GLUCOSE-TRANSPORT IN L6 MYOBLASTS OVEREXPRESSING GLUT1 AND GLUT4

The roles of the glucose transporter isoforms, GLUT1 and GLUT4, in med iating insulin-stimulated glucose transport were investigated by stabl y overexpressing the transporters in L6 myoblasts. Levels of GLUT1 and GLUT4 in myoblasts from the cell lines having the highest content of these transporters were approximately 16- and 30-fold higher, respecti vely, than levels in nontransfected cells. The basal rate of 2-deoxy[H -3]glucose uptake was severalfold higher in cells overexpressing GLUT1 than in the parent L6 myoblasts or in control cell lines that were ge nerated by transfecting cells with expression vectors lacking transpor ter insert. The basal rate was not elevated in any of the lines expres sing GLUT4. The net increase in 2-deoxy[H-3]glucose uptake produced by insulin was larger in both the GLUT1 and GLUT4 cells than in the cont rol cells. Insulin increased uptake in GLUT4 cells by as much as 6-fol d; whereas, the fold increase over basal uptake produced by insulin in GLUT1 cells was comparable to that (2-fold) observed in the control m yocytes. Thus, both GLUT1 and GLUT4 can mediate insulin-stimulated glu cose transport in L6 myoblasts, although GLUT4 is needed to observe la rge percentage increases comparable to those observed in skeletal musc le fibers in vivo. In contrast to insulin, the protein phosphatase inh ibitors, okadaic acid and calyculin A, inhibited glucose transport in cells expressing either GLUT1 or GLUT4. Calyculin A, which produced a half-maximum effect at 10 nM, was approximately 100 times more potent than okadaic acid in decreasing both basal and insulin-stimulated 2-de oxyglucose uptake. Inhibition of uptake by calyculin A was associated with a decrease in the cell surface concentration of both GLUT1 and GL UT4. These results indicate that increased protein phosphorylation can lead to inhibition of transport mediated by both GLUT1 and GLUT4.