Abstract
To investigate the potential role of trimeric GTP-binding proteins regulating GLUT4 translocation in adipocytes, wild type and constitutively active G(q) (G(q)/Q209L), G(i) (G(i)/Q205L), and G(s) (G(s)/Q227L) alpha subunit mutants were expressed in 3T3L1 adipocytes. Although expression of neither the wild type nor G(i)/Q205L and G(s)/Q227L alpha subunit mutants had any effect on the basal or insulin-stimulated translocation of a co-expressed GLUT4-enhanced green fluorescent protein (EGFP) fusion protein, expression of G(q)/Q209L resulted in GLUT4-EGFP translocation in the absence of insulin. In contrast, microinjection of an inhibitory G(q)/G(11) alpha subunit-specific antibody but not a G(i) or G(s) alpha subunit antibody prevented insulin-stimulated endogenous GLUT4 translocation. Consistent with a required role for GTP-bound G(q)/G(11), expression of the regulators of G protein signaling (RGS4 and RGS16) also attenuated insulin-stimulated GLUT4-EGFP translocation. To assess the relationship between G(q)/G(11) function with the phosphatidylinositol 3-kinase dependent pathway, expression of a dominant-interfering p85 regulatory subunit, as well as wortmannin treatment inhibited insulin-stimulated but not G(q)/Q209L-stimulated GLUT4-EGFP translocation. Furthermore, G(q)/Q209L did not induce the in vivo accumulation of phosphatidylinositol-3,4,5-trisphosphate (PIP(3)), whereas expression of the RGS proteins did not prevent the insulin-stimulated accumulation of PIP(3). Together, these data demonstrate that insulin stimulation of GLUT4 translocation requires at least two independent signal transduction pathways, one mediated through the phosphatidylinositol 3-kinase and another through the trimeric GTP-binding proteins G(q) and/or G(11).
Highlights
In contrast to insulin, various other stimuli display insulinomimetic properties and can induce the translocation of GLUT4 to the plasma membrane
Expression of the Constitutively Active Gq ␣ Subunit Stimulates GLUT4 Translocation—Recently several studies have shown that receptor agonists coupled to Gi and Gq/G11 can result in GLUT4 translocation in both transfected fibroblasts and 3T3L1 adipocytes [50, 55, 56]
To determine the potential involvement of the Gq/G11 ␣ subunit as an intermediate in a potential signal transduction cascade leading to GLUT4 translocation, we examined the effect of Gq/WT and constitutively active Gq/Q209L ␣ subunit expression (Fig. 1)
Summary
Various other stimuli display insulinomimetic properties and can induce the translocation of GLUT4 to the plasma membrane. Insulin stimulation resulted in the translocation of the GLUT4-EGFP protein to the plasma membrane detected as a continuous rim of cell surface fluorescence in both GLUT4-EGFP transfected and Gq/WT co-transfected cells (Fig. 1A, panel b). Cells expressing Gq/Q209L resulted in the constitutive association of the GLUT4-EGFP protein with the plasma membrane in the absence of insulin (Fig. 1A, panel c).
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