Partial purification from rat adipocyte plasma membranes of a chemical mediator which simulates the action of insulin on pyruvate dehydrogenase.

Abstract

This study demonstrates that the plasma membrane of the rat adipocyte releases an insulin-sensitive material which appears to mediate the stimulation of mitochondrial pyruvate dehydrogenase by insulin.This chemical mediator is partially characterized.The quantity of the chemical mediator spontaneously released from the plasma membrane is dependent on the buffer system used during the subcellular fractionation.SUpernatant from plasma membranes prepared in phosphate buffer failed to stimulate pyruvate dehydrogenase, whereas the supernatant of plasma membranes prepared in Tris buffer or Tris buffer and EDTA stimulated enzyme activity by 47 +-7.6% and 352 f 968, respectively.Insulin treatment of phosphate-prepared membranes caused the mediator to be released into the supernatant as determined by pyruvate dehydrogenase activation.Repeated incubation and centrifugation depleted the ws-prepared plasma membranes of the mediator.This mediator in the supernatant was stable at pH 7.0 and produced a linear dose response of pyruvate dehydrogenase in the presence or absence of ATP.Gel filtration of the supernatant on Sephadex 625 or G15 revealed only one fraction in the molecular weight range of 1000-1500, which stimulated pyruvate dehydrogenase.This same low molecular weight fraction from supernatant of insulin-treated membranes contained a greater quantity or activity of the chemical mediator than did control samples.Both the supernatant and the active fraction were shown to stimulate pyruvate dehydrogenase by activating the pyruvate dehydrogenase phosphatase and not by inhibiting the cyclic AMP-independent kinase.The phosphatase activation was demonstrated by the ability of the active material to increase pyruvate dehydrogenase activity in the absence of ATP and by the ability of NaF, a phosphatase inhibitor, to block this stimulation.The involvement of the kinase was eliminated by experiments in the presence of ATP showing 1) that NaF completely blocked the stimulation of pyruvate dehydrogenase, and 2) that the increase in pyruvate dehydrogenase induced by dichloroacetic acid, a kinase inhibitor, was additive to the stimulation caused by the mediator in the supernatant or in the Sephadex-active fraction.This insulin-sensitive material from the adipocyte plasma membrane is so far indistinguishable