The Journal of Nutrition

Biochemical, Molecular, and Genetic Mechanisms

Liver Choline Dehydrogenase and KidneyBetaine-Homocysteine MethyltransferaseExpression Are Not Affected by Methionineor Choline Intake in Growing Rats1

Sandy Slow and Timothy A. Garrow*

Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana IL 61801

Abstract

Choline dehydrogenase (CHDH) and betaine-homocysteine methyltransferase (BHMT) are 2 enzymes involved in choline

oxidation. BHMT is expressed at high levels in rat liver and its expression is regulated by dietary Met and choline. BHMT is

also found in rat kidney, albeit in substantially lower amounts, but it is not known whether kidney BHMT expression is

regulated by dietary Met or choline. Similarly, CHDH activity is highest in the liver and kidney, but the regulation of its

expression by diet has not been thoroughly investigated. Sprague Dawley rats (;50 g) were fed, for 9 d in 2 3 3 factorial

design (n¼8), an L-amino acid–defined diet varying in L-Met (0.125, 0.3, or 0.8%) and choline (0 or 25 mmol/kg diet). Liver and

kidney BHMT and CHDH were assessed using enzymatic, Western blot, and real-time PCR analyses. Liver samples were

also fixed for histological analysis. Liver BHMT activity was 1.3-fold higher in rats fed the Met deficient diet containing

choline, which was reflected in corresponding increases in mRNA content and immunodetectable protein. Independent of

dietary choline, supplemental Met increased hepatic BHMT activity;30%. Kidney BHMT and liver CHDH expression were

refractory to these diets. Some degree of fatty liver developed in all rats fed a choline-devoid diet, indicating that supple-

mental Met cannot completely compensate for the lack of dietary choline in growing rats. J. Nutr. 136: 2279–2283, 2006.

Introduction

Betaine (Bet;2 N,N,N-trimethylglycine) is an intermediate in thecholine oxidation pathway. The first committed step for the for-mation of Bet is catalyzed by choline dehydrogenase (CHDH;EC 1.1.99.1), which localizes to the matrix side of the innermitochondrial membrane. It is an FAD-dependent enzyme that isthought to be connected to ubiquinone (coenzyme Q) in the elec-tron transport chain (1,2). CHDH catalyzes the conversion ofcholine to Bet aldehyde, which is further oxidized to Bet bythe mitochondrial matrix enzyme, Bet aldehyde dehydrogenase(BADH; EC 1.2.1.8). Once formed, Bet leaves the mitochondriaand can be demethylated by betaine-homocysteine methyltrans-ferase (BHMT: EC 2.1.1.5), the third enzyme in the cholineoxidation pathway. BHMT catalyzes the transfer of a methylgroup from Bet to homocysteine (Hcy), forming dimethylglycine(DMG) and methionine (Met), respectively. Following theBHMT reaction, the last 2 reactions of choline oxidationconvert DMG to glycine and are catalyzed by the mitochondrialenzymes dimethylglycine dehydrogenase and sarcosine dehy-drogenase, respectively.

BHMT has a major role in the overall conversion of Hcy toMet (3), and more recently it has been shown that in vivoinhibition of BHMT in mice causes hyperhomocysteimia and areduction in liver S-adenosylmethionine (4). There is substantialinterest in the regulation of Hcy and Met metabolism, becausean elevated level of plasma total Hcy is thought to be anindependent risk factor for the development of coronary, ce-rebral, and peripheral vascular disease (5,6). BHMT is abundantin mammalian liver (0.5–2% of soluble liver protein), and it isalso found in the kidney of some species, particularly humans,pigs, and guinea pigs (7–9), but substantially lower levels arefound in rat kidney (.95% lower specific activity than rat liver).Dietary Met and methyl donor (Bet and choline) intake havebeen shown to modulate liver BHMT activity and gene expres-sion. Specifically, diets deficient inMet with excess methyl donorhave significantly increased hepatic BHMT activity and expres-sion in rats (10–12). However, there are few data concerning theeffect of supplemental Met on hepatic BHMT, nor has thenutritional regulation of kidney BHMT been examined.

CHDH is primarily active in the liver and kidney of mammals(1) but, relative to other respiratory chain enzymes, it has notbeen extensively studied. Little is known about its kineticproperties and whether expression or flux through the enzyme isinfluenced by diet and/or physiological state. We postulated that,because CHDH is the first step in the oxidation of choline andprovides the Bet that BHMTuses, both dietary Met and choline

1 This material is based upon work supported by the NIH (DK52501 to T.A.

Garrow).2 Abbreviations used: BADH, Bet aldehyde dehydrogenase; Bet, betaine; BHMT,

Betaine-homocysteine methyltransferase; CHDH, choline dehydrogenase;

DMG, dimethylglycine; Hcy, homocysteine; MGB, minor groove binding.

* To whom correspondence should be addressed. E-mail: tagarrow@uiuc.edu.

0022-3166/06 $8.00 ª 2006 American Society for Nutrition. 2279Manuscript received 16 March 2006. Initial review completed 12 April 2006. Revision accepted 13 June 2006.

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availability may affect CHDH expression in a manner similar tothat of BHMT. Indeed, Schneider and Vance (13) reported thatrats fed a choline deficient diet have a 30–41% reduction in themitochondrial oxidation of choline to betaine.

We sought to determine whether BHMT and CHDH are co-ordinately regulated by dietary Met and choline, how supple-mental Met affects hepatic BHMT, and whether these nutrientsare involved in the regulation of kidney BHMT expression.

Materials and Methods

Diets and animal protocol

The experimental protocol was approved by the University of Illinois

Institutional Animal Care and Use Committee. The nutrient levels in theL-amino acid–defined diets used in this study, except for L-Met, L-cystine,

and choline, were at the levels recommended for growing rats by the

American Institute of Nutrition (AIN-93G) and were prepared by Dyets.

The diets varied in L-Met and choline concentrations (Table 1). Thedietary treatments were chosen based on the requirements for growing

rodents as described by the National Research Council (14,15). The

recommended level of Met in a purified diet was defined as 6 g/kg diet

(0.6%), where half of theMet could be replaced with cystine (0.3%Met;0.3% cystine). All diets used in this study contained 3 g cystine/kg diet

(0.3%) and diets varied in Met by the following 3 levels: 1.25 g Met/kg

diet (0.125%), termed deficient; 3 g Met/kg diet (0.3%), termed ade-quate; and 8 g Met/kg diet (0.8%), termed supplemental. Whereas the

recommendations for the total sulfur amino acid (Met 1 cystine) con-

centrations of a purified diet for maximum growth in rodents has been

subsequently reported to be 7.4 g/kg diet (16), the levels as defined in thisstudy (deficient, adequate, and supplemental) are still applicable and

have been used in previous studies, both in this laboratory and others, to

investigate the affect of these nutrients on BHMT expression (10–12).

Similarly, the recommended intake of choline for the growing rodent hasbeen defined as 10 mmol/kg diet. Two levels of choline were used in this

study (0 mmol/kg diet, termed devoid and 25 mmol/kg diet, termed

supplemental).All diets contained 10 g/kg diet of succinylsulfathiazole (Sigma), an

antibiotic that was used to inhibit the microbial metabolism of choline in

the gastrointestinal tract, but, because of its use, all diets were supple-

mented with menadione sodium bisulfite (50 mg/kg diet; Sigma).The feeding trial was conducted using 3-wk old Sprague-Dawley rats

(n ¼ 48; Harlan), which were housed in standard shoebox housing (3/

cage) in a light (12-h light/12-h dark) and temperature (23�C) controlledroom. All rats were given free access to a Met and choline adequate diet(3 g/kgand10mmol/kgofdiet, respectively) for 3d to allowadjustment to

the purified diet. Following the adjustment period, the rats were divided

into 6 groups of 8, such that mean body weight did not differ among

groups. Ratswere housed individually in hanging stainless-steel cages. Allrats had free access to water and were provided 1 of the 6 diets described

above. The duration of the feeding study (9 d) was based on previ-

ous reports investigating the effect of these nutrients on BHMT expres-sion, where the typical feeding study duration ranged from 5 to 16 d

(10–12).

When rats are fed diets severely restricted in any essential amino acid,

they voluntarily decrease their food intake by up to 40–50% (g/d). There-fore, food intake among the groups fed the adequate and supplemental

Met diets (3.0 and 8.0 g/kg diet, respectively) were restricted to the mean

food intake of the groups fed the diets deficient in Met (1.25 g/kg diet).

Two groups consumed the Met deficient diets ad libitum; intakes did notdiffer between these groups. For each rat, food intake and weight were

recorded daily. Rats were killed via CO2 inhalation at the end of the trial

period and their liver and kidneys excised, flash frozen in liquid nitrogen,and stored at 280�C until analysis.

Histochemistry

Liver samples were fixed in 10% buffered formalin (12 h) and stored in

70% ethanol until embedding in paraffin. Paraffin blocks were sectioned

into 3-mm slices, mounted onto glass slides, and stained with hematox-ylin and eosin.

Assay procedures

Enzyme assays. Liver BHMT specific activity was determined as

described by Garrow (17). Kidney BHMT was measured the sameway, except that the concentration and specific activity of the 14C-Bet

(250 mmol/L; 0.5 mCi) was changed to increase the assay sensitivity.

Liver mitochondrial CHDH activity was measured as described previ-

ously (18).

Western analysis. Liver and kidney homogenates were probed forBHMT protein by the procedure described by Delgado-Reyes and

Garrow (18). Liver mitochondria were probed for CHDH protein using

the same procedure, except 5 mg total protein were loaded onto the gels

and primary antibodies (polyclonal) were prepared from rabbitsimmunized with CHDH peptides conjugated to bovine serum albumin

(T. A. Garrow, unpublished data).

Relative quantification of BHMT and CHDH expression by TaqMan

real-time PCR. Rat liver and kidney total RNA isolation was performedusing the SV total RNA system kit (Promega). First strand cDNA syn-

thesis was performed using total RNA, random hexamer primers, and

MultiScribe reverse transcriptase from the TaqMan reverse transcriptase

reagents (ABI). The oligonucleotide primers and TaqMan minor groovebinding (MGB) probes used for real-time PCR amplification were

as follows: BHMT 5#CGGGCAGACCGTACAATCC 3# (sense primer),

5#-CTTTCGTCACTCCCCAAGCA-3# (antisense primer), 5#-6FAM-

CGATGTCCAAGCCGG-3# (MGB probe); and CHDH 5#-AAGGAC-GGCCAGAGCCACAA-3# (sense primer), 5#-GCCCCCGCTCAGGA-

TCA-3# (antisense primer), 5#-6FAM-CTTACGTCAGCAGGGAG-3#(MGB probe). The ribosomal 18S gene was used as the endogenous

control (TaqMan 18S ribosomal control kit; ABI). Real-time PCR wasperformed in triplicate on an ABI Prism 7700 sequence detection system

(384-well plate). The relative abundance of amplified cDNA was

calculated using the standard curve method, where an independent ratliver cDNA sample was diluted (a total of 6, 10-fold serial dilutions

producing concentrations ranging from 100 ng to 10 pg cDNA) and real-

time PCR performed in triplicate to generate the standard curve for each

gene (BHMT, CHDH, and 18S). Results were calculated as mean relativeBHMT or CHDH expression/18S mRNA values.

For BHMT and CHDH enzyme activity and mRNA expression

analyses, treatment group 4 (0.3% Met; 25 mmol/kg of choline) was

assigned the relative value of 1 and was used as the control group.Although a true control diet would contain both adequate Met (0.3%)

and adequate choline (10 mmol/kg diet) we chose not to include this

treatment group in the present study because previous results showedthat liver BHMT activity (our positive control for dietary treatment) is

refractory to changes in choline intake when dietary Met is adequate or

supplemental (10,12). Rather than adding another level of choline

(10 mmol/kg diet) in our study, we opted to use only 2 levels of choline(devoid or supplemental) because we knew these diets should affect liver

BHMTexpression and also simplify our experimental design to a 2-way

ANOVA.

Statistics

Tests for significant differences among the 6 groups were performed by

2-way ANOVA, using the general linear model procedure of SAS/STAT

(SAS). When significant main effects were obtained, the post-hoc

Bonferroni correction for multiple pair-wise comparisons was applied.Differences were considered significant at P # 0.05. Data are expressed

as means 6 SEM.

Results

Growth. Rats fed theMet-deficient diets (groups 1 and 2) gainedless weight (P , 0.001) than those fed the diets containingadequate (groups 3 and 4) and supplemental (groups 5 and 6)Met (Table 1). The gain-to-food intake ratios indicated that the0.125% Met diets were severely deficient, although all ratsgained weight. The growth performance of rats fed the Met-adequate diet devoid of choline (group 3) was significantly lower

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than that of rats fed the diet with supplemental levels of Met andcholine (group 6), but it did not significantly differ in rats fed theMet-adequate diet containing supplemental choline (group 4) orin those fed the diet containing excess Met but devoid of choline(group 5; Table 1).

Liver and kidney BHMT.We expected that the diets used in thisstudy would result in a range of hepatic BHMT activities(10–12), which we observed (Fig. 1A). Rats fed theMet-deficientdiet containing supplemental choline (group 2) had higherBHMT activities (P , 0.01) than the other groups. BHMTactivity was 1.3-fold higher in this group than in rats fed theMet-adequate diet containing supplemental choline (group 4),which is consistent with previous findings (11,12). Liver BHMTactivity also was ;30% greater in rats fed the diets containingsupplemental Met (groups 5 and 6) than in those fed the Met-adequate diets (groups 3 and 4). Nonetheless, BHMT activitiesof rats fed the Met-supplemented diets were significantly lowerthan those consuming the diet deficient in Met with supplemen-tal choline (Fig. 1A). As expected (10,12), adding choline toeither the Met-adequate or Met-supplemented diets did notaffect hepatic BHMT activity. Liver BHMT protein contentmirrored activity levels (Fig. 1B).

Liver relative BHMT mRNA expression was also higher inrats fed the Met-deficient diet containing supplemental cholinethan in those fed theMet-adequate diet containing supplementalcholine (1.7-fold; P ¼ 0.032). The variation of BHMT mRNAmeasured within groups precluded us from detecting anyadditional significant differences.

Rat kidney BHMT activity was not significantly affected bythe diet treatments (Fig. 1C). Moreover, kidney BHMT mRNAexpression (data not shown) and immunodetectable proteincontent did not differ among the groups (Fig. 1D). Within-groupvariationof kidneyBHMTexpressionwasmuch greater than thatof liver BHMTexpression.

Liver CHDH. Although statistically there was a significant effectof methionine on CHDH activity, and a choline-methionineinteraction, the magnitude of the differences was small (Fig. 2A)and probably not physiologically important. Indeed, the differ-ences in activity were not reflected in CHDH protein concen-trations (Fig. 2B) or hepatic CHDHmRNA expression (data notshown).

Liver lipids. Fatty liver was observed in all rats fed the dietsdevoid of choline (Fig. 3A,C,E). However, the severity of fattyinfiltration decreased as the Met concentration of the dietincreased. It is important that even supplemental Met (8 g/kg

diet) did not completely prevent lipids from accumulating in theliver, indicating that supplemental Met cannot completely re-place the need for choline to prevent the development of fattyliver in young growing rats.

Discussion

Previous studies have shown that the specific activity and ex-pression of hepatic BHMT varies with the dietary intake of sulfuramino acids, choline, and Bet (10–12,19). Specifically, the greatestchanges have been in animals fed Met-deficient diets in combina-tion with excess methyl donor (choline or Bet), with the up-regulation increasing with the severity of Met-restriction and thedegree of methyl donor supplementation. These findings wereconfirmed in this study. There was a#1.7-fold increase in hepaticBHMTactivity, protein content, andmRNA expression in rats feda Met-deficient diet containing supplemental choline (0.125%Met; 25mmol choline/kg diet) over those fed aMet-adequate dietwith supplemental choline (0.3% Met; 25 mmol choline/kg diet)(Fig. 1A,B). Inaddition, rats feddiets containing supplementalMet

Figure 1 Hepatic (A) and kidney (C) BHMT activity in rats fed diets varying in

Met and choline concentrations for 9 d. Groups: 1) Met-deficient, choline

devoid; 2) Met-deficient, supplemental choline; 3) Met-adequate, choline

devoid; 4) Met-adequate, supplemental choline; 5) Supplemental Met, choline

devoid; and 6) Supplemental Met, supplemental choline. The BHMT activity of

treatment group 4 was assigned the relative value of 1, and all other values are

expressed relative to this. Values are means 6 SE, n ¼ 8. Means without a

common letter differ (P , 0.05). A representative Western blot of diet-induced

changes in hepatic (B) and kidney (D) BHMT protein content.

TABLE 1 Diets and growth performance of rats fed diets

varying in Met and choline1

Group Met Choline Weight gain2 Feed efficiency

g/kg mmol/kg g/ 9 d g gain/kg feed

1 1.25 0 25.0 6 1.2c 265.0 6 14.4c

2 1.25 25 20.2 6 2.6c 245.9 6 26.1c

3 3 0 32.8 6 0.6b 379.1 6 7.8b

4 3 25 37.0 6 1.0a,b 424.2 6 11.9a,b

5 8 0 35.6 6 1.4a,b 410.0 6 15.9a,b

6 8 25 39.7 6 1.8a 457.5 6 21.2a

1 Values are means 6 SEM, n ¼ 8. Means in a column without a common letter differ

(P , 0.05).2 Mean initial weight was 63 g.

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(0.8%, groups 5 and 6) had increased hepatic BHMT activity(;30–40%). These activity changes were also mirrored byincreased protein concentrations, as detected by Western blot(Fig. 1B). The results for liver BHMTexpressionwere as expected,

based on previouswork fromour laboratory (11,12) and of others(10,19). Hence, quantifying liver BHMT expression in this studyserved as a positive control for the dietary treatments.

This report is, to our knowledge, the first to have evaluatedthe effect of these nutrients on kidney BHMT activity. WhereasBHMT is expressed primarily in the liver of rats (7,8) it is presentin the kidney, with.95% lower specific activity than that of theliver (T. A. Garrow, unpublished results). Our data show thatkidney BHMT is refractory to dietary Met and choline intake(Fig. 1C). The differential tissue regulation of BHMT betweenliver and kidney is likely to be a means of controlling tissue Betconcentrations. Bet is not only a methyl donor, but it is accu-mulated as an osmoprotectant in a variety of cells, particularlythe kidneymedulla, where it protects the cells from damage fromhigh salt and urea concentrations. Thus, the primary role of Betin the kidney is likely to be as an osmoprotectant, rather than amethyl donor. Indeed, in guinea pigs, high sodium chlorideintake decreases both kidney and liver BHMTactivity by#50%(18).

CHDHmediates the first and committed step in the oxidationof choline to Bet. To date, very few data are available regardingthe potential regulation of mammalian CHDH expression bydiet. In Chesapeake Bay and Atlantic oysters, it has been re-ported that CHDH cannot function rapidly enough to saturateBADH, thus CHDH activity might be the rate-limiting step ofendogenous Bet synthesis. CHDH has also been shown to becompetitively inhibited by both Bet aldehyde and Bet (20).CHDH may have an important role in modulating Bet concen-trations and, if so, its activity could modulate flux throughBHMT.We sought to determine whether hepatic CHDH expres-sion is modulated by dietary Met and choline availability in amanner similar to BHMT. However, our results indicate thatCHDH is not affected by dietary Met and choline. No dif-ferences in mRNA levels (not shown) or protein concentration(Fig. 2B) were observed in any of the treatment groups. Al-though there was a significant interaction between Met andcholine on hepatic CHDH activity, the effect was small (Fig. 2A)and probably not physiologically important. Supporting ourfindings, Wong and Thompson also found no effect on eitherhepatic CHDH or BADH activity in rats fed a choline deficientdiet for 2 d and only a small reduction when choline deficiencywas prolonged for up to 13 d (21).

Schneider and Vance (13) reported that the oxidation ofcholine to Bet was reduced in mitochondria isolated from ratsfed choline deficient diets, compared with those fed choline suf-ficient diets. In addition, mitochondria from phosphatidyletha-nolamine N-methyltransferase and multidrug-resistant protein-2double knockout mice have little ability to oxidize choline toBet, as reported by Li et al. (22). It is possible the reduction incholine oxidation measured in these studies was the result of areduction of CHDH expression, which would conflict with theresults reported here and those of Wong and Thompson (21).However, no specific measurement of CHDH activity or protein,or mRNA content were reported in the studies by Schneider andVance (13) or Li et al. (22). One possibility that might explainthese seemingly disparate results is that the rate-limiting step inthe oxidation of choline to Bet formation could be its transportinto the mitochondria, rather than CHDH activity. Whereascholine can enter mitochondria via a high capacity nonsaturablediffusion process, which is dependent on a high membranepotential, there is also a low-capacity high-affinity choline trans-porter in the inner membrane in rat mitochondria. Porter et al.(23) and Kaplan et al. (24) have estimated that at physiolog-ical choline concentrations the transport-mediated process is

Figure 3 Liver sections from rats fed diets varying in Met and choline

concentrations for 9 d. Sections were stained with hematoxylin and eosin. One

rat from each treatment group is shown. Group 1: Met-deficient, choline devoid

diet (A); group 2: Met-deficient, supplemental choline (B); group 3: Met-adequate,

choline devoid (C ), group 4: Met-adequate, supplemental choline (D); group 5:

supplemental Met, choline devoid (E); group 6: supplemental Met, supplemental

choline (F). Rats fed diets devoid of choline (A,C,E) have fatty liver as indicated by

the arrows. The severity of fatty infiltration decreased as the methionine concen-

tration of the diets increased.

Figure 2 Hepatic CHDH activity (A) in rats fed diets varying in Met and

choline concentrations for 9 d. The CHDH activity of group 4 was assigned the

relative value of 1, and all other treatment groups are expressed relative to this

group. Values are means 6 SE, n ¼ 4. Means without a common letter differ

(P , 0.05). A representative Western blot of diet-induced changes in hepatic

CHDH protein content (B).

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dominant (90%) and that the rate-limiting step in the mito-chondrial oxidation of choline to Bet might be its transport intothe organelle. Taken together, these results indicate that the ex-pression of the mitochondrial choline transporter could be in-fluenced by dietary choline, an idea that warrants furtherinvestigation.

Interestingly, fatty infiltration in the liver was observed in allrats fed the choline-devoid diets (Fig. 3A,C,E). The lipotropiceffect of supplementalMetwas not enough to completely preventfatty infiltration, although it markedly reduced lipid accumula-tion compared with the Met-deficient diet. This suggests that inyoung, growing rats, choline is essential because de novo syn-thesis via sequential methylation of phosphatidylethanolamine isnot sufficient to meet metabolic demands.

In summary, although both dietary Met and choline avail-ability markedly affect both hepatic BHMTactivity and expres-sion, these nutrients have negligible affects on kidney BHMTorliver CHDH expression in rats.

Acknowledgments

We thank Jana Strakova for her technical assistance with therats in this study and Mathew Wallig for assistance with thehistopathology.

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