Supply Of Methyl Groups Research Articles

Enhanced lincomycin production by co-overexpression of metK1 and metK2 in Streptomyces lincolnensis.

Streptomyces lincolnensis is generally utilized for the production of lincomycin A (Lin-A), a clinically useful antibiotic to treat Gram-positive bacterial infections. Three methylation steps, catalyzed by three different S-adenosylmethionine (SAM)-dependent methyltransferases, are required in the biosynthesis of Lin-A, and thus highlight the significance of methyl group supply in lincomycin production. In this study, we demonstrate that externally supplemented SAM cannot be taken in by cells and therefore does not enhance Lin-A production. Furthermore, bioinformatics and in vitro enzymatic assays revealed there exist two SAM synthetase homologs, MetK1 (SLCG_1651) and MetK2 (SLCG_3830) in S. lincolnensis that could convert L-methionine into SAM in the presence of ATP. Even though we attempted to inactivate metK1 and metK2, only metK2 was deleted in S. lincolnensis LCGL, named as ΔmetK2. Following a reduction of the intracellular SAM concentration, ΔmetK2 mutant exhibited a significant decrease of Lin-A in comparison to its parental strain. Individual overexpression of metK1 or metK2 in S. lincolnensis LCGL either elevated the amount of intracellular SAM, concomitant with 15% and 22% increase in Lin-A production, respectively. qRT-PCR assays showed that overexpression of either metK1 or metK2 increased the transcription of lincomycin biosynthetic genes lmbA and lmbR, and regulatory gene lmbU, indicating SAM may also function as a transcriptional activator. When metK1 and metK2 were co-expressed, Lin-A production was increased by 27% in LCGL, while by 17% in a high-yield strain LA219X.

Lysine-specific demethylase 1 activation by vitamin B2 attenuates efficacy of apatinib for proliferation and migration of gastric cancer cell MGC-803.

B vitamins play an essential role in the biosynthesis of nucleotides, replication of DNA, supply of methyl-groups, growth and repair of cells, aberrancies of which have all been implicated in carcinogenesis. Although the potential role of vitamin B in relation to the risk of cancer, including breast, and colorectal cancer, has been investigated in several observational studies, the mechanism of action is still unclear. In this study, vitamin B2 exhibited efficient activation of LSD1 by occupying the active sites where FAD stands. Interestingly, vitamin B2 significantly downregulated expression of CD86, a sensitive surrogate biomarker of LSD1 inhibition, and showed marked activation of gastric cancer cell migration and invasion. Meanwhile, vitamin B2 induced activation of LSD1 may attenuate the proliferation inhibition, and anti-migration effects of apatinib in gastric cancer cells. These findings suggested that vitamin B supplementation may interfere with the efficacy of apatinib in patients with gastric cancer.

Metabolic Imbalance of Homocysteine and Hydrogen Sulfide in Kidney Disease.

Homocysteine (Hcy) and hydrogen sulfide (H2S) are important molecules produced during the metabolism of sulfur-containing amino acids. Hcy metabolism is central to the supply of methyl groups that are essential for biological function. Hcy can be either regenerated to methionine or metabolized to cysteine, a precursor for glutathione synthesis. Cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) play a crucial role in metabolizing Hcy to cysteine through the transsulfuration pathway. These two enzymes are also responsible for H2S generation through desulfuration reactions. H2S, at physiological levels serves as a gaseous mediator and has multifaceted effects. Metabolic imbalance of Hcy and H2S has been implicated in pathological conditions including oxidative stress, inflammation, cardiovascular and cerebral dysfunction, fatty liver disease and ischemiareperfusion injury. Organs such as liver, kidney, gut and pancreas contain all the enzymes that are required for Hcy metabolism. The kidney plays an important role in removing Hcy from the circulation. Hyperhomocysteinemia, a condition of elevated blood Hcy level, is a common clinical finding in patients with chronic kidney disease (CKD) or acute kidney injury (AKI), the latter is often caused by ischemia-reperfusion. This paper reviews exiting literatures regarding (1) the role of kidney in regulating Hcy and H2S metabolism; (2) disruption of sulfur-containing amino acid metabolism during ischemiareperfusion; (3) impact of metabolic imbalance of Hcy and H2S on kidney function. Better understanding of molecular mechanisms that regulate Hcy and H2S metabolism under physiological and pathophysiological conditions will help improve therapeutic strategies for patients with kidney disease or other organ injuries.

Alterations of methionine metabolism in hepatocarcinogenesis: the emergent role of glycine N-methyltransferase in liver injury.

The methionine and folate cycles play a fundamental role in cell physiology and their alteration is involved in liver injury and hepatocarcinogenesis. Glycine N-methyltransferase is implicated in methyl group supply, DNA methylation, and nucleotide biosynthesis. It regulates the cellular S-adenosylmethionine/S-adenosylhomocysteine ratio and S-adenosylmethionine-dependent methyl transfer reactions. Glycine N-methyltransferase is absent in fast-growing hepatocellular carcinomas and present at a low level in slower growing HCC ones. The mechanism of tumor suppression by glycine N-methyltransferase is not completely known. Glycine N-methyltransferase inhibits hepatocellular carcinoma growth through interaction with Dep domain-containing mechanistic target of rapamycin (mTor)-interacting protein, a binding protein overexpressed in hepatocellular carcinoma. The interaction of the phosphatase and tensin homolog inhibitor, phosphatidylinositol 3,4,5-trisphosphate-dependent rac exchanger, with glycine N-methyltransferase enhances proteasomal degradation of this exchanger by the E3 ubiquitin ligase HectH. Glycine N-methyltransferase also regulates genes related to detoxification and antioxidation pathways. It supports pyrimidine and purine syntheses and minimizes uracil incorporation into DNA as consequence of folate depletion. However, recent evidence indicates that glycine N-methyltransferase targeted into nucleus still exerts strong anti-proliferative effects independent of its catalytic activity, while its restriction to cytoplasm prevents these effects. Our current knowledge suggest that glycine N-methyltransferase plays a fundamental, even if not yet completely known, role in cellular physiology and highlights the need to further investigate this role in normal and cancer cells.

Vitamin B12.

The biosynthesis of B12, involving up to 30 different enzyme-mediated steps, only occurs in bacteria. Thus, most eukaryotes require an external source of B12, and yet the vitamin appears to have only two functions in eukaryotes: as a cofactor for the enzymes methionine synthase and methylmalonylCoA mutase. These two functions are crucial for normal health in humans, and in particular, the formation of methionine is essential for providing methyl groups for over 100 methylation processes. Interference with the methionine synthase reaction not only depletes the body of methyl groups but also leads to the accumulation of homocysteine, a risk factor for many diseases. The syndrome pernicious anemia, characterized by lack of intrinsic factor, leads to a severe, sometimes fatal form of B12 deficiency. However, there is no sharp cutoff for B12 deficiency; rather, there is a continuous inverse relationship between serum B12 and a variety of undesirable outcomes, including neural tube defects, stroke, and dementia. The brain is particularly vulnerable; in children, inadequate B12 stunts brain and intellectual development. Suboptimal B12 status (serum B12<300pmol/L) is very common, occurring in 30%-60% of the population, in particular in pregnant women and in less-developed countries. Thus, many tens of millions of people in the world may suffer harm from having a poor B12 status. Public health steps are urgently needed to correct this inadequacy.

Hypermethylation in the promoter of the MTHFR gene is associated with diabetic complications and biochemical indicators

BackgroundDNA methylation is an epigenetic mechanism for regulating the transcription of many genes and has been linked to the development of various diseases. A promising gene to investigate is methylenetetrahydrofolate reductase (MTHFR), since the enzyme methylenetetrahydrofolate reductase (MTHFR) promotes methyl radical synthesis in the homocysteine cycle and can provide methyl groups for DNA methylation. In addition, several studies have correlated gene polymorphisms of this enzyme with a greater risk of diabetes, but little is known regarding the relationship between epigenetic changes in this gene and diabetes and its complications. The aim of this study was to investigate the relationship between methylation profile in the MTHFR gene promoter and biochemical, inflammatory and oxidative stress markers in individuals with type 2 diabetes (T2DM) who have been diagnosed for 5–10 years with or without diabetic retinopathy (DR) and nephropathy (DN).MethodsSpecific PCR for methylation (MSP) was used to analyze MTHFR methylation profile in leucocytes DNA. Biochemical markers (glycemia, glycated hemoglobin, total cholesterol, LDL, HDL, triglycerides, serum creatinine), inflammatory markers (C-reactive protein and alpha-1 acid glycoprotein) and oxidative stress (total antioxidant and malonaldehyde) were determined in peripheric blood samples and microalbuminuria in 24 h urine samples. The X2 and Mann–Whitney statistical tests were performed and p < 0.05 were considered significant.ResultsThe hypermethylated profile was most frequently observed in individuals with retinopathy (p < 0.01) and was associated with higher total cholesterol and LDL levels (p = 0.0046, 0.0267, respectively). Individuals with DN and hypermethylated profiles had higher levels of alpha-1 acid glycoprotein (p = 0.0080) and total antioxidant capacity (p = 0.0169) compared to subjects without complications.ConclusionsHypermethylation in the promoter of the MTHFR gene is associated with the occurrence of DR and with biochemical, inflammatory and oxidative stress parameters in the context of chronic complications

Global Proteome Response to Deletion of Genes Related to Mercury Methylation and Dissimilatory Metal Reduction Reveals Changes in Respiratory Metabolism in Geobacter sulfurreducens PCA

Geobacter sulfurreducens PCA can reduce, sorb, and methylate mercury (Hg); however, the underlying biochemical mechanisms of these processes and interdependent metabolic pathways remain unknown. In this study, shotgun proteomics was used to compare global proteome profiles between wild-type G. sulfurreducens PCA and two mutant strains: a ΔhgcAB mutant, which is deficient in two genes known to be essential for Hg methylation and a ΔomcBESTZ mutant, which is deficient in five outer membrane c-type cytochromes and thus impaired in its ability for dissimilatory metal ion reduction. We were able to delineate the global response of G. sulfurreducens PCA in both mutants and identify cellular networks and metabolic pathways that were affected by the loss of these genes. Deletion of hgcAB increased the relative abundances of proteins implicated in extracellular electron transfer, including most of the c-type cytochromes, PilA-C, and OmpB, and is consistent with a previously observed increase in Hg reduction in the ΔhgcAB mutant. Deletion of omcBESTZ was found to significantly increase relative abundances of various methyltransferases, suggesting that a loss of dissimilatory reduction capacity results in elevated activity among one-carbon (C1) metabolic pathways and thus increased methylation. We show that G. sulfurreducens PCA encodes only the folate branch of the acetyl-CoA pathway, and proteins associated with the folate branch were found at lower abundance in the ΔhgcAB mutant strain than the wild type. This observation supports the hypothesis that the function of HgcA and HgcB is linked to C1 metabolism through the folate branch of the acetyl-CoA pathway by providing methyl groups required for Hg methylation.

Genetic animal models to decipher the pathogenic effects of vitamin B12 and folate deficiency.

Vitamin B12 and folate are essential micronutrients that provide methyl groups for cellular methylations through the so-called one-carbon metabolism. Deficits in the absorption and transport or defects of the enzymes can lead to human pathogenesis comprising hematologic, neural, gastrointestinal, hepatic, renal, cardiovascular and developmental manifestations. One-carbon metabolism is a complex, multistep and multi-organ metabolism, and the understanding of the mechanisms at work have benefited from human inborn errors and population studies, as well as from nutritional animal models. Since 15 years, a wide variety of genetically engineered mice has been developed and has proved to be useful to decipher the underlying mechanisms. These genetically engineered mice targetall the genes that are important for the intestinal absorption, cellular transport and metabolism of vitamin B12 and folate, which are detailed in this article. In conclusion, these mouse models represent valuable experimental paradigms for human pathogenesis. Since no animal model recapitulates the full spectrum of a human disease, researchers have to choose the one that is the most relevant for their specific needs, and this review may help in this respect.

One‐Carbon Metabolic Kinetics in Human Disease Models

The polymorphisms in MTHFR have been associated with altered risk of numerous human cancers whereas GNMT is commonly diminished in human hepatoma. Folate‐mediated one‐carbon metabolism is an important therapeutic target of many human diseases including cancer. We extensively investigated how gene‐nutrient interactions may modulate human cancer risk in 2 major folate metabolic genes: MTHFR and GNMT. To further understand the metabolic roles of these genes in human disease progression, the biochemical impacts of gene alterations were carefully investigated: methyl group supply, global DNA methylation, nucleotide biosynthesis, DNA damage, and partitioning of the folate dependent 1‐carbon groups. The distinct model systems used included: EB virus‐transformed lymphoblasts expressing human polymorphic genotypes; liver‐derived transformed cell‐lines with and without overexpression of the target gene; and cells with stabilized inhibition using shRNA. We discovered that the genotype with reduced MTHFR function significantly reduces folate‐dependent remethylation under folate restriction, but it assists purine synthesis when folate is adequate. The advantage of de novo purine synthesis found in the MTHFR TT genotype may account for the protective effect of MTHFR in human hematological malignancies. Furthermore, GNMT affects transmethylation kinetics, S‐adenosylmethionine synthesis, and facilitates the conservation of methyl groups by limiting homocysteine remethylation fluxes. Restoring GNMT assists methylfolate‐dependent reactions and ameliorates the consequences of folate depletion. GNMT expression in vivo improves folate retention and bioavailability in the liver. Loss of GNMT impairs nucleotide biosynthesis. Over‐expression of GNMT enhances nucleotide biosynthesis and improves DNA integrity by reducing uracil misincorporation in DNA both in vitro and in vivo. Our systematic series of studies give new insights into the underlying mechanisms by which MTHFR and GNMT may participate in human tumor prevention and progression.Support or Funding InformationMOST 104‐2320‐B005‐010‐MY3; MOST104‐2911‐I005‐301, Ministry of Education, Taiwan, R.O.C. under the ATU plan

Maternal Choline Supplementation Modulates Maternal and Fetal Choline Metabolism and Downregulates Inflammatory Gene Expression in a Mouse Model of Placental Insufficiency

A properly functioning placenta allows efficient transfer of nutrients from mother to fetus and promotes fetal growth. Decreased placental efficiency is commonly associated with intrauterine growth restriction, preeclampsia, and miscarriage. Choline is an essential nutrient that is a precursor for several molecules with crucial roles in fetal development: betaine, an osmolyte and methyl donor; acetylcholine, a neurotransmitter and signaling molecule; and phosphatidylcholine (PC), the most prevalent membrane phospholipid. We previously showed that maternal choline supplementation (MCS) in the Dlx3+/− mouse, a model of placental insufficiency, increases fetal growth in early gestation and lowers expression of pro‐angiogenic genes implicated in preeclampsia. To explore the mechanisms contributing to these effects, we measured choline metabolites in the maternal liver and placentas of Dlx3+/− female mice consuming 1X (control), 2X or 4X the recommended intake levels of choline during gestation. Additionally, we measured expression of inflammatory genes that are elevated in preeclampsia and indicative of placental dysfunction. Dams were sacrificed at embryonic days E10.5, E12.5, E15.5, and E18.5. Data were analyzed using linear mixed models with dam as a random factor. MCS increased betaine concentrations in the maternal liver at all four time points (p&lt;.01) and prevented a decline in maternal hepatic betaine stores in late pregnancy. 4X choline also increased phosphocholine, a precursor of PC, at E15.5 (p=.014), and decreased glycerophosphocholine, a product of PC breakdown, at E12.5 (p=.025). In the placenta, MCS increased betaine concentrations at E10.5 and 12.5 (p=.005 and .041, respectively). These data suggest that effects of MCS may be primarily driven by betaine, potentially through increased supply of methyl groups for one‐carbon reactions including DNA methylation. This is supported by data demonstrating widespread effects of choline on gene expression. In Dlx3+/− placentas, choline decreased (p&lt;0.05) expression of pro‐inflammatory genes Il1b (at E12.5, 15.5 and 18.5), Tnf‐α (at E12.5) and NfκB (at E15.5). Further studies are ongoing to identify downstream effects of increased betaine on placental DNA methylation and growth factor signaling pathways.Support or Funding InformationFunding provided by USDA‐NIFA 2012‐67017‐30176

Energy and Protein Supplementation Does Not Affect Protein and Amino Acid Kinetics or Pregnancy Outcomes in Underweight Indian Women

Background: In India, the prevalence of low birth weight is high in women with a low body mass index (BMI), suggesting that underweight women are not capable of providing adequate energy and protein for fetal growth. Furthermore, as pregnancy progresses, there is increased need to provide methyl groups for methylation reactions associated with the synthesis of new proteins and, unlike normal-BMI American women, low-BMI Indian women are unable to increase methionine transmethylation and remethylation rates as pregnancy progresses from trimester 1 to 3. This also negatively influences birth weight.Objective: The aim was to determine the effect of dietary supplementation with energy and protein from 12 ± 1 wk of gestation to time of delivery compared with no supplement on pregnancy outcomes, protein kinetics, and the fluxes of the methyl group donors serine and glycine.Methods: Protein kinetics and serine and glycine fluxes were measured by using standard stable isotope tracer methods in the fasting and postprandial states in 24 pregnant women aged 22.9 ± 0.7 y with low BMIs [BMI (in kg/m2) ≤18.5] at 12 ± 1 wk (trimester 1) and 30 ± 1 wk (trimester 3) of gestation. After the first measurement, subjects were randomly assigned to either receive the supplement (300 kcal/d, 15 g protein/d) or no supplement.Results: Supplementation had no significant effect on any variable of pregnancy outcome, and except for fasting state decreases in leucine flux (125 ± 7.14 compared with 113 ± 5.06 μmol ⋅ kg−1 ⋅ h−1; P = 0.04) and nonoxidative disposal (110 ± 6.97 compared with 101 ± 3.69 μmol ⋅ kg−1 ⋅ h−1; P = 0.02) from trimesters 1 to 3, it had no effect on any other leucine kinetic variable or urea, glycine, and serine fluxes.Conclusion: We conclude that in Indian women with a low BMI, supplementation with energy and protein from week 12 of pregnancy to time of delivery does not improve pregnancy outcome, whole-body protein kinetics, or serine and glycine fluxes.

Abstract 197: Statin Therapy Is Associated With Higher MTHFR Methylation Levels in a Stroke Cohort

Introduction: Epigenetics refers to changes in gene function that occur by gene methylation, histone modification or RNA mechanisms. Many of these are heritable, but can also be altered by lifestyle and medication. Genome-wide hypomethylation is associated with atherosclerosis and hyperhomocysteinemia, and specific genes can be hyper or hypomethylated in vascular disease. Statins have pleiotropic effects on the vasculature, and may affect DNA methylation. Hypothesis: The methylation levels of genes implicated in vascular disease are altered by treatment with common cardiovascular medications. Methods: 189 individuals from a well characterised stroke cohort and controls were included. 44 were established on statin therapy, 49 on aspirin and 28 on an ACE inhibitor. The following genes implicated in vascular disease were investigated: LINE1, SOD3, ALOX15, ERa, MTHFR , PITX2, IFNGR1, TNFa, IL10, and INS . Methylation status was assessed by bisulphite conversion of DNA followed by PCR and pyrosequencing. Each methylation site was analysed individually and mean methylation level across all sites within a gene calculated. Statistical analysis for the effect of drug therapy was based on logistic regression and ANOVA. Results: One significant association was found: subjects on statin therapy had higher MTHFR methylation levels than those not receiving statin (p=0.002). Statistical analysis adjusted for stroke effect (p=0.025) remained significant. Correction for multiple testing was not performed as the candidate genes are linked in pathways. There was no effect of aspirin or ACE inhibitor on methylation status of any of the genes tested. Discussion: MTHFR plays an important role in homocysteine and folate metabolism, providing methyl groups for DNA methylation. High levels of homocysteine are a risk factor for cardiovascular disease and there is published evidence that statin therapy results in lower plasma homocysteine levels. This finding requires further investigation in a larger cohort with a defined dose and duration of treatment. If this finding is replicated, there may be value in exploring the mechanisms linking statins, MTHFR methylation and homocysteine levels and the implications for primary and secondary prevention of vascular events.

Regulation of Folate-Mediated One-Carbon Metabolism by Glycine N-Methyltransferase (GNMT) and Methylenetetrahydrofolate Reductase (MTHFR).

Folate-mediated one-carbon metabolism is an important therapeutic target of human diseases. We extensively investigated how gene-nutrient interactions may modulate human cancer risk in 2 major folate metabolic genes, MTHFR and GNMT. The biochemical impacts of MTHFR and GNMT on methyl group supply, global DNA methylation, nucleotide biosynthesis, DNA damage, and partitioning of the folate dependent 1-carbon group were carefully studied. The distinct model systems used included: EB virus-transformed lymphoblasts expressing human MTHFR polymorphic genotypes; liver-derived GNMT-null cell-lines with and without GNMT overexpression; and HepG2 cells with stabilized inhibition of MTHFR using shRNA, GNMT wildtype, heterozygotous (GNMT(het)) and knockout (GNMT(nul)) mice. We discovered that the MTHFR TT genotype significantly reduces folate-dependent remethylation under folate restriction, but it assists purine synthesis when folate is adequate. The advantage of de novo purine synthesis found in the MTHFR TT genotype may account for the protective effect of MTHFR in human hematological malignancies. GNMT affects transmethylation kinetics and S-adenosylmethionine (adoMet) synthesis, and facilitates the conservation of methyl groups by limiting homocysteine remethylation fluxes. Restoring GNMT assists methylfolate-dependent reactions and ameliorates the consequences of folate depletion. GNMT expression in vivo improves folate retention and bioavailability in the liver. Loss of GNMT impairs nucleotide biosynthesis. Over-expression of GNMT enhances nucleotide biosynthesis and improves DNA integrity by reducing uracil misincorporation in DNA both in vitro and in vivo. The systematic series of studies gives new insights into the underlying mechanisms by which MTHFR and GNMT may participate in human tumor prevention.

Dietary l-methionine supplementation mitigates gamma-radiation induced global DNA hypomethylation: Enhanced metabolic flux towards S-adenosyl-l-methionine (SAM) biosynthesis increases genomic methylation potential

The objective of this study was to examine the effect of 60Co-gamma (γ) radiation on modulation of genomic DNA methylation, if any, of mice maintained (6weeks) on normal control diet (NCD) and l-methionine supplemented diet (MSD). To elucidate the possible underlying mechanism(s), we exposed the animals to γ-radiation (2, 3 and 4Gy) and investigated the profile of downstream metabolites and enzymes involved in S-adenosyl-l-methionine (SAM) generation. Liver samples were also subjected to histopathological examinations. Compared to NCD fed and irradiated animals, hepatic folate, choline and l-methionine levels decreased moderately, while hepatic SAM levels increased in MSD fed and irradiated animals. Under these conditions, a marked modulation of methionine adenosyltransferase (MAT) and l-methionine synthase (MSase) activities was observed. Concomitant with increase in liver SAM pool, increased DNA methyltransferase (dnmt) activity in MSD fed mice indicated enhanced metabolic flux towards DNA methylation. Further results showed that genomic DNA methylation and 5-methyl-2′-deoxy cytidine residues were maintained at normal levels in MSD fed and irradiated mice compared to NCD fed and irradiated animals. In conclusion, our results suggest that increasing supply of preformed methyl groups, via dietary l-methionine supplementation might significantly increase methylation potential of radiation stress compromised DNA methylation cycle.

Responses to betaine and inorganic sulphur of sheep in growth performance and fibre growth

Sulphur-containing amino acids (SAA) are essential and usually the first limiting amino acids for growth, milk and wool production. The keratin fibre that grows from epidermal tissue is rich in SAA. The rate of fibre growth and its S content are influenced by the availability of SAA. Betaine is a dietary source for a labile methyl group and actively participates in methionine metabolism by donating methyl groups for the remethylation of homocysteine to methionine. Ruminants are capable of synthesizing SAA from inorganic S sources, and most bacteria in the rumen can use inorganic S to meet their requirements for growth. The objective of this study was to examine whether betaine and an inorganic sulphur supplement could provide methyl groups and sulphur amino acids in a way that growth performance and wool production of ewes and lambs are improved. Treatments performed included betaine supplementation, sulphate supplementation and betaine plus sulphate supplementation with five replications for each treatment. The dry matter intake of the ewes was affected by betaine plus sulphate supplementation (p < 0.05). In the ewes, betaine plus sulphate supplementation increased (p < 0.05) the wool growth rate, wool yield, staple length and wool sulphur concentration, while decreasing wool wax and wool yellowness (p < 0.05). In the lambs, wool growth rate, wool yield, fibre diameter, staple length, staple strength, wool sulphur concentration, wool wax and fibre percentage did not differ (p > 0.05) between treatments. In the ewes, plasma methionine concentration increased (p < 0.05) with betaine plus sulphate treatment. No corresponding difference (p > 0.05) was observed in plasma methionine concentration in the lambs. It can be concluded that betaine plus sulphate supplementation has the potential to change wool characteristics in the ewes, while these compounds were without any effect on growth and wool production of the lambs. Combining the two supplements was advantageous.

Maternal choline modifies fetal liver copper, gene expression, DNA methylation, and neonatal growth in the tx-j mouse model of Wilson disease

Maternal diet can affect fetal gene expression through epigenetic mechanisms. Wilson disease (WD), which is caused by autosomal recessive mutations in ATP7B encoding a biliary copper transporter, is characterized by excessive hepatic copper accumulation, but variability in disease severity. We tested the hypothesis that gestational supply of dietary methyl groups modifies fetal DNA methylation and expression of genes involved in methionine and lipid metabolism that are impaired prior to hepatic steatosis in the toxic milk (tx-j) mouse model of WD. Female C3H control and tx-j mice were fed control (choline 8 mmol/Kg of diet) or choline-supplemented (choline 36 mmol/Kg of diet) diets for 2 weeks throughout mating and pregnancy to gestation day 17. A second group of C3H females, half of which were used to cross foster tx-j pups, received the same diet treatments that extended during lactation to 21 d postpartum. Compared with C3H, fetal tx-j livers had significantly lower copper concentrations and significantly lower transcript levels of Cyclin D1 and genes related to methionine and lipid metabolism. Maternal choline supplementation prevented the transcriptional deficits in fetal tx-j liver for multiple genes related to cell growth and metabolism. Global DNA methylation was increased by 17% in tx-j fetal livers after maternal choline treatment (P < 0.05). Maternal dietary choline rescued the lower body weight of 21 d tx-j mice. Our results suggest that WD pathogenesis is modified by maternal in utero factors, including dietary choline.

Stability of DNA Methylation Patterns in Mouse Spermatogonia Under Conditions of MTHFR Deficiency and Methionine Supplementation1

Little is known about the conditions contributing to the stability of DNA methylation patterns in male germ cells. Altered folate pathway enzyme activity and methyl donor supply are two clinically significant factors that can affect the methylation of DNA. 5,10-Methylenetetrahydrofolate reductase (MTHFR) is a key folate pathway enzyme involved in providing methyl groups from dietary folate for DNA methylation. Mice heterozygous for a targeted mutation in the Mthfr gene (Mthfr(+/-)) are a good model for humans homozygous for the MTHFR 677C>T polymorphism, which is found in 10% of the population and is associated with decreased MTHFR activity and infertility. High-dose folic acid is administered as an empirical treatment for male infertility. Here, we examined MTHFR expression in developing male germ cells and evaluated DNA methylation patterns and effects of a range of methionine concentrations in spermatogonia from Mthfr(+/-) as compared to wild-type, Mthfr(+/+) mice. MTHFR was expressed in prospermatogonia and spermatogonia at times of DNA methylation acquisition in the male germline; its expression was also found in early spermatocytes and Sertoli cells. DNA methylation patterns were similar at imprinted genes and intergenic sites across chromosome 9 in neonatal Mthfr(+/+) and Mthfr(+/-) spermatogonia. Using spermatogonia from Mthfr(+/+) and Mthfr(+/-) mice in the spermatogonial stem cell (SSC) culture system, we examined the stability of DNA methylation patterns and determined effects of low or high methionine concentrations. No differences were detected between early and late passages, suggesting that DNA methylation patterns are generally stable in culture. Twenty-fold normal concentrations of methionine resulted in an overall increase in the levels of DNA methylation across chromosome 9, suggesting that DNA methylation can be perturbed in culture. Mthfr(+/-) cells showed a significantly increased variance of DNA methylation at multiple loci across chromosome 9 compared to Mthfr(+/+) cells when cultured with 0.25- to 2-fold normal methionine concentrations. Taken together, our results indicate that DNA methylation patterns in undifferentiated spermatogonia, including SSCs, are relatively stable in culture over time under conditions of altered methionine and MTHFR levels.

Cereal foods are the major source of betaine in the Western diet – Analysis of betaine and free choline in cereal foods and updated assessments of betaine intake

Betaine and its precursor choline are important components of one-carbon metabolism, remethylating homocysteine into methionine and providing methyl groups for DNA methylation. Cereals are the main source of betaine in the diet, though there is little literature available on the content of betaine in cereal products, nor on betaine intake from cereals. Betaine and free-choline concentrations were measured by liquid-chromatography with tandem mass spectrometry in a wide range of commercially available cereal foods and cereal fractions. Whole grain wheat and related fractions were the best overall common source of betaine, while the pseudocereal quinoa had the highest amount of betaine measured (3900μg/g). Based on estimates of dietary intake data cereal foods provide approximately 60–67% of betaine in Western diets, and 20–40% of betaine in South-East Asian diets. Average intake of betaine was 131mg/d, well below those used in intervention studies using betaine to lower blood homocysteine.

Influence of lactation and choline intake on one‐carbon biomarkers in women

ObjectiveWe sought to quantify the effects of lactation and maternal choline intake on maternal indicators of one‐carbon metabolism.MethodsLactating (n = 28; 30‐d post‐partum) and nonlactating (NL, n = 21) women were randomized to choline intakes of 480 or 930 mg/d for 10 weeks. Blood and urine were collected at baseline, study midpoint, and study end for analysis of one carbon metabolites.ResultsCompared to NL women, lactating women exhibited elevated plasma choline (p=0.04) and decreased urinary dimethylglycine losses (p=0.008). Within lactating women, a higher choline intake yielded greater plasma betaine (p=0.015), greater plasma dimethylglycine (p=0.037), greater plasma methylglycine (p=0.036), and decreased plasma cystathionine (p=0.012). However, higher choline intake did not affect plasma concentrations of homocysteine, cysteine, methionine, or glycine.ConclusionsLactation and choline intake influence biomarkers of 1‐C metabolism with some evidence of a greater demand for choline during lactation and a positive effect of a higher choline intake on methyl group supply.

Wilson's Disease: Changes in Methionine Metabolism and Inflammation Affect Global DNA Methylation in Early Liver Disease

Hepatic methionine metabolism may play an essential role in regulating methylation status and liver injury in Wilson's disease (WD) through the inhibition of S-adenosylhomocysteine hydrolase (SAHH) by copper (Cu) and the consequent accumulation of S-adenosylhomocysteine (SAH). We studied the transcript levels of selected genes related to liver injury, levels of SAHH, SAH, DNA methyltransferases genes (Dnmt1, Dnmt3a, Dnmt3b), and global DNA methylation in the tx-j mouse (tx-j), an animal model of WD. Findings were compared to those in control C3H mice, and in response to Cu chelation by penicillamine (PCA) and dietary supplementation of the methyl donor betaine to modulate inflammatory and methylation status. Transcript levels of selected genes related to endoplasmic reticulum stress, lipid synthesis, and fatty acid oxidation were down-regulated at baseline in tx-j mice, further down-regulated in response to PCA, and showed little to no response to betaine. Hepatic Sahh transcript and protein levels were reduced in tx-j mice with consequent increase of SAH levels. Hepatic Cu accumulation was associated with inflammation, as indicated by histopathology and elevated serum alanine aminotransferase (ALT) and liver tumor necrosis factor alpha (Tnf-α) levels. Dnmt3b was down-regulated in tx-j mice together with global DNA hypomethylation. PCA treatment of tx-j mice reduced Tnf-α and ALT levels, betaine treatment increased S-adenosylmethionine and up-regulated Dnmt3b levels, and both treatments restored global DNA methylation levels. Reduced hepatic Sahh expression was associated with increased liver SAH levels in the tx-j model of WD, with consequent global DNA hypomethylation. Increased global DNA methylation was achieved by reducing inflammation by Cu chelation or by providing methyl groups. We propose that increased SAH levels and inflammation affect widespread epigenetic regulation of gene expression in WD.