Insulin stimulation of pyruvate dehydrogenase in an isolated plasma membrane-mitochondrial mixture occurs by activation of pyruvate dehydrogenase phosphatase.

Abstract

The direct addition of insulin or concanavalin A to a mixture of plasma membranes and mitochondria from rat adipocytes stimulated mitochondrial pyruvate dehydrogenase activity through a plasma membrane-generated second messenger (Seals, J. R., and Jarett, L. (1980) h.NutL Acad Sci U S. A. 77, 77-81).The insulin or concanavalin A effects were observed with ATP present in the assay; therefore, the stimulation of pyruvate dehydrogenase could have occurred by activation of pyruvate dehydrogenase phosphatase or by inhibition of pyruvate dehydrogenase kinase.The present study was designed to distinguish between these two mechanisms.The stimulation of pyruvate dehydrogenase by insulin or concanavalin A was not significantly different in the presence of ATP compared to its absence, suggesting that activation occurred through pyruvate dehydrogenase phosphatase.The mechanism was further investigated by studying the effects of NaF, a phosphatase inhibitor, and dichloroacetic acid, a kinase inhibitor.NaF at 50 l l l ~ completely blocked the ability of insulin or concanavalin A to stimulate pyruvate dehydrogenase activity in the presence or absence of ATP.In the presence of ATP, insulin or concanavalin A plus concentrations of dichloroacetic acid which produced maximal inhibition of kinase activity had additive effects on stimulation of pyruvate dehydrogenase activity.These data demonstrate that the stimulation of pyruvate dehydrogenase in this subcellular system by insulin or concanavalin A occurred through a plasma membrane-generated second messenger which activated the phosphatase but did not inhibit the kinase.Insulin has been shown to stimulate mitochondrial pyruvate dehydrogenase activity in rat epididymal adipose tissue in