Endothelin-1-induced GLUT4 translocation is mediated via Galpha(q/11) protein and phosphatidylinositol 3-kinase in 3T3-L1 adipocytes.
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 Galpha(q/11) function by single-cell microinjection using anti-Galpha(q/11) antibody or RGS2 protein (a GTPase activating protein for Galpha(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 Galpha(q/11) antibody or RGS2 protein, respectively. Pretreatment of cells with the Galpha(i) inhibitor (pertussis toxin) or microinjection of a Gbetagamma inhibitor (glutathione S-transferase-beta-adrenergic receptor kinase (GST-BARK)) did not inhibit ET-1-induced GLUT4 translocation, indicating that Galpha(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 p110alpha subunit (5.5-fold), and microinjection of anti-p110alpha or PKC-lambda antibodies inhibited ET-stimulated GLUT4 translocation. Finally, we found that Galpha(q/11) formed immunocomplexes with the type-A endothelin receptor and the 110alpha subunit of PI 3-kinase and that ET-1 stimulation enhances tyrosine phosphorylation of Galpha(q/11). These results indicate that: 1) ET-1 signaling to GLUT4 translocation is dependent upon Galpha(q/11) and PI 3-kinase; and 2) Galpha(q/11) can transmit signals from the ET(A) receptor to the p110alpha subunit of PI 3-kinase, as does insulin, subsequently leading to GLUT4 translocation.
Imamura
T
T
Department of Medicine, University of California, San Diego, La Jolla, California 92093-0673, USA.
Ishibashi
K
K
Dalle
S
S
Ugi
S
S
Olefsky
J M
JM
D016475:3T3 Cells; D017667:Adipocytes / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D019332:Endothelin-1 / Q000494:pharmacology*; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D019204:GTP-Binding Proteins / Q000378:metabolism*; D051275:Glucose Transporter Type 4; D051379:Mice; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D009124:Muscle Proteins; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction
D019332:Endothelin-1; D051275:Glucose Transporter Type 4; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; C495693:Slc2a4 protein, mouse; D019869:Phosphatidylinositol 3-Kinases; D019204:GTP-Binding Proteins; D043802:GTP-Binding Protein alpha Subunits, Gq-G11