With combined advances in genetic screening and identification of clinical markers of prediabetes, diagnosis for future risk of insulin-dependent diabetes mellitus (IDDM) is close to practical reality. Critically missing are effective therapeutic strategies to preserve pancreatic beta-cell function once prediabetic individuals are identified. This application is based on the contention that elucidation of the molecular mechanisms operative during the early stages of beta-cell destruction would contribute greatly to the development of such intervention therapies. Our research is focused on understanding the phenotypic and functional changes that occur in beta-cells in response to interferon-gamma (IFNgamma), a potent T cell-derived cytokine present in insulitic lesions and required for IDDM development. Corroborating clinical findings, we have demonstrated that two major alterations occur in IFN-gamma-treated beta- cells - - diminished glucose responsiveness and induction of the major histocompatibility complex (MHC) class I antigen - processing pathway. The overall objective of the present proposal is to determine whether these two events are linked through an interactive mechanism. Our underlying hypothesis is that intracellular insulin content is diminished in IFNgamma-treated beta-cells because pre-proinsulin is diverted from the normal secretory pathway and used as a donor of antigenic peptides for MHC class I assembly. Experiments are designed to characterize, at the molecular level, the subunit composition and subcellular location of the proteolytic complex thought to generate class I peptides in IFNgamma- treated beta-cells. To determine if a cause-effect relationship exists between induction of this low molecular mass polypeptide (LMP) complex and diminished beta-cell function, glucose-stimulated insulin biosynthesis will be measured in beta-cells in which LMP gene expression is either extinguished or constitutively elevated. In addition, the endogenous peptides bound to beta-cell MHC class I molecules will be isolated and sequenced to determine if pre-proinsulin is a major peptide donor. Neither insulin RNA expression nor insulin secretory granule exocytosis are altered by IFNgamma. The inhibitory effect of IFNgamma on beta-cell function does however require gene transcription. Thus, IFNgamma's effects must be contributed by induced factors that either block translation of insulin mRNA, enhance insulin degradation, or both. To resolve these possibilities, studies are included that will distinguish IFNgamma's effects on biosynthesis, degradation, and trafficking of pre-proinsulin in glucose-stimulated beta-cells. As a reduction in glucose utilization might selectively block pre-proinsulin translation, glucose uptake and metabolism are also compared in control and IFNgamma-treated beta-cells. Collectively, these studies will define molecular mechanisms likely operative in the prediabetic beta-cell, thereby providing information critical for bridging the gap between diagnosis and treatment of IDDM.
