I. Investigations will be continued on the processing, compartmental storage, and secretion of insulin and its precursors using pulse-chase experiments in normal islets and cultured insulinoma cells. Major attention will be placed on the regulation, physiologic significance and biochemical and morphologic identity of the sorting processes which direct newly synthesized insulin for preferential secretion. In addition, we find hamster insulinoma cells produce large quantities of """"""""preproinsulinlike peptides;"""""""" these lines will be used as a model for studying normal and abnormal processing and miss-sorting of protein precursors. The peptides will be chemically identified and their distribution, either into the secretory system or as membrane protein, determined. Simultaneous processing, storage, and secretion of endogenous and exogenous insulin and its precursors will be compared in similar cell lines containing the added human insulin gene to evaluate the genetic requirements for post translational processing. II. Studies of the storage forms of insulin, particularly as they relate to possible co-stored proteins and to the zinc ion, will be expanded. Proteins co-secreted with insulin will be identified by SDS-PAGE and HPLC. The kinetics of their release will be compared to insulin in perifused islets and the possibility that some may be retained as cell membrane components will be tested. Studies on zinc metabolism will particularly focus on the mechanism of zinc entry into granules, zinc-65Zn exchange with granular and extragranular compartments, and the possible transient secretion of zinc or other insulin complexes. III. New techniques that permit extended perfusion of pancreas or perifusion of islets in a non-recirculating system will be employed to establish: 1) if there are advantages of this approach over standard, static culture of islets; 2) how controlled ocillations of secretagogues or modulators of islet function affect insulin production and secretion; and 3) the nature of insensitivity or """"""""toxicity"""""""" generated by prolonged exposure of islet cells to glucose or other secretagogues.
