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 p70S6k phosphorylation is glucose-dependent, while the phosphorylation of AKT and MAPK did not differ in the presence or absence of glucose. Further, phosphorylation of p70S6k was dependent upon NOX enzymes. Finally, glucose was required for full stimulation of p70S6k 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 p70S6k phosphorylation.
Trans‐plasma membrane electron transport (tPMET), a process common to multiple cell types, transfers electrons from the cytosol to the extracellular space. The objective of this study was to determine the mechanism of tPMET in muscle cells. C2C12 myotubes and water‐soluble tetrazolium salt‐1 (WST‐1), a cell impermeable electron acceptor, were utilized along with metabolic inhibitors to elucidate the mechanism of tPMET. Upon addition of superoxide dismutase to extracellular media, WST‐1 reduction by myotubes was decreased by ~70%, indicating that the release of extracellular superoxide is the main mechanism of tPMET in C2C12 myotubes. The addition of GKT137831, a NADPH oxidase (NOX) 1 and 4 inhibitor, suppressed WST‐1 reduction while the addition of GSK2795039, a NOX2 inhibitor, did not decrease WST‐1 reduction. Preliminary western blot analysis showed the presence of NOX1 and NOX2 protein but not NOX4 protein. In the absence of glucose, tPMET was significantly decreased, thus indicating that tPMET is a glucose dependent process. The addition of the hexokinase inhibitor, 2‐deoxy‐D‐glucose, also suppressed tPMET as did the addition of glucose‐6‐phosphate dehydrogenase inhibitor, dehydroepiandrosterone. In contrast, glycolytic inhibitors including the 6‐phosphofructo‐2‐kinase inhibitor, 3PO, and enolase inhibitor, sodium fluoride, did not affect WST‐1 reduction. This indicates that the process of tPMET is dependent upon the pentose phosphate pathway and not glycolysis. In conclusion, these data suggest a glucose‐sensing model of tPMET in which presence of extracellular glucose provides substrate for NADPH production and subsequent NOX1‐dependent release of superoxide.Support or Funding InformationThis work was supported by the United States Public Health Service award R15DK102122 from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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