Evidence that insulin activates an intrinsic plasma membrane protease in generating a secondary chemical mediator

Three lines of evidence are presented which support the hypothesis that the binding of insulin to rat adipocyte plasma membranes activates a membrane protease which results in the production of a soluble factor that stimulates pyruvate dehydrogenase activity when added to mitochondria. First, low concentrations of trypsin (0.01 to 0.1 microgram/ml) mimic the action of insulin on plasma membranes in this system. Second, inhibitors of trypsin-like proteases (soy trypsin inhibitor, ovomucoid) block insulin action on the plasma membrane, as do exogenous protease substrates which are esters of arginine, presumably by preventing the normal interaction of the protease and its endogenous membrane substrate. Third, digestion by proteases (0.1 mg/ml of trypsin, chymotrypsin, or papain) of the soluble material released from plasma membranes after insulin treatment blocks the stimulation of pyruvate dehydrogenase, suggesting that the mediator may be a peptide released by a proteolytic reaction.