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Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions

Elevated Tissue Betaine Contents in Developing Rats Are Due to Dietary Betaine, Not to Synthesis^1,2

Department of Biochemistry, Memorial University of Newfoundland, St. John's, NL, A1B 3X9, Canada

^* To whom correspondence should be addressed. E-mail: jbrosnan{at}mun.ca.

	ABSTRACT

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
The time course of betaine accumulation and activities of enzymes involved in betaine metabolism were studied in developing rats. In study 1, pups weaned on a nonpurified diet had a transient increase in liver and kidney betaine content followed by a decline after 42–56 d. In study 2, dams and, following weaning, pups were fed an AIN-93G (betaine-free) or an AIN-93G betaine-supplemented diet (0.3%) to determine the source of the transient increase in betaine levels previously observed. In study 2, only rats fed betaine had an increase in plasma betaine concentration. Similarly, liver and kidney betaine contents increased postweaning; however, betaine levels returned to that found in rats fed a betaine-free diet by 49 d of age. The dietary content of betaine fed to dams did not affect pup betaine. The activities of choline dehydrogenase, an enzyme of betaine synthesis, and betaine:homocysteine methyltransferase (BHMT), which is the only known betaine-consuming enzyme in mammals, were also measured in study 2. Liver BHMT activity decreased after weaning, whereas liver and kidney choline dehydrogenase activity increased with age, possibly reaching a plateau by 42 d of age. We conclude that the transient increase in betaine reflects high dietary betaine and not a change in endogenous betaine synthesis.

	Introduction

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

View larger version (24K): [in this window] [in a new window]   FIGURE 1  Summary of the pathways and compartmentalization of betaine metabolism within mammalian hepatocytes. Transporters are listed numerically and enzymatic steps are designated by letters. Betaine can be synthesized from choline, obtained from the diet, or via lipid (including phospholipid) metabolism. Choline enters the mitochondrion via the choline transporter (1), which has been identified but not characterized or studied in detail (6,7). Once in the mitochondrial matrix, choline is metabolized to betaine by 2 sequential and physiologically irreversible enzymatic dehydrogenation steps, first by the FAD-linked choline dehydrogenase (A; EC 1.1.99.1), which is associated with the inner mitochondrial membrane, followed by the NAD-linked matrix enzyme betaine aldehyde dehydrogenase (B; EC 1.2.1.8) (8). Betaine cannot be further metabolized within the mitochondrion and requires transport out to the cytosol. It has been suggested this is via passive diffusion (28); however, this transport step has not been studied in detail. As such, a "?" has been included with step 2. Betaine can also enter the hepatocyte from the extracellular fluid via 2 routes (5), the betaine:-aminobutyrate transporter (3) or amino acid transport system A (4). Betaine in the cytosol can be metabolized to dimethyglycine (DMG) via betaine:homeocysteine methyltransferase (C; EC 2.1.1.5), producing methionine in the process (8). The DMG is rapidly oxidized in mitochondria, but the transporter(s) of DMG into the mitochondrion (5) are not well understood; thus an "?" is included on this step as well.

With respect to developmental changes in betaine metabolism, the rate of betaine formation by adult rat liver slices and mitochondria is much higher than that found in fetal and very young (7 d old) rats (15). The decline in circulating choline levels in concert with increased capacity for choline oxidation to betaine suggests synthesis from choline may be the source of betaine accumulated in the liver (14). Interestingly, betaine excretion becomes elevated after weaning in rats, peaking after 28 d, followed by a rapid decline at 40 d of age. It was hypothesized that the source of this urinary betaine was dietary choline and, although no clear explanation of why betaine excretion changes over time in rats, lack of BHMT activity was considered unlikely (16).

The origin of the accumulated betaine remains unclear and we undertook the present study to determine whether the accumulated betaine is of dietary or endogenous (synthesis) origin. We examined the time course of betaine accumulation in tissues in developing rats whose dams were maintained on either a betaine-containing or betaine-free diet and, following weaning, pups raised on betaine-containing or betaine-free diets to determine the source of the accumulated betaine. Until now, there have been no long-term studies, to our knowledge, comparing betaine-free and supplemented diets over the duration of early development. The activities of betaine-metabolizing enzymes were also determined to test the hypothesis that betaine synthetic capacity correlates with betaine concentration in the developing rat. In addition, the developmental pattern of enzymes of betaine metabolism and the effects of betaine supplementation on their pattern may illustrate if these enzymes play a role in the transitory nature of betaine accumulation.

	Materials and Methods

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
Animals and sampling procedures

All animals used were Sprague-Dawley rats obtained from the Memorial University of Newfoundland Animal Care Unit. All procedures and animal husbandry was approved by and conducted in accordance to protocols established by Memorial University of Newfoundland's Institutional Animal Care Committee.

Study 1 (nonpurified diet)

Rats that were 7, 14, 21, 28, 35, 42, and 56 d of age were obtained from Memorial University of Newfoundland Animal Care Unit. Rats consumed nonpurified diet (Purina 5008) ad libitum except for 7-, 14-, and 21-d-old pups, which were still nursing from dams fed that diet. On the day of organ collection, rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (65 mg/kg). Kidneys, liver, heart, brain, and muscle were removed, in that order, freeze-clamped, ground into a powder, and stored at –80°C until assays were performed. Muscle and heart from more than 1 rat (ages 7 and 14 d only) were combined to obtain enough tissue to perform enzyme assays and betaine analysis. Betaine was analyzed by NMR.

Study 2 (AIN 93G with or without 0.3% added betaine)

    Animals and tissue sampling. Ten pregnant (5-d gestation) rats were obtained from the Memorial University of Newfoundland Animal Care Unit and started on AIN 93G diets (17) with (n = 5) or without (n = 5) added 0.3% betaine (20 µmol/g of diet). Dams were fed the same diet after the pups were born. Pups were weaned on d 21 or 22 and fed either the same diet as the dam or the alternative diet (dam +betaine, pup –betaine; dam +betaine, pup +betaine; dam –betaine, pup +betaine; dam –betaine, pup –betaine). Liver, whole kidney, and arterial blood was taken from pups aged 13–15 d, 20–22 d (prior to weaning), 27–30 d, 34–36 d, 41–43 d, and 48–50 d. For simplicity, these sample periods will be referred to by the median sample day, for example 13–15, 20–22, and 27–30 are referred to as d 14, 21, and 28, respectively. Liver and kidneys were freeze-clamped and assayed for CHDH and BHMT activity and betaine. Plasma was obtained by centrifuging blood at 3000 x g; 15 min at room temperature and frozen –80°C until analyzed. Betaine in plasma was measured by HPLC and by ¹H NMR for liver and kidney extracts as described below.

    Enzyme assays. A piece of previously frozen liver or kidney was ground into a fine powder with liquid nitrogen. Samples were diluted 1:5 with 50 mmol/L potassium phosphate buffer (pH 7.0) and homogenized with a Polytron (Brinkman Instruments). An aliquot of the crude homogenate was reserved for the choline dehydrogenase assay. The rest of the homogenate was centrifuged at 16,000 x g; 30 min at 4°C and the supernatant was removed and used to measure BHMT activities. BHMT (0.3 mg protein) was assayed using previously described methods (18). CHDH was assayed following Haubrich and Gerber (9) with modified assay conditions, which included: 0.15 mg protein, 2 mmol/L [methyl-¹⁴C] choline chloride (0.2 µCi), and 1 mmol/L phenazine methosulfate. Assay volume was 50 µL and samples were incubated for 7.5 min. Protein concentrations were determined by the Biuret method using bovine serum albumin as a standard.

    Betaine determinations. Betaine levels in the liver and kidneys were measured by ¹H NMR (Buker AVANCE 500 MHz). Briefly, previously frozen tissues were ground into a fine powder and homogenized in 6% perchloric acid (1:5). Homogenates were centrifuged at 16,000 x g; 30 min at 4°C and the supernatant collected. Sample pH was adjusted to 9 by adding 20% KOH. The sample was centrifuged at 16,000 x g; 5 min to pellet the precipitate. The supernatant was diluted 1:1 with 100 mmol/L sodium phosphate buffer (pH 9). 65 µL D₂O (Cambridge Isotope Laboratories) and 6.5 µL 109 mmol/L sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DSS; Cambridge Isotope Laboratories) was added to 585 µL sample in phosphate buffer (pH 9). Samples were transferred to a 5-mm NMR tube and 1-dimensional NMR spectra were acquired on each sample at ambient probe temperature as described by Lundberg et al. (19). The resonance of DSS was set to a chemical shift of 0.00 ppm. We calculated betaine concentrations from a standard curve using the ratio of peak height of betaine (9 protons at 3.25 ppm):peak height of DSS (9 protons at 0.00 ppm).

Betaine levels in the plasma were measured using HPLC, because we thought that NMR might not be sensitive enough to detect betaine levels in plasma. The HPLC method is based on Ganzera et al. (20) with minor modifications. First, 50 µL plasma was added to 50 µL of 100 mmol/L KH₂PO₄. This sample was added to 900 µL derivatizing solution containing 66 mg 18-Crown-6 and 1390 mg 2,4'-dibromoacetophenone in 100 mL acetonitrile. Samples were heated at 80°C for 60 min, allowed to cool for 5 min, and centrifuged at 2000 x g; 5 min at room temperature. Samples were analyzed on a Waters HLPC system equipped with a LC spectrophotometer, a 717 Wisp autosampler, and a 600E system controller and were quantitated using Millennium software. Separation was achieved using a Supelcosil LC-SCX column (250 x 4.6 mm, 5 µm) and a mobile phase consisting of a 70:30 mix of methanol and 25 mmol/L choline chloride (pH 2) with a flow rate of 0.8 mL/min and a detection wavelength of 454 nm. Results obtained by the 2 analytical techniques for betaine quantification yielded comparable results for samples from the same animal.

    Plasma Hcy. Samples were prepared and analyzed for plasma Hcy by HPLC as described by Vester and Rasmussen (21).

Statistical analysis

For the initial exploratory experiment (study 1), means were compared with 1-way ANOVA followed by Tukey's post hoc test. Comparisons between groups in Study 2 were assessed by general linear model with the independent factors being the maternal and pups' diet and the pups' age as well as the interaction between these factors. When significant interaction was found between treatments, pairwise comparisons were made using Tukey's post hoc test. The relationship between plasma betaine concentration and tissue betaine concentrations was analyzed by linear regression. Growth rates in study 2 were determined by fitting an exponential growth curve to the mass data for individual rats. For all statistical analyses, P < 0.05 was considered significant with the exception of growth rates that were compared by overlapping 95% CI. When necessary, prior to conducting ANOVA, data were log transformed to normalize residuals. Values presented are means ± SD.

	Results

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
Study 1 (nonpurified diet)

The nonpurified diet contained 20 µmol/g betaine (mean of 3 separate determinations measured by NMR). Tissue betaine was low until pups were weaned, at which point liver and kidney betaine contents increased markedly (Fig. 2). Betaine levels in liver and kidney peaked at d 28 (1 wk postweaning) to d 42 and began declining by d 56. Heart betaine had a small but sustained increase, whereas brain betaine content decreased continually during the experiment (Fig. 2).

View larger version (11K): [in this window] [in a new window]   FIGURE 2  Tissue betaine content in developing rats weaned to nonpurified diet. Values are means ± SD, n = 3. Means without a common letter differ, P < 0.05 (ANOVA).

Having shown that Sprague-Dawley rats had a temporally transient increase in betaine when fed a nonpurified diet, which is a substantial source of betaine, we conducted all further experiments on rats fed a purified diet.

    Growth rates. The diets did not affect the growth rate of rats as determined by fitting data to an exponential curve (P < 0.001; r² 0.93 for all; data not shown). The growth rates of pups in the 4 groups ranged from 4.8 to 5.5%/d.

    Betaine contents. From d 14 to 21, prior to weaning, there was no effect of treatment on betaine in plasma, liver, or kidney (Fig. 3). Following weaning, plasma, liver, and kidney betaine increased in the pups fed a betaine-supplemented diet irrespective of the mothers' diet. Throughout the experiment, only the pups' diet affected betaine accumulation, whereas the mothers' diet did not affect betaine content in the pups. From d 42 to 49, liver betaine content was not affected by the pups' diets (Fig. 3B). Similarly, following a plateau, plasma (Fig. 3A) and kidney (Fig. 3B) betaine content decreased and the groups did not differ on d 49.

View larger version (12K): [in this window] [in a new window]   FIGURE 3  Plasma (A), liver (B), and kidney (C) betaine contents in developing rats exposed or not to dietary betaine before and/or after birth. Values are means ± SD, n = 5. *Pups fed betaine-free and -supplemented diets differ at that time, P < 0.05. Maternal diet did not affect tissue betaine content. –/–, dam (0% betaine)/pup (0% betaine); +/–, dam (0.3% betaine)/pup (0% betaine); –/+, dam (0% betaine)/pup (0.3% betaine); +/+, dam (0.3% betaine)/pup (0.3% betaine).

View larger version (13K): [in this window] [in a new window]   FIGURE 4  Liver BHMT (A), liver CHDH (B), and kidney CHDH (C) in developing rats exposed or not to dietary betaine before and/or after birth. Values are means ± SD, n = 5. In all groups combined, means without a common letter differ, P < 0.05. *Pups fed betaine-free and -supplemented diets differ at that time, P < 0.05. The same pattern of difference was found when data were expressed as µmol·min–1·g wet mass–1.

View larger version (23K): [in this window] [in a new window]   FIGURE 5  Correlations between plasma and liver (A) or kidney (B) betaine content in developing rats exposed or not to dietary betaine before and/or after birth.

View larger version (13K): [in this window] [in a new window]   FIGURE 6  Hepatic total betaine contents in developing rats exposed or not to dietary betaine before and/or after birth. Values are means ± SD, n = 5. *Pups fed betaine-free and -supplemented diets differ at that time, P < 0.05.

	Discussion

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

View larger version (10K): [in this window] [in a new window]   FIGURE 7  Plasma Hcy concentrations in developing rats exposed or not to dietary betaine before and/or after birth. Values are means ± SD, n = 3–5. In all groups combined, means without a common letter differ, P < 0.05.

Betaine feeding and subsequent elevation of betaine levels had little effect on enzymes of betaine metabolism (Fig. 4). A number of studies have shown activation of liver BHMT with increased dietary betaine or the betaine precursor choline (10,23–25); however, many of these studies required a methionine-deficient diet to display large increases in BHMT activity. The consistent pattern between liver and kidney CHDH, and among treatments within each tissue, indicates a strong developmental trend of increasing choline oxidative capacity, consistent with previous studies (14,15).

A substantial concentration gradient has been demonstrated between the extracellular fluid and the intracellular fluid of the liver and kidney (Fig. 5) and the tissue content rise in parallel with increasing plasma betaine; however, it is unclear if increased plasma betaine is due to insufficient betaine removal via BHMT or if the transporters responsible for betaine uptake into the liver and kidney are below saturation. In the later case, the increasing plasma concentration may result in an increased rate of inward transport and establish a higher intracellular concentration.

Betaine levels decreased in the supplemented rats by the end of the study to the same level as that found in rats fed the betaine-free diet (Fig. 3). This was not coincident with an increase in BHMT activity, which remained constant from d 35 to 49 (Fig. 4A). An increase in transport capacity into the hepatocyte would also fail to explain the decline in betaine, because even in the betaine-free diet where intracellular betaine concentrations would be in the mmol/L range, the intracellular betaine concentration in the liver would be expected to greatly exceed the Michaelis constant for BHMT, 48 µmol/L in rat liver (26). Although daily food consumption was not determined in the current study, it is well established that food intake decreases with age. For example, Chen and Nyomba (27) reported an 30% decrease in daily food intake (as percent body mass) from d 21–28 to d 42–49. The liver is the primary tissue of betaine metabolism and removal and it may be expected that liver betaine levels would decline in advance of plasma if flux through hepatic BHMT is the limiting site of betaine metabolism. Consistent with a decrease in dietary intake and a subsequently reduced requirement to metabolize betaine, the liver betaine levels decline before plasma or kidney levels. However, the present study indicates that there is still need for further study of hepatic betaine metabolism and handling, particularly in developing animals where marked changes occur.

	ACKNOWLEDGMENTS

 
We thank Dr. V. Booth for assistance with NMR techniques.

	FOOTNOTES

 
¹ Supported by the Canadian Institutes of Health Research (operating grant to J.T.B. and post-doctoral fellowship to J.R.T.) and the Canadian Diabetes Association in honor of the late Neil M. Miller.

² Author disclosures: K. A. Clow, J. R. Treberg, M. E. Brosnan, and J. T. Brosnan, no conflicts of interest.

³ Abbreviations used: BHMT, betaine-homocysteine methyltransferase; CHDH, choline dehydrogenase; DMG, dimethylglycine; DSS, 2,2-dimethyl-2-silapentane-5-sulfonate; Hcy, homocysteine; ZDF fa/fa, Zucker diabetic fatty rats (homozygous); ZDF fa/?, Zucker diabetic fatty rats (heterozygous).

Manuscript received 21 April 2008. Initial review completed 27 May 2008. Revision accepted 8 July 2008.

	LITERATURE CITED

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 

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This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Clow, K. A. Articles by Brosnan, J. T. Search for Related Content PubMed PubMed Citation Articles by Clow, K. A. Articles by Brosnan, J. T.