Control of Ketogenesis and Fatty‐Acid Synthesis at the Mitochondrial Branch‐Point for Acetyl‐CoA in the Chick Liver Cell: Effect of Adenosine 3′,5′‐Monophosphate

Previous work has shown that dibutyryl adenosine 3′,5′‐monophosphate (Bt2cAMP) markedly activates ketogenesis from fatty acids released during the mobilization of cytoplasmic triacylglycerol‐rich vesicles in the chick liver cell. In the present investigation, activation of the formation of [14C]acetoacetate and 3‐[14C]hydroxybutyrate from [1‐14C]oleate by the cyclic nucleotide was shown to occur at two mitochondrial sites: (a) the l‐(–)‐carnitine‐dependent translocation of the fatty acid across the inner mitochondrial membrane and (b) the intramitochondrial metabolism of acetyl‐CoA. The hypothesis was tested that cAMP regulates ketogenesis and fatty acid synthesis by controlling acetyl‐CoA flux at the mitochondrial branch‐point leading into the 3‐hydroxy‐3‐methylglutaryl‐CoA cycle or to citrate formation. Bt2cAMP caused an immediate activation of acetoacetate formation and a concomitant inhibition of fatty acid synthesis from acetate. Evidence is presented that these effects are due to a cyclic‐nucleotide‐induced fall in the mitochondrial concentration of oxaloacetate. An assay for oxaloacetate, sufficiently sensitive to analyze extracts of cells in monolayer culture, was employed. With this assay, Bt, cAMP was shown to cause a > 90% decrease in cellular oxaloacetate concentration. The calculated level to which intramitochondrial oxaloacetate fell was below the Km for oxaloacetate in the citrate‐synthase‐catalyzed reaction and, therefore, was sufficient to curtail citrate formation. Lactate, a precursor of oxaloacetate and an antiketogenic agent, reversed the effects of Bt2cAMP. Under conditions where glucose was sole precursor of oxaloacetate and citrate, the activation of ketogenesis and the inhibition of fatty acid synthesis by Bt2cAMP were reversed by lactate. Lactate also reversed the Bt2cAMP‐induced decrease in oxaloacetate and citrate concentration. The fact that lactate enters the pathway between glucose and oxaloacetate indicated that the site of inhibition by Bt, cAMP lies above pyruvate in the glycolytic pathway. Based on these and previous results it appears that, by inhibiting glycolysis, cAMP exerts long‐range effects on two major pathways in the chick hepatocyte, ketogenesis and fatty acid synthesis.