Intracellular signalling mechanisms which mediate the multiple actions of insulin on cellular metabolism are not understood. Recent studies have demonstrated that a soluble factor or factors can be released from isolated plasma membranes or generated in intact cells by insulin. Such factor(s) have been shown to activate the known insulin-sensitive enzyme pyruvate dehydrogenase (PDH) in isolated mitochondria and decrease the sensitivity of cAMP-dependent protein kinase to activation by cAMP. The factor or factors thus produced may play a key role in mediating the intracellular actions of insulin. We provide evidence in this proposal that the PDH activating factor(s) produced by insulin-receptor interaction in adipocyte plasma membranes is a peptide (M.W. less than 10,000) released from a membrane precursor by the action of a protease. The studies proposed seek to purify, isolate and define the structure of the PDH activating factor in order to gain insight into its structure-function relationships. Standard molecular sieve and ion exchange chromatography of the supernatant from an incubation of insulin and adipocyte plasma membranes will be performed. Fractions containing PDH-activating activity will be labeled by iodination (125I) and other labeling reagents and electrophoresed on 20% acrylamide gels. In order to evaluate the role of the factor in insulin action, other enzyme systems regulated by phosphorylation-dephosphorylation and known to be sensitive to insulin in intact cells will be surveyed for possible modulation by the factor(s) in cell homogenates. Attempts to raise antibodies to purified factor(s) will be made using rabbits and guinea pigs as well as mice. If successful in the latter, monoclonal antibodies will be prepared. These antibodies will be employed to identify the membrane precursor of the PDH activating factor, using standard immunoprecipitation techniques routinely employed in the laboratory.
