Glucose and NADPH Oxidase Dependent Trans‐plasma Membrane Electron Transport

Abstract

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.