Activation of pyruvate dehydrogenase by direct addition of insulin to an isolated plasma membrane/mitochondria mixture: Evidence for generation of insulin's second messenger in a subcellular system

Abstract

The addition of insulin [I] to a mixture of plasma membrane and mitochondrial fractions from rat adipocytes results in a decrease in the phosphorylation of a mitochondrial protein identified as the .alpha. subunit of pyruvate dehydrogenase [pyruvate:lipoamide oxidoreductase (decarboxylating and acceptor-acetylating), EC 1.2.4.1]. A corresponding increase in pyruvate dehydrogenase activity apparently can be effected by I treatment of this preparation. Incubation of the plasma membrane/mitochondria mixture with ATP inhibited pyruvate dehydrogenase activity as measured in a subsequent enzyme assay. The presence of I during this incubation with ATP resulted in a 24.5% stimulation of enzyme activity compared to incubation without I (n = 9, P < 0.001). The effect was specific for biologically active I and was I dose-dependent in the physiological range of I. Supermaximal doses of I produced reduced effects. An I effect of similar magnitude could also be observed when the plasma membrane/mitochondria mixture was incubated without ATP. Two I mimickers, concanavalin A and antibody to I receptor, stimulated pyruvate dehydrogenase by 30.4% (n = 6, P < 0.001) and 28.1% (n = 8, P < 0.001), respectively. Both of these agents also produced reduced effects at supermaximal concentrations. The effects of all 3 agents required plasma membranes and could not be produced by treatment of mitochondria alone. Evidently a mechanism common to all 3 agents is responsible for transmitting the stimulation from the plasma membrane to the mitochondrial components of the mixture.