The overall goal of this proposal is to develop a deeper understanding of the mechanisms whereby the prostaglandin E2 (PGE2)-receptor complex interacts with guanine nucleotide regulatory proteins (G-proteins) to regulate insulin gene expression and insulin secretion. The five specific aims are: (1) to identify which pertussis toxin substrates mediate the inhibitory effects of PGE2, somatostatin and epinephrine on insulin secretion; (2) to ascertain whether the decrease in insulin mRNA observed in HIT cells treated with PGE2, somatostatin or epinephrine is due to a decrease in the rate of transcription of the insulin gene, an increased rate of degradation of insulin mRNA or both; to ascertain whether decreased induction of insulin gene expression by its cyclic AMP response element is involved; and to ascertain which G-protein subspecies mediate these effects of PGE2, somatostatin and epinephrine; (3) to ascertain whether the decrements in insulin mRNA and insulin content in later passages of HIT cells associated with glucose desensitization are caused by direct and paradoxically inhibitory effects of glucose on insulin gene transcription and to establish whether glucose-driven endogenous PGE2 synthesis plays a role in these decrements; (4) to determine whether increments in levels of Gs mRNA and the 45 kDa form of Gs which occur in later passages of HIT cells are due to high glucose concentrations in, or low levels of insulin secreted into, the media, or both; and (5) to ascertain whether the changes in Gs mRNA and the 45 kDa form of Gs associated with serial passages of HIT cells in high glucose and low insulin concentrations are due to an increase in the rate of Gs gene transcription, decrease in the rate of Gs mRNA degradation, or both. The methods used to achieve these specific aims will involve several means of identification of G-protein subspecies; introduction into HIT cells of specific antisera directed against G- proteins; nuclear run-on assays and CAT reporter experiments to study insulin gene and Gs gene transcription; actinomycin D and cyclohexamide experiments to study mRNA degradation; studies of static and phasic insulin secretion; studies of PGE2 synthesis; measurement of GLUT-2 levels, glucose transport, and glucose kinase activity; and long term experiments (6-12 months) in which HIT cells are cultured under various conditions of high and low concentrations of glucose and insulin. At the conclusion of these studies we hope to have established the degree to which there is """"""""cross- talk"""""""" between the G-protein gene and the insulin gene that governs insulin gene transcription and insulin secretion as well as the roles that these mechanisms play in glucose desensitization (glucotoxicity) of the beta cell.
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