Pfli.igers Archiv
PfliigersArch. 369, 167-175 (1977)
EuropeanJoumal of Physiology 9 by Springer-Verlag1977
The Effect of Anoxia on Nitrogen Metabolism in the Isolated Perfused Rat Liver ROBERTO PARRILLA x, MICHAEL N. GOODMAN 2, and CORNELIUS J. TOEWS 2 1Departmentof Metabolism,InstituteG. Marafi6n,C.S.I.C.,Madrid, Spain 2Joslin ResearchLaboratory,Department of Medicine, Harvard MedicalSchool, Boston, Massachusetts02215, U.S.A.
Summary. The nitrogen metabolism was studied in the perfused rat liver under a variety of conditions. Whether pyruvate, alanine or glutamine was the substrate, the total nitrogen balance of the liver could be accounted for by proteolysis, alanine, glutamine, urea and ammonia production, and substrate uptake. When alanine was not added as a substrate, it was produced by the liver at a rate that was a function of the hepatic alanine concentration. Anoxia resulted in a dramatic increase in hepatic alanine production which was accounted for largely by an increased rate of proteolysis. The increased alanine production was probably largely derived from glutamate. However, a decrease in the hepatic glutamate content can not account for the increased alanine output indicating that glutamate had to be synthetized intramitochondrially and transferred to the cytosol. It is suggested then that under certain metabolic conditions, particulary anoxia, the increased alanine production may act as an alternative Shuttle mechanism for transferring reducing equivalents from the mitochondria to the cytosol. The mitochondrial [NAD + ]/[NADH] ratios were virtually the same when calculated from the glutamate or the fl-hydroxybutyrate dehydrogenase reaction, suggesting that both enzymes are in equilibrium with the same mitochondrial pool of nicotinamide nucleotides. Anoxia increased hepatic proteolysis in the presence of pyruvate. These results in the intact organ are in disagreement with previous experiments in subcellular systems, which suggested that proteolysis was decreased by anoxia and increased by the addition of ATP. Key words: Hepatic nitrogen metabolism - Proteolysis - Mitochondrial transfer of reducing equivalents - Cellular redox state.
INTRODUCTION Alanine is an important hepatic gluconeogenic precursor (Ross et al., 1967; Parrilla et al., 1971) and its flux and concentration probably plays a major role in hepatic gluconeogenesis under physiological conditions (Felig et al., 1970). Alanine is both preferentially released by muscle (London et al., 1965; Pozefsky et al., 1969) and preferentially extracted by the splachnic circulation (Carlsten et al., 1967; Felig et al., 1969). This finding has led to the postulation that alanine is probably one of the major molecules (Mallette et al., 1969) serving to transport ammonia and three carbon moieties from muscle to liver for urea synthesis and gluconeogenesis. In the course of experiments carried out with the perfused liver preparation it was found that under certain circumstances the liver produces alanine. Increased alanine production has been reported in vivo under conditions of ammonia overloading (Brosnan et al., 1967) and an increase in hepatic alanine levels has been reported with acute hepatic ischaemia (Brosnan et al., 1970). In both cases this increase in hepatic alanine was interpreted as an alternative route of ammonia detoxification under situations where the rate of nitrogen delivery to the liver exceeded the capacity of the urea cycle to synthetize urea. The experiments reported here were designed to study the metabolic situations which would result in a reversal of the normal alanine flux from the extra to the intracellular hepatic spaces. Special emphasis has been placed on the source of the carbon and nitrogen moieties for alanine production. Since glutamate dehydrogenase (EC 1.4.1.2; 1.4.1.4) plays a central role in the hepatic nitrogen metabolism (Braunstein and Asarkh, 1945) and, since it is a [NAD+]/[NADH] linked enzyme, attention has been directed to changes in the redox state as a way of regulating nitrogen metabolism in the per-
168
fused isolated liver. Recent reports do not agree on whether or not glutamate dehydrogenase is a NADlinked enzyme and whether or not it is equilibrated through the same mitochondrial pool of nicotinamide nucleotides with /3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) (Brosnan et al., 1970; Williamson et al., 1967; Chamalaun and Tager, 1970). The experiments reported here, in agreement with previous work using also the isolated perfused rat liver preparation (Parrilla and Goodman, 1974), indicate that /~-hydroxybutyrate and glutamate dehydrogenase may share the same intramitochondrial pool of nicotinamide nucleotides.
MATERIALS AND METHODS Reagents. All reagents were of the highest quality available commercially. The enzymes were purchased from Boehringer-Mannheim Corp. Radioactive compounds were obtained from New England Nuclear. Stripped E. coli tRNA was obtained from General Biochemicals (Chagrin Falls, Ohio, U.S.A.).
Animals and Perfusion Technique. 18 h fasted male Sprague-Dawley rats (180-200 g) were used in all experiments. After anesthesia with intraperitoneal sodium pentobarbital (3 mg/100 g body weight), the abdomen was opened, the portal vein cannulated in situ, and the liver perfused (30 ml/min) with warm oxygenated Krebs Ringer bicarbonate buffer (Krebs and Henseleit, 1932). The liver was then excised from the animal, trimmed of adherent tissue, and transferred to the perfusion apparatus maintained at 37~C. The livers were perfused with 100 ml of recirculating hemoglobin free buffer containing 4 g/100 ml of bovine serum albumin (Cohn Fraction V). Prior to the experiment the albumin was dissolved in a small volume of bicarbonate buffer and dialyzed against two changes of the same buffer for 18 h at 4~ All albumin and buffer solutions were filtered through a Millipore filter (0.45 lam pore size) just prior to the perfusion. The pH of the perfusion medium was kept at 7.4 by exposing it to a humidified mixture of 95 % 02 and 5 ~ CO2. The perfusion apparatus and glassware were similar to that described by Miller et al. (1951). Further details on the perfusion technique have been reported elsewhere (Goodman et al., 1973). When pyruvate and alanine were used as substrates, their concentration in the perfusion medium was kept constant by the continuous infusion of concentrated solutions of these substrates. In anoxia studies, the livers were perfused for a 20 rain control period with a 95 % 02/5 ~ CO~ gas mixture, followed by perfusion for 15 rain with a 95 % N2/5 % CO2 gas mixture. At 35 rain the gas mixture was switched back to 95 % 02/5 % CO2 and the perfusion continued for another 25 min. Preliminary studies indicated that a 15 min anoxia period assured that a new steady state was attained for the parameters measured, as well as the fact that almost complete recovery could be obtained after oxygen replacement. Control livers were perfused with a 95 % 02/5 % CO2 mixture for 60 min.
Sampling of Medium and Analytical Procedures. Perfusate samples for the ammonia and urea determinations were collected under a thin layer of toluene to prevent losses of ammonia. Urea was measured by the Auto Analyzer (Technicon Corporation, Tarrytown, New York, U.S.A.). Ammonia was determined by the procedure of Seligson and Seligson (1951). Perfusate samples taken at various times were deproteinized with 2 volumes of cold 6 % (w/v) perchloric acid. After centrifugation, the protein-free supernatant
Pflfigers Arch. 369 (1977) was neutralized to pH 6 - 7 with 5 M KzCO3. All procedures were carried out at 4 ~C. Liver biopsies taken at either 20, 35 or 60 min were immediately frozen in aluminum clamps (Wollemberger et al., 1960) precooled in liquid nitrogen or in a bath of solid COz in methanol. The frozen tissue was weighed and rapidly homogeneized in 6 volumes of cold 6 ~ perchloric acid (w/v) and 40 ~ ethanol (v/v). After centrifugation, the protein free supernatant was brought to pH 6 with 5 M K2CO3 in 0.5 M triethanolamine. All perfnsate and tissue metabolites were measured fluorimetrically. Glutamate was measured according to Bernt and Bergmeyer (1963) and glutamine was measured as glutamate after acid hydrolysis. Lactate (Hohorst, 1963), pyruvate (Bticher et al., 1963), e-ketoglutarate (Bergmeyer and Bernt, 1963), fi-hydroxybutyrate and acetoacetate (Williamson et al., 1962) were also measured fluorimetricaUy according to previously described methods. The concentrations of valine and alanine were determined by an isotopic dilution method involving aminoacylation of tRNA (Parrilla et al., 1973). [Free NAD+]/[Free NADH] ratios were calculated according to Holzer et al. (1956). The lactate dehydrogenase (EC 1.1.1.27) system was used for calculation of cytosolic [Free NAD+]/[Free NADH] ratio and the fl-hydroxybutyrate and glutamate dehydrogenase systems were used for calculating mitochondrial [Free NAD+]/[Free NADH] ratio. The equilibrium constants reported by Krebs and Veech (1968) were used for the calculations. The vertical bars in the graphs represent :the standard error of the mean. Each point on the graphs represents the mean from at least 6 livers. All the data are expressed per gram of fresh liver.
RESULTS The changes in the rate of alanine production is a function of perfusate pyruvate concentration as shown in Figure 1. Rates of alanine production were calculated during the time interval 0 - 3 0 min of perfusion. When no pyruvate was added to the perfusate, the hepatic alanine production was very low 3 + 0.2 jxmoles/g h. However, as the perfusate pyruvate concentration was increased, the alanine production increased as a sigmoidal function of the perfusate pyruvate concentration. These high rates of alanine production were maintained for about 1 h, after which it usually declined. The relatively low rates of alanine production obtained at physiological pyruvate concentrations (50-100 gM) suggest that hepatic alanine production would not be expected under normal physiological conditions. When the livers were perfused at different glutamine concentrations (Fig. 2), the alanine production again increased as a sigmoidal function of the perfusate glutamine concentration. Maximal rates of hepatic alanine production were not obtained until the perfusate glutamine concentration was at least 20 raM. Unlike the alanine production with pyruvate, the alanine production with glutamine as substrate appeared not to be linear with time (Fig. 3). The alanine production was considerably more rapid during the last 30 rain of the perfusion. The lag in alanine production during the first 30 min was associated with a corresponding decrease in the rate of hepatic glutamine uptake.
R. Parrilla et al. : Hepatic Nitrogen Metabolism
169
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Similar sigmoidal relationships between hepatic urea and ammonia (unpublished results) production and perfusate amino acid concentration have been observed (Mallette et al., 1969). The effect of hepatic anoxia on alanine and valine production and on the hepatic ATP concentration is shown in Table 1. Anoxia increased hepatic alanine production when pyruvate or glutamine were the substrates, although the changes were less marked with glutamine as substrate. The anoxia, as might be expected, resulted in a dramatic decrease in the hepatic ATP concentration. Since valine can not be significantly transaminated, deaminated or oxidized by the liver (Mortimore and Mondon, 1970), the rate of its hepatic release can be taken to reflect the rate of proteolysis. Under control conditions the rate of hepatic proteolysis as reflected by hepatic valine production, in agreement with previous reports (Parrilla and Goodman, 1974) was higher in the absence of any nitrogen donor, and glutamine displayed the lowest values. These results suggest that an inverse relationship may exist between the rate of proteolysis and the nitrogen supply to the organ (Table 1). During anoxia the rate of hepatic proteolysis seem to increase with all three substrates. However, the only statistically significant effect was when pyruvate was the substrate which is graphically illustrated in Figure 5,
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Fig. 2. Effects of perfusate glutamine concentration on hepatic alanine production. The glutamine concentrations were maintained constant +_ 15 ~o, by continuous infusion of glutamine. Each point represents the mean +__ S.E. of at least 6 livers
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PfliigersArch. 369, 167-175 (1977)
EuropeanJoumal of Physiology 9 by Springer-Verlag1977
The Effect of Anoxia on Nitrogen Metabolism in the Isolated Perfused Rat Liver ROBERTO PARRILLA x, MICHAEL N. GOODMAN 2, and CORNELIUS J. TOEWS 2 1Departmentof Metabolism,InstituteG. Marafi6n,C.S.I.C.,Madrid, Spain 2Joslin ResearchLaboratory,Department of Medicine, Harvard MedicalSchool, Boston, Massachusetts02215, U.S.A.
Summary. The nitrogen metabolism was studied in the perfused rat liver under a variety of conditions. Whether pyruvate, alanine or glutamine was the substrate, the total nitrogen balance of the liver could be accounted for by proteolysis, alanine, glutamine, urea and ammonia production, and substrate uptake. When alanine was not added as a substrate, it was produced by the liver at a rate that was a function of the hepatic alanine concentration. Anoxia resulted in a dramatic increase in hepatic alanine production which was accounted for largely by an increased rate of proteolysis. The increased alanine production was probably largely derived from glutamate. However, a decrease in the hepatic glutamate content can not account for the increased alanine output indicating that glutamate had to be synthetized intramitochondrially and transferred to the cytosol. It is suggested then that under certain metabolic conditions, particulary anoxia, the increased alanine production may act as an alternative Shuttle mechanism for transferring reducing equivalents from the mitochondria to the cytosol. The mitochondrial [NAD + ]/[NADH] ratios were virtually the same when calculated from the glutamate or the fl-hydroxybutyrate dehydrogenase reaction, suggesting that both enzymes are in equilibrium with the same mitochondrial pool of nicotinamide nucleotides. Anoxia increased hepatic proteolysis in the presence of pyruvate. These results in the intact organ are in disagreement with previous experiments in subcellular systems, which suggested that proteolysis was decreased by anoxia and increased by the addition of ATP. Key words: Hepatic nitrogen metabolism - Proteolysis - Mitochondrial transfer of reducing equivalents - Cellular redox state.
INTRODUCTION Alanine is an important hepatic gluconeogenic precursor (Ross et al., 1967; Parrilla et al., 1971) and its flux and concentration probably plays a major role in hepatic gluconeogenesis under physiological conditions (Felig et al., 1970). Alanine is both preferentially released by muscle (London et al., 1965; Pozefsky et al., 1969) and preferentially extracted by the splachnic circulation (Carlsten et al., 1967; Felig et al., 1969). This finding has led to the postulation that alanine is probably one of the major molecules (Mallette et al., 1969) serving to transport ammonia and three carbon moieties from muscle to liver for urea synthesis and gluconeogenesis. In the course of experiments carried out with the perfused liver preparation it was found that under certain circumstances the liver produces alanine. Increased alanine production has been reported in vivo under conditions of ammonia overloading (Brosnan et al., 1967) and an increase in hepatic alanine levels has been reported with acute hepatic ischaemia (Brosnan et al., 1970). In both cases this increase in hepatic alanine was interpreted as an alternative route of ammonia detoxification under situations where the rate of nitrogen delivery to the liver exceeded the capacity of the urea cycle to synthetize urea. The experiments reported here were designed to study the metabolic situations which would result in a reversal of the normal alanine flux from the extra to the intracellular hepatic spaces. Special emphasis has been placed on the source of the carbon and nitrogen moieties for alanine production. Since glutamate dehydrogenase (EC 1.4.1.2; 1.4.1.4) plays a central role in the hepatic nitrogen metabolism (Braunstein and Asarkh, 1945) and, since it is a [NAD+]/[NADH] linked enzyme, attention has been directed to changes in the redox state as a way of regulating nitrogen metabolism in the per-
168
fused isolated liver. Recent reports do not agree on whether or not glutamate dehydrogenase is a NADlinked enzyme and whether or not it is equilibrated through the same mitochondrial pool of nicotinamide nucleotides with /3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) (Brosnan et al., 1970; Williamson et al., 1967; Chamalaun and Tager, 1970). The experiments reported here, in agreement with previous work using also the isolated perfused rat liver preparation (Parrilla and Goodman, 1974), indicate that /~-hydroxybutyrate and glutamate dehydrogenase may share the same intramitochondrial pool of nicotinamide nucleotides.
MATERIALS AND METHODS Reagents. All reagents were of the highest quality available commercially. The enzymes were purchased from Boehringer-Mannheim Corp. Radioactive compounds were obtained from New England Nuclear. Stripped E. coli tRNA was obtained from General Biochemicals (Chagrin Falls, Ohio, U.S.A.).
Animals and Perfusion Technique. 18 h fasted male Sprague-Dawley rats (180-200 g) were used in all experiments. After anesthesia with intraperitoneal sodium pentobarbital (3 mg/100 g body weight), the abdomen was opened, the portal vein cannulated in situ, and the liver perfused (30 ml/min) with warm oxygenated Krebs Ringer bicarbonate buffer (Krebs and Henseleit, 1932). The liver was then excised from the animal, trimmed of adherent tissue, and transferred to the perfusion apparatus maintained at 37~C. The livers were perfused with 100 ml of recirculating hemoglobin free buffer containing 4 g/100 ml of bovine serum albumin (Cohn Fraction V). Prior to the experiment the albumin was dissolved in a small volume of bicarbonate buffer and dialyzed against two changes of the same buffer for 18 h at 4~ All albumin and buffer solutions were filtered through a Millipore filter (0.45 lam pore size) just prior to the perfusion. The pH of the perfusion medium was kept at 7.4 by exposing it to a humidified mixture of 95 % 02 and 5 ~ CO2. The perfusion apparatus and glassware were similar to that described by Miller et al. (1951). Further details on the perfusion technique have been reported elsewhere (Goodman et al., 1973). When pyruvate and alanine were used as substrates, their concentration in the perfusion medium was kept constant by the continuous infusion of concentrated solutions of these substrates. In anoxia studies, the livers were perfused for a 20 rain control period with a 95 % 02/5 ~ CO~ gas mixture, followed by perfusion for 15 rain with a 95 % N2/5 % CO2 gas mixture. At 35 rain the gas mixture was switched back to 95 % 02/5 % CO2 and the perfusion continued for another 25 min. Preliminary studies indicated that a 15 min anoxia period assured that a new steady state was attained for the parameters measured, as well as the fact that almost complete recovery could be obtained after oxygen replacement. Control livers were perfused with a 95 % 02/5 % CO2 mixture for 60 min.
Sampling of Medium and Analytical Procedures. Perfusate samples for the ammonia and urea determinations were collected under a thin layer of toluene to prevent losses of ammonia. Urea was measured by the Auto Analyzer (Technicon Corporation, Tarrytown, New York, U.S.A.). Ammonia was determined by the procedure of Seligson and Seligson (1951). Perfusate samples taken at various times were deproteinized with 2 volumes of cold 6 % (w/v) perchloric acid. After centrifugation, the protein-free supernatant
Pflfigers Arch. 369 (1977) was neutralized to pH 6 - 7 with 5 M KzCO3. All procedures were carried out at 4 ~C. Liver biopsies taken at either 20, 35 or 60 min were immediately frozen in aluminum clamps (Wollemberger et al., 1960) precooled in liquid nitrogen or in a bath of solid COz in methanol. The frozen tissue was weighed and rapidly homogeneized in 6 volumes of cold 6 ~ perchloric acid (w/v) and 40 ~ ethanol (v/v). After centrifugation, the protein free supernatant was brought to pH 6 with 5 M K2CO3 in 0.5 M triethanolamine. All perfnsate and tissue metabolites were measured fluorimetrically. Glutamate was measured according to Bernt and Bergmeyer (1963) and glutamine was measured as glutamate after acid hydrolysis. Lactate (Hohorst, 1963), pyruvate (Bticher et al., 1963), e-ketoglutarate (Bergmeyer and Bernt, 1963), fi-hydroxybutyrate and acetoacetate (Williamson et al., 1962) were also measured fluorimetricaUy according to previously described methods. The concentrations of valine and alanine were determined by an isotopic dilution method involving aminoacylation of tRNA (Parrilla et al., 1973). [Free NAD+]/[Free NADH] ratios were calculated according to Holzer et al. (1956). The lactate dehydrogenase (EC 1.1.1.27) system was used for calculation of cytosolic [Free NAD+]/[Free NADH] ratio and the fl-hydroxybutyrate and glutamate dehydrogenase systems were used for calculating mitochondrial [Free NAD+]/[Free NADH] ratio. The equilibrium constants reported by Krebs and Veech (1968) were used for the calculations. The vertical bars in the graphs represent :the standard error of the mean. Each point on the graphs represents the mean from at least 6 livers. All the data are expressed per gram of fresh liver.
RESULTS The changes in the rate of alanine production is a function of perfusate pyruvate concentration as shown in Figure 1. Rates of alanine production were calculated during the time interval 0 - 3 0 min of perfusion. When no pyruvate was added to the perfusate, the hepatic alanine production was very low 3 + 0.2 jxmoles/g h. However, as the perfusate pyruvate concentration was increased, the alanine production increased as a sigmoidal function of the perfusate pyruvate concentration. These high rates of alanine production were maintained for about 1 h, after which it usually declined. The relatively low rates of alanine production obtained at physiological pyruvate concentrations (50-100 gM) suggest that hepatic alanine production would not be expected under normal physiological conditions. When the livers were perfused at different glutamine concentrations (Fig. 2), the alanine production again increased as a sigmoidal function of the perfusate glutamine concentration. Maximal rates of hepatic alanine production were not obtained until the perfusate glutamine concentration was at least 20 raM. Unlike the alanine production with pyruvate, the alanine production with glutamine as substrate appeared not to be linear with time (Fig. 3). The alanine production was considerably more rapid during the last 30 rain of the perfusion. The lag in alanine production during the first 30 min was associated with a corresponding decrease in the rate of hepatic glutamine uptake.
R. Parrilla et al. : Hepatic Nitrogen Metabolism
169
30
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[GLUTAMINE] (raM)
[PYRUVATE] (mM) Fig. 1. Hepatic alanine production as a function of the perfusate pyruvate concentration. The pyruvate concentration was kept constant _+ 1 5 ~ at 1, 2.5, 5 and 10 m M by infusion of sodium _ pyruvate during the perfusion. Each point represents the m e a n _+ S.E. of at least 6 livers
Similar sigmoidal relationships between hepatic urea and ammonia (unpublished results) production and perfusate amino acid concentration have been observed (Mallette et al., 1969). The effect of hepatic anoxia on alanine and valine production and on the hepatic ATP concentration is shown in Table 1. Anoxia increased hepatic alanine production when pyruvate or glutamine were the substrates, although the changes were less marked with glutamine as substrate. The anoxia, as might be expected, resulted in a dramatic decrease in the hepatic ATP concentration. Since valine can not be significantly transaminated, deaminated or oxidized by the liver (Mortimore and Mondon, 1970), the rate of its hepatic release can be taken to reflect the rate of proteolysis. Under control conditions the rate of hepatic proteolysis as reflected by hepatic valine production, in agreement with previous reports (Parrilla and Goodman, 1974) was higher in the absence of any nitrogen donor, and glutamine displayed the lowest values. These results suggest that an inverse relationship may exist between the rate of proteolysis and the nitrogen supply to the organ (Table 1). During anoxia the rate of hepatic proteolysis seem to increase with all three substrates. However, the only statistically significant effect was when pyruvate was the substrate which is graphically illustrated in Figure 5,
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Fig. 2. Effects of perfusate glutamine concentration on hepatic alanine production. The glutamine concentrations were maintained constant +_ 15 ~o, by continuous infusion of glutamine. Each point represents the mean +__ S.E. of at least 6 livers
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