Cell type-selective secretome profiling in vivo.

Secreted polypeptides are a fundamental axis of intercellular and endocrine communication. However, a global understanding of the composition and dynamics of cellular secretomes in intact mammalian organisms has been lacking. Here, we introduce a proximity biotinylation strategy that enables labeling, detection and enrichment of secreted polypeptides in a cell type-selective manner in mice. We generate a proteomic atlas of hepatocyte, myocyte, pericyte and myeloid cell secretomes by direct purification of biotinylated secreted proteins from blood plasma. Our secretome dataset validates known cell type-protein pairs, reveals secreted polypeptides that distinguish between cell types and identifies new cellular sources for classical plasma proteins. Lastly, we uncover a dynamic and previously undescribed nutrient-dependent reprogramming of the hepatocyte secretome characterized by the increased unconventional secretion of the cytosolic enzyme betaine-homocysteine S-methyltransferase (BHMT). This secretome profiling strategy enables dynamic and cell type-specific dissection of the plasma proteome and the secreted polypeptides that mediate intercellular signaling.

Simultaneous administration of EZH2 and BET inhibitors inhibits proliferation and clonogenic ability of metastatic prostate cancer cells.

Androgen deprivation therapy (ADT) is a common treatment for recurrent prostate cancer (PC). However, after a certain period of responsiveness, ADT resistance occurs virtually in all patients and the disease progresses to lethal metastatic castration-resistant prostate cancer (mCRPC). Aberrant expression and function of the epigenetic modifiers EZH2 and BET over activates c-myc, an oncogenic transcription factor critically contributing to mCRPC. In the present work, we tested, for the first time, the combination of an EZH2 inhibitor with a BET inhibitor in metastatic PC cells. The combination outperformed single drugs in inhibiting cell viability, cell proliferation and clonogenic ability, and concomitantly reduced both c-myc and NF-kB expression. Although these promising results will warrant further in vivo validation, they represent the first step to establishing the rationale that the proposed combination might be suitable for mCRPC treatment, by exploiting molecular targets different from androgen receptor.

Profiling the Influence of Gene Variants Related to Folate-Mediated One-Carbon Metabolism on the Outcome of In Vitro Fertilization (IVF) with Donor Oocytes in Recipients Receiving Folic Acid Fortification.

Nutritional status and gene polymorphisms of one-carbon metabolism confer a well-known interaction that in pregnant women may affect embryo viability and the health of the newborn. Folate metabolism directly impacts nucleotide synthesis and methylation, which is of increasing interest in the reproductive medicine field. Studies assessing the genetic influence of folate metabolism on IVF treatments have currently been performed in women using their own oocytes. Most of these patients seeking to have a child or undergoing IVF treatments are advised to preventively intake folate supplies that restore known metabolic imbalances, but the treatments could lead to the promotion of specific enzymes in specific women, depending on their genetic variance. In the present study, we assess the influence of candidate gene variants related to folate metabolism, such as Serine Hydroxymethyltransferase 1 SHMT1 (rs1979276 and rs1979277), Betaine-Homocysteine S-Methyltransferase BHMT (rs3733890), Methionine synthase reductase MTRR (rs1801394), Methylenetetrahydrofolate reductase MTHFR (rs1801131 and rs1801133), methionine synthase MTR (rs12749581), ATP Binding Cassette Subfamily B Member 1 ABCB1 (rs1045642) and folate receptor alpha FOLR1 (rs2071010) on the success of IVF treatment performed in women being recipients of donated oocytes. The implication of such gene variants seems to have no direct impact on pregnancy consecution after IVF; however, several gene variants could influence pregnancy loss events or pregnancy maintenance, as consequence of folic acid fortification.

Interaction analysis of gene variants related to one-carbon metabolism with chronic hepatitis B infection in Chinese patients.

BACKGROUND: The risk of chronic hepatitis B (CHB) infection is influenced by aberrant DNA methylation and altered nucleotide synthesis and repair, possibly caused by polymorphic variants in one-carbon metabolism genes. In the present study, we investigated the relationship between polymorphisms belonging to the one-carbon metabolic pathway and CHB infection. METHODS: A case-control study using 230 CHB patients and 234 unrelated healthy controls was carried out to assess the genetic association of 24 single nucleotide polymorphisins (SNPs) determined by mass spectrometry. RESULTS: Three SNPs, comprising rs10717122 and rs2229717 in serine hydroxymethyltransferase1/2 (SHMT2) and rs585800 in betaine-homocysteine S-methyltransferase (BHMT), were associated with the risk of CHB. Patients with DEL allele, DEL.DEL and DEL.T genotypes of rs10717122 had a 1.40-, 2.00- and 1.83-fold increased risk for CHB, respectively. Cases inheriting TA genotype of rs585800 had a 2.19-fold risk for CHB infection. The T allele of rs2229717 was less represented in the CHB cases (odds ratio = 0.66, 95% confidence interval = 0.48-0.92). The T allele of rs2229717 was less in patients with a low hepatitis B virus-DNA level compared to the control group (odds ratio = 0.49, 95% confidence interval = 0.25-0.97) and TT genotype of rs2229717 had a significant correlation with hepatitis B surface antigen level (p = 0.0195). Further gene-gene interaction analysis showed that subjects carrying the rs10717122 DEL.DEL/DEL.T and rs585800 TT/TA genotypes had a 2.74-fold increased risk of CHB. CONCLUSIONS: The results of the present study suggest that rs10717122, rs585800 and rs2229717 and gene-gene interactions of rs10717122 and rs585800 affect the outcome of CHB infection, at the same time as indicating their usefulness as a predictive and diagnostic biomarker of CHB infection.

Transcriptomic Analysis Reveals an Altered Hcy Metabolism in the Stria Vascularis of the Pendred Syndrome Mouse Model.

Purpose: Slc26a4-/- mice exhibit severer defects in the development of the cochlea and develop deafness, while the underlying mechanisms responsible for these effects remain unclear. Our study was to investigate the potential mechanism linking SLC26A4 deficiency to hearing loss. Materials and Methods: RNA sequencing was applied to analyze the differential gene expression of the stria vascularis (SV) from wildtype and Slc26a4-/- mice. GO and KEGG pathway analysis were performed. Quantitative RT-PCR was applied to validate the expression of candidate genes affected by Slc26a4. ELISA and immunofluorescence technique were used to detect the homocysteine (Hcy) level in serum, brain, and SV, respectively. Results: 183 upregulated genes and 63 downregulated genes were identified in the SV associated with Slc26a4 depletion. Transcriptomic profiling revealed that Slc26a4 deficiency significantly affected the expression of genes associated with cell adhesion, transmembrane transport, and the biogenesis of multicellular organisms. The SV from Slc26a4-/- mice exhibited a higher expression of Bhmt mRNAs, as well as altered homocysteine (Hcy) metabolism. Conclusions: The altered expression of Bhmt results in a dramatic change in multiple biochemical reactions and a disruption of nutrient homeostasis in the endolymph which may contribute to hearing loss of Slc26a4 knockout mouse.

Betaine-homocysteine S-methyltransferase deficiency causes increased susceptibility to noise-induced hearing loss associated with plasma hyperhomocysteinemia.

Betaine-homocysteine S-methyltransferases (BHMTs) are methionine cycle enzymes that remethylate homocysteine; hence, their malfunction leads to hyperhomocysteinemia. Epidemiologic and experimental studies have revealed a correlation between hyperhomocysteinemia and hearing loss. Here, we have studied the expression of methionine cycle genes in the mouse cochlea and the impact of knocking out the Bhmt gene in the auditory receptor. We evaluated age-related changes in mouse hearing by recording auditory brainstem responses before and following exposure to noise. Also, we measured cochlear cytoarchitecture, gene expression by RNA-arrays and quantitative RT-PCR, and metabolite levels in liver and plasma by HPLC. Our results indicate that there is an age-dependent strain-specific expression of methionine cycle genes in the mouse cochlea and a further regulation during the response to noise damage. Loss of Bhmt did not cause an evident impact in the hearing acuity of young mice, but it produced higher threshold shifts and poorer recovery following noise challenge. Hearing loss was associated with increased cochlear injury, outer hair cell loss, altered expression of cochlear methionine cycle genes, and hyperhomocysteinemia. Our results suggest that BHMT plays a central role in the homeostasis of cochlear methionine metabolism and that Bhmt2 up-regulation could carry out a compensatory role in cochlear protection against noise injury in the absence of BHMT.-Partearroyo, T., Murillo-Cuesta, S., Vallecillo, N., Bermúdez-Muñoz, J. M., Rodríguez-de la Rosa, L., Mandruzzato, G., Celaya, A. M., Zeisel, S. H., Pajares, M. A., Varela-Moreiras, G., Varela-Nieto, I. Betaine-homocysteine S-methyltransferase deficiency causes increased susceptibility to noise-induced hearing loss associated with plasma hyperhomocysteinemia.

Taurine alleviates repression of betaine-homocysteine S-methyltransferase and significantly improves the efficacy of long-term betaine treatment in a mouse model of cystathionine ß-synthase-deficient homocystinuria.

Classical cystathionine ß-synthase-deficient homocystinuria (HCU) is a life-threatening inborn error of sulfur metabolism. Treatment for pyridoxine-nonresponsive HCU involves lowering homocysteine (Hcy) with a methionine (Met)-restricted diet and betaine supplementation. Betaine treatment efficacy diminishes significantly over time due to impairment of betaine-Hcy S-methyltransferase (BHMT) function. Little is known regarding the regulation of BHMT in HCU. Using a betaine-responsive preclinical mouse model of HCU, we observed that this condition induces a 75% repression of BHMT mRNA, protein and enzyme activity, and significant depletion of hepatic betaine levels. BHMT repression was proportional to plasma Hcy levels but was not observed in mouse models of homocystinuria due to either methylenetetrahydrofolate reductase or Met synthase deficiency. Both Met supplementation and chemically induced glutathione depletion exacerbated hepatic BHMT repression in HCU mice but not wild-type (WT) controls. Conversely, cysteine treatment normalized hepatic BHMT expression in HCU mice but had no effect in WT control animals. Taurine treatment induced BHMT expression in HCU mice by 5-fold and restored maximal lowering of Hcy levels during long-term betaine treatment with a concomitant normalization of inflammatory cytokine expression and a significantly improved coagulative phenotype. Collectively, our findings indicate that adjuvantial taurine treatment has the potential to significantly improve clinical outcomes in HCU.-Maclean, K. N., Jiang, H, Phinney, W. N., Keating, A. K., Hurt, K. J., Stabler, S. P. Taurine alleviates repression of betaine-homocysteine S-methyltransferase and significantly improves the efficacy of long-term betaine treatment in a mouse model of cystathionine ß-synthase-deficient homocystinuria.

Hepatic one-carbon metabolism enzyme activities and intermediate metabolites are altered by prepartum body condition score and plane of nutrition in grazing Holstein dairy cows.

Precalving feeding level and body condition score (BCS) alter postcalving energy balance and oxidant status of dairy cows. We hypothesized that the reported benefits of a controlled restriction precalving depend on precalving BCS. The objective was to identify alterations in activity and intermediates of the hepatic one-carbon metabolism, transsulfuration, and tricarboxylic acid pathways. Twenty-eight pregnant and nonlactating grazing dairy cows of mixed age and breed (Friesian, Friesian × Jersey) were randomly allocated to 1 of 4 treatment groups in a 2 × 2 factorial design: 2 prepartum BCS categories [4.0 (thin, BCS4) and 5.0 (optimal, BCS5); 10-point scale], by managing cows in late lactation to achieve the 2 groups at dry-off, and 2 levels of energy intake during the 3 wk preceding calving (75 or 125% of estimated requirements), obtained via allowance (m2/cow) of fresh pasture composed of mostly perennial ryegrass and white cover. Average (± standard deviation) age was 6 ± 2, 6 ± 3, 5 ± 1, and 7 ± 3 yr for BCS4 fed 75 and 125%, and BCS5 fed 75 and 125%, respectively. Breed distribution (average ± standard deviation) for the 4 groups was 79 ± 21, 92 ± 11, 87 ± 31, and 74 ± 23% Friesian, and 17 ± 20, 8 ± 11, 13 ± 31, and 25 ± 23% Jersey. Liver tissue was collected by biopsy at -7, 7, and 28 d relative to calving. Tissue was used for 14C radio-labeling assays to measure betaine-homocysteine S-methyltransferase, 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR), and cystathionine-ß-synthase (CBS) activity. Liver metabolomics was undertaken using a targeted liquid chromatography with tandem mass spectrometry-based profiling approach. After initial liquid chromatography separation, mass spectra were acquired under both positive and negative ionization, whereas multiple reaction monitoring was used to measure target compound signal response (peak area count). Enzyme activity and metabolite peak area count were normalized with the homogenate protein concentration. Repeated measures analysis of variance via PROC MIXED in SAS (SAS Institute Inc., Cary, NC), with BCS, feeding, and time as fixed effects, and cow as random effect was used. All enzyme activities were affected by time, with betaine-homocysteine S-methyltransferase activity peaking at 7 d, whereas CBS and MTR activity decreased postpartum. Overall, thin cows had greater MTR activity, whereas cows fed 125% requirements had greater CBS activity. An interaction was detected between BCS and feeding for CBS activity, as thin cows fed 125% of requirements had greater overall activity. Compared with liver from BCS4 cows, BCS5 cows had overall greater betaine, glycine, butyrobetaine/acetylcholine, serine, and taurine concentrations. The same metabolites, plus choline and N-N-dimethylglycine, were overall greater in liver of cows fed 75% compared with those fed 125% of requirements. An interaction of BCS and feeding level was detected for the aforementioned metabolites plus methionine, cystathionine, cysteinesulfinate, and hypotaurine, due to greater overall concentrations in BCS5 cows fed 75% of requirements compared with other groups. Overall, differences in hepatic enzyme activity and intermediate metabolites suggest that both BCS and feeding level can alter the internal antioxidant system (e.g., glutathione and taurine) throughout the periparturient period. Further studies are needed to better understand potential mechanisms involved.

Association between the BHMT gene rs3733890 polymorphism and the efficacy of oral folate therapy in patients with hyperhomocysteinemia.

Oral folate is currently the most common treatment for hyperhomocysteinemia (HHcy), which seriously threatens human health, but its efficacy is unsatisfactory. Betaine-homocysteine methyltransferase (BHMT) is a key enzyme that regulates Hcy metabolism. We investigated the association between the BHMT rs3733890 and the efficacy of oral folate therapy for HHcy in the Chinese Han population and analysed the effects of gene-environmental interactions on the efficacy. Blood samples were collected from 1071 eligible patients at baseline, and these individuals received subsequent folate treatment for 90 days. A total of 638 patients included in the final analysis were grouped into the treatment success group or the treatment failure group based on posttreatment Hcy levels. Hcy concentrations were measured by fluorescence polarization immunoassay. Time-of-flight mass spectrometry (MassArray system) was used to assess the genotype of BHMT rs3733890. Stratified analyses based on additive models and generalized multifactor dimensionality reduction were used to explore gene-environmental interactions. The genotype distribution presented distinct differences in the two groups. The mutant genotype and allele had significantly increased risk of treatment failure (p < 0.05). Furthermore, synergistic effects of the BHMT rs3733890 polymorphism with environmental risk factors (smoking, drinking, past history) on the efficacy of therapy were also found. However, future, large well-designed studies, as well as mechanistic studies, are still needed to validate our findings.

Long non-coding RNA Bhmt-AS attenuates hepatic gluconeogenesis via modulation of Bhmt expression.

Dysregulation of gluconeogenesis contributes to the pathogenesis of metabolic disease, such as type-2 diabetes. The role of long non-coding RNAs (lncRNAs) in the pathogenesis of diabetes has recently received increased attention. In the present study, we identified a novel lncRNA, betaine-homocysteine methyltransferase-antisense (Bhmt-AS), and examined its expression patterns under pathophysiological conditions. Our results revealed that the expression of Bhmt-AS was significantly increased in the livers of fasted and db/db mice and was induced by gluconeogenic hormonal stimuli. The Bhmt-AS was also shown to be a concordant regulator of Bhmt expression. Functionally, depletion of Bhmt-AS suppressed hepatic glucose production both in vivo and in vitro. Adenovirus-mediated hepatic knockdown of Bhmt-AS improved pyruvate tolerance, glucose tolerance, and insulin sensitivity. Furthermore, overexpression of Bhmt restored the decreased glucose production caused by knockdown of Bhmt-AS in primary hepatocytes. Taken together, we uncovered a novel antisense lncRNA (Bhmt-AS) that is co-expressed with Bhmt and concordantly and specifically regulates Bhmt expression both in vitro and in vivo to regulate hepatic gluconeogenesis.

Association between BHMT and CBS gene promoter methylation with the efficacy of folic acid therapy in patients with hyperhomocysteinemia.

Both betaine homocysteine methyltransferase (BHMT) and cystathionine ß-synthase (CBS) are major enzymes in the metabolism of plasma homocysteine (Hcy). Abnormal methylation levels of BHMT and CBS are positively associated with Hcy levels. The present study is performed to explore the association between the methylation levels in the promoter regions of the BHMT and CBS genes and the efficacy of folic acid therapy in patient with hyperhomocysteinemia (HHcy). A prospective cohort study recruiting HHcy (Hcy ≥ 15 µmol/L) patients was performed. The subjects were treated with oral folic acid (5 mg/d) for 90 days, and the patients were divided into the success group (Hcy < 15 µmol/L) and the failure group (Hcy ≥ 15 µmol/L) according to their Hcy levels after treatment. In the logistic regression model with adjusted covariates, the patients with lower total methylation levels in the BHMT and CBS promoter regions exhibited 1.627-fold and 1.671-fold increased risk of treatment failure compared with higher methylation individuals, respectively. Similarly, subjects who had lower methylation levels (

Dietary Egg Protein Prevents Hyperhomocysteinemia via Upregulation of Hepatic Betaine-Homocysteine S-Methyltransferase Activity in Folate-Restricted Rats.

BACKGROUND: Hyperhomocysteinemia is associated with increased cardiovascular disease risk. Whole eggs contain several nutrients known to affect homocysteine regulation, including sulfur amino acids, choline, and B vitamins. OBJECTIVE: The aim of this study was to determine the effect of whole eggs and egg components (i.e., egg protein and choline) with respect to 1) homocysteine balance and 2) the hepatic expression and activity of betaine-homocysteine S-methyltransferase (BHMT) and cystathionine ß-synthase (CBS) in a folate-restricted (FR) rat model of hyperhomocysteinemia. METHODS: Male Sprague Dawley rats (n = 48; 6 wk of age) were randomly assigned to a casein-based diet (C; n = 12), a casein-based diet supplemented with choline (C + Cho; 1.3%, wt:wt; n = 12), an egg protein-based diet (EP; n = 12), or a whole egg-based diet (WE; n = 12). At week 2, half of the rats in each of the 4 dietary groups were provided an FR (0 g folic acid/kg) diet and half continued on the folate-sufficient (FS; 0.2 g folic acid/kg) diet for an additional 6 wk. All diets contained 20% (wt:wt) total protein. Serum homocysteine was measured by HPLC and BHMT and CBS expression and activity were evaluated using real-time quantitative polymerase chain reaction, Western blot, and enzyme activity. A 2-factor ANOVA was used for statistical comparisons. RESULTS: Rats fed FR-C exhibited a 53% increase in circulating homocysteine concentrations compared with rats fed FS-C (P < 0.001). In contrast, serum homocysteine did not differ between rats fed FS-C and FR-EP (P = 0.078). Hepatic BHMT activity was increased by 45% and 40% by the EP (P < 0.001) and WE (P = 0.002) diets compared with the C diets, respectively. CONCLUSIONS: Dietary intervention with egg protein prevented elevated circulating homocysteine concentrations in a rat model of hyperhomocysteinemia, due in part to upregulation of hepatic BHMT. These data may support the inclusion of egg protein for dietary recommendations targeting hyperhomocysteinemia prevention.

Accumulation of rare coding variants in genes implicated in risk of human cleft lip with or without cleft palate.

Cleft lip with/without cleft palate (CLP) is a common craniofacial malformation with complex etiologies, reflecting both genetic and environmental factors. Most of the suspected genetic risk for CLP has yet to be identified. To further classify risk loci and estimate the contribution of rare variants, we sequenced the exons in 49 candidate genes in 323 CLP cases and 211 nonmalformed controls. Our findings indicated that rare, protein-altering variants displayed markedly higher burdens in CLP cases at relevant loci. First, putative loss-of-function mutations (nonsense, frameshift) were significantly enriched among cases: 13 of 323 cases (~4%) harbored such alleles within these 49 genes, versus one such change in controls (p = 0.01). Second, in gene-level analyses, the burden of rare alleles showed greater case-association for several genes previously implicated in cleft risk. For example, BHMT displayed a 10-fold increase in protein-altering variants in CLP cases (p = .03), including multiple case occurrences of a rare frameshift mutation (K400 fs). Other loci with greater rare, coding allele burdens in cases were in signaling pathways relevant to craniofacial development (WNT9B, BMP4, BMPR1B) as well as the methionine cycle (MTRR). We conclude that rare coding variants may confer risk for isolated CLP.

Short communication: The effect of increasing concentrations of different methionine forms and 2-hydroxy-4-(methylthio)butanoic acid on genes controlling methionine metabolism in primary bovine neonatal hepatocytes.

The d-isomer of Met cannot be used directly by the mammary gland in dairy cows; instead, it is transformed into l-Met, the proteogenic isomer, in the liver and other extramammary tissues. It remains unclear whether different Met forms and a Met hydroxy analog, 2-hydroxy-4-(methylthio)butanoic acid (HMB), are metabolized and function similarly in the liver. The objective of the present study was to examine the regulation of key genes in Met regeneration, transulfuration, and transmethylation pathways in response to increasing doses of different Met forms. Hepatocytes isolated from 4 calves between 4 and 7 d old were maintained as monolayer cultures for 24 h before addition of treatments. Treatments of (0, 10, 20, 40 µM) d-Met, l-Met, dl-Met, dl-HMB, or a 1:1 mixture of dl-Met and dl-HMB were added to Met-free medium in triplicate. After 24 h, cell lysates were collected for quantification of gene expression by quantitative PCR, and mRNA abundance was normalized to the mean of 3 reference genes. Data were analyzed with PROC MIXED of SAS 9.3 (SAS Institute Inc., Cary, NC). Analyses of covariance confirmed equivalent slopes of Met form, and the final model included form, dose, and random effect of calf within form. Data are reported as least squares means ± standard error. No main effect of Met form was observed for any genes examined. The enzymes encoded by betaine-homocysteine methyltransferase (BHMT) and 5-methyltetrahydrofolate-homocysteine methyltransferase use betaine and 5-methyltetrahydrofolate, respectively, to regenerate Met from homocysteine. Increasing concentration of Met did not alter 5-methyltetrahydrofolate expression, but decreased BHMT expression. Expression of glycine N-methyltransferase, the enzyme that controls transmethylation flux from S-adenosyl-methionine, was not affected by Met concentration. Methionine concentration had no effect on expression of cystathionine ß-synthase, a key enzyme for the transulfuration pathway. The decrease in BHMT expression indicates a decreased need for cellular Met regeneration with increasing Met concentration, independent of Met form. The lack of differences among Met forms on regulating genes examined indicates that all Met forms similarly reduced genes controlling Met regeneration and metabolism in primary bovine hepatocytes.

Hepatic 1-carbon metabolism enzyme activity, intermediate metabolites, and growth in neonatal Holstein dairy calves are altered by maternal supply of methionine during late pregnancy.

Maternal supply of methyl donors such as methionine (Met) during late pregnancy can affect offspring growth and development. The objective was to investigate the effect of postruminal Met supply during late pregnancy on 1-carbon, Met cycle, and transsulfuration pathways in the calf liver. During the last 28 d of pregnancy, cows were individually fed a control diet or the control diet plus rumen-protected dl-Met (MET; 0.09% dry matter intake). Liver samples obtained from calves (n = 14/group) at 4, 14, 28, and 50 d of age were used for metabolomics, real-time PCR, and enzyme activity analyses. Genes associated with 1-carbon metabolism, DNA methylation, and the cytidine 5'-diphosphocholine-choline pathway were analyzed via real-time PCR. Activity of betaine homocysteine methyltransferase, cystathionine ß-synthase, and 5-methyltetrahydrofolate homocysteine methyltransferase (MTR) was analyzed using 14C isotopes. Data were analyzed using a mixed model that included the fixed effects of maternal treatment, day, and their interaction, and the random effect was calf within maternal diet. Calves born to dams offered MET tended to have greater birth body weight and had overall greater body weight during the first 9 wk of life. However, no differences were detected for daily feed intake and average daily gain between groups. Concentrations of betaine and choline, reflecting Met cycle activity, at d 14 through 28 were greater in MET calves. Transsulfuration pathway intermediates also were altered in MET calves, with concentrations of cysteine sulfinic acid and hypotaurine (d 4 and 14) and taurine being greater (d 4, 14, 28, and 50). Despite the lack of differences in daily feed intake, the greater concentrations of the tricarboxylic acid cycle intermediates fumarate and glutamate along with NAD/NADH in MET calves indicated enhanced rates of energy metabolism. Although activity of betaine homocysteine methyltransferase was greater in MET calves at d 14, cystathionine ß-synthase was lower and increased at d 14 and 28, where it was greater compared with the control diet. Activity of MTR was lower at d 4 and 50 in MET calves. Among gene targets measured, MET calves had greater overall expression of MTR, phosphatidylethanolamine N-methyltransferase, and choline kinase α and ß. An interaction of maternal diet by time was detected for mRNA abundance of DNA methyltransferase 3α (involved in de novo methylation) due to greater values at d 4 and 14 in MET calves. Overall, the data indicate that enhanced postruminal supply of Met to cows during late pregnancy may program hepatic metabolism of the calf in the context of maintaining Met homeostasis, phosphatidylcholine and taurine synthesis, DNA methylation, and energy metabolism. These alterations potentially result in better efficiency of nutrient use, hence conferring the calf a physiologic advantage during a period of rapid growth and development. The precise biologic mechanisms remain to be established.

Hepatic betaine-homocysteine methyltransferase and methionine synthase activity and intermediates of the methionine cycle are altered by choline supply during negative energy balance in Holstein cows.

Although choline requirements are unknown, enhanced postruminal supply may decrease liver triacylglycerol (TAG) storage and increase flux through the methionine cycle, helping cows during a negative energy balance (NEB). The objective was to investigate effects of postruminal choline supply during NEB on hepatic activity of betaine-homocysteine methyltransferase (BHMT), methionine synthase (MTR), methionine adenosyltransferase, transcription of enzymes, and metabolite concentrations in the methionine cycle. Ten primiparous rumen-cannulated Holstein cows (158 ± 24 d postpartum) were used in a replicated 5 × 5 Latin square design with 4-d treatment periods and 10 d of recovery (14 d/period). Treatments were unrestricted intake with abomasal infusion of water (A0), restricted intake (R; 60% of net energy for lactation requirements to induce NEB) with abomasal infusion of water (R0) or R plus abomasal infusion of 6.25, 12.5, or 25 g/d of choline ion. Liver tissue was collected on d 5 after the infusions ended, blood on d 1 to 5, and milk on d 1 to 4. Statistical contrasts were A0 versus R0 (CONT1) and tests of linear (L), quadratic (Q), and cubic (C) effects of choline dose. Plasma choline increased with R (CONT1) and choline (L). Although R decreased milk yield (CONT1), choline increased milk yield and liver phosphatidylcholine (PC), but decreased TAG (L). No differences were observed in plasma PC or very-low-density lipoprotein concentrations with R or choline. Activity and mRNA abundance of BHMT were greater with R (CONT1) and increased with choline (L). Although activity of MTR was lower with R (CONT1), it tended to increase with choline (L). No effect of R was detected for activity of methionine adenosyltransferase, but it changed cubically across dose of choline. Those responses were associated with linear increases in the concentrations of liver tissue (+13%) and plasma methionine concentrations. The mRNA abundance of CPT1A, SLC22A5, APOA5, and APOB, genes associated with fatty acid oxidation and lipoprotein metabolism, was upregulated by choline (Q). Overall, enhanced supply of choline during NEB increases hepatic activity of BHMT and MTR to regenerate methionine and PC, partly to help clear TAG. The relevance of these effects during the periparturient period merits further research.

Genetic and epigenetic regulation of BHMT is associated with folate therapy efficacy in hyperhomocysteinaemia.

BACKGROUND AND OBJECTIVES: Hyperhomocysteinaemia (HHcy) is an independent risk factors for several disorders, including cardiovascular disease. The understanding of the relationship among genetic, epigenetic and the efficacy of folate therapy for HHcy remain unclear. This study aim to investigate whether betaine-homocysteine methyltransferase (BHMT) single-nucleotide polymorphisms (SNPs) and DNA methylation are related to the efficacy of folate therapy for HHcy and whether BHMT DNA methylation mediates the SNP-folate therapy efficacy association. METHODS AND STUDY DESIGN: A total of 638 patients with HHcy were involved in this prospective cohort study. Logistic and linear regression was used to explore associations among SNPs, DNA methylation, and folate therapy efficacy. Finally, mediation analysis was performed to investigate whether DNA methylation of BHMT mediates the association between SNPs and folate therapy efficacy. RESULTS: BHMT rs3733890 was significantly associated with folate therapy efficacy (p<0.05). BHMT and BHMT_1 DNA methylation level was significantly associated with folate therapy efficacy (p=0.017 and p=0.028). DNA methylation of BHMT and BHMT_1 mediated 34.84% and 33.06% of the effect of rs3733890 on folate therapy efficacy, respectively. CONCLUSIONS: There has a consistent interrelationship among BHMT genetic variants, methylation levels of BHMT, and folate therapy efficacy. BHMT and BHMT_1 DNA methylation proportionally mediated the effects of rs3733890 SNPs on the efficacy of folate therapy for HHcy.

[Effects of methionine on the activity of cystathionine-ß-synthase].

OBJECTIVE: To investigate the effects of methionine on the activity of cystathionine-ß-synthase. METHODS: A total of 56 male rats of the Wistar were randomly divided into 7 groups: 10% casein(10 C) group, 40% casein(40 C) group, 10 C+0.75% L-methionine(10 CM) group, 10 C+amino acid mixture(10 CAA) group, 10 CAA-methionine(10 CAA-Met) group, 10 C+ essential amino acid(10 C+EAA) group, and 10 C+ non-essential amino acid(10 C+NEAA) group, with 8 rats in each group for 10 days. RESULTS: The plasma homocysteine concentration significantly increased from(17.1±0.3)µmol/L to(50.7±4.8)µmol/L and(40.5±3.9)µmol/L in rats fed 10 CM and 10 C+EAA diets(P<0.01). Supplementation with methionine induced hyperhomocysteinemia. Compared to 10 C, the activity of hepatic cystathionine-ß-synthase(CBS) were significantly increased in the experimental group except for 10 CM(P<0.05). The activity of hepatic CBS was the largest increases in diets with 40 C and the smallest increases in 10 C+NEAA. The activity of hepatic betaine-homocysteine S-methyltransferase(BHMT) were increased in the experimental group except for 10 CAA-Met and 10 C+NEAA(P<0.05). CONCLUSION: The increased CBS activity induced by high protein diets is determined by high amino acid intake rather than methionine supplemention.

Betaine homocysteine S-methyltransferase emerges as a new player of the nuclear methionine cycle.

The paradigm of a cytoplasmic methionine cycle synthesizing/eliminating metabolites that are transported into/out of the nucleus as required has been challenged by detection of significant nuclear levels of several enzymes of this pathway. Here, we show betaine homocysteine S-methyltransferase (BHMT), an enzyme that exerts a dual function in maintenance of methionine levels and osmoregulation, as a new component of the nuclear branch of the cycle. In most tissues, low expression of Bhmt coincides with a preferential nuclear localization of the protein. Conversely, the liver, with very high Bhmt expression levels, presents a main cytoplasmic localization. Nuclear BHMT is an active homotetramer in normal liver, although the total enzyme activity in this fraction is markedly lower than in the cytosol. N-terminal basic residues play a role in cytoplasmic retention and the ratio of glutathione species regulates nucleocytoplasmic distribution. The oxidative stress associated with d-galactosamine (Gal) or buthionine sulfoximine (BSO) treatments induces BHMT nuclear translocation, an effect that is prevented by administration of N-acetylcysteine (NAC) and glutathione ethyl ester (EGSH), respectively. Unexpectedly, the hepatic nuclear accumulation induced by Gal associates with reduced nuclear BHMT activity and a trend towards increased protein homocysteinylation. Overall, our results support the involvement of BHMT in nuclear homocysteine remethylation, although moonlighting roles unrelated to its enzymatic activity in this compartment cannot be excluded.

Altered methylation of specific DNA loci in the liver of Bhmt-null mice results in repression of Iqgap2 and F2rl2 and is associated with development of preneoplastic foci.

Folate B12-dependent remethylation of homocysteine is important, but less is understood about the importance of the alternative betaine-dependent methylation pathway-catalyzed by betaine-homocysteine methyltransferase (BHMT)-for establishing and maintaining adequate DNA methylation across the genome. We studied C57Bl/6J Bhmt (betaine-homocysteine methyltransferase)-null mice at age 4, 12, 24, and 52 wk (N = 8) and observed elevation of S-adenosylhomocysteine concentrations and development of preneoplastic foci in the liver (increased placental glutathione S-transferase and cytokeratin 8-18 activity; starting at 12 wk). At 4 wk, we identified 63 differentially methylated CpGs (DMCs; false discovery rate < 5%) proximal to 81 genes (across 14 chromosomes), of which 18 were differentially expressed. Of these DMCs, 52% were located in one 15.5-Mb locus on chromosome 13, which encompassed the Bhmt gene and defined a potentially sensitive region with mostly decreased methylation. Analyzing Hybrid Mouse Diversity Panel data, which consisted of 100 inbred strains of mice, we identified 97 DMCs that were affected by Bhmt genetic variation in the same region, with 7 overlapping those found in Bhmt-null mice (P < 0.001). At all time points, we found a hypomethylated region mapping to Iqgap2 (IQ motif-containing GTPase activating protein 2) and F2rl2 (proteinase-activated receptor-3), 2 genes that were also silenced and underexpressed, respectively.-Lupu, D. S., Orozco, L. D., Wang, Y., Cullen, J. M., Pellegrini, M., Zeisel, S. H. Altered methylation of specific DNA loci in the liver of Bhmt-null mice results in repression of Iqgap2 and F2rl2 and is associated with development of preneoplastic foci.