Perinuclear localization and insulin responsiveness of GLUT4 requires cytoskeletal integrity in 3T3-L1 adipocytes

The GLUT4 glucose transporter resides mostly in perinuclear membranes in un stimulated 3T3-L1 adipocytes and is acutely translocated to the cell surfac e in response to insulin. Using a novel method to purify intracellular GLUT 4-enriched membranes, we identified by mass spectrometry the intermediate f ilament protein vimentin and the microtubule protein alpha -tubulin as comp onents of these membranes. Immunoelectron microscopy of the GLUT4-containin g membranes also revealed their association with these cytoskeletal protein s, Disruption of intermediate filaments and microtubules in 3T3-L1 adipocyt es by microinjection of a vimentin-derived peptide of the helix initiation 1A domain caused marked dispersion of perinuclear GLUT4 to peripheral regio ns of the cells. Inhibition of the microtubule-based motor dynein by brief cytoplasmic acidification of cultured adipocytes also dispersed perinuclear GLUT4 and inhibited insulin-stimulated GLUT4 translocation to the cell sur face. Insulin sensitivity was restored as GLUT4 was again concentrated near the nucleus upon recovery of cells in physiological buffer. These data sug gest that GLUT4 trafficking to perinuclear membranes of cultured adipocytes is directed by dynein and is required for optimal GLUT4 regulation by insu lin.