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 micrograms/ml) and insulin. BFA did not affect maximal rates of either 2-deoxyglucose or 3-O-methyl-glucose transport or the insulin:glucose transport dose-response curve but did increase basal transport by approximately 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.
Highlights
Insulin activates glucose transport by recruiting Glut4 glucose transporters from an intracellular pool to plasma membrane (PM)
Isolated rat adipocytes were preincubated with increasing concentrations of brefeldin A (BFA) (0 –10 g/ml) for 30 min and treated in the absence and presence of a maximally effective concentration (100 ng/ml) of insulin for 30 min at 37 °C
BFA blocks constitutive secretion by preventing the formation of non-clathrin-coated vesicles required for vesicular transport through Golgi cisternae [10] as well as clathrin-coated vesicles that bud from trans-Golgi network [18]
Summary
Vol 270, No 50, Issue of December 15, pp. 30199 –30204, 1995 Printed in U.S.A. (Received for publication, August 10, 1995, and in revised form, October 5, 1995). Most intracellular Glut was localized to sub-PM vesicles by immunoelectron microscopy in basal cells, and BFA did not affect insulin-mediated recruitment of immunogold-labeled Glut to PM. 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 Glut to PM; and 3) insulin action is predominantly mediated by a BFA-insensitive pool of intracellular Glut, which localizes to sub-PM vesicles. Smith et al [7] have shown in rat adipocytes that most intracellular Glut undergoing insulin-mediated translocation derive from vesicles adjacent to the endofacial surface of plasma membrane (PM) or PM invaginations. If insulinresponsive Glut derive from the Golgi network, BFA should inhibit insulin-stimulated glucose transport and Glut translocation
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