Translocation of the Glucose Transporter (GLUT4) to the Cell Surface in Permeabilized 3T3-L1 Adipocytes: Effects of ATP, Insulin, and GTPγS and Localization of GLUT4 to Clathrin Lattices

• Published in: The Journal of cell biology Vol. 117; no. 6; pp. 1181 - 1196
• Main Authors: Robinson, Linda J., Pang, Suhong, Harris, Diana S., Heuser, John, James, David E.
• Format: Journal Article
• Language: English
• Published: New York, NY Rockefeller University Press 01-06-1992; The Rockefeller University Press
• Subjects: 3T3 Cells; Adenosine Triphosphate - physiology; Adipocytes; Adipose Tissue - metabolism; Animals; ATP; Biological and medical sciences; Biological Transport; Brown adipocytes; Cell Membrane - metabolism; Cell Membrane Permeability; Cell membranes; Cell physiology; cell surface; Cells; clathrin; Clathrin - metabolism; effects on; Facilitative glucose transport proteins; Fibroblasts; Fluorescent Antibody Technique; Fundamental and applied biological sciences. Psychology; glucose transporter; Glucose Transporter Type 4; GLUT4 protein; GTP; Guanosine 5'-O-(3-Thiotriphosphate) - physiology; Insulin; Insulin - physiology; Kinetics; Lawns; localization; Mice; Molecular and cellular biology; Monosaccharide Transport Proteins - metabolism; Muscle Proteins; Physiological regulation; Secretion; Secretion. Exocytosis; translocation; Adipocyte; GTP; Analog; Plasma membrane; Permeabilizing; Stimulation; Exocytosis; Immunofluorescence; Electron microscopy; Mechanism of action; Carrier protein
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.117.6.1181

Insulin stimulates the movement of two glucose transporter isoforms (GLUT1 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 immunofluo-rescence microscopy we observed increased PM labeling for GLUT1 (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 flat 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 GTPγS 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 GTPγS were added together, in the presence of ATP, PM GLUT4 levels were similar to levels observed when either insulin or GTPγS was added individually. Addition of GTPγS was able to overcome this ATP dependence of insulin-stimulated GLUT4 movement. GTPγS had no effect on constitutive secretion of adipsin in permeabilized cells. In addition, there was no effect of insulin or GTPγS 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.