The roles of ATF3, an adaptive-response gene, in pancreatic islet beta-cell stress response and function

The focus of the research performed in this dissertation is on ATF3, a stress inducible transcription factor that plays a central role in the biology of beta-cells. The hypothesis of this dissertation is that ATF3 functions by contributing to the expression of target genes that strongly influence beta-cell survival and function. Previous studies demonstrated that activating transcription factor 3 (ATF3) is a stress-inducible gene and pro-apoptotic in various cell types, including beta-cells. These results support a model that ATF3 plays a role in the pathogenesis of diabetes, partly by its apoptotic effect in beta-cells. Based on this information, I set out to investigate the potential functional significance of ATF3 expression in beta-cells exposed to two physiologically relevant stress paradigms; islet transplantation and high fat diet induced type 2 diabetes.;In chapter 2, I tested whether ATF3 plays a role in the beta-cell stress response to islet transplantation. In the course of this study, I compared wild type (WT) and ATF3 knockout (KO) islets in vitro using stress paradigms relevant to islet transplantation: isolation, inflammation, and hypoxia. I also compared the WT and KO islets in vivo using a syngeneic mouse transplantation model. As a result, I found that ATF3 was induced in all three stress paradigms and played a deleterious role in islet survival, as evidenced by the lower viability of WT islets than KO islets. ATF3 regulated various downstream target genes in a stress-dependent manner. These target genes can be classified into two functional groups: (a) apoptosis (NOXA, bNIP3), and (b) immuno-modulation (TNFalpha, IL1beta, IL6, and CCL2/MCP-1). In vivo, ATF3 KO islets performed better than WT islets after transplantation, as evidenced by better glucose homeostasis in recipients, and reduced caspase 3 activation and macrophage infiltration in the KO grafts. These results identified a role for ATF3 in islet graft rejection by contributing to islet apoptosis and inflammatory responses at the graft sites. Therefore, we feel that silencing ATF3 may provide therapeutic benefits in islet transplantation.;In chapter 3 I tested whether the insertion of the Arg-Gly-Asp (RGD) amino acid sequence, a potent cell adhesion motif, into a capsid protein of the non-immunogenic AAV was capable of improving its islet infection efficiency. Work presented in chapter 3 demonstrates that the RGD modification improves AAV infection efficiency of murine, non-human primate and human islets in vitro. Furthermore, infecting islets with the AAV1/RGD vector did not compromise their ability to function in vivo, as transplant recipients of both non-infected and AAV1/RGD infected islets demonstrated similar ability to control blood glucose levels. These findings are significant in the context of islet transplantation as they identify AAV1/RGD as an improved non-immunogenic viral vector and leaves open the possibility for its use in many therapeutic applications.;In chapter 4, I tested whether ATF3 plays a role in the development of type 2 diabetes by comparing wild type (WT) and ATF3 knockout (KO) mice in high fat diet (HFD)-induced model of diabetes. To our surprise, I found that ATF3 played a protective role in the development of T2DM, as evidenced by reduced glycemic control and serum insulin levels in KO mice compared to WT. No differences in HFD induced insulin resistance, inflammation, and compensation in pancreatic islet mass were identified between WT and KO mice, thus suggesting a role for ATF3 in islet function. Subsequent in vitro studies using gain and loss of function approaches to examine steady-state mRNA levels, reporter activity, in vivo DNA binding, and in vivo transcription, strongly indicated that insulin is a target gene directly up-regulated by ATF. Thus, ATF3 plays a role in both beta-cell apoptosis (from previous work) and function (from this work). Results described in chapter 4 are consistent with a diabetes model that, before beta-cell apoptosis becomes an issue, expression of ATF3 is beneficial and helps beta cells to cope with higher metabolic demand.;In conclusion, work presented in this dissertation offers insight into the significance of ATF3 expression during two distinct and separate stress contexts, islet transplantation and high fat diet induced diabetes. In each case, potential mechanisms by which ATF3 may function are identified. Furthermore, this dissertation identifies the RGD modified AAV1 viral vector as a highly infectious and non-immunogenic gene therapy tool that may contribute towards efforts aimed at improving outcomes from clinical islet transplantation. (Abstract shortened by UMI.)...