Strain-dependent dysregulation of one-carbon metabolism in male mice is associated with choline- and folate-deficient diet-induced liver injury

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1 The FASEB Journal Research Communication Strain-dependent dysregulation of one-carbon metabolism in male mice is associated with choline- and folate-deficient diet-induced liver injury Igor P. Pogribny,*,1 Kristy Kutanzi,* Stepan Melnyk, Aline de Conti,* Volodymyr Tryndyak,* Beverly Montgomery,* Marta Pogribna,* Levan Muskhelishvili, John R. Latendresse, S. Jill James, Frederick A. Beland,* and Ivan Rusyn *Division of Biochemical Toxicology and Toxicologic Pathology Associates, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, Arkansas, USA; Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA; and Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, North Carolina, USA ABSTRACT Dysregulation of one-carbon metabolism-related metabolic processes is a major contributor to the pathogenesis of nonalcoholic fatty liver disease (NAFLD). It is well established that genetic and genderspecific variations in one-carbon metabolism contribute to the vulnerability to NAFLD in humans. To examine the role of one-carbon metabolism dysregulation in the pathogenesis and individual susceptibility to NAFLD, we used a population-based mouse model where male mice from 7 inbred were fed a choline- and folate-deficient (CFD) diet for 12 wk. Strain-dependent down-regulation of several key one-carbon metabolism genes, including methionine adenosyltransferase 1 (Mat1a), cystathionine- -synthase (Cbs), methylenetetrahydrofolate reductase (Mthfr), adenosyl-homocysteinase (Ahcy), and methylenetetrahydrofolate dehydrogenase 1 (Mthfd1), was observed. These changes were strongly associated with interstrain variability in liver injury (steatosis, necrosis, inflammation, and activation of fibrogenesis) and hyperhomocysteinemia. Mechanistically, the decreased expression of Mat1a, Ahcy, and Mthfd1 was linked to a reduced level and promoter binding of transcription factor CCAAT/enhancer binding protein (CEBP ), which directly regulates their transcription. The strain specificity of diet-induced dysregulation of one-carbon metabolism suggests that Abbreviations: -SMA, -smooth muscle actin; Ahcy, adenosylhomocysteinase; Cbs, cystathionine- -synthase; CEBP, CCAAT/ enhancer binding protein ; CFD, choline- and folate-deficient; ChIP, chromatin immunoprecipitation; cis-eqtl, cis-expression quantitative trait locus; Ddit3, DNA-damage-inducible transcript 3; ER, endoplasmic reticulum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GRP78, glucose-regulated protein 78; Mat1a, methionine adenosyltransferase 1 ; Mthfr, methylenetetrahydrofolate reductase; Mthfr, methylenetetrahydrofolate reductase; NAFLD, nonalcoholic fatty liver disease; qpcr, quantitative polymerase chain reaction; qrt-pcr, quantitative reverse transcription-polymerase chain reaction; SAH, S-adenosyl-l-homocysteine; SAM, S-adenosyl-l-methionine; SOD1/2, superoxide dismutase 1/2 interstrain variation in the regulation of one-carbon metabolism may contribute to the differential vulnerability to NFLD and that correcting the imbalance may be considered as preventive and treatment strategies for NAFLD. Pogribny, I. P., Kutanzi, K., Melnyk, S., de Conti, A., Tryndyak, V., Montgomery, B., Pogribna, M., Muskhelishvili, L., Latendresse, J. R., James, S. J., Beland, F. A., Rusyn, I. Strain-dependent dysregulation of one-carbon metabolism in male mice is associated with choline- and folate-deficient diet-induced liver injury. FASEB J. 27, (2013). Key Words: strain differences methyl donor deficiency gene expression homocysteinemia NAFLD One-carbon metabolism comprises cellular folate and methionine cycles and is dependent on a number of pathways and biochemical reactions that facilitate transfer of one-carbon units in a wide range of biological molecules (1, 2). There are 2 major factors that may cause disease by compromising the proper functioning of one-carbon metabolism. First, dietary deficiency in methyl donors and cofactors (methionine, choline, folate, B vitamins, etc.) leads to fatty liver and liver damage in humans (3 5) and animals (6 9). The key role of one-carbon metabolism in nonalcoholic fatty liver disease (NAFLD) has been further demonstrated in mouse knockout studies where deletion of genes responsible for the methyl donor supply and cycling resulted in fatty liver injury and, in some instances, hepatocarcinogenesis (10 13). Second are heritable genetic risk factors, such as genetic alterations in genes encoding enzymes involved 1 Correspondence: Division of Biochemical Toxicology, NCTR, 3900 NCTR Rd., Jefferson, AR 72079, USA. igor.pogribny@fda.hhs.gov doi: /fj This article includes supplemental data. Please visit to obtain this information /13/ FASEB 2233

2 in the cellular one-carbon metabolic network. In humans, polymorphisms in phosphatidylethanolamine N- methyltransferase (PEMT; ref. 14) and folate metabolism genes (15) have been shown to be associated with NAFLD and other diseases. Furthermore, a recent report by Corbin et al. (16) demonstrated that genetic polymorphisms in choline and one-carbon metabolism genes were significantly correlated with the severity of hepatic steatosis in humans. Our previous studies using a choline- and folate-deficient model in mice, found that major interstrain differences in the severity of liver injury are observed and were associated with strainspecific effects on hepatic lipid metabolism (17) and epigenetic phenotype (18). Several molecular mechanisms are recognized as associated with the development of liver disease (6, 19) induced by dietary deficiency of methyl-group donors, including altered one-carbon and lipid metabolism, aberrant cell signaling, the induction of endoplasmic reticulum stress and apoptosis, and epigenetic changes. Notable differences exist in the severity of liver injury induced by dietary methionine, choline, and folate deficiency between genders (20), among species (9), and even within the same species (17, 18, 21); however, the determinants of this differential variability have not been fully investigated. On the basis of our previous observation that interstrain differences in susceptibility to and severity of NAFLD in mice are associated with dysregulation of hepatic lipid metabolism (17) and because of a well-established linkage between lipid and one-carbon metabolism (20, 22), we hypothesized that differential vulnerability to liver injury induced by methyl-group donor deficiency in mice may also involve strain-specific effects on one-carbon metabolism. To test this hypothesis, we fed a choline- and folatedeficient (CFD) diet to panel of 7 inbred mouse strains that are parental lines in the Collaborative Cross (23). This diet consistently induces fat-related liver injury resembling histopathological features of human NAFLD (24). We found that interstrain variability in liver injury (steatohepatitis and activation of a profibrogenic response) was associated with hyperhomocysteinemia and changes in the expression of one-carbon metabolism genes, particularly with distinct strain-dependent down-regulation of methionine adenosyltransferase 1 (Mat1a), cystathionine- -synthase (Cbs), methylenetetrahydrofolate reductase (Mthfr), adenosylhomocysteinase (Ahcy), and methylenetetrahydrofolate dehydrogenase 1 (Mthfd1). The underlying mechanism associated with a reduced expression of these genes was attributed to a down-regulation of transcription factor CCAAT/enhancer binding protein (CEBP ), which directly regulates transcription of Mat1a, Ahcy, and Mthfd1. MATERIALS AND METHODS Animals and experimental design Male A/J, C57BL/6J, C3H/HeJ, CAST/EiJ, 129S1/SvImJ, PWK/PhJ, and WSB/EiJ mice (6 wk of age) were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). These strains were selected because they provide an excellent representation of genetic diversity (25). Specifically, genetic analyses have demonstrated that this panel of strains captured almost 90% of the randomly distributed known genetic variation existing in laboratory mice originating from M. musculus (26). The in-life portion of this study, tissue collection protocols, and results of histopathological (Supplemental Fig. S1) and clinical chemistry analyses are detailed in Tryndyak et al. (17). Methyl donor-deficient experimental groups were fed a diet low in methionine (0.17% w/w) and lacking choline and folic acid (diet ; CFD, iron supplemented, and l-amino acid defined diet; Dyets, Bethlehem, PA, USA) for 12 wk. Mice in the methyl donor-adequate control groups received the same diet supplemented with 0.4% methionine, 0.3% choline bitartrate, and 2 mg/kg folic acid for 12 wk. All experimental procedures were reviewed and approved by the National Center for Toxicological Research Animal Care and Use Committee. Determination of methionine, S-adenosyl-L-methionine (SAM), S-adenosyl-L-homocysteine (SAH), and homocysteine content Levels of methionine, SAM, SAH, and homocysteine in the livers and homocysteine in plasma of experimental animals were measured by high-performance liquid chromatography (HPLC) coupled with coulometric electrochemical detection, as described previously (27, 28). RNA extraction and quantitative reverse transcriptionpolymerase chain reaction (qrt-pcr) Total RNA was extracted from liver tissue using RNeasy Mini kits (Qiagen, Valencia, CA, USA), according to the manufacturer s instructions. Total RNA (2 g) was reverse transcribed using random primers and high-capacity cdna reverse transcription kits (Applied Biosystems, Foster City, CA, USA), according to the manufacturer s protocol. cdna was analyzed in a 96-well plate assay format using the 7900HT Fast Real- Time PCR System (Applied Biosystems). Each plate contained 1 experimental gene and a housekeeping gene. All primers for the gene expression analysis were obtained from Applied Biosystems. The cycle threshold (C t ) for each sample was determined from the linear region of the amplification plot. The C t values for all genes were determined relative to the endogenous control glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The C t values were calculated using treated group means relative to strain-matched control group means. The fold-change data were calculated from the C t values. All qrt-pcr reactions were conducted in triplicate, and experiments were repeated twice. Western blot analysis of protein levels Liver tissue lysates were prepared by homogenization of 50 mg of tissue in 500 l of lysis buffer (50 mm Tris-HCl, ph 7.4; 1% Nonidet P-40; 0.25% sodium deoxycholate; 150 mm NaCl; 1 mm EDTA; 1 mm PMSF; 1 g/ml each of aprotinin, leupeptin, and pepstatin; 1 mm Na 3 VO 4 ; and 1 mm NaF), sonication, and incubation at 4 C for 30 min, followed by centrifugation at 10,000 g at 4 C for 20 min. Extracts containing equal quantities of proteins were separated by SDS-PAGE on 8 15% polyacrylamide gels and transferred to PVDF membranes. Membranes were probed with primary antibodies against CEBP (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), glucose-regulated protein 78 (GRP78; 1:500; Santa Cruz Biotechnology), superoxide dismutase 1 (SOD1; 2234 Vol. 27 June 2013 The FASEB Journal POGRIBNY ET AL.

3 1:2000; Abcam, Cambridge, MA, USA), SOD2 (1:2000; Abcam), and catalase (1:2000; Abcam). Horseradish peroxidasecoupled donkey anti-rabbit and anti-mouse secondary antibody (Santa Cruz Biotechnology) was used for visualization. Chemiluminescence detection was performed with Immobilon Western Chemiluminescent HRP Substrate (Millipore Corp., Billerica, MA, USA) and measured directly with a UVP BioSpectrum Imaging system (UVP, Upland, CA, USA). Equal protein loading was confirmed by immunostaining against GAPDH (1:5000; Sigma-Aldrich, St. Louis, MO, USA). Chromatin immunoprecipitation (ChIP) assay A ChIP assay was used to evaluate levels of CEBP protein bound to CEBP binding consensus sequences in the Mat1a, Ahcy, and Mthfd1 gene promoters of A/J and WSB/EiJ mouse livers. Briefly, formaldehyde crosslinking and the ChIP assay, with primary antibodies against CEBP protein (Santa Cruz Biotechnology), were performed by using a ChIP assay kit (Millipore), according to the manufacturer s protocol. Immunoprecipitated DNA was then purified by phenol/chloroform extraction and analyzed by quantitative PCR (qpcr) with the primers for the Mat1a, Ahcy, and Mthfd1 gene promoters. The level of immunoprecipitated DNA relative to input DNA was determined using the C t method, and the results are presented as fold change. Immunohistochemistry The extent of apoptosis in liver sections of control mice and mice fed the CFD diet was evaluated with TUNEL assay, using an ApopTag peroxidase in situ apoptosis detection kit (Millipore), according to the manufacturer s protocol. To detect the expression of -smooth muscle actin ( - SMA), formalin-fixed paraffin-embedded liver sections were deparaffinized and rehydrated. Endogenous peroxidases were inhibited by incubation with freshly prepared 3% hydrogen peroxide containing 0.1% sodium azide for 10 min at room temperature. Nonspecific staining was blocked with 0.5% casein. The sections were then incubated with 0.7 g/ml mouse monoclonal anti- -SMA (Biosensis, Thebarton, SA, Australia) for 1 h at room temperature. After incubation with the primary antibody, tissue sections were incubated with goat anti-mouse peroxidase-conjugated IgG F(ab=) 2 fragments (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) at a dilution of 1:200 for 30 min at room temperature. Staining was developed in 3,3=-diaminobenzidine for 5 min, and the tissue sections were then counterstained with hematoxylin and mounted with Permount (Fisher Scientific, Hampton, NH, USA). For a negative control, 0.7 g/ml mouse IgG (Jackson ImmunoResearch Laboratories) replaced the primary antibody. All sections were examined by light microscopy (BX40; Olympus, Tokyo, Japan). Stained sections were scanned, and digital images were obtained with an Aperio Scanscope system (Aperio Technologies, Vista, CA, USA). The proportions of TUNEL or -SMA-positive areas were evaluated using the positive pixel count algorithm (Aperio Technologies). Statistical analyses Results are presented as means sd. Data were analyzed by 1-way ANOVA, with pair-wise comparisons being made by the Student-Newman-Keuls method. When necessary, the data were natural log transformed before conducting the analyses to maintain a more equal variance or normal data distribution. Pearson product-moment correlation coefficients were used to determine the strength of association between levels of methionine, SAM, SAH, homocysteine, and gene expression and histopathology scores. Values of P 0.05 were considered significant. RESULTS Levels of methionine, SAM, SAH, and homocysteine Feeding the CFD diet for 12 wk resulted in substantial strain-independent changes in the liver levels of methionine, SAM, SAH, and homocysteine (Fig. 1A). Specifically, the levels of methionine in the livers of mice fed the CFD diet decreased by 30% in all strains. Similarly, the hepatic levels of SAM were reduced in all but the C3H/HeJ mice, while liver SAH did not change, except in the PWK/PhJ mice. The hepatic homocysteine levels were significantly increased in all but the CAST/EiJ mice fed the CFD diet. Notably, diet-induced pronounced elevation in the plasma homocysteine levels, 4.2 to 10.6 times greater than in control mice, was observed in all strains, and it was the only 1-carbon metabolite that significantly correlated (r 0.584, P 0.001) with the extent of liver injury among strains (Fig. 1C). In contrast, the levels of plasma homocysteine did not differ among mouse strains fed the control diet. Expression of one-carbon metabolism genes To evaluate further the effect of the CFD diet on one-carbon metabolism, the expression of genes involved in the functioning of the one-carbon metabolic network was examined. Fig. 2A shows that feeding the CFD diet resulted in strain-dependent changes in the expression of Mat1a, Ahcy, Cbs, Cth, Bhmt, Mtr, Mtrr, Mthfr, and Mthfd1 genes. The most noticeable changes were distinct strain-dependent differences in the expression of Mat1a, Ahcy, Cbs, Mtrr, Mthfr, and Mthfd1, as the magnitude of the effect on transcription of these genes strongly correlated with the degree of liver injury (Supplemental Fig. S2). A hierarchical clustering analysis of the expression changes in Mat1a, Ahcy, Cbs, Cth, Bhmt, Mtrr, Mthfr, and Mthfd1 genes shows that dietinduced effects in 129S1/SvImJ, PWK/PhJ, and WSB/ EiJ strains are similar and distinct from those in other strains (Fig. 2B). Levels and transcriptional activity of CEBP protein Ikeda et al. (29) have demonstrated that transcription factor CEBP plays an important role in regulating expression of the mouse Mat1a gene. We performed computational analysis of the transcription factor binding sites of other one-carbon metabolism-related genes and identified the existence of putative CEBP binding sequences in the promoter region of mouse Ahcy and Mthfd1 genes (Fig. 3A). Hence, the effect of the cholineand folate-deficient diet on the level of CEBP protein in the livers of mice was examined. Feeding the CFD diet resulted in a strain-dependent decrease in liver levels of 1-CARBON METABOLISM AND NONALCOHOLIC FATTY LIVER 2235

4 Figure 1. Liver content of methionine, SAM, SAH, homocysteine and plasma homocysteine in mice fed a CFD diet. A) Levels of methionine, SAM, SAH, and homocysteine content in the liver (nmol/mg protein) and homocysteine in plasma ( M) in mice fed the CFD diet. Levels of metabolites did not differ among mouse strains fed the control diet. Values are means sd, n 5. Bars with no letter in common differ significantly. *P 0.05 vs. control mice fed control diet. B) Correlation plots of total liver pathology scores and the methionine, SAM, SAH, and homocysteine content in the liver and homocysteine in plasma in mice fed the CFD diet. Each symbol represents an individual animal in the CFD diet group of the respective mouse strain. CEBP (Fig. 3B), an effect, which strongly correlated with the degree of liver injury (r 0.50, P 0.01) and with the expression of Mat1a (r 0.44, P 0.01), Ahcy (r 0.38, P 0.05), and Mthfd1 (r 0.40, P 0.05) across a panel of strains. A ChIP analysis was performed on the livers of A/J and WSB/EiJ mice, strains that were least and most, respectively, prone to the CFD diet-induced liver injury. These experiments (Fig. 3C) showed that in the livers of WSB/ EiJ mice fed the CFD diet, the amount of CEBP protein 2236 Vol. 27 June 2013 The FASEB Journal POGRIBNY ET AL.

5 Figure 2. Expression of selective one-carbon metabolism genes in the liver of mice fed the CFD diet. A) Expression of Mat1a, Ahcy, Cbs, Cth, Bhmt, Mtrr, Mthfr, and Mthfd1 genes was determined by qrt-pcr, as detailed in Materials and Methods. Results are presented as average fold change in the expression of each gene in the liver of mice fed the CFD diet relative to that in control groups, which were assigned a value of 1. Values are means sd, n 5. Bars with no letter in common differ significantly. *P 0.05 vs. control mice fed control diet. B) Unsupervised hierarchical clustering analysis (HCA) illustrating differences in the expression (fold change relative to strain control) of Mthfr, Mtrr, Mat1a, Mthfd1, Cbs, and Ahcy in the liver among A/J, C57BL/6J, C3H/HeJ, CAST/EiJ, and 129S1/SvImJ, PWK/PhJ, and WSB/EiJ mice fed the CFD diet. Unsupervised HCA was performed using 1-way ANOVA with cutoff value of P Color bar identifies highest-expressed (red) and lowest-expressed (green) genes. bound at the promoter region of Ahcy and Mthfd1 genes was significantly reduced, as compared to control animals of this strain. Furthermore, substantially lower CEBP protein binding at the promoter region at Mat1a, Ahcy, and Mthfd1 genes was observed in WSB/EiJ strain as compared to the CFD diet-fed A/J mice. 1-CARBON METABOLISM AND NONALCOHOLIC FATTY LIVER 2237

6 Figure 3. Regulation of Mat1a, Ahcy, and Mthfd1 gene expression by CEBP transcription factor. A) Presence of putative CEBP binding sequences in the promoter region of mouse Mat1a, Ahcy, and Mthfd1 genes. B) Western blot analysis of CEBP protein in the liver of control mice and mice fed the CFD diet. Results are presented as average fold change in the level of CEBP protein in the liver of mice fed the CFD diet relative to that in control groups, which were assigned a value of 100%. Bars with no letter in common differ significantly. C) Level of CEBP protein at promoter region of Mat1a, Ahcy, and Mthfd1 genes in the liver of A/J and WSB/EiJ mice, strains that exhibit the smallest and greatest extent of pathological changes, respectively. A ChIP assay was performed with primary antibodies against CEBP protein. Purified DNA from CEBP -enriched fragments and from input DNA was amplified by qpcr with primers for mouse Mat1a, Ahcy, and Mthfd1 gene promoters. Data are presented as fold change relative to control mice after normalization to input DNA. Values are means sd, n 5. *P 0.05 vs. control mice fed control diet. Endoplasmic reticulum (ER) and oxidative stress response ER and oxidative stress play key roles in the development and progression of NAFLD (30 32). In addition, there is an established link between alterations in one-carbon metabolism, the induction of both ER and oxidative stress, and liver injury (2, 33 35). Thus, both ER and oxidative stress responses were evaluated in the livers of mice fed the CFD diet. A strain-dependent induction of ER stress was observed as a result of the CFD diet in all strains except for A/J, as indicated by a marked up-regulation of ER and oxidative stress-related protein GRP78 (Fig. 4A) and an increased expression of stress-inducible and proapoptotic DNA-damage-inducible transcript 3 (Ddit3, Gadd153, Chop10) gene (Fig. 4B). Both of these ER stress markers were significantly associated with the severity of liver injury across strains. Fig. 4C shows that the extent of apoptosis, one of the pathological consequences of sustained ER and oxidative stress and a key pathogenic event in NAFLD (30, 36), was significantly higher in the livers of strains prone to diet-induced liver injury (129S1/SvImJ, PWK/ PhJ, and WSB/EiJ), as compared to A/J, C57BL/6J, C3H/HeJ, and CAST/EiJ strains. Figure 5 shows that in mice fed CFD diet, a downregulation of antioxidant enzymes SOD1, SOD2, and catalase was also observed, in accord with our previously published observations (17). These observations, coupled with the finding of the increase in liver apoptosis (Fig. 4C), suggest that a strain-dependent down-regulation of antioxidant enzymes may also play a role in the extent of liver injury. Progression of liver injury to fibrogenesis Although in most individuals with fatty liver, NAFLD is without clinical manifestations, hepatic steatosis is considered as an early manifestation of liver dysfunction that may progress to steatohepatitis, fibrosis, cirrhosis, and liver cancer (37). We examined whether liver injury caused by 12 wk of choline and folate deficiency progressed to initial stages of fibrosis. A strain-dependent progression of fatty liver injury to liver fibrosis was observed (Fig. 6), as evidenced by the presence of activated hepatic stellate cells, a well-established marker for hepatic fibrogenesis (38). Figure 6A shows representative immunohistochemical staining for -SMA, an important marker of activated stellate cells in the liver sections from A/J, 129S1/SvImJ, and WSB/EiJ mice fed control and CFD diet, strains that exhibited significantly divergent liver injury. In the liver sections of mice fed the control diet, -SMA positivity was confined to the vascular smooth muscle cells only (Fig. 6A). In the livers of CFD diet-fed mice, positive staining was also observed in the cytoplasm of myofibroblast-like cells. While few -SMA-positive myofibroblasts were found in A/J mice, these cells were diffusely scattered throughout all liver tissue sections in 129S1/SwimJ and WSB/EiJ mice. Image analysis demonstrated that in the liver sections of the CFD diet-fed 129S1/SwimJ and WSB/EiJ mice, the proportion of the -SMA-positive area was, respectively, 16 to 20 times greater than either the corresponding controls or A/J mice fed the CFD diet (Fig. 6B). The data obtained by immunohistochemical study were confirmed by qrt-pcr (Supplemental Fig. S3), which demonstrated that the CFD diet resulted in a 2238 Vol. 27 June 2013 The FASEB Journal POGRIBNY ET AL.

7 Figure 4. ER stress response and apoptosis in the livers of mice fed the CFD diet. A) Western blot analysis of ER and oxidative stress-related protein GRP78 and correlation plots of total liver pathology scores and the levels of GRP78 protein in the liver of mice fed control or the CFD diet. Results are presented as average percentage change in mice fed the CFD diet relative to that in control groups, which were assigned a value of 100%. B) Expression of Ddit3 (Gadd153, Chop10) gene and correlation plots of total liver pathology scores and the expression of Ddit3 gene in the liver of mice fed the CFD diet. Results are presented as average fold change in Ddit3 expression in liver of mice fed the CFD diet relative to that in control groups, which were assigned a value of 1. C) Extent of apoptosis in the liver of mice fed control or CFD diet. Results are presented as average percentage change in mice fed the CFD diet relative to that in control groups, which were assigned a value of 100%. Values are means sd, n 5. Bars with no letter in common differ significantly. *P 0.05 vs. control mice fed control diet. strain-dependent increase of the expression of fibrosisrelated genes Acta2, Col1a1, Mmp2, Mmp9, Timp1, S100a4, Ccr1, and Cx3cr1, with the greatest up-regulation in WSB/EiJ mice. DISCUSSION One-carbon metabolism, an integral component of several interdependent metabolic processes, plays an important role in health and pathology. Specifically, impaired functioning of one-carbon metabolism is a critical early event in the pathogenesis of nonalcoholic and alcoholic liver diseases (2, 5, 39). Furthermore, dietary deficiency in methyl-group donors in animal models has been shown to alter one-carbon metabolism and trigger development of liver injury that resembles NAFLD in humans (24); however, the extent of liver injury varies substantially among and within species (7, 18). Similarly, accumulated evidence indicates clearly that genetic variations and gender substantially influence the incidence of NAFLD in the general human population (40). A greater understanding of the interindividual differences in susceptibility to NAFLD is critical for identifying susceptible individuals and for preventing and treating the disease (2). Investigating the molecular determinants of differential susceptibility to NAFLD in humans is generally impractical and very complex, and interpreting the findings without validation in animal studies is challenging. In contrast, relevant population-based mouse models can overcome many of the limitations of human-only studies (41) and provide testable hypotheses for confirmation in human cohorts (42). In this study, we demonstrate that feeding a panel of 7 genetically diverse strains of inbred male mice a CFD diet leads to a significant deregulation in the function of the hepatic one-carbon metabolism. This was evidenced by an altered level of key one-carbon cycle metabolites, strain- and injury-dependent hyperhomocysteinemia, and dysregulated expression of one-carbon-metabolism genes in the livers of CFD mice. Nota- 1-CARBON METABOLISM AND NONALCOHOLIC FATTY LIVER 2239

8 Figure 5. Level of SOD1, SOD2, and catalase proteins in the livers of control mice and mice fed the CFD diet. A) Pprotein level of SOD1, SOD2, and catalase in the livers of control mice and mice fed the CFD diet was determined by Western blot analysis. Results are presented as average fold change in the level of each protein in the livers of mice fed the CFD diet relative to that in control groups, which were assigned a value of 100%. Values are means sd, n 5. Bars with no letter in common differ significantly. *P 0.05 vs. control mice fed control diet. B) Correlation plots of total liver pathology scores and the levels of SOD1, SOD2, and catalase, respectively. bly, molecular alterations in one-carbon metabolism were well-documented, especially in the expression of Mat1a, Ahcy, Cbs, Mthfr, and Mthfd1, genes that play a central role in the proper maintenance of one-carbon metabolism (1). To examine whether basal gene expression of these transcripts could be affected by genetic variants, we considered the possibility that cisexpression quantitative trait loci (cis-eqtls) may be present. Permutation (n 1000)-based interval mapping was performed using WebQTL ( work.org/webqtl/main.py) on the mouse diversity panel of the liver gene expression data reported in Harrill et al. (43). These genes have no cis-eqtls, indicating that genetic variants among strains do not affect their basal level of expression. However, we found that the expression of these genes was lower in the livers of mouse strains prone to CFD diet-induced liver injury, suggesting that down-regulation of Mat1a, Ahcy, Cbs, Mtrr, Mthfr, and Mthfd1 may be a factor determining the magnitude of NAFLD-associated liver injury and account for the differences in interindividual disease severity and sensitivity. More importantly, the results of the present study indicate that low expression of these genes may be a determining factor that affects progression of NAFLD from simple uncomplicated steatosis to nonalcoholic steatohepatitis and then to fibrosis. This was evidenced by marked straindependent activation of fibrogenesis and is supported by clinical and experimental evidence demonstrating an importance of genetic variations in one-carbon genes (4, 16, 44), or gene knockouts, e.g., Mat1a, Bhmt, and Cbs, in the pathogenesis of NAFLD (reviewed in ref. 2). One of the pathophysiological consequences of dysregulation of one-carbon metabolism is hyperhomocysteinemia (34, 45, 46), which has been associated with the pathogenesis of alcoholic and nonalcoholic fatty liver injury (47, 48). Here, we demonstrated that feeding mice a CFD diet caused a marked increase of the homocysteine levels in the livers and, especially, plasma. There are several pathological consequences of hyperhomocysteinemia, all of which may contribute to the development of NAFLD. First, elevated homocysteine has been linked to induction of a hepatic ER response (46). Indeed, we show that feeding a CFD diet leads to homocysteine-related liver injury and the induction of ER stress, in accord with previous observations with alcohol-induced liver injury models (34, 46). It should be noted, however, that recent reports have demonstrated that an activated ER stress response may not have a primary role in the pathogenesis of NAFLD induced by methyl-group donor deficiency (49, 50). Second, hyperhomocysteinemia due to deficiency in Mat1, Cbs, or Mthfr may cause downstream dysregulation of genes involved in hepatic lipid homeostasis, including down-regulation of peroxisome proliferator receptor- (PPAR ). The results of our previous study demonstrating that feeding mice a CFD diet resulted in a profound alteration in the cellular lipid metabolism driven by a down-regulation of the PPAR -regulated lipid catabolic pathway (17) provides additional support for this suggestion. Third, hyperhomocysteinemia may lead to oxidative stress, and, more importantly, alter the oxidative stress response through down-regulation of the antioxidant enzymes (51). Indeed, observations reported in this study and those published 2240 Vol. 27 June 2013 The FASEB Journal POGRIBNY ET AL.

9 several findings demonstrating a critical role of DNA methylation in silencing of Mat1a, Ahcy, and Mthfr genes (29, 55). In summary, data presented herein indicate that dysregulated one-carbon metabolism, primarily a diminished expression of key one-carbon genes, is a driving force that promotes the development and progression of NAFLD, and, more importantly, determines interstrain susceptibility and severity of NAFLD in mice. However, future studies are needed to determine whether the strain-specific one-carbon metabolism gene expression alterations provoke the malfunctioning of encoded proteins. The results of these studies may provide a strong support for developing preventive and treatment strategies for NAFLD through correction of these one-carbon metabolism disturbances. The views expressed in this article do not necessarily represent those of the U.S. Food and Drug Administration. REFERENCES Figure 6. Immunohistochemical analysis of the expression of -SMA protein. A) Representative examples of control liver section and liver sections from A/J, 129S1/SvImJ, and WSB/ EiJ mice fed the CFD diet. B) Percentage of -SMA-positive area in A/J, 129S1/SvImJ, and PWK/PhJ control mice and mice fed the CFD diet. *Significantly different from control mice fed a control diet (mean sd; n 5). Bars with different letters indicate a significant difference between strains. elsewhere (17) lend credence to this mechanism and its role in strain-dependent responses. Another important finding of the present study is the down-regulation of the transcription factor CEBP in the livers of mice fed a CFD diet and the demonstration of its critical role in the regulation of the expression of the Mat1a, Ahcy, and Mthfd1 genes. These are 3 essential genes that intimately interconnect transmethylation, transsulfuration, and folate-dependent DNA synthesis pathways in one-carbon metabolism (52 54), and the down-regulation of any of these genes may impair the entire one-carbon metabolic pathway. The significant positive correlation between the levels of CEBP protein and the expression of Mat1a, Ahcy, and Mthfd1 genes suggests its direct role in their down-regulation; however, we cannot exclude the possibility that under methyl-group donor-deficient conditions single nucleotide polymorphisms existing in these genes or an altered DNA methylation pattern in the promoter region of Mat1a, Ahcy, and Mthfd1 may also contribute to their lower expression. This suggestion is based on 1. Tibbetts, A. S., and Appling, D. R. (2010) Compartmentalization of Mammalian folate-mediated one-carbon metabolism. Annu. Rev. Nutr. 30, Corbin, K. D., and Zeisel, S. H. 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