Partial purification from hepatoma cells of an intracellular substance which mediates the effects of insulin on pyruvate dehydrogenase and low Km cyclic AMP phosphodiesterase

Abstract

A substance capable of stimulating the activities of pyruvate dehydrogenase and low K m cyclic AMP phosphodiesterase was prepared from H4-II-EC3′ hepatoma cells by acid extraction and partially purified by molecular exclusion chromatography. The material thus prepared by gel chromatography was found to stimulate the activities of these enzymes in a concentration-dependent manner. The amount or activity of the pyruvate dehydrogenase stimulating factor was increased in cells which had been treated with physiological concentrations of insulin (0.2 mU/ml). Increasing the concentration of insulin increased the amount or activity of the factor generated. High concentrations of insulin did not cause a reversal of the effects of insulin. The stimulation of pyruvate dehydrogenase activity by the factor was eliminated when sodium fluoride (75 m m) was present in the enzyme assay, implying that activation was mediated by the pyruvate dehydrogenase phosphatase. The enzyme-stimulating factor isolated from hepatoma cells shares a number of important characteristics with the putative second messenger of insulin prepared from other cell types: (1) it is heat and acid stable, (2) it has a similar apparent molecular weight, (3) it is generated in an insulin-dependent manner, (4) it stimulates the activity of pyruvate dehydrogenase by a fluoride-sensitive mechanism, and (5) it elutes from the anion-exchange resin AG 1-X8 at an ionic strength of 0.4 m. These findings suggest that the stimulator of pyruvate dehydrogenase and of low K m cyclic AMP phosphodiesterase isolated from hepatoma cells has chemical properties identical with those of the putative second messenger of insulin action isolated from a number of other insulin-sensitive tissues.