GLUT4 translocation precedes the stimulation of glucose uptake by insulin in muscle cells: potential activation of GLUT4 via p38 mitogen-activated protein kinase

We previously reported that SB203580, an inhibitor of p38 mitogen-activated protein kinase (p38 MAPK), attenuates insulin-stimulated glucose uptake wi thout altering GLUT4 translocation. These results suggested that insulin mi ght activate GLUT4 via a p38 MAPK-dependent pathway. Here we explore this h ypothesis by temporal and kinetic analyses of the stimulation of GLUT4 tran slocation, glucose uptake and activation of p38 MAPK isoforms by insulin. I n L6 myotubes stably expressing GLUT4 with an exofacial Myc epitope, we fou nd that GLUT4 translocation (t(1/2) = 2.5 min) preceded the stimulation of 2-deoxyglucose uptake (t(1/2) = 6 min). This segregation of glucose uptake from GLUT4 translocation became more apparent when the two parameters were measured at 22 degreesC. Preincubation with the p38 MAPK inhibitors SB20219 0 and SB203580 reduced insulin-stimulated transport of either 2-deoxyglucos e or 3-0-methylglucose by 40-60%. Pretreatment with SB203580 lowered the ap parent transport V-max of insulin-mediated 2-deoxyglucose and 3-0-methylglu cose without any significant change in the apparent K-m for either hexose. The IC50 values for the partial inhibition of 2-deoxyglucose uptake by SB20 2190 and SB203580 were 1 and 2 muM respectively and correlated with the IC5 0 for full inhibition of p38 MAPK by the two inhibitors in myotubes (2 and 1.4 muM, respectively). Insulin caused a dose- (EC50 = 15 nM) and time- (t( 1/2) = 3 min) dependent increase in p38 MAPK phosphorylation, which peaked at 10 min (2.3 +/-0.3-fold). In vitro kinase assay of immunoprecipitates fr om insulin-stimulated myotubes showed activation of p38 alpha (2.6 +/-0.3-f old) and p38 beta (2-3 +/-0.2-fold) MAPK. These results suggest that activa tion of GLUT4 follows GLUT4 translocation and that both mechanisms contribu te to the full stimulation of glucose uptake by insulin. Furthermore, activ ation of GLUT4 may occur via an SB203580-sensitive pathway, possibly involv ing p38 MAPK.