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Vitamin B12 insufficiency induces cholesterol biosynthesis by limiting s-adenosylmethionine and modulating the methylation of SREBF1 and LDLR genes.

Adaikalakoteswari A, Finer S, Voyias PD, McCarthy CM, Vatish M, Moore J, Smart-Halajko M, Bawazeer N, Al-Daghri NM, McTernan PG, Kumar S, Hitman GA, Saravanan P, Tripathi G - Clin Epigenetics (2015)

Bottom Line: The induction of cholesterol biosynthesis was associated with reduced s-adenosylmethionine (AdoMet)-to-s-adenosylhomocysteine (AdoHcy) ratio, also known as methylation potential (MP).We therefore studied whether reduced MP could lead to hypomethylation of genes involved in the regulation of cholesterol biosynthesis.This finding was further confirmed by the addition of the methylation inhibitor, 5-aza-2'-deoxycytidine, which resulted in increased SREBF1 and LDLR expressions and cholesterol accumulation in vitamin B12-sufficient conditions.

View Article: PubMed Central - PubMed

Affiliation: Division of Metabolic and Vascular Health, Clinical Sciences Research Laboratories, Warwick Medical School, University Hospital Coventry and Warwickshire, University of Warwick, Clifford Bridge Road, Coventry, CV2 2DX UK.

ABSTRACT

Background: The dietary supply of methyl donors such as folate, vitamin B12, betaine, methionine, and choline is essential for normal growth, development, and physiological functions through the life course. Both human and animal studies have shown that vitamin B12 deficiency is associated with altered lipid profile and play an important role in the prediction of metabolic risk, however, as of yet, no direct mechanism has been investigated to confirm this.

Results: Three independent clinical studies of women (i) non-pregnant at child-bearing age, (ii) in early pregnancy, and (iii) at delivery showed that low vitamin B12 status was associated with higher total cholesterol, LDL cholesterol, and cholesterol-to-HDL ratio. These results guided the investigation into the cellular mechanisms of induced cholesterol biosynthesis due to vitamin B12 deficiency, using human adipocytes as a model system. Adipocytes cultured in low or no vitamin B12 conditions had increased cholesterol and homocysteine levels compared to control. The induction of cholesterol biosynthesis was associated with reduced s-adenosylmethionine (AdoMet)-to-s-adenosylhomocysteine (AdoHcy) ratio, also known as methylation potential (MP). We therefore studied whether reduced MP could lead to hypomethylation of genes involved in the regulation of cholesterol biosynthesis. Genome-wide and targeted DNA methylation analysis identified that the promoter regions of SREBF1 and LDLR, two key regulators of cholesterol biosynthesis, were hypomethylated under vitamin B12-deficient conditions, and as a result, their expressions and cholesterol biosynthesis were also significantly increased. This finding was further confirmed by the addition of the methylation inhibitor, 5-aza-2'-deoxycytidine, which resulted in increased SREBF1 and LDLR expressions and cholesterol accumulation in vitamin B12-sufficient conditions. Finally, we observed that the expression of SREBF1, LDLR, and cholesterol biosynthesis genes were increased in adipose tissue of vitamin B12 deficient mothers compared to control group.

Conclusions: Clinical data suggests that vitamin B12 deficiency is an important metabolic risk factor. Regulation of AdoMet-to-AdoHcy levels by vitamin B12 could be an important mechanism by which it can influence cholesterol biosynthesis pathway in human adipocytes.

No MeSH data available.


Related in: MedlinePlus

Effect of vitamin B12on methylation of cholesterol regulatory genes. Genome-wide DNA methylation analysis of cholesterol regulatory genes was performed in adipocytes cultured in customised media, control, LB, and NoB. (A) SREBF-1 and (B) LDLR from Illumina. Validation by BS-pyrosequencing was also done in the same samples and methylation status confirmed for (C) SREBF-1 and (D) LDLR. All experiments were performed as triplicates. Values are mean ± SEM. *P ≤ 0.05; **P ≤ 0.01, ***P ≤ 0.001, P value compared to control.
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Fig4: Effect of vitamin B12on methylation of cholesterol regulatory genes. Genome-wide DNA methylation analysis of cholesterol regulatory genes was performed in adipocytes cultured in customised media, control, LB, and NoB. (A) SREBF-1 and (B) LDLR from Illumina. Validation by BS-pyrosequencing was also done in the same samples and methylation status confirmed for (C) SREBF-1 and (D) LDLR. All experiments were performed as triplicates. Values are mean ± SEM. *P ≤ 0.05; **P ≤ 0.01, ***P ≤ 0.001, P value compared to control.

Mentions: Of the genes identified from the genome-wide dataset, probes at two targets; SREBF1 and LDLR showed differential methylation (reaching genome-wide significance) across experimental groups. The SREBF1 probe (cg27407935; chr17:17723235) showed a quantitative decrease in DNA methylation across from the control to LB to NoB, with mean β values for sample triplicates as follows: control, 0.47; LB, 0.41; NoB, 0.40 (F = 85.8, P value < 0.0001) (Figure 4A). This probe lies within the gene body of SREBF1 at a DNAse I hypersensitivity site and RNA Pol2 binding site (suggested by ENCODE data), suggesting a putative role in transcriptional regulation. (Additional file 1: Figure S4A). The LDLR probe (cg22971501; chr19:11199476) also showed relative hypomethylation across LBF and NoB with corresponding mean β values of 0.65, 0.61, and 0.60 (F = 14.41, P value < 0.0001) (Figure 4B). The LDLR probe lays 1,500 bp upstream of the 5′UTR at the gene promoter (Additional file 1: Figure S4B). For both SREBF1 and LDLR genes, the hypomethylation sites are close to the transcription start site (TSS) and are located near binding sites for PPARγ and C/EBPα (Additional file 1: Figure S4 A and B). These binding sites have been obtained from ChIP-seq data published elsewhere [28,29]. There was no significant difference in the mean beta values at SREBF2.Figure 4


Vitamin B12 insufficiency induces cholesterol biosynthesis by limiting s-adenosylmethionine and modulating the methylation of SREBF1 and LDLR genes.

Adaikalakoteswari A, Finer S, Voyias PD, McCarthy CM, Vatish M, Moore J, Smart-Halajko M, Bawazeer N, Al-Daghri NM, McTernan PG, Kumar S, Hitman GA, Saravanan P, Tripathi G - Clin Epigenetics (2015)

Effect of vitamin B12on methylation of cholesterol regulatory genes. Genome-wide DNA methylation analysis of cholesterol regulatory genes was performed in adipocytes cultured in customised media, control, LB, and NoB. (A) SREBF-1 and (B) LDLR from Illumina. Validation by BS-pyrosequencing was also done in the same samples and methylation status confirmed for (C) SREBF-1 and (D) LDLR. All experiments were performed as triplicates. Values are mean ± SEM. *P ≤ 0.05; **P ≤ 0.01, ***P ≤ 0.001, P value compared to control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4356060&req=5

Fig4: Effect of vitamin B12on methylation of cholesterol regulatory genes. Genome-wide DNA methylation analysis of cholesterol regulatory genes was performed in adipocytes cultured in customised media, control, LB, and NoB. (A) SREBF-1 and (B) LDLR from Illumina. Validation by BS-pyrosequencing was also done in the same samples and methylation status confirmed for (C) SREBF-1 and (D) LDLR. All experiments were performed as triplicates. Values are mean ± SEM. *P ≤ 0.05; **P ≤ 0.01, ***P ≤ 0.001, P value compared to control.
Mentions: Of the genes identified from the genome-wide dataset, probes at two targets; SREBF1 and LDLR showed differential methylation (reaching genome-wide significance) across experimental groups. The SREBF1 probe (cg27407935; chr17:17723235) showed a quantitative decrease in DNA methylation across from the control to LB to NoB, with mean β values for sample triplicates as follows: control, 0.47; LB, 0.41; NoB, 0.40 (F = 85.8, P value < 0.0001) (Figure 4A). This probe lies within the gene body of SREBF1 at a DNAse I hypersensitivity site and RNA Pol2 binding site (suggested by ENCODE data), suggesting a putative role in transcriptional regulation. (Additional file 1: Figure S4A). The LDLR probe (cg22971501; chr19:11199476) also showed relative hypomethylation across LBF and NoB with corresponding mean β values of 0.65, 0.61, and 0.60 (F = 14.41, P value < 0.0001) (Figure 4B). The LDLR probe lays 1,500 bp upstream of the 5′UTR at the gene promoter (Additional file 1: Figure S4B). For both SREBF1 and LDLR genes, the hypomethylation sites are close to the transcription start site (TSS) and are located near binding sites for PPARγ and C/EBPα (Additional file 1: Figure S4 A and B). These binding sites have been obtained from ChIP-seq data published elsewhere [28,29]. There was no significant difference in the mean beta values at SREBF2.Figure 4

Bottom Line: The induction of cholesterol biosynthesis was associated with reduced s-adenosylmethionine (AdoMet)-to-s-adenosylhomocysteine (AdoHcy) ratio, also known as methylation potential (MP).We therefore studied whether reduced MP could lead to hypomethylation of genes involved in the regulation of cholesterol biosynthesis.This finding was further confirmed by the addition of the methylation inhibitor, 5-aza-2'-deoxycytidine, which resulted in increased SREBF1 and LDLR expressions and cholesterol accumulation in vitamin B12-sufficient conditions.

View Article: PubMed Central - PubMed

Affiliation: Division of Metabolic and Vascular Health, Clinical Sciences Research Laboratories, Warwick Medical School, University Hospital Coventry and Warwickshire, University of Warwick, Clifford Bridge Road, Coventry, CV2 2DX UK.

ABSTRACT

Background: The dietary supply of methyl donors such as folate, vitamin B12, betaine, methionine, and choline is essential for normal growth, development, and physiological functions through the life course. Both human and animal studies have shown that vitamin B12 deficiency is associated with altered lipid profile and play an important role in the prediction of metabolic risk, however, as of yet, no direct mechanism has been investigated to confirm this.

Results: Three independent clinical studies of women (i) non-pregnant at child-bearing age, (ii) in early pregnancy, and (iii) at delivery showed that low vitamin B12 status was associated with higher total cholesterol, LDL cholesterol, and cholesterol-to-HDL ratio. These results guided the investigation into the cellular mechanisms of induced cholesterol biosynthesis due to vitamin B12 deficiency, using human adipocytes as a model system. Adipocytes cultured in low or no vitamin B12 conditions had increased cholesterol and homocysteine levels compared to control. The induction of cholesterol biosynthesis was associated with reduced s-adenosylmethionine (AdoMet)-to-s-adenosylhomocysteine (AdoHcy) ratio, also known as methylation potential (MP). We therefore studied whether reduced MP could lead to hypomethylation of genes involved in the regulation of cholesterol biosynthesis. Genome-wide and targeted DNA methylation analysis identified that the promoter regions of SREBF1 and LDLR, two key regulators of cholesterol biosynthesis, were hypomethylated under vitamin B12-deficient conditions, and as a result, their expressions and cholesterol biosynthesis were also significantly increased. This finding was further confirmed by the addition of the methylation inhibitor, 5-aza-2'-deoxycytidine, which resulted in increased SREBF1 and LDLR expressions and cholesterol accumulation in vitamin B12-sufficient conditions. Finally, we observed that the expression of SREBF1, LDLR, and cholesterol biosynthesis genes were increased in adipose tissue of vitamin B12 deficient mothers compared to control group.

Conclusions: Clinical data suggests that vitamin B12 deficiency is an important metabolic risk factor. Regulation of AdoMet-to-AdoHcy levels by vitamin B12 could be an important mechanism by which it can influence cholesterol biosynthesis pathway in human adipocytes.

No MeSH data available.


Related in: MedlinePlus