Folate deprivation reduces homocysteine remethylation in a human intestinal epithelial cell culture model: role of serine in one-carbon donation

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1 Am J Physiol Gastrointest Liver Physiol 286: G588 G595, First published November 13, 2003; /ajpgi Folate deprivation reduces homocysteine remethylation in a human intestinal epithelial cell culture model: role of serine in one-carbon donation Justin H. Townsend, Steven R. Davis, Amy D. Mackey, and Jesse F. Gregory III Food Science and Human Nutrition Department, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida Submitted 21 October 2003; accepted in final form 7 November 2003 Townsend, Justin H., Steven R. Davis, Amy D. Mackey, and Jesse F. Gregory III. Folate deprivation reduces homocysteine remethylation in a human intestinal epithelial cell culture model: role of serine in one-carbon donation. Am J Physiol Gastrointest Liver Physiol 286: G588 G595, First published November 13, 2003; /ajpgi Little is known about homocysteine metabolism in intestine. To address this question, we investigated homocysteine metabolism under conditions of folate adequacy and folate deprivation in the Caco-2 cell line, a model of human intestinal mucosal cells. Caco-2 cells were cultured in media enriched with [3-13 C]serine and [U- 13 C 5 ]methionine tracers, and enrichments of intracellular free amino acid pools of these amino acids as well as homocysteine, cystathionine, and cysteine were measured by using gas chromatography/mass spectrometry. Homocysteine transsulfuration plus folate-dependent and total remethylation were quantified from these amino acid enrichments. Homocysteine remethylation accounted for 19% of the intracellular free methionine pool in cells cultured with supplemental folate, and nearly all one-carbon units used for remethylation originated from the three carbon of serine via folate-dependent remethylation. Labeling of cystathionine and cysteine indicated the presence of a complete transsulfuration pathway in Caco-2 cells, and this pathway produced 13% of the intracellular free cysteine pool. Appearance of labeled homocysteine and cystathionine in culture medium suggests export of these metabolites from intestinal cells. Remethylation was reduced by one-third in folate-restricted cell cultures (P 0.001), and only 50% of the one-carbon units used for remethylation originated from the three carbon of serine under these conditions. In conclusion, the three carbon of serine is the primary source of one-carbon units used for homocysteine remethylation in folate-supplemented Caco-2 cell cultures. Remethylation is reduced as a result of folate restriction in this mucosal cell model, and one-carbon sources other than the three carbon of serine contribute to remethylation under this condition. methionine; methylation; Caco-2; transsulfuration; serine FOLATE DEFICIENCY AND GENETIC polymorphisms of several enzymes involved in one-carbon metabolism are linked to cardiovascular disease and certain cancers and particularly colorectal cancer (5, 6, 16, 18a). These associations are likely due to the effects of altered folate metabolism on homocysteine remethylation, DNA synthesis, and DNA methylation (3, 11, 29). Reduced homocysteine remethylation can result in elevated plasma homocysteine concentration, which is an independent risk factor for cardiovascular disease (19). Altered DNA metabolism is implicated in the etiology of cancers (20). Folate serves as a metabolic carrier of one-carbon units (8). In homocysteine remethylation, folate transports methyl groups as 5-methyltetrahydrofolate (5-CH 3 THF) to methionine synthase, the ubiquitous homocysteine methyltransferase that converts homocysteine to methionine in all cells (Fig. 1, reactions 6 8). Consequently, folate deficiency might alter homocysteine metabolism by limiting the availability of 5-CH 3 THF for the remethylation reaction (28). The methyl groups used by methionine synthase to convert homocysteine to methionine could originate from glycine, sarcosine, dimethylglycine, histidine, formate, or the three carbon of serine (8). The latter amino acid is thought to be the primary donor of one-carbon units for homocysteine remethylation (1), and a recent human metabolic tracer study supports this hypothesis (9). Homocysteine also can be remethylated in a folate-independent manner by betaine-homocysteine methyltransferase by using betaine as a methyl donor (Fig. 1, reaction 4) (15). However, limited expression of betaine-homocysteine methyltransferase (liver and kidney only) renders most tissues dependent on methionine synthase activity for homocysteine remethylation (10, 35). Although one-carbon metabolism is well studied in liver and kidney, relatively little is known about these pathways in mammalian intestine. Intestinal mucosa contains methionine synthase activity (21), but intestinal homocysteine remethylation capacity is restricted compared with liver and kidney due to the absence of betaine-homocysteine methyltransferase (31). This limitation renders the intestine more susceptible than liver and kidney to the homocysteine-elevating effects of folate depletion. However, the small intestine is one of only four tissues that contains both cystathionine -synthase and -cystathionase, the two enzymes of the transsulfuration pathway (Fig. 1, reactions 8 and 9) (24). Transsulfuration is the only route of homocysteine catabolism, and expression of this pathway enables intestinal cells to catabolize some of the homocysteine that it produces. Whether expression of this pathway renders the intestine self-sufficient with regard to homocysteine metabolism is uncertain, because few quantitative data exist regarding the rates of one-carbon cycle reactions in this tissue. The present investigation was designed to improve our understanding of homocysteine metabolism in intestinal cells and to assess the sensitivity of those processes to folate restriction. We hypothesized that homocysteine metabolism would consist of transsulfuration and folate-dependent remethylation and that serine would be the primary one-carbon donor for remethylation in intestinal cell cultures. Consequently, we hypothesized that homocysteine remethylation Address for reprint requests and other correspondence: J. F. Gregory III, Food Science and Human Nutrition Dept., PO Box , Gainesville, FL ( jfgy@mail.ifas.ufl.edu). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. G /04 $5.00 Copyright 2004 the American Physiological Society

2 HOMOCYSTEINE METABOLISM IN INTESTINAL CELLS G589 Fig. 1. Pathways of homocysteine metabolism. Methionine metabolism through the methylation cycle by methionine adenosyltransferase (1), S- adenosylmethionine-dependent methyltransferases (SAM; 2), and S-adenosylhomocysteine hydrolase (SAH; 3) produces homocysteine. Homocysteine may be remethylated via betaine-homocysteine methyltransferase (4) or through the folate-dependent pathway via methionine synthase (5). The latter reaction relies heavily on the generation of one-carbon units from the three carbon of serine via the sequential activities of serine hydroxymethyltransferase (6) and methylenetetrahydrofolate reductase (7). Homocysteine catabolism occurs through transsulfuration (8 and 9). In this pathway, cystathionine -synthase (8) catalyzes the condensation reaction of homocysteine with serine to generate cystathionine, which is catabolized into cysteine and -ketobutyrate by -cystathionase (9). would be reduced during folate restriction. These aspects of homocysteine metabolism were investigated in the Caco-2 cell line, which is an established model of human intestinal epithelial cells. Homocysteine metabolism was evaluated from the enrichments of one-carbon cycle metabolites after incubation in media containing added [3-13 C]serine and [U- 13 C 5 ]methionine. MATERIALS AND METHODS Materials. The Caco-2 human colon carcinoma cell line was obtained from American Type Culture Collection (Manassas, VA). Culture media and media supplements were obtained from GIBCO/ Invitrogen (Carlsbad, CA) or from Sigma (St. Louis, MO). The [U- 13 C 5]methionine, [3-13 C]serine, and [ 2 H 3]leucine tracers were obtained from Cambridge Isotope Laboratories (Woburn, WA). AG50W-X8 cation-exchange resin was obtained from Bio-Rad Laboratories (Hercules, CA). All other chemicals were obtained from Sigma or Fisher Scientific (Pittsburgh, PA). Culture conditions. Cells were cultured similarly to the method described by Fleet and Wood (13). Cells were cultured in DMEM supplemented with (in mm): 1 sodium pyruvate, 0.1 nonessential amino acids, 4 L-glutamine, and 25 HEPES, with 10% FBS and an antibiotic-antimycotic solution. Folate contents of media were determined by using the Quantaphase II B12/folate radiobinding assay (Bio-Rad). Folate-supplemented culture medium contained 9 M total folate (mainly as folic acid), whereas nonsupplemented culture medium contained 0.9 nm folate derived from the FBS. Concentrations of free methionine, serine, leucine, and cysteine in the medium were calculated to be (in mm) 0.183, 0.466, 0.720, and 0.466, respectively. Cell cultures were incubated at 37 C ina5%co 2 atmosphere, and culture media were changed every 2 3 days. Experiments were performed on cell passages Approximately cells were plated in each well of a three-well plate, and experiments were conducted 7 9 days postconfluence. Cell culture media were changed 24 h before experiments after cultures were washed three times with sterile PBS to completely remove folate-adequate medium. Tests for the presence of mycoplasmal contamination were not performed. Homocysteine metabolism in folate-adequate and folate-deprived Caco-2 cell cultures. Homocysteine metabolism experiments were initiated by the addition of enriched medium that contained [U- 13 C 5]methionine, [3-13 C]serine, and [ 2 H 3]leucine tracers at concentrations specified below to the cell cultures. The initial time course covered 24 h with time points of 0, 4, 8, and 24 h. Amino acid tracers were added to the culture medium to achieve an isotopic enrichment of 50% for methionine, serine, leucine, and cysteine. The second time course covered 4 h with time points of 0, 0.5, 1, 2, and 4 h with amino acid tracers added to achieve an isotopic enrichment of 20% for methionine, serine, and leucine. Cell and medium samples were taken from three culture wells at each time point. Effects of folate restriction on homocysteine metabolism also were investigated. Cells were cultured in DMEM for days 1 and 2 postconfluence, and cultures were then divided into two groups: cells cultured in medium that contained no added folic acid (folate-restricted cultures) for days 3 7 postconfluence and cells maintained in folate-supplemented medium (control cultures) over that time course. Cells were refreshed with the appropriate medium (folate-restricted or control) 24 h before tracer experiments. Experiments were initiated by the addition of amino acid tracers ([U- 13 C 5]methionine, [3-13 C]serine, and [ 2 H 3]leucine) to the medium to achieve an isotopic enrichment of 20%. Cell and media samples were collected at 0, 0.5, 1, 2, 4, and 8 h after the addition of tracers to measure amino acid enrichments. Collection of free intracellular amino acids. At each experimental time point, 500 l of culture medium were collected, and cells were washed with PBS to remove remaining medium. Cells were mechanically lifted by scraping with a plastic spatula and collected in PBS (500 l). Cell samples were acidified with 50 l 66% (wt/vol) trichloroacetic acid and homogenized with a Polytron homogenizer. Cell homogenates were centrifuged (14,000 g for 15 min at 4 C), and the supernatants were isolated and stored at 80 C before amino acid isolation. Culture medium samples were processed and stored similarly to cell samples with the exclusion of homogenization. To isolate free amino acids, 500 l of acidified supernatants were loaded onto columns packed with 0.5 ml of AG50W-X8 cation exchange resin (Bio-Rad Laboratories) in acid form. Columns were washed with water (10 1 ml), followed by elution of amino acids into reaction vials by using 3 ml of 3 M NH 4OH. Samples were dried in a SpeedVac (ThermoSavant, Holbrook, NY) at ambient temperature and then refrigerated overnight. Amino acids were esterified by reacting dry samples with 500 l of a 5:1 mixture of 1-propanol/acetyl chloride and 10 l of ethanethiol for1hat110 C. Samples were dried under N 2, and the n-propyl amino acid esters were derivatized by reaction with 100 l heptafluorobutyric anhydride and 10 l ethanethiol for 1hat60 C. The reaction mixtures were dried under N 2, and the resulting N-heptafluorobutyryl-n-propyl ester derivatives were solubilized in 100 l ethyl acetate and stored at 20 C until analysis. Isotopic enrichment was determined by gas chromatograph-mass spectrometry (Finnigan-Thermoquest Voyager, San Jose, CA). Chromatography was performed at a helium flow rate of 1.0 ml/min using a 30-m poly(5% diphenyl/95% dimethylsiloxane)-fused silica capillary column (Equity 5; Supelco, Bellefonte, PA), using 0.3- to 2.0- l injections and an injector temperature of 250 C. The initial oven temperature was held at 95 C for 3 min, increased to 130 C at 3.5 C/min, held at 130 C for 10 min, raised to a final temperature of 250 C at 8 C/min, and held at 250 C for 10 min. Negative chemical ionization/mass spectrometry was performed with methane as the reagent gas at a source temperature of 150 C and electron energy of 70 ev. The abundance of specific ions was determined by selected ion monitoring at the following mass/charge ratios: serine, ; leucine, ; methionine, ; homocysteine, ; and cystathionine, Isotopic enrichments are expressed as molar ratios of labeled/nonlabeled isotopomers after correction for the natural abundance of stable isotopes (34). Kinetic analyses. Isotopic enrichment data are plotted as the means SE (n 3) at each time point. Plateau enrichments were

3 G590 HOMOCYSTEINE METABOLISM IN INTESTINAL CELLS determined by fitting enrichment data to single exponential curves defined by the equation E E f (1 e kt ). In this equation, E is the enrichment at time t (h), whereas E f and k are the enrichment at infinity (i.e., plateau enrichment) and rate constant (h) from the fitted curve, respectively. Because this equation did not fit the cystathionine enrichment data of the folate restriction experiments, these data were fitted to a sigmoidal curve defined by the equation E E f(1 e kt ) c, where c is an empirical constant related to the rate of change in Y over time. Homocysteine remethylation and transsulfuration kinetics were calculated by using data from within individual culture dishes from time points at the plateau as well as from plateau enrichments derived from curve fitting. Behavior of the [U- 13 C 5]methionine and [3-13 C]serine tracers in one-carbon metabolism is illustrated in Fig. 1. As the [U- 13 C 5]methionine tracer is metabolized in the methylation cycle, one 13 C-labeled atom is lost in the S-adenosylmethionine-dependent methyltransferase reactions (Fig. 1, reactions 1 and 2). Consequently, homocysteine produced from this tracer contains four 13 C-labeled carbons (Fig. 1, reaction 3). Remethylation of [ 13 C 4]homocysteine with an unlabeled methyl group from 5-CH 3THF or betaine generates [ 13 C 4]methionine (Fig. 1, reactions 4 and 5). The fraction of the total methionine pool that had been remethylated (RM total) was calculated from enrichment data at plateau for the unmetabolized methionine tracer (Ep Met M 5) and remethylated methionine (Ep Met M 4) RM total (Ep Met M 4)/[(Ep Met M 4) (Ep Met M 5)] This calculation is based on the dual tracer principle for methionine kinetics reported by Storch et al. (34). As the [3-13 C]serine tracer is metabolized in the folate cycle, the 13 C-labeled three carbon is transferred to tetrahydrofolate (Fig. 1, reaction 6), reduced by MTHFR (Fig. 1, reaction 7), and subsequently transferred to homocysteine via methionine synthase to generate [ 13 C 1]methionine (Fig. 1, reaction 5). The fraction of the methionine pool derived from folate-dependent remethylation by using the three carbon of serine (RM from serine) was calculated from the plateau enrichments of the [3-13 C]serine tracer (Ep Ser M 1) and [ 13 C 1]methionine (Ep Met M 1) as RM from serine (Ep Met M 1)/(Ep Ser M 1) The percentage of remethylation (%RM from serine) using onecarbon units derived from the three carbon of serine is calculated from RM total and RM from serine %RM from serine (RM from serine/rm total) 100 This calculation is equivalent to that reported by Schalinske and Steele (26) for radioisotope studies of serine s role in homocysteine remethylation in rat liver. Combined use of serine and methionine tracers allows measurements related to transsulfuration of homocysteine as well (18, 22). In this protocol, [3-13 C]serine can be condensed with homocysteine by cystathionine -synthase to form [ 13 C 1]cystathionine in the transsulfuration pathway (Fig. 1, reaction 8). Cleavage of [ 13 C 1]cystathionine by -cystathionase yields [ 13 C 1]cysteine and -ketobutyrate (Fig. 1, reaction 9). The percentage of the cystathionine pool produced by transsulfuration (%Csn from TS) and the percentage of the cysteine pool produced by transsulfuration (%Cys from TS) were calculated by using the plateau enrichments of [ 13 C 1]cystathionine (Ep Csn M 1), [ 13 C 1]cysteine (Ep Cys M 1), and Ep Ser M 1 as and %Csn from TS (Ep Csn M 1/Ep Ser M 1) 100 %Cys from TS (Ep Cys M 1/Ep Ser M 1) 100 Data were analyzed by t-test or two-way ANOVA (17) with folate concentration and incubation time as the main factors. Multiple Fig. 2. Twenty-four-hour enrichment vs. time curves. A: enrichment curves of the [3-13 C]serine tracer and tracer-derived [ 13 C 1]methionine and [ 13 C 1]cystathionine. B: enrichment curves of the [U- 13 C 5]methionine tracer and tracer-derived [ 13 C 4]homocysteine and [ 13 C 4]methionine. Data are presented as means SE (n 3). comparisons were conducted by using the Student-Newman-Keuls method. All data are presented as means SE. RESULTS Homocysteine metabolism in folate-adequate Caco-2 cell cultures. Plots of isotopic enrichment vs. time for intracellular amino acids in 24- and 4-h time courses indicated that labeled intracellular precursor pools reached plateau enrichment at 1 h posttreatment (Figs. 2 and 3). Plateau enrichments derived by fitting data to a single exponential curve are presented in Table 1. The fraction of the intracellular free methionine pool formed by remethylation of homocysteine was and % in the 4h(1 4 h data combined; n 9) and 24 h (4- to 24-h data combined; n 9) time courses, respectively. The fraction of the intracellular free methionine pool that was formed through folate-dependent remethylation using the three carbon of serine was and % in the 4h(2- and 4-h data combined; n 6) and the 24 h (4- to 24-h data combined; n 9) time courses, respectively. Consequently, serine provided 108 and 82% of the one-carbon units used for remethylation in the 4- and 24-h cell culture experiments, respectively.

4 Fig. 3. Four-hour enrichment vs. time curves. A: enrichment vs. time curves for the [3-13 C]serine tracer and tracer-derived [ 13 C 1]methionine, [ 13 C 1] cystathionine, and [ 13 C 1]cysteine. B: enrichment vs. time curves for the [U- 13 C 5]methionine tracer and tracer-derived [ 13 C 4]homocysteine, [ 13 C 4] cystathionine, and [ 13 C 4]methionine. Data are presented as means SE (n 3). Labeling of intracellular pools of cystathionine and cysteine ([ 13 C 4 ]cystathionine from [U- 13 C 5 ]methionine and [ 13 C 1 ] cystathionine and [ 13 C 1 ]cysteine from [3-13 C]serine) indicate the existence of the transsulfuration pathway in Caco-2 cells (Table 1, Figs. 2 and 3). Cystathionine reached plateau between 4 and 24 h (Fig. 2A), and transsulfuration accounted for % of the cystathionine pool (n 9). Similar results were obtained from curve fit data for the 4-h time course (95%; Table 1). [ 13 C 1 ]cysteine enrichment was quantifiable in the 4-h time course only (Fig. 3A) in which % of the intracellular free cysteine pool was derived from transsulfuration (n 9). [ 13 C 4 ]homocysteine, [ 13 C 1 ]cystathionine, and [ 13 C 4 ]cystathionine appeared in the media after the addition of amino acid tracers. Extracellular enrichments were similar to intracellular enrichments for all isotopomers analyzed; i.e., [3-13 C]serine, [U- 13 C 5 ]methionine, [ 13 C 4 ]methionine, [ 13 C 1 ]methionine, [ 13 C 4 ]homocysteine, [ 13 C 4 ]cystathionine, and [ 13 C 1 ]cystathionine (data not shown). Effects of folate deprivation on homocysteine metabolism in Caco-2 cell cultures. Folate restriction did not affect the enrichment patterns of the [3-13 C]serine or [U- 13 C 5 ]methionine HOMOCYSTEINE METABOLISM IN INTESTINAL CELLS tracers (Fig. 4). However, significantly reduced enrichments of [ 13 C 1 ]methionine (P for individual 1- to 4-h time points, P for plateau enrichments derived from curvefitting), [ 13 C 4 ]methionine (P 0.01 for individual 1- to 4-h time points, P 0.02 for plateau enrichments derived from curve-fitting), and [ 13 C 1 ]cystathionine [P for individual 1- to 4-h time points, nonsignificant (P 0.08) for plateau enrichments derived from curve-fitting] were measured in folate-restricted cultures (Table 2). Folate-restricted cell cultures had a lower fraction of the methionine pool remethylated ( vs %, P 0.001, Fig. 5A) and a lower fraction of the methionine pool remethylated by folate-dependent remethylation from the three carbon of serine ( vs %, P 0.001, Fig. 5B) than control cell cultures (4- and 8-h data combined; n 6). The three carbon of serine accounted for only 53% of the one-carbon units used for remethylation in folate-restricted cultures compared with 100% in control cultures (Fig. 5C). Labeling of [ 13 C 1 ] cystathionine relative to [3-13 C]serine also was significantly lower at the 4- and 8-h time points (n 6) in the folaterestricted cells compared with control cells ( vs %, P 0.001; Fig. 6). Similar results for indices of remethylation and cystathionine labeling were obtained from curve-fit data (not shown). DISCUSSION Previous studies demonstrated that the activities of enzymes of the methionine cycle (21, 24) and the transsulfuration pathway (7, 24) are present in isolated rat enterocytes and rat intestinal mucosa. To the best of our knowledge, this is the first investigation of the kinetics of the homocysteine remethylation and transsulfuration pathways in an intestinal cell model of any species. This report also contains the first direct evidence that folate deprivation alters homocysteine metabolism in these cells. Because the Caco-2 cell model displays many characteristics of absorptive intestinal cells, similar results are expected in mammalian enterocytes and intestinal mucosa. An assumption of this tracer model is that all of the [ 13 C 1 ]methionine measured is produced from remethylation of homocysteine with one-carbon units derived from Table 1. Plateau enrichments of stable isotope-labeled amino acids in Caco-2 cell cultures after being cultured in medium containing [U- 13 C 5 ]methionine and [3-13 C]serine Labeled Amino Acid Plateau Enrichment, mol %excess Trial A G591 Trial B [ 2 H 3]leucine [ 13 C 1]serine [ 13 C 1]methionine [ 13 C 1]cystathionine [ 13 C 1]cysteine [ 2 H 2]cysteine [ 13 C 5]methionine [ 13 C 4]methionine [ 13 C 4]homocysteine [ 13 C 4]cystathionine Data (means SE) are derived by fitting time course data to a single exponential curve. See MATERIALS AND METHODS for details. Trial A, 20% initial enrichment, 4-h time course; Trial B, 50% initial enrichment, 24-h time course.

5 G592 HOMOCYSTEINE METABOLISM IN INTESTINAL CELLS Fig. 4. Enrichment curves of labeled amino acids from control of folate-restricted Caco-2 cell cultures. A: [3-13 C]serine tracer-derived enrichment curves from control cultures. B: [U- 13 C 5]methionine tracer-derived enrichment curves from control cultures. C: [3-13 C]serine tracer-derived enrichment curves from folaterestricted cultures. D: [U- 13 C 5]methionine tracer-derived enrichment curves from folaterestricted cultures. Data are presented as means SE (n 3). [3-13 C]serine. This may be overestimation, because the metabolism of the [U- 13 C 5 ]methionine tracer via the choline synthesis/degradation pathways and polyamine synthesis pathway followed by recycling of a labeled methyl group and its subsequent use for remethylation can also yield [ 13 C 1 ]methionine. These pathways are not believed to be quantitatively significant in overall methionine metabolism, however, and are ignored in this tracer model. The importance of these alternative pathways of [ 13 C 1 ]methionine metabolism is the subject of a current investigation in our laboratory. Table 2. Plateau enrichments of stable isotope-labeled amino acids in Caco-2 cells cultured in medium with or without supplemental folate (control and folate-restricted, respectively) for 4 days followed by incubation in culture medium containing [U- 13 C 5 ]methionine and [3-13 C]serine Labeled Amino Acid Plateau Enrichment, mol % excess Supplemented Nonsupplemented [ 2 H 3]leucine* [ 13 C 1]serine* [ 13 C 1]methionine* [ 13 C 1]cystathionine [ 13 C 5]methionine* [ 13 C 4]homocysteine* [ 13 C 4]methionine* [ 13 C 4]cystathionine *Data (means SE) are derived by fitting time course data from Fig. 4 to a single exponential curve. See MATERIALS AND METHODS for details. Data (means SE) are derived by fitting time course data from Fig. 4 to a sigmoidal curve. See MATERIALS AND METHODS for details. Significantly different from folate-adequate cultures; P Remethylated homocysteine comprised a significant fraction (18 19%) of the free methionine pool in the Caco-2 cell cultures, although exogenous methionine was accessible in the culture medium. These results indicate the importance of the methylation cycle to methionine metabolism in Caco-2 cell cultures. Significant remethylation activity is understandable in this cell type as a consequence of the sizable methionine and cysteine requirements of the intestine for protein synthesis, as well as for maintenance of methylation reactions in tissues with a large cell turnover rate. Reaction pathways that generate one-carbon units from betaine, dimethylglycine, sarcosine, glycine, and histidine are present in the liver and kidney (8). Although these compounds have been implicated as significant contributors of one-carbon groups for remethylation (26, 27, 37, 39), the three carbon of serine provided nearly all of the one-carbon units for homocysteine remethylation in folate-adequate Caco-2 cells (Figs. 2 and 5C). This result was predictable, considering that the intestine does not express betaine-homocysteine methyltransferase, has little expression of glycine N-methyltransferase or the glycine cleavage system, and is not known to express the enzymes of histidine catabolism (31, 38, 39). A similar dependence of one-carbon metabolism on serine might exist in most tissues other than the liver and kidney. For example, the three carbon of serine accounted for 95% of one-carbon groups in the human MCF-7 breast cancer cell line, whereas one-carbon group production from glycine cleavage was undetectable (14). Furthermore, 80% of one-carbon units used for total body remethylation were estimated to be derived from the three carbon of serine in our recent studies (9) with healthy humans, which suggest that tissues other than the liver and kidney

6 HOMOCYSTEINE METABOLISM IN INTESTINAL CELLS G593 Fig. 5. Homocysteine remethylation in folate-adequate and folate-restricted Caco-2 cell cultures. A: total fraction of intracellular free methionine remethylated (mean SE; n 3 at each time point). B: fraction of intracellular free methionine remethylated by folate-dependent remethylation using the three carbon of serine (mean SE; n 3 at each time point). C: percentage of total remethylation that used one-carbon units obtained from the three carbon of serine. Data are presented as means SE (n 6). *Significantly lower than control cultures (P 0.001); **significantly lower than control cultures and total remethylation in folate-restricted cultures (P 0.001). Fig. 6. Fraction of cystathionine synthesized from the [3-13 C]serine tracer. Data are presented as means SE (n 6). *Significantly lower than control cultures (P 0.001). primarily depend on serine to maintain remethylation. It seems plausible that the 10 20% of the one-carbon units used for total body remethylation that were not derived from the three carbon of serine were produced in the liver and kidney from the breakdown of choline, histidine, and glycine. It has been known for decades that folate restriction alters one-carbon metabolism and specifically the use of the three carbon of serine in one-carbon reactions (2). However, little is published on the effects of folate restriction on one-carbon metabolism in the intestine. Folate deficiency can reduce the colonic S-adenosylmethionine-to-S-adenosylhomocysteine ratio (30), and folate depletion alters certain aspects of DNA metabolism related to the one-carbon cycle (6, 11). This is the first report of reduced total remethylation ( 33%) and reduced folate-dependent remethylation from serine ( 60%) caused by folate restriction (Fig. 5, A and B). The 50% of unaccountable remethylation in folate-restricted cultures (i.e., not derived from the three carbon of serine) argues that one or more of the normally minor onecarbon donor pathways was more active under conditions of folate inadequacy than folate adequacy. Under folate-deficient conditions, glycine, histidine, betaine, dimethylglycine, and sarcosine may become important in intestinal one-carbon metabolism. This seems counterintuitive, because betaine-homocysteine methyltransferase is not expressed in intestine, and all other donors also require folate for their use in one-carbon metabolism. It is possible, however, that one or more of these pathways is more resistant to folate deficiency than the serinedependent pathway. For example, histidine was estimated to provide 20% of the one-carbon units for total body remethylation in rats, and its use was not hindered in folate deficiency (27). An alternative explanation for the unaccountable onecarbon donation to remethylation is that this cell line might express the enzymes of one-carbon metabolism in a pattern different from intact intestine or intestinal mucosa. Other than a report that the transsulfuration pathway has a role in cysteine metabolism of isolated rat enterocytes (7), the significance of the transsulfuration pathway s presence in the intestine is not well defined. The transsulfuration flux observed in Caco-2 cells was greater than anticipated based on in vitro

7 G594 HOMOCYSTEINE METABOLISM IN INTESTINAL CELLS mucosal cystathionine -synthase and -cystathionase enzyme activities relative to those observed in kidney and liver (24). We found transsulfuration to be quantitatively significant in homocysteine catabolism and cysteine production (13% of cysteine flux) in Caco-2 cells. Finkelstein (12) suggested that tissues containing the transsulfuration pathway enzymes utilize this pathway for cysteine production in support of their large demands for glutathione. In support of this hypothesis, transsulfuration produced nearly half of the cysteine and was used for a significant fraction of glutathione production in the HepG2 human hepatoma cell line, and transsulfuration flux was induced under oxidative conditions that consume glutathione (23). Increased transsulfuration fluxes also could be a function of the high methionine content of the medium, because subsequent overproduction of S-adenosylmethionine might activate the transsulfuration pathway. However, there is little evidence that upregulation of transsulfuration flux by methionine occurs in extrahepatic tissues (12). A functional transsulfuration system could empower the intestine to assist the liver in the catabolism of homocysteine, similar to the role of the kidney (4). Homocysteine removal capacity of the intestine might be less significant than that of the kidneys, however, because the activities of the transsulfuration pathway enzymes of rat mucosa in vitro were 20 40% of that found in rat kidney (24). As yet, there is no evidence that the intestine imports homocysteine from the plasma, but the high transsulfuration activity in this organ suggests that it could assist in homocysteine catabolism. In contrast, the discovery that media recovered from Caco-2 cell cultures contained [ 13 C]homocysteine and [ 13 C]cystathionine, which are produced intracellularly from the methionine and serine tracers, suggests that the intestine might contribute to the plasma homocysteine load. It also suggests that the transsulfuration pathway may not have the capacity to handle excess homocysteine, which it might produce. More research is needed to determine the role of the intestine in regulating the homocysteine concentration of the plasma. Reduced labeling of cystathionine in Caco-2 cells cultured in low-folate medium could be interpreted in two ways. If the cystathionine pool size of the folate-restricted cell cultures was similar to or smaller than that found in control cultures, then these data suggest that transsulfuration flux was reduced in folate restriction. This could be due to an overall reduction of methylation cycle flux in these cells, but the similar rates of homocysteine labeling between folate-restricted and control cells argue against this possibility. An alternative interpretation is that the labeled cystathionine produced under these conditions is diluted in a greater overall cystathionine pool size in folate-restricted cultures. An argument for this possibility is that folate restriction is associated with elevated plasma cystathionine concentrations in humans and rats (25, 32, 33) and might indicate that transsulfuration pathway activity is elevated in response to reduced remethylation capacity. Without knowledge of the intracellular cystathionine pool size, however, we cannot ascertain the mechanism behind the slower labeling of the cystathionine pool with folate restriction at this time. In conclusion, with the combined [3-13 C]serine and [U- 13 C 5 ]methionine tracer model we found significant activity of the remethylation and transsulfuration pathways in the Caco-2 model of absorptive intestinal cells. The three carbon of serine provides nearly all of the one-carbon units used for remethylation in folate-supplemented cultures. Remethylation was reduced with folate restriction, and under these conditions the three carbon of serine provided only 50% of the onecarbon units used for remethylation. Transsulfuration flux was significant and provided 13% of cellular cysteine in folatesupplemented Caco-2 cell cultures. Export of homocysteine and cystathionine from these cells might indicate that the intestine contributes to the plasma load of these metabolites, although these cells can catabolize them via transsulfuration. Further studies are needed to determine the identity and quantitative significance of the donors of one-carbon groups for remethylation during folate restriction as well as to determine whether intestinal cells have an impact on the plasma homocysteine load. GRANTS This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK (to J. F. Gregory III) and the University of Florida University Scholars Program (to J. H. Townsend). This paper is Florida Agricultural Experiment Station Journal Series No. R REFERENCES 1. Appling DR. Compartmentation of folate-mediated one-carbon metabolism in eukaryotes. FASEB J 5: , Arnstein HRV and Stankovic V. The effect of certain vitamin deficiencies on glycine biosynthesis. Biochemistry 62: , Blount BC, Mack MM, Wehr CM, MacGregor JT, Hiatt RA, Wang G, Wickramasinghe SN, Everson RB, and Ames BN. Folate deficiency causes uracil misincorporation into human DNA and chromosome breakage: implications for cancer and neuronal damage. Proc Natl Acad Sci USA 94: , Bostom AG, Brosnan JT, Hall B, Nadeau MR, and Selhub J. Net uptake of plasma homocysteine by the rat kidney in vivo. Atherosclerosis 116: 59 62, Boushey CJ, Beresford SA, Omenn GS, and Motulsky AG. A quantitative assessment of plasma homocysteine as a risk factor for vascular disease. Probable benefits of increased folic acid intakes. JAMA 274: , Choi SW and Mason JB. Folate status: effects on pathways of colorectal carcinogenesis. J Nutr 132, Suppl: 2413S 2418S, Coloso RM and Stipanuk MH. Metabolism of cyst(e)ine in rat enterocytes. J Nutr 119: , Cooke RJ. Folate metabolism. In: Homocysteine in Health and Disease, edited by Carmel R and Jacobsen DW. Cambridge, UK: Cambridge University Press, 2001, p Davis SR, Stacpoole PW, Williamson J, Kick LS, Quinlivan EP, Coats BS, Shane B, Bailey LB, and Gregory JF III. Tracer-derived total and folate-dependent homocysteine remethylation and synthesis rates in humans indicate that serine is the main one-carbon donor. Am J Physiol Endocrinol Metab 286: E272 E279, Delgado-Reyes CV, Wallig MA, and Garrow TA. Immunohistochemical detection of betaine-homocysteine S-methyltransferase in human, pig, and rat liver and kidney. Arch Biochem Biophys 393: , Duthie SJ, Narayanan S, Brand GM, Pirie L, and Grant G. Impact of folate deficiency on DNA stability. J Nutr 132, Suppl: 2444S 2449S, Finkelstein JD. Metabolism of homocysteine: pathways and regulation. Eur J Pediatr 157, Suppl: S40 S44, Fleet JC and Wood RJ. Specific 1,25(OH) 2 D 3-mediated regulation of transcellular calcium transport in Caco-2 cells. Am J Physiol Gastrointest Liver Physiol 276: G954 G958, Fu TF, Rife JP, and Schirch V. The role of serine hydoxymethyltransferase isozymes in one-carbon metabolism in MCF-7 cells as determined by 13 C NMR. Arch Biochem Biophys 393: 42 50, Garrow TA. Betaine-dependent remethylation. In: Homocysteine in Health and Disease, edited by Carmel R and Jacobsen DW. Cambridge, UK: Cambridge University Press, 2001, p

8 HOMOCYSTEINE METABOLISM IN INTESTINAL CELLS G Giovannucci E. Epidemiological studies of folate and colorectal neoplasia: a review. J Nutr 132, Suppl: 2350S 2355S, Glantz SA. Primer of biostatistics. New York: McGraw-Hill, 2002, p Gregory JF III, Cuskelly GJ, Shane B, Toth JP, Baumgartner TG, and Stacpoole PW. Primed, constant infusion with [ 2 H 3]serine allows in vivo kinetic measurement of serine turnover, homocysteine remethylation, and transsulfuration processes in human one-carbon metabolism. Am J Clin Nutr 72: , a.Homocysteine Studies Collaboration. Homocysteine and risk of ischemic heart disease. A meta analysis. JAMA 288: , Jacques PF, Bostom AG, Williams RR, Ellison RC, Eckfeldt JH, Rosenberg IH, Selhub JS, and Rozen R. Relation between folate status, a common mutation in methylenetetrahydrofolate reductase, and plasma homocysteine concentrations. Circulation 93: 7 9, Jones PA and Gonzalgo ML. Altered DNA methylation and genome stability: a new pathway to cancer? Proc Natl Acad Sci USA 94: , Keating JN, Weir DG, and Scott JM. Demonstration of methionine synthetase in intestinal mucosal cells of the rat. Clin Sci (Lond) 69: , Liu SM, Mata G, Figliomeni S, Powell BC, Nesci A, and Masters DG. Transsulfuration, protein synthesis rate and follicle mrna in the skin of young Merino lambs in response to infusions of methionine and serine. Br J Nutr 83: , Mosharov E, Cranford MR, and Banerjee R. The quantitatively important relationship between homocysteine metabolism and glutathione synthesis by the transsulfuration pathway and its regulation by redox changes. Biochemistry 39: , Mudd SH, Finkelstein JD, Irreverre F, and Laster L. Microassays and tissue distribution of three enzymes of the pathway. J Biol Chem 240: , Mudd SH, Levy HL, and Abeles RH. A derangement in B12 metabolism leading to homocystinemia, cystathionemia, and methylmalonic aciduria. Biochem Biophys Res Commun 35: , Schalinske KL and Steele RD. Quantitiation of carbon flow through the hepatic folate-dependent one-carbon pool in rats. Arch Biochem Biophys 271: 49 55, Schalinske KL and Steele RD. Quantification of the carbon flow through the folate-dependent one-carbon pool using radiolabeled histidine: effect of altered thyroid and folate status. Arch Biochem Biophys 328: , Selhub J. Homocysteine metabolism. Annu Rev Nutr 19: , Selhub J, Jacques PF, Wilson PWF, Rush D, and Rosenberg RH. Vitamin status and intake as primary determinants of homocysteinemia in an elderly population. JAMA 270: , Sohn KJ, Stempak JM, Reid S, Shirwadakar S, Mason JB, and Kim YI. The effect of dietary folate on genomic and p53-specific DNA methylation in rat colon. Carcinogenesis 24: 81 90, Sowden MP, Collins HL, Smith HC, Garrow TA, Sparks JD, and Sparks CE. Apolipoprotein B mrna and lipoprotein secretion are increased in McArdle RH-7777 cells by expression of betaine-homocysteine S-methyltransferase. Biochem J 341: , Stabler SP, Lindenbaum J, Savage DG, and Allen RH. Elevation of serum cystathionine levels in patients with cobalamin and folate deficiency. Blood 81: , Stabler SP, Sampson DA, Wang LP, and Allen RH. Elevations of serum cystathionine and total homocysteine in pyridoxine-, folate-, and cobalamin-deficient rats. J Nutr Biochem 8: , Storch KJ, Wagner DA, Burke JF, and Young VR. Quantitative study in vivo on methionine cycle in humans using [methyl- 2 H 3]- and [1-13 C]methionine. Am J Physiol Endocrinol Metab 255: E322 E331, Sunden SLF, Renduchintala MS, Park EI, Miklasz SD, and Garrow TA. Betaine-homocysteine methyltransferase expression in porcine and human tissues and chromosomal localization of the human gene. Arch Biochem Biophys 345: , Wendel U and Bremner H. Betaine in the treatment of homocystinuria due to 5,10-methylentetrahydrofolate reductase deficiency. Eur J Pediatr 142: , Yeo EJ and Wagner DA. Tissue distribution of glycine N-methyltransferase, a major folate-binding protein in liver. Proc Natl Acad Sci USA 91, Yoshida T and Kikuchi G. Major pathways of serine and glycine metabolism in various organs of the rat and cock. J Biochem (Tokyo) 73: , Downloaded from by guest on July 31, 2012