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J Biol Chem, Vol. 274, Issue 53, 37755-37762, December 31, 1999

Separation and Partial Characterization of Three Distinct Intracellular GLUT4 Compartments in Rat Adipocytes
SUBCELLULAR FRACTIONATION WITHOUT HOMOGENIZATION ^*

Wan Lee^§, Jiwon Ryu^§, Ricardo P. Souto^¶, Paul F. Pilch^¶, and Chan Y. Jung^§

From the  Biophysics Laboratory, Veterans Affairs Medical Center, and the ^§ Department of Physiology and Biophysics, School of Medicine, State University of New York, Buffalo, New York 14215 and the ^¶ Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118

Insulin recruits GLUT4 from an intracellular location to the plasma membrane in rat adipocytes. The process involves multiple intracellular compartments and multiple protein functions, details of which are largely unknown partly due to our inability to separate individual GLUT4 compartments. Here, by hypotonic lysis, differential centrifugation, and glycerol density gradient sedimentation, we separated intracellular GLUT4 compartments in rat adipocytes into three fractions: plasma membrane-containing fraction T and plasma membrane-free fractions H and L. The GLUT4 contents in fractions T, H, and L were ~25, 56, and 18% of total GLUT4, respectively, in basal adipocytes and 55, 42, and 3-4% in insulin-stimulated adipocytes. The plasma membrane GLUT4 contents estimated separately further revealed that intracellular GLUT4 in fraction T amounts to ~20% in both basal and insulin-stimulated adipocytes. Organelle-specific marker and membrane traffic-related protein distribution data suggested that intracellular GLUT4 in fraction T represents sorting endosomes, whereas GLUT4 in fractions H and L represents storage endosomes and exocytic vesicles, respectively. The subcellular fractionation without homogenization described here should be useful in identifying the role of the individual GLUT4 compartments and the associated proteins in insulin-induced GLUT4 recruitment in rat adipocytes.

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^* This work was supported in part by National Institutes of Health Grants RO1 DK13376 (to C. Y. J.) and RO1 DK30425 (to P. F. P.) and by the Buffalo Veterans Affairs Medical Center (to C. Y. J.).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.

To whom correspondence should be addressed: Biophysics Lab., VA Medical Center, 3495 Bailey Ave., Buffalo, NY 14215. Tel.: 716-862-6540; Fax: 716-862-6526; E-mail: cyjung@acsu.buffalo.edu.

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Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.

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