Insulin is the major hormone involved in overall glucose homeostasis, and abnormalities of insulin action are central to the pathogenesis of a variety of disease states. Consequently, detailed knowledge of the basic events underlying insulin's ability to interact with cells will lead to a better understanding of the mechanisms of disease states in which insulin action is abnoral. Therefore, the overall goals of this research proposal are to study the relationships between insulin binding to its cell surface receptors and the events which occur following this initial step. 1) To accomplish this, we plan studies of the insulin receptor intinerary. This includes studies of internalization of insulin receptor complexes, dissociation of insulin from receptors within endosomal vesicles, physical segregation of insulin from receptor and then recycling, degradation, and sequestration of the internalized receptor. Concomitant studies of intracellular insulin degradation will also be conducted. We will also examine subcellular localization to determine where in the cell these events occur. 2) Secondly, we will turn our attention to studies of adipocyte glucose transport. We plan experiments to isolate and purify glucose transporters (GT), and to raise polyclonal and monoclonal antibodies against GT. We plan to test the hypothesis that insulin induces covalent modification of GT (phosphorylation) and that this subserves an important physiologic role. Additionally, GT number and structure will be assessed in various physiologic conditions, in hepatocytes compared to adipocytes, and in plasma membrane versus low density membrane compartments. 3) Studies of the recently identified insulin receptor kinase activity will also be carried out. We plan to examine autophosphorylation as well as exogenous substrate phosphorylation in cell free systems and in intact cells, in an attempt to further understand the potential role of this kinase activity in normal insulin action as well as pathophysiologic conditions. 4) Finally, we will examine the mechanisms of abnormal insulin action in three models of post-binding cellular insulin resistance. These include studies of cells with down regulated insulin receptors and decreased maximal glucose transport activity, studies of cells with normal insulin binding but decresed insulin sensitivity, and studies of neuraminidase treated cells with normal insulin binding and decreased insulin stimulated glucose transport. The studies outlined in this proposal should provide needed information which will further our understanding of the cellular actions of this important hormone.
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