Disorders of protein metabolism in the liver are central to the pathogenesis of diabetes and other disease states that affect nutrient homeostatis. Development of rational approaches to treatment of these disorders requires knowledge of the basic events involved in the regulation of hepatic protein metabolism. Therefore, the overall goal of this project is to provide a better understanding of the mechanisms by which hormones and nutrients regulate translational and transcriptional events in the protein synthetic pathway in liver. Studies of translational control are to focus on peptide-chain initiation using deficiency of single essential amino acids to induce an inhibition in formation of 40S initiation complexes, and diabetes to induce an inhibition subsequent to 40S initiation complex formation. Studies of transcriptional control are to focus on the mechanism of insulin action on gene transcription.
The specific aims of the studies are: (1) to investigate the mechanism of inhibition of peptide-chain initiation in livers deprived of single essential amino acids, particularly the role of eukaroytic initiation factor eIF-1; (2) to investigate the mechanism of inhibition of peptide-chain initiation in livers of diabetic animals, particularly the mount of mRNA and the role of initiation factors involved in mRNA binding; (3) to further characterize rat liver eIF-2, particularly with respect to its differences with the reticulocyte-derived factor; (4) to further purify and characterize the guanine nucleotide exchange factor from rat liver; and (5) to further define the actions of insulin on the relative abundance of mRNAs in primary cultures of rat hepatocytes. The studies are to eploy a number of experimental systems including the perfused liver, suspensions of isolated hepatocytes, primary cultures of hepatocytes, and a cell-free protein-synthesizing system derived from isolated hepatocytes. They are also to utilize a number of methodologies including protein purification, immunochemical identification and quantitation of proteins, one- and two-dimensional gel electrophoresis, quantitation of mRNAs by solution and dot-blot hybridization using specific cDNAs, ligand binding assays, protein kinase and phosphatase assays, and gene transcription assays. Overall, the studies desribed in this proposal should help identify mechanisms by which hormones and nutrients regulate protein synthesis. They should also provide new insights into biochemical events involved in peptide-chain initiation and gene expression.
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