Production by plasma membranes of a chemical mediator of insulin action.

Purified rat adipocyte plasma membranes incubated with insulin produce a soluble chemical mediator that stimulates pyruvate dehydrogenase when added to isolated mitochondria, or glycogen synthase when added to cell homogenates. The mediator appears to be a peptide and has been characterized by conventional chromatographic methods including gel filtration, ion exchange, and hydroxylapatite chromatography. These studies reveal that an insulin-dependent bioactive component, which is small and negatively charged at pH 7.4, can be eluted from Dowex 1 x 4 by 0.3-0.4 N NaCl or from hydroxylapatite by 0.05-0.15 M potassium phosphate. The mediator has also been partially purified by high-pressure liquid chromatography. A molecular sieving matrix produces a peak of insulin-dependent bioactivity that corresponds to a peak of absorbance at 210 nm (apparent Mr of 2000) and is increased by insulin. Reversed-phase high-pressure liquid chromatography indicates that the insulin-dependent bioactivity is of a hydrophilic nature. Previous studies showed that release of mediator from plasma membranes in response to insulin was blocked by inhibitors of serine proteases and esters of arginine. In addition, bioactivity of the insulin-treated plasma membrane supernatant could be destroyed by protease treatment. In the present experiments, pretreatment of intact adipocytes with serine protease inhibitors blocked the action of subsequently added insulin on several intracellular enzyme systems. These observations have been summarized in the following working model of one mode of insulin action. The binding of insulin to its receptor activates a membrane protease or alters an endogenous membrane substrate, resulting in the increased release into the cell of a small peptide fragment by proteolytic cleavage. The released peptide is proposed to modulate several cellular enzymes such as pyruvate dehydrogenase and glycogen synthase by interacting with phosphoprotein phosphatase or protein kinase activities, or both.