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Nicotinamide methylation and hepatic energy reserve: a study by liver perfusion in vitro
Journal of Hepatology 1995; 23: 461-470 Printed in Denmark AN rights reserved
Copyright 0 Journal
of Hepotology 1995
Journal of Hepatology ISSN 0168-8278
Nicotinamide methylation and hepatic energy reserve: a study by liver perfusion in vitro Rosario
Cuomo, Rossella Pumpo, Giancarlo
Sarnelli, Gaetano
Capuano
and Gabriele
Cattedra di Gastroenterologia, Facoltci di Medicina, Universitd degli Studi di Napoli ‘%ederico II”,
Budillon
Napoli, Italy
Background/Aims: The synthesis of pyridine nucleotides from nicotinamide requires adenosine triphosphate. In man when exogenous nicotinamide is poorly utilized in this synthesis, the excess follows a dissipative metabolic pathway and is excreted in urine as N-methyhricotinamide. In human cirrhosis N-methyhticotinamide serum levels are higher than normal, in basal condition and after nicotinamide oral load. The aim of this study was to verify N-methyhricotinamide production in relation to hep atic content of adenosine triphosphate during in vitro perfusion of rat liver, in normal conditions and after adenosine triphosphate depletion by metabolic stress. Metlrods: “Stress” was obtained by pre-washing with saline for 15 min before the perfusion with nutritive medium. Results: The adenosine triphosphate decrease in the stressed liver was 38% after pre-washing with saline and 80% at the end of nutritive perfusion. In control liver the corresponding decreases were 1% after pre-washing with nutritive medium and 65% at the end of perfusion with the same medium. The total nic-
otinamide adenine dinucleotide decreases were 44% and 56% in the stressed liver, and 19% and 52% in control the liver. The output levels of N-methyhticotinamide at 90 min of rat liver nutritive perfusion were 31.5024.72 nmol/g for normal liver and 6640213.17 for stressed liver (p
of pyridine nucleotides (NAD-P) from nicotinamide (NAM), via nicotinamide mononucleotide, involves energy supply by adenosinetriphosphate (ATP) and phosphoribosyl pyrophosphate (PRPP) (14). In fact, the administration of NAM in rats induces cellular ATP depletion of liver (5). When an oral load of NAM in man is not completely utilized in nucleotide synthesis, the excess follows a dissipative metabolic pathway and is excreted in urine as N-methylnicotinamide (NMN), and its oxi-
dation product 2- or 4-pyridone-5-carboxamide (1,68). Because nicotinamide is also a potent inhibitor of ADP-ribosyltransferase (9), and poly-ADP-ribosyle ensures DNA repair, NAM methylation prevents the inhibition of poly-ADP ribosylation, so assuring the preservation of normal DNA (10-12). Therefore, NAM methylation plays an important protective function in man. We recently showed that N-methylnicotinamide production is increased in basal condition and after NAM oral load in patients with cirrhosis compared to healthy subjects (13,14). This could depend on the cell energy crisis present during the cirrhotic phase of liver disease (15,16), which affects many metabolic pathways (e.g., galactose and fructose metabolism) (17-19). We postulate that in case of energy failure, pyridine
T”
Received
SYNTHESIS
7 October 1994; revised 20 February; accepted 3 March 1995
Prof. Gabriele Budillon, Cattedra di Gastroenterologia, Facolta di Medicina e Chirurgia, Universitadi Napoli “Federico II”, Via S. Pansini, 5, 80131 Correspondence:
Napoli,
Italy
Key words: Adenine triposphate; Liver; Liver perfusion; Methylation; Nicotinamide-adenine dinucleotide; N-methyhdcotinamide. 0 Journal of Hepatology.
465
R. Cuomo et al.
nucleotide catabolism to NAM is no longer counteracted by NAD resynthesis, and NAM is diverted to the methylation reaction and subsequent urinary excretion to avoid its toxic effects. In an attempt to verify this hypothesis, we studied the relationship between the energy level of the liver (hepatic ATP) and the spontaneous production of NMN in experiments of rat liver in vitro perfusion using a model of metabolic “stress”.
IS-YIN PrBPERmSDN
Materials and Methods PERFUSION
Animals
Twenty-eight Sprague-Dawley rats (Charles-River, Calco, Como, Italy) weighing 225-250 g, housed in plastic cages under controlled temperature and light conditions (12 h light/dark cycle), were used. Rats were fed ad libitum until killed. Experimental
liver perfusion
study
The perfusion was performed in a “closed” recirculating system contained within a 37°C thermostatically controlled plexiglass cabinet (mod. EC 3B, Passoni, Milano, Italy). Perfusion medium consisted of KrebsRinger bicarbonate buffer supplemented with 3% bovine serum albumin (Cohn fraction V, Sigma Chemical Co., St. Louis, USA), 0.1% glucose and heparin 4 mg/ ml (20). The pH of perfusate was adjusted to 7.45 with 0.1 N NaOH. The volume of the perfusion medium was 90 ml at the beginning of the experiment. The perfusion medium supplied to the liver by a peristaltic pump (Watson-Marlow 502S, Cellai, Milano, Italy) at a constant rate of 2 ml/g of liver * min’ was continu466
Ta (4)
(4) Tm j
design
Four rats (basal group; B) were killed by cervical dislocation and the livers were excised, weighed, and tissue samples were immediately freeze-clamped with tongs cooled to the temperature of liquid nitrogen and stored at - 135°C until required. Twenty-four rats were anesthetized with diethyl ether, the livers were isolated by the usual surgical technique, and the biliary duct was cannulated. About 15 min elapsed between cannulating the portal vein and starting recirculation in the perfusion apparatus. In the meantime, to obtain metabolic stress, the liver was washed with saline alone (stress group; S) or with perfusion medium (normal group; N). The livers of four rats of group N (T,,N) and four of group S (T$) were excised and weighed, and tissue samples were treated after the washing period as described for group B. At this time liver samples were also examined by histology. The other livers were weighed and tissue samples were treated as above at perfusion times of 45 and 90 min (four livers at any time; see Fig. 1). Isolated
TImI
(4) TM
t
Ta (4)
Fig. 1. Experimental schedule. Basal: Livers examined immediately after the animals were killed (N.4). Normal: Livers treated with a 15-min pre-perfusion washing with nutritive medium. Stressed: Livers treated with a 15-min preperfusion washing with saline alone. ( ): Number of livers examined at the different perfusion times.
ously gassed with a mixture of 95% 02/5% CO2 (5 Y g of liver * min-‘) while it passed through a thin-walled silicone tube (Dow-Corning Co, Medical Products, Midland, Michigan, USA) 7 m long. Saline solution was supplemented with heparin 4 mg/ml. Samples of medium were collected after they had passed through the liver at 1, 3, 5, 10, 15, 20, 30, 45, 60 and 90 min and stored at -20°C until analysis. Samples of bile were collected in pre-weighed tubes and measured by gravimetric volume, assuming a density of 1 at the end of perfusion periods (see Fig. 1). The following vitality parameters were monitored in each liver perfusion experiment: 1) aspartate aminotransferase (AST) released in outflow samples; 2) glucose and urea concentration in medium; 3) bile production; 4) pH of perfusate at the beginning and at 30,45 and 90 min of perfusion. Biochemical
assay
Medium NMN concentrations were assessed by a fluorimetric technique in which an alkaline condensation reaction of NMN with methyl-ethylketone forms the fluorescent product (21,22). Glucose, urea and AST in the medium were evaluated by kit tests (Boehringer Mannheim, Milano, Italy). Adenosine triphosphate, total NAD (NAD+NADH) and protein content were measured in liver tissue samples. Adeno-
Nicotinamide methylation and hepatic energy reserve
sine triphosphate was measured by the luciferin-luciferase procedure (23) using an assay kit (Boehringer Mannheim, Milano, Italy). The NAD and NADH were extracted by a 70% buffered ethanol solution (24,25) and measured by a combined bioluminescent assay (Bio-Orbit Oy, Turku, Finland). Protein concentration was determined by the Bio-Rad protein assay kit with bovine serum albumin as a standard (26). Statistical
analysis
Results are expressed as the mean2S.E.M. Data were analyzed for the significance of differences with ANOVA. When ANOVA indicated a probability of less than 0.05 for the null hypothesis, the Tukey-Kramer multiple comparison test was performed to determine which values differed significantly. The ratio of NADH/NAD between different groups was evaluated by the test for linear trend. Spearman’s rank correlation test was performed to assess the relationship between the NMN level of perfusate and ATP and NAD(H) content of liver. All statistical calculations and graphs were obtained with Instat 2.0 and Prism
E
-rs-15
0
45
90
Fig. 3. Methylnicotinamide 750 [
(41
andpercentage
release in the perfusion medium decrease (nmol/g of liver) of ATP and total
NAD against the basal value in ‘tzormal” (A, 0, 0) and “stressed” liver (A, 0, W). The curve of methylnicotinamide release of ‘stressed” liver appears significantly higher (Two-way ANOVA; p
software packages (GraphPad Software, Sorrento Valley Road, San Diego, CA, USA).
Results c E wr Jo =
(4)
20 15
J. (4)
10, s
5’
3
0 60
c3 Y:
40 v
20 L .
Fig. 2. Vitality parameters during “normal” (0) and ‘Stressed” (m) rat liver perfusion (see Fig.l). * ~~0.05 vs normal.
The histology did not reveal any alteration in the liver after the “stress” treatment. The vitality parameters recorded during perfusion were similar in “stress” and control liver (see Fig. 2). As found by others (20), the glucose and urea concentration in the perfusion medium, and bile production, increased slowly. A mild AST release in the medium, higher for stressed livers, was observed only at 90 min (pcO.05). The nucleotide percentage variations per g liver are reported in Fig. 3. The 15 min pre-washing of the liver with saline solution without nutrients, used as “stress” mechanism, decreased the hepatic ATP level by 38% and total NAD by 44% (versus 1% and 19% respectively of the control liver .pre-washed with nutritive medium). This induced increased production of NMN (Fig. 3). In the recycling closed system used for the perfusion study, NMN con467
R. Cuomo et al. TABLE 1 ATP, NAD(H) and protein content of “normal” and “stressed” rat liver in basal condition and at different times of perfusion (see Fig. 1) N
ATP (nmol/mg of protein) Normal
4 4 4 4
Basal TO T45 T90
Stressed
3.65+0.28 3.9O-cO.46 2.70-r-0.50 3.45LO.51 2.02kO.36 1.92kO.258 1.28?0.2Om
NAD+NADH (nmol/mg of protein) Normal
NADHINAD Normal
Stressed
8.82k0.71 7.93eo.45 5.99?0.96* 6.51 k0.44 4.95?0.26$ 6.44-cO.70*11 5.0010.40$
Stressed
0.29+-0.42# 0.21r0.41 0.23t0.26# 0.21 to.27 0.19~0.10# 0.19~0.20 0.17~0.14#
Protein (mg/g of liver) Normal
Stressed
216.1212.6 199.623.5 18O.lk3.6 174.0t17.0 196.4t14.1 174.9? 14.0 170.6215.4
* p<0.05, t ~~0.01, $ p
centration increased continuously in the medium, and was significantly higher in the “stress” than in the control experiments. The NMN levels at 90 min were 31.50~4.72 nmol/g and 66.40~13.17 for normal and stressed liver, respectively. The hepatic concentrations of ATP and NAD(H) as mnol/mg of protein and the NADH/NAD ratio are reported in Table 1. The NADH/NAD ratio decreased linearly during the perfusion. In both control and stress experiments the liver content of ATP and NAD(H), after the initial drop higher in the stressed liver caused by the pre-washing, decreased even further during the nutritive perfusion. The total percentage decrease of ATP at the end of the perfusion period (T90) versus the basal value was 65% and 80% in the control and stressed liver, respectively; the NAD decrease was 52% and 56% respectively (see Fig. 3). The NMN output during perfusion was significantly correlated with the ATP decrease measured in the liver at 45 and 90 min.
100
1
I
0
~0.9297 p~o.001
l
I
I
I
I
25
50
75
100
ATP DECREASE (%I Fig. 4. Relationship between methylnicotinamide (NMN) production at the end of rat liver perfusion and decrease of hepatic ATP content (as percentage) at 45 and 90 min of perfusion versus basal level of “normal” (0) and “stressed” (a) rat livers.
468
Discussion The “stress” model with saline pre-washing was selected for this study because this treatment is less noxious than others and, unlike other systems, it achieves only partial depletion of liver ATP (27,28). The aim was to avoid morphological and functional alterations of the parenchyma so severe as to compromise the methylating function being studied. In addition, liver perfusion with nutritive medium, as performed by us, is viewed as continuous depletion of the internal resources of the isolated organ (29). In fact, the ATP concentration of the control liver during the 90 min of perfusion progressively decreased to reach that of the stressed liver at 90 min. It was the initial ATP content of stressed liver, lower than normal, that induced higher production of NMN, which is the terminal catabolite resulting from NAM not utilized in cell pyridine nucleotide synthesis (30,31). The final amount of NMN released up to 90 min in the medium of the recycling system of perfusion was twofold higher in stressed (66.40~ 13.17 nmol/g of liver) than in normal liver (31.5024.72). No direct quantitative relationship was observed between the hepatic level of NAD and NMN production, the latter being also dependent on the catabolic rate of NADP (3). Furthermore, the other intermediate metabolites (1,3CL32) arising from the catabolism of the two nucleotides were not determined. N-methylnicotinamide is a water-soluble terminal catabolite that does not accumulate inside the cells because of its immediate extracellular diffusion and removal from the organism by renal excretion (1). Therefore, the perfusate increase of NMN cannot be considered an epiphenomenon secondary to cell injury, but, on the contrary, may be seen as the result of a dissipative metabolic function, that protects the cell from NAM toxicity (5). The increased output of NMN from the stressed liver suggests an increase of substrate available to the methylating enzyme, nicotinamidemethyltransferase, although an activation of this enzyme cannot be excluded (33). Nicotinamide-methyl-
Nicotinamide methylation and hepatic energy reserve
transferase uses S-adenosyl-methionine as cosubstrate (34). This substance seems to be primarily used in NAM methylation reactions, thus becoming unavailable for other methylating reactions in conditions of liver damage (35). The methylation of NAM has been studied in various experimental conditions (5,33,36-41), and NMN has been implicated in cellular proliferation (33,37,41). The activity of the methylating enzyme increases markedly in the regenerating rat liver after partial hepatectomy (33). Increases in nicotinamide-methyltransferase activity have also been detected in livers of rats and mice bearing different kinds of tumors and Ehrlich ascites tumors (42). In contrast, the activity of nicotinamide-methyltransferase in the tumor itself has been generally reported to be lower than in the corresponding host liver, except for one type of the Morris hepatoma (34) and Walker 256 carcinosarcoma (38). Both NAM and its methylated product NMN can variously affect in vitro the hepatic cell growth and the variations of intracellular ATP provide the basis of their effects (33,41). our study shows that NAM In conclusion, methylation is increased in liver damage induced by hypoxia and metabolic stress. This finding appears to be inversely related to the ATP content of the liver. The investigations of NMN production (before and after NAM load), as shown in a previous study of human cirrhosis (13), may represent an interesting tool with which to explore some aspects of the energy failure of diseased liver in man.
Acknowledgements This study was supported by a grant from the Ministero dell’Universit8 e della Ricerca Scientifica e Tecnologica (MURST 60%, 1992).
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R. Cuomo et al. 23. Lundin A, Thore A. Analytical information obtainable by evaluation of the time course of firefly bioluminescence in the assay of ATF! Anal Biochem 1975; 66: 4763. 24. Karp MT, Raunio RP, Lovgren TNE. Simultaneous extraction and combined bioluminescent assay of NADH. Anal Biochem 1983; 128: 175-80. 25. Karp MT, Vuorinen PI. Simultaneous extraction and combined bioluminescent assay of oxidized and reduced nicotinamide adenine nucleotide. In: Methods of Enzymology, Vol. 122. New York: Academic Press Inc, 1986: 14652. 26. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 248-54. 27. Jaeschke H, Smith CV, Mitchell JR. Reactive oxygen species during ischemia-reflow injury in isolated perfused rat liver. J Clin Invest 1988; 81: 1240-6. 28. Kurokawa T, Nonami T, Harada A, Nakao A, Sugiya MAS, Ozawa T, Takagi H. Effects of prostaglandin El on the recovery of ischemia-induced liver mitochondrial dysfunction in rats with cirrhosis. Stand J Gastroenterol 1991; 26: 269-74. 29. Guaitani A, Gualano M, Villa P, Stramentinoli G, Bartosek I. Kinetics of S-adenosyl-L-methionine in isolated perfused rat liver and its effects on microsomal enzyme activity. Eur J Drug Metab Pharmacokinet 1979; 4: 187-92. 30. Dietrich LS, Fuller L, Farinas B. Effects of excessive doses of (-pyridylcarbinol and nicotinate on the synthesis and degradation of pyridine nucleotides in rat tissues. In: Gey KF, Carlson LA eds. Metabolic Effects of Nicotinic Acid and its Derivatives. Switzerland: Hans Huber Publishers 1971; 87-96. 31. McCreanor GM, Bender DA. The metabolism of high intakes of tryptophan, nicotinamide and nicotinic acid in the rat. Br J Nutr 1986; 56: 577-86. 32. Bender DA, Magboul BI, Wynick D. Probable mechanisms of regulation of the utilization of dietary tryptophan, nicotinamide and nicotinic acid as precursors of nicotinamide nucleotides in rat. Br J Nutr 1982; 48: 119-27.
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33. Hoshino J, Kiihne U, Krbger H. Methylation of nicotinamide in rat liver cytosol and its correlation with hepatocellular proliferation. Biochem Biophys Acta 1982; 719: 518-26. 34. Cantoni G. S-adenosylmethionine methyltransferase: methyl acceptor systems. In: Colowick SP, Kojdan NO eds. Methods in Enzymology, ~012. New York: Academic Press, 1957: 25763. 35. Geubel AP, Mairlot MC, Buchet JP Dive C, Laauwerys R. Abnormal methylation capacity in human liver cirrhosis. Int J Clin Pharmacol Res 1988; VIII: 177-22. 36. Seifert R, Hoshino J, Kriiger H. Nicotinamide methylation tissue distribution, developmental and neoplastic changes. Biochem Biophys Acta 1984; 801: 259-64. 37. Johnson GS. Metabolism of NAD and N’-methylnicotinamide in growing and growth-arrested cells. Eur J Biothem 1980; 112: 635-41. 38. Clark BR, Murai JT, Pomeranz A, Mills PA, Halpern RM, Smith RA. Altered distribution and excretion of Nr-methylnicotinamide in rats with Walkers 256 carcinosarcoma. Cancer Res 1975; 35: 1727-33. 39. Johnson GS, Chiang PK. 1-Methylnicotinamide and NAD metabolism in normal and transformed normal rat kidney cells. Arch Biochem Biophys 1981; 210: 263369. 40. Patterson JI, Harfer AE. Effect of tryptophan intake on oxidation of (7o-i4C) tryptophan and urinary excretion of N’methylnicotinamide in the rat. J Nutr 1982; 112: 766-75. 41. Hoshino J, Kiihne U, Krliger H. Enhancement of DNA synthesis and cell proliferation by 1-methylnicotinamide in rat liver cells in culture: implication for its in viva role. Biochem Biophys Res Commun 1982; 105: 1446-52. 42. Hanazawa Y, Sato K, Kuroiwa N, Ogawa M, Kuriyama A, Anasagi M, Kato N, Moriyama Y, Horitsu K, Fujimura S. Characterization of nicotinamide methyltransferase in livers of mice bearing Ehrlich ascites tumors: preferential increase of activity. Tumor Biol 1994; 15: 7-16.
Copyright 0 Journal
of Hepotology 1995
Journal of Hepatology ISSN 0168-8278
Nicotinamide methylation and hepatic energy reserve: a study by liver perfusion in vitro Rosario
Cuomo, Rossella Pumpo, Giancarlo
Sarnelli, Gaetano
Capuano
and Gabriele
Cattedra di Gastroenterologia, Facoltci di Medicina, Universitd degli Studi di Napoli ‘%ederico II”,
Budillon
Napoli, Italy
Background/Aims: The synthesis of pyridine nucleotides from nicotinamide requires adenosine triphosphate. In man when exogenous nicotinamide is poorly utilized in this synthesis, the excess follows a dissipative metabolic pathway and is excreted in urine as N-methyhricotinamide. In human cirrhosis N-methyhticotinamide serum levels are higher than normal, in basal condition and after nicotinamide oral load. The aim of this study was to verify N-methyhricotinamide production in relation to hep atic content of adenosine triphosphate during in vitro perfusion of rat liver, in normal conditions and after adenosine triphosphate depletion by metabolic stress. Metlrods: “Stress” was obtained by pre-washing with saline for 15 min before the perfusion with nutritive medium. Results: The adenosine triphosphate decrease in the stressed liver was 38% after pre-washing with saline and 80% at the end of nutritive perfusion. In control liver the corresponding decreases were 1% after pre-washing with nutritive medium and 65% at the end of perfusion with the same medium. The total nic-
otinamide adenine dinucleotide decreases were 44% and 56% in the stressed liver, and 19% and 52% in control the liver. The output levels of N-methyhticotinamide at 90 min of rat liver nutritive perfusion were 31.5024.72 nmol/g for normal liver and 6640213.17 for stressed liver (p
of pyridine nucleotides (NAD-P) from nicotinamide (NAM), via nicotinamide mononucleotide, involves energy supply by adenosinetriphosphate (ATP) and phosphoribosyl pyrophosphate (PRPP) (14). In fact, the administration of NAM in rats induces cellular ATP depletion of liver (5). When an oral load of NAM in man is not completely utilized in nucleotide synthesis, the excess follows a dissipative metabolic pathway and is excreted in urine as N-methylnicotinamide (NMN), and its oxi-
dation product 2- or 4-pyridone-5-carboxamide (1,68). Because nicotinamide is also a potent inhibitor of ADP-ribosyltransferase (9), and poly-ADP-ribosyle ensures DNA repair, NAM methylation prevents the inhibition of poly-ADP ribosylation, so assuring the preservation of normal DNA (10-12). Therefore, NAM methylation plays an important protective function in man. We recently showed that N-methylnicotinamide production is increased in basal condition and after NAM oral load in patients with cirrhosis compared to healthy subjects (13,14). This could depend on the cell energy crisis present during the cirrhotic phase of liver disease (15,16), which affects many metabolic pathways (e.g., galactose and fructose metabolism) (17-19). We postulate that in case of energy failure, pyridine
T”
Received
SYNTHESIS
7 October 1994; revised 20 February; accepted 3 March 1995
Prof. Gabriele Budillon, Cattedra di Gastroenterologia, Facolta di Medicina e Chirurgia, Universitadi Napoli “Federico II”, Via S. Pansini, 5, 80131 Correspondence:
Napoli,
Italy
Key words: Adenine triposphate; Liver; Liver perfusion; Methylation; Nicotinamide-adenine dinucleotide; N-methyhdcotinamide. 0 Journal of Hepatology.
465
R. Cuomo et al.
nucleotide catabolism to NAM is no longer counteracted by NAD resynthesis, and NAM is diverted to the methylation reaction and subsequent urinary excretion to avoid its toxic effects. In an attempt to verify this hypothesis, we studied the relationship between the energy level of the liver (hepatic ATP) and the spontaneous production of NMN in experiments of rat liver in vitro perfusion using a model of metabolic “stress”.
IS-YIN PrBPERmSDN
Materials and Methods PERFUSION
Animals
Twenty-eight Sprague-Dawley rats (Charles-River, Calco, Como, Italy) weighing 225-250 g, housed in plastic cages under controlled temperature and light conditions (12 h light/dark cycle), were used. Rats were fed ad libitum until killed. Experimental
liver perfusion
study
The perfusion was performed in a “closed” recirculating system contained within a 37°C thermostatically controlled plexiglass cabinet (mod. EC 3B, Passoni, Milano, Italy). Perfusion medium consisted of KrebsRinger bicarbonate buffer supplemented with 3% bovine serum albumin (Cohn fraction V, Sigma Chemical Co., St. Louis, USA), 0.1% glucose and heparin 4 mg/ ml (20). The pH of perfusate was adjusted to 7.45 with 0.1 N NaOH. The volume of the perfusion medium was 90 ml at the beginning of the experiment. The perfusion medium supplied to the liver by a peristaltic pump (Watson-Marlow 502S, Cellai, Milano, Italy) at a constant rate of 2 ml/g of liver * min’ was continu466
Ta (4)
(4) Tm j
design
Four rats (basal group; B) were killed by cervical dislocation and the livers were excised, weighed, and tissue samples were immediately freeze-clamped with tongs cooled to the temperature of liquid nitrogen and stored at - 135°C until required. Twenty-four rats were anesthetized with diethyl ether, the livers were isolated by the usual surgical technique, and the biliary duct was cannulated. About 15 min elapsed between cannulating the portal vein and starting recirculation in the perfusion apparatus. In the meantime, to obtain metabolic stress, the liver was washed with saline alone (stress group; S) or with perfusion medium (normal group; N). The livers of four rats of group N (T,,N) and four of group S (T$) were excised and weighed, and tissue samples were treated after the washing period as described for group B. At this time liver samples were also examined by histology. The other livers were weighed and tissue samples were treated as above at perfusion times of 45 and 90 min (four livers at any time; see Fig. 1). Isolated
TImI
(4) TM
t
Ta (4)
Fig. 1. Experimental schedule. Basal: Livers examined immediately after the animals were killed (N.4). Normal: Livers treated with a 15-min pre-perfusion washing with nutritive medium. Stressed: Livers treated with a 15-min preperfusion washing with saline alone. ( ): Number of livers examined at the different perfusion times.
ously gassed with a mixture of 95% 02/5% CO2 (5 Y g of liver * min-‘) while it passed through a thin-walled silicone tube (Dow-Corning Co, Medical Products, Midland, Michigan, USA) 7 m long. Saline solution was supplemented with heparin 4 mg/ml. Samples of medium were collected after they had passed through the liver at 1, 3, 5, 10, 15, 20, 30, 45, 60 and 90 min and stored at -20°C until analysis. Samples of bile were collected in pre-weighed tubes and measured by gravimetric volume, assuming a density of 1 at the end of perfusion periods (see Fig. 1). The following vitality parameters were monitored in each liver perfusion experiment: 1) aspartate aminotransferase (AST) released in outflow samples; 2) glucose and urea concentration in medium; 3) bile production; 4) pH of perfusate at the beginning and at 30,45 and 90 min of perfusion. Biochemical
assay
Medium NMN concentrations were assessed by a fluorimetric technique in which an alkaline condensation reaction of NMN with methyl-ethylketone forms the fluorescent product (21,22). Glucose, urea and AST in the medium were evaluated by kit tests (Boehringer Mannheim, Milano, Italy). Adenosine triphosphate, total NAD (NAD+NADH) and protein content were measured in liver tissue samples. Adeno-
Nicotinamide methylation and hepatic energy reserve
sine triphosphate was measured by the luciferin-luciferase procedure (23) using an assay kit (Boehringer Mannheim, Milano, Italy). The NAD and NADH were extracted by a 70% buffered ethanol solution (24,25) and measured by a combined bioluminescent assay (Bio-Orbit Oy, Turku, Finland). Protein concentration was determined by the Bio-Rad protein assay kit with bovine serum albumin as a standard (26). Statistical
analysis
Results are expressed as the mean2S.E.M. Data were analyzed for the significance of differences with ANOVA. When ANOVA indicated a probability of less than 0.05 for the null hypothesis, the Tukey-Kramer multiple comparison test was performed to determine which values differed significantly. The ratio of NADH/NAD between different groups was evaluated by the test for linear trend. Spearman’s rank correlation test was performed to assess the relationship between the NMN level of perfusate and ATP and NAD(H) content of liver. All statistical calculations and graphs were obtained with Instat 2.0 and Prism
E
-rs-15
0
45
90
Fig. 3. Methylnicotinamide 750 [
(41
andpercentage
release in the perfusion medium decrease (nmol/g of liver) of ATP and total
NAD against the basal value in ‘tzormal” (A, 0, 0) and “stressed” liver (A, 0, W). The curve of methylnicotinamide release of ‘stressed” liver appears significantly higher (Two-way ANOVA; p
software packages (GraphPad Software, Sorrento Valley Road, San Diego, CA, USA).
Results c E wr Jo =
(4)
20 15
J. (4)
10, s
5’
3
0 60
c3 Y:
40 v
20 L .
Fig. 2. Vitality parameters during “normal” (0) and ‘Stressed” (m) rat liver perfusion (see Fig.l). * ~~0.05 vs normal.
The histology did not reveal any alteration in the liver after the “stress” treatment. The vitality parameters recorded during perfusion were similar in “stress” and control liver (see Fig. 2). As found by others (20), the glucose and urea concentration in the perfusion medium, and bile production, increased slowly. A mild AST release in the medium, higher for stressed livers, was observed only at 90 min (pcO.05). The nucleotide percentage variations per g liver are reported in Fig. 3. The 15 min pre-washing of the liver with saline solution without nutrients, used as “stress” mechanism, decreased the hepatic ATP level by 38% and total NAD by 44% (versus 1% and 19% respectively of the control liver .pre-washed with nutritive medium). This induced increased production of NMN (Fig. 3). In the recycling closed system used for the perfusion study, NMN con467
R. Cuomo et al. TABLE 1 ATP, NAD(H) and protein content of “normal” and “stressed” rat liver in basal condition and at different times of perfusion (see Fig. 1) N
ATP (nmol/mg of protein) Normal
4 4 4 4
Basal TO T45 T90
Stressed
3.65+0.28 3.9O-cO.46 2.70-r-0.50 3.45LO.51 2.02kO.36 1.92kO.258 1.28?0.2Om
NAD+NADH (nmol/mg of protein) Normal
NADHINAD Normal
Stressed
8.82k0.71 7.93eo.45 5.99?0.96* 6.51 k0.44 4.95?0.26$ 6.44-cO.70*11 5.0010.40$
Stressed
0.29+-0.42# 0.21r0.41 0.23t0.26# 0.21 to.27 0.19~0.10# 0.19~0.20 0.17~0.14#
Protein (mg/g of liver) Normal
Stressed
216.1212.6 199.623.5 18O.lk3.6 174.0t17.0 196.4t14.1 174.9? 14.0 170.6215.4
* p<0.05, t ~~0.01, $ p
centration increased continuously in the medium, and was significantly higher in the “stress” than in the control experiments. The NMN levels at 90 min were 31.50~4.72 nmol/g and 66.40~13.17 for normal and stressed liver, respectively. The hepatic concentrations of ATP and NAD(H) as mnol/mg of protein and the NADH/NAD ratio are reported in Table 1. The NADH/NAD ratio decreased linearly during the perfusion. In both control and stress experiments the liver content of ATP and NAD(H), after the initial drop higher in the stressed liver caused by the pre-washing, decreased even further during the nutritive perfusion. The total percentage decrease of ATP at the end of the perfusion period (T90) versus the basal value was 65% and 80% in the control and stressed liver, respectively; the NAD decrease was 52% and 56% respectively (see Fig. 3). The NMN output during perfusion was significantly correlated with the ATP decrease measured in the liver at 45 and 90 min.
100
1
I
0
~0.9297 p~o.001
l
I
I
I
I
25
50
75
100
ATP DECREASE (%I Fig. 4. Relationship between methylnicotinamide (NMN) production at the end of rat liver perfusion and decrease of hepatic ATP content (as percentage) at 45 and 90 min of perfusion versus basal level of “normal” (0) and “stressed” (a) rat livers.
468
Discussion The “stress” model with saline pre-washing was selected for this study because this treatment is less noxious than others and, unlike other systems, it achieves only partial depletion of liver ATP (27,28). The aim was to avoid morphological and functional alterations of the parenchyma so severe as to compromise the methylating function being studied. In addition, liver perfusion with nutritive medium, as performed by us, is viewed as continuous depletion of the internal resources of the isolated organ (29). In fact, the ATP concentration of the control liver during the 90 min of perfusion progressively decreased to reach that of the stressed liver at 90 min. It was the initial ATP content of stressed liver, lower than normal, that induced higher production of NMN, which is the terminal catabolite resulting from NAM not utilized in cell pyridine nucleotide synthesis (30,31). The final amount of NMN released up to 90 min in the medium of the recycling system of perfusion was twofold higher in stressed (66.40~ 13.17 nmol/g of liver) than in normal liver (31.5024.72). No direct quantitative relationship was observed between the hepatic level of NAD and NMN production, the latter being also dependent on the catabolic rate of NADP (3). Furthermore, the other intermediate metabolites (1,3CL32) arising from the catabolism of the two nucleotides were not determined. N-methylnicotinamide is a water-soluble terminal catabolite that does not accumulate inside the cells because of its immediate extracellular diffusion and removal from the organism by renal excretion (1). Therefore, the perfusate increase of NMN cannot be considered an epiphenomenon secondary to cell injury, but, on the contrary, may be seen as the result of a dissipative metabolic function, that protects the cell from NAM toxicity (5). The increased output of NMN from the stressed liver suggests an increase of substrate available to the methylating enzyme, nicotinamidemethyltransferase, although an activation of this enzyme cannot be excluded (33). Nicotinamide-methyl-
Nicotinamide methylation and hepatic energy reserve
transferase uses S-adenosyl-methionine as cosubstrate (34). This substance seems to be primarily used in NAM methylation reactions, thus becoming unavailable for other methylating reactions in conditions of liver damage (35). The methylation of NAM has been studied in various experimental conditions (5,33,36-41), and NMN has been implicated in cellular proliferation (33,37,41). The activity of the methylating enzyme increases markedly in the regenerating rat liver after partial hepatectomy (33). Increases in nicotinamide-methyltransferase activity have also been detected in livers of rats and mice bearing different kinds of tumors and Ehrlich ascites tumors (42). In contrast, the activity of nicotinamide-methyltransferase in the tumor itself has been generally reported to be lower than in the corresponding host liver, except for one type of the Morris hepatoma (34) and Walker 256 carcinosarcoma (38). Both NAM and its methylated product NMN can variously affect in vitro the hepatic cell growth and the variations of intracellular ATP provide the basis of their effects (33,41). our study shows that NAM In conclusion, methylation is increased in liver damage induced by hypoxia and metabolic stress. This finding appears to be inversely related to the ATP content of the liver. The investigations of NMN production (before and after NAM load), as shown in a previous study of human cirrhosis (13), may represent an interesting tool with which to explore some aspects of the energy failure of diseased liver in man.
Acknowledgements This study was supported by a grant from the Ministero dell’Universit8 e della Ricerca Scientifica e Tecnologica (MURST 60%, 1992).
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