Control mechanisms of gluconeogenesis and ketogenesis. II. Interactions between fatty acid oxidation and the citric acid cycle in perfused rat liver.

Previous work has shown that gluconeogenesis from a variety of substrates is stimulated by oleate. This paper reports data on the effect of oleate on tissue levels of coenzyme A derivatives, ketone bodies, citric acid cycle intermediates, and adenine nucleotides in rat livers perfused with alanine, lactate, or pyruvate. The effect of oleate on the activity of the citric acid cycle with lactate as substrate has been assessed from measurements of the oxygen consumption, and the rates of production or utilization of lactate, pyruvate, glucose, acetoacetate, β-hydroxybutyrate, and urea. Oleate increased the acetyl-CoA content of liver, supporting the suggestion that this intermediate is a physiological regulator of pyruvate carboxylase activity. The oxygen uptake of the liver was increased by oleate, while the ATP: ADP ratio was decreased. Since adenine nucleotides are largely extramitochondrial, an increase of ADP in both mitochondrial and extramitochondrial spaces is indicated. Calculations of the energy requirement for gluconeogenesis and fatty acid activation compared with the extra oxygen consumed upon addition of lactate or oleate to the liver provided P:0 ratios in vivo of 2.9 and 2.8, respectively, showing that oleate in vivo has no uncoupling effect. With lactate as substrate, flow rates of 135 and 147 µmioles per 100 g, body weight, per hour through the citric acid cycle were calculated in the absence and presence of oleate. Flux through the pyruvate dehydrogenase step was inhibited 80% by oleate. Despite the fact that oleate addition did not change the activity of the citric acid cycle markedly, the tissue level of α-ketoglutarate decreased with all substrates tested. After oleate addition, the citrate content increased in the presence of lactate or pyruvate, but decreased in the presence of alanine. It is suggested that the observed fall of α-ketoglutarate and rise of citrate occur largely in the cytosol. Assuming equilibrium of NAD dehydrogenases in mitochondria and cytosol, the intracellular distribution of malate and oxalacetate was calculated from tissue β-hydroxybutyrate to acetoacetate and lactate to pyruvate ratios. These calculations indicate that oxalacetate is largely extramitochondrial. In the presence of pyruvate, large concentration gradients of malate from mitochondria to cytosol are established. Control of the citric acid cycle is discussed in relation to changes of known allosteric modulators of citrate synthase and isocitrate dehydrogenase, and possible compartmentation of intermediates. It is concluded that citrate accumulation is regulated by the mitochondrial oxalacetate concentration, which itself is controlled by the pyruvate carboxylase activity and the NAD oxidation-reduction potential. Feedback between the respiratory chain and mitochondrial isocitrate dehydrogenases is mediated by ADP, NADH, and NADPH. It appears that the distribution of citric acid cycle intermediates in the mitochondrial and cytosolic compartments is greatly affected both by the pyruvate supply and the rate of β oxidation.