Interrelationships between gluconeogenesis and ureogenesis in isolated hepatocytes

DL-Cycloserine (5 to 10 mM), when added to isolated hepatocytes, is shown to inhibit aspartate aminotransferase predominantly in the cytosol. At low concentration (0.1 to 0.2 mM) it selectively inhibits alanine aminotransferase. Aminooxyacetate (0.5 mM) irreversibly inhibits all transaminase activity provided the inhibitor is added prior to substrate addition. Neither cycloserine nor aminooxyacetate caused appreciable inhibition of gluconeogenesis from pyruvate, but both inhibitors greatly decreased gluconeogenesis from lactate. These observations confirm earlier findings that malate efflux from mitochondria predominates with pyruvate as glucose precursor while aspartate efflux predominates with lactate as substrate. Urea synthesis from ammonia by isolated hepatocytes was limited by endogenous ornithine. Half-maximal stimulation of urea synthesis in the presence of pyruvate was obtained with 0.5 mM exogenous ornithine. Measurements of citrulline accumulation showed (a) that carbamyl phosphate formation was not rate limiting and (b) that the apparent K(m) of argininosuccinate synthetase for citrulline was about 1.5 mM. Urea formation in the presence of ornithine and either lactate or pyruvate was limited by the activity of argininosuccinate synthetase. Urea synthesis was completely inhibited by aminooxyacetate, but was largely unaffected by cycloserine. These data show that aspartate required for urea synthesis must be generated in the mitochondria. Accumulation of aspartate showed that its rate of formation was not limiting for urea synthesis. Cycloserine addition in the absence of ornithine stimulated urea formation from ammonia. This effect was caused by an increase of intracellular citrulline levels because of inhibition of ornithine transaminase. Ammonia addition to hepatocytes incubated with oleate and either lactate or pyruvate caused an inhibition of gluconeogenesis and accumulation of amino acids (primarily alanine, glutamate, and aspartate). Decreased gluconeogenesis correlated with lowered malate levels, suggesting limitation of flux through P-enolpyruvate carboxykinase by a fall of the oxalacetate concentration. Pyruvate carboxylase flux was also inhibited, possibly because of elevated mitochondrial glutamate levels. On the other hand, pyruvate dehydrogenase was stimulated by ammonia addition as a result of the decreased mitochondrial NADH/NAD ratio. Ammonia addition with ornithine increased malate production in the cytosol as a consequence of increased urea formation, and released inhibition of flux through pyruvate carboxylase and P-enolpyruvate carboxykinase when pyruvate was added as substrate. With lactate as substrate, high rates of urea synthesis induced by ornithine did not stimulate gluconeogenesis, and malate levels remained low because of constraints imposed by maintenance of near-equilibrium of cytosolic aspartate aminotransferase. No evidence was obtained in the present experiments that transport of anions across the mitochondrial membrane provided a rate-limiting step in the pathways of either gluconeogenesis or urea synthesis.