Effect of phenylephrine on pyruvate dehydrogenase in fasting rat livers

Previous estimates of flux through the pyruvate‐dehydrogenase complex were made by measuring ¹⁴CO₂ generated from oxidation of [1‐¹⁴C]pyruvate, assuming a 1:1 stoichiometry. However, this method fails to discriminate between ¹⁴CO₂ produced from pyruvate dehydrogenase and ¹⁴CO₂ generated from phospho‐enolpyruvate carboxykinase and citric‐acid‐cycle dehydrogenases. While some previous reports have attempted to correct for the additional ¹⁴CO₂ production by comparing ¹⁴CO₂ generated by [1‐¹⁴C]pyruvate with [2‐¹⁴C]pyruvate or [3‐¹⁴C]pyruvate, the estimates are flawed by failure to determine the radioactivity and distribution of the ¹⁴C label in the oxalacetate pool. The present method circumvents these problems by utilizing [1,4‐¹⁴C]succinate to radiolabel the oxalacetate pool and by directly measuring the specific radioactivity of malate. The results demonstrate that flux through the pyruvate‐dehydrogenase complex is negligible compared to the other reactions which generate ¹⁴CO₂ from [1‐¹⁴C]lactate in the fasted state. Phenylephrine did not significantly alter this result in the fasted state. However, ¹⁴CO₂ production via the pyruvate‐dehydrogenase complex is large (∼ 11.5 nmol · min⁻¹· mg mitochondrial protein⁻¹) compared to ¹⁴CO₂ production via phosphoenolpyruvate carboxykinase and citric‐acid‐cycle dehydrogenases (∼ 6.4 nmol · min⁻¹· mg⁻¹) when the pyruvate‐dehydrogenase complex is activated, in the fed state with 1 mM dichloroacetate.