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 micrograms/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 Vmax 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 Km (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, pactamycin, 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.

Sugar Transport Regulation: Comparative Characterization of the Effect of NADH CoQ Reductase Deficiency in Two Cell Culture Systems

“…The data in Table IIT2 clearly indicate that the normal cells are insulin‐responsive, exhibiting a significantly increased rate of 2‐DG transport (>2‐fold) after short‐term insulin exposure whereas the respiration‐deficient cells exhibited no significant increase (one‐way ANOVA; P < 0.05; n = 5) in 2‐DG transport post‐insulin exposure. Previously, we have shown that the increase in glucose transport in response to insulin is the result of translocation of GLUT 1 (6). Thus, it is apparent that no transporter translocation is taking place in these respiration‐deficient cells in response to insulin.…”

“…Fibroblasts from confluent monolayers of 850‐cm 2 roller bottles (Falcon) were treated, and plasma membranes were isolated according to a modified method of Buchanan (6, 13, 14). In short, after hypotonic lysis, a 27,000 g spin yielded a crude plasma membrane–enriched pellet (i.e., P1 fraction) from six roller bottles of human fibroblasts or from one roller bottle of hamster fibroblasts.…”

“…Prior to electrophoresis, samples were solubilized in 2% sodium dodecyl sulfate (SDS) (Pierce Chemical Co., Rockford, IL), 10% glycerol, 1 m M EDTA, 0.002% bromphenol blue, and 100 m M TrisHCl (pH 6.8). Electrophoresis was performed as described previously (6, 7, 14). Purified human erythrocyte glucose transporter was prepared as described by others (17) and used as a control GLUT 1 in Western analysis.…”

“…In mammalian cells, sugar transport is mediated by a family of structurally related glucose transporter (GT) proteins (1) that are regulated by a number of physiological conditions (2). The mechanisms involved in sugar transport regulation include: (i) the translocation of GTs between internal membranes and the active plasma membrane site, (ii) altered synthesis and/or degradation of the GT, and (iii) modification and/or modulation of the intrinsic or catalytic activity of the GT (3–6).…”

Different Forms of Vanadate on Sugar Transport in Insulin Target and Nontarget Cells

“…Prior to electrophoresis, samples were solubilized in 2% sodium dodecyl sulfate (SDS) (Pierce Chemical Co), 10% glycerol, 1 mmol/L EDTA, 0.002% bromphenol blue, 100 mmol/L tris-HCl (pH 6.8). Electrophoresis was performed as described previously in [24, 25, 26]. Proteins were blotted onto nitrocellulose paper (0.2 μm, Schleicher and Schull, Ontario, Canada) that was blocked with 1% BSA (1 hour at 20°C).…”