merck & Co., Speaker Bureau Member, Warner Chilcott. EML: Principal Investigator, Amgen, Advisory Group Member, Amgen, Speaker Bureau Member, Amgen, Principal Investigator, Eli Lilly & Company, Advisory Group Member, Eli Lilly & Company, Speaker Bureau Member, Eli Lilly & Company, Principal Investigator, Merck & Co., Principal Investigator, GlaxoSmithKline, Speaker Bureau Member, Novartis Pharmaceuticals, Advisory Group Member, Merck & Co., Consultant, GlaxoSmithKline, Speaker Bureau Member, Warner Chilcott. PRH: Employee, Amgen, Employee, Amgen. MAB: Investigator, Amgen, Speaker, Amgen, Speaker, Astra Zeneca, Investigator, Eli Lilly & Company, Speaker, Eli Lilly & Company, Speaker, GlaxoSmithKline, Investigator, Merck & Co., Speaker, Novartis Pharmaceuticals, Investigator, Roche Pharmaceuticals, Investigator, Proctor & Gamble, Investigator, Takeda. BD: Employee, Amgen, Employee, Amgen. MG: Employee, Amgen, Employee, Amgen. CO: Employee, Amgen, Employee, Amgen. RBW: Employee, Amgen, Employee, Amgen. PDM: Principal Investigator, Amgen, Scientific Board Member, Amgen, Consultant, Amgen, Speaker, Amgen, Scientific Board Member, Proctor & Gamble, Principal Investigator, Proctor & Gamble, Consultant, Proctor & Gamble, Speaker, Proctor & Gamble, Scientific Board Member, Roche Pharmaceuticals, Principal Investigator, Roche Pharmaceuticals, Consultant, Roche Pharmaceuticals, Speaker, Roche Pharmaceuticals, Scientific Board Member, Eli Lilly & Company, Principal Investigator, Eli Lilly & Company, Consultant, Eli Lilly & Company, Speaker, Eli Lilly & Company, Scientific Board Member, Merck & Co., Principal Investigator, Merck & Co., Consultant, Merck & Co., Speaker, Merck & Co., Scientific Board Member, Novartis Pharmaceuticals, Principal Investigator, Novartis Pharmaceuticals, Consultant, Novartis Pharmaceuticals, Speaker, Novartis Pharmaceuticals, Scientific Board Member, Sanofi, Principal Investigator, Sanofi, Consultant, Sanofi, Speaker, Sanofi, Speaker, GlaxoSmithKline, Advisory Group Member, GlaxoSmithKline, Consultant, GlaxoSmithKline, Advisory Group Member, Novartis Pharmaceuticals, Advisory Group Member, Amgen, Advisory Group Member, Roche Pharmaceuticals, Advisory Group Member, Eli Lilly & Company, Advisory Group Member, Merck & Co., Advisory Group Member, Proctor & Gamble, Advisory Group Member, Sanofi, Principal Investigator, Takeda.

OR10-6 5779 6.0000 A MANAGEMENT TRENDS AFTER 8 YEARS OF DENOSUMAB: FOLLOW-UP AFTER A ONE-YEAR OBSERVATIONAL PHASE OF THE PHASE 2 EXTENSION STUDY 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 15th 1:00:00 PM OR10 2222 11:30:00 AM Osteoporosis Oral


Jessica Sparks Lilley*1, Irene Marta Predazzi2, William Scott Bush2, Mac Rae F Linton3, Sergio Fazio4 and Scott M Williams5
1Vanderbilt Children's Pediat, Nashville, TN, 2Vanderbilt University School of Medicine, 3Vanderbilt Univ Sch of Med, Nashville, TN, 4Vanderbilt Univ, Nashville, TN, 5Dartmouth Medical School

 

Introduction:  The roles of genetic and environmental contributions to plasma lipid levels are complex and not fully understood. Heritability estimates are about 40-50% for HDL,1 but only 10-12% of the total variance has been accounted for by individual genes.2 Part of the missing variance can be attributed to epigenetic influences since the maternal environment has been shown to affect fetal outcomes in a variety of disease processes, from diabetes to cancer to psychiatric illness.  Maternal hypercholesterolemia is thought to affect children’s cardiovascular outcomes in adulthood, though animal and epidemiologic studies have produced conflicting results.  We recently have determined that parental lipid parameters strongly influence levels of LDL-C, HDL-C, and TG in the offspring, even when correcting for known confounders.3 These results, however, do not account for the impact of potential shared environment. The Framingham Heart Study (FHS) provides a means to examine a large cohort of parents and their offspring. Though parents and offspring no longer shared a household at the time of their inclusion in the study, parents most likely lived together at the time of their enrollment and can serve as internal controls for the effects of shared environment.   

Objective:To determine the impact of the environment on lipid parameters of couples in the Framingham Heart Study.

Methods: We examined the second and third generations of the FHS (n= 9,219 participants) and performed linear regression analyses comparing lipid levels of mothers and fathers from Generation 2 to their offspring from Generation 3. To compare the effect of shared environment to the effect of shared genetics, we then analyzed correlation between parents from Generation 2, who were unrelated but shared a living environment. Correlations (R2) of lipid traits were computed and adjusted for BMI, age, smoking status, and menopausal status.

Results: The relationship of parent to offspring lipid traits was highly significant, even when adjusted for confounders. The relationship of parent-to-child LDL was the most significant with R2=0.089; the comparison of father to mother (parent-parent) LDL was much lower with R2=0.010.  The total variance of offspring’s HDL explained by parental HDL was 5%, while parent-to-parent comparison accounted for only 0.8% of observed variance.  As expected, triglycerides were the most variable lipid with little of the variance explained either by parent of origin or shared environment (R2 0.013 and 0.003, respectively). The p-values for each comparison were highly significant (p<1e-4) aside from parent-parent TG (p=0.449). 

Conclusions:There is a significant relationship between lipid phenotypes of parents and offspring, whereas the influence of environment is small but statistically significant. These results support a dominant role of genetics over environment in determining serum lipid levels.

 

Nothing to Disclose: JSL, IMP, WSB, MRFL, SF, SMW

OR05-1 9078 1.0000 A ENVIRONMENTAL EFFECTS ON PLASMA LIPIDS IN THE FRAMINGHAM COHORT 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Cardiovascular Endocrinology Saturday, June 15th 1:00:00 PM OR05 2229 11:30:00 AM Lipids: Regulation & Mechanism of Disease Oral


Kathryn CB Tan*, Joanne KY Lam, Sammy WM Shiu and Ying Wong
University of Hong Kong, Hong Kong

 

Several proatherogenic modifications of LDL have been detected in humans. In addition to oxidative modification, LDL is also subjected to post-translational protein modification like carbamylation in vivo. Carbamylation is a spontaneous non-enzymatic modification of lysine or the terminal protein amino acids of apolipoprotein B by urea-derived isocyanate, and carbamylated LDL (cLDL) is an important proatherogenic isoform of LDL in conditions like uraemia. Recent evidence has shown that cLDL is also found in healthy individuals and can be formed by urea-independent mechanism. The objective was to investigate plasma cLDL level in type 2 diabetes and its association with myeloperoxidase (MPO) which can catalyse protein carbamylation. 

264 diabetic patients not on lipid lowering agents and 200 non-diabetic controls were recruited. Plasma cLDL concentration was measured using an in-house sandwich ELISA using polyclonal rabbit anti-human cLDL antibody. Plasma MPO and high sensitivity C-reactive protein (CRP) was measured by ELISA and immunoturbidimetric assay respectively. 

Plasma LDL cholesterol and apolipoprotein B levels were similar between diabetic patients and controls. However, plasma cLDL level was higher in diabetic subjects than controls [327.6 ng/mg (231.0 – 501.8) vs 302.4 ng/mg (215.6 – 425.6) respectively, median (interquartile range), p<0.01], and this remained significant even after excluding subjects with elevated urea level. Both plasma MPO and CRP were also significantly increased in diabetic subjects (p<0.01). Plasma cLDL correlated with plasma MPO (r=0.40, p<0.001) and urea (r=0.23, p<0.01) but not with CRP in diabetic subjects. On linear regression analysis, plasma MPO remained an independent determinant of plasma cLDL even after adjusting for age, gender, body mass index, HbA1c and urea (partial correlation r=0.31, p<0.001) in subjects with diabetes.

In conclusion, plasma level of cLDL is increased in patients with type 2 diabetes and is partly related to MPO-induced carbamylation. Carbamylated LDL is pro-atherogenic and may contribute to the increased cardiovascular risk of diabetic subjects.

 

Nothing to Disclose: KCT, JKL, SWS, YW

OR05-2 3964 2.0000 A Carbamylation of LDL in Type 2 Diabetes Mellitus 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Cardiovascular Endocrinology Saturday, June 15th 1:00:00 PM OR05 2229 11:30:00 AM Lipids: Regulation & Mechanism of Disease Oral


Jun Zhu*1 and Farid F Chehab2
1University of Califonia, San Francisco, CA, 2Univ of California, San Francisco, CA

 

We recently identified that the C7orf58 gene (also called CPED1) was disrupted by a chromosome translocation in an obese female with mental retardation and coarse features. To gain insights into the pathophysiology of this novel gene, we generated whole body knockout mice for the C7orf58 mouse homolog A430107O13Rik. On an inbred C57BL/6J genetic background, homozygous disruption of C7orf58 resulted in early embryonic lethality. However, heterozygous knockout mice were viable and normal, but approximately 20% of them display by 7 days of age, ichthyosis, alopecia and a severely compromised skin barrier function. Moreover, they display peripheral lipoatrophy and die invariably by 2-3 weeks of age. On the mixed 129-C57BL/6J genetic background, homozygosity for the null allele also results in embryonic lethality and a similar proportion of heterozygous knockout mice exhibit the same features as those on the C57BL/6J background. In addition, they develop kyphosis and survive for about 5 months. Histological skin analysis from heterozygous knockout mice with ichthyosis of both strains uncovered extensive keratinization of the surface epithelium and the epithelium lining the hair follicles. In the dermis, markedly decreased or absence of adipocytes was replaced by a proliferative epidermis. Also, electron microscopy revealed the presence of multiple abnormal lamellar bodies in the epidermis. Analyses of epidermal and dermal lipids from knockout mice with ichthyosis of both genetic strains revealed prominent accumulation only in the epidermis, of an unknown lipid that we unveiled by mass spectrometry to consist of 5alpha-cholesta-8,14-dien-3beta-ol, an intermediary sterol synthesized during the late steps of cholesterol biosynthesis. Furthermore, saturated very long chain fatty acids (22:0, 24:0, 26:0) and their respective conjugated ceramides were 4-10 fold higher in knockout than control mice. The likely role of C7orf58 in the distant cholesterol biosynthesis pathway is consistent with bioinformatics studies, proposing that it consists of a hydrolase/esterase. Taken together, our studies demonstrate that perturbed epidermal lipid biosynthesis associated with packaging of abnormal lipids into lamellar bodies result in the secretion of detrimental lipids to the stratum corneum, resulting in ichthyosis and its sequelae. Thus, the susceptibility of the epidermis to a systemic reduction of C7orf58 levels supports a critical role for C7orf58 in epidermal lipid homeostasis.

 

Nothing to Disclose: JZ, FFC

OR05-3 7315 3.0000 A Cutaneous Lipid Abnormalities In C7orf58 Knockout Mice Reveal A Critical Role For C7orf58 In Epidermal Lipid Homeostasis And The Late Steps Of Cholesterol Biosynthesis 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Cardiovascular Endocrinology Saturday, June 15th 1:00:00 PM OR05 2229 11:30:00 AM Lipids: Regulation & Mechanism of Disease Oral


Katya B. Rubinow*1, Valerie Z Wall2, Joel Nelson2, Daniel Mar2, Karol Bomsztyk2, Bardia Askari2, Marvin Lai2, Kelly D Smith2, Myoung Sook Han3, Anuradha Vivekanandan-Giri4, Subramaniam Pennathur4, Carolyn Albert5, David A Ford5, Roger J Davis6 and Karin E Bornfeldt2
1University of Washington, Seattle, WA, 2University of Washington, 3HHMI/UMASS Med Schl, 4University of Michigan, 5Saint Louis University School of Medicine, 6HHMI/UMASS Med Schl, Worcester, MA

 

Background:Acyl-CoA synthetase 1 (ACSL1) mediates inflammatory effects in macrophages, and myeloid cell-targeted ACSL1 deficiency protects mice from early atheroma formation in models of type 1 diabetes.  In insulin target tissues, ACSL1 plays a predominant role in fatty acid β-oxidation, and its transcription is mediated by peroxisome proliferator-activated receptor (PPAR) α and γ.  However, its regulation and biological role in macrophages remain largely unknown.

Methods: We investigated the specific signals resulting in ACSL1 induction in macrophages to determine whether PPAR agonists or alternative stimuli regulate ACSL1 expression in this cell type.  Next, we selectively examined signal transduction pathways involved in ACSL1 expression through use of pharmacological inhibitors, siRNA, and genetic knockout mice.  Finally, we determined whether ACSL1 deficiency altered phospholipid composition in lipopolysaccharide (LPS)-stimulated macrophages.

Results: PPAR agonists do not stimulate ACSL1 expression in macrophages, whereas LPS, IFN-γ, TNFα, and Gram-negative pathogens significantly induce ACSL1 mRNA and protein.  LPS-induced ACSL1 expression requires TLR4-TRIF-mediated signaling, but the effects of Escherichia coli (E. coli) on ACSL1 expression are TRIF-independent.  IFN-γ induction of ACSL1 partially depends on JNK1/2 signaling, but JNK1/2 deficiency has no effect on LPS- or E. coli-mediated induction of ACSL1.   ACSL1 expression is required for maximal LPS-induced turnover of phospholipid species.

Conclusion: The regulation and function of ACSL1 differ substantially in macrophages and insulin target tissues. Multiple pathways involved in inflammatory responses contribute to the induction of ACSL1 in macrophages, and the relative contribution of each implicated pathway depends on the specific inflammatory stimulus.  ACSL1 in macrophages is required for LPS-stimulated turnover of several phospholipid species.  These findings indicate a novel role for ACSL1 in innate immune function and, further, illustrate an interesting paradigm in which the same enzyme confers distinct biological effects in different cell types.  Moreover, these disparate functions are paralleled by differences in the pathways that regulate its expression. Future studies are necessary to determine how ACSL1-dependent flux of phospholipid species contributes functionally to host defense and other facets of innate immune activity. 

 

Nothing to Disclose: KBR, VZW, JN, DM, KB, BA, ML, KDS, MSH, AV, SP, CA, DAF, RJD, KEB

OR05-4 7454 4.0000 A Acyl-CoA Synthetase 1 is Induced by Gram-Negative Bacteria and Lipopolysaccharide and is Required for Phospholipid Turnover in Stimulated Macrophages 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Cardiovascular Endocrinology Saturday, June 15th 1:00:00 PM OR05 2229 11:30:00 AM Lipids: Regulation & Mechanism of Disease Oral


Fang Yan*1, Jiajun Zhao2, Ling Gao2, Chao Xu3, Haiqing Zhang4, Chunxiao Yu5 and Yongfeng Song6
1Provincial Hospital Affiliated to ShanDong University, Endo, 2Shandong Provincial Hospital affiliated to Shandong University, Jinan, China, 3Shandong Provincial Hospital affiliated to Shandong University, Department of Endocrinology and Metabolism, China ;, Jinan, Shandong, 4Shandong Provincial Hospital, Endo, 5Provincial Hospital affiliated to Shandong University, China, 6Shandong Provincial Hospital, Endo, Jinan, Shandong Province

 

Objective:

Subclinical hypothyroidism (SCH) has been associated with increased plasmic triglyceride (TG) in recent clinical studies, but these reports are controversial. Up to date, it is unclear for the underlying mechanism of this association. Our previous study has found TSH receptors exist in the hepatocytes. Here we focused on TSH, which is elevated in serum of SCH, to explore the effect of TSH on TG synthesis in the liver.

Methods:

We firstly set up Tshr-/- and Tshr +/+ mouse models, and tested in vivo if there was difference of liver TG contents and the expression of the lipogenic gene and protein using oil-red O staining, real-time PCR, western-blotting, immunohistochemistry and immunofluorescence, respectively. Next, we chosed HepG2 cells, which was widely used in hepatocytic research, as a model in vitro. After treating the cells with TSH or PPARα agonist in the presence or absence of PPARα antagonist, we observed that TSH played direct effects on a panel of molecules relative to TG synthesis such as SREBP1c, a main regulator of triglyceride synthesis.

Results:

(1)         Compared with the control littermate (Tshr+/+), the liver TG content of Tshr-/- (supplemented with T4) mice declined to 52.6% (p < 0.05), accompanied with a decrease in the expression of nuclei PPARα and mature SREBP1c (48.9% and 71.1% vs. control, respectively, both p < 0.05). It suggests that the changes depends on liver TSH receptor.

(2)         TSH induced dose- and time-dependent increase in the levels of PPARα, SREBP1c as well as their downstream molecules in HepG2 cells. Compared to the control, the levels of PPARα and SREBP1c were elevated by approximately 1 fold, respectively (both p < 0.05) when 4μM TSH treated for 48h. Similarly, double-immunofluorescence showed that TSH stimulated stronger fluorescence of both PPARα and SREBP1c in the nuclei and plasma. As a consequence, intracellular TG elevated 21.7% (p< 0.05) relative to control, which was reconfirmed by oil-red O staining.

(3)         The PPARα agonist, fenofibrate, increased the hepatocytic expression of mature SREBP1c protein in dose- and time-dependent manners. 100 μM fenofibrate for 48h triggered mature SREBP1c protein a 3.5-fold increase of the control (p< 0.05).

(4)         When PPARα was immunoprecipitated, endogenous SREBP-1c mature protein was present in the complex with PPARα. SREBP-1c was also detected in reciprocal coimmunoprecipitation of PPARα. That implies there exist a direct intercombine between PPARα and SREBP1c proteins.

(5)         When treatment of MK886 to block PPARα activation prior to TSH, the TSH-inducing changes above were inversed, and showed the decreased expression of the activated PPARα, SREBP-1c mature form as well as intercellular TG content.  It suggests that PPARα mediated the TG synthetic role of TSH by direct interaction with SREBP1c mature protein.

Conclusion:

TSH increases the TG content in liver cells through PPARα/SREBP1c signaling pathway.

 

Nothing to Disclose: FY, JZ, LG, CX, HZ, CY, YS

OR05-5 8642 5.0000 A Hepatic role of TSH in the control of triglyceride synthesis through PPAR/SREBP1c pathway 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Cardiovascular Endocrinology Saturday, June 15th 1:00:00 PM OR05 2229 11:30:00 AM Lipids: Regulation & Mechanism of Disease Oral


Jian Valentine Zhang*
Chinese Academy of Sciences (CAS), ShenZhen, China

 

EFFECT OF NOVEL CMKLR1 SMALL MOLECULE ANTAGONIST ON STEATOSIS AND INFLAMMATION OF NONALCOHOLIC FATTY LIVER DISEASE IN MICE

Xu ZHANG1#, Qin-ce SUN1#, Shu-Hua Mu1, Yong-jun LIU1, Ping Ma1, Wen-juan XU1, Jian ZHANG*1,2

 

  1. Shenzhen Institute of Advance Technology, Chinese Academy of Sciences, and 2. ShenZhen Innovative Pharmacology and Biotherapy Pre-clinical Test Public Service Platform, ShenZhen, 518055, China

# These authors contribute equally to this work.

*Address for correspondence: Tel: (86) 0755-86582290; Fax: (86) 0755-86585222; E-mail: jian.zhang@siat.ac.cn

Objectives:Worldwide, the incidence of nonalcoholic fatty liver disease (NAFLD) has increased dramatically throughout the last three decades. Chemokine-like receptor-1 (CMKLR1) is a chemoattractant receptor that binds chemerin, a proteolytically regulated chemoattractant. CMKLR1 is expressed by macrophages, subsets of dendritic cells, natural killer cells and microglia.Some studies previously suggested a close association between Chemokine-like receptor 1 (CMKLR1) and non-alcoholic fatty liver disease (NAFLD). This study was to investigate the role of a novel small molecule CMKLR1 antagonist in steatosis induced by oleic acid (OA) in vitro and the progression of NAFLD in vivo mice model. 

Methods:We set up in vitro OA-induced steatosis Hepa1-6 cells model and in vivo high-fat induced NAFLD mice model. A novel small molecule CMKLR1 antagonist was used for interferring the progression of the lipid accumulation in Hepa1-6 cells and high-fat induced NAFLD in mice. The gene expression of Chemerin and CMKLR1, lipid metabolism-associated factors AdipoR, LDLR, ADRP, PPAR-α, PPAR-γ, PPAR-δ, HMGCR, HSL, and inflammatory factors, such as IL-6 and TNF-α were analyzed by real-time PCR. The serum TC, TG, AST and ALT levels were also measured by Elisa.

 Results:The expression of Chemerin and CMKLR1 mRNA level is increased after OA induction in Hepa1-6 cells, compared with the control group. The CMKLR1 mRNA level is upregulated in the liver of NAFLD mice model induced by high-fat diet compared with normal feeding mice. Lipid droplet accumulation, and ADRP, PPAR-γ, PPAR-δ, HMGCR, IL-6 and TNF-α mRNA levels were inhibited by the small molecule CMKLR1 antagonist treatment in OA-induced Hepa1-6 cells and in high-fat diet induced mice model. In high-fat diet mice, serum TC, TG, AST and ALT levels were suppressed by the small molecule CMKLR1 antagonist.

 Conclusions:Above data showed that the small molecule CMKLR1 antagonist inhibits the progression of NAFLD both in vitro and in vivo model, implying that chemerin/CMKLR1 signaling may play an important role in the pathogenesis of NAFLD, not only as regulator of lipid metabolism, but also as mediator of the inflammatory process. This suggests that the small molecule CMKLR1 antagonist might be a novel therapeutic target for the NAFLD.

 

Nothing to Disclose: JVZ

OR05-6 6186 6.0000 A EFFECT OF NOVEL CMKLR1 SMALL MOLECULE ANTAGONIST ON STEATOSIS AND INFLAMMATION OF NONALCOHOLIC FATTY LIVER DISEASE IN MICE 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Cardiovascular Endocrinology Saturday, June 15th 1:00:00 PM OR05 2229 11:30:00 AM Lipids: Regulation & Mechanism of Disease Oral


Robert L Sorenson*1, Nicholas Bhagroo2, Laurence Stout2 and T. Clark Brelje2
1Univ of Minnesota, Minneapolis, MN, 2University of Minnesota, Minneapolis, MN

 

Islets undergo changes to meet the increased need for insulin during pregnancy, including increased beta cell proliferation.  Placental lactogen and/or prolactin (PRL) regulate many of these changes.  Since lipids are elevated in pregnancy, we examined the effect of prolactin and oleic acid on islet beta cell proliferation (BrdU labeling) and islet mass in vitro.

  PRL treatment results in a 5.5±0.5-fold increase in BrdU labeling, oleate a 3.5±0.5-fold increase and synergizes with PRL to a 14.2±1.5-fold increase (n=16, p<0.01).  Similar results were obtained when using Ki-67 or PCNA to determine cell proliferation and further confirmed with Western analysis of PCNA.  Synergy between PRL and oleate was seen with both neonatal and adult islets.  Unsaturated fatty acids (FA) palmitoleate (18-fold) and linoleate (13-fold) also induce synergy, but not the saturated FA palmitate.  While synergy was seen with oleate and PRL, it was not observed with oleate and GH, GLP-I, EGF or EGF+IGF.

  To examine the effects of PRL and oleate on islet mass, islets were cultured for 14 days and each islet photographed on alternate days.  The volume was determined to estimate the growth rate.  Neonatal islet growth was -0.4±.5%/day for control, 3.7±0.8%/day for PRL, 0.4±0.8%/day for oleate and 7.2±1.3%/day for PRL+oleate (n=8, p<0.01).  This represents a 16 day doubling time for the PRL/oleate treated islets.   Adult islet growth was -0.5±0.3%/day for control, 1.4±0.3%/day for PRL, 0.1±0.2%/day for oleate and 3.2±0.5%/day for PRL+oleate (n=7, p<0,05).   This represents a 32 day doubling time for the PRL/oleate treated adult islets.  Approximately two thirds of the islet growth is from an increase in cell volume and one third from cell number.

   Inhibition of FA oxidation (etomoxir) had no effect on cell division or synergy between PRL and oleate.  In contrast, inhibition of fatty acylCoA synthetase (Triacin C) reduced cell division by approximately 50% in all groups (control, PRL, oleate and oleate/PRL) without affecting the synergy seen between PRL and oleate.  Similarly, PKCζ inhibition and MEK inhibition (U0126) reduced cell division in all groups without affecting synergy (n=5, p<0.05).

  The results suggest that the increase in β cell growth during pregnancy represents the combined effects of PRL/PL and FAs however, the mechanisms for synergy between prolactin and oleate remain unclear.

 

Nothing to Disclose: RLS, NB, LS, TCB

OR13-1 7343 1.0000 A Synergy between Oleic Acid and Prolactin in Beta Cell Growth: Adaptation of Islets to Pregnancy 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Diabetes & Glucose Metabolism Saturday, June 15th 1:00:00 PM OR13 2246 11:30:00 AM Systemic Regulation of Islet Development & Function Oral


Lynley Pound*1, Sarah Michelle Comstock2, Ashley Kostrba3, Diana Lynn Takahashi3, India Tindale2, Peter Blundell2 and Kevin L. Grove4
1Oregon National Primate Research Center, Beaverton, OR, 2OHSU/ONPRC, Beaverton, OR, 3Oregon Health and Science University/ONPRC, Beaverton, OR, 4Oregon National Primate Research Center, Oregon Health & Science University, Beaverton, OR

 

Recent studies have indicated that children exposed to maternal obesity in utero have an increased risk of developing obesity and type 2 diabetes later in life. The present study assessed the impact of a maternal high fat diet (HFD) during pregnancy versus post-weaning HFD on pancreas development. Adult female Japanese Macaques were placed on a control (CTR) or HFD for 4-7 years. Pregnancies were either terminated in the early third trimester by C-section or were allowed to progress to natural birth. At weaning, these offspring were either maintained on the maternal diet or switched to the opposing diet to produce four post-weaning groups. Juvenile offspring were sent to necropsy at 1 year of age. Pancreata from both fetal and juvenile offspring were processed for biochemical and morphological analyses.

Maternal diet (HFD vs. CTR) did not alter fetal offspring body weight, glycemia, pancreatic mass, or circulating glucagon levels. HFD offspring displayed decreased circulating C-peptide levels but normal insulin levels, indicative of decreased insulin secretion and a reduction in insulin clearance.  Maternal diet had no effect on offspring islet mass, islet proliferation or β-cell mass, but HFD fetal offspring displayed a significant reduction in α-cell mass. Furthermore, HFD exposure in utero resulted in a significant reduction in islet capillary density and sympathetic innervation.

Juvenile animals exposed to both HFD in utero and postweaning displayed an increase in body weight, fasting insulin and insulin secretion during a glucose tolerance test at 1 year of age compared to animals on the CTR diet. HFD consumption during the postweaning period resulted in expansion of both β- and α-cell mass. HFD-fed offspring that had been exposed to the HFD in utero, however, failed to display this expansion in α-cell mass indicating that maternal HFD exposure causes reduced α-cell plasticity. Furthermore, maternal HFD exposure resulted in a significant reduction in islet capillary density in the juvenile offspring. Overall, our results suggest that HFD consumption during pregnancy leads to an adaptive islet response in the α-cell. Furthermore, our data suggest that exposure to a HFD in utero results in impaired islet vascularization in the fetus and that this effect is sustained later in life. Because of the important role of vascularization of the islet in its morphology and function, these data may provide a novel mechanism by which maternal HFD consumption leads to increased risk of type 2 diabetes.

 

Nothing to Disclose: LP, SMC, AK, DLT, IT, PB, KLG

OR13-2 6361 2.0000 A Maternal High Fat Diet Consumption Alters Islet Vasculature and Innervation in the Nonhuman Primate 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Diabetes & Glucose Metabolism Saturday, June 15th 1:00:00 PM OR13 2246 11:30:00 AM Systemic Regulation of Islet Development & Function Oral


Amita Bansal*1, Jane M Alsweiler1, Kristin L Connor2, Mike Dragunow1, Jane E Harding1 and Frank H Bloomfield3
1University of Auckland, Auckland, New Zealand, 2Mount Sinai Hospital, Toronto, ON, Canada, 3Gravida: National Centre for Growth & Development, Auckland, New Zealand

 

Background: Fetal exposure to a hyperglycemic environment predisposes offspring to impaired glucose tolerance (IGT) in adulthood. Preterm babies also commonly are hyperglycemic and treated with insulin, and are at risk of IGT in adulthood. Objective: We investigated in preterm lambs whether neonatal hyperglycemia has long-term effects on β-cell mass and on key genes involved in insulin secretion and hepatic insulin sensitivity, and whether restoration of euglycemia with insulin treatment reverses these effects. Methods: Pancreata and liver tissue were collected at 12 months of age from lambs born at term (148d; term controls) or preterm (137d) (n=12-13 per group), following antenatal glucocorticoids as given clinically. Preterm lambs were randomised to saline controls, hyperglycemic (50% dextrose infused intravenously for 12d to maintain blood glucose concentration (BGC) at 10-12 mmol/L), or insulin treated hyperglycemic (hyperglycemia treated with insulin to achieve euglycemia (BGC= 4-6 mmol/L)) groups. ß-cell mass and islet size were determined using Metamorph automated quantitative analysis of pancreatic sections triple-immunofluorescent stained for insulin, glucagon & somatostatin proteins. Data were analysed by 2-way ANOVA, with post-hoc Tukey’s test if p<0.05. mRNA levels, calculated as fold change with 99% confidence intervals, were determined by qPCR normalised to 3 housekeeping genes. Results: All preterm lamb groups had similar ß-cell mass which was approximately half that in term lambs, with significantly smaller islets (p<0.05). Preterm birth also reduced pancreatic mRNA expression of igf-2, glucokinase (gck) and insulin. Hyperglycemia further reduced mRNA expression of pdx1, igf-I and gck; this was not reversed with insulin treatment. Preterm birth increased hepatic mRNA expression of PEPCK and PPAR-α but not of SREBP1. Hyperglycemia reduced mRNA expression of GLUT2 and PPAR-α; insulin treatment restored GLUT2 and PPAR-α mRNA expression to levels of preterm controls. Conclusions: Preterm birth in sheep leads to reduced β-cell mass in adulthood, independent of neonatal hyperglycemia or its treatment with insulin. Preterm birth also altered expression of key genes involved in insulin secretion and hepatic insulin sensitivity; this was exacerbated by hyperglycemia and partially reversed with insulin treatment. These findings may inform mechanisms underlying IGT in adults born preterm.

 

Nothing to Disclose: AB, JMA, KLC, MD, JEH, FHB

OR13-3 6628 3.0000 A Preterm Birth in Sheep Alters Adult Pancreatic Beta-Cell Mass and Expression of Key Genes Involved in Insulin Secretion and Hepatic Insulin Sensitivity 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Diabetes & Glucose Metabolism Saturday, June 15th 1:00:00 PM OR13 2246 11:30:00 AM Systemic Regulation of Islet Development & Function Oral


Mary N Morcos1, Daniel T Meier2, Rebecca L Hull2 and Steven E Kahn*2
1Virginia Mason Medical Center, Seattle, WA, 2VA Puget Sound Health Care System and University of Washington, Seattle, WA

 

Islet amyloid deposition and beta-cell loss are pathological hallmarks of human type 2 diabetes. To study islet amyloid, transgenic mice expressing the unique component of these deposits, human islet amyloid polypeptide (hIAPP), were produced as rodent IAPP is not amyloidogenic. When fed a high fat (HF) diet, hIAPP transgenic mice develop amyloid deposits morphologically identical to those in human type 2 diabetes and these are accompanied by a reduction in beta-cell number. Given the strong dietary association with the development of type 2 diabetes, we sought to determine whether addition of sucrose to a HF diet exacerbates islet amyloid deposition and beta-cell loss above that observed with a HF diet alone. Thus, hIAPP transgenic mice were for 12 months fed diets containing 15% (low fat, LF, n=8) or 60% (high fat, HF, n=11) of calories derived from fat. A third group of hIAPP transgenic mice were fed the HF diet and received drinking water supplemented with 20% sucrose (HFHS, n=11). At the end of the 12-month period, mice were euthanized and islet amyloid deposition and beta-cell area quantified by thioflavin S and insulin staining, respectively. Compared to the LF group, mice in the HF and HFHS groups weighed more (LF: 38.8±2.6, HF: 68.2±3.9, HFHS: 62.5±2.7 g; p<0.001 for HF and HFHS vs LF). The increase in body weight was associated with an increase in islet area (LF: 19862±3158, HF: 58519±13048, HFHS: 42841±5822 µm2; p<0.05 for HF and HFHS vs LF). The proportion of the islet comprised of beta-cells (beta-cell area/islet area) tended to decrease in the HF (52.9±3.0%) and decreased in the HFHS (51.5±2.3%) groups compared to the LF group (58.4±0.9%; p=0.13 for HF vs LF and p<0.05 for HFHS vs LF). In keeping with amyloid replacing beta-cells, islet amyloid was observed in mice on HF and HFHS diets, but not in mice on the LF diet. Amyloid severity (% islet area occupied by amyloid) among the HF and HFHS groups was 6.7±2.2% and 10.1±2.6%, respectively (p<0.05 for HF and HFHS vs LF). Islet area (p=0.3), beta-cell area (p=0.73) and amyloid severity (p=0.35) did not differ between HF and HFHS fed mice. In conclusion, in hIAPP transgenic mice a HF diet results in increased islet amyloid deposition and beta-cell loss. However, addition of high concentrations of sucrose does not exacerbate this effect. Thus, calories derived from fat may be more deleterious than those from sucrose in determining islet amyloid deposition and beta-cell loss in type 2 diabetes.

 

Disclosure: SEK: Advisory board, Scientific symposium speaker, Merck & Co., Advisory Group Member, Lupin Pharmaceuticals, Advisory Group Member, Jansen Pharmaceuticals, Advisory Group Member, Intarcia, Advisory Group Member, GlaxoSmithKline, Advisory Group Member, Eli Lilly & Company, Advisory Group Member, Elcelyx, Advisory Group Member, Bristol-Myers Squibb, Advisory board, Study steering committee, Scientific symposium speaker, Boehringer Ingelheim, Advisory Group Member, Novo Nordisk, Advisory Group Member, Receptos. Nothing to Disclose: MNM, DTM, RLH

OR13-4 6026 4.0000 A Dietary Sucrose Does Not Exacerbate The Effect of Dietary Fat to Promote Islet Amyloid Deposition and Beta-Cell Loss in Human Islet Amyloid Polypeptide (hIAPP) Transgenic Mice 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Diabetes & Glucose Metabolism Saturday, June 15th 1:00:00 PM OR13 2246 11:30:00 AM Systemic Regulation of Islet Development & Function Oral


Undine Schubert*, Barbara Ludwig, Henning Morawietz and Stefan Richard Bornstein
University Hospital Carl Gustav Carus, Dresden, Germany

 

Background

For restoration of β-cell function in diabetic patients, intraportal islet transplantation has evolved into a viable treatment option. However, several factors hamper a widespread application and long-term success: chronic hypoxia, exposure to an inappropriate microenvironment and suppression of regenerative and proliferative potential by the need of potent immunosuppressive agents.

The idea of the adrenal tissue as an alternative niche for pancreatic islets is derived from (1) the fact that both are endocrine tissues with similar microenvironment, (2) the unique feature of extensive vascularization of the adrenal, (3) anti-apoptotic and pro-proliferative effects of various signalling molecules within the adrenal, and (4) the advantage of a local anti-inflammatory and immunosuppressive microenvironment.

Method and Results

For analysis of in vitro islet viability and function a co-culture system of adrenal cells and pancreatic islets was established. Pancreatic islets and adrenal cells were isolated from Wistar rats and co-cultured using inserts for up to 7 days and sequentially assayed for viability, insulin secretion and reactive oxygen species (ROS). The co-culture setting did not significantly impact on islet viability, insulin content and secretion of pancreatic islets and even allowed for long-term culture.

For in vivo studies, Streptozotocin induced diabetic NOD-SCID mice were used as islet recipients (n=6). For islet transplantation, the adrenal was exteriorized via retroperitoneal incision and 300 islets were injected through the upper pole of the gland. Blood glucose levels were measured daily throughout the observation period of 30 days. Most animals showed a fast decrease in blood glucose and reached normoglycemia within 2 days. On day 5 an intraperitoneal glucose tolerance test was performed and four out of five animals reached target blood glucose levels. Upon removing of the islet graft by unilateral adrenalectomy, the animals showed an immediate recurrence of hyperglycemia. Immunostaining of insulin revealed intense cytosolic staining.

Conclusion

Our studies demonstrated that co-localization of adrenal cells and isolated islets allows for long-term culture of islets in vitro and intra-adrenal islet transplantation is a technically feasible and a functionally promising approach to restore normoglycemia in a diabetic mouse model. This novel concept might allow reducing the islet mass that is needed to reverse diabetes through ameliorated engraftment and minimized islet loss due to dense vascularisation and the endocrine microenvironment might be beneficial for long term survival and function of transplanted islets.

 

Nothing to Disclose: US, BL, HM, SRB

OR13-5 6947 5.0000 A The Adrenal Gland and Pancreatic Islets. A beneficial Endocrine Alliance? 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Diabetes & Glucose Metabolism Saturday, June 15th 1:00:00 PM OR13 2246 11:30:00 AM Systemic Regulation of Islet Development & Function Oral


Ramamani Arumugam*1, Donald Edward Fleenor2, Jennifer Moss1, Danhong Lu1 and Michael Freemark1
1Duke University Medical Center, Durham, NC, 2Duke Univ Med Ctr, Durham, NC

 

Background: While immune interventions have reduced the rate of decline in insulin secretion in type 1 diabetic (T1D) patients they have failed to increase human beta cell mass or endogenous insulin production. Since beta cell mass is largely depleted by diagnosis, the effective treatment of T1D will require induction of beta cell regeneration in combination with suppression of autoimmunity.   Regeneration can be achieved through replication of pre-existing beta cells and/or neogenesis from cellular precursors.  But the rates of beta cell replication and neogenesis in human adolescents and adults are far lower than in human fetuses, infants, and toddlers.  This makes endogenous regeneration of beta cells in older children and adults with T1D difficult to achieve. 

Hypothesis: Beta cell replication can be induced in adult rodent islets by treatment with prolactin (PRL) or placental lactogen (PL).  Conversely, a deletion of PRL receptors (PRLRs) reduces pancreatic beta cell mass.  We hypothesized that over-expression of PRLRs would stimulate replication of adult rat islets cultured in the presence of fetal bovine serum (FBS), which contains endogenous bovine PRL (~50 ng/ml) and PL (10-20 ng/ml).

Methods: We transfected adult rat islets with an adenovirus expressing the rat PRLR.  Control islets were treated with adenoGFP.  The islets were incubated in medium with 10% FBS.

Results: After 72 hr in culture, PRLR receptor mRNA and protein levels were increased ~50 fold.  Receptor protein was localized primarily to the plasma membranes of islet beta cells. 

Over-expression of PRLRs stimulated a 3.2-fold increase in islet thymidine incorporation (p<0.001). The induction of DNA synthesis was accompanied by striking increases (2-3 fold, p<0.001) in the mRNA levels of cyclins A2, B1, B2, and CDK1, and lesser and variable increases in cyclin D1 and FoxM1 mRNAs (20-50%).  Conversely, there were 20-50% decreases in expression of cyclin E1, p21, menin, and Bcl6 mRNAs.  Cyclin D2 mRNA did not change, but D2 protein levels were mildly increased. Expression of MafA, a determinant of beta cell maturation, increased 45% (p<0.001) but contrary to expectations, Tph1 mRNA levels declined and BclXL mRNA did not change.  However there was a 3.4-fold increase (p<0.001) in the mRNA levels of the anti-apoptotic protein PTTG1 (securin).

Conclusion: Over-expression of the PRLR in adult rat islets markedly increases DNA synthesis and regulates the expression of critical cell cyclins, cell cycle inhibitors, MafA, and PTTG1, a novel anti-apoptotic protein. 

Implications: Our findings suggest a potential therapeutic approach for T1D by which targeted up-regulation of PRLR expression could increase beta cell replication without imposing systemic risks from treatment with PRL or placental lactogen.

 

Nothing to Disclose: RA, DEF, JM, DL, MF

OR13-6 5413 6.0000 A OVER-EXPRESSION OF PROLACTIN RECEPTORS IN ADULT RAT ISLETS PROMOTES DNA SYNTHESIS THROUGH REGULATION OF CELL CYCLE GENE EXPRESSION 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Diabetes & Glucose Metabolism Saturday, June 15th 1:00:00 PM OR13 2246 11:30:00 AM Systemic Regulation of Islet Development & Function Oral


Carol Huang*1, Saara Rawn1 and Jay C Cross2
1University of Calgary, Calgary, AB, Canada, 2University of Calgary

 

Pregnancy is characterized by an increase in maternal insulin resistance to shunt nutrients to the growing fetus.  The pregnant mother needs to adapt to this increased metabolic demand in order to maintain the health of the maternal-fetal unit.  The placenta likely plays a significant role in this context as it produces a large number of metabolically active hormones, including the placental lactogens (PLs).   PLs act through the prolactin receptor (Prlr), and signaling through Prlr is important in maintaining normal blood glucose in the pregnant dams since transgenic mice with a heterozygous deletion of Prlr exhibited glucose intolerance during pregnancy.  In early pregnancy, pituitary Prl secretion increases significantly.  By mid-gestation, Prl secretion declines while production of the PLs from the placenta begins. However, since both Prl and PLs signal through Prlr, whether both Prl and PLs are required for maintenance of normal glucose homeostasis during pregnancy is unknown.  To determine the distinctive function of Prl and PLs during pregnancy, we compared the maternal physiology and fetal outcomes of the ligand-null (i.e. Prl-/-) and receptor-null (i.e. Prlr-/-) mice.  Phenotypic difference between the pregnant Prl-/- and Prlr-/- mice should represent the actions of PLs. Here, we extended our previous finding of impaired glucose tolerance in the Prlr+/- mice and found that the Prlr-/- mice, but not the Prl-/- mice, had higher blood glucose than Prlr+/+ mice during pregnancy, suggesting that PLs but not Prl is important for regulation of glucose homeostasis. Next, we analyzed their metabolomes.  Serum metabolite profiles of pregnant Prlr+/- mice and the Prlr+/+ mice during pregnancy could be readily separated using multivariate statistical analysis, and we found that the Prlr+/- mice had higher TMAO, acetate, betaine, taurine, cholate, and o-phosphocholine  level than wild type mice.  As compared to Prl-/- mice (which has normal blood glucose during pregnancy), Prlr-/- mice had a higher TMAO level but levels of other metabolites were comparable between the two groups.  When we compared other maternal physiologic parameters, we did not find any difference in maternal blood pressure or spleen size between mutant and wild type mice.  Prl and PLs levels also seem to have no impact on fetal growth, as the fetal crown-rump length, body mass index, and litter size were comparable amongst the various genotypes. In conclusion, our data support PLs, not Prl, as one of the main hormones that regulate maternal glucose homeostasis and other metabolites during pregnancy.

 

Nothing to Disclose: CH, SR, JCC

OR02-1 7554 1.0000 A Placental lactogens regulate maternal glucose homeostasis during pregnancy 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Diabetes & Glucose Metabolism Saturday, June 15th 1:00:00 PM OR02 2257 11:30:00 AM Diabetes in Pregnancy Oral


Jacinda Mawson Nicklas*1, Ellen W. Seely2 and Chloe Zera3
1University of Colorado School of Medicine, Aurora, CO, 2Brigham & Women's Hospital, Boston, MA, 3Brigham and Women's Hospital, Boston, MA

 

Background: Hospital discharge data are often used to determine incidence of gestational diabetes (GDM) at state and national levels. However, previous studies demonstrate substantial variability in accuracy of GDM discharge diagnoses. We assessed accuracy of GDM discharge diagnoses at the Brigham and Women’s Hospital (BWH) in Boston, MA for the year 2010 and reviewed cases to determine patterns of clinician diagnosis of GDM. Methods: We identified cases assigned ICD-9 codes for GDM (648.80, 648.81, 648.83) during 2010 using the Brigham Integrated Computing System and the Research Patient Data Registry, a centralized database, to extract glucose loading test (GLT) and oral glucose tolerance test (OGTT) data. For cases that did not meet OGTT criteria for GDM, we conducted medical chart review to determine what led to assignment of a GDM delivery code. In 2010, BWH followed the American College of Obstetrics and Gynecology (ACOG) recommendations for GDM testing: screening with a 1 hour GLT, and administering 3 hour oral glucose tolerance tests OGTT when the GLT result was ≥140 mg/dl. Two or more abnormal values on OGTT indicated GDM by Carpenter-Coustan (CC) criteria. Results: Of 7883 deliveries at BWH in 2010, five percent (n=362) were coded as GDM. Among these 362 cases, 210 (58%) had OGTT results available meeting CC criteria. 28 (8%) women had a clinician diagnosis of GDM based upon a GLT result ≥200, without an OGTT. 76 (21%) women underwent GLT testing and then received a clinician diagnosis of GDM without a GLT≥200 and without failing an OGTT. Of these 76, 24 did OGTT testing and had 1 or 0 abnormal values but were given a clinician diagnosis of GDM, usually after demonstrating elevated home fingerstick glucose (FSG) values, and 52 never completed an OGTT, either because they could not tolerate the test (n=6), they declined (n=5), or they were given a clinician diagnosis of GDM based on FSG and/or a history of GDM (n=41). 24/362 (7%) received a clinician diagnosis of GDM without GLT or OGTT testing based upon a history of GDM or impaired glucose tolerance, and/or elevated FSG. For 7 (2%) cases we could not determine whether they had GLT/OGTT testing and for 8 (2%) cases the provider indicated abnormal OGTT or GDM but results were not available. We identified only 6 (2%) coding errors for cases without evidence of GDM in medical records and 3 (1%) clinician errors where OGTTs were misread. Conclusions: At our institution in 2010, 36% of women assigned GDM delivery codes did not meet OGTT criteria for GDM by ACOG guidelines, with 33% receiving clinician diagnoses of GDM outside of standard protocols. Only 3% of all cases were errors. Further investigations of prevalence should address the heterogeneity of GDM diagnosis patterns and studies of the impact of adoption of new diagnosis criteria for GDM should take into consideration that clinician diagnosis outside of published guidelines may be common.

 

Nothing to Disclose: JMN, EWS, CZ

OR02-2 5668 2.0000 A Patterns of clinician diagnosis leading to the assignment of ICD-9 discharge codes for gestational diabetes 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Diabetes & Glucose Metabolism Saturday, June 15th 1:00:00 PM OR02 2257 11:30:00 AM Diabetes in Pregnancy Oral


Geetha Mukerji*1, Jennifer Price2, Faith Delos-Reyes2, Sarah McTavish3 and Lorraine Lucille Lipscombe4
1University of Toronto, Toronto, ON, Canada, 2Women's College Hospital, Toronto, Canada, 3Women's College Research Institute, Toronto, Canada, 4Women's College Hospital, Toronto, ON, Canada

 

Introduction: Women with Gestational Diabetes Mellitus (GDM) are at high risk for future type 2 diabetes and cardiovascular disease (CVD) which can be prevented with lifestyle modification. However, prevention programs in this population have had limited success due to poor adherence. Programs need to be more customized to address the unique needs and barriers faced by new mothers.

Objective: To determine the feasibility of a pilot home-based lifestyle program to improve fitness among women with recent GDM. 

Methods: The study population included women 18 years or older with a pre-pregnancy BMI ≥ 25 kg/m2 during their first postpartum year after GDM. Participants were enrolled between July 2012 to January 2013 into a 6-month home-based lifestyle program, delivered by a kinesiologist trained in motivational interviewing, at the Women’s College Hospital Cardiovascular Prevention and Education Program. The intervention consisted of an individualized exercise prescription based on baseline fitness levels with regular telephone support. Outcomes [exercise capacity (EC) on a graded exercise treadmill test (metabolic equivalents, METS), body mass index (BMI), waist circumference (WC)] were assessed at baseline, 3 months and 6 months.

Results: We present interim data from this ongoing study. Of the 47 women contacted, 21 (45%) were enrolled and 17 (36%) attended the first visit (mean age 38 ± 8 SD years; BMI 35 ± 8 SD kg/m2; postpartum 7 ± 2 SD months). Mean baseline EC was 10.4 ± 1.0 SE METS, with 41% of women at <85% age-predicted EC. For the 8 women who have completed their 3 month visit, EC increased by a mean 0.53 ± 0.47 SE METs (p=0.29), and by 2.0 ± 1.15 METS (p<0.01) among those at <85% age-predicted EC (n=3).  Mean WC decreased by 2.75 ± 0.84 SE cm (p<0.05) with no significant change in BMI (n=8). The retention at 3 months is 100% with 88% reporting excellent or very good program satisfaction.

Conclusions: Our preliminary findings show that a customized lifestyle program in women with recent GDM is feasible with good adherence rates. Interim results indicate early improvements in EC, especially among those with lower baseline fitness levels, and significant reduction in WC after 3 months. Further analysis of the complete cohort at 6 months may demonstrate greater impact.  As increases in EC is associated with a lower risk of all-cause mortality and CVD events, these findings provide evidence for prevention programs designed for women with recent GDM.

 

Nothing to Disclose: GM, JP, FD, SM, LLL

OR02-3 5319 3.0000 A Postpartum Lifestyle Intervention in Women with Gestational Diabetes Mellitus: a pilot feasibility study 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Diabetes & Glucose Metabolism Saturday, June 15th 1:00:00 PM OR02 2257 11:30:00 AM Diabetes in Pregnancy Oral


Stephanie R Thorn*1, Sean A Newsom1, Rachel C Janssen1, Rebecca M Aikens1, Karalee C Baquero2, Diana Lynn Takahashi2, Kevin L. Grove3 and Jed Friedman1
1University of Colorado Anschutz Medical Campus, Aurora, CO, 2Oregon Health and Science University/ONPRC, Beaverton, OR, 3Oregon National Primate Research Center, Oregon Health & Science University, Beaverton, OR

 

Maternal obesity or high fat (HF) diet consumption produces adverse metabolic outcomes in the offspring.  To study the mechanisms involved, we have used a non-human primate (Japanese macaques) model where mothers are fed a HF diet beginning at 3 yr of age and continuing through their subsequent pregnancies.  Previously, we reported that maternal HF diet during pregnancy results in increased lipid accumulation, oxidative stress, and apoptosis in the fetal liver.   Here, we tested whether maternal resveratrol supplementation, a potent anti-oxidant, would ameliorate the harmful effects of maternal HF diet on the developing fetal liver.  HF diet mothers received resveratrol (HF+RESV, 0.37% diet) throughout pregnancy.  Fetuses were studied in early third trimester by c-section from maternal HF+RESV (n=7), HF (n=18), and CON (n=14) groups.  Liver TG content was 3-fold higher in HF compared to CON fetuses and was reduced in HF+RESV fetuses to concentrations similar to CON fetuses.  Hepatic citrate synthase activity, a marker of mitochondria number, was decreased by ~10% (P<0.05) in HF and HF+RESV (P=0.06) compared to CON fetuses and correlated with hepatic triglyceride content (r= -0.42, P<0.005).  Protein expression of SIRT3, a key regulator of mitochondria acetylation and function, was decreased in HF compared to CON and HF+RESV fetal livers and correlated with fetal liver TG accumulation (r= -0.47, P<0.01), citrate synthase activity (r= 0.61, P<0.005) and expression of the anti-oxidant MnSOD (r= -0.76, P<0.001).  Interestingly, protein expression of MnSOD and phosphorylation of EIF2α, JNK, and NFKB (markers of cell stress and inflammation) were increased in HF fetal liver and remained higher in HF+RESV compared to CON fetal livers.  SIRT1 protein expression, a target of resveratrol, was increased by ~15% in HF+RESV compared to CON fetal livers and circulating resveratrol concentrations were elevated in HF+RESV compared to CON maternal and fetal plasma (maternal: 0.499 ± 0.36 vs 0.131 ± 0.04 ng/ml; fetal: 0.749 ± 0.57 vs. 0.134 ± 0.02 ng/ml) indicative of placental transfer to the fetus.  These results indicate that maternal RESV supplementation during HF diet reduces fetal hepatic TG accumulation. The beneficial effects of RESV may be due to direct effects on the fetal liver or improved effects on maternal and placental metabolism.   Several stress related proteins remained activated in HF+RESV liver, suggesting that increased lipid supply or maternal or placental derived inflammatory signals during maternal HF diet exposure remain capable of activating these pathways in the fetal liver despite lower triglyceride content.  Consequently, maternal RESV supplementation may be an important intervention to improve fetal outcomes during maternal HF diet exposure.

 

Nothing to Disclose: SRT, SAN, RCJ, RMA, KCB, DLT, KLG, JF

OR02-4 9070 4.0000 A Maternal resveratrol supplementation reverses fetal hepatic lipid accumulation during maternal high fat diet exposure in non-human primate: Effect on mitochondrial activity and stress signals 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Diabetes & Glucose Metabolism Saturday, June 15th 1:00:00 PM OR02 2257 11:30:00 AM Diabetes in Pregnancy Oral


Annie Yang*1, Grace Xiong1, Hang Lee1, Catherine Kim2 and Rhonda Bentley-Lewis1
1Massachusetts General Hospital, Boston, MA, 2University of Michigan, Ann Arbor, MI

 

Background: Metabolomics is the science of systematically examining products of biochemical pathways in order to identify biomarkers predictive of metabolic diseases, such as type 2 diabetes. Metabolomics has been used to elucidate the response to an oral glucose tolerance test (OGTT) in normoglycemic and glucose intolerant subjects; however, this response has not been examined specifically in women with a history of GDM.

Objectives: To examine the metabolite response to an OGTT, i.e. change from fasting to 2-hour post-challenge, in women with a history of GDM and to determine if insulin resistance is associated with these changes.

Methods: We analyzed metabolomic profiles of 39 non-diabetic, non-pregnant women with a history of GDM within 3 years. Women provided information on family history of type 2 diabetes, race, parity, smoking, and breastfeeding. A 75 g-oral glucose load was given and fasting and 2-hr plasma samples were collected. Gas chromatography-mass spectrometry was employed to construct metabolite profiles on 23 amino acid (AA) or AA derivatives. Stepwise regression analyses (forward variable inclusion criteria p < 0.15) and correlations (p < 0.05) were performed to examine associations between metabolites and clinical measures.

Results: The women were 35 ± 4 years old (mean ± SD) and 69% white. They had mean fasting (90 ± 10 mg/dl) and 2-hr (125 ± 38 mg/dl) glucose levels and were insulin resistant as reflected by body mass index (BMI; 28.4 ± 5.6 kg/m2), glucose:insulin ratio (G/I, 5.5 ± 2.1) and homeostatic model assessment – insulin resistance (HOMA-IR; 6.08 ± 12.9). The levels of twenty AAs increased significantly in response to the glucose load (p < 0.0001) and G/I was most significantly associated with tyrosine levels (r = -0.43, p = 0.007).  Of the clinical parameters, breastfeeding, race, and G/I were associated with several AA levels (beta coefficients, b = 8.59 ± 1.0, -23.54 ± 2.4, and -3.43 ± 0.7 respectively, p values ranging < 0.002 to < 0.05), but the most significant association was between parity and change in cystine levels (b = 1.257, p = 0.0009).

Conclusions: Among insulin-resistant women with a recent history of GDM, breastfeeding, race, and G/I were most strongly associated with metabolite profile changes following the OGTT. Parity and cystine levels had the most significant association. Confirmation of these relationships and closer examination of the specific pathways implicated in these associations are warranted.

 

Nothing to Disclose: AY, GX, HL, CK, RB

OR02-5 7055 5.0000 A Metabolomic Response to an Oral Glucose Challenge in Women with a History of Gestational Diabetes Mellitus 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Diabetes & Glucose Metabolism Saturday, June 15th 1:00:00 PM OR02 2257 11:30:00 AM Diabetes in Pregnancy Oral


Melissa Kallas-Koeman*1, Jason M Kong1, Jennifer Klinke2, Sonia Butalia3, Abhay Lodha3, Kenneth Lim1, Qiuli Duan4 and Lois E Donovan3
1University of British Columbia, Vancouver, BC, Canada, 2University of British Columbia, New Westminster, BC, Canada, 3University of Calgary, Calgary, AB, Canada, 4Alberta Health Services, Calgary, AB, Canada

 

Background and objective: Pregnancy in women with type 1 diabetes is high risk, but it is unknown whether insulin pump therapy can reduce this risk. Our objective was to compare glycemic control and maternal-fetal outcomes between women with type 1 diabetes managed on the insulin pump versus multiple daily injections of insulin.

Methods: In a retrospective cohort study, we reviewed 387 consecutive pregnancies in women with type 1 diabetes who attended specialized clinics at three centres between 2006-2010. We assessed the average A1c per trimester, metabolic complications, gestational hypertension, weight gain, and cesarean section rate in the mother, and rate of infants large for gestational age (>90%ile), preterm delivery, neonatal hypoglycemia, special/intensive care nursery admission, jaundice requiring phototherapy, congenital anomalies, and perinatal mortality in the offspring.

Results: Women who used the insulin pump (129/387) were older (31.5±4.3 vs. 29.6±5.2 years, p<0.001) and had higher rates of preconception care (45.3 vs. 31.8%, p=0.009), a longer duration of diabetes (17.0±6.6 vs. 12.8±8.4 years, p<0.001), smoked less in pregnancy (5.3 vs. 21.4%, p<0.001), and had more retinopathy (17.1 vs. 8.5%, p=0.006). Among 113 completed pregnancies in women on pumps and 218 in women on multiple daily injections (including 1 and 2 stillbirths, respectively), there was a significant difference in glycemic control in the first trimester (mean A1c 6.9±0.7% vs. 7.6±1.4%, p<0.001) which persisted until the third trimester (mean A1c 6.5±0.5% vs. 6.8±0.9%, p=0.002). Despite tighter glycemic control, women on the pump did not have an increased rate of severe hypoglycemia (8.0 vs. 7.6%, p=0.90). Pump therapy was not associated with an increased risk of diabetic ketoacidosis (1.8 vs. 3.0%, p=0.72). Cesarean section rate was comparable at 69.0 vs. 64.2% (p=0.38), respectively, though women on the pump delivered significantly more large-for-gestational-age infants, at 55.1 vs. 39.2% (p=0.007). The remaining maternal-fetal outcomes were similar.

Conclusions: In this largest retrospective comparison of this population to date, women using the insulin pump in pregnancy had better glycemic control without an increased risk of severe hypoglycemia or diabetic ketoacidosis.

 

Nothing to Disclose: MK, JMK, JK, SB, AL, KL, QD, LED

OR02-6 7090 6.0000 A Insulin Pump Use in Pregnancy is Associated with Better Glycemic Control Without Increasing the Rate of Severe Hypoglycemia or Diabetic Ketoacidosis in Women with Type 1 Diabetes 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Diabetes & Glucose Metabolism Saturday, June 15th 1:00:00 PM OR02 2257 11:30:00 AM Diabetes in Pregnancy Oral


Horacio Novaira*1, Gloria E Hoffman2, Yongbum Koo3, Andrew Wolfe*1 and Sally Radovick4
1Johns Hopkins University School of Medicine, Baltimore, MD, 2Morgan State University, Baltimore, MD, 3Inje University, Gimhai, Korea, Republic of (South), 4Johns Hopkins School of Medicine, Baltimore, MD

 

Introduction: The Kiss-Gpr54 signaling pathway in the GnRH neuron is thought to be critical for both the onset of puberty as well as the attainment of normal reproductive function. Our working model includes the hypothesis that kiss neurons synapse directly on GnRH neurons and influence GnRH gene expression and release by directly activating Gpr54 on the membranes of GnRH neurons. However, Gpr54 is present in other hypothalamic cells that may synapse with GnRH neurons, and no animal models are available to test the hypothesis of direct kiss regulation on GnRH neurons. In addition, only complete Gpr54 KO animals have been generated, which do not experience normal pubertal development and are infertile. In this study, we directly test the hypothesis that kiss neurons regulate GnRH expression through synapses that release kiss and activate Gpr54 on the plasma membrane of GnRH neurons.

Aim: To define further GnRH neuronal signaling by kiss-Gpr54 and resulting physiological outcomes.

Methods and results: A GnRH neuron-specific Gpr54 knockout (GnRH-Gpr54KO) mouse model was generated and reproductive development and fertility was assessed.  Exon 2 of Gpr54 was surrounded by LoxP sites in a targeting construct that also included a Frt flanked neomycinr selection cassette. The targeting construct was electroporated into ES cells, and six positive targeted clones were obtained. The Gpr54 “floxed” animals were crossed to GnRH-Cre mice. Cre recombinase expression in GnRH neurons produced a cell-specific Gpr54 KO. A delay in pubertal onset in females was observed. The mean day of vaginal opening in wild-type littermates was 27 (±0.20) days versus 39 (±1.4) days in GnRH-Gpr54KO mice (n=5, P≤0.001). The first day in estrus and estrous cycles were monitored by vaginal lavages. Absence of the first day in estrus and estrous cyclicity were observed in the GnRH-Gpr54KO female mice. In addition, infertility was observed in both female and male GnRH-Gpr54KO mice.

Conclusion: Taken together, these data provide in vivo evidence that Gpr54 in GnRH neurons is critical for reproductive development and fertility. This work provides new insight into the physiological role of Gpr54 in mediating GnRH neuronal function and mammalian reproduction.

 

Disclosure: SR: Ad Hoc Consultant, CVS/Caremark, Speaker, Novo Nordisk. Nothing to Disclose: HN, GEH, YK, AW

OR04-1 8914 1.0000 A Reproductive abnormalities associated with deletion of Gpr54 in mouse GnRH neurons 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Neuroendocrinology and Pituitary Saturday, June 15th 1:00:00 PM OR04 2288 11:30:00 AM GnRH & Gonadotroph Biology & Signaling Oral


Sayeda Nasrin Alam*, Cadence True, Margaret Flynn Lippincott, Yee-Ming Chan and Stephanie Beth Seminara*
Massachusetts General Hospital, Boston, MA

 

Humans with mutations in the neurokinin B (NKB) signaling pathway (TAC3 and TACR3) have hypogonadotropic hypogonadism although these patients frequently demonstrate reversal of their GnRH-deficient state. Tacr3-/- mice exhibit normal timing of sexual maturation but have abnormal estrous cycling and subfertility. As Tac2-/- mice (neurokinin B deficient) have yet to be described, we sought to characterize the reproductive phenotype of these animals, and hypothesized that they would be less severely affected than their receptor counterparts.Targeted deletion of all exons of Tac2 was achieved by homologous recombination on a C57Bl/6/129Sv background strain by the Texas A&M Institute for Genomic Medicine. Mice were weighed and examined daily from P21-P36 for evidence of sexual maturation. Daily vaginal smears were done from day of vaginal opening to P90.

The Tac2 deficient state was confirmed by an absence of NKB immunoreactivity (Novus Biological, 1:1000) in the arcuate nucleus. Compared to controls, Tac2-/- females demonstrated delayed vaginal opening (P 25.1 ± 0.6 vs. P 27.4 ± 0.9; p<0.05) and time to first estrus (P 31.3 ± 1.3 vs. P 41.9 ± 2.7; p<0.01). In addition, Tac2-/- females had significantly longer cycle lengths (4.4 ± 0.2 days vs. 12.4 ± 0.9 days; p<0.001). Despite the abnormalities in estrous cyclicity, all Tac2-/- females achieved pregnancy when placed in a cage with WT or Tac2-/- males. Although the time to preputial separation, anogenital distance and testicular weight were all suggestive of an abnormal reproductive phenotype in  Tac2-/- males, these differences were not statistically different from littermate controls.   

 Tac2-/- females have delayed time to vaginal opening, delayed first estrous, and abnormal cycling compared to WT littermates, but seemingly robust fertility. Whereas both Tac2-/- and Tacr3-/- mice have abnormal estrus cycling, only Tac2-/- mice have significant delays in sexual maturation.  Though subtle background differences between different B6/129 hybrid lines cannot be excluded, this unexpected difference in the timing of sexual maturation between Tac2-/- and Tacr3-/- female animals suggests that neurokinin B may signal through multiple tachykinin receptors to modulate the hypothalamic-pituitary-gonadal axis. Tac2-/- mice have parallels to humans with reversible hypogonadotropism, with defects in sexual maturation but recovery of reproductive function and fertility in adulthood.

 

Nothing to Disclose: SNA, CT, MFL, YMC, SBS

OR04-2 8975 2.0000 A Delayed sexual maturation in mice lacking neurokinin B mirrors the phenotype of human patients with mutations in the neurokinin B pathway 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Neuroendocrinology and Pituitary Saturday, June 15th 1:00:00 PM OR04 2288 11:30:00 AM GnRH & Gonadotroph Biology & Signaling Oral


Robert L. Goodman*, Katrina L Porter, John M Connors and Stan M Hileman
West Virginia University School of Medicine, Morgantown, WV

 

Neurokinin B (NKB) is critical for reproductive function in humans, and agonists to the NKB-specific receptor, NK3R, can stimulate LH secretion in several species. Indirect evidence supports the proposal that NKB acts via arcuate nucleus (ARC) kisspeptin neurons to stimulate LH secretion, but most of this work has been done with the NK3R agonist, senktide.  This study had two aims.  First, we directly tested if NKB acts in the ARC, and compared the effects of NKB and senktide on episodic LH secretion in ovariectomized (OVX) ewes. Second, we tested whether afferent input to the ARC from neurons containing orphanin-FQ (OFQ), an inhibitory opioid, controls LH pulses using an antagonist to the OFQ receptor (UFP-101). Ewes (n=6) were OVX and bilateral chronic guide tubes were stereotaxically implanted to target the dorsal edge of the ARC. Starting two weeks later, crystalline drugs, tamped into the lumen of 22 gauge tubing, were administered via the guide tubes. Blood samples were collected for LH measurement every 10 min from 3 h before to 4h  after insertion of tubing that was either empty (controls) or contained NKB, senktide, or UFP-101.  At the end of sampling, microimplants were removed and this protocol was repeated three more times, with four days between replicates, until all ewes received all four treatments in a randomized order.  Histological analysis of microimplantation sites indicated successful targeting of the ARC in 5 of 6 ewes.  In these 5 ewes, clear LH pulses were evident before and during NKB treatment, and NKB significantly decreased interpulse interval (IPI) from 54 ± 2 min (pre) to 42 ± 3 min (during treatment).  In contrast, no change in pulse frequency occurred with control (IPI pre: 55 ± 6; during: 56 ± 5 min) or with UFP-101 (IPI pre: 54 ± 5; during: 55 ± 4 min) treatment. Senktide produced a prolonged increase in LH concentrations lasting 3.5 ± 0.4 hrs, during which discrete LH pulses were difficult to detect. These results demonstrate that NKB can act in the ARC of OVX ewes to increase LH pulse frequency.  Because UFP-101 had no effect, it is unlikely that OFQ plays an important role within the ARC to control pulsatile LH secretion. Local administration of senktide to the ARC clearly produced a different pattern of LH release than NKB.  Whether this pattern reflects continuous GnRH release or very high frequency pulsatile secretion remains to be determined.

 

Nothing to Disclose: RLG, KLP, JMC, SMH

OR04-3 7279 3.0000 A NEUROKININ B (NKB) AND THE NKB RECEPTOR AGONIST, SENKTIDE, ACT IN THE OVINE ARCUATE NUCLEUS TO PRODUCE DIFFERENT PATTERNS OF LH RELEASE 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Neuroendocrinology and Pituitary Saturday, June 15th 1:00:00 PM OR04 2288 11:30:00 AM GnRH & Gonadotroph Biology & Signaling Oral


Tyler CarlLee, Michael Cozart, Mohsin M Syed, Noor Akhter, Anessa C. Haney, Angela K. Odle, Melody Lyn Allensworth and Gwen V. Childs*
University of Arkansas for Medical Sciences, Little Rock, AR

 

Leptin regulates appetite and metabolism and is permissive for the onset of puberty and reproduction. Defining a role for leptin in the regulation of pituitary gonadotropes has been challenging (1,2).  To determine its significance to gonadotropes, we crossed mice bearing LEPRexon17 loxP/loxP with mice bearing Cre-recombinase (Cre) driven by the LHβ promoter (Cre-LHβ) (3), thereby ablating LEPR signaling selectively in gonadotropes. Organ genotyping proved no extra-pituitary expression in females. Mutant males showed extra-pituitary Cre-expression in the testes, because LH is expressed in that organ (4-6). This caused global deletion of LEPR in a subset of progeny from mutant males, resulting in a db/db-like phenotype. Therefore, experimental animals derived only from Cre-LHβ bearing females were used in this study. Serum LH and GH were lower in 19 deletion mutant males (ng/ml--0.9±0.2 LH; 3.5±1 GH) compared with 26 littermate controls (ng/ml--2.2±0.5 LH; 11.2±2.8 GH) p<0.01; with no differences in follicle stimulating hormone (FSH).  In vitro studies showed that, after 1 h exposure to 0-30 nM GnRH, cultures from mutants secreted 63% less LH and 55% less FSH basally than controls and showed a blunted response to 0.1-1 nM GnRH with 30-64% less LH and 27% less FSH. Mutant and control cultures secreted similar levels of LH and FSH in response to 10-30 nM GnRH.  Breeding studies detected the impact of gonadotrope-specific loss of LEPR on reproduction. The timing of puberty, first litter, or time between litters was normal in male and female mutants. Litters (n=9) from mutant (Cre-LHβ+) males and Cre-negative females averaged 8.8±0.7 pups/ litter, which was not different from the average seen in 6 litters from the same-strain control animals (FVB.129S--9.3±0.7 pups/litter).  In contrast, 9 litters from mutant Cre-LHβ+ females and Cre negative males averaged only 5.5 ±1.4 pups/ litters, which was significantly lower than those from the mutant males or the control strain (p<0.03).  No further reduction was seen in 9 litters from two mutant parents (6.2±1 pups/litter). These studies suggest that leptin’s signaling to pituitary gonadotropes helps maintain serum levels of LH and GH, in vivo. No obvious reproductive or metabolic phenotype was seen in mutant males, (which expressed Cre only in the pituitary and testes). However, mutant females, (which expressed Cre only in the pituitary) clearly show a 40% reduction in number of pups/litter. The impact of lack of leptin signaling is also seen dramatically in vitro by the relatively low basal gonadotropin secretion and blunted responses to physiological doses of GnRH. Perhaps, when the timing of development and metabolic conditions are right, leptin signaling optimizes trafficking or storage of gonadotropins to facilitate responses to physiologically relevant pulses of GnRH and insure reproductive success.

 

Nothing to Disclose: TC, MC, MMS, NA, ACH, AKO, MLA, GVC

OR04-4 9236 4.0000 A The Gonadotrope as a Metabolic Sensor: Ablation of Leptin Receptor (LEPR) Signaling in Gonadotropes Blunts Responses to Gonadotropin Releasing Hormone (GnRH), Reducing Serum Luteinizing hormone (LH), Growth hormone (GH) and Litter Size 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Neuroendocrinology and Pituitary Saturday, June 15th 1:00:00 PM OR04 2288 11:30:00 AM GnRH & Gonadotroph Biology & Signaling Oral


Joelle Cohen-Tannoudji*, Violaine Simon, Chantal Denoyelle, Muhammad Ishaq and Ghislaine Garrel
University Paris 7, France

 

We have recently shown using a rat model of central lipid overload combined with in vitro studies on gonadotrope cells that unsaturated fatty acids (UFA) directly act on rodent pituitary to stimulate Lhb subunit gene expression and LH secretion, providing the first demonstration of a pituitary “lipid sensing” (1). Unexpectedly, we report here that UFA regulate FSH expression in an opposite manner. Indeed, kinetics studies using linoleate in primary cultures of rat pituitary cells revealed that the Lhb transcript increase was concomitant with a dramatic decrease of Fshb transcript. A 4 h-treatment with 200 microM linoleate dose-dependently reduced Fshb transcript levels in LbetaT2 gonadotrope cell line and rat pituitary cell cultures, with a maximum of 60 to 80% inhibition depending on the cell model. In contrast, the saturated fatty acid palmitate was ineffective. Because activin and Bone-Morphogenetic Protein 2 (BMP2) are major regulators of Fshb gene expression in gonadotrope cells, we wondered whether linoleate could interfere with the signaling of these hormones. Interestingly, in both cellular models, linoleate counteracted by about 50% the strong stimulatory effect of activin and BMP2 on Fshb gene expression. Analysis of the underlying mechanisms in LbetaT2 cells revealed that linoleate treatment decreased Smad recruitment by activin and BMP2. This was evidenced by a reduced phosphorylation of Smad2/3 and Smad1/5/8 in response to activin and BMP2, respectively. Linoleate also impaired basal phosphorylation level of Smad2/3. Altogether, this suggests that linoleate regulate Smad-specific phosphatases in gonadotrope cells. We also demonstrated that linoleate significantly increased (~50%) follistatin transcript levels, suggesting that neutralization of activin and BMP2 by an excess of follistatin contributes to reduced Fshb gene expression. This works highlights for the first time, the ability of UFA to differentially regulate the expression of the two gonadotropin beta-subunit genes. This suggests that UFA may disturb reproductive function by altering the balance between gonadotropin hormones. Altogether, our study underlines the relevant role of pituitary in the sensing of nutritional information and especially nutrients.

 

Nothing to Disclose: JC, VS, CD, MI, GG

OR04-5 8314 5.0000 A Unsaturated fatty acids strongly inhibit Fshb gene expression by counteracting Activin and BMP2 signaling in rat pituitary and LβT2 gonadotrope cell line 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Neuroendocrinology and Pituitary Saturday, June 15th 1:00:00 PM OR04 2288 11:30:00 AM GnRH & Gonadotroph Biology & Signaling Oral


Jerome Fortin*1, Ulrich Boehm2, Chuxia Deng3 and Daniel J. Bernard1
1McGill University, Montreal, QC, Canada, 2University of Saarland School of Medicine, Homburg, Germany, 3NIH, Bethesda, MD

 

In mammals, follicle-stimulating hormone (FSH), a dimeric protein produced by pituitary gonadotrope cells, is required for proper reproductive function. Activins, members of the TGFβ family, are potent and selective regulators of FSH synthesis. Thus far, experiments in cell lines have shown that activins stimulate the transcription of the FSH β (FSHβ/Fshb) subunit via the effector proteins, SMAD2 and SMAD3, downstream of heteromeric type I/type II receptor complexes. Receptor-regulated SMAD2/3 partner with the obligatory co-SMAD, SMAD4, and bind to the Fshb promoter in cooperation with other transcription factors, such as forkhead box L2 (FOXL2), to activate Fshb transcription. We recently made the unexpected observation that mice lacking the full-length forms of both SMAD2 and SMAD3 in gonadotropes have normal FSH levels and fertility. To test whether any SMAD-dependent signaling is required for FSH synthesis, we crossed mice carrying floxed alleles of Smad4 (Smad4fl/fl) with GnRHR-IRES-Cre (GRIC) mice, which express Cre recombinase exclusively in gonadotropes, to generate Smad4-conditional knockout mice (hereafter S4cKO). S4cKO males had reduced testes weights and sperm production, whereas females had smaller ovaries and reduced fertility compared with control littermates. The subfertility in females was secondary to impaired ovarian follicle maturation beyond the pre-antral stage but not due to intrinsic ovarian dysfunction, as S4cKO animals ovulated normally in response to exogenous gonadotropins. Hypogonadism in S4cKO mice was largely explained by FSH deficiency in both males and females. The phenotype was particularly striking in S4cKO males, where serum FSH and pituitary Fshb mRNA levels were reduced by 90%. In addition, S4cKO males had elevated pituitary expression of the GnRH receptor (Gnrhr) and reduced expression of the gonadotropin α subunit (Cga). Pituitary activin receptor (Acvr1b, Acvr2), luteinizing hormone β subunit (Lhb), and Foxl2 mRNA levels were normal. Cultured pituitary cells from S4cKO mice showed dramatically lower basal and activin-stimulated Fshb transcription compared with cells from control mice. A similar, but milder effect was observed upon ex vivo (acute) deletion of Smad4 in pituitary cells from Smad4fl/fl mice. Together, these results demonstrate that SMAD signaling in gonadotropes is necessary for Fshb transcription, FSH synthesis, and fertility in vivo. The receptor-regulated SMAD(s) in this system remain to be identified.

 

Nothing to Disclose: JF, UB, CD, DJB

OR04-6 6458 6.0000 A SMAD signaling in gonadotropes is critical for normal FSH synthesis and fertility in mice 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Neuroendocrinology and Pituitary Saturday, June 15th 1:00:00 PM OR04 2288 11:30:00 AM GnRH & Gonadotroph Biology & Signaling Oral


Rui LI*1, Minho Chae2, Miao Sun2, Shino Murakami1 and W Lee Kraus2
1University of Texas Southwestern Medical Center, Dallas, TX, 2UT Southwestern Medical Center, Dallas, TX

 

Identification, Regulation, and Function of Antisense Transcription in the Estrogen Response in Breast Cancer Cells

Rui Li, Minho Chae, Miao Sun, Shino Murakami, and W. Lee Kraus

1 Signaling and Gene Regulation Laboratory, Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, TX 75390.

2 Division of Basic Research, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, TX, 75390.

            Estrogen signaling plays key roles in a wide array of physiological processes and disease states.  Many of these effects are through the gene regulatory actions of estrogen, which acts through estrogen receptor protein to induce genome-wide alterations in the expression of protein-coding and non-coding genes.  We are using a variety of gene-specific and genomic approaches in MCF-7 human breast cancer cells to examine the effects of estrogen signaling on the production of antisense transcripts.  We have used Global Run-On and sequencing (GRO-seq), a genomic approach that maps the location and direction of all active RNA polymerases, combined with de novo transcript calling, to identify about ~1,200 antisense transcription units.  We have also used RNA-seq to detect the steady-state RNAs that are produced from these transcription units, as well as their exon/intron structure.  Little is known about antisense transcription in mammalian cells and the mechanisms by which it may affect sense gene expression or biological processes are poorly understood. Our results indicate that sense and antisense transcription are largely co-regulated in similar direction and magnitude upon estrogen treatment. From gene specific studies, we have found two novel antisense RNAs originating from the MYC locus, which may be implicated in breast cancer biology. We are characterizing these antisense RNAs by cloning and ASO-mediated knockdown.  We are also examining their potential biological roles in human breast cancer cells.  These studies will shed new light on the structure, function, and regulation of antisense transcripts.

 

Nothing to Disclose: RL, MC, MS, SM, WLK

OR06-1 7481 1.0000 A Identification, Regulation, and Function of Antisense Transcription in the Estrogen Response in Breast Cancer Cells 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Nuclear Receptors and Steroid Hormone Action Saturday, June 15th 1:00:00 PM OR06 2302 11:30:00 AM Molecular Mechanisms of Estrogen Action and Androgen Synthesis Oral


Matti Poutanen*, Paivi Jarvensivu, Taija Saloniemi and Niina Saarinen
University of Turku, Turku, Finland

 

Hydroxysteroid (17beta) dehydrogenase 1 (HSD17B1) converts low active estrogen, estrone, to highly active estradiol with high catalytic efficiency. Altered HSD17B1 expression has been associated with several hormone dependent diseases, including endometriosis, ovarian epithelial cancer, and breast cancer. We have developed a transgenic mouse line over-expressing human HSD17B1 (HSD17B1TG mice). These mice have increased HSD17B1 activity in multiple tissues including mammary gland. HSD17B1TG females show extensive lobuloalveolar development at the age of 4 months forward that resembles the structure present in mammary gland of pregnant  mice. Serum prolactin levels were found to be higher in HSD17B1TG female mice than that measured in virgin wild type (WT) mice.  HSD17B1TG mice also develop hyperplastic alveolar nodules (HANs), and later on, part of the mice develop mammary cancers. Accordingly, HANs along with inflammatory cell infiltration and increased mammary epithelial cell proliferation is observed in mammary glands of WT female mice transplanted with HSD17B1TG mammary epithelium. However, there was no difference in epithelial cell estrogen receptor alpha or estrogen receptor beta expression between the TG and WT mammary glands transplanted to a WT females. These data indicate the involvement of increased mammary epithelial HSD17B1 expression in the development of hyperplastic mammary lesions, putative precursors of mammary cancer.

 

Nothing to Disclose: MP, PJ, TS, NS

OR06-2 6862 2.0000 A Hydroxysteroid (17beta) dehydrogenase 1 Expression Leads to Inflammation Assisted Epithelial Cell Proliferation in Mouse Mammary Gland Tissue 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Nuclear Receptors and Steroid Hormone Action Saturday, June 15th 1:00:00 PM OR06 2302 11:30:00 AM Molecular Mechanisms of Estrogen Action and Androgen Synthesis Oral


Mathew M Cherian* and David J Shapiro
University of Illinois, Urbana, IL

 

It is widely assumed that estrogen receptor α (ERα) is progressively activated as it is saturated with estrogens at concentrations approaching its KD of approximately 1 nM. However, clinical data from postmenopausal women indicates that the circulating estrogen concentration is often below 1 picomolar (pM) and the risk of developing breast cancer increases significantly with serum estrogen concentrations in the range of 1-10 picomolar. We propose an alternative model for ERα action in which only a tiny fraction of the ERα pool needs to be bound to 17β-estradiol (E2) in order to elicit key biological effects. After rigorously excluding estrogen from the medium, proliferation of ERα containing MCF-7 and T47D breast cancer cells and BG-1 ovarian cancer cells is completely dependent upon addition of E2. Under these conditions, very low 5-20 picomolar E2, likely to occupy only a tiny fraction of the available ERα, is sufficient to elicit near-maximal proliferation of all 3 cell lines. These low concentrations of E2 also induce anchorage-independent cell growth and colony formation in soft agar. The ERα antagonists TPSF and ICI 182,780 block induction of cell proliferation by pM E2, indicating that E2 induction of cell proliferation is mediated through ERα. While pM E2 induces some ERα target genes, such as pS2 and progesterone receptor (PR), other E2-ERα regulated genes require nanomolar E2 for induction. The wide variation in the concentration of E2 required to regulate expression of different ERα regulated genes provides a novel approach to identifying E2-ERα regulated genes critical for cell proliferation. Fos mRNA provides an example as it is robustly induced by 10 nM E2, but is not induced at the picomolar E2 concentrations that maximally stimulate cell proliferation. We suggest that key actions of ERα do not result from progressive saturation of ERα with estrogens, but rather require achieving a threshold level of liganded ERα that often represents a minute fraction of the total receptor population.  These observations alter our understanding of the E2-ERα-regulated transcriptome and of the effects of endogenous estrogens and xenoestrogens in cancer.

 

Nothing to Disclose: MMC, DJS

OR06-3 7656 3.0000 A An Alternative Model for Estrogen Receptor α Action: Minimal Occupancy of ERα by Picomolar Concentrations of 17 β-Estradiol Stimulates Gene Transcription and Cell Proliferation 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Nuclear Receptors and Steroid Hormone Action Saturday, June 15th 1:00:00 PM OR06 2302 11:30:00 AM Molecular Mechanisms of Estrogen Action and Androgen Synthesis Oral


Erin Scott*1, Quanguang Zhang1, Ruimin Wang2, Gangadhara Reddy Sareddy3, Ratna Vadlamudi3 and Darrell Wayne Brann1
1Georgia Regents University, Augusta, GA, 2Hebei United University, Tangshan, China, 3UTHSCSA, San Antonio, TX

 

Women who enter menopause prematurely have a doubled lifetime risk of cognitive decline and dementia. The mechanism underlying this phenomenon is unclear, but dramatic and prolonged deprivation of the neuroprotective ovarian hormone 17beta-estradiol (E2) is thought to play a key role. To further investigate this issue, we utilized short-term and long-term E2 deprivation models in which 3-month-old female Sprague Dawley rats were bilaterally ovariectomized and treated with vehicle or a low, physiological dose of E2 either immediately (short-term E2 deprivation - STED) or 10 weeks later (long-term E2 deprivation - LTED). One week after initiation of vehicle or E2 treatment, animals were subjected to 10 minutes of global cerebral ischemia (GCI) and sacrificed following reperfusion. The results revealed that LTED females had dramatically increased hippocampal expression of the Alzheimer’s disease (AD)-related proteins beta-amyloid, beta-amyloid cleaving enzyme 1 (BACE1), and hyperphosphorylated tau following GCI. We hypothesized that the increase in AD-related proteins may be mediated by activation of c-Jun N-terminal kinase (JNK), since JNK activation can lead to enhanced BACE1 expression and hyperphosphorylation of tau and since we previously showed that E2 can markedly attenuate JNK activation in STED, but not LTED, rats. In support of a role for JNK in post-ischemic AD-protein induction and cell death, our studies demonstrated that administration of a JNK inhibitor 15 minutes prior to GCI ameliorated hippocampal AD protein induction and exerted strong neuroprotection in LTED animals. Intriguingly, further work using mass spectrometry analysis and co-immunoprecipitation identified JNK and its upstream regulator, Mitogen-Activated Protein Kinase Kinase 7 (MKK7), as potential interacting proteins of the novel estrogen receptor co-regulator, Proline-, Glutamic Acid-, and Leucine- Rich Protein 1 (PELP1), which has been implicated as a key mediator of E2’s genomic and nongenomic signaling. Along these lines, PELP1 interaction with JNK and MKK7 appears functionally important, as in vivo knockdown of hippocampal PELP1 reversed E2’s attenuation of post-ischemic JNK activation and reversed E2 neuroprotection. Taken as a whole, these studies shed new light on the mechanisms of E2 signaling and neuroprotection and provide important insight into the detrimental effects of LTED on the brain.

 

Nothing to Disclose: ES, QZ, RW, GRS, RV, DWB

OR06-4 4969 4.0000 A Long-Term Estrogen Deprivation Enhances JNK-Mediated AD Protein Induction after Global Cerebral Ischemia: Potential Role for PELP1 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Nuclear Receptors and Steroid Hormone Action Saturday, June 15th 1:00:00 PM OR06 2302 11:30:00 AM Molecular Mechanisms of Estrogen Action and Androgen Synthesis Oral


Ali A Pedram*1, Mahnaz Razandi2, Kenneth Steven Korach3, Ramesh Narayanan4, James T Dalton5 and Ellis R Levin6
1Univ of CA-Irvine, Long Beach, CA, 2Long Beach VAMC, Long Beach, CA, 3NIH-NIEHS, Research Triangle Pk, NC, 4GTX Inc, Memphis, TN, 5GTx, Inc, Memphis, TN, 6University of California-Irvine/Long Beach VAMC, Long Beach, CA

 

Hypertension in humans induces cardiac hypertrophy that can progress to heart failure as a common cause of death. We and others have shown that estrogen acting through ERβ prevents cardiac hypertrophy as induced by several agents in mice or in genetic rat models. However, thinking translationally, estrogen promotes breast and uterine proliferation and cancer from engaging ERα. We therefore evaluated whether a non-steroidal, ERβ agonist (β-LGND) could prevent cardiovascular pathology induced by the administration of angiotensin II (AngII), the most common cause of these disorders in humans. Angiotensin II was administered by osmotic minipump, placed under the skin of ovariectomized wild type (WT) and ERβKO mice, at a rate of 0.7mg/kd/day in 10ml of saline for 3 weeks (n=6 for each group). In some mice, AngII + β-LGND (0.5mg) or β-LGND alone was infused for comparison. As additional controls, Ang II or an equivalent volume of saline was infused in the WT and ERβKO mice that each received an estradiol (E2) pellet placed under the skin, producing physiological blood concentrations of the steroid. AngII produced a strong hypertensive response in all mice over the 3 week period, where blood pressure rose on average to 153/94, compared to 121/83 from saline infusion (control).Either E2 or β-LGND significantly and comparably reduced blood pressure to a mean of 130/86 in the WT mice co-infused with AngII (*p<0.05 by ANOVA + Scheffe’s test and all SDs were less than 10%). In contrast, neither estrogenic compound prevented AngII-induced hypertension in ERβKO mice. β-LGND and E2 each caused a ~65% reduction in the size and weight of the left ventricle of WT mice infused with AngII, but again not seen in ERβKO mice. Parameters of cardiac hypertrophy were comparably stimulated by AngII in both WT and ERbKO mice left ventricles including reversal of the myosin heavy chain α/β proteins ratio, brain natriuretic protein production, and ERK activation, the latter an important signal for hypertrophy. For each aspect, β-LGND or E2 induced a comparable and significant reversal of the AngII effect, but only in WT mice. β-LGND and E2 also stimulated the gene and protein for MCIP1, a protein that blocks calcineurin function that is essential to hypertrophy, seen only in the ventricles from WT mice. AngII induced a 6-fold increase in cardiac fibrosis that was ~93% inhibited by β-LGND or E2 only in WT mice. This correlated to β-LGND or E2 inhibition in WT mice of AngII- stimulated collagen I deposition in the ventricles. Overall, a new ERβ-selective agonist shows great promise to inhibit both the underlying vascular impetus (hypertension) as well as the direct cardiac effects of AngII that promote cardiac hypertrophy/fibrosis thus justifying consideration for clinical trial.

 

Disclosure: RN: Researcher, GTx. JTD: Chief Scientific Officer, GTx, Inc., Chief Scientific Officer, GTx, Inc.. Nothing to Disclose: AAP, MR, KSK, ERL

OR06-5 3374 5.0000 A An ERß Selective Agonist Inhibits Angiotensin-Induced Cardiovascular Pathology 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Nuclear Receptors and Steroid Hormone Action Saturday, June 15th 1:00:00 PM OR06 2302 11:30:00 AM Molecular Mechanisms of Estrogen Action and Androgen Synthesis Oral


Varun Sondhi*1, Jiayan Liu2, Susan Matthew2, David J Mangelsdorf3 and Richard J. Auchus4
1UT Southwestern Medical Center, 2University of Michigan, 3U.T. Southwestern Medical Center, Dallas, TX, 4University of Michigan, Ann Arbor, MI

 

Cytochrome P450c17 (CYP17A1, steroid 17α-hydroxylase/17,20-lyase) is the sole enzyme capable of converting 21-carbon progestins to 19-carbon androgens. The 17,20-lyase activity is more vulnerable to mutations than the 17-hydroxylase activity and requires cytochrome b5 (CYB5A) for maximal activity. The physiologic importance of CYB5A in human physiology has been validated by the description of two kindred with 46,XY DSD due to CYB5A deficiency, in which 17,20-lyase activity is selectively impaired, leading to testosterone but not cortisol deficiency. To study the biochemical consequences of CYB5A deficiency in an intact animal and in steroidogenic tissue, we generated mice lacking Cyb5a in the Leydig cell (LCb5KO), crossing Cyb5a(lox/lox) and Cre(+/Sf1) animals. The LCb5KO animals were born in a normal Mendelian ratio. We examined the male mice and observed normal fertility with no overt phenotype. Testicular histology revealed no differences between LCb5KO and WT animals. Homogenates from LCb5KO testes had normal progesterone (P)-to-17α-hydroxyprogesterone (17OHP) conversion but low 17OHP-to-androstenedione (A) and testosterone (T) metabolism. The ratio of the hydroxylase to lyase activity was observed to be 1.7 in the WT and 4.5 (3-fold higher) in the LCb5KO testes due to deficient lyase activity in the knockout animals. Addition of recombinant CYB5A to in vitro assays had no effect on the WT lyase activity or the hydroxylase/lyase ratio but restored the LCb5KO lyase activity and ratio to the level of WT animals. We examined the effects of Cyb5a knockout on steroid production using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Exogenous hCG administration gave a large increase in serum steroids for both the LCb5KO and WT animals. In the LCb5KO animals, this rise was accompanied by the accumulation of 17OHP in serum, which led to a 17OHP/(A+T) ratio of 8.3%, compared to 0.19% in the WT animals, a 44-fold increase. These data demonstrate the physiological significance of Cyb5a in the Cyp17a1 lyase reaction. We demonstrate that the decrease in Cyp17a1 lyase activity in LCB5KO animals can be rescued by the addition of exogenous CYB5A. The residual Cyp17a1 lyase activity in LCB5KO mice, unlike human beings, is sufficient to maintain androgens at a level that leads to normal reproductive phenotype. Importantly, we found that 17OHP is undetectable by LC-MS/MS in WT mice but accumulates in the LCb5KOs upon hCG stimulation.

 

Nothing to Disclose: VS, JL, SM, DJM, RJA

OR06-6 5220 6.0000 A Impaired 17,20-lyase activity in mice lacking cytochrome b5 in testicular Leydig cells 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Nuclear Receptors and Steroid Hormone Action Saturday, June 15th 1:00:00 PM OR06 2302 11:30:00 AM Molecular Mechanisms of Estrogen Action and Androgen Synthesis Oral


Erin C Hanlon*1, Kara Stuhr2, Rachel Leproult1, Esra Tasali3, Harriet de Wit1, Cecilia J Hillard2 and Eve Van Cauter1
1University of Chicago, Chicago, IL, 2Medical College of Wisconsin, Milwaukee, WI, 3The University of Chicago, Chicago, IL

 

Evidence from laboratory and epidemiologic studies suggest that insufficient sleep duration may be a contributing factor to the epidemic of obesity. The EC system, a pharmacotherapeutic target for obesity treatment, mediates the hedonic control of feeding, and modulates reward mechanisms. Stimulation of the EC system could be involved in the risk in overeating associated with sleep loss. The roles of sleep and circadian rhythmicity in modulating circulating EC lipids are not known as previous studies have only assessed single time points or narrow time intervals. We examined the 24-h profile of (2-AG), and its structural analog 2-oleoylglycerol (2-OG), which does not bind cannabinoid receptors, in healthy subjects and determined the impact of recurrent partial sleep restriction.

In a randomized cross over design, nine healthy subjects (age:23 ± 1yrs; BMI:23.6±0.7kg/m2) were studied in the laboratory with controlled energy expenditure and caloric consumption.  Blood sampling was performed for 24-h following two nights of normal sleep (2300-0730) or partial sleep restriction (0100-0530).  Samples taken at 60min intervals were assayed with liquid chromatography electrospray ionization-mass spectrometry (LC-ES-MS), for detection of 2-AG and 2-OG.

Both 2-AG and 2-OG display clear circadian rhythms with a nadir around mid sleep and peak levels in the early afternoon. Following sleep restriction, the amplitude of the rhythm is significantly increased for both lipids (2-AG: 151 ±24 vs 126 ±24pmol/ml, p = 0.01; 2-OG 894±120 vs 709±85pmol/ml, p = 0.005), due to an increase in peak levels (2-AG: 386 ±60 vs 335 ±61 pmol/ml, p = 0.008; 2-OG: 3329±284 pmol/ml vs 3138 ±255 pmol/ml, p = 0.08). Mean 24-h levels of 2-AG and 2-OG were highly correlated during the normal sleep condition (r = 0.80, p < 0.05) and during the restriction condition (r = 0.84, p < 0.05). Moreover, despite the two conditions being separated by at least one month, mean 24-h levels were highly correlated between the normal and restricted sleep sessions for both 2-AG (r = 0.98,  p < 0.001) and 2-OG (r = 0.83, p = 0.005), indicating within-subject reproducibility.

This study provides the first demonstration of a robust circadian rhythm of human plasma EC levels and reveals that sleep restriction results in increased amplitude of the mid-sleep to early afternoon rise of both 2-AG and 2-OG.  Elevation of peak daytime levels of EC may contribute to the risk of overeating associated with sleep deprivation.

 

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

OR09-1 7333 1.0000 A Circadian rhythm of circulating endocannabinoid (EC), 2-arachidonoylglycerol (2-AG), concentrations following normal and restricted sleep 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Obesity, Adipocyte Biology and Appetite Saturday, June 15th 1:00:00 PM OR09 2306 11:30:00 AM Obesity: Physiologic Responses to Energy Balance Disruption Oral


Elizabeth Anne Thomas*, Jamie L Bechtell, Janine Higgins and Marc-Andre Cornier
University of Colorado Denver, Aurora, CO

 

Breakfast skipping has been associated with higher BMI and metabolic derangements.  However, studies showing such associations have been either population studies or small studies done primarily in lean individuals. This randomized, cross-over trial was designed to investigate the metabolic responses to skipping breakfast in overweight and obese women. 9 women (29.2±2 years, BMI 31.4±2.7 kg/m²) were studied on 2 separate days approximately 1 month apart.  During the study visits they either ate breakfast (B), which contained 25% of daily energy intake (EI) or had no breakfast (NB), then consumed a standard lunch meal 4 hours later, which contained 35% of daily EI. Blood sampling for insulin, glucose, free fatty acids (FFA) and triglycerides (TG) was performed every 30 minutes for 3 hours following the lunch meal. Although pre-lunch insulin did not differ between conditions, the insulin total area under the curve (AUC) was higher in the NB compared to B condition (12322±3093 vs 8882±2803 µIU/mLx180 min, p=0.001). Similarly, pre-lunch glucose did not differ by condition, but glucose total AUC was higher in NB compared to B (20775±1194 vs 18126±1123 mg/dLx180 min, p=0.004). Pre-lunch FFA were higher in NB compared to B (705±121 vs 287±180 µEq/L, p=0.0002).The total AUC for FFA was higher in the NB compared to B condition (33980±8026 vs 25692±6503 µEq/Lx180 min, p=0.026). The incremental AUC for FFA was also greater in the NB compared to B condition (-92980±16973 vs -26008±27764 µEq/Lx180 min, p<0.001), suggesting it was not the pre-lunch FFA driving the increased AUC. Pre-lunch TG were lower in NB compared to B (85.7±37.8 vs 121.4±39.7 mg/dL, p=0.00954). TG total AUC was lower in the NB compared to B condition (17352±8484 vs. 24060±7147 mg/dLx180 min, p=0.0293), but the incremental AUC did not differ by condition, suggesting the difference was driven by the pre-lunch TG levels. The results of this study indicate that, in obese women, skipping breakfast results in acute relative insulin resistance, as indicated by the increased insulin AUC in NB. Additionally, the increased FFA AUC in NB suggests insulin resistance at the level of adipose tissue, as lipolysis is not appropriately down-regulated despite the higher insulin levels. In summary, these results indicate that skipping breakfast may contribute to the development of insulin resistance, which may predispose to further metabolic derangements and possibly progression to type 2 diabetes mellitus.

 

Nothing to Disclose: EAT, JLB, JH, MAC

OR09-2 8750 2.0000 A Metabolic Effects of Skipping Breakfast in Obese Women 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Obesity, Adipocyte Biology and Appetite Saturday, June 15th 1:00:00 PM OR09 2306 11:30:00 AM Obesity: Physiologic Responses to Energy Balance Disruption Oral


Natacha Germain1, Bogdan Galusca1, Dominique Caron-Dorval1, Dominique Grouselle2, Estelle Pujos-Guillot3, Jean-Francois Martin3, Yves Boirie3, James S Minnion4, Stephen R Bloom5, Jacques P Epelbaum6 and Andre Bruno Estour*1
1University Hospital, Saint-Etienne, France, 2UMR894 INSERM, Paris, France, 3INRA, Clermont-Ferrand, France, 4Imperial College London, London, United Kingdom, 5Imperial College London, United Kingdom, 6INSERM UMR-S 894, Université Paris Descartes, Paris, France

 

Context:Constitutional thinness (CT) is a rare condition of natural low bodyweight in both genders, with preserved menstruation in females, no biological marker of undernutrition, a bodyweight gain desire and an anorexigenic hormonal profile. CT can be considered as the opposite of obesity, where some patients appear to resist bodyweight loss.

Objectives: We tested the hypothesis that CT would be resistant to bodyweight gain.

Design:A 4-week fat overfeeding (630 kcal excess), performed in an ambulatory position, compared 8 CT women (BMI<17.5kg/m²) and 8 female controls (BMI 18.5-25kg/m²): Bodyweight, food intake, urinary metabolomics profiles, body composition, energy expenditure (EE) and appetite regulatory hormones profile after test meal were monitored before and after dietary intervention.

Results: Four weeks of fat overfeeding failed to increase bodyweight in CTs (+0.225 kg ± 0.180; controls: +0.725 kg ± 0.268, p=0.26 and 0.03 vs. baseline respectively) despite a well-documented compliance with dietary intervention: dietary records, lipid assessment, fat oxidation and metabolomics. Appetite regulatory hormones exhibited an overall anorexigenic adaptive response in CTs with significant increase in post-meal Peptide YY and Glucagon like Peptide type 1, inefficient to counteract the supervised overfeeding but may partly explain CTs eating behavior and play a role in bodyweight gain resistance. Resting EE increased in CTs only with no increase in activity energy expenditure, explaining partially the bodyweight gain resistance. Over all, CTs displayed a paradoxical positive energy balance after overfeeding contrasting with the bodyweight gain resistance. This gap in energy balance could suggest specific EE in CTs.

Conclusion:This study proposes CT as a human model of bodyweight gain resistance in a fat overfeeding paradigm.

 This study is registered in Clinical Trial.gov, no. NCT01224561

 

Nothing to Disclose: NG, BG, DC, DG, EP, JFM, YB, JSM, SRB, JPE, ABE

OR09-3 9035 3.0000 A Constitutional thinness: a bodyweight gain resistance human model with specific appetite, energetic and metabolomics responses to fat overfeeding 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Obesity, Adipocyte Biology and Appetite Saturday, June 15th 1:00:00 PM OR09 2306 11:30:00 AM Obesity: Physiologic Responses to Energy Balance Disruption Oral


Daniel Li Tu Chen*1, Dorit Samocha-Bonet1, Carsten Liess2, Mike Trenell3, Don Chisholm1 and Jerry R Greenfield4
1Garvan Institute of Medical Research, Sydney, Australia, 2Philips Healthcare, Hamberg, Germany, 3Newcastle University, Newcastle, United Kingdom, 4Garvan Institute of Medical Research, Darlinghurst, Australia

 

Background: While insulin resistance and obesity coexist, some obese individuals remain insulin-sensitive (up to 30%). The metabolic factors that co-segregate with, and hence may explain, insulin-sensitivity in obesity have not been fully elucidated. While visceral adiposity correlates positively with insulin resistance, it remains unclear whether insulin-sensitive obesity is characterized by low visceral adiposity. Most previous studies classified obese individuals as insulin-sensitive and insulin-resistant based on surrogate measures, rather than the gold standard hyperinsulinaemic-euglycaemic clamp. The present study examined the metabolic features of insulin-sensitive obesity.

Methods: Fifty three obese subjects (34 female) aged 52 ± 11 years were studied. Anthropometric and clinical metabolic data were measured. Participants underwent hyperinsulinaemic-euglycaemic clamp (insulin infusion rate 80 mU/m2/min) with indirect calorimetry.  Subjects in the top tertile of glucose infusion rate (GIR, normalized for fat-free mass [FFM]) were deemed obese insulin-sensitive (Obsen), with separate cut-offs for men and women. Obese insulin-resistant (Obres) individuals were those in the bottom two tertiles of GIR/FFM. Body composition and abdominal fat distribution were measured by DXA and MRI (at L4/L5), respectively.

Results: Age (P=0.42) and BMI (34.9 ± 3.0 vs. 36.5 ± 4.6 kg/m2; P=0.16) were similar in Obsen and Obres groups. By definition, insulin sensitivity was higher in Obsen subjects (121 ± 26 vs. 76 ± 19 µmol/min/kgFFM). Obsen subjects had significantly lower waist-to-hip ratio (0.87 ± 0.1 vs. 0.93 ± 0.1; P=0.04), HbA1c (5.3 ± 0.3 vs. 5.5 ± 0.3 %; P=0.01) and systolic blood pressure (119 ± 10 vs. 127 ± 14 mmHg; P=0.04). Despite similar total body fat (45.8 ± 10.4 vs. 46.6 ± 10.4 kg; P=0.78) and FFM (50.2 ± 11.2 vs. 54.1 ± 11.5 kg; P=0.24), Obsen subjects had lower central abdominal fat (3.1 ± 0.6 vs. 3.6 ± 0.7 kg; P=0.02). There was no difference in subcutaneous fat (517 ± 146 vs. 524 ± 131 cm2; P=0.87) between the groups. However, Obsen had significantly lower visceral fat (213 ± 49 and 288 ± 80 cm2; P<0.001) and visceral-to-subcutaneous fat ratio (0.47 ± 0.26 vs. 0.59 ± 0.25; P=0.03) than Obres subjects. Obsen subjects were more metabolically flexible (as defined by change in respiratory quotient from baseline to clamp steady state) than Obres subjects (0.18 ± 0.05 vs. 0.14 ± 0.05; P=0.01).

Conclusions: Obese insulin-sensitive adults are characterized by lower HbA1c, systolic blood pressure, abdominal adiposity and visceral fat compared to equally-obese but insulin-resistant subjects. Whether insulin-sensitive obese humans remain insulin-sensitive life-long, and whether their insulin-sensitive phenotype reduces their risk of developing diabetes, cardiovascular disease and certain cancers, is yet to be determined.

 

Nothing to Disclose: DLTC, DS, CL, MT, DC, JRG

OR09-4 6535 4.0000 A Metabolic Features of Insulin-Sensitive Obesity 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Obesity, Adipocyte Biology and Appetite Saturday, June 15th 1:00:00 PM OR09 2306 11:30:00 AM Obesity: Physiologic Responses to Energy Balance Disruption Oral


Moahad Saeed Dar*1, William Chapman1, John R Pender1, Almond Jerkins Drake III2 and Joseph A Houmard3
1Brody School of Medicine, Greenville, NC, 2ECU Brody School of Medicine, Greenville, NC, 3East Carolina University, Greenville, NC

 

Roux-en-Y gastric bypass (RYGB) is an effective treatment for morbid obesity, insulin resistance and type 2 diabetes. However, a recent cohort study noted a 2.7 fold increased risk for hypoglycemia post-RYGB (Marsk et al; 2010). Post-RYGB hypoglycemia typically occurs 1-2 hours after meals and is clinically defined as a fingerstick blood sugar < 70 mg/dl. This complication adversely impacts the emotional and physical well being of patients and current medical/surgical treatments are often ineffective due to a poor understanding of the mechanism(s) causing hypoglycemia.  Accordingly, we performed minimal model testing on 7 hypoglycemic subjects to determine if an increase in whole body insulin sensitivity and/or insulin secretion compared to controls was contributing to the development of hypoglycemia.
    In terms of insulin secretion, the mean acute insulin response to glucose (AIRg) in the post-RYGB hypoglycemic cohort (age 44.3± 9.3 yr, BMI 32.1±4.7,N=7) was 254±199 mu/L·min compared to 380±40 mu/L·min in overweight, exercise trained  cross-sectional controls (age 51.4 + 0.9 y, BMI 28.6 + 0.4 kg/m2, N=43). In terms of insulin sensitivity, the mean insulin sensitivity index (Si) from minimal model testing for the post-RYGB hypoglycemic cohort  was 7.5± 5.03 mU·l-1·min-1 compared to 4.1±0.5 mU·l-1·min-1 in the euglycemic post-RYGB control (age 39.8± 3.1y, BMI 27.1±0.9 kg/m2,N=11). Interestingly, the preliminary data suggested two distinct subgroups of hypoglycemic patients: 1)  high insulin sensitivity, low insulin secretion (Si 9.9 ± 3.6 mU·l-1·min-1,AIRg 173±113 mu/L·min, N=5); 2) low insulin sensitivity, high insulin secretion (Si 1.56 ± 0.28 mU·l-1·min-1, AIRg 455±271 mu/L·min, N=2).
     In conclusion, there appear to be distinct differences in whole body insulin sensitivity and insulin secretion among patients experiencing post-RYGB hypoglycemia which may explain the inconsistent clinical response to current medical/surgical treatments. These findings suggest that assessing whole body insulin sensitivity and insulin secretion in post-RYGB hypoglycemic patients may better inform future treatment strategies for such patients.

 

Nothing to Disclose: MSD, WC, JRP, AJD III, JAH

OR09-5 4559 5.0000 A Hypoglycemia after Roux-en-Y Gastric Bypass: Is Extreme Insulin Sensitivity the Cause? 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Obesity, Adipocyte Biology and Appetite Saturday, June 15th 1:00:00 PM OR09 2306 11:30:00 AM Obesity: Physiologic Responses to Energy Balance Disruption Oral


Andreas Pettersson*1, Carl-Johan Olsson2, Lars Nyberg2 and Tommy Olsson3
1Umeå University, UMEA, Sweden, 2Umeå University, Umeå, Sweden, 3Umeå University, Umea, Sweden

 

Obesity and type 2-diabetes is associated with impaired cognitive functions. To examine whether altered diet may improve memory and alter brain activity we performed functional magnetic resonance imaging (fMRI) during an episodic memory test before and after a diet intervention.

Overweight (BMI > 27) postmenopausal women (mean age 61) were randomized to a Paleolithic diet (PD; 30E% protein, 30E% carbohydrates, 40E% fat, with a high content of mono- and polyunsatured fatty acids, n=9) or a diet according to the Nordic nutrition recommendations (NNR, 15E% P, 55E% C, 30E% F, n=11). At baseline and 6 months we measured anthropometrics, body fat, glucose tolerance and blood lipids. fMRI scans were used to examine functional brain response of episodic memory. Inside the scanner the subjects were instructed to memorize unknown pairs of faces and names presented on a screen (encoding). Subsequently the faces were presented once again together with three letters, one corresponded with the initial letter of the name of the face. The retrieval task was to indicate the correct letter.

As there were no differences between groups in anthropometrics or fMRI data they are treated as one group. BMI decreased from 32.1 kg/m2 at baseline to 29.2 kg/m2 at 6 months, body fat and serum cholesterol decreased as well but there were no effects on glucose tolerance. 

Memory performance improved after the intervention. Brain activity increased in several regions during encoding, including insula and superior temporal gyrus; both regions are important for identification and matching of faces. These changes correlated with decreased waist to hip ratio. Brain activity also increased in superior frontal gyrus which is important for self-reference; this can increase the ability to sustain new eating habits.

In contrast, during retrieval, brain activity decreased in the inferior and superior frontal gyrus. These regions are associated with retrieval effort of episodic memories, suggesting more efficient retrieval after weight loss. In addition a region related to retrieval success (medial temporal gyrus) increased its activity after the weight loss, further indicating improved memory performance.

In summary, diet-induced weight loss leads to improved memory performance associated with a more efficient brain function indicated by increased recruitment of brain resources during the encoding phase of the episodic task followed by decreased need of resources during the retrieval phase.

 

Nothing to Disclose: AP, CJO, LN, TO

OR09-6 7376 6.0000 A Diet-induced weight loss improves episodic memory and alters brain activity in overweight postmenopausal women 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Obesity, Adipocyte Biology and Appetite Saturday, June 15th 1:00:00 PM OR09 2306 11:30:00 AM Obesity: Physiologic Responses to Energy Balance Disruption Oral


Andrea Armani*1, Vincenzo Marzolla1, Francesca Cinti2, James Morgan3, Greg Cranston3, Antonella Antelmi1, Giulia Carpinelli4, Rossella Canese4, Carmelo Quarta5, Uberto Pagotto6, Giuseppe Rosano1, Saverio Cinti2, Andrea Fabbri7, Morag Jennifer Young3 and Massimiliano Caprio1
1IRCCS San Raffaele Pisana, Rome, Italy, 2University of Ancona, 3Prince Henry's Institute of Medical Research, Clayton VIC, Australia, 4Istituto Superiore di Sanità, Rome, Italy, 5University of Bologna, Bologna, Italy, 6Alma Mater University of Bologna S.Orsola Hospital, Bologna, Italy, 7University of Tor Vergata, Rome, Italy

 

We have previously shown that knock-down of mineralocorticoid receptor (MR) expression as well as its antagonism result in a potent antiadipogenic activity on murine and human preadipocytes. In this study responses to MR antagonists were investigated in a model of diet-induced obesity. Female 10-week-old C57bl6 mice were fed with normal chow or a high fat (HF) diet for 12 weeks. Mice fed HF were concomitantly treated for 12 weeks with drospirenone (DRSP, 6 mg/Kg/day), a potent MR antagonist with progestative properties, or spironolactone (SPIRO, 20 mg/kg/day). Mice fed HF showed a significant increase in total body weight, fat mass, mean adipocyte size, expression of WAT markers and showed impaired glucose tolerance after intraperitoneal plasma glucose tolerance test. DRSP and SPIRO prevented weight gain and white fat mass expansion induced by HF in parametrial, perivescical, and inguinal depots without affecting interscapular fat pad weight.  Magnetic Resonance Imaging confirmed that MR antagonists blocked the HF-driven expansion of abdomino-pelvic fat volume. Importantly, both DRSP and SPIRO prevented the impaired glucose tolerance in mice fed HF, countered HF-induced up-regulation of WAT markers transcripts and adipocyte hypertrophy. Indeed, treatment with DRSP and SPIRO markedly increased the number of UCP1-positive brown-like adipocytes interspersed in WAT depots analysed, suggesting that MR antagonism promotes browning of adipose tissue. Accordingly, Magnetic Resonance Spectroscopy analysis in vivo performed in inguinal fat showed a significant increase in the percentage of water in mice treated with MR antagonists, reflecting an increase in number of brown-like adipocytes, which are richer in water. In addition, PET/CT analysis showed increased glucose uptake in visceral abdomino-pelvic and interscapular fat in MR antagonists-treated mice, confirming a notable rise in activity of metabolically active brown fat. Finally, 2 days treatment of murine primary preadipocytes extracted from inguinal fat with MR antagonists up-regulated PRDM-16, a determinant of brown fat-like gene program, whereas 6 days treatment increased UCP-1 expression, suggesting a specific involvement of adipocyte MR in browning of AT observed in vivo. In conclusion, we show that MR antagonism counters metabolic dysfunctions induced by HF in mice, at least in part by activation of browning in WAT. Indeed MR antagonism has promise as a novel approach to treat metabolic syndrome

.

 

Nothing to Disclose: AA, VM, FC, JM, GC, AA, GC, RC, CQ, UP, GR, SC, AF, MJY, MC

OR08-1 6932 1.0000 A Mineralocorticoid Receptor Antagonism Counters Metabolic Dysfunctions Induced By High Fat Diet in Mice, Through Active Browning of Adipose Tissue 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Obesity, Adipocyte Biology and Appetite Saturday, June 15th 1:00:00 PM OR08 2307 11:30:00 AM Obesity: Novel Mechanisms of Body Weight Regulation Oral


Stephanie M Correa*1, David W Newstrom1, Clement C Cheung1, James Philip Warne1, Pierre Flandin2, Andrew A Pierce3, Allison Xu1, John L Rubenstein2 and Holly A Ingraham1
1University of California San Francisco, San Francisco, CA, 2University of California San Francisco, 3University of California San Francisco, CA

 

The ventromedial hypothalamus (VMH) plays important roles in reproduction, anxiety, aggression, ingestive behavior, and energy expenditure.  Nkx2-1 (Ttf-1) is a homeobox transcription factor that is expressed in hypothalamic progenitors beginning at embryonic day 9 and persists in adult VMH neurons.  Sf1 (Nr5a1) is one of the earliest markers of VMH neurons and its expression overlaps with Nkx2-1 only in the VMH.  To determine Nkx2-1 function in VMH neurons, we conditionally ablated Nkx2-1 in the developing VMH using Sf1-driven CRE recombinase (Nkx2-1Sf1Cre).  We and found that female mutant mice were significantly heavier than control littermates when fed normal chow.  In contrast, body weights of male mutants were normal.  Increased body weight in Nkx2-1Sf1Cre females was attributed to higher body fat deposition in visceral and subcutaneous depots but no changes in brown fat mass or thermogenesis.  Metabolic analyses revealed that obesity in Nkx2-1Sf1Cre females was due to reduced energy expenditure rather than increased food intake; Nkx2-1Sf1Cre females exhibit a specific deficit in locomotor activity.  Expression analyses of Nkx2-1Sf1Cre female VMH revealed a significant and selective decrease of NKX2-1 and ERa in the ventrolateral VMH (VMHvl), whereas NKX2-1 and ERa were unchanged in the neighboring arcuate nucleus.  Despite the loss of ERa expression in the VMH, female Nkx2-1 Sf1Cre mice exhibited normal fertility.  To further demonstrate that Nkx2-1 neurons in the VMHvl regulate physical activity, we performed pharmacogenetic experiments using Designer Receptors Activated by Designer Drugs (DREADDs).  Artificially activating Nkx2-1 neurons in the VMHvl of Nkx2-1Cre female mice resulted in higher VO2 consumption and increased movement, demonstrating that these specialized VMH neurons regulate physical activity.  We propose that estrogen-responsive Nkx2-1 neurons in the VMH regulate physical activity in female mice and that loss of these neurons underlies the “couch-potato” phenotype observed in mutant females.  If successful, research efforts to define this unique population of hypothalamic neurons should provide new potential strategies for decreasing sedentary behavior and improving metabolic health in women.

 

Nothing to Disclose: SMC, DWN, CCC, JPW, PF, AAP, AX, JLR, HAI

OR08-2 7526 2.0000 A A Population of Nkx2-1 Neurons in the Ventromedial Hypothalamus (VMH) Mediates Sex-Specific Obesity and Sedentary Behavior in Mice 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Obesity, Adipocyte Biology and Appetite Saturday, June 15th 1:00:00 PM OR08 2307 11:30:00 AM Obesity: Novel Mechanisms of Body Weight Regulation Oral


Miho Yamashita*1, Veronica Otero-Corchon1, Thomas L Saunders2, Lev Fedorov3 and Malcolm James Low1
1University of Michigan Medical School, Ann Arbor, MI, 2University of Michigan, Ann Arbor, MI, 3Oregon Health & Science University, Portland, MI

 

Hypothalamic proopiomelanocortin (POMC) neurons in the arcuate nucleus (ARC) are heterogeneous for their afferent and efferent signaling properties. Discrete or partially overlapping subpopulations respond to leptin, insulin and 5-HT and utilize GABA or glutamate as a neurotransmitter. Little is known about the functional consequences of this diversity at the systems level. To address the physiological function of subpopulations of POMC neurons in an unbiased fashion, we are using somatic chimeras in which each mouse has stochastically determined juxtapositions of genetically distinct POMC neurons. As proof of principle, we first generated a series of chimeras by aggregation of morulae pairs derived from transgenic mice expressing either a green (EGFP) or red (tDimer-dsRed) fluorescent protein in ARC POMC neurons and pituitary corticotrophs and melanotrophs. Nine chimeras exhibited unique distribution patterns of fluorescently labeled cells in the pituitary gland and throughout the ARC, with no cellular co-expression of the two fluorescent tags.  The percentage of neuronal chimerism ranged from 10-90%. We next crossed a mutant ArcPomcfneo allele containing a neomycin cassette in the vicinity of the neuronal-specific upstream enhancers, which silences Pomc expression in the ARC but not pituitary, to the transgenic strain with cell autonomous expression of EGFP in all POMC cells. These mice were used to generate a novel embryonic stem (ES) cell line that is homozygous for both the ArcPomcfneo and PomcEGFPtg alleles. Initial production of chimeras by the aggregation of these ES cells with morulae from PomcdsRedtg/tg mice was inefficient, but demonstrated the expected result of mixed neuronal populations in the ARC; green neurons that lack POMC peptide immunoreactivity and red neurons that normally express POMC. Microinjection of the ArcPomcfneo/fneo:PomcEGFPtg/tg ES cells into blastocysts derived from the PomcdsRedtg/tg mice mice has yielded 13 chimeras to date. Each mouse is being phenotyped longitudinally by evaluations of body weight, fat mass, length, food intake, refeeding response, locomotor activity, O2 consumption, and corticosterone, terminating with a detailed histological analysis of the ARC and POMC projections throughout the brain. A multivariate analysis will determine whether the number and spatial localization of subpopulations of Pomc expressing and non-expressing POMC neurons can predict the final metabolic and obesity outcome of the mice.

 

Nothing to Disclose: MY, VO, TLS, LF, MJL

OR08-3 8603 3.0000 A Chimera Analysis of Pomc Neuron Function Utilizing Combinations of Fluorescently Tagged Wild-Type and Arcuate Nucleus-Specific Pomc Deficient Mouse Embryos 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Obesity, Adipocyte Biology and Appetite Saturday, June 15th 1:00:00 PM OR08 2307 11:30:00 AM Obesity: Novel Mechanisms of Body Weight Regulation Oral


Jian Valentine Zhang*
Chinese Academy of Sciences (CAS), ShenZhen, China

 

NOVEL CMKLR1 SMALL MOLECULE ANTAGONIST REGULATES ADIPOGENESIS AND OBESITY

Qin-ce SUN * 1,Shu-hua MU * 1, Xu ZHANG 1, Yong-jun LIU1, Ping MA1, Wen-juan XU 1,Jian V Zhang1,2#

1 Institute of Biomedicine and Biotechnology, CAS and 2 Shenzhen Engineering Laboratory of Single-molecule Detection and Instrument Development, China

*These authors contributed equally to this work.

#Address for correspondence:  Shenzhen Institute of Advance Technology, Chinese Academy of Sciences,  518055, China. Tel: (86) 0755-86582290; Fax: (86) 0755-86585222; E-mail: jian.zhang@siat.ac.cn

Objectives:Obesity as a new epidemic disease has been associated with excessive adipocyte number and increasing size in vivo. Actually, the relevance of adipokines from adipose tissue and adipogenesis have attracted lots of attention. Previous studies have shown that novel adipokine chemerin and its receptor CMKLR1 play an important role during adipogenesis. The objective of this study is to investigate whether the novel CMKLR1 small molecule antagonist has any physiological function and its possible involvement in adipogenesis and obesity.

 Methods:The gene expression of chemerin, CMKLR1, the adipocyte markers (C/EBPα, FABP4, PPARγ, HSL, ATGL), and inflammatory factors (IL-6 and TNF-α) in 3T3-L1 preadipocyte and adipocyte tissues was analyzed by real-time PCR. The lipid accumulation was identified by Oil red O staining. The cellular incorporation of oil red O was measured using a spectrophotometer at 500 nm. The serum cholesterol (TC), triglycerides (TG) and low-density lipoprotein cholesterol (LDL-C) levels were detected by ELISA kit. The effects of CMKLR1 antagonist compound on adipogenesis and obesity were evaluated in mouse 3T3-L1 preadipocyte and mice of 6-week old fed on high-fat-diet for 8-week.

 Results: Chemerin and CMKLR1 mRNA were increased during 3T3-L1 preadipocyte differentiation, and these changes were also detected in the adipose tissue of obesity mouse induced by high-fat diet. C/EBPα, FABP4, PPARγ, HSL, ATGL, IL-6 and TNF-α mRNA levels were also upregulated. Administration of the novel CMKLR1 small molecule antagonist suppressed 3T3-L1 preadipocyte differentiation and the progression of obesity. The mRNA expression of adipocyte markers and inflammatory factors were decreased by the compound both in vitro and in vivomodel. Treatment of the compound produced a dose-dependent reduction of the lipid accumulation in 3T3-LI preadipocyte differentiation. Besides, the weight gain of mice fed on high-fat-diet was significantly lower than that of mice without CMKLR1 antagonist i.p. injection. These changes were also observed in serum TG, TC and LDL after CMKLR1 antagonist treatment in mice.

 Conclusions: Taken together, CMKLR1 antagonist compound protects against obesity in both in vitro and  in vivo model, suggesting CMKLR1 and its ligand chemerin maybe the potential therapeutic target for obesity.

 

Nothing to Disclose: JVZ

OR08-4 6214 4.0000 A NOVEL CMKLR1 SMALL Molecule Antagonist Regulates Adipogenesis and Obesity 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Obesity, Adipocyte Biology and Appetite Saturday, June 15th 1:00:00 PM OR08 2307 11:30:00 AM Obesity: Novel Mechanisms of Body Weight Regulation Oral


Shannon Liu1, Hongzhi Miao1, Liang Sheng1, Thomas L Saunders2, Ormond A MacDougald2, Ronald J Koenig3 and Bin Xu*4
1University of Michigan, 2University of Michigan, Ann Arbor, MI, 3Univ of Michigan Med Ctr, Ann Arbor, MI, 4The Univ of Michigan, Ann Arbor, MI

 

We have recently shown that the Steroid Receptor RNA Activator (SRA), promotes adipocyte differentiation and improves insulin-stimulated glucose uptake in adipocytes in vitro through multiple mechanisms, such as coactivating transcriptional activity of PPARΥ, promoting S-phase entry during mitotic clonal expansion, increasing phosphorylation of Akt and FOXO1 in response to insulin, and inhibiting expression of adipocyte-related inflammatory genes. To assess SRA function in vivo, we have generated a whole mouse SRA gene knockout (SRA-/-). Here, we show that the SRA gene is an important regulator of fat mass and function. SRA is expressed at higher levels in both brown and white adipose tissues (BAT, WAT) than other organs in wild type mice. SRA-/- mice are resistant to diet-induced obesity, weighing on average 6 g less after 14 weeks on a high fat diet. Body composition analysis showed the whole mouse fat content decreased from 28% (wild type) to 17% (SRA-/-). This lean phenotype of SRA-/- mice is associated with decreased expression of a subset of WAT genes including C/EBPα and FABP4, as well as decreased expression of the inflammatory genes TNFα and CCL2. The SRA-/- mice are more insulin sensitive, as evidenced by reduced fasting insulin despite unchanged blood glucose, as well as lower blood glucoses in response to IP glucose and insulin. In addition, the livers of SRA-/- mice had fewer lipid droplets after high fat feeding, and the expression of lipogenesis-associated genes was decreased. At the end of the high fat feeding, energy expenditure (CLAMS analysis) and food intake showed no significant change. In summary, we have created a whole mouse SRA knockout, and found that these mice are resistant to diet-induced obesity, with decreased fat, decreased expression of a subset of adipocyte genes, and improved insulin sensitivity. These data indicate an important role for SRA in adipose tissue biology in vivo.

 

Nothing to Disclose: SL, HM, LS, TLS, OAM, RJK, BX

OR08-5 7630 5.0000 A SRA Gene Knockout Protects Against Diet-Induced Obesity and Improves Glucose Tolerance 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Obesity, Adipocyte Biology and Appetite Saturday, June 15th 1:00:00 PM OR08 2307 11:30:00 AM Obesity: Novel Mechanisms of Body Weight Regulation Oral


David Joel DiSilvestro*1, Ouliana Ziouzenkova2 and Kichoon Lee1
1The Ohio State University, Columbus, OH, 2Ohio State Univ, Columbus, OH

 

Obesity affects about 30% of Americans causing health care costs to rise.  Novel treatment methods are needed to combat the obesity problem when tradition methods, such as diet and exercise, fail.  Therapies involving the implantation of thermogenic cells into adipose tissue induce the host’s immune response and may cause the host to compensate weight loss with increased food consumption.  To overcome this problem, we encapsulated thermogenic cells in a porous alginate-poly-l-lysine (APL) membrane that enables long-term cell survival in the host’s tissue.  We hypothesized that injecting these capsules, which contain thermogenic and appetite suppressing cells, into adipose tissue would increase energy metabolism and stimulate weight loss. To test this, we generated two sets of thermogenic capsules. We knocked out raptor in 3T3-L1 cells (RKO).  To control appetite, we stably transfected an aliquot of RKO with leptin and amylin to generate a new cell line (LAR).  We determined that RKO and LAR were prime candidates for thermogenic capsules because these lines had greater glucose uptake than 3T3-L1 cells in vitro.  To test in vivo, 12 week old wild-type female mice were fed a high-fat (HF) diet (45% kcal from fat) for 132 days and then injected with cellular or acellular APL capsules.  The control group (Empty­­SV) received acellular capsule injections into both subcutaneous (S) and visceral (V) fat pads.  The second group (RVEmptyS) received RKO capsule injections into the visceral fat pads and acellular capsule injections in the subcutaneous fat pads. The final group (RVLARS) received RKO ­­capsules in the visceral fat and LAR capsules in the subcutaneous fat pads.  After 51 days on a HF diet, the percent weight gain of the RVLARS was significantly less than the percent weight gain of the EmptySV; likewise, the percent weight gain of the RVEmptyS was less than the percent weight gain of the Empty­­SV.  These results show the efficacy of these capsules for increasing thermogenesis and reducing appetite in the attenuation of weight gain induced by a HF diet.

 

Nothing to Disclose: DJD, OZ, KL

OR08-6 7340 6.0000 A Encapsulated Thermogenic and Appetite Suppressing Fibroblasts Attenuate Weight Gain in Mice 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Obesity, Adipocyte Biology and Appetite Saturday, June 15th 1:00:00 PM OR08 2307 11:30:00 AM Obesity: Novel Mechanisms of Body Weight Regulation Oral


Judith L. Ross*1, Martha D Bardsley1, Harvey Kushner2, Karen Kowal3 and David Roeltgen4
1duPont Hospital for Children, 2Biomedical Research Company, Philadelphia, PA, 3Thomas Jefferson University/duPont Hospital for Children, Philadelphia, PA, 4University of Pennsylvania, South Seaville, NJ

 

Background: KS is a common genetic disorder (1/700 males) defined by the karyotype 47,XXY. The behavioral/social phenotype in KS includes increased social problems, anxiety, and somatic symptoms and may result in part from testicular failure and androgen deficiency in childhood. Androgen replacement is standard in adolescent and adult KS males but has not been used in childhood. The effects of treatment with prepubertal androgen replacement in KS are unknown.  In this study, we examined the effects of early low-dose androgen on self-image and quality of life in prepubertal boys with KS ages 5-12 years.
Objective: To determine the effects of Oxandrolone (Ox) versus Placebo (Pl) treatment for two years on self-image and quality of life in boys with KS.
Design/Methods: In this 2-year, double-blind, placebo-controlled clinical trial (2005-2011, NCT00348946), 93 boys with KS (95% 47,XXY), ages 4-12 y, were randomized to two groups: Ox (0.06 mg/kg (N=46)) and Pl (N=47). The evaluation at baseline (BL), one, and two years included two child self-report questionnaires: the Revised Childrens’ Manifest Anxiety Scale (RCMAS, ages 6-19 y), and the Children’s Depression Inventory (CDI, ages 6-17 y), and one Parent questionnaire: the Child Behavior Checklist (CBCL, ages 2-18 y). Statistical analysis included ANCOVA for change at 2-years from BL, adjusting for baseline differences in age.
Results: 80 (86%) subjects completed the 2-year study. Mean ages at study completion were (Ox) 9.1±2.1 years (n=39) vs. (Pl) 10.2 ±2.6 years (n=41), indicating the Ox group was slightly younger (P=0.03). At 2-years, on the RCMAS, the Ox group had significantly more positive Total (P=0.01) and Worry (P=0.02) scale scores and on the CDI, better outcomes on the Interpersonal Problems (P=0.003) and negative self esteem (P=0.0003) scales vs no significant changes from baseline in the Pl group. On the CBCL, at 2-years vs baseline, the Ox group had significantly better self-image scores for Problem Behaviors (P=0.02), Internalizing (P=0.02), Withdrawn (P=0.002), Anxious (P=0.04), and Somatic Problems (P=0.003) scales vs no significant changes from baseline in the Pl group. In contrast, neither the Ox nor the Pl groups had any significant changes at two years in CBCL hyperactive or aggressive behavior scales.  No major safety issues were identified.
Conclusions: This unique double-blind, randomized clinical trial demonstrates that childhood low-dose androgen treatment for two years in boys with KS had a positive impact on children’s self-esteem and social function and on parental perception of their children’s symptoms of anxiety, depression, and somatic problems. In contrast, there were no group differences in delinquent or aggressive problem behaviors. Treatment was well tolerated. Therefore, low-dose, childhood androgen replacement appears to have a positive impact on self-image in boys with KS.


 

Nothing to Disclose: JLR, MDB, HK, KK, DR

OR11-1 3407 1.0000 A Effect of Childhood Low-Dose Androgen Treatment on Self Image and Quality of Life in Boys with Klinefelter Syndrome (KS): Results of a Two-Year, Placebo-Controlled Clinical Trial 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Pediatric Endocrinology Saturday, June 15th 1:00:00 PM OR11 2330 11:30:00 AM Pediatric Endocrinology Oral


Jose Atilio Canas*1, Judith L. Ross2, Martha V Taboada3, Kaitlin Sikes1, Ligeia Damaso3, Jobayer Hossain4, Michael Phillip Caulfield5, Samuel S Gidding6 and Nelly Mauras1
1Nemours Children's Clinic, Jacksonville, FL, 2Thomas Jefferson University/duPont Hospital for Children, Philadelphia, PA, 3Nemours Children's Hospital, Orlando, FL, 4Alfred I duPont Hospital for Children, Wilmington, DE, 5Quest Diagnostics Nichols Institute, San Juan Capistrano, CA, 6AIfred I duPont Hospital for Children, Wilmington, DE

 

Background: Dysglycemia, inflammation, and elevations in LDL-C and small dense lipoprotein (Lp) fractions increase the risk for early development of atheromatous plaque and micro- and macrovascular disease. These factors are especially concerning in children with T1DM.

Specific Aim: To investigate whether statin use in children with T1DM and elevated LDL-C has an acceptable safety profile and whether statins improve LDL-C and other atherogenic Lp concentrations.

 

Methods: Children with T1DM were recruited after appropriate consent for a multiarm study of cardiovascular risk: 80 children with LDL-C >90 mg/dl were targeted for a randomized clinical trial (RCT) and 28 non-diabetic, normal LDL-C, age-matched, lean children studied as controls. A 3-month run-in phase of intensification of diabetes and dietary management was followed by randomization to atorvastatin (Lipitor®, 10 mg titrated up to 20 mg) or placebo (PL) for 6 months. Safety parameters and adverse events (AEs) were monitored and Lp subfractions measured by a novel ion mobility assay (Quest Diagnostics).

 

Results: 82 children were recruited (39M/43F, mean age 15 ± 0.3 (SE) yr, mean diabetes duration 6.9 ± 0.4 yr, HbA1C 8.7 ± 0.2%, BMI 22.9 ± 0.4 kg/m2, with normal BP, and mean LDL-C 110 ± 3.0 mg/dl).  After a 3 month run-in, 40 dropped out - 49% due to improved LDL-C (<90 mg/dl) and others for miscellaneous reasons; none due to AEs. Lp subfractions were significantly higher in T1DM than in controls, particularly for most non-HDL subfractions. Mixed model analysis showed a significant decrease in total mean non HDL-C (nmol/L) at 6 months in the statins group (-554 ± 258) vs. PL (+529 ± 265, p=0.007). Lp subparticles (LDL-Large & Total, VLDL-Small & Medium, and ApoB) and the ApoB/A1 ratio significantly decreased in the statins group and remained the same or increased in PL. HbA1C was constant at 3 and 6 months post-randomization in both groups (p=NS). Both groups had a small, comparable number of AEs, with 1 SAE unrelated to statins. Creatine kinase and liver function tests were unchanged in both groups.

Conclusion: Atorvastatin significantly lowered LDL-C and atherogenic Lp subparticles in children with T1DM and elevated LDL-C. The drug was well tolerated and safe for 6 months. Ongoing studies on the relationship of Lp subparticles and dysglycemia measures will provide insight into the early development of cardiovascular disease in childhood T1DM.

 

 

Disclosure: MPC: Employee, Quest Diagnostics. Nothing to Disclose: JAC, JLR, MVT, KS, LD, JH, SSG, NM

OR11-2 5895 2.0000 A Atorvastatin Safely Reduces Non-HDL Cholesterol in Children with Type I Diabetes (T1DM) and High LDL-C: Preliminary Results 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Pediatric Endocrinology Saturday, June 15th 1:00:00 PM OR11 2330 11:30:00 AM Pediatric Endocrinology Oral


Faisal S Malik*1, Rajendu Srivastava2, Russell Localio3, Lisa McLeod3, Ron Keren3, Xianqun Luan3, Sanjay Mahant4, Samir S Shah5, Karen M Wilson6, Joel S Tieder1 and For the Pediatric Research in Inpatient Settings (PRIS) Network7
1University of Washington and Seattle Children's Hospital, Seattle, WA, 2University of Utah Health Sciences Center, Salt Lake City, UT, 3Children's Hospital of Philadelphia, Philadelphia, PA, 4University of Toronto, Toronto, ON, 5Cincinnati Children's Hospital Medical Center, Cincinatti, OH, 6Children's Hospital Colorado, Aurora, CO, 7PRIS Network

 

Background: Hospital readmission for diabetic ketoacidosis (DKA) is preventable, yet it remains extremely common. Our goal was to examine patient characteristics associated with higher rates of readmission for DKA at major U.S. children’s hospitals.

Methods: Multi-center retrospective cohort study of children 2-18 years of age admitted to 38 freestanding children’s hospitals participating in the Pediatric Health Information Systems (PHIS) database between January 2004 and December 2009 with a discharge diagnosis code for DKA and that received insulin. We used a logistic regression model to evaluate patient characteristics and the odds of readmission at 30 and 365 days, and predictive margins to measure the average chance of readmission for children with each characteristic. Covariates included age, public insurance, complex care conditions, mental health conditions, and severity of illness at the index visit.

Results: Of the 24,890 children with DKA, 55.3% were female, 7.3% were <5 years, 57.4% were >12 years, 43.1% had government insurance, and 9.4% had mental health condition(s). Readmission rate for DKA was 2.8% at 30 days and 20.3% at 365 days after discharge. Overall, higher risk of readmission was associated with age >12 years (30 days: OR, 1.8; 95% CI, 1.4-2.3; 365 days: OR 2.6; 95% CI, 2.3-2.9), public insurance (30 days: OR, 1.8; 95% CI, 1.4-2.3; 365 days: OR 1.7; 95% CI, 1.6-2.0), and a diagnosis of a mental health condition (30 days: OR, 1.8; 95% CI, 1.4-2.3; 365 days: OR 1.7; 95% CI, 1.6-2.0).  Children <5 years were less likely to be readmitted for DKA (30 days: OR, 0.2; 95% CI, 0.1-0.4; 365 days: OR 0.3; 95% CI, 0.2-0.4).  Females >12 years of age and with public insurance had a 40% average chance for readmission compared to 2.1% for boys under age 5 with private insurance at 365 days.

Conclusion: In a large sample of freestanding children’s hospitals, adolescents, females, and the publically insured had the highest risk for hospital readmission at both 30 and 365 days. These data may be used to target interventions for DKA prevention toward the most vulnerable populations.

 

Nothing to Disclose: FSM, RS, RL, LM, RK, XL, SM, SSS, KMW, JST, FTPRIISN

OR11-3 7939 3.0000 A Patient Characteristics Associated with Readmission for Diabetic Ketoacidosis at U.S. Children's Hospitals 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Pediatric Endocrinology Saturday, June 15th 1:00:00 PM OR11 2330 11:30:00 AM Pediatric Endocrinology Oral


Carmen L Soto-Rivera*1, Lisa A Scoppettuolo1, Jamin Alexander1, Alexandra Oldershaw1, David Wypij2, Michael G Gaies3 and Michael S D Agus1
1Boston Children's Hospital, Boston, MA, 2Harvard School of Public Health, Boston, MA, 3University of Michigan's C.S. Mott Children's Hospital, MI

 

Background: There are expected alterations in thyroid function after pediatric cardiopulmonary bypass (CPB) that are associated with prolongation of recovery. Thyroid changes in the setting of tight glycemic control (TGC) after pediatric cardiac surgery have not been described in the setting of a trial with a low hypoglycemia rate. We tested the hypothesis that TGC would be associated with earlier normalization of thyroid function compared with standard care (STD), or permissive hyperglycemia, in children undergoing cardiac surgery.

Methods: In this two-center, prospective, randomized trial, we studied 980 children, birth to 36 months of age, undergoing cardiac surgery with CPB. Patients were randomly assigned to either TGC or STD in the cardiac intensive care unit. Thyroid stimulating hormone (TSH), total thyroxine (T4), total triiodothyronine (T3) and thyroid hormone binding ratio (THBR) were obtained on post-operative days (POD) 2, 7 and 14 for subjects in both groups. All hormones were assayed by electrochemiluminescent immunoassay. Subjects having at least one pair of POD 2-7 or POD 2-14 values were studied.

Results: 260 patients were still on study on POD 7, of which 191 patients (99 TGC, 92 STD) had thyroid function data through POD 7 and 67 (30 TGC, 37 STD) through POD 14. The great majority of patients had POD 2 values that were lower than age-specific normal ranges for T4 (98%), T3 (98%), and TSH (85%), while 76% patients were within normal range for THBR. Values on POD 2 were similar for both groups (all p>0.20). By POD 7, the TGC group had higher T4 [median 6.4 (interquartile range 4.4-9.0) vs 5.3 (4.1-7.1) mcg/dL, p=0.02] and a greater change from POD 2 [2.4 (0.2-4.3) vs 1.3 (−0.2-2.8) mcg/dL, p=0.02] compared with the STD group. By POD 14, the TGC group had higher T3 [104.5 (79-137) vs 74 (60-112) ng/dL, p=0.02] and a greater change from POD 2 [26 (9-68) vs 5 (−10-42) ng/dL, p=0.02] compared with the STD group. There was no difference in TSH or THBR between groups at POD 7 or POD 14.

Conclusion: TGC resulted in earlier normalization of thyroid hormone concentrations in children who are post-operative from cardiac surgery with CPB. This finding differs from previous reports of TGC increasing peripheral inactivation of thyroid hormone mimicking a fasting response, which could be related to the very low rates of hypoglycemia in our cohort. Our findings are specific for pediatric cardiac surgery patients and the impact of TGC on other critically ill children warrants further investigation.

 

Disclosure: MSDA: Consultant, Roche Diagnostics, Consultant, Medtronic Minimed. Nothing to Disclose: CLS, LAS, JA, AO, DW, MGG

OR11-4 8683 4.0000 A Effect of Tight Glycemic Control on Thyroid Function after Pediatric Cardiac Surgery 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Pediatric Endocrinology Saturday, June 15th 1:00:00 PM OR11 2330 11:30:00 AM Pediatric Endocrinology Oral


Kathryn E Ackerman*1, Lisa Pierce2, Gabriela Guereca2, Meghan Slattery3, Mark Goldstein4 and Madhusmita Misra2
1Massachusetts General Hospital and Harvard Medical School, Boston, MA, 2Massachusetts General Hospital/Harvard Medical School, Boston, MA, 3Massachusetts General Hospital, Boston, MA, 4Masschusetts General Hospital

 

Background:  Stress fractures of the foot, tibia and femur are common in endurance athletes. Although studies have described microarchitecture of the distal tibia in athletes, there are few data regarding hip structure and strength in athletes and non-athletes. Hip structural analysis (HSA) using dual energy x-ray absorptiometry (DXA) is a validated technique to assess hip geometry and strength parameters while avoiding the radiation associated with quantitative CT.

Objectives: Our objective was to compare hip geometry and strength estimates in oligo-amenorrheic athletes (AA), eumenorrheic athletes (EA) and non-athletes (NA) using HSA. We hypothesized that AA would have impaired geometry compared with EA.

Methods: We enrolled 55 AA, 24 EA and 23 NA between 14-22 years of normal weight. Athletes ran ≥20 miles/week or were engaged in weight-bearing sports for ≥4 hours/week, whereas NA were not engaged in any organized sports and exercised for <2 hours/week. DXA was used to assess hip bone density and for HSA.

Results: Although subjects were of normal weight, BMI was lower in AA compared with EA and NA (20.1±2.2, 22.4±2.4 and 21.7±2.5 kg/m2, p<0.0001). Groups did not differ for height. Hip Z-scores were lower in AA and NA than EA (p= 0.002).  However, a larger proportion of AA compared with EA and NA had hip Z-scores <-1 (30.9% vs. 4.2 and 17.4%, p=0.01). Reported caloric intake did not differ among groups, however, reported energy expenditure was higher in athletes compared with non-athletes. 25(OH)D levels were highest in AA, followed by EA and NA (p<0.0001). At the narrow neck, trochanteric region and femoral shaft, subperiosteal width, cross sectional moment of inertia (CSMI) (estimate of resistance to bending forces) and section modulus (Z) (index of strength of bending) were higher in EA than NA, whereas AA did not differ from NA. Cross sectional area (CSA) (estimate of resistance to axial forces) was lower in AA and NA than EA. In addition, at the trochanteric region, endocortical width was higher in EA than NA, whereas AA did not differ from NA. Groups did not differ for cortical thickness, buckling ratio and hip axis length. Subjects with hip Z<-1 had lower CSA, CSMI, Z and cortical thickness, and higher buckling ratio at the narrow neck and trochanteric region than subjects with Z>-1, whereas subperiosteal and endocortical width did not differ. On multivariate analysis, after controlling for hip Z-scores, differences persisted between EA and NA for most parameters. Lean mass correlated with most measures of HSA, and differences between groups were lost after adjusting for lean mass.

Conclusions: In an eugonadal state, athletic activity confers benefits at the hip for most geometric parameters independent of areal bone density. This advantage is lost in AA, who do not differ from non-athletes for most parameters, and fare worse than eumenorrheic, estrogen-replete athletes for cross-sectional area.

 

Nothing to Disclose: KEA, LP, GG, MS, MG, MM

OR11-5 3572 5.0000 A Hip Structural Analysis in Adolescent and Young Adult Oligo-amenorrheic and Eumenorrheic Athletes and Non-athletes 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Pediatric Endocrinology Saturday, June 15th 1:00:00 PM OR11 2330 11:30:00 AM Pediatric Endocrinology Oral


Daniel Kelberman*1, Emma Webb1, Angham N Almutair2, Chiara Bacchelli1, Estelle Chanudet1, Francesco Lescai1, Cynthia Lilian Andoniadou3, Abdul Hameed Al Banyan2, Abdulrahman Al Swaid2, Mohammad Alrifai2, Mohammed AlBalwi2, Neda Mousavy-Gharavy1, Biljana Lukovic4, Derek Burke5, Simon Heales4, Mark J McCabe1, Tessa Kasia1, Robert Kleta6, Elia Stupka7, Philip L Beales1, Dorothy A Thompson4, Kling W Chong4, Fowzan Alkuraya8, Juan Pedro Martinez-Barbera1, Jane C Sowden1 and Mehul Tulsidas Dattani1
1UCL Institute of Child Health, London, United Kingdom, 2King Abdulaziz Med City, Riyadh, Saudi Arabia, 3UCL, London, United Kingdom, 4Great Ormond Street Hospital for Children NHS Foundation Trust, London, United Kingdom, 5Great Ormond Street Hospital for Children NHS Foundation Trust, London`, United Kingdom, 6UCL Centre for Nephrology, London, United Kingdom, 7San Raffaele Scientific Institute, Milan, Italy, 8King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia

 

Introduction Mutations affecting hypothalamic development in humans have been identified in genes that affect isolated domains of hypothalamic function leading to restricted phenotypes, such as obesity or hypogonadotrophic hypogonadism. We describe the first human cases of diabetes insipidus and combined pituitary hormone deficiency due to a mutation in a gene regulating hypothalamic development.

Results Six affected individuals from a highly consanguineous pedigree presented with cortisol deficiency and central diabetes insipidus. Four also presented with or developed central hypothyroidism and three showed an abnormal growth curve, with maintenance of linear growth in conjunction with obesity. Despite initial cerebral sparing, progressive microcephaly was present in all patients in association with global developmental delay and seizures (onset 5 days to 2.5 years) as well as  hydronephrosis, vesicoureteric reflux and a neurogenic bladder. Using a combination of whole genome homozygosity mapping coupled with exome sequencing we have identified a single novel homozygous variant, c.1373_1374insTC, segregating between affected family members. This mutation resides in a transcription factor essential for normal hypothalamic development and is predicted to result in a frameshift and consequent loss of function. Moreover, levels of the mutant transcript were significantly reduced in patient fibroblasts compared to controls. We demonstrate high levels of expression in the hypothalamus, telencephalon and renal tract in the developing human embryo identical to that previously observed in the mouse.

Conclusion We describe a mutation in a transcription factor known to regulate development of the paraventricular, supraoptic and anterior periventricular nuclei in mice. The affected patients display several features of hypothalamic insufficiency, including obesity, diabetes insipidus, ACTH and TSH deficiency. Oxytocin and somatostatin are also likely to be deficient. The growth pattern of three of these children, the first human cases of probable somatostatin deficiency, is significantly abnormal with increased weight appearing to be the main driver of linear growth.

 

Nothing to Disclose: DK, EW, ANA, CB, EC, FL, CLA, AHA, AA, MA, MA, NM, BL, DB, SH, MJM, TK, RK, ES, PLB, DAT, KWC, FA, JPM, JCS, MTD

OR11-6 3455 6.0000 A ARNT2 Deficiency Causes a Multisystem Disorder with Hypothalamic Insufficiency 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Pediatric Endocrinology Saturday, June 15th 1:00:00 PM OR11 2330 11:30:00 AM Pediatric Endocrinology Oral


Bu Beng Yeap*1, Helman Alfonso2, Paul Chubb3, David J Handelsman4, Graeme J Hankey1, Jonathan Golledge5 and Leon Flicker1
1University of Western Australia, Perth, Australia, 2Curtin University, Perth, Australia, 3Fremantle Hospital, Fremantle, Australia, 4University of Sydney, Sydney NSW, Australia, 5James Cook University, Townsville, Australia

 

Context

In men testosterone (T) levels decline with increasing age, and previous studies have found that lower T levels predict increased mortality in older men. Whether reduced circulating T contributes to poorer health outcomes or reflects the presence of pre-existing disease remains debated. Furthermore the associations of its metabolites dihydrotestosterone (DHT) and estradiol (E2) with mortality are poorly defined.

Objective

We assessed the associations of circulating T, DHT and E2 with all-cause mortality in older men.

Participants

Community-dwelling men aged 70-89 years resident in Perth, Western Australia.

Main outcome measures

Plasma T, DHT and E2 were assayed using liquid chromatography-tandem mass spectrometry in early morning samples collected in 2001-04 from 3,690 men. Sex hormone-binding globulin (SHBG) and luteinising hormone (LH) were measured by immunoassay. Deaths to 31 December 2010 were ascertained using the Western Australian Data Linkage System. Cox proportional hazards regression was performed to assess associations of hormones with all-cause mortality. Adjustments were made for age, education, smoking, body mass index, waist:hip ratio, hypertension, dyslipidemia, diabetes, creatinine, cancer and cardiovascular disease.

Results

There were 974 deaths (26.4%) at a median follow-up of 7.1 years. Men who died had lower baseline levels of T (mean±SD: 12.8±5.1 vs 13.2±4.8 nmol/L, p=0.013), DHT (1.4±0.7 vs 1.5±0.7 nmol/L, p=0.002) and E2 (71.6±29.3 vs 74.0±29.0 pmol/L, p=0.022) compared to surviving men. After adjusting fully for covariates, there were U-shaped associations of T (reference lowest quartile, Q1: hazard ratio [HR] for Q2=0.82, 95% confidence interval [CI]=0.69-0.98, p=0.033; HR for Q3=0.78, 95% CI= 0.65-0.94, p=0.010) and DHT (reference Q1: HR for Q3=0.76, 95% CI=0.63-0.91, p=0.003) with all-cause mortality. E2 was not associated with mortality in the regression analysis. When either SHBG or LH were included with T in multivariable models, the association of T with death from any cause remained significant.

Conclusions

Older men with mid-range levels of T and DHT exhibited the lowest rates of death from any cause, after adjustment for other risk factors. T predicted mortality independently of LH and SHBG. Optimal androgen exposure may be a contributing factor or robust biomarker for survival. Interventional studies are needed to clarify whether modulating T levels would improve male longevity.

 

Nothing to Disclose: BBY, HA, PC, DJH, GJH, JG, LF

OR12-1 6806 1.0000 A Differential associations of plasma testosterone, dihydrotestosterone and estradiol measured using liquid chromatography-tandem mass spectrometry with all-cause mortality in a population-based cohort of older men 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Reproductive Endocrinology Saturday, June 15th 1:00:00 PM OR12 2349 11:30:00 AM Reproductive Hormones: Vital Effects Evidenced In Human Cohorts & Experimental Models Oral


Andre B. Araujo*1, Robert Chang2, Shan Chen2, Nicholas Dagincourt2, Gretchen Chiu1, Elizabeth Suarez2, Rachael Gerber2, Julia Akeroyd2, Carrie G. Wager2 and Benedetta Bartali2
1New England Research Institutes, Watertown, MA, 2New England Research Institutes, Inc., Watertown, MA

 

As men age, testosterone (T) levels decline, estradiol (E2) levels either decline or remain stable, and sex hormone-binding globulin (SHBG) levels increase. Compared with T, E2 has a stronger influence on bone strength, an observation confirmed in our Boston Area Community Health/Bone (BACH/Bone) Survey. There are, however, relatively few population-based studies of men which have examined the longitudinal associations of T, E2, and SHBG with bone loss. The objective of this study was to examine the relation of total T (TT), total E2 (TE2), and SHBG with bone loss at the femoral neck. We used data from the BACH/Bone Survey, a population-based cohort study of 1219 racially diverse men. Subjects were examined at baseline and follow-up visits. Femoral neck bone mineral density (fnBMD) was assessed by dual x-ray absorptiometry (DXA) using the same scanner at baseline and follow-up. Multivariable linear regression was used to examine the independent associations of baseline TT, SHBG (both by immunoassay), and TE2 (mass spectrometry) concentrations (quartiles (Q) and continuous) with percent (%) change in fnBMD, controlled for baseline fnBMD, age, race/ethnicity, income, smoking, type 2 diabetes mellitus, self-rated health, and DXA-derived total body fat and lean mass. A total of 692 men (70% of eligible men) with mean±SD baseline age 51±12y completed follow-up examinations 7.0±0.6y later. Mean±SD TT was 425±169ng/dL, TE2 was 24.01±9.71pg/mL, and SHBG was 35.34±17.09nmol/L. Estimated (relative standard error; RSE) % decline in fnBMD was 5.0(0.2)% during follow-up (annualized decline, 0.71%/y). Mean fnBMD declines were 5.7(0.5)% in Q1 of TT compared with 6.2(0.5)% among men in Q4; for TE2, fnBMD declined by 5.0(0.6)% in Q1 vs. 5.8(0.6)% in Q4; for SHBG, fnBMD declined by 5.1(0.5)% in Q1 vs. 6.0(0.5)% in Q4. In a multivariable model with TT, TE2, SHBG, and covariates, SHBG (Q, p=0.08, continuous, p=0.01) but not TT (Q, p=0.15, continuous, p=0.27) or E2 (Q, p=0.66, continuous, p=0.52) was significantly associated with change in fnBMD. Based on this model, decline in fnBMD was 1.0(0.4)% greater with each SD increase in SHBG. The SHBG-associated decline in fnBMD appeared to be most pronounced in black men, with each SD increase in SHBG associated with an additional 1.6% decrease in fnBMD (vs. white men), but age did not modify the relation of SHBG with decline in fnBMD. In conclusion, declines in fnBMD in this relatively young and racially diverse population-based cohort of men are consistent with previous studies. These data support mounting evidence for SHBG either having an independent effect on, or being a marker of, health outcomes of relevance to the health of aging men.

 

Disclosure: ABA: Principal Investigator, Abbott Laboratories, Principal Investigator, GlaxoSmithKline, Consultant, Lilly USA, LLC. Nothing to Disclose: RC, SC, ND, GC, ES, RG, JA, CGW, BB

OR12-2 8607 2.0000 A Sex hormones in relation to bone loss at the femoral neck among racially/ethnically diverse middle-aged men 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Reproductive Endocrinology Saturday, June 15th 1:00:00 PM OR12 2349 11:30:00 AM Reproductive Hormones: Vital Effects Evidenced In Human Cohorts & Experimental Models Oral


Karine Braun1, Irène Netchine2, Paula Borrego1, Hélène Bony-Trifunovic1, Corinne Fouveaut3, Chrystel Leroy3, Christine Grapin4, Yara Rhayem3, Catherine Dodé3 and Najiba Lahlou*5
1CHU d'Amiens, AMIENS, France, 2Hopital Trousseau-Université Pierre et Marie Curie, PARIS, France, 3Hopital Cochin-Université Paris-Descartes, PARIS, France, 4Hopital Robert-Debre-Université Paris-Diderot, PARIS, France, 5Paris-Descartes University , APHP, Cochin, PARIS

 

Context. The persistance of Mullerian duct syndrome (PMDS) is a rare condition, clinically expressed as cryptorchidism and inguinal hernias. Mutations in the anti-Mullerian hormone gene (AMH) account for nearly half of reported cases. Very scarse data are available regarding testis function in the affected subjects.

Subjects. We have encountered 3 novel mutations, together with already known ones, in 4 infants operated on for inguinal hernia and bilateral cryptorchidism. Mullerian remnants were discovered during surgery and were not removed in order to preserve testis vascularization. Histological examination of testes on tissue samples collected during surgery showed seminiferous tubules containing germ cells. One boy, born from consanguineous parents, harbored the homozygous mutation p.W121X, c.363G>A. Three unrelated patients had the following genotypes: p.G74W / p.R302Q; p.Y167C / c.343-344delCT and p.E382X / p.E382X, respectively.

Methods. FSH and LH were measured by means of time resolved fluoroimmunoassay on Wallac Delfia autoanalyzer (PerkinElmer, Courtaboeuf, France). Testosterone was measured by mass spectrometry on Quattro Premier equipment (Waters, Saint-Quentin en Yvelines, France). Insulin-like peptide 3 (INSL3) was measured by fluoroimmunoassay (Phoenix reagents, Burlingame CA), inhibin B (INHB) and AMH by means of chemiluminescence immunoassay (Anshlabs reagents, Webster TX). Assays were performed on samples collected before and / or after surgery in these 4 infants.

Results. In all boys AMH was undetectable. FSH and LH, testosterone, INSL3 and INHB levels were within normal ranges for age. Gonadotropins and testis hormones exhibited the normal minipubertal postnatal changes, with higher levels of FSH (1.4-2.6 IU/L), LH (2.58-3.17 IU/L), testosterone (2.47-2.6 nmol/L), INSL3 (68-96 pg/ml) and inhibin B 187-221 pg/ml) at 3-5 months of age than afterwards (0.08-0.44 IU/L, 0.04-0.54 IU/L, 0.04-0.45 nmol/l, 38-40 pg/ml, and 110-117 pg/ml, respectively).

Conclusion. These data give evidence of normal pituitary-testis function in infants with PMDS despite cryptorchidism and surgical manipulation. Given the uncertainty regarding fertility of  adult males harboring a mutation in the AMH gene, a prolonged and close follow up of such patients should likely be beneficial to assess their gonadal prognosis.

 

Nothing to Disclose: KB, IN, PB, HB, CF, CL, CG, YR, CD, NL

OR12-3 8693 3.0000 A PHENOTYPE GENOTYPE CORRELATIONS IN 4 YOUNG MALE PATIENTS HARBORING MUTATIONS IN THE AMH GENE : HORMONAL EVIDENCE OF NORMAL LEYDIG AND SERTOLI CELL COMPLEMENTS 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Reproductive Endocrinology Saturday, June 15th 1:00:00 PM OR12 2349 11:30:00 AM Reproductive Hormones: Vital Effects Evidenced In Human Cohorts & Experimental Models Oral


Olayiwola O. Oduwole*, Hellevi Peltoketo, Jennifer H. Steel and Ilpo T. Huhtaniemi
Imperial College London, London, United Kingdom

 

The gonadotropins LH and FSH are essential regulators of male and female fertility. Lack of these hormones or their cognate receptors result in a wide range of hypogonadal phenotypes and infertility in humans and mice. Constitutive activation of the gonadotropin receptors also disturbs the function of the pituitary-gonadal axis. Transgenic expression of an activating mouse FSH receptor mutant (D580H) in granulosa cells, under the anti-Müllerian hormone promoter, leads to ovarian hyperstimulation with increased estradiol biosynthesis, premature loss of follicles, ovulation disturbances, hemorrhagic ovarian cysts and ovarian teratomas1. Interestingly, transgenic males, expressing the mutant FSH receptor in Sertoli cells, remain largely normal. To explore in more detail the effects of amplified FSH action we now crossed the mFSHRD580H transgenic mice with the hypogonadal LH receptor knockout (LuRKO) mice, presenting as homozygotes with hypogonadism and infertility2.

The phenotype of female mFSHRD580H x LuRKO crossbreeds mimicked closely that of LuRKO females with hypogonadism and arrested follicular maturation. The double-mutant males, however, presented with rescue of fertility, with normal testes size and spermatogenesis, and partially recovered seminal vesicle size. Their puberty, as monitored by balano-preputial separation, was delayed, but after reaching the full sexual maturity they were able to sire litters similar to wild-type males. Histological analysis of the testes of adult mice revealed seminiferous tubuli with normal diameter, but total absence of discernible Leydig cells in the interstitial space. The delayed puberty and the partially recovered size of the seminal vesicles were apparently due to incomplete rescue of testosterone production. Our findings suggest that excessive FSH action, in the absence of LH-stimulated Leydig cell function, is able to induce in Sertoli cells paracrine factor(s) that stimulate the rudimentary Leydig cells to produce testosterone. This is sufficient to maintain normal spermatogenesis, but insufficient for fully normal extragonadal androgen actions.

 

Nothing to Disclose: OOO, HP, JHS, ITH

OR12-4 6959 4.0000 A A constitutively active mutant FSH receptor is able to restore fertility in male mice with hypogonadal LH receptor null background 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Reproductive Endocrinology Saturday, June 15th 1:00:00 PM OR12 2349 11:30:00 AM Reproductive Hormones: Vital Effects Evidenced In Human Cohorts & Experimental Models Oral


Evan Easton, Pawan Puri, William H. Walker and Jeyasuria Pancharatnam*
University of Pittsburgh, Pittsburgh, PA

 

Testosterone targets the androgen receptor (AR) to produce the factors that are required, for male secondary sexual characteristic development, prostate function, germ cell development and survival, muscle growth and other AR related functions. We are developing a transgenic mouse model for AR in which wildtype AR expression can be replaced by a functionally modified AR transgene. The AR gene is present on the X chromosome, which makes genetic manipulation of this gene in the male difficult, as there is only a single copy of the gene. In this pilot study we developed a mouse transgenic model of AR using a bacterial artificial chromosome (BAC) construct containing the entire AR gene including 40 kb each of 5' and 3' regulatory sequence. A recombinereed internal ribosome entry site (IRES) and EGFP cassette was inserted in the 3' UTR of the AR transgene thus allowing co-expression of EGFP and AR. To produce this final construct, we combined two BACs with the relevant 5’ and 3’ sequences by recombineering. Pronuclear injection of the BAC (236 kb) resulted in 9 founders and 5 of these founders expressed EGFP in appropriate AR expressing tissues.

Our interest in the non-classical action of AR in the Sertoli cell led us to test the AR construct in a SCARKO (Sertoli cell specific knockout of AR) background to determine whether AR expression by the BAC transgene was able to rescue the SCARKO phenotype. The 5 founders showed different degrees of rescue with 4 founders having spermatogenesis completely rescued whereas 1 founder displayed a partial rescue to the early elongated spermatid stage of development in 10% of seminiferous tubules. In one founder strain having complete rescue, 4 male mice produced offspring in 6 out of 8 matings. These mice provide the first in vivo model in which an AR transgene is capable of rescuing AR function in a null background. Furthermore, these initial studies provide proof of principle that the same strategy can be employed to create additional transgenic mouse models in which endogenous AR is replaced with AR mutants that selectively activate the classical or non-classical testosterone signaling pathway. Analysis of these planned model mice will identify the processes and factors regulated by the classical or non-classical action of testosterone in vivo as well as provide needed information regarding the molecular and cellular mechanisms by which testosterone acts in Sertoli cells to maintain spermatogenesis and fertility.

 

Nothing to Disclose: EE, PP, WHW, JP

OR12-5 9241 5.0000 A Spermatogenic Rescue of the SCARKO Mouse Model Using a Recombineered 240 kb Androgen Receptor (AR) Transgene 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Reproductive Endocrinology Saturday, June 15th 1:00:00 PM OR12 2349 11:30:00 AM Reproductive Hormones: Vital Effects Evidenced In Human Cohorts & Experimental Models Oral


Fang-Ju Wu*
National Yang-Ming University, Taipei, Taiwan

 

Bone morphogenetic proteins (BMPs) have been known to be indispensable for the reproductive system. A previous study indicates that targeted depletion of Bmp8b causes male infertility due to the reduction of germ cells before puberty, indicative of its importance in the maintenance of spermatogenesis. However, no direct signaling pathway of BMP8 in cells has been clarified in vitro due to the lack of functional BMP8 proteins. Therefore, we aimed to unveil the BMP8 downstream signaling and clarify its function in the testis. Firstly, we generated recombinant BMP8 protein and found that the bioactive BMP8 protein mediates the BMP signaling by promoting the phosphorylation of Smad1/5/8. Overexpression of either ALK3 or 6 in cells enhances the BMP8 signaling, indicating they are the type I receptors of BMP8. In addition, shRNA knockdown experiments demonstrated that ACVR2A and BMPR2 can serve as the type II receptors for BMP8. BMP8 is shown to be expressed in the testis of 4-day-old mice by immunoblotting and its expression is further localized at spermatogonia by immunocytochemical staining. By isolating and culturing neonatal mouse spermatogonia, we found that BMP8 induces differentiation of spermatogonia by increasing Kit expression through the Smad1/5/8 pathway. We also found that the expression levels of self-renewal-related genes such as Bcl6b, Etv5 and Lhx1, which are up-regulated by Sertoli cell-derived GDNF, are down-regulated upon the addition of BMP8. These results conclude that BMP8 participates in the maintenance of spermatogenesis by promoting spermatogonia differentiation through the Smad1/5/8 pathway.

 

Nothing to Disclose: FJW

OR12-6 4713 6.0000 A BMP8 maintains spermatogenesis by promoting spermatogonia differentiation through Smad1/5/8 pathway 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Reproductive Endocrinology Saturday, June 15th 1:00:00 PM OR12 2349 11:30:00 AM Reproductive Hormones: Vital Effects Evidenced In Human Cohorts & Experimental Models Oral


Youn Hee Jee*1, Francesco S. Celi2, Electron Kebebew3, Maureen Sampson4, David B. Sacks4, Alan Remaley4 and Jeffrey Baron5
1NICHD, NIH, Bethesda, MD, 2National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD, 3National Cancer Institute, NIH, Bethesda, MD, 4Clinical Center, NIH, Bethesda, MD, 5NIH, Bethesda, MD

 

Background:The primary preoperative method for distinguishing malignant from benign thyroid nodules is fine-needle aspiration (FNA) cytology, but it is frequently inconclusive. Midkine is a heparin binding growth factor which is preferentially expressed in papillary thyroid carcinoma (PTC). We hypothesized that midkine concentration in PTC would be higher than in benign lesions and therefore its measurement in FNA samples might aid in the preoperative diagnosis of thyroid nodules.

Methods: 44 subjects (age 46 ± 11 y, 9 male) underwent thyroidectomy providing a histological diagnosis. Of these, 24 also underwent preoperative FNA. FNA needle contents were expressed for cytology and then the needle was washed with buffer for immunoassay. For subjects without preoperative FNA samples, FNA was performed ex vivo on surgically excised tissue. Midkine and thyroglobulin were measured using a high-sensitivity sandwich ELISA and chemiluminescent immunoassay, respectively. Samples with thyroglobulin < 50 ng/mL were excluded on the assumption of insufficient tissue sampling. Values from multiple passes (1-4/nodule) were averaged.

Results:Midkine concentration was higher in 11 PTCs (4 classical, 4 tall cell variant, 3 follicular variant) than in 40 benign nodules (34 adenomatoid nodules including 4 hyperplastic, 4 follicular and 2 Hurthle cell adenomas) (0.5 ± 0.2 vs 0.08 ± 0.03 ng/mL, mean ± SEM, p = 0.004). To adjust for tissue content and to enhance differences between benign and malignant samples, midkine was normalized to thyroglobulin. Midkine/thyroglobulin ratio in PTC was greater than in benign nodules (297 ± 163 vs 1.5 ± 0.4 ng/mg, p<0.001). Using a threshold of 10 ng/mg, the sensitivity and specificity of the midkine/thyroglobulin ratio for diagnosis of PTC were 64% and 100%, respectively. The area under the ROC curve was 0.84. All follicular variant PTCs had low midkine/thyroglobulin (1.0 ± 0.6 ng/mg). Plasma midkine concentrations did not differ between subjects with benign nodules and with PTC (0.26 ± 0.03 ng/mL vs 0.19 ± 0.02 ng/mL, p = NS).

Conclusion:In FNA samples, the midkine/thyroglobulin ratio in PTC is greater than in benign thyroid nodules. Because FNA cytology is often non-diagnostic, the midkine/thyroglobulin ratio may represent a novel adjunctive diagnostic tool to help identify malignancies. Larger studies are warranted to confirm the diagnostic utility for PTC and investigate the utility for other thyroid cancers.

 

Nothing to Disclose: YHJ, FSC, EK, MS, DBS, AR, JB

OR14-1 6510 1.0000 A Midkine Concentration in FNA Washout: a Novel Diagnostic Approach to Identify Papillary Thyroid Carcinoma 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Thyroid/HPT Axis Saturday, June 15th 1:00:00 PM OR14 2365 11:30:00 AM Thyroid Cancer: Insights into Diagnosis & Treatment Oral


Brian C Netzel*1, Robert L Taylor1, Ravinder J. Singh1 and Stefan Karl Gunther Grebe2
1Mayo Clinic, Rochester, MN, 2Mayo Clinic and Foundation, Rochester, MN

 

Introduction: Serum thyroglobulin (Tg) testing is a key part of thyroid cancer follow-up. However, Tg auto-antibodies (TgAB) can lead to false-low/false-negative measurements in standard immunometric assay. Recently, it has been shown that proteomic Tg analysis by mass spectrometry (MS) overcomes TgAB interferences. Tg, and any TgAB, in patient serum are digested using proteases. Tg-proteotypic peptides can then be monitored and quantified to give true Tg concentrations regardless of TgAB status. However, 1st generation Tg MS assays showed suboptimal detection sensitivity. To improve this careful consideration must be given to peptide selection and specimen preparation, as these will affect assay sensitivity and specificity.

Materials and Methods: 250mL of serum was selectively depleted of middle and low molecular weight proteins. After re-suspension, specimens were reduced, alkylated and digested with trypsin for 16h. Following trypsin inactivation, we used protein-G paramagnetic beads (Life Technologies, Carlsbad, CA) with bound anti-peptide monoclonal antibodies against the peptide sequence FSPDDSAGASALLR (SISCAPA Assay Technologies, Vancouver, BC) for further purification, before analyis on an API 5000 triple quadrupole mass spectromter (AB SCIEX, Foster City, CA). MS analysis was performed in positive electrospray ionization (ESI) mode, monitoring three transitions for both the proteotypic peptide and its internal standard.

Results: Compared with a 1st generation Tg MS assay, which we have previously described (1), the choice of a different peptide target combined with target peptide enrichment reduced the limits of detection (LOD) and quantification (LOQ) 5-10 fold,  from 2 and 5ng/mL to 0.2 and 1ng/mL, respectively. Method agreement between the two generations of MS assays (N = 48) was excellent (slope: 1.01, y-intercept: 0.40, R2: 0.991). Comparison with the Beckman DXI immunoassay (N = 70) in TgAB-negative patients showed a slope of 0.95, intercept 1.2 and R2 0.91. In TgAb positive specimens (N= 42) the corresponding figures were slope: 1.44, y-intercept: 0.02, and R2: 0.97.  Imprecision (CV) was 4.1 – 7.1% (Tg range 2 – 100ng/mL). Spiked recovery (% predicted) across the detectable range was 87-110%.

Conclusion: Peptide selection and specimen preparation are crucial for optimal analytical sensitivity and specificity of Tg by MS. Factors to be considered include ionization potential of individual peptides, ease of specific fragmentation, and absence of peptide polymorphisms in the target peptide that might render it invisible to MS detection. Based on these principles, our 2nd generation Tg MS assay now achieves a LOD and LOQ that make it suitable as a viable alternative for modern immunometric assays, when TgAB interference is suspected.

 

Nothing to Disclose: BCN, RLT, RJS, SKGG

OR14-2 8806 2.0000 A A 2nd generation mass spectrometric serum thyroglobulin (Tg) assay achieves analytical performance similar to many immunometric Tg assays and is immune to Tg autoantibody interferences 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Thyroid/HPT Axis Saturday, June 15th 1:00:00 PM OR14 2365 11:30:00 AM Thyroid Cancer: Insights into Diagnosis & Treatment Oral


David Viola*1, Laura Valerio1, Pinuccia Faviana1, Eleonora Molinaro1, Elisa Sensi1, Laura Agate1, Gabriele Materazzi1, Fulvio Basolo2, Paolo Miccoli1, Paolo Vitti3 and Rossella Elisei1
1University of Pisa, Pisa, Italy, 2University of Pisa, Italy, 3University Hospital, Pisa, Italy

 

Differentiated thyroid cancer (DTC) is the most common endocrine malignancy and commonly metastasizes to locoregional lymph nodes (up to 65%). Despite its lymphotropism that is considered a risk factor for persistent/recurrent disease the clinical benefit of prophylactic central compartment lymph node dissection (pCCND) in DTC is still controversial. This treatment seems to reduce DTC recurrence rates and mortality. However the lack of prospective randomized trials and a higher rate of surgical complications represent the major concerns. Moreover the increasing incidence of DTC that from some authors are considered indolent arises the necessity to better define the optimal treatment for DTC patients (pts).

The aim of this prospective randomized controlled study was to evaluate the advantages and disadvantages of pCCND, in particular: the outcome, the rate of surgical complications and the possible predictors of central compartment lymph node metastases of DTC pts treated with either total thyroidectomy (TTx) or TTx and pCCND.

A total of 169 DTC pts without evidence of preoperative/intraoperative lymph node metastases (N0) were randomly assigned to TTx, (Group-A, n=84) or TTx with pCCND (Group-B, n=85).

The two groups did not differ for the epidemiological and clinical-pathological features but, as expected, only from a high prevalence of microscopic central compartment lymph node metastases (N1a) in Group-B (50%). After a mean follow-up of 3.5 years no difference was observed in the outcome of the two groups. Group-A pts were treated with a higher number of 131I courses (p=0.0017) than Group-B while a higher prevalence of permanent hypoparathyroidism was observed in Group-B (p=0.046). Among Group-B, N1a pts had a higher prevalence of extrathyroid extension (p=0.002) and advanced stage (p=0.046), no other predictors of central compartment lymph node metastases were found. Moreover pCCND “upstaged” 3.5% of pts and in 1.2% affected radioiodine treatment decision.

In conclusion no preoperative features of DTC could suggest for or against central compartment lymph node dissection. Total thyroidectomy alone was as effective as total thyroidectomy with prophylactic central compartment lymph node dissection with a lower rate of surgical complications but the necessity of a higher number or radioiodine courses. Neck dissection will increase the number of patients that according to the European and American guidelines need to be treated with radioiodine.

 

Nothing to Disclose: DV, LV, PF, EM, ES, LA, GM, FB, PM, PV, RE

OR14-3 6264 3.0000 A Advantages and disadvantages of prophylactic central compartment lymph node dissection for differentiated thyroid cancer: the first randomized controlled study from a single referral center 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Thyroid/HPT Axis Saturday, June 15th 1:00:00 PM OR14 2365 11:30:00 AM Thyroid Cancer: Insights into Diagnosis & Treatment Oral


Carol Li*1, Patricia A. Han1, Kathleen C. Lee1, Louis C. Lee2, Amy C. Fox3, Toni Beninato4, Michele Thiess1, Thomas J Sebo5, Geoffrey B Thompson2, Clive S. Grant2, Thomas J Giordano3, Paul G. Gauger3, Gerard M. Doherty3, Thomas J. Fahey III4, Justin Bishop1, James R. Eshleman1, Christopher B Umbricht1, Eric B. Schneider1 and Martha A. Zeiger1
1Johns Hopkins University School of Medicine, Baltimore, MD, 2Mayo Clinic, Rochester, MN, 3University of Michigan Health Systems, Ann Arbor, MI, 4Weill Cornell Medical Center, New York, NY, 5Mayo Clinic College of Medicine, Rochester, MN

 

Does BRAF V600E Mutation Predict Aggressive Features in Classic Papillary Thyroid Cancer? Results from Four Endocrine Surgery Centers

Introduction: Existing evidence is controversial regarding the association between BRAF mutation status and central lymph node metastases (CLNM) in patients with papillary thyroid cancer (PTC) and therefore, whether to use BRAF as an indication for central lymph node dissection (CLND). Importantly, no study has incorporated multiple endocrine surgery practices that perform routine CLND for PTC and thus have patients who are truly evaluable for the presence of CLNM. 

Methods: Under IRB approval, consecutive patients with classical variant PTC who underwent total thyroidectomy and CLND as part of routine surgical practices at 4 tertiary endocrine surgery centers between January 2009 and December 2011 were retrospectively reviewed. BRAF mutation status was determined by pyrosequencing. Standard descriptive and bivariable analyses examined demographic, patient, and tumor-related factors. Multivariable logistic regression controlling for gender, age≥45, tumor size >2 cm, extrathyroidal extension (ETE), surgical margin involvement, lympho-vascular invasion (LVI) and multifocality examined the odds of CLNM associated with positive BRAF status.

Results:  A total of 315 individuals (70 males and 245 females) from the 4 centers, of whom 239 underwent prophylactic and 76 underwent therapeutic CLND were eligible for study.  253 (80.3%) patients were positive for the BRAF mutation.  59.3% of BRAF positive vs. 51.6% of BRAF negative patients had CLNM (p=0.27). Bivariable analysis also demonstrated no significant relationship between positive BRAF mutation and gender, age, tumor size, multifocality, LVI, surgical margin involvement, lateral lymph node metastasis, ETE, or TNM stage. In the multivariate analysis, positive BRAF mutation was not associated with CLNM. Multivariate analysis including only factors potentially available preoperatively (BRAF mutation, age, gender, and size) also showed no significant correlation between BRAFmutation and CLNM. 

Conclusion: This is the first multi-institutional study that included only patients who underwent CLND as part of routine surgical practice and examined the association of BRAF mutation status and aggressive features of PTC. Our results show that in patients with classical PTC, BRAF mutation is not an independent predictor of CLNM and therefore may not be useful for decisions regarding initial surgical management. Prospective studies are therefore needed before BRAF mutation analysis should be incorporated into a surgical algorithm.

 

Disclosure: MAZ: Principal Investigator, Veracyte, Inc.. Nothing to Disclose: CL, PAH, KCL, LCL, ACF, TB, MT, TJS, GBT, CSG, TJG, PGG, GMD, TJF III, JB, JRE, CBU, EBS

OR14-4 4117 4.0000 A Does BRAF V600E Mutation Predict Aggressive Features in Classic Papillary Thyroid Cancer? Results from Four Endocrine Surgery Centers 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Thyroid/HPT Axis Saturday, June 15th 1:00:00 PM OR14 2365 11:30:00 AM Thyroid Cancer: Insights into Diagnosis & Treatment Oral


Ramona Dadu*1, Mimi I-Nan Hu2, Mouhammed Amir Habra2, Steven G Waguespack2, Anita K Ying2, Naifa L Busaidy2 and Maria E Cabanillas2
1Baylor College of Medicine, Houston, TX, 2The University of Texas MD Anderson Cancer Center, Houston, TX

 

Background:  Treatment with a TKI is considered for metastatic, progressive, iodine-refractory DTC. However, patients will eventually develop progressive disease (PD) or intolerability leading to drug discontinuation. Sorafenib is often used as first line therapy in DTC. For papillary thyroid cancer patients treated with first line sorafenib, previously reported median overall survival (OS) and progression free survival (PFS) were 92 and 54 weeks, respectively(1). Treatment with TKIs with similar mechanism of action after sorafenib failure is common in clinical practice, but no data are available with regards to benefit of sequential treatment. We hypothesize that salvage TKIs use after sorafenib failure represents a viable treatment option in selected patients. With the increasing use of TKIs in clinical practice, this information will be important to physicians who are facing these challenging cases.

Methods: We performed a retrospective analysis of DTC patients who received salvage therapy with TKIs after first line sorafenib failure. Sorafenib failure was defined as PD or unacceptable toxicity leading to drug discontinuation. The objective was to compare the median time on treatment with sorafenib and second line TKIs and to assess the median OS.

Results:  Twenty-one adult patients were identified (12 female, 9 male): 12 follicular, 8 papillary and 1 poorly differentiated carcinoma. Median age at diagnosis was 54 years (range 39-74). The majority had T3 and T4 tumors at diagnosis; median tumor size was 3.5 cm (range 1.8-9.5). Prior treatment included surgery (95%), radioactive iodine (100%), and radiation to the neck (33%). All patients had RAI refractory disease. Prior to sorafenib start, neck disease was present in 17/21 (81%) patients; most common sites of distant metastases included lung in all and bone in 9/21 (43%) patients. Median time from diagnosis to sorafenib start was 48 months (range 4-136). Sorafenib hold and dose reduction was needed in 13/21 (62%) and 10/21 (58%) patients, respectively. The reason for sorafenib discontinuation was PD in 19/21 (90%) and toxicity in 2/21 (10%) patients. Salvage therapy included sunitinib (8), pazopanib (2), vemurafenib (2) and investigational TKIs (9). Median time on treatment with sorafenib and salvage therapy was 46 and 61 weeks respectively (p=0.26). Estimated median OS was 166 weeks.

Conclusions:  Duration of treatment is comparable between first and second line therapy suggesting an added clinical benefit of salvage TKI use after sorafeib failure. The median OS of our group of patients receiving multiple line TKIs after sorafenib failure is higher than previously reported on patients receiving first line sorafenib.  This small retrospective study supports the potential utility of salvage TKI use after sorafenib failure, but further study, including a comparison against a control group and a prospective study, will be required.

 

Disclosure: MAH: Clinical Researcher, Eisai. NLB: Clinical Researcher, Bayer, Inc.. MEC: Clinical Researcher, Exelixis, Inc., Clinical Researcher, Eisai, Clinical Researcher, Roche Diagnostics, Advisory Group Member, Exelixis, Inc., Advisory Group Member, Eisai. Nothing to Disclose: RD, MINH, SGW, AKY

OR14-5 6648 5.0000 A Salvage therapy with tyrosine kinase inhibitors (TKIs) for differentiated thyroid carcinoma (DTC) after first line sorafenib failure 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Thyroid/HPT Axis Saturday, June 15th 1:00:00 PM OR14 2365 11:30:00 AM Thyroid Cancer: Insights into Diagnosis & Treatment Oral


Karen Gomez-Hernandez1, Sonia Cheng2, Shereen Z Ezzat2, Sylvia L. Asa2 and Ozgur Mete*2
1University Health Network, University of Toronto, Toronto, ON, Canada, 2University Health Network, Toronto, ON, Canada

 

The diagnosis and classification of papillary carcinoma (PTC) is challenging because of variable criteria. There are limited data on the relationship between somatic mutational profiling and tumor behaviour. In this study, we aimed to identify genetic signatures of the major morphologic thyroid cancer histotypes and their relationships with pTNM status and MACIS scoring.

DNA from formalin-fixed paraffin-embedded sections of 131 PTCs was subjected to a panel of 23 multiplexed assays interrogating 286 mutations in 23 genes on a MassARRAY platform (Sequenom). Mutation calling was determined using TyperAnalyzer software and manual analysis. Mutational status was compared with histological architecture, pTNM stage, MACIS score, and recurrence rate.

There were 68 mutations in 65 tumors: BRAFV600E (n: 40), BRAFK601E (n:1), EGFRS768I (n: 2); HRASQ61K (n:5), KRASG12C (n:4), KRASG12D (n:1), KRASQ61R (n: 2), NRASQ61K (n:3), NRASQ61R (n:8), STK11W332 (n:1) and STK11F354L (n: 1). Three samples revealed more than one mutation. Classical (papillary) and follicular architecture was seen in 67 and 64 PTCs, respectively. Complete pathologic data was available in 128 patients allowing complete pTNM stage and MACIS assignment. Thirteen patients received were not followed at our institution after surgical and radioactive iodine treatment; the remaining 115 patients had a mean follow up of 6.26 years. Our results demonstrate that the BRAFV600E mutation correlates strongly with classical architecture (p=0.001). While BRAFV600E mutation was associated with increasing frequency of lymph-nodal involvement and hence pTNM score (p=0.002), this relationship was abolished when histologic architecture was accounted for. Moreover, within the classic variant PTC group, BRAF status was not associated with pTNM or MACIS scores. In contrast, FV-PTC was more frequently associated with the RAS mutations (p=0.05). However, RAS mutations were not associated with clinical outcomes including staging and recurrence.

Our results highlight the importance of morphologic classification of PTC tissue architecture. Tumors with classical papillary architecture are more likely to feature a predominantly BRAFV600E signature, however, they display more aggressive clinical behavior independent of mutation status. In contrast, FV-PTCs are more frequently associated with an RAS signature whose contribution to the relatively more benign behavior of this tumor type requires longer-term outcome studies.

 

Nothing to Disclose: KG, SC, SZE, SLA, OM

OR14-6 7901 6.0000 A DISTINCT GENOTYPIC SIGNATURES CORRELATE WITH MORPHOLOGY BUT DO NOT INDEPENDENTLY PREDICT BEHAVIOUR IN PAPILLARY THYROID CANCER 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Thyroid/HPT Axis Saturday, June 15th 1:00:00 PM OR14 2365 11:30:00 AM Thyroid Cancer: Insights into Diagnosis & Treatment Oral


Irida Kastrati*1, Emanuele Canestrari2, Rajyasree Emmadi2 and Jonna Frasor1
1University of Illinois at Chicago, Chicago, IL, 2University of Illinois at Chicago

 

Pro-inflammatory cytokines and the NFkB pathway may contribute to more aggressive ER+ breast cancers through cooperative ER-NFkB crosstalk. We have shown that these factors work together to synergistically induce a specific gene signature associated with cell survival, the luminal B subtype of breast tumors, and poor outcome following endocrine therapy. Bioinformatic analysis has suggested that multiple genes in this ER-NFkB crosstalk signature are also putative targets of miR-181a and miR-181b, two miRs clustered in a polycistronic fashion within a long non-coding (lnc) RNA transcript. Previous studies have suggested that miR-181a/ b are down-regulated by E2 and that the lncRNA transcript may be ER regulated. However, reports on the function of miR-181a/b in breast cancer are contradictory. Therefore, we asked how these miRs are regulated by ER and NFkB, if they affect ER-NFkB crosstalk, and how they influence ER+ breast cancer biology. Treatment of MCF-7 breast cancer cells with estradiol (E2) resulted in ER recruitment to the lncRNA promoter, reduced levels of the lncRNA transcript, and a reduction in mature miR-181a/b levels. These effects are ER-dependent because pretreatment with ICI 182,780 or siER reversed the inhibitory actions of E2. Interestingly, the combination of E2 and TNFa caused further down-regulation of miR-181a/b transcripts compared to E2 alone, in an NFkB-dependent manner. We next overexpressed miR-181a/b mimics in MCF-7 cells and found that they reduced the expression of several genes that are up-regulated by ER-NFkB crosstalk. Previous work from our lab has indicated that ER-NFkB crosstalk can promote cell survival, therefore we examined miR-181a/b role on cell viability. Indeed, overexpression miR-181a/ b mimics results in reduced viability in an additive manner. A potential role for miR-181b in cancer stem cell biology was suggested by our finding that expression of miR-181b is reduced while ER-NFkB crosstalk genes are increased in MCF-7 cells grown as mammospheres compared to standard 2D culture. We also examined expression of miR-181a and b in human breast tumors and observed an inverse correlation between miR-181a/b levels and risk of recurrence, based on the OncotypeDx assay. Taken together, our findings suggest that the down-regulation of miR-181a/b by ER and NFkB may be detrimental in ER+ breast cancer, potentially through the increased ER and NFkB crosstalk, cell survival, and cancer stem cell properties, all of which may increase the risk of recurrence.

 

Nothing to Disclose: IK, EC, RE, JF

OR07-1 7836 1.0000 A An Estrogen Receptor / NFkappaB / miR-181a/b Feedforward Loop Promotes Breast Cancer Cell Survival and is Associated with Increased Risk of Recurrence 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Cancer/Neoplasia Saturday, June 15th 1:00:00 PM OR07 2375 11:30:00 AM New Players in Hormonal Control of Breast & Prostate Cancer Oral


Katarzyna Anna Ludwik* and Deborah Ann Lannigan
Vanderbilt University, Nashville, TN

 

Approximately 65% of breast cancers are estrogen receptor alpha (ERalpha)-positive and are treated with anti-estrogen therapy. However, half of these patients fail to respond to treatment and therefore, there is an ongoing effort to identify better prognostic markers of responsiveness to anti-estrogen therapy. Phosphorylation of ERalpha at Ser-167 (pSer-167) has been found to correlate with longer progression free survival of patients treated with anti-estrogens [3]. Furthermore, in these tumors high levels of pSer-167 positively correlated with active RSK [2]. We have previously found that RSK phosphorylates Ser-167 [1]. We investigated the relationship between active RSK and the responsiveness of ERalpha-positive breast cancer to anti-estrogen therapy. We found that in 25% of ERalpha-positive breast tumors active RSK was localized to the nucleus in regions of high ERalpha expression.  In response to mitogen RSK2 is activated and accumulates in the nucleus of the human breast cell line, MCF-7. Inhibition of RSK activity decreased pSer-167 levels, which indicates that RSK is the predominant kinase that phosphorylates ERalpha.  RSK2 physically associates with ERalpha, and therefore, we determined whether ERalpha could regulate RSK2 nuclear accumulation.  Silencing of ERalpha decreased mitogen-induced RSK2 nuclear accumulation by ~35%.  Similarly, anti-estrogen treatment caused an ~35% decrease in RSK2 nuclear accumulation. Anti-estrogens also decreased the levels of cyclin D1, a potent oncogene, by ~40%. RSK2 silencing or inhibition caused a similar reduction in cyclin D1 expression. Forced RSK2 nuclear localization, achieved by fusing RSK2 with a nuclear localization signal, was sufficient to induce cyclin D1 expression in the absence of mitogen.  Moreover, forced nuclear localization of RSK2 prevented the decrease in cyclin D1 levels caused by 4-hydroxy tamoxifen treatment.  Taken together, these data show that the ERalpha –mediated regulation of cyclin D1 levels occurs through a RSK2-dependent pathway. We propose that patients with high pSer-167 levels have high levels of nuclear RSK2 and that responsiveness to anti-estrogen therapy is a consequence of an ERalpha-dependent decrease in nuclear RSK2 accumulation, which results in reduced cyclin D1 expression and inhibition of tumor growth.

 

Nothing to Disclose: KAL, DAL

OR07-2 7492 2.0000 A Reduced nuclear accumulation of the Ser/Thr protein kinase, RSK2, explains responsiveness to anti-estrogen therapy in patients that have phosphorylation of estrogen receptor alpha at Ser-167 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Cancer/Neoplasia Saturday, June 15th 1:00:00 PM OR07 2375 11:30:00 AM New Players in Hormonal Control of Breast & Prostate Cancer Oral


María Celeste Diaz Flaqué*1, Natalia M Galigniana1, Cecilia J Proietti1, Rosalia Ines Cordo Russo2, Florencia Mercogliano1, Franco Izzo2, Mercedes Tkach1, Roxana Schillaci1 and Patricia Virginia Elizalde2
1Instituto de Biología y Medicina Experimental, CONICET, Buenos Aires, Argentina, 2Instituto de Biología y Medicina Experimental (IBYME-CONICET), Buenos Aires, Argentina

 

The molecular mechanisms through which the progesterone receptor (PR) controls breast cancer growth and response to endocrine treatments remain a major clinical challenge. PR acts as a transcription factor (TF) and as an activator of signaling pathways through a rapid or nongenomic mechanism. Furthermore, it participates in an extensive and bidirectional crosstalk with growth factors and estrogen receptor (ER) signaling. In the present work, we demonstrated that progestin treatment of breast cancer cells induces the rapid phosphorylation of c-Jun and c-Fos, (AP-1 TF), via p42/p44 MAPKs. AP-1 activation leads to the assembly at the proximal cyclin D1 promoter of a multi-component complex which functions as an enhaceosome, where AP-1 is loaded at its response element (TRE), PR is tethered to AP-1, the signal transducer and activator of transcription 3 (Stat3) is co-recruited to its binding sites located close to the TRE, and ErbB-2 is simultaneously loaded.  This complex drives in vitro and in vivo progestin-induced breast cancer growth. Here, we explored the clinical significance of PR and AP-1 nuclear interaction in breast tumors through a retrospective study in a cohort of 99 PR+ primary invasive breast carcinomas. Unexpectedly, evaluation of nuclear co-localization of PR and p-c-Jun by immunofluorescence and confocal microscopy was a marker of good prognosis and better overall survival (OS) in patients treated with tamoxifen (TAM), an anti-ER therapy. We found that 81 tumors showed nuclear colocalization of both proteins and was significantly associated with the absence of nodal metastasis. Kaplan-Meier survival analysis revealed that colocalization correlated with better OS both in the total cohort and in the subgroup of patients that received TAM (n=85). A rationale for these clinical findings was provided by our demonstration that TAM inhibited progestin-induced AP-1 transcriptional activation in BT474-HR breast cancer cells which are Herceptin resistant and sensitive to the antiproliferative effects of TAM. Our findings offer novel insight into the major clinical challenge of endocrine resistance, revealing that PR transcriptional complexes assembled via coordinated rapid and transcriptional PR actions govern breast cancer growth. These complexes are disrupted by TAM, rendering breast cancer cells sensitive to therapy.

 

Nothing to Disclose: MCD, NMG, CJP, RIC, FM, FI, MT, RS, PVE

OR07-3 4510 3.0000 A Responsiveness to Tamoxifen is associated with the assembly of an AP-1/Stat3/ErbB-2 non classical PR Transcriptional Complex in breast cancer patients 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Cancer/Neoplasia Saturday, June 15th 1:00:00 PM OR07 2375 11:30:00 AM New Players in Hormonal Control of Breast & Prostate Cancer Oral


Nicholas D'Amato*1, Hui Lyu1, Bolin Liu1, Haihua Gu1, Andrew Protter2, Anthony Elias3 and Jennifer K Richer1
1University of Colorado Anschutz Medical Campus, Aurora, CO, 2Medivation Inc., San Francisco, CA, 3Univ of Colorado Anschutz Medical Campus, Aurora, CO

 

Introduction: The androgen receptor (AR) is expressed in approximately 60% of Her2+ breast cancers. Activated AR elicits transcriptional upregulation of Her3. Her3 can heterodimerize with Her2, is essential for growth of Her2+ tumors, and has been implicated in therapeutic resistance to tamoxifen, paclitaxel, and trastuzumab. Enzalutamide (ENZA) is an anti-androgen that impairs nuclear entry of liganded AR, binds to AR with higher affinity than bicalutamide, and was recently approved for treatment of castrate-resistant prostate cancer.

Hypothesis: ENZA will enhance the efficacy of trastuzumab in Her2+ breast cancer lines by inhibiting Her3 expression.

Methods:We tested both estrogen receptor positive and negative Her2+ (amplified or overexpressing without amplification) breast cancer cell lines for androgen-induced upregulation of Her3 protein and inhibition of proliferation with trastuzumab or ENZA alone as compared to the two drugs combined. Resistance to trastuzumab was generated in two Her2+ breast cancer cell lines (SKBR3 and BT474) and these lines were also tested.

Results: Dihydrotestosterone (DHT) induced an increase in total Her3 and phospho-Her3 in some Her2+ BC cell lines and this effect was inhibited by the addition of ENZA.  The combination of ENZA and trastuzumab inhibited proliferation more effectively than either agent alone in the ER-/Her2+ MDA-MB-453, SUM185E, and SKBR3 cell lines. Interestingly, ENZA inhibited proliferation in MDA-MB-453 and SUM185PE cells (Her2 overexpression without amplification) as well or better than trastuzumab, whereas trastuzumab showed a greater inhibitory effect than ENZA in SKBR3 cells (Her2 amplified). In the ER+/AR+/Her2 amplified BT474 cell line, the combination of ENZA and trastuzumab inhibited proliferation significantly more than either drug alone. In the trastuzumab resistant lines, where trastuzumab alone is ineffective, ENZA combined with trastuzumab significantly inhibited proliferation.

Conclusions:  Our results suggest that ENZA may serve as an effective therapeutic in Her2+ breast cancer when combined with Her2-directed therapies such as trastuzumab, pertuzumab, or T-DM1. Furthermore, in tumors resistant to Her2 directed therapy, ENZA may be useful alone or in combination with anti-Her3 therapy. Targeting AR with ENZA in patients with Her2+ disease may result in therapeutic benefit and warrants clinical investigation.

 

Disclosure: AP: Employee, Medivation. Nothing to Disclose: ND, HL, BL, HG, AE, JKR

OR07-4 6452 4.0000 A Targeting Androgen Receptor in Her2-Driven Breast Cancer 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Cancer/Neoplasia Saturday, June 15th 1:00:00 PM OR07 2375 11:30:00 AM New Players in Hormonal Control of Breast & Prostate Cancer Oral


Michael D. Nyquist*1 and Yingming Li2
1University of Minnesota, Minneapolis, MN, 2University of Minnesota, Minneapolis

 

Understanding mechanisms by which advanced prostate cancer (PCa) progresses to a castration resistant PCa (CRPC) phenotype is crucial for tailoring targeted therapies for individual patients.  Constitutively active androgen receptor (AR) splice variants that lack the ligand binding domain drive androgen independent growth in cell lines and xenograft models of PCa and their overexpression in CRPC metastases is associated with poor prognosis.  However, the mechanism of AR splice variant overexpression in CRPC is not completely understood.  We have shown that diverse genomic rearrangements within the AR gene are linked to AR splicing alterations in PCa cell lines, xenografts, and CRPC metastases.  To test whether these AR gene rearrangements drive AR splice variant synthesis and a CRPC phenotype, we developed transcription activator-like effector nucleases (TALENs) for precision genome engineering in androgen-sensitive PCa cells. Engineering of specific AR gene rearrangements induced splicing alterations and efficient expression of AR splice variants.  Functionally, these genome-engineered cells displayed constitutive, ligand-independent AR transcriptional activity and an androgen-independent growth profile.  These fundamental properties of CRPC were blocked following siRNA-mediated knock-down of truncated AR variants.  Overall, these data demonstrate that AR gene rearrangements induce fundamental aspects of the CRPC phenotype including constitutive, ligand-independent, AR splice variant activity.  Overall, these data advance the concept of AR splice variants as key drivers of advanced disease, and provide new genome-engineered models that could be used to develop agents targeted to these AR-derived species.

 

Nothing to Disclose: MDN, YL

OR07-5 5654 5.0000 A Creation of androgen independent prostate cancer cell lines using targeting nucleases 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Cancer/Neoplasia Saturday, June 15th 1:00:00 PM OR07 2375 11:30:00 AM New Players in Hormonal Control of Breast & Prostate Cancer Oral


Gail P Risbridger*1, Ashlee K Clark1, Anna V Taubenberger2, Zhen Chea1, John Pedersen3, Mark Frydenberg1, Mitchell G Lawrence1, Dietmar W Hutmacher2, Renea A Taylor1 and Stuart J Ellem1
1Monash University, Melbourne, Australia, 2Queensland University of Technology, Australia, 3Tissupath Pathology Services, Melbourne, Australia

 

The stromal microenvironment is a heterogeneous mix of cells that regulates the differentiation and function of prostate cancer cells. It comprises cancer-associated fibroblasts (CAFs) and immune cells, with the latter including mast cells, a resident population that is expanded in the peri-tumoral microenvironment. There is a current need for human models to study stromal cell interactions, especially immune cells, and their impact on cancer development and progression. Although tissue recombination is commonly used to study the functional effects of CAFs, the method is not quantitative, is lengthy, technically challenging, and the xenografts are grown in immune suppressed hosts thereby excluding the immune contribution. Therefore, we have developed a novel bioengineered cellularized matrix co-culture model, to investigate and quantify multiple cellular interactions between human tumour stromal fibroblasts and mast cells on epithelial cell morphology and motility. Patient-matched CAFs and non-malignant prostatic fibroblasts (NPFs) from moderate grade (Gleason Score 7) or more aggressive tumors (Gleason Score 8-9 or CRCP) were cultured with epithelia in the cellularised matrix and quantitative cell morphometric analyses of epithelial cell shape factor, spread area or orientation were measured. Surprisingly, CAFs induce morphological changes in epithelial cells independent of the Gleason grade of the tumor of origin; all CAFs regardless of grade induced epithelial changes of the same magnitude. However, when human mast cells were included in the co-culture system, there was further and significant potentiation of the effects of CAFs on epithelial cells features. Further mechanistic studies also revealed that estrogen (via ERalpha) mediates the synergism of CAFs and mast cells. These data, using a quantitative method to study the tumourigenic properties of human prostate cancer stroma, show a complex interplay between CAFs and mast cells orchestrated by estrogen action at the tumour interface.

 

Nothing to Disclose: GPR, AKC, AVT, ZC, JP, MF, MGL, DWH, RAT, SJE

OR07-6 7958 6.0000 A MODELING ESTROGEN DRIVEN FIBROBLAST-IMMUNE CELL INTERACTIONS IN THE HUMAN PROSTATE CANCER MICROENVIRONMENT 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Cancer/Neoplasia Saturday, June 15th 1:00:00 PM OR07 2375 11:30:00 AM New Players in Hormonal Control of Breast & Prostate Cancer Oral


Richard J. Auchus*1, Elizabeth Osborne Buschur1, Gary D Hammer1, Alice Yoonju Chang2, Carole Andrea Ramm1, David Anthony Madrigal1, George Wang3, Martha Gonzalez3, Steven Xu3, Hans Smit4, Italo Poggesi5 and Margaret K. Yu6
1University of Michigan, Ann Arbor, MI, 2Mayo Clinic, Rochester, MN, 3Janssen Research & Development, Raritan, NJ, 4Janssen Research & Development, Beerse, Belgium, 5Jan-Cilag, Cologno Monzese, Italy, 6Janssen Research & Development, Los Angeles, CA

 

In the treatment of women with congenital adrenal hyperplasia due to 21OHD, control of androgen excess without iatrogenic Cushing syndrome remains an elusive goal. Recent cohort studies show that glucocorticoid overtreatment is common in adults with 21OHD, leading to comorbidities and impaired quality of life, but therapeutic alternatives to glucocorticoids for androgen excess management are lacking. AA is a potent inhibitor of cytochrome P450c17 (CYP17A1) used in combination with prednisone for prostate cancer treatment. We hypothesized that AA added to physiologic HC and FC would correct androgen excess in women with 21OHD without causing hypertension and hypokalemia.

This phase 1 open-label, multiple-dose, intrasubject, sequential dose-escalation study enrolled 6 women with classic 21OHD and serum androstenedione (AD)>1.5× normal (>345 ng/dL), who were taking 20 mg/d HC plus FC. AA oral suspension was taken for 6 days, starting at 100 mg QAM with dose escalations after ≥7-day washouts, until >80% of participants had normalization (<230 ng/dL) of the morning AD before HC and AA, mean of AD on Days 6 and 7 (mAD, primary end point). Secondary end points include 17-hydroxyprogesterone, total testosterone (T), urine androsterone and etiocholanolone glucuronides (T metabolites), electrolytes, and safety.

At 100 mg/d AA, mAD normalized in 50% of participants, with a reduction from a median baseline of 764 ng/dL to median mAD of 254 ng/dL. The primary end point was met at 250 mg/d AA, as mAD normalized in 5/6 (83%) participants, with a decrease from a median baseline of 664 ng/dL to mAD of 126 ng/dL. After the Day 6 AA dose, AD fell to a median nadir of 66 and 38 ng/dL by 8 h at 100 and 250 mg/d, respectively. Serum T and urine T metabolites fell in parallel to AD. At 250 mg/d AA, T decreased from a median baseline of 89 ng/dL to a median of 28 ng/dL (69% fall) for mean of T on Days 6 and 7 (mT). AA exposure was strongly and significantly negatively correlated with mAD and mT. The correlation coefficients between AUC24and mAD and mT were -0.73 (p=0.007) and -0.72 (p=0.008), respectively. The one participant who did not normalize mAD had the lowest drug exposure, without evidence of dose proportionality. AA was safe and well tolerated. No adverse events of hypertension or hypokalemia were observed.

Based on data generated in this dose-escalation study, AA added to replacement HC and FC normalizes androgen excess in this population of women with 21OHD.

 

Disclosure: RJA: Principal Investigator, Jansen Pharmaceuticals. CAR: Investigator, Jansen Pharmaceuticals. DAM: Investigator, Jansen Pharmaceuticals. GW: Owner, Johnson &Johnson, Employee, Janssen Research & Development. MG: Employee, Jansen Pharmaceuticals. SX: Employee, Janssen Research & Development, Owner, Johnson &Johnson. HS: Employee, Johnson &Johnson, Owner, Johnson &Johnson. IP: Employee, Jansen Pharmaceuticals. MKY: Employee, Janssen Research & Development, Owner, Janssen Research & Development. Nothing to Disclose: EOB, GDH, AYC

LB-OR-1 9612 1.0000 A Marked Androgen Reduction in Adult Women With Classic 21-Hydroxylase Deficiency (21OHD) Treated With Abiraterone Acetate (AA) Added to Physiologic Hydrocortisone (HC) and Fludrocortisone (FC) 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Miscellaneous/Other Saturday, June 15th 1:00:00 PM LB-OR 2669 11:30:00 AM Late-Breaking Oral Session Oral


Diana M. Cittelly*1, Patricia S Steeg2 and Carol A Sartorius3
1University of Colorado Anschutz Medical Campus, Aurora, CO, 2Center for Cancer Research, National Cancer Institute, Bethesda, MD, 3Univ of Colorado Anschutz Medical Campus, Aurora, CO

 

Ten to 16% of patients with breast cancer develop symptomatic brain metastases and ~80% die within one year of diagnosis. Symptomatic brain metastases are more prevalent in breast tumors overexpressing Human Epidermal Growth Factor Receptors 1 (HER1, EGFR) or 2 (HER2) and triple-negative (TN) tumors (lacking estrogen receptor (ER), progesterone receptor (PR) and HER2)1. Although the majority of breast cancer cells colonizing the brain lack ER, evidence suggests estrogens still play an important role in brain metastases: 1) brain metastases are more frequent in pre-menopausal women with higher circulating estrogen levels2, 2) estrogens accelerate the growth of ER tumor xenografts by modulating the tumor microenvironment3 and 3) estrogens are important in the response to brain-injury by astrocytes4 cells that surround and infiltrate brain metastases. We hypothesize that estrogens act in a paracrine manner on reactive astrocytes surrounding breast cancer cells in the brain to promote brain metastatic colonization. Results:  We demonstrate using global gene expression arrays and RT-PCR analysis that 17-β-estradiol (E2) upregulates Egf and TGF-α in primary mouse astrocytes, and that tamoxifen blocks this effect. The brain-seeking sub-line of human MDA-MB-231 TN breast carcinoma cells (231Br-EV) exposed to concentrated supernatant from E2-treated astrocytes had increased p-EGFR (Tyr1068) compared to cells exposed to ethanol vehicle (EtOH), or E2+tamoxifen treated astrocytes. Furthermore, E2 compared to EtOH treated astrocytic supernatant significantly increased migration and invasion of 231Br-Ev cells and a HER2 overexpressing subline (231Br-HER2) and tamoxifen blocked this effect. These data suggest that astrocytic EGFR-ligands activate EGFR and increase migration and invasion in brain metastatic cells. Global gene expression profiling of 231BrEV cells co-cultured with E2- or EtOH-stimulated-astrocytes showed that E2-treated astrocytes increased expression of metastatic mediators Matrix-metalloproteinase-9 (MMP9) and S100 Calcium-binding protein A4 (S100A4) in brain metastatic cells. To determine whether estrogen affects brain colonization in vivo, 231BrEV cells were injected into the left cardiac ventricle of ovariectomized female nude mice supplemented with placebo (n=5) or 1mg E2 continuous release pellets (n=5). Macrometastases were detected by MRI in 3/5 (60%) estrogen-depleted placebo mice compared to 5/5 mice (100%) supplemented with E2, supporting the hypothesis that E2 promotes brain metastatic colonization. Conclusion: We demonstrate a novel role for estrogen in the promotion of brain metastasis, by acting on E2-responsive astrocytes. Since aromatase inhibitors and anti-estrogens used to treat ER+ tumors can cross the blood brain barrier, our data suggests anti-endocrine therapies could be useful for treatment of ER- metastatic brain tumors.

 

Nothing to Disclose: DMC, PSS, CAS

LB-OR-2 9688 2.0000 A Estrogen signaling through astrocytes promotes brain metastasis colonization, migration and invasion of ER-negative breast cancer cells 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Miscellaneous/Other Saturday, June 15th 1:00:00 PM LB-OR 2669 11:30:00 AM Late-Breaking Oral Session Oral


Shin Ichiro Takahashi*1, Takayuki Yano2, Yosuke Yoneyama2, Toshiaki Matsui2, Hiroshi Okajima2, Kazuhiro Chida3 and Fumihiko Hakuno3
1Univ of Tokyo, Tokyo, Japan, 2The University of Tokyo, Graduate School of Agriculture and Life Sciences, Tokyo, Japan, 3Graduate School of Agriculture and Life Sciences, The University of Tokyo, Tokyo, Japan

 

Macropinocytosis, which is the major system of endocytosis responsible for liquid-phase uptake in a number of cell types, has been reported to be regulated by various growth factors. Binding of insulin-like growth factors (IGFs) to their specific receptors on the plasma membrane induces activation of receptor intrinsic tyrosine kinase. Activated receptor then phosphorylates insulin receptor substrates (IRSs), leading to downstream signal activation. These events exert a variety of IGF bioactivities. The present study is undertaken to elucidate the effects of IGFs on macropinocytosis and the molecular mechanism to regulate it. First, we examined whether IGF-I stimulation induces macropinocytosis in MCF-7 human breast cancer cells. Based on time-lapse microscopy analysis, we found that IGF-I treatment induced macropinocytosis. In addition, serum starved cells were incubated with Texas-Red conjugated-dextran in the presence or absence of IGF-I and uptake of dextran was analyzed. The results revealed that dextran uptake was enhanced by IGF-I stimulation. On the other hand, uptake of Alexa546 conjugated-transferrin, which represents receptor-mediated endocytosis was not enhanced by IGF-I stimulation, suggesting that IGF-I specifically activates macropinocytosis. To investigate the roles of IRS-1 in macropinocytosis, we repressed endogenous IRS-1 levels in MCF-7 cells using siRNA and assessed pinocytosis. We found that IRS-1 knockdown decreased macropinocytosis. Recently, we have shown that IRSs form  high-molecular-mass complexes and have identified Rabankyrin-5 as a novel IRS-1-associated protein by yeast-two hybrid screening using IRS-1 as a bait and placenta cDNA library as a prey. Rabankyrin-5 is an effector of the small GTPase Rab5 and plays an important role in macropinocytosis. We then examined the interaction between IRS and Rabankyrin-5 by co-immunoprecipitation assay. FLAG-IRS-1/2 and GFP-Rabankyrin-5 were co-transfected in HEK293T cells and the lysates were subjected to immunoprecipitation with anti-FLAG antibody. As a result, Rabankyrin-5 was co-immunoprecipitated with IRS-1, but not with IRS-2. IRS-1 was co-localized with Rabankyrin-5 around the plasma membrane where actin was polymerized in response to IGF-I stimulus. Using time-lapse microscopy, we also found that IGF-I induced formation of Rabankyrin-5-rich vesicles at protrusion and endocytosis of these vesicles. In cells overexpressing Rabankyrin-5 and the dominant negative form of Rab5, IGF-I caused actin polymerization, but not vesicle formation. Taken together, we concluded that IRS-1-Rabankyrin-5 complex plays some roles in IGF-I-induced vesicle formation through Rab5 activation leading to macropinocytosis.

 

Nothing to Disclose: SIT, TY, YY, TM, HO, KC, FH

LB-OR-3 9704 3.0000 A Roles of Insulin Receptor Substrate Complexes Containing Rabankyrin-5 in Insulin-Like Growth Factor-I-Induced Macropinocytosis 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Miscellaneous/Other Saturday, June 15th 1:00:00 PM LB-OR 2669 11:30:00 AM Late-Breaking Oral Session Oral


Fumihiko Furuya*, Hiroki Shimura, Sayaka Ichijo, Masashi Ichijo and Tetsuro Kobayashi
University of Yamanashi, Chuo, Yamanashi, Japan

 

One goal of diabetic regenerative medicine is to instructively convert mature pancreatic exocrine cells into insulin-producing cells. We recently reported that ligand-bound thyroid hormone receptor α (TRα) plays a critical role in expansion of the β-cell mass during postnatal development. Herein, we used adenovirus vector that expresses TRα driven by the amylase 2 promoter (AdAmy2TRα) to induce the reprogramming of pancreatic acinar cells into insulin-producing cells. Treatment with L-3,5,3-triiodothyronine  increases the association of TRα with the p85α subunit of Phosphatidylinositol 3-Kinase (PI3K), leading to the phosphorylation and activation of Akt and the expression of Pdx1, Ngn3 and MafA in purified acinar cells. Analyses performed with the lectin-associated cell lineage tracing system and the Cre/loxP-based direct cell lineage tracing system indicate that newly synthesized insulin-producing cells originate from elastase-expressing pancreatic acinar cells. Insulin-containing secretory granules were identified in these cells by electron microscopy. The inhibition of p85α expression by siRNA or the inhibition of PI3K by LY294002 prevents the expression of Pdx1, Ngn3 and MafA and the reprogramming to insulin-producing cells. In immunodeficient mice with streptozotocin-induced hyperglycemia, treatment with AdAmy2TRα leads to the reprogramming of pancreatic acinar cells to insulin-producing cells in vivo. Our findings suggest that ligand-bound TRα plays a critical role in β-cell regeneration during postnatal development via activation of PI3K signaling.

 

Nothing to Disclose: FF, HS, SI, MI, TK

LB-OR-4 9705 4.0000 A Ligand-Bound Thyroid Hormone Receptor Contributes to Reprogramming of Pancreatic Acinar Cells 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Miscellaneous/Other Saturday, June 15th 1:00:00 PM LB-OR 2669 11:30:00 AM Late-Breaking Oral Session Oral


Raymond E Soccio*1, Eric R Chen1, Fenfen Wang1, Joanna R DiSpirito1, Heewoong Lim2, Kyoung-Jae Won2 and Mitchell A Lazar2
1Perelman School of Medicine at the University of Pennsylvania, 2Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA

 

The nuclear receptor PPARγ is the master regulator of fat cell development, and is implicated in type 2 diabetes both as the target of anti-diabetic thiazolidinedione drugs and in genome-wide association studies.  Using chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) to describe genome-wide binding regions (cistromes) for PPARγ in cultured human SGBS adipocytes, we previously found that only ~10% of binding sites from mouse 3T3-L1 adipocytes were retained at syntenic genomic locations in the human cells.  Deeper sequencing tripled the number of high confidence binding sites in each species but confirmed the low degree of binding site retention across species.  However, these cell culture models may not reflect the binding of PPARγ in intact adipose tissue.  To address this, PPARγ ChIP-seq was performed in subcutaneous white adipose tissue (WAT) from obese humans and lean C57Bl/6 mice.  Similar to the findings in cultured adipocytes, ~14% of the human adipose PPARγ cistrome was retained in mouse adipose tissue.  Comparing the WAT cistromes to cultured adipocytes, within each species ~40% of WAT binding regions were present in the respective cell culture model.  These data strongly indicated that the most clinically relevant PPARγ cistrome is not from animal or cell culture models, but from actual human fat.  Therefore, ChIP-seq was performed on four additional WAT samples, revealing marked variability among subjects.   The PPARγ cistrome of each subject overlapped by only ~30% with that of the initial subject, and in each cistrome more than half of identified binding sites were unique to that subject.  This variability is much greater than we observe in replicate WAT cistromes from inbred lean C57BL/6 male mice, likely due to technical challenges with obese human adipose tissue as well as heterogeneity in age, sex, body weight, genotype, and other clinical factors.  Analysis of the PPARγ cistromes from multiple subjects revealed three classes of human adipose PPARγ binding sites: “core” sites identified in nearly all subjects, which typically show strongest binding; “variable” binding sites that are found in multiple subjects, but are typically weaker than the core sites and not detected in every subject; and “sporadic” subject-specific binding sites, typically the weakest and of uncertain relevance.  Preliminary analysis of single nucleotide polymorphisms (SNPs) falling in PPARγ binding sites suggests that genotype may account for some of the observed variability, and these genetic differences have the potential to affect metabolic phenotypes.

 

Disclosure: MAL: Ad Hoc Consultant, Pfizer, Inc., Ad Hoc Consultant, BioU, LLC, Ad Hoc Consultant, Merck & Co., Ad Hoc Consultant, Madrigal Pharmaceuticals, Ad Hoc Consultant, Lycera, Corp., Board Member, Novartis Pharmaceuticals, Board Member, Eli Lilly & Company, Ad Hoc Consultant, Vanda Pharmaceuticals. Nothing to Disclose: RES, ERC, FW, JRD, HL, KJW

LB-OR-5 9754 5.0000 A PPARγ Binding Sites in Human White Adipose Tissue 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Miscellaneous/Other Saturday, June 15th 1:00:00 PM LB-OR 2669 11:30:00 AM Late-Breaking Oral Session Oral


Pratima Basak1, Sumanta Chatterjee1, Steve Weger1, Ketan Badiani2, James R Davie1, Leigh C Murphy3 and Afshin Raouf*1
1University of Manitoba, Winnipeg, MB, Canada, 2CancerCare Manitoba, Winnipeg, MB, Canada, 3University of Manitoba

 

The developmentally regulated and imprinted gene, H19,  is a long non-coding RNA that is expressed exclusively from the maternal allele. High expression of H19 gene in many malignant tumors including breast tumors has been reported. While previous observations have suggested that estrogen signaling enhances the expression of H19 gene in breast cancer, we and others have found that in normal human and mouse mammary glands H19 is expressed predominantly in the Estrogen Receptor alpha (ERα) negative, basal epithelial cells. In this study, we examined if estrogen regulation of H19 gene is specfic to ERα  positive breast cancer cells. To this end, we isolated purified ERα positive luminal progenitors and ERα negative, undifferentiation bipotential progenitors from normal human breast reduction samples and placed them in matrigel cultures under estrogen-depleted growth conditions. Using quantitative PCR, we observed that the progeny of the bipotential progenitors exhibited the highest expression of the H19 gene compared to the progeny of the luminal progenitors. However, addition of 17-beta estradiol to the progeny of the uminal progenitors, increased the expression of H19 gene by 4.5 fold (p = 0.04). This observation suggests that estrogen regulation of H19 gene expression is not unique to ERα  positive breast cancer cells and that H19 may have key roles in regulating the normal proliferation and differentiation of ERα positive luminal progenitors. Using ERa  positive breast cancer cells we have further determined that estrogen treatment at 10nM requires at least 10 hr to significantly increase H19 expression (2 fold, p=0.002) which reaches its apex after 24hr (4.5 fold p=0.02). Interestingly, H19 expression subsides after 48hr of exposure to estrogen. Using gain of function studies and loss of function studies, as well as specific ERα or ERβ agonists, we demonstrate that ERα alone regulates the expression of H19. Moreover, using the transcriptional inhibitor actinomycin D and the protein synthesis inhibitor cycloheximide we found that estrogen-induced up regulation of H19 requires activation of transcription but not de novo protein synthesis, suggesting that ERα directly activates the transcription of H19 gene. Using chromatin immunoprecipitation technique we have identified 5 ER response-element half-sites within the H19 promoter to which ERα is bound after estrogen treatment. Our data therefore, suggest that estrogen signaling through the ERα, up regulates H19 gene expression in both normal and malignant human breast epithelial cells and that H19 may play a role in regulating the normal proliferation and differentiation of luminal progenitors. Understanding the molecular mechanisms that underlie estrogen’s regulation of proliferation in both normal and malignant ERα positive breast cells may lead to the identification of early diagnostic markers to detect breast cancers at an early stage.

 

Nothing to Disclose: PB, SC, SW, KB, JRD, LCM, AR

LB-OR-6 9575 6.0000 A Erα Regulates the Expression of H19, a Long Non-Coding RNA, in Normal Human Luminal and Malignant Breast Epithelial Cells 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Miscellaneous/Other Saturday, June 15th 1:00:00 PM LB-OR 2669 11:30:00 AM Late-Breaking Oral Session Oral


Toshihiro Goto*, Jun Nagamine, Yasushi Hiramine, Yuko Saida, Tomoko Iwama, Yoshihiro Horiuchi, Hitoshi Suda, Nobuyuki Kasai, Toru Negishi, Atsushi Tsuchida, Ryuji Hiramatsu and Hiroshi Kato
Dainippon Sumitomo Pharma Co. Lt, Osaka, Japan

 

Background and aims:

Production of the glucocorticoid cortisol is regulated by 11beta-hydroxysteroid dehydrogenase type 1 (11bHSD1) in the adipose tissue and liver, and its excess is associated with accumulation of visceral fat and development of insulin resistance in human. 11bHSD1 (-/-) mice fed a high-fat diet exhibit reduced visceral fat accumulation and improved insulin sensitivity in adipose tissue. We investigated in this study the effects of a novel selective (IC50:3.8 nM) 11bHSD1 inhibitor, DSP-0011, on triglycerides (TG) accumulation in human adipocytes, body fat mass and insulin sensitivity in diet-induced-obese (DIO) mice. In addition, we developed a DIO common marmoset (C. marmoset) model, and evaluated the effect of our compound on fat distribution in the model.

Materials and methods:

The effect of DSP-0011 on TG accumulation in human adipocytes was evaluated using the Oil Red O staining method. The in vivo effects of DSP-0011 were assessed by conversion of prednisone to prednisolone instead of conversion of endogenous glucocorticoids.DIO mice were administered DSP-0011 (10, 30 and 100 mg/kg) once daily for 10 weeks and subjected to Oral Glucose Tolerance Test (OGTT) after the final dosing. The weight of visceral fat was also measured in these mice. DIO C. marmosets were administered DSP-0011 (200 mg/kg) once daily for 4 weeks, and the volumes of visceral and subcutaneous fat were measured by micro CT. The concentration of plasma adiponectin was also determined by LC-MS.

Results:

DSP-0011 reduced TG accumulation in human adipocytes as indicated by decreased Oil Red O stained area (IC50: 230 nM). In vivo, DSP-0011 dose-dependently inhibited 11bHSD1-mediated conversion of prednisone to prednisolone in both mice (94% inhibition at 6 hr after administration of DSP-0011 at 30 mg/kg) and C. marmosets (76% inhibition at 6 hr after administration of DSP-0011 at 200 mg/kg). In DIO mice, DSP-0011 dose-dependently decreased both the area under the curve of plasma insulin level and the weight mesenteric fat. In DIO C.marmosets, DSP-0011 decreased visceral fat mass with little effects on body weight and food intake. Furthermore, DSP-0011 increased plasma total adiponectin.

Conclusion:

Our results show that DSP-0011 can ameliorate insulin resistance not only in DIO mice but also in DIO C. marmosets mainly by reduction of visceral fat mass. DSP-0011 is therefore expected to treat metabolic syndrome caused by visceral fat accumulation. The DIO model in nonhuman primate C. marmosets could be useful to predict clinical efficacy more accurately.

 

Nothing to Disclose: TG, JN, YH, YS, TI, YH, HS, NK, TN, AT, RH, HK

FP03-3 3562 4.0000 SAT-4 A Effect of DSP-0011, a Novel Selective 11beta-Hydroxysteroid Dehydrogenase Type 1 Inhibitor, On Visceral Adiposity in Diet-Induced-Obese Mice and Common Marmosets 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 1-25 2200 1:45:00 PM Glucocorticoid Actions & HPA Axis Poster


Krystle Anne Frahm* and Stuart Allen Tobet
Colorado State University, Fort Collins, CO

 

The Paraventricular Nucleus of the Hypothalamus (PVN) is a dense collection of neurons that play key roles in maintaining homeostasis and initiating stress responses. It is also characterized by a dense matrix of blood vessels compared to surrounding brain regions. Glucocorticoids have been shown to alter the neural circuitry of the hypothalamic-pituitary-adrenal axis that includes the PVN, but whether blood vessel density or the blood-brain barrier (BBB) are also impacted is unknown. The current study investigated whether glucocorticoid signaling regulates the development of the unique vasculature within the PVN. We focused on BBB permeability and pericytes as an important cellular component of a functional BBB. Pregnant mice on an FVB background were injected with 0.1mg/kg/day of dexamethasone or vehicle during embryonic days (E) 11-17 when there is major neuronal development of the PVN (McClellan et al., 2010). On P20, brains were immersion fixed in 4% paraformaldehyde and vascular endothelial cells were visualized by immunoreactive platelet endothelial cell adhesion molecule (PECAM) to determine blood vessel density. Alternatively, mice were perfused transcardially with heparin PBS containing fluorescein isothiocyanate (FITC) followed by 4% paraformaldehyde to view a compromised BBB for extravascular leakage. For changes in BBB composition, we examined immunoreactive desmin, an intermediate filament in pericytes. Results showed significant decreases in blood vessel length and total immunoreactivity in fetal dex- compared to vehicle-treated mice across the entire PVN and in the rostral and mid PVN regions particularly. For branch points, there was a significant decrease in the rostral and mid regions of the PVN in dex-treated compared to vehicle-treated mice. To investigate BBB competency, leakage of FITC from the perfused vasculature was determined following confocal microscopy. There was PVN-specific increased leakage in the mid region for dex-treated compared to vehicle-treated mice (p < 0.05), corroborated by increased desmin-immunoreactivity (p < 0.05). There was also a strong trend (p < 0.09) for an increase in FITC leakage in the rostral PVN due to dex treatment. Alterations in the vasculature of the PVN may impair neuronal function just as neuron specific alterations would. In general, alterations in the functioning of neurovascular units may provide a novel mechanism for fetal antecedent programming that may influence adult disorder.

 

Nothing to Disclose: KAF, SAT

FP03-4 5695 5.0000 SAT-5 A Prenatal Dexamethasone Impacts Blood Vessel Density and Blood-Brain Barrier Competency Within the Paraventricular Nucleus of the Hypothalamus 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 1-25 2200 1:45:00 PM Glucocorticoid Actions & HPA Axis Poster


Aiqing Li1, Rowan Samuel Hardy*2, Shihani Stoner1, Jan Tuckermann3, Markus J Seibel1 and Hong Zhou1
1ANZAC Research Institute, University of Sydney, Sydney, Australia, 2The University of Birmingham, Birmingham, United Kingdom, 3Leibniz Inst for Age Rsrch, Jena, Germany

 

Glucocorticoid signalling is essential during embryonic lung development, with both the global and epithelial glucocorticoid receptor (GR) null mice presenting with lung atelectasis and post natal lethality. In this study we examined the role of glucocorticoid signalling within mesenchymal tissues. To study the role of the GR in mesenchymal tissues during embryogenesis we crossed GRflox mice with Dermo1-Cre mice to generate GRDermo1 mice, where the GR gene was conditionally deleted within mesenchymal cells. Organ development between E14.5 through to birth was determined by histological staining and MRI performed at E18.5. Specific mesenchymal cell populations were assessed by immunohistochemistry and quantitative RT-PCR. GRDermo1 mice displayed severe pulmonary atelectasis, defective abdominal wall formation and postnatal lethality. GRDermo1 mice failed to progress from the canalicular to saccular stage of lung development, evidenced by the presence of immature air sacs, thickened interstitial mesenchyme and an underdeveloped vascular network between E14.5 and E18.5 (lung tissue to alveolar space; GRDermo1, 90.3% vs WT, 79.6%, p< 0.001). Interstitial fibroblast numbers were expanded within GRDermo1 mice compared to WT littermates (50.1 vs 21.1 cells/ 50mm2; P<0.005). However, myofibroblasts, vascular smooth muscle and endothelial cells were shown to be present in normal numbers. Analysis of their functionality revealed that myofibroblasts from GRDermo1 mice possessed significantly reduced elastin synthesis. In contrast epithelial lining cells of immature saccules were poorly differentiated. Reduced elastin and collagen deposits were also noted in connective tissues adjacent to the umbilical hernia. This study demonstrates that eliminating the GR in cells of the mesenchymal lineage results in marked effects on interstitial fibroblast function, including a significant decrease in elastin synthesis. This results in lung atelectasis and postnatal lethality, as well as additional and hitherto unrecognized developmental defects in abdominal wall formation. In addition, altered glucocorticoid signalling in the mesenchyme indirectly attenuates normal lung epithelial differentiation.

 

Nothing to Disclose: AL, RSH, SS, JT, MJS, HZ

4054 7.0000 SAT-7 A Disruption of Mesenchymal Glucocorticoid Signaling Attenuates Embryonic Lung Development and Results in Post Natal Lethality in Mice 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 1-25 2200 1:45:00 PM Glucocorticoid Actions & HPA Axis Poster


Luisa Fernanda Gonzalez*1, Derrick Tint2, Selma Feldman Witchel1, Paula Monaghan-Nichols2 and Donald Benedict DeFranco2
1Children's Hospital of Pittsburgh of UPMC, Pittsburgh, PA, 2University of Pittsburgh School of Medicine, Pittsburgh, PA

 

Background: Corrective or palliative surgery with cardiopulmonary bypass (CPB) and hypothermic circulatory arrest are utilized to treat infants with complex congenital heart disease (CHD). Glucocorticoids (GC), typically dexamethasone (Dex) or methylprednisolone (MP), are used to attenuate the ischemic injury and inflammatory responses in infants undergoing CPB (1). With improved survival rate among infants with CHD, greater attention is being directed to the quality of life of survivors. Impaired neurologic development after surgical repair of CHD remains is a major common adverse outcome. Yet, no consistent improvements in long-term neurodevelopmental outcomes have been achieved. Although neurogenesis occurs mainly in utero, peri-operative steroid exposure might protect the CNS or, alternatively, might have deleterious effects on gliogenesis since gliogenesis continues during postnatal human brain development along with the formation of myelin and synapses.  To evaluate the effect of GCs on neuronal development, we developed an in vitro model system of murine cerebral cortical neural stem cells (NSC) that adopt distinct differentiation fates as they mature in culture. The in vivo temporal order of neuron, astrocyte, and oligodendrocyte production is preserved in the neurosphere cultures. 

Results: We have examined the impact of GCs or hypothermia (HT) on cell fate specification, using NSCs exposed to Dex or HT only during their proliferative phase and then withdrawn from hormone or changed to normothermia upon the initiation of differentiation. The number and type of cells produced from each lineage were examined following 5-14 days of differentiation using indirect immunofluorescence to detect markers of neurons (neuron specific class III beta-tubulin) and glia, (glial fibrillary acidic protein). Preliminary results showed that GC treatment during proliferation alters the fate of NSCs leading to enhancement of neurogenesis at a time when untreated cells were mainly adopting a glial identity. Furthermore, Dex alters neurogenic fate as evidenced by the increased production of bipolar neurons and decreased multi-polar neurons generated upon differentiation of early passage progenitors. Hypothermia alone did not lead to these changes in neurogenic fate.

Conclusion: Future studies will include more detailed analysis of GC effects on genes responsible for triggering gliogenesis and examination of hormone effects on NSCs with distinct cell fates.

 

Nothing to Disclose: LFG, DT, SFW, PM, DBD

7708 8.0000 SAT-8 A Glucocorticoid and hypothermia exposure of neural stem cells during proliferation alters their differentiation potential 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 1-25 2200 1:45:00 PM Glucocorticoid Actions & HPA Axis Poster


Sandhya Khurana*1, Phong Nguyen2, Julie Grandbois2, Heather Peltsch2, Amanda Hollingsworth2, Krishnan Venkataraman3 and T.C. Tai1
1Northern Ontario School of Medicine, Sudbury, ON, Canada, 2Laurentian University, Sudbury, ON, Canada, 3Huntington University, Sudbury, ON, Canada

 

Human health and disease are influenced not only by the genetic make-up of an individual, but also to a great degree by environmental factors. The prenatal environment can be a significant determinant of long-term health outcomes. An adverse fetal milieu such as undernourishment or exposure to environmental insults can have long-term developmental consequences impacting adult health, a phenomenon known as fetal programming. Epidemiological data suggests that conditions in utero can be linked to the development of diseases such as hypertension, diabetes and other pathophysiological conditions in adulthood. Studies suggest that fetal programming of adult diseases is mediated by glucocorticoids (GCs); either endogenous (ex. maternal stress), or exogenous (ex. synthetic GCs administered to aid in the development of the premature babies). Indeed, prenatal exposure to high levels of GCs can predispose the offspring to hypertension. Glucocorticoids regulate catecholamine biosynthesis and are critical for blood pressure homeostasis, with elevated levels leading to hypertension. The purpose of this study was to examine whether prenatal exposure to elevated GC levels influences the development of adult hypertension via alterations in epinephrine synthesis. We investigated the impact of prenatal GC exposure on the post-natal regulation of the gene encoding for phenylethanolamine N-methyltransferase (PNMT), the enzyme involved in the biosynthesis of the catecholamine, epinephrine. PNMT has been linked to hypertension and is elevated in animal models of hypertension. Pregnant Wistar-Kyoto dams were injected with either 100 μg/kg/day of the synthetic glucocorticoid dexamethasone (DEX) or saline in the third trimester of pregnancy. Blood pressure and weights of the offspring were measured (5 to 17 weeks of age), at which point the animals were sacrificed and tissues collected. The results show that systolic, diastolic and mean arterial blood pressures were elevated in male WKY rat offspring born to DEX injected dams (+15%). Adrenal PNMT mRNA was elevated (1.5-fold) in prenatally DEX-exposed rats at 17 weeks.  Additionally, analyses of the transcriptional regulators of the PNMT gene show that prenatal GC exposure increased mRNA levels of Egr-1, AP-2 and GR.  A corresponding increased expression of PNMT protein, along with an elevation of Egr-1, AP-2, and GR protein levels were also observed. Furthermore, GMSA showed increased binding of Egr-1 and GR to the PNMT promoter in DEX exposed animals. These results suggest that prenatal GC exposure increases adrenal PNMT gene expression via altered transcriptional regulation. The study exemplifies the influence of in utero stress on PNMT and thereby epinephrine biosynthesis as a potential mechanism by which elevated prenatal GC levels may program for hypertension later in life.

 

Nothing to Disclose: SK, PN, JG, HP, AH, KV, TCT

7570 9.0000 SAT-9 A ROLE OF ADRENAL PHENYLETHANOLAMINE N-METHYLTRANSFERASE IN GLUCOCORTICOID-MEDIATED FETAL PROGRAMMING OF HYPERTENSION 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 1-25 2200 1:45:00 PM Glucocorticoid Actions & HPA Axis Poster


Laura Louise Gathercole*1, Craig L Doig2, Jonathan Mark Hazlehurst1, Paul Michael Stewart2, Gareth Geoffrey Lavery1 and Jeremy W Tomlinson3
1University of Birmingham, Birmingham, United Kingdom, 2University of Birmingham, United Kingdom, 3Centre for Endocrinology, Diabetes and Metabolism, School of Clinical and Experimental Medicine, University of Birmingham, United Kingdom

 

Patients with glucocorticoid (GC) excess develop insulin resistance and central obesity. We have demonstrated that GCs have tissue-specific effects on insulin sensitivity in humans, causing resistance in skeletal muscle but sensitivity in subcutaneous adipose tissue. The molecular mechanisms that underpin these differences remain poorly understood. Over the last decade small non-coding RNAs (microRNAs-miRNAs) controlling protein expression have been identified, representing an additional regulatory layer to the control of metabolism through the regulated expression of enzymes, transcription factors and signalling components. miRNAs are readily detected in human serum and altered miRNA profiles have been linked to metabolic disease.

In order to identify GC regulated miRNAs blood was extracted from 10 healthy volunteers under four treatment conditions. Volunteers were fasted for 12h and infused with either saline or hydrocortisone (0.2mg/kg/h) this was followed by 4h of insulin infusion (100mU/m2.min). Samples were taken after fasting (+/- hydrocortisone) and after insulin infusion (+/- hydrocortisone). RNA was extracted and used in miRNA array analysis, providing full coverage of mirBASE17, including 1750 known human miRNAs. Expression of the most regulated miRNAs was measured by real-time PCR in human liver, adipose and muscle samples.

In the fasting state, hydrocortisone treatment significantly altered serum levels of 7 miRNAs, including some with predicted metabolic targets. Compared to fasting saline, the combination of hydrocortisone and insulin regulated 16 miRNAs, interestingly increasing miR-195 (associated with hypertension) and miR-144 (inhibition of insulin receptor substrate 1 [IRS1]). Compared to insulin alone, hydrocortisone regulated 25 miRNAs, interestingly increasing miR-637 (involved in adipocyte differentiation) and miR-145 (inhibition of IRS1 and 2). The 10 most highly regulated miRNAs were all expressed in the three key metabolic target tissues (liver, adipose and muscle). This study has identified novel profiles of GC regulated miRNAs in human serum associated with insulin sensitivity, a number of which have predicted and demonstrated metabolic targets. These data will allow us to investigate the endocrine regulation of miRNAs and their role in metabolic homeostasis and highlights potential miRNA targets that may underpin the tissue-specific effects of GCs on insulin action.

 

Nothing to Disclose: LLG, CLD, JMH, PMS, GGL, JWT

6983 10.0000 SAT-10 A A Serum MicroRNA Profile Potentially Associated with Glucocorticoid Mediated Insulin Resistance 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 1-25 2200 1:45:00 PM Glucocorticoid Actions & HPA Axis Poster


Maryam Nasiri*1, Iwona Bujalska2, Paul M Stewart1, Laura Louise Gathercole1 and Jeremy W Tomlinson3
1University of Birmingham, Birmingham, United Kingdom, 2University of Birmingham, United Kingdom, 3Centre for Endocrinology, Diabetes and Metabolism, School of Clinical and Experimental Medicine, University of Birmingham, United Kingdom

 

Patients with glucocorticoids (GC) excess develop central obesity, insulin resistance and hepatic steatosis in up to 20% of cases. Current dogma suggests that GCs cause insulin resistance in all tissues. However, we have previously demonstrated that GCs induce insulin sensitisation in adipose tissue in vitro, whilst causing insulin resistance in skeletal muscle. In rodent hepatocytes, GCs enhance insulin stimulated lipogenesis but studies in human hepatocytes have not been performed and the cellular mechanisms underpinning these observations have not been determined.

Cryopreserved human hepatocytes were purchased from Celsis in Vitro Technologies (Baltimore, USA). All donors were healthy, male non-diabetic, on no regular medications. Cells were incubated with variable doses of cortisol (0-1000 nM) for 24h in the presence and absence of insulin (5 nM). Insulin signalling gene expression levels were quantified by real-time PCR and western blotting was performed to determine total and phospho PKB/akt protein expression levels. De novo lipogenesis (DNL) was measured by 1-[14C] acetate incorporation in triglyceride.

GC receptor, IRS1/2, Insulin receptor, AKT1/2, ACC1/2, CPT1, DGAT and FAS were all expressed in primary cultures. Incubation with cortisol alone (0-1000 nM) or in combination with insulin did not alter expression of insulin signalling, steroid hormone regulatory genes or those involved in lipid metabolism. However, whilst cortisol treatment did not alter total PKB/akt protein levels, phosphorylation of PKB/akt at serine 473 after insulin stimulation increased following cortisol pre-treatment in a dose dependant manner (1.23-fold [100nM], 1.68-fold [250nM], 2.44-fold [1000nM] vs. control n=4 p<0.05).

Insulin alone (5nM, 24h) had a modest impact upon acetate incorporation into lipid (129.1±13.0%), however, co-incubation with cortisol significantly enhanced insulin stimulated lipogenesis (43.9±12.7% [250nM], 66.13±9.8% [1000nM] vs. control (23.61±10.7%), p<0.05).

We have demonstrated that in primary human hepatocytes GC treatment enhances insulin signalling through increased serine phosphorylation of PKB/akt and that GCs and insulin can act synergistically to promote lipogenesis. Whilst translation to the clinical setting is crucially important, this mechanism may be fundamental in explaining the interaction between GCs and insulin to drive lipogenesis. Furthermore, this may contribute to the pathogenesis of non-alcoholic fatty liver disease with GC excess.

 

Nothing to Disclose: MN, IB, PMS, LLG, JWT

8327 11.0000 SAT-11 A Glucocorticoids Enhance Insulin Sensitivity in Human Hepatocytes 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 1-25 2200 1:45:00 PM Glucocorticoid Actions & HPA Axis Poster


Taiyi Kuo*, Tzu-Chieh Chen and Jen-Chywan Wang
University of California at Berkeley, Berkeley, CA

 

Excess exogenous and endogenous glucocorticoids cause metabolic syndrome, which is a constellation of metabolic risk factors that include insulin resistance, dyslipidemia, hypertension and central obesity.  Metabolic syndrome significantly increases the risks of Type 2 diabetes and cardiovascular diseases.  Glucocorticoids convey their signal through an intracellular glucocorticoid receptor (GR).  From genome-wide chromatin-immunoprecipitation sequencing and gene expression analysis in mouse C2C12 myotubes and 3T3-L1 adipocytes, Pik3r1 (a.k.a. p85a) topped the list of importance as one of the potential primary targets of GR capable of negatively regulating the insulin/insulin growth factor-1 (IGF-1) signaling.  The fact that, dexamethasone (DEX, a synthetic glucocorticoid) treatment induced newly synthesized Pik3r1 transcript in nuclear run-on and loosened nucleosome structure near the GRE of Pik3r1 in micrococcal nuclease assay, further confirmed that Pik3r1 is a primary target of GR.  Previously we reported that, overexpressing Pik3r1 mimicked DEX-induced myotube atrophy in C2C12 myotubes.  We further found that, decreasing the level of Pik3r1 and treating the cells with DEX have compromised DEX-induced myotube atrophy and DEX-decreased protein synthesis, through decreased atrogenes’ expressions and restored activities of insulin/IGF-1 signaling molecules, respectively.  These data demonstrated that Pik3r1 mediates glucocorticoid action in myotube atrophy and insulin resistance in vitro.  To validate Pik3r1’s effect in vitro, skeletal muscle-specific and adipose tissue-specific Pik3r1 knockout mice (mPik3r1-KO and aPik3r1-KO, respectively) were generated.  Our preliminary data show that, mPik3r1-KO had improved DEX-induced insulin insensitivity and aPik3r1-KO had reduced DEX-induced lipolysis.  More detailed studies are on the way comparing the metabolic effects of DEX in wild type, mPik3r1-KO and aPik3r1-KO mice.  Overall, our findings have established Pik3r1 as a critical mediator of excess glucocorticoid-induced metabolic disorders.

 

Nothing to Disclose: TK, TCC, JCW

7710 12.0000 SAT-12 A The Role of Pik3r1 in Excess Glucocorticoid-induced Metabolic Disorders 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 1-25 2200 1:45:00 PM Glucocorticoid Actions & HPA Axis Poster


Xiantong Zou*1, Antonella Pellicoro2, Prakash Ramachandran2, Rebecca Aucott2, Michelle Clarkson1, Andrew McBride1, Scott Webster1, John Iredale2, Brian R Walker1 and Zoi Michailidou2
1University of Edinburgh, Edinburgh, United Kingdom, 2University of Edinburgh

 

Background: Liver fibrosis and cirrhosis are characterized by accumulation of extracellular matrix from activated hepatic stellate cells (HSCs). Glucocorticoids (GCs) limit HSC activation in vitro. Tissue GC levels are regulated by 11β-hydroxysteroid dehydrogenase-1 (11βHSD1) which converts inactive 11-dehydrocorticosterone (DHC) into active corticosterone. We hypothesized that 11βHSD1 modulates HSC fibrotic responses.

Method:  Liver fibrotic responses were studied at intervals for up to 8 days after a 12 week period of twice weekly intraperitoneal carbon tetrachloride (CCL4) administration in male C57Bl6 mice with or without global 11βHSD1 deletion (KO) or administration (in food) of the selective murine 11βHSD1 inhibitor, UE2316. Immunohistochemistry, qPCR, Western blot and flow cytometry were conducted in liver. The fibrotic response of mouse HSCs to glucocorticoid treatment and 11βHSD1 inhibition was studied in in vitro.

Results: 11βHSD1 KO mice were protected from CCl4-induced hepatocyte injury (with lower serum transaminases), and had a similar inflammatory response to injury (macrophage counts and cytokine transcripts), yet had more profound fibrosis, with higher fibrillar collagen staining (A.U. of picrosirus red staining: KO 1.3±0.1 v.s. wild type 1.0 ±0.1 p=0.048 and A.U. of Collagen 1 staining: KO 1.2 ±0.1 v.s. wild type 1.0 ±0.1,  p=0.039) and increased pro-fibrotic gene transcripts (aSMA 4-fold, p<0.04; Col1 ~2-fold, p<0.03; Timp1 8-fold, p<0.02) compared with wild type controls. Similar results were obtained in mice treated with the 11βHSD1 inhibitor UE2316 from the start of CCl4 administration. However, administration of UE2316 only during the 8 day recovery phase showed no effects on fibrosis, indicating that loss of 11βHSD1 during liver injury is important. In HSCs in vitro, both corticosterone and DHC inhibited aSMA, Col1 and MMP expression, with effects of DHC prevented by UE2316. HSCs from 11βHSD1 KO mice were prone to activation in vitro.

Conclusion: 11βHSD1 deficiency causes increased activation of HSCs following chemical injury and promotes liver fibrosis. Effects of 11βHSD1 inhibition, a potential therapy in metabolic syndrome, on tissue repair appear context-specific, with beneficial anti-fibrotic effects in adipose, anti-inflammatory effects in atherosclerosis, and improved tissue repair after myocardial infarction perhaps being offset by adverse outcomes in liver.

 

Nothing to Disclose: XZ, AP, PR, RA, MC, AM, SW, JI, BRW, ZM

5770 13.0000 SAT-13 A 11βHSD1 DEFICIENCY OR INHIBITION INCREASES SUSCEPTIBILITY TO LIVER FIBROSIS BY ACTIVATING HEPATIC STELLATE CELLS 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 1-25 2200 1:45:00 PM Glucocorticoid Actions & HPA Axis Poster


Zhenguang Zhang*1, Agnes Elizabeth Coutinho2, Patrick Hadoke2, Donald Salter3, Jonathan Robert Seckl3 and Karen Elizabeth Chapman1
1University of Edinburgh, Edinburgh, United Kingdom, 2Univ of Edinburgh, Edinburgh Scotland, United Kingdom, 3University of Edinburgh

 

11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) amplifies local glucocorticoid action by converting inactive glucocorticoids (cortisone and 11dehydro-corticosterone, in humans and rodents respectively) into the active forms (cortisol and corticosterone). 11β-HSD1 is expressed in most types of immune cells and its global deficiency in mice worsens acute inflammation, including experimental (K/BxN serum transfer) arthritis. To dissect the contribution of macrophages to the acute inflammatory phenotype of global 11β-HSD1-deficiency, mice with selective 11β-HSD1 deficiency in macrophages were generated, LysM-Cre Hsd11b1flox/flox (MKO) mice. Hsd11b1flox/flox littermates were used as controls. MKO mice have an 82% decrease in 11β-HSD1 reductase activity in peritoneal resident macrophages.

Inflammatory arthritis (induced by intra-peritoneal injection of 125μl of K/BxN serum) was used to investigate the role of macrophage 11β-HSD1 in the inflammatory response of MKO and control littermates (n=6-7, 7-10 weeks old). Mice globally deficient in 11β-HSD1 (n=3) were also used in the experiment. Clinical scoring was conducted daily for 21 days. Initial inflammation (d1-8) did not differ between MKO and control littermates. However, during the resolution phrase (d8-21), MKO mice exactly recapitulated the worse inflammatory phenotype of global 11β-HSD1-deficiency with a significant interaction between genotypes (MKO and control littermates) and time by repeated 2 way-ANOVA (p<0.0001).  A Bonferroni post-hoc test showed that the clinical score in MKO mice was significantly higher than the littermate controls between d13-18. . Area-under-the-curve (d13-21) exhibited a significant higher inflammation in the MKO than control littermates as well (86.6±14.7 versus 60.1±13.4; p<0.005). H&E staining on d21 ankle joints revealed pronounced fibroplasia, predominantly in the supporting mesenchyme associated with the tenosynovium.

This study highlights the importance of intracellular amplification of active glucocorticoid levels in macrophages to facilitate the resolution of inflammation. Deficiency in or inhibition of 11β-HSD1 may result in a dysregulated healing response with increased fibroproliferation.

 

Nothing to Disclose: ZZ, AEC, PH, DS, JRS, KEC

4377 14.0000 SAT-14 A Impaired resolution of inflammation in macrophage-specific 11β-hydroxysteroid dehydrogenase type 1 deficient mice 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 1-25 2200 1:45:00 PM Glucocorticoid Actions & HPA Axis Poster


Zaki K Hassan-Smith*1, Stuart Andrew Morgan2, Mark Sherlock3, Nicola Crabtree1, Iwona Bujalska2, Mark Cooper2, Jeremy W Tomlinson4, Gareth Geoffrey Lavery1 and Paul Michael Stewart2
1University of Birmingham, Birmingham, United Kingdom, 2University of Birmingham, United Kingdom, 3Trinity College Dublin, Ireland, 4Centre for Endocrinology, Diabetes and Metabolism, School of Clinical and Experimental Medicine, University of Birmingham, United Kingdom

 

Myopathy, characterized by muscle atrophy and reduced strength, is a key discriminatory feature of Cushing’s Syndrome. 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) functions as a reductase converting inactive cortisone to active cortisol in humans, and inactive 11-dehydrocorticosterone (11DHC) to active corticosterone (CORT) in mice, thus amplifying local glucocorticoid (GC) action. We hypothesize that in muscle 11β-HSD1 mediated GC generation contributes to age associated sarcopenia. To investigate this we assessed 6-week old male wildtype (WT) mice treated with CORT (100μg/mL), 11DHC (100μg/mL) or vehicle via drinking water for 5 weeks, and young (26 wks) and aged (112 wks) WT and 11β-HSD1KO mice. Grip strength and tissue weights were assessed as markers of muscle function with muscle mRNA expression profiles completed using fluidigm arrays. In WT quadriceps, both CORT and 11DHC increased expression of key muscle atrophy genes including FOXO1, FOXO3a, MuRF1, atrogin-1, myostatin, GSK3β, and GADD45a. This was paralleled by decreased quadriceps weight and grip strength compared to vehicle treated mice. WT mice at 112 wks of age shared a common atrophy gene expression profile with CORT treated mice, and demonstrated an age-dependent decrease in grip strength. However, aged 11β-HSD1KO mice were protected from the atrophy associated gene expression profile and retained a muscle mass and grip strength phenotype similar to that of a young WT. In human clinical studies we assessed muscle function using Leonardo jump plate mechanography, and carried out fluidigm gene expression profiling on percutaneous vastus lateralis muscle biopsies in 70 patients ranging from 20-80 years of age. This revealed again an increase in FOXO1, FOXO3a and GADD45a expression with age. Furthermore, there was an inverse relationship between maximum peak power and maximal velocity during upward movement and expression of muscle atrophy genes including FOXO1, FOXO3a, myostatin and GADD45a. In summary, we have identified a muscle gene expression profile common to both GC and age associated myopathy, which is not seen in aged 11β-HSD1KO mice. Furthermore, human studies have identified the importance of these genes in the muscle aging phenotype. These data suggest that muscle 11β-HSD1 could offer a novel therapeutic target in the treatment of both GC-associated myopathy and sarcopenia.

 

Nothing to Disclose: ZKH, SAM, MS, NC, IB, MC, JWT, GGL, PMS

9014 15.0000 SAT-15 A Identification of an 11 Beta-Hydroxysteroid Dehydrogenase Type 1 Regulated Gene Expression Profile Common to Glucocorticoid and Age Associated Myopathies 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 1-25 2200 1:45:00 PM Glucocorticoid Actions & HPA Axis Poster


Erika Siofra Harno*1, Elizabeth Claire Cottrell2, Brian G. Keevil3, Andrew Vincent Turnbull4, Brendan Leighton5 and Anne White2
1University Of Manchester, Manchester, United Kingdom, 2University of Manchester, Manchester, United Kingdom, 3Univ Hospital of South Manchester, Manchester, United Kingdom, 4AstraZeneca, Macclesfield, United Kingdom, 5AstraZeneca, Macclesfield

 

Metabolic syndrome shares many clinical features with Cushing’s syndrome, including central obesity and insulin resistance, implicating excess local glucocorticoids in its etiology. Active glucocorticoids (cortisol/corticosterone in humans/rodents) can be regenerated from inactive forms [cortisone/11-dehydrocorticosterone (11-DHC)] by the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) in tissues including liver, adipose tissue and brain. The aim of this study was to investigate whether inactive 11-DHC can induce a metabolic syndrome-like phenotype in mice via 11β-HSD1.

11-DHC was administered in drinking water for 5 weeks to wild-type mice (WT, 25µg/ml and 50µg/ml) and mice lacking 11β-HSD1 globally (GKO, 25µg/ml and 50µg/ml) or only in the liver (LKO, 25µg/ml). 11-DHC raised circulating corticosterone in both WT (25.2±5.2 vs. 169.1±32.2 nM) and LKO mice (20.2±2.6 vs. 162.7±44.6 nM), but not in GKO mice (23.9±3.9 vs. 13.6±2.4 nM). This indicates that corticosterone regenerated from 11-DHC enters the circulation and that liver 11β-HSD1 is not the main source of the regenerated steroid. Treatment with 11-DHC increased body weight and adiposity in WT mice, but not in LKO or GKO mice. This suggests that regenerated corticosterone can drive body weight gain. However, given the LKO data it is corticosterone regenerated by the liver which acts on fat tissue to increase adiposity. Food intake was increased (12.5±1.9%) when 11-DHC was administered to WT mice but not when administered to GKO mice. 11-DHC treatment also led to insulin resistance in WT mice, but not in LKO or GKO mice, suggesting that corticosterone regenerated by hepatic 11β-HSD1 is important in induction of insulin resistance. In contrast, circulating corticosterone derived from 11-DHC influenced HPA axis function, as increased corticosterone in WT and LKO mice resulted in reductions in ACTH, POMC and adrenal weight. These HPA axis markers were unaffected by 11-DHC treatment in GKO mice.

Together, these results demonstrate that 11β-HSD1 regenerated corticosterone can cause a metabolic syndrome-like phenotype in mice. While extra-hepatic (likely adipose tissue) 11β-HSD1 is largely responsible for the regeneration of corticosterone that enters the circulation, liver 11β-HSD1 appears key in the regulation of insulin sensitivity and body weight.

 

Disclosure: ESH: Researcher, Astra Zeneca. AVT: Employee, Astra Zeneca, Employee, Astra Zeneca. BL: Employee, Astra Zeneca, Employee, Astra Zeneca. Nothing to Disclose: ECC, BGK, AW

5068 16.0000 SAT-16 A Increased 11-dehydrocorticosterone causes a metabolic syndrome–like phenotype in mice: a monopoly by 11β-HSD1? 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 1-25 2200 1:45:00 PM Glucocorticoid Actions & HPA Axis Poster


Peter J Duncan* and Michael J Shipston
The University of Edinburgh, Edinburgh, Scotland

 

Anterior pituitary corticotroph cells are an integral component of the hypothalamic-pituitary-adrenal (HPA) axis which governs the physiological stress response. In response to a stressor, CRH and AVP from the hypothalamus stimulate ACTH release from corticotrophs. ACTH, in turn, releases glucocorticoids from the adrenal gland which negatively feedback to inhibit ACTH secretion. Corticotroph cells are electrically excitable and fire single-spike action potentials as well as showing complex bursting patterns. The aim of this project was to establish whether glucocorticoid negative feedback changes the electrical properties of murine corticotroph cells.

Corticotrophs were acutely isolated from male mice (aged 2-5 months) constitutively expressing GFP under control of the POMC promoter (POMC-GFP). Electrophysiological recordings were obtained using the perforated patch clamp technique in the current clamp configuration. Under basal conditions, cells had a resting membrane potential of -53.7 ±1.5mV (n = 7, Data are Means ± SEM) and showed low frequency spontaneous action potentials (0.34 ±0.14Hz). Stimulation with physiological concentrations of CRH and AVP (0.2nM and 2nM respectively) results in an increase in firing frequency and a transition from single spikes to bursting-like behaviour.

Cells pre-treated for 1.5 hours with corticosterone (100nM) were significantly (p < 0.01) hyperpolarised compared with controls (-62.9 ±2.2mV) under basal conditions (n = 8). Although CRH and AVP could depolarise resting membrane potential in corticosterone treated cells, this was still significantly (p < 0.05) hyperpolarised (-55.7 ±2.6mV) compared with controls treated with CRH and AVP. Basal firing rate was lower in cells treated for 1.5 hours (0.12 ±0.1Hz) and although CRH/AVP was still able to increase firing frequency (0.49 ±0.13Hz), it was significantly (p < 0.05) reduced compared with control cells exposed to CRH and AVP. Furthermore, in corticosterone pre-treated cells, CRH and AVP failed to induce a significant transition from single spikes to bursting behaviour.

To summarise, treatment of corticotrophs with corticosterone causes an overall suppression of both spontaneous and CRH/AVP-evoked firing frequency. Interestingly, corticosterone treated cells fail to transition from single spike to complex bursting patterns. This highlights a potential mechanism for corticosterone negative feedback although molecular targets remain to be defined.

 

Nothing to Disclose: PJD, MJS

6409 17.0000 SAT-17 A Stress Regulates Electrical Excitability of Murine Corticotroph Cells 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 1-25 2200 1:45:00 PM Glucocorticoid Actions & HPA Axis Poster


Le Min*1, Victor M. Navarro2, Oluwaseun Adeola3, Shuyun Xu4, Jiangfeng Mao5, Rona S. Carroll6 and Ursula B Kaiser7
1Brigham and Women's, Boston, MA, 2Harvard Medical School and Brigham and Women's Hospital, Boston, MA, 3Brigham and Women’s Hospital, Boston, MA, 4Brigham & Womens Hosp, Boston, MA, 5Peking Union Medical College Hospital, Beijing, China, 6Brigham and Women's Hospital/Harvard Med School, Boston, MA, 7Brigham and Women's Hospital and Harvard Medical School, Division of Endocrinology/Diabetes, Boston, MA

 

Hypercortisolemia, as occurs in in a wide variety of medical conditions such as Cushing syndrome, depression, anorexia nervosa, metabolic syndrome and chronic glucocorticoid use, can lead to hypogonadism, irregular menses, and infertility.  The mechanisms by which hypercortisolemia results in impaired reproductive function are largely unknown, but independent inhibitory actions on hypothalamic Kiss1 mRNA expression, on pulsatile GnRH release, and on GnRH-stimulated LH release have all been implicated.  Glucocorticoid receptors (GR) are expressed in GnRH neurons, but the mechanisms by which glucocorticoids act at the level of the GnRH neurons to regulate GnRH release remain to be determined.  Kisspeptin receptors (KISS1R) are expressed in GnRH neurons as well and play a critical role in the regulation of GnRH secretion. Disruption of KISS1R signaling results in dysregulation of the hypothalamic-pituitary-gonadal (HPG) axis.  Glucocorticoids have been shown to inhibit cellular signaling pathways; for example, blunting insulin receptor signaling to contribute to insulin resistance. Hence, we hypothesized that glucocorticoids may similarly inhibit KISS1R signaling to contribute to hypercortisolemia-associated hypogonadotropic hypogonadism. We tested this hypothesis through a series of pharmacological and functional in vitro and in vivo studies.  Using the immortalized GT1-7 GnRH neuronal cell line, with stable expression of KISS1R, we found that treatment with 100 nM dexamethasone for 48 hours blunted the kisspeptin-10 (KP10)-stimulated intracellular calcium response. Furthermore, mice pretreated with dexamethasone (1 mg/kg intraperitoneally for 24 hours) showed decreased KP10-stimulated (50 pmol, intracerebroventricularly) LH release compared to controls.  This effect was reversed by co-administration of the GR antagonist, mifepristone (25 mg/kg intraperitoneally). In summary, our data suggest that glucocorticoid-induced dysregulation of the HPG axis occurs in part through an inhibitory effect on KISS1R signaling. This study will help to elucidate the mechanisms underlying reproductive dysfunction in patients with hypercortisolemic states.

 

Nothing to Disclose: LM, VMN, OA, SX, JM, RSC, UBK

8619 18.0000 SAT-18 A Glucocorticoids inhibit kisspeptin-stimulated LH secretion by disrupting KISS1R signaling 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 1-25 2200 1:45:00 PM Glucocorticoid Actions & HPA Axis Poster


Bence Tamas Ács1, Istvan Liko2, Karolina Feldman3, Henriett Butz3, Karoly Racz4 and Attila Patocs*5
1SemmeLweis University, Budapest, Hungary, 2Richter Ltd, Budapest, Hungary, 3Semmelweis University, Budapest, Hungary, 4Semmelweis Univ, Budapest, Hungary, 5MTA TKI, Budapest, Hungary

 

Introduction: Glucocorticoids act through glucocorticoid receptor (GR). Two major isoforms of the GR, GRa and GRβ have been described. GRβ, which differs in its C-terminal region from GRα, does not bind ligands and it has been shown to act as a dominant-negative inhibitor of transactivation produced by GRα. The potential pathological significance of GRβ has been proposed in studies showing increased GRβ expression in steroid resistant states.

Aims: To develop an in vitro model for evaluation of the GRβ-related transcriptional regulation relevant in the development of steroid resistance.

Material and Methods: Caco-2 intestinal cell line was used for developing of the GRß stable expressing cell line (Caco-Grβ). GRß was cloned form the GRa isoform. Clonal selection was performed with neomycin treatment. The GRβ expression was confirmed by quantitative real-time PCR and Western blot analysis. The glucocorticoid-mediated signaling transduction pathway was evaluated using pGRE-SEAP (Clontech) system after treatment of the basic Caco-2 and Caco-GRβ cell lines with dexamethasone (Dex, 100 nmol).  Gene expression profiles of both the basic Caco-2 and the Caco-2-GRß were evaluated using Agilent44K cDNA microarrays under basal condition and after Dex treatment. Pathways affected by differentially expressed genes were evaluated by Ingenuity pathway analysis (IPA).

Results: The GRα/GRβ ratios on the mRNA level were 1/0.6 and 1/0.001 in Caco-2GRβ cells and wild type Caco-2 cells, respectively. Luciferase activity measured after Dex treatment in Caco-GRβ cell lines was approximately 50% of that measured in basic Caco-2 cells. Dex treatment affected the expression of 151 transcripts (88 up and 63 down) in basic Caco-2 and only 16 transcripts in Caco-GRβ cells. 1182 transcripts were differentially expressed (279 under- and 903 overexpressed) in Caco-2-GRß cells compared to the basic Caco-2 cell line. IPA revealed that these transcripts are involved in the following pathways: “cell death and survival”, “cell morphology”, “cancer”, “cell-to-cell signaling”, “connective tissue disorders”, “cell mediated immune response”, “gastrointestinal and endocrine and immunological diseases”.

Conclusions: In Caco-2 cells, similarly to HeLa cells, GRβ may posses a dominant-negative effect of GRα mediated gene transcription. Therefore, Caco-2GRβ cell line may be a useful model for studying the pathomechanism of steroid resistance in inflammatory bowel disease. Forced expression of GRβ may affect the sort of cells through GR-independent mechanisms.

 

Nothing to Disclose: BTÁ, IL, KF, HB, KR, AP

9062 19.0000 SAT-19 A Overexpression of the glucocorticoid receptor-beta isoform in Caco-2 cell line causes resistance against glucocorticoids 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 1-25 2200 1:45:00 PM Glucocorticoid Actions & HPA Axis Poster


Nancy L Krett*1, Jun Qian1, Weimin Xiao2, Cristian Coarfa3, Preethi Gunaratne2 and Steven T. Rosen1
1Northwestern University, Chicago, IL, 2University of Houston, Houston, TX, 3Baylor College of Medicine, Houston, TX

 

Glucocorticoids (GC) are a cornerstone of treatment for myeloma, a hematologic malignancy of B cells.  However, patients develop resistance to this therapy in part due to loss of glucocorticoid receptor (GR) expression.  Despite the long term clinical use of GCs, little is known about mechanisms which induce cell death or post-receptor mechanisms to obviate resistance to GCs. Our overall goal is to define the direct targets of GC actions which induce cell death and identify druggable targets of GC signaling pathways that can be used to overcome resistance to GC in the absence of a functional GR. The induction of apoptosis by GCs primarily involves trans-repression of growth inducing genes but may also involve trans-activation.  Mechanisms of trans-activation involve binding to glucocorticoid response elements (GRE) as dimers leading to transcription.  Mechanisms for GR-mediated trans-repression include GR tethering to and inhibiting growth inducing transcription factors (TFs) through protein-protein interactions; by interactions with growth inducing TFs at composite GREs or by direct DNA binding to newly described negative GREs (nGRE).  The TFs which interact with GR have not been identified in myeloma and are likely cell specific.  In addition, global GR-chromatin interactions have not been investigated in myeloma. Chromatin immunoprecipitation combined with massively parallel sequencing (ChIP-seq) of GR in conjunction with bioinformatics platforms to integrate ChIP-seq data with GR-induced changes in gene expression have been used to globally elucidate the primary targets of GC actions in myeloma cell lines.  GC sensitive MM.1S myeloma cell line was treated with 1 micromolar GC for 2 hours prior to ChIP of GR.  As negative controls we performed GR ChIP on MM.1S cells with no drug treatment as well as GR negative MM.1RL cells with and without GC treatment.  In the massively parallel sequencing, we observed 8,689 GR peaks in the GC-treated MM.1S cells compared to 177 peaks in the untreated MM.1S cells and less than 26 peaks in either of the MM.1RL treatments indicating specific binding with GC treatment.  We identified GR binding sites in 44 of 503 genes repressed by GCs including genes for growth inducing kinases, cytokines and inhibitors of apoptosis.  We identified GR binding sites in 118 of 310 genes up-regulated by GC treatment including GILZ, inducers of apoptosis and tumor suppressors.  These data will provide the basis of downstream target identification for drug development.

 

Disclosure: STR: Speaker, Alios Therapeutics, Inc., Speaker Bureau Member, Celgene, Expert Medical Testimony, Hunt Suedhoff & Kalamaros, LLP, Review of Medical records, Flubrite & Jawarski LLP, Speaker, The Medal Group Corporation, Scientific Board Member, Cephalon, Inc., Speaker, Studio ER Congresi - The Triumph Group, Advisory Group Member, Millennium Pharmaceutical, Inc, Consultant, Prostraken, Inc., Advisory Group Member, Seattle Genetics, Stipend for attendence related expenses, The CM Group. Nothing to Disclose: NLK, JQ, WX, CC, PG

8612 20.0000 SAT-20 A Genomic mapping of glucocorticoid receptor binding in myeloma 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 1-25 2200 1:45:00 PM Glucocorticoid Actions & HPA Axis Poster


Marc Simard*, Lesley Hill and Geoffrey L Hammond
University of British Columbia, Vancouver, BC, Canada

 

Background: Corticosteroid-binding globulin (CBG) is a serine proteinase inhibitor family member encoded by SERPINA6. It is the major plasma binding protein of glucocorticoids and regulates cortisol bioavailability in humans. Cleavage of CBG by neutrophil elastase also disrupts its steroid-binding activity. Most genetic deficiencies that alter the production or cortisol-binding properties of CBG have so far been identified in patients with various clinical conditions. Large-scale human genome sequencing projects offer new opportunities to discover functional variants of CBG, and we have now characterized all currently known SERPINA6 non-synonymous single nucleotide polymorphisms (SNPs) that might alter CBG function and/or production.

Methods: The NCBI dbSNP, the NHLBI GO Exome Sequencing Project and the 1000 Genomes Project databases were screened for SERPINA6 non-synonymous SNPs. Expression plasmids bearing the identified SNPs were generated and recombinant proteins were produced, concentrated and semi-purified by fast performance liquid chromatography for ELISA and cortisol-binding activity measurements. Loss of steroid-binding was determined after elastase cleavage. Production of CBG variants were determined by comparing amounts of CBG secreted and retained by Chinese hamster ovary cells by Western blotting, in relation to CBG mRNA levels.

Results:  Analyses of 32 previously uncharacterized SERPINA6 non-synonymous SNPs, revealed eight with abnormal CBG production and/or function. Marked decreases in cortisol-binding affinity were observed for CBG H36Q, H36R, and H111Y variants, whereas CBG I201V and I301F variants showed a moderate decrease in cortisol-binding affinity. CBG I70N and R282N showed very low secretion, while no secretion was observed for CBG P268Q. In addition, CBG I201V and I301F variants displayed residual steroid-binding activity after neutrophil elastase cleavage when compared to wild-type CBG.

Conclusion: We have identified eight new CBG variants with either abnormal steroid-binding properties, reduced production/secretion, or altered cortisol-binding capacity after elastase cleavage. These results reveal the importance of specific residues for CBG function and add to a growing number of naturally-occurring human CBG variants that need to be considered in clinical evaluations of diseases associated with abnormalities in cortisol levels or activity.

 

Nothing to Disclose: MS, LH, GLH

7229 21.0000 SAT-21 A Naturally Occurring Variants of Human Corticosteroid-Binding Globulin 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 1-25 2200 1:45:00 PM Glucocorticoid Actions & HPA Axis Poster


Matthew J Maiden*1, Iain J. Clarke2, Marianne J Chapman1, Guy L Ludbrook3, Tim R Kuchel4 and David J Torpy5
1Royal Adelaide Hospital, Australia, 2Monash University, Melbourne, Australia, 3University of Adelaide, Australia, 4SAHMRI, Gilles Plains, Australia, 5Royal Adelaide Hospital, Adelaide, Australia

 

Title:Early hydrocortisone replacement in septic shock – a randomised controlled pre-clinical trial.

Introduction: Several studies have illustrated that “physiological dose” hydrocortisone (HC) reduces need for vasopressors in patients with established septic shock. However the effect of HC on mortality has been variable and its role in septic shock remains controversial(1,2). The response to HC may depend on timing of administration and some clinical studies suggest benefit when it is given soon after recognition of sepsis(3,4). However it is unknown to what extent HC provided early in sepsis will attenuate septic pathophysiology.

Subjects and Setting:Prospective blinded randomised placebo controlled trial in a validated 24-hour ovine model of septic shock.

Methods: Animal ethics approval was obtained from all institutions. Sixteen ewes were anaesthetised for insertion of carotid arterial line, pulmonary artery catheter, tracheostomy and insertion of cannulae in the coronary sinus, renal vein, hepatic vein, and femoral vein. Sepsis was induced by intravenous E.coli (108 organisms/kg). Animals remained sedated, ventilated and received protocol guided management of parenteral fluids and noradrenaline (NorA) to maintain mean arterial pressure of 75mmHg. Two hours following injection of E.coli, sheep received continuous infusion of HC (0.1mg/kg/hr) or placebo (Pbo) for 24 hours. Primary outcome was the total dose of NorA over 24-hours. Secondary outcomes included haemodynamics, metabolic status, renal function and immunological parameters.

Results: Study groups were evenly matched at baseline (t0) and all animals developed a hyperdynamic septic response. After 24-hours (t24), plasma cortisol levels fell in the Pbo group (nmol/L; t0 395±26 vs. t24 168±43; p<0.01) but were maintained near baseline in the HC group (t0 339±21 vs. t24298±37; p=0.13). There was no difference in the amount of NorA required for the Pbo and HC groups (mg/kg; 208 ±160 vs. 167 ±101; p=0.73). Two animals in the Pbo group died (p=0.14). There was no treatment effect on any haemodynamic variable, temperature, pH, lactate, organ-specific oxygen extraction, renal function, interleukin-6 or haematological variable.

Conclusion: In a 24-hour model of septic shock, HC infusion did not reduce NorA requirement nor alter any other measured physiological variable. Clinical trials of early HC replacement would need to measure longer-term outcomes and require large sample size.

 

Nothing to Disclose: MJM, IJC, MJC, GLL, TRK, DJT

5058 22.0000 SAT-22 A Early hydrocortisone replacement in septic shock – a randomised controlled pre-clinical trial 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 1-25 2200 1:45:00 PM Glucocorticoid Actions & HPA Axis Poster


Menno Hoekstra*, Vanessa Frodermann, Tim van den Aardweg, Ronald J. van der Sluis and Johan Kuiper
LACDR, Leiden, Netherlands

 

Hyperlipidemic apolipoprotein E knockout (APOE KO) mice show an enhanced basal level and action of anti-inflammatory glucocorticoids. Here we determined the impact of removal of the glucocorticoid function in APOE KO mice on two inflammation-associated pathologies, endotoxemia and atherosclerosis.

Mice received bilateral adrenalectomy (ADX) or control surgery. ADX mice exhibited decreased basal corticosterone levels (13±4 ng/ml vs 176±15 ng/ml: P<0.001), leukocytosis (WBC count: 10.0±0.4 x 10E9/L vs 6.5±0.5 x 10E9/L; P<0.001) and an increased spleen weight (123±7 mg vs 92±6 mg; P<0.01). Plasma total cholesterol levels were decreased by 35% (P<0.001) in ADX mice. This could be attributed to a decrease in pro-atherogenic very-low-density lipoproteins (VLDL) as a result of a diminished hepatic VLDL secretion rate (-24%; P<0.05). FACS analysis on blood leukocytes revealed increased B-lymphocyte numbers (56±5% vs 46±3%; P<0.01). T-lymphocyte populations in blood appeared to be more immature (CD62L+: 26±2% vs 19±1% for CD4 T-cells, P<0.001 and 58±7% vs 47±4% for CD8 T-cells, P<0.05), which coincided with CD4/CD8 double positive thymocyte enrichment (93.0±0.2% vs 91.1±1.4%; P<0.05). Exposure to lipopolysaccharide failed to increase corticosterone levels in ADX mice, which was associated with a 3-fold higher (P<0.05) TNF-alpha response. In contrast, the development of initial fatty streak lesions and progression to advanced collagen-containing atherosclerotic lesions was unaffected.

In conclusion, our studies show that adrenalectomy in APOE KO mice induces leukocytosis and enhances the susceptibility for endotoxemia. The adrenalectomy-associated rise in white blood cells, however, does not alter atherosclerotic lesion development probably due to a compensatory decrease in the pro-atherogenic lipids.

 

Nothing to Disclose: MH, VF, TV, RJV, JK

8114 23.0000 SAT-23 A Leukocytosis and Enhanced Susceptibility to Endotoxemia but not Atherosclerosis in Adrenalectomized APOE Knockout Mice 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 1-25 2200 1:45:00 PM Glucocorticoid Actions & HPA Axis Poster


Aristotle Panayiotopoulos*1, Divya Khurana2, Sheila Perez3, Amrit Pal S Bhangoo4, Steven Nigel Ghanny5 and Svetlana B Ten6
1SUNY Downstate Med Ctr, Brooklyn, NY, 2MMC, Brooklyn, NY, 3Maimonides Medical Center, Brooklyn, NY, 4Miller Children's Hospital, Long Beach, CA, 5Hackensack UMC, Hackensack, NJ, 6Maimonides Med Ctr, Brooklyn, NY

 

Background:

The cortisol response to oral glucose load in PCOS and metabolic syndrome is not well understood.  Decreased cortisol release after OGTT has been noted in obese subjects, which correlated with degree of insulin resistance and obesity.  We believe that glucocorticoid (GC) sensitivity is associated with cortisol response in OGTT, as shown in comparison to in vitro GC binding assay.

Methods:

We recruited 15 patients, 12 females with PCOS and 3 males with metabolic syndrome.  GC sensitivity was evaluated by F-Dex binding assays. GC index (GCI) was calculated as area under curve (AUC) (Normal value 300 ±40, calculated from 12 control patients).  A lower GCI represents decreased level of GC sensitivity.  A 2-hour OGTT was performed on same patients where serum cortisol levels were obtained at 30-minute intervals.  Cortisol index (CI) was calculated as AUC.

Results:

A positive correlation between GCI and CI was noted, indicating that lower GC sensitivity in a patient, the less of a cortisol response to OGTT and lower AUC (R=0.77, p<0.0001).  Cortisol response was statistically different between GC sensitivity groups at 60, 90, and 120 minutes (Serum cortisol (mcg/dl): GC resistant patients at 60’ 11 ± 4.5, 90’ 7 ± 1.9, 120’ 7 ± 1.6; GC Sensitive patients at 60’ 21 ±10, 22 ± 12, 12 ± 6)

Conclusions:

Low cortisol response during OGTT was associated with increased GC resistance. One suggested model can be cortisol metabolism enzymes such 11β-HSD1 in hepatocytes, which regulates glucose metabolism.

 

Nothing to Disclose: AP, DK, SP, APSB, SNG, SBT

7857 24.0000 SAT-24 A Lower Cortisol Response after OGTT Related to Glucocorticoid Sensitivity 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 1-25 2200 1:45:00 PM Glucocorticoid Actions & HPA Axis Poster


Hidenori Fukuoka*1, Takehito Takeuchi1, Yushi Hirota1, Genzo Iguchi1 and Yutaka Takahashi2
1Kobe University Hospital, Kobe, Japan, 2Kobe University Graduate School of Medicine, Kobe, Japan

 

Background; Recently, it has been reported that venous thromboembolism (VTE) is frequent in patients with Cushing’s syndrome (CS). The risk of VTE is high especially after surgical treatment of the disease. To perform selective thromboprophylaxis, it is important to clarify useful prediction marker for VTE in pre-operative patients with CS. It is well known that elevated serum D-dimer is closely associated with VTE.

Objectives; The aim of this study was to clarify the prediction marker of VTE in pre-operative patients with CS. 

Design and Methods; Twenty-seven pre-operative patients with CS and Subclinical CS due to pituitary adenoma, adrenal adenoma, or ectopic ACTH syndrome, who diagnosed at Kobe University Hospital from 2002 to 2012, were included in this study. Patients were divided to two groups; D-dimer (+); serum D-dimer levels ³a 1 mg/ml (n = 11) or D-dimer (-); serum D-dimer levels < 1mg/ml (n = 16), and compared various risk factors related to VTE. Patients who already treated with anti-coagulant drugs were excluded.

Results; Serum cortisol levels at mid-night and after low-dose dexamethasone suppression test (LDDST) were significantly higher in D-dimer (+) than in D-dimer (-) (26.6 ± 10.8 vs 14.8 ± 6.7 mg/dl, p < 0.01 and 23.0 ± 6.8 vs 14.6 ± 7.4 mg/dl, p < 0.01, respectively), while cortisol levels at morning and 24 hour urinary free cortisol (UFC) levels did not show differences (22.4 ± 11.7 vs 16.1 ± 5.6 mg/dl, p = 0.12 and 512 ± 446 vs 230 ± 253 mg/24h, p = 0.09, respectively). The aPTT were significantly shortened in D-dimer (+) than in D-dimer (-) (23.8 ± 3.3 vs 29.0 ± 7.9 sec, p < 0.05). Linear regression analysis revealed that both cortisol levels at mid-night and after LDDST were negatively correlated with the aPPT (r = - 0.45, p < 0.05 and r = - 0.53, p < 0.05, respectively).

Conclusion; High serum cortisol levels at mid-night and after LDDST, both of which negatively correlated with the aPTT, were associated with elevated levels of D-dimer rather than morning cortisol levels or UFC levels. These results suggest that a sustained exposure to high cortisol levels at night increases susceptibility to thrombogenesis in CS.

 

Nothing to Disclose: HF, TT, YH, GI, YT

7145 25.0000 SAT-25 A High Serum Cortisol Levels at Mid-night and After LDDST are Associated with Elevated Levels of D-dimer in Patients with Cushing's Syndrome 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 1-25 2200 1:45:00 PM Glucocorticoid Actions & HPA Axis Poster


Richard J Ross*1, Martin J Whitaker2, Miguel Debono3, Hiep Huatan4, Deborah P. Merke5 and Wiebke Arlt6
1University of Sheffield, United Kingdom, 2University of Sheffield, 3University of Sheffield, Sheffield, United Kingdom, 4Diurnal Limited, 5National Institutes of Health, Bethesda, MD, 6University of Birmingham, Birmingham, United Kingdom

 

It is not possible with current immediate release oral formulations of hydrocortisone to fully replicate the circadian profile of endogenous cortisol. Physiological hormone replacement is important for patients with congenital adrenal hyperplasia as it addresses the critical overnight build-up of androgens and for patients with adrenal insufficiency as it may circumvent early morning fatigue.  Previous attempts to develop a circadian product of hydrocortisone, using a modified release tablet formulation, have suffered from the drawback of reduced bioavailability (Debono et al, JCEM 2009,94,1548-54). This was hypothesized to be due to the reduced lumenal exposure of hydrocortisone in the lower gut which is required to confer an extended drug delivery profile. We now report the development of a new, modified release, multiparticulate formulation of Chronocort®, which replicates the circadian profile of cortisol without compromising bioavailability. Pharmacokinetic evaluation of Chronocort® in healthy male adult volunteers, in whom cortisol secretion was supressed with dexamethasone, showed excellent comparability to the circadian profile of cortisol over the dose range of 20mg to 30mg. Following dosing of 30mg Chronocort®( 20mg at 23:00h and 10mg at 07:00h), the mean Tmax was achieved at 8h post-dosing with a corresponding mean Cmax of 665nmol/L (24 ug/dl). It was further observed that the splitting of the 30mg dose into a toothbrush regimen (last thing at night and first thing in the morning), the mean cortisol serum levels for the afternoon were consistently maintained above 200nmol/L (7ug/dl), in line with the endogenous cortisol profile observed in healthy controls. Over the evaluated dose range of 5mg to 30mg, Chronocort® exhibited dose linearity with a relative bioavailability (estimated from the AUC0-t  ratio of Chronocort® to hydrocortisone immediate release) of greater than 100%. We conclude from this study that Chronocort® has the potential to replicate the physiological circadian profile of cortisol without compromising the bioavailability of hydrocortisone.

 

Disclosure: RJR: Founder, Diurnal. MJW: Board Member, Diurnal. HH: Board Member, Diurnal. DPM: Clinical Researcher, Diurnal. WA: Consultant, Diurnal. Nothing to Disclose: MD

6072 1.0000 SAT-26 A Pharmacokinetic characterisation of Chronocort® - A new modified release oral formulation of hydrocortisone that mimics the endogenous circadian profile of cortisol 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 26-40 2201 1:45:00 PM Glucocorticoid Actions & Disease Poster


Irina Bancos*1, Ravinder J. Singh1, Sandra c Bryant2, Kristi Mielke2, Jolaine Hines2, Neena Natt1, Todd B Nippoldt1 and Dana Z Erickson1
1Mayo Clinic, Rochester, MN, 2Mayo Clinic

 

Context: While 10% of total cortisol is free and considered biologically active, measurements of total cortisol levels after cosyntropin stimulation are used in current clinical practice in evaluation of adrenal insufficiency. Total cortisol comprises cortisol bound to proteins (such as CBG and albumin) and free cortisol. As there is a high inter- and intraindividual variability of binding proteins influenced by disease states or medications, measurement of free cortisol fraction can potentially improve diagnosis of adrenal insufficiency, especially in the borderline cases.

Aim: Our aim was to investigate the relationship between free cortisol and total cortisol levels in subjects undergoing 250 mcg Cosyntropin stimulation test for suspected or established adrenal insufficiency.

Methods: After signing the consent form, 296 consecutive subjects undergoing Cosyntropin stimulation testing at the outpatient Endocrine testing center underwent total and free cortisol measurements at baseline, 30 and 60 minutes.  Free cortisol levels were measured by equilibrium dialysis followed by liquid chromatography tandem mass spectrometry, an in-house developed assay. Adrenal insufficiency was defined as failure to reach a level of 18 mg/dl for total cortisol. ROC (receiver-operator characteristic) analysis was used to identify the most optimal cut-point of free cortisol values to distinguish between the group with confirmed adrenal insufficiency and the group without adrenal insufficiency.

Results: Of the 296 patients participating in this study (mean age of 48.8 ± 17.1, 77 males (26%)), 42 subjects (14%) were classified as adrenally insufficient. Causes of adrenal insufficiency included: steroid use (52%), Addison’s (12%),  adrenalectomy for a cortisol producing adenoma (7%), medications or sleep apnea (5%), idiopathic secondary ACTH deficiency (2%), pituitary lesion (4%), Cushing’s disease with pituitary adenoma resection (2%), Sheehan syndrome (2%), unknown (12%). ROC curves for free cortisol were derived with a 60 minute cutpoint at 1190 ng/dl (CI 95% 1030-1210) providing the best AUC of 0.99 (CI 95% 0.98-0.99), p<0.0001. Patients on oral estrogens (n=43, 15%) had higher baseline total cortisol levels but no difference in baseline free cortisol concentrations when compared to non-estrogen group. Both 30 min. and 60 min. free and total stimulated cortisol levels were similar across estrogen and non-estrogen groups. In the group receiving oral estrogen and classified as adrenally sufficient,  4 of 37 patients (10.8%)  would have been defined as  adrenally insufficient using the 60 min. free cortisol cut-off defined by only those patients not using estrogen replacement (1170 ng/dl) .

Conclusion: Free cortisol measurement is a potential useful tool for determination of adrenal insufficiency, especially in borderline cases.

 

Disclosure: DZE: Advisory Group Member, Ipsen. Nothing to Disclose: IB, RJS, SCB, KM, JH, NN, TBN

6100 2.0000 SAT-27 A FREE CORTISOL MEASUREMENT BY EQUILIBRIUM DIALYSIS FOLLOWED BY LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 26-40 2201 1:45:00 PM Glucocorticoid Actions & Disease Poster


Dominika Ewa Nanus*1, Andrew Filer1, Benjamin Fisher1, Peter Taylor2, Paul Michael Stewart1, Christopher Buckley1, Iain McInnes3, Mark Cooper1 and Karim Raza1
1University of Birmingham, Birmingham, United Kingdom, 2University of Oxford, United Kingdom, 3University of Glasgow, Glasgow, United Kingdom

 

Abnormalities in endogenous glucocorticoid synthesis or metabolism have been linked to the development of rheumatoid arthritis (RA). Within the synovium of patients with RA, synovial fibroblasts generate active corticosteroids through expression of the glucocorticoid metabolising enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). In vitro, this enzyme is strongly up-regulated by pro-inflammatory cytokines such as tumour necrosis factor α (TNFα) and IL-1β. In this study, we determined the relationship between inflammation and glucocorticoid metabolism in vivo, in a clinical study of patients with inflammatory arthritis treated with anti-TNFα therapy. Urine samples were collected from RA (n=20) and psoriatic arthritis (PsA) (n=20) patients as part of a multicentre study assessing responses to infliximab and etanercept and from healthy controls (HC, n=51). Systemic measures of glucocorticoid metabolism were assessed by gas chromatography/mass spectrometry at week 0, 4 and 12 after anti-TNFα therapy and calculated as the tetrahydrocortisol+5αTHF /tetrahydrocortisone ((THF+5αTHF)/THE) and the cortols/cortolones ratios. Clinical data including DAS28 and CRP were also collected. Urinary (THF+5αTHF)/THE (1) and cortols/cortolones (2) ratios were significantly higher in RA and PsA patients prior to treatment compared to HC (1: RA, 1.22(0.93-1.3), P=0.005; PsA, 1.05(0.87-1.41), P=0.02; HC, 0.91(0.75-1.05); 2: RA, 0.66(0.51-0.75), P=0.0001; PsA, 0.60(0.51-0.66), P=0.0005; HC, 0.48(0.41-0.54)). The elevated (THF+5αTHF)/THE ratio fell following anti-TNFα therapy at 4 weeks for RA (1.22(0.93-1.30) vs 0.94(0.81-1.23), P=0.017) and at 12 weeks for PsA (1.05(0.87-1.41) vs 0.96(0.72-1.23), P=0.018). A similar observation was made for the cortols/cortolones ratio at 4 weeks and 12 weeks for RA (0.66(0.51-0.75) vs 0.55(0.51-0.62), P= 0.004 and 0.66(0.51-0.75) vs 0.56(0.50-0.62), P= 0.03). In patients with RA there was a positive correlation between the 12 week change in DAS28 score (1), CRP (2) and the 12 week change in the cortols/cortolones ratio (1: r=0.64, P= 0.003; 2: r=0.45, P= 0.048). This study demonstrates, for the first time, that the increased 11β-HSD1 activity seen in patients with inflammatory arthritis is causally related to the underlying inflammatory disease. Furthermore, the changes in related glucocorticoid metabolising enzymes suggest that there is a coordinated change in glucocorticoid metabolism which promotes higher tissue glucocorticoid levels.

 

Nothing to Disclose: DEN, AF, BF, PT, PMS, CB, IM, MC, KR

7088 3.0000 SAT-28 A TNFα regulates in vivo cortisol metabolism in inflammatory arthritis 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 26-40 2201 1:45:00 PM Glucocorticoid Actions & Disease Poster


Peter Kamenický**1, Alban Redheuil*2, Charles Roux2, Sylvie Salenave3, Laurent Macron2, Christel Jublanc4, Christiane Ajzenberg5, Zainab Raissouni2, Arshid Azarine2, Jacques Young1, Nadjia Kachenoura6, Sylvie Brailly-Tabard1, Elie Mousseaux7 and Philippe Chanson8
1CHU de Bicêtre, APHP, Le Kremlin Bicêtre, France, 2HEGP, APHP, Paris, France, 3AP-HP, Hôpital de Bicêtre, Service d'Endocrinologie et des Maladies de la Reproduction, Le Kremlin Bicêtre, France, 4CHU Pitié-Salpêtrière, APHP, Paris, France, 5CHU Henri Mondor, APHP, Creteil, France, 6UMR_S 678, INSERM, Paris, France, 7Université Paris Descartes, Faculté de Médecine & AP-HP, Hôpital Européen George Pompidou, Service de Radiologie Cardiovasculaire, Paris, France, 8Univ 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:Cardiomyopathy in Cushing’s syndrome (CS) has been evaluated in few echocardiograpic studies showing left ventricular (LV) remodeling and dysfunction, but has not yet been analyzed by cardiac magnetic resonance imaging (cMRI), currently presenting the gold standard in heart imaging.

Objective:To investigate the impact of CS on cardiac structure and function by cMRI.

Design:Prospective case-control study in a tertiary referral endocrine center and cardiovascular imaging center.

Patients: Seventeen consecutive patients with newly diagnosed Cushing’s syndrome (16F/1M) and 17 age and sex-matched normontensive volunteers were studied. Patient’s median 24-hour urine free cortisol excretion was 371 μg/24h (range 102-3205).

Results: BMI, systolic blood pressure and heart rate were higher in patients with CS, whereas body surface area and diastolic blood pressure were not different between the groups. Compared to controls, patients had similar median end-diastolic LV volumes but decreased LV stroke volumes (67 mL vs 81 mL, P=0.02) and ejection fractions (52% vs 66%, P<0.001) with increased end-systolic volumes (P=0.01), indicative of reduced systolic LV performance. Both end-diastolic and end-systolic left atrial (LA) volumes were comparable between groups but LA ejection fraction was markedly reduced in patients compared to controls (64% vs 113%, P<0.001) pointing to severe diastolic LV dysfunction. Right ventricular parameters were comparable to LV determinants with increased end-systolic volumes (P=0.004), reduced stroke volumes (61 mL vs 76 mL, P=0.004) and ejection fraction (48% vs 62%, P<0.001). End-diastolic LV segmental thickness was increased in patients compared to controls in the basal (11.7 vs 7.9 mm, P<0.0001), mid-LV (10.7 vs 7.0 mm, P<0.0001) and apical (9.0 vs6.1 mm, P<0.0001) short axis planes, demonstrating global LV hypertrophy. This was not due to increased LV after-load since the proximal aortic stiffness was not different between the groups. One patient had dilated cardiomyopathy with normal coronary angiography and deeply altered LV systolic function (EF < 30%). Delayed gadolinium enhancement indicated myocardial fibrosis in 3 patients.

Conclusion: Patients with Cushing’s syndrome present with global cardiac hypertrophy associated with significantly reduced systolic performance and diastolic dysfunction of the left and right ventricles, compensated by higher heart rate. Reversibility of these observations is currently being evaluated.

 

Nothing to Disclose: PK, AR, CR, SS, LM, CJ, CA, ZR, AA, JY, NK, SB, EM, PC

7504 4.0000 SAT-29 A Cardiomyopathy in Cushing's syndrome revisited by cardiac magnetic resonance imaging 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 26-40 2201 1:45:00 PM Glucocorticoid Actions & Disease Poster


Andrea Rebellato1, Andrea Grillo2, Francesca Dassie3, Nicoletta Sonino4, Chiara Martini5, Agostino Paoletta6, Renzo Carretta2, Pietro Maffei5 and Francesco Fallo*1
1Univ of Padova, Padova, Italy, 2Trieste University Hospital, Trieste, Italy, 3Padova University Hospital, Padova, Italy, 4University of Padova, Padova, Italy, 5Padua University Hospital, Padua, Italy, 6Cittadella Civic Hospital, Padova, Italy

 

Cushing’s syndrome is associated with high cardiovascular morbidity and mortality. Blood pressure (BP) variability within a 24-hour period is increasingly recognized as both a marker and a risk factor for cardiovascular disease, either in hypertensive cohorts or in general populations. The aim of our study was to investigate short-term systolic (SBP) and diastolic (DBP) variability indexes in Cushing’s syndrome. Twenty-one newly diagnosed patients with Cushing’s syndrome (18F, 3M; mean age 48±13 years; 17 pituitary-dependent Cushing’s disease and 4 adrenal adenoma) underwent 24-hour ambulatory BP monitoring (ABPM) and evaluation of cardiovascular risk factors. Based on ABPM, patients were divided into 8 normotensive (NORCUSH) and 13 hypertensive (HYPCUSH) patients, and were compared with 16 normotensive age-, sex-, BMI- and ABPM-matched control subjects (NORCTR). Short-term SBP/DBP variability was derived from ABPM and calculated as the following: (1) Standard Deviation (SD) of 24-hour, daytime, and nighttime SBP/DBP; (2) weighted SD of 24-hour SBP/DBP; (3) average real variability (ARV), i.e., the average of the absolute differences between consecutive SBP/DBP measurements over 24 hours. In comparison with controls, patients with Cushing’s syndrome, either normotensive or hypertensive, had higher SD of 24-hour, daytime, and nighttime SBP/DBP, as well as higher weighted SD and ARV of 24-hour SBP/DBP (NORCUSH and HYPCUSH vs NORCTR, P <0.0001 for all indexes). There was no difference between NORCUSH and HYPCUSH in urinary cortisol levels, in absolute BP and variability BP measures, and in the prevalence of diabetes, hyperlipidemia and obesity. Conclusion: Short-term BP variability is increased in patients with Cushing’s syndrome, independent of BP elevation. It may represent an additional cardiovascular risk factor in this disease. The role of excess cortisol in BP variability has to be further clarified.

 

Nothing to Disclose: AR, AG, FD, NS, CM, AP, RC, PM, FF

4189 5.0000 SAT-30 A SHORT-TERM BLOOD PRESSURE VARIABILITY IS INCREASED IN CUSHING'S SYNDROME 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 26-40 2201 1:45:00 PM Glucocorticoid Actions & Disease Poster


Ah Reum Khang*, A Ram Hong, Kyong Yeun Jung, Jung Hee Kim, Sang Wan Kim and Seong Yeon Kim
Seoul National University College of Medicine, Seoul, Korea, Republic of (South)

 

Measurement of plasma adrenocorticotrophic hormone (ACTH) is the first step to distinguish adrenal Cushing’s syndrome from Cushing’s disease according to the current guidelines. However, plasma ACTH levels were within normal range in about 30% of patients with adrenal Cushing’s syndrome due to the variability of ACTH assay. The high-dose dexamethasone suppression tests (HDST) and serum dehydroepiandrosterone sulfate (DHEA-S) had been used to make the differential diagnosis of Cushing’s syndrome. The aim of the present study was to assess the utility of plasma ACTH levels in differential diagnosis of Cushing’s syndrome and compare with HDST and serum DHEA-S.

We conducted a retrospective and multicenter study from January 2000 to May 2012. A total of 109 patients with endogenous Cushing’s syndrome were recruited. We excluded 17 patients with ectopic Cushing’s syndrome, recurrent Cushing’s syndrome, cyclic Cushing’s syndrome and subclinical Cushing’s syndrome. Serum cortisol or 24 hour urine free cortisol (UFC) after HDST (2mg per 6 hours for 2 days) and serum DHEA-S levels were measured.

Ninety-two patients were included in final analysis. Of them, 57 patients had adrenal Cushing’s syndrome and 35 patients had Cushing’s disease. Median age was 40 years in both groups. In receiver operating characteristic analysis, the area under the curve (AUC) of plasma ACTH, DHEA-S and percent suppression of serum cortisol or UFC after HDST were 0.954, 0.841, 0.950 and 0.997, repectively (all P <0.001). The cut-off value of plasma ACTH and percent suppression of serum cortisol or UFC after HDST was 24 pg/ml (normal range, 10 ~ 60 pg/ml), 33.3% and 61.6%. The sensitivity and specificity of plasma ACTH were 84.2%, 94.3%, and those of serum cortisol or UFC after HDST were 95.8%, 90.6% and 97.9%, 96.7%, respectively. Three patients with adrenal incidentalomas and non-suppressed plasma ACTH levels were successfully diagnosed with HDST.

In the present study, there was an overlap in plasma ACTH levels between patients with adrenal Cushing’s syndrome and those with Cushing’s disease. The high dose dexamethasone suppression test may be useful in etiological diagnosis of Cushing’s syndrome, especially when plasma ACTH levels alone were inconclusive.

 

Nothing to Disclose: ARK, ARH, KYJ, JHK, SWK, SYK

7826 6.0000 SAT-31 A The Limited Value of Plasma Adrenocorticotrophic Hormone to Differentiate Adrenal Cushing's Syndrome from Cushing's Disease 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 26-40 2201 1:45:00 PM Glucocorticoid Actions & Disease Poster


Cornelie D. Andela*1, Steven J.A. van der Werff2, J. Nienke Pannekoek1, Susan M. van den Berg2, Onno C. Meijer1, Mark A. Buchem1, Serge A.R.B. Rombouts1, Roos C. van der Mast2, Johannes A. Romijn3, Jitske Tiemensma4, Nienke R. Biermasz1, Nic J.A. van der Wee1 and Alberto M. Pereira2
1Leiden University Medical Center, Netherlands, 2Leiden University Medical Center, The Netherlands, 3Academic Medical Center, Amsterdam, Netherlands, 4University of California Merced

 

Context: Patients in long-term remission of Cushing’s disease (CD) have persistent impairments in psychological and cognitive functioning. It is unknown whether, and to what extent, these impairments are also accompanied by structural abnormalities in the brain.

Objective: To investigate structural changes in the brain of patients after long-term remission of CD.

Design: A cross-sectional study.

Patients and Methods: In 25 patients in long-term remission of CD and 25 matched healthy controls, grey matter volume of the regions of interest (hippocampus, amygdala, anterior cingulate cortex) and the whole brain were examined, using 3T Magnetic Resonance Imaging and a voxel based morphometry (VBM) approach. In addition, we assessed psychological and cognitive functioning using validated questionnaires, and clinical severity using the Cushing's syndrome Severity Index.

Results: Compared to controls, patients had significantly smaller grey matter volumes of areas in the anterior cingulate cortex (on average 14%, P<0.05) and significantly greater volumes in the left posterior lobe of the cerebellum (on average 34%, P<0.05). There were no significant differences in grey matter volumes in the hippocampus and amygdala. As expected CD patients demonstrated more depressive symptoms (P=0.005), more anxiety (P=0.003) and more apathy (P=0.002) compared to controls, but the differences in grey matter volumes were not associated with clinical severity, nor with measures of psychological or cognitive dysfunction.

Conclusion: Patients in long-term remission of CD showed structural abnormalities in the brain, with smaller ACC volumes and an asymmetric enlargement of the posterior lobe of the cerebellum, in the presence of increased depressive symptoms, anxiety, and apathetic behavior. This indicates incomplete recovery of the central nervous system despite successful treatment for CD.

 

Nothing to Disclose: CDA, SJAV, JNP, SMV, OCM, MAB, SARBR, RCV, JAR, JT, NRB, NJAV, AMP

8244 7.0000 SAT-32 A Smaller grey matter volumes of the anterior cingulate cortex (ACC) and greater cerebellar volumes in patients despite long-term remission of Cushing's disease: a case control study 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 26-40 2201 1:45:00 PM Glucocorticoid Actions & Disease Poster


Diego Federico Cobice*1, Logan MacKay2, Andrew McBride1, Pat Langridge Smith2, Scott Webster1, Brian R Walker1 and Ruth Andrew1
1University of Edinburgh, Edinburgh, United Kingdom, 2Scottish Instrumentation and Resource Centre for Advanced Mass Spectrometry, Edinburgh, United Kingdom

 

Intracellular inter-conversion of steroids has potent local effects on receptor activation, providing therapeutic targets in diseases as diverse as breast cancer (aromatase inhibition), prostate hyperplasia (5α-reductase inhibition), type 2 diabetes (11β-HSD1 inhibition to reduce glucocorticoid regeneration in liver and adipose), and cognitive impairment (11β-HSD1 inhibition in brain). However, the field is hampered by inadequate methods to assess steroids within tumours or tissue sub-regions. Here, we address this challenge using mass spectrometry imaging (MSI) to assess effects of 11β-HSD1 on conversion of inert 11-dehydrocorticosterone (A) to active corticosterone (B) within sub-regions of murine brain.

Four groups of mice (male, 12 weeks) were studied: 11β-HSD-/- mice (KO) and C57Bl/6 (WT) controls (n=6/group); C57Bl/6 mice (n=3/group) 1h after UE2316 (11β-HSD1 inhibitor; 20mg/kg oral) or vehicle. Steroids and UE2316 were imaged following on-tissue derivatisation with Girard T (GirT) in brain sections (10µm) embedded in gelatin to obtain mass spectral images (150-1000amu, positive ion, spatial resolution 200µm) of m/z 460.31698±0.025 (GirT B), m/z 458.30133±0.025 (GirT A), and m/z 390.08377±0.025 (UE2316) following Matrix assisted Laser Desorption Ionisation using a 12T SolariX Fourier transform Ion cyclotron MS (MALDI-FTICR-MS). Confirmatory quantitation in tissue homogenates was by Liquid Chromatography tandem Mass Spectrometry. Data are mean±SEM, *p<0.05.

Non-derivatised neutral steroids were poorly detected by MSI. Signals were boosted (104 fold) by formation of GirT hydrazones. α-Cyano-4-hydroxycinnamic acid, applied by spray coating, was selected as the matrix producing the best signal to noise.

Glucocorticoids were detected in highest abundance in cortex and hippocampus. In 11β-HSD1 deficiency, the B/A ratio was reduced (KO vs WT; cortex 5.1±0.4* vs 10.1±0.6; hippocampus 3.7±0.4* vs 6.9±0.9; amygdala 1.9±0.5* vs 4.4±0.8), with confirmatory LC/MS values (whole brain) of 5.4±0.7* (KO) vs 11.9±1.6 (WT). Likewise agreement between MSI and LC/MS was observed in plasma (B/A: MSI 8.2±0.5 vs 11.0±1.2; LC/MS 7.3±0.8 vs 11.3±1.1). With 11β-HSD1 inhibition, MSI-detected levels of UE2316 peaked in brain 1h post-dose, with similar reduction in B/A ratio (of 32% cortex, 52% hippocampus, and 58% amygdala; (* significant effect of treatment). Lower ratios in KOs were mainly driven by increased amounts of 11-dehydrocorticosterone.

MSI is a powerful new tool to study the regional variation in steroids within tissues with a resolution <200µm. We have demonstrated its utility for pharmacokinetic/pharmacodynamic analysis during development of novel 11β-HSD1 inhibitors for Alzheimer's disease. MSI has great potential to enhance (patho) physiological studies of steroid metabolism in many tissues and for multiple steroids.

 

Nothing to Disclose: DFC, LM, AM, PL, SW, BRW, RA

6893 8.0000 SAT-33 A Monitoring effects of 11β-hydroxysteroid dehydrogenase-1 deficiency or inhibition on Region-Specific Corticosteroid Regeneration in Brain using Mass Spectrometry Imaging 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 26-40 2201 1:45:00 PM Glucocorticoid Actions & Disease Poster


Sigridur Bjornsdottir*1, Marianne Oksnes2, Magnus Isaksson3, Roy Miodini Nilsen4, Eystein Sverre Husebye5, Kristian Lovas5, Olle Kämpe6, Anna-Lena Hulting7, Thomas Nystrom8 and Sophie Bensing9
1Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden, Sweden, 2Institute of Medicine, Haukeland University Hospital, Bergen, Norway, 3Uppsala University, 4Haukeland University hospital, Bergen, Norway, 5University of Bergen, Bergen, Norway, 6Karolinska Institutet, Stockholm, Sweden, 7Karolinska Univ Hosp, Stockholm, Sweden, 8Karolinska Institutet, Department of Clinical Science and Education, Division of Internal Medicine, Södersjukhuset AB, Stockholm, Sweden., 9Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden

 

Background

Conventional glucocorticoid replacement therapies result in unphysiological variation in plasma cortisol levels; concern has been raised regarding long-term metabolic consequences. Glucocorticoid replacement is technically feasible by continuous subcutaneous hydrocortisone infusion (CSHI), which can mimic the normal diurnal cortisol rhythm. The aim of this study was to compare insulin sensitivity in patients with Addison’s disease (AD) on CSHI vs. three daily doses of oral hydrocortisone (OHC).

 

Design, Subjects, Measurements

This was an open randomised, cross-over trial, comparing 3 months of weight adjusted OHC with 3 months on CSHI in 33 AD patients. Treatment A was OHC with weight-adjusted doses as suggested by Mah et al (1). Treatment B was CSHI using Solu-Cortef® (50mg/ml) with a body surface area adjusted dose (10mg/m2/24hrs). Patients were examined at baseline and after 2 and 3 months of each treatment. Insulin sensitivity was determined after 2 month of each treatment in 15 Swedish patients using the euglycemic hyperinsulinemic clamp technique (40mU/m2). Whole-body insulin sensitivity (glucose disposal), M value, was calculated from the amount of glucose infused during the last 30 min of the clamp divided by body weight (kg) and period (min) and expressed as mg/kg/min. We also assessed fasting insulin sensitivity, in all 33 patients, using HOMA and the insulin resistance index (HOMA-IR) calculated as ((fasting plasma glucose [mmol/L] x fasting serum insulin [μU/mL]) /22.5). Statistical analyses were performed with linear mixed effects models with random intercepts.

 

Results

Twenty-five women and 8 men mean±SD, age 48±12 and AD duration 12±10 years participated in the study. The median dose of OHC was 0.23 mg/kg/day, (range; 0.2-0.5) and 0.28 mg/kg/24h, (0.24-0.5) in CSHI.

Body Mass index (BMI) and log HOMA index slightly increased during CSHI (p for trend 0.037 and 0.011), but compared with OHC no significant difference was found (p for interaction 0.085 and 0.19). No difference was found in insulin sensitivity, M-value (p= 0.59). There were no treatment differences over time in waist-hip ratio, (p for interaction 0.24) systolic or diastolic blood pressure (p for interaction 0.73 and 0.94).

Conclusion

CSHI replacement over a three-month period does not influence antropometric measures, blood pressure, or insulin sensitivity, compared with OHC. More randomized trials on long-term effects of CSHI replacement on cardiovascular parameters are needed.

 

Nothing to Disclose: SB, MO, MI, RMN, ESH, KL, OK, ALH, TN, SB

7244 9.0000 SAT-34 A Insulin sensitivity in patients with Addison's disease: A randomised cross-over trial comparing conventional glucocorticoid replacement therapy with continuous subcutaneous hydrocortisone infusion therapy 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 26-40 2201 1:45:00 PM Glucocorticoid Actions & Disease Poster


Ashwini Mallappa*1, Lori-Ann Daley1, Carol Van Ryzin1, Richard J Ross2 and Deborah P. Merke1
1National Institutes of Health, Bethesda, MD, 2University of Sheffield, Sheffield, United Kingdom

 

Background: Classic congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is an autosomal recessive disorder resulting in cortisol and aldosterone deficiency and androgen excess. Treatment goals include replacement of the deficient hormones and suppression of excess androgens. There is no standard treatment regimen for adults with classic CAH. Glucocorticoid regimens are individualized and a variety of short- and long-acting glucocorticoids are used. Close monitoring of clinical and biochemical parameters is essential to avoid complications of over- or under-treatment.  We typically measure hormone levels prior to morning dose of medications for patients receiving hydrocortisone, aiming for mildly elevated morning 17-hydroxyprogesterone levels (1).  However, the optimal manner in which to monitor hormone levels remains unclear, especially for patients receiving longer-acting glucocorticoids.  Here, we present 24-hour serial sampling data from adult patients with classic CAH.

Methods: Sixteen adults with the classic form of CAH were admitted to our inpatient unit for serial hormone profiling. Serial sampling was done on their home regimen of glucocorticoid (dexamethasone, prednisone, or hydrocortisone) and fludrocortisone.  Every 2 hour blood samples were obtained for adrenocorticotrophic hormone (ACTH), 17-hydroxyprogesterone, androstenedione and glucocorticoid levels.

Results: Preliminary results of serial hormone profiling show extreme fluctuations in hormone levels in relation to the timing of the medications. Patients exhibited persistence of an endogenous diurnal variation in their hormone levels, despite being on glucocorticoid medication. The timing of peak hormone levels could be predicted based on the half-life of the medication.

Conclusion: Twenty-four hour hormone profiling reveals many of the complexities involved in the management of CAH. Randomly obtained hormone levels provide limited information. Diurnal fluctuations and hormonal variation in relation to the timing and half-life of glucocorticoid medications need to be considered when evaluating hormone levels. An optimal timing for the measurement of hormones in patients with CAH can be determined based on their treatment regimen, although some patient-to-patient variability remains.

 

Disclosure: RJR: Founder, Diurnal. DPM: Clinical Researcher, Diurnal. Nothing to Disclose: AM, LAD, CV

7932 10.0000 SAT-35 A Timing is Everything: Hormonal Evaluation of Patients with Congenital Adrenal Hyperplasia 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 26-40 2201 1:45:00 PM Glucocorticoid Actions & Disease Poster


Paolo Beck-Peccoz*1, Serena Palmieri1, Valentina Morelli2, Antonio Salcuni3, Cristina Eller-Vainicher1, Elisa Cairoli1, Volha V. Zhukouskaya1, Alfredo Scillitani3 and Iacopo Chiodini1
1University of Milan, Fondazione IRCCS Cà Granda-Ospedale Maggiore Policlinico, Milan, Italy, 2University of Milan, Fondazione IRCCS Ca’ Granda-Ospedale Maggiore Policlinico, Milan, Italy, 3Casa Sollievo della Sofferenza Hospital, San Giovanni Rotondo (FG), Italy

 

Introduction. In overt hypercortisolism, GH reserve is decreased and normalized after surgery. In patients with subclinical hypercortisolism (SH), GH secretion has been not fully investigated. In particular, no data about the GH reserve after recovery from SH are available. We assessed GH secretory reserve in AI patients with and without SH and, in the former, before and after surgery.

Methods. We enrolled 24 patients with adrenal incidentalomas, 12 with SH (SH+, 8F/4M, 58.3±6.5 yrs) and 12 without SH (SH-; 11F/1M, 61.8±10.6 yrs). SH was diagnosed in the presence of  ≥2 out of: 1 mg overnight dexamethasone suppression test between 1.8 and 5 µg/dL, urinary free cortisol between 70 and 140 µg/24hrs (n.v. 10-70) and ACTH levels <10 pg/mL. We assessed GH secretion by GHRH+Arginine test (GHRH-ARG) and basal serum IGF-I levels, expressed as SDS (IGF-I SDS, corrected for age). Body mass index (BMI) and insulin resistance index (HOMA-IR) were also evaluated. Eight SH+ patients, who underwent adrenalectomy, were re-evaluated after the restoration of a normal cortisol secretion.

Results.  Age, gender, BMI, HOMA-IR, IGF-I and mean basal GH values were comparable between SH+ and SH- patients. After GHRH+ARG stimulus, the mean GH peak levels (GH-P) and the GH response measured as Area Under Curve (GH-AUC) were significantly lower in SH+ than in SH- Group (15.2±8.1 vs 44.5±30.9 µg/L, P=0.004; 1417.6±802.9 vs 4027.8±2475.7 µg/L/120 min, P=0.002, respectively), even after adjusting for age and BMI. One SH- and 2 SH+ patients had a completely blunted response to GHRH+ARG (GH-P 5.2 µg/L, 5.4 µg/L and 7.7 µg/L, BMI 27.6, 25.6 and 28.2 respectively). In SH+ patients after adrenalectomy GH-P levels and GH-AUC significantly increased as compared to pre-treatment values (23.7±16.3 vs 15.8±10.2 µg/L, P=0.04; 2548.8±1982.2 vs 1617.6±910.8 µg/L/120 min, P=0.072), while IGF-I levels, though increased, did not reach the statistical significance. The two SH+ patients with completely blunted response to GHRH+ARG at baseline, regained a normal response post-adrenalectomy (GH-P 11.6 µg/L and 11.8 µg/L). In the multivariate linear regression analysis, the GH-AUC and the GH-P levels were negatively associated with UFC after adjusting for age and BMI (β= -0.39, P=0.02 and β= -0.4, P=0.02 respectively).

Conclusion. GH secretion appears to be decreased in SH patients and normalizes after recovery. The evaluation of GH secretion could be used as an additional parameter for the diagnosis of SH.

 

Nothing to Disclose: PB, SP, VM, AS, CE, EC, VVZ, AS, IC

8577 11.0000 SAT-36 A GH reserve in subclinical hypercortisolism: evaluation pre- and post-adrenalectomy 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 26-40 2201 1:45:00 PM Glucocorticoid Actions & Disease Poster


Walter Bonfig*1 and Hans Peter Schwarz2
1University Children's Hospital, TU Munich, Munich, Germany, 2University of Bern, Lugnorre, Switzerland

 

Introduction: Newborns and infants with salt-wasting congenital adrenal hyperplasia (CAH)  require relatively high doses of fludrocortisone (FC) and may therefore be at risk for hypertension.

Objective: To evaluate blood pressure, FC doses and plasma renin activity (PRA) in infants with CAH due to 21-hydroxylase deficiency.

Patients & Methods: 33 patients (18f/15m), who were diagnosed with classical CAH by newborn-screening, were prospectively followed until the age of 4 years. Only two patients were diagnosed with simple virilizing CAH, 31 patients had salt-wasting CAH. All patients were initially treated with both hydrocortisone (HC) and FC. Mean start of HC and FC treatment was day 9.8±9.2 postnatally. Mean daily HC dose ranged from 8.6 to 12.3 mg/m²/day over the study period.

Results: Mean birth weight and length were 0.39±0.8 SDS and 1.0±0.7 SDS, respectively. From three to 18 months of age, mean daily FC dose was approximately 0.1±0.05 mg. At 24 months mean daily FC dose could be decreased to 0.05±0.02 mg. Initially, at three months of age, mean PRA was elevated (PRA 16.6±26.6 ng/ml/h) and normalized by the age of six months (PRA 5.3±13.1 ng/ml/h). The lowest mean PRA was measured at 24 months of age (PRA 1.8±2.7 ng/ml/h). Mean blood pressure was normal at three and six months of age (RR sytolic 100±25 mmHg and 105±21 mmHg, RR diastolic 66±19 mmHg and 68±16 mmHg). At twelve and at 18 months of age mean RR was highest (RR sytolic 117±20 mmHg and 118±28 mmHg, RR diastolic 77±21 mmHg and 81±26 mmHg). At 36 and at 48 months RR was back to the mean initial RR levels (RR systolic 100±15 mmHg and 99±17 mmHg, RR diastolic 61±12 mmHg and 63±12 mmHg). BMI-SDS was elevated at three months of age (mean BMI-SDS +0.55±1.1) and was below average between six and 24 months of age (mean BMI-SDS at six months –0.3±1.1, at twelve months –0.4±1.3, at 18 months –0.3±0.9 and at 24 months –0.2±0.9). At 36 months of age BMI-SDS was around average (0.1±0.9 SDS) and increased further at 48 months (0.4±0.77 SDS).

Conclusion: Elevated RR was found at 12 and at 18 months of age in children with classical CAH under HC and FC treatment. Between 18 and 24 months of age mean FC dose could be decreased from 0.1 mg/day to 0.05 mg/day. The changing mineralocorticoid sensitivity in infants is a risk factor for the development of hypertension in patients with CAH, who are treated with FC. Therefore measurement of PRA or renin and blood pressure at regular intervals are essential.

 

Nothing to Disclose: WB, HPS

3665 12.0000 SAT-37 A Blood Pressure, Fludrocortisone Dose and Plasma Renin Activity in Infants with Classical Adrenal Hyperplasia due to 21-Hydroxylase Deficiency 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 26-40 2201 1:45:00 PM Glucocorticoid Actions & Disease Poster


Esra Hatipoglu*, Asli Sezgin Caglar, Erkan Caglar, Murat Tuncer, Ahmet Sadi Gundogdu and Pinar Kadioglu
Cerrahpasa Medical School, University of Istanbul, Istanbul, Turkey

 

Purpose:Despite the well-known association of exogenous corticosteroid replacement and peptic ulcer disease, ulcerogenic and other upper gastrointestinal system (GIS) effects of endogenous hypercortisolism has not been based on an evidence from a study, yet. There is not a controlled study to represent the exact frequency of peptic ulcer disease (PUD)  and helicobacter pylori infection in presence of high endogenous cortisol levels. The objective of the current study is to evaluate the relationship of endogenous cushing syndrome with  H.pylori infection and PUD.

Methods: In this cross-sectional, comparative study 20 active cases with ACTH dependent Cushing syndrome (CS) (19 with pituitary Cushing disease and 1 with ectopic CS; 13 with new diagnosis and 7 with recurrence) were included in the study. Gender and age matched 100 non-cushingoid dispeptic patients composed the control group (CG). All cases were questionned about secondary causes of PUD, including their medications and upper GI endoscopy was performed to all. Biopsy was taken from 5 different points ; 2 samples from antrum, 2 samples from from corpus and 1 sample from fundus.

Results: In patients with CS median time of diagnosis was 2 [IQR:1-24] monthes,time between the onset of symptoms to the diagnosis of CS was 24 [IQR:12-51] monthes, basal cortisol levels was 21 [IQR:13.6-26.55] µg/dL and basal ACTH levels was 49.9 [IQR:21.31-93.17] pg/mL. Usage of proton pump inhibitor was not different between the groups (p=0.21). Candida esophagitis was present in 10% of patients CS and none of  the CG (p=0.001). Frequency of pangastritis was higher in CS than it was in CG (55% vs 5%, p<0.001). Antral gastritis was higher in CG ( 45% in CS and 81% in CG, p=0.001). Frequency of stomach ulcer, and bulbitis were not different between two groups (peptic ulcer 0% in CS and 3% in CG, p=0.43; bulbitis 20% in CS and 11% in CG, p=0.26). Duedonal ulcer (0% vs 2%, p=0.52) and duodenitis (0% vs 3%, p=0.43) were also not different. Biopsy results showed no difference for active gastritis (55% vs 67.7%, p=0.27), atrophic gastritis (0% vs 5%, p=0.3) and H.pylori infection (60% vs 68%, p=0.48) between CS and CG.  Inactive gastritis was higher in CS (40% vs 17%, p=0.02).

Conclusion: Despite the increased pangastritis in endoscopy of patients with CS, biopsy confirmation of active gastritis did not accompany this finding so in case of increased endogenous cortisol, endoscopic appearence of Upper GIS may not be parallel to the  histopathologic changes. Although endoscopic finding of pangastritis and histologic verification of inactive gastritis were higher in CS, peptic ulcer frequency was not increased on the contrary to what was expected. Endogenous cortisol excess may have different effects on GIS from  exogenously taken glucocorticoids.

 

Nothing to Disclose: EH, ASC, EC, MT, ASG, PK

4819 13.0000 SAT-38 A Is Endogenous Cortisol Excess Really Associated with Peptic Ulcer Disease ? 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 26-40 2201 1:45:00 PM Glucocorticoid Actions & Disease Poster


Angela E Taylor*1, Niki Karavitaki2, Mark Foster3, Sibylle Meier2, Donna M O'Neil1, John Komninos2, Dimitra A Vassiliadi1, Christopher J Mowatt1, Janet M Lord4, John A. H. Wass5 and Wiebke Arlt1
1University of Birmingham, Birmingham, United Kingdom, 2Oxford Centre for Diabetes, Endocrinology and Metabolism, Oxford, United Kingdom, 3University Hospital Birmingham, Birmingham, United Kingdom, 4MRC ARUK Centre for Musculoskeletal Ageing, School of Immunity and Infection, University of Birmingham, Birmingham, UK, Birmingham, United Kingdom, 5Oxford Centre for Diabetes, Endocrinology and Metabolism, Churchill Hospital, Oxford, United Kingdom

 

Patients with adrenal insufficiency (AI) require adjustment of hydrocortisone (HC) dose replacement to avoid life-threatening adrenal crisis during illness, surgery and trauma. However, studies assessing the optimal perioperative HC cover in patients with AI are lacking and suggested doses have been selected on empirical rather than rational grounds, with huge variability in choice of doses and administration modes in routine practice. 

The aim of this study was to compare cortisol levels achieved by currently recommended HC doses to those achieved under real life stress conditions such as elective surgery and acute trauma.

To this end, we firstly studied patients undergoing elective surgery (n=23) or surgery after acute trauma (n=25) in comparison to healthy controls (n=86).  Severity of the operation was graded as either minor (day case procedure, minimal blood loss, <1h duration) or major (complex surgery, significant expected blood loss).  Secondly, we investigated a group of ten patients with chronic autoimmune adrenal failure on four study days: 50mg HC orally every 6hrs, 50mg HC im every 6hrs, 50mg iv injection every 6hrs, and 200mg HC per continuous iv infusion.  Serum samples were collected over a 24hr period and analyzed by liquid chromatography/tandem mass spectrometry.

Results demonstrated that cortisol levels peaked in both minor and major elective surgery between 2 and 4 hours after induction of anaesthesia (minor, median(range) 431(249-570)nmol/L, major 611(165-1379)nmol/L). Cmax values for trauma surgery patients were 433(337-585)nmol/L for minor and 363(203-1504)nmol/L for major surgery. All these values were significantly lower than Cmax observed after HC administration via any administration mode with median values ranging from 836-1440nmol/L. However, cortisol levels decreased to Cmin 277(64-398), 289(148-458), 173(118-375)nmol/L after administration of HC via oral, im or iv injection, respectively, which is below the required range.  

By contrast, continuous infusion of HC yielded steady state cortisol concentrations after one hour, with Cmax 836(661-1073) and Cmin(388-617nmol/L). These data indicate that an HC dose of 200mg per 24hr will only be required in AI patients undergoing major surgery or severe inflammatory stress. Furthermore, the pharmacokinetic results for the different modes of HC administration clearly indicate that steroid stress dose cover in these situations should be administered by continuous i.v. infusion.

 

Nothing to Disclose: AET, NK, MF, SM, DMO, JK, DAV, CJM, JML, JAHW, WA

6162 14.0000 SAT-39 A Prevention of Adrenal Crisis in Stress: Serum cortisol during elective surgery, acute trauma surgery and during 'stress dose cover' hydrocortisone replacement in adrenal insufficiency 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 26-40 2201 1:45:00 PM Glucocorticoid Actions & Disease Poster


Marie Bicikova*, Lucie Sosvorova, Jana Kubatova, Ludmila Macova, Richard Hampl and Luboslav Starka
Institute of Endocrinology, Prague, Czech Republic

 

Two decades ago scientists were interested mainly in the activity of 11β-hydroxysteroid dehydrogenase (11β-HSD) type II and to its mineralocorticoid receptors protective activity. Hand in hand with the study of neuroactive steroids and neurosteroids (NAS) it was discovered how necessary is the knowledge of activities both 11β-HSD isoenzymes I and II in various disease. It was documented that 11β-HSD is responsible not only for oxido-reductive transformations of glucocorticoids but also for metabolism of dehydroepiandosterone  (DHEA) and its 7-hydroxylated metabolites known as potent NAS. Recent results indicate that abnormal levels of DHEA and its 7-hydroxylated metabolites may play a negative role in neurodegenerative diseases.

Our new study focused on a possible role of 11βHSD and above named NAS in cerebrospinal fluid of patients with hydrocephalus, enabling prediction and consequent targeted treatment of dementia, which, after initial relief, appears in most of the patients. We predict the crucial role of 11β-HSD in the pathogenesis of hydrocephalus.

Similar principle of disturbed levels of 7-hydroxylated DHEA metabolites (produced mainly in glial cells) could be caused by the disturbances in 11β-HSD activity in another very serious disease – multiple sclerosis.

   The above mentioned steroids act as regulators of local cortisol activity due to their competition in the cortisol-cortisone balance mediated by 11β-HSD. 7-Hydroxy-dehydroepiandrosterone is marketed as anti-obesity dietary supplement, though no clinical study has appeared until now. The aim of our other most recent project was to contribute to the discovery of new hormonal factors involved in the formation and development of obesity in children.

Currently, we are studying the role 11β-HSD in men with fertility disturbances where enzyme activity could be negatively affected by endocrine disruptors resulting in extensive glucocorticoid activity.  The consequence of it is the inhibition of gene expression of LH receptors in Leydig cells and the disorders in testosterone and sperm production. We hope that results of our studies summarized here could lead to a new therapeutic approaches of disorders outlined above.

The works are supported by the grants IGA NT/13369, NT/12349-4, NT 13542-3 from the Czech Ministry of Health.

 

 

Nothing to Disclose: MB, LS, JK, LM, RH, LS

3856 15.0000 SAT-40 A Significance of determination of 11beta-hydroxysteroid dehydrogenase 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 26-40 2201 1:45:00 PM Glucocorticoid Actions & Disease Poster


Dganit Dinour1, Etty Osher2, Elena Dumin3, Naftali Stern2 and Karen Michele Tordjman*4
1Sheba Medical Center, Ramat Gan, Israel, 2Tel Aviv Sourasky Medical Center, Tel Aviv, Israel, 3Rambam Medical Center, Haifa, Israel, 4Tel Aviv Medical Center, Tel Aviv, Israel

 

Case presentation: A 47 year-old man presented with recently discovered hypertension. He had a positive family history of hypertension. His past medical history included papillary thyroid cancer, and hypercholesterolemia. He had been polyuric (5-7 liters/d) since the age of 18. Office blood pressure was 145/100 mmHg. While a 24-hr ambulatory blood pressure (BP) recording revealed an average BP of 176/100 mmHg under 5 drugs (ACE inhibitor, calcium channel blocker, beta blocker, alpha blocker, and hydralazine). Laboratory evaluation revealed normal kidney function, however serum K+ was consistently between 3.0-3.5 mEq/l. He denied licorice ingestion. Renal artery stenosis, pheochromocytoma, and Cushing's syndrome, were ruled out. Plasma ACTH was normal. However, he repeatedly had unmeasurable plasma renin activity with low plasma aldosterone levels. Given the longstanding history of polyuria, a putative diagnosis of a mild form of apparent mineralocorticoid excess (AME) was entertained.
Methods: Gas chromatography-mass spectrometry of urinary steroid metabolites was performed. Subsequently, genomic DNA was isolated from peripheral blood cells. The coding sequence and splice-sites of HSD11B2 (AME), SCNN1A (ENaCα), SCNN1B (ENaCβ), SCNN1G (ENaCγ), and NR3C2 (MR) were amplified by PCR and directly sequenced.
Results: A normal urinary cortisol to cortisone (THF+5αTHF/THE) ratio excluded typical AME, while a mildly elevated THF+alloTHF/F ratio, indicative of a decreased A ring reduction, suggested AME type 2. Both typical AME and AME type 2 have been ascribed to mutations in the HSD11B2 gene. However, sequencing of the entire coding region of the gene failed to reveal any mutation. Furthermore, sequencing of the 3 genes composing the epithelial sodium channel complex ENaC ruled out the possibility of Liddle's syndrome. Finally, the search for a possible activating mutation in the MR gene was also negative. Spironolactone therapy succeeded in controlling both the hypertension and the hypokalemia.
Conclusions: Sequencing of 3 candidate genes failed to uncover the molecular basis for this case of adult-onset mild AME-like syndrome. This raises the possibility that a previously unidentified factor, possibly one that regulates the function of the HSD11B2 or that of the MR gene, might be the culprit, thus widening the spectrum of conditions that mimic mineralocorticoid excess hypertension. The patient’s whole genome sequencing will next be undertaken.

 

Nothing to Disclose: DD, EO, ED, NS, KMT

6321 1.0000 SAT-53 A "Triple-negative" apparent mineralocorticoid excess 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 53-73 2207 1:45:00 PM Primary Aldosteronism & Mineralocorticoid Excess Poster


Namita Hattangady*1, Fumitoshi Satoh2, Ryo Morimoto2, Hironobu Sasano2, Yasuhiro Nakamura2, Franco Mantero3, Maria Verena Cicala3, Raffaele Pezzani3, Beatrice Rubin3, Hirotaka Shibata4, Isao Kurihara5, Richard J. Auchus1, Tobias Else1, Thomas J Giordano1 and William E Rainey1
1University of Michigan, Ann Arbor, MI, 2Tohoku University Hospital, Sendai, Japan, 3University of Padova, Padova, Italy, 4Keio University, School of Medicine, Tokyo, Japan, 5School of Medicine, Keio University, Tokyo, Japan

 

Introduction: Primary aldosteronism (PA) causes almost 8% of hypertension and is often caused by adrenal aldosterone-producing adenomas (APA).  Recently, somatic mutations in the KCNJ5 potassium channel were shown to cause dysregulation of aldosterone production in APA. The objective of our study was to compare the transcriptomes of normal adrenals (NA) and APA with/without KCNJ5 mutations, and to determine the prevalence of KCNJ5 mutations in an expanded cohort of NA and APA.

Methods: RNA was isolated from 111 APA and 28 NA tissue samples. Sanger sequencing was performed on cDNA to categorize APA as harboring wild type (WT) or mutant KCNJ5 for prevalence studies. Further, RNA from 61 APA (33 KCNJ5 mutants, 28 WT APA) and 28 NA was analyzed using Illumina microarray. Statistical analyses included unpaired T test (p≤0.001; all APA vs. NA) and One Way ANOVA (p≤0.001; WT APA vs. KCNJ5 mutant APA vs. NA) and included genes up-regulated by ≥1.5-fold.

Results: KCNJ5 mutations had an overall prevalence of 44 % in APA, with a higher prevalence in women (59 %) than men (27 %). Microarray comparison of all APA samples with NA revealed a significant increase in the expression of 294 transcripts. Comparison of NA separately with KCNJ5 mutant and WT tumors revealed a larger number of differentially expressed transcripts in mutant tumors (344 vs. 147 genes, respectively). Furthermore, comparison KCNJ5 mutant and WT APA indicated 63 differentially expressed genes. In all these analyses, CYP11B2 (aldosterone synthase) appeared as the top-most upregulated gene in APA (5-fold in WT and 15-fold in KCNJ5 mutant APA vs. NA; 3-fold in KCNJ5 mutant vs. WT APA). KCNJ5 expression was higher 2-fold in all APA (as compared to NA), and also 2-fold higher in KCNJ5 mutant vs. WT APA. Other interesting genes upregulated in both KCNJ5 mutant and WT APA include selective G protein coupled receptors including MC2R, GPRC5C and HTR4, as well as genes normally expressed in the adrenal glomerulosa including PCP4, PLD5 and HOPX.

Conclusion: Our results indicate considerable overlap between the transcriptome changes in WT and KCNJ5 mutant APA, suggesting similar downstream genetic alternations in the formation of APA and/or the dysregulation of aldosterone secretion. However, the findings also suggest that KCNJ5 mutant APA have increased capacity to produce aldosterone when compared to WT APA. Functional analyses of candidate genes could increase understanding of the molecular mechanisms of PA.

 

Nothing to Disclose: NH, FS, RM, HS, YN, FM, MVC, RP, BR, HS, IK, RJA, TE, TJG, WER

6352 2.0000 SAT-54 A Genomic Approaches to Understanding Primary Aldosteronism 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 53-73 2207 1:45:00 PM Primary Aldosteronism & Mineralocorticoid Excess Poster


Gian Paolo Rossi*1, Teresa M. Seccia1, Claudio Letizia2, Maria Verena Cicala3, Laura Zinnamosca2, Maniselvan Kuppusamy1, Maurizio Cesari1, Susanna Sciomer4, Maurizio Iacobone1, Franco Mantero1 and Achille C. Pessina1
1University of Padova, Padova, Italy, 2University 'La Sapienza', Rome, Italy, 3University of Padua, Padova, Italy, 4University 'La Sapienza', Italy

 

Context. Considering the detrimental cardiovascular (CV) effects of aldosterone, APA patients carrying somatic mutations in the  selectivity filter of KCNJ5 K+ channel, which were found to be associated with higher plasma aldosterone secretion from aldosterone producing adenomas (APA), might develop a more prominent CV damage than those without such mutations.

Objective. To test this hypothesis we compared the echocardiographic changes between APA patients with(mutAPA) and without (wtAPA) the G151R, G151E, L168R, and T158A mutations.

Design. From a cohort of 250 consecutive PA patients, we identified 170 patients who had an unequivocal diagnosis of APA by the four corners criteria, and high-quality echocardiographic data.  Of them 106 who had comprehensive clinical and KCNJ5 sequencing information and outcome data at long-term follow-up, were analyzed using the rest as controls.

Results. The KCNJ5 mutations were about two-fold more prevalent in women than in men and overall involved 18.8% of the APA.  At baseline the mutAPA patients were similar to the wtAPA patients for systolic and diastolic blood pressure and need for antihypertensive medications, in spite of higher plasma aldosterone (PAC,70.0 (41.9 -98.1) ng/dl vs 44.5 (38.2 - 50.7), p< 0.0001), aldosterone-renin-ratio (ARR, 513 (381 - 1571)ng/dl/ng/ml/h vs 169 (72 - 266), p< 0.0001), and left ventricular mass index (LVMI, 60±7.4 mg/h2.7vs 49±3.6, p=0.004).

At long-term follow-up after adrenalectomy the mutAPA showed a greater fall of LVMI than the wtAPA (14.9±3.9 mg/m2vs 6.4±1.2, p=0.007), despite a similar fall of BP and a similar  normalization of PAC and ARR.

Conclusions. In APA patients the occurrence of the somatic KCNJ5 mutations implies higher PAC, ARR, and LVMI, and a greater decrease of LVMI after adrenalectomy than in the wild type APA patients. However, the presence of these mutations did not compromise the chances of being cured from the hyperaldosteronism and the high blood pressure.

 

Nothing to Disclose: GPR, TMS, CL, MVC, LZ, MK, MC, SS, MI, FM, ACP

7132 3.0000 SAT-55 A SOMATIC MUTATIONS IN THE KCNJ5 GENE AFFECT CARDIAC REMODELING AND REGRESSION OF LEFT VENTRICULAR HYPERTROPHY INPRIMARY ALDOSTERONISM DUE TO ALDOSTERONE-PRODUCING ADENOMA 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 53-73 2207 1:45:00 PM Primary Aldosteronism & Mineralocorticoid Excess Poster


Takumi Kitamoto*1, Sachiko Suematsu2, Yoko Matsuzawa2, Jun Saito2, Masao Omura1 and Tetsuo Nishikawa2
1Yokohama Rosai Hosp, Kanagawa, Japan, 2Yokohama Rosai Hosp, Yokohama, Japan

 

Introduction: Aldosterone-producing adenoma (APA) is a common disease of surgically curable hypertension. The KCNJ5 gene mutations had been found in some cases of APA. Our objective was to investigate characteristics of clinical features in Japanese patients of APA with/without mutations of the KCNJ5 gene.

Subjects and Methods: We definitely diagnosed 60 APA patients, according to the Guidelines of Japan Endocrine Society and lateralized unilateral APA by ACTH-AVS. We sequenced KCNJ5 cDNA in 60 samples of APA tissues, and retrospectively compared clinical data between each group.

Results: There were 42 (70 %) cases with two somatic mutations of the KCNJ5 gene (p.G151R and p.L168R) among 60 patients with APA. The age was significantly younger in the mutated group than in the wild type group, although BMI, BP, duration of hypertension, serum K level, Cr clearance rate, and tumor size were not different between two groups. The basal levels of plasma aldosterone concentration (PAC) and urinary aldosterone excretion were significantly higher in the mutated group than the wild type group, while plasma renin activity (PRA) was not different between two groups. The ratio of PAC to cortisol 30 or 60 min after ACTH stimulation, and lateralization index of ACTH-AVS for detecting the laterality of the main lesion were significantly higher in the mutated group than in the wild type group, respectively.  

Conclusion: The present data clearly demonstrated high incidence (70%) of mutations of the KCNJ5 gene in Japanese APA patients. Patients in the mutated group showed much higher aldosterone levels than the wild type group, suggesting that the existence of the KCNJ5 gene mutations may induce stronger autonomous production of aldosterone, and also younger cases of APA may possess the KCNJ5 gene anomalies. Moreover, we can easily differentiate the mutated group from the wild type group by examining the response of aldosterone to rapid ACTH test and ACTH-AVS.

 

Nothing to Disclose: TK, SS, YM, JS, MO, TN

8073 4.0000 SAT-56 A Clinical characteristics of Japanese patients with aldosterone-producing adenoma showing the KCNJ5 gene mutations 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 53-73 2207 1:45:00 PM Primary Aldosteronism & Mineralocorticoid Excess Poster


Kazumi Iino*1, Yutaka Oki2, Etsuko Hamada3, Miho Yamashita3, Kosuke Yogo1, Shoko Shibata3, Toshihiro Ohishi1, Keisuke Kakizawa1 and Masato Maekawa3
1Hamamatsu Univ Sch of Med, Hamamatsu, Japan, 2Hamamatsu Univ Schl of Med, Hamamatsu, Shizuoka, Japan, 3Hamamatsu Univ Sch of Medicine, Hamamatsu, Japan

 

[Background] Several reports have shown that severe hypertension in primary aldosteronism (PA), which is often resistant to plural antihypertensive medicines, may carry higher risk of arteriosclerosis in comparison with that of essential hypertension (E-HT). Previously we reported that IMT levels tended to be higher in PA than in E-HT, and hyperaldosteronism might be one of independent risk factors of arteriosclerosis or cardiovascular disease. [Subjects/Method] PA patients (n=145) and background (except the age)-matched E-HT patients (n=37; f/m=17/22) were entered to this study. All of the PA patients were diagnosed as Aldosterone Producing Adenoma (APA) (n=63; f/m=27/36) or Idiopathic hyperaldosteronism (IHA) (n=82; f/m=53/29) based on their data of adrenal venous sampling. They underwent carotid ultrasonography and their maximum IMT was measured. Risk reductions 12 month after PA treatment were examined with the use of IMT (in 25 APA and 30 IHA) and biochemical markers including high-sensitivity C-reactive protein (HS-CRP), nitrotyrosine and adiponectin (in 4 APA and 7 IHA). [Result] IMT in PA was 1.00±0.42 mm, which was evidently thicker than that of age-matched normal controls. While there was a significant difference in age, an unequivocal contributing factor of IMT, between PA and E-HT (54.14±10.6 and 58.21±7.93 respectively), no difference was recognized between their IMTs (E-HT; 1.05±0.34mm). Significant improvement of IMT after control of hyperadosteronism was observed in both APA (1.00±0.92 vs. 0.94±0.23mm) and IHA (0.95±0.31 vs. 0.88±0.23mm). The apparent change of the three biomarkers could not be identified because of the small number examination. [Discussion] From our results, it can be concluded that, with the same degree of blood pressure, PA tends to develop severe IMT thickness, and that hyperaldosteronism may be one of independent risk factors of arteriosclerosis. We also demonstrated that the proper control of hyperaldosteronism state makes a contribution to the risk reduction in this study. Based on the two possible mechanisms of the arteriosclerosis, one is the secondary action of hypertension and the other is the direct action of aldosterone, the active treatments for both blood pressure and hyperaldosteronism itself, are of importance in prevention of cardiovascular event in PA patients. Biomarkers of PA patients did not reveal significant change before and after the treatment. More samples may be required for the conclusion.

 

Nothing to Disclose: KI, YO, EH, MY, KY, SS, TO, KK, MM

6561 5.0000 SAT-57 A Risk reductions of arteriosclerosis by normalization of hyperaldosteronism state. -Evaluation with the use of both intima-media thickness (IMT) of carotid artery and biochemical markers- 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 53-73 2207 1:45:00 PM Primary Aldosteronism & Mineralocorticoid Excess Poster


Mitra Lynn Rauschecker*1, Christopher Tate Sibley2, Charalampos Lyssikatos3, Elena Belyavskaya4, Constantine A Stratakis3 and Smita Baid Abraham5
1NIH, Bethesda, MD, 2NIH/CC, Bethesda, MD, 3National Institutes of Health (NIH), Bethesda, MD, 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, National Institutes of Health, Bethesda, MD

 

Background: Compared to patients with essential hypertension and healthy controls (HC), patients with primary aldosteronism (PA) demonstrate an increased rate of carotid wall thickening, as assessed by carotid intima-media thickness on ultrasound (1).  There are no studies, however, evaluating carotid wall volume, or the presence of lipid-rich cores, which can only be measured by carotid MRI, in patients with PA. Carotid MRI findings are reported to be highly correlated with atherosclerosis risk factors in asymptomatic subjects and were predictive of prior cardiovascular events in subjects undergoing carotid endarterectomy.  Compared with carotid ultrasound, carotid MRI has less user variability (2, 3).  In order to evaluate the degree of atherosclerosis in patients with PA, measurements of carotid wall volume (CWV) were obtained by MRI. We report three cases of PA in which carotid MRI revealed increased arterial wall thickness as compared with HC.

Methods: Patients were diagnosed with PA on the basis of an elevated aldosterone/plasma renin activity ratio (ARR) in conjunction with a positive confirmatory test. Participants underwent high resolution black blood carotid MR imaging at 3 Tesla using carotid coils, with semi-automated contouring to quantify wall volume. PA patients were compared with age-matched HC who had undergone carotid MRI at the NIH. CWV for the PA patients and HC were analyzed using an unpaired t-test.

Results: Mean CWV for the PA group (n=4) was 587.2 mm(95% confidence interval (CI) for the mean 393.6 to 780.8) while mean CWV for the HC group (n=4) was 469.9 (95% CI for the mean 418.1 to 521.6). The difference between the two groups was statistically significant (P<0.05).

Conclusion:  We demonstrate that patients with PA, compared to HC, have increased carotid artery thickness, which suggests an increased risk of atherosclerosis and cardiovascular events.

 

Nothing to Disclose: MLR, CTS, CL, EB, CAS, SBA

7567 6.0000 SAT-58 A Arterial Thickening and Atherosclerosis as seen on Carotid MRI in Patients with Primary Aldosteronism 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 53-73 2207 1:45:00 PM Primary Aldosteronism & Mineralocorticoid Excess Poster


Mitra Lynn Rauschecker*1, Andreas G. Moraitis2, Charalampos Lyssikatos3, Elena Belyavskaya4, Smita Baid Abraham5 and Constantine A Stratakis3
1NIH, Bethesda, MD, 2NICHD/NIH, Bethesda, MD, 3National Institutes of Health (NIH), Bethesda, MD, 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, National Institutes of Health, Bethesda, MD

 

Background:  Confirmatory testing is necessary to make the diagnosis of primary aldosteronism (PA).  However, the available sensitivity and specificity data on these tests is limited and each has technical challenges (1). The two most commonly performed tests are the normal saline suppression test (SST) and the oral salt load test (OST).  In the SST, it is not infrequent to see false negative results.  In the OST, it is difficult to attain a 24-hour urine sodium level >200 mmol/24-hr.  We evaluated the accuracy of the SST, OST, and a “modified SST” (MSST).

Methods: Patients were suspected of having PA on the basis of an elevated aldosterone/plasma renin activity ratio (ARR). Confirmatory testing was performed. During the SST, patients received 2 L normal saline over four hours (hr).  Plasma aldosterone levels were collected hourly. A plasma aldosterone value of >10 ng/dl after 4 hr of saline infusion was considered positive for PA. For the OST, patients received 2 grams NaCl three times a day for three days, and on the third day, performed a 24-hr urine collection for aldosterone and sodium. Urine aldosterone (UA)>12 mcg/24-hr with a urine Na (UNa)>200 mmol/24-hr was considered positive. For the MSST, a 24-hr urine collection for aldosterone and sodium was collected starting on the day of the SST; UA>12 mcg/24-hr with a UNa>200 mmol/24-hr was considered positive, as with the OST.

Results: Six PA patients underwent SST, OST, and MSST. Four of six patients had negative SST. Of those four patients, two patients had a positive OST, while all four patients had a positive MSST. Of the two patients without a positive OST, the test was uninterpretable, as UNa was not >200 mmol/24-hr, while all four patients with positive MSST had UNa>200 mmol/24-hr. Patients with an uninterpretable OST and negative SST will undergo repeat OST to confirm the diagnosis.

Conclusion: We demonstrate that the MSST may be an alternative to OST and SST in the diagnosis of PA. The benefits of the MSST include ease for patients, as no oral salt tablets are required. Additionally, MSST appeared to have fewer false negative results as compared with SST.

 

Nothing to Disclose: MLR, AGM, CL, EB, SBA, CAS

8680 7.0000 SAT-59 A A Modified Saline Suppression Test to Confirm the Diagnosis of Primary Aldosteronism 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 53-73 2207 1:45:00 PM Primary Aldosteronism & Mineralocorticoid Excess Poster


Jacques W.M. Lenders*1, Tanja Dekkers2, Jaap Deinum3, Leo J. Schultze Kool2, Dirk Blondin4, Oliver Vonend5, Ad RMM Hermus6, Mirko Peitzsch7, Lars Rump4, Gerald Antoch4, Fred C.G.J. Sweep2, Stefan Richard Bornstein8, Holger Willenberg5 and Graeme Eisenhofer8
1Radboud University Medical Center, Nijmegen, Netherlands, 2Radboud University Nijmegen Medical Center, Nijmegen, Netherlands, 3Radboud university medical center, Nijmegen, Netherlands, 4University Hospital Duesseldorf, Dusseldorf, Germany, 5University Hospital Duesseldorf, Duesseldorf, Germany, 6Radboud University Medical Center, Nijmegen, 7Technische Universit, Dresden, Germany, 8University Hospital Carl Gustav Carus, Dresden, Germany

 

Context: More reliable parameters than cortisol are needed for assessing correct catheter positioning during adrenal vein sampling (AVS). Plasma metanephrine represents one such alternative.

Objective: To determine the utility of adrenal venous (AV) plasma concentrations of metanephrine to establish correct catheter positioning during AVS.

Design and methods: We included 86 AVS procedures: 52 ACTH-stimulated and 34 non-stimulated sequential procedures. Plasma cortisol, metanephrine, normetanephrine, epinephrine and norepinephrine concentrations were measured in AV and peripheral venous (PV) samples. AVS success rates, according to cortisol AV:PV selectivity indices of 2.0 and 3.0, were compared with that for metanephrine using a selectivity index (SI) determined by ROC curve analysis.

Results:Among AVS procedures assessed as selective using a cortisol SI of 3.0, the median AV:PV plasma metanephrine ratio was 6-fold higher than that for cortisol (94.0 versus 15.5, P<0.0001). There were significant positive relationships between AV-PV ratios for cortisol and metanephrine for ACTH-stimulated samplings, but not for non-stimulated samplings. ROC curve analysis indicated a plasma metanephrine SI cut-off of 10. There was 96% concordance in AVS success rates determined by cortisol (SI=3.0) and metanephrine (SI=10) in ACTH-stimulated AVS. Without stimulation, the concordance was 82% and 59% at respective cortisol-derived SIs of 2.0 and 3.0; AVS success rates determined by metanephrine (91%) were higher (P<0.01) than those determined by cortisol at an SI of 3.0 (56%), but not 2.0 (79%).

Conclusions: Metanephrine provides an alternative analyte to cortisol for sensitive assessment of AVS selectivity that appears particularly advantageous in sampling performed without ACTH stimulation.

 

Nothing to Disclose: JWML, TD, JD, LJS, DB, OV, ARH, MP, LR, GA, FCGJS, SRB, HW, GE

4272 8.0000 SAT-60 A Plasma metanephrine for assessing the selectivity of adrenal venous sampling 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 53-73 2207 1:45:00 PM Primary Aldosteronism & Mineralocorticoid Excess Poster


Maria Verena Cicala*1, Anna Patalano2, Monica Salvà2, Diego Miotto2, Beatrice Rubin2, Raffaele Pezzani1, Barbara Mariniello3 and Franco Mantero2
1University of Padua, Padova, Italy, 2University of Padova, Padova, Italy, 3Univ of Padova, Padova, Italy

 

Role of adrenal vein sampling in primary aldosteronism. Impact of different diagnostic criteria on subtype diagnosis.

 

Cicala MV1, Patalano A1, Salvà M1, Miotto D1, Rubin B1, Pezzani R1, Mariniello B1, Mantero F1.

1Endocrinology Unit, Department of Medicine, University of Padova, Padova, Italy.

In patients with primary aldosteronism (PA), adrenal vein sampling (AVS) is considered the gold standard to distinguish between unilateral and bilateral autonomous production of aldosterone, while diagnostic imaging tests by CT scan or MRI are often inconclusive for the diagnosis. To date agreement is lacking on the best criteria indicating successful cannulation and lateralization.

The aim of the study was to evaluate the impact of different diagnostic criteria for the successful cannulation and lateralization on subtype diagnosis and to compare the difference of the findings between adrenal CT scan and AVS.

Seventy-four patients with confirmed PA underwent AVS. The different diagnosis of PA subtypes reached using AVS data assessed by more permissive (type 1) and strict (type 2) criteria were compared. Al patients performed CT scan before AVS and imaging results were compared with results of AVSs (using both criteria).

Using Type 1 criteria AVSs were successful in 86% of patients, and  in only 64,5% using type 2 criteria. Type 1 criteria led to a higher rate of diagnosis of unilateral PA (85% of successful procedures) than type 2 (75%). There was considerable disparity in the diagnosis reached, with a concordance in only 45% of patients. In conclusion more permissive criteria for successful cannulation and lateralization on AVS can lead to incorrect diagnosis and accordingly to inappropriate treatment options. In the selected group of patients with successful AVS, CT findings correlated with AVSs findings in 58,5% of patients using type 1 criteria and in 47,5% using type 2 criteria. Finale diagnosis was based on histological results in 36 patients (49%) which underwent adrenalectomy based on AVSs findings. On the basis of CT findings alone 17% of patients from the first group and 32,5% of patients of the second group probably would have been incorrectly bypassed as candidates for adrenalectomy. CT scanning lacks sensitivity and specificity and should be followed by AVS, which is the only reliable means of differentiating unilateral from bilateral PA and lateralizing APAs preoperatively. However, there are still controversies to be solved by large prospective studies on the criteria to adopt for defining the most appropriate cut off for both correct cannulation and lateralization.

 

Nothing to Disclose: MVC, AP, MS, DM, BR, RP, BM, FM

6837 9.0000 SAT-61 A Role of adrenal vein sampling in primary aldosteronism. Impact of different diagnostic criteria on subtype diagnosis 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 53-73 2207 1:45:00 PM Primary Aldosteronism & Mineralocorticoid Excess Poster


Jalaja Joseph*1, Charalampos Lyssikatos2, Andreas G. Moraitis3, Mitra Lynn Rauschecker4, Smita Baid Abraham5 and Constantine A Stratakis6
1National Institutes of Health, Bethesda, MD, 2Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH), Bethesda, MD, 3University of Michigan, Ann Arbor, MI, 4NIH, Bethesda, MD, 5Natl Institutes of Hlth, Bethesda, MD, 6National Institutes of Health (NIH), Bethesda, MD

 

Background: Adrenal venous sampling (AVS) 1 is considered as the gold standard in distinguishing unilateral from bilateral adrenal disease, in patients with primary hyperaldostreronism (PA). Although it is a relatively safe procedure, few centers in the US perform this procedure successfully. The technical difficulty in cannulating the right adrenal vein dictates the success rate. This procedure is associated with minimal complications varying from 5-10 % to less than 0.2% in centers depending on the volume of cases performed 2.  Adrenal hemorrhage from thrombosis or transection of the vein is a complication that may be seen in PA patients.

We report the case of a 56-year-old African American female who was diagnosed with hypertension at the age of 21 after she presented with hypertension. Since then, she has been on multiple medications with inadequate control. Hypokalaemia was noted at the age of 51 years. Laboratory evaluation revealed an aldosterone of 16.5ng/dL (normal 1-21ng/dL) and plasma rennin activity less than 0.15ng/ml/hr (normal <= 0.6-3) with an aldosterone-renin ratio of 1104. Her medical history was also significant for obesity, temporal lobe epilepsy, vitamin D insufficiency and secondary hyperparathyroidism. The patient underwent two inconclusive saline suppression tests, with plasma aldosterone of 5.2 and 7.6 ng/dL. 24 hour urine aldosterone level during the second saline suppression was elevated at 21mcg/24h (normal 2-20 mcg/24h) and the sodium excretion was appropriate at 251mmol/24 h.  MRI of the adrenals showed nodular hyperplasia of the adrenal glands, most notably in the lateral limb of the left adrenal. Subsequently, the patient underwent AVS. Two hours status post procedure, the patient complained of severe right upper quadrant pain radiating to the right shoulder. Examination was significant for rigidity and rebound tenderness in the right upper quadrant. Laboratory evaluation was significant for normal hematocrit and CT scan of the abdomen revealed right adrenal hemorrhage. Patient was managed conservatively and with repeat serial imaging; by 6 months, there was an almost complete resolution of the hemorrhage.

Conclusion: Adrenal hemorrhage from transection of adrenal vein is an uncommon complication after adrenal venous sampling. Most of the time, patients require non-invasive monitoring and pain control. There are very few cases reported in the literature and physicians need to be aware of this potential complication of AVS.

 

Nothing to Disclose: JJ, CL, AGM, MLR, SBA, CAS

6427 10.0000 SAT-62 A Bleeding Adrenal: Adrenal Hemorrhage as a Complication of Adrenal Venous Sampling 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 53-73 2207 1:45:00 PM Primary Aldosteronism & Mineralocorticoid Excess Poster


Akiyo Tanabe* and Atsuhiro Ichihara
Tokyo Women's Medical University, Tokyo, Japan

 

The prevalence of primary aldosteronism (PA) has been reported to be high in the patients with drug-resistant hypertension and/or hypertension with spontaneous or diuretic-induced hypokalemia. In these typical PA cases, renal function could deteriorate because of severe hypertension and hypokalemia, and some patients consequently fell into end-stage renal disease (ESRD). Serum potassium concentration (s-K) level is increased to normal range, and plasma aldosterone concentration (PAC) level and plasma renin activity (PRA) level tend to be increased in patients with PA in ESRD. There are limitations in washing out of interfering antihypertensive medications or in using contrast medium in the imaging study. Therefore the detection and diagnosis of PA in ESRD are often difficult. Herein we describe the clinical characteristics of 4 patients with aldosterone producing adenoma (median of 50 years) in ESRD on hemodialysis (HD). The cause of renal insufficiency were polycystic kidney disease in 1 patient, IgA chronic nephropathy in 1 patient, nephrosclerosis due to hypertension in 2 patients. Hypertension preceded renal insufficiency in all patients. Mean interval between first diagnoses of renal insufficiency to initiation of HD was 7 years and all patients showed drug-resistant hypertension after initiation of HD. S-K levels before each HD treatment were low (4.1±0.7 mEq/L) in spite of renal insufficiency and those were low after each HD treatment (3.1±0.1 mEq/L). PAC levels were 48-2350 ng/dl and they did not change before and after HD. PRA levels were 0.4-6.0 ng/ml/h and they were decreased after HD. PAC levels and PAC responsiveness to ACTH loading in tumor- and contralateral-side adrenal veins were similar to those in non-ESRD patients in selective adrenal sampling (AVS). All patients were performed adrenalectomy and PAC levels after tumor removal were normalized (6-8 ng/dl) in 3 patients, decreased from 2350 to 141 ng/dl in 1 patient. S-K levels after HD (4.7±0.5 mEq/L) were increased in all patients. Blood pressure was slightly improved and the number of antihypertensive medicines was decreased in 2 patients after adrenalectomy. Those findings suggest that AVS is useful in localization of APA with ESRD as well as APA without ESRD. It is important to pay attention on hypokalemia and low PRA level after HD in order to prevent failing to detect typical PA in patients with ESRD.

 

Nothing to Disclose: AT, AI

5276 11.0000 SAT-63 A The diagnosis and treatment of primary aldosteronism associated with end-stage renal disease 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 53-73 2207 1:45:00 PM Primary Aldosteronism & Mineralocorticoid Excess Poster


Kristen Anne Hyland* and Jennifer Marie Perkins
Duke University Medical Center, Durham, NC

 

Background:  Contralateral reversible suppression of the renin-aldosterone axis can rarely be seen after unilateral adrenalectomy for Conn’s syndrome without risk factors or prevention. 

Clinical Case:

A 43 year old male with history of hypertension and hypokalemia presented for evaluation of his type 2 diabetes. He had an elevated aldosterone of 20 ng/dL (<21) and a plasma rennin activity (PRA) of <0.6 ng/mL/hr (<0.6-3). He underwent confirmatory testing with a saline suppression test and aldosterone was 6.9 ng/dL, sodium 139 mmol/L (135-145), potassium 3.2 mmol/L (3.5-5.0), PRA <0.6 ng/mL/h. This was deemed indeterminate. A 24-hour urine aldosterone after salt loading was performed showing an aldosterone of 12.82 ng/dL (<12), sodium of 275 meq/volume, and creatinine of 1,549 mg/TV. An adrenal CT showed a 1.0 cm x 1.4 nodule in the right adrenal gland consistent with adenoma. Patient then underwent adrenal venous sampling (AVS) confirming a > 4:1 ratio of hypersecretion of aldosterone from the right adrenal gland. The right adrenal gland was removed laparoscopically with pathology that showed: adrenal adenoma. Patient did well postoperatively.

Eighteen days after surgery, his sodium was 134 mmol/L with potassium of 5.3 mmol/L. Follow-up panel 48 days after surgery sodium 131 mmol/L with potassium of 7.4 mmol/L with blood pressure of 129/76 mmHg and pulse 70 beats per minute. Cortisol stimulation test performed with initial cortisol of 6.3 mcg/dL (5.0-25.0) that stimulated to 25.5 mcg/dL after administration of 250mcg of cosyntropin IM with ACTH of 21 pg/mL (15-66). Aldosterone was <4.0 ng/dL and PRA was suppressed at <0.6 ng/mL/h. He was started on fludrocortisone therapy with resolution of the hyperkalemia with subsequent titration for blood pressure control. He has been able to taper down his fludrocortisone doses over the subsequent 3 months.

Conclusion:  It is rare to have contralateral suppression of aldosterone production following adrenalectomy for primary hyperaldosteronism. This case represents prolonged suppression of both aldosterone and renin suggesting decreased adrenal mass and suppressed juxtaglomerular apparatus. It is pertinent to follow potassium after adrenalectomy for primary hyperaldosteronism.

 

Nothing to Disclose: KAH, JMP

6396 12.0000 SAT-64 A Persistent Hyperkalemia Status-Post Adrenalectomy for Primary Hyperaldosteronism 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 53-73 2207 1:45:00 PM Primary Aldosteronism & Mineralocorticoid Excess Poster


Eu Jeong Ku*1, Kyeong Seon Park1, Jung Hee Kim1, Ah Reum Kang2, Sang Wan Kim1 and Seong Yeon Kim1
1Seoul National University College of Medicine, Seoul, Korea, Republic of (South), 2Seoul National University College of Medicine

 

Unilateral aldosterone excess may suppress zona glomerulosa function in contralateral adrenal gland. Adrenalectomy is the first recommended treatment for unilateral aldosterone excess. There have been a few reports regarding hyperkalemia after adrenalectomy due to suppressed contralateral zona glomerulosa insufficiency. The aim of the present study was to analyze the clinical risk factors associated with postoperative hyperkalemia in patients with unilateral aldosterone excess after adrenalectomy.

The study was conducted retrospectively by medical records review in Seoul National University hospital from 2000 and 2012. Eighty-eight patients who underwent adrenalectomy were included. Hyperkalemia was defined as serum potassium greater than 5.0 mmol/L. Clinical risk factors included blood pressure, plasma renin activity (PRA), plasma aldosterone concentration (PAC), serum potassium, serum creatinine, glomerular filtration rate (GFR), number of antihypertensive mediations and use of mineralocorticoid antagonist.

Fourteen of 88 patients (18%) developed postoperative hyperkalemia. In 7 of patients, hyperkalemia was documented only once and return to normal range spontaneously. Prolonged postoperative hyperkalemia more than 3 months was observed in seven patients. Postoperative hyperkalemic patients did not show significant difference in PRA and PAC compared with normokalemic patients. The patient with persistent hyperkalemia were significantly older at diagnosis (59.4 ± 7.3 vs. 45.1 ± 11.1 yr, P = 0.006) and had elevated creatinine (1.51 ± 0.89 vs. 0.93 ± 0.21 mg/dl, P < 0.001) and lower GFR (53.5 ± 10.9 ml/min vs. 81.3 ± 20.1 ml/min, P < 0.001) than normokalemic patients. The incidence of postoperative hyperkalemia was not different between a mineralocorticoid antagonist users (n= 74) and non-users (n = 14).

Persistent postoperative hyperkalemia occurs in 7.9% of adrenalectomized patients with unilateral aldosterone excess. Older age and preoperative renal function were associated with postoperative hyperkalemia. Use of mineralocorticoid antagonists did not prevent postoperative hyperkalemia in our study.

 

Nothing to Disclose: EJK, KSP, JHK, ARK, SWK, SYK

7855 13.0000 SAT-65 A Clinical Risk Factors Predicting Postoperative Hyperkalemia in Patients with Unilateral Aldosterone Excess after Adrenalectomy 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 53-73 2207 1:45:00 PM Primary Aldosteronism & Mineralocorticoid Excess Poster


Ryo Morimoto*1, Masataka Kudo1, Yoshitsugu Iwakura1, Ken Matsuda1, Yoshikiyo Ono1, Masahiro Nedzu2, Kazumasa Seiji1, Kei Takase1, Yoichi Arai1, Yasuhiro Nakamura1, Hironobu Sasano1, Sadayoshi Ito1 and Fumitoshi Satoh1
1Tohoku University Hospital, Sendai, Japan, 2Tohoku University Hospital

 

Background: Primary aldosteronism (PA) due to bilateral aldosterone-producing adenomas is considered to be one of surgically curable subtypes. Bilateral adrenal tumors detected by computed tomography scan in patiens with PA should be functionally differentiated between 'aldosterone-producing adenoma (APA)' and clinically non-functioning adenoma. We therefore performed adrenal venous sampling (AVS) to show aldosterone hypersecretion from drainage veins of 'APA' and suppressed secretion of aldosterone from non-tumor adrenal drainers, which makes it possible to preoperative diagnosis of bilateral APA, but not bilateral hyperplasia with clinically non-functioning bilateral adenomas.

Clinical case: Three patients (two males) were referred to investigate PA with bilateral adrenal nodules. Mean age and blood pressure were 50+/-1.53 years and 151.3/96.7 mmHg, respectively with anti-hypertensive medication. Mean baseline aldosterone and plasma renin activity (PRA) were 14.3 ng/dl and 0.40 ng/ml/h, respectively, and captopril challenge tests showed mean ARR of 67.6 ng/dl per ng/ml/h, while 1mg overnight dexamethasone suppression tests confirmed no autonomous secretion of cortisol in all cases. CT showed bilateral adrenal nodules with mean size of 10.7 mm of right and 8.0 mm of left. In AVS, we defined hypersecretion of aldosterone as higher aldosterone/cortisol ratio obtained from tumor drainer than that from peripheral vein, while we regarded lower aldosterone/cortisol ratio obtained from non-tumor drainer as suppressed secretion of aldosterone from attached non-tumor adrenal tissues. AVS of the three cases showed hypersecretion of aldosterone from each tumor drainer and suppressed aldosterone secretion from non-tumor drainer, which seemed consistent with clinical diagnosis of bilateral APA. Based on both AVS and CT findings, non-tumor adrenal sparing bilateral adrenalectomy was performed, and histopathological diagnosis was confirmed. Postoperative evaluation showed mean aldostereone and PRA were 3.67 and 0.75, respectively and temporary replacement of glucocorticoid was withdrawn in all cases with mean duration of 249 days. After surgery, hypertension was so improved that one patient became free from medication and the others reduced the number of anti-hypertensives to one each.

Conclusion: Bilateral APA could be preoperatively differentiated from bilateral hyperplasia with bilateral adrenal nodules by AVS with specific sampling from drainers of both APA and non-tumor attached adrenal tissue.

 

Nothing to Disclose: RM, MK, YI, KM, YO, MN, KS, KT, YA, YN, HS, SI, FS

9205 14.0000 SAT-66 A Non-tumor sparing bilateral adrenalectomy for bilateral aldosterone-producing adenomas; AVS-based approach 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 53-73 2207 1:45:00 PM Primary Aldosteronism & Mineralocorticoid Excess Poster


Rajeev Sharma*1, Jocelyne Georges Karam2 and Elizabeth Sedlis Singer2
1SUNY Downstate Medical Center, Brooklyn, NY, 2Maimonides Medical Center, Brooklyn, NY

 

Background: Primary hyperaldosteronism is a known but commonly overlooked cause of hypertension in general population. Herein, we describe a case with atypical presentation, so called intermittent hyperaldosteronism.

Case report: 25 year-old Chinese man with no medical history was admitted with generalized muscle weakness and fatigue of few weeks duration. On presentation, BP was 160/104, and pulse 74. History was negative for polyuria, vomiting or diarrhea. No use of diuretics, liquorice, excessive soda, laxatives or OTC’s. Laboratory tests showed K 1.8 mEq/L, Na 142, Cl 98, HCO3- 32, Mg 2.1, normal renal and thyroid functions. Urinary K+ was 22 mmol/L.  EKG showed U waves in lateral leads. Potassium was replaced aggressively. CT abdomen showed 1.5 cm enhancing right adrenal nodule. Renin/aldosterone was sent on two occasions before and after K replacement. Overnight 1 mg dexamethasone suppression test and plasma metanephrines were normal. Patient was discharged with K tablets, hydralazine and verapamil. The initial aldosterone level was 5 ng/dL with plasma renin 0.08 ng/ml/hr and ARR 62 (Serum K 3.4). Repeat aldosterone level after K replacement (K- 4.4) was 4 ng/dL. Patient did not fulfill the diagnostic criteria of primary hyperaldosteronism (both aldosterone>15 and ARR>20) and was lost to follow up.

Three months later, the patient returned to the ED with dizziness, pre-syncope and weakness. He reported not taking his medications.  BP was elevated (152/103) and serum K 2.8 mEq/L. Random urinary K was 69. Serum aldosterone was 35 and plasma renin 0.14 with ARR 250 (Serum K- 3.1).  An IV saline suppression test failed to suppress the aldosterone (Serum K- 4.2; Aldosterone: pre-test-34, post-test-30; Renin: pre-test- 0.12, post-test- 0.06). He was diagnosed with primary hyperaldosteronism and discharged on only spironolactone 25 mg daily. One week later in the clinic his serum K was 4.4 mEq/L and BP 130/90. He was referred to the surgeon for right adrenalectomy.

Conclusion: As per our literature search, this is the first reported case of intermittent hyperaldosteronism in an adult patient; there is only one published case of a pediatric patient with this entity. (1) In patients with clinical, radiological and laboratory features suggestive of hyperaldosteronism a repeat aldosterone/renin levels might be warranted due to possible intermittent secretion of aldosterone from the adrenal adenoma.

Reference:

  1. Rogoff D, Bergada I, Venara M, Chemes H, Heinrich JJ, Barontini M. Intermittent hyperaldosteronism in a child due to an adrenal adenoma. Eur J Pediatr. 2001; 160(2): 114-6.

 

Nothing to Disclose: RS, JGK, ES

7744 15.0000 SAT-67 A Intermittent hyperaldosteronism. Is this an entity? 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 53-73 2207 1:45:00 PM Primary Aldosteronism & Mineralocorticoid Excess Poster


Isao Kurihara*, Hirotaka Shibata, Kazutoshi Miyashita, Hideyo Oguchi, Kenichi Yokota, Ayano Murai-Takeda, Yuko Mitsuishi, Rie Jo, Takako Ohyama, Toshifumi Nakamura, Akiko Tanikawa and Hiroshi Itoh
School of Medicine, Keio University, Tokyo, Japan

 

[Introduction] A large number of basic and clinical researches have been demonstrating the pro-inflammatory effects of aldosterone. Aldosterone plays a crucial role in the pathogenesis of cardiovascular diseases, which is accompanied by elevation of several inflammatory biomarkers, such as TNF-α, MCP, CRP, and PAI-1. These aldosterone actions mostly go through mineralocorticoid receptor (MR); therefore, the MR blockade could effectively alleviate these complications. Primary aldosteronism (PA) is thoroughly chased and commonly diagnosed recently because of prevailing awareness of its poor prognosis. Cardio-renoprotective effects of MR antagonist have been well-documented, but the benefit for skin disease was rarely discussed. Here we report an impressive case where MR antagonist was truly effective to induce cure for intractable leg ulcer.

[Clinical Case] A 54-year-old man primarily visited a dermatologist 2 years ago because an egg-sized ulcer lesion emerged on his left leg. He was obese (BMI 27.3) and had history of hypertension for more than 10 years, but no history of diabetes mellitus. Screening of markers for collagen disease and coagulopathy was all negative, and CT angiography image in his lower extremities revealed no obstructive or stenotic vascular lesion. Livedo vasculitis was suspected and 30 mg/day PSL was initiated, but new ulcer lesions emerged and were progressively expanded. His blood pressure (BP) was poorly controlled on multi-antihypertensive drugs; therefore, he was consulted to our department for endocrinological evaluation. Active renin concentration was 2.1 pg/mL and serum aldosterone level was 110 pg/mL, indicating screening-positive for PA.  The 24-h urinary aldosterone under salt-loading test was extremely high (31.4 μg) and other confirmatory tests also led to diagnosis as PA. Abdominal CT image revealed a low-density mass in the left adrenal, but the patient chose medication as treatment for PA, so we started 100 mg/day eplerenone. Months later his leg ulcer lesions strikingly got reduced in size concomitantly with better BP control, and epithelialization are mostly completed in the current status.

[Discussion] This type of skin ulcer was originally reported as “arteriosclerotic ulcer of Martorell” in 1945. The underlying mechanism is supposed to be arterial ischemia caused by uncontrolled severe hypertension and lifestyle-related diseases. Although it is not conclusive whether MR blockade or BP control contributes to disappearance of skin lesion, MR antagonist was notably effective to heal the intractable skin lesion. This case suggests the potential benefit of MR antagonism on ulcerative skin disease such as peripheral artery disease (PAD) and diabetic gangrene.

 

Nothing to Disclose: IK, HS, KM, HO, KY, AM, YM, RJ, TO, TN, AT, HI

8345 16.0000 SAT-68 A Medical Treatment for Primary Aldosteronism Healed Intractable Leg Ulcer : Involvement of Aldosterone in the Dermatological Disease 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 53-73 2207 1:45:00 PM Primary Aldosteronism & Mineralocorticoid Excess Poster


Rodis Paparodis*1, Subarna Mani Dhital2 and Yoram Shenker3
1University of Wisconsin - Madison, Madison, WI, 2Patan Academy of Health Sciences, Kathmandu, Nepal, 3University of Wisconsin, Madison, WI

 

Background: Aldosterone receptor antagonists (ARA) are the mainstay of medical management of aldosterone excess syndromes. Spironolactone (SPR) is an effective ARA, but causes painful gynecomastia in more than 50% of patients when doses >100mg daily are used. Eplerenone (EPL) is less potent and causes gynecomastia in <5%. Combination therapy of EPL and low dose SPR could be beneficial in patients intolerant of high dose SPR.

Cases presentation: We present two patients with PA treated with a combination of high dose EPL and low dose SPR, after they developed painful gynecomastia from high dose SPR.

1. A 60 year old man with HTN since the age of 46, on 4 medications, found to have PA due to bilateral hypersecretion of aldosterone by adrenal vein sampling (AVS). He was placed on SPR 100mg BID and developed painful gynecomastia. This was changed to EPL 50mg BID, but was inadequate to control the HTN or hypokalemia. SPR 25mg BID was added; the HTN got well controlled and the hypokalemia resolved. The latest K level off supplements was 3.8 mmol/L. He complains of rare breast discomfort, which is not serious enough to affect compliance.

2. A 71 year old man with resistant HTN and hypokalemia diagnosed with PA at the age of 55, with normal CT of the adrenal glands. He was placed on SPR 50mg BID, but developed painful gynecomastia and discontinued it. Subsequently he was placed on 5 antihypertensive medications and 80 meq of KCl daily, without controlling the HTN or hypokalemia. Follow up CT abdomen revealed a 1.2cm left adrenal adenoma, but due to patient preference, AVS was not performed. EPL was started at 50 mg BID, and all antihypertensives were discontinued, but the BP rose and the K dropped to 2.9 mmol/L. SPR was added at 25 mg BID, with prompt improvement in the blood pressure and K. Currently he is only on two additional antihypertensives. He denies any painful gynecomastia.

Conclusions: Spironolactone is the first line of therapy for medical management of aldosterone excess, but is associated with high incidence of painful gynecomastia. Eplerenone alone is frequently inadequate to control the mineralocorticoid excess, due to its low potency. The addition of low dose spironolactone in this patient population could decrease the number of medications needed to control the HTN and hypokalemia, without the onset of its common adverse effects, such as painful gynecomastia.

 

Nothing to Disclose: RP, SMD, YS

5001 17.0000 SAT-69 A Dual Therapy with Aldosterone Receptor Antagonists in Patients with Primary Hyperaldosteronism (PA) and Intolerance to High Dose Spironoloactone 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 53-73 2207 1:45:00 PM Primary Aldosteronism & Mineralocorticoid Excess Poster


Nimalie Jacintha Perera*
Royal Prince Alfred Hospital, Sydney NSW, Australia

 

Background:Blood Pressure (BP) tends to fall during pregnancy due to increased GFR, peripheral vasodilatation caused by vasorelaxation factors including prostaglandins and progesterone induced increased sodium excretion (aldosterone antagonist).

Clinical case:A 28 year women presented  15 weeks post partum with typical symptoms of  hyperthyroidism and headaches with hypertension (BP167/104 mmHg).

During pregnancy and post partum she was normotensive (BP110-130/60-80mmHg). Initial tests were suggestive of primary aldosteronism (PA) and thyroditis with hypokalemia (2.4-3.1mmol/L, n 3.5-5.0mmol/L), metabolic alkalosis (bicarbonate 35mmol/L, n 24-32mmol/L), suppressed plasma renin activity (<40fmol/L, n 130-2350fmol/L/sec) and raised aldosterone  (1053-1084pmol/L, n 80-1040pmol/L). TSH suppressed (<0.005mU/L, n 0.40-0.50mU/L) with increased fT4 (29.3pmol/L, n 9.0-19.0pmol/ L) and fT3 (9.6pmol/L, n 2.6-6.0pmol/L) with normal thyroid antibodies. Urine catecholamines, ACTH, cortisol (salivary, urine free cortisol) were normal. A saline infusion test on Verapamil 240mg/day (Lactation category B1, and less interference with renin-aldosterone levels) and  potassium supplementation to maintain potassium at 3.5-4.0mmol/L showed non suppression of aldosterone (post infusion 770pmol/L, with levels >277pmol/L suggesting PA very likely).  Non contrast CT (to avoid contrast/iodine with lactation and thyroiditis) showed a  2.4x1.5x2.2cm right adrenal adenoma (11-13HU) and subsequent adrenal vein sampling (during interrupted lactation) showed localisation to the right (right and left Adrenal vein aldosterone 810000 pmol/L and 2225 pmol/L with Aldosterone/Cortisol ratio 36 and 1.8 respectively). After laporoscopic resection of the adrenal adenoma, the potassium supplements and antihypertensive were ceased on day 5 and 7 respectively (BP124/76mmHg, Potassium  4.5mmol/L) . The thyroid function followed a typical course with a brief period of  hypothyroidism and euthyroid 2 weeks post surgery (TSH 2.34mU/L).

Conclusion:This case not only reminds us to investigate the development of hypertension during pregnancy, but also in the puerperium. With PA, BP changes variably during pregnancy. This patient was normotensive during pregnancy since the aldosterone concentration did not exceed pregnancy reference ranges to overwhelm or exceed the various physiological antihypertensive mechanisms operating during pregnancy including the aldosterone antagonist effect of progesterone. Previous reports (none post partum) have shown that excessive aldosterone release leads to the development of inflammatory cytokines and thyroid infiltration by inflammatory cells resulting in thyroiditis that improves with spiranolactone or surgical removal (as in this patient) of the adenoma.

 

Nothing to Disclose: NJP

4699 18.0000 SAT-70 A Primary Aldosteronism with Thyroiditis Unmasked during Post Partum Period 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 53-73 2207 1:45:00 PM Primary Aldosteronism & Mineralocorticoid Excess Poster


Julie T Hetherington*1, Elizabeth Lian Chua1 and Nimalie Jacintha Perera2
1University of Sydney, Sydney, Australia, 2Royal Prince Alfred Hospital, Sydney NSW, Australia

 

Investigation for primary hyperaldosteronism and concurrent post partum thyroiditis whilst continuing to breast feed (BF) is difficult and challenging for both the woman and the clinical team.

A 28 year old woman presented with palpitations, headache, BP 190/110 mmHg, p 120. No history of hypertension, normal to low BP in recent pregnancy, currently BF 4 month old child.

Lab results- hypokalaemia 2.4mmol/L (n 3.5-5.0 mmol/L), suppressed TSH <0.01 mIU/L, freeT4 28.9 pmol/L  (n 9.0-22.0 pmol/L), fT3 10.8 pmol/L (n 2.5-6.5pmol/L).

Initial care focused on normalizing K+ with supplements and treating hypertension. Potassium requirements were significant, titrated up to 14 supplements daily. A calcium channel blocker (verapamil) was a safe antihypertensive for use in lactation but also allowed ongoing assessment and interpretation plasma aldosterone. The aldosterone level was 1084 pmol/L (n 80-1040 pmol/L) with suppressed renin <40fmol/L. TFT’s normalised without treatment.

The Endocrine Clinical Nurse Consultant’s (CNC) role throughout the following months included explanation, education, psychological support and the diagnostic testing.

A saline load test (SLT) was performed. A potassium infusion was required to treat progressive hypokalaemia over the 4 hour test. In the presence of undetectable plasma renin, the aldosterone level was not suppressed, only decreasing from 1067 to 700 pmol/L at the end of the SLT (n < 135pmol/L). A non contrast CT scan was performed allowing BF to continue. It showed 2 cm adenoma of the right adrenal.

Adrenal vein sampling (AVS) was coordinated by the CNC. This involves an iodine load which potentially raises breast milk iodine content. Infant exposure to high iodine levels could cause transient hypothyroidism therefore expressed breast milk was analysed daily for iodine content before BF could recommence. AVS confirmed an aldosterone producing adenoma with levels of >800,000pmol/L from the right adrenal. The patient proceeded to successful right adrenalectomy with rapid improvement in K+and cessation of all medications.

This case required continual reflection on how every test or treatment may impact on the breast milk quality and quantity, as well as maternal and infant well being. The CNC was closely involved in timing of diagnostic tests and radiological scans. Continual monitoring of medical therapy was required to ensure that BF could be continued for as long as possible without compromising the woman’s clinical condition.

 

Nothing to Disclose: JTH, ELC, NJP

8494 19.0000 SAT-71 A A Juggling Act - Breast Feeding, Post Partum Thyroiditis and Conn's Syndrome: The Endocrine Nurse's Role in the Diagnosis and Treatment of a Complex Clinical Case 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 53-73 2207 1:45:00 PM Primary Aldosteronism & Mineralocorticoid Excess Poster


Hyma V Polimera*1, Shamsuddin Shaik1 and David Leh2
1St Lukes University Hospital, Bethlehem, PA, 2St Lukes University Hospital, bethlehem, PA

 

Introduction

Unusual case of adrenal masses in a normotensive patient

Clinical case             

A 37 year old female, no significant medical history, presented with sudden onset of right sided weakness and dysarthria. On presentation she was normotensive but exhibited expressive aphasia and complete right hemiplegia. Initial CT Head demonstrated a dense left MCA infarct with no hemorrhage. Echo was normal. Abdominal Ultra sonogram and CT revealed large bilateral cystic and solid adrenal masses, right measured 22x12.5x11cm, weighing 2058g and left measured 16x11.5x7.7cm, weighing 672g; highly suspicious for neoplastic disease.  A repeat CT head showed increasing edema and brainstem herniation which necessitated emergent decompressive frontotemporoparietal craniectomy and durotomy.

24-hour urine studies were metanephrine 130,896 µg (35-460 µg), normetanephrine 25,938 µg (110-1050 µg) and vanillylmandelic acid 127.2 mg (1.8-6.7 mg). TSH and serum calcitonin were normal. MEN2 screening for RET proto-oncogene mutation was negative. Hypercoagulable work up was negative. Patient was pretreated with phenoxybenzamine and had exploratory laparotomy and bilateral adrenalectomy. Pathology and immunohistochemical staining confirmed the diagnosis of bilateral benign pheochromocytomas. Patient was discharged with daily hydrocortisone and fludrocortisone. 24-hour urinary fractionated metanephrines measured 2 weeks post-surgery were normal.

The triad of episodic headache, sweating and tachycardia is infrequent; pheochromocytomas may present as paroxysmal hypertension, acute pulmonary edema, myocardial infarction or stroke. Plasma metanephrines were normal though urinary metanephrines were elevated. Patient was not on medication that might alter catecholamine metabolism.

Conclusion

Manifestations of catecholamine hypersecretion are common. Though adrenal masses were found incidentally in our patient, pheochromocytoma should be considered in young patients presenting with acute stroke.

 

Nothing to Disclose: HVP, SS, DL

7817 20.0000 SAT-72 A A Stroke of Bad Luck 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 53-73 2207 1:45:00 PM Primary Aldosteronism & Mineralocorticoid Excess Poster


Anne Marie Hannon*1, Ruth Casey2, Raquel O'Halloran1 and Domhnall Jude O'Halloran3
1Cork University Hospital, Cork, Ireland, 2Cork Univ Hospital, Cork, Ireland, 3Cork Univ Hosp, Cork, Ireland

 

Glucocorticoid Remediable Aldosteronism (GRA) is a rare autosomal dominant form of familial hyperaldosteronism (HA)  Patients with GRA, have ACTH-sensitive aldosterone production occurring in the zona fasciculate, due to a mutation in the promoter region of the gene for CYP11B1 and the coding sequences of CYP11B2 resulting in fusion of the two genes.

This patient first presented to the pediatric service at age 13, with palpitations and exertional chest pain. His blood pressure was found to be 170/100 and remained persistently elevated on follow up examination. His cardiovascular examination was normal, and there were no syndromic features. There was a strong family history of hypertension, which extended over at least three generations. The family history of hypertension was associated with early onset of diagnosis and a history of HA in two maternal cousins.

Biochemically evaluation showed a normal angiotensin:renin ratio and unremarkable 24 hour urinary catecholamine collections. Radiological investigation of his adrenal glands demonstrated no abnormality. Genetic testing confirmed the presence of a chimeric gene characteristic of GRA. This genetic defect was confirmed biochemically by a dexamethasone suppression test. The patient was commenced on amiloride, which resulted in excellent blood pressure control. This proven genetic defect prompted genetic screening of the entire family. To date, his mother has tested positive for GRA, and his three other siblings and extended family are undergoing investigation. In conclusion, this case highlights the importance of a thorough secondary workup in young patients presenting with symptomatic hypertension. Although a rare entity, GRA presents early in life and is associated with significant disease burden, related to hypertension. It is easily treated, therefore early genetic screening, enables early detection and management of at risk family members.

 

Nothing to Disclose: AMH, RC, RO, DJO

7411 21.0000 SAT-73 A A case of familial hyperaldosteronism presenting in childhood 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 53-73 2207 1:45:00 PM Primary Aldosteronism & Mineralocorticoid Excess Poster


Anne I Turner*1, Susan J Torres1, Emma Townsin1, Caryl A Nowson1, Alan J Tilbrook2 and Sisitha U Jayasinghe1
1Deakin University, Melbourne, Australia, 2South Australian Research & Deve, Roseworthy, SA, Australia

 

Previous research has shown that increased levels of adiposity can lead to increased cortisol response to stress (1, 2, 3).  Food intake has also been shown to activate the hypothalamo-pituitary adrenal axis (4, 5) but it is not clear if this activation is influenced by levels of adiposity.  We tested the hypothesis that overweight/obese men will have a greater cortisol response to food ingestion compared to lean men.

Lean (BMI=20-25 kg/m2; n=19) and overweight/obese (BMI=27-35 kg/m2; n=17) men aged 50-70 years were allowed to prepare their own lunch at 12:00 pm using bread, margarine, cheese, processed meat (ham or chicken), tomato, cucumber, nuts, fruit bars and a fruit juice box drink.  Records were made of foods consumed.  Energy and macronutrient intake were determined using Foodworks (version 6.0; Xyris Software, QLD).  Concentrations of cortisol were measured (by enzyme immunoassay) in samples of saliva collected every 15 min from 11:45 am to 2:00 pm with the exception of during lunch (12:15 pm) when no sample was collected.

Mean (±SEM) body weight and BMI were significantly higher in overweight/obese men compared to lean men (93.8±2.3 vs 69.7±1.6 kg and 30.6±0.6 vs 23.5±0.3 kg/m2, respectively; p<0.001 for both).  Lean and overweight/obese men did not differ significantly in their energy intake (2895±245 vs 3015±235 kJ) or macronutrient intake (Protein: 27.2±2.1 vs 29.9±2.5 g; Carbohydrate: 65.2±6.4 vs 73.0±5.4 g; Fat: 37.2±4.3 vs 35.7±4.3 g, respectively; p>0.05 for all).  For cortisol, repeated measures analysis of variance revealed a significant time*treatment interaction (p=0.008). Overweight/obese men responded to food intake with a significant elevation (51%) in salivary cortisol (time effect: p=0.005) whereas lean men did not have a significant elevation (5%) of cortisol (time effect: p=0.382).

While overweight/obese men had a significant cortisol response to food ingestion, lean men did not.  If overweight/obese men have an elevated cortisol response every time they ingest food, they may be more susceptible to the development of stress-related disease.

 

Nothing to Disclose: AIT, SJT, ET, CAN, AJT, SUJ

6683 1.0000 SAT-41 A Overweight and Obesity Influence Cortisol Response to Food Ingestion in Men 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 41-52 2209 1:45:00 PM HPA Axis & Disease States Poster


David J. Waters*1, Aimee H Maras2, Huiping Xu3, Seema S Kengeri2 and Emily C Chiang2
1Purdue University, West Lafayette, IN, 2Gerald P. Murphy Cancer Foundation, West Lafayette, IN, 3Indiana University School of Medicine, Indianapolis, IN

 

An age-related loss of hypothalamic-pituitary-adrenal (HPA) axis resilience can result in hypercortisolism, which has been linked to an array of adverse outcomes including cognitive impairment, progressive cancer growth, and clinical frailty.  To better understand the biological mechanisms and adaptive capacities that contribute to highly successful aging, we studied exceptionally long-lived Rottweiler dogs that, similar to human centenarians, had achieved a duration of longevity that exceeds by more than 30% their breed-specific norm.  We hypothesized that highly successful aging might be associated with a preservation of HPA resilience and life-long avoidance of hypercortisolism.  The HPA axis of 28 canine centenarians was evaluated by determining: basal plasma cortisol, aldosterone, and ACTH concentrations;  post-ACTH challenge plasma cortisol and aldosterone concentrations;  and urine cortisol : creatinine ratio.  Frailty burden was measured using a 13-item frailty index using assessments obtained from interviews with pet owners; one investigator conducted all interviews.  HPA axis evaluation revealed three categories of canine centenarians.  Forty-three percent of dogs had normal basal cortisol with youthful response to ACTH challenge.  Forty percent of dogs had low basal cortisol levels, yet preserved a youthful response to ACTH challenge.  Seventeen percent of dogs showed deterioration of the aldosterone arm of the HPA axis.  None of the canine centenarians had hypercortisolism.  Compared to dogs with normal basal cortisol, dogs with low basal cortisol had lower frailty burden, with strongest difference seen in measures of stamina and mobility (p=.07).  Taken together, our results from canine centenarians suggest that hypercortisolism resulting from impairment of the HPA axis is not an obligate phenotype intrinsic to the aging process.  Further, the discovery of an adaptive, low basal cortisol endophenotype in some of these dogs emphasizes the importance of studies that rely upon challenge, rather than basal measures, to determine how HPA axis resilience contributes to highly successful aging.  Pet dogs should provide a tractable model to study further the relationship between HPA axis resilience and clinical frailty.

 

Nothing to Disclose: DJW, AHM, HX, SSK, ECC

5370 2.0000 SAT-42 A Pathways of HPA Axis Resilience in Highly Successful Aging: Evaluation of Basal Plasma Cortisol, ACTH Challenge, and Frailty Burden in Dogs with Exceptional Longevity 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 41-52 2209 1:45:00 PM HPA Axis & Disease States Poster


Subarna Mani Dhital*1, Theresa M Duello2, Yoram Shenker3 and Ian Michael Bird4
1Patan Academy of Health Sciences, Kathmandu, Nepal, 2Univ Wisconsin-Madison Sch Med, Madison, WI, 3University of Wisconsin, Madison, WI, 4Univ of Wisconsin-Madison, Madison, WI

 

Background: Nitric oxide (NO) is known to modulate adrenal steroidogenesis (1). Because of short half-life of NO (2), presence of catalyzing enzyme in proximity to target tissue is important. The main isozyme of NOS expressed in adrenal cortex is eNOS with marked interspecies variation in zonal expression (3). In humans, expression of eNOS and functional modulation of steroidogenesis by NO has been demonstrated in zona glomerulosa (ZG) cells but has not been evaluated in zona fasciculata (ZF) and zona reticularis (ZR).   

Sample collection: Histologically normal-looking adrenal glands were collected from adrenalectomy specimens in 5 patients. Two adrenal glands were collected from Cushing’s disease patient. Tissues were processed immediately for immunohistochemistry (IHC) and western blot.

Methodology: IHC was performed for eNOS in paraffin-embedded sections of adrenal tissues. P450c17 IHC served as zonal marker for ZR and ZF and Cytochome b5 (b5) as ZR marker. In addition, western blot for eNOS was performed in dissected tissue blocks from different adrenal zones. Digital grayscale densitometry was performed to compare staining intensity and results analyzed with one-way ANOVA using Kruskall-Wallis test. P <0.05 was considered significant.

Results: Capsule-subtracted value of eNOS staining on IHC was significantly higher than medullary eNOS expression in ZG, ZF and ZR (p= 0.012) in normal adrenal glands.  These results also corresponded to eNOS expression pattern on western blot. Zonal differentiation was confirmed by appropriate expression patterns of P450c17 and b5 in both IHC and western blot. In adrenals from Cushing’s disease patient, ZF expression of eNOS was significantly lower than in ZG or ZR (p = 0.001).

Discussion: eNOS expression was demonstrated in ZG, ZF and ZR of histologically normal-looking human adrenals using IHC and western blot. The presence of eNOS in adrenal cortex points to the potential role for NO in local modulation and fine-tuning of adrenal steroidogenesis. Inhibitory role of NO has been reported in human ZG cells (4). But these results point to its relevance in ZF and ZR as well. Low expression of eNOS in adrenals from Cushing’s disease patient further support its functional relevance because lowered inhibition of steroidogenic enzymes by decreased NO production fits the profile of increased cortisol output from ZF.

 

Nothing to Disclose: SMD, TMD, YS, IMB

4371 3.0000 SAT-43 A Zonal Expression of Endothelial Nitric Oxide Synthase (eNOS) in Normal Human Adrenal Cortex and Functional Insights from Comparison with Adrenals in Cushing's Disease 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 41-52 2209 1:45:00 PM HPA Axis & Disease States Poster


Richard Joseph Blythe*, Ismail Sami Elkashif and Mohamed Abdel Aziz Malik
Scunthorpe Centre for Diabetes and Endocrinology, North Lincolnshire, United Kingdom

 

Introduction

Insulin induced hypoglycaemia or Insulin Tolerance Test (ITT) is considered the gold standard assessment of the hypothalamic-pituitary-adrenal axis (HPA). Both synthetic adrenocorticotropic hormone (short synacthen test, SST) and glucagon provocation (glucagon stimulation test, GST) are considered as safe and less labour intense alternative to ITT. Much controversy surrounds the accuracy of these two screening tests when compared to ITT with different levels of sensitivities and specificities reported.

As part of ongoing audit we examined the agreement of test results from SST, GST and ITT performed for investigation of clinically suspected HPA axis dysfunction.

Method

Between March 2009 and March 2012 thirty two patients underwent two or more assessment of the HPA axis with SST, GST or ITT. Patients were included in the audit if they had at least two of the three screening tests and their full medical notes could be retrieved. The agreement between the three tests results were evaluated using Cohen’s Kappa coefficient.

Results

32 patients underwent ITT of whom 31 had pre-screening SST and/or GST. Twenty one were females and average age was 42.9.

26 patients underwent both SST and ITT, 16 patients underwent GST and ITT, and 13 had SST and GST.  

Calculated kappa coefficient between tests for SST and ITT were 0.013 (95%CI -0.143 to 0.168),  -0.250 (95% CI -0.250 to 0.145) for GST and ITT, and 0.130 (95% CI -0.130 to 0.403) for SST and GST.

Discussion

Taking Kappa threshold of < 0.40 or even <0.20 to indicate poor agreement, our audit data show poor or no agreement between the three tests used for HPA axis assessment. Most importantly our results indicate no agreement between SST/GST and the gold standard ITT.

Recommendation

In patients with relative or absolute contraindication to ITT, pre-tests probability and clinical assessment should form an integral part of interpreting SST and GST results when used in the evaluation of the HPA axis.

 

Nothing to Disclose: RJB, ISE, MAAM

7199 4.0000 SAT-44 A Agreement between Short Synacthen and Glucagon Stimulation Tests with the gold standard Insulin Tolerance Test in assessment of the hypothalamic-pituitary axis 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 41-52 2209 1:45:00 PM HPA Axis & Disease States Poster


Animesh N Sharma*, Jean Wigham, Sue Weist and Johannes D Veldhuis
Mayo Clinic, Rochester, MN

 

Background: Type 1 diabetes mellitus (T1DM) is a pro-inflammatory, pro-oxidative stress state which, with its attendant hyperglycemia, likely disrupts adaptive neuroendocrine control. Preliminary studies of the hypothalamic-pituitary-adrenal axis have shown that ACTH and cortisol secretion may be exaggerated in animal models with artificially induced diabetes. These findings, if confirmed in humans, would be important since altered ACTH and cortisol secretion could further dysregulate glucose homeostasis.

Objective: To test the hypothesis that endogenous ACTH-cortisol dose-responsive drive is significantly different in adolescents and young adults with type 1 diabetes mellitus (T1DM) compared with that in healthy individuals

Methods: This was a pilot study of 11 volunteers with type 1 diabetes mellitus (T1DM) and 10 controls, ages 16-30. Volunteers underwent a screening visit followed by overnight testing with frequent blood sampling (every 10 minutes for ACTH and cortisol and every 60 minutes for blood glucose) from 10 pm to 8 am.T1DM volunteers maintained their home insulin regimen.

Results: Mean cortisol, but not ACTH, concentrations were lower in the T1DM group compared with controls (p=0.05). This was in part due to lower deconvolved total cortisol secretion (p=0.02) and not burst mass or pulse frequency. Analytical dose-response estimates identified that the T1DM group had lower ACTH efficacy (maximal cortisol secretion), P=0.009, greater EC50 (ACTH concentration driving half-maximal cortisol secretion), P=0.04, and increased ACTH sensitivity (ACTH-cortisol slope), P=0.03. Linear regression showed a strong correlation between ACTH efficacy and sensitivity with c-peptide levels (R=0.06, P=0.004 and R= -0.43, P=0.04, respectively), further confirming our findings. ACTH and cortisol approximate entropy (a measure of secretion regularity) and cortisol-ACTH cross-approximate entropy (a measure of feedback synchrony) did not differ between the 2 groups.

Discussion: The hypothalamic-pituitary-adrenal axis in our group with T1DM was characterized by decreased ACTH efficacy and increased EC50, thus explaining decreased cortisol concentrations compared with healthy individuals. T1DM volunteers also had increased ACTH sensitivity, which was likely compensatory to the lower cortisol secretion. The sum of these findings may help explain altered glucose homeostasis in T1DM as well as impact stress-adaptive changes in this group of individuals.

 

Nothing to Disclose: ANS, JW, SW, JDV

5375 5.0000 SAT-45 A Overnight and Early Morning Cortisol Secretion and ACTH Potency is Reduced in Adolescents and Young Adults with Type 1 Diabetes Mellitus: A Pilot Study 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 41-52 2209 1:45:00 PM HPA Axis & Disease States Poster


Naoki Hattori*1 and Akira Shimatsu2
1College of Pharmaceutical Scie, Kusatsu-City, Shiga, Japan, 2National Hospital Organization Kyoto Medical Center, Kyoto, Japan

 

The aetiology of macroprolactin is heterogeneous but mainly a complex of prolactin (PRL) with immunoglobulin (Ig)-G, particularly anti-PRL autoantibodies. However, it is unknown why such autoantibodies develop in certain subjects. This study aims to elucidate the possible involvement of post-translational modifications of PRL molecule in the pathogenesis of macroprolactin in some patients with rheumatoid arthritis (RA).

Subjects and Methods.  The prevalence of macroprolactinaemia was screened by polyethylene glycol (PEG) method in 238 patients with RA (200 women and 38 men, 60.0 ± 12.5 years) and 647 control subjects (514 women and 133 women). The relationships of macroprolactinaemia with RA disease markers such as matrix metalloproteinase-3 (MMP-3), C-reactive protein (CRP), erythrocyte sedimentation ratio (ESR), g-globulin and anti-cyclic citrullinated peptides (CCP) antibody were examined.

Results.  Fifteen patients with RA had macroprolactinaemia and the prevalence (6.3%) was significantly (P=0.042) higher that in young (younger than 50 years) control group (n=458; 35.6 ± 7.2 years; 3.1%) but not different (P=0.66) from that in aged (older than 50 years) control group (n=189; 55.4 ± 4.2 years; 5.3%). Serum MMP-3 levels were elevated in 124 patients (52.1%) with RA and the prevalence of macroprolactinaemia in patients with elevated MMP-3 levels (12/124 patients; 9.68%) was significantly (P=0.025) higher than those with normal MMP-3 levels (3/114 patients; 2.63%). Serum CRP and ESR levels were elevated in 107 and 142 patients, respectively, and the prevalence of macroprolactinaemia was not significantly different between patients with and without elevated levels of these markers. There were no significant correlations of the levels of macroprolactin with those of g-globulin and RA-specific anti-CCP autoantibody. Serum free PRL concentrations after removing macroprolactin components were significantly (P<0.001) higher in RA patients (142 ± 65 mIU/l) than in age-matched elderly controls (86 ± 59 mIU/l).

Conclusion.  Ageing is a chronic inflammatory process and the chances for proteins acquiring post-translational modifications may increase. PRL is a substrate of MMP-3, producing vasoinhibins. It may be possible that newly developed epitopes by MMP-3 trigger immune response producing anti-PRL autoantibodies in some patients with RA, and that such post-translational modifications may contribute to the increased prevalence of macroprolactinaemia in elderly subjects.

 

Nothing to Disclose: NH, AS

3608 6.0000 SAT-46 A Prolactin and macroprolactin in patients with rheumatoid arthritis 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 41-52 2209 1:45:00 PM HPA Axis & Disease States Poster


Paraskevi Salpea*1, Anelia Dafinova Horvath2, Edra London1, Annalisa Vetro3, Alison Manning4, Evgenia Gourgari5, Margaret Farmar Keil6, Antonella Forlino3, Orsetta Zuffardi3 and Constantine A Stratakis1
1National Institutes of Health (NIH), Bethesda, MD, 2The George Washington University, Washington, DC, 3University of Pavia, Pavia, Italy, 4Brigham and Women's Hospital, Boston, MA, 5Georgetown University Hospital, Washington, DC, 6Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH)., Bethesda, MD

 

Carney Complex (CNC) is an autosomal dominant multiple endocrine neoplasia syndrome characterized by spotty skin pigmentation, cardiac and other myxomas, and different types of endocrine tumors. PRKAR1A gene encodes for the type 1A regulatory subunit of protein kinase A and inactivating mutations have been shown to cause Carney complex (~70% of CNC patients). Most of these mutations consist of single base substitutions, small deletions, insertions or combined rearrangements, all of them not exceeding 15bp.

In this study we report large PRKAR1A gene deletions in 7 CNC patients. The deletions were identified by microarray-based Comparative Genomic Hybridization (array-CGH) and confirmed by Sanger sequencing of the boundaries and, in one case of complete deletion of the PRKAR1A gene - through Fluorescent in situ Hybridization (FISH). Quantitative PCR showed that these deletions lead to decreased PRKAR1A mRNA levels. Thus, we show that deletions of PRKAR1A cause CNC through haploinsufficiency, which is the molecular mechanism of the disease in the vast majority of the PRKAR1A point mutation carriers.

These deletions spread through all functional R1a domains and with significantly decreased PRKAR1A expression compared to normal. Interestingly preliminary clinical data indicated that these patients with extended PRKAR1A deletions also shared a different phenotype.

Until now only several cases of CNC patients with large PRKAR1A deletions have been described. In this study we present new cases that extend PRKAR1A mutational spectrum. Our data suggest that testing for large PRKAR1A alterations might need to be considered for routine genetic diagnosis of CNC.

 

Nothing to Disclose: PS, ADH, EL, AV, AM, EG, MFK, AF, OZ, CAS

9219 7.0000 SAT-47 A Large Deletions of the PRKAR1A Gene in Carney Complex 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 41-52 2209 1:45:00 PM HPA Axis & Disease States Poster


Sabine M. Staufenbiel*1, Anne T. Spijker2, Manja A. Koenders2, Bernet M. Elzinga3 and Elisabeth F.C. van Rossum4
1Erasmus MC, Rotterdam, Netherlands, 2PsyQ, The Hague, Netherlands, 3Leiden University, Leiden, Netherlands, 4Erasmus MC, University Medical Center Rotterdam, Rotterdam, Netherlands

 

Stress is thought to be one of the main factors negatively impacting the course of disease in patients with bipolar disorder (BD) with its effects being predominantly mediated by cortisol. Cortisol in scalp hair has recently been identified as a retrospective biomarker for long-term cortisol exposure in endocrine research. Hair provides the opportunity to measure mean cortisol levels of months to years in retrospect, in contrast to the short time frame covered by serum and saliva cortisol. It is hypothesized that the hypothalamic-pituitary-adrenal (HPA) axis’ activity is increased after the experience of major life events. This study aimed to investigate the association between the exposure to life events and hair cortisol concentrations (HCC) in BD patients.

Hair samples were collected in 96 patients with BD. The proximal 3 cm of hair were used for analysis. Cortisol was extracted from the hair with methanol, and cortisol levels were measured using a competitive enzyme-linked immunoassay (ELISA). The occurrence of 61 major life events in the period that was represented in the hair sample (i.e. 3 months) was assessed with the Paykel Life Events Scale.

HCC were significantly higher in patients that experienced life events compared to the patients who did not (35.1 pg/mg [95% CI: 29.5-41.7] vs. 26.4 pg/mg [95% CI: 22.7-30.8], p=0.01). HCC were also associated with the total number of events (β=0.03, p=0.04). When the life events were divided into positive or negative events (according to the individual assessment), higher HCC were only found after the occurrence of events that were perceived as negative (36.4 pg/mg [95% CI: 29.7-44.7] vs. 27.5 pg/mg [95% CI: 24.0-31.5], p=0.06) and were also associated with the number of negative events (β=0.05, p=0.01), but were not found after the occurrence of events that were experienced as positive (31.7 pg/mg [95% CI: 24.3-41.3] vs. 32.2 pg/mg [95% CI: 27.7-37.4], p=0.2).

The occurrence of recent negative major life events, but not of positive major life events, is associated with an increase in HCC in BD patients. This suggests that the individual interpretation of a stressful event as a pleasant or unpleasant occurrence influences the bodily stress response. This may have consequences for the course of disease in BD patients. Furthermore, this study provides further insight in the neurobiological stress response in BD patients, which is of importance for the development of novel intervention strategies.

 

Nothing to Disclose: SMS, ATS, MAK, BME, EFCV

5321 8.0000 SAT-48 A Recent Major Life Events Increase Hair Cortisol Concentrations in Patients with Bipolar Disorder 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 41-52 2209 1:45:00 PM HPA Axis & Disease States Poster


Julia Claire Kowalczyk*1, Eirini Meimaridou1, Tatiana V Novoselova1, Leonardo Guasti1, Rathi Prasad1, Xingen Lei2, Philippe Touraine3, Li F Chan1, Paul Chapple1, Peter James King1, Adrian J L Clark4 and Louise A Metherell1
1WHRI, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom, 2Cornell University, New York, NY, 3AP-HP, Hôpital Pitié-Salpêtrière, Paris, France, 4St George's University of London, London, United Kingdom

 

Familial Glucocorticoid Deficiency is an autosomal recessive disorder characterised by resistance to ACTH of the adrenal cortex, leading to isolated glucocorticoid deficiency and life-threatening hypoglycaemia. Recently defects in nicotinamide nucleotide transhydrogenase (NNT) have been shown to cause FGD in 10% of cases(1).  NNT ensures the maintenance of the high reduced to oxidized glutathione (GSH/GSSG) ratio necessary for detoxification of reactive oxygen species (ROS) by enzymes such as the glutathione peroxidases and peroxiredoxins. In a FGD patient with unknown aetiology, we identified a homozygous mutation p.R130-L133del in glutathione peroxidase 1 (GPX1). The mutation was heterozygous in his parents and an unaffected sibling.  Gpx1-/- mice are phenotypically normal but show increased sensitivity to oxidative stress (2). Adrenals from Gpx1-/- mice showed no gross morphological changes and corticosterone levels were not significantly different to their wild-type counterparts (in contrast to the Nnt mutants).  Knockdown of GPX1 in H295R cells reduced total GPX activity to 50% and the cells were less viable when exposed to oxidative stress but GSH/GSSG ratios were unchanged and cortisol production unaffected. Furthermore sequencing of >100 FGD patients did not reveal further GPX1 mutations. We therefore hypothesized that there could be a second gene defect present in this proband. Whole exome sequencing revealed a homozygous stop gain mutation, p.Q67X, in peroxiredoxin 3 (PRDX3) in this patient, the change was heterozygous in his parents but also homozygous in the unaffected brother, raising the possibility that both gene defects are necessary to cause FGD.  The PRDX3 mutation is predicted to be loss-of-function since the early truncation removes a residue critical for the enzyme’s activity.

The glutathione peroxidases and peroxiredoxins work simultaneously to reduce hydrogen peroxide, preventing cellular damage.  The adrenal cortex has a particularly harsh oxidative environment due to steroidogenic enzyme activity, increasing its sensitivity to redox changes. Previous studies have implicated GPX1 and PRDX3 as regulators of steroidogenesis by modulation of ROS levels (3, 4).  We identified a patient with two homozygous mutations in these antioxidant genes. Our studies show that loss of PRDX3 alone is insufficient to cause FGD and suggest that mutation in GPX1, either alone or in combination with PRDX3, may tip the redox balance to cause the disorder.

 

Nothing to Disclose: JCK, EM, TVN, LG, RP, XL, PT, LFC, PC, PJK, AJLC, LAM

8291 9.0000 SAT-49 A Digenic inheritance of mutations in antioxidant pathway genes causing Familial Glucocorticoid Deficiency? 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 41-52 2209 1:45:00 PM HPA Axis & Disease States Poster


Eva Boonen*1, Philippe Meersseman1, Hilke Vervenne1, Geert Meyfroidt1, Johannes D Veldhuis2 and Greet Van den Berghe1
1KU Leuven, Leuven, Belgium, 2Mayo Clinic, Rochester, MN

 

Background: We recently documented that reduced cortisol metabolism, via suppressed expression and activity of A-ring reductases and 11b-HSD2, substantially contributes to hypercortisolism during critical illness, and that morning ACTH levels are low (1). We hypothesized that reduced cortisol breakdown elevates circulating cortisol, which could suppress pulsatile ACTH secretion via negative-feedback, in turn suppressing pulsatile cortisol release. To test this hypothesis, we compared the dynamics of nocturnal ACTH and cortisol time series in critically ill patients and demographically matched healthy controls.

Methods: Blood was sampled every 10 minutes between 21:00h and 06:00h from 40 critically ill patients and 8 healthy controls, after excluding interfering drugs and pre-existing risk factors for HPA-axis dysfunction. Plasma ACTH and cortisol concentrations were quantified by radioimmunoassay.  ACTH and cortisol secretion profiles were constructed by multiple parameter deconvolution analysis, using cortisol half-lives as previously determined (1). In addition, approximate entropy (ApEn), an estimation of secretory irregularity, Cross ApEn, a quantitation of ACTH and cortisol synchrony, and the ACTH-cortisol dose-response relationships were calculated.

Results: Mean nocturnal plasma cortisol levels were 2.6-fold higher in patients than controls (P<0.0001) while mean nocturnal plasma ACTH levels were comparable (P=0.42). Deconvolution analysis revealed a reduced pulsatile ACTH secretion in critically ill patients to 69% of normal (P=0.03) explained by a decreased ACTH mass per burst to 72% of normal (P=0.02) while ACTH pulse frequency (P=0.50) and ACTH basal secretion (P=0.80) remained unaltered. Deconvolution analysis of the cortisol time series also revealed a reduced pulsatile cortisol secretion in patients to 46% of controls (P=0.005) explained by a reduced cortisol mass per burst to 60% of normal (P=0.03) with a comparable number of cortisol pulses (P=0.35) and a similar cortisol basal secretion (P=0.80).
Approximate entropy of the ACTH and cortisol time series was higher in patients than controls (P<0.03), indicating more irregularity in the secretory patterns. ACTH/cortisol crossApEns were also higher in patients than controls (P≤0.001) suggesting reduced synchrony between both hormones. Dose-response estimates were overall similar in patients and controls.

Conclusions: In the presence of reduced cortisol breakdown, pulsatile ACTH-driven cortisol secretion was found to be suppressed during critical illness, in line with negative feedback inhibition brought about by elevated circulating cortisol. Furthermore, the increased irregularity and the reduced synchrony between ACTH and cortisol secretory patterns support non ACTH-dependent mechanisms active during critical illness, which should be further investigated.

 

Nothing to Disclose: EB, PM, HV, GM, JDV, GV

6419 10.0000 SAT-50 A Reduced ACTH-driven cortisol secretion during critical illness 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 41-52 2209 1:45:00 PM HPA Axis & Disease States Poster


Chris Martin Roffe*1, Philipp Grimminger2, Craig Ramsey3, Zoltan Seres3 and Martin Bidlingmaier4
1Immunodiagnostic Systems Ltd., Boldon, United Kingdom, 2Medizinische Klinik und Poliklinik IV, Ludwig-Maximilian University, Munich, Germany, 3Immunodiagnostic Systems Ltd, United Kingdom, 4Klinikum der Universitaet, Munich, Germany

 

Background: Abnormal changes in cortisol levels due to hypothalamic, pituitary or adrenal malfunction, such as in Cushing’s syndrome and Addison’s disease, can lead to severe metabolic imbalance. Current diagnostic guidelines recommend the measurement of cortisol in blood following stimulation or suppression stress tests, but also the measurement of integrated 24hour cortisol secretion in urine or of late night salivary cortisol.. We present here the development of a competitive immunoassay for the measurement of cortisol,,on a chemiluminescence analyzer (IDS-iSYS). 

Method: Cortisol in the sample competes with biotinylated cortisol for binding to a monoclonal anti-cortisol-acridinium conjugate in a first incubation. Streptavidin-coupled magnetic particles are added and after further incubation and washing, the bound anti-cortisol-acridinium is measured whereby chemiluminescence generated is inversely proportional to the cortisol concentration in the sample. Total time to first result is 8 minutes.  IDS-iSYS assay results were correlated against commercially available assays for serum (Roche, Cobas), urine and saliva (IBL, EIA).

Results: The assay range covering all matrices was determined at 0.05-75µg/dL with an analytical sensitivity of 0.02ug/dL. Inter-assay precision CVs were between  3.6% - 8.9% at concentrations 0.5 to 54µg/dL. For the same samples intra-assay precisions were between 2.9% - 5.9%. The assay showed linearity between 86-103% at 0.2µg/dL to 48.4µg/dL Matched serum and plasma samples were run on the IDS-iSYS showing excellent correlation (plasma = 0.92x serum + 0.95, R2=0.95, n=35). In serum samples from two external quality assessment schemes, UK NEQAS and the German DGKL, the iSYS showed excellent correlation to the Roche assay (y=0.98x + 1.69, R2=0.99, n=15; and y=0.90x + 1.86, R2=0.96, n=15). Additionally the iSYS had excellent agreement to the target gold standard mass spectrometry values of the DGKL samples (y=1.02x + 16.41, R2=0.99). Good correlation to the IBL assays was observed with urine and saliva samples (y=0.92x + 1.34, R2=0.95, n=10; and y=0.90x + 0.09, R2=0.97, n=28).

 Conclusion: The data presented above demonstrate that the fully automated IDS-iSYS cortisol assay potentially presents a new, accurate and reliable immunoassay for all commonly used sample types across a wide assay range. The extremely short time to first result makes the assay suitable also for use during diagnostic procedures like adrenal vein sampling.

 

Disclosure: CMR: Employee, Immuno Diagnostic Systems Ltd. PG: Employee, Immuno diagnostic Systems Ltd. CR: Employee, Immuno Diagnostic Systems Ltd. ZS: Employee, Immuno Diagnostic Systems Ltd. MB: Consultant, Immunodiagnostic Systems Ltd.

8190 11.0000 SAT-51 A A new chemiluminescence immunoassay for the determination of serum, plasma, urine and saliva cortisol on the IDS-iSYS Automated System 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 41-52 2209 1:45:00 PM HPA Axis & Disease States Poster


Vasiliki Michopoulos*1, Kathy Reding2, Mark E Wilson*3 and Donna Toufexis4
1Emory University School of Medicine, 2Emory University, 3Emory University, Atlanta, GA, 4University of Vermont

 

Psychosocial stress is a precipitating factor in the emergence of a range of adverse health outcomes in women, including anxiety and depression.  Psychopathology is more prevalent in women than men, suggesting that individual vulnerability to stress-induced disorders is modulated by gonadal hormones including estradiol (E2).  While exposure to chronic psychosocial stress in females can result in the dysregulation of the limbic-hypothalamic-pituitary-adrenal (LHPA) axis and altered behavioral and physiological sensitivity to E2, it remains unclear whether exposure to a social stressor alters E2’s ability to modulate the activity of the LHPA axis. The current study was conducted to assess whether exposure to a chronic psychosocial stressor mediated by social subordination in ovariectomized female rhesus monkeys alters E2’s dose-dependent ability to modulate the LHPA axis following an acute stressor.  Ten socially dominant and 13 subordinate female rhesus monkeys were studied under four conditions: placebo, 2 µg/kg/d of E2, 4 µg/kg/d of E2, and 8 µg/kg/d of E2 for two weeks.  On day 12 of each phase, animals were exposed to the acute stressor of an approach-avoidance (AA) task where blood samples were collected prior to and following stressor exposure to assess basal levels and stress-induced changes in cortisol levels.   Our results show that subordinate females have lower basal cortisol levels than dominant females in the placebo, no-E2 treatment condition.  Treatment with E2 in subordinate females at all doses was sufficient to increase basal cortisol levels to those seen in dominant females.  Furthermore, while exposure to the AA task significantly elevated cortisol levels, it did so independent of social status.  Importantly, the change in cortisol levels due to the AA task was decreased by the 4 µg/kg/d of E2 compared to all other study conditions, but independent of social status.  These data indicate that the social subordination alters E2’s ability to modulate basal LHPA axis activity and suggests that future studies are necessary to determine how hormone replacement with E2 in postmenopausal women interacts with stress background to influence LHPA activity.

 

Nothing to Disclose: VM, KR, MEW, DT

8656 12.0000 SAT-52 A Social subordination and estradiol effects on basal and stress-induced stress axis activity in female rhesus monkeys 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Adrenal/HPA Axis Saturday, June 15th 3:45:00 PM SAT 41-52 2209 1:45:00 PM HPA Axis & Disease States Poster


Corina Galesanu*1, Veronica Mocanu2, Vlad Gorduza2 and Valentin Zaharia2
1University of Medicine and Pharmacy, IASI, Romania, 2University of Medicine and Pharmacy

 

Objective: Turner Syndrome (TS) is caused by complete or partial or complete absence of the 2nd X chromosome. Many women with TS remain undiagnosed. Adult patients  with TS present osteoporosis in 25% and high risk of fractures.

Aim: To follow the bone mineral density (BMD) and fractures at adults with TS diagnosticated after 25 years old.

Materials and methods: Five adult women with TS, mean age 39 years old (27-45) were verified for osteoporosis by BMD (measured by proximal femur and lumbar spine by dual-energy-X ray absorptiometry-DXA: Hologic). The diagnosis of TS was confirmed by karyotype in women with short stature, absent puberty or history of irregular menses, congenital heart disease (2 cases), renal anomaly (2 cases).The DXA-BMD was measured at the moment of diagnosis and under HRT (hormone replacement therapy) 1 mg Estradiol + 2 mg Drospirenone at every 12 months for three years and more. Mean hight of women was 143.0 cm and mean  weight 41.0 kg (BMI=20kg/m2). At lumbar spine, the mean BMD at the base line was 0.824±0.08 g/cm2 (T-score: -2.0)  and decreases at 0.713±0.08g/cm2 (T-score:-3.0). At total hip, the mean BMD at baseline was 0.701±0.06 g/m2 (T-score: -2:3) and decreases at 0.645±0.06g/cm2  (T-score: -2.4) after 3 years. One case with lombar fracture (L2) under HRT at 39 years.

Discussions: Bone mass and strength achieved at the and of growth period =peak of bone mass (PBM). Increase PBM by one standard deviation would reduce the fracture risk by 50% after menopause. Sex hormones and IGF 1 system are implicated in the bone pubertal maturation and bone mineral mass acquisition. The absence of rhGH-treatment in the childhood for height gain and a late age of  HRT were found to be associated with lower BMD in TS.

Conclusions: The GH-IGF 1 system is essential for harmonious skeleton development. The late diagnosis in adult life at TS, no rhGH-treatment in childhood and no estrogens replacement during pubertal maturation is associated with low bone mass and continuu decreases of BMD and high osteoporotic fractures risk.

 

Nothing to Disclose: CG, VM, VG, VZ

5301 1.0000 SAT-199 A Turner Syndrome in adults – no peak bone mass with high risk of osteoporotic fracture 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 15th 3:45:00 PM SAT 199-223 2216 1:45:00 PM Disorders of Bone & Calcium Homeostasis: Case Reports Poster


Sira Korpaisarn, Pattana Sornmayura, Niramol Chanplakorn, Objoon Trachoo, Pattarana Sae-Chew, Rangsima Aroonroch and Chutintorn Sriphrapradang*
Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand

 

Introduction

We report on a case of unsuspected parathyroid lesion whose two FNA resembled thyroid lesions.

Clinical case

A 31-year-old man presented with a right neck mass. Palpation of the thyroid glands showed a 3-cm nodule in the right lobe and FNA was performed. The cytology was reported as “intrathyroidal lymphoidal hyperplasia”. After observation for 5 years, the size of nodule was enlarged. Repeated FNA was done with result of Hurthle cell lesion. Surgeon decided to perform right lobectomy. His preoperative laboratory investigations revealed normal thyroid function tests, elevated serum calcium of 13.5 mg/dL (reference range, 8.5-10.1 mg/dL), and increased parathyroid hormone (PTH) level of 1,859 pg/mL (reference range, 15-65 pg/mL). Then he was referred to endocrinologists. On ultrasound examination, a 2.8-cm heterogenous hypervascular mass was found inferior to right lobe of thyroid gland. MIBI scan showed increased uptake corresponding to ultrasound findings. Careful systemic history taking revealed that he had been suffered from chronic knee pain for 7 years. Therefore, parathyroid carcinoma was suspected, and then the en bloc resection was performed. The pathology result was parathyroid carcinoma. Immunohistochemical (IHC) staining of tumor cells revealed positive immunoreactivity for PTH but negative for thyroglobulin. In addition, patient had ossifying fibroma at the right mandible since he was 10 years old. The genetic analyses were done in patient’s blood and parathyroid tissue and found somatic frameshift mutation of CDC73 gene in exon1 (c.70delG) caused premature stop codon in amino acid 26 (p.Glu24Lysfs*2). The final diagnosis was parathyroid carcinoma with hyperparathyroidism-jaw tumor syndrome. FNA cytology of parathyroid can mimic thyroid lesion. The oxyphil cells and chief cells present in the parathyroid resembled Hurthle cells and lymphocytes respectively. PTH assay in FNA is also useful in differentiating parathyroid from thyroid tissue.

Conclusions

It is difficult to distinguish parathyroid lesions from thyroid lesions based on solely FNA cytomorphologic diagnosis because of their morphologic similarities.Therefore, it is important to consider and relate the whole information from careful clinical history taking, laboratory, imaging studies, and FNA. PTH assay in FNA specimen and IHC staining can definitely distinguish between parathyroid and thyroid lesions.

 

Nothing to Disclose: SK, PS, NC, OT, PS, RA, CS

5048 2.0000 SAT-200 A Fine-Needle Aspiration Cytology of Parathyroid Carcinoma Mimic Hurthle Cell Thyroid Neoplasm 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 15th 3:45:00 PM SAT 199-223 2216 1:45:00 PM Disorders of Bone & Calcium Homeostasis: Case Reports Poster


Benjamin Adam Dennis* and Rene J Harper
Georgia Regents University, Augusta, GA

 

Introduction:

Hypercalcemia mediated by 1,25-dihydroxyvitamin D has a broad differential and may present biochemically before the etiology of excessive 1α-hydroxylase is known.  The underlying diagnosis in such cases may remain elusive even after extensive evaluation.  We present a case of hypercalcemia due to splenic sarcoidosis that resolved after splenetomy and review evaluation and management issues in such cases, including the emerging role of PET/CT.

Clinical Case

A 65 year old Caucasian male presented with headache, weakness, gait instability, cognitive impairment, and an unintentional 20 lb weight loss over the previous two months.  There was no prior history of hypercalcemia.  Initial evaluation revealed serum a calcium of 14.1 mg/dL (8.6-10.2), albumin 4.0 g/dL (3.5-5.5), creatinine 1.5 mg/dL (previous baseline 1.2), parathyroid hormone 8.48 pg/mL (15-65), 25-hydroxyvitamin D  45 ng/mL, and 1,25-dihydroxyvitamin D 117 pg/mL (18-72).  Three months prior to presentation he was initiated on ergocalciferol 50,000 IU weekly for twelve weeks for vitamin D deficiency with a 25-hydroxyvitamin D level of 13 ng/mL.

PTHrP, serum/urine protein electrophoresis, HIV, TSH, cortisol, and angiotensin converting enzyme were normal.  CT scan of the chest, abdomen, and pelvis showed only mild splenomegaly, 1 cm mediastinal lymph nodes, and a renal cyst.  Bone scan was negative.  Bronchoscopic lung biopsy and bone marrow biopsy were negative.

Patient was treated with intravenous fluids and bisphosphonates with only transient reduction in serum calcium.  We initiated prednisone 30 mg daily which resulted in rapid correction of serum calcium and 1,25-dihydroxyvitamin D levels.  A PET/CT was performed which revealed intense FDG uptake in the spleen concerning for splenic lymphoma.  Laparoscopic splenectomy was performed yielding a 14 cm spleen without gross nodularity but extensive non-casseating granulomas seen on histology.  His calcium remained normal after surgery without requiring corticosteroids.

Conclusion:

Splenic sarcoidosis causing hypercalcemia has been rarely reported.  Correction of vitamin D deficiency precipitated an underlying hypercalcemic disorder.  This case is unique in that the spleen lacked typical nodularity on cross sectional imaging seen in previously reported cases.  Our report adds to an emerging literature documenting the potential value of FDG/PET CT in localizing otherwise occult 1,25-dihydroxvitamin D medicated hypercalcemia.

 

Nothing to Disclose: BAD, RJH

5435 3.0000 SAT-201 A Splenic Sarcoidosis without Focal Nodularity: A Case of 1,25-Dihydroxyvitamin D-Mediated Hypercalcemia localized with FDG PET/CT 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 15th 3:45:00 PM SAT 199-223 2216 1:45:00 PM Disorders of Bone & Calcium Homeostasis: Case Reports Poster


Andy Cheng*1, Reza Bashtar1, Anthony Francis Firek2 and Kevin Anthony Codorniz1
1Loma Linda University Medical Center, Loma Linda, CA, 2Jerry L Pettis Veterans Administration, Loma Linda, CA

 

Background: Calcium abnormalities are common complications from rhabdomyolysis. Hypocalcemia is typically seen during the acute phase of rhabdomyolysis, thought to be caused by i) precipitation of calcium phosphate from hyperphosphatemia due to acute kidney injury and ii) dystrophic calcium deposition into damaged muscles. Hypercalcemia is less common and is seen during the recovery phase of rhabdomyolysis. Several etiologies for delayed hypercalcemia have been proposed, including mobilization of calcium deposits out of the recovering muscles, secondary hyperparathyroidism, increase in calcitriol, and resolution of hyperphosphatemia.

Purpose: We present a case that highlights mobilization of calcium deposits out of the recovering muscles as the principal mechanism for severe delayed hypercalcemia in a patient with rhabdomyolysis.

Hospital course: 23 year old male admitted after found down from suspected hit-and-run motor vehicle accident, with subsequent rhabdomyolysis, compartment syndrome (ultimately requiring amputation), intubation, cardiac arrest, and acute kidney injury requiring hemodialysis (HD) for 3 weeks. Renal function improved and the patient was taken off HD, but he then developed progressive hypercalcemia. Pt was started on calcitonin and then pamidronate with increasingly aggressive fluid resuscitation. Despite these measures, over the next several days calcium persistently increased to peak 17.1 mg/dL corrected (8.9-10.3 mg/dL). Pt experienced fatigue, nausea and vomiting but showed no other signs/symptoms of hypercalcemia, and EKG was unchanged. Labs measured at hypercalcemic peak included PTH < 2.5 pg/mL (10-65 pg/mL), Vit 1,25(OH)2D <8 (18-72 pg/mL) and phosphate 6.3 mg/dL (2.4-4.7 mg/dL). Technetium pyrophosphate scan discovered extensive calcium deposition in the left thigh muscles and left amputation stump. Calcium level normalized briefly with HD but then continued rising to 13.4 mg/dL, requiring a second day of HD. Hypercalcemia resolved several days later. The total episode of hypercalcemia was 16 days in duration.

Discussion: This case demonstrates that mobilization of calcium deposits out of recovering muscles is a primary mechanism for hypercalcemia during recovery phase of rhabdomyolysis-induced acute kidney injury. This mobilization phase may continue for over 2 weeks. Calcitonin and pamidronate are ineffective therapies for this syndrome since their mechanisms of action are unrelated to calcium release from recovering muscles.

 

Nothing to Disclose: AC, RB, AFF, KAC

5848 4.0000 SAT-202 A Severe Hypercalcemia Presenting During Recovery Phase of Rhabdomyolysis-Induced Acute Kidney Injury 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 15th 3:45:00 PM SAT 199-223 2216 1:45:00 PM Disorders of Bone & Calcium Homeostasis: Case Reports Poster


Mohamad Hosam Horani*1, Omar Salim2, Khalid Salim2, Joseph Abdo3 and Gautam Chore4
1Alsham Endocrinology, Chandler, AZ, 2Midwestern University, AZ, 3Mercy Gilbert Hospital, Gilbert, AZ, 4Desert Kidney, Phoenix, AZ

 

Introduction: Hypercalcemic crisisis a serious and potentially life-threatening complication of markedly increased serum calcium concentrations most commonly due to severe primary sporadic hyperparathyroidism (HPT).

Case Study: A 50 year old female with PMHx of HTN and GERD presented to ED complaining of lethargy, HA, and balance problems. She was found to be severely hypercalcemic at 26.1 mg/dL (nl: 8.4-10.2) and in acute renal failure with a creatinine of 1.8 mg/dL (nl: .6-1.2) and albumin of 3.5 g/dL (nl: 3.5-5.5). She was aggressively hydrated and given furosemide, calcitonin, and pamidronate. Her calcium did not improve, so she underwent hemodialysis. PTH level was 2,090 pg/mL (nl: 230-630). The patient received a CT / thyroid US revealing left sided parathyroid and thyroid nodules. Surgery consult performed a neck exploration with left inferior parathyroidectomy and left thyroid lobectomy. Pathology reveals a parathyroid adenoma and adenomatous and hyperplastic nodularity of thyroid with no evidence of malignancy.

Postoperatively, the patient recovers, but noted with worsening nonoliguric acute renal failure with creatinine of 4.7 mg/dL. Calcium had improved drastically, and actually found low at 7 mg/dL. Patient was started on calcium supplements. PTH levels improved and normalized. Repeat TSH / Free T4 were unremarkable. Patient was later discharged after a 13 day hospital course.

Discussion: The prevalence of PHPT is approximately 1:1000, most commonly manifested as an asymptomatic disease. Acute PHPT is rare and can precipitate a parathyroid crisis, characterized by life-threatening hypercalcemia that can cause muscle weakness, headache, neurological symptoms, and renal failure as described in the patient.It should be suspected in acutely ill patients complaining of muscular weakness, gastrointestinal and cerebral symptoms.

Elective parathyroidectomy is the most appropriate treatment for PHPT.  Despite emergency parathyroidectomies to treat hypercalcaemic crisis, mortality rates remain high

Conclusion: The hypercalcemic crisis of hyperparathyroidism due to a parathyroid adenoma is a rare endocrine emergency that is fatal without surgical intervention. Despite emergency parathyroidectomies, mortality rates remain high. Even postoperatively, the danger of a rapid decline of serum calcium contributes to complications. Making a definite diagnosis and performing an early parathyroidectomy within 48 hours are then required, especially in patients exhibiting poor medical response.

 

Nothing to Disclose: MHH, OS, KS, JA, GC

7228 5.0000 SAT-203 A "A Midlife Crisis" Parathyroid Crisis Secondary to Parathyroid Adenoma 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 15th 3:45:00 PM SAT 199-223 2216 1:45:00 PM Disorders of Bone & Calcium Homeostasis: Case Reports Poster


Moe Thuzar*, Vasant V Shenoy and Kunwarjit Singh Sangla
Townsville Hospital, Australia

 

Metabolic Consequences of proton pump inhibitor therapy – A case report

Thuzar M., 1 Shenoy V.V.,2 Sangla KS.3

1. Senior Registrar, 2. Staff Endocrinologist & Senior Lecturer, 3. Director & Senior Lecturer, Dept. of Endocrinology, The Townsville Hospital & James Cook University, Townsville, QLD 4814, Australia.

Background:Proton pump inhibitors (PPI) are among the most commonly prescribed medications and available over the counter in many countries.  There has been  case reports of hypomagnesemia related to PPI use (1). Hypomagnesemia is well known to cause hypocalcemia but only a few cases have reported the possible underlying mechanism in the setting of PPI use.

Clinical Case: A 64-year-old woman presented with severe generalised lethargy, muscle cramps and paraesthesia. She had reduced oral intake for two weeks prior to presentation as she was recovering from a lower respiratory tract infection. She had been on omeprazole 20mg for over 5 years for symptoms of gastroesophageal reflux.

Clinical examination revealed normal body mass index, euvolemic status and muscular twitches with positive Trousseau’s sign on blood pressure measurement. ECG revealed a prolonged QTc (QTc=560 msec) and blood tests showed severe hypocalcaemia with serum total calcium corrected for albumin of 1.4 (N = 2.15 - 2.55) mmol/L and ionised calcium of 0.8 (N =1.15-1.35) mmol/L. She also had severe hypomagnesaemia with serum magnesium of 0.24 (N = 0.7 - 1.1) mmol/L and hypokalaemia with serum potassium of 3 (N =3.5-5.1) mmol/L. Serum phosphate level was normal. There was no diarrhoea, diuretic use, alcohol excess or other obvious aetiology for the hypomagnesaemia and hypocalcaemia.

24 hour urine calcium and magnesium were both low at <0.31 (N = 1.2 - 10) mmol/24hr, and 0.30 (N = 0.6 - 12.5) mmol/24hr respectively. Serum intact PTH level was inappropriately normal at 6 (N = 1 - 7) pmol/L. 25-hydroxy vitamin D was adequate at 91 (N = 50 - 150) nmol/L.

She was initially given 1g (4 mmol) of magnesium intravenously over 30 min followed by intravenous calcium replacement 2g (180 mmol). This was followed by infusions of 5 g (20 mmol) magnesium and 10 g of calcium over the next 24 hours and oral replacement thereafter with oral calcium and magnesium. Serum potassium was also replaced orally. The serum magnesium normalised on day 2, but dropped again to 0.56 (N = 0.7-1.1) mmol/L, on day 4 despite ongoing oral supplements, necessitating further intravenous replacement. Omeprazole was ceased on day 4 of admission with subsequent maintenance of normal serum magnesium, calcium and potassium levels. 

Clinical lesson:Severe hypocalcemia in patients with PPI-induced hypomagnesemia is associated with inadequate PTH secretion. PPI needs to be considered as a possible cause in this kind of presentation and discontinuation is important to prevent recurrence. All clinicians should be aware of this potential serious adverse effect and rationalise use of PPI.

 

Nothing to Disclose: MT, VVS, KSS

8739 6.0000 SAT-204 A METABOLIC CONSEQUENCES OF PROTON PUMP INHIBITOR THERAPY - A CASE REPORT 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 15th 3:45:00 PM SAT 199-223 2216 1:45:00 PM Disorders of Bone & Calcium Homeostasis: Case Reports Poster


Supamit Ukarapong*1, Shankar Srinivas Ganapathy1, Jaime Haidet1, Bradley James Van Sickle1 and Gary David Berkovitz2
1Akron Children's Hospital, Akron, OH, 2University of Miami, Miller School of Medicine, Miami, FL

 

Background:  Hypophosphatasia is a condition in which mutation of the tissue nonspecific alkaline phosphatase (ALPL) gene results in subnormal alkaline phosphatase activity and abnormal skeletal development.  Infantile hypophosphatasia is associated with severe skeletal abnormalities.  By contrast childhood hypophosphatasia is a milder condition that may present with premature loss of deciduous teeth, but which is also associated with skeletal deformities and fractures.  In addition to abnormalities of dentition, radiographic signs in childhood hypophosphatasia include osteopenia and rachitic changes with lucent or streak like defects extending to the metaphyses creating "tongues of hypolucency" appearance.  The severe forms of hypophosphatasia usually result from homozygous or compound heterozygous mutations of ALPL.  Childhood forms of hypophosphatasia are typically the result of heterozygous mutations.  We describe a case of childhood hypophosphatasia caused by homozygous mutation in ALPL gene.    

Clinical case:  The subject presented at 13 years of age for evaluation of left knee pain that persisted over several months.  He had a history of loss of four lower incisors in childhood but was not evaluated for skeletal abnormalities.  Parents denied consanguinity.  The patient’s height and linear growth rate were normal.    The left knee had full range of motion and there were no joint deformities.  Radiograph of left knee indicated two oval radiolucent lesions in the femoral metaphysis.  A similar oval radiolucent area was also present in the humerus.  An MRI of lower extremities indicated mild soft tissue swelling of left knee joint and an increased signal in the distal metaphysis and distal diaphysis both femurs.  The ESR was abnormally elevated and serum alkaline phosphatase activity (17 U/L; normal 42-362) was subnormal.  Both thyroid function and serum zinc level were normal.   Serum pyridoxal 5’-phosphate was abnormally elevated (258 mcg/L, normal range 5-50).   Sequence analysis of ALPLgene indicated a homozygous missense mutation, c.1077 C>G (p. Ile359Met).

 Conclusion:  This case adds to information about the broad spectrum of symptoms, radiographic findings, and gene mutation in childhood hypophosphatasia.  Pain, joint swelling and elevated ESR were mostly likely due to inflammation caused by accumulation of inorganic pyrophosphate.  Oval radiolucent skeletal lesions are an unusual radiographic finding in childhood hypophosphatasia, and indeed were initially considered suspicious for malignancy until biochemical analysis indicated the diagnosis.  Another case of homozygous mutations of ALPL was described recently in a child with a mild form of hypophosphatasia (1), but to the best of our knowledge this finding is extremely rare.  The case we report also indicates that the effect of homozygous mutation in ALPL may be modified by other factors and result in a mild form of hypophosphatasia.

 

Nothing to Disclose: SU, SSG, JH, BJV, GDB

5027 7.0000 SAT-205 A Childhood Hypophosphatasia with A Homozygous Mutation of ALPL Gene 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 15th 3:45:00 PM SAT 199-223 2216 1:45:00 PM Disorders of Bone & Calcium Homeostasis: Case Reports Poster


Katarzyna G Zarnecki*1, John Charlson2, Barbara Wilson2 and Joseph L Shaker3
1Medical College of Wisconsin, Brookfield, WI, 2Medical College of Wisconsin, 3Medical College of Wisconsin, Milwaukee, WI

 

Osteosclerosis (OS) refers to the thickening of trabecular bone.  OS may be associated with hepatitis C, fluoride toxicity, hypervitaminosis A or D, plasma-cell dyscrasias, hyperparathyroidism, Paget's disease, osteopetrosis, skeletal dysplasias, mastocytosis, and sclerotic metastases.

We report a 46-yo woman with a history of invasive lobular breast carcinoma (T1cN0M0) treated with bilateral mastectomy and tamoxifen.  Four years later, she developed lower extremity pain and weight loss.  A PET/CT, skeletal survey, and DXA were consistent with diffuse OS.  A bone scan revealed diffuse increased uptake in the humeral heads, femoral heads, long bones, and spine.  Radiographs 3 years earlier did not show OS confirming that OS was recently acquired.

 Serum biochemistries revealed elevated total (~ 3X upper normal) and bone specific (~7X upper normal) alkaline phosphatase.  The serum PTH was initially mildly elevated but a subsequent level was normal.  Serum calcium, phosphorus and creatinine, as well as serum/urine protein electrophoresis, hepatitis C serology, plasma fluoride, biochemical tests for mastocytosis, serum vitamin A, 25(OH)D and PTHrP were normal.  A bone marrow biopsy revealed OS and absent hematopoietic bone marrow.   Malignant cells were not present and there was no increase in mast cells.  The pancytokeratin stain was negative. 

During her evaluation, new, multiple, up to 1 cm firm dermal nodules were noted.  Biopsy of a skin lesion revealed metastatic poorly differentiated adenocarcinoma most consistent with lobular breast cancer. CSF analysis revealed leptomeningeal carcinoma.

Although it is possible that the OS was directly due to osteoblastic (OB) metastases, the diffuse nature of the OS, as well as the absence of cancer cells and pancytokeratin staining in bone marrow biopsy raises the possibility of a paraneoplastic phenomenon.  We speculate that metastases in the bone marrow or elsewhere were producing a cytokine that stimulated OB bone formation.  In a prior report of recurrent breast cancer presenting with diffuse OS, the marrow was positive for pancytokeratin consistent with the presence of cancer cells (1).

Breast cancer may present with diffuse OS. This may be due to the presence of metastases, cytokine production, or both.

1. Eck et al, Endocrine Practice 2008.

 

Nothing to Disclose: KGZ, JC, BW, JLS

6726 8.0000 SAT-206 A Diffuse Osteosclerosis as a Presentation of Recurrent Breast Cancer 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 15th 3:45:00 PM SAT 199-223 2216 1:45:00 PM Disorders of Bone & Calcium Homeostasis: Case Reports Poster


Jordanna E Kapeluto*, David Edward Harris, Breay W Paty and David L Kendler
University of British Columbia, Vancouver, BC, Canada

 

Patients with Primary Biliary Cirrhosis (PBC) may have, in addition to vitamin D malnutrition, defects in conversion of cholecalciferol to 25OHD resulting in severe metabolic bone disease and osteomalacia (OM). We report a case of PBC presenting with severe osteoporosis (OP), responding to cholecalciferol therapy.

A 69 year-old Vancouver female was hospitalized with a 16-month history of fatigue, diffuse pain and weakness. She had non-steatorrheic diarrhea for 1 year, took no calcium or vitamin D supplements and sparse sun exposure.

Serum calcium and phosphate were normal; creatinine was elevated at 112umol/L; ALP and GGT were elevated at 648U/L and 258U/L. 25OHD was low (<20nmol/L) and PTH was elevated (147pmol/L).

Investigations during hospitalization included normal 1,25 dihydroxy vitamin D (150pmol/L), elevated anti-mitochondrial antibodies (1:320), negative TTG antibodies, and normal bilirubin (6umol/L). Diagnosis of PBC was made and ursodiol initiated, but not tolerated due to diarrhea. Cholecalciferol 120000IU was given followed by 4000IU and 1000mg elemental calcium daily. 

Bone mineral density (BMD) was measured 3 and 6 months after discharge, with symptoms improved. Spine (L1-L3), total hip and femoral neck bone densities were 0.703g/cm2 (T-score -2.9), 0.566g/cm2 (T-score -3.1) and 0.495g/cm2 (T-score -3.2) respectively. 25OHD was 96nmol/L (sufficient). 

Six months post discharge, PTH declined to 25.7pmol/L. Spine, total hip and femoral neck BMD increased to 20.4%, 17.8% and 28.1% respectively.

This case reports an association of PBC with extremely low 25OHD levels and secondary hyperparathyroidism, in the absence of malabsorption and hepatic hydroxylation defects. We provide a time course for the separate resolution of the low 25OHD, elevated PTH and BMD. Although we do not have bone biopsy confirmation, the marked increases in BMD due to remineralization of osteoid suggest diagnosis of OM. Multifactorial cause of hypovitaminosis is likely, relating to lack of sun exposure and Vit D supplementation. Monitoring DXA is helpful as an index of bone mineralization. Bisphosphonate or other antiresorptive therapies would be contraindicated prior to correction of Vit D deficiency; antiresorptive therapy is likely unnecessary prior to optimization of BMD response to calcium and Vit D.

The association of PBC with OP and OM is well reported in the context of Vit D deficiency. This would suggest the utility of 25OHD and BMD testing in this population.

 

Disclosure: DLK: Advisory Group Member, Pfizer, Inc., Principal Investigator, Pfizer, Inc., Speaker, Eli Lilly & Company, Investigator, Eli Lilly & Company, Consultant, Merck & Co., Investigator, Novartis Pharmaceuticals, Consultant, Novartis Pharmaceuticals, Speaker, Novartis Pharmaceuticals, Speaker, Amgen, Investigator, Amgen, Consultant, Amgen, Speaker, Warner Chilcott, Speaker, GlaxoSmithKline, Investigator, Johnson &Johnson. Nothing to Disclose: JEK, DEH, BWP

7887 9.0000 SAT-207 A Osteomalacia Associated with Primary Biliary Cirrhosis and Response to Cholecalciferol Therapy: A Case Report 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 15th 3:45:00 PM SAT 199-223 2216 1:45:00 PM Disorders of Bone & Calcium Homeostasis: Case Reports Poster


Aristotle Panayiotopoulos*1, Divya Khurana2, Amrit Pal S Bhangoo3 and Svetlana B Ten4
1SUNY Downstate Med Ctr, Brooklyn, NY, 2MMC, Brooklyn, NY, 3Miller Children's Hospital, Long Beach, CA, 4Maimonides Med Ctr, Brooklyn, NY

 

Background:

Hypophosphotemic rickets (HR) is a caused by a number of genetic mutations resulting in decrease reabsorption of phosphorus by the proximal renal tubule along with inhibition of 1α-hydroxlase leading to low levels of 1,25 (OH)2-vitamin D. Current standard treatment includes phosphate replacement and 1,25 (OH)2-vitamin D.  Complications to this therapy include renal insufficiency, and secondary hyperparathyroidism. Calcimimetics have been introduced in management as it modulates the calcium sensing receptor (CaR) resulting in decrease of parathyroid hormone (PTH) secretion and lowering phosphorus product.  

Design:

Two cases of patients with HR had been managed with phosphorus replacement and calcitriol, but serum phosphorus levels were hard to normalize even on high doses of phosphorus replacement.  Cinacalcet at 30 mg twice daily was started, and serum measurements of calcium, phosphorus, and PTH was compared before starting and after 1week treatment.     

Results:

Case 1 is an 11yo boy who presented at our clinic at 7yrs of age with history of leg bowing and sleep apnea due to facial bone malformation, dental caries, and bone pain. Patient was shown to have X-linked hypophosphatemic rickets with confirmed missense mutation of PHEX gene.  On calcitriol 1.5mcg daily and phosphorus 30mg/kg/day divided in 4 doses phosphorus was never above 2.5mg/dL, while PTH was 70pg/dL and Calcium of 9.6mg/dL.  One week after starting cinacalcet, phosphorus improved to 3.1 along with a decrease in PTH to 12 and calcium of 9.

Case 2 is of a 6yo girl who presented at 2yrs of age with leg bowing, frontal bossing, and bone pain.  Genetic analysis for cause of HR pending.  Patient was on calcitriol 1mcg daily along with phosphorus 18mg/kg/day divided in 4 doses.  Cinacalcet was started and one week later phosphorus improved from 2.6 to 3.5, PTH went from 20 to 12, while calcium changed from 9.3 to 9.1.

Conclusion:

Addition of cinacelcet to Phosphorus and Calcitriol had an immediate effect on decreasing phosphorus replacement requirements along with reduction in PTH levels.  Thus calcimimetics such as cinacalcet can play a quick and significant role in better managing disease process of HR and its sequelae.

 

Nothing to Disclose: AP, DK, APSB, SBT

7890 10.0000 SAT-208 A Cinacalcet in Management of Hypophosphotemic Rickets 1 week after Treatment 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 15th 3:45:00 PM SAT 199-223 2216 1:45:00 PM Disorders of Bone & Calcium Homeostasis: Case Reports Poster


Alina Elperin*1, Richard A Prinz2, Janardan Khandekar3, Igor Jovanovic3 and Romy Jill Block4
1Northshore University Health System, 2NorthShore University HealthSystem, Evanston, IL, 3NorthShore University Health System, 4Northshore University Healthsystem, Highland Park, IL

 

Background: Recurrent hyperparathyroidism after parathyroidectomy is defined as recurrent hypercalcemia > 6 months after initial surgery. Parathyromatosis is a difficult to diagnose and treat cause of recurrent hyperparathyroidism. It is defined as multiple rests of hypercellular, hyperfunctioning parathyroid tissue. While parathyromatosis is well documented in patients with renal disease, little is known about parathyromatosis in primary hyperparathyroidism. We present a case series of three patients with persistent hypercalcemia due to parathyromatosis.

Case 1: A 35yo female had persistent hypercalcemia after 4 parathyroidectomies. Her labs were: Ca-11.3mg/dL (normal 8.5-10.3 mg/dL), PTH-251pg/mL (normal 12-88 pg/mL). Sestamibi showed abnormal uptake and CT scan of the chest showed multiple nodules in the mediastinum. She underwent 3 more surgeries to remove this abnormal tissue. Pathology each time confirmed parathyromatosis. Despite 7 surgeries, the patient is hypercalcemic (12-13 mg/dL) and requires Cinacalcet and periodic IV Zoledronic Acid. She is currently undergoing familial genetic testing.

Case 2: A 41yo female had persistent hypercalcemia. Her labs were Ca-11.3 mg/dL, PTH-61.2pg/mL. Sestamibi showed single gland uptake. She underwent a parathyroidectomy and pathology showed multi-gland hyperplasia.  Postoperatively, she remained hypercalcemic.  Repeat Sestamibi was normal but a neck CT showed a lesion in the mediastinum. She underwent her second parathyroidectomy with pathology consistent with parathyromatosis.  She remained hypercalcemic: Ca-10.5 mg/dL, PTH-100pg/mL and she began medical management with Cinacalcet.

Case 3: A 47yo female had recurrent hypercalcemia despite 4 parathyroidectomies. At presentation, her labs were:  Ca-16.5mg/dL, PTH-317pg/mL. Sestamibi showed abnormal uptake in the thyroid and neck CT revealed 2 nodules in the mediastinum. She had her 5th neck exploration. Pathology was consistent with parathyromatosis. She remained normocalcemic for a year then her hypercalcemia recurred:  Ca-13.3 mg/dL, PTH-65pg/ml. She was given IV Zoledronic Acid and started on Cinacalcet.

Conclusion: We present a case series of 3 patients with recurrent or persistent primary hyperparathyroidism from parathyromatosis. These patients have debilitating symptoms despite multiple operations.  Medical therapy to control symptoms of hypercalcemia in parathyromatosis is limited and minimally effective.

 

Nothing to Disclose: AE, RAP, JK, IJ, RJB

5724 11.0000 SAT-209 A Parathyromatosis: a rare cause of recurrent, debilitating hypercalcemia with limited treatment options 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 15th 3:45:00 PM SAT 199-223 2216 1:45:00 PM Disorders of Bone & Calcium Homeostasis: Case Reports Poster


Divya Seval*1 and Sanjay Navin Mediwala2
1Baylor College of Medicine, 2Michael E. DeBakey V A Medical Center, Houston, TX

 

Congenital Hypoparathyroidism Presenting In Adulthood

Divya Seval, MD and Sanjay Mediwala, MD

Department of Medicine, Division of Diabetes, Endocrinology and Metabolism, Baylor College of Medicine, Michael E DeBakey VA Medical Center, Houston, TX 77030

Introduction: Microdeletion of chromosome 22q11 encompasses a spectrum of highly variable clinical phenotypes including Digeorge Syndrome, velocardiofacial syndrome, and conotruncal anomaly face syndrome. While majority of patients are diagnosed in childhood, there remains a small cohort of patients with milder phenotypes who present with late onset symptoms in their adult years. We present a case of hypocalcemia in an adult subsequently diagnosed as DiGeorge Syndrome.

Objectives: (1) Present a case of 22q11 deletion syndrome presenting in adulthood, (2) discuss recommended evaluation and management, and (3) review of the literature of 22q11 deletions presenting in adulthood.

Clinical Case: A 39 year old woman presented to the emergency room after experiencing a witnessed generalized seizure. She had two previous seizures over the prior two years, but had not sought medical care. Her medical history was significant for multiple hospital admissions for schizoaffective disorder. She had no family history of genetic disorders. On physical exam, she was short in stature with small hooded eyelids, and exhibited a positive Chovstek’s signs with the remainder of the exam unremarkable. Lab values: corrected calcium 6.0 mg/dl (n 8.7-10.5), phosphorus 5.2 mg/dl (n 2.5-4.6), and intact PTH 11 pg/ml (n 12-88). She was treated with calcium, vitamin D, and calcitriol to correct the hypocalcemia. Given the constellation of hypoparathyroidism, dysmorphic facial features and psychiatric illness a 22q11 deletion syndrome was suspected. Fluorescent in situ hybridization (FISH) confirmed the diagnosis. An echocardiogram showed no evidence of congenital heart disease and lymphocyte counts were within normal range. Following initiation of treatment, she remained normocalcemic without seizure activity.  

Clinical Lessons: 22q11 deletion presents with highly variable clinical features which can lead to delayed diagnosis in the adult population. Adults typically present with palate anomalies, learning difficulties, psychiatric illness, and hypocalcemia.

Conclusion: Given the highly variable presentation of 22q11 deletion, increased suspicion is warranted in adults presenting with PTH-deficient hypocalcemia, learning difficulties and dysmorphic facial features. Early recognition of 22q11 deletion in the adult population will facilitate planning for long term management including genetic counseling for these patients and their families.

 

Nothing to Disclose: DS, SNM

6652 12.0000 SAT-210 A Congenital Hypoparathyroidism Presenting In Adulthood 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 15th 3:45:00 PM SAT 199-223 2216 1:45:00 PM Disorders of Bone & Calcium Homeostasis: Case Reports Poster


Nagashree Gundu Rao* and Richard J Santen
University of Virginia, Charlottesville, VA

 

Leukemic  hypocalcemia driven by  sclerotic bone lesions

Nagashree Gundu Rao, Richard J. Santen. University of Virginia Medical Center, Charlottesville, VA.

Background:Metastatic sclerotic bone lesions leading to hypocalcemia and hypophosphatemia, are very unusual findings in AML.

Case:A 70 caucasian woman with known history of osteopenia, and recently diagnosed AML, was admitted for induction chemotherapy with Idarubicin and Cytarabine. Her clinical course was complicated by neutropenia, typhilitis, and gangrene of her toes bilaterally (secondary to AML-related disseminated intravascular coagulation). She complained of diffuse bone pain, involving her lower extremities and back. She reported prior use of calcium and vitamin D supplements, but denied history of hypocalcemia or fractures. She had been treated with bisphosphonates for osteopenia, for a total duration of 8 years, but was on drug holiday since the previous year.  Physical examination was remarkable for gangrene involving 3 toes bilaterally. Her corrected serum calcium (for albumin of 1.5 mg/dl) was 7.5 (8.5-10.5 mg/dl), ionized calcium 3.7 (4.5-5.5 mg/dl), phosphorus 1.6 (2.3-4.7 mg/dl) and magnesium 1.8 (1.6-2.6 mg/dl). Additional laboratory testing revealed 24h urine calcium of 189 mg/dl,  normal 25,OH vitamin D level (33; 25-80 ng/ml), with appropriately elevated PTH (390.8; 10-70 pg/ml)  and 1,25 dihydroxy vitamin D (117; 18-78 pg/ml) levels. Her alkaline phosphatase was significantly elevated at 2013 U/L and exclusively bone specific (i.e. 97%). Due to accompanying typhilitis and contraindications to oral medications, calcium and phosphorus were replaced intravenously. CT of the abdomen and pelvis demonstrated multiple sclerotic bone lesions throughout the axial and appendicular skeleton, suspicious for sclerotic bone lesions secondary to AML. A bone scan revealed enhanced radiotracer uptake consistent with increased bone formation in the sternum, right 1st rib, lower thoracic and lumbar spine, sacrum, iliac bones, femoral necks, and shaft of right femur. She required calcium supplementation for 3 weeks post induction chemotherapy, after which her hypocalcemia resolved, without any further treatment.

Discussion: This case describes a rare case of hypocalcemia and hypophosphatemia, likely due to increased calcium and phosphorus deposition in bone as a result of accelerated osteogenesis, stimulated by the leukemic cells. The pathophysiology is similar to the hypocalcemia observed in prostate cancer patients with extensive blastic metastases. Resolution of hypocalcemia and hypophosphatemia often occurs in association with response to chemotherapy. Bone marrow biopsy can reveal fibrosis and thickened bony trabeculae, with evidence of extensive calcification noted on tetracycline labeling.

 

Disclosure: RJS: Advisory Group Member, Pfizer, Inc.. Nothing to Disclose: NG

8701 13.0000 SAT-211 A Leukemic Hypocalcemia Driven by Sclerotic Bone Lesions 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 15th 3:45:00 PM SAT 199-223 2216 1:45:00 PM Disorders of Bone & Calcium Homeostasis: Case Reports Poster


Gil L P Afonso*, Ricardo A Guerra, Kelly M Ferro, Mariana R N Couto, Daniel D R Paulo, Livia M Santos and Evandro S Portes
Hospital do Servidor Publico Estadual de Sao Paulo

 

Introduction: Hypomagnesemia induced by proton-pump inhibitor (PPI) is a rare condition, first described by Epsien in 20061.Although calcium (Ca) is considered to be the major regulator of PTH secretion, a number of studies have demonstrated that magnesium (Mg) can modulate PTH secretion in a manner similar to Ca. Especially, it has been suggested that intracellular Mg depletion impairs the ability of the parathyroid to secrete PTH resulting in a fall in the serum PTH levels, and subsequently a fall in the serum Ca concentration.The mechanism by which PPI drugs reduce the intestinal absorption of Mg is not understood.

Case Report: A 66-year-old woman presented with paresthesias for 6 months. She was on PPI therapy since 2000 (omeprazole 40mg daily) for peptic ulcer disease. On admission the laboratory findings showed hypocalcemia (7.3 mg/dL, normal range 8,5 – 10 mg/dL), hypomagnesemia (0,6 mg/dL, normal range 1,5 – 2,0 mg/dL)  and a low parathyroid hormone (11.4 pg/mL, normal range 12 – 65 pg/mL). Serum vitamin D3 level was normal. A 24-hour urinary magnesium excretion was low, suggesting a total body Mg deficiency due to extra renal Mg wasting. We started intravenous calcium and magnesium supplementation. In seven days the calcium, magnesium and PTH level normalized and her symptoms slowly resolved. We interpreted these results as severe hypomagnesemia with secondary hypoparathyroidism. After 12 days she was discharged with oral magnesium supplements. One month later the magnesium was discontinued. Within 2 weeks a dramatic drop in the serum magnesium and calcium followed. After, we discontinued the magnesium supplements and the omeprazole. The serum magnesium and calcium level did not change in four weeks. Because of increasing dyspeptics symptoms we let her resume the omeprazole. Within four weeks the serum Mg level dropped to 1.4 mg/dL. The pattern we observed in our patient fits nicely with the theory of proton pump inhibitor induced intestinal magnesium loss that was suggested in others publications.

Conclusion: This patient, in addition to others reported previously, suggest that PPI therapy can cause severe, symptomatic hypomagnesemia; and withdrawal of PPI therapy results in resolution of this problem.The prevalence of PPI-induced hypomagnesemia is not known. The need for long-term PPI therapy in patients should be kept under regular review, however if PPI therapy is required on a long-term basis, then we suggest that the serum magnesium should be checked annually, or if the patient feels unwell.

 

Nothing to Disclose: GLPA, RAG, KMF, MRNC, DDRP, LMS, ESP

8202 14.0000 SAT-212 A Case Report: Hypomagnesemic hypoparathyroidism associated with the use of proton-pump inhibitors 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 15th 3:45:00 PM SAT 199-223 2216 1:45:00 PM Disorders of Bone & Calcium Homeostasis: Case Reports Poster


Manikya Kuriti*1, Vinay gopal Nidadavolu2 and Mamta Shah3
1St.Elizabeth Medical Center, Boston, MA, 2University Of Connecticut, hartford, CT, 3University of connecticut, HARTFORD, CT

 

BACKGROUND - Zometa(Zoledronic Acid) is one of the most common medication used for malignant hypercalcemia. While its considered a relatively safe drug in these settings, severe electrolyte abnormalities have been reported rarely. We hereby present a case of severe hypocalcemia from the use of Zometa.

CASE - 66 yr old African American female with no past medical history comes to the ED for non-specific fever, malaise, sore throat and loss of weight for a few weeks. She was found have abnormal lymphocytes and an elevated calcium of 11.8 mg/dl (corrected - 12.6) [Normal =9-10.5 mg/dL] on the day of admission. She was eventually diagnosed to have Acute Adult T-cell Leukemia/ Lymphoma from HTLV-1 infection. The persistently elevated calcium levels resulted in the oncologist prescribing Zometa 4 mg/ IV on the third day of admission after the diagnosis. Her calcium decreased to around 8 in the next few days. Patient was started on Hyper CVAD regimen for leukemia and was sent home after she finished the induction chemotherapy.

Patient returned back in 3 weeks for chemotherapy. Repeat calcium done on admission showed a level of 6.6(corrected – 7.2) for which she was started on oral calcium tablets 500mg daily along with 1 gram of intravenous calcium gluconate.

On day 4 of the admission a rapid response was called for new onset confusion, shivering and “not feeling herself”. She had abnormal sensations in her extremities. No motor deficit was noticed but she had diffusely suppressed deep tendon reflexes. A concern for a stroke/encephalitis was expressed in the setting of neutropenia from the chemotherapy and patient was about to be wheeled down for a CT/MRI Head. A BP check incidentally showed a classic trousseau’s sign. Further examination also revealed a classic Chvostek’s sign. EKG showed pathognomonic short QRS intervals, prolonged QTc and bradycardia. A gram of intravenous calcium chloride showed immediate effect and patients confusion improved immediately. The total calcium came back at 5.2 (corrected 6) along with phosphorus of 1.6 [Normal = 2.4 - 4.1(mg/dL)]. The vitamin D level was 9 [Normal = 40-100 nmol/L]. PTH was 50 [Normal = 10 - 55 (pg/mL)]. She received a total of 22 grams of calcium gluconate (or equivalent) in the hospital along with oral calcium and intravenous vitamin D50,000 units for 3 days.

CONCLUSION - While zometa is a very effective and relatively safe medication to use in the setting of malignant hypercalcemia, its essential to check baseline vitamin D levels and repleting it before giving it and also monitor calcium levels regularly after giving the medication to prevent severe metabolic abnormalities that have been rarely reported to occur with its use.

 

Nothing to Disclose: MK, VGN, MS

8838 15.0000 SAT-213 A Severe hypocalcemia after use of Zometa for malignant hypercalcemia 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 15th 3:45:00 PM SAT 199-223 2216 1:45:00 PM Disorders of Bone & Calcium Homeostasis: Case Reports Poster


Naweed Alzaman*1, Anastassios G Pittas2, Miriam O'Leary2 and Lisa Ceglia2
1Tufts Med Ctr, Boston, MA, 2Tufts Medical Center, Boston, MA

 

Background: Transient hypocalcemia post-thyroidectomy is not uncommon and risk increases with extensive neck surgery. We present a case of severe and prolonged hypocalcemia post-total thyroidectomy complicated by thoracic duct injury.

Clinical case: A 58-year-old man presented with multiple thyroid nodules and enlarged cervical lymph nodes. Fine needle aspiration of the thyroid nodules was consistent with medullary thyroid carcinoma (MTC) and calcitonin level was elevated at 470 ng/L (<13.8). He underwent total thyroidectomy with central and left lateral lymph node dissection. Surgical pathology confirmed MTC. Both inferior parathyroids were removed during the surgery, which was also complicated by thoracic duct injury. On postoperative day 0, serum ionized calcium (iCa) level was 3.9 mg/dL (4.2-5.2) and parathyroid hormone (PTH) was undetectable <3pg/ml (11-80). After receiving 2 ampoules of intravenous (iv) calcium gluconate (186 mg of elemental calcium), he was started on oral calcium carbonate (CaCO3) 3 g, calcitriol 0.5 µg, and vitamin D3 1000 IU daily. Although iCa levels initially responded to oral therapy, on postoperative day 5, he developed symptoms of tetany (peri-oral numbness and Chvostek’s sign), confusion, and dysarthria. Serum iCa was 3.3 mg/dL. He was transferred to intensive care and started on a continuous iv calcium infusion. Oral treatment was titrated to CaCO3 4 g, calcitriol 4 µg, and vitamin D250,000 IU daily. Symptoms of tetany resolved as serum iCa improved but remained 3.7-4.2 mg/dL. In search for a cause of refractory hypocalcemia, it was noted that it was concurrent with a moderate output of chyle from the thoracic duct injury measuring 110 cc every 12 hours.  Analysis of the chyle revealed a calcium concentration of 5.4 mg/dL (0.95-1.5). A medium chain fatty acid diet and subcutaneous octreotide were initiated with a subsequent decrease in chyle output and a rise in iCa level to 4.6 mg/dL. As chyle output decreased to <15 cc/day, iCa levels remained stable off of iv calcium.

Clinical lesson: Hypoparathyroidism and a chyle leak are potential complications following total thyroidectomy and left lateral lymph node dissection. This case of refractory hypocalcemia was attributed primarily to the chyle leak, which has an electrolyte composition similar to that of plasma. In patients with extensive neck dissection and chyle leak, calcium levels should be monitored and may need aggressive replacement to prevent hypocalcemia.

 

Nothing to Disclose: NA, AGP, MO, LC

8871 16.0000 SAT-214 A Post-thyroidectomy hypocalcemia exacerbated by chyle leak 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 15th 3:45:00 PM SAT 199-223 2216 1:45:00 PM Disorders of Bone & Calcium Homeostasis: Case Reports Poster


Uzma Zohra Shafqat*1, David Bleich2 and Maya P Raghuwanshi3
1UMDNJ, Parsippany, NJ, 2UMDNJ-NJ Med School, Newark, NJ, 3UMDNJ-NJ Med Sch, Newark, NJ

 

Hypercalcemia has been associated with significant morbidity and mortality; its treatment in patients with renal failure has been challenging.  Here, we report a case of hypercalcemia secondary to silicone implants, resulting in chronic kidney disease and successful therapy with Denosumab.

THE CASE: 47 yr Puerto Rican woman was referred to Endocrinology for hypercalcemia with calcium levels varying from 11.0 mg/dl to 14.5 mg/dl for the last 5 yrs associated with recurrent nephrolithiasis requiring lithotripsy, urethral strictures, and stent placement.  The stents were replaced frequently due to clogging and recurrent urinary tract infections.  Consequently she developed nephrocalcinosis and chronic kidney disease requiring intermittent dialysis.  An extensive workup revealed that hypercalcemia was due to granuloma formation from silicone injections 15 yrs earlier into her thighs and buttocks for cosmetic reasons. She was treated with IV hydration, calcitonin and IV pamidronate on her previous hospital admissions. Oral bisphosphonate therapy led to renal insufficiency so she was treated with prednisone up to 240mg/day in tapering doses that resulted in reduction of serum calcium from 15.2mg/dl to 9.9mg/dl in one month.  However, long term low dose Prednisone (10mg/day) failed to keep her in remission and subsequent high dose steroids caused psychosis. Treatment with subcutaneous Denosumab 60 mg normalized her serum calcium into the range of 9.5mg/dl -10.5 mg/dl for the next four months. She was re-admitted to the hospital with a calcium level of 13.5mg/dl and the second dose of Denosumab was given with the same response.

DISCUSSION: Denosumab is a human monoclonal antibody directed against RANK-L that blocks osteoclastogenesis. It is indicated for post menopausal osteoporosis, prevention of skeletal related events in solid tumors and fracture prevention in prostate cancer patients on GnRH agonists. Rare case reports indicate that is has been used for malignancy associated hypercalcemia. In our patient with refractory hypercalcemia, renal failure, and steroid intolerance Denosumab treatment lowered serum calcium into the normal range.  We believe this is the first evidence of Denosumab use in a patient with granulomatous hypercalcemia.

 

Nothing to Disclose: UZS, DB, MPR

4075 17.0000 SAT-215 A GRANULOMATOUS HYPERCALCEMIA DUE TO SILICONE IMPLANTS: SUCCESSFUL TREATMENT WITH DENOSUMAB 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 15th 3:45:00 PM SAT 199-223 2216 1:45:00 PM Disorders of Bone & Calcium Homeostasis: Case Reports Poster


Victoria Mendoza*1, Maria Isabel Garcia2, Irma Hernandez2, Alex Francisco Hernandez2, Guadalupe Vargas3, Baldomero Gonzalez3 and Moises Mercado Atri4
1HECMN IMSS, Mexico City, Mexico, 2Hospital de Especialidades, CMN S. XXI,IMSS, 3Hospital de Especialidades, Centro Médico Nacional Siglo XXI, IMSS, Mexico, Mexico, 4UNAM CMN IMSS, Mexico DF, Mexico

 

Background and objectives: Thyroid abnormalities are known to occur in the context of primary hyperparathyroidism (PHPT). Such thyroid conditions include benign thyroid nodules and lesss frequently, thyroid carcinoma. The more frequent use of mimimally invasive approaches to treat PHPT, many of these thyroid conditions would be missed. Our purpose was to stablish the prevalence of thyroid abnormalities in the context of PHPT. 

Patients and methods: We analyzed 120 patients with PHPT treated at our center with in the past three years, in whom preoperatively thyroid ultrasonography was available, besides parathyroid Tecnetium-sestamibi scintigraphy. The precise location of the parathyroid lesion, as well as hystopathological details of the thyroid and the parathyroid conditions were evaluated.

Results: Of the 120 analyzed cases, 103 (86%) were women and 17 (14%) were men, with an age range of 18-80. Preoperative USG found thyroid structural abnormalities in 61 of these patients. Benign appearing lesions, smaller than 1 centimeter, were found in 27 cases, whereas in 34, lesions greater than 1 centimeter, with USG features suspicious for malignancy were found. These 34 cases underwent thyroid surgical exploration (hemithyroidectomy or neal total thyroidectomy) and the final hystopathological diagnosis were the following: nodular hyperplasia in 13, follicular adenoma in 4, chronic thyroiditis in 5, normal thyroid in 4 and papillary thyroid carcinoma in 8. 

Conclusions: We found a prevalence of 51% of thyroid abnormalities within the context of PHPT. 6% of these patients harbored papillary carcinoma of the thyroid. Thyroid ultrasound is mandatory in the preoperative evaluation of PHPT.

 

Nothing to Disclose: VM, MIG, IH, AFH, GV, BG, MM

9303 18.0000 SAT-216 A INCIDENTAL THYROID DISEASE FOUND IN THE CONTEXT OF PRIMARY HYPERPARATHYROIDISM 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 15th 3:45:00 PM SAT 199-223 2216 1:45:00 PM Disorders of Bone & Calcium Homeostasis: Case Reports Poster


Udaya Manohar Kabadi*
University of Iowa, Clive, IA

 

Background

 25 OH vitamin (vit) D deficiency is reported to occur frequently in Primary Hyperthyroidism (Hyperpara). However it is plausible that 25 OH Vit D is decreased due to its enhanced conversion into 125 OH vit D by Parathyroid Hormone (PTH).

Objective

To examine significance of low 25 OH vit D in primary hyperpathyroidism

Methods

 Serum concentrations of calcium (Ca),mg/dl; PTH, pg/ml; 25OH vit D, ng/ml and 125 OH vit D, pg/ml were determined after an overnight fast in 20 consecutive Subjects (14 men and 6 women, age 52±6 years) with established diagnosis of Primary Hyperthyroidism, 10 age matched (49 ± 4) healthy volunteers, 7 men and 3 women (N) with normal 25 OH Vit D concentrations (20-80 ng/dl) and 10 subjects ( 50 ± 4 years) with vit D deficiency, 25 OH vit D<20 ng/dl( Low D). Comparisons were conducted between groups by Student’s ‘t’ test. Serum levels were also determined in 3 subjects with Hyperpara following vit D supplementation and 6 subjects after 3-4 months following surgery.

 Correlations were also assessed by linear regression analyses between PTH on one aspect and Calcium, 25 OH vit D, 125 OH vit D and 125 OH vit D/25OH vit D (125/25) ratio in subjects with Hyperpara.  

Results

 Serum Ca, PTH and 125 OH vit D concentrations were significantly higher in Hyperpara (11.2±0.3, 128±20, 79±6) than N (9.5±0.2, 46±5, 52±5; p<0.001 for all comparisons) and Low D (9.1± 0.2, 51± 6, 28±4; p<0.001for all comparisons) whereas 25 OH vit D was significantly lower (18 ±3) than N (47±5; p<0.001) and not Low D (15± 2). In 6 subjects after 3-4 months following surgery, Ca and125 OH vit D levels declined to normal whereas 25OH vit D rose to normal with a normalization of PTH. In 3 subjects with Hyperpara and low 25 OH vit D, weekly Ergocalciferol 50000 units supplementation induced a further rise in 125 OH vit D and Ca without significant lowering of PTH. Significant positive correlations were documented in subjects with Hyperpara between Serum PTH and Ca (r=0.74), 125 OH vit D (r=0.52) and 125/25 ratio (r=0.46) as well as between 125/25 and Ca(r=0.47) whereas a significant negative correlation (r= -0.43) was observed between PTH and 25 OH vit D (p<0.01 for all correlations).

 Conclusion

 Low serum 25 OH vit D in Primary hyperparathyroidism may be secondary to enhanced conversion to 125 OH vit D by circulating high PTH rather than a ’true’ deficiency and may normalize following surgery with normalization of PTH. Vit D supplementation is likely to exacerbate hypercalcemia.

 

Nothing to Disclose: UMK

5806 19.0000 SAT-217 A Low 25 OH Vitamin D in Primary Hyperparathyroidism: Enhanced Conversion into 125 OH Vitamin D, Not ‘True' Deficiency 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 15th 3:45:00 PM SAT 199-223 2216 1:45:00 PM Disorders of Bone & Calcium Homeostasis: Case Reports Poster


Krystal L Cleven*1 and Alyson K. Myers2
1North Shore University Hospital/Long Island Jewish Medical Center, Manhasset, NY, 2Northwell Health, Manhasset, NY

 

Background: In most cases of hypercalcemia of malignancy, there is an elevation in PTH, PTHrP, or calcitriol (Jacobs and Bilezikian, 2005). This case represents an uncommon etiology for hypercalcemia in malignancy.

Clinical Case: A 71 year-old Trinidadian woman with diabetes mellitus, hypothyroidism, HTN, and CAD presented with confusion, lethargy, back pain, and constipation for one week. On exam the patient was mildly confused with weakness and depressed reflexes. The patient’s initial labs showed severe hypercalcemia: total calcium 15.2mg/dL, ionized calcium 1.76mmol/L. Two months prior the patient was normocalcemic with a calcium of 9mg/dL. Further work-up of the hypercalcemia demonstrated an inappropriately normal PTH (25pg/mL) in the setting of Vitamin D deficiency (vitamin 25-OH 19.8ng/mL), with a low-normal calcitriol (14.9pg/mL) and TSH 0.23 µIU/m. The remaining labs were normal: PTHrP (<0.74pmol/L), cortisol 15µg/dL, magnesium 1.6mg/dL, phosphorus 3mg/dL, alkaline phosphatase 133 U/L, albumin 4.5g/dL, and SPEP/UPEP were negative. A bone marrow biopsy also showed no plasma cells. In addition, the patient’s creatinine and hemoglobin were normal at 0.99 mg/dL (GFR 66mL/min/1.73m2) and 12.8g/dL, respectively. A thyroid/parathyroid sonogram and bone scan both showed no abnormalities. A skeletal survey showed multiple focal lucencies notable in the distal right femur, pelvis, left humerus, and skull. These focal lucencies were acute changes not seen on a CT head or chest x-ray completed 2 months prior. The patient's labs did not explain the cause of her lytic lesions, thus a bone biopsy was done of the right iliac crest which showed a reactive non-neoplastic process. In this patient, with severe PTH-independent hypercalcemia, a low-normal calcitriol, and negative PTHrP and SPEP/UPEP, atypical causes of malignant hypercalcemia were considered. The patient's history of residing in the Caribbean for 30 years, an area endemic for Human T-lymphotropic virus Type I (HTLV-1), made Adult T-cell leukemia/lymphoma (ATL) a part of the differential diagnosis. Antibodies for HTLV-1 were positive. To confirm the diagnosis of ATL, flow cytometry was completed on the peripheral blood, which demonstrated evidence of a T-cell lymphoproliferative disorder. The patient’s hypercalcemia was refractory to five doses of bisphosphonate therapy but improved after treatment of the primary malignancy with Zidovudine and Interferon alfa-2b.

Conclusion: This case of hypercalcemia of malignancy highlights unique diagnostic and treatment challenges in PTH-independent hypercalcemia without a concomitant elevation of PTHrP or calcitriol.

 

Nothing to Disclose: KLC, AKM

8514 20.0000 SAT-218 A A rare case of hypercalcemia: a PTH-independent malignancy in a patient with a normal PTHrP and low-normal calcitriol 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 15th 3:45:00 PM SAT 199-223 2216 1:45:00 PM Disorders of Bone & Calcium Homeostasis: Case Reports Poster


Jennifer Tinloy1 and Pamela Taxel*2
1Pennsylvania State University, 2University of Connecticut Health Center

 

Skeletal metastases can change skeletal morphology through dysregulation of the normal bone remodeling process. They  are typically diagnosed through CT, MRI, and PET; although,these imaging modalities are typically not sensitive enough to detect early, asymptomatic bone metastases. We report the unusual case of a significant increase in BMD in a solitary vertebrae in an asyptomatic patient who recently completed 5 years of aromatase inhibitor therapy for hormone recptor-positive breast cancer. 

A 71 yo woman with a 2.1 cm infiltrating ductal ER/PR+ breast  cancer (stage IIb), underwent a right breast lumpectomy, axillary node dissection, radiation, and 4 cycles of chemotherapy.  A post-op PET scan showed no evidence of metastases and she was started on anti-estrogen therapy  with anastrozole (aromatase inhibitor) for 5 years. A baseline bone mineral density (BMD) revealed normal spine and hip BMD with mild-moderate degenerative changes L1-4. Serial BMDs from 2007-2010 showed an overall 10% decline in the spine (Table). She was maintained on clacium and vitamin D for bone health, and declined anti-resorptive medication with bisphosphonate.  Anastrozole was completed in June, 2012 and a repeat BMD revealed a significant increase in BMD at the L-spine of over 15% and L1 increase of 55% with new diffuse L1 sclerotic changes. The patient reported no back pain, fevers/chills. On exam, there was no pain/tenderness over L-spine. A plain film showed an L1 radiodense vertebral body, and a PET scan showed a mixed osteoblastic and osteolytic process involving L1. The differential diagnosis included metastatic bony lesion, compression fracture, acquired bone tumor, and osteomyelitis. A bone biopsy confirmed recurrence of the primary tumor.

Clinicians need to carefully evaluate BMD images for unusual gains or losses in cancer patients, as they may herald the onset of asymptomatic metastatic disease.

 

Nothing to Disclose: JT, PT

3355 21.0000 SAT-219 A Detection of an Asymptomatic Early Skeletal Metastasis on routine Dual-energy X-ray absorptiometry (DEXA) scan: A case report 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 15th 3:45:00 PM SAT 199-223 2216 1:45:00 PM Disorders of Bone & Calcium Homeostasis: Case Reports Poster


Arianne S Umali* and Thelma D Crisostomo
Makati Medical Center, Makati, Philippines

 

Introduction: Glucocorticoids (GC) are commonly prescribed for their anti-inflammatory and immunosuppressive properties in the treatment of a variety of pediatric conditions, including rheumatic conditions, leukemia, and organ transplantation. Therapeutic use of GC is by far the most common form of GC-induced osteoporosis.

Clinical case: A 44-year old woman was admitted due to multiple joint pains. She was diagnosed with Juvenile Idiopathic Arthritis (JIA) at the age of two, was prescribed Prednisone 5mg daily with improvement of symptoms, however was lost to follow-up but continued taking Prednisone 5-10mg daily for 42 years. On admission, she had cushingoid facies, hypertrichosis, buffalo hump, central obesity, stunted growth (height of 123cm, compatible with a 7 year-old), fusiform deformities of fingers, a swollen and tender right knee, and tenderness on both shoulder joints, right elbow and hips.

Xrays showed rheumatoid arthritis of the shoulders, hands and knees; cervical spondylosis and disc disease, C2-C3 and C3-C4; osteoporosis of the lumbosacral spine with compression deformity of most of the thoracic vertebrae; and avascular necrosis of both hips. BMD of the hips and lower spine showed severe or established osteoporosis (Z score of -3.5), moderate wedge deformity of T11. She was shifted to hydrocortisone for two days prior to plasma cortisol determination, which were elevated: 8am – 1,320nmol/L, 9am – 936nmol/L (n AM values: 138-690nmol/L), 11pm – 527nmol/L (n PM values: half of AM). ACTH (4.708pg/mL), IFG-I (149ng/mL), GH (2.783ng/mL), iPTH (44.934pg/mL), Phosphorus (3.82mg/dL), RF (11.7IU/mL), ESR (13mm/hr) and anti-CCP (1U/mL) were normal. Hypocalcemia (8.38mg/dL, n 8.6-10.2mg/dL), Vitamin D deficiency (15.9ng/mL, n ≥30ng/mL), and elevated CRP-LX (23.73mg/L, n <5mg/L) were noted. To rule out any genetic factor that may have contributed to her stunted growth, karyotyping was requested which was normal. She was discharged on Alendronate+Cholecalciferol 70mg/5600IU/tab one tablet once a week, Calcium carbonate 600mg/cap one capsule OD, Vitamin D3 2000IU/cap one capsule BID, and Prednisone 5mg/tab one tablet OD. Plans for this patient are to slowly taper her off from steroids and discontinue, if her ACTH stimulation test is normal, and start her on Methotrexate for her JIA.

Conclusion: Prolonged steroid use, even in small doses, can cause stunted growth due to premature closure of the epiphyses, severe osteoporosis and avascular necrosis.

 

Nothing to Disclose: ASU, TDC

8273 22.0000 SAT-220 A The Effect of Chronic Steroid Use on Bone Growth and Development 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 15th 3:45:00 PM SAT 199-223 2216 1:45:00 PM Disorders of Bone & Calcium Homeostasis: Case Reports Poster


Sidra Azim*, Amit Bhargava, Farheen Kassim Dojki and Faryal Sardar Mirza
University of Connecticut, Farmington, CT

 

Background: Strontium ranelate (SrR), used for the treatment of osteoporosis (OP) in Europe and Australia, is not FDA approved or available in United States (US). Here, strontium citrate (SrC) is available as a nutritional supplement. Data on effects of SrC on bone are lacking, but recently we are seeing an increase in its self administration by patients to treat OP “naturally.” We report changes in bone mineral density (BMD) in a patient using SrC.

Clinical Case: 72-year-old female with history of OP was treated with weekly alendronate for 10 years till 2006, when she was changed to an every other week regimen. Alendronate was stopped in 2007. Patient started self administrating SrC (680mg/day) in 2006 and continued to take calcium (600mg bid) and vitamin D (2000units/day). She remained physically active. Serum calcium and creatinine remained normal, and 25OH Vitamin D remained in the 32-66ng/ml range. Urine N-telopeptide (NTX) was monitored as a bone resorption marker (22-28 NTX units) and bone specific alkaline phosphatase (12-13.7mcg/L) as a bone formation marker. She continued SrC on her own till 2012, when she was advised to stop taking it due to concerns about accumulation. Total hip mean BMD increased 2.7% in the first 2 years, to a maximum of 9.2% in 6 years (1.53%/yr). Femoral neck mean BMD increased by 9% in 6 years (1.52%/yr). L2-L4 BMD increased by 30% in 6 years (5%/yr), which was difficult to interpret in view of degenerative changes.

Conclusion: There are no data available on the use of SrC to treat OP. SrR has been shown in vivo and in vitro to decrease bone resorption by inhibiting osteoclasts and stimulate bone formation by activating osteoblasts. Sr is a heavier element than calcium, and although less than 1% incorporates into bone, it can cause possible overestimation of BMD by 10%. We see a steady increase in our patient’s BMD. Whether it is due to a true increase or an increased attenuation of x-rays by Sr is not clear. One study assessing bone Sr levels after SrC use showed a 6-7x increase in bone Sr signal, but there was no analysis of a corresponding effect on BMD. Furthermore, whether the increase seen in BMD with SrC will translate into fracture risk reduction is not known. SrC is increasingly being used by patients for osteoporosis self treatment. Further studies are needed to delineate its effect on bone health, fracture reduction and its safety.

 

Nothing to Disclose: SA, AB, FKD, FSM

6667 23.0000 SAT-221 A CHANGE IN BONE MINERAL DENSITY IN A PATIENT WITH OSTEOPOROSIS TAKING OVER THE COUNTER STRONTIUM CITRATE 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 15th 3:45:00 PM SAT 199-223 2216 1:45:00 PM Disorders of Bone & Calcium Homeostasis: Case Reports Poster


Yanal Masannat* and Christine Irene Oakley
Joan C Edwards School of Medicine at Marshall university, Huntington, WV

 

Introduction

Osteoporotic hip fractures have devastating effects on quality of life and are associated with increased morbidity and mortality.  Bisphosphonates have been used to suppress bone turnover, and remain the first-line therapy for osteoporosis given their proven benefits to reduce fracture rates. However, these medications should not be used indiscriminately or indefinitely given the potential for adverse effects, including the most recently described possible association with atypical femoral fractures. We report a case of a 78 year old female with osteopenia on alendronate for three years duration who presented after suffering an atraumatic left subtrochanteric femur fracture.   

Clinical case

78 years old female with history significant for osteopenia. She was initiated on alendronate 70mg weekly three years ago for fracture prevention due to high risk status.  She presented with acute onset left hip pain without preceding trauma, but admitted to experiencing milder hip discomfort for the preceding month. Her x-ray demonstrated a complete subtrochanteric fracture of the left femur that was transverse in orientation laterally and oblique configuration medially. Her clinical course and radiographic features were consistent with an atypical femur fracture. She had a successful open reduction and internal fixation of the fracture. Evaluation revealed vitamin D deficiency for which she was initiated on ergocalciferol 50,000 units daily for three days then weekly thereafter. Her oral bisphosphonate was discontinued. 

Conclusion

Bisphosphonates remain the first-line treatment for osteoporosis and are efficacious at reducing fracture risk. Although the risk of atypical femur fractures is much less than the risk  of untreated osteoporotic fractures, physicians should appropriately choose bisphosphonate therapy for patients, and be aware of prevention strategies to minimize the risk of developing atypical fractures.  These strategies include identifying high-risk patients for fractures using WHO FRAX calculator, employing drug holidays for those on prolonged bisphosphonate therapy with stable disease,  educating patients to report new-onset thigh and groin pain that could be indicative of impending atypical femoral fractures, and consider early investigations such as bone scans and/or MRI to identify these fractures at an early stage.

 

Nothing to Disclose: YM, CIO

4252 24.0000 SAT-222 A A case of atypical femoral fracture associated with alendronate therapy 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 15th 3:45:00 PM SAT 199-223 2216 1:45:00 PM Disorders of Bone & Calcium Homeostasis: Case Reports Poster


Yara Rhayem1, Catherine Le-Stunff2, Agnes Linglart3, Caroline Silve4 and Eric Laurent Clauser*5
1INSERM U970, Paris, France, 2INSERM U986, Hôpital Bicêtre, Bicêtre, France, 3INSERM U986, Service d'Endocrinologie Pédiatrique, Hôpital Bicêtre, Bicêtre, France, 4INSERM U986, Hôpital Bicêtre, Le Kremlin Bicêtre, France, 5Département de, Paris, France

 

Acrodysostosis is a rare genetic disease characterized by a skeletal dysmorphic syndrome. One genetic cause in patients with acrodysostosis and multihormonal resistance (PTH, PTHRP, TSH) (ADOHR) was recently identified as mutations of the PRKAR1Agene (1), the major regulatory subunit of cAMP dependent protein kinase (PKA), a pivotal actor of the cAMP signaling pathway. PRKAR1A binds to the catalytic subunit (PRKAC1A) inhibiting its enzymatic activity. The binding of cAMP to the domains A and B of PRKAR1A activates the enzyme. To date, 9 PRKAR1A mutations located in cAMP binding domains have been identified associated with ADOHR (2). The functional characterization of 2 of them suggests an inhibitory effect of the PRKAR1A mutants on PKA activity (1,3).

To further document the functional consequences of PRKAR1A mutations on cAMP/PKA signaling and the molecular basis of the phenotype observed in the patients, 5 novel mutations of the cAMP domain B (Q285R, G289E, A328V, R335L, Q372X) were characterized. These 5 mutations were all de novo and identified in unique ADOHR patients.

Studies were performed using western blot analysis (WB), BRET (bioluminescence resonance energy transfer) technology and Cre-luciferase reporter assays (Cre-Luc RA) after transient expression of mutant proteins in HEK293 cells.

All 5 PRKAR1A mutant proteins are expressed (WB). As shown by BRET saturation experiments, the affinity of mutant proteins for PRKAC1A was similar (BRET50 = 0.42 to 0.57) to that observed for WT protein (BRET50 = 0.42). This BRET signal is reduced after addition of a cAMP analog for both the WT and mutant PRKAR1A proteins, reflecting the dissociation between the catalytic and regulatory subunits; however, the dose–response curve for all mutant proteins was significantly shifted to the right (EC50 = 2 to 70nM) as compared to WT PRKAR1A (EC50 = 1nM), indicating a decreased sensitivity to cAMP of the mutant proteins.  Cre-Luc RA showed significantly lower CRE–luciferase stimulation with forskolin for mutant as compared to WT PRKAR1A (P<0.01). Interestingly the extent to which the signals for both BRET analysis and Cre-Luc RA were altered varied as a function of the mutations.

These studies further indicate that PRKAR1A mutations identified in ADOHR are dominant gain of function mutations, acting as dominant negative for PKA function and partially inactivating the catalytic subunit. The mutation/phenotype correlations are under investigation.

YR and CLS contributed equally to the work

 

Nothing to Disclose: YR, CL, AL, CS, ELC

7050 25.0000 SAT-223 A Functional characterization of PRKAR1A mutants causing acrodysostosis with hormonal resistance 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 15th 3:45:00 PM SAT 199-223 2216 1:45:00 PM Disorders of Bone & Calcium Homeostasis: Case Reports Poster


Valentina Micheletti1, Maria Vittoria Davi'*2, Maria Teresa Valenti1, Luca Dalle Carbonare1 and Giuseppe Francia1
1Internal Medicine sect D, University of Verona, Verona, Italy, 2Medicina Generale e Malattie Aterotrombotiche, University of Verona, Verona, Italy

 

Background: a high prevalence of vertebral fractures, despite normal bone mineral density (BMD), is reported in acromegalic patients in both the active and inactive phases of the disease. Aim of the study: to analyze the prevalence of vertebral fractures, bone turnover, BMD and histomorphometric parameters including bone microarchitecture in a group of acromegalic patients. Patients and methods: 42 acromegalic patients (26 males, 16 females, mean age 58 ±12,5 and 58,8 ±13,8 years), divided in 2 subgroups: one with active disease and one in remission (13, 29 patients respectively) underwent bone turnover evaluation (serum calcium, serum phosphate, PTH, 25- hydroxyvitamin (OH) D , ALP, CTX, urinary calcium/creatinine) and bone densitometry by Dual-Energy X-Ray Absorptiometry (DXA). Vertebral fractures were assessed by morphometric analysis during DXA. In 4 patients  iliac crest bone biopsy was performed.Results: the prevalence of vertebral fractures was 64% in the whole population, 69% in the active group, 59% in the remission group. Among fractured patients, vertebral t-score was normal in 59% and it showed osteoporosis in only 11%. Vertebral BMD was higher in fractured patients compared to non-fractured  (mean SD 1,108 ±0,175 vs 0,961± 0,118 g/cm2, p=0,009),  also considering the active (1,147±0,241 vs 1,107±0,169 g/cm2) and inactive subgroups (1,093 ±0,15 vs 0,945 ±0,106g/cm2, p0,01). Disease duration was significantly higher in fractured compared to non-fractured patients (respectively 8,67 ±6,65 and 6,93 ±3,41 years, p<0,05). As regards bone metabolism parameters, urinary calcium/creatinine was significantly higher in fractured patients compared to non-fractured patients (0,125 ±0,096 vs 0,074 ±0,031, p<0,01). 25-OH vitamin D was low in both groups (fractures 24,93 ±11,5 ng/ml, non-fractured patients 25,02 ±7,28 ng/ml). Hypogonadism was a risk factor for vertebral fractures in active patients (p=0,04). The preliminary results regarding histomorphometric evaluation showed a greater cortical thickness and  an increased porosity compared to that of normal subjects (cortical width 1882±250 vs 816±291 μm, cortical porosity index 10 ±4% vs 5,79 ±2,76%). IGF-1 positively correlated with porosity index (p<0,05).Conclusions: our data confirm a high prevalence of vertebral fractures in acromegalic patients despite a normal BMD. This apparent paradox could be explained by an increased cortical thickness associated with  abnormal cortical porosity, mediated by IGF-1.

 

Nothing to Disclose: VM, MVD, MTV, LD, GF

FP10-4 9159 3.0000 SAT-226 A Bone Fragility in Acromegalic Patients: A Mystery Almost Solved? 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 15th 3:45:00 PM SAT 224-247 2223 1:45:00 PM Osteoporosis I Poster


Christopher Recknor*1, Christian Roux2, Pei-Ran Ho3, Jesse Hall3, Henry G Bone III4, Sydney Bonnick5, Joop van den Bergh6, Irene Ferreira7, Rachel B Wagman8 and Jacques P Brown9
1United Osteoporosis Centers, Gainesville, GA, 2Paris Descartes University, Paris, France, 3Amgen Inc., Thousand Oaks, CA, 4Michigan Bone & Mineral Clinic, Detroit, MI, 5Clinical Research Center of North Texas, Denton, TX, 6VieCuri Medical Centre and Maastricht University, Netherlands, 7Amgen Ltd, Cambridge, United Kingdom, 8Amgen, Thousand Oaks, CA, 9CHU de Québec Research Centre, Laval University, Québec City, QC, Canada

 

Poor adherence to bisphosphonate (BP) therapy in osteoporosis is both common and associated with poor outcomes and increased treatment costs (1,2). Although improved compliance has been shown with monthly vs weekly dosing regimens (3), there is no evidence to suggest that cycling through BP agents offers therapeutic benefit, as assessed by BMD. In two randomized, open-label studies (TTI and TTR), postmenopausal women aged ≥55 yrs previously treated with, but suboptimally adherent to, BP therapy received denosumab (DMAb) 60mg SC Q6M, ibandronate (IBN) 150mg PO QM or risedronate (RIS) 150mg PO QM for 12 months; DMAb treatment was associated with greater increases in bone mineral density (BMD) than either IBN or RIS (4,5). We evaluated whether these comparative differences were similarly observed in a subset of subjects who had BMD data recorded at baseline and month 12, stratified by high risk for fracture, defined by previous fragility fracture. In TTI, 399 subjects received DMAb and 368 received IBN, and in TTR, 405 subjects received DMAb and 404 received RIS. In TTI, 31% (237/767) and in TTR, 35% (280/809) of subjects had a prior fracture. There were no significant differences in BMD at baseline between treatment groups or prior fracture subgroups. In TTI, BMD increases were greater with DMAb than IBN at the total hip (TH), femoral neck (FN) and lumbar spine (LS) in subjects with prior fracture (2.4% vs 0.6%, 2.2% vs 0.1% and 4.2% vs 1.6%, respectively; p<0.0001 for all) and without prior fracture (2.2% vs 1.3%, 1.5% vs 1.0% and 4.1% vs 2.2%; p<0.05 for all). There was no significant difference in treatment effect between subjects with or without fracture at the TH or LS (pinteraction>0.1) while at the FN, DMAb seemed to have a greater treatment effect in those with a prior fracture (pinteraction=0.002 ). In TTR, BMD increases were greater with DMAb than RIS at the TH, FN and LS in subjects with prior fracture (2.2% vs 0.5%, 1.4% vs 0.3% and 3.4% vs 1.4%; p<0.0001 for all) and without fracture (1.9% vs 0.3%, 1.5% vs -0.3% and 3.4% vs 1.4%; p<0.01 for all). There was no significant difference in treatment effect between subjects with or without fracture at any measured site (pinteraction>0.1 for all). In subjects who were suboptimally adherent with an oral BP, transitioning to DMAb provided greater gains in BMD at all key skeletal sites measured compared with transitioning to either IBN or RIS. Our findings suggest that the magnitude of treatment effect is not significantly influenced by classification of high risk, as deofined by prior fragility fracture.

 

Disclosure: CR: Speaker, Novartis Pharmaceuticals, shareholder, Ion Med Systems. CR: Investigator, Eli Lilly & Company, Advisory Group Member, Novartis Pharmaceuticals, Board Member, Novartis Pharmaceuticals, Board Member, Merck & Co., Speaker, Merck & Co., Investigator, Merck & Co., Board Member, Amgen, Speaker, Amgen, Advisory Group Member, Amgen, Investigator, Amgen, Advisory Group Member, UCB. PRH: Employee, Amgen, Employee, Amgen. JH: Employee, Amgen, Employee, Amgen. HGB III: Advisory Group Member, Merck & Co., Advisory Group Member, Amgen, Investigator, Tarsa, Investigator, Novartis Pharmaceuticals, Investigator, Merck & Co., Investigator, Amgen, Advisory Group Member, Tarsa. SB: Speaker Bureau Member, Amgen, Investigator, Wyeth, Investigator, Takeda, Investigator, Merck & Co., Investigator, Amgen, Speaker Bureau Member, Novartis Pharmaceuticals. JV: Investigator, Nycomed, Investigator, Eli Lilly & Company, Investigator, Amgen. IF: Employee, Amgen, Employee, Amgen. RBW: Employee, Amgen, Employee, Amgen. JPB: Advisory Group Member, Amgen, Investigator, Warner Chilcott, Investigator, Takeda, Investigator, Roche Pharmaceuticals, Investigator, Servier, Investigator, Pfizer Global R&D, Investigator, Novartis Pharmaceuticals, Investigator, Merck & Co., Investigator, Eli Lilly & Company, Investigator, Amgen, Advisory Group Member, Eli Lilly & Company, Advisory Group Member, Merck & Co., Speaker Bureau Member, Amgen, Speaker Bureau Member, Eli Lilly & Company, Speaker Bureau Member, Merck & Co., Speaker Bureau Member, Novartis Pharmaceuticals.

FP10-3 5604 4.0000 SAT-227 A Comparative Treatment Outcomes In Patients With Prior Fracture Previously Treated With A Bisphosphonate: Results From The Denosumab/Ibandronate and Denosumab/Risedronate Trials 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 15th 3:45:00 PM SAT 224-247 2223 1:45:00 PM Osteoporosis I Poster


Julia Michelsen1, Henri Wallaschofski2, Nele Friedrich1, Christin Spielhagen1, Rainer Rettig1, Till Ittermann1, Matthias Nauck1 and Anke Hannemann*1
1University Medicine Greifswald, 2University Medicine Greifswald, Greifswald, Germany

 

Objective: Bone turnover markers (BTMs) reflect the metabolic activity of bone tissue and can be used to assess fracture risk and monitor treatment of osteoporosis. To adequately interpret BTMs, method-specific reference intervals are needed. We aimed to determine reference intervals for serum concentrations of two bone formation markers: procollagen type 1 N-terminal propepetide (PINP), and bone-specific alkaline phosphatase (BAP), and one bone resorption marker: C-terminal telopeptide of type 1 collagen (CTX).

Material and Methods: We established a healthy reference population from the participants of the first follow-up of the Study of Health in Pomerania. Serum PINP, BAP, and CTX concentrations were measured on the IDS-iSYS Automated System (Immunodiagnostic Systems, Frankfurt am Main, Germany). The reference interval was defined as the central 95% range. We determined age-specific reference intervals for PINP and CTX in men by quantile regression. Age-independent reference intervals were determined for BAP in men and for all three BTMs in pre- and postmenopausal women.

Results: In 1107 men, upper and lower reference limits for PINP and CTX decreased over the observed age range of 25-79 years (reference intervals for 25-29 year-old men: PINP 31.1-95.9 ng/mL and CTX: 0.12-0.83 ng/mL; 75-79 year-old men: PINP 15.7-68.1 ng/mL and CTX: 0.05-0.58 ng/mL). The reference interval for BAP was age independent (25-79 year-old men: 7.5-26.1 ng/mL). In 544 premenopausal women, the reference limits for PINP, BAP and CTX were lower (PINP:17.3-76.3 ng/mL; BAP: 5.8-23.8 ng/mL; CTX: 0.05-0.67 ng/mL) than in 498 postmenopausal women (PINP:17.3-100.9 ng/mL; BAP: 7.5-31.6 ng/mL; CTX: 0.08-1.05 ng/mL). Women taking sex hormones had lower BTMs than women not taking sex hormones.

Conclusion: Adult reference intervals for serum PINP, BAP, and CTX concentrations measured on the IDS-iSYS Automated System were successfully established in a population-based cohort free of bone-related diseases.

 

Nothing to Disclose: JM, HW, NF, CS, RR, TI, MN, AH

6165 7.0000 SAT-230 A Reference Intervals for Serum Concentrations of Three Bone Turnover Markers (PINP, BAP, CTX) in Male and Female Adults 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 15th 3:45:00 PM SAT 224-247 2223 1:45:00 PM Osteoporosis I Poster


Kerstin Landin-Wilhelmsen*1, Emily Amundson2 and Penelope Trimpou3
1Section for Endocrinology, Institution of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden, 2Section for Cardiology, Borås, Sweden, 3Sahlgrenska University Hospital, Gothenburg, Sweden

 

Aim: The aim was to study the effect of growth hormone (GH) treatment on self-reported quality of life (QoL) and fractures in postmenopausal osteoporosis at 10 years´ follow-up after given treatment1. The hypothesis was that GH improved bone mass and QoL long time after discontinuation of GH2.

Patients: Eighty women 50-70 years with osteoporosis and ongoing estrogen hormone replacement (HRT) were studied and compared with an age-matched random population sample of women (n=120), the WHO MONICA project, Gothenburg.

Methods: A double blind, placebo controlled study was performed. Patients were randomized to GH 1.0 Unit (U) or GH 2.5U (Genotropin®) daily during 3 years or corresponding volumes of placebo subcutaneously. All received calcium 750 mg and vitamin D 400 units and followed-up during 10 years. Bone mineral density (BMD) and content (BMC) were measured with DXA annually until 6 years and then every second year. QoL was estimated with the Short Form (SF-36) annually. The patients were examined annually while the controls were examined at start and after 10 years by the same staff. The same methods were used on all occasions.

Results: GH increased BMD and BMC dose dependently in all regions (p=0.01 GH 1.0 U and p=0.0006 GH 2.5U vs placebo) until 5 years. Thereafter BMD and BMC declined in both GH groups and did not differ from the placebo group. At 10 years, all groups had BMD and BMC similar to baseline. After 10 years, the amount of patients who fractured decreased from 56% to 28% (p=0.0003), evenly distributed between groups. Serum IGF-1 was lower in patients who fractured during follow-up (p=0.01). No one dropped out. Six of the oldest patients died (2 high dose GH, 1 low dose GH and 3 placebo). HRT use decreased from 100% to 41% while 23% had started with bisphosphonates and 3% had received teriparatide among patients. In controls, the women who fractured increased from 8% to 32% (p=0.0008), HRT use decreased from 40% to 8% while use of bone specific agents increased from 0 to 4% only. QoL did not change during GH-treatment or during the 10-year follow-up in the patients and did not differ compared with controls.

Conclusion: GH-treatment was beneficial for the bone mass and fracture outcome after 10 years but did not affect QoL in postmenopausal women with osteoporosis. The QoL was similar to women in the general population. The fracture incidence decreased in treated women with osteoporosis and increased in the population, where bone specific agents were rarely used, at follow-up.

 

Nothing to Disclose: KL, EA, PT

8361 8.0000 SAT-231 A EFFECT OF GROWTH HORMONE TREATMENT ON FRACTURES AND QUALITY OF LIFE IN POSTMENOPAUSAL OSTEOPOROSIS — A 10-YEAR FOLLOW-UP STUDY 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 15th 3:45:00 PM SAT 224-247 2223 1:45:00 PM Osteoporosis I Poster


Navneet Mangat*1, Shuko Lee2 and Jan M Bruder3
1UTHSCSA, San Antonio, TX, 2South Texas Veterans Health Care System, San Antonio, TX, 3Univ of TX HSC at San Antonio, San Antonio, TX

 

Osteoporosis in Men Receiving Androgen Deprivation Therapy for Prostate Cancer: How Should We Screen? 

Mangat NM, Lee S, Bruder JM

Introduction: Androgen deprivation therapy (ADT), used in the treatment of prostate cancer, is associated with accelerated bone loss and increased risk of fractures. It is recommended that men over the age of 50 years with osteoporosis diagnosed by bone mineral density (BMD) with a T-score of < -2.5 at the spine, total hip (TH), femoral neck (FN) or distal 1/3 radius (DR) or a fracture be considered for pharmacological therapy. If a patient has low bone density (T-score -1.0 to -2.5), then the FRAX® calculator is used to estimate an individual’s 10 year fracture risk probability. Predetermined cutoffs are used to guide therapy. Although not routinely measured, the BMD at DR will diagnose osteoporosis in more men with prostate cancer. Newer data has also suggested that in men with prostate cancer, the FRAX® calculator used without BMD identifies more men needing treatment than calculations with the BMD. Objective: The aim of our study was to determine if the FRAX® calculator used without BMD identifies the same individuals as BMD at the spine, TH, FN or DR and to compare the number of men identified for treatment vs. no treatment by each measurement. Materials & Methods: The BMD at the spine, TH, FN, and DR were measured as part of routine care in 510 men with prostate cancer, treated with ADT. The FRAX® calculator was used without BMD to estimate each individual’s 10 year fracture risk probability. Comparisons of BMD and the FRAX® calculator were done using the Pearson's Chi-Square test to determine the reliability (sensitivity and specificity) and positive (PPV) and negative predictive values (NPV). Results: The patient population had a mean age of 74.2 +8.6 with 62% Caucasian, 27% Hispanic and 12% Black. The mean height was 67.7+2.8 in and mean weight was 183.4+36.9 lbs. The mean BMD at the spine, TH, FN and DR was 0.60+0.009, 0.75+0.15, 0.92+0.20, and 0.73+0.14 g/cm2, respectively. The mean T-score at the spine, TH, FN and DR were -0.49+1.87, -1.56+1.54, -0.82+1.11, and -1.44+1.9, respectively. BMD at the spine and hip identified 140 men with osteoporosis, which increased to 216 men with the addition of the DR. 314 patients were identified to be at risk for fracture by using the FRAX® calculator without BMD. Of those 216 individuals diagnosed with osteoporosis by BMD, the FRAX® calculator identified 158 (73%) to be at risk for fracture, missed 58 men (30%), and identified an additional 156 men needing treatment. The accuracy of the 2 methods indicated a sensitivity of 73%, specificity 47%, PPV 50%, and NPV 30%. Conclusion: In men with prostate cancer, treated with ADT, using the FRAX® calculator without BMD identifies more individuals requiring therapy for osteoporosis than BMD alone. There was no association between BMD and the FRAX® calculator in identifying men needing treatment.

 

Nothing to Disclose: NM, SL, JMB

3986 9.0000 SAT-232 A Osteoporosis in Men Receiving Androgen Deprivation Therapy for Prostate Cancer: How Should We Screen? 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 15th 3:45:00 PM SAT 224-247 2223 1:45:00 PM Osteoporosis I Poster


Louis J Gooren*1, Ahmad Haider2, Abdulmaged M Traish3, Gheorghe Doros4, Ulrich Meergans5 and Farid Saad6
1VU University Medical Center, Amsterdam, Netherlands, 2Private Urology Practice, Bremerhaven, Germany, 3Boston Univ Sch of Med, Boston, MA, 4Boston University School of Public Health, Boston, MA, 5DRK-Krankenanst. Wesermuende Seepark-Klinik, Langen, Germany, 6Bayer Pharma AG, Berlin, Germany

 

Introduction: Numerous studies point to the significance of normal serum testosterone to maintain bone mineral density (BMD) at various stages of life. Testosterone deficiency leads to loss of BMD and testosterone treatment has a beneficial effect. This study investigated the effects of normalizing serum testosterone on BMD in 36 men with osteoporosis who had consulted an orthopedic surgeon and who were diagnosed as testosterone deficient. 
Methods: Cumulative, prospective, registry study of men (mean age: 54.89 ± 6.31 years) with testosterone levels below 12.1 nmol/L. Their T scores were -2.99±0.4 (minimum -3.90, maximum -2.60). They received parenteral testosterone undecanoate 1000 mg/12 weeks following an initial 6-week interval for up to five years. After one year, 36 men were included in the registry, after two years, 32 men, after three years, 26 men, after four years, 10 men, after five years, 4 men. The declining numbers do not reflect drop-out rates but are a result of the registry design. New patients are consecutively entered once they have completed one year of treatment.
Results: Over the 5 year period there was a significant improvement of the T-score in these men. The mean T-score decreased from -2.99±0.4 (minimum: -3.90; maximum: -2.60) at baseline to -2.62±0.34 at 12 months, -2.29±0.32 at 24 months, -2.03±0.35 at 36 months, -1.75±0.2 at 48 months and -1.58±0.19 at 60 months without reaching a plateau. The improvement was progressive: each year of testosterone treatment led to a significant further improvement of the T-score (p<0.0001 vs baseline and vs previous year).
Conclusions: Normalizing of serum testosterone leads to an improvement of bone mineral density and this improvement is progressive throughout the time period of testosterone administration.

 

Disclosure: AH: Speaker, Bayer Pharma AG, Speaker, Takeda. AMT: Investigator, Bayer Pharma AG. GD: statistical analyses, Bayer Pharma AG. FS: Employee, Bayer Pharma AG. Nothing to Disclose: LJG, UM

5775 10.0000 SAT-233 A Progressive improvement of T-scores in middle-aged men with osteoporosis upon long-term treatment with testosterone 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 15th 3:45:00 PM SAT 224-247 2223 1:45:00 PM Osteoporosis I Poster


Dileep K Atluri*1 and Pratibha P Raghavendra2
1University Hospitals of Cleveland, 2University Hospitals, Case Medical Center, Cleveland, OH

 

Effect of topical testosterone on bone mineral density in men: a systematic review and meta-analysis

Abstract:

Introduction:  Testosterone deficiency puts aging men at higher risk of osteoporosis. Several forms of testosterone supplementation are currently available to clinician. The effect of topical testosterone supplementation on bone mineral density remains unclear.

Objective: To systematically review available randomized controlled trials and conduct meta analysis to evaluate the effect of topical testosterone on bone mineral density in men.

Data sources: Pubmed and Cochrane central register of controlled trials

Study eligibility criteria: Randomized controlled trials comparing topical testosterone with placebo in adult male patients, Minimum duration of therapy 6 months.

Study appraisal and synthesis methods: 4 randomized, placebo controlled trials were included. Primary outcomes assessed were bone mineral density (BMD) at lumbar spine and femoral neck. Meta analysis was conducted using Revman 5 software. Random effects model was used in meta analysis and standardized mean difference (SMD) was used as a summary statistic.

Results: 4 studies including 289 patients were included. In trials of topical testosterone vs placebo, allocation to topical testosterone did not significantly increase BMD.

Limitations: Some of the included studies did not specify intention to treat (ITT) population and had significant loss to follow-up. Duration of therapy varied from 6 months to 36 months. Additional studies addressing the same question may have been missed in the search. Patients with different levels of hypogonadism/pre treatment testosterone concentration were included.

Conclusions: Trials with clinical data such as bone fracture incidence are needed.

 

Nothing to Disclose: DKA, PPR

9304 11.0000 SAT-234 A Effect of topical testosterone on bone mineral density in men: a systematic review and meta-analysis 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 15th 3:45:00 PM SAT 224-247 2223 1:45:00 PM Osteoporosis I Poster


Giulia Brigante, Daniele Santi, Chiara Diazzi, Sara De Vincentis, Giulia Ferrannini, Bruno Madeo, Manuela Simoni, Cesare Carani and Vincenzo Rochira*
Chair and Unit of Endocrinology & Metabolism, Department of Biomedical, Metabolic and Neural Sciences, University of Modena & Reggio Emilia, Modena, Italy

 

Background: GH-IGF-1 status is important for bone health. Acromegaly affects bone status, but less is known on the role of treatments for acromegaly on bone mineral density (BMD). Pegvisomant (Peg) is effective in treating acromegaly by reducing IGF-1. As serum GH is not influenced by Peg, it is not known if residual, direct GH effects on bone (not IGF-1 mediated) are preserved during treatment. Methods: To evaluate the effects of Peg on BMD, we compared 5 patients treated with Peg (alone or in combination) to 6 patients treated with Somatostatin Analogues (SA) and to 7 patients surgically cured, not under medical therapy. All the patients had normal serum IGF-1. BMD was measured by DEXA (Hologic-QDR-2000 densitometer, Inc., Waltham, MA). A t-score of ≤ 1 and ≤ 2.5 at lumbar spine (L1-L4) and at femoral neck was used for diagnosis of osteopenia and osteoporosis, respectively .

Results: Mean age of subjects (seven males and nine females) was 60.7 ± 9.8 yrs. At lumbar spine, 40% of Peg-patients, 33.3% of SA-patients, and 60% of not-treated patients had osteopenia; none of the Peg-patients, and 16.7% of SA-patients, and none of not-treated patients were osteoporotic. Considering the femoral neck, 60% of Peg-patients, 33% of SA-patients, and 60% of not-treated patients had osteopenia; 20% of Peg-patients and none of the other two groups were osteoporotic.

Conclusions: The percentage of osteoporotic/osteopenic acromegalic patients seems to be lower than that reported in literature. Peg seems to protect bone at lumbar spine, but this protective effect does not seem to be exerted at femoral level where, indeed, patients treated with Peg present lower densitometric values. Patients surgically cured, not under medical therapy, have higher rate of lumbar osteopenia. No data are available on bone quality, a parameter that is usually altered in acromegaly.

 

Nothing to Disclose: GB, DS, CD, SD, GF, BM, MS, CC, VR

7044 12.0000 SAT-235 A EFFECTS OF TREATMENT FOR ACROMEGALY ON BONE MINERAL DENSITY (BMD): IS PEGVISOMANT PROTECTIVE ON LUMBAR BMD? 2013-06-18 San Francisco 2013-06-13 The Endocrine Society's 95th Annual Meeting & Expo Bone, Calciotropic Hormones & Vitamin D Saturday, June 15th 3:45:00 PM SAT 224-247 2223 1:45:00 PM Osteoporosis I Poster


Antonio Mancini*1, Sebastiano Raimondo1, Francesco Ciro Tamburrelli1, Chantal Di Segni1, Mariasara Persano1, Roberto Festa2, Andrea Silvestrini1, Elisabetta Meucci1, Luca Tiano2, Gian Paolo Littarru2 and Alfredo Pontecorvi3
1Catholic University of the Sacred Heart, Rome, Italy, 2Polytechnic University of the Marche, Ancona, Italy, 3Endocrinologia e Malattie del Metabolismo, Università Cattolica del Sacro Cuore, Rome, Italy, Italy

 

Male idiopathic osteoporosis is underestimated, despite its clinical and social importance; the underlying biochemical mechanisms are still poorly understood. Previously, we demonstrated low plasma Total Antioxidant Capacity (TAC) in hypogonadal patients. The aim of this study was to investigate oxidative stress as risk factor for bone fracture and its relationships with endocrine milieu. We enrolled 31 males (36-72 ys), affected by back pain/spine fracture due to trivial trauma and 10 healthy controls (30-48 ys). TAC was determined by  colorimetric assay, using the system H2O2-metmyoglobin as source of radicals and a chromogen (ABTS); the latency time (LAG) in the accumulation of ABTS.+, spectroscopically detectable, is proportional to antioxidants concentration. Coenzyme Q10 (CoQ10), lipophilic antioxidant, was also determined, by electrochemical method, to assess it oxidized and reduced forms, in 16 out of 31 patients. The following parameters were evaluated: testosterone, estradiol, insulin, IGF-1, PRL, FT3, FT4, TSH, PTH, Vitamin D, osteocalcin, beta-cross laps. Statistical evaluation was performed using Mann-Whitney test. The prevalence of IGF-1 defects (52.8±15.3 ng/ml) was 5/31 (suggesting GH deficiency, GHD, confirmed by GHRH+arginine test). Hypogonadism (mean testosterone levels 2.03±0.46 ng/ml) was present in 4/31. The 22 patients left did not show alterations in the hormonal parameters studied. Despite LAG levels did not differ between patients and controls (72.7±8.5 vs 75.0±6.0 sec), 12 out of 31 patients had low LAG levels (between 50 and 60 sec) irrespective of hormonal milieu. Moreover, we found significantly lower Vitamin D levels in hypogonadal subjects, than in patients with GHD and patients with normal hormonal parameters (10.7±5.8 ng/ml vs 19.7±17.7 and 22.7±9.7 respectively). Again, despite lower levels of total CoQ10  in GHD patients vs hypogonadal and other patients (0.54±0.28 ug/ml, vs 0.71±0.25 and 0.82±0.30, respectively), a higher ratio ox/red  CoQ10  was observed in patients with normal hormone milieu (16.67±1.75 vs 11.00±1.87 in GHD), similar to that of hypogonadal subjects (16.50±2.65%), suggesting that oxidative stress per se can be present in such a condition, irrespective of h