Endothelin-1-induced GLUT4 translocation is mediated via Gα(q/11) protein and phosphatidylinositol 3-kinase in 3T3-L1 adipocytes

Abstract

Endothelin-1 (ET-1) can stimulate insulin-responsive glucose transporter (GLUT4) translocation in 3T3-L1 adipocytes (Wu-Wong, J. R., Berg, C. E., Wang, J., Chiou, W. J., and Fissel, B. (1999) J. Biol. Chem. 274, 8103-8110), and in the current study, we have evaluated the signaling pathway leading to this response. First, we inhibited endogenous Gα(q/11) function by single- cell microinjection using anti-Gα(q/11) antibody or RGS2 protein (a GTPase activating protein for Gα(q)) followed by immunostaining to quantitate GLUT4 translocation in 3T3-L1 adipocytes. ET-1-stimulated GLUT4 translocation was markedly decreased by 70 or 75% by microinjection of Gα(q/11) antibody or RGS2 protein, respectively. Pretreatment of cells with the Gα(i) inhibitor (pertussis toxin) or microinjection of a Gβγ inhibitor (glutathione S- transferase-β-adrenergic receptor kinase (GST-BARK)) did not inhibit ET-1- induced GLUT4 translocation, indicating that Gα(q/11) mediates ET-1 signaling to GLUT4 translocation. Next, we found that ET-1-induced GLUT4 translocation was inhibited by the phosphatidylinositol (PI) 3-kinase inhibitors wortmannin or LY294002, but not by the phospholipase C inhibitor U-73122. ET-1 stimulated the PI 3-kinase activity of the p110α subunit (5.5- fold), and microinjection of anti-p110α or PKC-λ antibodies inhibited ET- stimulated GLUT4 translocation. Finally, we found that Gα(q/11) formed immunocomplexes with the type-A endothelin receptor and the 110α subunit of PI 3-kinase and that ET-1 stimulation enhances tyrosine phosphorylation of Gα(q/11). These results indicate that: 1) ET-1 signaling to GLUT4 translocation is dependent upon Gα(q/11) and PI 3-kinase; and 2) Gα(q/11) can transmit signals from the ET(A) receptor to the p110α subunit of PI 3- kinase, as does insulin, subsequently leading to GLUT4 translocation.