Abstract
It is well established that insulin stimulation of glucose uptake requires the translocation of intracellular localized GLUT4 protein to the cell surface membrane. This plasma membrane-redistributed GLUT4 protein was partially co-localized with caveolin as determined by confocal fluorescent microscopy but was fully excluded from lipid rafts based upon Triton X-100 extractability. Cholesterol depletion with methyl-beta-cyclodextrin, filipin, or cholesterol oxidase resulted in an insulin-independent increase in the amount of plasma membrane-localized GLUT4 that was fully reversible by cholesterol replenishment. This basal accumulation of cell surface GLUT4 occurred due to an inhibition of GLUT4 endocytosis. However, this effect was not specific since cholesterol extraction also resulted in a dramatic inhibition of clathrin-mediated endocytosis as assessed by transferrin receptor internalization. To functionally distinguish between caveolin- and clathrin-dependent endocytic processes, we took advantage of a dominant-interfering caveolin 1 mutant (Cav1/S80E) that specifically disrupts caveolae organization. Expression of Cav1/S80E, but not the wild type (Cav1/WT) or Cav1/S80A mutant, inhibited cholera toxin B internalization without any significant effect on transferrin receptor endocytosis. In parallel, Cav1/S80E expression increased the amount of plasma membrane-localized GLUT4 protein in an insulin-independent manner. Although Cav1/S80E also decreased GLUT4 endocytosis, the extent of GLUT4 internalization was only partially reduced ( approximately 40%). In addition, expression of Cav1/WT and Cav1/S80A enhanced GLUT4 endocytosis by approximately 20%. Together, these data indicate that the endocytosis of GLUT4 requires clathrin-mediated endocytosis but that the higher order structural organization of plasma membrane caveolin has a significant influence on this process.
Highlights
A large increase in the rate of GLUT4 exocytosis coupled with a smaller decrease in the rate of GLUT4 endocytosis [6, 7]
Plasma Membrane-localized GLUT4 Partially Associates with Large Caveolin Clusters—Previously, we and others have observed that differentiated 3T3L1 adipocytes assemble large clusters of individual caveolae that are discernible by confocal fluorescent microscopy [25, 30, 36]
The organization of these proteins is distinct from marker proteins that do not associate in lipid raft microdomains such as the transferrin receptor, which recycles from the plasma membrane through clathrin-coated pits, and the clathrin coat protein itself (Fig. 1A, panels d–i)
Summary
A large increase in the rate of GLUT4 exocytosis coupled with a smaller decrease in the rate of GLUT4 endocytosis [6, 7]. This effect was not specific since cholesterol extraction resulted in a dramatic inhibition of clathrin-mediated endocytosis as assessed by transferrin receptor internalization.
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