Essential role of insulin receptor substrate-2 in insulin stimulation of Glut4 translocation and glucose uptake in brown adipocytes

Insulin and insulin-like growth factor I signals are mediated via phosphory lation of a family of insulin receptor substrate (IRS) proteins, which may serve both complementary and overlapping functions in the cell. To study th e metabolic effects of these proteins in more detail, we established brown adipocyte cell lines from wild type and various IRS knockout (KO) animals a nd characterized insulin action in these cells in vitro. Preadipocytes deri ved from both wild type and TRS-S KO mice could be fully differentiated int o mature brown adipocytes, In differentiated IRS-2 KO adipocytes, insulin-i nduced glucose uptake was decreased by 50% compared with their wild type co unterparts. This was the result of a decrease in insulin-stimulated Glut4 t ranslocation to the plasma membrane. This decrease in insulin-induced gluco se uptake could be partially reconstituted in these cells by retrovirus-med iated reexpression of IRS-2, but not overexpression of IRS-1, Insulin signa ling studies revealed a total loss of IRS-2-associated phosphatidylinositol (PI) 3-kinase activity and a reduction in phosphotyrosine-associated PI 3- kinase by 30% (p < 0.05) in the RO cells. The phosphorylation and activity of Akt, a major downstream effector of PI 3-kinase, as well as Akt-dependen t phosphorylation of glycogen synthase kinase-3 and p70S6 kinase were not a ffected by the lack of IRS-2; however, there was a decrease in insulin stim ulation of Akt associated with the plasma membrane. These results provide e vidence for a critical role of IRS-2 as a mediator of insulin-stimulated Gl ut4 translocation and glucose uptake in adipocytes, This occurs without eff ects in differentiation, total activation of Akt and its downstream effecte rs, but may be caused by alterations in compartmentalization of these downs tream signals.