Available online at www.sciencedirect.com

ScienceDirect

Journal of Nutritional Biochemistry 25 (2014) 329–336

Betaine attenuates hepatic steatosis by reducing methylation of the MTTP promoterand elevating genomic methylation in mice fed a high-fat diet☆

Li-jun Wanga,1, Hong-wei Zhangb,1, Jing-ya Zhoua, Yan Liua, Yang Yanga, Xiao-ling Chena, Cui-hong Zhua,Rui-dan Zhengc, Wen-hua Linga, Hui-lian Zhua,⁎

aFaculty of Nutrition, School of Public Health, Sun Yat-Sen University, 510080 Guangzhou, People's Republic of ChinabDepartment of Hepatobiliary Surgery, Sun Yat-Sen Memorial Hospital, University of Sun Yat-Sen, 510120 Guangzhou, People's Republic of China

cResearch and Therapy Center for Liver Disease, the Affiliated Dongnan Hospital of Xiamen University, 363000 Zhangzhou, People's Republic of China

Received 14 August 2013; received in revised form 8 November 2013; accepted 16 November 2013

Abstract

Aberrant DNA methylation contributes to the abnormality of hepatic gene expression, one of the main factors in the pathogenesis of nonalcoholic fatty liverdisease (NAFLD). Betaine is a methyl donor and has been considered to be a lipotropic agent. However, whether betaine supplementation improves NAFLD via itseffect on the DNA methylation of specific genes and the genome has not been explored. Male C57BL/6 mice were fed either a control diet or high-fat diet (HFD)supplemented with 0%, 1% and 2% betaine in water (wt/vol) for 12 weeks. Betaine supplementation ameliorated HFD-induced hepatic steatosis in a dose-dependent manner. HFD up-regulated FAS and ACOX messenger RNA (mRNA) expression and down-regulated PPARα, ApoB and MTTP mRNA expression;however, these alterations were reversed by betaine supplementation, except ApoB. MTTP mRNA expression was negatively correlated with the DNAmethylation of its CpG sites at−184,−156,−63 and−60. Methylation of these CpG sites was lower in both the 1% and 2% betaine-supplemented groups than inthe HFD group (averages; 25.55% and 14.33% vs. 30.13%). In addition, both 1% and 2% betaine supplementation significantly restored the methylation capacity [S-adenosylmethionine (SAM) concentration and SAM/S-adenosylhomocysteine ratios] and genomic methylation level, which had been decreased by HFD (0.37%and 0.47% vs. 0.25%). These results suggest that the regulation of aberrant DNA methylation by betaine might be a possible mechanism of the improvements inNAFLD upon betaine supplementation.© 2014 Elsevier Inc. All rights reserved.

Keywords: Nonalcoholic fatty liver disease; Betaine; DNA methylation; Microsomal triglyceride transfer protein; Methylation capacity

1. Introduction

Nonalcoholic fatty liver disease (NAFLD) is a complex disease andis likely the result of both nutritional and genetic disorders. Althoughthe causes of human NAFLD are not clearly understood, it has beenwell documented in animal models that high-fat and carbohydratediets and low-methyl-donor diets (e.g., methionine- and choline-deficient diets) are important pathogenic factors of NAFLD [1]. NAFLD

Abbreviations: FAS, fatty acid synthase; ACC, acetyl-CoA carboxylase;PPARα, peroxisomal proliferator-activated receptor alpha; ACOX, acyl-CoAoxidase; MTTP, microsomal triglyceride transfer protein; ApoB, apolipopro-tein B; SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine.

☆ Funding sources: This work was jointly supported by grants from theNational Natural Science Foundation of China (81072302 and 81273050);Science and Technology Project of Guangzhou, China (No. 201300000148)and Danone Institute China Diet and Nutrition Research and Communication(2011).

⁎ Corresponding author. School of Public Health, Sun Yat-sen University(Northern Campus), 74 Zhongshan Road II, Guangzhou 510080, People'sRepublic of China.

E-mail address: zhuhl@mail.sysu.edu.cn (H. Zhu).1These authors contributed equally to this work.

0955-2863/$ - see front matter © 2014 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.jnutbio.2013.11.007

is initially characterized by the aberrant accumulation of triglyceride(TG) within the hepatocytes. The liver contains a full complement ofenzymes involved in fatty acid metabolism, including fatty acidsynthesis, uptake, oxidation and transport, and the disorderedexpression of the genes encoding for these enzymes will definitelyresults in TG deposits [2,3].

Increasing evidence indicates that many chronic diseases, includ-ing NAFLD, are affected by aberrant DNA methylation [4]. Diet is oneof main factors that affect DNA methylation. Stidley et al. [5] havereported that increasing the intake of multivitamins and folatedecreases the DNA methylation levels of gene promoters in cellsthat were exfoliated from the airway epithelium of smokers. Inmousemodels, a methyl-deficient diet has been confirmed to perturb DNAmethylation by causing a profound loss of genomic methylation andleading to specific hyper- or hypomethylation of gene promoters inthe liver [6]. DNA methylation has a specific effect on geneexpression: hypermethylation of the CpG islands in the promoterregion of gene directly represses gene expression, and hypomethyla-tion of the global DNA affects genomic stability and integrity. Thus, itis important to realize that DNA methylation is an interface for thecausal relationship between nutrition and genetics. Abnormal geneexpression in livers of mice fed a high-fat diet (HFD) has been

330 L.-J. Wang et al. / Journal of Nutritional Biochemistry 25 (2014) 329–336

demonstrated [7]; however, the role of aberrant DNA methylation inthe pathogenesis of NAFLD is largely unknown.

DNA methylation depends on the availability of methyl groupsfrom S-adenosylmethionine (SAM). Dietary methyl groups derivefrom foods containing folate, choline and betaine, and the intake ofthese compounds is closely interconnected to SAM synthesis. Dietarydepletion of these methyl donors has been shown to decrease thehepatocellular SAM concentration [6], while dietary supplementationincreased the concentration [8,9]. The association between folate/choline and the DNA methylation of the genome or of specific geneshas been well documented [5,6,10,11]; however, studies on theassociation between betaine and DNA methylation are still limited.

The hepatoprotective effect of betaine has been reported in miceand rats fed with either an HFD [12–14] or a high-sucrose diet [8] andin patients with nonalcoholic steatohepatitis [15,16]. Although theoverall results are promising, the mechanisms relating to betaine inNAFLD mostly focus on insulin resistance and oxidative stress. Otherpotential nutritional strategies to alleviate hepatic steatosis, forexample, supplementation with berberine [17], zinc [18] and grapeseed proanthocyanidins [19], have been shown to regulate theabnormal expression of key genes involved in lipid metabolism.Recent research in our laboratory in a dyslipidemia mouse model hasshown that betaine supplementation might alleviate hepatic TGaccumulation and oxidative stress by decreasing the DNA methyla-tion of PPARα promoter and up-regulating its gene expression [20].However, a careful and comprehensive analysis of the expression andmethylation of lipid metabolism-related genes by betaine supple-mentation in NAFLD has yet to be performed.

To fill these gaps, we used an NAFLD mouse model induced by anHFD to investigate (a) whether the alleviation of NAFLD by betainesupplementation is related to regulation of the abnormal expressionof lipid metabolism-related genes in the liver, (b) whether betaine-mediated gene expression is associated with the DNA methylationof gene promoters and the genome and (c) the underlyingmechanisms involved.

2. Materials and methods

2.1. Animal model and experiment protocols

Male C57BL/6 mice (7 weeks) weighing 14.5–17.8 g were obtained from theanimal center of Guangdong Province with the certificate number SCXK (Guang Zhou)2008-0002. The study was approved by the Institutional Animal Care and UseCommittee of Sun Yat-Sen University. All mice were acclimated on a control diet (CD)and water ad libitum in temperature and humidity-controlled rooms with 12-h light/dark cycle for 1 week before the experiment. Thereafter, 24 mice were randomlydivided into four groups (n=6) and fed a CD, an HFD, and an HFD supplemented witheither 1% or 2% betaine (HFD+1%B and HFD+2%B) in the drinking water (wt/vol). Boththe CD (D12450B) and the HFD (D12451) were purchased from Research Diets (NewBrunswick, NJ, USA). Betaine was obtained from Danisco A/S (Denmark). The bodyweight and food and water consumption of the mice were recorded weekly. All micehad free access to food and water for 12 weeks before sacrifice. At the end ofexperiment, whole blood was collected to separate the serum. Visceral fat (includingthe mesenteric fat pad, epididymal fat pad and perirenal fat pad) and the liver wererapidly excised, weighed and frozen in liquid nitrogen and stored at –80°C.

2.2. Serum biochemical parameters

The serum biochemical parameters were determined using commercially availablekits (Biosino Bio-Technology and Science Inc., Beijing, China) on an automaticbiochemistry analyzer (A25Biosystem, Barcelona, Spain). The measured parametersincluded alanine aminotransferase (ALT), aspartate transaminase (AST) and TG.

2.3. Measurement of hepatic steatosis

The liver TG content was measured using a commercially available kit fromApplygen Technologies Inc. (E1003-2). For the histology analysis, the liver samplesfrom each mouse were divided into several sections, one of which was subsequentlyfixed in 10% formalin and embedded in paraffin blocks. Another section was directlyembedded in O.C.T. compound. The sections from the paraffin blocks were sliced andstained with hematoxylin and eosin to evaluate the pathologic structure of the

hepatocytes, and the sections from the O.C.T. compound were sliced and stained withoil red O to examine the lipid droplets.

2.4. Determination of the betaine and choline levels in the serum

Thirty microliters of either the serum or standards was treated with 100 μlacetonitrile containing 10 μM internal standards, and the samples were centrifuged at13,000×g for 10 min to precipitate the proteins. The supernatants were injected intospin columns and centrifuged at 3000×g for 2 min to filter impurities. Finally, thesupernantants were collected and analyzed by high-performance liquid chromatog-raphy-tandem mass spectrometry (HPLC-MS) [21].

2.5. Determination of the homocysteine level in the serum

Sixty microliters of either the serum or standards was treated with 10 μl tris-(2-carboxyethyl)-phosphine hydrochloride (TCEP) (120 mg/ml) solution and 30 μl2-mercaptopropionylglycine (MPG) (10 μM), mixed thoroughly and incubated at 37°Cfor 30 min. After the addition of 90 μl trichloroacetic acid (TCA) (100 g/L with 1 mMEDTA), the mixtures were immediately centrifuged at 13,000×g for 10 min toprecipitate the proteins. Fifty microliters of the supernatants was incubated with 125 μlborate buffer (0.125 M, pH 9.5, with 4 mM EDTA), 10 μl NaOH solution (1.55 M) and50 μl 3-diazole-4-sulfonic acid (SBD-F) solution (1 g/L in borate buffer) at 60°C for60 min after being thoroughly mixed. The samples were then placed on ice for 10 min,followed by (10 μl) injection into the HPLC system for analysis [22].

2.6. Determination of the SAM and S-adenosylhomocysteine concentrations in the liver

We used the method of Wang et al. [23] to determine the hepatic SAM and S-adenosylhomocysteine (SAH) concentrations. The supernatants were directly injectedinto a Waters HPLC system equipped with an e2695 Separation Module, 2998Photodiode Array Detector, UV LC spectrophotometer and Hypersil BDS C18 reversed-phase analytical column.

2.7. RNA isolation and real-time polymerase chain reaction analysis

Total RNAwas isolated from the liver with the TRIzol reagent (Invitrogen, Carlsbad,CA, USA) according to the manufacturer's instruction. The concentration and quality ofthe total RNA were assessed by spectrophotometer, and its integrity was tested onagarose gels. Two-microgram RNA was reverse transcribed to synthetize single-stranded complementary DNA using a commercial kit (Invitrogen). A quantitativeanalysis of the gene expression was performed by real-time polymerase chain reaction(RT-PCR) in an Applied Biosystems 7500 fast real-time PCR system. Primers of targetgenes and β-actin are described in Supplementary Table 1.

2.8. Measurement of the DNA methylation level of gene promoters and the genome

Genomic DNA was isolated from the livers of mice using the TIANamp GenomicDNA Kit (TIANGEN, Beijing, China), and DNA bisulfite conversion was performed asdetailed in our previous publication [20]. The chemically modified DNA wassubsequently used as a template for a direct sequencing method to determine themethylation level of the selected CpG sites in the target gene promoters [24]. Briefly,the sequence was retrieved from NCBI at http://www.ncbi.nlm.nih.gov/gene?. Primerswere designed online via methprimer (http://www.urogene.org/methprimer/), andthe sequences are presented in Supplementary Table 1. The product of target genepromoters was amplified by PCR. For each 25 μl PCR reaction, 2.5 μl 10× buffer, 2 μldNTPs, 0.125 μl Taq Hotstart polymerase (Takara), 1 μl 10 μM forward primer, 1 μl 10μM reverse primer, 2 μl DNA template and 16.375 μl water were used. The size andquality of the PCR products (5 μl) were verified on 2% agarose gels, and the remainswere purified and directly sequenced.

The genomic methylation levels for the liver tissue were determined using theMethylFlash Methylated DNA Quantification Kit (Fluorometry, Epigentek) according tothe manufacturer's instructions.

2.9. Western immunoblot analysis

Protein from the mouse liver samples was extracted with the RIPA buffer (BioTeke,Beijing, China) containing a protease inhibitor cocktail and phenylmethanesulfonylfluoride. A BCA-100 Protein Quantitative Analysis Kit was used to measure the proteinconcentrations. After denaturation, the protein samples were subjected to sodiumdodecyl sulfate polyacrylamide gel electrophoresis and blotted onto nitrocelluloseblotting membranes (PALL, New York, NY, USA). Nonspecific binding sites wereblocked with 8% skim milk in Tris-buffered saline containing 0.1% Tween-20 for 1.5 hand then incubated with primary antibodies against MTTP (Santa Cruz Biotechnology,Santa Cruz, CA, USA) and GAPDH as a control overnight at 4°C. Subsequently, thenitrocellulose membranes were incubated with either secondary goat antirabbit (anti-GAPDH) or donkey antigoat (anti-MTTP) conjugated with horseradish peroxidase. Theprotein bands were detected by ECL (Thermo Fisher, Waltham, MA, USA), and asemiquantitative analysis was performed using the Quantity One package (Bio-Rad,Hercules, CA, USA).

Table 1Effect of betaine supplementation on the visceral weight and serum biochemicalparameters in mice fed with HFD

Parameter CD HFD HFD+1%B HFD+2%B

Liver weight (g) 1.02±0.05 1.06±0.04 1.02±0.07 1.13±0.05Visceral fat weight (g) 0.59±0.06 1.10±0.15 ⁎ 0.78±0.14 0.70±0.08Serum TG (mmol/L) 0.98±0.10 1.14±0.21 0.57±0.13## 0.65±0.02#

Serum AST (U/L) 135.8±6.47 158.4±3.54 ⁎ 135.0±6.16# 138.1±4.00Serum ALT(U/L) 34.4±2.56 46.1±3.20 ⁎ 36.0±2.42 42.2±1.73

C57BL/6 mice were divided into four different groups (n=6) and respectively fed a CD,an HFD and an HFD supplemented with either 1% or 2% betaine (HFD+1%B and HFD+2%B) in the drinking water (wt/vol) for 12 weeks.

⁎ Pb.05 vs. CD group.# Pb.05.## Pb.01 vs. HFD group.

331L.-J. Wang et al. / Journal of Nutritional Biochemistry 25 (2014) 329–336

2.10. Cell culture experiment

HepG2 cells were incubated with 60 mg/L oleic acid (OA) for 24 h to induce themodel of hepatocellular steatosis. In addition, HepG2 cells were incubated in a mediumwith 60 mg/L OA containing 5 or 10 mmol/L betaine or 2.5 or 5 mmol/L 5-aza-2′-deoxycytidine (5-aza-2′-dC) for 24 h. The degree of hepatocellular steatosis wasevaluated by oil red O–hematoxylin staining. Cells were washed by phosphate-buffered saline, and RNA was extracted using the TRIzol reagent for further RT-PCRanalysis. The primers for MTTP and β-actin are listed in Supplementary Table 1.

2.11. Statistic analysis

All results were presented as the means±standard error (S.E.M.). After testing fornormality, one-way analysis of variance was used to compare the mean differencesamong the four groups and Tukey's HSD (honest significant difference) test was usedfor multiple comparisons. Spearmen correlation was conducted to assess therelationship between the DNA methylation of the MTTP promoter and the MTTPmessenger RNA (mRNA) expression. Linear correlation was used to evaluate the dose-dependent effect of betaine on other parameters. A two-tailed Pb.05 was consideredstatistically significant.

3. Results

3.1. Effect of betaine supplementation on body weight, visceral weightand serum biochemical parameters in the HFD-fed mice

Changes in body weight, food and water consumption wererecorded weekly during the experiment. Mice in the HFD groupweighed significantly more than those in the CD group at weeks 2–12,while betaine supplementation had no significant effect on the bodyweight of HFD-fed mice (Fig. 1). No significant differences wereobserved in the water or food consumption among the four groups(data not shown). The visceral fat weight in the betaine-supplemen-ted groups was decreased compared to the HFD group; the liverweight did not show significant differences among the groups. Theserum TG level did not differ between the CD and HFD groups but wassignificantly decreased in the betaine-supplemented groups(Table 1). These data suggested that betaine supplementationreduced the visceral fat accumulation and serum TG level in theHFD-fed mice.

3.2. Betaine supplementation alleviated hepatic steatosis in theHFD-fed mice

Histological analysis of livers stained with hematoxylin–eosin(HE) and oil red O demonstrated that betaine-supplementedmice did

Fig. 1. Betaine supplementation had no effect on the body weight of mice fed with HFD.C57BL/6 mice were divided into four different groups (n=6) and respectively fed a CD,an HFD and an HFD supplemented with either 1% or 2% betaine (HFD+1%B and HFD+2%B) in the drinking water (wt/vol) for 12 weeks. Data are presented as the means±S.E.M. *Pb.05, **Pb.01 for HFD vs. CD.

not show any sign of lipid droplet accumulation within thehepatocytes, which is commonly observed in the livers of HFD-fedmice (Fig. 2A). Consistently, a significant attenuation by betainesupplementation on the HFD effects of increased hepatic TG content(Fig. 2B) and serum ALT and AST levels (Table 1) was observed. Theseresults indicated that betaine supplementation alleviated HFD-elicited hepatic steatosis and liver injury.

3.3. Betaine supplementation reversed the HFD-induced abnormalexpression of genes involved in hepatic lipid metabolism

HFD resulted in a ~1.5-fold elevation in FAS and ACOX expression,and this increase was inhibited by betaine supplementation (Fig. 3A,B). PPARα, MTTP and ApoB expression levels were significantlydecreased by HFD, but betaine supplementation significantly up-regulated both the PPARα and MTTP expression levels (Fig. 3B, C).Consistent with the alteration of the MTTP mRNA levels, the Westernimmunoblot analysis showed that the MTTP protein level in betaine-supplemented groupswas increased relative to that in the HFD group;however, the difference was only significant in the HFD+2%B group(Fig. 3D). These data indicated that betaine supplementation revisedHFD-induced dysregulation of both the mRNA and protein expressionlevels of lipid metabolism-related genes.

3.4. Betaine supplementation revised the HFD-inducedhypermethylation of the MTTP promoter

The methylation levels of four CpG sites located at −184, −156,−63 and −60 in the promoter region of the MTTP gene werequantified. The representative sequencing chromatograms for eachCpG site of each group are shown in Supplementary Fig. 1. Overall,HFD significantly increased the averagemethylation level of theMTTPpromoter, but this effect was inhibited by betaine supplementation.When the methylation status was analyzed individually at the fourCpG sites, the DNA methylation of all sites was increased by HFD, andthere was a significant decrease in the DNA methylation in thebetaine-supplemented groups compared to the HFD group (Fig. 4A). Adose-dependent relationship was found between betaine supple-mentation and DNA methylation at the −184 (r=−0.567, P=.001),−156 (r=−0.416, P=.022),−63 (r=−0.407, P=.026) and−60 (r=−0.454, P=.012) CpG sites. Moreover, the expression level of MTTPmRNA displayed a significantly inverse correlation with the DNAmethylation level of MTTP promoter at four CpG sites (Fig. 4B–F). Inaddition, the DNA methylation level of the CpG sites in the FAS, ACC,PPARα, ACOX and ApoB promoter regions was also analyzed by directsequencing; however, none of these genes were efficiently modifiedby HFD and betaine. These results suggested that the betaine-induced

Fig. 2. Betaine alleviated hepatic steatosis and TG accumulation in mice fed with HFD. C57BL/6 mice were divided into four different groups (n=6) and respectively fed a CD, an HFDand an HFD supplemented with either 1% or 2% betaine (HFD+1%B and HFD+2%B) in the drinking water (wt/vol) for 12 weeks. A: Histological analysis of the liver tissue. Photographsare at 400× magnification. B: Hepatic TG content. Data are presented as the means±S.E.M. *Pb.05 vs. CD group, ##Pb.01 vs. HFD group.

332 L.-J. Wang et al. / Journal of Nutritional Biochemistry 25 (2014) 329–336

up-regulation of MTTP expression could be attributed to the decreasein DNA methylation of its promoter.

3.5. Betaine supplementation improved the methylation capacity inthe HFD-fed mice

HFD reduced the hepatic SAM concentration, which was 3.70 and4.89 times higher in the HFD+1%B and HFD+2%B groups, respec-tively, than in the HFD group (Fig. 5A). There was a parallel increasein the hepatic SAM/SAH ratios with SAM concentration by betainesupplementation. However, there was no significant difference in thehepatic SAH concentration among the four groups (data not shown).The serum choline level decreased by 24.7% in the HFD group andonly increased by 1% betaine supplementation. The serum betainelevels were similar in the HFD and CD groups but increased by 1.2and 2.5 times in the HFD+1%B and HFD+2%B groups, respectively,relative to the HFD group. HFD significantly elevated the level of

serum homocysteine (Hcy), which was reduced by betaine supple-mentation in a dose-dependent manner (r=−0.815, Pb.001)(Fig. 5B). Compared with CD, the HFD caused a reduction in theglobal DNA methylation level by 26.8%; betaine supplementationsignificantly increased the genomic methylation in a dose-dependentmanner (r=0.736, Pb.001) (Fig. 5C). These results demonstratedthat betaine supplementation reversed HFD-induced impairment ofmethylation capacity and obviously increased the genomic methyl-ation in the liver.

3.6. Betaine supplementation increased the SAM/SAH ratio and theMTTP expression level in HepG2 cells

Compared with the 60-mg/L OA group, the betaine-supplementedgroups displayed a significantly increased SAM/SAH ratio in a dose-dependent manner (r=0.811, P=.008) (Fig. 6A). Treatment withbetaine or 5-aza both resulted in an increase in the MTTP expression

Fig. 3. Betaine supplementation reversed the abnormal expression of lipidmetabolism-related genes inmice fedwith HFD. C57BL/6mice were divided into four different groups (n=6)and respectively fed a CD, an HFD and an HFD supplemented with either 1% or 2% betaine (HFD+1%B and HFD+2%B) in the drinking water (wt/vol) for 12 weeks. A: The mRNAexpression of genes in hepatic lipogenesis. B: The mRNA expression of genes in hepatic fatty acid oxidation. C: The mRNA expression of genes in hepatic TG transport. D–E:Quantitation of Western blots ofMTTP protein expression, normalized by corresponding GAPDH expression in liver tissues. Data are presented as the means±S.E.M. *Pb.05, **Pb.01 vs.CD group, #Pb.05, ##Pb.01 vs. HFD group. FAS, fatty acid synthase; ACC, acetyl-CoA carboxylase; PPARα, peroxisomal proliferator-activated receptor alpha; ACOX, acyl-CoA oxidase;MTTP, microsomal TG transfer protein; ApoB, apolipoprotein B.

333L.-J. Wang et al. / Journal of Nutritional Biochemistry 25 (2014) 329–336

level (Fig. 6B). These data indicated that betaine supplementationmight not only increase the methylation capacity in the cell but alsoexert the same hypomethylated effect as 5-aza to increase the MTTPmRNA expression level.

Fig. 4. Betaine supplementation reversed the hypermethylation of the MTTP promoter in mrespectively fed a CD, an HFD (HFD) and an HFD supplemented with either 1% or 2% betaimethylation levels at four CpG sites and the average DNA methylation level of the MTTP promand the DNAmethylation level at the−184,−156,−63 and−60 CpG sites. Data are presentedmicrosomal TG transfer protein.

4. Discussion

The results of the present study demonstrated that betainesupplementation normalized the abnormal expression of the FAS,

ice fed with HFD. C57BL/6 mice were divided into four different groups (n=6) andne (HFD+1%B and HFD+2%B) in the drinking water (wt/vol) for 12 weeks. A: DNAoter in the liver. B–F: The linear association between the MTTP mRNA expression levelas the means±S.E.M. *Pb.05, **Pb.01 vs. CD group, #Pb.05, ##Pb.01 vs. HFD group.MTTP,

Fig. 5. Betaine supplementation improved the methylation capacity of mice fed with HFD. C57BL/6 mice were divided into four different groups (n=6) and respectively fed a CD, anHFD and an HFD supplemented with either 1% or 2% betaine (HFD+1%B and HFD+2%B) in the drinking water (wt/vol) for 12 weeks. A: Hepatic SAM concentrations and SAM/SAHratios. B: Serum betaine, choline and Hcy levels. C: Genomic methylation levels in the liver. Data are presented as the means±S.E.M. *Pb.05, **Pb.01 vs. CD group, #Pb.05, ##Pb.01 vs.HFD group.

334 L.-J. Wang et al. / Journal of Nutritional Biochemistry 25 (2014) 329–336

ACOX, PPARα and MTTP genes induced by a HFD. Furthermore, theMTTP mRNA expression level was negatively associated with theDNA methylation level of its promoter. Betaine supplementationdecreased the DNA methylation of MTTP promoter and increased thegenomic methylation in a dose-dependent manner compared to theHFD. These DNA methylation changes by betaine supplementationcontributed to the improvement of hepatic steatosis.

4.1. Betaine and fatty acid synthesis, oxidation and transport

Betaine is initially utilized in feed industry to improve carcass leangain and reduce total fat in pigs [25]. Consistent with the mechanism,

Fig. 6. Betaine supplementation increased the SAM/SAH ratios andMTTP expression levels in Hedifferent concentrations of betaine (0 mM, 5mM, 10mM) or 5-aza-dC (2.5 mM, 5 mM), togethHepG2 cells. Data are presented as the means±S.E.M. #Pb.05, ##Pb.01 vs. 60-mg/L OA group.

our results also found that betaine supplementation decreasedvisceral fat weight but had no significant effect on body weight inthe HFD-fed mice, possibly due to the increase of lean body mass.Betaine has been tested as a lipotropic agent in many animal models[8,12–14] and clinical trials [15,16], and it displayed similarlybeneficial effects on HFD-fed mice in our study. Betaine supplemen-tation significantly improved liver function. In accord with thealleviation of hepatic steatosis observed in the histological analysis,betaine dose dependently decreased hepatic TG content.

It is a prerequisite for the initiation and progression of NAFLD thatTG deposits in the liver. The homeostasis of hepatic TG depends on thedynamic balance of multiple pathways, including de novo synthesis,oxidation and very low-density lipoprotein (VLDL) transport. Defects

pG2 cells. HepG2 cells were cultured in Dulbecco's modified Eagle's medium containinger with 60mg/L OA. A: SAM/SAH ratio in HepG2 cells. B:MTTPmRNA expression level in

335L.-J. Wang et al. / Journal of Nutritional Biochemistry 25 (2014) 329–336

in one or more pathways could lead to hepatic steatosis; thus,correcting this defect is a promising strategy for the treatment ofhepatic steatosis. FAS and ACC are key rate-limiting enzymes in denovo synthesis, and their activities determine the ability of OAsynthesis. Although ACC expression was not significantly changed,FAS expression was significantly decreased in the betaine-supple-mented mice compared to that of the HFD-fed mice, indicating thatbetaine supplementation partially inhibits the HFD-induced excessivesynthesis of fatty acids. The oxidation of fatty acids occurs mainly inthe mitochondria. When β-oxidation in the mitochondria is impairedby fatty acid overload, the alternative peroxisomal β-oxidation andmicrosomal ω-oxidation pathways will be activated. ACOX is the firstand rate-limiting enzyme in the peroxisomal β-oxidation pathway.Our study discovered an increase of ACOX expression in the HFD-fedmice, indicating that β-oxidation in peroxisome was compensativelyenhanced by HFD, but this condition failed to occur when feedingwith betaine. PPARα serves as a key regulator of the hepatic lipidoxidation-related genes [26]. PPARα-null mice are characterized byfatty acid oxidation disorders and have been demonstrated as a usefulmodel for fatty liver for decades [2]; in addition, PPARα agonists havebeen shown to attenuate hepatic steatosis [27]. In the present study,PPARα was dramatically down-regulated in the HFD-fed mice andbetaine supplementation increased its expression, indicating that up-regulation by betaine may promote the PPARα-mediated response,which was inhibited by an HFD. MTTP plays a vital role in lipoproteinassembly. In 1992, Wetterau et al. [28] demonstrated that defects inMTTP led to the lack of TG transfer activity and caused abetalipopro-teinemia. Consistent with later studies, we found a down-regulationof MTTP expression in the HFD group, and the contribution of down-regulated MTTP expression to fatty liver was further demonstratedby decreased amount of TG in the VLDL particles [17]. Moreover,ApoB is an important component for MTTP binding; we also founddecreased ApoB mRNA expression in the HFD group. Betainesupplementation up-regulated MTTP mRNA expression in a dose-dependent manner. Generally speaking, betaine supplementationcould counteract the HFD-induced dysregulation of gene expression,as shown by the down-regulation of genes involved in de novosynthesis and up-regulation of genes involved in fatty acid oxidationand VLDL–TG transfer.

4.2. Betaine and DNA methylation

Gene expression generally exhibits a negative relationship withthe DNA methylation of CpG islands in the gene promoter region.Sookoian et al. [29] reported that an increase of PPARGC1Amethylation correlated with decreased mRNA expression in theliver and contributed to insulin resistance in NAFLD patients. In HFD-fed mice, the increased methylation of the PPARγ promoteraccompanied by the decreased expression of PPARγmRNA in visceraladipose tissues was associated with the pathogenesis of metabolicsyndrome [30]. These data suggest that correcting the aberrant DNAmethylation of specific genes is a promising strategy for the treatmentof hepatic steatosis. In the present study, we observed that the DNAmethylation levels at specific CpG sites of the MTTP promoter (−184,−156,−63,−60) were significantly increased in the liver of HFD-fedmice; however, betaine supplementation effectively decreased theHFD-induced hypermethylation. We further observed an inversecorrelation between the DNA methylation of MTTP promoter and itsmRNA expression. These results suggest that betaine exerts adecreasing effect on DNA methylation of MTTP promoter. This inturn up-regulates the MTTP mRNA and protein expression, whichfurther enhances MTTP function in the assembly of VLDL, ultimatelyrelieving hepatic steatosis by transporting TG in form of VLDL–TG.

In normal tissues, the majority of CpG islands in the promoterregion are methylation-free, while repetitive sequences and inter-

spread CpG dinucleotides in the genome are heavily methylated [31].Conversely, hypermethylation of gene-specific promoters and globalDNA hypomethylation could simultaneously present in tumor tissues[6]. However, this aberrant DNA methylation status has never beenreported in NAFLD. Here, we first found that HFD caused hyper-methylation of the MTTP promoter and global DNA hypomethylationin mice. More importantly, betaine supplementation markedlyincreased global DNA methylation in a dose-dependent manner.Genomic hypomethylation has been associated with aberrantchromosomal structure, resulting in genomic instability and abnor-mal gene expression [32]. The improvement on the dysregulation oflipid metabolism genes by betaine supplementation in NAFLD mayrelate to the effect of betaine on increasing global DNA methylation.

4.3. Betaine, methyl group metabolism and DNA methylation

Although the mechanisms for the betaine effect on DNAmethylation of gene promoters and the genome are not clearlyunderstood, it is possible that they are related to the function ofbetaine as a methyl donor. Betaine, oxidized from choline in vivo,serves as a methyl group for the conversion of Hcy to methionine(Met), which is further converted to SAM. SAM is a universal methyldonor for numerous methylation reactions, including DNA methyla-tion, and its concentration indicates the methylation capacity of thecell. In mammals, methyl donors such as betaine and choline arederived from diet; thus, adequate dietary betaine and choline intake isnecessary to maintain SAM concentrations and the DNA methylationcapacity in the cell [6,8,9].

In the present study, betaine supplementation effectively revisedthe HFD-induced hypomethylation, most likely because of itsenhancement on methylation capacity. Indeed, betaine supplemen-tation markedly increased the hepatic SAM concentrations and SAM/SAH ratios in a dose-dependent manner compared to HFD. Similarchanges also emerged for the SAM/SAH ratios in HepG2 cells thatwere incubated with OA and betaine. In addition to the crucial role ofSAM in DNA methylation, disorders of the other metabolites (e.g.,choline, Hcy) in methyl group metabolism also promote aberrantgenomic methylation. Rodents fed a choline-deficient diet displayedprofound loss of genomic methylcytosine [6,33]. HFD can reducecholine bioavailability for the host and mimic the choline-deficientdiet contribution to NAFLD [34]. We found that betaine supplemen-tation significantly normalized the HFD-induced reduction in serumcholine. Moreover, both human subjects and animal models withhyperhomocysteine (HHcy) from genetic defects or dietary depletionof methyl donors displayed a diminished methylation capacity andglobal DNA hypomethylation [11,35]. Our studies and those of otherresearchers have found that HFD increases the Hcy level in theserum [17,36], and betaine supplementation dose dependentlyattenuated HFD-induced HHcy. Together, these findings supportthe effect of betaine on increasing the global DNA methylation level,which predominantly results from revising the HFD-inducedimpairment of the methylation capacity via the normalization ofmethyl group metabolism.

Until now, many mechanisms of demethylation in mammals havebeen proposed but without conclusive evidence [37,38]. Themechanisms behind the hypomethylated effect of betaine on theMTTP promoter are also difficult to define. One possible explanationcould be considered. DNAmethylation is catalyzed by a family of DNAmethyltransferases (DNMTs) that transfer methyl group from SAM.Rats fed a methyl donor-deficient diet frequently displayed anincreased expression of DNMTs, possibly as a compensatory mech-anism for the diminished methylation capacity [39,40]. In the presentstudy, HFD significantly caused reduced methylation capacitythrough the loss of choline and SAM. Furthermore, HFD has beenshown to up-regulate DNMT1 expression and increase the

336 L.-J. Wang et al. / Journal of Nutritional Biochemistry 25 (2014) 329–336

methylation level of gene promoters [41]. Given that betainesupplementation effectively revises the disordered methyl groupmetabolism and enhances the methylation capacity in our study, wededuced that the feedback of DNMTs by HFD could be counteractedand the DNA methylation levels of gene promoters could benormalized by betaine supplement. In HepG2 cells incubated withOA, treatmentwith either betaine or 5-aza both showed an increase inMTTP expression. 5-Aza is a hypomethylating agent, and it hypo-methylates DNA by inhibiting DNMTs. Therefore, the demethylatedeffect of betaine on theMTTP promoter is more likely to be associatedwith the repression of DNMTs.

5. Conclusion

In summary, betaine supplementation reversed the hypermethy-lation of the MTTP promoter and the genomic hypomethylationinduced by anHFD via the regulation ofmethyl groupmetabolism andconsequently improved NAFLD. The modified DNA methylation ofboth the genome and specific genes by betaine is a new andunreported mechanism in the process of NAFLD, and these findingsreveal a novel role for betaine in the treatment of NAFLD.

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jnutbio.2013.11.007.

References

[1] Yoshihisa T, Yurie S, Toshio F. Animal models of nonalcoholic fatty liverdisease/nonalcoholic steatohepatitis. World J Gastroenterol 2012;18:2300–8.

[2] Leone TC, Weinheimer CJ, Kelly DP. A critical role for the peroxisome proliferator-activated receptor alpha (PPARalpha) in the cellular fasting response: thePPARalpha-null mouse as a model of fatty acid oxidation disorders. Proc NatlAcad Sci U S A 1999;96:7473–8.

[3] Zhu L, Baker SS, Liu W, Tao MH, Patel R, Nowak NJ, et al. Lipid in the livers ofadolescents with nonalcoholic steatohepatitis: combined effects of pathways onsteatosis. Metabolism 2011;60:1001–11.

[4] Li YY. Genetic and epigenetic variants influencing the development of nonalco-holic fatty liver disease. World J Gastroenterol 2012;18:6546–51.

[5] Stidley CA, Picchi MA, Leng S,Willink R, Crowell RE, Flores KG, et al. Multivitamins,folate, and green vegetables protect against gene promoter methylation in theaerodigestive tract of smokers. Cancer Res 2010;70:568–74.

[6] Tryndyak VP, Han T, Muskhelishvili L, Fuscoe JC, Ross SA, Beland FA, et al. Couplingglobal methylation and gene expression profiles reveal key pathophysiologicalevents in liver injury induced by a methyl-deficient diet. Mol Nutr Food Res2011;55:411–8.

[7] Kim S, Sohn I, Ahn JI, Lee KH, Lee YS, Lee YS. Hepatic gene expression profiles in along-term high-fat diet-induced obesity mouse model. Gene 2004;340:99–109.

[8] Song Z, Deaciuc I, Zhou Z, Song M, Chen T, Hill D, et al. Involvement of AMP-activated protein kinase in beneficial effects of betaine on high-sucrose diet-induced hepatic steatosis. Am J Physiol Gastrointest Liver Physiol 2007;293:G894–902.

[9] Achon M, Alonso-Aperte E, Reyes L, Ubeda N, Varela-Moreiras G. High-dose folicacid supplementation in rats: effects on gestation and the methionine cycle. Br JNutr 2000;83:177–83.

[10] Niculescu MD, Zeisel SH. Diet, methyl donors and DNA methylation: interactionsbetween dietary folate, methionine and choline. J Nutr 2002;132:2333S–5S.

[11] Jacob RA, Gretz DM, Taylor PC, James SJ, Pogribny IP, Miller BJ, et al. Moderatefolate depletion increases plasma homocysteine and decreases lymphocyte DNAmethylation in postmenopausal women. J Nutr 1998;128:1204–12.

[12] Wang Z, Yao T, Pini M, Zhou Z, Fantuzzi G, Song Z. Betaine improved adipose tissuefunction in mice fed a high-fat diet: a mechanism for hepatoprotective effect ofbetaine in nonalcoholic fatty liver disease. Am J Physiol Gastrointest Liver Physiol2010;298:G634–42.

[13] Kwon DY, Jung YS, Kim SJ, Park HK, Park JH, Kim YC. Impaired sulfur-amino acidmetabolism and oxidative stress in nonalcoholic fatty liver are alleviated bybetaine supplementation in rats. J Nutr 2009;139:63–8.

[14] Kathirvel E, Morgan K, Nandgiri G, Sandoval BC, Caudill MA, Bottiglieri T, et al.Betaine improves nonalcoholic fatty liver and associated hepatic insulinresistance: a potential mechanism for hepatoprotection by betaine. Am J PhysiolGastrointest Liver Physiol 2010;299:G1068–77.

[15] Abdelmalek MF, Sanderson SO, Angulo P, Soldevila-Pico C, Liu C, Peter J, et al.Betaine for nonalcoholic fatty liver disease: results of a randomized placebo-controlled trial. Hepatology 2009;50:1818–26.

[16] Abdelmalek MF, Angulo P, Jorgensen RA, Sylvestre PB, Lindor KD. Betaine, apromising new agent for patients with nonalcoholic steatohepatitis: results of apilot study. Am J Gastroenterol 2001;96:2711–7.

[17] Chang X, Yan H, Fei J, Jiang M, Zhu H, Lu D, et al. Berberine reduces methylation ofthe MTTP promoter and alleviates fatty liver induced by a high-fat diet in rats.J Lipid Res 2010;51:2504–15.

[18] Kang X, Zhong W, Liu J, Song Z, McClain CJ, Kang YJ, et al. Zinc supplementationreverses alcohol-induced steatosis in mice through reactivating hepatocytenuclear factor-4alpha and peroxisome proliferator-activated receptor- alpha.Hepatology 2009;50:1241–50.

[19] Quesada H, Del BJ, Pajuelo D, Diaz S, Fernandez-Larrea J, PinentM, et al. Grape seedproanthocyanidins correct dyslipidemia associated with a high-fat diet in rats andrepress genes controlling lipogenesis and VLDL assembling in liver. Int J Obes(Lond) 2009;33:1007–12.

[20] Wang L, Chen L, Tan Y, Wei J, Chang Y, Jin T, et al. Betaine supplement alleviateshepatic triglyceride accumulation of apolipoprotein E deficient mice via reducingmethylation of peroxisomal proliferator-activated receptor alpha promoter.Lipids Health Dis 2013;12:34.

[21] Holm PI, Ueland PM, Kvalheim G, Lien EA. Determination of choline, betaine, anddimethylglycine in plasma by a high-throughput method based on normal-phasechromatography-tandem mass spectrometry. Clin Chem 2003;49:286–94.

[22] Nolin TD, McMenamin ME, Himmelfarb J. Simultaneous determination of totalhomocysteine, cysteine, cysteinylglycine, and glutathione in human plasma byhigh-performance liquid chromatography: application to studies of oxidativestress. J Chromatogr B Analyt Technol Biomed Life Sci 2007;852:554–61.

[23] Wang W, Kramer PM, Yang S, Pereira MA, Tao L. Reversed-phase high-performance liquid chromatography procedure for the simultaneous determina-tion of S-adenosyl-L-methionine and S-adenosyl-L-homocysteine in mouse liverand the effect of methionine on their concentrations. J Chromatogr B Biomed SciAppl 2001;762:59–65.

[24] Jiang M, Zhang Y, Fei J, Chang X, Fan W, Qian X, et al. Rapid quantification of DNAmethylation bymeasuring relative peak heights in direct bisulfite-PCR sequencingtraces. Lab Invest 2010;90:282–90.

[25] Matthews JO, Southern LL, Higbie AD, Persica MA, Bidner TD. Effects of betaine ongrowth, carcass characteristics, pork quality, and plasma metabolites of finishingpigs. J Anim Sci 2001;79(3):722–8.

[26] Rakhshandehroo M, Hooiveld G, Muller M, Kersten S. Comparative analysis ofgene regulation by the transcription factor PPARalpha between mouse andhuman. PLoS One 2009;4:e6796.

[27] Fischer M, You M, Matsumoto M, Crabb DW. Peroxisome proliferator-activatedreceptor alpha (PPARalpha) agonist treatment reverses PPARalpha dysfunctionand abnormalities in hepatic lipid metabolism in ethanol-fed mice. J Biol Chem2003;278:27997–8004.

[28] Wetterau JR, Aggerbeck LP, Bouma ME, Eisenberg C, Munck A, Hermier M, et al.Absence of microsomal triglyceride transfer protein in individuals withabetalipoproteinemia. Science 1992;258:999–1001.

[29] Sookoian S, Rosselli MS, Gemma C, Burgueno AL, Fernandez GT, Castano GO, et al.Epigenetic regulation of insulin resistance in nonalcoholic fatty liver disease:impact of liver methylation of the peroxisome proliferator-activated receptorgamma coactivator 1alpha promoter. Hepatology 2010;52:1992–2000.

[30] Fujiki K, Kano F, Shiota K, Murata M. Expression of the peroxisome proliferatoractivated receptor gamma gene is repressed by DNA methylation in visceraladipose tissue of mouse models of diabetes. BMC Biol 2009;7:38.

[31] Brena RM, Huang TH, Plass C. Quantitative assessment of DNA methylation:potential applications for disease diagnosis, classification, and prognosis in clinicalsettings. J Mol Med (Berl) 2006;84:365–77.

[32] Chen RZ, Pettersson U, Beard C, Jackson-Grusby L, Jaenisch R. DNA hypomethyla-tion leads to elevated mutation rates. Nature 1998;395:89–93.

[33] Wainfan E, Dizik M, Stender M, Christman JK. Rapid appearance of hypomethy-lated DNA in livers of rats fed cancer-promoting, methyl-deficient diets. CancerRes 1989;49:4094–7.

[34] Dumas ME, Barton RH, Toye A, Cloarec O, Blancher C, Rothwell A, et al. Metabolicprofiling reveals a contribution of gut microbiota to fatty liver phenotype ininsulin-resistant mice. Proc Natl Acad Sci U S A 2006;103:12511–6.

[35] Kim JM, Hong K, Lee JH, Lee S, Chang N. Effect of folate deficiency on placental DNAmethylation in hyperhomocysteinemic rats. J Nutr Biochem 2009;20:172–6.

[36] Bravo E, Palleschi S, Aspichueta P, Buque X, Rossi B, Cano A, et al. High fat diet-induced non alcoholic fatty liver disease in rats is associated with hyperhomo-cysteinemia caused by down regulation of the transsulphuration pathway. LipidsHealth Dis 2011;10:60.

[37] Kangaspeska S, Stride B, Metivier R, Polycarpou-Schwarz M, Ibberson D,Carmouche RP, et al. Transient cyclical methylation of promoter DNA. Nature2008;452:112–5.

[38] Ooi SK, Bestor TH. The colorful history of active DNA demethylation. Cell2008;133:1145–8.

[39] Ghoshal K, Li X, Datta J, Bai S, Pogribny I, Pogribny M, et al. A folate- and methyl-deficient diet alters the expression of DNA methyltransferases and methyl CpGbinding proteins involved in epigenetic gene silencing in livers of F344 rats. J Nutr2006;136:1522–7.

[40] KovachevaVP,Mellott TJ, Davison JM,WagnerN, Lopez-Coviella I, SchnitzlerAC, et al.Gestational choline deficiency causes global and Igf2 geneDNAhypermethylation byup-regulation of Dnmt1 expression. J Biol Chem 2007;282:31777–88.

[41] de Assis S, Warri A, Cruz MI, Laja O, Tian Y, Zhang B, et al. High-fat or ethinyl-oestradiol intake during pregnancy increases mammary cancer risk in severalgenerations of offspring. Nat Commun 2012;3:1053.