THE CAPACITY OF REDUCING-EQUIVALENT SHUTTLES LIMITS GLYCOLYSIS DURINGETHANOL OXIDATION

The inhibition of glycolysis during ethanol oxidation has been examine d in isolated hepatocytes from fasted rats. Glycolytic flux was measur ed by determining the rate of release of tritium from [6-H-3]glucose. During ethanol oxidation, the rate of glycolysis was inhibited 80% in freshly prepared hepatocytes, in which shuttle intermediates are deple ted, but was depressed only about 20% in the presence of asparagine, a condition under which activity of the malate/aspartate shuttle was re stored to normal levels. The inhibition of glycolysis was also partial ly released by addition of pyruvate and when alcohol dehydrogenase act ivity was depressed by 4-methylpyrazole. Titrations with this inhibito r revealed inverse linear relationships between the rates of glycolysi s and ethanol oxidation. For any given rate of ethanol oxidation, glyc olytic flux was lowest and the [lactate]/[pyruvate] ratio highest in t he presence of aminooxyacetate, an inhibitor of the malate/aspartate s huttle, whereas flux was highest and the ratio lowest in the presence of asparagine. During these titrations with 4-methylpyrazole the inhib ition of ethanol oxidation and concomitant restoration of glycolysis w ere accompanied by a decline in the [lactate]/[pyruvate] ratio, a subs tantial fall in the rate of reducing-equivalent transfer from cytoplas m to mitochondria and an increase in lactate accumulation. These findi ngs imply that the reducing equivalents generated during ethanol oxida tion compete with those arising in glycolysis for transfer to the mito chondria. This competition leads to an inhibition of aerobic glycolysi s, and at the same time contributes to a rise in cytoplasmic NADH and fall in NAD(+) that results in depression of anaerobic glycolysis. All osteric inhibition of 6-phosphofructo-1-kinase due to a decrease in th e concentration of fructose 2,6-bisphosphate did not appear to play a primary role in the inhibition of glycolysis by ethanol. Ethanol oxida tion had no effect on glucose phosphorylation as measured with [2-H-3] glucose, but induced a substantial increase in cycling between glucose and glucose 6-phosphate.