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Am J Physiol Endocrinol Metab 281: E72-E80, 2001;
0193-1849/01 $5.00
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Vol. 281, Issue 1, E72-E80, July 2001

Glucose uptake and metabolism by cultured human skeletal muscle cells: rate-limiting steps

Laureta M. Perriott1,2, Tetsuro Kono3, Richard R. Whitesell3, Susan M. Knobel3, David W. Piston3, Daryl K. Granner1,2,3, Alvin C. Powers1,2, and James M. May1,2,3

1 Department of Veterans Affairs Medical Center; and Departments of 2 Medicine and 3 Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

To use primary cultures of human skeletal muscle cells to establish defects in glucose metabolism that underlie clinical insulin resistance, it is necessary to define the rate-determining steps in glucose metabolism and to improve the insulin response attained in previous studies. We modified experimental conditions to achieve an insulin effect on 3-O-methylglucose transport that was more than twofold over basal. Glucose phosphorylation by hexokinase limits glucose metabolism in these cells, because the apparent Michaelis-Menten constant of coupled glucose transport and phosphorylation is intermediate between that of transport and that of the hexokinase and because rates of 2-deoxyglucose uptake and phosphorylation are less than those of glucose. The latter reflects a preference of hexokinase for glucose over 2-deoxyglucose. Cellular NAD(P)H autofluorescence, measured using two-photon excitation microscopy, is both sensitive to insulin and indicative of additional distal control steps in glucose metabolism. Whereas the predominant effect of insulin in human skeletal muscle cells is to enhance glucose transport, phosphorylation, and steps beyond, it also determines the overall rate of glucose metabolism.

transport-metabolism coupling; glucose transport; hexokinase; rate-determining step; substrate specificity


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