Akt mediates insulin induction of glucose uptake and up-regulation of GLUT4 gene expression in brown adipocytes

Insulin acutely stimulated glucose uptake in rat primary brown adipocytes i n a PI3-kinase-dependent but p70S6-kinase-independent manner. Since Akt rep resents an intermediate step between these kinases, this study investigated the contribution of Akt to insulin-induced glucose uptake by the use of a chemical compound, ML-9, as well as by transfection with a dominant-negativ e form of Akt (Delta Akt). Pretreatment with ML-9 for 10 min completely inh ibited insulin stimulation of (1) Akt kinase activity, (2) Akt phosphorylat ion on the regulatory residue Ser473 but not on Thr308, and (3) mobility sh ift in Akt1 and Akt2. However, ML-9 did not affect insulin-stimulated PI3-k inase nor PKC zeta activities. In consequence, ML-9 precluded insulin stimu lation of glucose uptake and GLUT4 translocation to plasma membrane (determ ined by Western blot), without any effect on the basal glucose uptake. More over, Delta Akt impaired insulin stimulation of glucose uptake and GFP-tagg ed GLUT4 translocation to plasma membrane in transiently transfected immort alised brown adipocytes and HeLa cells, respectively. Furthermore, ML-9 tre atment for 6 h down-regulated insulin-induced GLUT4 mRNA accumulation. with out affecting GLUT1 expression, in a similar fashion as LY294002. Indeed. c o-transfection of brown adipocytes with Delta Akt precluded the transactiva tion of GLUT4-CAT promoter by insulin in a similar fashion as a dominant-ne gative form of PI3-kinase, Our results indicate that activation of Akt may be an essential requirement for insulin regulation of glucose uptake and GL UT4 gene expression in brown adipocytes, (C) 2001 Federation of European Bi ochemical Societies. Published by Elsevier Science B.V. All rights reserved .