Decreased Flux through Pyruvate Dehydrogenase by Thiol Oxidation during t-Butyl Hydroperoxide Metabolism in Perfused Rat Liver

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Abstract
Addition of tert-butyl hydroperoxide [t-BHP] to isolated perfused rat liver leads to a decreased flux through pyruvate dehydrogenase, shown by a decreased 14CO2 release from [1-14C]pyruvate. The effect is observed at rates of infusion of t-BHP exceeding 0.7 .mu.mol/min per g liver in normal livers and at significantly lower rates in glutathione(GSH)-depleted livers. The effect is absent in livers from Se-deficient rats in which the hepatic Se-dependent GSH peroxidase [EC 1.11.1.9] activity is very low, indicating that reduction of t-BHP by GSH peroxidase is a necessary prerequisite for the inhibition. With isolated mitochondria, decreased 14CO2 release from [1-14C]pyruvate during t-BHP metabolism correlates with decreased GSH and increased GSSG [oxidized glutathione] contents, respectively. The addition of various disulfide compounds, including GSSG, inhibits activity of the enzyme in mitochondrial extracts. In both mitochondria and perfused liver, t-BHP-mediated decreased of pyruvate dehydrogenase flux is relieved by thiol reductants. The active (dephospho)form of pyruvate dehydrogenase as measured in freeze-stopped liver samples is actually increased from 46 to 72% during t-BHP metabolism. The tissue levels of ATP and ADP and perfusate .beta.-hydroxybutyrate/acetoacetate ratio are not markedly perturbed by addition of the hydroperoxide (10 min). The decreased flux through pyruvate dehydrogenase during t-BHP metabolism probably results from oxidation of critical thiol group(s) of the enzyme complex consequential to a decrease in mitochondrial GSH/GSSG.