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

To use primary cultures of human skeletal muscle cells to establish defects in glucose metabolism that underlie clinical insulin resistance, it is nec essary to define the rate-determining steps in glucose metabolism and to im prove the insulin response attained in previous studies. We modified experi mental conditions to achieve an insulin effect on 3-O-methylglucose transpo rt that was more than twofold over basal. Glucose phosphorylation by hexoki nase limits glucose metabolism in these cells, because the apparent Michael is-Menten constant of coupled glucose transport and phosphorylation is inte rmediate between that of transport and that of the hexokinase and because r ates of 2-deoxyglucose uptake and phosphorylation are less than those of gl ucose. The latter reflects a preference of hexokinase for glucose over 2-de oxyglucose. Cellular NAD(P) H autofluorescence, measured using two-photon e xcitation microscopy, is both sensitive to insulin and indicative of additi onal distal control steps in glucose metabolism. Whereas the predominant ef fect of insulin in human skeletal muscle cells is to enhance glucose transp ort, phosphorylation, and steps beyond, it also determines the overall rate of glucose metabolism.