The primary goal of this proposal is to elucidate the cellular mechanisms of insulin action in target tissues. The project will concentrate on the biochemical characterization and identification of the putative mediators of insulin action. These are defined as a class of substances which are produced in cells or cell membranes in response to insulin and acutely reproduce the effects of insulin on cellular enzymes. The chemical identities of the mediators will be pursued. They will be detected by assaying the modulation of key insulin sensitive enzymes, such as pyruvate dehydrogenase, acetyl CoA carboxylase and adenylate cyclase. Purification methods which have been developed for these substances will be scaled up to obtain sufficient material for chemical analysis using sensitive techniques, such as NMR, mass spectrometry and others. If necessary, the mediators will be further purified on HPLC. Chemical derivatization, followed by HPLC and assay of biological activity, will be used to identify the functionality of each substance. The mechanism of insulin stimulated generation of these substances will be studied, including the contribution of insulin-receptor phosphorylation to initiating the release of the mediators from the plasma membrane. These studies will be carried out by introducing agents which may modify receptor phosphorylation and quantitating the effects of these agents on mediator generation. In addition, the production and metabolism of the mediators will be monitored in cultured hepatoma cells. These cells offer a highly responsive system in which to study the generation and processing fo the mediators by following the incorporation of radioactive precursors into insulin sensitive fractions with identical enzyme modulating activities. Furthermore, the biological activities of the mediators on intact cells will be evaluated in order to determine whether distinct mechanisms are involved in the short- and long-term actions of insulin. It is expected that elucidation of the processes by which this second messenger is generated, processed and its effects exerted will contribute to understanding the hormonal regulation of cellular metabolism and perhaps uncover the critical post receptor defect in insulin resistance.
