Nj Edgell

Regulation of Protein Kinase B and Glycogen Synthase Kinase-3 by Insulin and β-Adrenergic Agonists in Rat Epididymal Fat Cells ACTIVATION OF PROTEIN KINASE B BY WORTMANNIN-SENSITIVE AND -INSENSITIVE MECHANISMS

Previous studies using L6 myotubes have suggested that glycogen synthase kinase-3 (GSK-3) is phosphorylated and inactivated in response to insulin by protein kinase B (PKB, also known as Akt or RAC) (Cross, D. A. E., Alessi, D. R., Cohen, P., Andjelkovic, M., and Hemmings, B. A. (1995) Nature 378, 785-789). In the present study, marked increases in the activity of PKB have been shown to occur in insulin-treated rat epididymal fat cells with a time course compatible with the observed decrease in GSK-3 activity. Isoproterenol, acting primarily through β3-adrenoreceptors, was found to decrease GSK-3 activity to a similar extent (approximately 50%) to insulin. However, unlike the effect of insulin, the inhibition of GSK by isoproterenol was not found to be sensitive to inhibition by the phosphatidylinositol 3′-kinase inhibitors, wortmannin or LY 294002. The change in GSK-3 activity brought about by isoproterenol could not be mimicked by the addition of permeant cyclic AMP analogues or forskolin to the cells, although at the concentrations used, these agents were able to stimulate lipolysis. Isoproterenol, but again not the cyclic AMP analogues, was found to increase the activity of PKB, although to a lesser extent than insulin. While wortmannin abolished the stimulation of PKB activity by insulin, it was without effect on the activation seen in response to isoproterenol. The activation of PKB by isoproterenol was not accompanied by any detectable change in the electrophoretic mobility of the protein on SDS-polyacrylamide gel electrophoresis. It would therefore appear that distinct mechanisms exist for the stimulation of PKB by insulin and isoproterenol in rat fat cells.

The hormonal regulation of pyruvate dehydrogenase complex

The pyruvate dehydrogenase complex has a central role in the regulation of mammalian metabolism as it represents the point-of-no-return in the utilization of carbohydrate. This article summarizes our studies into how signalling systems initiated by hormones binding to cell surface receptors can reach the pyruvate dehydrogenase system which is located within the inner mitochondrial membrane. One class of hormones which activate pyruvate dehydrogenase are those that increase cytoplasmic Ca2+. A wide range of studies on isolated enzymes, separated mitochondria and intact cell preparations have shown that the activation is due to the stimulation of pyruvate dehydrogenase phosphatase. Two other intramitochondrial dehydrogenases which regulate the citrate acid cycle are activated in parallel and this is an important means of balancing the supply of ATP to increasing cell demand. Insulin is also able to activate pyruvate dehydrogenase, but this is restricted to fat and other cells capable of lipogenesis. Insulin acts by stimulating pyruvate dehydrogenase phosphatase, but the activation does not involve alterations in Ca2+. The signalling pathway involved has not been established, but it appears to be quite distinct from those involved in many other actions of insulin.