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Diabetes Mellitus is characterized by high blood sugar and is caused by resistance to (type 2) or insufficiency of (type 1) the pancreatic β-cell hormone insulin. Most commonly, type 2 diabetes is associated with obesity whereas type 1 diabetes is largely a result of immune-mediated destruction of the β-cell. One rare but significant cause of type 2 diabetes is excess blood glucocorticoid levels (Cushing’s syndrome). High circulating glucocorticoids potently induce metabolic disorders including peripheral insulin resistance in key metabolic tissues (muscle, liver and fat) as well as directly suppressing β-cell function and can precipitate type 2 diabetes. However, in common forms of metabolic syndrome (visceral obesity, type 2 diabetes, increased cardiovascular disease risk) it appears that amplification of local tissue glucocorticoid action by increased levels of the intracellular enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), particularly in adipose tissue, is a key driver of the adverse metabolic phenotype rather than altered circulating glucocorticoid levels. 11β-HSD1 is also elevated in pancreatic islets from obese rodents. This thesis aimed to determine the role of 11β-HSD1 in pancreatic islets (β-cells) under normal conditions and its potential pathogenic role in the development of diabetes. We first determined that 11β-HSD1 acted primarily as a reductase (amplifying glucocorticoid action) in pancreatic islets. We then determined that islet 11β-HSD1 transcription is under the control of the promoters that express in other tissues like liver. Islet 11β-HSD1 is significantly regulated by factors relevant to the diabetic state; high glucose and insulin suppressed whereas fatty acids and TNFα increased 11β-HSD1 activity. To test whether the high islet 11β-HSD1 found in obese rodents was directly diabetogenic, we generated transgenic mice specifically overexpressing β-cell 11β-HSD1 under the mouse insulin promoter (MIP-HSD1 mice) in a mouse strain prone to develop β-cell failure when subjected to diabetic challenge (eg. chronic high fat feeding). Unexpectedly, MIP-HSD1tg/+ mice (expressing ~2 fold elevated 11β-HSD1 activity) exhibited markedly improved β-cell insulin secretory responses, whereas MIP-HSD1tg/tg mice had partially impaired β-cell insulin secretory function in vivo and in vitro. Moreover, MIP-HSD1tg/+ mice completely resisted the mild hyperglycaemia induced by multiple-low doses of the β-cell toxin streptozotocin (40mg/kg i.p. for 5 days) and partially resisted the profound hyperglycaemia induced by a single high dose of streptozotocin (180mg/kg). Notably, MIP-HSD1tg/+ mice exhibited lower macrophage infiltration (MAC-2) and higher T-regulatory cell (Foxp3) infiltration after these challenges with evidence of increased insulin-positive cells and maintenance of normal levels of proliferation-competent β-cells. Overall, MIP-HSD1tg/tg exhibited a partial protection from the streptozotocin challenge. Modestly increased 11β-HSD1 expression in β-cells unexpectedly supports compensatory insulin hypersecretion preventing type 2 diabetes and protects β-cells from inflammatory mediated damage in the setting of type 1 diabetes. Above a protective threshold, elevated β-cell 11β-HSD1 may result in β-cell dysfunction and diabetes. These findings have important implications for the currently advocated therapeutic strategies to inhibit 11β-HSD1 in the context of obesity and diabetes.

Pancreatic beta-cells secrete insulin in response to increase in blood glucose concentration with a rapid first phase and slower, sustained second phase. This secretion pattern is similar in entire pancreas, isolated islets of Langerhans and single beta-cells and it is disrupted in type 2-diabetes. Insulin stored in secretory vesicles has to undergo preparatory steps upon translocation to the plasma membrane which include docking and priming before being released by exocytosis. A better understanding of the molecules involved in these steps is required to determine the rate limiting factors for sustained secretion. Here these processes were studied in real time using total internal reflection fluorescence microscopy, which enables observation of insulin granules localized at the plasma membrane. A pool of granules morphologically docked at the plasma membrane was found to be depleted upon repeated stimulations. Recovery of the docked pool of granules took tens of minutes and became rate limiting for sustained secretion. Shorter depolarization stimuli did not deplete the docked pool and allowed rapid recovery of releasable granules. When a new granule arrived at the plasma membrane, docking was initiated by de novo formation of syntaxin/munc18 clusters at the docking site. Two-thirds of the granules which arrived at the plasma membrane failed to recruit these proteins and hence failed to dock. Priming involved recruitment of several other proteins including munc13, SNAP25 and Cav1.2 channels. Exocytosing granules were in close proximity to Ca2+ influx sites with high degree of association with Cav1.2 channels. This is because of the association of these channels to exocytosis site through syntaxin and SNAP25. During exocytosis the assembled release machinery disintegrated and the proteins at the release site dispersed. Syntaxin dispersal was initiated already during fusion pore formation rather than after release during exocytosis. This was studied using a newly developed red fluorescent probe - NPY-tdmOrange2 which was the most reliable pH sensitive red granule marker to label insulin granules. Overall these data give new insights into the molecular mechanisms involved in biphasic insulin secretion. Disturbances in the secretion at the level of granule docking and fusion may contribute to the early manifestations of type-2 diabetes.

This thesis gives insight into the way that transcription of the insulin gene is regulated by nutrients. This is achieved primarily by characterising a MAP kinase pathway which links glucose metabolism to the activation of a beta cell transcription factor IUF1. An understanding of the precise mechanisms by which nutrients control beta cell function may be invaluable for the development of artificial cell lines that can be used for gene replacement therapy. A study of the E2 element of the rat II promoter illustrated that at least three factors bound to the region. These were identified as IUF1 (complex D5), USF (complex D4) and an uncharacterised factor D3. IUF1 is a beta cell specific transcription factor that has been implicated previously in glucose responsive insulin gene transcription. IUF1 binds to the insulin promoter in response to high levels of extracellular glucose. USF has been shown to be involved in the carbohydrate responsive transcription of various hepatic genes. The recently characterised stress activated (Reactivating Kinase) MAP kinase pathway was clearly shown to be involved in mediating the link between glucose metabolism within the beta cell and the binding activity of IUF1. Phosphorylation of the factor serves to induce an alteration in protein structure, which converts the factor to an active form that shows a high affinity for its DNA binding site, thus activating transcription. The RK pathway may prove to be a crucial link between nutrient metabolism and the activity of other physiological processes.

Our study aims to investigate the role of Syntaxin-3 in glucose stimulated insulin secretion (GSIS) and how it regulates the recruitment to plasma membrane and/or exocytotic fusion of insulin granules. We examined endogenous Syn-3 function by down-regulating its expression using siRNA/lenti-shRNA, which impaired GSIS. Although Syn-3 depleted cells showed no change in the number and fusion of docked granules, there was a reduction in newcomer granules and their subsequent exocytotic fusion. We then examined the effects of overexpressing Syn-3-WT, which enhanced biphasic GSIS. Since open conformation (OF) Syn-1A was reported to enhance exocytosis by promoting SNARE complex formation, we constructed OF Syn-3. Exogenous OF Syn-3 had no effect on secretion as it is unable to be trafficked to insulin granules. Taken together, we conclude that Syn-3 facilitates mobilization of newcomer insulin granules to the plasma membrane, to contribute to both first and second phase of GSIS in pancreatic beta cells.

Our study aims to investigate the role of Syntaxin-3 in glucose stimulated insulin secretion (GSIS) and how it regulates the recruitment to plasma membrane and/or exocytotic fusion of insulin granules. We examined endogenous Syn-3 function by down-regulating its expression using siRNA/lenti-shRNA, which impaired GSIS. Although Syn-3 depleted cells showed no change in the number and fusion of docked granules, there was a reduction in newcomer granules and their subsequent exocytotic fusion. We then examined the effects of overexpressing Syn-3-WT, which enhanced biphasic GSIS. Since open conformation (OF) Syn-1A was reported to enhance exocytosis by promoting SNARE complex formation, we constructed OF Syn-3. Exogenous OF Syn-3 had no effect on secretion as it is unable to be trafficked to insulin granules. Taken together, we conclude that Syn-3 facilitates mobilization of newcomer insulin granules to the plasma membrane, to contribute to both first and second phase of GSIS in pancreatic beta cells.

Indiana University-Purdue University Indianapolis (IUPUI) / Diabetes mellitus is a complex metabolic disease that currently affects an estimated 422 million people worldwide, with incidence rates rising annually. Type 1 diabetes (T1D) accounts for 5-10% of these cases. Its complications remain a major cause of global deaths. T1D is characterized by autoimmune destruction of β-cell mass. Efforts to preserve and protect β-cell mass in the preclinical stages of T1D are limited by few blood-borne biomarkers of β-cell destruction. In healthy β-cells, insulin secretion requires soluble n-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) complexes and associated accessory regulatory proteins to promote the docking and fusion of insulin vesicles at the plasma membrane. Two target membrane (t)-SNARE proteins, Syntaxin 1/4 and SNAP25/23, and one vesicle-associated (v)-SNARE protein, VAMP2, constitute the SNARE core complex. SNARE complex assembly is also facilitated by the regulatory protein, Double C2-domain protein β (DOC2B). I hypothesized that DOC2B deficiency may underlie β-cell susceptibility to T1D damage; conversely , overexpression of DOC2B may protect β-cell mass. Indeed, with regard to DOC2B abundance, my studies show reduced levels of DOC2B in platelets and islets of prediabetic rodents and new-onset T1D humans. Remarkably, clinical islet transplantation in T1D humans restores platelet DOC2B levels, indicating a correlation With regard to protection/functional effects, DOC2B deficiency enhances susceptibility to T1D in mice, while overexpression of DOC2B selectively in β-cells protects mice from chemically induced T1D; this correlates with preservation of functional β-cell mass. Mechanistically, overexpression of DOC2B and the DOC2B peptide, C2AB, protects clonal β-cell against cytokine or thapsigargin-induced apoptosis and reduces ER stress; this is dependent on C2AB’s calcium binding capacity. C2AB is sufficient to enhance glucose stimulated insulin secretion (GSIS) and SNARE activation in clonal β-cells to the same extent as full-length DOC2B. In summary, these studies identify DOC2B as a potential biomarker and novel therapeutic target for prevention/management of T1D.

OBJECTIVE: Type 2 diabetes (T2D), the most common type of diabetes characterized by high blood glucose and insulin resistance, results from both genetic and environmental factors. Our lab has proposed that chronic excess nutrients induce insulin hypersecretion from the pancreatic ß-cell, contributing to hyperinsulinemia, a prequel to T2D. Normal glucose-stimulated insulin secretion (GSIS) is oscillatory, a feature that is lost in patients with T2D. In this thesis we examine the oscillatory secretion profiles of clonal pancreatic ß-cells cultured in normal and excess nutrients that mimic conditions of T2D. We also begin to examine oscillations in protein phosphorylation that may contribute to normal ß-cell metabolism and GSIS, but if altered might potentially lead to impaired insulin secretion. METHODS: Nutrient regulation of oscillatory insulin release was studied in clonal pancreatic β-cells (INS-1) cultured in multiwell plates in both low (4 mM) and high (11 mM) glucose. Insulin secretion was stimulated in cells from multiwell plates one well at a time at 30 sec intervals and sampled simultaneously at the end of the timecourse. Insulin secretion and insulin content were measured using a homogenous time-resolved fluorescence (HTRF) insulin kit (Cisbio). Protein was extracted from these same cells for analysis of time-dependent phosphorylation by western blot using specific antibodies. Protein phosphorylation was detected using SuperSignal West Femto chemiluminescence reagent (ThermoFisher) and imaged on an iBright Imaging System (Invitrogen). RESULTS: Insulin secretion from INS-1 cells grown in separate plates and in 4 mM glucose oscillated with a period of 8.2  0.5 min compared to 5.0  0.5 min in cells cultured at 11 mM glucose. The amplitude of oscillations was 40.4  11.5 and 14.6  1.5 for cells cultured in 4 and 11 mM glucose respectively. Oscillations in secretion from cells cultured in 4 and 11 mM glucose in the same plate were not different in period but different in amplitude due in part to reduced insulin content. Oscillation in the phosphorylation patterns of acetyl-CoA carboxylase (ACC) and myristoylated alanine rich C kinase substrate (MARCKS) were measured in cells cultured in 4 mM glucose and both exhibited a peak in phosphorylation that occurred at the nadir of the insulin oscillation between peaks of insulin release. CONCLUSION: Insulin secretion from pancreatic ß-cells is affected by nutrient status as excess nutrients decrease the amplitude of oscillations in insulin release. The period of oscillations can also be affected. Oscillations in protein phosphorylation are consistent with both ACC and MARCKS contributing to normal GSIS. These initial studies provide evidence of the suitability of this model system to correlate oscillations in protein activity to exocytosis. Future studies focused on the effects of low and high glucose will potentially reveal new important therapeutic targets that may help prevent/reverse/ameliorate insulin hypersecretion leading to insulin resistance and T2D.

Secretion of appropriate amounts of insulin from pancreatic β-cells is crucial for glucose homeostasis. The β-cells release insulin in response to glucose and other nutrients, hormones and neurotransmitters, which trigger intracellular signaling cascades, that result in exocytotic fusion of insulin-containing vesicles with the plasma membrane. Increases of the intracellular concentration of calcium ions ([Ca2+]i) trigger exocytosis, whereas the messenger cyclic adenosine monophosphate (cAMP) amplifies various steps of the secretion process. The protein Epac2 mediates some effects of cAMP, but little is known about its regulation in β-cells. In this study, the spatio-temporal dynamics of Epac2 was investigated in insulin-secreting MIN6-cells and primary β-cells using various cell signaling biosensors and live-cell fluorescence microscopy approaches. Increases in the cAMP concentration triggered translocation of Epac2 from the cytoplasm to the plasma membrane. Oscillations of cAMP induced by glucose and the insulin-releasing hormone GLP-1 were associated with cyclic translocation of Epac2. Analyses of Epac2 mutants showed that the high-affinity cyclic nucleotide-binding domain and Ras-association domains were crucial for the translocation, whereas neither the DEP domain, nor the low-affinity cAMP-binding domain were required for membrane binding. However, the latter domain targeted Epac2 to insulin granules at the plasma membrane, which promoted their priming for exocytosis. Depolarization-induced elevations of [Ca2+]i also stimulated Epac2 translocation, but the effects were complex and in the presence of high cAMP concentrations, [Ca2+]i increases often reduced membrane binding. The stimulatory effect of Ca2+ was mediated by increased Ras activity, while the inhibitory effect reflected reduced concentrations of the membrane phospholipid PtdIns(4,5)P2. Anti-diabetic drugs of the sulfonylurea class, suggested to directly activate Epac2, induced translocation indirectly by depolarizing β-cells to increase [Ca2+]i. Epac2 is an activator of Rap GTPases, and its translocation increased Rap activity at the plasma membrane. It is concluded that the subcellular localization of Epac2 is controlled by a complex interplay between cAMP, Ca2+ and PtdIns(4,5)P2 and that the protein controls insulin release by binding to the exocytosis machinery. These results provide new insights into the regulation of β-cell function and may facilitate the development of new anti-diabetic drugs that amplify insulin secretion.

Pancreatic beta-cells regulate blood glucose by secreting insulin in response to nutrients. The development of Type 2 Diabetes (T2D) is characterized by elevated insulin secretion in the fasted state and a failure to adequately respond to nutrient influx, particularly glucose. Current dogma states that insulin resistance is the initiating event in the development of T2D, with compensation by beta-cells necessary to maintain glucose homeostasis. An alternative model, which will be a central theme throughout this thesis, is that hypersecretion of insulin is the initiating and sustaining event in the development of T2D. The underlying cause of insulin hypersecretion is unclear. Determining this is important in order to test this alternative model as a viable target for prevention and treatment of T2D. Because of the association between obesity and hyperinsulinemia, we hypothesized that exposure of the β-cell to high levels of nutrients stimulates insulin hypersecretion. We found that chronic incubation of β-cells in high glucose and/or oleate, which mimics nutrient conditions in obesity, lowered the half-maximal response for glucose to stimulate insulin secretion. The degree of the left-shift correlated with lipid stores. We determined that heightened sensitivity of granule exocytosis to Ca2+ was driving this left-shift. Thus glucose, while not necessarily abnormal in obesity, may cause hypersecretion of insulin due to altered sensitivity of the β-cell to this secretagogue. Iron stores are increased in obesity and are predictive of T2D development. We found that iron acutely stimulated both basal and glucose-stimulated insulin secretion (GSIS) in a reactive oxygen species dependent manner. Interestingly, iron did not increase insulin secretion via Ca2+ influx. Thus, both iron and glucose/oleate induce insulin hypersecretion via an aspect of the triggering pathway that is not Ca2+, the putative triggering signal. Previous work in our laboratory documented that exogenous mono-oleoyl-glycerol, an endogenous lipid signaling molecule and food additive, increases basal insulin secretion. We found that inhibition of monoacylglycerol lipase, which increases cellular monacylglycerol species, reduced GSIS, possibly via a reduction in long-chain CoA. Collectively, our works supports the hypothesis that chronic exposure to high nutrient levels drives insulin hypersecretion in obesity. / 2018-06-15T00:00:00Z