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J. Biol. Chem., Vol. 279, Issue 20, 21598-21605, May 14, 2004
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GLUT4 Overexpression or Deficiency in Adipocytes of Transgenic Mice Alters the Composition of GLUT4 Vesicles and the Subcellular Localization of GLUT4 and Insulin-responsive Aminopeptidase*
From the aDivision of Endocrinology, Diabetes and Metabolism, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215, fInstitute for Molecular Bioscience and the Department of Physiology and Pharmacology, University of Queensland, St. Lucia, Queensland 4072, and kGarvan Institute of Medical Research, 384 Victoria Road, Darlinghurst, New South Wales 2010, Australia
The majority of GLUT4 is sequestered in unique intracellular vesicles in the absence of insulin. Upon insulin stimulation GLUT4 vesicles translocate to, and fuse with, the plasma membrane. To determine the effect of GLUT4 content on the distribution and subcellular trafficking of GLUT4 and other vesicle proteins, adipocytes of adipose-specific, GLUT4-deficient (aP2-GLUT4-/-) mice and adipose-specific, GLUT4-overexpressing (aP2-GLUT4-Tg) mice were studied. GLUT4 amount was reduced by 80-95% in aP2-GLUT4-/- adipocytes and increased 
10-fold in aP2-GLUT4-Tg adipocytes compared with controls. Insulin-responsive aminopeptidase (IRAP) protein amount was decreased 35% in aP2-GLUT4-/- adipocytes and increased 45% in aP2-GLUT4-Tg adipocytes. VAMP2 protein was also decreased by 60% in aP2-GLUT4-/- adipocytes and increased 2-fold in aP2-GLUT4-Tg adipocytes. IRAP and VAMP2 mRNA levels were unaffected in aP2-GLUT4-Tg, suggesting that overexpression of GLUT4 affects IRAP and VAMP2 protein stability. The amount and subcellular distribution of syntaxin4, SNAP23, Munc-18c, and GLUT1 were unchanged in either aP2-GLUT4-/- or aP2-GLUT4-Tg adipocytes, but transferrin receptor was partially redistributed to the plasma membrane in aP2-GLUT4-Tg adipocytes. Immunogold electron microscopy revealed that overexpression of GLUT4 in adipocytes increased the number of GLUT4 molecules per vesicle nearly 2-fold and the number of GLUT4 and IRAP-containing vesicles per cell 3-fold. In addition, the proportion of cellular GLUT4 and IRAP at the plasma membrane in unstimulated aP2-GLUT4-Tg adipocytes was increased 4- and 2-fold, respectively, suggesting that sequestration of GLUT4 and IRAP is saturable. Our results show that GLUT4 overexpression or deficiency affects the amount of other GLUT4-vesicle proteins including IRAP and VAMP2 and that GLUT4 sequestration is saturable.
Received for publication, November 10, 2003 , and in revised form, February 8, 2004.
* This work was supported in part by National Institutes of Health Grant DK43051 and an American Diabetes Association Mentor-based Fellowship Grant (to B. B. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
b These authors contributed equally to this study.
c Supported by the Swedish Foundation for the Study of Diabetes, the Swedish Society for Medical Research, and the Helmuth Hertz Foundation.
d Supported by a Student Fellowship from the Endocrine Society.
e Supported by National Institutes of Health NRSA Grant DK09903 and Research Career Development Award K01 DK62212.
g Present address: Bristol-Myers Squibb, Princeton, NJ 08543.
h Supported by the Association de Langue Francaise d'Etudes du Diabetes et des Anomalies du Metabolisme.
i Present address: Dept. of Cardiovascular and Metabolic Diseases, Pfizer Global Research and Development, Groton, CT 06340.
j Supported by the Biomedical Sciences Exchange Program.
l To whom correspondence should be addressed: Endocrine Division-Research North 325E, Beth Israel Deaconess Medical Center, 99 Brookline Ave., Boston, MA 02215. Tel.: 617-667-5422; Fax: 617-667-2927; E-mail: bkahn{at}bidmc.harvard.edu.
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