Saturable and nonsaturable hexose uptake in cultured human skin fibroblasts

Abstract

The saturable transport of 2-deoxy-D-glucose across the cell membrane of cultured human skin fibroblasts was measured in sparse and confluent cultures. The contribution of nonsaturable sugar uptake to total sugar uptake was monitored by determining L-glucose uptake. The uptake of 2-deoxy-D-glucose was studied as a function of time and substrate concentration. Greater than 70% of transported 2-deoxy-D-glucose was phosphorylated after incubation for 2 min or less at all substrate concentrations employed (0.1 to 3.0 mM), and phosphorylation paralleled sugar uptake at these time intervals. Experiments with cytochalasin B demonstrated that an inhibition of transport was always paralleled by an equal inhibition of sugar phosphorylation.The kinetic constants for the uptake and phosphorylation of 2-deoxy-D-glucose and the inhibition of transport by competing sugars and cytochalasin B were calculated from Line-weaver-Burk plots. The Km and Vmax for saturable sugar uptake were calculated for sparse and confluent cultures after subtracting the contribution of nonsaturable sugar uptake. The resulting Km values for sugar uptake in the sparse and confluent cultures were 1.21 ± 0.04 and 0.88 ± 0.2 mM respectively. The corresponding Vmax values were 15.5 ± 1 nmol/mg protein∙min−1 for the sparse cultures and 10.1 ± 1 nmol/mg protein∙min−1 for the confluent cultures. In both sparse and confluent cultures, the Ki values for the competitive inhibition of sugar transport by D-glucose and 3-O-methyl-D-glucose were 0.8 and 2.7 mM respectively; the Ki value for the noncompetitive inhibition of sugar transport by cytochalasin B was 0.5 μM. The Km values for sugar phosphorylation by cell-free homogenates of sparse and confluent cultures were 0.57 ± 0.1 and 0.6 ± 0.1 mM respectively, while their respective Vmax values were 160 ± 53 and 139 ± 43 nmol/mg protein∙min−1.The data are in agreement with the concept that in cultured human skin fibroblasts sugar transport is the rate-limiting step in 2-deoxy-D-glucose metabolism and that phosphorylation is distinct from transport.