Action of insulin receptor substrate-3 (IRS-3) and IRS-4 to stimulate translocation of GLUT4 in rat adipose cells

The insulin receptor initiates insulin action by phosphorylating multiple i ntracellular substrates. Previously, we have demonstrated that insulin rece ptor substrates (IRS)-1 and -2 can mediate insulin's action to promote tran slocation of GLUT4 glucose transporters to the cell surface in rat adipose cells. Although IRS-1, -2, and -4 are similar in overall structure, IRS-3 i s approximate to 50% shorter and differs with respect to sites of tyrosine phosphorylation. Nevertheless, as demonstrated in this study, both IRS-3 an d IRS-4 can also stimulate translocation of GLUT4. Rat adipose cells were c otransfected with expression Vectors for hemagglutinin (HA) epitope-tagged GLUT4 (GLUT4-HA) and human IRS-1, murine IRS-3, or human IRS-4. Overexpress ion of IRS-1 led to a 2-fold increase in cell surface GLUT4-HA in cells inc ubated in the absence of insulin; overexpression of either IRS-3 or IRS-4 e licited a larger increase in cell surface GLUT4-HA. Indeed, the effect of I RS-3 in the absence of insulin was approximate to 40% greater than the effe ct of a maximally stimulating concentration of insulin in cells not overexp ressing IRS proteins. Because phosphatidylinositol (PI) 3-kinase is essenti al for insulin-stimulated translocation of GLUT4 we also studied a mutant I RS-3 molecule (IRS-3-F4) in which Phe was substituted for Tyr in all four Y XXM motifs (the phosphorylation sites predicted to bind to and activate PI 9-kinase). Interestingly, overexpression of IRS-3-F4 did not promote transl ocation of GLUT4-HA, but actually inhibited the ability of insulin to stimu late translocation of GLUT4-HA to the cell surface. Our data suggest that I RS-3 and IRS-4 are capable of mediating PI 3-kinase-dependent metabolic act ions of insulin in adipose cells, and that IRS proteins play a physiologica l role in mediating translocation of GLUT4.