Key words: citrulline synthesis, immunohistochemistry, liver, ornithine aminotransferase, small intestine.

Transcription

1 J. Biochem. 116, (1994) Changes in Ornithine Metabolic Enzymes Induced by Dietary Protein in Small Intestine and Liver: Intestine-Liver Relationship in Ornithine Supply to Liver' Takeo Matsuzawa,*.2 Tatsuhiko Kobayashi,* Kazuhiro Tashiro,t and Masao Kasaharat Departments of *Biochemistry and tpathology, School of Medicine, Fujita Health University, Toyoake, Aichi Received for publication, October 12, 1993 Compared with the activity obtained with a high-protein diet in rats, a low-protein diet doubled the activity of ornithine aminotransferase [EC ] (OAT), a key enzyme for citrulline synthesis, in the small intestine. The induction of ornithine aminotransferase in the small intestine by the low-protein diet and its suppression by the high-protein diet, and the converse in the liver, were immunohistochemically verified with anti-oat antiserum. The immunohistochemical studies revealed that ornithine aminotransferase molecules localized in the villous surface epithelia, but not in the cryptic epithelia, were most responsive to the changes in dietary conditions, these results indicating that intestinal ornithine aminotransferase may be involved in the ornithine supply to the liver, with the reversal of the enzyme reaction occurring with a low-protein diet. Reconstituted model experiments on citrulline synthesis revealed that the addition of ornithine carbamoyltransferase and carbamoyl phosphate was essential to overcome the unfavorable equilibrium of the reverse reaction, and the further addition of glutamate dehydrogenase and ammonia resulted in a stimulating effect. Key words: citrulline synthesis, immunohistochemistry, liver, ornithine aminotransferase, small intestine. This work was supported by a Grant-in-Aid from Fujita Health University. Parts of this study have been reported at the 5th FAOB Congress in Seoul (1989). 2 To whom correspondence should be addressed. Abbreviations: ArgSuc, argininosuccinate; CarbP, carbamoyl phosphate; Crea, creatine; GDH, glutamate dehydrogenase; GSA, glutamic y-semialdehyde; GuaAc, guanidinoacetate; Fum, fumarate; OAT, ornithine aminotransferase; OCT, ornithine carbamoyltransferase; akg, 2-oxoglutarate; P5C,,l'-pyrroline-5-carboxylate. Ornithine is the nitrogen carrier in the urea cycle in the mammalian liver. The indispensability of ornithine for carbamoyl phosphate synthesis was first pointed out by Krebs et al. in liver perfusion experiments (1); other investigations with isolated mitochondria have since shown that ornithine, as well as N-acetylglutamate and arginine, is essential for carbamoyl phosphate synthesis (2-4). The hepatic ornithine content is increased by ammonia, which decreases the hepatic level of 2-oxoglutarate; by branched chain amino acids, which inhibit ornithine degradation; and by arginine and proline, derived from dietary protein, both of these being ornithine precursors (5, 6). In the mucosal epithelial cells of the small intestine in both starved and fed rats, glutamine or glutamate given through the blood or lumen is a better respiratory fuel than glucose, and is converted into citrulline, proline, and ornithine (7-9). Citrulline may be further converted into arginine in the kidney (10, 11), and it then supplies ornithine in the liver, as shown in Fig. 1. Ornithine aminotransferase and pyrroline-5-carboxylate synthetase are both key enzymes for the citrulline synthesis pathway in the small intestine (12-14). Hepatic ornithine, measured after livers were freezeclamped, had increased proportionally to the dietary protein level, suggesting the maintenance of a basal level of about 150 nmol ornithine/g liver when the dietary protein was extrapolated to zero (15). Sanada et al. (16) reported that intestinal ornithine aminotransferase activity was not affected by dietary protein level. However, to clarify the mechanism of ornithine supply to the liver after the feeding of a low protein diet, we investigated whether the activities of the four ornithine metabolic enzymes, ornithine aminotransferase (OAT) [EC ], 1-pyrroline-5-carboxylate dehydrogenase (P5CDH) [EC ], pyrroline-5- carboxylate reductase (P5CR) [EC ], and glutamine synthetase (GLNS) [EC ], in the small intestine and liver were responsive to the dietary protein level. We found that ornithine aminotransferase in the small intestine was increased by the low-protein diet compared with the high-protein diet, determined on the basis of enzyme activity and immunohistochemical staining. Herein we report changes in the activity of ornithine metabolic enzymes in the small intestine and liver in response to dietary protein levels, and related findings about the reversal of the ornithine aminotransferase reaction, which works for citrulline synthesis in the small intestine. MATERIALS AND METHODS Animals and Feeding Conditions-All rats were Wistar males; they were fed ad libitum and kept under a daily controlled 12-h light/dark lighting cycle. Rats used for tissue enzyme assays weighed about 80 g and received a high- or low-protein diet for 4 weeks. Rats used for hepatic Vol. 116, No. 4,

2 722 T. Matsuzawa et al. Fig. 1. Metabolic pathway of ornithine supply to the liver through the kidney, beginning from citrulline synthesis in the small intestine. 1, glutaminase; 2, urease; 3, P5C synthetase; 4, P5C dehydrogenase (P5CDH) [EC ]; 5, P5C reductase (P5CR) [EC ; 6, proline oxidase; 7, ornithine aminotransferase (OAT); 8, glutamate dehydrogenase (GDH); 9, carbamoylphosphate synthetase; 10, ornithine carbamoyltransferase (OCT); 11, argininosuccinate synthetase; 12, argininosuccinate lyase; 13, arginase; 14, glutamine synthetase (GLNS); 15, glycine amidinotransferase. metabolite assays weighed g and received the high- or low-protein diet for a week before use. Both the high- and low-protein diets, which contained 70 and 5% of milk-casein and 23 and 88%, respectively, of cornstarch, plus adequate amounts of vegetable oil, vitamins, and minerals, were purchased from Oriental Yeast Company (Tokyo). When animals received the low-protein diet, small amounts of powdered cheese and small dried sardines were added to stimulate their appetite. Enzyme Assays-At 1-week intervals, the rats were anesthetized with ether and decapitated; the tissues were then removed for sample preparation. The small intestines were slit and washed well with saline solution. The upper one-third of the small intestine was separated and the mucosal portion prepared as the assay sample. The supernatant obtained after centrifugation at 10,000 rpm of the 0.25% Triton-treated homogenate was used for the assay of ornithine aminotransferase activity (17). DL-Pyrroline-5- carboxylate was chemically synthesized (18). Because of its cold ]ability, the cytosol fraction used for determining pyrroline-5-carboxylate reductase activity was prepared at room temperature (19). The 1-pyrroline-5-carboxylate dehydrogenase activity was determined in the Tritontreated mitochondrial fraction (20). Glutamine synthetase was measured by determining y-glutamylhydroxamate formation in the cytosolic fraction (21). Protein concentration was determined by the biuret method (22). Metabolite Assays-The livers were rapidly freezeclamped after the rats were decapitated, and the powdered frozen liver (2 g) was homogenized in 5% HC1O4i then centrifuged at low speed. The supernatant was neutralized at ph 7.5 with KOH, and KC1O4 was precipitated in the cold. Ornithine was assayed by our enzymatic method (15). Immunohistochemistry-Rabbit anti-oat antiserum was raised as reported previously (23). Rats fed the 5% casein (low-protein) or 70% casein (high-protein) diet for one to 4 weeks were anesthetized with ether and the livers and small intestines were rapidly removed. The tissues were slit and immediately fixed with 10% neutralized formalin (ph 7.5) for 48 h and then cut into a few blocks. The blocks were washed with tap water for 12 h, then dehydrated with alcohol, and the alcohol was removed with xylene; the blocks were then processed routinely, and embedded in paraffin. Paraffin sections (3- to 5-,um thick) were deparaffinized, washed with tap water, and treated with 3% H2O2 for 30 min in a moist chamber to block endogenous peroxidase activity. We employed the ABC method and used Dakopatts-AB complex (Dako, Santa Barbara, CA, U.S.A.). After being washed with 0.05 M Tris/HC1 buffer (ph 7.5) (TBS), the sections were treated with normal blocking serum for 20 min; the blocking serum was then removed, and the sections were incubated with the anti - OAT serum, diluted 1,000-fold with TBS, in a moist chamber for 20 min at room temperature. After being washed with TBS, the sections were then incubated with the second biotinylated anti-rabbit IgG, washed with TBS, f urther incubated with avidin-biotinylated peroxidase complexes, washed again with TBS, and then stained with 0.05% 3,3'-diaminobenzidine tetrahydrochloride plus 0.01% H2O2 in 0.05 M Tris/HCI buffer (ph 7.5) for 5-10 min. The sections were then washed with water, and -ounterstained with methylgreen for nuclear staining, after which they were dehydrated, and mounted in Harleco synthetic resin. RESULTS Changes Induced in Ornithine Metabolic Enzymes by Dietary Protein-Changes in the levels of the four enzymes involved in the metabolism of ornithine in the liver, small intesti ne, and kidney, were noted after animals were fed with the low- or high-protein diets for 4 weeks (Figs. 2 and 3). The high-protein diet strongly induced ornithine amino - transferase in the liver, in accordance with previous reports (24, 25) ; the specific activity was times higher in the high-protein diet group than in the low -protein group. In contrast, in duplicate experiments, ornithine aminotrans-f erase activity in the small intestine was doubled by feedi ng animals with the low-protein diet; the enzyme activity was f ound to be increased by feeding and decreased by fasting (Fig. 1S). The specific activity in both groups on the third week declined markedly due to loss of appetite, which led to fasti ng, as shown in Fig. 2A. Kidney ornithine aminotrans -f erase showed no significant difference between the t wo groups, as noted previously (16) ; rather, it showed a gradual increase with growth. Brain ornithine aminotrans -f erase showed the same specific activity, about 3 mu/mg J. Biochem.

3 Ornithine Metabolic Enzymes in Small Intestine and Liver 723 protein, in both groups throughout the experimental period (data not shown). Feeding with the high-protein diet doubled the activity of 1-pyrroline-5-carboxylate dehydrogenate in the liver and small intestine; the specific activity of the liver enzyme was much higher than that of the intestinal enzyme. 1-Pyrroline-5-carboxylate dehydrogenase activity in the kidney was very low in both groups (Fig. 2B). Levels of glutamine synthetase in the liver were moderate, and no significant difference was observed between the two groups; the activity of this enzyme in the small intestine was very low. However, feeding with the low-protein diet significantly increased the specific activity of kidney glutamine synthetase compared with that obtained with the high-protein diet (Fig. 3A). Brain glutamine synthetase activity was very high (about 40 mu/mg protein), regardless of the dietary protein level (data not shown). Pyrroline-5-carboxylate reductase activity in the liver of rats fed the low-protein diet was double that in the high-protein diet group. This enzyme activity in the small intestine was gradually decreased by the lowprotein diet, but was unchanged by the high-protein diet (Fig. 3B). Immunohistochemical Observations-In the rats fed standard lab chow, strong immunostaining (brown granules) with anti-oat serum was observed in the villous and cryptic epithelia in the small intestine and in a few rows of perivenous and periportal hepatocytes in the liver. In this study, we visually compared the intensity of immunostaining in the livers and small intestines of the rats fed the lowand high-protein diets over 4 weeks. Feeding with the low-protein diet caused strong immunostaining in the villous surface epithelia, but not in the cryptic epithelia in the small intestine, from the first through the fourth week. In the liver of rats fed the low-protein diet, however, immunostaining was observed only in a single row of periportal and perivenous hepatocytes (Fig. 4, A and C). In contrast, feeding with the high-protein diet resulted in a marked decrease in immunostaining of the villous epithelia in the small intestine after the second week. Immunostaining in the liver, however, increased in response to the high-protein diet and had extended to all lobular hepatocytes by the first week (Fig. 4, B and D). Reversal of Ornithine Aminotransferase Reaction-As noted in the introduction, hepatic ornithine level increased Fig. 2. Changes in ornithine aminotransferase (A) and 1-pyrroline-5-carboxylate dehydrogenase (B) activity in the liver, small intestine, and kidney in rats fed the low- and highprotein diets. The average body weight gain of rats was 3.4 and 6.3 g/d in the low- and high-protein diet groups, respectively. Light dotted columns, low-protein diet group; dark shadowed columns, high-protein diet group. The values are means±sd (bars) (n=3-5) for duplicate experiments. Significant differences of values between the two groups: *p<0.05, **p<0.01, and ***p<0.001, No asterisks are shown where the difference is not significant. Fig. 3. Changes in glutamine synthetase (A) and pyrroline-5- carboxylate reductase (B) activity in the liver, small intestine, and kidney in rats fed the low- and high-protein diets. All conditions are the same as those described in the legend to Fig. 2. Vol. 116, No. 4, 1994

4 724 T. Matsuzawa et a i J. Biochem.

5 Ornithine Metabolic Enzymes in Small Intestine and Liver 725 TABLE I. Reconstituted model system for citrulline synthesis. The reaction mixture contained 20 mm potassium phosphate buffer (ph 7.5), 280,u M L-P5C, 10 mm glutamate, 563 ji M NADPH, 3 mm NH,CI, 2 mm ADP, 1 mm carbamoyl phosphate, 10 mu of rat liver ornithine aminotransferase, 40 mu of bovine liver glutamate dehydrogenase, and 60 mu of rat liver ornithine carbamoyltransferase in a final volume of 500 kl. The reaction was started by addition of P5C, incubated for 60 min at 37C and terminated by addition of hydrochloric or perchloric acid. Citrulline and ornithine were determined by a modified method of Archibald (34) and our method (15), respectively. Rat liver ornithine aminotransferase was purified (35), and rat liver ornithine carbamoyltransferase was expressed using an insect cell -baculovirus system and purified (36). 'The values are mean ±SD (n=3). 'The values are mean (n=2). in proportion to the dietary protein level, as shown in Fig. 2S, in which the intercept of the line on the ordinate gave a basal level of hepatic ornithine of about 150 nmol/g liver. To maintain this basal level of ornithine with the zeroprotein diet, the intestinal ornithine aminotransferase reaction may have been reversed for citrulline synthesis in the intestinal mucosa. In the reversal of this reaction, the low affinity for L-glutamate (26) and the unfavorable equilibrium constant of about 20 at ph 8 are known to be limiting factors. However, we found that the reaction easily proceeded in the presence of carbamoyl phosphate and ornithine carbamoyltransferase in a reconstituted model system that mimicked the in vivo enzyme activity ratios of mitochondrial matrix enzymes involved in citrulline synthesis in the small intestine (OAT : GDH : OCT=1 : 2 : 3 in the case of the low-protein diet where the specific activity of OAT was about 40 mu/mg protein, that of GDH was 84 mu/mg and that of OCT was 120 mu/mg; and OAT : GDH : OCT - 1 : 4 : 6 in the case of the high-protein diet where the specific activity of OAT was about 20 mu/mg protein, but those of GDH and OCT were unchanged). Further addition of ammonia and glutamate dehydrogenase resulted in some stimulatory effect. In the absence of ornithine carbamoyltransferase, only a small amount of ornithine was formed compared with the citrulline synthesized in the complete system. However, the citrulline synthesis was roughly dependent upon the activity of ornithine aminotransferase in situ. When the activity of ornithine aminotransferase was doubled, citrulline synthesis increased 1.5 times (Table I). In addition, the ornithine aminotransferase of the small intestine was indistinguishable from those of the liver and kidney by Western blot analysis (Fig. 3S). Fig. 4. Immunohistomierographs of ornithine aminotransferase in the small intestine and liver. A: Small intestine of rat fed the low-protein diet, second week (a, villous surface epithelia; b, cryptic epithelia). Magnification x100. Bar, 100,um; B: Small intestine of rat fed the high-protein diet, second week (longitudinal section of duodenum). Magnification x 66. Bar, 100,u m. C: Liver of rat fed the low-protein diet, fourth week (perivenous area). Magnification x 100. Bar, 100,um. D: Liver of rat fed the high-protein diet, fourth week (a, perivenous area; b, liver cell cords). Magnification 66. Bar, 100 u m. DISCUSSION Biochemical evaluation of changes in enzyme activity often leads to ambiguous conclusions because of the lack of knowledge of the histological localization of the target enzymes in the tissues. Here, we found that the increase of ornithine aminotransferase activity in the liver induced by the high-protein diet was apparently due to the increase in the number of hepatocytes expressing the enzyme, from a few rows of perivenous and periportal hepatocytes in animals fed the low-protein diet to all lobular hepatocytes in the animals fed the high-protein diet. In the small intestine, in contrast, the localization of ornithine aminotransferase was rather heterogenous; it was localized in both the villous and cryptic epithelia; and intense immunostaining induced by the low-protein diet was found particularly in the villous surface epithelia throughout the first to fourth weeks, whereas the reduced immunostaining induced by the high-protein diet was observed exclusively in the villous epithelia in the third to fourth weeks. The changes in immunostaining induced by the dietary protein level in the crypts, which are the center of villous epithelial renewal, were not so marked as those in the villous epithelia. We believe that this activity and histochemical changes in ornithine aminotransferase in the villous epithelia are compatible with the possible regulation of citrulline synthesis in the small intestine, operating to maintain the basic and dietary protein-dependent ornithine levels in the liver. The ratios of the specific activity of ornithine aminotransferase to that of 1-pyrroline-5-carboxylate dehydrogenase, OAT/P5CDH, were in the liver and 3-30 in the small intestine during the first to fourth weeks in animals fed the low-protein diet, whereas the ratios of the specific activity of ornithine aminotransferase to that of pyrroline-5-carboxylate reductase, OAT/P5CR, were in the liver and 2-8 in the small intestine during the first to fourth weeks in the same animals. These findings suggest the reversal of the ornithine aminotransferase reaction is more likely to occur in the small intestine than in the liver. Strong immunostaining with anti-oat serum has been observed in the villous surface epithelia and cryptic epithelia in the human duodenum, as well as in the rat small intestine (27). The above view is thus also applicable to the human small intestine. Glutamine is supplied from the muscle to the small intestine for citrulline synthesis; however, we found that, in animals fed the low-protein diet, the kidney was the other glutamine donor. The high activity of pyrroline-5-carboxylate reductase in the liver in animals fed the low-protein diet, with respect to the cold-inactivation (28) and NADP+ supply, appeared to be relevant to the activation of the pentose phosphate pathway and fatty acid synthesis. Hepatic ornithine is derived from arginine that is synthesized in the extrahepatic tissues. Citrulline synthesized from glutamate in the small intestine is converted into arginine in the kidney, arginine is supplied to various tissues and then converted into ornithine in the liver, as shown in Fig. 1. The synthesis of citrulline from glutamate is carried out by the reversal of the ornithine aminotransferase reaction. This reversal, despite the unfavorable equilibrium constant (about 20), provides the only known pathway of de novo ornithine synthesis in mammalian cells, %' , No. 4, 1994

6 726 T. Matsuzawa et al. as pointed out by Valle and Simell (29), and is probably responsible for the nonessentiality of arginine in human adult and infant nutrition. In these experiments, we have presented some evidence of the reversal of this enzyme reaction, based on the reconstituted system mimicking the in vivo enzyme activity ratios of ornithine aminotransferase, glutamate dehydrogenase, and ornithine carbamoyltransferase, using the substrates pyrroline-5-carboxylate, glutamate, ammonia, and carbamoyl phosphate. The addition of ornithine carbamoyltransferase and carbamoyl phosphate was essential for citrulline synthesis in the reconstituted system. Without addition of ornithine carbamoyltransferase, only a small amount of ornithine was formed. Further addition of ammonia and glutamate dehydrogenase resulted in some stimulating effect on citrulline synthesis. The citrulline synthesis was also dependent upon the activity of ornithine aminotransferase added. When the amount of ornithine aminotransferase was doubled, the citrulline synthesis increased 1.5 times. Therefore, the low-protein diet may result in increased citrulline synthesis in the small intestine. Assuming that 1 g tissue of the small intestine contains roughly 40 mg protein, whole mitochondria occupy about 16% of the tissue volume, the volume of the inner membrane plus matrix is about 80% of the volume of whole mitochondria, and the matrix space is about 50% of the volume of inner membrane plus matrix (33), the present experimental conditions were at least 625-fold diluted compared with in vivo conditions. Matrix enzymes might be densely packed in the matrix space and protein-protein interactions presumably play a crucial role in citrulline synthesis in the small intestine. Ammonia is an activator of glutaminase and carbamoyl phosphate synthesis (30), and it is probable that it is an essential component in citrulline synthesis in the small intestine, since 15 to 30% of the urea synthesized daily in rats and humans circulates between the liver and small intestine and, in the small intestine, urea is degraded into ammonia and CO, by urease derived from the intestinal flora (31, 32). The authors are grateful to Prof. M. Mori, Department of Molecular Genetics, Kumamoto University School of Medicine, for supplying rat OCT cdna and Associate Prof. H. Ogawa of our University for rat OCT expression. We also thank Dr. M. Nishii and Misses H. Sobue, R. Okumura, S. Honda, and S. Nakanishi for their technical assistance. REFERENCES 1. Krebs, H.A., Hems, R., and Lund, P. (1973) Some regulatory mechanisms in the synthesis of urea in the mammalian liver. Adv. Enzyme Regul. 11, Glasgow, A.M. and Chase, H.P. 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7 Ornithine Metabolic Enzymes in Small Intestine and Liver Valle, D. and Simell, 0. (1989) The hyperornithinemias in Metabolic Basis of Inherited Disease (Scriber, C.R., Beaudet, A.L., Sly, W.S., and Valle, D., eds.) pp , McGraw-Hill, New York 30. Haeussinger, H., Meijer, A.J., Gerok, W., and Sies, H. (1988) Hepatic nitrogen metabolism and acid-base homeostasis in ph Homeostasis (Haeussinger, D., ed.) pp , Academic Press, New York 31. Takebe, S., Kobashi, K., Hase, J., and Koizumi, T. (1983) Kinetic study on ureolysis in rats. Chem. Pharm. Bull. 31, Walser, M. and Bodenlos, L.J. (1959) Urea metabolism in man. J. Clin. Invest. 38, Srere, P.A. (1982) The structure of the mitochondrial inner membrane-matrix compartment. Trends Biochem. Sci. 7, Oginsky, E.L. (1957) Isolation and determination of arginine and citrulline. II. Citrulline in Methods in Enzymology (Colowick, S.P. and Kaplan, N.O., eds.) Vol. III, pp , Academic Press, New York 35. Matsuzawa, T., Katsunuma, T., and Katunuma, N. (1968) Crystallization and properties of rat liver ornithine transaminase. Biochem. Biophys. Res. Commun. 32, Lusty, C.J., Jilka, R.L., and Nietsch, E.H. (1979) Ornithine transcarbamylase of rat liver. J. Biol. Chem. 254, Supplemental Materials Fig. is. Changes in ornithine aminotransferase activity in the small intestine depending upon food intake. A: With full stomach; B: With partially filled stomach; C: With empty stomach. Values are means ±SD (bars) (n=5-6). Significant differences of values between the two columns: * p<0.05 and ** p<0.01. Fig. 3S. Western blot analysis of rat ornithine aminotransferase from the small intestine. Liver (12.5ƒÊU)(A), kidney (7.4ƒÊU)(B) and small intestine (10ƒÊU)(C) ornithine aminotransferase were subjected to 10% SDS-PAGE, followed by transfer to nitrocellulose membrane. The membrane was incubated with anti-rat kidney OAT IgG (1.3 mg/ml 1% gelatin in 20mM Tris/HCl (ph 7.5) including 500mM NaCl and 0.02% Tween 20 (TTBS)) for 2 h after blocking with 3% gelatin in TTBS, washed with TTBS, incubated with goat anti-rabbit IgG conjugated peroxidase (MBL) for one h, washed with TTBS, and then visualized by using diaminobenzidine tetrahydrochloride (DAB). Fig. 2S. Dependence of hepatic ornithine concentration on dietary protein level. Values are means±sd (bars) (n=5). Vo1. 116, No. 4, 1994