Activation of cell surface glucose transporters measured by photoaffinity labeling of insulin-sensitive 3T3-L1 adipocytes.
Several studies have demonstrated that the intrinsic catalytic activity of cell surface glucose transporters is highly regulated in 3T3-L1 adipocytes expressing GLUT1 (erythrocyte/brain) and GLUT4 (adipocyte/skeletal muscle) glucose transporter isoforms. For example, inhibition of protein synthesis in these cells by anisomycin or cycloheximide leads to marked increases in hexose transport without a change in the levels of cell surface glucose transporter proteins (Clancy, B. M., Harrison, S. A., Buxton, J. M., and Czech, M. P. (1991) J. Biol. Chem. 266, 10122-10130). In the present work the exofacial hexose binding sites on GLUT1 and GLUT4 in anisomycin-treated 3T3-L1 adipocytes were labeled with the cell-impermeant photoaffinity reagent [2-3H]2-N-[4-(1-azitrifluoroethyl)benzoyl]-1,3-bis- (D-mannos-4-yloxy)-2-propylamine [( 2-3H] ATB-BMPA) to determine which isoform is activated by protein synthetic blockade. As expected, a 15-fold increase in 2-deoxyglucose uptake in response to insulin was associated with 1.7- and 2.6-fold elevations in plasma membrane GLUT1 and GLUT4 protein levels, respectively. Anisomycin treatment of cultured adipocytes for 5 h produced an 8-fold stimulation of hexose transport but no increase in the content of glucose transporters in the plasma membrane fraction as measured by protein immunoblot analysis. Cell surface GLUT1 levels were also shown to be unaffected on 3T3-L1 adipocytes in response to anisomycin using an independent method, the binding of an antiexofacial GLUT1 antibody to intact cells. In contrast, anisomycin fully mimicked the action of insulin to stimulate (about 4-fold) the radiolabeling of GLUT1 transporters specifically immunoprecipitated from intact 3T3-L1 adipocytes irradiated after incubation with [2-3H] ATB-BMPA. Photolabeling of GLUT4 under these conditions was also significantly enhanced (1.8-fold) by anisomycin treatment, but this effect was only 15% of that caused by insulin. These results suggest that: 1) the photoaffinity reagent [2-3H]ATB-BMPA labels those cell surface glucose transporters present in a catalytically active state rather than total cell surface transporters as assumed previously and 2) inhibition of protein synthesis in 3T3-L1 adipocytes stimulates sugar transport primarily by enhancing the intrinsic catalytic activity of cell surface GLUT1, and to a lesser extent, GLUT4 proteins.
Harrison
S A
SA
Program in Molecular Medicine, University of Massachusetts Medical Center, Worcester 01605.
Clancy
B M
BM
Pessino
A
A
Czech
M P
MP
D016475:3T3 Cells; D000273:Adipose Tissue / Q000378:metabolism*; D000345:Affinity Labels; D000818:Animals; D000841:Anisomycin / Q000494:pharmacology; D001386:Azides / Q000378:metabolism; D015153:Blotting, Western; D002384:Catalysis; D003513:Cycloheximide / Q000494:pharmacology; D004187:Disaccharides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006027:Glycosides; D006601:Hexoses / Q000378:metabolism; D007328:Insulin / Q000494:pharmacology; D051379:Mice; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D010777:Photochemistry; D011437:Propylamines; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000378:metabolism
D000345:Affinity Labels; D001386:Azides; D004187:Disaccharides; D006027:Glycosides; D006601:Hexoses; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D011437:Propylamines; D011956:Receptors, Cell Surface; C065412:2-N-(4-(1-azitrifluoroethyl)benzoyl)-1,3-bis-(mannos-4-yloxy)-2-propylamine; D000841:Anisomycin; D003513:Cycloheximide