The Effects of Brefeldin A on the Glucose Transport System in Rat Adipocytes
IMPLICATIONS REGARDING THE INTRACELLULAR LOCUS Of INSULIN-SENSITIVE Glut4 (*)
- From the (1) Department of Medicine, Medical University of South Carolina, and the Ralph H. Johnson Veteran Affairs Medical Center, Charleston, South Carolina 29425 and the
- (2) Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104
- § To whom correspondence should be addressed: Division of Endocrinology, Medical University of South Carolina, 171 Ashley Ave., Charleston, SC 29425. Tel.: 803-792-2529; Fax: 803-792-4114; tim_garvey{at}smtpgw.musc.edu.
Abstract
Insulin activates glucose transport by recruiting Glut4 glucose transporters from an intracellular pool to plasma membrane
(PM). To localize intracellular translocating Glut4, we studied the effects of brefeldin A (BFA), which disassembles Golgi
and prevents trans-Golgi vesicular budding, on the glucose transport system. Isolated rat adipocytes were treated with and
without both BFA (10 μg/ml) and insulin. BFA did not affect maximal rates of either 2-deoxyglucose or 3-O-methylglucose transport or the insulin:glucose transport dose-response curve but did increase basal transport by 
2-fold (p < 0.05). We also measured Glut4 in PM, low (LDM) and high density microsome subfractions. In basal cells, BFA increased PM
Glut4 by 58% concomitant with a 18% decrease in LDM (p < 0.05). Insulin alone increased PM Glut4 by 3-fold concomitant with a 56% decrease in LDM. BFA did not affect insulin-induced
changes in Glut4 levels in PM or LDM. Most intracellular Glut4 was localized to sub-PM vesicles by immunoelectron microscopy
in basal cells, and BFA did not affect insulin-mediated recruitment of immunogold-labeled Glut4 to PM. In summary, 1) in basal
cells, BFA led to a small increase in glucose transport activity and redistribution of a limited number of transporters from
LDM to PM; 2) BFA did not affect insulin's ability to stimulate glucose transport or recruit normal numbers of LDM Glut4 to
PM; and 3) insulin action is predominantly mediated by a BFA-insensitive pool of intracellular Glut4, which localizes to sub-PM
vesicles. Thus, the major translocating pool of Glut4 in rat adipocytes does not involve trans-Golgi.
Footnotes
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↵* This work was supported by National Institutes of Health Grants DK-38765 (to W. T. G.), DK-28143 (to L. J.), and DK-19525 and by grants from the Department of Veterans Affairs and the American Heart Association. (to W. T. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 The abbreviations used are:
- IEM
-
immunoelectron microscopy
- BFA
-
brefeldin A
- 2-DOG
-
2-deoxyglucose
- HDM
-
high density microsome
- LDM
-
low density microsome
- 3-OMG
-
3-O-methylglucose
- PM
-
plasma membrane.
-
- Received August 10, 1995.
- Revision received October 5, 1995.
- © 1995 by The American Society for Biochemistry and Molecular Biology, Inc.
