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 ␤ 3 -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.

An important effect of insulin after a carbohydrate meal is to increase the conversion of glucose into fatty acids in fat, liver and mammary cells. Both short-term and long-term mechanisms are involved. A main interest of this laboratory for a number of years has been the short-term mechanisms involved in insulin signalling in rat epididymal fat cells, and this will be the subject of this brief article. Three different steps in the pathway are activated in parallel within 5 min of exposing these cells to insulin, and the overall result is a 10-fold increase in the rate of fatty acid synthesis. These steps are: glucose transport across the cell membrane, pyruvate dehydrogenase (PDH) in the mitochondria, and the cytoplasmic enzyme acetyl-CoA carboxylase (ACC) [l]. It is now becoming clear that different signalling pathways are involved although, as with other metabolic actions of insulin, important gaps in our knowledge remain. Temporal regulation of lipid homoeostasis is effected at several levels. Rapid modulation of cellular rates of lipolysis, fatty acid oxidation and lipogenesis occurs by changes in t h e activities of Abbreviations used: PPAR, peroxisomal proliferatoractivated receptor; PPRE, peroxisomal proliferator-key enzymes in the pathways of lipid metabolism. response element; DR, direct repeat; aP,, adipocyte activities may be allosterically, fatty acid-binding protein; LPI,, lipoprotein lipase; or by hormone-or catecholamine-induced NIDDM, non-insulin-dependent diabetes. changes in phosphorylation state. In the longer Volume 25

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