The effects of sulfhydryl modifying reagents on nonhormonal and hormonally regulated hexose transport in cultured human skin fibroblasts

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Abstract
The effects of various sulfhydryl modifying reagents on hexose transport in cultured human skin fibroblasts were studied. H2O2 was observed to have no effect on 2‐deoxy‐D‐glucose transport in serum‐starved glucose‐fed cells. The elevation of hexose transport rates in cells by glucose deprivation, insulin, or serum stimulation rendered them sensitive to H2O2. Hexose transport in glucose‐deprived cells was inhibited 51–55% by 1–2 mM H2O2, while hexose transport in insulin or serum‐stimulated glucose‐fed cells was inhibited 45% and 46%, respectively. H2O2 inhibition was blocked or reversed by 8 mM dithiothreitol. N‐ethyl‐maleimide (NEM), a permeant, sulfhydryl reagent, elicited effects on hexose transport similar to those effected by H2O2 (i.e., in glucose‐deprived and insulin‐stimulated cells, inhibition of hexose transport was 44% and 23%, respectively). Impermeant sulfhydryl reagents such as dithio(bis)nitrobenzoic acid (DTNB) and N‐iodoacetyl‐N'‐(5‐sulfo‐1‐naphthylethylenediame (1,5,‐I‐AEDANS)) had no inhibitory effect on hexose transport under any conditions (i.e., glucose‐fed, glucose‐deprived, and insulin‐stimulated cells). DTNB and 1,5‐I‐AEDANS afforded no protection from the action of H2O2 on hexose transport. The data suggest that the sensitive sites are thiol in nature and are located at an intramembrane or intracellular site and probably not exofacial.