Lipid rafts are required for GLUT4 internalization in adipose cells

It has been recently reported that insulin recruits a novel signaling machi nery to lipid rafts required for insulin-stimulated GLUT4 translocation [Ba umann, A., Ribon, V., Kanzaki, M., Thurmond, D. C., Mora, S., Shigematsu, S ., Bickel, P. E., Pessin, J. E. & Saltiel, A. R. (2001) Nature 407, 202-207 , 2000; Chiang, S. H., Baumann, C. A., Kanzaki, M., Thurmond, D. C., Watson , R. T., Neudauer, C. L., Macara, I. G., Pessin, J. E. & Saltiel, A. R. (20 01) Nature 410, 944-948]. We have assessed the role of lipid rafts on GLUT4 traffic in adipose cells. High GLUT4 levels were detected in caveolae from adipocytes by two approaches, the mechanical isolation of purified caveola e from plasma membrane lawns and the immunogold analysis of plasma membrane lawns followed by freeze-drying. The role of lipid rafts in GLUT4 traffick ing was studied by adding nystatin or filipin at concentrations that specif ically disrupt caveolae morphology and inhibit caveolae function without al tering clathrin-mediated endocytosis. These caveolae inhibitors did not aff ect the insulin-stimulated glucose transport. However, they blocked both th e GLUT4 internalization and the down-regulation of glucose transport trigge red by insulin removal in 3T3-L1 adipocytes. Our data indicate that lipid r afts are crucial for GLUT4 internalization after insulin removal. Given tha t high levels of GLUT4 were detected in caveolae from insulin-treated adipo se cells, this transporter may be internalized from caveolae or caveolae ma y operate as an obligatory transition station before internalization.