Glucose-dependent trans-plasma membrane electron transport and p70S6k phosphorylation in skeletal muscle cells

The reduction of extracellular oxidants by intracellular electrons is known as trans-plasma membrane electron transport (tPMET). The goal of this study was to characterize a role of tPMET in the sensing of glucose as a physiological signal. tPMET from C2C12 myotubes was monitored using a cell-impermeable extracellular electron acceptor, water-soluble tetrazolium salt-1 (WST-1). Superoxide dismutase in the incubation medium or exposure to an NADPH oxidase (NOX) isoform 1/4 inhibitor suppressed WST-1 reduction by 70%, suggesting a role of NOXs in tPMET. There was a positive correlation between medium glucose concentration and WST-1 reduction, suggesting that tPMET is a glucose-sensing process. WST-1 reduction was also decreased by an inhibitor of the pentose phosphate pathway, dehydroepiandrosterone. In contrast, glycolytic inhibitors, 3PO and sodium fluoride, did not affect WST-1 reduction. Thus, it appears that glucose uptake and processing in the pentose phosphate pathway drives NOX-dependent tPMET. Western blot analysis demonstrated that p70(S6k) phosphorylation is glucose-dependent, while the phosphorylation of AKT and MAPK did not differ in the presence or absence of glucose. Further, phosphorylation of p70(S6k) was dependent upon NOX enzymes. Finally, glucose was required for full stimulation of p70(S6k) by insulin, again in a fashion prevented by NOX inhibition. Taken together, the data suggest that muscle cells have a novel glucose-sensing mechanism dependent on NADPH production and NOX activity, culminating in increased p70(S6k) phosphorylation.