Interaction of oxamate with the gluconeogenic pathway in rat liver

Oxamate, a structural analog of pyruvate, known as a potent inhibitor of lactic dehydrogenase, lactic dehydrogenase, produces an inhibition of gluconeogenic flux in isolated perfused rat liver or hepatocyte suspensions from low concentrations of pyruvate (less than 0.5 mM) or substrates yielding pyruvate. The following observations indicate that oxamate inhibits flux through pyruvate carboxylase: accumulation of substrates and decreased concentration of all metabolic intermediates beyond pyruvate; decreased levels of aspartate, glutamate, and alanine; and enhanced ketone body production, which is a sensitive indicator of decreased mitochondrial free oxaloacetate levels. The decreased pyruvate carboxylase flux does not seem to be the result of a direct inhibitory action of oxamate on this enzyme but is secondary to a decreased rate of pyruvate entry into the mitochondria. This assumption is based on the following observations: Above 0.4 mM pyruvate, no significant inhibitory effect of oxamate on gluconeogenesis was observed. The competitive nature of oxamate inhibition is in conflict with its effect on isolated pyruvate carboxylase which is noncompetitive for pyruvate. Fatty acid oxidation was effective in stimulating gluconeogenesis in the presence of oxamate only at concentrations of pyruvate above 0.4 mM. Since only at low pyruvate concentrations its entry into the mitochondria occurs via the monocarboxylate translocator, from these observations it follows that pyruvate transport across the mitochondrial membrane, and not its carboxylation, is the first nonequilibrium step in the gluconeogenic pathway. In the presence of oxamate, fatty acid oxidation inhibited gluconeogenesis from lactate, alanine, and low pyruvate concentrations (less than 0.5 mM), and the rate of transfer of reducing equivalents to the cytosol was significantly decreased. Whether fatty acids stimulate or inhibit gluconeogenesis appears to correlate with the rate of flux through pyruvate carboxylase which ultimately seems to rely on pyruvate availability. Unless adequate rates of oxaloacetate formation are maintained, the shift of the mitochondrial NAD couple to a more reduced state during fatty acid oxidation seems to decrease mitochondrial oxaloacetate resulting in a decreased rate of transfer of carbon and reducing power to the cytosol.