Stimulation of glucose transport is among the most important metabolic actions of insulin. Studies in adipose cells have demonstrated that insulin stimulates its receptor to phosphorylate tyrosine residues in IRS-1, leading to activation of phosphatidylinositol 3-kinase, which plays a necessary role in mediating the translocation of the insulin-responsive glucose transporter GLUT4 to the cell surface. Akt is a serine-threonine kinase recently identified as a direct downstream target of phosphatidylinositol 3-kinase. A previous study in 3T3-L1 cells showed that overexpression of a constitutively active mutant of Akt is sufficient to recruit GLUT4 to the cell surface. Since effects of overexpression of signaling molecules in tissue culture models do not always reflect physiological function, we have overexpressed a dominant inhibitory mutant of Akt in rat adipose cells to investigate the effects of inhibiting endogenous Akt in a physiologically relevant insulin target cell. Cells were transfected with either wild type (Akt-WT), constitutively active (Akt-myr), or dominant inhibitory (Akt-K179A) forms of Akt, and effects of overexpression of these constructs on insulin-stimulated translocation of a cotransfected epitope-tagged GLUT4 were studied. Overexpression of Akt-WT resulted in significant translocation of GLUT4 to the cell surface even in the absence of insulin. Interestingly, overexpression of Akt-myr resulted in an even larger effect that was independent of insulin. More importantly, overexpression of Akt-K179A (kinase-inactive mutant) significantly inhibited insulin-stimulated translocation of GLUT4. Taken together, our data suggest that Akt is not only capable of stimulating the translocation of GLUT4 but that endogenous Akt is likely to play a significant physiological role in insulin-stimulated glucose uptake in insulin targets such as muscle and adipose tissue.

Insulin receptor substrates-1 and -2 (IRS-1 and -2) are important substrates of the insulin receptor tyrosine kinase. Previous studies have focused upon the role of IRS-1 in mediating the actions of insulin. In the present study, we demonstrate that IRS-2 can mediate translocation of the insulin responsive glucose transporter GLUT4 in a physiologically relevant target cell for insulin action. Co-immunoprecipitation experiments performed on cell lysates derived from freshly isolated rat adipose cells incubated in the presence or absence of insulin indicated that twice as much phosphatidylinositol 3-kinase was associated with endogenous IRS-1 as with IRS-2 after insulin stimulation. When rat adipose cells in primary culture were transfected with expression vectors for IRS-1 or IRS-2, we observed 40-fold overexpression of human IRS-1 or murine IRS-2. In addition, anti-phosphotyrosine immunoblotting experiments confirmed that the recombinant substrates were phosphorylated in response to insulin stimulation. To examine the role of IRS-2 in insulin-stimulated translocation of GLUT4, we studied the effects of overexpression of IRS-1 and -2 on translocation of a co-transfected epitopetagged GLUT4 (GLUT4-HA). Overexpression of IRS-1 or IRS-2 in adipose cells resulted in a significant increase in the basal level of cell surface GLUT4 (in the absence of insulin). Interestingly, at maximally effective concentrations of insulin (60 nM), the level of cell surface GLUT4 in cells overexpressing IRS-1 or -2 significantly exceeded the maximal recruitment observed in the control cells (160 and 135% of control, respectively; p < 0.003). Our data directly demonstrate that IRS-2, like IRS-1, is capable of participating in insulin signal transduction pathways leading to the recruitment of GLUT4. Thus, IRS-2 may provide an alternative pathway for critical metabolic actions of insulin.

The insulin receptor initiates insulin action by phosphorylating multiple intracellular substrates. Previously, we have demonstrated that insulin receptor substrates (IRS)-1 and -2 can mediate insulin's action to promote translocation 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 is approximately 50% shorter and differs with respect to sites of tyrosine phosphorylation. Nevertheless, as demonstrated in this study, both IRS-3 and IRS-4 can also stimulate translocation of GLUT4. Rat adipose cells were cotransfected with expression vectors for hemagglutinin (HA) epitope-tagged GLUT4 (GLUT4-HA) and human IRS-1, murine IRS-3, or human IRS-4. Overexpression of IRS-1 led to a 2-fold increase in cell surface GLUT4-HA in cells incubated in the absence of insulin; overexpression of either IRS-3 or IRS-4 elicited a larger increase in cell surface GLUT4-HA. Indeed, the effect of IRS-3 in the absence of insulin was approximately 40% greater than the effect of a maximally stimulating concentration of insulin in cells not overexpressing IRS proteins. Because phosphatidylinositol (PI) 3-kinase is essential for insulin-stimulated translocation of GLUT4, we also studied a mutant IRS-3 molecule (IRS-3-F4) in which Phe was substituted for Tyr in all four YXXM motifs (the phosphorylation sites predicted to bind to and activate PI 3-kinase). Interestingly, overexpression of IRS-3-F4 did not promote translocation of GLUT4-HA, but actually inhibited the ability of insulin to stimulate translocation of GLUT4-HA to the cell surface. Our data suggest that IRS-3 and IRS-4 are capable of mediating PI 3-kinase-dependent metabolic actions of insulin in adipose cells, and that IRS proteins play a physiological role in mediating translocation of GLUT4.

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