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 without altering GLUT4 translocation. These results suggested that insulin might activate GLUT4 via a p38 MAPK-dependent pathway. Here we explore this hypothesis by temporal and kinetic analyses of the stimulation of GLUT4 translocation, glucose uptake and activation of p38 MAPK isoforms by insulin. In L6 myotubes stably expressing GLUT4 with an exofacial Myc epitope, we found 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 degrees C. Preincubation with the p38 MAPK inhibitors SB202190 and SB203580 reduced insulin-stimulated transport of either 2-deoxyglucose or 3-O-methylglucose by 40-60%. Pretreatment with SB203580 lowered the apparent transport V(max) of insulin-mediated 2-deoxyglucose and 3-O-methylglucose without any significant change in the apparent K(m) for either hexose. The IC(50) values for the partial inhibition of 2-deoxyglucose uptake by SB202190 and SB203580 were 1 and 2 microM respectively, and correlated with the IC(50) for full inhibition of p38 MAPK by the two inhibitors in myotubes (2 and 1.4 microM, respectively). Insulin caused a dose- (EC(50)=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 from insulin-stimulated myotubes showed activation of p38 alpha (2.6+/-0.3-fold) and p38 beta (2.3+/-0.2-fold) MAPK. These results suggest that activation of GLUT4 follows GLUT4 translocation and that both mechanisms contribute to the full stimulation of glucose uptake by insulin. Furthermore, activation of GLUT4 may occur via an SB203580-sensitive pathway, possibly involving p38 MAPK.
Somwar
R
R
Programme in Cell Biology, Hospital for Sick Children, 555 University Avenue, Toronto, ON, Canada, M5G 1X8.
Kim
D Y
DY
Sweeney
G
G
Huang
C
C
Niu
W
W
Lador
C
C
Ramlal
T
T
Klip
A
A
D000818:Animals; D002460:Cell Line; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005947:Glucose / Q000031:analogs & derivatives / Q000378:metabolism*; D051275:Glucose Transporter Type 4; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D007527:Isoenzymes; D020935:MAP Kinase Signaling System / Q000502:physiology; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D009124:Muscle Proteins; D018482:Muscle, Skeletal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D021381:Protein Transport / Q000187:drug effects; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases
D017362:Cyclic AMP Response Element-Binding Protein; D004791:Enzyme Inhibitors; D051275:Glucose Transporter Type 4; D007328:Insulin; D007527:Isoenzymes; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; C495692:SLC2A4 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D005947:Glucose