Fatty liver and fibrosis in glycine N-methyltransferase knockout mice is prevented by nicotinamide

Authors


  • Potential conflict of interest: Nothing to report.

Abstract

Deletion of glycine N-methyltransferase (GNMT), the main gene involved in liver S-adenosylmethionine (SAM) catabolism, leads to the hepatic accumulation of this molecule and the development of fatty liver and fibrosis in mice. To demonstrate that the excess of hepatic SAM is the main agent contributing to liver disease in GNMT knockout (KO) mice, we treated 1.5-month-old GNMT-KO mice for 6 weeks with nicotinamide (NAM), a substrate of the enzyme NAM N-methyltransferase. NAM administration markedly reduced hepatic SAM content, prevented DNA hypermethylation, and normalized the expression of critical genes involved in fatty acid metabolism, oxidative stress, inflammation, cell proliferation, and apoptosis. More importantly, NAM treatment prevented the development of fatty liver and fibrosis in GNMT-KO mice. Because GNMT expression is down-regulated in patients with cirrhosis, and because some subjects with GNMT mutations have spontaneous liver disease, the clinical implications of the present findings are obvious, at least with respect to these latter individuals. Because NAM has been used for many years to treat a broad spectrum of diseases (including pellagra and diabetes) without significant side effects, it should be considered in subjects with GNMT mutations. Conclusion: The findings of this study indicate that the anomalous accumulation of SAM in GNMT-KO mice can be corrected by NAM treatment leading to the normalization of the expression of many genes involved in fatty acid metabolism, oxidative stress, inflammation, cell proliferation, and apoptosis, as well as reversion of the appearance of the pathologic phenotype. (HEPATOLOGY 2010)

Expression of glycine N-methyltransferase (GNMT) is predominant in hepatocytes, where it comprises about 1% of the total soluble protein, but is also found in other tissues such as pancreas and prostate.1 GNMT catalyzes the conversion of glycine into sarcosine (methylglycine), which is then oxidized to regenerate glycine (Fig. 1). The function of this futile cycle is to catabolize excess S-adenosylmethionine (SAM) synthesized by the liver after an increase in methionine concentration (for example, after a protein-rich meal) to maintain a constant SAM/S-adenosylhomocysteine (SAH) ratio and avoid aberrant methylation reactions.1, 2 Accordingly, individuals with GNMT mutations that lead to inactive forms of the enzyme have elevated blood levels of methionine and SAM, but the concentration of total homocysteine (the product of SAH hydrolysis) is normal.3, 4GNMT knockout (KO) mice recapitulate the situation observed in individuals with mutations of the GNMT gene5, 6 and have elevated methionine and SAM both in serum and liver. These findings indicate that the hepatic reduction in total transmethylation flux caused by the absence of GNMT cannot be compensated by other methyltransferases that are abundant in the liver, such as guanidinoacetate N-methyltransferase, phosphatidylethanolamine N-methyltransferase, or nicotinamide N-methyltransferase (NNMT), and that this situation leads to the accumulation of hepatic SAM and increased transport of this molecule to the blood.

Figure 1.

Schematic representation of hepatic methionine metabolism. The first steps in hepatic methionine (Met) metabolism are conversion to SAM (SAMe) through a reaction catalyzed by MAT, and transfer of the methyl group of SAM to numerous methyl acceptors with formation of SAH. Although there are a large number of SAM-dependent methyltransferases, the reaction that quantitatively contributes most to the transmethylation flux is the methylation of glycine by GNMT (a reaction activated by SAM) to form N-methyl-glycine (Me-Gly).1, 2 Accordingly, livers of GNMT-KO mice show a marked increase in the concentration of SAM and aberrant methylation reactions of DNA and proteins.6 NAM administration may be used to reduce the elevated levels of hepatic SAM in GNMT-KO mice. NAM is methylated to form N-methylnicotinamide (Me-NAM) by the enzyme NNMT. SAH is subsequently hydrolyzed to homocysteine (Hcy) by SAH hydrolase (SAHH), which is an important metabolic hub. Homocysteine can be remethylated to regenerate Met, through either methionine synthase (MS, a pathway inhibited by SAM) and betaine homocysteine methyltransferase (BHMT), or used for the synthesis of cysteine and α-ketobutyrate as result of its transsulfuration. The transsulfuration pathway involves two enzymes: cystathionine β-synthase (which is activated by SAM) and cystathionine γ-lyase (CGL). Cysteine is then used for the synthesis of GSH, while α-ketobutyrate penetrates the mitochondria where is further metabolized. This coordinated modulation by SAM of the flux of homocysteine through the remethylation and transsulfuration pathways maximizes the production of cysteine, and consequently of GSH, after a methionine load minimizing the regeneration of this amino acid.

The marked steatosis and fibrosis observed in GNMT-KO mice6 suggests that GNMT prevents these two processes both directly, by avoiding the excessive accumulation of SAM in the hepatocytes, and indirectly, by preventing the activation of stellate cells through an unidentified mechanism. In addition, the development of hepatocellular carcinoma (HCC) in GNMT-KO mice6-8 suggests that GNMT prevents carcinogenesis directly by avoiding the hypermethylation of DNA and histones of specific and critical carcinogenesis pathways, such as the Ras and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) inhibitors suppressor of cytokine signaling-1 (SOCS1-3), cytokine-inducible SH2-containing protein, RASSF1, and RASFF4.6GNMT is silenced in human HCC and is down-regulated in the livers of patients at risk of developing HCC, such as patients with hepatitis C virus-induced and alcohol-induced cirrhosis.9, 10 The identification of several individuals with mutations of GNMT as having mild to moderate liver disease with elevated serum aminotransferases3, 4 further suggests that GNMT plays a critical role in human liver health and its silencing can lead to disease.

Using the yeast two-hybrid strategy, Rual et al.11 observed interactions between GNMT and a variety of proteins, including arrestin-3 and β-arrestin-1, two proteins involved in the regulation of G protein-coupled receptors and mitogen-activated protein kinase. This finding raises the question of whether the effect of GNMT silencing on the development of fatty liver, fibrosis, and carcinogenesis is triggered only by the increase in cellular SAM or involves the interaction of GNMT with other proteins. In the present study, we show that treatment of GNMT-KO mice with nicotinamide (NAM), a substrate of NNMT, an enzyme mainly expressed in the liver that removes NAM by converting it to N-methylnicotinamide,12 leads to the normalization of hepatic SAM content and prevention of fatty liver and fibrosis formation.

Abbreviations:

5mC, 5-methyl-cytosine; α-SMA, α-smooth muscle actin; ADFP, adipose differentiation-related protein; COL1A1, pro-α1-collagen type I; CYP2E1, cytochrome P4502E1; CYP39A1, cytochrome P45039A1; CYP4A10, cytochrome P4504A10; CYP4A14, cytochrome P4504A14; CD36, fatty acid translocase CD36; ERK, extracellular signal-regulated kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GNMT, glycine N-methyltransferase; GSH, glutathione; HCC, hepatocellular carcinoma; IL6, interleukin-6; iNOS, inducible nitric oxide synthase; JAK, Janus kinase; KO, knockout; MAT, methionine adenosyltransferase; NAM, nicotinamide; NNMT, nicotinamide N-methyltransferase; PARP, poly(ADP-ribose) polymerase; PCR, polymerase chain reaction; PPAR, peroxisome proliferator-activated receptor; RASSF1A, Ras-association domain family/tumor suppressor-1A; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; SEM, standard error of the mean; SIRT1, sirtuin-1; SOCS1, suppressor of cytokine signaling-1; STAT, signal transducer and activator of transcription; TIMP-1, TIMP tissue inhibitor of metalloproteinase-1; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling; UCP2, uncoupling protein-2; WT, wild-type.

Materials and Methods

Study Design.

Wild-type (WT) and GNMT-KO mice were fed a standard diet (Teklad irradiated mouse diet 2014; Harlan, Madison, WI) and housed in a temperature-controlled animal facility with 12-hour light/dark cycles. At 1.5 months of age, GNMT-KO (n = 20) and WT (n = 5) mice were treated for 6 weeks with NAM (50 μM dissolved in drinking water, which was replaced weekly) (Sigma-Aldrich) before sacrifice. For control groups, we used WT (n = 15) and GNMT-KO mice (n = 10) of the same age. At the time of sacrifice, livers were rapidly split into several pieces; some were snap-frozen for subsequent RNA or protein extraction, and others were formalin-fixed for histological and immunohistochemical analysis. Serum samples were also collected for determination of alanine aminotransferase and aspartate aminotransferase activity. Animals were treated humanely, and all procedures were in compliance with our institutions' guidelines for the use of laboratory animals.

Histological and Immunohistochemical Analysis.

Sections from formalin-fixed liver tissue were stained with hematoxylin-eosin or with Sirius Red for collagen visualization. For α-smooth muscle actin (α-SMA) immunostaining and apoptosis detection, frozen liver tissue sections were fixed with 4% paraformaldehyde for 15 minutes at room temperature, followed by treatment with 3% hydrogen peroxide in methanol for 10 minutes. The sections were then incubated with 150 mM sodium citrate for 2 minutes followed by washes in phosphate-buffered saline. For α-SMA immunolabeling, anti-α-SMA Cy3-conjugated antibody (Sigma) was applied overnight at 4°C. For apoptosis detection, fluorescein isothiocyanate-conjugated terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) enzyme was applied overnight at 4°C (in situ cell death detection kit, Roche). Washing in ultrapure H2O and then in phosphate-buffered saline terminated the reaction. Nuclei were then labeled with Hoechst, and the cover slips were mounted in Citifluor mounting medium.

Quantitative Real-Time Polymerase Chain Reaction.

Total RNA was isolated using the RNeasy Mini Kit (Qiagen) including DNase treatment on column. Total RNA (1.5 μg) was retrotranscribed with Super Script III (Invitrogen) in the presence of random primers and oligodeoxythymidylic acid following the manufacturer's instructions. Real-time polymerase chain reaction (PCR) was performed using the BioRad iCycler thermalcycler. Five microliters of a 1/20 dilution were used in each PCR reaction in a total reaction volume of 30 μL using iQ SYBR Green Super Mix (BioRad), and all reactions were performed in duplicate. PCR was performed with the primers described in Supporting Table 1. After checking specificity of the PCR products with the melting curve, cycle threshold values were extrapolated to a standard curve performed simultaneously with the samples and data were then normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. Validation of GAPDH as a housekeeping gene for normalizing RNA expression was performed after measuring the expression of messenger RNA encoding GAPDH, actin, and actin related protein (ARP) using quantitative real-time PCR in matched NAM-treated and untreated tissues obtained from 16 liver samples. GAPDH expression was not significantly different between these two groups.

Protein Isolation and Western Blot Analysis.

Tissue was homogenized in lysis buffer (10 mM Tris/HCl [pH 7.6], 5 mM ethylene diamine tetraacetic acid, 50 mM NaCl, 1% Triton X-100, complete protease inhibitor cocktail, and 50 mM NaF). Samples were centrifuged (15,000g, 1 hour, 4°C) and supernatants were collected. Protein lysates (30 μg, determined by BCA [Rockford, IL]) were electrophoresed on sodium dodecyl sulfate-polyacrylamide gels and transferred onto nitrocellulose membranes. After blocking, membranes were incubated overnight at 4°C with specific antibodies against Ras-association domain family/tumor suppressor-1A (RASSF1A) from Biosciences (eBiosciences, Inc., San Diego, CA); pSTAT3 (Tyr705), pJAK2 (Tyr1007/1008), extracellular signal-regulated kinase (ERK) 1/2, pERK1/2 (Thr202/Tyr204), and pRAF1 (Ser338) from Cell Signaling Technology (Danvers, MA); STAT3, Ki-67, and cyclin D1 from Santa Cruz Biotechnology (Santa Cruz, CA); phospho-histone 3 (Ser10) from Millipore (Billerica, MA); and SOCS1 from Novus Biologicals (Littleton, CO). This was followed by 1-hour incubation with goat anti-mouse (Santa Cruz Biotechnology) or goat anti-rabbit (Bio-Rad, Hertfordshire, United Kingdom) secondary antibodies conjugated to horseradish peroxidase. Immunoreactive proteins were detected using Western Lightning chemiluminescence reagent (PerkinElmer, Boston, MA).

Global DNA Methylation.

Global DNA methylation was assessed using two different methods. In the first method, 5-methyl-cytosine (5mC) genomic content was determined by way of high-performance capillary electrophoresis.13 In brief, genomic DNA samples were boiled and were then treated with nuclease P1 (Sigma) for 16 hours at 37°C and with alkaline phosphatase (Sigma) for an additional 2 hours at 37°C. After hydrolysis, total cytosine and 5mC content were measured by way of capillary electrophoresis using a P/ACE MDQ system (Beckman Coulter). Relative 5mC content was expressed as the percentage of total cytosine content (methylated and nonmethylated). Each sample was assayed in triplicate. The second method, which was used to assess global DNA methylation, was based on the use of HpaII methylation-sensitive restriction endonucleases that leave a 5′-guanine overhang after DNA cleavage, with subsequent single-nucleotide extension with radiolabeled [3H]dCTP.14 The extent of [3H]dCTP incorporation after restriction enzyme treatment is directly proportional to the number of unmethylated (cleavage) CpG sites.

Hepatic SAM, SAH, and Glutathione Levels.

Liver specimens were homogenized in 0.4 M perchloric acid on ice for 5 minutes and centrifuged at 1,000g for 15 minutes at 4°C. The aqueous layer was quantitatively removed, neutralized with 3 M KOH, and centrifuged at 3,000g for 10 minutes at 4°C. SAM and SAH concentrations were determined by way of liquid chromatography/mass spectrometry using a Waters ACQUITY-UPLC system coupled to a Waters Micromass LCT Premier mass spectrometer equipped with a Lockspray ionization source as described.15 The amount of glutathione (GSH) was determined using the Sigma GSH kit.

Statistical Analysis.

The Student t test was used to evaluate statistical significance. Values of P < 0.05 were considered statistically significant.

Results

Nicotinamide Treatment Reduces Hepatic SAM Content in GNMT-KO Mice.

Our previous studies showed that SAM levels of both liver5 and serum6 of GNMT-KO mice are much higher than in WT animals. This is accompanied by development of liver injury and eventually by development of HCC.9 In order to prove that the pathological phenotype is a result of the elevated levels of SAM in the liver, we sought to reduce the elevated levels by administration of NAM and evaluate whether this would reverse the appearance of the pathologic phenotype. The enzyme NNMT uses SAM to form N-methylnicotinamide, which is excreted in the urine (Fig. 1). In order to verify this hypothesis, NAM was added to the drinking water of 1.5-month-old GNMT-KO and WT mice for 6 weeks, and at the end of this period the hepatic SAM content was determined. As demonstrated,5 SAM content in the livers of 3-month-old GNMT-KO animals was about 40-fold higher than in WT animals (Table 1). As hypothesized, the livers of NAM-treated GNMT-KO animals exhibited markedly lower SAM levels than untreated GNMT-KO mice. The administration of NAM to WT animals had no significant effect on hepatic SAM content. This result is consistent with GNMT's role as a SAM buffer. SAM is an allosteric regulator of GNMT.1 Accordingly, when the hepatic content of SAM increases, as a result of its augmented synthesis or reduced catabolism, GNMT activity is stimulated; when the content of SAM diminishes, as a result of a decrease in its synthesis or increased consumption, GNMT activity is reduced. The amount of hepatic SAH in GNMT-KO mice was similar to that of WT animals (Table 1). However, in the livers of NAM-treated GNMT-KO mice, SAH content was about 1.7-fold higher than that of untreated animals. The administration of NAM to WT animals had no significant effect on hepatic SAH content. It is remarkable that the levels of hepatic GSH are similar in the WT and GNMT-KO animals in spite of the significant reduction in transmethylation reactions. This is probably due to the activation by SAM of cystathionine β-synthase (the first enzyme linking homocysteine with GSH synthesis) as well as the inhibition by SAM of homocysteine remethylation2 (Fig. 1). In WT and GNMT-KO mice, NAM administration had no significant effect on hepatic GSH content (Table 1).

Table 1. Effect of NAM Administration on Hepatic SAM, SAH, and GSH Content in WT and GNMT-KO Mice
 WTGNMT-KOWT+NAMGNMT-KO +NAM
  • Data are presented as the mean ± SEM of at least five independent experiments.

  • *

    P < 0.05 (GNMT-KO versus WT mice).

  • P < 0.05 (GNMT-KO versus GNMT-KO +NAM mice).

SAM, nmol/g tissue87.8 ± 8.93,245 ± 134*63.8 ± 14.494.4 ± 9.7
SAH, nmol/g tissue41.9 ± 6.536.3 ± 8.638.3 ± 6.962.2 ± 13.4
GSH, μmol/g tissue13.8 ± 0.814.4 ± 0.312.4 ± 0.214.5 ± 0.2

Nicotinamide Treatment Prevents Liver Injury, Steatosis, and Fibrosis in GNMT-KO Mice.

Next, we determined the levels of serum aminotransferases in NAM-treated GNMT-KO mice. We have previously demonstrated that both serum alanine aminotransferase and aspartate aminotransferase are increased in GNMT-KO mice compared with WT animals.6 We observed that in NAM-treated GNMT-KO mice, serum aminotransferase levels were significantly reduced when compared with untreated GNMT-KO animals (aspartate aminotransferase, 55.3 ± 4.1 U/L [WT], 266.3 ± 27 U/L [GNMT-KO], 61.8 ± 9.5 U/L [NAM-treated GNMT-KO]; alanine aminotransferase, 24.5 ± 3 U/L [WT], 177.8 ± 10.4 U/L [GNMT-KO], 35.6 ± 1.4 U/L [NAM-treated GNMT-KO]; NAM-treated GNMT-KO versus untreated GNMT-KO for both aminotransferases [n = 5; P < 0.05]).

Furthermore, histological examination revealed that the livers of 3-month-old GNMT-KO mice treated with NAM lacked signs of steatosis or fibrosis. As reported,6 3-month-old GNMT-KO mice exhibited extensive accumulation of liver fat (hematoxylin-eosin staining) and fibrosis (Sirius Red staining and α-SMA immunohistochemical analysis) (Fig. 2). In contrast, NAM-treated GNMT-KO mice showed no signs of steatosis or fibrosis (Fig. 2). Liver histology was normal in NAM-treated WT animals.

Figure 2.

Hepatic steatosis and fibrosis in GNMT-KO mice after NAM administration. (A) Livers of 3-month-old GNMT-KO mice treated with NAM during the last 6 weeks lacked signs of hepatic steatosis (white droplets) compared with GNMT-KO livers. NAM administration in GNMT-KO mice also prevented (B) collagen deposition (Sirius red staining) and (C) α-SMA expression, both of which are indicative of liver fibrosis. At least 10 animals per group were examined Original magnification ×40 (Zeiss AX10 microscope). H&E, hematoxylin-eosin stain; SR, Sirius Red stain.

Nicotinamide Treatment Normalizes Hepatic Gene and Protein Expression in GNMT-KO Mice and Prevents Apoptosis.

Consistent with the high SAM levels, the liver expression of methionine adenosyltransferase 2A (MAT2A), a gene whose expression is inhibited by SAM,16 was markedly reduced in GNMT-KO mice but was normal in NAM-treated KO animals (Fig. 3E). Similarly, the livers of 3-month-old GNMT-KO mice showed marked alterations in the expression of critical genes involved in lipid metabolism (fatty acid translocase CD36 [CD36], adipose differentiation-related protein [ADFP], peroxisome proliferator-activated receptor-α [PPARα], and PPARγ), oxidative stress and inflammation (cytochrome P4502E1 [CYP2E1], cytochrome P45039A1 [CYP39A1], cytochrome P4504A10 [CYP4A10], cytochrome P4504A14 [CYP4A14], uncoupling protein-2 [UCP2], PPARγ, interleukin-6 [IL6], and inducible nitric oxide synthase [iNOS]), and extracellular matrix regulation (pro-α1-collagen type I [COL1A1], TIMP tissue inhibitor of metalloproteinase-1 [TIMP-1], α-SMA). Furthermore, the treatment of GNMT-KO mice with NAM prevented completely (CD36, ADFP, CYP4A10, CYP4A14, CYP39A1, UCP2, IL6, iNOS, COL1A1, α-SMA) or largely (PPARα, PPARγ, CYP2E1, TIMP-1) the abnormal expression of these genes in the liver (Fig. 3A-E). NAM administration had no significant effect on the expression of these genes in WT mice (Fig. 3A-E), indicating that the effect of NAM on gene expression in GNMT-KO mice is mediated by its capacity to reduce hepatic SAM content. The hepatic expression of sirtuin-1 (SIRT1), a NAD+-dependent protein deacetylase that is an important regulator of energy metabolism modulating many aspects of glucose and lipid homeostasis,17 was similar in WT and GNMT-KO mice and was not modified by NAM administration (Fig. 3A). Finally, the livers of 3-month-old GNMT-KO mice showed marked apoptosis as demonstrated by poly(ADP-ribose) polymerase (PARP) cleavage and TUNEL immunostaining, which was also prevented in NAM-treated GNMT-KO animals (Fig. 4A,B).

Figure 3.

Gene expression in GNMT-KO mice after NAM administration. NAM administration in GNMT-KO prevented alterations in the expression of critical genes involved in (A) lipid metabolism (CD36, ADFP, PPARα, and SIRT1), (B) oxidative stress (CYP4A10, CYP4A14, CYP39A1, and CYP2E1), (C) inflammation (PPARγ, UCP2, iNOS, and IL6), (D) extracellular matrix regulation (COL1A1, TIMP-1 and α-SMA), and (E) MAT2A. Gene expression was assessed with real-time PCR using specific primers, and values were normalized with GAPDH messenger RNA expression. Each bar represents the mean ± standard error of the mean (SEM) of at least five independent experiments. Results are expressed in arbitrary units (a.u). *P < 0.05 (GNMT-KO versus WT mice). **P < 0.05 (GNMT-KO versus GNMT-KO + NAM mice).

Figure 4.

Hepatic apoptosis in GNMT-KO mice after NAM administration. (A) Upper: Western blot of liver extracts from WT, GNMT-KO, and GNMT-KO mice after NAM administration, incubated with the indicated antibodies. Data are representative of an experiment performed three times. Bottom: Densitometric changes (mean ± SEM) of PARP cleavage expressed in arbitrary units (a.u). *P < 0.05, (GNMT-KO versus WT mice). **P < 0.05 (GNMT-KO versus GNMT-KO+NAM mice). (B) Livers of 3-month-old GNMT-KO mice treated with NAM showed a decrease in TUNEL-positive cells compared with GNMT-KO livers. The number of TUNEL-positive cells was counted and expressed as the percentage of Hoechst nuclei per field in liver specimens from GNMT-KO mice and GNMT-KO mice treated with NAM. Data (mean ± SEM) are the average of five experiments performed independently. *P <0.05, GNMT-KO versus GNMT-KO+NAM mice.

Nicotinamide Treatment Prevents DNA Hypermethylation and the Activation of Ras and JAK/STAT Pathways in GNMT-KO Mice.

We have reported the existence of global DNA hypermethylation in the livers of GNMT-KO mice.6 Global DNA methylation, assayed both by the quantification of 5mC groups (Fig. 5A) and by the measurement of the number of unmethylated CpG sites (Fig. 5B), was significantly reduced in the livers of GNMT-KO animals treated with NAM. This is consistent with the observation that NAM administration induces a marked reduction of hepatic SAM content as well as an increase in SAH content.

Figure 5.

Global DNA methylation and activation of Ras and JAK/STAT pathways in GNMT-KO mouse liver after NAM administration. (A,B) Quantification of global DNA methylation in genomic DNA samples isolated from livers of WT, GNMT-KO mice, and GNMT-KO mice treated with NAM. (A) Total cytosine and 5mC content was determined in genomic DNA, and relative 5mC content, expressed as the percentage of total cytosine content (methylated and nonmethylated), was determined. Data represent the mean ± SEM from five animals per subgroup. (B) The DNA methylation status was determined by measuring the number of DNA unmethylated CpG sites using the cytosine extension assay. Two micrograms of DNA per sample were subjected to restriction digestion with the HpaII endonuclease. Lower incorporation of [3H]dCTP means a higher DNA methylation status. Data represent the mean ± SEM from five animals per subgroup. *P < 0.05 (GNMT-KO versus WT mice). **P < 0.05 (GNMT-KO versus GNMT-KO+NAM mice). (C,D) Western blot analysis of activated Ras (Ras-GTP), the RAS inhibitor RASSF1A, the Ras downstream effectors, pRAF and pERK1/2, activated JAK2 (pJAK2), the JAK inhibitor SOCS1, and the JAK downstream effectors pSTAT3, cyclin D1, pHistone 3, and Ki67. The down-regulation of RASSF1A and SOCS1 expression in GNMT-KO mice compared with WT mice was prevented by NAM administration. The induction of Ras-GTP, pRAF1, pERK1/2, pJAK2, pSTAT3, and cyclin D1 and of the mitotic markers pHistone 3 and Ki67 in GNMT-KO livers was prevented or markedly reduced by NAM administration in GNMT-KO mice as compared with untreated knockout animals. Bottom: Densitometric changes (mean ± SEM) of the mentioned proteins expressed in arbitrary units (a.u). *P < 0.05 (GNMT-KO versus WT mice). **P < 0.05 (GNMT-KO versus GNMT-KO+NAM mice).

Next, we examined the Ras and JAK/STAT signaling pathways. We have shown the persistent activation of the Ras and JAK/STAT signaling pathways through suppression of Ras and JAK/STAT inhibitors such as RASSF1A and SOCS1 in GNMT-KO mouse liver.6 In the present study, we observed that NAM administration to GNMT-KO mice prevented the hepatic suppression of RASSF1A and SOCS1 protein expression (Fig. 5C,D). Concomitant with this normalization of RASSF1A protein expression, we observed that the livers of NAM-treated GNMT-KO mice exhibited markedly lower expression of Ras and downstream effectors of Ras involved in cell proliferation and survival (including pRAF1 and pERK1/2) than untreated knockout animals (Fig. 5C). Ras activity, assessed by immunoprecipitation with anti-pan Ras antibody and probed with anti-RAF1 antibody, was markedly increased in GNMT-KO mice liver but much less elevated in NAM-treated GNMT-KO mice (Fig. 5C). Similarly, pERK1/2 content increased more than 15-fold in GNMT-KO livers and only seven-fold in NAM-treated KO livers (Fig. 5C). The levels of pRAF1 were elevated in GNMT-KO mice compared with WT animals but were similar in WT and NAM-treated GNMT-KO mice (Fig. 5C). Similarly, concurrent with the normalization of SOCS1 protein expression, we observed that whereas the liver protein levels of pSTAT3 and of the downstream mitotic markers effectors pHistone 3 and Ki-67 were significantly elevated in both GNMT-KO mice groups compared with WT animals, induction in the NAM-treated group was significantly lower than in the untreated group (Fig. 5D). The protein levels of activated JAK2 tyrosine kinase (pJAK2) and cyclin D1 were elevated in GNMT-KO mice compared with WT animals but were similar in WT and NAM-treated GNMT-KO mice (Fig. 5D).

Discussion

SAM is synthesized by methionine adenosyltransferase (MAT). In mammals, there are three isoforms of MAT (MATI, MATII, and MATIII) that are encoded by two genes (MAT1A and MAT2A). MATI and MATIII are tetrameric and dimeric forms, respectively, of the same subunit (α1) encoded by MAT1A, whereas the MATII isoform is a tetramer of a different subunit (α2) encoded by MAT2A. Adult differentiated liver expresses predominantly MAT1A, whereas extrahepatic tissues and fetal liver express MAT2A.2, 18 The prevalent liver form, MATIII, has lower affinity for its substrates, is activated by methionine, and has higher Vmax, contrasting with the other two enzymes.2, 18 Based on the differential properties of hepatic MAT isoforms, it has been postulated that MATIII is the truly liver-specific isoform.2 Under normal conditions, MATI synthesizes most SAM required by the hepatic cells (as MATII does outside the liver). However, after an increase in methionine concentration (for example, after a protein-rich meal), conversion to the high-activity MATIII occurs, and methionine excess is eliminated.2 This leads to an accumulation of SAM and to the activation of GNMT, the main enzyme involved in hepatic SAM catabolism (Fig. 1).1 Consequently, the excess of SAM is eliminated and converted to homocysteine through SAH. Once formed, the excess of homocysteine is used for methionine regeneration or the synthesis of cysteine and α-ketobutyrate as result of its transsulfuration.2, 18 Cysteine is then used for the synthesis of GSH as well as other sulfur-containing molecules such as taurine, while α-ketobutyrate penetrates the mitochondria, where it is further metabolized.

Consistent with this model, MAT1A-KO animals, despite overexpressing MAT2A in the liver,19 have high blood methionine and reduced hepatic SAM, whereas GNMT-KO mice show increased liver SAM.6GNMT-KO mice, which are neither obese nor diabetic, spontaneously develop fatty liver and fibrosis 3 months after birth, and about 5 months later develop HCC.6 In GNMT-KO mice, the expression of hepatic MAT2A was down-regulated, but its levels were normal in NAM-treated animals. This finding is consistent with the changes observed in hepatic SAM content, because MAT2A expression is inhibited when the concentration of SAM increases.16 Three-month-old GNMT-KO mice showed induction of CD36, a fatty acid translocase whose elevated expression is sufficient to increase hepatic fatty acid uptake,20 and of ADFP, a lipid droplet protein whose deficiency confers resistance to fatty liver development,21 as well as an increase in the expression of PPARγ, a transcription factor whose overexpression in the liver induces steatosis,22 and a reduction in the expression of hepatic PPARα, a lipid-activated transcription factor primarily expressed in the liver, which has been shown to activate β-oxidation of fatty acids.23 On a related note, we have observed that the activation of adenosine monophosphate-activated protein kinase, a main regulator of cellular energy stores that activates fatty acid oxidation, is blocked in GNMT-KO mice due to the inhibitory effect of SAM accumulation on this pathway.24, 25

SIRT1 is an important modulator of lipid homeostasis. It has been shown that SIRT1 transgenic mice are more resistant to develop fatty liver than WT animals in response to a high-fat diet,26 and that mice with a hepatocyte-specific deletion of SIRT1 are more prone to develop steatosis than WT animals when administered a high-fat diet,27 indicating that this protein deacetylase is relevant in preventing fatty liver. From this perspective, and because the expression of SIRT1 is not altered in GNMT-KO mice, it seems that this protein deacetylase is not playing a significant role in the generation of steatosis in this animal model. Moreover, the finding that the administration to GNMT-KO mice of NAM, an inhibitor of SIRT1 activity,28 prevented rather than aggravated the abnormal expression of CD36, ADFP, PPARα, and PPARγ further supports the conclusion that the development of steatosis in GNMT-KO mice is independent of SIRT1. This may be due to the low dose of NAM used in the present experiments (50 μM) compared with the high concentration used to inhibit SIRT1 activity in culture cells (5 mM).29

One of the factors associated with the development and progression of steatosis is the oxidative stress originated by toxic lipid peroxidation catalyzed by CYP2E1, the main enzyme involved in the NAM adenine dinucleotide phosphate (reduced form)-dependent reduction of oxygen leading to lipid peroxidation.30 The CYPs constitute a super-family of heme-containing microsomal mono-oxygenases that play a central role in the detoxification of xenobiotics, as well as in the metabolism of endogenous compounds, including fatty acids. CYP2E1 expression and activity is up-regulated in SAM-deficient, MAT1A-KO mouse liver.31 In contrast, CYP2E1, as well as expression of CYP39A1, an oxygenase catalyzing the rate-limiting step of bile acid synthesis,32 are reduced in GNMT-KO mouse liver, but the expression of two alternative fatty acid hydroxylases (CYP4A10 and CYP4A14, the two major CYP4A genes) is markedly induced. It has been demonstrated that CYP4A enzymes are key intermediates of an adaptive response to perturbation of hepatic lipid metabolism. Thus, in CYP2E1-KO mice, lipid peroxidation induced by the accumulation of hepatic fatty acids in response to a methyl-deficient diet is mediated by the up-regulation of CYP4A10 and CYP4A14 expression.33 SAM is known to be an inhibitor of CYP2E1 activity,34 and, although the Ki is relatively high, it is likely that at the elevated concentration of SAM present in GNMT-KO mouse liver, a direct effect of this molecule on CYP2E1 activity may be also responsible for the induction of CYP4A genes. Again, normalization of SAM content in GNMT-KO mice through NAM treatment prevented the abnormal expression of CYP2E1, CYP39A1, CYP4A10 and CYP4A14.

Additionally, NAM treatment prevented the abnormal expression of critical genes involved in the generation of oxidative stress (UCP2, PPARγ, IL6, and iNOS) and liver fibrosis (COL1A1, TIMP-1, and α-SMA) and prevented apoptosis (determined both by PARP cleavage and TUNEL immunostaining). These findings agree with the observation that in whole blood stimulated with endotoxin, NAM is an anti-inflammatory agent inhibiting PARP activation, iNOS expression, and the stimulation of proinflammatory cytokines such as IL6 and iNOS.35 Whether NAM also reduced cellular SAM content in this experimental setting is not known. Finally, NAM administration prevented global DNA hypermethylation, normalized the expression of RASSF1A and SOCS1 tumor suppressors, which are frequently inactivated by promoter methylation in human HCC,36 and inhibited or markedly reduced the activation of the Ras and JAK/STAT proliferative pathways in GNMT-KO mice, agreeing with the hypothesis that NAM exerts its therapeutic activity primarily through reduction of hepatic SAM content.

These results indicate that deletion of GNMT is associated with an increased expression of genes inducing steatosis (CD36, ADFP, PPARγ, CYP4A10, CYP4A14, UCP2) and also with a reduction in the expression of PPARα, a major activator of fatty acid oxidation, and that these changes are prevented by NAM administration. Moreover, these findings indicate that the hepatic reduction in total transmethylation flux caused by deletion of GNMT and the concomitant accumulation of SAM can be compensated by NNMT if exogenous NAM is provided. Additionally, our results indicate that NAM administration to GNMT-KO mice prevents global DNA hypermethylation as well as the abnormal expression of numerous genes involved in fatty acid metabolism, oxidative stress, fibrosis, apoptosis, and proliferation observed in untreated animals. More significantly, NAM treatment not only normalized the expression of all these genes and proteins in GNMT-KO mice, it also prevented the development of fatty liver and fibrosis. The mechanism by which GNMT deletion leads to fibrosis is not known; it is possible that increased lipid accumulation and apoptosis in GNMT-KO hepatocytes activate hepatic stellate cells,37, 38 the central mediators of liver fibrogenesis. Accordingly, NAM may attenuate fibrogenesis by preventing hepatic fat accumulation and apoptosis by lowering SAM content. At present, however, alternative direct effects of NAM on stellate cell activation cannot be excluded.

In conclusion, these results rule out the possibility that the interaction of GNMT with other target proteins, such as arrestin-3 and β-arrestin-111 may play a critical role in the initiation of liver disease in GNMT-KO mice. Because GNMT expression is down-regulated in patients with cirrhosis and HCC, and because some individuals with GNMT mutations have spontaneous liver disease,3, 4, 9, 10 the clinical implications of the present findings are obvious, at least with respect to the latter group. NAM has been used for many years to treat a broad spectrum of diseases without significant side effects.39, 40 Our findings suggest that individuals with GNMT mutations are likely to benefit from NAM treatment.

Acknowledgements

We thank Begoña Rodríguez for technical assistance.

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