Gene Response Elements, Genetic Polymorphisms and Epigenetics Influence the Human Dietary Requirement for Choline

Summary

Recent progress in the understanding of the human dietary requirement for choline highlights the importance of genetic variation and epigenetics in human nutrient requirements. Choline is a major dietary source of methyl-groups (one of choline's metabolites, betaine, participates in the methylation of homocysteine to form methionine); also choline is needed for the biosynthesis of cell membranes, bioactive phospholipids and the neurotransmitter acetylcholine. A recommended dietary intake for choline in humans was set in 1998, and a portion of the choline requirement can be met via endogenous de novo synthesis of phosphatidylcholine catalyzed by phosphatidylethanolamine N-methyltransferase (PEMT) in the liver. Though many foods contain choline, many humans do not get enough in their diets. When deprived of dietary choline, most adult men and postmenopausal women developed signs of organ dysfunction (fatty liver, liver or muscle cell damage, and reduces the capacity to handle a methionine load, resulting in elevated homocysteine). However, only a portion of premenopausal women developed such problems. The difference in requirement occurs because estrogen induces expression of the PEMT gene and allows premenopausal women to make more of their needed choline endogenously. In addition, there is significant variation in the dietary requirement for choline that can be explained by common polymorphisms in genes of choline and folate metabolism. Choline is critical during fetal development, when it alters DNA methylation and thereby influences neural precursor cell proliferation and apoptosis. This results in long term alterations in brain structure and function, specifically memory function.

EFFECTS OF A LOW CHOLINE DIET IN HUMANS

Choline is a dietary component essential for normal function of all cells (1). It is the major source of methyl-groups in the diet (one of choline's metabolites, betaine, participates in the methylation of homocysteine to form methionine) (2). In addition, choline is used for the biosynthesis of cell membranes, bioactive phospholipids, and the neurotransmitter acetylcholine (1). The functional consequences of dietary choline deficiency in humans include the development of fatty liver (3, 4) (hepatosteatosis; occurs because a lack of phosphatidylcholine limits the export of excess triglyceride from liver in lipoproteins (5, 6)), liver damage (3, 7 – 10) (elevated serum aminotransferases; secondary to apoptosis (7, 11, 12) and muscle damage (10, 13) (elevated creatine phosphokinase in blood; occurs because muscle membranes are more fragile and because of induction of apoptosis (13)).

In 1998 the US Institute of Medicine's Food and Nutrition Board established an Adequate Intake (AI) and Tolerable Upper Limit (UL) for choline (14).

CHOLINE, FOLATE AND METHIONINE METABOLISM ARE INTERRELATED

Choline, methionine and folate metabolism interact at the point that homocysteine is converted to methionine (Fig. 1). Thus, any requirement for dietary choline must be considered in relation to these other nutrients. Homocysteine can be methylated to form methionine (15) by two parallel pathways, both of which lower homocysteine concentrations (16). In the first, vitamins B₁₂ and folic acid are involved in a reaction catalyzed by methionine synthase (17). Deficiency of these nutrients (18, 19), or single nucleotide polymorphisms in the genes for the enzymes involved in this pathway (17, 19, 20), can result in elevated plasma homocysteine concentrations. In addition, tetrahydrofolate is needed to scavenge one-carbon groups when betaine is metabolized (21). The alternative pathway for the methylation of homocysteine to form methionine is catalyzed by betaine homocysteine methyltransferase (BHMT) (22), an enzyme whose activity has been reported to increase in rats during methionine excess (23). Betaine, derived from dietary choline by the action of choline dehydrogenase (CHDH), is the methyl group donor in this reaction and supplemental oral betaine can lower plasma homocysteine concentrations (24, 25).

Figure 1.

Choline metabolism and links to methionine and folate metabolism. The pathways described are all present in the liver, with other tissues having one or more of these pathways. PEMT, phosphatidylethanolamine-N-methyltransferase; CHDH, choline dehydrogenase; BHMT, betaine homocysteine methyltransferase; MTHFR, methylene tetrahydrofolate reductase; MTHFD, methylene tetrahydrofolate dehydrogenase.

Perturbing metabolism of one of the methyl-donors results in compensatory changes in the other methyl-donors due to the intermingling of these metabolic pathways (26 – 28). Rats treated with the anti-folate, methotrexate, had diminished pools of choline metabolites in liver (27, 29). Rats ingesting a choline-deficient diet had diminished tissue concentrations of methionine and S-adenosylmethionine (30) and doubled plasma homocysteine concentrations (31). We recently reported that humans who are depleted of choline have diminished capacity to methylate homocysteine and developed elevated homocysteine concentrations in plasma after a methionine loading test (2).

DIETARY INTAKE AND ENDOGENOUS SYNTHESIS OF CHOLINE

Excellent sources of dietary choline include liver, eggs and wheat germ (32, 33). In foods, choline is found free and as choline esters. Though it is likely that these forms are fungible, there is some evidence that they may have different bioavailability (34) because the lipid-soluble forms bypass the liver when absorbed from the diet while the water soluble forms enter the portal circulation and are mostly absorbed by the liver. It is not clear whether normal dietary patterns deliver the recommended amounts of choline for all people. Shaw and colleagues, studying pregnant women in California observed intakes of choline in 25% of the population that were less than needed to prevent birth defects in their fetuses (35, 36).

The only source of choline other than diet is from the de novo biosynthesis of phosphatidylcholine catalyzed by phosphatidylethanolamine-N-methyltransferase (PEMT) in liver. This enzyme uses S-adenosylmethionine as a methyl donor and forms a new choline moiety (37). When fed a diet deficient in choline, Pemt −/− mice developed fatty liver, severe liver damage and died; a choline supplemented diet prevented this (38) and reversed hepatic damage if begun early enough (39). The PEMT pathway is not just a minor pathway that backs up the cytidine diphosphocholine pathway for phosphatidylcholine biosynthesis. Pemt −/− mice have lower choline pools in liver despite being fed sufficient or supplemental amounts of dietary choline (40), suggesting that choline production by PEMT is a significant source of choline relative to dietary intake. When Pemt is deleted in mice, plasma homocysteine concentrations fall 50% and, when it is over expressed, plasma homocysteine concentrations increase 40% (41, 42), demonstrating that PEMT activity is a very major consumer of S-adenosylmethionine (and thereby a producer of homocysteine).

ESTROGEN RESPONSE ELEMENTS AND THE REQUIREMENT FOR CHOLINE

Premenopausal women, relative to males and postmenopausal women, are resistant to developing organ dysfunction when fed a low choline diet (10). The classic actions of estrogen occur through its receptors ERα and ERβ which bind as homodimers or heterodimers to estrogen response elements (EREs) in the promoters of many estrogen-responsive genes (43). The consensus ERE (PuGGTCAnnnTGACCPy) (43) and some imperfect ERE half site motifs (ERE1/2) bind with ERα and ERβ (44 – 46). There are multiple EREs in the promoter region(s) of the PEMT gene (47) and estrogen, at doses bracketing physiological concentrations in humans (0 − 100 nmol/l), causes a marked up-regulation in PEMT mRNA expression and enzyme activity in human hepatocytes (47). Thus, premenopausal women have an enhanced capacity for de novo biosynthesis of choline moiety.

During pregnancy, estradiol concentration rises from approximately 1 nM to 60 nM at term (48, 49), suggesting that capacity for endogenous synthesis of choline should be highest during period when females need to support fetal development. Pregnancy and lactation are times when demand for choline is especially high; transport of choline from mother to fetus (50, 51) depletes maternal plasma choline in humans (52). It is interesting that despite enhanced capacity to synthesize choline, the demand for this nutrient is so high that stores are depleted during pregnancy. Pregnant rats had diminished total liver choline compounds compared to non-mated controls and become as sensitive to choline-deficient diets as were male rats (53). Because milk contains a great deal of choline, lactation further increases maternal demand for choline resulting in further depletion of tissue stores (53, 54). These observations suggest that women depend on high rates of endogenous biosynthesis of choline induced by estrogen, as well as on dietary intake of choline to sustain normal pregnancy. It is biologically plausible that, during evolution, appropriate mechanisms for inducing endogenous choline biosynthesis (via PEMT) were developed to assure that young women are less susceptible to dietary choline deficiency and have adequate stores of choline prior to, and during pregnancy. Pemt −/− mice abort pregnancies around 9 − 10 days gestation unless fed supplemental choline (personal observation; (55)). A better understanding of why there is variation in dietary choline requirements might be important for identifying women at greater risk for choline deficiency during pregnancy. This is a real possibility, as the data of Shaw and colleagues show that women in the USA vary enough in dietary choline intake (from <300 mg/d to >500 mg/d) to influence the risk that they will have a baby with a birth defect (35). Choline nutriture during pregnancy is especially important because it influences brain development in the fetus (56 – 68) and because it is important for maintaining normal plasma homocysteine concentrations during pregnancy (69). High maternal homocysteine concentrations are associated with increased incidence of birth defects (70).

GENE POLYMORPHISMS AND DIETARY CHOLINE REQUIREMENTS

Though some humans develop fatty liver, as well as liver and muscle damage when fed a low choline diet, others do not. Even among premenopausal women who should be resistant to choline deficiency, a significant number develop organ dysfunction when deprived of choline (10). Genetic variation likely underlies these differences in dietary requirements. From what we understand about choline metabolism, several pathways influence how much choline is required from diet and single nucleotide polymorphisms (SNPs) in genes for these pathways might be of importance. Specifically, polymorphisms in the folate pathways could limit the availability of methyltetrahydrofolate and thereby increase use of choline as a methyl donor. Polymorphisms in the PEMT gene might alter endogenous synthesis of choline and polymorphisms in other genes of choline metabolism could influence dietary requirements by changing the utilization of choline moiety.

We developed a clinical methodology for phenotyping individuals with respect to their susceptibility to developing organ dysfunction when fed a low choline diet (2, 10, 13, 71). In a 95-day repeated measure, within subject study with graded repletion, adult men and women (pre- and post-menopausal) ages 18 − 70 were admitted to the General Clinical Research Center and fed a standard diet containing a known amount of choline (550 mg/70 kg/d; baseline). On day 11 subjects were placed on a diet containing <50 mg choline/day for up to 42 days. Blood and urine were collected to measure various experimental parameters of dietary choline status and markers of organ dysfunction (serum creatine phosphokinase (CPK), alanine amino transferase (ALT), aspartate amino transferase (AST)) and liver fat level was assessed by MRI. If at some point during the depletion period, functional markers indicated organ dysfunction associated with choline deficiency, subjects were switched to a diet containing 137 mg/70 kg/d choline (low or normal folate) for 10 days. If the functional markers indicated that deficiency status persisted, subjects were then switched to a diet containing 275 mg/70 kg/d choline (low or normal folate) for 10 days. If markers of organ dysfunction did not indicate repletion at this time, subjects were switched to a diet containing 413 mg/70 kg/d choline (low or normal folate) for 10 days. If still not repleted, subjects were fed a diet containing at least 550 mg/70 kg/d choline (low or normal folate) until repleted.

We examined whether major genetic variants of folate metabolism modify susceptibility of humans to choline deficiency (72). Premenopausal women who were carriers of the very common 5,10-methylenetetrahydrofolate dehydrogenase-1958A (MTHFD1; rs2236225) gene allele were more than 15 times as likely as non-carriers to develop signs of choline deficiency (P < 0.0001) on the low-choline diet. Sixty-three percent of our study population had at least one allele for this SNP. Two reactions, mediated by methylenetetrahydrofolate dehydrogenase and methenyltetrahydrofolate cyclohydrolase can convert 10-formyl tetrahydrofolate to 5,10-methylene tetrahydrofolate. While the formation of 5-methyl tetrahydrofolate is practically irreversible in vivo, the interconversion of 5,10-methylene tetrahydrofolate and 10-formyl tetrahydrofolate is closer to equilibrium (73). This means that 5,10-methylene tetrahydrofolate may be directed either toward homocysteine remethylation or away from it. The MTHFD1 G1958A polymorphism may thus affect the delicately balanced flux between 5,10-methylene tetrahydrofolate and 10-formyl tetrahydrofolate and thereby influence the availability of 5-methyl THF for homocysteine remethylation. This would increase demand for choline as a methyl-group donor. It is of interest that the risk of having a child with a neural tube defect increases in mothers with the G1958A SNP in MTHFD1 (74).

As noted earlier PEMT encodes for a protein responsible for endogenous formation of choline. We identified an SNP in the promoter region of the PEMT gene (−744 G−>C; rs12325817) for which 18 of 23 carriers of the C allele (78%) developed organ dysfunction when fed a low choline diet (odds ratio 25, P = 0.002) (75). Given the sexual differences in the effect of PEMT rs12325817, it is possible that this SNP alters the estrogen responsiveness of the promoter. The frequency of this variant allele was 0.74. A SNP in the PEMT coding region (+5465 G−>A; rs7946) results in a 30% loss of function and is associated with increased risk for nonalcoholic fatty liver disease (76) but was not associated with susceptibility to choline deficiency (75). The first of two SNPs in the coding region of the choline dehydrogenase gene (CHDH; +318 A−>C; rs9001) had a protective effect on susceptibility to choline deficiency, while a second CHDH variant (+432 G−>T; rs12676) was associated with increased susceptibility to choline deficiency (75). A SNP in the betaine:homocysteine methyltransferase gene (BHMT; +742 G−>A; rs3733890) was not associated with susceptibility to choline deficiency (75). Identification of common polymorphisms that affect dietary requirements for choline could enable us to identify individuals for whom we need to assure adequate dietary choline intake.

CHOLINE AND BRAIN DEVELOPMENT IN UTERO

Large amounts of choline are delivered to the fetus across the placenta, where choline transport systems pump it against a concentration gradient (51). Plasma or serum choline concentrations are 6−7-fold higher in the fetus and newborn than they are in the adult (77, 78). High levels of choline circulating in the neonate presumably ensure enhanced availability of choline to tissues. There is a novel form of PEMT in neonatal rat brain that is extremely active (37); this isoform is not present in adult brain. Furthermore, in the brains of newborn rats, S-adenosylmethionine concentrations are 40 − 50 nmol/g of tissue (79) – levels probably sufficient to enable the neonatal form of phosphatidylethanolamine-N-methyltransferase to maintain high rates of activity.

Maternal dietary choline supplementation or choline deficiency during late pregnancy in rodents were associated with significant and irreversible changes in hippocampal function in the adult rodent, including altered long term potentiation (LTP) (64, 80, 81) and altered memory (65, 66, 82 – 85). More choline (about 4 times dietary levels) during days 11 − 17 of gestation in the rodent increased hippocampal progenitor cell proliferation (56, 60), decreased apoptosis in these cells (56, 60), enhanced LTP in the offspring when they were adult animals (64, 80, 81) and enhanced visuospatial and auditory memory by as much as 30% in the adult animals through out their lifetimes (65, 66, 82, 83, 85 – 87). Indeed, adult rodents decrement in memory as they age, and offspring exposed to extra choline in utero do not show this ‘senility’ (83, 86). Mothers fed choline deficient diets during late pregnancy have offspring with diminished progenitor cell proliferation and increased apoptosis in fetal hippocampus (56, 60), insensitivity to LTP when they were adult animals (81), and decremented visuospatial and auditory memory (85). The effects of perinatal choline supplementation on memory were initially found using radial-arm maze tasks and the Sprague-Dawley rat strain, but other laboratories have found similar results using other spatial memory tasks, such as the Morris water maze (88, 89), using passive avoidance paradigms (90), using measures of attention (91), using other strains of rats such as Long-Evans (92 – 94), and using mice (90). The effects of choline supplementation in utero were also detected in studies on effects of fetal alcohol exposure, where supplementation with choline attenuated behavioral alterations but not motor abnormalities (95, 96). Thus, choline supplementation during a critical period in pregnancy causes lifelong changes in brain structure and function.

EPIGENETICS AND THE EFFECTS OF CHOLINE

The effects of choline on neural tube closure and on brain development likely are mediated by changes in the expression of genes. Dietary choline deficiency decreases S-adenosylmethionine concentrations in tissues (30, 97), with resulting hypomethylation of DNA (98, 99). DNA methylation occurs at cytosine bases that are followed by a guanosine (5′-CpG-3′ sites) (100) and influences many cellular events, including gene transcription, genomic imprinting and genomic stability (101 – 103). In mammals, about 60 − 80% of CpG sites in DNA are methylated, while most CpGs within CpG islands are not (104). When this modification occurs in promoter regions, gene expression is altered (105); increased methylation is associated with gene silencing or reduced gene expression (104). In choline deficient cells in culture, and in fetal rodent brains from mothers fed choline deficient diets, methylation of the CDKN3 gene promoter is decreased, resulting in over expression of this gene which inhibits cell proliferation (106, 107). This change in gene promoter methylation likely alters neurogenesis in the hippocampus for life – prenatal choline supplementation in rats resulted in increased neurogenesis that was still detectable at 7 months of age (108). There are other examples where maternal diets high in methyl groups had permanent effects on their offspring. Feeding pregnant Pseudoagouti Avy/a mouse dams a choline methyl-supplemented diet altered epigenetic regulation of agouti expression in their offspring, as indicated by increased agouti/black mottling of their coats (109, 110). In another example, there was increased DNA methylation of the fetal gene axin fused (Axin(Fu)) after methyl donor supplementation of female mice before and during pregnancy which reduced by 50% the incidence of tail kinking in Axin(Fu)/+ offspring. It is clear that the dietary manipulation of methyl donors (either deficiency or supplementation) can have a profound impact upon gene expression and, by consequence, upon the homeostatic mechanisms that ensure the normal function of physiological processes.

Whether these findings in rodents apply as well to humans is not known. Of course human and rat brains mature at different rates, with rat brain comparatively more mature at birth than is the human brain. In humans, the architecture of the hippo-campus continues to develop after birth, and by 4 years of age it closely resembles adult structure (111). This area of brain is one of the few areas in which nerve cells continue to multiply slowly throughout life (112, 113).

CONCLUSION

Understanding dietary choline requirement is an exercise in understanding nutrigenomics. Endogenous biosynthesis is induced by estrogen response elements, DNA methylation is influenced by the availability of choline, and common genetic polymorphisms have major effects on the dietary requirement for this nutrient. These interactions have important health significance because this nutrient is important for brain development and for preventing birth defects. In addition, choline deficiency can be a cause of fatty liver, an extremely common problem in adults (114). Choline deficiency has other health consequences – it is associated with liver and muscle damage and with exaggerated plasma homocysteine rise after a methionine load (2). Elevated plasma homocysteine is an independent risk factor for cardiovascular disease and stroke in humans (115 – 117).