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© 2004 The American Society for Nutritional Sciences J. Nutr. 134:2504-2508, October 2004

Biochemical and Molecular Actions of Nutritients

Dietary Nucleotides Enhance the Liver Redox State and Protein Synthesis in Cirrhotic Rats1

María José Pérez*, Fermín Sánchez-Medina, Maribel Torres{dagger}, Angel Gil and Antonio Suárez2

Department of Biochemistry and Molecular Biology, University of Granada, Granada, Spain; * Department of Physiology and Pharmacology, University of Salamanca, Salamanca, Spain; and {dagger} Department of Experimental Biology, University of Jaen, Spain

2To whom correspondence should be addressed. E-mail: asuarez{at}ugr.es.


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Cirrhosis is characterized by altered lipid and protein metabolism and an excessive accumulation of extracellular matrix components. The aim of this work was to determine the effect of dietary nucleotide intake on the intracellular pools of nucleic acids and nucleotides, hepatic redox state, and protein synthesis during cirrhosis. Rats were given 300 mg/L thioacetamide (TAA) in drinking water and were fed diets without (TAA–Nt) or with nucleotides (Nt) (TAA+Nt, 3 g each of AMP, inosine 5'-monophosphate, CMP, GMP, and UMP per kg diet) for 4 mo. The degree of liver histological injury was less in group TAA+Nt than in TAA–Nt. The intake of nucleotides significantly increased the hepatic concentration of total nucleotides, adenine nucleotides, and ATP+ADP+AMP. Interestingly, the concentration of CDP-choline, a nucleotide necessary for phospholipid synthesis, was significantly higher in TAA+Nt than in TAA–Nt. The hepatic pyruvate:lactate (P = 0.075) and acetoacetate:ß-hydrodybutyrate (P < 0.05) ratios, indicators of cytosolic and mitochondrial redox states, were lower in TAA–Nt than in TAA+Nt. The total protein concentration was higher in the livers of TAA+Nt than in TAA–Nt. Although there were no differences in the expression of the albumin gene, the hepatic albumin concentration was significantly higher in TAA+Nt than in TAA–Nt. These data indicate that the reduction of liver injury in nucleotide-supplemented rats may be due to the increased intracellular availability of key metabolic nucleotides, the restoration of mitochondrial function, and the augmentation of protein synthesis.

KEY WORDS: • dietary nucleotides • thioacetamide • cirrhosis • redox state • protein synthesis

Liver cirrhosis is associated with important metabolic changes, including altered amino acid, carbohydrate, lipid, vitamin, and mineral metabolism. The inability to adequately use glucose and fat as tissue fuels increases protein catabolism, which is the limiting factor in satisfying the nutritional requirements of patients with cirrhosis. Adequate nutritional support is essential for the regeneration of damaged hepatocytes in liver cirrhosis and contributes to decreased morbidity of the disease in humans (1).

Nucleotides are conditionally essential nutrients that modulate lipid metabolism, immune function, and intestinal microbiota and have a reparative effect in pathological conditions that demand intense nucleic acid and protein synthesis, such as intensive growth and repair of certain tissues (2). Our research group studied the reparative effects of dietary nucleotides in rats with experimental liver cirrhosis induced by thioacetamide (TAA),3 a model that has a number of metabolic and histological alterations similar to those found in the human disease. Histological analysis of liver sections in TAA-induced cirrhotic rats showed increased hepatocyte binuclearity and reduced extension of liver damage in rats fed nucleotides (3). Dietary supplementation with nucleotides normalized protein concentration and serum amino acids as well as linoleic and arachidonic acid concentrations in liver microsomes (4). We recently demonstrated that reduced hepatic fibrosis in rats fed a nucleotide-supplemented diet is due to significantly lower prolyl-4-hydroxylase activity, reduced hepatic protein concentration and TIMP-1 gene expression, and markedly increased total collagenase activity (5).

These results suggest that exogenous nucleotides play an important role in the repair and regeneration of cirrhotic liver. Because deprivation of nucleotides significantly reduces the hepatic protein synthesis rate (6), a mechanism that may be responsible for tissue improvement is that incorporation of exogenous nucleotides into intracellular nucleotides pools would increase nucleic acid and protein synthesis, promoting cell growth and proliferation under pathological conditions. However, to date no data concerning the effect of exogenous nucleotides on the concentration of total nucleotides and nucleic acids and on protein synthesis in liver cirrhosis have been reported. To address these questions, we determined the nucleotide concentration of liver, hepatic redox state, and hepatic albumin mRNA and protein concentration in TAA-induced cirrhotic rats fed nucleotides.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
    Materials. Female Wistar rats, with an initial weight of 150–170 g, were supplied by the Animal Service of the University of Granada. Semipurified diets were supplied by Abbott Laboratories, and nonpurified standard rat diet was purchased from Panlab (Diet No. A04). Both diets were stored at 4°C under nitrogen.

    Experimental design. All rats were treated in accordance with the recommendations of the American Physiological Society (Council of Europe, 1982). Adult female Wistar rats were housed in wire-bottom cages with a 12-h light/dark cycle. The rats were divided into 3 groups of 10 rats each and were fed for 4 mo: 2 of these groups consumed ad libitum 300 mg/L TAA in drinking water and the other group was given water without TAA for 4 mo. One TAA-treated group was fed a semipurified diet (AIN-93) (7) (group TAA–Nt) while the other TAA-treated group was pair-fed the same semipurified diet supplemented with 0.3 g each of AMP, inosine 5-monophosphate (IMP), CMP, GMP, and UMP per kg diet (group TAA+Nt). Group R was fed the standard nonpurified diet and served as the reference group to obtain the values of healthy rats to verify that the experimental groups were cirrhotic. After 4 mo of treatment, the rats were maintained for another 10 d under the same conditions, but without TAA administration, to eliminate its acute effects. Rats were deprived of food for 12 h, anesthetized with a 250 g/L solution of urethane at a dose of 1 mL/100 g body wt, and killed by terminal bleeding. The livers were removed and a slice of the right lobule was fixed in formaldehyde solution and embedded in paraffin. The rest was frozen in liquid nitrogen and stored at –80°C.

    Liver histology. Slices of hepatic tissue were fixed in 3% glutaraldehyde and postfixed in 1.5% osmium tetroxide. Ultrathin sections of 50 nm were double stained with uranyl acetate and lead citrate and examined as previously described (3).

    Enzyme activities. Enzyme activities were determined in serum or crude homogenates and units were expressed per milligram of protein in the supernatant. Aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT) were assayed in serum and liver by the methods described in Bergmeyer's handbook (8). Liver glucose-6-phosphatase was determined by measuring the phosphorus released in the presence of glucose 6-phosphate (9). The protein concentration was determined by the Bradford method (10).

    Cellular redox state. Liver redox state was determined in freeze-clamped samples by the calculation of acetoacetate:hydroxybutyrate and pyruvate:lactate ratios. Liver acetoacetate and ß-hydroxybutyrate concentrations (11) and the lactate concentration (12) were determined as described previously. Pyruvate concentration was determined using a diagnostic kit purchased from Sigma-Aldrich S.A. (Ref. No. 726).

    Acid-soluble nucleotides. Total free nucleotides were determined in freeze-clamped samples by ion-exchange HPLC (13).

    Liver total DNA and RNA concentrations. Liver DNA was measured on liver homogenates using a fluorimetric assay (14), and liver RNA was measured by the method of Fleck and Munro (15). Calf liver DNA and RNA type IV were used as standards.

    Northern blot analysis. Total RNA was extracted from ~100 mg of fresh rat liver tissue as described previously (16). Ten micrograms of denatured total RNA was electrophoresed, transferred to nylon filters, and fixed with a UV crosslinker. The filters were sequentially hybridized with an albumin ({alpha}-32P)dCTP-labeled cDNA probe, with an 18S rRNA ({alpha}-32P)dCTP-labeled oligonucleotide (5'-CAT GGT AGG CAC GGC GAC TAC CAT-3'), and with a ({alpha}-32P)dCTP-labeled rat glyceraldehyde-3-phosphate dehydrogenase cDNA probe to validate Northern blot results.

    Liver albumin. Liver concentration of albumin was quantified by an indirect sandwich ELISA as previously described (5), using an anti-rat albumin sheep antibody and purified albumin as standard. Western blot analysis was used to confirm the results obtained by ELISA (17). Rat albumin and equal amounts of total liver protein per sample were detected using the same anti-rat albumin antibody and the chemiluminescence light detection kit from Amersham-Pharmacia.

    Statistical analysis. Results are means ± SEM, n = 10. An unpaired Student's t test was used to compare the TAA-treated TAA–Nt and TAA+Nt groups. Differences with P-values < 0.05 were considered significant. All data were analyzed using BMDP software, PC 90 version (BMDP Statistical Software).


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
    Histology. Chronic intoxication with orally administered TAA affected the ultrastructure of hepatocytes in the TAA–Nt group. The cytoplasm contained few and small mitochondria and they appeared to be electron-dense (Fig. 1A). Numerous lipid droplets were seen in the cytoplasm. Nuclei became edematous and increased in size until they occupied a large proportion of cell volume and the nuclear envelope was electron-dense (Fig. 1B). The nucleoli were enlarged, granular, and vacuolated. The rough endoplasmic reticulum (ER) became disaggregated and many of its cisternae were dispersed, dislocated, and altered in length.



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  		FIGURE 1 Electron micrographs of liver in cirrhotic rats fed diets with (TAA+Nt, A and B) or without (TAA–Nt, C and D) nucleotides. N, nucleus; Nu, nucleolus; M, mitochondria; R, endoplasmic reticulum; L, lipid droplets. Original magnification X2200. Scale bar = 1 µm.

 
In contrast, the TAA+Nt had less histological injury than TAA–Nt. Normal-appearing mitochondria and large amounts of ER were observed in the hepatocyte cytoplasm in TAA+Nt (Fig. 1C). The shape and size of the nucleus and nucleoli were normal in TAA+Nt (Fig. 1D). Lipid droplets were substantially smaller in TAA+Nt than in TAA–Nt.

    Biochemical analyses. Liver ALAT and ASAT activities and serum ASAT activity did not differ between the groups. Liver G-6-Pase activitiy was higher and serum ALAT activity was lower in TAA+Nt than in TAA–Nt (P < 0.01, Table 1).


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  		TABLE 1 Serum and liver biochemistry of healthy (R) and orally thioacetamide-treated rats fed diets with (TAA+Nt) or without (TAA–Nt) nucleotides1

 
The hepatic acetoacetate:ß-hydroxybutyrate ratio was higher (P < 0.05, Fig. 2A) and the pyruvate:acetoacetate ratio tended to be higher (P = 0.075, Table 1) in TAA+NT than in TAA–NT.



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  		FIGURE 2 Hepatic acetoacetate:ß-hydroxybutyrate ratio (A), total nucleotide concentration (B), and albumin protein (C) in healthy (R) and cirrhotic rats fed diets with (TAA+Nt) or without (TAA–Nt) nucleotides. Values are means ± SEM, n = 10. Different letters indicate differences between groups, P < 0.05.

 
The liver protein concentration was higher in TAA+Nt than in TAA–Nt (P < 0.05, Table 1). Hepatic DNA and RNA and 18S RNA concentrations did not differ between the groups (Table 1).

Except for UDP-glucose levels (Table 1), the concentrations of total nucleotides (Fig. 2B), adenine nucleotides, CDP-choline, and the sum of ATP+ADP+AMP were higher in TAA+Nt than in TAA–Nt (P < 0.05, Table 1).

TAA+Nt had a greater hepatic albumin concentration (P < 0.01 Table 1; Western blot in Fig. 2C) although the level of albumin mRNA did not differ between the groups (Table 1).


   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Our group and others extensively studied the beneficial effects of nucleotides in the repair and restoration of the structure and functioning of liver after injury or partial hepatectomy (4,5,18–20). The mechanism by which exogenous nucleotides exert their beneficial effect has yet to be established. In this paper, we present evidence suggesting that exogenous nucleotides are incorporated into intracellular pools and enhance hepatocyte functionality and protein synthesis.

Oral ingestion of TAA generates experimental cirrhosis in rats, a model that resembles human cirrhosis because both share a number of metabolic and histological alterations (4,5). Transmission electron microscopic analysis of liver sections (Fig. 1) showed group TAA–Nt hepatocytes with edematous nuclei, vacuolated nucleoli, disaggregated ER, and cytoplasm containing lipid droplets and few mitochondria compared with group TAA+Nt hepatocytes. Serum and liver ALAT and ASAT activities confirmed the establishment of a TAA-induced cirrhotic process in rats (Table 1).

Because alterations in nuclear size and ER aggregation could be related to changes in nucleic acid, total nucleotide concentration, and ER integrity, we measured total liver DNA, RNA, 18S rRNA, total nucleotide, ATP+ADP+AMP, adenine nucleotide, UDP-glucose and CDP-choline values, and G-6-Pase activity. Our group recently reported that exogenous nucleotides are actively incorporated into intracellular pools (21) and enhance DNA synthesis and proliferation of fetal hepatocytes (22). Under our experimental conditions, we did not detect significant differences in DNA, 18S rRNA, and RNA concentrations in livers of rats fed diets with or without nucleotides. However, the concentration of total nucleotides was substantially higher in TAA+Nt than in TAA–Nt (Fig. 2A). These results agree with those of Palombo et al. (20), who reported that dietary nucleotides contributed to maintain the hepatocyte concentrations of ATP in cold ischemic rats. Furthermore, hepatic G-6-Pase activity, a marker of ER integrity, was significantly higher in TAA+Nt than in TAA–Nt. These data indicate that the intake of nucleotides in the diet (1.5 g/kg diet) is enough to restore intracellular levels of free nucleotides and to enhance ER integrity in hepatocytes of cirrhotic rats.

Hepatic CDP-choline concentrations were higher in TAA+Nt than in TAA–Nt (P < 0.01, Table 1). Arnaud et al. (21) showed that exogenous nucleotides increased CDP-choline concentration in cultured fetal hepatocytes. CDP-choline is a nucleotide necessary for phospholipid synthesis and lipoprotein assembly. The elevation in CDP-choline levels by nucleotide dietary supplementation may explain the absence of intracellular lipid droplets (Fig. 1).

The mitochondria in hepatocytes of cirrhotic rats were altered and their number was reduced. To study the impact of nucleotide intake on mitochondrial function, we measured pyruvate:lactate (Table 1) and acetoacetate:hydroxybutyrate (Fig. 2B) ratios as indicators of cytosolic and mitochondrial redox states. Both ratios were lower in TAA–Nt than in TAA+Nt, an indication of an altered electron transport chain. The concentrations of ATP+ADP+AMP and adenine nucleotides (Table 1), which are the carriers of electrons and the final acceptors of the energy generated during the mitochondrial energy coupling process, were lower in TAA–Nt than in TAA+Nt. Hernández-Muñoz et al. (23) obtained similar results in rats with carbon tetrachloride-induced cirrhosis and found a correlation between cellular redox state and collagen metabolism. These biochemical alterations in hepatocytes of TAA–NT rats may cause uncoupling of the electron transport chain and oxidative phosphorylation, which would result in NADH and lactate accumulation and in an insufficient energy synthesis rate. We hypothesize that incorporation of preformed nucleotides enhances the efficacy of oxidative phosphorylation, electron transport, and the turnover between oxidized and reduced forms of NAD, which would stimulate the production and storage of energy in hepatocytes.

Most of the energy generated by a cell is used to drive protein synthesis. Our group previously reported that deprivation of dietary nucleotides produced significant reductions in hepatic protein synthesis (6). To determine the effect of dietary nucleotides on liver protein synthesis during cirrhosis, we determined the hepatic protein concentration and used albumin as a marker of hepatic protein synthesis because it is one of the major proteins produced and secreted by the liver. Hepatic total protein and albumin concentrations were significantly higher in rats fed nucleotides. Since the expression of the albumin gene did not differ between the groups, these data suggest that dietary nucleotide intake by cirrhotic rats enhances the efficiency of protein synthesis in the liver.

This is the first report that dietary nucleotides increase the intracellular pool of nucleotides and improve the redox state and the efficiency of protein synthesis in cirrhotic rats. Based on these results, we conclude that the intake of nucleotides can improve liver function and reduce the extent of cirrhosis in rats. We contend that these results justify a study on the efficacy of nucleotide-supplemented formula in cirrhotic patients.


   FOOTNOTES
 
1 This work was supported by Research Project ATRI-656, cofinanced by Abbott Laboratories and the University of Granada in Spain. Back

3 Abbreviations used: ASAT, aspartate aminotransferase; ALAT, alanine aminotransferase; ER, rough endoplasmic reticulum; G-6-Pase, glucose-6-phosphatase; IMP, inosine 5'-monophosphate; R, healthy reference group; TAA, thioacetamide. TAA–Nt, cirrhotic rats fed semipurified diet; TAA+Nt, cirrhotic rats fed semipurified diet supplemented with 0.3 g each of AMP, IMP, CMP, GMP, and UMP per kg diet. Back

Manuscript received 8 April 2004. Initial review completed 17 May 2004. Revision accepted 25 June 2004.


   LITERATURE CITED
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 

1. Moriwaki, H., Tajika, M., Miwa, Y., Kato, M., Yasuda, I., Shiratori, Y., Okuno, M., Kato, T., Ohnishi, H. & Muto, Y. (2000) Nutritional pharmacotherapy of chronic liver disease: from support of liver failure to prevention of liver cancer. J. Gastroenterol. 35:13-17.

2. Sanchez-Pozo, A. & Gil, A. (2002) Nucleotides as semi-essential nutritional components. Br. J. Nutr. 87:S135-S137.

3. Fontana, L., Moreira, E., Torres, M. I., Fernández, I., Ríos, A., Medina, F. S. & Gil, A. (1996) Serum amino acid changes in rats with thioacetamide-induced liver cirrhosis. Toxicology 106:197-206.[Medline]

4. Fontana, L., Moreira, E., Torres, M. I., Fernández, I., Ríos, A., Medina, F. S. & Gil, A. (1998) Dietary nucleotides correct plasma and liver microsomal fatty acid alterations in rats with liver cirrhosis induced by oral intake of thioacetamide. J. Hepatol. 28:662-669.[Medline]

5. Pérez, M. J., Suárez, A., Gómez-Capilla, J. A., Sánchez-Medina, F. & Gil, A. (2002) Dietary-nucleotide supplementation reduces thioacetamide-induced liver fibrosis in rats. J. Nutr. 132:652-657.[Abstract/Free Full Text]

6. López-Navarro, A. T., Ortega, M. A., Peragón, J., Bueno, J. D., Gil, A. & Sánchez-Pozo, A. (1996) Deprivation of dietary nucleotides decreases protein synthesis in the liver and small intestine in rats. Gastroenterology 110:1760-1769.[Medline]

7. American Institute of Nutrition (1993) AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J. Nutr. 123:1939-1951.

8. Bergmeyer, H. U. (1974) Methods of Enzymatic Analysis 1974 Academic Press New York, NY.

9. Fiske, C. H. & Subbarow, Y. (1925) The colorimetric determination of phosphorus. J. Biol. Chem. 66:375-400.[Free Full Text]

10. Bradford, M. M. (1979) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254.

11. Williamson, D. H. & Mellanby, J. (1974) Acetoacetate. Bergmeyer, H. U. eds. Methods of enzymatic analysis 1974:1840-1843 Academic Press New York, NY. .

12. Noll, F. (1984) Lactate. Bergmeyer, H. U. eds. Methods of enzymatic analysis 1984:582-588 Academic Press New York, NY. .

13. Perret, D. (1993) Nucleotides, nucleosides and bases. Holman, R. B. Cross, A. J. Joseph, M. H. eds. High performance liquid chromatography in neuroscience research 1993:165-163 Wiley London, UK. .

14. Labarca, C. & Paigen, K. (1980) A simple, rapid, and sensitive DNA assay procedure. Anal. Biochem. 102:344-352.[Medline]

15. Fleck, A. & Munro, H. N. (1962) The precision of ultraviolet absorption measurements in the Schmidt-Thannhauser procedure for nucleic acid estimation. Biochim. Biophys. Acta 55:571-583.[Medline]

16. Chomczynski, P. & Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162:156-159.[Medline]

17. Towbin, H. & Gordon, J. (1984) Immunoblotting and dot immunobinding-current status and outlook. J. Immunol. Methods 72:313-340.[Medline]

18. Ohyanagi, H., Nishimatsu, S., Kanbara, Y., Usami, M. & Saitoh, Y. (1989) Effects of nucleosides and a nucleotide on DNA and RNA syntheses by the salvage and de novo pathway in primary monolayer cultures of hepatocytes and hepatoma cells. J. Parenter. Enter. Nutr. 13:51-58.[Abstract]

19. Ogoshi, S., Iwasa, M., Mizobuchi, S., Iwasa, Y., Martiz, A. & Tamiya, T. (1990) Effect of a nucleoside-nucleotide mixture on protein metabolism in rats given total parenteral nutrition after 70% hepatectomy. Tanaka, T. Okada, A. eds. Nutritional Support in Organ Failure 1990:309-317 Elsevier Amsterdam. .

20. Palombo, J. D., Bowers, J. L., Clouse, M. E. & McCullough, A. (1993) Hepatic utilization of exogenous nucleotide precursor for restoration of ATP after cold ischemia in rats. Am. J. Clin. Nutr. 57:420-427.[Abstract/Free Full Text]

21. Arnaud, A., Fontana, L., Angulo, A. J., Gil, A. & Lopez-Pedrosa, J. M. (2003) Exogenous nucleosides alter the intracellular nucleotide pool in hepatic cell cultures. Implications in cell proliferation and function. Clin Nutr. 22:391-399.[Medline]

22. Saez-Lara, M. J., Manzano, M., Angulo, A. J., Torres, M. I., Gómez-Llorente, C., Suárez, A., Gil, A. & Fontana, L. (2004) Exogenous nucleotides stimulate proliferation of rat fetal hepatocytes. J. Nutr. 134:1309-1313.[Abstract/Free Full Text]

23. Hernández-Muñoz, R., Díaz-Muñoz, M. & Chagoya de Sánchez, V. (1994) Possible role of cell redox state on collagen metabolism in carbon tetrachloride-induced cirrhosis as evidenced by adenosine administration to rats. Biochim. Biophys. Acta 1200:93-99.[Medline]





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