Regulation of Insulin-stimulated GLUT4 Translocation by Munc18c in 3T3L1 Adipocytes*
- Debbie C. Thurmond‡,
- Brian P. Ceresa§,
- Shuichi Okada,
- Jeffrey S. Elmendorf¶,
- Kenneth Coker and
- Jeffrey E. Pessin‖
Abstract
Insulin stimulates glucose transporter (GLUT) 4 vesicle translocation from intracellular storage sites to the plasma membrane in 3T3L1 adipocytes through a VAMP2- and syntaxin 4-dependent mechanism. We have observed that Munc18c, a mammalian homolog of the yeast syntaxin-binding protein n-Sec1p, competed for the binding of VAMP2 to syntaxin 4. Consistent with an inhibitory function for Munc18c, expression of Munc18c, but not the related Munc18b isoform, prevented the insulin stimulation of GLUT4 and IRAP/vp165 translocation to the plasma membrane without any significant effect on GLUT1 trafficking. As expected, overexpressed Munc18c was found to co-immunoprecipitate with syntaxin 4 in the basal state. However, these complexes were found to dissociate upon insulin stimulation. Furthermore, endogenous Munc18c was predominantly localized to the plasma membrane and its distribution was not altered by insulin stimulation. Although expression of enhanced green fluorescent protein-Munc18c primarily resulted in a dispersed cytosolic distribution, co-expression with syntaxin 4 resulted in increased localization to the plasma membrane. Together, these data suggest that Munc18c inhibits the docking/fusion of GLUT4-containing vesicles by blocking the binding of VAMP2 to syntaxin 4. Insulin relieves this inhibition by inducing the dissociation of Munc18c from syntaxin 4 and by sequestering Munc18c to an alternative plasma membrane binding site.
Footnotes
-
↵* This work was supported in part by Research Grants DK33823 and DK25925 from the National Institutes of Health.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.
-
↵‡ Supported by Postdoctoral Fellowship Training Grant DK09813 from the National Institutes of Health.
-
↵§ Present address: Dept. of Cell Biology, Scripps Institute, La Jolla, CA 92037.
-
↵¶ Supported by Postdoctoral Fellowship Training Grant 398234 from the Juvenile Diabetes Foundation.
-
↵‖ To whom correspondence should be addressed. Tel.: 319-335-7823; Fax: 319-335-7886; E-mail: jeffrey-pessin{at}uiowa.edu.
- Abbreviations:
- GLUT
-
glucose transporter
- eGFP
-
enhanced green fluorescent protein
- IRAP/vp165
-
insulin-responsive aminopeptidase
- v-SNARE
-
vesicle SNAP receptor
- t-SNARE
-
target membrane SNAP receptor
- DMEM
-
Dulbecco’s modified Eagle’s medium
- CHO/IR
-
Chinese hamster ovary cells expressing the human insulin receptor
- PAGE
-
polyacrylamide gel electrophoresis
- PBS
-
phosphate-buffered saline.
-
- Received August 11, 1998.
- Revision received September 28, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
