Vesicle-associated Membrane Protein 2 Plays a Specific Role in the Insulin-dependent Trafficking of the Facilitative Glucose Transporter GLUT4 in 3T3-L1 Adipocytes*
- From the ‡Centre for Molecular and Cellular Biology and ‖Department of Physiology and Pharmacology, University of Queensland, St. Lucia 4072, Australia and the §Division of Biochemistry and Molecular Biology, University of Glasgow, Glasgow G12 8QQ, Scotland
Abstract
Vesicle-associated membrane protein 2 (VAMP2) has been implicated in the insulin-regulated trafficking of GLUT4 in adipocytes. It has been proposed that VAMP2 co-localizes with GLUT4 in a postendocytic storage compartment (Martin, S., Tellam, J., Livingstone, C., Slot, J. W., Gould, G. W., and James, D. E. (1996) J. Cell Biol. 134, 625–635), suggesting that it may play a role distinct from endosomal v-SNAREs (solubleN-ethylmaleimide-sensitive factor attachment protein receptors) such as cellubrevin that are also expressed in adipocytes. The present study examines the effects of recombinant glutathioneS-transferase (GST) fusion proteins encompassing the entire cytoplasmic tails of VAMP1, VAMP2, and cellubrevin on insulin-stimulated GLUT4 translocation in streptolysin O permeabilized 3T3-L1 adipocytes. GST-VAMP2 inhibited insulin-stimulated GLUT4 translocation by ∼35%, whereas GST-VAMP1 and GST-cellubrevin were without effect. A synthetic peptide corresponding to the unique N terminus of VAMP2 also inhibited insulin-stimulated GLUT4 translocation in a dose-dependent manner. This peptide had no effect on either guanosine 5′-3-O-(thio)triphosphate-stimulated GLUT4 translocation or on insulin-stimulated GLUT1 translocation. These results imply that GLUT4 and GLUT1 may undergo insulin-stimulated translocation to the cell surface from separate intracellular compartments. To confirm this, adipocytes were incubated with a transferrin-horseradish peroxidase conjugate to fill the itinerant endocytic system after which cells were incubated with H2O2 and diaminobenzidine. This treatment completely blocked insulin-stimulated movement of GLUT1, whereas in the case of GLUT4, movement to the surface was delayed but still reached similar levels to that observed in insulin-stimulated control cells after 30 min. These results suggest that the N terminus of VAMP2 plays a unique role in the insulin-dependent recruitment of GLUT4 from its intracellular storage compartment to the cell surface.
Footnotes
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↵* This work was supported by grants from the National Health and Medical Research Council of Australia and Glaxo Wellcome (to D. E. J.) and by the British Diabetic Association and The Wellcome Trust (to G. W. G.).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.
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↵¶ A Lister Institute of Preventive Medicine Research Fellow.
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↵** A Wellcome Trust Professorial Research Fellow. To whom correspondence should be addressed. Tel.: 61-7-33654986; Fax: 61-7-33654388; E-mail: D.James{at}cmcb.uq.edu.au.
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↵1 The abbreviations used are: SNARE, solubleN-ethylmaleimide-sensitive factor attachment protein receptor; DAB, 3,3′-diaminobenzidine; GSV, GLUT4 storage vesicle; HRP, horseradish peroxidase; LDM, low density microsome(s); PM, plasma membrane(s); SSV, small synaptic vesicle; Tf, transferrin; Tf-HRP, transferrin-HRP conjugate; VAMP, vesicle-associated membrane protein; SNAP, synaptosome-associated protein; DMEM, Dulbecco’s modified Eagle’s medium; SLO, streptolysin; GTPγS, guanosine 5′-3-O-(thio)triphosphate; PBS, phosphate-buffered saline; BSA, bovine serum albumin.
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↵2 Rea, S., and James, D. E., (1997) Diabetes 46, 1667–1677.
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↵3 L. B. Martin, A. Shewan, C. A. Millar, G. W. Gould, and D. E. James, unpublished observation.
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- Received September 10, 1997.
- Revision received October 29, 1997.
- The American Society for Biochemistry and Molecular Biology, Inc.
