Betaine-homocysteine methyltransferase promotes adipocyte commitment and insulin resistance via p38 MAPK/Smad signaling.

OBJECTIVE: Betaine-homocysteine methyltransferase (Bhmt) belongs to the family of methyltransferases and is involved in the one-carbon metabolic cycle, which is associated with the risk of diabetes and adiposity. This study aimed to explore whether Bhmt participated in the development of obesity or its associated diabetes, as well as the mechanism involved. METHODS: The expression levels of Bhmt were examined in stromal vascular fraction cells and mature adipocytes in obesity and nonobesity. Knockdown and overexpression of Bhmt in C3H10T1/2 cells were used to investigate Bhmt's function in adipogenesis. Bhmt's role in vivo was analyzed using an adenovirus-expressing system and a high-fat diet-induced obesity mouse model. RESULTS: Bhmt was highly expressed in stromal vascular fraction cells rather than mature adipocytes of adipose tissue and was upregulated in adipose tissue in obesity and C3H10T1/2-commited preadipocytes. Overexpression of Bhmt promoted adipocyte commitment and differentiation in vitro and exacerbated adipose tissue expansion in vivo, with a concomitant increase in insulin resistance, whereas Bhmt silencing exhibited opposite effects. Mechanistically, Bhmt-induced adipose expansion was mediated by stimulating the p38 MAPK/Smad pathway. CONCLUSIONS: The findings of this study highlight the obesogenic and diabetogenic role of adipocytic Bhmt and propose Bhmt as a promising therapeutic target for obesity and obesity-related diabetes.

Vegetable Oil-Peroxidation Product 'Hydroxynonenal' Causes Hepatocyte Injury and Steatosis via Hsp70.1 and BHMT Disorders in the Monkey Liver.

Hsp70.1 has a dual function as a chaperone protein and lysosomal stabilizer. In 2009, we reported that calpain-mediated cleavage of carbonylated Hsp70.1 causes neuronal death by inducing lysosomal rupture in the hippocampal CA1 neurons of monkeys after transient brain ischemia. Recently, we also reported that consecutive injections of the vegetable oil-peroxidation product 'hydroxynonenal' induce hepatocyte death via a similar cascade in monkeys. As Hsp70.1 is also related to fatty acid ß-oxidation in the liver, its deficiency causes fat accumulation. The genetic deletion of betaine-homocysteine S-methyltransferase (BHMT) was reported to perturb choline metabolism, inducing a decrease in phosphatidylcholine and resulting in hepatic steatosis. Here, focusing on Hsp70.1 and BHMT disorders, we studied the mechanisms of hepatocyte degeneration and steatosis. Monkey liver tissues with and without hydroxynonenal injections were compared using proteomics, immunoblotting, immunohistochemical, and electron microscopy-based analyses. Western blotting showed that neither Hsp70.1 nor BHMT were upregulated, but an increased cleavage was observed in both. Proteomics showed a marked downregulation of Hsp70.1, albeit a two-fold increase in the carbonylated BHMT. Hsp70.1 carbonylation was negligible, in contrast to the ischemic hippocampus, which was associated with ~10-fold increments. Although histologically, the control liver showed very little lipid deposition, numerous tiny lipid droplets were seen within and around the degenerating/dying hepatocytes in monkeys after the hydroxynonenal injections. Electron microscopy showed permeabilization/rupture of lysosomal membranes, dissolution of the mitochondria and rough ER membranes, and proliferation of abnormal peroxisomes. It is probable that the disruption of the rough ER caused impaired synthesis of the Hsp70.1 and BHMT proteins, while impairment of the mitochondria and peroxisomes contributed to the sustained generation of reactive oxygen species. In addition, hydroxynonenal-induced disorders facilitated degeneration and steatosis in the hepatocytes.

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.

Betaine consumption as a new clinical approach to treatment and prophylaxis of folate-related pathologies.

The most important pathway in the development of folate-related pathologies is an increase in the level of homocysteine (HC). HC, a cytotoxic and neurotoxic amino acid (when its level is ≥12 µmol/L), is 1 of the most widely studied compounds in cardiology, neurobiology, oncology, and embryology for the last 20 years. Given its toxicity, the processes of endogenous detoxification of HC are of particular interest to medicine. To date, the most studied pathway is that of remethylation (the conversion of HC to methionine), with the participation of B12- and B9-dependent methionine synthase. Less studied is remethylation with the participation of the choline derivatives betaine and betaine-HC-S-methyltransferase (BHMT). Therefore, the aim of this review was to conduct a theoretical analysis of available information regarding the contribution of betaine metabolism, its enzyme, and its genetic polymorphism to folate metabolism disturbances, and the development of folate-related pathologies. This review emphasizes the potential clinical significance of 2 factors that can influence the remethylation reaction of HC: the use of betaine and identifying the BHMT gene variants and their impact on the risk for developing certain folate-related pathologies, and treatment options. Moreover, with a high level of methylation of the BHMT gene and in the presence of its low-function variants (eg, rs3733890), it is necessary to use betaine as an additional methyl donor, especially during folate therapy. More clinical research is needed to identify the effects of the different BHMT gene variants on the individual risk for folate-related pathologies to better assess the clinical significance, the need for genetic testing, and betaine consumption.

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.

One-carbon metabolism and global DNA methylation in mothers of individuals with Down syndrome.

Down syndrome (DS) is the most common chromosomal disorder, resulting from the failure of normal chromosome 21 segregation. Studies have suggested that impairments within the one-carbon metabolic pathway can be of relevance for the global genome instability observed in mothers of individuals with DS. Based on the association between global DNA hypomethylation, genome instability, and impairments within the one-carbon metabolic pathway, the present study aimed to identify possible predictors, within the one-carbon metabolism, of global DNA methylation, measured by methylation patterns of LINE-1 and Alu repetitive sequences, in mothers of individuals with DS and mothers of individuals without the syndrome. In addition, we investigated one-carbon genetic polymorphisms and metabolites as maternal predisposing factors for the occurrence of trisomy 21 in children. Eighty-three samples of mothers of children with DS with karyotypically confirmed free trisomy 21 (case group) and 84 of mothers who had at least one child without DS or any other aneuploidy were included in the study. Pyrosequencing assays were performed to access global methylation. The results showed that group affiliation (case or control), betaine-homocysteine methyltransferase (BHMT) G742A and transcobalamin 2 (TCN2) C776G polymorphisms, and folate concentration were identified as predictors of global Alu DNA methylation values. In addition, thymidylate synthase (TYMS) 28-bp repeats 2R/3R or 3R/3R genotypes are independent maternal predisposing factors for having a child with DS. This study adds evidence that supports the association of impairments in the one-carbon metabolism, global DNA methylation, and the possibility of having a child with DS.

The BHMT-betaine methylation pathway epigenetically modulates oligodendrocyte maturation.

Research into the epigenome is of growing importance as a loss of epigenetic control has been implicated in the development of neurodegenerative diseases. Previous studies have implicated aberrant DNA and histone methylation in multiple sclerosis (MS) disease pathogenesis. We have previously reported that the methyl donor betaine is depleted in MS and is linked to changes in histone H3 trimethylation (H3K4me3) in neurons. We have also shown that betaine increases histone methyltransferase activity by activating chromatin bound betaine homocysteine S-methyltransferase (BHMT). Here, we investigated the role of the BHMT-betaine methylation pathway in oligodendrocytes. Immunocytochemistry in the human MO3.13 cell line, primary rat oligodendrocytes, and tissue from MS postmortem brain confirmed the presence of the BHMT enzyme in the nucleus in oligodendrocytes. BHMT expression is increased 2-fold following oxidative insult, and qRT-PCR demonstrated that betaine can promote an increase in expression of oligodendrocyte maturation genes SOX10 and NKX-2.2 under oxidative conditions. Chromatin fractionation provided evidence of a direct interaction of BHMT on chromatin and co-IP analysis indicates an interaction between BHMT and DNMT3a. Our data show that both histone and DNA methyltransferase activity are increased following betaine administration. Betaine effects were shown to be dependent on BHMT expression following siRNA knockdown of BHMT. This is the first report of BHMT expression in oligodendrocytes and suggests that betaine acts through BHMT to modulate histone and DNA methyltransferase activity on chromatin. These data suggest that methyl donor availability can impact epigenetic changes and maturation in oligodendrocytes.

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.

Pharmacologic downregulation of protein arginine methyltransferase1 expression by adenosine dialdehyde increases cell senescence in breast cancer.

We investigated the role of protein arginine methylation (PAM) in estrogen receptor (ER)-positive breast cancer cells through pharmacological intervention. Tamoxifen (TAM) or adenosine dialdehyde (ADOX), independently, triggered cell cycle arrest and down-regulated PAM, as reduced protein arginine methyltransferase1 (PRMT1) mRNA and asymmetric dimethylarginine (ADMA) levels. Synergistic effect of these compounds elicited potent anti-cancer effect. However, reduction in ADMA was not proportionate with the compound-induced down-regulation of PRMT1 mRNA. We hypothesized that the disproportionate effect is due to the influence of the compounds on other methyltransferases, which catalyze the arginine dimethylation reaction and the diversity in the degree of drug-protein interaction among these methyltransferases. In silico analyses revealed that independently, ADOX or TAM, binds with phosphatidylethanolamine-methyltransferase (PEMT) or betaine homocysteine-methyl transferase (BHMT); and that the binding affinity of ADOX with PEMT or BHMT is prominent than TAM. These observations suggest that in breast cancer, synergistic effect of ADOX + TAM elicits impressive protective function by regulating PAM; and plausibly, restoration of normal enzyme activities of methyltransferases catalyzing arginine dimethylation could have clinical benefits.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) dysregulates hepatic one carbon metabolism during the progression of steatosis to steatohepatitis with fibrosis in mice.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a persistent environmental contaminant, induces steatosis that can progress to steatohepatitis with fibrosis, pathologies that parallel stages in the development of non-alcoholic fatty liver disease (NAFLD). Coincidently, one carbon metabolism (OCM) gene expression and metabolites are often altered during NAFLD progression. In this study, the time- and dose-dependent effects of TCDD were examined on hepatic OCM in mice. Despite AhR ChIP-seq enrichment at 2 h, OCM gene expression was not changed within 72 h following a bolus dose of TCDD. Dose-dependent repression of methionine adenosyltransferase 1A (Mat1a), adenosylhomocysteinase (Achy) and betaine-homocysteine S-methyltransferase (Bhmt) mRNA and protein levels following repeated treatments were greater at 28 days compared to 8 days. Accordingly, levels of methionine, betaine, and homocysteic acid were dose-dependently increased, while S-adenosylmethionine, S-adenosylhomocysteine, and cystathionine exhibited non-monotonic dose-dependent responses consistent with regulation by OCM intermediates and repression of glycine N-methyltransferase (Gnmt). However, the dose-dependent effects on SAM-dependent metabolism of polyamines and creatine could not be directly attributed to alterations in SAM levels. Collectively, these results demonstrate persistent AhR activation disrupts hepatic OCM metabolism at the transcript, protein and metabolite levels within context of TCDD-elicited progression of steatosis to steatohepatitis with fibrosis.

Betaine restores epigenetic control and supports neuronal mitochondria in the cuprizone mouse model of multiple sclerosis.

Methionine metabolism is dysregulated in multiple sclerosis (MS). The methyl donor betaine is depleted in the MS brain where it is linked to changes in levels of histone H3 trimethylated on lysine 4 (H3K4me3) and mitochondrial impairment. We investigated the effects of replacing this depleted betaine in the cuprizone mouse model of MS. Supplementation with betaine restored epigenetic control and alleviated neurological disability in cuprizone mice. Betaine increased the methylation potential (SAM/SAH ratio), levels of H3K4me3, enhanced neuronal respiration, and prevented axonal damage. We show that the methyl donor betaine and the betaine homocysteine methyltransferase (BHMT) enzyme can act in the nucleus to repair epigenetic control and activate neuroprotective transcriptional programmes. ChIP-seq data suggest that BHMT acts on chromatin to increase the SAM/SAH ratio and histone methyltransferase activity locally to increase H3K4me3 and activate gene expression that supports neuronal energetics. These data suggest that the methyl donor betaine may provide neuroprotection in MS where mitochondrial impairment damages axons and causes disability.

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.

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.

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.

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.

Alleviation of paraquat-induced oxidative lung injury by betaine via regulation of sulfur-containing amino acid metabolism despite the lack of betaine-homocysteine methyltransferase (BHMT) in the lung.

Betaine is a methyl donor utilized in regeneration of methionine from homocysteine in a metabolic reaction catalyzed by betaine-homocysteine methyltransferase (BHMT), an enzyme mostly localized in the liver. However, we recently showed that the metabolism of sulfur-containing amino acids in the kidney was also influenced by betaine, which is attributable to elevation of renal methionine availability resulting from an increase in its supply through blood. In this study we investigated the change in pulmonary sulfur-containing amino acid metabolism by betaine and its potential beneficial effect on paraquat (PQ)-induced lung injury. Male rats were provided with betaine for 2 weeks prior to an intratracheal instillation of PQ (0.3 mg/500 µl kg-1). Two weeks after PQ exposure, histopathological assessment revealed severe fibrotic lesions accompanied with elevation of 4-hydroxyproline in the lung, which were all prevented effectively by betaine supplementation. PQ-induced DNA fragmentation in lymphocytes, reduction of oxidant scavenging capacity, expression of heme oxygenase 1 and inducible nitric oxide synthase in the lung, and elevation of serum transforming growth factor beta 1 were also inhibited. PQ instillation increased cysteine, but depleted glutathione in the lung. Betaine supplementation before PQ exposure suppressed the cysteine accumulation and increased the glutathione synthesis. The polyamine synthesis, which requires decarboxylated S-adenosylmethionine as a substrate, was also increased significantly. The results suggest that betaine may enhance pulmonary antioxidant capacity by modulating the metabolism of sulfur-containing amino acids and related substances despite the lack of BHMT in the lung.

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.

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.

Methionine and choline supply alter transmethylation, transsulfuration, and cytidine 5'-diphosphocholine pathways to different extents in isolated primary liver cells from dairy cows.

Insufficient supply of Met and choline (Chol) around parturition could compromise hepatic metabolism and milk protein synthesis in dairy cows. Mechanistic responses associated with supply of Met or Chol in primary liver cells enriched with hepatocytes (PHEP) from cows have not been thoroughly ascertained. Objectives were to isolate and culture PHEP to examine abundance of genes and proteins related to transmethylation, transsulfuration, and cytidine 5'-diphosphocholine (CDP-choline) pathways in response to Met or Chol. The PHEP were isolated from liver biopsies of Holstein cows (160 d in lactation). More than 90% of isolated cells stained positively for the hepatocyte marker cytokeratin 18. Cytochrome P450 (CYP1A1) mRNA abundance was only detectable in the PHEP and liver tissue compared with mammary tissue. Furthermore, in response to exogenous Met (80 µM vs. control) PHEP secreted greater amounts of albumin and urea. Subsequently, PHEP were cultured with Met (40 µM) or Chol (80 mg/dL) for 24 h. Compared with control or Chol, mRNA and protein abundance of methionine adenosyltransferase 1A (MAT1A) and phosphatidylethanolamine methyltransferase (PEMT) were greater in PHEP treated with Met. The mRNA abundance of S-adenosylhomocysteine hydrolase (SAHH), betaine-homocysteine methyltransferase (BHMT), and sarcosine dehydrogenase (SARDH) was greater in Met-treated PHEP compared with control or Chol. Compared with control, greater expression of 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR), betaine aldehyde dehydrogenase (BADH), and choline dehydrogenase (CHDH) was observed in cells supplemented with Met and Chol. However, Chol led to the greatest mRNA abundance of CHDH. Abundance of choline kinase α (CHKA), choline kinase ß (CHKB), phosphate cytidylyltransferase 1 α (PCYT1A), and choline/ethanolamine phosphotransferase 1 (CEPT1) in the CDP-choline pathway was greater in PHEP treated with Chol compared with control or Met. In the transsulfuration pathway, mRNA and protein abundance of cystathionine ß-synthase (CBS) was greater in PHEP treated with Met compared with control or Chol. Similarly, abundance of cysteine sulfinic acid decarboxylase (CSAD), glutamate-cysteine ligase, catalytic subunit (GCLC), and glutathione reductase (GSR) was greater in response to Met compared with control or Chol. Overall, these findings suggest that transmethylation and transsulfuration in dairy cow primary liver cells are more responsive to Met supply, whereas the CDP-choline pathway is more responsive to Chol supply. The relevance of these data in vivo merit further study.