Regulation of Protein Kinase B and Glycogen Synthase Kinase-3 by Insulin and β-Adrenergic Agonists in Rat Epididymal Fat Cells

doi:10.1074/jbc.272.12.7713

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*

From the ^‡ Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, BS8 1TD and the
^¶ Department of Biosciences, University of Kent, Canterbury, CT2 7NJ, United Kingdom

^§ To whom correspondence should be addressed. Tel.: +44 117 9287432; Fax: +44 117 9288274.

Abstract

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.

Footnotes

↵^∥ Recipient of an MRC Postgraduate Studentship.
↵* This work was supported in part by a Project Grant from the Wellcome Trust (to C. G. P.), by a Program Grant from the Medical Research Council (to R. M. D. and Dr. J. M. Tavaré), and Group Support from the British Diabetic Association. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

GSK-3

glycogen synthase kinase-3

GS

glycogen synthase

MAP kinase

mitogen-activated protein kinase

MBP

myelin basic protein

PKB

protein kinase B

p70^S6k

p70 ribosomal S6 protein kinase

p90^rsk

p90 ribosomal S6 protein kinase

PI 3′-kinase

phosphatidylinositol 3′-kinase

cpt cAMP

chlorophenylthio-cAMP

db-cAMP

dibutyryl cAMP

PAGE

polyacrylamide gel electrophoresis

MOPS

4-morpholinepropanesulfonic acid.
↵² G. I. Welsh, J. C. Patel, and C. G. Proud, submitted for publication.
- Received October 2, 1996.
- Revision received November 21, 1996.