481
Bioehimica et Biophysica Acta, 586 (1979) 481--491 © Elsevier/North-Holland Biomedical Press
BBA 28995
AMMONIA O V E R L O A D I N G IN HEPATOCYTES ISOLATED F R O M L I V E R OF FETAL AND A D U L T RATS
ENRIQUE CHICO, JESUS S. OLAVARRfA and IGNACIO NONEZ DE CASTRO *
Centro de Biologia Molecular. Departamento de Bioqulmica, C.S.I.C. Facultad de Ciencias, Universidad Aut6noma de Madrid, Cantoblanco, Madrid 34 (Spain) (Received February 26th, 1979)
Key words: Ammonia overloading; Ammonia detoxification; Amino acid synthesis; Urea cycle; Lactate production
Summary Ammonia overloading was investigated during glucose and fructose metabolism in isolated hepatocytes under a variety of metabolic conditions. In all assay conditions, the glycolytic flux and oxygen uptake was not modified b y 10 mM ammonia. In hepatocytes isolated from rats fed as libitum, the presence of ammonia caused a decrease in the production of lactate (pyruvate); this effect was not observed in anaerobic incubations, in hepatocytes isolated from starved animals, or in fetal hepatocytes. In spite of an overproduction of urea, ammonia detoxification also takes place by the synthesis of alanine, glutamate and aspartate. Addition of 1 mM aminooxyacetate, an inhibitor of aminotransferases, to the incubation medium prevents the formation of these amino acids, and also prevents the decrease of lactate in hepatocytes isolated from fed animals.
Introduction The early work of Schoenheimer [ 1 ] demonstrated that glutamate, aspartate, glutamine and asparagine were the most heavily labeled amino acids of rat protein when lSN-labelled ammonium compounds were administered. Nordmann et al. [2] investigated the effect of intraperitoneal administration of nonlethal doses of ammonium chloride to rats. They found an increase in the hepatic levels of glutamate, alanine and aspartate, whereas glutamine, pyruvate and citrate decreased. Since aminooxyacetate prevents ammonia detoxification by the production o f amino acids and urea, transamination reactions play an im* To w h o m
c o r r e s p o n d e n c e s h o u l d b e addressed.
482 portant role in the metabolism of ammonia by liver [3]. As Braunstein [4] originally reported, the transamination reactions constitute an important crossroad between the metabolism of alanine, aspartate and glutamate and the a-ketoacid precursors in the citric acid cycle. Brosnan and Williamson [5] reported the time course of the changes in the concentration of hepatic metabolites in response to a non-toxic load of ammonia and also the changes in the cellular redox state caused by ammonia. The process of ureogenesis in isolated rat hepatocytes has been studied by many authors [6,7]. Urea synthesis from ammonia is limited by endogenous ornithine. Addition of ammonia to hepatocytes causes an inhibition of gluconeogenesis from lactate or pyruvate [8]. Likewise, during ureogenesis induced by the ammonia treatment an oxidation of the cells takes place increasing the mitochondrial NADP/NADPH ratio [9,10]. Paush et al. [11] reported an induction of de novo pyrimidine synthesis in rat liver after the administration of a m m o n i u m ion in amounts surpassing the capacity of the urea cycle. More recently, Skaper et al. [ 12], using liver homogenates and minces, have found an enhancement of uridine nucleotide biosynthesis through the action of mitochondrial carbamoyl phosphate synthase, concomitant with a decrease in the phosphoribosyl pyrophosphate available for purine synthesis. The study of regulation of metabolism in isolated hepatocytes avoids the influence of extrahepatic effects of hormones and permits analysis of the response of cells in a variety of metabolic conditions. In the present paper, a systematic investigation is reported using hepatocytes isolated from fed and starved rats, in order to ascertain the effect of ammonia overloading on glucose or fructose metabolism. Since h y p o x i a causes an impairment of the urea cycle in liver, experiments were carried out during anaerobiosis. The response to ammonia of inmature fetal hepatocytes isolated from 19
Bioehimica et Biophysica Acta, 586 (1979) 481--491 © Elsevier/North-Holland Biomedical Press
BBA 28995
AMMONIA O V E R L O A D I N G IN HEPATOCYTES ISOLATED F R O M L I V E R OF FETAL AND A D U L T RATS
ENRIQUE CHICO, JESUS S. OLAVARRfA and IGNACIO NONEZ DE CASTRO *
Centro de Biologia Molecular. Departamento de Bioqulmica, C.S.I.C. Facultad de Ciencias, Universidad Aut6noma de Madrid, Cantoblanco, Madrid 34 (Spain) (Received February 26th, 1979)
Key words: Ammonia overloading; Ammonia detoxification; Amino acid synthesis; Urea cycle; Lactate production
Summary Ammonia overloading was investigated during glucose and fructose metabolism in isolated hepatocytes under a variety of metabolic conditions. In all assay conditions, the glycolytic flux and oxygen uptake was not modified b y 10 mM ammonia. In hepatocytes isolated from rats fed as libitum, the presence of ammonia caused a decrease in the production of lactate (pyruvate); this effect was not observed in anaerobic incubations, in hepatocytes isolated from starved animals, or in fetal hepatocytes. In spite of an overproduction of urea, ammonia detoxification also takes place by the synthesis of alanine, glutamate and aspartate. Addition of 1 mM aminooxyacetate, an inhibitor of aminotransferases, to the incubation medium prevents the formation of these amino acids, and also prevents the decrease of lactate in hepatocytes isolated from fed animals.
Introduction The early work of Schoenheimer [ 1 ] demonstrated that glutamate, aspartate, glutamine and asparagine were the most heavily labeled amino acids of rat protein when lSN-labelled ammonium compounds were administered. Nordmann et al. [2] investigated the effect of intraperitoneal administration of nonlethal doses of ammonium chloride to rats. They found an increase in the hepatic levels of glutamate, alanine and aspartate, whereas glutamine, pyruvate and citrate decreased. Since aminooxyacetate prevents ammonia detoxification by the production o f amino acids and urea, transamination reactions play an im* To w h o m
c o r r e s p o n d e n c e s h o u l d b e addressed.
482 portant role in the metabolism of ammonia by liver [3]. As Braunstein [4] originally reported, the transamination reactions constitute an important crossroad between the metabolism of alanine, aspartate and glutamate and the a-ketoacid precursors in the citric acid cycle. Brosnan and Williamson [5] reported the time course of the changes in the concentration of hepatic metabolites in response to a non-toxic load of ammonia and also the changes in the cellular redox state caused by ammonia. The process of ureogenesis in isolated rat hepatocytes has been studied by many authors [6,7]. Urea synthesis from ammonia is limited by endogenous ornithine. Addition of ammonia to hepatocytes causes an inhibition of gluconeogenesis from lactate or pyruvate [8]. Likewise, during ureogenesis induced by the ammonia treatment an oxidation of the cells takes place increasing the mitochondrial NADP/NADPH ratio [9,10]. Paush et al. [11] reported an induction of de novo pyrimidine synthesis in rat liver after the administration of a m m o n i u m ion in amounts surpassing the capacity of the urea cycle. More recently, Skaper et al. [ 12], using liver homogenates and minces, have found an enhancement of uridine nucleotide biosynthesis through the action of mitochondrial carbamoyl phosphate synthase, concomitant with a decrease in the phosphoribosyl pyrophosphate available for purine synthesis. The study of regulation of metabolism in isolated hepatocytes avoids the influence of extrahepatic effects of hormones and permits analysis of the response of cells in a variety of metabolic conditions. In the present paper, a systematic investigation is reported using hepatocytes isolated from fed and starved rats, in order to ascertain the effect of ammonia overloading on glucose or fructose metabolism. Since h y p o x i a causes an impairment of the urea cycle in liver, experiments were carried out during anaerobiosis. The response to ammonia of inmature fetal hepatocytes isolated from 19
