The pancreatic β−cell secretes insulin through a combination of triggering and amplification pathways in response to glucose. Glucose metabolism is known to increase intracellular calcium and trigger insulin secretion from the β−cell while fatty acid (FA), an essential component of glucose stimulated insulin secretion (GSIS), is known to amplify secretion through varied mechanisms. Orlistat, a lipase inhibitor, blocks GSIS although the mechanism has not been clearly elucidated. We show that orlistat can also inhibit basal insulin secretion. FA can prevent/reverse orlistat-induced inhibition of secretion but fails to amplify GSIS after orlistat treatment. Here we test the hypothesis that FA is required to maintain normal plasma membrane potential and calcium influx in the β−cell. Clonal pancreatic β−cells (INS-1 832/13) were cultured in RPMI media containing 11 mM glucose and 10% FBS. Insulin secretion was measured over 2 hours with and without FA and orlistat (200 µM) using homogeneous time resolved fluorescence insulin assay (HTRF, Cisbio) and reported as ng/million cells. Intracellular calcium was measured in single cells using fura-2 AM. Single cell membrane potential was measured with virally expressed ARC Lite protein (Montana molecular). High glucose (8 mM) stimulated insulin secretion at least 3-fold over basal glucose (2 mM) and addition of FA enhanced GSIS. Orlistat almost completely abolished GSIS. This inhibition was mostly prevented in the presence of FA. Orlistat blocked calcium influx required for triggering insulin release while addition of FA recovered normal calcium homeostasis. The monoacylglycerol lipase inhibitor JZL 184 reduced both calcium influx and the increase in plasma membrane potential induced by KCl. FA recovered plasma membrane potential inhibited by JZL 184. Our results suggest that reducing β−cell intracellular FA availability by lipase inhibition blocks GSIS by preventing the glucose-induced rise in plasma membrane potential required to induce β−cell calcium influx through voltage dependent calcium channels (VDCC). FA is thus required to maintain normal nutrient metabolic coupling to insulin secretion. Whether the effect of FA to modulate plasma membrane potential plays a role in β−cell insulin hypersecretion resulting from excess nutrients requires further investigation.

Background: Hypogonadism has been described in association with HIV infection and anti-retroviral therapy (ART). Furthermore, hypogonadism is associated with comorbidity and a reduced quality of life. The prevalence of hypogonadism in HIVinfected South African men is unknown. We examined the prevalence of hypogonadism in HIV-infected men before and 12 months after the initiation of ART. Methods: This is a sub-study of the McHAART Study designed to investigate the metabolic complications of ART. HIV-infected men attending the Crossroad Community Healthcare Clinic were conveniently sampled prior to commencing ART and then followed for 6-months, 12-months and 24-months. Here we report on total testosterone, LH, FSH, SHBG and free testosterone measured on blood samples taken at 08h00 prior to initiating ART (baseline) and then again at 12 months on ART. Results: There were 44 patients at baseline and 30 patients at the 12-month visit. There were no participants with hypogonadism at baseline or after 12 months on ART. Testosterone levels or change in testosterone levels were not associated with alcohol intake, bone miner density, body mass index, waist circumference, fasting glucose, HOMA IR, HOMA β, fasting cholesterol or blood pressure at baseline or 12 months. Conclusions: There were no cases of hypogonadism in HIV-infected ART-naïve men and there were no cases of hypogonadism in HIV-infected men on ART for 12 months in this study population. Testosterone levels or change in testosterone levels were not associated with any measures of body composition, glucose metabolism, lipids or bone mineral density.

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Bird abundance in North America has declined by almost 30% in just the past 50 years. It is crucial that we understand the many factors contributing to declines, including the impacts of environmental contaminants. Mercury is a persistent and global neurotoxic contaminant of aquatic systems that will continue to increase in the environment as anthropogenic activities and climate change accelerate its emission. The impact of mercury on songbirds, a large and diverse but declining taxon, is not well understood. I sought to better understand the effects of mercury exposure on songbirds by studying its impact on the environmentally sensitive hypothalamic-pituitary-adrenal axis, which controls the stress response mediated by the hormone corticosterone. Using adult, captive-bred zebra finches as a model songbird, I tested the effect of an environmentally relevant level of dietary mercury on corticosterone in both blood and feathers. I also tested the effect of an additional stressor – chronic, unpredictable food deprivation – both on its own and in combination with mercury exposure, to better estimate how mercury might impact birds differently when another challenge occurred simultaneously. Baseline corticosterone was measured in the blood. Corticosterone was also measured from primary feathers grown during the treatment period, and mercury was measured in those same feathers, to test whether this minimally invasive technique might be useful in assessing stress due to mercury exposure in the field. The resultant baseline blood corticosterone levels were best explained by models including treatment, time elapsed between disturbance and sampling, sex, and an individual’s change in body mass over the study period. On average, birds under mercury stress alone or in combination with food stress had higher baseline blood corticosterone levels than controls. Interestingly, birds in the dual stress group did not exhibit the expected gradual increase in blood corticosterone levels in the first three minutes after disturbance. This was the opposite of all other groups, in which birds sampled longer after the initial disturbance of researchers entering the room had higher corticosterone levels. Feather corticosterone was not correlated with blood corticosterone. While treatment group alone was not related to feather corticosterone, the interaction between treatment group and pre-treatment feather corticosterone predicted corticosterone in replacement feathers. In individuals that were not exposed to mercury, there was a positive correlation between original and replacement feather corticosterone. However, mercury exposure disrupted this association. Chronic mercury exposure appears to elevate baseline corticosterone levels in adult songbirds, which could be detrimental to their health. Faced with combined mercury and food stressors, the stress response may have been suppressed, as suggested by the lack of an increase in corticosterone with time since disturbance. An effect of mercury on feather corticosterone was only observed when considering both treatment group and feather corticosterone before mercury exposure, so caution should be exercised in using feather corticosterone as a bioassay for contaminant or food stress in the field. Further study of stress-induced corticosterone is necessary to better understand the effects seen here, and the modulating roles of feather color and size, also examined here, must be considered.

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INTRODUCTION: Diabetes Mellitus affects 415 million people worldwide. It causes hyperglycemia due to impaired insulin production or action. It has been known for a long time that insulin secretion oscillates in vivo and in vitro. These oscillations in insulin release are impaired in diabetic patients. Oscillations in insulin secretion are driven by oscillations in metabolic coupling factors including the ATP/ADP ratio and intracellular Ca2+. The Aim of this thesis is to determine whether phosphorylation of proteins regulating beta-cell lipid metabolism correlates with oscillations in insulin secretion. METHODS: INS-1 cells were cultured in 4 and 11 mM glucose in 48-well plates. Insulin secretion was initiated with 12 mM glucose at timed intervals to generate an oscillation profile over 22 min. Media was collected and insulin was assayed by fluorescence based HTRF insulin assay. Cell protein was extracted with SDS-PAGE sample buffer, separated by electrophoresis and transferred to PVDF membrane for western blotting after SDS PAGE electophoresis. Phosphorylated and unphosphorylated acetyl-CoA carboxylase (ACC) and AMP-activated protein kinase (AMPK) were detected with specific rabbit antibodies (Cell Signaling). Protein bands were detected on a GE LAS-4000 gel imager using enhanced chemiluminescence. Bands were analyzed using ImageJ software (Schneider, Rasband and Eliceiri 2012). RESULTS: Insulin oscillations were detected over the 22 min time course with at least three resolved peaks of insulin secretion for cells cultured in either 4 or 11 mM glucose. The oscillations were of a 5 min period under both culture conditions while the amplitude was 10-20 fold higher in 4 mM glucose cells. The amplitude was dependent on the insulin content of the cells such that when normalized to insulin content the average insulin secretion was well matched between the high and low glucose conditions. Oscillations in pACC/ACC and pAMPK/AMPK ratios were detected in cells cultured in both 4 mM and 11 mM glucose. In cells cultured at 4 mM glucose the pACC/ACC ratio oscillated with a similar period to insulin but was slightly left shifted such that pACC peaked before insulin. This correlation was not as strictly adhered to in cells cultured at high glucose. Oscillations in pAMPK/AMPK tracked well with those of pACC/ACC in cells cultured at both 4 and 11 mM. pAMPK/AMPK peaks were left-shifted relative to peaks in insulin secretion in cells cultured at 4 mM glucose while they seemed to be coincident with insulin peaks in cells cultured at 11 mM glucose. CONCLUSION: Oscillations in insulin secretion are accompanied by oscillations in ACC and AMPK phosphorylation to regulate lipid signals that amplify normal glucose-stimulated insulin secretion. Chronic excess nutrients may alter changes in ACC and AMPK phosphorylation resulting in impaired oscillations in insulin secretion. Regulation of lipid signals in the pancreatic beta-cell may provide therapeutic benefit in the treatment of hyperinsulinemia, insulin resistance and Type 2 diabetes.