Characterization of a pyruvate dehydrogenase activator released by adipocyte plasma membranes in response to insulin.

The supernatant from purified rat adipocyte plasma membranes incubated with insulin and pelleted by centrifugation contains a substance that stimulates pyruvate dehydrogenase when added to purified mitochondria.Plasma membranes incubated in the absence of insulin also released a factor that stimulated pyruvate dehydrogenase, but at a slow rate.The rate of production of a stimulating factor was increased with increasing concentrations of insulin so that at lOO-microunits/ ml of insulin, maximum stimulation occurred within 1 min of incubation of membranes and hormone.The maximum level of stimulation was the same at all concentrations of insulin.The increment of stimulation by insulin compared to control was found to be greater when plasma membranes were prepared in phosphate rather than Tris buffer.Incubation of membranes beyond the point required to produce maximum stimulation resulted in progressive loss of stimulation, with this reversal occurring more rapidly with higher levels of insulin.Dilution of this nonstirnulatory supernatant resulted in renewed capability to activate pyruvate dehydrogenase.The insulin-dependent stimulator of pyruvate dehydrogenase present in plasma membrane supernatant was stable under moderate conditions of pH and temperature for short periods of time up to 24 min.These observations indicate that a stable soluble enzyme regulator is released from plasma membranes in response to insulin, but that other regulating factors may also be present.The chromatographic properties of the insulin-dependent activator were determined by gel filtration, ion exchange, and hydroxylapatite chromatography.The activator exhibited an apparent molecular size of 2000-4000 daltons.It displayed an isoelectric point of 4.5, so that at pH 7.4, it possessed a negative charge and bound to anion exchange resin from which it was eluted with 0.3-0.4M NaCl.The activator also bound to hydroxylapatite and was eluted with 0.1-0.2M phosphate.In conjunction with previous data demonstrating destruction of the activator by proteases (Seals, J. R., and Czech, M. P. (1980)