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Endothelin-1-induced GLUT4 Translocation Is Mediated via Gαq/11 Protein and Phosphatidylinositol 3-Kinase in 3T3-L1 Adipocytes*

  • Takeshi Imamura
    Footnotes
    Affiliations
    From the Department of Medicine, University of California, San Diego, La Jolla, California 92093-0673
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  • Ken-ichi Ishibashi
    Affiliations
    From the Department of Medicine, University of California, San Diego, La Jolla, California 92093-0673
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  • Stephane Dalle
    Affiliations
    From the Department of Medicine, University of California, San Diego, La Jolla, California 92093-0673
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  • Satoshi Ugi
    Affiliations
    From the Department of Medicine, University of California, San Diego, La Jolla, California 92093-0673
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  • Jerrold M. Olefsky
    Correspondence
    To whom correspondence should be addressed: Dept. of Medicine (0673), University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0673. Tel.: 858-534-6651; Fax: 858-534-6653
    Affiliations
    From the Department of Medicine, University of California, San Diego, La Jolla, California 92093-0673
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  • Author Footnotes
    * This work was supported in part by National Institutes of Health Grant DK-33651 and the Veterans Affairs Medical Research Service.
    ‡ Recipient of an American Diabetes Association Mentor-based Fellowship Award.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:November 19, 1999DOI:https://doi.org/10.1074/jbc.274.47.33691
      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 Gαq/11 function by single-cell microinjection using anti-Gαq/11 antibody or RGS2 protein (a GTPase activating protein for Gαq) 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 Gαq/11 antibody or RGS2 protein, respectively. Pretreatment of cells with the Gαi inhibitor (pertussis toxin) or microinjection of a Gβγ inhibitor (glutathioneS-transferase-β-adrenergic receptor kinase (GST-BARK)) did not inhibit ET-1-induced GLUT4 translocation, indicating that Gαq/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 Gαq/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 Gαq/11. These results indicate that: 1) ET-1 signaling to GLUT4 translocation is dependent upon Gαq/11 and PI 3-kinase; and 2) Gαq/11 can transmit signals from the ETAreceptor to the p110α subunit of PI 3-kinase, as does insulin, subsequently leading to GLUT4 translocation.
      ET
      endothelin
      ETA-R
      type-A endothelin receptor
      RGS
      regulators of G protein signaling
      GPCR
      G protein-coupled receptor
      GLUT4
      insulin-responsive glucose transporter isoform
      GST
      glutathioneS-transferase
      BARK
      β-adrenergic receptor kinase
      2-DOG
      2-deoxy-d-glucose
      PI 3-kinase
      phosphatidylinositol 3-kinase
      PKC
      protein kinase C
      PLC
      phospholipase C
      PBS
      phosphate-buffered saline
      PTX
      pertussis toxin
      p110
      catalytic subunit of PI 3-kinase
      Endothelin-1 (ET-1)1 is a 21-amino acid polypeptide hormone, produced mainly in cardiac myocytes and vascular endothelial cells (
      • Schiffrin E.L.
      • Touyz R.M.
      ). ET-1 binds to the ETA receptor, which is a G protein-coupled receptor (GPCR) that is expressed in many tissues and cultured cells including 3T3-L1 adipocytes (
      • Douglas S.A.
      • Ohlstein E.H.
      ,
      • Wu-Wong J.R.
      • Berg C.E.
      • Wang J.
      • Chiou W.J.
      • Fissel B.
      ). A prominent function of ET-1 is to increase the contractility of cardiac and vascular smooth muscles in order to regulate systemic hemodynamics between vessel capacity and the content of the systemic circulation (
      • Schiffrin E.L.
      • Touyz R.M.
      ). It was reported that plasma ET-1 levels were markedly elevated in patients with heart failure (
      • Miyauchi T.
      • Goto K.
      ). Recently, it has also been found that type 2 diabetic patients with microvascular complications have increased levels of plasma ET-1 (
      • Sármán B.
      • Tóth M.
      • Somogyi A.
      ,
      • Kawamura M.
      • Ohgawara H.
      • Naruse M.
      • Suzuki N.
      • Iwasaki N.
      • Naruse K.
      • Hori S.
      • Demura H.
      • Omori Y.
      ), and there was a report that ET-1 can modulate insulin-stimulated PI 3-kinase activity in vascular smooth muscle cells (
      • Jiang Z.Y.
      • Zhou Q.L.
      • Chatterjee A.
      • Feener E.P.
      • Myers Jr., M.G.
      • White M.F.
      • King G.L.
      ). These findings raise the possibility that ET-1 might be involved in the insulin resistance associated with hypertension and the vascular complications of type 2 diabetes mellitus.
      It has also been found that acute treatment of 3T3-L1 adipocytes with ET-1 leads to an increase in GLUT4 translocation to the cell surface as well as increased glucose uptake (
      • Wu-Wong J.R.
      • Berg C.E.
      • Wang J.
      • Chiou W.J.
      • Fissel B.
      ). However, the signaling pathway mediating this ET-1-induced increase in glucose transport is poorly understood. In the current study, we show that, in 3T3-L1 adipocytes, the G protein αq/11 (Gαq/11) is necessary for ET-1-induced GLUT4 translocation and that ET-1-induced Gαq/11 signaling to GLUT4 translocation is mediated through a PI 3-kinase dependent mechanism.

      Acknowledgments

      We thank Dr. John R. Hepler (Washington University, St. Louis, MO) for providing the RGS2 expression vector, Dr. David W. Rose (University of California, San Diego, CA) for technical assistance on microinjection, and Elizabeth Hansen and Augustus P. Lestick for editorial assistance.

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