Insulin-stimulated GLUT4 translocation in adipocytes is dependent upon cortical actin remodeling

Rhodamine-labeled phalloidin staining of morphologically differentiated 3T3 L1 adipocytes demonstrated that F-actin predominantly exists juxtaposed to and lining the inner face of the plasma membrane (cortical actin) with a sm aller amount of stress fiber and/or ruffling actin confined to the cell bot tom in contact with the substratum. The extent of cortical actin disruption with various doses of either latrunculin B or Clostridium difficile toxin B (a Rho family small GTP-binding protein toxin) directly correlated with t he inhibition of insulin-stimulated glucose uptake and GLUT4 translocation. The dissolution of the cortical actin network had no significant effect on proximal insulin receptor signaling events including insulin receptor auto phosphorylation, tyrosine phosphorylation of insulin receptor substrate and Cb1, or serine/threonine phosphorylation of Akt. Surprisingly, however, st abilization of F-actin with jasplakinolide also resulted in a dose-dependen t inhibition of insulin-stimulated glucose uptake and GLUT4 translocation. In vivo time-lapse confocal fluorescent microscopy of actin-yellow fluoresc ent protein demonstrated that insulin stimulation initially results in cort ical actin remodeling followed by an increase in polymerized actin in the p eri-nuclear region. Importantly, the insulin stimulation of cortical actin rearrangements was completely blocked by treatment of the cells with latrun culin B, C. difficile toxin B, and jasplakinolide. Furthermore, expression of the dominant-interfering TC10/T31N mutant completely disrupted cortical actin and prevents any insulin-stimulated actin remodeling. Together, these data demonstrate that cortical actin, but not stress fibers, lamellipodia, or filopodia, plays an important regulatory role in insulin-stimulated GLU T4 translocation. In addition, cortical F-actin does not function in a stat ic manner (e.g. barrier or scaffold), but insulin-stimulated dynamic cortic al actin remodeling is necessary for the GLUT4 translocation process.