Thesis (Ph. D. in Cell and Developmental Biology)--Vanderbilt University, Dec. 2009. / Title from title screen. Includes bibliographical references.

No description available.

No description available.

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.

The key defects characteristic of hyperglycemia in T2D include increased hepatic glucose production, a diminution of insulin secretion, and an absolute impairment in peripheral insulin action. The objective of my thesis was to investigate the molecular mechanisms leading to fatty acid induced β-cell dysfunction and determine the role of a novel transcriptional coregulator in the regulation of hepatic glucose homeostasis. The first part of my work focused on the chronic effects of fatty acids on human pancreatic β-cell function. Using microarray technology I established an important role for fatty acids in the pathogenesis of β-cell dysfunction. Accordingly chronic exposure of islets to oleate resulted in a significant reduction in glucose-stimulated insulin secretion and to an increase in the rate of reactive oxygen species generation. Additionally, pre-treatment of human islets with oleate led to a significant increase in the rate of oxidation of this fatty acid and to a significant decrease in glucose oxidation. My data indicate that chronic exposure of human islets to fatty acids activates inflammatory and metabolic pathways that lead to oxidative stress. In addition, the first part of my work demonstrated that fatty acids induce oxidative stress in vitro an effect that is preventable to a large extent by the use of antioxidants. In this setting and recapitulating the human islet data, fatty acids are causally linked to impaired insulin secretion, and the induction of oxidative stress. Our report demonstrated that oxidative stress plays a key role in the decrease in β-cell function induced by chronic lipotoxicity. My work also demonstrated that fatty acids are causally linked to the induction of endoplasmic reticulum stress in human islets. Finally, in the second part of my work I provide novel evidence for the role of PHIP in the regulation of hepatic gluconeogenesis. My work is the first to demonstrate that PHIP suppresses hepatic gluconeogenesis in vitro and in vivo. PHIP is amongst the few proteins that have ever been reported to suppress gluconeogenesis to date. PHIP thus represents a novel target for pharmaceutical intervention of diabetes and the suppression of hepatic glucose production.

The key defects characteristic of hyperglycemia in T2D include increased hepatic glucose production, a diminution of insulin secretion, and an absolute impairment in peripheral insulin action. The objective of my thesis was to investigate the molecular mechanisms leading to fatty acid induced β-cell dysfunction and determine the role of a novel transcriptional coregulator in the regulation of hepatic glucose homeostasis. The first part of my work focused on the chronic effects of fatty acids on human pancreatic β-cell function. Using microarray technology I established an important role for fatty acids in the pathogenesis of β-cell dysfunction. Accordingly chronic exposure of islets to oleate resulted in a significant reduction in glucose-stimulated insulin secretion and to an increase in the rate of reactive oxygen species generation. Additionally, pre-treatment of human islets with oleate led to a significant increase in the rate of oxidation of this fatty acid and to a significant decrease in glucose oxidation. My data indicate that chronic exposure of human islets to fatty acids activates inflammatory and metabolic pathways that lead to oxidative stress. In addition, the first part of my work demonstrated that fatty acids induce oxidative stress in vitro an effect that is preventable to a large extent by the use of antioxidants. In this setting and recapitulating the human islet data, fatty acids are causally linked to impaired insulin secretion, and the induction of oxidative stress. Our report demonstrated that oxidative stress plays a key role in the decrease in β-cell function induced by chronic lipotoxicity. My work also demonstrated that fatty acids are causally linked to the induction of endoplasmic reticulum stress in human islets. Finally, in the second part of my work I provide novel evidence for the role of PHIP in the regulation of hepatic gluconeogenesis. My work is the first to demonstrate that PHIP suppresses hepatic gluconeogenesis in vitro and in vivo. PHIP is amongst the few proteins that have ever been reported to suppress gluconeogenesis to date. PHIP thus represents a novel target for pharmaceutical intervention of diabetes and the suppression of hepatic glucose production.

No description available.

No description available.

No description available.

INTRODUCTION Carnitine palmitoyltransferase I, which is expressed in the pancreas as the liver isoform (LCPTI), catalyzes the rate-limiting step in the transport of fatty acids into the mitochondria for their oxidation. To directly examine whether the availability of long-chain fatty acyl-CoA affects the regulation of insulin secretion in the Beta-cell, we infected INS(832/13) cells and rat islets with an adenovirus encoding a mutant form of LCPTI (Ad-LCPTI M593S) that is insensitive to its inhibitor malonyl-CoA. C75 is described as a potential drug for treatment of obesity and type 2 diabetes. First known as a synthetic inhibitor of fatty acid synthase, it has been also described as an activator of CPTI, increasing peripheral energy utilization and fatty acid oxidation in mice. To further investigate the C75/CPTI interaction, we have characterized the effects of C75 on CPTI in vitro and in vivo.OBJECTIVES 1) Study of the malonyl-CoA/CPTI interaction in the pancreatic Beta-cell and its involvement in glucose-stimulated insulin secretion (GSIS). 2) Construction of an INS stable cell line overexpressing LCPTI wt and LCPTI M593S. 3) Determine the effect of C75 on the CPTI activity and palmitate oxidation in pancreatic Beta-cells. RESULTS. In Ad-LCPTI M593S infected INS(832/13) cells LCPTI activity increased six-fold. This was associated with enhanced fatty acid oxidation, at any glucose concentration, and a 60% suppression of GSIS. In isolated rat islets in which LCPTI M593S was overexpressed, GSIS decreased 40%. At high glucose concentration, overexpression of LCPTI M593S reduced partitioning of exogenous palmitate into lipid esterification products, and decreased PKC activation. Moreover, LCPTI M593S expression impaired KATP channel-independent GSIS in INS(832/13) cells. INS-1 stable clones of LCPTIwt and LCPTImut were constructed, however none of them resulted in an increase in LCPTI protein expression compared to endogenous LCPTI nor in CPTI activity. Therefore, slight basal overexpression of LCPTI could probably be toxic for the cells, as a result of which only those cells that do not contain the LCPTI plasmids survived throughout cell passages. When INS(823/13) cells are incubated with C75, CPTI activity is inhibited, as is fatty acid oxidation. In vivo, a single intraperitoneal injection of C75 to mice produces a short-term inhibition of CPTI activity in mitochondria from liver and pancreas.DISCUSSION. The results with LCPTImut provide direct support for the hypothesis proposing that the malonyl-CoA/CPTI interaction is a component of a metabolic signalling network that controls insulin secretion. Overall, the findings with C75 provide compelling evidence that the drug is a potent inhibitor of CPTI.