525
Biochimica et Biophysica Acta; 582 (1979) 525--532
© Elsevier/North-Holland Biomedical Press
BBA 28782 THE I N T E R R E L A T I O N S H I P BETWEEN UREOGENESIS AND PROTEIN SYNTHESIS IN ISOLATED RAT-LIVER CELLS
H.E.S.J. HENSGENS and A.J. MEIJER Laboratory of Biochemistry, B.C.P. Jansen Institute, University of Amsterdam, Plantage Muidergracht 12, 1018 T V Amsterdam (The Netherlands)
(Received September 4th, 1978) Key words: Ureogenesis; Protein synthesis; Norvaline; Cycloheximide; (Hepatocyte)
Summary The relationship was studied between urea synthesis and protein synthesis in isolated hepatocytes obtained from fasted rats. 1. Addition of a physiological mixture of amino acids, in which arginine was replaced by ornithine, considerably increased ureogenesis and protein synthesis above the endogenous rates. 2. Cycloheximide inhibited protein synthesis by a b o u t 95% and stimulated urea synthesis by about 15% only (see Woodside, K.H. (1976) Biochim. Biophys. Acta 421, 70--79). 3. In the presence of norvaline, when flux through the urea cycle was inhibited by a b o u t 65%, the intraceUular arginine concentration increased severalfold, b u t there was no significant change in the rate of protein synthesis. Furthermore, increasing the concentrations of added amino acids had no effect on the intracellular level of arginine. Thus arginine is n o t rate limiting for protein synthesis under these experimental conditions. 4. A group of 11 amino acids added at low concentrations stimulated protein synthesis considerably b u t had little effect on ureogenesis. 5. It is concluded that protein synthesis proceeds independently of urea synthesis, the reason being that the rate of the former process is governed primarily by the availability of a group of exogenous amino acids that, at low concentrations, do not contribute significantly to urea synthesis. Introduction In vivo, the liver is supplied with a mixture of amino acids which are used for protein synthesis, gluconeogenesis and urea synthesis. The composition of the Abbreviation: PMAA, physiological m i x t u r e o f a m i n o acids.
526
amino acid mixture presented depends on the physiological condition prevailing; the latter also determines the amounts of different amino acids released by the liver [1]. For instance, a release of glutamine may occur enabling the kidney to regulate blood and urinary pH [2]. In perfused livers, a net o u t p u t of amino acids occurs until the amino acid composition of the perfusate approaches that of blood plasma [3]. In the absence of added amino acids a slow rate of urea formation from endogenously produced amino acids has been observed in perfused liver [4,5] and in isolated liver cells [6,7]. We have been engaged in an investigation of the regulation of urea synthesis in isolated rat-liver cells using ammonia or single amino acids as nitrogen source [ 7--11], and have n o w extended these studies to more physiological conditions by using a mixture of amino acids similar in composition to that found in the portal vein of the fasted rat. Since these amino acids are utilized n o t only for urea and glucose synthesis but also for protein synthesis, the question arises of whether there is any competitive relationship between the utilization of amino acids for protein synthesis and the conversion of the amino group of the amino acids to urea. The only report of this relationship is that by Woodside [12], who showed that in the perfused rat liver inhibition of protein synthesis by cycloheximide stimulated urea synthesis and giuconeogenesis. In our studies with isolated ratliver cells we have used not only cycloheximide but also norvaline to inhibit urea synthesis [13,14] and aminooxyacetate to inhibit transamination reactions [ 15--17 ]. Materials and Methods Cells were isolated from the livers of 18--24 h starved male Wistar rats (200 g), using a modification [7] of the Berry and Friend method [18]. Incubations were performed in sealed 25-ml Erlenmeyer flasks containing 2--4 ml KrebsRinger medium (pH 7.4) [19], 2% {w/v} dialysed serum albumin (fraction V, Sigma) and the additions indicated in the legends to the figures. The gas atmosphere was O2/CO2 ( 9 5 : 5, v/v). The cell concentration was 5--10 mg dry weight per ml. For the determination of metabolites, samples of the incubation medium were deproteinized with HC104 and the extracts neutralized as described in ref. 7. For measurement of protein synthesis, 1--2 pCi L-[U-l*C]leucine (10 Ci/ mol) was added to the incubations. In order to reduce the effect of isotope dilution by proteolysis, unlabelled L-leucine was present at 6 or 10 mM [20, 21]. At these concentrations, leucine had only slight inhibitory effect (maximally 15%) on urea synthesis under our experimental conditions (cf. refs. 13 and 14; see also ref. 22). The incorporation of L-[U-14C]leucine into trichloroacetic acid-insoluble material was used as a measure of protein synthesis. The incubations were terminated by addition of 0.8 ml sample to 4.0 ml 0.1 M L-leucine in Krebs-Ringer medium; 25 s later, 3 ml 30% trichloroacetic acid were added. The precipitates were washed twice with 5.0 ml 5% trichloroacetic acid, kept at 90°C for 30 min to unload [l*C]leucyl-tRNA, and washed again with 5.0 ml 5% trichloroacetic acid. The washed precipitate was dissolved in 1 ml hyamine hydroxide and kept at 55°C for 3 h. The dissolved precipitate
527 was counted in 10 ml toluene/ethanol (3 : 1, v/v), containing 2 mg PPO and 0.025 mg POPOP per ml, in a liquid scintillation counter. Leucine incorporation was calculated, assuming that the specific activity of leucyl-tRNA was identical to the specific activity of the [14C]leucine added [20,21]. The final concentrations of amino acids used in the incubations were approximately equal to, or multiples of, the normal plasma concentrations found in starved rats [3,23] and contained the following amino acids (pM): Asp (60), Ile (100), Leu (25), Lys (300), Met (40), Phe (50), Pro (100), Thr (180), Trp (70), Val (180), Ala (400), Asp (30), Glu (100), Gin (350), Gly (300), Cys (60), His (60), Set (200), Tyr (75) and Orn (100). Ornithine was used instead of arginine in order to avoid overestimation of the flux through the complete ornithine cycle. It was found that added arginine, even at high concentrations of up to 1 mM, was almost completely converted into ornithine and urea within 60 min. This is in accordance with the very high activity of arginase in the liver [24]. Glucose, ammonia and urea were determined by standard enzymic methods [25]. Intracellular concentrations of metabolites were obtained by centrifugation of cells through a layer of silicone oil into HC104 as described by Meijer et al [7]. Citrulline and arginine were determined with a Beckman Multichrom M amino acid analyser. Results
Protein synthesis and ureogenesis from physiological mixtures of amino acids Fig. 1 shows the time course of leucine incorporation into protein in the absence of added amino acids and in the presence of physiological mixtures of amino acids (PMAA). The rates of incorporation were approximately linear for at least 2 h. Leucine incorporation was stimulated by increasing the concentration of amino acids in the medium. The stimulation could not have been due to an energy effect, since it was also seen in the presence of 1 mM oleate or 10 mM lactate (results not shown). Fig. 2 shows that protein synthesis reached a plateau when the amino acid concentration was about four times the normal plasma concentration. These results agree well with those of Jefferson and Kornet [26] in the perfused rat liver, but are in conflict with those obtained by Peavy and Hansen [27], who found no effect of increasing the perfusate concentration of amino acids on the incorporation of labelled valine (5 mM) in perfusions of fasted rat liver. Peavy and Hansen [27] suggested that the difference between their results and those of Jefferson and Korner [26] was due to isotope dilution effects. However, in our experiments we observed the stimulatory effects of amino acids at leucine concentrations of 6 and 10 mM where the isotope dilution by leucine arising from proteolysis is minimized [20,21]. With L-[U-l*C]valine we found similar effects of amino acids on valine incorporation (results not shown). In contrast to protein synthesis, production of urea did not reach a plateau even at 10 times the normal plasma concentration of amino acids (Fig. 2). The rate of urea synthesis at low concentration of amino acids was of the same order of magnitude as the rate of protein synthesis under these conditions. Assuming, that the newly synthesized proteins contained 8% leucine, the total amino acid incorporation in the absence of added amino acids was
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F i g . 1. E f f e c t o f p h y s i o l o g i c a l m i x t u r e s o f a m i n o a c i d s ( P M A A ) o n p r o t e i n s y n t h e s i s . F o r e x p e r i m e n t a l d e t a i l s see t e x t . B e s i d e s t h e s m a l l a m o u n t o f l e u c i n e p r e s e n t in t h e m i x t u r e s o f a m i n o acids, 1 0 m M leucine w a s also p r e s e n t in all i n c u b a t i o n s . • •, no additions; o - - o 1 X PMAA; oo, 5 X PMAA. F i g . 2. E f f e c t o f c y c l o h c x i m i d e o n p r o t e i n s y n t h e s i s (a) a n d u r e a p r o d u c t i o n (b). F o r e x p e r i m e n t a l d e t a i l s see t e x t . B e s i d e s t h e s m a l l a m o u n t o f t h e l e u c i n e p r e s e n t in t h e m i x t u r e s o f a m i n o a c i d s 6 m M l e u c i n e w a s p r e s e n t in all i n c u b a t i o n s . 1 m M o l e a t e w a s also p r e s e n t in all i n c u b a t i o n s , o o, c o n t r o l ; o -o 20 p M c y c l o h e x i m i d e . T h e c o m p l e t e m i x t u r e o f a m i n o a c i d s w a s a d d e d as i n d i c a t e d .
12.5 X 2.6 = 32.5 gmol/h per g dry weight of cells. Urea production under these conditions was 35 gmol/h per g dry weight. At 1 × PMAA the corresponding values were 57.5 ~mol amino acids incorporated into protein and 80 gmol urea synthesized per h per g dry weight of cells.
Interrelationship between protein synthesis and ureogenesis Effect of cycloheximide, L-norvaline and aminooxyacetate. Because of the similarity in the rates of protein synthesis and urea production at 1 × PMAA, we investigated whether protein synthesis and urea synthesis compete for amino acids. Cycloheximide inhibited leucine incorporation by more than 95% (Fig. 2), in agreement with Woodside [12]. On the other hand, urea synthesis and also glucose synthesis were slightly but significantly stimulated by cycloheximide (Table I). A much greater stimulation than that observed here was found by Woodside in perfused liver from fed rats [12]. Our results and those of Woodside [12] suggest that the inhibition of protein synthesis increases the availability of amino acids for degradation to yield urea and glucose. On the other hand, inhibition of flux through the urea cycle by L-norvaline (Fig. 3a) had no effect on the rate of protein synthesis (Fig. 3b). The inhibition of urea synthesis by L-norvaline was accompanied by accumulation of NH3 (Fig. 3c). When aminooxyacetate was present, both urea synthesis and protein synthesis were strongly depressed (Fig. 3). Aminooxyacetate inhibits not only aspartate aminotransferase, which is necessary for urea synthesis, but also several other transaminases [15,16]. These results suggest that for optimal
529 TABLE I E F F E C T O F C Y C L O H E X I M I D E ON G L U C O N E O G E N E S I S A N D U R E O G E N E S I S F o r e x p e r i m e n t a l details see t e x t a n d Materials a n d M e t h o d s . Besides the s m a l l a m o u n t of leucinc present in t h e m i x t u r e of a m i n o acids, 6 m M leucine was also p r e s e n t in all i n c u b a t i o n s . T h e c o n c e n t r a t i o n of c y c l o h e x i m i d e w a s 20 pM. R e s u l t s are given as m e a n 2 S.E. n, n u m b e r of e x p e r i m e n t s . Stimulation (%) b y cycloheximide of
Addition
None 1 X PMAA
Glnconeogenesis
Ureogenesis
8.5 + 3.0 (n = 4) 22.5 + 6.0 (n = 4)
15.8 +- 1.5 (n = 4) 16.4 -+ 2.2 (n = 6)
rates of protein synthesis interconversion by transamination reactions of some of the amino acids in the hepatocyte is still required, despite the fact that all amino acids are present in the incubation medium. Possibly some of the added amino acids do not readily cross the plasma membrane. However, the possibility that aminooxyacetate also inhibits some reaction(s) of protein synthesis more directly cannot be excluded.
Is protein synthesis limited by the availability of arginine? Arginine is required not only for protein synthesis but is also an intermediate in the ornithine cycle. In our experiments the amino acid mixture used contained ornithine instead of arginine because of the very high arginase activity in the cells (see above). It was, therefore, important to check whether arginine was rate limiting for protein synthesis under our experimental conditions. Increasing the concentrations of added amino acids had no effect on the intracellular concentration of arginine (Fig. 4), despite the fact that protein synthesis increased under these conditions (see Fig. 3a). Furthermore, addition of nor-
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valine led to an increase in both intracellular and total arginine (Fig. 4) without influencing protein synthesis (see Fig. 3a). These results clearly demonstrate that the availability of arginine is not rate limiting for protein synthesis under our experimental conditions. It has been shown [13] that norvaline inhibits ornithine transcarbamylase. If this were the only site of interaction with the ornithine cycle, one would expect addition of norvaline to lead to a decrease in citrulline and arginine. The fact that both arginine and citrulline actually increased in concentration (see Fig. 4) suggests that norvaline has additional sites of interaction with the ornithine cycle.
Amino acid requirement for optimal rates of protein synthesis Jefferson and Korner [26] have shown that in the isolated perfused liver of the fed rat several amino acids present in the physiological mixture of amino acids are not essential for optimal stimulation o f protein synthesis. Fig. 5 shows that a group of 11 amino acids stimulated protein synthesis considerably but had little effect on urea synthesis and gluconeogenesis at one and four times the physiological concentration. This group of amino acids was the same as the one used by Jefferson and Korner [26], except that arginine was replaced by
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Fig. 5. R e q u i r e m e n t f o r e x o g e n o u s a m i n o a c i d s f o r p r o t e i n s y n t h e s i s . F o z e x p e r i m e n t a l d e t a i l s s e e t e x t o, c o m p l e t e m i x t u r e o f 2 0 a m i n o a c i d s ( P M A A ) ; • •, incomplete mixt u r e o f a m i n o a c i d s (valine, t r y p t o p h a n , t h r e o n l n e , p r o ] i n e , p h e n y l a l a n i n e , m e t h i o n i n e , ]ysine, l e u c i n e , i s o l e u c i n e , a s p a r a g i n e a n d o r n i t h i n e ) a t c o n c e n t r a t i o n s e q u a l t o t h o s e in P M A A . a n d l e g e n d s t o Fig. 2. o
ornithine, since addition of arginine overestimates ornithine cycle flux (see above). Discussion It is important that protein synthesis, a synthetic function, should be independent of urea synthesis, a disposal function; only in this way can the liver cell make optimal use of the available amino acids for protein synthesis. The results presented above lead us to conclude that in isolated rat-liver cells protein synthesis from a near physiological mixture of amino acids does, indeed, proceed independently of urea synthesis, the reason being that the rate of protein synthesis is governed primarily by the availability of certain amino acids that, at low concentrations, do not contribute significantly to urea synthesis. On the other hand the rate of urea synthesis is n o t independent of the rate of protein synthesis, since a diminished rate of protein synthesis leads to an increased availability of amino acids of which some are readily metabolized. Recent studies by Shigesada et al. [28] have stressed the importance of the level of N-acetylglutamate, an essential cofactor for carbamoylphosphate synthase, in regulating the rate of flux through the urea cycle. Furthermore, they have shown that the synthesis of N-acetylglutamate is stimulated by arginine. Under our experimental conditions the rate of urea synthesis is influenced b y the availability of amino acids, and not by the capacity of the urea cycle. This is indicated by the fact that ammonia does n o t accumulate unless the capacity of the urea cycle is decreased by adding norvaline. Furthermore, addition of ammonia or of high concentrations of certain amino acids leads to an immediate increase in the rate of urea synthesis [6,7,10,11] far in excess of those found in our experiments. The percentage stimulation of urea synthesis by cycloheximide in our experiments was smaller than that observed by Woodside [12]. This apparent dis-
532 crepancy may be due to a difference either in experimental technique or in the nutritional state of the animals, or to both. Whereas Woodside [12] used the isolated perfused liver (from fed rats}, we used isolated liver cells (obtained from starved rats}.
Acknowledgements The authors are grateful to Joseph Tager for suggestions and for critically reviewing the manuscript, to Elly Laanen for technical assistance and to Jan Post for carrying o u t the amino acid analyses. This study was supported by a grant to J.M. Tager from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.) under the auspices of the Netherlands Foundation for Chemical Research (S.O.N.).
References 1 2 3 4 5 6 7 8
9
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
Felig, P. (1975) Annu. Rev. Biochem. 4 4 , 9 3 3 - - 9 5 5 Addae, S.K. and Lotspeich, W.D. (1968) Am. J. Physiol. 2 1 5 , 2 6 9 - - 2 7 7 Schimassek, H. and Gerok, W. (1965) Biochem. Z. 3 4 3 , 4 0 7 - - 4 1 5 Hems, R., Ross, B.D., Berry, M.N. and Krebs, H.A. (1966) Biochem. J. 1 0 1 , 2 8 4 - - 2 9 2 Chamalaun, R.A.F.M. and Tager, J.M. (1970) Bioehim. Biophys. Acta 222, 119--134 Krebs, H.A., Lund, P. and Stubbs, M. (1976) in Gluconeogenesis (Hanson, R.W. and Mehlman, M., eds.), pp. 269--291, Wiley, New York Meijer, A.J., Gimpel, J.A., DeLeeuw, G.A., Tager, J.M. and Williamson, J.R. (1975) J. Biol. Chem. 250, 7 728--7738 Hensgens, H.E.S.J., Hensgens, L.A.M., Meijer, A.J., Gimpel, J.A. and Tager, J.M. (1976) in Use of Isolated Liver Cells and Kidney Tubules in Metabolic Studies (Tager, J.M., SSling, H.D. and Williamson, J.R., eds.), pp. 331--338, North-Holland Publ. Co., Amsterdam WiUiamson, J.R., Gimpel, J.A., Meijer, A.J., DeLeeuw, G.A. and Refino, C. (1976) in Use of Isolated Liver Cells and Kidney Tubules in Metabolic Studies (Tagar, J.M., SSling, H.D. and Wflliamson, J.R., eds.), pp. 339--349, North-Holland Publ. Co., Amsterda m Hensgens, H.E.S.J., MeiJer, A.J., Williamson, J.R., Gimpel, J.A. and Tager, J.M. (1978) Biochem. J. 170, 699--707 Meijer, A.J., Gimpel, J.A., DeLeeuw, G.A., Tager, J.M. and Williamson, J.R. (1978) J. Biol. Chem. 253, 2 3 0 8 - - 2 3 2 0 Woodside, K.H. (1976) Biochim. Biophys. Acta 421, 70--79 Marshall, M. and Cohen, P.P. (1972) J. Biol. Chem. 247, 1654--1668 Rognstad, R. (1977) Biochim. Biophys. Acta 4 9 6 , 2 4 9 - - 2 5 4 Wallach, D.P. (1960) Bioehem. Pharmacol. 5, 166--167 Hopper, S. and Segal, H.L. (1962) J. Biol. Chem. 237, 3 189--3195 Rognstad, R. and Katz, J. (1970) Biochem. J. 1 1 6 , 4 8 3 - - 4 9 1 Berry, M.N. and Friend, D.S. (1969) J. Cell Biol. 4 3 , 5 0 6 - - 5 2 0 Krebs, H.A. and Henseleit, K. (1932) Hoppe Seyler's Z. Physiol. Chem. 210, 33--66 Mortimore, G.E., Woodside, K.H. and Henry, J.E. (1972) J. Biol. Chem. 247, 2776--2784 Bezooyen, C.F.A., Grill, T. and Knook, D.L. (1977) Mech. Ageing Dev. 6 , 2 9 3 - - 3 0 4 Mendes-Mour~o, J., MeGivan, J.D. and Chappell, J.B. (1975) Biochem. J. 146, 457--464 Aikawa, T., Matsutaka, H., Y a m a m o t o , H., Okuda, T., Ishikawa, E., Kawano, T. and Matsumura, E. (1973) J. Biochem. (Tokyo) 74, 1003--1017 Schimke, R.T. (1963) J. Biol. Chem. 238, 1012--1018 Bergmeyer, H.U. (1970) Methoden der E n z y m a t i s e h e n Analyse, Verlag Chemie, Weinheim Jefferson, L.S. and Korner, A. (1969) Bioehem. J. 1 1 1 , 7 0 3 - - 7 1 2 Peavy, D.E. and Hansen, R.J. (1976) Biochem. J. 1 6 0 , 7 9 7 - - 8 0 1 Shigesada, K., Aoyagi, K. and Tatibana, M. (1978) Eur. J. Biochem. 85, 385--391
Biochimica et Biophysica Acta; 582 (1979) 525--532
© Elsevier/North-Holland Biomedical Press
BBA 28782 THE I N T E R R E L A T I O N S H I P BETWEEN UREOGENESIS AND PROTEIN SYNTHESIS IN ISOLATED RAT-LIVER CELLS
H.E.S.J. HENSGENS and A.J. MEIJER Laboratory of Biochemistry, B.C.P. Jansen Institute, University of Amsterdam, Plantage Muidergracht 12, 1018 T V Amsterdam (The Netherlands)
(Received September 4th, 1978) Key words: Ureogenesis; Protein synthesis; Norvaline; Cycloheximide; (Hepatocyte)
Summary The relationship was studied between urea synthesis and protein synthesis in isolated hepatocytes obtained from fasted rats. 1. Addition of a physiological mixture of amino acids, in which arginine was replaced by ornithine, considerably increased ureogenesis and protein synthesis above the endogenous rates. 2. Cycloheximide inhibited protein synthesis by a b o u t 95% and stimulated urea synthesis by about 15% only (see Woodside, K.H. (1976) Biochim. Biophys. Acta 421, 70--79). 3. In the presence of norvaline, when flux through the urea cycle was inhibited by a b o u t 65%, the intraceUular arginine concentration increased severalfold, b u t there was no significant change in the rate of protein synthesis. Furthermore, increasing the concentrations of added amino acids had no effect on the intracellular level of arginine. Thus arginine is n o t rate limiting for protein synthesis under these experimental conditions. 4. A group of 11 amino acids added at low concentrations stimulated protein synthesis considerably b u t had little effect on ureogenesis. 5. It is concluded that protein synthesis proceeds independently of urea synthesis, the reason being that the rate of the former process is governed primarily by the availability of a group of exogenous amino acids that, at low concentrations, do not contribute significantly to urea synthesis. Introduction In vivo, the liver is supplied with a mixture of amino acids which are used for protein synthesis, gluconeogenesis and urea synthesis. The composition of the Abbreviation: PMAA, physiological m i x t u r e o f a m i n o acids.
526
amino acid mixture presented depends on the physiological condition prevailing; the latter also determines the amounts of different amino acids released by the liver [1]. For instance, a release of glutamine may occur enabling the kidney to regulate blood and urinary pH [2]. In perfused livers, a net o u t p u t of amino acids occurs until the amino acid composition of the perfusate approaches that of blood plasma [3]. In the absence of added amino acids a slow rate of urea formation from endogenously produced amino acids has been observed in perfused liver [4,5] and in isolated liver cells [6,7]. We have been engaged in an investigation of the regulation of urea synthesis in isolated rat-liver cells using ammonia or single amino acids as nitrogen source [ 7--11], and have n o w extended these studies to more physiological conditions by using a mixture of amino acids similar in composition to that found in the portal vein of the fasted rat. Since these amino acids are utilized n o t only for urea and glucose synthesis but also for protein synthesis, the question arises of whether there is any competitive relationship between the utilization of amino acids for protein synthesis and the conversion of the amino group of the amino acids to urea. The only report of this relationship is that by Woodside [12], who showed that in the perfused rat liver inhibition of protein synthesis by cycloheximide stimulated urea synthesis and giuconeogenesis. In our studies with isolated ratliver cells we have used not only cycloheximide but also norvaline to inhibit urea synthesis [13,14] and aminooxyacetate to inhibit transamination reactions [ 15--17 ]. Materials and Methods Cells were isolated from the livers of 18--24 h starved male Wistar rats (200 g), using a modification [7] of the Berry and Friend method [18]. Incubations were performed in sealed 25-ml Erlenmeyer flasks containing 2--4 ml KrebsRinger medium (pH 7.4) [19], 2% {w/v} dialysed serum albumin (fraction V, Sigma) and the additions indicated in the legends to the figures. The gas atmosphere was O2/CO2 ( 9 5 : 5, v/v). The cell concentration was 5--10 mg dry weight per ml. For the determination of metabolites, samples of the incubation medium were deproteinized with HC104 and the extracts neutralized as described in ref. 7. For measurement of protein synthesis, 1--2 pCi L-[U-l*C]leucine (10 Ci/ mol) was added to the incubations. In order to reduce the effect of isotope dilution by proteolysis, unlabelled L-leucine was present at 6 or 10 mM [20, 21]. At these concentrations, leucine had only slight inhibitory effect (maximally 15%) on urea synthesis under our experimental conditions (cf. refs. 13 and 14; see also ref. 22). The incorporation of L-[U-14C]leucine into trichloroacetic acid-insoluble material was used as a measure of protein synthesis. The incubations were terminated by addition of 0.8 ml sample to 4.0 ml 0.1 M L-leucine in Krebs-Ringer medium; 25 s later, 3 ml 30% trichloroacetic acid were added. The precipitates were washed twice with 5.0 ml 5% trichloroacetic acid, kept at 90°C for 30 min to unload [l*C]leucyl-tRNA, and washed again with 5.0 ml 5% trichloroacetic acid. The washed precipitate was dissolved in 1 ml hyamine hydroxide and kept at 55°C for 3 h. The dissolved precipitate
527 was counted in 10 ml toluene/ethanol (3 : 1, v/v), containing 2 mg PPO and 0.025 mg POPOP per ml, in a liquid scintillation counter. Leucine incorporation was calculated, assuming that the specific activity of leucyl-tRNA was identical to the specific activity of the [14C]leucine added [20,21]. The final concentrations of amino acids used in the incubations were approximately equal to, or multiples of, the normal plasma concentrations found in starved rats [3,23] and contained the following amino acids (pM): Asp (60), Ile (100), Leu (25), Lys (300), Met (40), Phe (50), Pro (100), Thr (180), Trp (70), Val (180), Ala (400), Asp (30), Glu (100), Gin (350), Gly (300), Cys (60), His (60), Set (200), Tyr (75) and Orn (100). Ornithine was used instead of arginine in order to avoid overestimation of the flux through the complete ornithine cycle. It was found that added arginine, even at high concentrations of up to 1 mM, was almost completely converted into ornithine and urea within 60 min. This is in accordance with the very high activity of arginase in the liver [24]. Glucose, ammonia and urea were determined by standard enzymic methods [25]. Intracellular concentrations of metabolites were obtained by centrifugation of cells through a layer of silicone oil into HC104 as described by Meijer et al [7]. Citrulline and arginine were determined with a Beckman Multichrom M amino acid analyser. Results
Protein synthesis and ureogenesis from physiological mixtures of amino acids Fig. 1 shows the time course of leucine incorporation into protein in the absence of added amino acids and in the presence of physiological mixtures of amino acids (PMAA). The rates of incorporation were approximately linear for at least 2 h. Leucine incorporation was stimulated by increasing the concentration of amino acids in the medium. The stimulation could not have been due to an energy effect, since it was also seen in the presence of 1 mM oleate or 10 mM lactate (results not shown). Fig. 2 shows that protein synthesis reached a plateau when the amino acid concentration was about four times the normal plasma concentration. These results agree well with those of Jefferson and Kornet [26] in the perfused rat liver, but are in conflict with those obtained by Peavy and Hansen [27], who found no effect of increasing the perfusate concentration of amino acids on the incorporation of labelled valine (5 mM) in perfusions of fasted rat liver. Peavy and Hansen [27] suggested that the difference between their results and those of Jefferson and Korner [26] was due to isotope dilution effects. However, in our experiments we observed the stimulatory effects of amino acids at leucine concentrations of 6 and 10 mM where the isotope dilution by leucine arising from proteolysis is minimized [20,21]. With L-[U-l*C]valine we found similar effects of amino acids on valine incorporation (results not shown). In contrast to protein synthesis, production of urea did not reach a plateau even at 10 times the normal plasma concentration of amino acids (Fig. 2). The rate of urea synthesis at low concentration of amino acids was of the same order of magnitude as the rate of protein synthesis under these conditions. Assuming, that the newly synthesized proteins contained 8% leucine, the total amino acid incorporation in the absence of added amino acids was
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F i g . 1. E f f e c t o f p h y s i o l o g i c a l m i x t u r e s o f a m i n o a c i d s ( P M A A ) o n p r o t e i n s y n t h e s i s . F o r e x p e r i m e n t a l d e t a i l s see t e x t . B e s i d e s t h e s m a l l a m o u n t o f l e u c i n e p r e s e n t in t h e m i x t u r e s o f a m i n o acids, 1 0 m M leucine w a s also p r e s e n t in all i n c u b a t i o n s . • •, no additions; o - - o 1 X PMAA; oo, 5 X PMAA. F i g . 2. E f f e c t o f c y c l o h c x i m i d e o n p r o t e i n s y n t h e s i s (a) a n d u r e a p r o d u c t i o n (b). F o r e x p e r i m e n t a l d e t a i l s see t e x t . B e s i d e s t h e s m a l l a m o u n t o f t h e l e u c i n e p r e s e n t in t h e m i x t u r e s o f a m i n o a c i d s 6 m M l e u c i n e w a s p r e s e n t in all i n c u b a t i o n s . 1 m M o l e a t e w a s also p r e s e n t in all i n c u b a t i o n s , o o, c o n t r o l ; o -o 20 p M c y c l o h e x i m i d e . T h e c o m p l e t e m i x t u r e o f a m i n o a c i d s w a s a d d e d as i n d i c a t e d .
12.5 X 2.6 = 32.5 gmol/h per g dry weight of cells. Urea production under these conditions was 35 gmol/h per g dry weight. At 1 × PMAA the corresponding values were 57.5 ~mol amino acids incorporated into protein and 80 gmol urea synthesized per h per g dry weight of cells.
Interrelationship between protein synthesis and ureogenesis Effect of cycloheximide, L-norvaline and aminooxyacetate. Because of the similarity in the rates of protein synthesis and urea production at 1 × PMAA, we investigated whether protein synthesis and urea synthesis compete for amino acids. Cycloheximide inhibited leucine incorporation by more than 95% (Fig. 2), in agreement with Woodside [12]. On the other hand, urea synthesis and also glucose synthesis were slightly but significantly stimulated by cycloheximide (Table I). A much greater stimulation than that observed here was found by Woodside in perfused liver from fed rats [12]. Our results and those of Woodside [12] suggest that the inhibition of protein synthesis increases the availability of amino acids for degradation to yield urea and glucose. On the other hand, inhibition of flux through the urea cycle by L-norvaline (Fig. 3a) had no effect on the rate of protein synthesis (Fig. 3b). The inhibition of urea synthesis by L-norvaline was accompanied by accumulation of NH3 (Fig. 3c). When aminooxyacetate was present, both urea synthesis and protein synthesis were strongly depressed (Fig. 3). Aminooxyacetate inhibits not only aspartate aminotransferase, which is necessary for urea synthesis, but also several other transaminases [15,16]. These results suggest that for optimal
529 TABLE I E F F E C T O F C Y C L O H E X I M I D E ON G L U C O N E O G E N E S I S A N D U R E O G E N E S I S F o r e x p e r i m e n t a l details see t e x t a n d Materials a n d M e t h o d s . Besides the s m a l l a m o u n t of leucinc present in t h e m i x t u r e of a m i n o acids, 6 m M leucine was also p r e s e n t in all i n c u b a t i o n s . T h e c o n c e n t r a t i o n of c y c l o h e x i m i d e w a s 20 pM. R e s u l t s are given as m e a n 2 S.E. n, n u m b e r of e x p e r i m e n t s . Stimulation (%) b y cycloheximide of
Addition
None 1 X PMAA
Glnconeogenesis
Ureogenesis
8.5 + 3.0 (n = 4) 22.5 + 6.0 (n = 4)
15.8 +- 1.5 (n = 4) 16.4 -+ 2.2 (n = 6)
rates of protein synthesis interconversion by transamination reactions of some of the amino acids in the hepatocyte is still required, despite the fact that all amino acids are present in the incubation medium. Possibly some of the added amino acids do not readily cross the plasma membrane. However, the possibility that aminooxyacetate also inhibits some reaction(s) of protein synthesis more directly cannot be excluded.
Is protein synthesis limited by the availability of arginine? Arginine is required not only for protein synthesis but is also an intermediate in the ornithine cycle. In our experiments the amino acid mixture used contained ornithine instead of arginine because of the very high arginase activity in the cells (see above). It was, therefore, important to check whether arginine was rate limiting for protein synthesis under our experimental conditions. Increasing the concentrations of added amino acids had no effect on the intracellular concentration of arginine (Fig. 4), despite the fact that protein synthesis increased under these conditions (see Fig. 3a). Furthermore, addition of nor-
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F i g . 4. E f f e c t o f L - n o r v a l i n e o n t h e rate o f u r e a s y n t h e s i s , t o t a l N H 3 a n d o n i n t r a c e l l u l a r a n d t o t a l c o n centrations of citrulline and arginine. For experimental d e t a i l s , see t e x t a n d l e g e n d t o F i g . 2. o c, control; • •, 20 mM L-norvaline present.
valine led to an increase in both intracellular and total arginine (Fig. 4) without influencing protein synthesis (see Fig. 3a). These results clearly demonstrate that the availability of arginine is not rate limiting for protein synthesis under our experimental conditions. It has been shown [13] that norvaline inhibits ornithine transcarbamylase. If this were the only site of interaction with the ornithine cycle, one would expect addition of norvaline to lead to a decrease in citrulline and arginine. The fact that both arginine and citrulline actually increased in concentration (see Fig. 4) suggests that norvaline has additional sites of interaction with the ornithine cycle.
Amino acid requirement for optimal rates of protein synthesis Jefferson and Korner [26] have shown that in the isolated perfused liver of the fed rat several amino acids present in the physiological mixture of amino acids are not essential for optimal stimulation o f protein synthesis. Fig. 5 shows that a group of 11 amino acids stimulated protein synthesis considerably but had little effect on urea synthesis and gluconeogenesis at one and four times the physiological concentration. This group of amino acids was the same as the one used by Jefferson and Korner [26], except that arginine was replaced by
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Fig. 5. R e q u i r e m e n t f o r e x o g e n o u s a m i n o a c i d s f o r p r o t e i n s y n t h e s i s . F o z e x p e r i m e n t a l d e t a i l s s e e t e x t o, c o m p l e t e m i x t u r e o f 2 0 a m i n o a c i d s ( P M A A ) ; • •, incomplete mixt u r e o f a m i n o a c i d s (valine, t r y p t o p h a n , t h r e o n l n e , p r o ] i n e , p h e n y l a l a n i n e , m e t h i o n i n e , ]ysine, l e u c i n e , i s o l e u c i n e , a s p a r a g i n e a n d o r n i t h i n e ) a t c o n c e n t r a t i o n s e q u a l t o t h o s e in P M A A . a n d l e g e n d s t o Fig. 2. o
ornithine, since addition of arginine overestimates ornithine cycle flux (see above). Discussion It is important that protein synthesis, a synthetic function, should be independent of urea synthesis, a disposal function; only in this way can the liver cell make optimal use of the available amino acids for protein synthesis. The results presented above lead us to conclude that in isolated rat-liver cells protein synthesis from a near physiological mixture of amino acids does, indeed, proceed independently of urea synthesis, the reason being that the rate of protein synthesis is governed primarily by the availability of certain amino acids that, at low concentrations, do not contribute significantly to urea synthesis. On the other hand the rate of urea synthesis is n o t independent of the rate of protein synthesis, since a diminished rate of protein synthesis leads to an increased availability of amino acids of which some are readily metabolized. Recent studies by Shigesada et al. [28] have stressed the importance of the level of N-acetylglutamate, an essential cofactor for carbamoylphosphate synthase, in regulating the rate of flux through the urea cycle. Furthermore, they have shown that the synthesis of N-acetylglutamate is stimulated by arginine. Under our experimental conditions the rate of urea synthesis is influenced b y the availability of amino acids, and not by the capacity of the urea cycle. This is indicated by the fact that ammonia does n o t accumulate unless the capacity of the urea cycle is decreased by adding norvaline. Furthermore, addition of ammonia or of high concentrations of certain amino acids leads to an immediate increase in the rate of urea synthesis [6,7,10,11] far in excess of those found in our experiments. The percentage stimulation of urea synthesis by cycloheximide in our experiments was smaller than that observed by Woodside [12]. This apparent dis-
532 crepancy may be due to a difference either in experimental technique or in the nutritional state of the animals, or to both. Whereas Woodside [12] used the isolated perfused liver (from fed rats}, we used isolated liver cells (obtained from starved rats}.
Acknowledgements The authors are grateful to Joseph Tager for suggestions and for critically reviewing the manuscript, to Elly Laanen for technical assistance and to Jan Post for carrying o u t the amino acid analyses. This study was supported by a grant to J.M. Tager from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.) under the auspices of the Netherlands Foundation for Chemical Research (S.O.N.).
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Felig, P. (1975) Annu. Rev. Biochem. 4 4 , 9 3 3 - - 9 5 5 Addae, S.K. and Lotspeich, W.D. (1968) Am. J. Physiol. 2 1 5 , 2 6 9 - - 2 7 7 Schimassek, H. and Gerok, W. (1965) Biochem. Z. 3 4 3 , 4 0 7 - - 4 1 5 Hems, R., Ross, B.D., Berry, M.N. and Krebs, H.A. (1966) Biochem. J. 1 0 1 , 2 8 4 - - 2 9 2 Chamalaun, R.A.F.M. and Tager, J.M. (1970) Bioehim. Biophys. Acta 222, 119--134 Krebs, H.A., Lund, P. and Stubbs, M. (1976) in Gluconeogenesis (Hanson, R.W. and Mehlman, M., eds.), pp. 269--291, Wiley, New York Meijer, A.J., Gimpel, J.A., DeLeeuw, G.A., Tager, J.M. and Williamson, J.R. (1975) J. Biol. Chem. 250, 7 728--7738 Hensgens, H.E.S.J., Hensgens, L.A.M., Meijer, A.J., Gimpel, J.A. and Tager, J.M. (1976) in Use of Isolated Liver Cells and Kidney Tubules in Metabolic Studies (Tager, J.M., SSling, H.D. and Williamson, J.R., eds.), pp. 331--338, North-Holland Publ. Co., Amsterdam WiUiamson, J.R., Gimpel, J.A., Meijer, A.J., DeLeeuw, G.A. and Refino, C. (1976) in Use of Isolated Liver Cells and Kidney Tubules in Metabolic Studies (Tagar, J.M., SSling, H.D. and Wflliamson, J.R., eds.), pp. 339--349, North-Holland Publ. Co., Amsterda m Hensgens, H.E.S.J., MeiJer, A.J., Williamson, J.R., Gimpel, J.A. and Tager, J.M. (1978) Biochem. J. 170, 699--707 Meijer, A.J., Gimpel, J.A., DeLeeuw, G.A., Tager, J.M. and Williamson, J.R. (1978) J. Biol. Chem. 253, 2 3 0 8 - - 2 3 2 0 Woodside, K.H. (1976) Biochim. Biophys. Acta 421, 70--79 Marshall, M. and Cohen, P.P. (1972) J. Biol. Chem. 247, 1654--1668 Rognstad, R. (1977) Biochim. Biophys. Acta 4 9 6 , 2 4 9 - - 2 5 4 Wallach, D.P. (1960) Bioehem. Pharmacol. 5, 166--167 Hopper, S. and Segal, H.L. (1962) J. Biol. Chem. 237, 3 189--3195 Rognstad, R. and Katz, J. (1970) Biochem. J. 1 1 6 , 4 8 3 - - 4 9 1 Berry, M.N. and Friend, D.S. (1969) J. Cell Biol. 4 3 , 5 0 6 - - 5 2 0 Krebs, H.A. and Henseleit, K. (1932) Hoppe Seyler's Z. Physiol. Chem. 210, 33--66 Mortimore, G.E., Woodside, K.H. and Henry, J.E. (1972) J. Biol. Chem. 247, 2776--2784 Bezooyen, C.F.A., Grill, T. and Knook, D.L. (1977) Mech. Ageing Dev. 6 , 2 9 3 - - 3 0 4 Mendes-Mour~o, J., MeGivan, J.D. and Chappell, J.B. (1975) Biochem. J. 146, 457--464 Aikawa, T., Matsutaka, H., Y a m a m o t o , H., Okuda, T., Ishikawa, E., Kawano, T. and Matsumura, E. (1973) J. Biochem. (Tokyo) 74, 1003--1017 Schimke, R.T. (1963) J. Biol. Chem. 238, 1012--1018 Bergmeyer, H.U. (1970) Methoden der E n z y m a t i s e h e n Analyse, Verlag Chemie, Weinheim Jefferson, L.S. and Korner, A. (1969) Bioehem. J. 1 1 1 , 7 0 3 - - 7 1 2 Peavy, D.E. and Hansen, R.J. (1976) Biochem. J. 1 6 0 , 7 9 7 - - 8 0 1 Shigesada, K., Aoyagi, K. and Tatibana, M. (1978) Eur. J. Biochem. 85, 385--391
