Decreased vitamin B12 availability induces ER stress through impaired SIRT1-deacetylation of HSF1

Vitamin B12 (cobalamin) is a key determinant of S-adenosyl methionine (SAM)-dependent epigenomic cellular regulations related to methylation/acetylation and its deficiency produces neurodegenerative disorders by elusive mechanisms. Sirtuin 1 deacetylase (SIRT1) triggers cell response to nutritional stress through endoplasmic reticulum (ER) stress. Recently, we have established a N1E115 dopaminergic cell model by stable expression of a transcobalamin–oleosin chimera (TO), which impairs cellular availability of vitamin B12, decreases methionine synthase activity and SAM level, and reduces cell proliferation. In contrast, oleosin-transcobalamin chimera (OT) does not modify the phenotype of transfected cells. Presently, the impaired cellular availability of vitamin B12 in TO cells activated irreversible ER stress pathways, with increased P-eIF-2α, P-PERK, P-IRE1α, ATF6, ATF4, decreased chaperon proteins and increased pro-apoptotic markers, CHOP and cleaved caspase 3, through reduced SIRT1 expression and consequently greater acetylation of heat-shock factor protein 1 (HSF1). Adding either B12, SIRT1, or HSF1 activators as well as overexpressing SIRT1 or HSF1 dramatically reduced the activation of ER stress pathways in TO cells. Conversely, impairing SIRT1 and HSF1 by siRNA, expressing a dominant negative form of HSF1, or adding a SIRT1 inhibitor led to B12-dependent ER stress in OT cells. Addition of B12 abolished the activation of stress transducers and apoptosis, and increased the expression of protein chaperons in OT cells subjected to thapsigargin, a strong ER stress stimulator. AdoX, an inhibitor of methyltransferase activities, produced similar effects than decreased B12 availability on SIRT1 and ER stress by a mechanism related to increased expression of hypermethylated in cancer 1 (HIC1). Taken together, these data show that cellular vitamin B12 has a strong modulating influence on ER stress in N1E115 dopaminergic cells. The impaired cellular availability in vitamin B12 induces irreversible ER stress by greater acetylation of HSF1 through decreased SIRT1 expression, whereas adding vitamin B12 produces protective effects in cells subjected to ER stress stimulation.     

The role of folic acid and Vitamin B12 in genomic stability of human cells

Folic acid plays a critical role in the prevention of chromosome breakage and hypomethylation of DNA. This activity is compromised when Vitamin B12 (B12) concentration is low because methionine synthase activity is reduced, lowering the concentration of S-adenosyl methionine (SAM) which in turn may diminish DNA methylation and cause folate to become unavailable for the conversion of dUMP to dTMP. The most plausible explanation for the chromosome-breaking effect of low folate is excessive uracil misincorporation into DNA, a mutagenic lesion that leads to strand breaks in DNA during repair. Both in vitro and in vivo studies with human cells clearly show that folate deficiency causes expression of chromosomal fragile sites, chromosome breaks, excessive uracil in DNA, micronucleus formation and DNA hypomethylation. In vivo studies show that Vitamin B12 deficiency and elevated plasma homocysteine are significantly correlated with increased micronucleus formation. In vitro experiments indicate that genomic instability in human cells is minimised when folic acid concentration in culture medium is >227nmol/l. Intervention studies in humans show: (a) that DNA hypomethylation, chromosome breaks, uracil misincorporation and micronucleus formation are minimised when red cell folate concentration is >700nmol/l folate; and (b) micronucleus formation is minimised when plasma concentration of Vitamin B12 is >300pmol/l and plasma homocysteine is <7.5micromol/l. These concentrations are achievable at intake levels in excess of current RDIs i.e. more than 200-400microgram folic acid per day and more than 2microgram Vitamin B12 per day. A placebo-controlled study with a dose-response suggests that based on the micronucleus index in lymphocytes, an RDI level of 700microgram/day for folic acid and 7microgram/day for Vitamin B12 would be appropriate for genomic stability in young adults. Dietary intakes above the current RDI may be particularly important in those with extreme defects in the absorption and metabolism of these Vitamins, for which ageing is a contributing factor.     

Folate (vitamin B9) and vitamin B12 and their function in the maintenance of nuclear and mitochondrial genome integrity

Folate plays a critical role in the prevention of uracil incorporation into DNA and hypomethylation of DNA. This activity is compromised when vitamin B12 concentration is low because methionine synthase activity is reduced, lowering the concentration of S-adenosyl methionine (SAM) which in turn may diminish DNA methylation and cause folate to become unavailable for the conversion of dUMP to dTMP. The most plausible explanation for the chromosome-breaking effect of low folate is excessive uracil misincorporation into DNA, a mutagenic lesion that leads to strand breaks in DNA during repair. Both in vitro and in vivo studies with human cells clearly show that folate deficiency causes expression of chromosomal fragile sites, chromosome breaks, excessive uracil in DNA, micronucleus formation, DNA hypomethylation and mitochondrial DNA deletions. In vivo studies show that folate and/or vitamin B12 deficiency and elevated plasma homocysteine (a metabolic indicator of folate deficiency) are significantly correlated with increased micronucleus formation and reduced telomere length respectively. In vitro experiments indicate that genomic instability in human cells is minimised when folic acid concentration in culture medium is greater than 100nmol/L. Intervention studies in humans show (a) that DNA hypomethylation, chromosome breaks, uracil incorporation and micronucleus formation are minimised when red cell folate concentration is greater than 700nmol/L and (b) micronucleus formation is minimised when plasma concentration of vitamin B12 is greater than 300pmol/L and plasma homocysteine is less than 7.5μmol/L. These concentrations are achievable at intake levels at or above current recommended dietary intakes of folate (i.e. >400μg/day) and vitamin B12 (i.e. >2μg/day) depending on an individual's capacity to absorb and metabolise these vitamins which may vary due to genetic and epigenetic differences.     

The deficit in folate and vitamin B12 triggers liver macrovesicular steatosis and inflammation in rats with dextran sodium sulfate-induced colitis

The risks of nonalcoholic steatohepatitis (NASH) and deficiency in vitamin B12 and folate (methyl donor deficiency, MDD) are increased in inflammatory bowel disease (IBD). We investigated the influence of MDD on NASH in rats with DSS-induced colitis. Two-month-old male Wistar rats were subjected to MDD diet and/or ingestion of DSS and compared to control animals. We studied steatosis, inflammation, fibrosis, plasma levels of metabolic markers, cytokines and lipopolysaccharide, and inflammatory pathways in liver. MDD triggered a severe macrovesicular steatosis with inflammation in DSS animals that was not observed in animals subjected to DSS or MDD only. The macrovesicular steatosis was closely correlated to folate, vitamin B12, homocysteine plasma level and liver S-adenosyl methionine/S-adenosyl homocysteine (SAM/SAH) ratio. Liver inflammation was evidenced by activation of nuclear factor kappa B (NFκB) pathway and nuclear translocation of NFκB phospho-p65. MDD worsened the increase of interleukin 1-beta (IL-1β) and abolished the increase of IL10 produced by DSS colitis. It increased monocyte chemoattractant protein 1 (MCP-1). MDD triggers liver macrovesicular steatosis and inflammation through imbalanced expression of IL-1β vs. IL10 and increase of MCP-1 in DSS colitis. Our results suggest evaluating whether IBD patients with MDD and increase of MCP-1 are at higher risk of NASH.     

The relationships between vitamin B12 and folic acid and the effect of methionine on folate metabolism

The relationship between vitamin B12 and folate and the effect of methionine on folate metabolism during B12 deficiency in rats is best explained by the prevention of the accumulation of 5-methyl-H4PteGlu by vitamin B12 and/or methionine. Although several points remain to be clarified, the 'methyl trap' hypothesis provides the most satisfactory explanation for the relation between vitamin B12, methionine and folic acid. This concept is extended by the hypothesis that H4PteGlu is the most active substrate for pteroylpolyglutamate synthetase, and thus accounts for the effect of methionine or vitamin B12 increasing liver folate levels.     

Folate deprivation increases presenilin expression, gamma-secretase activity, and Abeta levels in murine brain: potentiation by ApoE deficiency and alleviation by dietary S-adenosyl methionine

Folate deficiency has been associated with age-related neurodegeneration. One direct consequence of folate deficiency is a decline in the major methyl donor, S-adenosyl methionine (SAM). We demonstrate herein that dietary deficiency in folate and vitamin E, coupled pro-oxidant stress induced by dietary iron, increased presenilin-1 expression, gamma-secretase activity, and Abeta levels in normal adult mice. These increases were potentiated by apolipoprotein E deficiency as shown by treatment of transgenic mice homozygously lacking murine apolipoprotein E. Dietary supplementation with SAM in the absence of folate attenuated or alleviated these deleterious consequences. These findings link nutritional and genetic risk factors for age-related neurodegeneration and underscore that dietary supplementation with SAM may be useful to augment therapeutic approaches.     

Polyamine concentrations in the brain of vitamin B12-deficient rats

To study the pathophysiology of the neuronal degeneration in vitamin B12 deficiency, we investigated the concentrations of the polyamines putrescine, spermidine, and spermine in brain regions and liver using high-performance liquid chromatography with fluorescence detection. Male Wistar rats were fed either a control or vitamin B12-deficient diet for 20 weeks. No remarkable behavioral changes were observed. Serum vitamin B12 and hepatic methionine concentrations were significantly lower and hepatic homocysteine was elevated in rats fed vitamin B12-deficient diet than in controls. Vitamin B12 deficiency was associated with decreased concentrations of spermidine, spermidine in liver and some regions of brain, although there were no observed abnormalities in behavior. These results suggest that vitamin B12 deficiency may play a role in neuronal degeneration through the disturbance of polyamine concentrations in rat brain.     

Effects of methionine synthase and methylenetetrahydrofolate reductase gene polymorphisms on markers of one-carbon metabolism

Genetic and nutritional factors play a role in determining the functionality of the one-carbon (1C) metabolism cycle, a network of biochemical reactions critical to intracellular processes. Genes encoding enzymes for methylenetetrahydrofolate reductase (MTHFR) and methionine synthase (MTR) may determine biomarkers of the cycle including homocysteine (HCY), S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH). MTHFR C677T is an established genetic determinant of HCY but less is known of its effect on SAM and SAH. Conversely, the relationship between MTR A2756G and HCY remains inconclusive, and its effect on SAM and SAH has only been previously investigated in a female-specific population. Folate and vitamin B₁₂ are essential substrate and cofactor of 1C metabolism; thus, consideration of gene–nutrient interactions may clarify the role of genetic determinants of HCY, SAM and SAH. This cross-sectional study included 570 healthy volunteers from Kingston, Ontario, Ottawa, Ontario and Halifax, Nova Scotia, Canada. Least squares regression was used to examine the effects of MTR and MTHFR polymorphisms on plasma HCY, SAM and SAH concentrations; gene–gene and gene–nutrient interactions were considered with the inclusion of cross-products in the model. Main effects of MTR and MTHFR polymorphisms on HCY concentrations were observed; however, no gene–gene or gene–nutrient interactions were found. No association was observed for SAM. For SAH, interactions between MTR and MTHFR polymorphisms, and MTHFR polymorphism and serum folate were found. The findings of this research provide evidence that HCY and SAH, biomarkers of 1C metabolism, are influenced by genetic and nutritional factors and their interactions.     

Effect of folate deficiency on placental DNA methylation in hyperhomocysteinemic rats

We report that the maternal folate status can influence folate-mediated one-carbon metabolism and DNA methylation in the placenta. Thirty-six female Sprague-Dawley rats were divided into the following three dietary groups: folate-supplemented (FS; 8 mg/kg folic acid, n=12), homocystine- and folate-supplemented (HFS; 0.3% homocystine and 8 mg/kg folic acid, n=12) and homocystine-supplemented and folate-deficient (HFD; 0.3% homocystine and no folic acid, n=12). The animals were fed their experimental diets from 4 weeks prior to mating until Day 20 of pregnancy (n=7-9 per group). The HFS diet increased the plasma homocysteine and placental DNA methylation but did not affect plasma folate, vitamin B-12, S-adenosyl methionine (SAM) or S-adenosyl homocysteine (SAH) levels, or the SAM/SAH ratio in the liver and placenta compared with the FS diet. The HFD diet induced severely low plasma folate concentrations, with plasma homocysteine levels increasing up to 100 micromol/L, and increased hepatic SAH and decreased placental SAM levels and SAM/SAH ratio in both tissues, with a concomitant decrease in placental DNA methylation. Placental DNA methylation was significantly correlated with placental (gamma=0.819), hepatic (gamma=0.7) and plasma (gamma=0.752) folate levels; plasma homocysteine level (gamma=-0.688); hepatic SAH level (gamma=-0.662) and hepatic SAM/SAH ratio (gamma=0.494). These results suggest that the maternal folate status in hyperhomocysteinemic rats influences the homeostasis of folate-mediated one-carbon metabolism and the methyl pool, which would, in turn, affect placental DNA methylation by altering the methylation potential of the liver.     

The Status of Vitamin B12 and Folate among Chinese Women: A Population-Based Cross-Sectional Study in Northwest China

Objective

To assess the status of the vitamin B12 and folate of Chinese women living in northwest China.

Methods

A population-based cross-sectional study was conducted in 2008 among Chinese women aged 10–49 years living in Shaanxi province of northwest China. A stratified multistage random sampling method was adopted to obtain a sample of 1170 women. The women were interviewed for collection of their background information and their plasma vitamin B12 and folate were measured with the immunoassay method. The status of both vitamins was evaluated and the prevalence of deficiency was estimated.

Results

The median value of the women was 214.5 pg/mL for vitamin B12 and 4.6 ng/mL for folate. The urban women had a significantly higher vitamin B12 (254.1 vs. 195.9 pg/mL) but lower folate (4.4 vs. 4.7 ng/mL) than rural women. Total prevalence of deficiency was 45.5% (95% CI: 42.6%∼48.4%) for vitamin B12 and 14.7% (95% CI: 12.6%∼16.8%) for folate. About 36% of women presented vitamin B12 deficiency alone, 5.2% belonged to folate deficiency alone and 9.5% was combined deficiency in both vitamins. More than 25% of the women were in marginal vitamin B12 status (200–299 pg/mL) and 60% in marginal status of folate (3–6 ng/mL). About 75.2% of rural women with folate deficiency were deficient in vitamin B12 and 46% for urban women. Quantile regression model found decreasing coefficient of folate status across 73 different quantiles of vitamin B12, which indicated that the women with folate deficiency had lower vitamin B12 significantly compared with those with no deficiency.

Conclusions

The deficiency of vitamin B12 and folate is still prevalent among the Chinese women in northwest China. Vitamin B12 deficiency could be more serious and the improvement of poor vitamin B12 status should be invoked when practicing the supplementation of folate against the neural tube defects in northwest China.     

Folic acid fortification: why not vitamin B12 also?

Folic acid fortification of cereal grains was introduced in many countries to prevent neural tube defect occurrence. The metabolism of folic acid and vitamin B12 intersect during the transfer of the methyl group from 5-methyltetrahydrofolate to homocysteine catalyzed by B12-dependent methioine synthase. Regeneration of tetrahydrofolate via this reaction makes it available for synthesis of nucleotide precursors. Thus either folate or vitamin B12 deficiency can result in impaired cell division and anemia. Exposure to extra folic acid through fortification may be detrimental to those with vitamin B12 deficiency. Among participants of National Health And Nutrition Examination Survey with low vitamin B12 status, high serum folate (>59 nmol/L) was associated with higher prevalence of anemia and cognitive impairment when compared with normal serum folate. We also observed an increase in the plasma concentrations of total homocysteine and methylmalonic acid (MMA), two functional indicators of vitamin B12 status, with increase in plasma folate under low vitamin B12 status. These data strongly imply that high plasma folate is associated with the exacerbation of both the biochemical and clinical status of vitamin B12 deficiency. Hence any food fortification policy that includes folic acid should also include vitamin B12.     

DNA methylase and demethylase activities are modulated by one-carbon metabolism in Alzheimer's disease models

Late-onset Alzheimer's disease seems to be a multi-factorial disease with both genetic and non-genetic, environmental, possible causes. Recently, epigenomics is achieving a major role in Alzheimer's research due to its involvement in different molecular pathways leading to neurodegeneration. Among the different epigenetic modifications, DNA methylation is one of the most relevant to the disease. We previously demonstrated that presenilin1 (PSEN1), a gene involved in amyloidogenesis, is modulated by DNA methylation in neuroblastoma cells and Alzheimer's mice in an experimental model of nutritionally altered one-carbon metabolism. This alteration, obtained by nutritional deficiency of B vitamins (folate, B12 and B6) hampered S-adenosylmethionine (SAM)-dependent methylation reactions. The aim of the present paper was to investigate the regulation of DNA methylation machinery in response to hypomethylating (B vitamin deficiency) and hypermethylating (SAM supplementation) alterations of the one-carbon metabolism. We found that DNA methylases (DNMT1, 3a and 3b) and a putative demethylase (MBD2) were differently modulated, in line with the previously observed changes of PSEN1 methylation pattern in the same experimental conditions.     

Plasma Homocysteine,Vitamin B12 and Folate Levels in Multiple System Atrophy: A Case-Control Study

Background

Multiple system atrophy (MSA) is a neurodegenerative disease, and its pathological hallmark is the accumulation of α-synuclein proteins. Homocysteine (Hcy) is an intermediate amino acid generated during the metabolism of methionine. Hcy may contribute to the pathogenesis of neurodegenerative disorders. Vitamin B12 and folate are cofactors necessary for the methylation of homocysteine.

Methods

This study compared the levels of serum Hcy, vitamin B12 and folate in patients with MSA with those in healthy people to reveal the possible association between MSA and plasma levels of Hcy, vitamin B12 and folate. We enrolled 161 patients with MSA and 161 healthy people in this study. The association between MSA and the levels of Hcy, vitamin B12 and folate were analyzed using binary logistic regression.

Results

The mean level of Hcy in patients with MSA was significantly higher than that in healthy controls (16.23 ± 8.09 umol/l vs 14.04 ± 4.25 umol/l, p < 0.05). After adjusting for age, sex and medical history, the odds ratio for Hcy was 1.07 (95% CI = 1.01–1.13, p < 0.05) for patients with MSA. Vitamin B12 and folate levels were not significantly different between patients with MSA and controls.

Conclusion

Our data suggest that higher levels of Hcy may be associated with an increased risk for MSA.     

Neural tube defects and maternal biomarkers of folate, homocysteine, and glutathione metabolism

BACKGROUND: Alterations in maternal folate and homocysteine metabolism are associated with neural tube defects (NTDs). The role played by specific micronutrients and metabolites in the causal pathway leading to NTDs is not fully understood. METHODS: We conducted a case-control study to investigate the association between NTDs and maternal alterations in plasma micronutrients and metabolites in two metabolic pathways: methionine remethylation and glutathione transsulfuration. Biomarkers were measured in a population-based sample of women who had NTD-affected pregnancies (n = 43) and a control group of women who had a pregnancy unaffected by a birth defect (n = 160). We compared plasma concentrations of folate, vitamin B(12), vitamin B(6), methionine, S-adenosylmethionine (SAM), s-adenosylhomocysteine (SAH), adenosine, homocysteine, cysteine, and reduced and oxidized glutathione between cases and controls after adjusting for lifestyle and sociodemographic factors. RESULTS: Women with NTD-affected pregnancies had significantly higher plasma concentrations of SAH (29.12 vs. 23.13 nmol/liter, P = .0011), adenosine (0.323 vs. 0.255 mumol/liter; P = .0269), homocysteine (9.40 vs. 7.56 micromol/liter; P < .001), and oxidized glutathione (0.379 vs. 0.262 micromol/liter; P = .0001), but lower plasma SAM concentrations (78.99 vs. 83.16 nmol/liter; P = .0172) than controls. This metabolic profile is consistent with reduced methylation capacity and increased oxidative stress in women with affected pregnancies. CONCLUSIONS: Increased maternal oxidative stress and decreased methylation capacity may contribute to the occurrence of NTDs. Further analysis of relevant genetic and environmental factors is required to define the basis for these observed alterations.     

Homocysteine Level and Mechanisms of Injury in Parkinson's Disease as Related to MTHFR,MTR, and MTHFD1 Genes Polymorphisms and L-Dopa Treatment

An elevated concentration of total homocysteine (tHcy) in plasma and cerebrospinal fluid is considered to be a risk factor for Alzheimer''s disease (AD) and Parkinson''s disease (PD). Homocysteine (Hcy) levels are influenced by folate concentrations and numerous genetic factors through the folate cycle, however, their role in the pathogenesis of PD remains controversial. Hcy exerts a neurotoxic action and may participate in the mechanisms of neurodegeneration, such as excitotoxicity, oxidative stress, calcium accumulation, and apoptosis. Elevated Hcy levels can lead to prooxidative activity, most probably through direct interaction with N-methyl-D-aspartate (NMDA) receptors and sensitization of dopaminergic neurons to age-related dysfunction and death. Several studies have shown that higher concentration of Hcy in PD is related to long-term administration of levodopa (L-dopa). An elevation of plasma tHcy levels can also reflect deficiencies of cofactors in remethylation of Hcy to methionine (Met) (folates and vitamin B12) and in its transsulfuration to cysteine (Cys) (vitamin B6). It is believed that the increase in the concentration of Hcy in PD can affect genetic polymorphisms of the folate metabolic pathway genes, such as MTHFR (C677T, A1298C and G1793A), MTR (A2756G), and MTHFD1 (G1958A), whose frequencies tend to increase in PD patients, as well as the reduced concentration of B vitamins. In PD, increased levels of Hcy may lead to dementia, depression and progression of the disease.     

Effects of nitrous oxide inactivation of vitamin B12 and of methionine on folate coenzyme metabolism in rat liver, kidney, brain, small intestine and bone marrow

The effects of nitrous oxide inactivation of the vitamin B12-dependent enzyme, methionine synthetase (EC 2.1.1.13), and of methionine on folate coenzyme metabolism were determined in rat liver, kidney, brain, small intestine and bone marrow cells. Nitrous oxide exposure led to an increase in the proportion of 5-methyltetrahydrofolate at the expense of other reduced folates in all tissues examined. Administration of methionine at levels up to 400 mg/kg resulted in the normalization of folate coenzyme patterns in liver as a result of the increased levels of S-adenosylmethionine. In other tissues examined, methionine had no effect on the levels of S-adenosylmethionine or S-adenosylhomocysteine, or on the distribution of folate coenzymes. These results are consistent with the methyl trap hypothesis as the explanation of the relationship between vitamin B12 and folate metabolism, and provide direct evidence that the sparing effect of methionine on folate metabolism is a phenomenon restricted to the liver.     

In vivo hydrolysis of S-adenosyl-L-methionine in Escherichia coli increases export of 5-methylthioribose

Escherichia coli can not synthesize methionine from 5-methylthioribose (MTR) but instead exports this sulfur-containing, energy-rich molecule into the surrounding medium. Transforming E. coli with plasmids that direct expression of the cloned coliphage T3 S-adenosyl-L-methionine (SAM) hydrolase (SAMase) induces the met regulon by cleaving the SAM co-repressor to form 5'-methylthioadenosine, which is then cleaved to produce MTR. To test the effect of in vivo SAMase activity on MTR production and its fate, cultures were incubated in the presence of [35S]methionine and [methyl-3H]methionine. Cells with SAMase activity produced significantly enhanced levels (up to 40-fold in some trials) of extracellular MTR -- the only radiolabeled compound released in significant amounts -- when compared with controls. SAM synthetase (metK) mutants transformed with SAMase expression vectors did not show this increase, verifying the path through SAM as the sole route to MTR production. SAMase expression had little or no effect on intracellular MTR pools, levels of radiolabeled macromolecules, or the transfer of methyl groups to compounds that could be precipitated by trichloroacetic acid. Thus, MTR appears to be a dead-end metabolite in E. coli, begging questions about how this has evolved, the mechanism of MTR export for the cell, and whether the release of MTR is important for some other activity.