# Molecular and cellular effects of vitamin B12 in brain, myocardium and liver through its role as co-factor of methionine synthase

Jean-Louis Guéant, Maatem Caillerez-Fofou, Shyuefang Battaglia-Hsu,

Jean-Marc Alberto, Jean-Noël Freund, Isabelle Dulluc, Charles Adjalla,

Florence Maury, Carole Merle, Jean-Pierre Nicolas, et al.

► To cite this version:

Jean-Louis Guéant, Maatem Caillerez-Fofou, Shyuefang Battaglia-Hsu, Jean-Marc Alberto, Jean-Noël Freund, et al. Molecular and cellular effects of vitamin B12 in brain, myocardium and liver through its role as co-factor of methionine synthase. Biochimie, 2013, 95 (5), pp.1033-1040. 10.1016/j.biochi.2013.01.020. hal-01704564

HAL Id: hal-01704564

https://hal.univ-lorraine.fr/hal-01704564v1

Submitted on 4 Mar 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
Molecular and cellular effects of vitamin B12 in brain, myocardium and
liver through its role as co-factor of methionine synthase

Jean-Louis Guéant<sup>1,2,*†</sup>, Maatem Caillerez-Fofou<sup>1,*</sup>, Shyuefang Battaglia-Hsu<sup>1</sup>, Jean-Marc
Alberto<sup>1</sup>, Jean-Noel Freund<sup>3</sup>, Isabelle Dulluc<sup>3</sup>, Charles Adjalla<sup>1</sup>, Florence Maury<sup>1</sup>, Carole
Merle<sup>1</sup>, Jean-Pierre Nicolas<sup>1</sup>, Fares Namour<sup>1</sup>, Jean-Luc Daval<sup>1</sup>

<sup>1</sup>Inserm U954, department of Nutrition-Genetics-Environmental Risk Exposure, University of Lorraine and University Hospital of Nancy, Vandoeuvre-lès-Nancy, France; <sup>2</sup>IRCCS, Oasi Maria SS-Institute for Research on Mental Retardation, Troina (EN), Italy (GA, PB, ME, AR, J-LG); <sup>3</sup>Inserm Unit 682, University Louis Pasteur, 3 Avenue Molière, 67200 Strasbourg, France. *equal contribution

Correspondence: †Jean-Louis Guéant, MD, DSc, Inserm U-954, Faculté de Médecine, 9 avenue de la Forêt de Haye, B.P. 184, 54500, Nancy-Vandœuvre, France. Phone: (33) 3 83 68 39 92. Fax: (33) 3 83 68 32 79. E-mail: jean-louis.gueant@medecine.uhp-nancy.fr

Abbreviations: B12: vitamin B12, cobalamin; DNA deoxyribonucleic acid; ERα, estrogen receptor alpha; ERRα, estrogen-related receptor alpha; HNF: hepatic nuclear factor; MTR: methionine synthase; MTRR: methionine synthase reductase; MTHFR: methylenetetrahydrofolate reductase; NAFLD: non-alcoholic fatty liver disease; NGF: nerve growth factor; PGC-1α: peroxisome proliferator-activated receptor gamma coactivator 1-alpha; PP2A: protein phosphatase; PPAR, peroxisome proliferator-activated receptor; PRMT, protein arginine methyltransferase; P53: tumor protein 53; RNA: ribonucleic acid; SAM: S-adenosylmethionine; SAH: S-adenosylhomocysteine; SIRT1: sirtuin (silent mating type information regulation 2 homolog) 1; TACE: tumor necrosis factor (TNF)-converting enzyme; TC: transcobalamin; THF: tetrahydrofolate.
**Abstract**

Vitamin B12 (cobalamin, cbl) is a cofactor of methionine synthase (MTR) in the synthesis of methionine, the precursor of the universal methyl donor S-Adenosylmethionine (SAM), which is involved in epigenomic regulatory mechanisms. We have established a neuronal cell model with stable expression of a transcobalamin-oleosin chimer and subsequent decreased cellular availability of vitamin B12, which produces reduced proliferation, increased apoptosis and accelerated differentiation through PP2A, NGF and TACE pathways. Anti-transcobalamin antibody or impaired transcobalamin receptor expression produce also impaired proliferation in other cells. Consistently, the transcription, protein expression and activity of MTR are increased in proliferating cells of skin and intestinal epitheliums, in rat intestine crypts and in proliferating CaCo2 cells, while MTR activity correlates with DNA methylation in rat intestine villi. Exposure to nitrous oxide in animal models identified impairment of MTR reaction as the most important metabolic cause of neurological manifestations of B12 deficiency. Early vitamin B12 and folate deprivation during gestation and lactation of a 'dam-progeny' rat model developed in our laboratory is associated with long-lasting disabilities of behaviour and memory capacities, with persisting hallmarks related to increased apoptosis, impaired neurogenesis and altered plasticity. We found also an epigenomic deregulation of energy metabolism and fatty acids beta-oxidation in myocardium and liver, through imbalanced methylation/acetylation of PGC-1alpha and decreased expression of SIRT1. These nutrigenomic effects display similarities with the molecular mechanisms of foetal programming. Beside deficiency, B12 loading increases the expression of MTR through internal ribosome entry sites (IRES) and down-regulates *MDR-1* gene expression. In conclusion, vitamin B12 influences cell proliferation, differentiation and
apoptosis in brain. Vitamin B12 and folate combined deficiency impairs fatty acid oxidation
and energy metabolism in liver and heart through epigenomic mechanisms related to
imbalanced acetylation/methylation. Some but not all of these effects reflect the upstream role
of vitamin B12 in SAM synthesis.

**Key words:** Vitamin B12, cobalamin, methionine synthase, epigenomics, foetal programming
This review is focused on the metabolic, molecular and cellular consequences of vitamin B12
deficiency in relation with its metabolic function as co-factor of methionine synthase. These
consequences undertake the cellular effects of vitamin B12 and methionine synthase on
proliferation and differentiation, and the consequences of vitamin B12 deficiency on brain
and on metabolic disorders in liver and heart, through epigenomic deregulation of PGC1-
alpha pathway.

# 1. Metabolic role of vitamin B12.

The metabolic role of vitamin B12 (cobalamin, Cbl) is highly targeted to only two pathways, in eukaryotic cells. MethylCbl (MeCbl) is the cofactor of methionine synthase in the cytoplasm and AdoCbl the cofactor of methylmalonyl coenzyme A mutase in the mitochondrion. Each of these two reactions plays a key role in cellular metabolism [1,2].

Methylmalonyl coenzyme A mutase is the latest step in the oxidative degradation of a number
of amino acids. It ensures the conversion of methylmalonic acid to succinyl Co-enzyme A,
which subsequently enters in the Krebs cycle. MeCbl plays a limiting role in the gearing
between folate pathways and the methionine cycle, through its role as a cofactor of
methionine synthase (MTR). The folate metabolism ensures the synthesis of the methyl group
of Methyl-tetrahydrofolate (MeTHF), which is used as a co-substrate in the reaction catalyzed
by MTR for the remethylation of homocysteine to methionine. In this reaction, MeCbl is the
transient acceptor of the methyl group at the interface between folate and methionine cycles,
where its role as a metabolic gearing is illustrated by the MeTHF trap hypothesis. In this trap,
vitamin B12 deficiency not only impairs the remethylation of homocysteine but also blocks
the conversion of MeTHF into THF, leading to accumulation of MeTHF. THF is a reduced
folate intermediate involved in one-carbon exchanges needed for DNA synthesis, and this
may explain that vitamin B12 deficiency impairs DNA synthesis by blocking adequate supply
of reduced folate. Vitamin B12 deficiency leads also to the accumulation of homocysteine, which may be responsible for a cellular stress, by mechanisms that have been largely documented in the literature [3].

Paradoxically, the decreased synthesis of methionine has deserved less attention than homocysteine accumulation, in the experimental and clinical studies dealing with metabolic and molecular consequences of vitamin B12 deficiency. The endogenous synthesis of methionine is a major event in the cell life, since this essential amino acid is the immediate precursor of S-adenosylmethionine (SAM), which is the universal donor in the transmethylation of many substrates [4]. Cellular methionine has a dual source, the remethylation pathway and the degradation of endogenous and food proteins. The importance of the remethylation pathway for maintaining an adequate cellular content of SAM is illustrated by experimental studies, which show that vitamin B12 deficiency influences directly the cellular concentration of SAM and S-adenosylhomocysteine (SAH) and produces a decrease of SAM/SAH ratio [5,6]. The main consequence of this decreased ratio is an impaired capacity of the cell to ensure transmethylation reactions. Among these reactions, the methylation of DNA, RNA, histones and co-regulators of nuclear receptors plays a key role in epigenetic and epigenomic mechanisms [4,7]. Consistently, B12 deficiency and decreased MTR activity are associated with global hypomethylation of DNA, in experimental models of vitamin B12 deficiency such as the gastrectomized rat [8]. Recent data of the literature show that vitamin B12 deficiency may produce many dramatic effects, including those on cell proliferation and differentiation, through epigenomic mechanisms related to the cellular homeostasis of methionine and SAM [9-11].

## 2. Consequences of the decreased bioavailability of vitamin B12 in experimental cell models
It is very difficult to obtain a cell deficiency using usual cell culture conditions, as vitamin B12 is present in the foetal calf serum in sufficient amount. To solve this problem, several experimental approaches have been proposed. Transcobalamin (TC) is the protein that displays the highest affinity for B12; it is constitutively secreted into plasma to bind the circulating vitamin B12, internalized into cells via receptor-mediated endocytosis, and subsequently addressed in cytosol and mitochondria [1]. Quadros et al. used two experimental strategies, by impairing TC-dependent cellular entry of vitamin B12 with either anti-TC monoclonal antibody or down regulation of the expression of cd320, the TC receptor, by siRNA [12,13]. These strategies have proven to be effective in blocking vitamin B12 uptake in cancer cells and inhibiting subsequently their proliferation. However, the use of siRNA or anti-TC has some limitations for studying the molecular consequences of impaired cellular availability of vitamin B12 over a long period of culture. To address this issue, we have developed a cell model made deficient in B12 through the stable expression of a chimeric protein associating TC with oleosin, a plant protein that is targeted to oil bodies via the endoplasmic reticulum [14]. The expression of oleosin is restricted to endoplasmic reticulum when the heterologous oleosin is expressed in mammalian cells [14]. We linked TC to the hydrophobic oleosin to sequester B12 in the cytosolic face of intracellular membranes. When C-terminal end of TC is merged with the N-terminal side of oleosin, the TO chimeric protein retains a high affinity for vitamin B12. In contrast, when N-terminal end of TC is merged with the C-terminal end of oleosin (OT construct), the OT chimeric protein expressed in the cell has no binding activity for B12. This OT construct provides therefore a valuable control condition for studying the consequences of impaired cellular availability of B12 in TO cells [6,14]. The stable expression of TO and OT in reticulum was obtained in Caco2 (colon carcinoma), NIE-115 (murine neuroblastoma cell line), HEK (human embryonic kidney), COS-7 (Green Monkey SV40-transfected kidney fibroblasts) and CHO (Chinese hamster
ovary cells). The expression of GFP-TO was observed in all cell lines and localized in endoplasmic reticulum [14]. The decreased cellular availability of B12 was evidenced by impaired conversion of cyano-Cbl to AdoCbl and MeCbl, decreased MTR activity, reduced SAM/SAH ratio, as well as increase in homocysteine and methylmalonic acid concentrations.

The common consequence of the TO transfection in these cells was a dramatic reduced rate of proliferation [14], which was reversed by adding B12 in the culture medium. Further investigations showed that vitamin B12 influences apoptosis and neuronal differentiation in NIE-115 cells, with an increased length of neurites. Vitamin B12 shortage reduced neuroblastoma cell proliferation and enhanced their differentiation through PP2A (protein phosphatase 2A) and proNGF pathways [6]. In proliferating cells, neurotrophin p75NTR intramembrane proteolysis was increased and associated with elevated Adam 10 and Adam 17, which belongs to the TACE secretase protein family (TNFα converting enzyme) [6]. The increase in TACE agreed with previous results obtained by Scalabrino et al. [15] in the brain of rats, which were rendered B12 deficient by gastrectomy. This TC-Oleosin model was next reproduced in vivo by targeted transfection of the constructs in rat dopaminergic neurons of substantia nigra. Vitamin B12 deficiency induced dopaminergic cell death via caspase-3 dependent apoptosis. Furthermore, in a metamphetamine administration test, B12 deficiency determined in rats an ipsilateral rotation reminiscent of Parkinson phenotype [16]. This suggests that vitamin B12 deficiency might aggravate Parkinson Syndrome.

**3. Methionine synthase is a marker of cell proliferation.**

MTR gene has 33 exons and encodes a protein with a predicted molecular weight of 140.4 kDa. The 3D structure has been resolved for bacterial MTR, but none is yet available for human MTR, except for a short C-terminal fragment [17]. MTR has 4 functional domains that bind Hcy, CH3-H4folate, Cbl, and AdoMet, respectively [18]. MTR is a modular protein with distinct regions for binding homocysteine, methyltetrahydrofolate, cobalamin, and
adénosylméthionine [17]. Activity of the enzyme requires the presence of a second protein, methionine synthase reductase (MTRR), which maintains the B12 prosthetic group in its active reduced state. Cbl plays a crucial role during the catalytic cycle, acting both as methyl donor and methyl acceptor. MTR generates MeCbl during the catalytic cycle of methionine synthesis. The demethylation of MeCbl leads to formation of cob(I)alamin, which is very sensitive to oxidation. Under the microaerophilic conditions of mammalian cells, the cofactor is oxidized about once in every 2,000 turnovers [19], resulting in the accumulation of inactive enzyme. MTRR catalyzes the regeneration of methylcobalamin in a reduced form, during the cycle. MTRR needs NADPH as a coenzyme and could serve also as a chaperone for MTR [20,21]. The association of single nucleotide polymorphisms of *MTRR 66A→G* with the gravity index of Crohn disease depends on SOD activity, reflecting probably that the role of MTRR becomes more crucial in conditions where oxidative stress is accentuated [22]. The *MTR* KO mice are lethal, with embryo development blocked at the stage of 20 somites. Moreover, some studies have found an association between the single nucleotide polymorphisms of *MTR 2756A→G* and *MTRR 66A→G* and birth defects [23,24], supporting a key role of the vitamin B12-dependent endogenous synthesis of methionine in human foetal development.

In the brain, MTR is expressed in neurones as well as glial cells, at least in hippocampus, cerebellum and subventrivular area [5]. The deficiency in vitamin B12 and folate produces a dramatic reduction of MTR expression, in contrast to cystathionine-beta-synthase [5]. MTR has a marked expression in the proliferation site of various epithelia such as crypts of intestinal villi and the basal layer of the Malpighi epithelium of the skin (Fig. 2) and conversely, its activity is reduced in the epithelium zones where proliferation is reduced, such as the apical side of intestinal villi and the layer of differentiated keratinocytes, in the skin (Fig. 2). Consistently, we observed an increase in MTR activity in the crypts with a very
sharp gradient along the crypto-villous axis, in rat intestinal explants (Fig. 3). The MTR activity was about 8 times higher in the crypts, where the proliferation rate is high, than in the apical part of the villi, where apoptosis produced exfoliation of enterocytes. This gradient mirrored that of DNA methylation (Fig. 3), illustrating the key role of MTR- and B12-dependent remethylation pathway on DNA methylation, in animal models with B12 deficiency [5]. These observations agree also with the dramatic influence of B12 cellular availability on cell proliferation, in cell models. To confirm this, we studied the transcription and protein level of MTR according to growth delay, in Caco2 human colon adenocarcinoma cells. This cell line display similarities with foetal enterocytes and has been largely used for studying the cellular metabolism of vitamin B12 in the intestine [25,26]. Semi-quantitative RT-PCR and northern blots showed a maximum gene transcription during the proliferative phase and a sharp decline between the 7th and 9th days (Fig. 4), when the cells enter into contact and stop to proliferate [25,26]. These results of MTR transcription were corroborated by a dramatic decline of MTR protein expression and activity, after the 10th day of growth delay (Fig. 5). In fact, the western blot of MTR detected two bands with respective Mr of 150 and 124 kDa, which may correspond to two splicing products, one of them being a truncated MTR without activity (Fig. 5). We hypothesize that this MTR splicing mechanism could regulate the expression of active MTR to adapt the need of the cell for SAM, in the proliferation and differentiation states, respectively.

**4. Manifestations of vitamin B12 deficiency in the human population.**

The vitamin B12 deficiency is a worldwide public health problem mainly observed during pregnancy or ageing, which is often associated with folate deficiency during these periods of life [27, 28]. It produces targeted and specific manifestations on the nervous system, digestive system and erythropoiesis, by molecular mechanisms that need to be better understood [27, 28]. Vitamin B12 deficiency, together with folate deficiency and increased homocysteine, is
also associated with complex neurologic and cardiovascular complex pathologies, such as Alzheimer and Parkinson diseases, thrombosis, stroke and myocardial infarction [29, 30]. Neurological effects of vitamin B12 are not identical to those of folate deficiency despite their complementary role in the one carbon metabolism [15]. In particular, vitamin B12 produces a peripheral and central myelopathy that is not observed in folate deficiency, with demyelination of the posterolateral columns of the spinal cord [15]. The lesion was thought to arise primarily from impairment of the AdoCbl-dependent methylmalonyl CoA mutase reaction, leading to the formation of abnormal odd-chain and branched-chain fatty acids and their incorporation into myelin with resultant demyelination.

# 5. Vitamin B12 deficiency in animal models, Manifestations and related mechanisms.

## 5.1. The animal models of vitamin B12 deficiency and impaired pathway of methionine synthase.
Studying the consequences of B12 deficiency at the tissue level needs the use of animal models. Neuropathy was a common manifestation in animal models of vitamin B12 deficiency, including monkeys, pigs and bats of the species Rousettus aegyptiacus fed a vitamin B12-deficient diet [31, 32]. Data from the Cb1 neuropathy induced by exposure to nitrous oxide in pig animal model did not substantiate the hypothesis of neurological manifestations related to impaired AdoCbl-dependent methylmalonyl CoA mutase reaction, but rather identified impairment of MTR reaction as the more important basic defect [32]. Methionine had the ability to delay the neuropathy in a context where the fall in the SAM/SAH ratio was believed to cause defective methylation and demyelination [32]. However, this hypothesis did not agree with the maintained SAM/SAH ratio and methylation of myelin lipid or basic protein in the fruit bat model with vitamin B12-related neuropathy [32]. The gastrectomized rat model produced vitamin B12 deficiency and a neuropathy with imbalanced expression of cytokine and growth factors, including increased tumor necrosis factor (TNF)-alpha and nerve growth factor (NGF) and decreased interleukin-6 and epidermal
growth factor, EGF. However, in our hands, this model had some limitations for studying the effects specifically related with vitamin B12 since it produced a more global malnutrition [8]. Other rodent adult models fed a deficient diet are not adapted as they can get a significant source of B12 from coprophagy.

The vitamin B12 deficiency observed during pregnancy or ageing is often associated with folate deficiency during these periods of life. This is the reason why we have developed an animal model “dam-progeny” in which rat dams were fed a diet deficient in vitamin B12 and folate during gestation and pregnancy. In order to reproduce clinical conditions, the dams received the deficient diet one month before mating, during pregnancy and the suckling period [5]. The consequences were then analyzed in the progeny from the neonatal and lactating periods until ageing. Maternal deficiency in methyl donors was shown to induce in the progeny growth retardation with small birth weight similar to the one observed in humans [5,28] and with hyperhomocysteinemia in line with decreased folate and vitamin B12 concentrations, along with reduced SAM/SAH ratio, indicative of altered methylation capacity [5,28].

Most of the nutritional animal models used for studying the molecular and cellular consequences of vitamin B12 deficiency have some limitations. Some of them undertake not only the consequences of the impaired remethylation pathway that depends on vitamin B12 and MeTHF, but also the consequences related to the roles of other isoforms of folate. They do not allow studying the consequences specifically related to the brain and other target tissues of interest. In this regard, transgenic models will be able to highlight unknown molecular mechanisms linked to B12 deficiency. Recently, CD320 KO mice have been produced in the C57Bl/6 N strain using heterozygous ES cells. The null TCblR/CD320 KO mouse was generated by cross breeding. The mice have a dramatic decrease in B12 and global DNA hypomethylation in brain from TCblR/CD320 KO mice, compared with control mice
[33]. This suggests that TCblR/CD320 has a more limiting role on vitamin B12 uptake in brain than in peripheral organs. Very recently, we have prepared Tamoxifen-inducible CreERTM mouse lines with conditional invalidation of MTR gene, to study the consequences of impaired MTR activity in specific target tissues during the early and late periods of fetal development. We expect that this model will help deciphering MTR invalidation specifically in each targeted organ. Tamoxifen-inducible CreERTM mouse lines will provide on-demand controlled invalidation.

**5.2. Manifestations in brain.** In the brain, early deficiency was associated with apoptosis and altered neurobehavioral development [5]. In weanling pups, at 21 days after birth, immuno-histochemistry revealed homocysteine accumulation in neurons and astrocytes in the hippocampus, cerebellum and striatum [5]. In these regions, most of homocysteine-positive cells had high p53 levels and fragmented DNA. Despite switching back to a post-weaning normal diet, behavioural disturbances were still present in rats aged 80 days [5]. These findings suggest a relation between the observed functional impairment and apoptosis associated with homocysteine accumulation in structures involved in motor (striatum, cerebellum) and memory (hippocampus) functions. A concomitant reduction in neurogenesis might enhance the deleterious consequences of vitamin B deficiency, since homocysteine accumulation was detected in the neurogenic sites of brains from pups and adult animals [5]. We observed a 48% reduction of the thickness of the CA1 pyramidal layer without noticeable gliosis, which persisted during ageing, in the rats, even when restoring a normal diet after weaning [34]. Neurogenesis was slightly affected in the dentate gyrus of deficient rats, but not in the subventricular zone. Consistently, early vitamin B deprivation during gestation and lactation was associated with long-lasting functional disabilities of exploratory behaviour and learning and memory capacities, at 80 days of age, long after switch to normal food. This suggested that vitamin B deprivation in early life produce persisting brain hallmarks in related
with definitive CA atrophy, by neurodegenerative mechanism. Troen et al. suggested that cerebral microvascular rarefaction can cause cognitive dysfunction, in the absence of or preceding neurodegeneration, in male C57BL6/J mice subjected to deficiency in folate, vitamin B12 and vitamin B6 [35]. They reported a rarefaction of hippocampal microvasculature without neurodegeneration or gliosis, with significantly impaired spatial learning and memory [35].

Hypoxia is frequent and constitutes a major cause of cerebral injury in newborns, during the neonatal period. We investigated the cellular and molecular mechanisms that control neuronal fate, in our rat model subjected to the combined effects of brief neonatal hypoxia and methyl donor deficiency. Newborn rats exposed only to brief hypoxia display normal neurobehavioral development and enhanced memory retrieval capacities, at 40 days of age, as compared to their non-exposed counterparts [36]. Brief hypoxia prevents significantly the neurobehavioral deficits caused by methyl donor deprivation. This rescue process originates, at least in part, from a reactive stimulation of neurogenesis [37].

**5.3. Manifestations in liver and heart.** Compared to the important literature on the consequences of vitamin B12 deficiency on brain, those related to metabolic disorders in liver and heart have deserved a much more limited interest. Recent clinical reports and population studies suggested a potential link of vitamin B12 metabolism with the patho-mechanisms of cardiomyopathies. Prenatal dilated cardiomyopathy can be the presenting manifestation of *CblC* deficiency, an inborn error of intracellular metabolism of vitamin B12 [38]. In the very recent years, we, and others have shown an association of Homocysteine and vitamin B12 with left ventricular mass and left ventricular systolic dysfunction [39,40]. Methyl donor deficiency induces heart hypertrophy, with increased myocardium thickness, increased cardiomyocyte size and impaired mitochondrial fatty acid oxidation, in our "dam-progeny" animal model [10]. Proteomics analyses revealed that peroxisome proliferator-activated
receptor gamma coactivator 1-alpha (PGC-1alpha), peroxisome proliferator-activated receptor alpha (PPARalpha) and estrogen-related receptor alpha (ERRalpha) were the contributors to the decreased expression of proteins involved in mitochondrial oxidative metabolism. Consistently, we have observed a decreased expression of PPAR-alpha and ERR-alpha and impaired activation of the co-regulator PGC-1alpha through hypomethylation and hyperacetylation related with decreased protein arginine methyltransferase 1 (PRMT1) and sirtuin 1 (SIRT1) and elevated SAH [10]. The link between the one carbon metabolism and impaired mitochondrial fatty oxidation was confirmed in two contrasted populations, patients undergoing coronarography and ambulatory elderly subjects from OASI cohort [41].

Hepatic steatosis is observed in patients with genetic disorders of vitamin B12 metabolism [42] and vitamin B12, folate, homocysteine are associated with NAFLD severity, in operated and non-operated obese [43-45]. Similar observations have been made in experimental models, illustrating the link between the one-carbon metabolism and non-alcoholic fatty liver disease (NAFLD). Mice fed a methionine and choline deficient diet develop a steatohepatitis [46,47], while methyl donor deficiency yields microvesicular liver steatosis with high lipid and triglyceride tissue concentrations, in rat pups [48]. NAFLD is regarded as the liver manifestation of metabolic syndrome, which results from convergent complex mechanisms, including the imbalance between lipid storage and oxidation and the foetal programming hypothesis [49,50,11]. In this respect, we showed that the deficit in carnitine synthesis and the impaired beta-oxidation of fatty acids play a major role in the pathogenesis of steatosis of newborn rats early exposed to methyl donor deficiency. In these animals, fatty acid oxidation and activity of respiratory chain complexes 1 and 2 are impaired through PGC-1-alpha hypomethylation and decreased expression of PPAR-alpha, ER-alpha and ERR-alpha [10]. On the other hand, neonatal hypoxia was shown to accentuate the perturbations of
homocysteine metabolism in the liver by reducing the transsulfuration pathway, increasing the methylation index SAM/SAH, and thus aggravating the steatosis [48].

The small birth weight of our experimental model is a potential predictor of metabolic syndrome, according to the fetal programming hypothesis [5,10,11,51]. The methyl donor deficiency produces a small birth weight and a dramatic injury of gastric mucosa that may participate to the patho-mechanisms of intra-uterine growth retardation and the subsequent consequences of foetal programming, through cellular stress, inflammation, altered mucosal organization and impaired release of ghrelin in blood [51]. In this context, liver and myocardium steatosis could be two early predisposing manifestations of metabolic syndrome, in which deregulation of lipid fuel homeostasis by PGC1-alpha deactivation and decreased expression of SIRT1 play a master role (Fig. 5) [7,9,10,11,52]. This deregulation could contribute to explain the links between vitamin B12, small birth weight and subsequent manifestations of metabolic syndrome, in various population studies [28,53,54].

## 6. Cellular and tissular effects of vitamin B12 loading.

Beside deficiency, vitamin B12 loading produces also significant changes in cells. The activity of MS is enhanced in cells cultured by supplementation with vitamin B12 [55]. B12 increases the expression level of MTR, through an internal ribosome entry site (IRES); mRNAs employ internal ribosome entry sites (IRES) as alternative translation initiation of the initiator AUG codon, in eukaryote cells. MTR mRNA contains an IRES element upstream of the initiator AUG, and its activity is modulated by vitamin B12 through an auxiliary protein.

Vitamin B12 is stored in high concentrations in the human liver and may participate in the regulation of hepatotropic virus functions. HCV is a single-strand RNA virus that possesses an internal ribosomal entry site (IRES) at the end of its genome [56,57]. B12 can inhibit the HCV internal ribosome entry site (IRES)-dependent translation of a reporter gene in vitro in a dose-dependent manner without significantly affecting the cap-dependent mechanism [56,57].
In patients with chronic HCV infection, vitamin B12 supplementation improved the overall rate of sustained viral response to pegylated interferon-α and ribavirin by 34%. Consistently with the in vitro data, the effect was particularly pronounced in patients infected with HCV genotype 1 and who had a high baseline viral load [58].

The addition of vitamin B12 can also decrease the expression of endogenous genes, which are not directly related to its own metabolism, by mechanisms than are not completely clarified. P-glycoprotein, produced by multidrug resistance-1 gene (*MDR-1*), is the main transporter expressed by cancer cells to eliminate anti-cancer drugs. The B12 load produces a decreased expression and functionality of P-glycoprotein, which significantly increases cytotoxicity of vinblastine, in HepG2 hepatocarcinoma cell line [59]. The mechanisms linking B12 to *MDR-1* gene expression involve phospholipase D, P-Akt, and Cox2. Vitamin B12 down-regulates *MDR-1* gene expression through increased SAM and phosphatidylethanolamine-methyltransferase (PEMT)-dependent phosphatidylcholine production and activation of phospholipase D activity, in HepG2 cells [60]. This observation points out phospholipase D as a potential target to improve chemotherapy efficacy through the down-regulation of *MDR-1*.

In conclusion, vitamin B12 deficiency influences cell proliferation and differentiation by molecular mechanisms that reflect nutrigenomic effects that deregulate cytokine and growth factor pathways, including those of NGF, EGF, TNF and TACE. Some of the manifestations on the nervous system, the liver and the heart are produced through epigenomic mechanisms related to imbalanced acetylation/methylation of co-regulators of nuclear receptors. Some but not all of these effects reflect the key role of vitamin B12 in SAM synthesis.
Acknowledgements: The studies performed by U954 and presented in this review were supported by Inserm, by the Region Lorraine and by a grant from the national agency for research (ANR Nutrivigène).

## References

[1] E.V. Quadros, Advances in the understanding of cobalamin assimilation and metabolism, Br. J. Haematol. 148 (2010) 195-204.

[2] R. Banerjee, S.W. Ragsdale, The many faces of vitamin B12: catalysis by cobalamin-dependent enzymes, Annu. Rev. Biochem. 72 (2003) 209-247.

[3] L. Papatheodorou, N. Weiss, Vascular oxidant stress and inflammation in hyperhomocysteinemia, Antioxid. Redox Signal. 9 (2007) 1941-1958.

[4] O.S. Anderson, K.E. Sant, D.C. Dolinoy, Nutrition and epigenetics: an interplay of dietary methyl donors, one-carbon metabolism and DNA methylation, J. Nutr. Biochem. 23 (2012) 853-859.

[5] S.A. Blaise, E. Nedelec, H. Schroeder, J.M. Alberto, C. Bossenmeyer-Pourié, J.L. Guéant, J.L. Daval, Gestational vitamin B deficiency leads to homocysteine-associated brain apoptosis and alters neurobehavioral development in rats, Am. J. Pathol. 170 (2007) 667-679

[6] S.F. Battaglia-Hsu, N. Akchiche, N. Noel, J.M. Alberto, E. Jeannesson, C.E. Orozco-Barrios, D. Martinez-Fong, J.L. Daval, J.L. Guéant, Vitamin B12 deficiency reduces proliferation and promotes differenciation of neuroblastoma cells and up-regulates PP2A, proNGF, and TACE, Proc. Natl. Acad. Sci. U.S.A. 106 (2009) 21930-21935.

[7] M.J. Holness, P.W. Caton, M.C. Sugden, Acute and long-term nutrient-led modifications of gene expression: potential role of SIRT1 as a central co-ordinator of short and longer-term programming of tissue function, Nutrition 26 (2010) 491-501
[8] L. Brunaud, J.M. Alberto, A. Ayav, P. Gérard, F. Namour, L. Antunes, M. Braun, J.P. Bronowicki, L. Bresler, J.L. Guéant, Vitamin B12 is a strong determinant of low methionine synthase activity and DNA hypomethylation in gastrectomized rats, Digestion. 68 (2003) 133-140.

[9] S. Pooya, S. Blaise, M. Moreno Garcia, J. Giudicelli, J.M. Alberto, R.M. Guéant-Rodriguez, E. Jeannesson, N. Gueguen, A. Bressenot, B. Nicolas, Y. Malthiery, J.L. Daval, L. Peyrin-Biroulet, J.P. Bronowicki, J.L. Guéant, Methyl donor deficiency impairs fatty acid oxidation through PGC-1α hypomethylation and decreased ER-α, ERR-α, and HNF-4α in the rat liver, J Hepatol. 57 (2012) 344-351.

[10] M.M. Garcia, R.M. Guéant-Rodriguez, S. Pooya, P. Brachet, J.M. Alberto, E. Jeannesson, F. Maskali, N. Gueguen, P.Y. Marie, P. Lacolley, M. Herrmann, Y. Juillière, Y. Malthiery, J.L Guéant, Methyl donor deficiency induces cardiomyopathy through altered methylation/acetylation of PGC-1α by PRMT1 and SIRT1, J Pathol. 225 (2011) 324-335.

[11] J. Joseph, Fattening by deprivation: methyl balance and perinatal cardiomyopathy, J Pathol. 225 (2011) 315-317.

[12] G.R. McLean, E.V. Quadros, S.P. Rothenberg, A.C. Morgan, J.W. Schrader, H.J. Ziltener, Antibodies to transcobalamin II block in vitro proliferation of leukemic cells, Blood 89 (1997) 235-242.

[13] S.C. Lai, Y. Nakayama, J.M. Sequeira, E.V. Quadros, Down-regulation of transcobalamin receptor TCb1R/CD320 by siRNA inhibits cobalamin uptake and proliferation of cells in culture, Exp. Cell. Res. 317 (2011) 1603-1607.

[14] L. Pons, S.F. Battaglia-Hsu, C.E. Orozco-Barrios, S. Ortiou, C. Chery, J.M. Alberto, M.L. Arango-Rodriguez, D. Dumas, D. Martinez-Fong, J.N. Freund, J.L. Gueant, Anchoring secreted proteins in endoplasmic reticulum by plant oleosin: the example of vitamin B12 cellular sequestration by transcobalamin, PLoS One. 4 (2009) e6325.
[15] G. Scalabrino, The multi-faceted basis of vitamin B12 (cobalamin) neurotrophism in adult central nervous system: Lessons learned from its deficiency, Prog Neurobiol. 88 (2009) 203-220.

[16] C.E. Orozco-Barrios, S.F. Battaglia-Hsu, M.L. Arango-Rodriguez, J. Ayala-Davila, C. Chery, J.M. Alberto, H. Schroeder, J.L.Daval, D. Martinez-Fong, J.L. Gueant, Vitamin B12-impaired metabolism produces apoptosis and Parkinson phenotype in rats expressing the transcobalamin-oleosin chimera in substantia nigra, PLoS One 4 (2009) e8268.

[17] R.G.Matthews, M. Koutmos, S. Datta, Cobalamin-dependent and cobamide-dependent methyltransferases, Curr. Opin. Struct. Biol. 18 (2008) 658-666.

[18] C.W. Goulding, D. Postigo, R.G. Matthews, Cobalamin-dependent methionine synthase is a modular protein with distinct regions for binding homocysteine, methyltetrahydrofolate, cobalamin, and adenosylmethionine, Biochemistry 36 (1997) 8082-8091.

[19] J.T. Drummond, S. Huang, R.M. Blumenthal, R.G. Matthews, Assignment of enzymatic function to specific protein regions of cobalamin-dependent methionine synthase from Escherichia coli, Biochemistry 32 (1993) 9290-9295.

[20] H. Olteanu, R. Banerjee, Human methionine synthase reductase, a soluble P-450 reductase-like dual flavoprotein, is sufficient for NADPH-dependent methionine synthase activation, J. Biol. Chem. 276 (2001) 35558-35563

[21] K. Yamada, R.A. Gravel, T. Toraya, R.G. Matthews, Human methionine synthase reductase is a molecular chaperone for human methionine synthase, Proc. Natl. Acad. Sci. U S A 103 (2006) 9476-9481

[22] L.Peyrin-Biroulet, R.M. Guéant-Rodriguez, M. Chen, J.P. Bronowicki, M.A. Bigard, J.L. Guéant, Association of MTRR 66A>G polymorphism with superoxide dismutase and disease activity in patients with Crohn's disease, Am. J. Gastroenterol. 103 (2008) 399-406.
[23] J.L. Guéant, R.M. Guéant-Rodriguez, G. Anello, P. Bosco, L. Brunaud, C. Romano, R. Ferri, A. Romano, M. Candito, B. Namour, Genetic determinants of folate and vitamin B12 metabolism: a common pathway in neural tube defect and Down syndrome?, Clin Chem Lab Med 41 (2003) 1473-1477.

[24] F. Li, D. Watkins, D.S. Rosenblatt, Vitamin B(12) and birth defects, Mol Genet Metab 98 (2009) 166-172.

[25] C.J. Dix, I.F. Hassan, H.Y. Obray, G. Wilson, The transport of vitamin B12 through polarized monolayers of Caco-2 cells, Gastroenterology 98 (1990) 1272-1279.

[26] L. Pons, M. Guy, D. Lambert, R. Hatier, J.L. Guéant, Transcytosis and coenzymatic conversion of [(57)Co]cobalamin bound to either endogenous transcobalamin II or exogenous intrinsic factor in caco-2 cells, Cell Physiol Biochem 10 (2000) 135-148.

[27] S.P. Stabler, R.H. Allen, Vitamin B12 deficiency as a worldwide problem, Annu Rev Nutr. 24 (2004) 299-326.

[28] C.S. Yajnik, U.S. Deshmukh, Fetal programming: maternal nutrition and role of one-carbon metabolism, Rev Endocr Metab Disord. 13 (2012) 121-127.

[29] J. Selhub, Public health significance of elevated homocysteine, Food Nutr Bull. 29 (2008) S116-S125.

[30] A.D. Smith, The worldwide challenge of the dementias: a role for B vitamins and homocysteine?, Food Nutr Bull. 29 (2008) S143-S172.

[31] S.V. Van Tonder, J. Metz, R. Green, Vitamin B12 metabolism in the fruit bat (Rousettus aegyptiacus). The induction of vitamin B12 deficiency and its effect on folate levels, Br J Nutr. 34 (1975) 397-410.

[32] J. Metz, Cobalamin deficiency and the pathogenesis of nervous system disease, Annu Rev Nutr. 12 (1992) 59-79.
[33] S. Fernàndez-Roig, S.C. Lai, M.M. Murphy, J. Fernandez-Ballart, E.V. Quadros, Vitamin B12 deficiency in the brain leads to DNA hypomethylation in the TCblR/CD320 knockout mouse. Nutr Metab (Lond) 9 (2012) 41.

[34] J.L. Daval, S. Blaise, J.L. Guéant, Vitamin B deficiency causes neural cell loss and cognitive impairment in the developing rat, Proc Natl Acad Sci U S A. 106 (2009) E1.

[35] A.M. Troen, M. Shea-Budgell, B. Shukitt-Hale, D.E. Smith, J. Selhub, I.H. Rosenberg, B-vitamin deficiency causes hyperhomocysteinemia and vascular cognitive impairment in mice, Proc Natl Acad Sci U S A. 105 (2008) 12474-12479.

[36] N. Martin, G. Pourié, C. Bossenmeyer-Pourié, R. Jazi, J.L. Guéant, P. Vert, J.L. Daval, Conditioning-like brief neonatal hypoxia improves cognitive function and brain tissue properties with marked gender dimorphism in adult rats, Semin Perinatol. 34 (2010) 193-200.

[37] S.A. Blaise, E. Nédélec, J.M. Alberto, H. Schroeder, S. Audonnet, C. Bossenmeyer-Pourié, J.L. Guéant, J.L. Daval, Short hypoxia could attenuate the adverse effects of hyperhomocysteinemia on the developing rat brain by inducing neurogenesis, Exp Neurol. 216 (2009) 231-238.

[38] I. De Bie, S.D. Nizard, G.A., Mitchell Fetal dilated cardiomyopathy: an unsuspected presentation of methylmalonic aciduria and hyperhomocystinuria, cblC type, Prenat Diagn 29 (2009) 266-270.

[39] R.M. Gueant-Rodriguez, Y. Juilliere, M. Nippert, I. Abdelmouttaleb, B. Herbeth, E. Aliot, N. Danchin, J.L. Gueant, Left ventricular systolic dysfunction is an independent predictor of homocysteine in angiographically documented patients with or without coronary artery lesions, J Thromb Haemost 5 (2007) 1209-1216.

[40] W. Herrmann, M. Herrmann, J. Joseph, S.C. Tyagi, Homocysteine, brain natriuretic peptide and chronic heart failure: a critical review, Clin Chem Lab Med 45 (2007) 1633-1644.
[41] R.M. Guéant Rodriguez, R. Spada, S. Pooya, E. Jeannesson, M.A. Moreno Garcia, G. Anello, P. Bosco, M. Elia, A. Romano, J.M. Alberto, Y. Juillière, J.L. Guéant, Homocysteine predicts increased NT-pro-BNP through impaired fatty acid oxidation, Int J Cardiol. (2012) Mar 27. [Epub ahead of print]

[42] P. Russo, J. Doyon, E. Sonsino, H. Ogier, J.M. Saudubray, A congenital anomaly of vitamin B12 metabolism: a study of three cases, Human Pathol 23 (1992) 504-512.

[43] M. Koplay, E. Gulcan, F. Ozkan, Association between serum vitamin B12 levels and the degree of steatosis in patients with nonalcoholic fatty liver disease, J Investig Med 59 (2011) 1137-1140.

[44] S. Hirsch, J. Poniachick, M. Avendaño, A. Csendes, P. Burdiles, G. Smok, J. C. Diazb, M. P. de la Maza, Serum folate and homocysteine levels in obese females with non-alcoholic fatty liver, Nutrition 21 (2005) 137-141.

[45] M. Gulsen, Z. Yesilova, S. Bagci, A. Uygun, A. Ozcan, C.N. Ercin, A. Erdil, S.Y. Sanisoglu, E. Cakir, Y. Ates, M.K. Erbil, N. Karaeren, K. Dagalp, Elevated plasma homocysteine concentrations as a predictor of steatohepatitis in patients with non-alcoholic fatty liver disease, J Gastroenterol Hepatol 20 (2005) 1448-1455.

[46] M.D. Weltman, G.C. Farrell, C. Liddle, Increased hepatocyte CYP2E1 expression in a rat nutritional model of hepatic steatosis with inflammation. Gastroenterology. 111 (1996) 1645-1653.

[47] E. Ip, G. Farrell, P. Hall, G. Robertson, I. Leclercq, Administration of the potent PPARalpha agonist, Wy-14,643, reverses nutritional fibrosis and steatohepatitis in mice, Hepatology 39 (2004) 1286-1296.

[48] S.A. Blaise, J.M. Alberto, S. Audonnet-Blaise, J.L. Guéant, J.L. Daval, Influence of preconditioning-like hypoxia on the liver of developing methyl-deficient rats, Am J Physiol Endocrinol Metab. 293 (2007) E1492-1502.
[49] N.M. de Alwis, C.P. Day, Non-alcoholic fatty liver disease: the mist gradually clears, J Hepatol. 48 (2008) S104-112.

[50] P. Rinaudo, E. Wang, Fetal programming and metabolic syndrome, Annu Rev Physiol. (2012) 107-130

[51] C. Bossenmeyer-Pourié, S. Blaise, G. Pourié, C. Tomasetto, S. Audonnet, S. Ortiou, V. Koziel, M.C. Rio, J.L. Daval, J.L. Guéant, B. Beck, Methyl donor deficiency affects fetal programming of gastric ghrelin cell organization and function in the rat, Am. J. Pathol. 176 (2010) 270-277.

[52] P.J. Fernandez-Marcos, J. Auwerx, Regulation of PGC-1α, a nodal regulator of mitochondrial biogenesis, Am J Clin Nutr. 93 (2011) 884S-990S.

[53] C.S. Yajnik, S.S. Deshpande, A.A. Jackson, H. Refsum, S. Rao, D.J. Fisher, D.S. Bhat, S.S. Naik, K.J. Coyaji, C.V. Joglekar, N. Joshi, H.G. Lubree, V.U. Deshpande, S.S. Rege, C.H. Fall, Vitamin B12 and folate concentrations during pregnancy and insulin resistance in the offspring: the Pune Maternal Nutrition Study, Diabetologia 51 (2008) 29-38.

[54] C.P. Stewart, P. Christian, K.J. Schulze, M. Arguello, S.C. Leclerq, S.K. Khatry, K.P.Jr West, Low Maternal Vitamin B-12 Status Is Associated with Offspring Insulin Resistance Regardless of Antenatal Micronutrient, J Nutr 141 (2011) 1912-1917.

[55] J.H. Mangum, B.K. Murray, J.A. North, Vitamin B12 dependent methionine biosynthesis in cultured mammalian cells, Biochemistry 8 (1969) 3496-3499.

[56] S.S. Takyar, E.J. Gowans, W.B. Lott WB, Vitamin B12 stalls the 80s ribosomal complex on the hepatitis C internal ribosome entry site, J. Mol. Biol. 319 (2002) 1-8.

[57] W.B. Lott, S.S. Takyar, J. Tuppen, D.H. Crawford, M. Harrison, T.P. Sloots, E.J Gowans, Vitamin B12 and hepatitis C: molecular biology and human pathology, Proc. Natl. Acad. Sci. U S A. 98 (2001) 4916-4921.
[58] A. Rocco, D. Compare, P. Coccoli, C. Esposito, A. Di Spirito, A. Barbato, P. Strazzullo, G. Nardone, Vitamin B12 supplementation improves rates of sustained viral response in patients chronically infected with hepatitis C virus, Gut (2012) [Epub ahead of print]

[59] V. Marguerite, M. Beri-Dexheimer, S. Ortiou, J.L. Guéant, M. Merten, Cobalamin potentiates vinblastine cytotoxicity through downregulation of mdr-1 gene expression in HepG2 cells, Cell. Physiol. Biochem. 20 (2007) 967-976.

[60] V. Marguerite, E. Gkikopoulou, J.M. Alberto, J.L. Guéant, M. Merten, Phospholipase D activation mediates Cobalamin-induced downregulation of Multidrug Resistance-1 gene and increase in sensitivity to vinblastine in HepG2 cells, Int. J. Biochem. Cell. Biol. (2012) Sep 29. pii: S1357-2725(12)00329-9. doi: 10.1016/j.biocel.2012.09.018. [Epub ahead of print]

[61] Freund JN, Jost B, Duluc I, Morel G. Ultrastructural study of intestinal lactase gene expression. Biol. Cell. 83(1995) 211-7.

## Figure legends

Figure 1: Nutrigenomic effects of vitamin B12 (cobalamin, Cbl). Vitamin B12 originates from food of animal origin while folate is mainly provided by vegetables and fruits. The nutrigenomic influence of vitamin B12 results from its role as co-factor of methionine synthase (MTR), which catalyses the synthesis of methionine. Methionine is the direct precursor of S-adenosylmethionine (SAM), the methyl donor involved in transmethylations reactions and related epigenomic mechanisms. Homocysteine (Hcy) originates from the hydrolysis of S-adenosyl-homocysteine (SAH), the product of transmethylation reactions. The co-substrate of Hcys, methyltetrahydrofolate (methylTHF) is synthesized by methyl-tetrahydrofolate-reductase
Figure 2: Immunhisto-chemical detection of methionine synthase (MTR) in the human skin. MTR is detected in cells from the proliferating layer while it is undetectable in mature keratinocytes. Inlet: MTR is detected in the cytoplasm of a proliferating cell.

Figure 3: DNA methylation and methionine synthase (MTR) activity in the crypt – villus axis of adult rat intestinal mucosa. The release of enterocytes from the crypt-villus axis was obtained by gentle shacking of mucosal pieces in presence of EDTA, as described previously [61]. A: DNA methylation was evaluated by enzymatic incorporation of tritiated-methyls, as described previously [8]. The global DNA methylation mirrored the incorporation of methyls. B: MTR activity in the crypt-apical axis. MTR activity was the highest in crypts, and was correlated with the global DNA methylation.

Figure 4: Transcription of *MTR* in Caco2 cells, according to growth delay. A: semi-quantitative RT-PCR showing a high level of transcripts in the proliferation stage, and a dramatic decrease of transcription when cells reach confluence. Total RNA was isolated from cultured liver tissue using RNeasy Plus extraction kit (Qiagen). First strand cDNA was synthesized from 2 µg of total RNA using oligo(dT)12-18 primers (Invitrogen) with SuperScript® II reverse transcriptase (Invitrogen). The transcripts were amplified from cDNA using specific primers (Table1) using a QuantiTect SYBR Green PCR Kit (Qiagen) and Lightcycler® 1.0 (Roche). B: Example of RT-PCR of *MTR* transcripts performed between day 3 and day 12 of growth delay. C: Northern blot of MTR according to growth delay, showing a decrease of the expression level of the 10kb transcript after 7 days (e.g. after the proliferation phase) and the presence of 7.5 and 6.5 kb transcripts at day 15, suggesting splicing of several isoforms of transcripts according to growth delay.
Figure 5. Protein expression and activity of methionine synthase (MTR), according to growth delay of Caco2 cells. A: Immunohistochemical detection of methionine synthase in cells at day 5 and day 15, showing a dramatic decrease of MTR expression when cells stop to proliferate. B: Similarly, western blot of cellular extract (using a polyclonal rabbit anti-serum) showed a dramatic decrease of MTR protein expression at day 15, compared to the proliferation phase (days 4, 7 and 8). C: the MTR activity decreased dramatically after day 12. The procedures of immunohistochemistry, western blotting and MTR activity were performed as described previously [5].

Figure 6. Molecular mechanisms explaining the link between methyl donor deficiency and energy metabolism. The deficiency in vitamin B12 and folate of the rat ‘dam-progeny’ model impairs fatty acid oxidation and complexes 1 and 2 of mitochondrion respiratory chain through a deregulation of epigenomic mechanisms related to the decreased expression of peroxisome proliferator-activated receptor alpha (PPARα), estrogen-related receptor alpha (ERRα) and hepatic nuclear factor 4 (HNF-4) and to the decreased methylation and/or increased acetylation of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), respectively. This imbalanced methylation/acetylation results from the decreased expression of PRMT1 arginine methyl-transferase, the increased cellular concentration of S-adenosylhomocysteine (SAH), which is a potent inhibitor of PRMT1 and the decreased expression of sirtuin 1 (SIRT1) deacetylase (adapted from reference 7).
<figure><img src="image_2.png" /></figure>

Food of animal
origin

Vitamin B12

X=
DNA
RNA
Histones
Proteins
Lipids
Xenobiotics

Epigenetics/
Epigenomics
<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>
A

<figure><img src="image_5.png" /></figure>

B

<figure><img src="image_6.png" /></figure>
A

<figure><img src="image_7.png" /></figure>

B

<figure><img src="image_8.png" /></figure>

C

<figure><img src="image_9.png" /></figure>
A

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

B

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

C

<figure><img src="image_14.png" /></figure>
<figure><img src="image_15.png" /></figure>