Glutaminase and Glutamine Synthetase Activities in Human Cirrhotic Liver and Hepatocellular Carcinoma Tetsuya Matsuno1 and Lori Goto

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(CANCER RESEARCH 52, 1192-1194, March 1, 1992] Glutaminase and Glutamine Synthetase Activities in Human Cirrhotic Liver and Hepatocellular Carcinoma Tetsuya Matsuno1 and lori Goto

Department of Measles Virus, National Institute of Health, Musashimurayama, Tokyo 208 [T.M.], and Third Department of Internal Medicine, Nihon University, Tokyo 173 [LG.], Japan

ABSTRACT Â¡tiesandpreparation of mitochondria were carried out within no more than 2 h after removal of the samples. Glutamine synthetase and glutaminase activities in human cirrhotic For measuring enzyme activities tissues were homogenized with liver tissues and hepatocellular carcinomas were determined for compar saline by Polytron (Kinematica, Lucerne, Switzerland) and the super- ison with normal liver tissues. In hepatocellular carcinoma, glutamine natants obtained after centrifugation at 800 x g for 5 min were used. synthetase activity was approximately one-third of that in normal liver, Glutamine synthetase (EC 6.3.1.2) was assayed by the sensitive method whereas no detectable change in the enzyme activity was observed in described previously (7). The reaction mixture for assaying glutaminase cirrhotic liver. Phosphate-dependent and phosphate-independent gluta (EC 3.5.1.2) were essentially as described by Matsuda et al. (8) except minase activities were increased approximately 20-fold and 6-fold, re that L-glutamine was replaced by [MC]glutamine (>250 mCi/mmol; spectively, both in the carcinoma and cirrhotic liver compared with those Amersham) (0.2 ^Ci/tube) and the total volume of the reaction mixture from normal liver, Oxypolarographic tests showed that the rate of was 50 n\. The reaction was carried out at 37Â°Cfor 30 min and glutamine oxidation in the tumor and cirrhotic liver mitochondria was terminated by adding cold ethanol. The radioactive glutamate formed about 5-fold higher than that in the liver mitochondria. The rate of was measured using DE81 paper (Whatman) according to the method glutamate oxidation in the liver mitochondria was comparable to that in of Martin (9). Glutamine transaminase (EC 2.6.1.15) was measured by the cirrhotic liver and tumor mitochondria. Glutamine oxidation was the method of Kupchik and Knox (10). Protein was determined by the inhibited by prior incubation of the mitochondria with 6-diazo-S-oxo-L- method of Smith et al. (11) using bicinchonic acid with bovine serum norleucine, which inhibited mitochondria! glutaminase. These results albumin as the standard. indicate that the product of glutamine hydrolysis, glutamate, is catabo- Mitochondria were prepared essentially as described (12). Oxygen lized in the tumor and cirrhotic liver mitochondria to supply ATP. consumption was measured polarographically at 30Â°Cby means of a In the liver and cirrhotic liver mitochondria, glutamate was oxidized Clark oxygen electrode (Central Sci. Co., Tokyo, Japan). Reactions via the routes of transamination and deamination. On the other hand, were started by the addition of 200 n\ of mitochondria! suspension (4 glutamate oxidation was initiated preferentially via a transamination mg protein/ml) in 0.25 M sucrose and 0.2 IHMEDTA to 600 ^1 of 16 pathway in the tumor mitochondria. HIMpotassium phosphate buffer (pH 7.4) containing 0.25 M sucrose, 8 IHMKC1, 1.6 HIMMgCl2, and 0.2 HIMEDTA. Then, about 3 min later, INTRODUCTION 100 n\ of 0.11 M glutamate or other respiratory substrates and 50 n\ of 5 HIMADP were added. Glutamine (99-100% pure) was obtained from Sigma. All other Glutamine catabolism is of considerable interest because of its link with neoplastic transformation (1-3). An earlier report reagents were of the highest grade commercially available. Aminooxy- acetic acid and bromofuroic acid were neutralized with NaOH. showed that glutaminase activities were increased in Morris hepatomas (4). Previously, we reported that the chicken hepatoma induced by the myelocytomatosis virus, MC29 strain, RESULTS AND DISCUSSION behaved like a trap for glutamine in blood and tissues of the tumor-bearing host (5). In the tumor tissue the activity of Glutamine synthetase and glutaminase activities in hepato glutamine synthetase was extremely low compared with that of cellular carcinomas and cirrhotic liver tissues are presented in normal liver, while the activities of glutaminase were high in Table 1 for comparison with normal liver tissues. The ATP- the tumor mitochondria. Similar results were obtained in var dependent formation of glutamine from glutamate and ammo ious hepatoma cells of human and rat origins in cultured form nia, catalyzed by glutamine synthetase, plays an important role or in ascitic form (6). These hepatomas showed a prominent in normal cellular glutamine metabolism and this enzyme ac glutamine oxidation activity. These results indicated that glu tivity is high in the liver. Glutamine synthetase activity was tamine hydrolysis may have an important role in the bioener- reported to be low in several lines of rat hepatomas (13, 14). getics of malignant cells. It was, therefore, of interest to deter Similar results were obtained in a series of hepatoma cells of mine these enzyme activities in human hepatocellular carcino human and rat origins (6). Our results shown in Table 1 address mas and cirrhotic liver tissues obtained at operation. additional indications that this enzyme activity is markedly decreased in human hepatocellular carcinomas thus far exam ined. On the other hand, decrease in glutamine synthetase MATERIALS AND METHODS activity was not detectable in cirrhotic liver tissues. Hepatocellular carcinomas and cirrhotic liver tissues were obtained Since prominent glutaminase activities were detectable in from Department of Surgery, Nihon University Surugadai Hospital, both the hepatoma and cirrhotic liver (Table 1), additional and Tokyo National Chest Hospital. Specimens of fresh liver obtained experiments on glutamine metabolism in these mitochondria at autopsy were supplied by Dr. T. Takahashi, Tokyo National Chest were undertaken. Fig. 1 shows representative oxypolarographic Hospital. The samples were transported in ice-cold Krebs-Henseleit tracings of oxygen consumption by liver and hepatocellular saline. The following procedures for the measurement of enzyme activ- carcinoma mitochondria with either glutamine or glutamate as substrates. Glutamine oxidation was prominent in the tumor Reeeived 8/14/91; accepted 12/13/91. The costs of publication of this article were defrayed in part by the payment mitochondria (Fig. le), while mitochondria from the liver of page charges. This article must therefore be hereby marked advertisement in showed a feeble glutamine oxidation (Fig. la). Both liver and accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1To whom requests for reprints should be addressed, at Department of Measles hepatoma mitochondria utilized glutamate well for oxidation Virus, National Institute of Health, Gakuen, Musashimurayama, Tokyo 208. (Fig. 1, b, d). Prior incubation of the tumor mitochondria with 1192

6-diazo-5-oxo-L-norleucine at 0Â°Cfor 5 min markedly blocked Liver glutam ine oxidation (Fig. le) at 5 HIMwhere both glutaminase activities in the mitochondrial homogenates were suppressed. Similar results were obtained with cirrhotic liver mitochondria (data not shown). Fig. 2 shows the typical representative oxypolarographic tracings of the effects of aminooxyacetatc and bromofuroate on o the oxygen consumption by liver, cirrhotic liver, and the tumor s mitochondria with glutamate as substrate. Oxygen uptake in the liver mitochondria was suppressed either by aminooxyacetate, an inhibitor of glutamate transaminase (15), or bromofu- 2 min

Table 1 Glutamine synthetase and glutaminase activities Cirrhotic liver activitySpecimenNormal Enzyme synthetase (nmol vglutamyl- (nmol glutamate formed 'min ' hydroxamate protein)"Pi-dependentmg formed/min/mg protein)9.80 Prindependent4.19 human Â±1.96* Â±1.59132.74Â±0.09 5.32 liver (n= 15) Cirrhotic liver 7.77Â±2.183.01 Â±60.03 35.11 Â±14.08 (n = ll) (P<0.00\)c (P< 0.001) Hepatocellular Â±0.71 88.97 Â±52.32 47.19 Â±32.28 carcinoma (P<0.01)Glutaminase (P< 0.001) (P< 0.001) Hepatocellular carcinoma (n=ll)Glutamine " Expressed as subtracted background due to glutamine hydrolysis during incubation without test material. * Mean Â±SD. ' Statistical analysis was carried out as using Student's / test.

Liver

Fig. 2. Effects of aminooxyacetate (AOA) and bromofuroate (Bf) on the oxidation of glutamate (glu) in human liver (a, b), cirrhotic liver (<â€¢,

o roate, an inhibitor of glutamate dehydrogenase (16) (Fig. 2, a, <Ã¬ b}. In the cirrhotic liver mitochondria, aminooxyacetate and bromofuroate inhibited the respiration, although the latter had 2min a little inhibitory effect on the cirrhotic liver mitochondria compared with that on the liver mitochondria (Fig. 2, c, d). On the other hand, bromofuroate up to the concentration of 8 HIM Hepatocellular carcinoma exerted little or no inhibitory effect on the respiration of the tumor mitochondria, while aminooxyacetate inhibited the res piration (Fig. 2,e,f). These results indicate that while glutaminase activities were detectable in normal liver, such activities became much higher in the cirrhotic liver and the carcinoma mitochondria, thus allowing them a better oxidation of glutamine. The rate-limiting step in glutamine-dependent oxygen uptake was obviously not glutamine oxidation per se because it was inhibited by 6-diazo- 5-oxo-L-norleucine, an inhibitor of glutaminase (17). Consist ent with the above interpretation was the finding of little or no glutamine transaminase in these mitochondria. Thus, it was concluded that the product of glutamine hydrolysis, glutamate, was catabolized in the tumor and cirrhotic liver mitochondria to supply ATP. Similar results were obtained with mitochondria from rat hepatoma induced by 3'-methyl-4-dimethylamino- -Â£ Ãˆ S O < azobenzene.2 Furthermore, as shown in Fig. 2, in the liver and ÃœJ cirrhotic liver mitochondria, glutamate was oxidized via the e routes of transamination and deamination. On the other hand, Fig. 1. Oxidative phosphorylation in human liver (a, b) and hepatocellular glutamate oxidation was initiated preferentially via the trans- carcinoma (c--c) mitochondria (Mi) with glutamine (din) or glutamate (K/H)as substrate. Incubation conditions were as described in "Materials and Methods." â€¢¿'Unpublishedobservations. 1193

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1992 American Association for Cancer Research. GLUTAMINE METABOLISM IN LIVER AND HEPATOMA amination pathway in the tumor mitochondria. These results transplantable hepatomatous growth induced by MC-29 virus. Int. J. Biochem., 18: 187-189, 1986. demonstrate the aberrations in the glutamine metabolism es 6. Matsuno, T., and Hirai, H. Glutamine synthetase and glutaminase activities pecially in the carcinoma tissues. in various hepatoma cells. Biochem. Int., 19: 219-225, 1989. 7. Matsuno, T., and Satoh, T. A sensitive assay for chicken glutamine synthe The cirrhotic liver still retained the liver function with respect tase. Int. J. Biochem., 17: 1369-1371, 1985. to glutamine synthetase which plays an important role in the 8. Matsuda, Y., Kuroda, Y.. Kobayashi, K., and Katsunuma, N. Comparative liver nitrogen metabolism. Prominent glutamine oxidation and study on glutamine, serine and glycine metabolism in ureotelic and uricotelic animals. J. Biochem. (Tokyo), 73: 291-298, 1973. low glutamine synthetase activity observed in the hepatocellular 9. Martin, D. W., Jr. Radioassay for enzyme production of glutamate from carcinoma tissue may be favorable to the bioenergetics of the glutamine. Anal. Biochem., 46: 239-243, 1972. tumor cells since the existence of an ATP-dissipating futile 10. Kupchik, H. /... and Knox, W. E. Phenylpynivate glutamine aminotransferase (rat kidney). Methods Enzymol., 17A: 951-954, 1970. cycle due to glutamine synthetase is negligible. 11. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., The present findings make us conclude that the imbalance in Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C. Measurement of protein using bincinchoninic acid. Anal. Biochem., the glutamine metabolism in the tumor cells is independent of ISO: 76-85, 1985. the nature of carcinogenic agent and the species. This conclu 12. Matsuno, T., Satoh, T., and Suzuki, H. Prominent glutamine oxidation sion confirms an earlier statement by Prajda (18) that "the activity in mitochondria of avian transplantable hepatoma induced by MC- 29 virus. J. Cell. Physiol., 128: 397-401, 1986. biochemical phenotype of liver cancer is independent both from 13. Sebolt, J. S., and Weber, G. Negative correlation of L-glutamine concentra the carcinogen and the species." tion with proliferation rate in rat hepatomas. Life Sci., 34: 301-306, 1984. 14. Deuel, T. F., Louie, M., and Morris, H. P. Abnormal dietary regulation of glutamine synthetase in Morris hepatomas. Adv. Exp. Med. Biol., 92: 665- REFERENCES 675, 1978. 15. Hopper, S., and Segal, H. L. Comparative properties of glutamic-alanine 1. Kovacevic, /.. and McGivan, J. D. Mitochondria! metabolism of glutamine transam inusc from several sources. Arch. Biochem. Biophys. Â¡OS:501-505, and glutamate and its significance. Physiol. Rev., 63: 547-605, 1983. 1957. 2. Zielke, H. R.. Zielke, C. L., and Ozand, P. T. Glutamine: a major energy 16. Caughey, W. S., Smiley, J. D., and I Id 1erman, L. L-Glutamic acid dehydrog- source of cultured mammalian cells. Fed. Proc., 43: 121-125, 1984. enase: structural requirements for substrate competition: effect of thyroxine. 3. Matsuno, T. Bioenergetics of tumor cells: glutamine metabolism in tumor J. Biol. Chem., 224: 591-607, 1957. cell mitochondria. Int. J. Biochem., 19: 303-307, 1987. 17. Hartman, S. C. Glutaminase (EscherÃchÃacoli).Methods Enzymology, 17A: 4. Katsunuma, N., Kuroda, Y., Sanada, Y., Towatari, T., Tornino, I., and 941-945, 1970. Morris, H. P. Anomalous distribution of glutaminase isozyme in various 18. Prajda, N. Biochemical phenotype in human and animal liver tumors. Prog. hepatomas. Adv. Enzyme Regul., 8: 281-287, 1970. Clin. Biol. Res. (Thirteenth International Cancer Congress), 132 (Part D): 5. Matsuno, T., and Satoh, T. Glutamine metabolism in the avian host bearing 79, 1983.

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1992 American Association for Cancer Research. Glutaminase and Glutamine Synthetase Activities in Human Cirrhotic Liver and Hepatocellular Carcinoma

Tetsuya Matsuno and Iori Goto

Cancer Res 1992;52:1192-1194.

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