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

Abstract

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.

Muscle glucose transport and phosphorylation in type 2 diabetic, obese nondiabetic, and genetically predisposed individuals

“…The finding that the defect in glucose phosphorylation exceeds that of inward glucose transport is consistent with previous studies from our laboratory that have demonstrated a severe defect in insulin-stimulated muscle hexokinase II activity, mRNA levels, and protein content (37,40,48,63,64). This observation is also consistent with prior studies demonstrating that, under insulin-stimulated conditions, post-glucose transport defects can be rate limiting for intracellular glucose metabolism (10,29,38,42,44,47,49,69). However, it should be emphasized that, even if the defect in hexokinase II were corrected, a severe defect in glucose transport most likely would remain (11) and become rate limiting.…”

Characterization of Glucose Uptake by Cultured Rat Podocytes

“…However, despite our expectations, in podocytes treated with insulin, 3 H-2DG uptake increased only 1.5-fold ( fi g. 2 a). The presence of various transport systems in podocytes could, at least in part, explain why the effect of insulin was relatively low as compared to other insulinsensitive cell types [18,19] . Although we have not quantitatively measured expression of individual GLUT forms, it cannot be ruled out that in our experimental conditions, in rat podocytes an insulin-sensitive transporter GLUT4, despite its high affi nity for glucose [20] , might contribute to a minor part of total glucose uptake.…”

“…The apparent S 0.5 value for total glucose transport, as calculated from the Hill equation, was 7.8 m M . This value, corresponding to Michaelis constant K m , may be resultant of the values for high-affi nity transporters (GLUT4, K m = 2-5 m M ; SGLT1, K m = 0.3-1.9 m M ) [2] and low-affi nity transporter GLUT2 (K m = 6-12 m M ) [18] . The Hill coeffi cient value h = 1.0 confi rms that glucose uptake into podocytes follows the Michaelis-Menten kinetics.…”

Zingiber officinale(Ginger): A Future Outlook on Its Potential in Prevention and Treatment of Diabetes and Prediabetic States

“…Skeletal muscle is the major site of glucose clearance in the body and accounts for over 75% of insulin-stimulated postprandial glucose disposal [66,67]. The rate-limiting step of glucose metabolism in skeletal muscle is glucose transport through the plasma membrane via GLUT4, one isoform of the 12-transmembrane domain sugar transporter family predominantly expressed in skeletal muscle [68,69]. It has been established that GLUT4 translocates from intracellular storage compartments to the cell surface in response to acute insulin stimulation or other stimuli [70].…”