Kinetics of GLUT1 and GLUT4 glucose transporters expressed in Xenopus oocytes

H. Nishimura, F. V. Pallardo, G. A. Seidner, S. Vannucci, I. A. Simpson, M. J. Birnbaum

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Abstract

The predominant mechanism by which insulin activates glucose transport in muscle and adipose tissue is by affecting the redistribution of the facilitated hexose carriers, GLUT1 and GLUT4, from an intracellular site to the plasma membrane. A quantitative analysis of this process has been hampered by the lack of reliable determinations for kinetic constants catalyzed by each of these isoforms. In order to obtain such information, each transporter was expressed in Xenopus oocytes by the injection of mRNA encoding rat GLUT1 or GLUT4. Equilibrium exchange 3-O-methylglucose uptake was measured and the data fitted to a two-compartment model, yielding K(m) = 26.2 mM and V(max) = 3.5 nmol/min/cell for GLUT1 and K(m) = 4.3 mM and V(max) = 0.7 nmol/min/cell for GLUT4. Measurement of the abundance of cell surface transporters was accomplished by two independent protocols: photolabeling with the impermeant hexose analog 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl- 1,3-bis(D-mannos-4-yloxy)-2-propylamine and subcellular fractionation of oocytes. Data obtained by either technique revealed that the ratio of plasma membrane GLUT1 to GLUT4 was about 4; this paralleled the relative maximal velocities for hexose transport, indicating that the turnover numbers for the two isoforms were the same. Moreover, measurement of the concentration of exofacially disposed transporters with 2-N-4-(1-azi-2,2,2- trifluoroethyl)benzoyl-1,3-bis(D-mannos-4-yloxy)-2-propylamine allowed calculation of the turnover number to be about 20,000 min-1. These data indicate that, at low substrate concentrations, the catalytic efficiency of GLUT4 is significantly greater than GLUT1. Extrapolation to mammalian systems suggests that GLUT4 is responsible for virtually all of the hexose uptake in insulin-responsive targets, particularly in the presence of hormone.

Original languageEnglish (US)
Pages (from-to)8514-8520
Number of pages7
JournalJournal of Biological Chemistry
Volume268
Issue number12
StatePublished - 1993

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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