Michelle E. Kimple, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): The heterotrimeric G protein, Gz, controls an unusual MAP kinase signaling cascade in neuroendocrine cells that it triggered by activation of the Ras family member, Rap1. Interestingly, one of the few places that Gz is expressed outside of the nervous system is in islet cells in the pancreas. In addition, we have recently found that a key regulator of Gz signaling, Rap1GAP, is also expressed in islet cells. Given the evidence for the importance of G protein-coupled receptors in regulation of multiple aspects of islet cell biology, this data hints at potentially very important roles for Gz in the physiology, and possible pathophysiology, of the pancreas. The experiments described in this proposal aim to develop molecular tools for dissecting the biology of Gz in the pancreatic islet cells, including the identification of reagents that directly inhibit the interaction between Gz and the Rap1 regulator Rap1GAP that we have identified, and then utilize these reagents and other experimental strategies to define the importance of the Gz/Rap1GAP interaction in signaling events important in islet cell biology. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): [unreadable] It is currently appreciated that both insulin resistance and beta-cell dysfunction are early and essential events in the development of type 2 diabetes. Our current knowledge of factors that influence beta-cell function is lacking, despite research in this field having been conducted for several decades. To this end, we have recently shown that the heterotrimeric G-protein alpha-subunit, G{alpha}z, modulates an endogenous signaling pathway that is inhibitory to glucose-stimulated insulin secretion in an insulinoma cell line [Kimple et al. (2005) J Biol Chem 280:31708]. These results led to the hypothesis that loss of G{alpha}z activity would result in increased insulin secretion and improved beta-cell function in vivo, possibly protecting against the development of type 2 diabetes. In support of this hypothesis, we have performed preliminary experiments in which G{alpha}z-null mice, when compared to wild-type littermate controls, display increased plasma insulin concentrations and correspondingly decreased blood glucose levels during glucose tolerance tests. Furthermore, the increased plasma insulin levels observed in G{alpha}z-null mice are likely a direct result of enhanced insulin secretion, as pancreatic islets isolated from G{alpha}z-null mice exhibit significantly higher glucose-stimulated insulin secretion than those from wild-type mice. To further address our hypothesis, and our understanding of the role of G{alpha}z signaling in insulin secretion and islet cell function, we propose the following Specific Aims: (1) to delineate the signaling pathways upstream and downstream of G{alpha}z that are important for its inhibition of insulin secretion, (2) to determine at which step in the stimulated secretion process G{alpha}z is acting, and (3) to determine whether loss of G{alpha}z is protective against the development of diabetes, both age-induced and high-fat diet-induced. The results of these studies are expected to yield important new insights into the regulation of insulin secretion and beta- cell function at a molecular level, and may point to G{alpha}z as a potential new target for therapeutics aimed at ameliorating beta-cell dysfunction in Type 2 diabetes. Relevance: This proposal aims to delineate the specific pathways by which a protein involved in the regulation of insulin secretion functions. How much insulin is secreted into the blood is one determinant of blood glucose levels; therefore, this project has direct relevance to diabetes. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Type 2 diabetes (T2D) is a costly and complex chronic illness and a serious public health problem. The children of today have an overall lifetime risk of developing diabetes of nearly 50%. Therefore, developing new methods for preventing T2D and identifying and properly treating T2D patients is very timely and of great significance. The pathophysiology of T2D is increasingly being linked with inflammatory molecules such as prostaglandin E2 (PGE2), a major arachidonic acid metabolite. Beta-cells themselves express all of the enzymes required for synthesis of PGE2, which is thought to act in an autocrine or paracrine fashion to regulate insulin secretion and possibly even beta-cell growth, proliferation, and survival. Even with the clear importance of this pathway in the normal and dysfunctional beta-cell, few studies have explored the impact of modulating the production or signaling of PGE2 at the level of the beta-cell, in part because of insufficient knowledge of th molecular mechanisms important in these pathways. Our long-term goal is to fully characterize the PGE2 synthesis and signaling pathways in the normal and diabetic beta-cell, determining steps that become dysfunctional in the diabetic state, and ultimately modulating these steps for preventative and therapeutic purposes. The overall objective of this work is to elucidate critical molecular interactions at distinct steps that have remained relatively uncharacterized, with the rationale that in gaining a complete understanding of this important beta-cell signaling pathway, we will be better able to target the dysfunctional beta-cell in T2D prevention and therapy using novel and innovative approaches. Our central hypothesis is that the PGE2 synthesis and signaling pathway is a critical mediator of diabetic beta-cell dysfunction and can be specifically targeted at one or more key steps besides the well-studied cyclooxygenase-2 (COX-2; or prostaglandin-endoperoxidase synthase 2, PTGS2) step. We will test our central hypothesis and, thereby, accomplish the objective of this application, by pursuing the following three specific aims: (1) Identify the role of arachidonic acid membrane incorporation and release in diabetic beta-cell dysfunction; (2) Determine the role of C-terminal splice variants of the EP3 isoform of the PGE2 receptor in coupling to G-protein signaling partners; and (3) Elucidate the signaling mechanisms downstream of EP3 in promoting beta-cell function, replication, and survival. With the completion of these aims, we anticipate a much more complete understanding of the pathway from arachidonic acid to PGE2 and other metabolites, including how PGE2 activation of its cellular receptor impacts on the diabetic beta-cell. Such results are anticipated to have an important positive impact on the field, as in delineating this long-known but relatively uncharacterized pathway we may be able to meld the discrepant results in the literature and reveal and confirm new targets for the prevention and therapy of T2D.