Inhibitors of protein synthesis cause increased hexose transport in cultured human fibroblasts by a mechanism other than transporter translocation

Abstract

We have investigated the effect of various inhibitors of protein synthesis on hexose transport in human skin fibroblasts using 2-deoxy-D-glucose (2-DG) and 3-0-methyl-D-glucose (3-OMG) to measure hexose transport. Exposure of glucose-fed, serum-free cultures to cycloheximide (CHX) (50 μg/ml) for 6 h resulted in increased 2-DG transport (3.81 ± .53 vs. 6.62 ± .88 nmoles/mg protein/2 min; n = 9) and 3-OMG transport (1.36 ±.66 vs. 3.18 ± .83 nmoles/mg protein/30 sec; n = 4) in the CHX exposed group. Under these conditions inhibition of protein synthesis was greater than 90%. This CHX induced transport increase was time dependent (approaching maximum within 1 h of exposure to CHX) and related to an increase in the V_max of hexose transport in the CHX exposed group (18.4 ± 2.4 vs. 4.8 ± 1.1 nmoles 2-DG/mg protein/min) with no difference in the transport K_m (1.55 ± .63 vs. 2.92 ± .59 mM). Further, the CHX induced increase in hexose transport was reversible. Exposure of human fibroblasts to inhibitors of protein synthesis with different mechanisms of action (e.g., puromycin, pactamy-cin, or CHX) all generated hexose transport increases in a concentration-dependent fashion correlating with their increasing inhibitory effects on protein synthesis. Nucleotidase enriched (i.e., plasma membrane) fractions of control and CHX-exposed cells showed no differences in D-glucose inhibitable cytochalasin B binding activity. Further, quantitative Western analysis of nucleotidase enriched fractions indicated CHX exposure resulted in no significant increase in glucose transporter mass compared with control plasma membrane fractions. Glucose deprived cells, however, which exhibited increased sugar transport comparable to the CHX-exposed group, did show increased glucose transporter mass in the plasma membrane fraction. The data indicate that inhibitors of protein synthesis can cause a significant elevation in hexose transport and that the hexose transporter mass in the isolated plasma membrane fractions did not reflect the whole cell transport change. It is suggested that a mechanism other than glucose transporter translocation to the plasma membrane may be involved in causing this sugar transport increase.