Abstract. Insulin stimulates the movement of two glucose transporter isoforms (GLUTI and GLUT4) to the plasma membrane (PM) in adipocytes. To study this process we have prepared highly purified PM fragments by gently sonicating 3T3-L1 adipocytes grown on glass coverslips. Using confocal laser immunofluorescence microscopy we observed increased PM labeling for GLUTI (2.3-fold) and GLUT4 (eightfold) after insulin treatment in intact cells. EM immunolabeling of PM fragments indicated that in the nonstimulated state GLUT4 was mainly localized to fiat clathrin lattices. Whereas GLUT4 labeling of clathrin lattices was only slightly increased after insulin treatment, labeling of uncoated PM regions was markedly increased with insulin. These data suggest that GLUT4 recycles from the cell surface both in the presence and absence of insulin. In streptolysin-O permeabilized adipocytes, insulin, and GTP3,S increased PM levels of GLUT4 to a similar extent as observed with insulin in intact cells. In the absence of an exogenous ATP source the magnitude of these effects was considerably reduced. Removal of ATP per se caused a significant increase in cell surface levels of GLUT4 suggesting that ATP may be required for intracellular sequestration of these transporters. When insulin and GTPvS were added together, in the presence of ATE PM GLUT4 levels were similar to levels observed when either insulin or GTP3,S was added individually. Addition of GTP3,S was able to overcome this ATP dependence of insulinstimulated GLUT4 movement. GTP7S had no effect on constitutive secretion of adipsin in permeabilized cells. In addition, there was no effect of insulin or GTP3~S on GLUT4 movement to the PM in noninsulin sensitive streptolysin-O-permeabilized 3T3-L1 fibroblasts overexpressing GLUT4. We conclude that the insulin-stimulated movement of GLUT4 to the cell surface in adipocytes may require ATP early in the insulin signaling pathway and a GTP-binding protein(s) at a later step(s). We propose that the association of GLUT4 with clathrin lattices may be important in maintaining the exclusive intracellular location of this transporter in the absence of insulin.

We have examined the expression and cellular location offacilitated glucose transporter proteins (GLUT1, -3, and -5) in a human colonic epithelial cell line (Caco-2) by using peptide-specific antibodies. A differential cellular distribution of these transporters was observed in differentiated (>14 days postconfluence) Caco-2 cells by immunofluorescence and immunoelectron microscopy. GLUTi was localized primarily to the basolateral membrane, whereas GLUT3 was predominantly localized to the apical membrane. GLUTS, which was detected in only =40% offully differentiated Caco-2 cells, was found primarily in the apical membrane but was also present in both basolateral and intracellular membranes. A Na+-independent glucose transport system in the apical membrane of Caco-2 cells has been described previously [Blais, A., Bissonnette, A. & Berteloot, A. (1987) (7), the insulin-regulated movement of GLUT4 from intracellular tubulo-vesicles to the cell surface (8), and the targeting of GLUT2 to the basolateral membrane in kidney and intestinal epithelial cells (9). By using GLUT1/GLUT4 chimeric proteins, it has recently been shown that the amino-terminal 19 amino acids of GLUT4 are both necessary and sufficient for intracellular sequestration (10).The purpose of the present studies was to examine the targeting of various glucose transporter isoforms in epithelial cells in order to establish a system that would enable a molecular dissection of polarized sorting of membrane proteins. Previous studies of glucose transport kinetics in a human colonic adenocarcinoma cell line (Caco-2) have indicated the presence of a facilitated transport system at the apical and basolateral surfaces (11). Using isoform-specific antibodies, we show in these studies that Caco-2 cells express high levels of GLUT1, GLUT3, and GLUT5. Importantly, each of these isoforms displays a unique subcellular distri- 7556The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.