Project Summary The sphingolipid ceramide [CER] has been shown to be an important mediator of signal transduction processes leading to a variety of cellular responses, including apoptosis. Despite the compelling experimental evidence to suggest that CER-dependent signaling mechanisms might underlie -cell dysfunction in in vitro and in vivo models of impaired insulin secretion, very little is known with regard to the precise modes of action of CER in the signaling events leading to metabolic dysregulation of the islet -cell. Our preliminary findings suggest that long-term exposure of INS 832/13 cells and primary rat islets to elevated glucose and lipids promote CER-dependent activation of an okadaic acid-sensitive protein phosphatase [CAPP] and the phagocytic NADPH-oxidase [NOX] leading to mitochondrial dysregulation. We also present preliminary evidence to indicate that these two pathways are accelerated in islets from the Zucker Diabetic Fatty [ZDF] rat, a widely accepted model for type 2 diabetes. Based on these data we hypothesize that an accumulation of intracellular CER, induced following chronic exposure of isolated -cells to glucose and lipids, causes mitochondrial dysfunction leading to cell demise. The three Specific Aims of the proposed studies are: [I] to demonstrate that glucolipotoxic conditions promote CER-mediated activation of the mitochondrial isoform of CAPP leading to dephosphorylation and inactivation of Bcl-2 culminating in the mitochondrial dysfunction of the islet -cell; [II] to demonstrate that glucolipotoxic conditions promote CER-mediated holoenzyme assembly and functional activation of NOX to result in the generation of ROS and the associated onset of mitochondrial dysfunction of the islet -cell; and [III] to precisely define the progression, and prevention of mitochondrial defects and metabolic dysfunction [identified under Aims I and II] by CER synthesis inhibitors in the ZDF rat islet. We will employ a number of biochemical, molecular biological, cell biological and immunological approaches to validate our hypothesis and accomplish our goals in INS 832/13 cells, primary rat islets and whole animals. It is hoped that data derived from the proposed studies will provide fresh insights into the regulatory roles of specific CER-sensitive signaling steps in the onset of mitochondrial dysfunction leading to the demise of the islet -cell under the duress of glucolipotoxic conditions. Our long-term goal is to develop specific therapeutic modalities to prevent the establishment of these cell defects and the onset of diabetes. Our proposed studies have direct relevance to the VA research and patient care missions. Available data clearly suggest that veterans are more likely than the general population to have diabetes, one of the major complications associated with obesity. According to the American Diabetes Association, greater than 7% of the U.S. population has diabetes, and the rate increases with age. Among veterans receiving VA health care, who are on average older than the general population, the rate is greater than 20%. According to the VA, 70% of the 7.5 million veterans who receive health benefits through the department are obese, and one in five has diabetes, which can lead to heart disease, high blood pressure and amputations. We envision that data derived from the proposed studies will provide fresh insights into regulatory roles of specific CER-sensitive signaling steps in the onset of mitochondrial dysfunction leading to the demise of the -cell under the duress of glucolipotoxic conditions. The data accrued from our studies might form the basis for the development of specific therapeutic modalities to prevent the establishment of these -cell defects and the onset of diabetes.