The effects of energetic steady state, pyruvate concentration, and octanoyl-(--)-carnitine on the relative rates of carboxylation and decarboxylation of pyruvate by rat liver mitochondria
Journal of Biological Chemistry
Rat liver mitochondria were incubated with controlled concentrations of pyruvate over a range of energetic and respiratory steady states, in the presence and absence of octanoyl-( -)-carnitine in order to evaluate conditions and effectors which perturb the absolute and relative rates of flux through pyruvate carboxylase and pyruvate dehydrogenase. Control experiments using saturating concentrations of pyruvate are also reported. With high [pyruvate] (10 m~) as sole substrate, carboxylation rate
... was very rapid in the resting state, and was diminished in a stepwise manner on stimulation of respiration with increasing amounts of ATPase. Carboxylase flux rates did not correlate with [acetyl-CoA] or acetyl-CoA/CoASH ratio, or with changes in the mitochondrial ATP/ADP until ATPase in excess of maximal respiratory stimulation was added. Octanoyl carnitine stimulated carboxylation further, and this rate was better sustained in stimulated respiratory states. This stimulation is correlated qualitatively with elevation of the acetyl-CoA/CoA ratio. With pyruvate alone, pyruyate dehydrogenase flux was maximally stimulated, Independent of the energetic state. The latter was suppressed by octanoyl carnitine in all respiratory states, but only marginally. When pyruvate was in low concentration (-50 to 500 PM), carboxylation was now relatively low in the resting state (concentration-dependent on pyruvate), and was abolished by stepwise stimulation of respiration. This was accompanied by increased production of ketone bodies. Octanoyl carnitine now stimulated pyruvate carboxylase, and suppressed pyruvate dehydrogenase manyfold, such that their relative rates were altered up to >1,000-fold under some conditions. These effects are correlated with changes (in the same direction) of the acetyl-CoA/CoASH, NADH/NAD+, and ATP/ADP ratios. However, with pyruvate alone, decrease in pyruvate carboxylation again is not correlated with decreased [acetyl-CoA] or decreased acetyl-CoA/CoA. Apparently, other factors such as ADP will decrease carboxylase flux, even in fact of constant or increasing [acetyl-CoA]. It is concluded that the energetic state of the liver cell, and especially the availability of fatty acids, can trigger a very effective coordinated switch in gating pyruvate carbon to oxaloacetate or acetyl-CoA. Endocrine signals may initiate this gating by altering these parameters.