Mutations in HNF1A cause Maturity Onset Diabetes of the Young type 3, the second most frequent form of diabetes caused by single gene mutation. We generated human stem cell-derived pancreatic endocrine cells with clinically pathogenic mutations in HNF1A and show that HNF1A deficiency impairs endocrine cell fate, insulin granule maturation and the secretion of insulin in response to glucose. Single-cell RNA sequencing reveals that HNF1A orchestrates a network of genes involved in β-cell fate, granule maturation, glucose metabolism, calcium ion binding and hormone exocytosis. In both patients and stem cell-derived β-cells, HNF1A deficiency altered the stoichiometry of secreted insulin to c-peptide. Sulfonylurea, used in the treatment of these patients, restored both insulin secretion and stoichiometry. The uncoupling of insulin and c-peptide secretion as described here questions the common practice of using c-peptide as a proxy to evaluate β-cell function. We also demonstrate that correction of the HNF1A mutations restores function, providing a path to cell-based replacement therapy.

Human and murine diabetes is characterized by pancreatic β-cell dedifferentiation, a process in which β-cells lose expression of markers of maturity and gain those of endocrine progenitors. Failing β-cells inappropriately metabolize lipids over carbohydrates and exhibit impaired mitochondrial oxidative phosphorylation. Therefore, pathways involved in mitochondrial fuel selection and catabolism may represent potential targets for the prevention or reversal of dedifferentiation. In chapter I of this dissertation, we isolated and functionally characterized failing β-cells from various experimental models of diabetes. We found a striking enrichment in the expression of aldehyde dehydrogenase 1 isoform A3 (Aldh1a3) as β-cells become dedifferentiated. Flow-sorted Aldh1a3-expressing (ALDH+) islet cells demonstrate impaired glucose-induced insulin secretion, are depleted of Foxo1 and MafA, and include a Neurogenin3-positive subset. RNA sequencing analysis demonstrated that ALDH+ cells are characterized by: (i) impaired oxidative phosphorylation and mitochondrial complex I, IV, and V; (ii) activated RICTOR; and (iii) progenitor cell markers. We propose that impaired mitochondrial function marks the progression from metabolic inflexibility to dedifferentiation in the natural history of β-cell failure. In chapter II of this dissertation, we report that cytochrome b5 reductase 3 (Cyb5r3) is a FoxO1-regulated mitochondrial oxidoreductase critical to β cell function. Expression of Cyb5r3 is greatly decreased in multiple murine models of diabetes, and in vitro Cyb5r3 knockdown leads to increased ROS generation and impairment of respiration, mitochondrial function, glucose-stimulated insulin secretion, and calcium mobilization. In vivo, mice with β-cell-specific ablation of Cyb5r3 (B-Cyb5r3) display impaired glucose tolerance with decreased insulin secretion, and their islets have significantly lower basal respiration and glucose-stimulated insulin secretion. B-Cyb5r3 β-cells lose expression of Glut2, MafA, and Pdx1 expression despite a compensatory increase in FoxO1 expression. Our data suggest that Cyb5r3 is a critical mediator of FoxO1’s protective response in β-cells, and that loss of Cyb5r3 expression is an early event in β-cell failure.

Endoplasmic reticulum (ER) stress has been implicated in pancreatic beta-cell loss contributing to diabetes mellitus, however the molecular mechanisms of ER stress-induced apoptosis are unclear. In the first project of this thesis, the contribution of ER stress in proinflammatory cytokine-mediated beta-cell dysfunction and apoptosis is examined. Although exogenous cytokine treatment did induce unfolded protein response (UPR) genes, increased chaperone capacity had no effect on apoptosis induction, insulin biosynthesis and insulin secretion. Thus, ER stress is most likely not an important pathway in cytokine toxicity under our experimental system. The second project develops a pathophysiological model of ER stress based on the mutant misfolded insulin of the Akita mouse. Microarray analysis was conducted and we observed early induction of ER chaperone and ER-associated degradation (ERAD) genes, followed by a large increase in pro-apoptotic genes with mutant insulin expression. A detailed analysis of the ER stress response in this system is presented.

Endoplasmic reticulum (ER) stress has been implicated in pancreatic beta-cell loss contributing to diabetes mellitus, however the molecular mechanisms of ER stress-induced apoptosis are unclear. In the first project of this thesis, the contribution of ER stress in proinflammatory cytokine-mediated beta-cell dysfunction and apoptosis is examined. Although exogenous cytokine treatment did induce unfolded protein response (UPR) genes, increased chaperone capacity had no effect on apoptosis induction, insulin biosynthesis and insulin secretion. Thus, ER stress is most likely not an important pathway in cytokine toxicity under our experimental system. The second project develops a pathophysiological model of ER stress based on the mutant misfolded insulin of the Akita mouse. Microarray analysis was conducted and we observed early induction of ER chaperone and ER-associated degradation (ERAD) genes, followed by a large increase in pro-apoptotic genes with mutant insulin expression. A detailed analysis of the ER stress response in this system is presented.

Thesis (Ph. D.)--School of Chemical Engineering, Georgia Institute of Technology, 2004. Directed by Athanassios Sambanis. / Includes bibliographical references (leaves 125-135).

Diabetes is a chronic disorder of the pancreas, where a decline in the insulin-producing β-cell population disrupts metabolic homeostasis. Pancreatic transplantation has shown to be effective in circumventing the problem of β-cell insufficiency. However, availability of donor islets remains an obstacle. Although progressive differentiation of embryonic stem cells (ESCs) to pancreatic β-cells is a solution, current protocols are wrought with inefficiencies. It is obvious that to realize ESC differentiation for therapy many steps need to be optimized, and this study describes improvement of Pdx1+pancreatic progenitor derivation, a critical determinant of pancreatic fate. The compounds melatonin and sPDZD2 have been suggested to act through the Protein Kinase A (PKA) pathway to exert transcriptional effects, and in particular sPDZD2 stimulates the expression of pancreatic genes in INS-1E rat pancreatic cells. This led to the hypothesis that the PKA-targeting characteristics of said molecules could be exploited for pancreatic specification through post-translational activation ofPdx1. hESCs were first induced to form definitive endoderm before treatment with melatonin and sPDZD2. Pdx1 expression induced by these molecules was then compared with levels triggered by known pancreatic progenitor inducer Indolactam V (ILV). A secondary objective of this study was to assess the endoderm induction potential of small molecules in hESCs, which claim to be potentially useful in differentiation. In this research, I show that small molecules are noticeably more challenging to use in the hESC context. Between the TGF-β pathwayactivatorsIDE-1 and 2, the latter is more potent at inducing endoderm formation, though it does not surpass the capabilities of Stauprimide, a molecule originally thought to only serve a priming purpose in mESCs.IDE-2 and Stauprimide consistently perform better than Activin A, the near universal factor for endoderm induction. Possible synergy between IDE-2 and Stauprimide was explored, but their combination appears detrimental to Sox17expression. Subsequent pancreatic differentiation was also inefficient, and my results affirm the immaturity of chemically-induced endoderm by contrasting with mainstream means of endoderm induction; levels of endoderm marker expression between the two methods are millions of folds apart. This work exposes the risks of using small molecules, and they necessitate proper characterization before being adopted for differentiation. Most favorably, both sPDZD2 and melatonin were able to trigger Pdx1 expression in STEMDiffTm derived definitive endoderm; 10 and 30folds respectively, comparable to the known Pdx1 inducer ILV (25 folds). I also reveal concentration-mediated differentiation and proliferative purposes of ILV and sPDZD2, which are highly reminiscent of the signaling mechanisms involved during pancreatic development. Preliminary quantification of Pdx1+ cells suggest that high concentrations of ILV and sPDZD2 favor self-renewal of Pdx1+ progenitors, whilst lower doses elevate Pdx1 expression. Demonstration of Pdx1 at both gene and protein expression levels was encouraging, but it remains uncertain if melatonin and sPDZD2 manipulate PKA signaling to exert Pdx1 promoting effects. My work supports the use of melatonin as a candidate for pancreatic differentiation, and suggests involvement of sPDZD2 in deriving and expanding progenitors during pancreatic organogenesis. / published_or_final_version / Biochemistry / Master / Master of Philosophy

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Type 1 diabetes is an autoimmune disease in which patients' insulin-secreting beta cells in pancreatic islets are destroyed by their own immune system, leading to unregulated blood glucose levels and severe complications. Its only treatment is intensive insulin therapy, which carries the risk of hypoglycemic episodes and can result in seizures, coma, and even death. Islet transplantation has recently become an alternative, albeit experimental, treatment for type 1 diabetes patients. More than one donor graft is usually required to render recipients insulin independent, making the shortage of donor tissue an extremely important challenge in islet transplantation. Identifying the cell type that has the ability to differentiate into islet-like tissue is an important area of study. In this study, I hypothesized that insulin secreting human islet-like clusters could be generated from pancreatic ductal cells, a potential pancreatic progenitor cell type. Islet-like clusters were generated using crude exocrine tissue from human cadaveric donors. This crude exocrine tissue contained a large number of ductal cells, as well as other pancreatic cell types. To evaluate insulin secretion by human islet-like clusters, a static incubation system was set up and tested using Min6 cells, a known insulin-secreting cell line. Using static incubation, significant increases in insulin secretion by islet-like clusters were observed when the clusters were exposed to higher glucose levels and GLP-1, a known insulin secretagogue. Presence of corresponding C-peptide secretion demonstrated that de novo insulin secretion occurred. Furthermore, basal insulin secretion increased as culture stages progressed. An attempt was made to generate islet-like clusters using ductal cells purified by fluorescent activated cell sorting or magnetic activated cell sorting. Nevertheless, it was difficult to ensure survival and proliferation of purified ductal cells. Further studies will be necessary to confirm the role of ductal cells in the generation of islet-like clusters using the crude exocrine tissue, as well as to identify factors that can promote ductal cells proliferation after cell sorting.

Type 2 diabetes is now recognized as a worldwide epidemic. Pancreatic beta-cell decompensation in the presence of insulin resistance is a major mechanism for the development of type 2 diabetes and may be triggered by mitochondrial dysfunction. Alcoholism is a known risk factor for type 2 diabetes. Excessive or chronic alcohol consumption leads to increased oxidative stress and mitochondrial dysfunction in beta-cells. Prohibitin is a multifunctional protein that also regulates mitochondrial biogenesis and function. Although it has anti-oxidant effects in some cell types, its role in pancreatic beta-cells is not known. This study has investigated the effects of prohibitin in ethanol treated pancreatic beta-cells using RINm5F and INS-1E cell lines. Prohibitin was found to be expressed in pancreatic beta-cells with localization to the nucleus and the perinuclear area. Ethanol increased the expression of prohibitin and induced its translocation from the nucleus to the mitochondria. Ethanol, through its metabolism by alcohol dehydrogenase (ADH), increased oxidative stress and altered mitochondrial membrane potential, decreased the activity of mitochondrial respiratory complexes I and IV, and uncoupled energy production with resulting reduction in ATP production. This was associated with activation of the proinflammatory enzyme c-Jun N-terminal kinase and proapoptotic proteins Bax and caspase-3, leading to beta-cell apoptosis. Ethanol also reduced glucose induced insulin secretion without alteration of the beta-cell transcription factors PDX-1 and MafA. Treatment with exogenous prohibitin or cellular overexpression of endogenous prohibitin attenuated ADH activity, prevented the deleterious effects of ethanol on mitochondrial function and reduced apoptosis, whereas prohibitin knockdown enhanced ethanol-induced apoptosis. In addition, prohibitin per se increased PDX-1 and MafA levels. Through the above mechanisms, prohibitin restored glucose induced insulin secretion in ethanol exposed beta-cells. In brief, ethanol causes mitochondrial dysfunction and induces apoptosis in beta-cells, which result in a reduction of insulin secretion; whereas prohibitin prevents mitochondrial dysfunction, apoptosis, and -cell failure by stabilizing mitochondrial complexes I and IV and inhibiting ADH activity during ethanol metabolism. In addition, prohibitin in itself increases the levels of beta-cell transcription factors. As a consequence, prohibitin maintains normal pancreatic beta-cell function and could be useful in diabetes prevention and treatment.

Type 2 diabetes is now recognized as a worldwide epidemic. Pancreatic beta-cell decompensation in the presence of insulin resistance is a major mechanism for the development of type 2 diabetes and may be triggered by mitochondrial dysfunction. Alcoholism is a known risk factor for type 2 diabetes. Excessive or chronic alcohol consumption leads to increased oxidative stress and mitochondrial dysfunction in beta-cells. Prohibitin is a multifunctional protein that also regulates mitochondrial biogenesis and function. Although it has anti-oxidant effects in some cell types, its role in pancreatic beta-cells is not known. This study has investigated the effects of prohibitin in ethanol treated pancreatic beta-cells using RINm5F and INS-1E cell lines. Prohibitin was found to be expressed in pancreatic beta-cells with localization to the nucleus and the perinuclear area. Ethanol increased the expression of prohibitin and induced its translocation from the nucleus to the mitochondria. Ethanol, through its metabolism by alcohol dehydrogenase (ADH), increased oxidative stress and altered mitochondrial membrane potential, decreased the activity of mitochondrial respiratory complexes I and IV, and uncoupled energy production with resulting reduction in ATP production. This was associated with activation of the proinflammatory enzyme c-Jun N-terminal kinase and proapoptotic proteins Bax and caspase-3, leading to beta-cell apoptosis. Ethanol also reduced glucose induced insulin secretion without alteration of the beta-cell transcription factors PDX-1 and MafA. Treatment with exogenous prohibitin or cellular overexpression of endogenous prohibitin attenuated ADH activity, prevented the deleterious effects of ethanol on mitochondrial function and reduced apoptosis, whereas prohibitin knockdown enhanced ethanol-induced apoptosis. In addition, prohibitin per se increased PDX-1 and MafA levels. Through the above mechanisms, prohibitin restored glucose induced insulin secretion in ethanol exposed beta-cells. In brief, ethanol causes mitochondrial dysfunction and induces apoptosis in beta-cells, which result in a reduction of insulin secretion; whereas prohibitin prevents mitochondrial dysfunction, apoptosis, and -cell failure by stabilizing mitochondrial complexes I and IV and inhibiting ADH activity during ethanol metabolism. In addition, prohibitin in itself increases the levels of beta-cell transcription factors. As a consequence, prohibitin maintains normal pancreatic beta-cell function and could be useful in diabetes prevention and treatment.