Interactions between glycolysis and mixed function oxidation: studies with 7-ethoxycoumarin in perfused livers from...

Interaction between glycolysis and mitochondrial oxidations to supply reducing equivalents at high rates for mixed function oxidation was evaluated in the perfused liver after treatment of rats with beta-naphthoflavone. Livers from fasted beta-naphthoflavone-treated rats were employed because rates of 7-ethoxycoumarin O-deethylation were constant (16 mumol/g/hr) for at least 1 hr of perfusion. Preinfusion with KCN, an inhibitor of oxidative phosphorylation, caused the rate of 7-ethoxycoumarin O-deethylation to decline by 60% over 30 min of perfusion. The decline in rates of mixed function oxidation in the intact liver was not due to a direct effect of KCN on cytochrome P-450, inasmuch as cyanide did not diminish rates of 7-ethoxycoumarin O-deethylation by isolated microsomes. Cyanide rapidly decreased hepatic oxygen uptake by 70% and increased rates of glycolysis (lactate plus pyruvate production) from less than 10 to over 60 mumol/g/hr. Rates of glycolysis and mixed function oxidation subsequently declined in parallel during infusion of KCN. Infusion of ethanol (20 mM), a known inhibitor of glycolysis, decreased the stimulation of glycolysis caused by KCN to 20 mumol/g/hr and lowered maximal rates of 7-hydroxycoumarin production to about 6 mumol/g/hr. Both mixed function oxidation and glycolysis also declined in parallel over 30 min of perfusion in the presence of ethanol and KCN. When cyanide infusion was terminated, rates of oxygen uptake returned rapidly to basal values; however, rates of mixed function oxidation remained low. In contrast, infusion of ethanol in the absence of cyanide had no effect on rates of mixed function oxidation. Infusion of glucose (30 mM) or pyruvate (1 mM) after KCN restored maximal rates of mixed function oxidation in parallel with increases in rates of glycolysis. In contrast to results obtained in livers from fasted rats, cyanide and ethanol had little effect on 7-ethoxycoumarin O-deethylation in livers from fed rats. Taken together, these results argue strongly that rates of mixed function oxidation in the intact livers of fasted rats are sustained by reducing equivalents derived from mitochondrial oxidations. Glycolysis can supply substrates needed for the transport of reducing equivalents from the mitochondria into the cytosol for mixed function oxidation. Because glycogen reserves are minimal in the fasted state, rates of glycolysis and mixed function oxidation declined in parallel during the infusion of cyanide, because reducing equivalents derived from mitochondria are not available.