Kinetic resolution of the separate GLUT1 and GLUT4 glucose transport activities in 3T3-L1 cells.
A bis-mannose-photolabel-displacement method has been developed for resolving the separate kinetic properties of the glucose transporters GLUT1 and GLUT4, which are both present in 3T3-L1 cells. We have quantified the cell-surface transporter abundance (Bmax.) for the two isoforms by displacing radiolabelled 2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]-1,3-bis-(D- mannos-4-yloxy)-2-propylamine (ATB-BMPA) by non-labelled ATB-BMPA. In cells acutely treated with insulin, the GLUT1 Bmax. was 0.19 microM and the GLUT4 Bmax. was 0.17 microM. In cells which were chronically treated with insulin, the GLUT1 Bmax. was increased by approximately 4-fold to 0.7 microM, whereas the GLUT4 was decreased by approximately 50% (Bmax. = 0.1 microM). However, this large increase in total concentrations of cell-surface transporters (the sum of GLUT1 and GLUT4 concentrations) was not reflected in a large increase in 3-O-methyl-D-glucose transport, suggesting that GLUT1 makes a smaller contribution to transport than does GLUT4. In acutely insulin-treated cells at 37 degrees C, the apparent kinetic parameters for 3-O-methyl-D-glucose transport were Vapp.max. = 0.52 mM.s-1 and Kapp.m = 12.3 mM. In chronically insulin-treated cells the Vapp.max. = 1.24 mM.s-1 and Kapp.m = 23.0 mM. We have measured the displacement of ATB-BMPA by different concentrations of 3-O-methyl-D-glucose to resolve the separate affinity constants of GLUT1 and GLUT4 for this transported ligand. In acute- and chronic-insulin-treated cells the GLUT1 Km for 3-O-methyl-D-glucose was approximately 20 mM, and the GLUT4 Km for 3-O-methyl-D-glucose was approximately 7 mM. An analysis of these data and the 3-O-methyl-D-glucose transport rates was carried out to calculate transport capacity (TK values) for the two isoforms at 37 degrees C. In acute- and chronic-insulin-treated cells the TK values were 0.36 x 10(4) mM-1.min-1 for GLUT1 and 1.13 x 10(4) mM-1.min-1 for GLUT4. Thus GLUT1 has an approximately 3-fold lower transport capacity than GLUT4 at low concentrations of transported sugar. The lower GLUT1 transport capacity was shown to be mainly due to the high Km of GLUT1. The calculated turnover numbers were 7.2 x 10(4) min-1 for GLUT1 and 7.9 x 10(4) min-1 for GLUT4.
Palfreyman
R W
RW
Department of Biochemistry, University of Bath, Claverton Down, U.K.
Clark
A E
AE
Denton
R M
RM
Holman
G D
GD
Kozka
I J
IJ
D019325:3-O-Methylglucose; D016475:3T3 Cells / Q000378:metabolism*; D000818:Animals; D001386:Azides / Q000493:pharmacokinetics; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability; D004187:Disaccharides / Q000493:pharmacokinetics; D006027:Glycosides; D007328:Insulin / Q000494:pharmacology; D007536:Isomerism; D007700:Kinetics; D008757:Methylglucosides / Q000493:pharmacokinetics; D051379:Mice; D009004:Monosaccharide Transport Proteins / Q000378:metabolism* / Q000493:pharmacokinetics; D011233:Precipitin Tests; D011437:Propylamines
D001386:Azides; D004187:Disaccharides; D006027:Glycosides; D007328:Insulin; D008757:Methylglucosides; D009004:Monosaccharide Transport Proteins; D011437:Propylamines; C065412:2-N-(4-(1-azitrifluoroethyl)benzoyl)-1,3-bis-(mannos-4-yloxy)-2-propylamine; D019325:3-O-Methylglucose