Betaine-homocysteine Methyltransferase Research Articles

Nutrient-Gene Interaction in Arsenic Metabolism

Arsenic (As) naturally occurs in water and its long-term exposure is linked with many health problems, including skin lesions and cancer. Nutrient intake and genetic polymorphisms involved in one-carbon metabolism, including the choline pathway, may explain inter-individual variation in As metabolism and elimination, possibly influencing susceptibility to As-related disease. To examine As metabolism and elimination efficiency in relation to intake of choline, betaine, and methionine, and choline dehydrogenase (CHDH) rs7626693 and betaine homocysteine methyltransferase (BHMT) rs3733890 genetic variants. Women (n=415) from As-exposed regions of Northern Mexico, were interviewed and provided biological samples. Nutrient intake was estimated using a validated semi-quantitative food frequency questionnaire. Urinary As species were determined with HPLC-ICP-MS and the relative proportions and ratios of As metabolism were calculated. Genetic polymorphisms were genotyped via allelic discrimination. Multivariate linear regression models were developed to evaluate the associations between nutrients, polymorphisms and urinary As parameters In gene-nutrient adjusted models, methionine was the only nutrient significantly associated with As metabolism. After adjusting for BHMT, methionine was associated with lower percentage of inorganic As (%iAs) and higher percentage of dimethylarsinic acid (%DMA) and total methylation index (DMA/iAs). Among women with the GG+AG genotypes of BHMT, methionine was additionally associated with lower percentage of monomethylarsonic acid (%MMA) and higher secondary methylation index (DMA/MMA). A statistically significant interaction between methionine and BHMT was detected. Conclusion. Nutrients may play a role in As metabolism, particularly within certain genetic variant subgroups, via one-carbon and choline pathways. Differences in nutrient intake and genetic polymorphisms may jointly influence As metabolism and elimination efficiency.

Betaine homocysteine methyltransferase (BHMT) as a specific and sensitive blood marker for acute liver injury

We developed a high-performance ELISA assay and measured serum BHMT levels in healthy individuals and patients with acute liver injury (ALI). The detection range of this ELISA assay was from 1.56 to 100 ng/ml. BHMT levels are significantly higher in ALI groups. In the healthy group (n = 244), the median value (interquartile range, IQR 0–56.40) was 1.83 ng/ml. In the ALI group (n = 42), the median value of BHMT was 748.48 ng/ml (IQR, 0–51095.92). ROC curve analysis demonstrated good sensitivity (0.86) and specificity (0.98). In addition, in five ALI cases with time course samples available, BHMT and ALT both followed the “rise and fall” temporal pattern with the disease progression. However, the slopes of BHMT curves were steeper than ALT curves. And in three out of the five cases, BHMT levels peaked 1 day earlier than ALT levels be a sensitive marker with good prognostic value.

Characterization of differentially expressed genes in liver in response to the rearing temperature of rainbow trout Oncorhynchus mykiss and their heritable differences.

To characterize thermal-responsive genes in fish, firstly, juvenile rainbow trout were reared in four different temperature conditions (average temperatures were 10, 14, 18, and 22°C, respectively) and differentially expressed genes were identified. Gene expression in the liver was analyzed by the differential display method, followed by validation using real-time PCR. Subsequently, to examine whether the identified genes show heritable differences, the gene expression levels were compared among juveniles of three genetically distinct lines of rainbow trout (a strain and two closed colonies) by rearing at two different temperature conditions (average 14 and 22°C). By rearing at 22°C, growth retardation was observed compared with fish reared at 14 and 18°C, and six genes were identified as differentially expressed genes in response to the rearing temperature in the gene expression analyses. With the increase in rearing temperature, gene expressions of a complement C1q and two ribosomal proteins were significantly up-regulated. On the other hand, three metabolic genes (betaine homocysteine methyltransferase, triosephosphate isomerase, and glucose-6-phosphatase) were down-regulated, indicating a metabolic depression due to high temperature. In the subsequent analyses, in response to the rearing temperature (14 and 22°C), there was a trend that the complement C1q and glucose-6-phosphatase genes showed different expression patterns among the three rainbow trout lines, suggesting heritable differences in these genes. Our study provides information on thermal-responsive genes in fish, and we anticipate it will facilitate further investigation in the thermal biology of fish.

Dietary methionine supplementation alters the expression of genes involved in methionine metabolism in salmonids

The objective of the present study was to investigate the effect of dietary methionine (Met) concentration and alternating feeding strategies in methionine delivery, on the mRNA transcript levels of genes involved in Met resynthesis (betaine-homocysteine methyltransferase, BHMT; S-adenosylhomocysteine hydrolase, SAHH) and net Met loss (taurine synthesis) (cystathionine beta-synthase, CBS) in Atlantic salmon (Salmo salar) liver. Salmon alevins (265 ± 3 mg) were distributed into 24 tanks (50 fish per tank; 3 replicates). The experimental diets were supplemented with l-methionine at 0, 1.9, 5.8, and 17.4 g/kg (M0, M1/3, M1, and M3, respectively). The M3 diet without glycine was prepared to examine Met toxicity (M3-G). These diets were provided via “mono-feeding strategy,” meaning fish were fed a designated single diet. This experiment also included alternative feeding groups with “duo-feeding” strategy: AF1 (fish fed M0 for 2 days followed by M1 for 1 day), AF2 (fish fed M0 for 2 days followed by M3 for 1 day), and AF3 (fish fed two meals of M0 followed by one meal of M3). Salmon fed M0 diet had smaller weight compared to all other groups. There was no effect of alternative feeding on the growth except with the M0 group. The highest expression of CBS gene was found in the M0, M1/3, and AF-1 groups compared to the M3 and M3-G groups. The expression of CBS gene in the M1 group was lower compared to the AF-1 group. The expression of SAHH gene was the highest in the M3 group compared to the M1, M1/3, and AF-3 groups. The highest expression of BHMT gene was found in the M0, M1/3, and AF-1 groups, indicating enhanced re-methylation of homocysteine by betaine to Met. The lowest BHMT expression in M1, M3, M3-G, AF-2, and AF-3 groups compared to other treatments can be indicative of downregulation of remethylation in the liver. The alternate provision of methionine in a form of combination of low and high methionine containing diets seemed to support its efficient utilization in the salmon liver.

The Heme Oxygenase System Selectively Modulates Proteins Implicated in Metabolism, Oxidative Stress and Inflammation in Spontaneously Hypertensive Rats

Although recent studies have underscored the role of the heme-oxygenase (HO) inducer hemin, on insulin-signaling and glucose metabolism, the underlying mechanisms are not completely understood. In this study, two-dimensional-gel electrophoresis, massspectrometry and MSACOT-analyses were used to identify and characterize novel proteins modulated by hemin in spontaneoushypertensive rat (SHR), a model of essential hypertension with insulin resistance/impaired glucose metabolism. In addition, the effects of hemin on endothelin-1 (ET-1), protein-tyrosine-phosphatase-1B (PTP-1B), atrial-natriuretic-peptide (ANP) and its surrogate-marker urinary cGMP, and inflammatory cytokines including TNF-α, IL-6 and IL-1β were investigated. In hemin-treated SHR, several proteins related to oxidative-stress and metabolism were modulated. Particularly, hemin enhanced aldolase- B, fumarylacetoacetate hydrolase, purine-nucleoside phosphorylase, adenosine-kinase, argininosuccinate synthetase and carbonic anhydrase-3 all of which are enzymes involved in glucose/energy metabolism and pH homeostasis. Similarly, hemin potentiated antioxidant pathways including, NADP(+)-dependant isocitrate-dehydrogenase, catalase, glutathione-S-transferase-Yb1 and hsp70, a pleiotropic agent that regulates protein-folding, oxidative/pro-inflammatory events. Hemin also increased enzymes implicated in cell-growth such as the nitrilase-protein-family, but reduced betaine-homocysteine methyltransferase, an enzyme associated with insulin resistance and dysfunctional glucose metabolism. Furthermore, hemin increased ANP and its surrogate marker, urinary cGMP, but reduced ET-1, PTP-1B, TNF-α, IL-6, IL-1β, whereas the HO-inhibitor, chromium-mesoporphyrin abolished the effects. The potentiation of ANP, urinary-cGMP, aldolade-B, fumarylacetoacetate hydrolase, purine-nucleoside phosphorylase, adenosine-kinase, argininosuccinate synthetase, carbonic anhydrase-3, hsp70 and the corresponding reduction of betaine-homocysteine methyltransferase, PTP-1B, TNF-α, IL-6, IL-1β, and ET-1 may be responsible for the improved glucose metabolism in hemin-treated animals. Collectively, these findings underscore the pleiotropic effects of the HO-system in cellular homeostasis with important roles in metabolism and defence.

Abstract P452: Pathway Analysis Of One-carbon Metabolism In Association With Acute Myocardial Infarction In Patients With Stable Angina Pectoris

Background: One-carbon metabolism (OCM) is involved in RNA, DNA synthesis and in epigenetic regulation. Pathway gene analysis is more likely to capture potential gene-disease associations comparing to single gene analysis. We investigated whether genetic variants in folate network as well as their interactions would predict subsequent acute myocardial infarction (AMI) in patients undergoing elective coronary angiography for stable angina pectoris (SAP). Methods: A total of 2381 subjects (98% Caucasian) recruited to the Western Norway B Vitamin Intervention Trial (WENBIT) in 2000-2004 were studied. Patients were followed until a subsequent AMI or end of 2006. We genotyped 18 commonly investigated single nucleotide polymorphisms (SNPs) in 13 genes in the OCM network. Predictable variants were selected based on both out-of-bag importance scores from random survival forests and their incremental effects from recursive nested analysis. We then assessed gene-gene interactions among those predictive variants with generalized logistic model and calculated hazard ratio (HR), confidence interval (CI) for each predictor in Cox proportional hazard models. Results: There are 204 (8.57%) patients who experienced an AMI throughout 4.9 years (median) follow-up. We observed no violation from Hardy-Weinberg equilibrium in 15 SNPs in our population. Four SNPs (rs1076991, rs1021737, rs1801133 and rs3733890) in folate network were identified as promising candidates for predicting AMI risk. Interestingly, only rs1076991 associated significantly with AMI (HR=1.30, 95% CI: 1.10-1.52), indicating the other SNPs primarily mediate their risk bygene-gene interactions. There was a strong and stable interaction between the dominant model of rs3733890 and dominant model of rs34489327 (P for interaction: 0.003; HR =2.23, 95% CI: 1.27-3.92, adjusted for age, gender, smoking, diabetes mellitus, hypertension, extent of CAD by angiography (0-3), apolipoprotein (Apo) A1 and B, left ventricular ejection fraction and estimated glomerular filtration rate). Further adjustment for vitamins involved in OCM (folate, B2, B6, B12), the substrates (betaine and homocysteine) and products (methionine and dimethylglycine) of the betaine--homocysteine S-methyltransferase (BHMT) had minimal influence on the estimate. Conclusions: Prior studies show that BHMT transcription is associated with co-transcription of Apo B and that the rs3733890 of this gene is associated with higher dimethylglycine levels. We have recently shown that elevated plasma dimethylglycine is associated with increased risk of AMI and improves risk prediction in patients with SAP independent of Apo B levels. The current investigation extends previous knowledge, suggesting that a genetic variant in BHMT may modulate the risk of AMI by interfering with thymidylate synthesis independent of plasma dimethylglycine.

Responses of the European flounder (Platichthys flesus) to a mixture of PAHs and PCBs in experimental conditions

A multibiomarker approach was developed to evaluate the juvenile European flounder responses to a complex mixture of 9 polycyclic aromatic hydrocarbons (PAHs) and 12 polychlorinated biphenyls (PCBs). Exposure was performed through contaminated food pellets displaying: (1) PAH and PCB levels similar to those detected in the heavily polluted Seine estuary, respectively in sediments and in flatfish and (2) ten times these concentrations. Several biomarkers of the immune system (e.g., lysozyme concentration and gene expression of complement component C3 and TNF-receptor), DNA damage (e.g., Comet assay), energetic metabolism (e.g., activity of cytochrome C oxidase), detoxification process (e.g., cytochrome P450 1A1 expression level: CYP1A1; betaine homocysteine methyl transferase expression level: BHMT) were investigated after 14 and 29 days of contamination, followed by a 14-days recovery period. After 29 days of contamination, the detoxification activity (CYP1A1 expression level) was positively correlated with DNA damages; the increase of the BHMT expression level could also be related to the detoxification process. Furthermore, after the recovery period, some biomarkers were still upregulated (i.e., CYP1A1 and BHMT expression levels). The immune system was significantly modulated by the chemical stress at the two concentration levels, and the lysozyme appeared to be the most sensitive marker of the mixture impact.

Alterations in the metabolomics of sulfur-containing substances in rat kidney by betaine

Earlier studies have shown that betaine administration may modulate the metabolism of sulfur amino acids in the liver. In this study, we determined the changes in the metabolomics of sulfur-containing substances induced by betaine in the kidney, the other major organ actively involved in the transsulfuration reactions. Male rats received betaine (1%) in drinking water for 2 weeks before killing. Betaine intake did not affect betaine-homocysteine methyltransferase activity or its protein expression in the renal tissue. Expression of methionine synthase was also unchanged. However, methionine levels were increased significantly both in plasma and kidney. Renal methionine adenosyltransferase activity and S-adenosylmethionine concentrations were increased, but there were no changes in S-adenosylhomocysteine, homocysteine, cysteine levels or cystathionine β-synthase expression. γ-Glutamylcysteine synthetase expression or glutathione levels were not altered, but cysteine dioxygenase and taurine levels were decreased significantly. In contrast, betaine administration induced cysteine sulfinate decarboxylase and its metabolic product, hypotaurine. These results indicate that the metabolomics of sulfur-containing substances in the kidney is altered extensively by betaine, although the renal capacity for methionine synthesis is unresponsive to this substance unlike that of the liver. It is suggested that the increased methionine availability due to an enhancement of its uptake from plasma may account for the alterations in the metabolomics of sulfur-containing substances in the kidney. Further studies need to be conducted to clarify the physiological/pharmacological significance of these findings.

Suppression effects of betaine-enriched spinach on hyperhomocysteinemia induced by guanidinoacetic acid and choline deficiency in rats.

Betaine is an important natural component of rich food sources, especially spinach. Rats were fed diets with betaine or spinach powder at the same level of betaine for 10 days to investigate the dose-dependent effects of spinach powder supplementation on hyperhomocysteinemia induced by guanidinoacetic acid (GAA) addition and choline deprivation. The GAA-induced hyperhomocysteinemia in rats fed 25% casein diet (25C) was significantly suppressed by supplementation with betaine or spinach, and it was completely suppressed by taking 11.0% spinach supplementation. The choline deprivation-induced enhancement of plasma homocysteine concentration in rats fed 25% soybean protein diet (25S) was markedly suppressed by 3.82% spinach. Supplementation with betaine or spinach partially prevented the effects of GAA on hepatic concentrations of methionine metabolites. The decrease in activity of betaine-homocysteine S-methyltransferase (BHMT) and cystathionine β-synthase (CBS) in GAA-induced hyperhomocysteinemia was recovered by supplementation with betaine or spinach. Supplementation with betaine or spinach did not affect BHMT activity, whereas it partially restored CBS activity in choline-deprived 25S. The results indicated that betaine or spinach could completely suppress the hyperhomocysteinemia induced by choline deficiency resulting from stimulating the homocysteine removal by both remethylation and cystathionine formation.

Homocysteine Homeostasis and Betaine-Homocysteine S-Methyltransferase Expression in the Brain of Hibernating Bats

Elevated homocysteine is an important risk factor that increases cerebrovascular and neurodegenerative disease morbidity. In mammals, B vitamin supplementation can reduce homocysteine levels. Whether, and how, hibernating mammals, that essentially stop ingesting B vitamins, maintain homocysteine metabolism and avoid cerebrovascular impacts and neurodegeneration remain unclear. Here, we compare homocysteine levels in the brains of torpid bats, active bats and rats to identify the molecules involved in homocysteine homeostasis. We found that homocysteine does not elevate in torpid brains, despite declining vitamin B levels. At low levels of vitamin B6 and B12, we found no change in total expression level of the two main enzymes involved in homocysteine metabolism (methionine synthase and cystathionine β-synthase), but a 1.85-fold increase in the expression of the coenzyme-independent betaine-homocysteine S-methyltransferase (BHMT). BHMT expression was observed in the amygdala of basal ganglia and the cerebral cortex where BHMT levels were clearly elevated during torpor. This is the first report of BHMT protein expression in the brain and suggests that BHMT modulates homocysteine in the brains of hibernating bats. BHMT may have a neuroprotective role in the brains of hibernating mammals and further research on this system could expand our biomedical understanding of certain cerebrovascular and neurodegenerative disease processes.

Relationships between genotypes and phenotypes in natural populations of the European flounder (Platichthys flesus) under different types of contamination in estuaries

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Investigations of single nucleotide polymorphisms in folate pathway genes in Chinese families with neural tube defects

AimsWe investigated the hypothesis that there are interactions between SNPs in folate metabolism pathway genes and environmental risk factors to the etiology of neural tube defects (NTDs). MethodIn 602 Chinese families, 609 aborted fetus tissues or blood samples were collected from NTD individuals, and 1106 parental blood samples were detected as controls. We analyzed 28 SNPs in 12 folate pathway genes. Folate supplementation, gestational diabetes mellitus (GDM) and medicine administration before and during pregnancy were investigated. Case–parental control study and transmission/disequilibrium tests were performed according to environmental cofactor stratification. ResultsAssociation between 5,10-methylenetetrahydrofolate reductase (MTHFR) 677C>T and NTDs was significant in all stratifications (all P<.05), and synergistic effects of no folate supplementation and GDM were shown on NTD occurrence. 5-Methyltetrahydrofolate–homocysteine methyltransferase (MTHM) 501A>G in case of GDM, and betaine–homocysteine methyltransferase (BHMT) 716G>A in case of no folate supplementation significantly associated with NTDs (both P<.05), whereas the two genotypes alone did not significantly associate with NTDs (both P>.05). ConclusionsMTHFR 677C>T genotype, especially in case of no folate supplementation and GDM, promotes NTD occurrence. MTHM 501A>G only in case of GDM, and BHMT 716G>A only in case of no folate supplementation contribute to the etiology of NTDs.

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

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

Splicing variants of the porcine betaine–homocysteine S-methyltransferase gene: Implications for mammalian metabolism

Betaine–homocysteine S-methyltransferase (BHMT) activity is only detected in the liver of rodents, but in both the liver and kidney cortex of humans and pigs; therefore, the pig was chosen as a model to define the spatial and temporal expression of BHMT during development. During fetal development, a total of ten splice variants of bhmt were expressed at varying levels across a wide range of porcine tissues. Two variants contained an identical ORF that encoded a C-terminal truncated form of BHMT (tBHMT). The bhmt transcripts were expressed at significant levels in the liver and kidney from day 45 of gestation (G45) onward. The transcripts encoding tBHMT represented 5–13% of the total bhmt transcripts in G30 fetus, G45 liver, and adult liver and kidney cortex. The dominant structural feature of wild type BHMT is an (βα)8 barrel, however, a modeled structure of tBHMT suggests that this protein would assume a horseshoe fold and lack methyltransferase activity. Low BHMT activity was detected in the G30 fetus, and slightly increased levels of activity were observed in the liver from G45 and G90 fetuses. The bhmt promoter contained three key CpG sites, and methylation of these sites was significantly higher in adult lung compared to adult liver. The data reported herein suggest that genomic DNA methylation and variation of the 5′ and 3′ UTRs of bhmt transcripts are key regulators for the level of BHMT transcription and translation.

Dynamic Regulation of Hepatic Lipid Droplet Properties by Diet

Cytoplasmic lipid droplets (CLD) are organelle-like structures that function in neutral lipid storage, transport and metabolism through the actions of specific surface-associated proteins. Although diet and metabolism influence hepatic CLD levels, how they affect CLD protein composition is largely unknown. We used non-biased, shotgun, proteomics in combination with metabolic analysis, quantitative immunoblotting, electron microscopy and confocal imaging to define the effects of low- and high-fat diets on CLD properties in fasted-refed mice. We found that the hepatic CLD proteome is distinct from that of CLD from other mammalian tissues, containing enzymes from multiple metabolic pathways. The hepatic CLD proteome is also differentially affected by dietary fat content and hepatic metabolic status. High fat feeding markedly increased the CLD surface density of perilipin-2, a critical regulator of hepatic neutral lipid storage, whereas it reduced CLD levels of betaine-homocysteine S-methyltransferase, an enzyme regulator of homocysteine levels linked to fatty liver disease and hepatocellular carcinoma. Collectively our data demonstrate that the hepatic CLD proteome is enriched in metabolic enzymes, and that it is qualitatively and quantitatively regulated by diet and metabolism. These findings implicate CLD in the regulation of hepatic metabolic processes, and suggest that their properties undergo reorganization in response to hepatic metabolic demands.

Proteomic Differences Between Hepatocellular Carcinoma and Nontumorous Liver Tissue Investigated by a Combined Gel-based and Label-free Quantitative Proteomics Study

Proteomics-based clinical studies have been shown to be promising strategies for the discovery of novel biomarkers of a particular disease. Here, we present a study of hepatocellular carcinoma (HCC) that combines complementary two-dimensional difference in gel electrophoresis (2D-DIGE) and liquid chromatography (LC-MS)-based approaches of quantitative proteomics. In our proteomic experiments, we analyzed a set of 14 samples (7 × HCC versus 7 × nontumorous liver tissue) with both techniques. Thereby we identified 573 proteins that were differentially expressed between the experimental groups. Among these, only 51 differentially expressed proteins were identified irrespective of the applied approach. Using Western blotting and immunohistochemical analysis the regulation patterns of six selected proteins from the study overlap (inorganic pyrophosphatase 1 (PPA1), tumor necrosis factor type 1 receptor-associated protein 1 (TRAP1), betaine-homocysteine S-methyltransferase 1 (BHMT)) were successfully verified within the same sample set. In addition, the up-regulations of selected proteins from the complements of both approaches (major vault protein (MVP), gelsolin (GSN), chloride intracellular channel protein 1 (CLIC1)) were also reproducible. Within a second independent verification set (n = 33) the altered protein expression levels of major vault protein and betaine-homocysteine S-methyltransferase were further confirmed by Western blots quantitatively analyzed via densitometry. For the other candidates slight but nonsignificant trends were detectable in this independent cohort. Based on these results we assume that major vault protein and betaine-homocysteine S-methyltransferase have the potential to act as diagnostic HCC biomarker candidates that are worth to be followed in further validation studies.

Nicotinamide supplementation induces detrimental metabolic and epigenetic changes in developing rats

Ecological evidence suggests that niacin (nicotinamide and nicotinic acid) fortification may be involved in the increased prevalence of obesity and type 2 diabetes, both of which are associated with insulin resistance and epigenetic changes. The purpose of the present study was to investigate nicotinamide-induced metabolic changes and their relationship with possible epigenetic changes. Male rats (5 weeks old) were fed with a basal diet (control group) or diets supplemented with 1 or 4 g/kg of nicotinamide for 8 weeks. Low-dose nicotinamide exposure increased weight gain, but high-dose one did not. The nicotinamide-treated rats had higher hepatic and renal levels of 8-hydroxy-2'-deoxyguanosine, a marker of DNA damage, and impaired glucose tolerance and insulin sensitivity when compared with the control rats. Nicotinamide supplementation increased the plasma levels of nicotinamide, N1-methylnicotinamide and choline and decreased the levels of betaine, which is associated with a decrease in global hepatic DNA methylation and uracil content in DNA. Nicotinamide had gene-specific effects on the methylation of CpG sites within the promoters and the expression of hepatic genes tested that are responsible for methyl transfer reactions (nicotinamide N-methyltransferase and DNA methyltransferase 1), for homocysteine metabolism (betaine-homocysteine S-methyltransferase, methionine synthase and cystathionine β-synthase) and for oxidative defence (catalase and tumour protein p53). It is concluded that nicotinamide-induced oxidative tissue injury, insulin resistance and disturbed methyl metabolism can lead to epigenetic changes. The present study suggests that long-term high nicotinamide intake (e.g. induced by niacin fortification) may be a risk factor for methylation- and insulin resistance-related metabolic abnormalities.

Effects of hyperhomocysteinemia and betaine–homocysteine S-methyltransferase inhibition on hepatocyte metabolites and the proteome

Both cardiovascular disease and liver injury are major public health issues. Hyperhomocysteinemia has been linked to cardiovascular diseases, and defects in methyl group metabolism, often resulting in hyperhomocysteinemia, are among the key molecular events postulated to play a role in liver injury. We employed proteomics and metabolomics analyses of human hepatocytes in primary cell culture to explore the spectrum of proteins and associated metabolites affected by the disruption of methyl group metabolism. We treated the hepatocytes with homocysteine (Hcy, 0.1mM and 2mM) to follow the impact of hyperhomocysteinemia, and in parallel, we used a specific inhibitor of betaine–homocysteine S-methyltransferase (BHMT) to extend our understanding of the physiological functions of the enzyme. The major effect of BHMT inhibition was a 50% decrease in S-adenosylmethionine levels. The treatments with Hcy resulted in multiple changes in the metabolite levels depending on the treatment modality. The BHMT inhibition and 0.1mM Hcy treatment induced only moderate changes in the hepatocyte proteome and secretome, while the changes induced by the 2mM Hcy treatment were extensive. Phosphatidylethanolamine carboxykinase and ornithine aminotransferase were up-regulated about two fold indicating an intervention into metabolism. Cellular proliferation was suspended, secretome composition was changed and signs of apoptosis were discernible. We have detected fibrinogen gamma dimers, which might have a role as a potentially new biomarker of early liver injury. Finally, we have demonstrated the failed maturation of apolipoprotein A1, which might be a new mechanism of disruption of cholesterol efflux from tissues.

Exercise prevents hyperhomocysteinemia in a dietary folate-restricted mouse model

Hyperhomocysteinemia is a condition that results from altered methyl group metabolism and is associated with numerous pathological conditions. A number of nutritional and hormonal factors have been shown to influence circulating homocysteine concentrations; however, the impact of exercise on homocysteine and methyl group balance is not well understood. Our hypothesis was that exercise represents an effective means to prevent hyperhomocysteinemia in a folate-independent manner. The purpose of this study was to determine the influence of exercise on homocysteine metabolism in a dietary folate-restricted mouse model characterized by moderate hyperhomocysteinemia. Female outbred mice (12 weeks old) were assigned to either a sedentary or free-access wheel exercise group. Following a 4-week acclimation period, half of the mice in each group were provided a folate-restricted diet for 7-weeks prior to euthanasia and tissue collection. As expected, folate-restricted sedentary mice exhibited a 2-fold increase in plasma total homocysteine concentrations; however, exercise completely prevented the increase in circulating homocysteine concentrations. Moreover, exercise reduced plasma homocysteine concentrations 36% within the group fed only the control diet. The prevention of hyperhomocysteinemia by exercise appears, at least in part, to be the result of increased folate-independent homocysteine remethylation owing to a 2-fold increase in renal betaine homocysteine S-methyltransferase. To our knowledge, this is the first report demonstrating the prevention of hyperhomocysteinemia by exercise in a dietary folate-restriction model. Future research will be directed at determining if exercise can have a positive impact on other nutritional, hormonal, and genetic models of hyperhomocysteinemia relevant to humans.

Alterations in hepatic metabolism of sulfur amino acids in non-obese type-2 diabetic Goto-Kakizaki rats

Elevated plasma homocysteine has been identified as a risk factor for cardiovascular disease and non-alcoholic liver disease, which are major complications of diabetes. Hence, hepatic homocysteine metabolism has become a major focus of diabetes research. However, little information is available regarding plasma homocysteine levels in non-obese diabetic animals. Therefore, we investigated the hepatic metabolism of sulfur-amino acids in non-obese type-2 diabetic Goto-Kakizaki rats. The experiments were performed using 9-week-old Goto-Kakizaki rats and age-matched Wistar rats. The major finding of this study is that homocysteine levels in the liver and plasma are maintained by a balance between the up-regulation of betaine homocysteine methyltransferase and the inhibition of cystathionine β-synthase in non-obese type-2 diabetic rats. Hepatic levels of cysteine and its metabolites, such as hypotaurine, taurine, and glutathione, were increased despite inhibition of the transsulfuration of homocysteine to cysteine. The elevated hepatic taurine and glutathione levels may be attributed to the up-regulation of cysteine dioxygenase expression and increased cysteine availability for glutathione synthesis. Inhibition of hepatic methionine adenosyltransferase activity in Goto-Kakizaki rats was associated with a decrease in hepatic S-adenosylmethionine, which serves as an allosteric activator of cystathionine β-synthase. The non-obese type-2 diabetic condition results in profound changes in hepatic sulfur-amino acid metabolism and raises the possibility that sulfur-amino acid metabolism may be regulated by obesity- as well as diabetes-associated factors. Further study to elucidate the pathological significance of sulfur-amino acid metabolism in chronic liver disease in type-2 diabetic animals is underway in this laboratory.