Folate Intake, Mthfr Genotype, and Sex Modulate Choline Metabolism in Mice

Abstract

Choline and folate are interrelated in 1-carbon metabolism, mostly because of their shared function as methyl donors for homocysteine remethylation. Folate deficiency and mutations of methylenetetrahydrofolate reductase (MTHFR) reduce the availability of a major methyl donor, 5-methyltetrahydrofolate, which in turn may lead to compensatory changes in choline metabolism. This study investigated the hypothesis that reductions in methyl group supply, either due to dietary folate deficiency or Mthfr gene deletion, would modify tissue choline metabolism in a sex-specific manner. Mthfr wild type (+/+) or heterozygous (+/−) knockout mice were randomized to a folate-deficient or control diet for 8 wk during which time deuterium-labeled choline (d9-choline) was consumed in the drinking water (~10 μmol/d). Mthfr heterozygosity did not alter brain choline metabolite concentrations, but it did enhance their labeling in males (P < 0.05) and tended to do so in females (P < 0.10), a finding consistent with greater turnover of dietary choline in brains of +/− mice. Dietary folate deficiency in females yielded 52% higher (P = 0.027) hepatic glycerophosphocholine, which suggests that phosphatidylcholine (PtdCho) degradation was enhanced. Labeling of the hepatic PtdCho in d3 form was also reduced (P < 0.001) in females, which implies that fewer of the dietary choline-derived methyl groups were used for de novo PtdCho biosynthesis under conditions of folate insufficiency. Males responded to folate restriction with a doubling (P < 0.001) of hepatic choline dehydrogenase transcripts, a finding consistent with enhanced conversion of choline to the methyl donor, betaine. Collectively, these data show that several adaptations in choline metabolism transpire as a result of mild perturbations in folate metabolism, presumably to preserve methyl group homeostasis.

Introduction

Choline, a water-soluble micronutrient, can be converted to the neurotransmitter acetylcholine, oxidized to the 1-carbon donor betaine, or metabolized to phosphatidylcholine (PtdCho).⁸ There are 2 pathways of PtdCho synthesis: the de novo pathway, which sequentially methylates phosphatidylethanolamine in a reaction performed by phosphatidylethanolamine N-methyltransferase (PEMT) using S-adenosylmethionine (SAM)-derived methyl groups, and the CDP-choline pathway where free choline is used to synthesize PtdCho and CTP:phosphocholine cytidylyltransferase catalyzes the rate-limiting reaction (1). PtdCho is a structural component of all cell membranes, a source of lipid-signaling molecules, and a constituent of lipoproteins (1, 2).

Choline oxidation to the methyl donor, betaine, is catalyzed by choline dehydrogenase (CHDH). In liver and kidney, the labile methyl groups associated with betaine are used to reform methionine from homocysteine in a reaction catalyzed by betaine homocysteine methyltransferase (BHMT) (3). Alternatively, 5-methyltetrahydrofolate (THF), the product of methyleneTHF reductase (MTHFR), is used as the methyl donor (4). Methionine is the precursor of the primary methyl donor SAM, which can subsequently be used by over 50 methyltransferases, including PEMT (5, 6).

Due to their intermingling in 1-carbon metabolism, the use of choline is dependent in part upon the availability of folate and vice versa (7). Deficiency in the folate-metabolizing enzyme, MTHFR, results in lower hepatic concentrations of phosphocholine (PCho; the storage form of choline) in mice (8) and yields lower plasma PtdCho in men with the MTHFR 677TT genotype (9). Similarly, dietary folate deficiency depletes brain membrane PtdCho in male rats (10) and yields diminished plasma PtdCho in women (11, 12). More recently, chronic administration of a folate-deficient diet to mice yielded lower hepatic betaine concentrations and steatosis, particularly in males (13). Males are more susceptible to choline deficiency than females, due in part to reduced synthesis of a new choline moiety through the PEMT pathway (14).

The primary aim of the current study was to further delineate the sex-specific metabolic and genomic effects of folate deficiency and a single gene deletion in the Mthfr gene on choline metabolism. Male and female mice with the Mthfr +/+ or +/− genotype were administered a control diet or a folate-deficient (FD) diet for 8 wk along with deuterium-labeled choline (d9-choline) in their drinking water. The three deuterated methyl group (d9-choline) d9-choline tracer enabled examination of the fate of dietary choline (d9-choline metabolites) and its methyl groups (d3-PtdCho) (Supplemental Fig. 1) as a function of folate deficiency and/or Mthfr genotype.

Methods and Materials

Mice and diets.

Protocols for all animal procedures were approved by the Institutional Animal Care and Use Committee at Cal Poly Pomona University and were conducted in accordance with the Guide for the Care and Use of Laboratory Animals. Mthfr heterozygote (+/−) BALB/c mice breeding pairs (n = 2) were a gift from Dr. Rima Rozen (McGill University, Montreal, Canada) and were generated as previously described (15). A mouse colony was established and maintained via heterozygous breeding in microisolator cages (Ancare) containing corn cob bedding (Harlan Teklad) in a temperature-controlled room (22–25°C and 70% humidity) with a 12-h-light/-dark cycle.

At weaning (3 wk of age), genotyping was performed (15) and Mthfr +/+ and +/− mice (female and male) were fed a nutritionally adequate, semipurified control diet (Harland Teklad, TD.06565) with all the necessary components recommended by the AIN (16), including 2 mg folic acid/kg diet (Supplemental Table 1). Throughout this period (wk 3 to 10), deionized water supplemented with a small amount of unlabeled free choline (1.6 mmol/L or ~10 μmol/d) was also provided. At 10 wk of age, Mthfr +/+ and +/− mice continued consuming the semipurified control diet sufficient in folic acid or were fed the FD diet that was identical to the control diet except that it lacked folic acid. During the treatment phase, unlabeled choline in the deionized water was replaced with d9-choline (Cambridge Isotopes Laboratory). Food and water were consumed ad libitum and body weights were recorded weekly. The percentage of total ingested choline derived from the labeled choline was ~20%, a calculation based on the known amounts of unlabeled and labeled choline present in the food and water along with reported food and water intakes of mice of similar genetic background (17).

After 8 wk of the diets (18 wk of age), mice were killed by exposure to CO₂. Blood was collected by cardiac puncture, placed on ice, and processed for plasma within 4 h. Tissues (liver, brain, and kidney) were removed, immediately frozen in liquid nitrogen, and stored at −80°C.

Measurements of plasma metabolites.

Blood was collected into EDTA-coated tubes and centrifuged at 1300 × g for 15 min at 4°C. Plasma was separated from blood cells and stored at −80°C. Liquid chromatography-MS was used to measure plasma choline, betaine, and dimethylglycine (DMG) (18) as well as plasma PtdCho, lysophosphatidylcholine, glycerophosphocholine (GPC), PCho, and sphingomyelin (19) with modifications based on our instrumentation (12, 20). Total plasma folate concentrations were microbiologically measured using Lactobacillus rhamnosus as the test organism (21).

Measurements of tissue metabolites.

Liquid chromatography-MS was used to measure unlabeled and labeled choline, betaine, and DMG (18) as well as PCho, GPC, PtdCho, and sphingomyelin (19) following their extraction from pulverized frozen tissue (10–20 mg) as previously detailed (12, 20).

Isotopic enrichment [labeled metabolite/(labeled + unlabeled metabolite)] was examined in each of the tissues and plasma to monitor the flow of dietary choline (d9-choline metabolites) and its methyl groups (d3-PtdCho) within and among relevant pools. In liver, metabolite enrichment ratios were examined as indicators of dietary choline use through a specific portion of a pathway (enrichment of product/enrichment of precursor) or its partitioning between pathways [enrichment of metabolite(s) in the CDP-choline compared with oxidative pathways]. For liver, we assumed that the majority of orally consumed d9-choline was taken up as d9-choline. Peripheral tissues, however, have access to secreted labeled PtdCho metabolites (i.e. lipoproteins) and thus multiple entry routes are feasible. As such, enrichment ratios were not examined in peripheral tissues.

Quantitative real-time RT-PCR.

Real-time reactions were carried out to analyze the gene expression of Ct α isoform (Ctα), Pemt, Chdh, Bhmt, and Mthfr in liver, kidney, and brain tissues. Total RNA was extracted from frozen tissues by using a NucleoSpin RNA II kit (Machery-Nagel). Turbo DNA-free DNase (Ambion) was applied to remove contaminated genomic DNA. Total concentration and quality of the isolated RNA were assayed with a NanoDrop ND-1000 instrument (Thermo). RNA with a 260:280 ratio above 1.8 was accepted. RT was performed with the ImProm-II Reverse Transcription system (Promega) according to the manufacturer’s instructions. Real-time PCR was carried out by the SYBR Green system in a Roche LightCycler 480 machine. Primers for the targeted genes were designed using Gene Runner Version 3.01 (Supplemental Table 2). The reaction conditions were as follows: 95°C for 5 min followed by 40 cycles with 15 s at 95°C, 20 s at 60°C, and 20 s at 72°C. Data are expressed as ΔΔCt (22) in which the expression level of the gene of interest is normalized by the expression level of the housekeeping gene [hypoxanthine-guanine phosphoribosyltransferase (Hprt-1)] as fold-change.

Western blotting.

Protein was extracted from 50 mg frozen tissue in lysis buffer with proteinase inhibitor cocktails (Sigma-Aldrich). Total protein concentrations were quantified by the Bradford assay (Thermo). The extracts were then separated by SDS-PAGE and transferred to Immobilon FL polyvinylidene difluoride membranes (Sigma-Aldrich). Primary antibodies included: Mthfr (gift from Dr. Rima Rozen, McGill University, Montreal, Canada), Pemt (gift from Dr. Dennis Vance, University of Alberta, Canada), and Gapdh (Abcam). Secondary antibodies were either IRDye 800CW goat anti rabbit or IRDye 680 goat anti mouse (LI-COR), depending on the primary antibodies. Target protein bands were visualized and quantified by the LI-COR Odyssey imaging system (LI-COR) and normalized by reference protein Gapdh as fold-change.

Statistical analysis.

To assess the sex-specific effects of folate intake and Mthfr genotype on choline metabolic indicators (i.e. choline metabolites, gene transcripts, and proteins), 1- or 2-factor ANOVA tests were conducted separately for females and males. Correlations between plasma folate concentrations and choline metabolites or gene expression were determined using the Pearson correlation coefficient (r). To further elucidate the established effect of sex on choline metabolism, differences in the choline metabolic indicators between male and female mice consuming the control diet were examined using Student’s t tests. Differences were considered significant at P 0.05, and 0.05 < P < 0.10 were indicative of trends. Attempts to correct for multiple analyses were not undertaken due to the hypothesis-driven nature of the study and the relatively small sample size. Values presented are means ± SEM. All analyses were conducted with SPSS (release 15.0 for Windows).

Results

Plasma total folate concentrations

At the end of this 8-wk feeding study (i.e. age 18 wk), plasma total folate concentrations were lower (P < 0.001) than in controls in the FD-treated females (19.7 ± 3.0 vs. 60.1 ± 5.7 nmol/L) and males (10.6 ± 1.9 vs. 63.6 ± 5.0 nmol/L). In male mice, the Mthfr genotype modified the response of plasma folate to folate treatment (P-interaction = 0.015). In those fed the control diet, +/− mice had lower (P = 0.027) folate concentrations (53 ± 6 nmol/L) than +/+ mice (74 ± 5 nmol/L), but in those fed the FD diet, +/− mice tended to have higher (P = 0.073) plasma folate (13.4 ± 2.5 nmol/L) than +/+ mice (6.7 ± 0.6 nmol/L).

Effects of folate intake and Mthfr genotype on hepatic choline indices

Female mice.

Folate deficiency yielded 52% higher (P = 0.027) GPC concentrations and 30% lower (P = 0.017) hepatic d3-PtdCho enrichment (Table 1). The enrichment ratio of d3-PtdCho:d9-choline was also lower (P = 0.049) in the FD group (Table 1). Chdh and Ct-α expression tended to be higher (P = 0.055) and lower (P = 0.054), respectively, in FD mice (Supplemental Fig. 2). Additionally, an inverse relationship was observed between plasma folate and hepatic free choline (r = −0.55; P = 0.026) (Supplemental Fig. 3). Collectively, these data suggest that folate deficiency promotes PtdCho degradation as a means of providing free choline for cellular methylation, but that it also attenuates the use of choline-derived methyl groups for PtdCho biosynthesis through the PEMT pathway.

TABLE 1.

Liver choline metabolites in female Mthfr +/+ or +/− mice fed a control or folate-deficient (FD) diet for 8 wk¹

	Control diet			FD diet			P (ANOVA)
	+/+	+/−	Total	+/+	+/−	Total	Diet	Genotype	Interaction
Metabolite concentration
Choline, nmol/g	303 ± 19	285 ± 24	294 ± 15	311 ± 28	371 ± 49	341 ± 28	0.16	0.52	0.24
Betaine, nmol/g	412 ± 76	349 ± 29	380 ± 40	392 ± 100	349 ± 22	371 ± 49	0.88	0.43	0.89
DMG, nmol/g	19.5 ± 2.9	24.2 ± 2.4	21.8 ± 1.9	20.6 ± 2.9	22.7 ± 1.6	21.6 ± 1.6	0.94	0.20	0.61
PCho, nmol/g	450 ± 173	478 ± 110	464 ± 97	389 ± 146	590 ± 120	501 ± 94	0.93	0.78	0.60
PtdCho, μmol/g	17.1 ± 1.3	15.1 ± 1.7	16.1 ± 1.1	17.3 ± 1.5	18.0 ± 1.6	17.6 ± 1.0	0.33	0.66	0.38
Sphingomyelin, μmol/g	1.5 ± 0.2	1.5 ± 0.2	1.5 ± 0.1	1.7 ± 0.2	1.7 ± 0.2	1.7 ± 0.1	0.70	0.56	0.63
GPC, nmol/g	636 ± 211	534 ± 21	573 ± 82	962 ± 89	799 ± 112	871 ± 75	0.027	0.29	0.80
Metabolite enrichment, %
d9-Choline	4.0 ± 0.2	4.6 ± 0.4	4.3 ± 0.2	3.7 ± 0.2	4.1 ± 0.3	3.9 ± 0.2	0.23	0.12	0.88
d9-Betaine	9.5 ± 0.8	11 ± 1.7	10 ± 0.9	9.2 ± 1.2	9.4 ± 1.2	9.0 ± 0.8	0.53	0.59	0.69
d6-DMG	19 ± 1.7	21 ± 1.6	20 ± 1.1	19 ± 2.1	18 ± 2.1	19 ± 1.4	0.46	0.82	0.39
d9-PtdCho	3.8 ± 0.5	4.0 ± 0.5	3.9 ± 0.3	3.0 ± 0.3	3.5 ± 0.2	3.3 ± 0.2	0.13	0.39	0.85
d3-PtdCho	3.1 ± 0.4	3.9 ± 0.5	3.5 ± 0.3	2.5 ± 0.4	2.5 ± 0.3	2.5 ± 0.2	0.017	0.30	0.29
d9-GPC	5.4 ± 1.5	4.2 ± 0.9	4.8 ± 0.8	5.3 ± 0.6	4.6 ± 1.0	4.9 ± 0.6	0.89	0.39	0.81
Enrichment ratio
d9-Betaine:d9-choline	2.4 ± 0.2	2.4 ± 0.3	2.4 ± 0.2	2.4 ± 0.2	2.2 ± 0.2	2.3 ± 0.1	0.90	0.56	0.70
d6-DMG:d9-choline	4.7 ± 0.4	4.7 ± 0.3	4.7 ± 0.2	5.1 ± 0.4	4.3 ± 0.3	4.7 ± 0.3	0.99	0.25	0.27
d6-DMG:d9-betaine	2.0 ± 0.2	2.1 ± 0.2	2.1 ± 0.1	2.1 ± 0.1	1.9 ± 0.1	2.0 ± 0.1	0.89	0.73	0.46
d9-Betaine:d9-PtdCho	2.7 ± 0.3	2.6 ± 0.2	2.6 ± 0.2	3.1 ± 0.4	2.8 ± 0.4	2.9 ± 0.3	0.34	0.53	0.60
d9-PtdCho:d9-choline	0.9 ± 0.1	0.9 ± 0.1	0.9 ± 0.1	0.8 ± 0.1	0.9 ± 0.1	0.8 ± 0.1	0.48	0.96	0.71
d3-PtdCho:d9-choline	0.8 ± 0.1	0.9 ± 0.1	0.8 ± 0.1	0.7 ± 0.1	0.6 ± 0.1	0.6 ± 0.1	0.049	0.93	0.46
d3-PtdCho:d9-PtdCho	0.9 ± 0.1	1.0 ± 0.1	0.9 ± 0.1	0.8 ± 0.1	0.7 ± 0.1	0.8 ± 0.1	0.13	0.84	0.24
d9-GPC:d9-PtdCho	1.4 ± 0.4	1.1 ± 0.2	1.2 ± 0.2	1.7 ± 0.4	1.3 ± 0.3	1.5 ± 0.2	0.35	0.30	0.93

¹Values are means ± SEM, = 5.

The Mthfr genotype did not affect measurements or enrichments of hepatic choline metabolites and the 2-way interaction term (folate × Mthfr) was not significant.

To further explore the lack of effect of the Mthfr genotype on hepatic choline metabolism, the transcript and protein levels of Mthfr were measured. No differences were detected in transcript or protein levels between mice with 1 or 2 copies of the gene. This indicates that by 18 wk of age (i.e. the end of the study), +/− mice were compensating for their gene deletion by overexpressing their single copy.

Male mice.

Similar to females, Chdh expression in males was higher (200%; P < 0.001) in the FD group than in the control group. However, folate deficiency did not modify hepatic concentration of the choline metabolites, their enrichment, or the enrichment ratios (Supplemental Table 3). In addition, none of the biomarkers of choline metabolism were affected by Mthfr genotype and the 2-way interaction term (folate × Mthfr) was not significant. Like female mice, no differences were detected in Mthfr expression between +/+ and +/− mice.

Effects of folate intake and Mthfr genotype on extrahepatic choline indices

Female mice.

Short-term dietary folate deficiency itself did not modify any of the extrahepatic indices of choline metabolism (Supplemental Tables 4–6). However, enrichment of plasma d3-PtdCho was 27% higher (P = 0.003) in Mthfr +/− than in +/+ mice and the brain metabolite enrichments of d9-choline (P = 0.08), d9-PtdCho (P = 0.06), d3-PtdCho (P = 0.05), and d9-sphingomyelin (P = 0.09) tended to be higher (Table 2). These data demonstrate that more of the dietary choline appeared in brain of the Mthfr +/− mice with evidence of enhanced turnover and recycling of the choline moiety (d9-metabolites). Because Bhmt is not expressed in adult brain, the higher d3-PtdCho enrichment in the Mthfr heterozygotes may reflect greater uptake of d3-PtdCho from plasma or its greater synthesis from d3-SAM (not measured in the present study).

TABLE 2.

Brain choline metabolite enrichment in female Mthfr +/+ or +/− mice fed a control or folate-deficient (FD) diet for 8 wk¹

	Control diet			FD diet			P (ANOVA)
	+/+	+/−	Total	+/+	+/−	Total	Diet	Genotype	Interaction
Metabolite enrichment, %
d9-Choline	3.7 ± 0.4	4.1 ± 0.3	3.9 ± 0.2	3.5 ± 0.3	4.4 ± 0.5	3.9 ± 0.3	0.89	0.08	0.58
d9-Betaine	5.1 ± 0.5	5.4 ± 0.4	5.2 ± 0.3	5.1 ± 0.3	6.2 ± 0.7	5.6 ± 0.4	0.41	0.18	0.40
d9-PCho	1.2 ± 0.3	2.7 ± 0.2*	2.0 ± 0.3	1.6 ± 0.2	1.6 ± 0.4	1.6 ± 0.2	0.22	0.027	0.017
d9-PtdCho	3.3 ± 0.3	3.7 ± 0.2	3.5 ± 0.2	3.2 ± 0.2	3.9 ± 0.4	3.5 ± 0.2	0.74	0.06	0.51
d3-PtdCho	1.9 ± 0.2	2.1 ± 0.1	2.0 ± 0.1	1.7 ± 0.1	2.1 ± 0.2	1.9 ± 0.1	0.66	0.05	0.46
d9-Sphingomyelin	3.1 ± 0.2	3.3 ± 0.2	3.2 ± 0.2	3.0 ± 0.2	3.5 ± 0.3	3.2 ± 0.2	0.86	0.09	0.53
d9-GPC	2.8 ± 0.7	3.6 ± 0.5	3.2 ± 0.4	3.0 ± 0.3	3.5 ± 0.8	3.2 ± 0.4	0.95	0.29	0.77

¹Values are means ± SEM, = 6–7. *Different from +/+, P < 0.001.

In addition, Mthfr genotype and folate intake interacted to influence a few of the choline metabolites in brain (Table 2) and kidney (Supplemental Table 5). However, no clear patterns emerged. The expression of the candidate genes (including Mthfr) were unaltered by folate deficiency and/or Mthfr heterozygosity (data not shown).

Male mice.

The effect of the Mthfr genotype was assessed in plasma, brain, and kidney of males consuming the control diet using a 1-factor ANOVA (peripheral tissues from FD mice were not available). In plasma, heterozygotes had higher enrichment levels of d9-sphingomyelin (P = 0.008), d9-lysoPtdCho (P = 0.016), and d9-PtdCho (P = 0.06). Similarly, the enrichment of the majority of choline metabolites in brain and kidney (including d9-choline) was greater in mice with the Mthfr +/− genotype (Table 3). Total concentrations of the choline metabolites (Supplemental Table 7) and expression of the candidate genes (including Mthfr) were unaltered by Mthfr heterozygosity.

TABLE 3.

Brain and kidney choline metabolite enrichment in male Mthfr +/+ or +/− mice fed a control diet for 8 wk¹

		Brain			Kidney
	+/+	+/−	P (ANOVA)	+/+	+/−	P (ANOVA)
Metabolite enrichment, %
d9-Choline	2.3 ± 0.1	3.7 ± 0.2	<0.001	2.8 ± 0.2	4.3 ± 0.3	0.004
d9-Betaine	3.7 ± 0.3	5.5 ± 0.6	0.023	4.0 ± 0.3	5.7 ± 0.4	0.005
d9-PCho	0.8 ± 0.1	1.6 ± 0.2	0.004	2.0 ± 0.3	2.9 ± 0.5	0.17
d9-PtdCho	2.4 ± 0.09	3.3 ± 0.2	0.002	3.2 ± 0.2	4.4 ± 0.3	0.003
d3-PtdCho	0.9 ± 0.07	1.2 ± 0.07	0.008	2.1 ± 0.3	2.5 ± 0.1	0.212
d9-Sphingomyelin	2.4 ± 0.06	3.0 ± 0.2	0.009	2.9 ± 0.2	4.3 ± 0.3	0.003
d9-GPC	2.1 ± 0.2	2.9 ± 0.5	0.24	3.1 ± 0.2	4.3 ± 0.4	0.014

¹Values are mean ± SEM, = 5–6.

Effects of sex on hepatic choline metabolism

Because genotype did not affect hepatic indicators of choline metabolism in this study, the effect of sex was investigated in mice consuming the control diet without regard to the presence of the gene deletion (Table 4). Compared with males, females displayed: 1) greater use of dietary choline as a methyl donor (i.e. higher enrichment ratios of d9-betaine:d9-choline and d6-DMG:d9-choline); 2) greater use of dietary choline for the biosynthesis of PtdCho via the PEMT pathway compared with the CDP-choline pathway (i.e. higher enrichment ratios of d3-PtdCho:d9-PtdCho and d3-PtdCho:d9-choline); and 3) enhanced degradation of PtdCho (i.e. higher concentrations of GPC).

TABLE 4.

Liver choline metabolites and gene transcripts in female and male mice fed a control diet for 8 wk¹

	Female	Male	P (ANOVA)
Metabolite concentration
Choline, nmol/g	294 ± 15	434 ± 26	<0.001
Betaine, nmol/g	380 ± 40	191 ± 17	<0.001
DMG, nmol/g	21.8 ± 1.9	20.2 ± 1.5	0.59
PCho, nmol/g	464 ± 97	629 ± 83	0.22
PtdCho, μmol/g	16.1 ± 1.1	20.8 ± 1.1	0.006
Sphingomyelin, μmol/g	1.5 ± 0.1	2.0 ± 0.3	0.12
GPC, nmol/g	595 ± 81	312 ± 72	0.02
Metabolite enrichment, %
d9-Choline	4.3 ± 0.2	2.8 ± 0.1	<0.001
d9-Betaine	10.1 ± 0.9	4.6 ± 0.4	<0.001
d6-DMG	20.0 ± 1.2	11.4 ± 0.6	<0.001
d9-PtdCho	3.9 ± 0.3	2.1 ± 0.2	<0.001
d3-PtdCho	3.5 ± 0.3	1.3 ± 0.1	<0.001
d9-GPC	4.8 ± 0.5	2.4 ± 0.6	ND2
Enrichment ratio
d9-betaine:d9-choline	2.4 ± 0.2	1.6 ± 0.1	<0.001
d6-DMG:d9-choline	4.7 ± 0.2	3.9 ± 0.4	0.09
d6-DMG:d9-betaine	2.1 ± 0.1	2.2 ± 0.2	0.54
d9-betaine:d9-PtdCho	2.6 ± 0.1	2.2 ± 0.2	0.21
d9-PtdCho:d9-choline	0.9 ± 0.06	0.8 ± 0.07	0.12
d3-PtdCho:d9-choline	0.8 ± 0.06	0.4 ± 0.04	<0.001
d3-PtdCho:d9-PtdCho	0.9 ± 0.08	0.6 ± 0.04	0.003
d9-GPC:d9-PtdCho	1.4 ± 0.2	0.8 ± 0.2	ND2
Gene transcript3
Ct-α	0.5 ± 0.03	0.4 ± 0.04	0.03
Chdh	0.05 ± 0.01	0.03 ± 0.01	0.11
Pemt	16.6 ± 0.8	10.8 ± 0.9	<0.001
Bhmt	21.1 ± 2.5	37.8 ± 3.5	0.001

¹Values are means ± SEM, = 9–10 except for d9-GPC, which was detected in only 2 of 10 male mice.

²Data were not analyzed statistically.

³Gene transcripts are expressed as fold-change relative to the housekeeping gene, hprt-1.

A higher enrichment of the choline metabolites in females than in males was also observed in plasma, brain, and kidney (Supplemental Table 8). The higher enrichment in females cannot be due entirely to pool size differences between genders, because tissue betaine concentrations were higher in females than in males. Another novel finding was the ~55 times higher expression of kidney bhmt transcripts in female than in male mice.

Discussion

Although it is recognized that folate and choline metabolism are inter-related (7), the implications of disturbances in folate metabolism on choline use remain largely unexplored. This study investigated the hypothesis that reductions in methyl group supply, either due to dietary folate deficiency or Mthfr single gene deletion, would modify tissue choline metabolism in a gender-specific manner. d9-Choline was used to monitor the contribution of dietary choline (d9-choline metabolites) and its methyl groups (d3-PtdCho) to choline homeostasis within and among relevant pools.

Mthfr heterozygosity enhances the appearance of dietary choline and its methyl groups in brain.

The higher d9 enrichment of the brain choline metabolites demonstrates that more dietary choline (either as free choline or a phospholipid derivative) appears in brain of Mthfr +/− mice compared with +/+ mice. The higher enrichment of d3-PtdCho in brain of heterozygotes may arise from its entry into brain or entry via a “non-choline" moiety such as methionine and SAM, which would be particularly advantageous given the absence of brain BHMT. The lack of effect of Mthfr heterozygosity on total brain choline concentrations shows that the system is in homeostasis, whereas the labeling reveals metabolic distinctions in how this homeostasis is achieved. Having more dietary-derived choline (and its methyl groups) in the tissues of Mthfr +/− mice may reflect an adaptation that enables provision of methyl groups in the face of diminished 5-methylTHF (15), the product of MTHFR. Because methyl groups are also consumed by the PEMT pathway for the production of PtdCho, the enhanced appearance of dietary-derived choline may also reduce the use of methyl groups in brain. Similar effects of the Mthfr genotype were observed in kidney of male mice.

Despite the significant effects of Mthfr heterozygosity on the labeling of the choline metabolites in brain (and kidney in males), differences in Mthfr transcripts between genotypes were not detected (i.e. an upregulation of the single Mthfr gene copy occurred in heterozygotes in every tissue examined). However, our measurements of gene expression represent a single time point in adult mice and thus do not necessarily reflect the expression profile during development. Previous work in this mouse model reported lower Mthfr transcript levels at 5 wk of age in +/− mice along with evidence of feedback upregulation of the enzyme, which was 60–70% of that in +/+ mice (15). Thus, although upregulation of the Mthfr single gene copy appears to occur with time, data from the present study indicate that some of its effects on dietary choline metabolism persist into adulthood.

The divergence between liver and plasma choline metabolite enrichment is unexpected given the prominent role of liver in the distribution of choline to other organs via lipoproteins (23). Specifically, the enrichment of the liver choline metabolites was unaffected by Mthfr heterozygosity, whereas plasma d3-PtdCho enrichment was higher in female heterozygotes and labeling of the d9-phospholipids was higher in male heterozygotes. The lack of concordance between the liver and plasma data likely results from the presence of several choline pools in liver. Specifically, it is the newly synthesized PtdCho that is incorporated into VLDL and subsequently exported to plasma (24). As such, plasma represents a pool of choline that is distinct, or does not completely mix with, other pools like membrane phospholipids. Further, the observed increase in isotope enrichment from the d9-choline precursor to several of its products (i.e. DMG) (Table 4) is consistent with the presence of more than 1 precursor pool for derivation of subsequent products. For example, when betaine enrichment is measured in liver, it represents the overall (mean) enrichment of the betaine pool(s). When multiple pools exist, the enrichment of 1 betaine subpool may be high (i.e. 15%) due to its rapid turnover (or its smaller size), whereas the enrichment of a second betaine subpool may be low (i.e. 2%) due to its slow turnover (and larger size). Our data are most consistent with a hypothesis in which DMG is derived primarily from the highly enriched betaine subpool. A similar finding (i.e. higher enrichment of plasma d6-DMG relative to d9-betaine) was observed by our research group in a human study in which men consumed d9-choline (20).

Dietary folate deficiency increased CHDH expression and PtdCho mobilization.

The FD diet yielded a diminished circulating folate concentration in both sexes. Notably, however, the folate impairment was not severe, because the dietary treatment was relatively short (8 wk) and antibiotics were not employed to prevent bacterial synthesis of folate species. In females, short-term dietary folate restriction yielded higher hepatic concentrations of the PtdCho-catabolic product, GPC, suggesting enhanced degradation of PtdCho. Enhanced degradation of PtdCho may represent a mechanism to provide extra methyl groups under conditions of reduced 5-methylTHF. The modest increases in Chdh and decreases in Ct-α gene expression are consistent with this working hypothesis. Interestingly, both d3-PtdCho and the ratio of d3-PtdCho:d9-choline were lower (P < 0.05) in FD mice, which suggests that fewer of the dietary choline-derived methyl groups were used by the PEMT reaction under conditions of folate insufficiency. This may be due to greater use of SAM by other cellular methylation reactions. Despite the occurrence of these metabolic changes during folate deficiency, total PtdCho concentrations remained unchanged, which implies that other adaptive mechanisms were engaged, such as reduced secretion of PtdCho in bile (25).

In males, folate deficiency yielded a doubling in Chdh expression, a finding that is consistent with enhanced oxidation of choline in the face of diminished 5-methyTHF (i.e. plasma folate, which consists primarily of 5-methylTHF, was 83% lower in FD males in the present study). However, no alterations in choline metabolite concentrations (or their enrichment) occurred in FD males and homeostasis was maintained.

Females, compared with males, favor the use of dietary choline as a methyl donor.

The higher d9 and d3 enrichments of the liver choline metabolites in females compared with males is consistent with a generally faster replacement of existing pools with dietary choline. Of particular interest is the greater use by females of dietary choline-derived methyl groups for synthesis of PtdCho through the PEMT pathway (i.e. higher d3-PtdCho enrichment and higher d3-PtdCho:d9-PtdCho enrichment ratio in females than in males). The greater use of dietary choline-derived methyl groups to synthesize a new choline moiety through PEMT in females may reflect the prominent role of this metabolic reaction in the generation of DHA-enriched PtdCho molecules (26, 27). Critical for fetal brain development (28), PtdCho-DHA is made available to tissue after its incorporation into VLDL (29, 30). The sex-specific differences in dietary choline metabolism may therefore be linked to the critical role of PEMT in DHA metabolism.

The higher concentration of GPC in liver of females is consistent with their greater degradation of PtdCho compared with males. The d9 labeling of GPC, which was detected in 9 of 10 females but in only 2 of 10 males, suggests that recycling of dietary choline (choline→PtdCho→choline) is also enhanced in female mice. This metabolic cycling of dietary choline may ensure a sufficient supply of free choline for cellular methylation reactions and, more specifically, PEMT.

In conclusion, dietary folate and Mthfr +/− genotype influenced the metabolism of choline by metabolically distinct routes. Compared with Mthfr +/+ mice, more of the dietary choline (and its methyl groups) appeared in brain tissue of +/− mice, possibly reflecting a metabolic adaptation to a single gene deletion that develops early in life and persists into adulthood. Short-term folate deficiency appeared to enhance hepatic use of choline as a methyl donor in both sexes, with evidence of greater PtdCho degradation and reduced use of dietary choline for the biosynthesis of PtdCho through the PEMT pathway in females. Sex was the strongest effecter of dietary choline metabolism, with females diverting more dietary choline toward cellular methylation reactions, including the biosynthesis of PtdCho through the PEMT pathway. Collectively, these data provide support of a higher dietary choline requirement in the presence of disturbances in folate metabolism (i.e. dietary folate deprivation and/or Mthfr gene disruption). These data also show that dietary choline is as an important source of labile methyl groups used in the production of PtdCho through the PEMT pathway, particularly in females.