Dietary Methyl Donors Research Articles

Restriction of dietary methyl donors limits methionine availability and affects the partitioning of dietary methionine for creatine and phosphatidylcholine synthesis in the neonatal piglet

Methionine is required for protein synthesis and provides a methyl group for >50 critical transmethylation reactions including creatine and phosphatidylcholine synthesis as well as DNA and protein methylation. However, the availability of methionine depends on dietary sources as well as remethylation of demethylated methionine (i.e., homocysteine) by the dietary methyl donors folate and choline (via betaine). By restricting dietary methyl supply, we aimed to determine the extent that dietary methyl donors contribute to methionine availability for protein synthesis and transmethylation reactions in neonatal piglets. Piglets 4–8 days of age were fed a diet deficient (MD−) (n=8) or sufficient (MS+) (n=7) in folate, choline and betaine. After 5 days, dietary methionine was reduced to 80% of requirement in both groups to elicit a response. On day 8, animals were fed [3H-methyl]methionine for 6h to measure methionine partitioning into hepatic protein, phosphatidylcholine, creatine and DNA. MD− feeding reduced plasma choline, betaine and folate (P<.05) and increased homocysteine ~3-fold (P<.05). With MD− feeding, hepatic phosphatidylcholine synthesis was 60% higher (P<.05) at the expense of creatine synthesis, which was 30% lower during MD− feeding (P<.05); protein synthesis as well as DNA and protein methylation were unchanged. In the liver, ~30% of dietary label was traced to phosphatidylcholine and creatine together, with ~50% traced to methylation of proteins and ~20% incorporated in synthesized protein. Dietary methyl donors are integral to neonatal methionine requirements and can affect methionine availability for transmethylation pathways.

Essential role of methyl donors in animal productivity

Dietary requirements for the methyl donors, choline, betaine and folate, in livestock species are poorly defined and have not been included in diet formulation software or simulation models for animals. A deficiency of methyl donors may promote an inflammatory state, which is significant for the livestock industry because chronic low-grade inflammation is widespread among livestock under commercial conditions. Furthermore, recent evidence showing that methyl donors activate adenosine monophosphate-activated protein kinase, an anti-inflammatory master switch, indicates that dietary methyl-donor supplementation could be used to prevent or ameliorate chronic inflammation and its sequelae in livestock, which include fatty liver disease in dairy cows, fatty liver and kidney syndrome in broilers, fatty liver haemorrhagic syndrome in layers, gut ulcers in pigs, liver abscesses in feedlot cattle, enteritis in poultry and susceptibility to heat stress in all species. Because of the complexity of interactions among methyl donors, a modelling approach inclusive of a supporting research effort will be required to harness the potential of methyl-donor supplementation in livestock production.

The Pediatric Methionine Requirement Should Incorporate Remethylation Potential and Transmethylation Demands

The metabolic demand for methionine is great in neonates. Indeed, methionine is the only indispensable sulfur amino acid and is required not only for protein synthesis and growth but is also partitioned to a greater extent to transsulfuration for cysteine and taurine synthesis and to >50 transmethylation reactions that serve to methylate DNA and synthesize metabolites, including creatine and phosphatidylcholine. Therefore, the pediatric methionine requirement must accommodate the demands of rapid protein turnover as well as vast nonprotein demands. Because cysteine spares the methionine requirement, it is likely that the dietary provision of transmethylation products can also feasibly spare methionine. However, understanding the requirement of methionine is further complicated because demethylated methionine can be remethylated by the dietary methyl donors folate and betaine (derived from choline). Intakes of dietary methyl donors are highly variable, which is of particular concern for newborns. It has been demonstrated that many populations have enhanced requirements for these nutrients, and nutrient fortification may exacerbate this phenomenon by selecting phenotypes that increase methyl requirements. Moreover, higher transmethylation rates can limit methyl supply and affect other transmethylation reactions as well as protein synthesis. Therefore, careful investigations are needed to determine how remethylation and transmethylation contribute to the methionine requirement. The purpose of this review is to support our hypothesis that dietary methyl donors and consumers can drive methionine availability for protein synthesis and transmethylation reactions. We argue that nutritional strategies in neonates need to ensure that methionine is available to meet requirements for growth as well as for transmethylation products.

Methyl donor supplementation in rats reverses the deleterious effect of maternal separation on depression-like behaviour

Adverse early life events are associated with altered stress responsiveness and metabolic disturbances in the adult life. Dietary methyl donor supplementation could be able to reverse the negative effects of maternal separation by affecting DNA methylation in the brain. In this study, maternal separation during lactation reduced body weight gain in the female adult offspring without affecting food intake, and altered total and HDL-cholesterol levels. Also, maternal separation induced a cognitive deficit as measured by NORT and an increase in the immobility time in the Porsolt forced swimming test, consistent with increased depression-like behaviour. An 18-week dietary supplementation with methyl donors (choline, betaine, folate and vitamin B12) from postnatal day 60 also reduced body weight without affecting food intake. Some of the deleterious effects induced by maternal separation, such as the abnormal levels of total and HDL-cholesterol, but especially the depression-like behaviour as measured by the Porsolt test, were reversed by methyl donor supplementation. Also, the administration of methyl donors increased total DNA methylation (measured by immunohistochemistry) and affected the expression of insulin receptor in the hippocampus of the adult offspring. However, no changes were observed in the DNA methylation status of insulin receptor and corticotropin-releasing hormone (CRH) promoter regions in the hypothalamus. In summary, methyl donor supplementation reversed some of the deleterious effects of an early life-induced model of depression in rats and altered the DNA methylation profile in the brain.

The effects of dietary methyl donors on methionine partitioning in the neonatal piglet

The metabolism of the indispensable amino acid methionine is critical during development. Methionine is used to synthesize protein for growth and, using the methionine cycle, it is the precursor of &gt;50 critical nutrients and contributes to epigenetic regulation. Therefore, the dietary methionine requirement must factor all the potential roles of methioine. Three major processes summarize the methionine cycle: transmethylation (TM), which transfers methyl groups to nutrient precursors and DNA; transsulfuration (TS), which represents methionine disposal; and remethylation (RM), which resynthesizes methionine using the dietary methyl donors folate and choline (via betaine). Dietary intakes of folate vary drastically, and choline intakes are often below the adequate intakes during pregnancy and in early life, which we hypothesized would influence the methionine requirement. To test our hypothesis, we fed 4- to 8-day-old neonatal piglets a low-methionine diet that was either deficient (MD−) or replete (MS+) in dietary methyl donors. We evaluated how methionine was balanced between the major TM reactions and protein synthesis. The MD− group exhibited marked differences in TM as creatine synthesis was ≈30% less (p &lt; 0.05) and phosphatidylcholine synthesis was ≈60% more (p &lt; 0.05) during MD− feeding. Interestingly, while MD− feeding did not affect liver protein synthesis, the methionine availability and protein synthesis were lower in skeletal muscle of the MD− versus MS+ animals (p &lt; 0.05). Furthermore, whole-body protein turnover was also reduced during MD− feeding (p &lt; 0.05), which is significant as protein turnover is especially critical during infancy. Next, we measured the effect of methyl donors on the rates of TM, TS, and RM. The rates of RM and TM were reduced by ≈75% in the MD− group (p &lt; 0.05), while TS was unchanged. To evaluate the effectiveness of individual methyl donors on RM, we “rescued” a second group of MD− animals with betaine (MD + B), folate (MD + F) or both (MD + FB). The rate of RM and TM increased by ≈2-fold after rescue (p &lt; 0.05) and reduced protein breakdown (p &lt; 0.05). These studies showed that dietary methyl donors affect neonatal methionine metabolism, which should be considered when defining the dietary requirements of methionine during development.

Mono-2-ethylhexyl phthalate (MEHP) alters histiotrophic nutrition pathways and epigenetic processes in the developing conceptus

Histiotrophic nutrition pathways (HNPs) are processes by which the organogenesis-stage conceptus obtains nutrients, amino acids, vitamins and cofactors required for protein biosynthesis and metabolic activities. Nutrients are captured from the maternal milieu as whole proteins and cargoes via receptor-mediated endocytosis in the visceral yolk sac (VYS), degraded by lysosomal proteolysis and delivered to the developing embryo (EMB). Several nutrients obtained by HNPs are required substrates for one-carbon (C1) metabolism and supply methyl groups required for epigenetic processes, including DNA and histone methylation. Increased availability of methyl donors has been associated with reduced risk for neural tube defects (NTDs). Here, we show that mono-2-ethylhexyl phthalate (MEHP) treatment (100 or 250μM) alters HNPs, C1 metabolism and epigenetic programming in the organogenesis-stage conceptus. Specifically, 3-h MEHP treatment of mouse EMBs in whole culture resulted in dose-dependent reduction of HNP activity in the conceptus. To observe nutrient consequences of decreased HNP function, C1 components and substrates and epigenetic outcomes were quantified at 24h. Treatment with 100-μM MEHP resulted in decreased dietary methyl donor concentrations, while treatment with 100- or 250-μM MEHP resulted in dose-dependent elevated C1 products and substrates. In MEHP-treated EMBs with NTDs, H3K4 methylation was significantly increased, while no effects were seen in treated VYS. DNA methylation was reduced in MEHP-treated EMB with and without NTDs. This research suggests that environmental toxicants such as MEHP decrease embryonic nutrition in a time-dependent manner and that epigenetic consequences of HNP disruption may be exacerbated in EMB with NTDs.

Abstract 893: Metabolomic profiling of APCΔ14/+ mice maintained on a methyl donor deficient diet reveals alterations to methionine and fatty acid metabolism associated with cancer protection

Abstract Our laboratory has previously demonstrated that administration of a diet deficient in methyl donor nutrients (folic acid, methionine, choline and vitamin B12) reduces tumor multiplicity by greater than 95% in ApcMin/+ mice. However, the mechanism by which this dramatic cancer protection is afforded is unknown. To begin to identify the metabolic consequences of methyl donor deficiency, we have conducted an LC/MS-based metabolic profiling analysis of colonic mucosa collected from ApcΔ14/+ mice fed a methyl donor deficient (MDD) diet for 17 weeks. Methyl donor deficiency causes significant alterations to a number of key metabolic pathways, including those involved in the methionine cycle, fatty acid oxidation, and tryptophan catabolism. We observed significant reductions in methionine (65% reduction, p&amp;lt;0.001) and betaine (70% reduction, p&amp;lt;0.001), accompanied by large increases in homocysteine (88.6-fold increase, p&amp;lt;0.001) and S-adenosylmethionine (17.2-fold increase, p&amp;lt;0.001), confirming that dietary methyl donor deficiency alters one-carbon metabolites in the colon. MDD mice were found to have reduced levels of acyl-carnitines and free carnitine (58% reduction, p&amp;lt;0.001). Additionally, we detected a reduction in the critical metabolic component, acetyl CoA, suggesting decreased transport of fatty acids into the mitochondria for use in fatty acid oxidation (FAO). Finally, we observed changes in the mucosal levels of tryptophan (32% reduction, p = 0.02), kynurenine (2.8-fold increase, p = 0.008) and indolepropionate (5.1-fold increase, p = 0.005), that were retained for seven weeks beyond active methyl donor deficiency. Taken together, these findings suggest that dietary methyl donor deficiency significantly disrupts the methionine cycle, inhibits fatty acid transport and oxidation, and may induce long-term changes to tryptophan metabolism. This study has improved our understanding of the molecular mechanisms underlying the cancer protection conveyed by dietary methyl donor deficiency and has identified metabolic pathways that are candidate targets for chemopreventive intervention. Citation Format: Matthew P. Hanley, Daniel W. Rosenberg. Metabolomic profiling of APCΔ14/+ mice maintained on a methyl donor deficient diet reveals alterations to methionine and fatty acid metabolism associated with cancer protection. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 893. doi:10.1158/1538-7445.AM2015-893

255 High Unsaturated Fat Diets and Unsaturated Visceral Fat Worsen Acute Pancreatitis (AP) Outcomes At Lower Body Mass Index (BMI)

Background & Aims: Environmental enteropathy (EE) is a subclinical intestinal condition highly prevalent in lowand middle-income countries characterized, in part, by malabsorption, intestinal villous atrophy, and crypt hypertrophy. The pathogenesis of EE remains unclear, however supplementation with folate (a key source of one carbon units for DNA methylation) is an effective adjunct therapy for tropical sprue, i.e., persistent diarrhea on a background of EE. To determine the extent to which methyl donor deficiency (MDD) provokes features of EE in mice, we evaluated mechanistic links between dietary methyl donor deficiency and intestinal crypt hypertrophy in mice and in mouse small intestinal crypt cultures (enteroids). Methods: We randomized dams to a standard diet or an isocaloric MDD diet lacking folate, choline, and betaine when pups were 10-days-old. We then randomized weanlings to their dams' diet on day of life 21. Mice were sacrificed and the jejunum was harvested at 6 weeks of age for both histology (n=6/group) and generation of enteroids. Results: Histological comparisons of the jejunum revealed longer crypts in MDD versus control mice, moreso in the distal (85.6 +/13.7 μm vs. 69.3 +/7.2 μm; P<0.0001), vs. proximal (88.8 +/17 μm vs 80.7 +/10.7 μm; P=0.04) portions of the small intestine. Enteroids from control and MDD mice were both viable in standard minigut media; however, all enteroids maintained in MDD media displayed alterations in crypt morphology and decreased epithelial proliferation. Enteroid crypt neck width was 1.3-fold greater in standard vs. MDD media (P<0.001). The number of crypt buds per enteroid was 1.6-fold higher in standard media vs. MDD media (p = 0.0007). The ratio of EdU-positive, or proliferating, cells to Hoechst-positive cells was 2.2-fold higher in standard media vs. MDD media (P<0.0001). Qualitatively, enteroids in MDD media displayed longer crypt domains vs. enteroids in standard media. Conclusions: Complementary in vivo and in vitro findings of altered small intestinal crypt in the setting of methyl donor deficiency suggest that methyl donor deficiency plays a role in the pathogenesis of environmental enteropathy. Further studies are needed to determine whether prolonged methyl donor deficiency promotes epigenetic changes of intestinal stem cells and whether methyl donor supplementation prevents or reverses these changes to promote intestinal epithelial homeostasis in global settings of poverty.

The effect of a methyl-deficient diet on the global DNA methylation and the DNA methylation regulatory pathways.

Methyl-deficient diets are known to induce various liver disorders, in which DNA methylation changes are implicated. Recent studies have clarified the existence of the active DNA demethylation pathways that start with oxidization of 5-methylcytosine (5meC) to 5-hydroxymethylcytosine by ten-eleven translocation (Tet) enzymes, followed by the action of base-excision-repair pathways. Here, we investigated the effects of a methionine-choline-deficient (MCD) diet on the hepatic DNA methylation of mice by precisely quantifying 5meC using a liquid chromatography-electrospray ionization-mass spectrometry and by investigating the regulatory pathways, including DNA demethylation. Although feeding the MCD diet for 1 week induced hepatic steatosis and lower level of the methyl donor S-adenosylmethionine, it did not cause a significant reduction in the 5meC content. On the other hand, the MCD diet significantly upregulated the gene expression of the Tet enzymes, Tet2 and Tet3, and the base-excision-repair enzymes, thymine DNA glycosylase and apurinic/apyrimidinic-endonuclease 1. At the same time, the gene expression of DNA methyltransferase 1 and a, was also significantly increased by the MCD diet. These results suggest that the DNA methylation level is precisely regulated even when dietary methyl donors are restricted. Methyl-deficient diets are well known to induce oxidative stress and the oxidative-stress-induced DNA damage, 8-hydroxy-2'-deoxyguanosine (8OHdG), is reported to inhibit DNA methylation. In this study, we also clarified that the increase in 8OHdG number per DNA by the MCD diet is approximately 10 000 times smaller than the reduction in 5meC number, suggesting the contribution of 8OHdG formation to DNA methylation would not be significant.

Pleiotropic effects of a methyl donor diet in a novel animal model.

Folate and other methyl-donor pathway components are widely supplemented due to their ability to prevent prenatal neural tube defects. Several lines of evidence suggest that these supplements act through epigenetic mechanisms (e.g. altering DNA methylation). Primary among these are the experiments on the mouse viable yellow allele of the agouti locus (Avy). In the Avy allele, an Intracisternal A-particle retroelement has inserted into the genome adjacent to the agouti gene and is preferentially methylated. To further test these effects, we tested the same diet used in the Avy studies on wild-derived Peromyscus maniculatus, a native North American rodent. We collected tissues from neonatal offspring whose parents were fed the high-methyl donor diet as well as controls. In addition, we assayed coat-color of a natural variant (wide-band agouti = ANb) that overexpresses agouti as a phenotypic biomarker. Our data indicate that these dietary components affected agouti protein production, despite the lack of a retroelement at this locus. Surprisingly, the methyl-donor diet was associated with defects (e.g. ovarian cysts, cataracts) and increased mortality. We also assessed the effects of the diet on behavior: We scored animals in open field and social interaction tests. We observed significant increases in female repetitive behaviors. Thus these data add to a growing number of studies that suggest that these ubiquitously added nutrients may be a human health concern.

Supplementation with methyl donors during lactation to high-fat-sucrose-fed dams protects offspring against liver fat accumulation when consuming an obesogenic diet.

Methyl donor supplementation has been reported to prevent obesity-induced liver fat accumulation in adult rats. We hypothesized that this protection could be mediated by perinatal nutrition. For this purpose, we assessed the response to an obesogenic diet (high-fat-sucrose, HFS) during adulthood depending on maternal diet during lactation. Female Wistar rats fed control diet during pregnancy were assigned to four postpartum dietary groups: control, control supplemented with methyl donors (choline, betaine, folic acid, vitamin B12), HFS and HFS supplemented with methyl donors. At weaning, the male offspring was transferred to a chow diet and at week 12th assigned to a control or a HFS diet during 8 weeks. The offspring whose mothers were fed HFS during lactation showed increased adiposity (19%, P<0.001). When fed the HFS diet as adults, offspring whose mothers were HFS supplemented had more body fat (23%, P<0.001) than those from HFS non-supplemented. However, they showed lower liver fat accumulation (-18%, P<0.001). Srebf1, Dnmt1 and Lepr liver mRNA levels increased after adulthood HFS feeding. In those animals HFS fed during adulthood, previous maternal HFS decreased Lepr and Dnmt1 expression levels when compared with c-HFS offspring, while the supplementation of control and HFS-fed dams, respectively, induced higher hepatic Mme and Lepr mRNA levels after adult HFS intake compared with hfs-HFS offspring. In conclusion, maternal HFS diet during lactation influenced the response to an obesogenic diet in the adult progeny. Interestingly, dietary methyl donor supplementation in lactating mothers fed an obesogenic diet reduced liver fat accumulation, but increased adipose tissue storage in adult HFS-fed offspring.

Methyl-donor supplementation in obese mice prevents the progression of NAFLD, activates AMPK and decreases acyl-carnitine levels.

Non-alcoholic fatty liver disease (NAFLD) results from increased hepatic lipid accumulation and steatosis, and is closely linked to liver one-carbon (C1) metabolism. We assessed in C57BL6/N mice whether NAFLD induced by a high-fat (HF) diet over 8 weeks can be reversed by additional 4 weeks of a dietary methyl-donor supplementation (MDS). MDS in the obese mice failed to reverse NAFLD, but prevented the progression of hepatic steatosis associated with major changes in key hepatic C1-metabolites, e.g. S-adenosyl-methionine and S-adenosyl-homocysteine. Increased phosphorylation of AMPK-α together with enhanced β-HAD activity suggested an increased flux through fatty acid oxidation pathways. This was supported by concomitantly decreased hepatic free fatty acid and acyl-carnitines levels. Although HF diet changed the hepatic phospholipid pattern, MDS did not. Our findings suggest that dietary methyl-donors activate AMPK, a key enzyme in fatty acid β-oxidation control, that mediates increased fatty acid utilization and thereby prevents further hepatic lipid accumulation.

Remethylation with dietary methyl donors contributes a significant proportion of methionine for the synthesis of protein and creatine in neonatal piglets (258.3)

Methionine is an essential amino acid that is in high demand in neonates for both protein synthesis and as the primary methyl donor for transmethylation (TM) reactions. The vast majority of methyl groups are used for synthesis of creatine (CR) and phosphatidylcholine (PC). The demethylated methionine can be remethylated by dietary methyl donors such as folate, choline and betaine, but to what extent is unclear. We hypothesized that these methyl donors provide a nutritionally significant proportion of available methionine for TM and protein synthesis. 4‐8 d old piglets were fed a diet devoid (n=8) or replete (n=7) of methyl donors at 80% of the methionine requirement; methionine and phenylalanine isotopes were infused to calculate flux to products. Whole body protein synthesis and breakdown were respectively, 15% (p&lt;0.05) and 20% (p&lt;0.04) lower during methyl deficiency, corresponding to a ~70% reduction in fractional protein synthesis (Ks) in muscle (p&lt;0.03) and unchanged Ks in liver. Methyl deficiency also led to 25% lower hepatic TM to creatine (p&lt;0.02), which was offset by a 2‐fold greater TM to PC (p&lt;0.03), due to choline deficiency which increases PC synthesis via TM. In conclusion, dietary methyl donors can re‐synthesize a substantial proportion of methionine that is used primarily for protein synthesis and potentially growth.Grant Funding Source: Supported by CIHR

Maternal Methyl Donors Supplementation during Lactation Prevents the Hyperhomocysteinemia Induced by a High-Fat-Sucrose Intake by Dams

Maternal perinatal nutrition may program offspring metabolic features. Epigenetic regulation is one of the candidate mechanisms that may be affected by maternal dietary methyl donors intake as potential controllers of plasma homocysteine levels. Thirty-two Wistar pregnant rats were randomly assigned into four dietary groups during lactation: control, control supplemented with methyl donors, high-fat-sucrose and high-fat-sucrose supplemented with methyl donors. Physiological outcomes in the offspring were measured, including hepatic mRNA expression and global DNA methylation after weaning. The newborns whose mothers were fed the obesogenic diet were heavier longer and with a higher adiposity and intrahepatic fat content. Interestingly, increased levels of plasma homocysteine induced by the maternal high-fat-sucrose dietary intake were prevented in both sexes by maternal methyl donors supplementation. Total hepatic DNA methylation decreased in females due to maternal methyl donors administration, while Dnmt3a hepatic mRNA levels decreased accompanying the high-fat-sucrose consumption. Furthermore, a negative association between Dnmt3a liver mRNA levels and plasma homocysteine concentrations was found. Maternal high-fat-sucrose diet during lactation could program offspring obesity features, while methyl donors supplementation prevented the onset of high hyperhomocysteinemia. Maternal dietary intake also affected hepatic DNA methylation metabolism, which could be linked with the regulation of the methionine-homocysteine cycle.

Behavioral and neurochemical effects of dietary methyl donor deficiency combined with unpredictable chronic mild stress in rats

Methyl donor deficiencies and chronic stress cause depression independently, but their interaction has never been thoroughly evaluated. In our study, methyl donor deficient diet and chronic stress condition consisted respectively of a B2, B9, B12, and choline-free diet and a chronic mild stress procedure. Rats were randomly assigned to six groups with three “diet” conditions (free-feeding, pair-fed and methyl donor deficient diet) and two “stress” conditions (no-stress and stress) and were evaluated in the open-field, the elevated plus-maze and the forced swimming test. After the behavioral evaluation, corticosterone and homocysteine plasma levels were measured and dopamine, DOPAC, serotonin, 5HIAA concentrations were evaluated in several brain areas. Rats given a methyl donor deficient diet for 11 weeks causing elevated plasma homocysteine levels were compared to pair-fed and free-feeding rats with or without unpredictable chronic mild stress. Regardless of stress environmental conditions, the methyl donor deficient diet decreased plasma corticosterone levels and caused disinhibition in the elevated plus-maze condition relative to both control groups. However, stress potentiated the effects of the deficient regimen on rearing in the open-field and climbing in the forced swim test. The dietary changes involved in behavior and plasma corticosterone could be caused by homocysteine-induced decreases in dopamine and 5-hydroxytryptamine metabolites in selective brain regions and it can be noted that regardless of stress-conditions, methyl donor deficient diet decreases DOPAC/dopamine and 5HIAA/serotonin ratios in striatum and hypothalamus and selectively 5HIAA/serotonin ratio in the sensorimotor cortex. Our experimental data is particularly relevant in the context of neuropsychiatric disorders frequently associated with folate deficiency and hyperhomocysteinemia.

Abstract ED02-02: Interaction between genetics, epigenetics, and environment in cancer prevention

Abstract A recent convergence of genomic and epigenomic findings has brought the study of DNA methylation to the forefront of cancer research. In this session I will review our knowledge to date and suggest future directions for translational research and clinical applications in two key areas – (i) dietary methyl donors and DNA methylation in cancer prevention, (ii) allele-specific DNA methylation mapping as a tool for improving the yield of true-positive signals from genome-wide association studies (GWAS) for cancer susceptibility. These topics will highlight the importance of cis- and trans-acting influences of the genetic background on epigenetic patterns and cancer susceptibility.

Effects of Methyl Donor Diets on Incisional Pain in Mice

BackgroundDietary supplementation with methyl donors can influence the programming of epigenetic patterns resulting in persistent alterations in disease susceptibility and behavior. However, the dietary effects of methyl donors on pain have not been explored. In this study, we evaluated the effects of dietary methyl donor content on pain responses in mice.MethodsMale and female C57BL/6J mice were treated with high or low methyl donor diets either in the perinatal period or after weaning. Mechanical and thermal nociceptive sensitivity were measured before and after incision.ResultsMice fed high or low methyl donor diets displayed equal weight gain over the course of the experiments. When exposed to these dietary manipulations in the perinatal period, only male offspring of dams fed a high methyl donor diet displayed increased mechanical allodynia. Hindpaw incision in these animals caused enhanced nociceptive sensitization, but dietary history did not affect the duration of sensitization. For mice exposed to high or low methyl donor diets after weaning, no significant differences were observed in mechanical or thermal nociceptive sensitivity either at baseline or in response to hindpaw incision.ConclusionsPerinatal dietary factors such as methyl donor content may impact pain experiences in later life. These effects, however, may be specific to sex and pain modality.

Transcriptomic and epigenetic changes in early liver steatosis associated to obesity: Effect of dietary methyl donor supplementation

Non-alcoholic fatty liver disease is a primary hepatic manifestation of obesity and an important adverse metabolic syndrome trait. Animal models of diet-induced obesity promote liver fat accumulation putatively associated with alterations in epigenetic profile. Dietary methyl donor-supplementation may protect against this disturbance during early developmental stages affecting the molecular basis of gene regulation. The aim of this study was to investigate the transcriptomic and epigenetic mechanisms implicated in liver fat accumulation as a result of an obesogenic diet and the putative preventive role of dietary methyl donors. Forty-eight male Wistar rats were assigned into four dietary groups for 8weeks; control, control methyl-donor-supplemented with a dietary cocktail containing betaine, choline, vitamin B12 and folic acid, high-fat-sucrose and high-fat-sucrose methyl-donor-supplemented. Liver fat accumulation induced by a HFS diet was prevented by methyl donor supplementation in HFS-fed animals. A liver mRNA microarray, subsequently validated by real time-qPCR, showed modifications in some biologically relevant genes involved in obesity development and lipid metabolism (Lepr, Srebf2, Agpat3 and Esr1). Liver global DNA methylation was decreased by methyl donor supplementation in control-fed animals. Methylation levels of specific CpG sites from Srebf2, Agpat3 and Esr1 promoter regions showed changes due to the obesogenic diet and the supplementation with methyl donors. Interestingly, Srebf2 CpG23_24 methylation levels (−167bp and −156bp with respect to the transcriptional start site) correlated with HDLc plasma levels, whereas Esr1 CpG14 (−2623bp) methylation levels were associated with body and liver weights and fat content. Furthermore HFS diet-induced liver fat accumulation was prevented by methyl donor supplementation. In conclusion, both obesogenic diet and methyl donor supplementation modified the mRNA hepatic profile as well as the methylation of specific gene promoters and total DNA.

The Metabolic Burden of Methyl Donor Deficiency with Focus on the Betaine Homocysteine Methyltransferase Pathway

Methyl groups are important for numerous cellular functions such as DNA methylation, phosphatidylcholine synthesis, and protein synthesis. The methyl group can directly be delivered by dietary methyl donors, including methionine, folate, betaine, and choline. The liver and the muscles appear to be the major organs for methyl group metabolism. Choline can be synthesized from phosphatidylcholine via the cytidine-diphosphate (CDP) pathway. Low dietary choline loweres methionine formation and causes a marked increase in S-adenosylmethionine utilization in the liver. The link between choline, betaine, and energy metabolism in humans indicates novel functions for these nutrients. This function appears to goes beyond the role of the nutrients in gene methylation and epigenetic control. Studies that simulated methyl-deficient diets reported disturbances in energy metabolism and protein synthesis in the liver, fatty liver, or muscle disorders. Changes in plasma concentrations of total homocysteine (tHcy) reflect one aspect of the metabolic consequences of methyl group deficiency or nutrient supplementations. Folic acid supplementation spares betaine as a methyl donor. Betaine is a significant determinant of plasma tHcy, particularly in case of folate deficiency, methionine load, or alcohol consumption. Betaine supplementation has a lowering effect on post-methionine load tHcy. Hypomethylation and tHcy elevation can be attenuated when choline or betaine is available.

Diet influences expression of autoimmune-associated genes and disease severity by epigenetic mechanisms in a transgenic mouse model of lupus.

Lupus flares occur when genetically predisposed individuals encounter appropriate environmental agents. Current evidence indicates that the environment contributes by inhibiting T cell DNA methylation, causing overexpression of normally silenced genes. DNA methylation depends on both dietary transmethylation micronutrients and ERK-regulated DNA methyltransferase 1 (DNMT-1) levels. We used transgenic mice to study the effect of interactions between diet, DNMT-1 levels, and genetic predisposition on the development and severity of lupus. A doxycycline-inducible ERK defect was bred into lupus-resistant (C57BL/6) and lupus-susceptible (C57BL/6 × SJL) mouse strains. Doxycycline-treated mice were fed a standard commercial diet for 18 weeks and then switched to a transmethylation micronutrient-supplemented (MS) or -restricted (MR) diet. Disease severity was assessed by examining anti-double-stranded DNA (anti-dsDNA) antibody levels, the presence of proteinuria and hematuria, and by histopathologic analysis of kidney tissues. Pyrosequencing was used to determine micronutrient effects on DNA methylation. Doxycycline induced modest levels of anti-dsDNA antibodies in C57BL/6 mice and higher levels in C57BL/6 × SJL mice. Doxycycline-treated C57BL/6 × SJL mice developed hematuria and glomerulonephritis on the MR and standard diets but not the MS diet. In contrast, C57BL/6 mice developed kidney disease only on the MR diet. Decreasing ERK signaling and methyl donors also caused demethylation and overexpression of the CD40lg gene in female mice, consistent with demethylation of the second X chromosome. Both the dietary methyl donor content and the duration of treatment influenced methylation and expression of the CD40lg gene. Dietary micronutrients that affect DNA methylation can exacerbate or ameliorate disease in this transgenic murine lupus model, and contribute to lupus susceptibility and severity through genetic-epigenetic interactions.