Regulation of Pyruvate Carboxylase in Rat Liver Mitochondria by Adenine Nucleotides and Short Chain Fatty Acids

The activity of pyruvate carboxylase of intact rat liver mitochondria was measured by improved methods under various experimental conditions. In agreement with previous work, elevated ADP concentrations in the medium caused a strong inhibition of the enzyme activity. A partial reversal of this inhibition was observed upon addition of octanoate or decanoate, but not with octanoyl‐ l ‐carnitine. Furthermore, atractylate completely reversed this inhibition whereas dinitrophenol or oligomycin increased it. In these experiments, no clear correlation between pyruvate carboxylase activity and the levels of adenine nucleotides of the whole medium was apparent. However, the measurement of the intramitochondrial adenine nucleotide levels revealed a close inverse relationship between ADP levels and pyruvate carboxylase activity, as fatty acids and atractylate lowered whereas oliogomycin elevated the intramitochondrial ADP levels. Other evidence indicates that the intramitochondrial ATP to ADP ratio may also be of importance in regulating the pyruvate carboxylase activity. In the experiments with octanoate, the intramitochondrial AMP was strongly elevated and the ADP plus ATP pool was smaller. In agreement with this finding, the amounts of rapidly exchanging adenine nucleotides were found to be smaller in the presence of octanoate than in its absence, as measured by the incorporation of added [ 14 C]ATP. The addition of octanoyl‐ l ‐carnitine caused only small changes in the intramitochondrial adenine nucleotide pattern and had also no significant effect on the incorporation of added [ 14 C]ATP into the mitochondria. The solubilized mitochondrial pyruvate carboxylase could also be inhibited by ADP but this inhibition could not be reversed by octanoate nor with high amounts of acetyl‐CoA. Oxygen uptake measurements with intact mitochondria showed a strong increase in the rate of respiration upon addition of octanoate or octanoyl‐ l ‐carnitine to the ADP supplemented system. This increase could be inhibited by oligomycin indicating that the stimulated respiration is coupled to an increased phosphorylation of ADP. It is concluded that the pyruvate carboxylase activity is regulated mainly by the level of intramitochondrial ADP. The lowering of the intramitochondrial ADP level and the resulting stimulation of pyruvate carboxylase by fatty acids is related on one hand to the production of intramitochondrial AMP by fatty acid activation resulting in a lowering of the ADP plus ATP pool, and on the other hand to the increased utilization of ADP for oxidative phosphorylation. The possible physiological implications of these findings are discussed.