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Biochemical, Molecular, and Genetic Mechanisms

Liver Choline Dehydrogenase and Kidney Betaine-Homocysteine Methyltransferase Expression Are Not Affected by Methionine or Choline Intake in Growing Rats¹

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

^* To whom correspondence should be addressed. E-mail: tagarrow{at}uiuc.edu.

	ABSTRACT

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
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 x 3 factorial design (n = 8), 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 supplemental Met cannot completely compensate for the lack of dietary choline in growing rats.

	Introduction

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

BHMT has a major role in the overall conversion of Hcy to Met (3), and more recently it has been shown that in vivo inhibition of BHMT in mice causes hyperhomocysteimia and a reduction in liver S-adenosylmethionine (4). There is substantial interest in the regulation of Hcy and Met metabolism, because an elevated level of plasma total Hcy is thought to be an independent risk factor for the development of coronary, cerebral, and peripheral vascular disease (5,6). BHMT is abundant in mammalian liver (0.5–2% of soluble liver protein), and it is also found in the kidney of some species, particularly humans, pigs, and guinea pigs (7–9), but substantially lower levels are found in rat kidney (>95% lower specific activity than rat liver). Dietary Met and methyl donor (Bet and choline) intake have been shown to modulate liver BHMT activity and gene expression. Specifically, diets deficient in Met with excess methyl donor have significantly increased hepatic BHMT activity and expression in rats (10–12). However, there are few data concerning the effect of supplemental Met on hepatic BHMT, nor has the nutritional 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 not been extensively studied. Little is known about its kinetic properties and whether expression or flux through the enzyme is influenced by diet and/or physiological state. We postulated that, because CHDH is the first step in the oxidation of choline and provides the Bet that BHMT uses, both dietary Met and choline availability may affect CHDH expression in a manner similar to that of BHMT. Indeed, Schneider and Vance (13) reported that rats fed a choline deficient diet have a 30–41% reduction in the mitochondrial oxidation of choline to betaine.

We sought to determine whether BHMT and CHDH are coordinately regulated by dietary Met and choline, how supplemental Met affects hepatic BHMT, and whether these nutrients are involved in the regulation of kidney BHMT expression.

	Materials and Methods

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
Diets and animal protocol

The experimental protocol was approved by the University of Illinois Institutional Animal Care and Use Committee. The nutrient levels in the L-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). The dietary 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 the Met 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 adequate; and 8 g Met/kg diet (0.8%), termed supplemental. Whereas the recommendations for the total sulfur amino acid (Met + cystine) concentrations 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 this study (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 has been 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).

The feeding trial was conducted using 3-wk old Sprague-Dawley rats (n = 48; Harlan), which were housed in standard shoebox housing (3/cage) in a light (12-h light/12-h dark) and temperature (23°C) controlled room. All rats were given free access to a Met and choline adequate diet (3 g/kg and 10 mmol/kg of diet, respectively) for 3 d to allow adjustment 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. Rats were housed individually in hanging stainless-steel cages. All rats 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 previous reports investigating the effect of these nutrients on BHMT expression, 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). Therefore, 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 not differ 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 –80°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-µm slices, mounted onto glass slides, and stained with hematoxylin and eosin.

Assay procedures

    Enzyme assays. Liver BHMT specific activity was determined as described by Garrow (17). Kidney BHMT was measured the same way, except that the concentration and specific activity of the ¹⁴C-Bet (250 µmol/L; 0.5 µCi) was changed to increase the assay sensitivity. Liver mitochondrial CHDH activity was measured as described previously (18).

    Western analysis. Liver and kidney homogenates were probed for BHMT protein by the procedure described by Delgado-Reyes and Garrow (18). Liver mitochondria were probed for CHDH protein using the same procedure, except 5 µg total protein were loaded onto the gels and primary antibodies (polyclonal) were prepared from rabbits immunized 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 performed using the SV total RNA system kit (Promega). First strand cDNA synthesis 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 groove binding (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'-AAGGACGGCCAGAGCCACAA-3' (sense primer), 5'-GCCCCCGCTCAGGATCA-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 was performed 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 rat liver 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 relative BHMT 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 showed that 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 BHMT expression 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 ± SEM.

	Results

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
    Growth. Rats fed the Met-deficient diets (groups 1 and 2) gained less weight (P < 0.001) than those fed the diets containing adequate (groups 3 and 4) and supplemental (groups 5 and 6) Met (Table 1). The gain-to-food intake ratios indicated that the 0.125% Met diets were severely deficient, although all rats gained weight. The growth performance of rats fed the Met-adequate diet devoid of choline (group 3) was significantly lower than that of rats fed the diet with supplemental levels of Met and choline (group 6), but it did not significantly differ in rats fed the Met-adequate diet containing supplemental choline (group 4) or in 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 this study would result in a range of hepatic BHMT activities (10–12), which we observed (Fig. 1A). Rats fed the Met-deficient diet containing supplemental choline (group 2) had higher BHMT activities (P < 0.01) than the other groups. BHMT activity was 1.3-fold higher in this group than in rats fed the Met-adequate diet containing supplemental choline (group 4), which is consistent with previous findings (11,12). Liver BHMT activity also was 30% greater in rats fed the diets containing supplemental Met (groups 5 and 6) than in those fed the Met-adequate diets (groups 3 and 4). Nonetheless, BHMT activities of rats fed the Met-supplemented diets were significantly lower than those consuming the diet deficient in Met with supplemental choline (Fig. 1A). As expected (10,12), adding choline to either the Met-adequate or Met-supplemented diets did not affect hepatic BHMT activity. Liver BHMT protein content mirrored activity levels (Fig. 1B).

View larger version (27K): [in this window] [in a new window]   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 ± SE, n = 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.

Rat kidney BHMT activity was not significantly affected by the diet treatments (Fig. 1C). Moreover, kidney BHMT mRNA expression (data not shown) and immunodetectable protein content did not differ among the groups (Fig. 1D). Within-group variation of kidney BHMT expression was much greater than that of liver BHMT expression.

    Liver CHDH. Although statistically there was a significant effect of methionine on CHDH activity, and a choline-methionine interaction, the magnitude of the differences was small (Fig. 2A) and probably not physiologically important. Indeed, the differences in activity were not reflected in CHDH protein concentrations (Fig. 2B) or hepatic CHDH mRNA expression (data not shown).

View larger version (23K): [in this window] [in a new window]   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 ± SE, n = 4. Means without a common letter differ (P < 0.05). A representative Western blot of diet-induced changes in hepatic CHDH protein content (B).

View larger version (182K): [in this window] [in a new window]   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 concentration of the diets increased.

	Discussion

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

This report is, to our knowledge, the first to have evaluated the effect of these nutrients on kidney BHMT activity. Whereas BHMT is expressed primarily in the liver of rats (7,8) it is present in the kidney, with >95% lower specific activity than that of the liver (T. A. Garrow, unpublished results). Our data show that kidney BHMT is refractory to dietary Met and choline intake (Fig. 1C). The differential tissue regulation of BHMT between liver and kidney is likely to be a means of controlling tissue Bet concentrations. Bet is not only a methyl donor, but it is accumulated as an osmoprotectant in a variety of cells, particularly the kidney medulla, where it protects the cells from damage from high salt and urea concentrations. Thus, the primary role of Bet in the kidney is likely to be as an osmoprotectant, rather than a methyl donor. Indeed, in guinea pigs, high sodium chloride intake decreases both kidney and liver BHMT activity by 50% (18).

CHDH mediates the first and committed step in the oxidation of choline to Bet. To date, very few data are available regarding the potential regulation of mammalian CHDH expression by diet. In Chesapeake Bay and Atlantic oysters, it has been reported that CHDH cannot function rapidly enough to saturate BADH, thus CHDH activity might be the rate-limiting step of endogenous Bet synthesis. CHDH has also been shown to be competitively inhibited by both Bet aldehyde and Bet (20). CHDH may have an important role in modulating Bet concentrations and, if so, its activity could modulate flux through BHMT. We sought to determine whether hepatic CHDH expression is modulated by dietary Met and choline availability in a manner similar to BHMT. However, our results indicate that CHDH is not affected by dietary Met and choline. No differences in mRNA levels (not shown) or protein concentration (Fig. 2B) were observed in any of the treatment groups. Although there was a significant interaction between Met and choline on hepatic CHDH activity, the effect was small (Fig. 2A) and probably not physiologically important. Supporting our findings, Wong and Thompson also found no effect on either hepatic CHDH or BADH activity in rats fed a choline deficient diet for 2 d and only a small reduction when choline deficiency was prolonged for up to 13 d (21).

Schneider and Vance (13) reported that the oxidation of choline to Bet was reduced in mitochondria isolated from rats fed choline deficient diets, compared with those fed choline sufficient diets. In addition, mitochondria from phosphatidylethanolamine N-methyltransferase and multidrug-resistant protein-2 double knockout mice have little ability to oxidize choline to Bet, as reported by Li et al. (22). It is possible the reduction in choline oxidation measured in these studies was the result of a reduction of CHDH expression, which would conflict with the results 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 and Vance (13) or Li et al. (22). One possibility that might explain these seemingly disparate results is that the rate-limiting step in the oxidation of choline to Bet formation could be its transport into the mitochondria, rather than CHDH activity. Whereas choline can enter mitochondria via a high capacity nonsaturable diffusion process, which is dependent on a high membrane potential, there is also a low-capacity high-affinity choline transporter in the inner membrane in rat mitochondria. Porter et al. (23) and Kaplan et al. (24) have estimated that at physiological choline concentrations the transport-mediated process is dominant (90%) and that the rate-limiting step in the mitochondrial oxidation of choline to Bet might be its transport into the organelle. Taken together, these results indicate that the expression of the mitochondrial choline transporter could be influenced by dietary choline, an idea that warrants further investigation.

Interestingly, fatty infiltration in the liver was observed in all rats fed the choline-devoid diets (Fig. 3A,C,E). The lipotropic effect of supplemental Met was not enough to completely prevent fatty infiltration, although it markedly reduced lipid accumulation compared with the Met-deficient diet. This suggests that in young, growing rats, choline is essential because de novo synthesis via sequential methylation of phosphatidylethanolamine is not sufficient to meet metabolic demands.

In summary, although both dietary Met and choline availability markedly affect both hepatic BHMT activity and expression, these nutrients have negligible affects on kidney BHMT or liver CHDH expression in rats.

	ACKNOWLEDGMENTS

 
We thank Jana Strakova for her technical assistance with the rats in this study and Mathew Wallig for assistance with the histopathology.

	FOOTNOTES

 
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.

Manuscript received 16 March 2006. Initial review completed 12 April 2006. Revision accepted 13 June 2006.

	LITERATURE CITED

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18. Delgado-Reyes CV, Garrow TA. High sodium chloride intake decreases betaine-homocysteine S-methyltransferase expression in guinea pig liver and kidney. Am J Physiol Regul Integr Comp Physiol. 2005;288:182–7.

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