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.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20238号 / 医博第4197号 / 新制||医||1019(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 河本 宏, 教授 小杉 眞司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM

No description available.

Type 2 Diabetes (T2D) affects over 415 million people globally and is characterised by cellular stresses including: poor glucose homeostasis, dyslipidaemia, inflammation, hypoxia and ER stress. Studies in mice have shown that exposure to these stresses influences beta cell differentiation status as well as cell survival and may explain the extent of beta cell mass loss that is seen in the disease. To date, studies of altered beta cell differentiation have largely been confined to murine models. I used the EndoC-bH1 human beta cell line, along with human pancreatic tissue sections, to better characterise this mechanism in human disease. To elucidate these mechanisms, I firstly established a humanised version of cell culture techniques for the EndoC βH1 cell model and assessed the influence on cell function. Secondly, I evaluated the effects of the diabetic microenvironment on beta cell differentiation and gene expression patterns. Finally, I investigated whether a diabetomimetic microenvironment induced differences in microRNA regulation in the cells. I found that the humanised EndoC-βH1 culture techniques improved glucose sensitive insulin release in the cell model. EndoC-βH1 cells exposed to a Diabetic microenvironment showed some degree of transdifferentiation and this may be due to dysregulation of splicing factor expression. These effects may be compounded by altered microRNA regulation in response to these cell stresses. These data suggest that altered gene regulation caused by a diabetic microenvironment may alter gene regulation to produce a reversible delta-like phenotype in human beta cells.

Diabetes epidemic is underway. Beta cell dysfunction (BCF) and loss of beta cell mass (BCM) are known to be key events in its progression. Currently, there are no reliable techniques to estimate or follow the loss of BCM, in vivo. Non-invasive imaging and quantification of the whole BCM in the pancreas, therefore, has a great potential for understanding the progression of diabetes and the scope for early diagnosis for Type 2 diabetes. Glucagon-like peptide-1 receptor (GLP-1R) is known to be selectively expressed on the pancreatic beta cells and overexpressed on the insulinoma, a pancreatic neuroendocrine tumor (PNET). Therefore, this receptor is considered to be a selective imaging biomarker for the beta cells and the insulinoma. Exendin-4 is a naturally occurring analog of GLP-1 peptide. It binds and activates GLP-1R with same the potency and engages in the insulin synthesis, with a longer biological half-life. In this thesis, Exendin-4 precursor, DO3A-VS-Cys40-Exendin-4 labeled with [68Ga], [68Ga]Ga-DO3A-VS-Cys40-Exendin-4 ([68Ga]Exendin-4), was evaluated in different species models, namely, immune deficient nude mice, rats, pigs, non-human primate (NHP), and clinically in one insulinoma patient by positron emission tomography (PET), for its potential in beta cell imaging and its quantification as well as for visualizing the insulinoma. From internal dosimetry, the possible number of repetitive [68Ga]Exendin-4-PET/CT scans was estimated. Pancreatic uptake and insulinoma tumor uptake of [68Ga]Exendin-4 were confirmed to be mediated by the specific binding to the GLP-1R. Pancreatic GLP-1R could be visualized and semi-quantified, for diabetic studies, except in rats. Nonetheless, we found conflicting results regarding the GLP-1R being a selective imaging biomarker for the beta cells. PET/CT scan of the patient with [68Ga]Exendin-4 has proven to be more sensitive than the clinical neuroendocrine tracer, [11C]5-HTP, as  it could reveal small metastatic tumors in liver. The kidney was the dose-limiting organ in the entire species model, from absorbed dose estimation. Before reaching a yearly kidney limiting dose of 150 mGy and a whole body effective dose of 10 mSv, 2–4 [68Ga]Exendin-4 PET/CT scans be performed in an adult human, which enables longitudinal clinical PET imaging studies of the GLP-1R in the pancreas, transplanted islets, or insulinoma, as well as in healthy volunteers enrolled in the early phase of anti-diabetic drug development studies.

Complications in pregnancy elevate fetal norepinephrine (NE) concentrations. Previous studies in NE-infused sheep fetuses revealed that sustained exposure to high NE resulted in lower expression of α2-adrenergic receptors in islets and increased insulin secretion responsiveness after acutely terminating the NE infusion. In this study, we determined if the compensatory increase in insulin secretion after chronic elevation of NE is independent of hyperglycemia in sheep fetuses and whether it is persistent in conjunction with islet desensitization to NE. After an initial assessment of glucose-stimulated insulin secretion (GSIS) at 129 ± 1 days of gestation, fetuses were continuously infused for seven days with NE and maintained at euglycemia with a maternal insulin infusion. Fetal GSIS studies were performed again on days 8 and 12. Adrenergic sensitivity was determined in pancreatic islets collected at day 12. NE infusion increased (P < 0.01) fetal plasma NE concentrations and lowered (P < 0.01) basal insulin concentrations compared to vehicle-infused controls. GSIS was 1.8-fold greater (P < 0.05) in NE-infused fetuses compared to controls at both one and five days after discontinuing the infusion. Glucose-potentiated arginine-induced insulin secretion was also enhanced (P < 0.01) in NE-infused fetuses. Maximum GSIS in islets isolated from NE-infused fetuses was 1.6-fold greater (P < 0.05) than controls, but islet insulin content and intracellular calcium signaling were not different between treatments. The half-maximal inhibitory concentration for NE was 2.6-fold greater (P < 0.05) in NE-infused islets compared to controls. These findings show that chronic NE exposure and not hyperglycemia produce persistent adaptations in pancreatic islets that augment β-cell responsiveness in part through decreased adrenergic sensitivity.

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

Zinc levels in pancreatic islets are amongst the highest in the body and reduction in its levels in the pancreas has been associated with diabetes. The link between zinc, diabetes and islet dysfunction has recently been reiterated by genome-wide association studies that identified an islet cell membrane zinc transporter, SLC30A8 (ZnT8), as one of the risk loci for type 2 diabetes. Here we begin to elucidate the molecular mechanisms linking ZnT8 and type 2 diabetes by characterizing global and beta cell specific ZnT8 knockout mice. Our results associate absence of ZnT8 with a reduction in zinc sequestration into insulin vesicles, abnormal insulin granule morphology, down regulation of insulin processing enzymes, abnormal insulin secretion, elevated plasma proinsulin levels and diet-induced obesity and insulin resistance. Furthermore, we observed differential zinc uptake properties by two human ZnT8 variants. We report here that the W325 variant of ZnT8 is more efficient in mediating zinc transport than the at risk variant, R325. Cumulatively, these results suggest that ZnT8 is crucially important for zinc transport and zinc-insulin crystallization in insulin granules of the pancreatic beta cell.

GABA and GABA type A receptor (GABAAR) are expressed in pancreatic β-cells and comprise an autocrine signaling system. How the GABA-GABAAR system is regulated is unknown. In this study, I investigated insulin’s effect on this system in the INS-1 β-cell line. I found that GABA evoked current (IGABA) in INS-1 cells, resulting in membrane depolarization. Perforated-patch recordings showed that pre-treatment of insulin or zinc-free insulin suppressed IGABA in INS-1 cells (p < 0.01). Radioimmunossay showed that GABA (30 μM) increased C-peptide secretion from INS-1 cells, which was blocked by GABAAR antagonist picrotoxin, indicating that GABA increased insulin secretion through activation of GABAAR. However, insulin significantly reduced the stimulatory effect of GABA on C-peptide secretion (p < 0.05). These data suggest that GABA released from β-cells positively regulates insulin secretion via GABAAR activation, and that insulin negatively regulates the β-cell secretory pathway likely via inhibiting the GABA-GABAAR system in β-cells.

GABA and GABA type A receptor (GABAAR) are expressed in pancreatic β-cells and comprise an autocrine signaling system. How the GABA-GABAAR system is regulated is unknown. In this study, I investigated insulin’s effect on this system in the INS-1 β-cell line. I found that GABA evoked current (IGABA) in INS-1 cells, resulting in membrane depolarization. Perforated-patch recordings showed that pre-treatment of insulin or zinc-free insulin suppressed IGABA in INS-1 cells (p < 0.01). Radioimmunossay showed that GABA (30 μM) increased C-peptide secretion from INS-1 cells, which was blocked by GABAAR antagonist picrotoxin, indicating that GABA increased insulin secretion through activation of GABAAR. However, insulin significantly reduced the stimulatory effect of GABA on C-peptide secretion (p < 0.05). These data suggest that GABA released from β-cells positively regulates insulin secretion via GABAAR activation, and that insulin negatively regulates the β-cell secretory pathway likely via inhibiting the GABA-GABAAR system in β-cells.