Analysis of Regulatory Factors for Urea Synthesis. by Isolated Perfused Rat Liver

Transcription

1 J. Biochem., 77, (1975) Analysis of Regulatory Factors for Urea Synthesis by Isolated Perfused Rat Liver II. Comparison of Urea Synthesis in Livers of Rats Subjected to Different Dietary Conditions Takeyori SAHEKI,1 Michio TSUDA, Tomi TANAKA, and Nobuhiko KATUNUMA Department of Enzyme Chemistry, Institute for Enzyme Research, School of Medicine, Tokushima University, Tokushima, Tokushima 770 Received for publication, August 6, 1974 Capacities for urea synthesis and amino acid patterns in the perfused livers isolated from rats fed low and high-protein diets were compared. Urea formation with ammonium chloride as the nitrogen source in perfused livers isolated from rats fed on a 70% casein diet was rapid and the efficiency of conversion of ammonia to urea was 97.9%. However, that in livers isolated from rats fed on a 5% casein diet was much slower and the efficiency of conversion of ammonia to urea was only 36.1%. The ratios of the rate of urea formation from ammonium chloride to activity of ornithine transcarbamylase [EC ] in the perfused livers of rats fed on 5 and 70% casein diets were calculated. The ratio of the former condition was much lower than that of the latter. The ratios reached nearly the same level by the addition of ornithine and N-acetylglutamate, the addition of which to the perfusate caused marked elevation of the ratios in both cases. In the perfused livers from rats fed on a 5% casein diet a considerable portion of the ammonia added to the perfusate was fixed into an amino or an amide group of amino acids such as alanine, aspartate, and glutamine. On the other hand, in the perfused livers from rats fed on a 70% casein diet most of the ammonia added was converted to urea. The regulation of urea synthesis and the relation between anabolism and catabolism of amino acids in rat livers subjected to different dietary conditions were compared. It has been shown by Schimke (1) that the content in the liver of all urea cycle enzymes 1 Present address : Department of Biochemistry, School of Medicine, Tokai University, Isehara, Kanagawa was directly proportional to the daily consump tion of protein. It has also been reported that the content of ornithine and N-acetylglutamate in the liver is also affected by alterations in the dietary intake of protein (2, 3). How- Vol. 77, No. 3,

2 672 T. SAHEKI, M. TSUDA, T. TANAKA, and N. KATUNUMA. ever, it remains uncertain to what degree alter ations in the content of these substances in the liver actually influences urea synthesis and which factors are critical to this influence in vivo. Furthermore, several investigators have reported that ammonia added to the perfusate was converted almost quantitatively to urea under various experimental conditions (4-6). However, since the substitution of ammonium salts for non-essential amino acids permits the growth of rats (7), the question arises as to how ammonia is channeled into amino acid synthesis in the perfused liver. We have reported studies on urea synthesis with ammonia or glutamine as nitrogen sources in isolated perfused rat livers and on the effects of several compounds on it (8). The follow ing two questions are discussed in this paper : whether these substances, which have a pro moting effect on urea synthesis in the perfused liver, are actually effective in the regulation of the activity of urea cycle in vivo, and whether the fate of ammonia in the perfused liver is affected by the dietary history of the liver donor. MATERIALS AND METHODS Animals - In all perfusion experiments adult male Wister rats weighting between 150 and 220 g were used. They were fed ad libi tum on a 5% casein diet or a 70% casein diet for 4 to 24 days. The 5 and 70% casein diets used for the experiments consisted of 5 or 70 g of casein and 7 or 72 g of corn starch, re spectively, 1 g of vitamin mixture, 3 g of salt mixture, 15 g of sucrose, 2 g of cellulose powder, 0.15 g of choline mixture, and 2 ml of oil mixture. Perfusions were always begun between 10 and 11 o'clock. A low protein liver and high protein liver represent the livers isolated from rats which were fed on a 5% casein diet and a 70% casein diet, respectively, in the paper. Materials-Casein, Vitamin mixture, corn starch, choline hydrochloride, and oil mixture (mixture of bean oil and liver oil) and salt mixture were obtained from Tanabe Amino Acid Research Foundation, Osaka (Japan). Cellulose powder was from Toyo Roshi Kaisha Ltd., Tokyo (Japan). L-Ornithine-6-HC1 was from Kyowa Hakko Kogyo Co., Ltd., Tokyo (Japan). Carbamylphosphate was from Boehringer Mannheim, Mannheim (Germany). Perfusion Technique - Perfusion procedures, apparatus, and medium have been described previously (8). Analysis of Medium-Urea and ammonia. in the perfusate were determined as described previously (8). Amino acid analysis in the medium was performed as follows ; 3% per chloric acid in a final volume was added to the medium and the supernatant was neutralized after centrifugation ; the supernatant was then used for the analysis. The rate of urea for mation was expressed as umoles urea formed/ min per g wet weight of liver. Analysis of Liver-At the end of the per fusion, a selection of the liver (medial lobe) was rapidly frozen in dry ice/aceton and ex tracted with 5 volume of 6% perchloric acid. After concentration by liophilization, amino acids in the neutralized perchloric acid extract were determined using an amino acid analyzer (Yanako, Kyoto, Japan), the determination of urea, ammonia, glutamine, and glutamate was performed as described previously (8). Enzyme Activity-The activity of ornithine transcarbamylase [EC ] was measured according to the method of Schimke (1). After about 2 hr perfusion a part of the liver was removed and homogenized with deionized water. The homogenate, deluted 1,000 fold with water, was used for determination of the enzyme activity. Assays were designed for a total volume of 2 ml at 37?. The reaction mix ture consisted of 0.05 M Tris-HC1 buffer, ph 8.0, 10 mm ornithine, 5 mm carbamyl phos phate, and a suitable amount of the enzyme solution. The reaction was stopped by the addition of 1 ml of 6% perchloric acid. After centri fugation, citrulline in the supernatant was determined by the sensitive method described by Hunninghake and Grisolia (9), using di acetylmonoxime and semidine. The activity of ornithine transcarbamylase was expressed as umoles of citrulline formed/min per g wet. weight of liver. J. Biochem.

3 UREA SYNTHESIS BY ISOLATED PERFUSED RAT LIVER. II 673 RESULTS Using perfused high and low protein livers, the formation of urea with ammonia as the substrate was observed. In high protein livers, the ammonia added to the perfusate decreased rapidly and linearly with time as shown in Fig. 1 (a). The ratio of the rate of urea for mation to the rate of ammonia removal was 0.91, calculated as nitrogen equivalent. Dur ing the period of rapid urea production following addition of the substrate (see Fig. 1), 249 }22.5 pmoles of urea accumulated with a reduction of 600 timoles in the ammonium chloride. (The method for this calculation was shown in Part 1 of this paper). On the other hand, the reduction of the ammonia level with the low protein livers showed essentially a first order kinetics pattern, and the rate of urea formation was much slower than that of ammonia removal (Fig. 1b). Only 108 }29.0 pmoles of urea were formed in total, in spite of the addition of 600 pmoles ammonia to the perfusate. These different yields in urea formation might be explained as the consequence of the regulatory action of some factors or compo nents of the urea cycle. In order to elucidate these differences in the urea yields from am monia, the correlation between the rates of urea formation from ammonia and activites of ornithine transcarbamylase in the perfused low and high protein livers was compared. Ornithine transcarbamylase was taken as a representative enzyme of urea cycle activity, since all urea cycle enzymes change their ac tivities together and in nearly constant ratio in response to varying dietary protein intake, as reported by Schimke (1). The ratio of urea production to ornithine transcarbamylase ac tivity can be considered as a standard for the efficiency of the cycle. For example, under conditions giving the same ratio, it could be considered that the cycle enzymes are in the same state of activation and that the activators are present in the same concentration. It can be clearly seen in Fig. 2 that the ratios in low protein livers were generally lower than those in high protein livers. By the addition of sufficient amounts of ornithine and N-acetyl glutamate to the perfusate, the ratios were in- Fig. 1. Urea formation and ammonia removal by perfused livers from rats fed on 5 or 70% casein diets. Fig. 1 (a) ; the liver, weighting 9.0 g, was from a rat fed on a 5% casein diet for 11 days. 600 umoles of ammonium chloride were added to the perfusate (60 ml) at the time indicated. The ammonia of urea formed ( ü) were corrected for the content in the perfusate at 30 min and the imoles ammonia found ( ~) in the perfusate. Abscissa ; time of perfusion (min). Vol. 77, No. 3, 1975

4 674 T. SAHEKI, M. TSUDA, T. TANAKA, and N. KATUNUMA creased and maintained the same values in both livers. This means that low protein livers contain smaller amounts of ornithine and/or N-acetylglutamate, which are activators of urea synthesis, than high protein livers do and that the urea cycles in both livers are not saturated with ornithine and/or N-acetylglutamate. As Fig. 2. Ratios of the rate of urea formation from ammonia as substrate to the activity of ornithine transcarbamylase in perfused livers from rats fed on. 5 and 70% casein diets. Livers from rats fed on 5 or 70% casein diets for 4 to 14 days were used. 600, pmoles of ammonium chloride were added to the perfusate 30 min after the perfusion started. 150, moles of ornithine were added at the same time ammonium chloride addition and 1,200 pmoles of N-acetylglutamate were added at time 0 (the onset of perfusion). The volume of the perfusion medium was 60 ml. After 2 hr perfusion a portion of the liver was removed and the enzyme activity was measured. Ordinate ; ratio of the rate of urea formation, expressed as pmoles of urea formed per min per g of liver, to ornithine transcarbamylase activity, expressed as pmoles of citrulline formed per min per g of liver. "Ammonium chloride" indicates addition of ammonium chloride and " Am monium chloride +Orn + N-AcGlu " indicates addition of ammonium chloride with ornithine and N-acetyl glutamate. TABLE I. Urea formation from ammonia by perfused livers. Perfusions were performed with livers of rats fed on 5, 10, 40, and 70% casein diets, respectively, for 4 to 14 days. There were no clear differences. in the rate of urea formation between 4 and 14 days feeding. Perfusions were performed as described in the legends of Figs. 1 and 2. The amount of urea formed was calculated on the assumption that the amount of urea formed from exogenous substrate is equal to the amount of urea accumulated in the perfusate during the period of rapid urea formation induced by the addition of substrate (see Fig. 1 of the preceeding paper). The volume of the recirculating medium was 60 ml. Values are given as means } standard error of the mean. The data on fasted rats were from Saheki and Katunuma (8). J. Biochem.

5 UREA SYNTHESIS BY ISOLATED PERFUSED RAT LIVER. II 675 mentioned above, in perfused high protein livers the ratio of the rate of urea formation to the rate of ammonia removal was 0.91, calculated as nitrogen equivalent, and 294 }22.5 pmoles of urea were formed when 600 Đmoles am monium chloride were added to the perfusate as shown in Table I. This means that am monia removed was converted almost quan titatively to urea. Similar results were obtained in our perfusion experiments using TABLE II. Changes in urea and amino acid nitrogen content in the perfusate between 30 and 115 min after the onset of perfusion. Livers from the rats fed on a 5% casein diet for 1 to 2 weeks were used. Perfusions were performed as described in the legend of Fig. 1. Ammonium chloride was added 33 min after the start of perfusion. 2 ml of the perfusate was taken at 30 min and at 115 min respectively, and the differences in the content of urea or amino acids over this 85 min interval are presented. Each value represents the mean } standard error of the mean except the values indicated with an asterisk, which represent the average of two experiments. starved rats (8), and have also been reported by other investigators (4-6). On the other hand, when low protein livers were used in the perfusion experiments, the rate of urea formation was very slow in spite of rapid ammonia removal. It is noteworthy that in the case of per fusion of low protein livers, the proportion of ammonia converted to urea is increased from 36.1% to 63.2% by the addition of ornithine and N-acetylglutamate (Table I). The fact that the conversion of ammonia to urea by low protein livers was very low suggests that a considerable proportion of the TABLE III. Amino acid concentration in the liver after perfusion with or without ammonia. Livers from rats fed on a 5% casein diet for 1 to 2 weeks were used. 600 Đmoles of ammonia chloride were added, as indicated, 33 min after the start of per fusion and a portion of perfused liver was excised and frozen with dry ice/acetone after 115 min. After deproteinization with 5 volumes of 6% per chloric acid, the amino acid concentrations were determined with an amino acid analyzer as described in "MATERIALS AND METHODS." Each value represents the mean } standard error of the mean except the values indicated with an asterisk, which represent the average of two experiments. Vol. 77, No. 3, 1975

6 676 T. SAHEKI, M. TSUDA, T. TANAKA, and N. KATUNUMA ammonia removed was converted to compounds other than urea. Changes in the amounts of amino acid nitrogen were calculated from the concentrations of amino acids in the perfusate before and after addition of ammonium chlo ride. As shown in Table II, the concentra tions of glutamine, alanine, and aspartate in creased on the addition of ammonium chloride in the perfusate of low protein livers. The increase in glutamate content of the perfusate was observed irrespective of ammonia addition. Changes in the amounts of the other amino acids following addition of ammonia were not significant. In the perfused low protein liver, as in the perfusate, the concentrations of aspartate, alanine, and glutamine were considerably higher when ammonia was added. Almost no difference in the concentrations of the other amino acids was observed (Table III). In the case of high protein liver, amino plus amide nitrogen increased to same extent irrespective of ammonia addition. Thus a considerable proportion of the am monia added to the perfusate of low protein liver was fixed to specific amino acids, some portion of which remained in the perfusate or in the liver and some was presumably con verted to protein and other compounds. DISCUSSION The direct relationship between the daily pro tein intake and the levels of urea cycle en zymes was recognized by Schimke (1). Fur ther, he concluded (1 0) that, as no alteration in the concentration of urea cycle substrates, ornithine, arginine, and citrulline, occurred under the conditions where the rates of urea excretion were altered, these substrates are not regulatory factors in urea synthesis. Katu numa et al. (2) have shown that the ornithine level in liver was elevated following an increase of ammonia concentration. In the pres ent experiments too, the hepatic concentration of ornithine in rats fed on a 70% casein diet (Table IV). No significant differences in the intracellular concentration of arginine or citrulline was found under the two different dietary conditions. It has been reported recently by Shigesada and Tatibana (3) that the intrahepatic concentration of N-acetylglutamate corresponds to the increases in dietary protein intake. However, it was not clear whether the changes of the N-acetylglutamate and orni thine levels exert a real influence upon urea synthesis or not. In our experiments the dif ferences between the rates of urea formation from ammonia by low and high protein livers were much grater than the differences in the activities of urea cycle enzymes represented by ornithine transcarbamylase. This discrep ancy suggests that factors other than the amounts of urea cycle enzymes may play an important role in determining the flux through the urea cycle in vivo. The possible candidates are ornithine and N-acetylglutamate, which follows from the fact that the difference in the ratios of the rate of urea formation to the ac tivity of ornithine transcarbamylase between livers prepared from rats fed on 5 and 70% casein diets disappears on the addition of orni thine plus N-acetylglutamate. These com pounds also protect against mortality and against increase in the blood ammonia level in rats injected intraperitoneally with ammonia (11, 12). From the observed hepatic content of ornithine, its intracellular concentration is estimated at 0.3 to 0.6 ~ 10-3 M, which is lower than the Km value (1.8x10-8m) of ornithine transcarbamylase for ornithine at ph 7.5 (13 ). Shigesada and Tatibana (3) reported that the average concentration of N-acetylglutamate in rat liver mitochondria is 1 to 2 ~ 10-4 M, with in the range of the reported Ka value (1.1 ~ 10-4 M) of carbamylphosphate synthase [EC ] for this activator (14 ). These data also support our contentions that urea cycle enzymes in liver are not saturated with orni thine and N-acetylglutamate, that the urea synthesis system in rat liver has a capacity for further amplification by alterations in their concentration, and that these alterations finely regulate the in vivo activity of the urea cycle which is coarsely regulated by alterations in the content of urea cycle enzymes. One of the factors that decides whether ammonia is converted to urea for excretion or is synthesized to utilizable amino acids may be the amount of ornithine and/or N-acetyl- J. Biochem.

7 UREA SYNTHESIS BY ISOLATED PERFUSED RAT LIVER. II 677 glutamate in the liver, because formation of urea from ammonia in perfused livers from rats fed on a 5% casein diet increases from 36.1 to 63.2% on the addition of ornithine plus N-acetylglutamate. It has been reported by Underwood and Newsholme (15) that ammonia activates phos phofructokinase [EC ] of rat liver. Abrahams and Younathan (16) have shown that the physiological concentration of am monia activates rabbit skeletal muscle phos phofructokinase, thereby stimulating glycolysis. They discussed the possibility that the increase in tissue concentrations of ammonia under anoxia might be contributing to the "Pasteur effect." It is conceivable that the supply of ammonia acceptors, such as pyruvate, a-keto glutarate, and oxaloacetate, increases the capac ity to assimilate ammonia. It is well known that nitrogen balance can be attained by rats fed on 7 to 10% casein diets. In our perfusions of livers from rats fed on a low casein diet, the total increases of amino acids and urea nitrogen in the liver and the perfusate were smaller than the amounts of ammonia nitrogen added to the perfusate. The difference may result from a net incorporation of amino acid nitrogen into TABLE IV. Comparison of amino acid concentration in the liver of rats fed on 5 and 70% casein diets. Rats were sacrificed by cervical fracture at noon after being fed ad libitum on a 5 or 70% casein diet for two weeks. All of the stomachs were full of chow. A portion of the liver was rapidly frozen with dry ice/acetone. After deproteinization with 5 vol. of 6% perchloric acid, the amino acid concentration were determined with an amino acid analyzer. The results are given as the means } standard error of the mean of the results from three animals. Vol. 77, No. 3, 1975

8 678 T. SAHEKI, M. TSUDA, T. TANAKA, and N. KATUNUMA protein. The hepatic concentrations of nonessential amino acids such as alanine, aspar tate, glycine, and serine, are rather higher in rats fed on a low protein diet than in those fed on a high protein diet (Table IV). Of course, the concentration of essential amino acids is higher in high protein liver than in low protein liver. The rats fed on a high protein diet utilize the non-essential amino acids preferentially as an energy source, so that they maintain a higher concentration of essential amino acids and a lower concentration of non-essential amino acids. The inverse re lation is observed in rats fed on a low protein diet. This latter pattern was observed typical ly in the perfused liver supplied with ammonia. It is suggested that this amino acid pattern may result from the conversion of ammonia to non-essential amino acids in the livers of rats which have received a low protein diet in vivo. Glycine and serine are exceptions to this generalization. No increases in their concen tration in perfused liver or perfusate were ob served. It has been reported that the enzymes which synthesize serine or glycine from glucose carbon and amino acid nitrogen, such as glu tamate or glutamine, exist in rat liver, and that the activities of the enzymes of the phos phorylated pathway are enhanced by a low protein diet (17). Thus one might also have expected serine and glycine to increase in the perfusate and the liver. This suggests either that synthesis of serine and glycine in the per fused liver requires other factors or that they are converted rapidly into other compounds. However, as mentioned before, the concentra tions of glycine and serine as well as those of alanine and aspartate, in livers, in situ, of rats fed on a low protein diet, are much higher than those in the livers of rats fed on a high pro tein diet. This may indicate an input of serine and glycine from extrahepatic tissues. The authors are greatly indebted to Dr. R. Wohlhueter, Dr. Y. Sanada, and Dr. Y. Matsuda for discussion and critical reading of the manuscript, to Mr. H. Miyai for his achievement of amino acid analysis, and to Mrs. E. Inai for her assistance with the preparation of the manuscript. REFERENCES 1. Schimke, R.T. (1962) J. Biol. Chem. 237, Katunuma, N., Okada, M., & Nishii, Y. (1966) in Advances in Enzyme Regulation (Weber, G., ed.) Vol. 4, pp , Pergamon Press, Oxford and New York 3. Shigesada, K. & Tatibana, M. (1971) J. Biol. Chem. 246, Hems, R., Ross, B.D., Berry, M.N., & Krebs, H.A. (1966) Biochem. J. 101, Chamalaun, R.A.F.M. & Tager, J.M. (1970) Biochim. Biophys. Acta 222, Barak, A.J. & Beckenhauer, H.C. (1969) Proc.. Soc. Exptl. Biol. Med. 131, Rose, W.C. & Smith, L.C. (1949) J. Biol. Chem. 181, Saheki, T. & Katunuma, N. (1975) J. Biochem. 77, Hunninghake, D. & Grisolia, S. (1966) Anal. Biochemistry 16, Schimke, R.T. (1963) J. Biol. Chem. 238, Greenstein, J.P., Winits, M., Gullino, P., Birnbaum, S.M., & Otev, M.C. (1956) Arch. Biochem. Biophys. 64, Chiosa, L., Niculescu, V., Bonciocat, C., & Stascu, C. (1965) Biochem. Pharmacol. 14, Raijman, L. (1974) Biochem. J. 138, Marshall, M., Metzenberg, R.L., & Cohen, P.P. (1961) J. Biol. Chem. 236, Underwood, A.H. & Newsholme, E.A. (1965) Biochem. J. 95, Abrahams, S.L. & Younathan, E.S. (1971) J. Biol. Chem. 246, Fallon, H.J., Hackney, E.J., & Byrne, W.L. (1966) J. Biol. Chem. 241, J. Biochem-