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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 <25^th, 25-50^th, and 51-75^th percentiles maintained stable (or decreasing body fat), whereas those in the >75-90^th and >90^thpercentiles 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 (Ca_v) 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 Ca_v mutations, compared to only 2/50 patients in a less selected Dutch cohort, and 0/42 of the controls. The function of the Ca_v 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 Ca_v 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 Ca_vproteins themselves to be more abundant in ZG than ZF, and in the APAs of ZG-like phenotype.

The Na⁺,K⁺-ATPase and Ca_v 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.

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.

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.

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.

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.

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.

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.

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.

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.

 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.

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.

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.

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.

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. 

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.

Xu ZHANG^1#, Qin-ce SUN^1#, Shu-Hua Mu¹, Yong-jun LIU¹, Ping Ma¹, Wen-juan XU^1, 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.

  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.

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.

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.

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.

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.

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.

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.

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 neomycin^r 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.

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.

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.

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.

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

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 (Ob_sen), with separate cut-offs for men and women. Obese insulin-resistant (Ob_res) 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 Ob_sen and Ob_res groups. By definition, insulin sensitivity was higher in Ob_sen subjects (121 ± 26 vs. 76 ± 19 µmol/min/kgFFM). Ob_sen 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), Ob_sen 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, Ob_sen 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 Ob_res subjects. Ob_sen subjects were more metabolically flexible (as defined by change in respiratory quotient from baseline to clamp steady state) than Ob_res 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.

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.

.

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

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.

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.

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.

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.

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.

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.

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.

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.

The phenotype of female mFSHR^D580H 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.

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.

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.

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 1^st 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 2^nd 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.