Insulin induction of SOCS-2 and SOCS-3 mRNA expression in C2C12 skeletal muscle cells is mediated by Stat5

Previously, by a yeast 2-hybrid screen, we identified signal transducer and activator of transcription 5b (Stat5b) as a substrate of the insulin recep tor (IR). We demonstrated that refeeding of fasted mice leads to rapid acti vation of Stat5 proteins in liver, skeletal muscle, and fat, suggesting tha t Stat5b is a physiological target of insulin. Here, we show that injection of glucose or insulin into fasted mice leads to robust activation of both Stat5a and Stat5b in skeletal muscle. In C2C12 myotubes, we find that insul in stimulates tyrosine phosphorylation of Stat5a and Stat5b by 3-5-fold. Th is degree of Stat5 activation in vitro is significantly lower than what we observe in vivo and inversely correlates with IRS-1/2 levels. We can recapi tulate robust insulin activation of Stat5 in C2C12 cells by stable overexpr ession of the human IR (hIR). To identify insulin-activated genes that are Stat5 targets, we also overexpressed an IR mutant (LA-hIR) that signals nor mally for mitogen-activated protein kinase- and phosphatidylinositol 3-kina se-dependent pathways but is deficient in Stat5 signaling in response to in sulin. We demonstrate that insulin induces the expression of SOCS-2 mRNA in the wild type hIR but not in the LA-hIR-overexpressing cells. The inductio n of SOCS-3 by insulin is reduced but not lost in the LA-hIR cells. Therefo re, our results suggest that insulin induction of SOCS-2, and in part SOCS- 3 mRNA expression, is mediated by Stat5 and can be independent of mitogen a ctivated protein kinase and phosphatidylinositol 3-kinase-signaling pathway s.