Jean-Marc Alberto

Methyl donor deficiency induces cardiomyopathy through altered methylation/acetylation of PGC-1α by PRMT1 and SIRT1.

Cardiomyopathies occur by mechanisms that involve inherited and acquired metabolic disorders. Both folate and vitamin B12 deficiencies are associated with left ventricular dysfunction, but mechanisms that underlie these associations are not known. However, folate and vitamin B12 are methyl donors needed for the synthesis of S‐adenosylmethionine, the substrate required for the activation by methylation of regulators of energy metabolism. We investigated the consequences of a diet lacking methyl donors in the myocardium of weaning rats from dams subjected to deficiency during gestation and lactation. Positron emission tomography (PET), microscope and metabolic examinations evidenced a myocardium hypertrophy, with cardiomyocyte enlargement, disturbed mitochondrial alignment, lipid droplets, decreased respiratory activity of complexes I and II and decreased S‐adenosylmethionine:S‐adenosylhomocysteine ratio. The increased concentrations of triglycerides and acylcarnitines were consistent with a deficit in fatty acid oxidation. These changes were explained by imbalanced acetylation/methylation of PGC‐1α, through decreased expression of SIRT1 and PRMT1 and decreased S‐adenosylmethionine:S‐adenosylhomocysteine ratio, and by decreased expression of PPARα and ERRα. The main changes of the myocardium proteomic study were observed for proteins regulated by PGC‐1α, PPARs and ERRα. These proteins, namely trifunctional enzyme subunit α‐complex, short chain acylCoA dehydrogenase, acylCoA thioesterase 2, fatty acid binding protein‐3, NADH dehydrogenase (ubiquinone) flavoprotein 2, NADH dehydrogenase (ubiquinone) 1α‐subunit 10 and Hspd1 protein, are involved in fatty acid oxidation and mitochondrial respiration. In conclusion, the methyl donor deficiency produces detrimental effects on fatty acid oxidation and energy metabolism of myocardium through imbalanced methylation/acetylation of PGC‐1α and decreased expression of PPARα and ERRα. These data are of pathogenetic relevance to perinatal cardiomyopathies. Copyright

Methyl donor deficiency impairs fatty acid oxidation through PGC-1α hypomethylation and decreased ER-α, ERR-α, and HNF-4α in the rat liver.

BACKGROUND & AIMSnFolate and cobalamin are methyl donors needed for the synthesis of methionine, which is the precursor of S-adenosylmethionine, the substrate of methylation in epigenetic, and epigenomic pathways. Methyl donor deficiency produces liver steatosis and predisposes to metabolic syndrome. Whether impaired fatty acid oxidation contributes to this steatosis remains unknown.nnnMETHODSnWe evaluated the consequences of methyl donor deficient diet in liver of pups from dams subjected to deficiency during gestation and lactation.nnnRESULTSnThe deprived rats had microvesicular steatosis, with increased triglycerides, decreased methionine synthase activity, S-adenosylmethionine, and S-adenosylmethionine/S-adenosylhomocysteine ratio. We observed no change in apoptosis markers, oxidant and reticulum stresses, and carnityl-palmitoyl transferase 1 activity, and a decreased expression of SREBP-1c. Impaired beta-oxidation of fatty acids and carnitine deficit were the predominant changes, with decreased free and total carnitines, increased C14:1/C16 acylcarnitine ratio, decrease of oxidation rate of palmitoyl-CoA and palmitoyl-L-carnitine and decrease of expression of novel organic cation transporter 1, acylCoA-dehydrogenase and trifunctional enzyme subunit alpha and decreased activity of complexes I and II. These changes were related to lower protein expression of ER-α, ERR-α and HNF-4α, and hypomethylation of PGC-1α co-activator that reduced its binding with PPAR-α, ERR-α, and HNF-4α.nnnCONCLUSIONSnThe liver steatosis resulted predominantly from hypomethylation of PGC1-α, decreased binding with its partners and subsequent impaired mitochondrial fatty acid oxidation. This link between methyl donor deficiency and epigenomic deregulations of energy metabolism opens new insights into the pathogenesis of fatty liver disease, in particular, in relation to the fetal programming hypothesis.

Homocysteinylation of neuronal proteins contributes to folate deficiency-associated alterations of differentiation, vesicular transport, and plasticity in hippocampal neuronal cells

Despite the key role in neuronal development of a deficit in the methyl donor folate, little is known on the underlying mechanisms. We therefore studied the consequences of folate deficiency on proliferation, differentiation, and plasticity of the rat H19‐7 hippocampal cell line. Folate deficit reduced proliferation (17%) and sensitized cells to differentiation‐associated apoptosis (+16%). Decreased production (–58%) of S‐adenosylmethionine (the universal substrate for transmethylation reactions) and increased expression of histone deacetylases (HDAC4,6,7) would lead to epigenomic changes that may impair the differentiation process. Cell polarity, vesicular transport, and synaptic plasticity were dramatically affected, with poor neurite outgrowth (–57%). Cell treatment by an HDAC inhibitor (SAHA) led to a noticeable improvement of cell polarity and morphology, with longer processes. Increased homocysteine levels (+55%) consecutive to folate shortage produced homocysteinylation, evidenced by coimmunoprecipitations and mass spectrometry, and aggregation of motor proteins dynein and kinesin, along with functional alterations, as reflected by reduced interactions with partner proteins. Prominent homocysteinylation of key neuronal proteins and subsequent aggregation certainly constitute major adverse effects of folate deficiency, affecting normal development with possible long‐lasting consequences.—Akchiche, N., Bossenmeyer‐Pourié, C., Kerek, R., Martin, N., Pourié, G., Koziel, V., Helle, D., Alberto, J.‐M., Ortiou, S., Camadro, J.‐M., Léger, T., Guéant, J.‐L., Daval, J.‐L. Homocysteinylation of neuronal proteins contributes to folate deficiency‐associated alterations of differentiation, vesicular transport, and plasticity in hippocampal neuronal cells. FASEB J. 26, 3980–3992 (2012). www.fasebj.org

Short hypoxia could attenuate the adverse effects of hyperhomocysteinemia on the developing rat brain by inducing neurogenesis.

Gestational deficiency in methyl donors such as folate and vitamin B12 impairs homocysteine metabolism and can alter brain development in the progeny. Since short hypoxia has been shown to be neuroprotective in preconditioning studies, we aimed to investigate the effects of brief, non-lesioning neonatal hypoxia (100% N2 for 5 min) on the developing brain of rats born to dams fed either a standard diet or a diet lacking vitamins B12, B2, folate and choline until offsprings weaning. While having no influence on brain accumulation of homocysteine and concomitant apoptosis in 21-day-old deficient pups, exposure to hypoxia reduced morphological injury of the hippocampal CA1 layer. It also markedly stimulated the incorporation of bromodeoxyuridine (BrdU) in permissive areas such as the subventricular zone and the hippocampus followed by the migration of new neurons. Scores in a locomotor coordination test (days 19-21) and learning and memory behavior in the eight-arm maze (days 80-84) were found to be significantly improved in rats exposed to hypoxia in addition to the deficient diet. Therefore, by stimulating neurogenesis in rat pups, brief neonatal hypoxia appeared to attenuate the long-term effects of early exposure to a deficiency in nutritional determinants of hyperhomocysteinemia.

Early methyl donor deficiency alters cAMP signaling pathway and neurosteroidogenesis in the cerebellum of female rat pups

Early deficiency of the methyl donors folate and vitamin B12 produces hyperhomocysteinemia and cognitive and motor disorders in 21-day-old rat pups from dams fed a diet deficient in methyl donors during gestation and lactation. These disorders are associated with impaired neurogenesis and altered synaptic plasticity in cerebellum. We aimed to investigate whether these disorders could be related to impaired expression of neurosteroidogenesis-associated proteins, key regulator receptors, and some steroid content in the cerebellum. The methyl donor deficiency produced a decreased concentration of folate and vitamin B12, along with accumulation of homocysteine in Purkinje cells in both sexes, whereas the S-adenosylmethionine/S-adenosylhomocysteine ratio was reduced only in females. The transcription level and protein expression of StAR, aromatase, ERα, ERβ, and LH receptors were decreased only in females, with a marked effect in Purkinje cells, as shown by immunohistochemistry. Consistently, reduced levels of estradiol and pregnenolone were measured in cerebellar extracts of females only. The decreased expression levels of the transcriptional factors CREB, phospho-CREB, and SF-1, the lesser increase of cAMP concentration, and the lower level of phospho-PKC in the cerebellum of deficient females suggest that the activation of neurosteroidogenesis via cAMP-mediated signaling pathways associated with LHR activation would be altered. In conclusion, a gestational methyl donor deficiency impairs neurosteroidogenesis in cerebellum in a sex-dependent manner.

Interaction between methionine synthase isoforms and MMACHC: characterization in cblG-variant, cblG and cblC inherited causes of megaloblastic anaemia

The cblG and cblC disorders of cobalamin (Cbl) metabolism are two inherited causes of megaloblastic anaemia. In cblG, mutations in methionine synthase (MTR) decrease conversion of hydroxocobalaminxa0 (HOCbl) to methylcobalamin, while in cblC, mutations in MMACHC disrupt formation of cob(II)alamin (detected as HOCbl). Cases with undetectable methionine synthase (MS) activity are extremely rare and classified as cblG-variant. In four cblG-variant cases, we observed a decreased conversion of cyanocobalamin to HOCbl that is also seen in cblC cases. To explore this observation, we studied the gene defects, splicing products and expression of MS, as well as MS/MMACHC protein interactions in cblG-variant, cblG, cblC and control fibroblasts. We observed a full-size MS encoded by MTR-001 and a 124 kDa truncated MS encoded by MTR-201 in cblG, cblC, control fibroblasts and HEK cells, but only the MTR-201 transcript and inactive truncated MS in cblG-variant cells. Co-immunoprecipitation and proximity ligation assay showed interaction between truncated MS and MMACHC in cblG-variant cells. This interaction decreased 2.2, 1.5 and 5.0-fold in the proximity ligation assay of cblC cells with p.R161Q and p.R206W mutations, and HEK cells with knock down expression of MS by siRNA, respectively, when compared with control cells. In 3D modelling and docking analysis, both truncated and full-size MS provide a loop anchored to MMACHC, which makes contacts with R-161 and R-206 residues. Our data suggest that the interaction of MS with MMACHC may play a role in the regulation of the cellular processing of Cbls that is required for Cbl cofactor synthesis.

Homocysteine Increases Methionine Synthase mRNA Level in Caco-2 Cells

Background: Methionine synthase (MTR) synthesizes methionine from homocysteine, using cobalamin as a cofactor and 5-methyltetrahydrofolate as a cosubstrate. Aim: To determine the influence of homocystine (Hcy, dimer of homocysteine) in the presence of either cobalamin or methionine on the transcription and the activity of methionine synthase in Caco-2, a human adenocarcinoma cell line. Methods: Methionine synthase activity and quantification of its mRNA by real-time RT-PCR were determined in cells cultivated under four differents conditions: Hcy with cobalamin (Hcy+ Cbl+), Hcy with methionine (Hcy+Met+), methionine with Cbl (Met+ Cbl+) and methionine only (Met+). Results: Activity (nmol/h/mg protein) was maximal in cells cultivated in Hcy+Cbl+ (2.45 ± 0.35), compared to cells cultivated in Hcy+Met+ (0.18 ± 0.01, p<0.001), in Met+ Cbl+ (1.60 ± 0.06, p<0.05), and in Met+ (0.40 ± 0.05, p<0.001), suggesting an adaptation of the cells to requirement in synthesized methionine. The mRNA level of MTR in Hcy+ Cbl+ and Hcy+Met+ (2.82 ±0.49 and 3.33 ± 0.48 AU, respectively ) was about 2.5 / 3.0-fold higher than that in Met+ Cbl+ and in Met+ (1.00 ±0.13 and 1.20 ±0.20 AU, respectively, p<0.001). Conclusion: Methionine synthase expression of Caco-2 cell is under a transcriptional control influenced by Hcy.

Production of Elastin-Derived Peptides Contributes to the Development of Nonalcoholic Steatohepatitis

Affecting more than 30% of the Western population, nonalcoholic fatty liver disease (NAFLD) is the most common liver disease and can lead to multiple complications, including nonalcoholic steatohepatitis (NASH), cancer, hypertension, and atherosclerosis. Insulin resistance and obesity are described as potential causes of NAFLD. However, we surmised that factors such as extracellular matrix remodeling of large blood vessels, skin, or lungs may also participate in the progression of liver diseases. We studied the effects of elastin-derived peptides (EDPs), biomarkers of aging, on NAFLD progression. We evaluated the consequences of EDP accumulation in mice and of elastin receptor complex (ERC) activation on lipid storage in hepatocytes, inflammation, and fibrosis development. The accumulation of EDPs induces hepatic lipogenesis (i.e., SREBP1c and ACC), inflammation (i.e., Kupffer cells, IL-1β, and TGF-β), and fibrosis (collagen and elastin expression). These effects are induced by inhibition of the LKB1-AMPK pathway by ERC activation. In addition, pharmacological inhibitors of EDPs demonstrate that this EDP-driven lipogenesis and fibrosis relies on engagement of the ERC. Our data reveal a major role of EDPs in the development of NASH, and they provide new clues for understanding the relationship between NAFLD and vascular aging.

H023 Altération du métabolisme lipidique dans le tissue myocardique des ratons de 21 jours, carencés en donneurs de méthyles au cours de l’allaitement

Contexte L’homocysteine est un facteur de risque des maladies cardiovasculaires. Les perturbations des activites du cycle de remethylation de l’homocysteine, liees a une carence de groupements methyles, seraient probablement impliquees dans la pathogenese de ces maladies. Objectif Etudier les repercutions du regime carence en donneurs de methyles sur le myocarde des ratons nes de meres carencees, et determiner les effets sur les activites enzymatiques du cycle de l’homocysteine, et sur le metabolisme lipidique. Methodes Modele : Rats Wistar alimentes avec un regime carence un mois avant la mise en accouplement et poursuivi jusqu’au sevrage. Analyses Biochimiques : Dosages vitaminiques, activites enzymatiques, metabolites du cycle de l’homocysteine, concentration en carnitine, BNP et recepteurs d’adenosine. Voies de signalisation. Proteines AKT/PKB (hypertrophie) Analyses Fonctionnelles. Fraction d’ejection (FEVG) par miniPET et mesure de la pression arterielle. Resultats La carence maternelle diminue les concentrations plasmatiques en vitamines B9 et B12 et une hyperhomocysteinemie. On observe une augmentation des taux de SAM et de SAH ainsi que de l’activite MS. Histologie : Hypertrophie myocardique. Augmentation significative du taux de BNP plasmatique en absence d’une dysfonction systolique chez le rat carence. Parallelement il y a une augmentation du recepteur A2A. Il existe un effet ambivalent de l’hyperhomocysteinemie sur les acylcarnitines avec une augmentation au niveau plasmatique et une diminution au niveau tissulaire chez le rat carence. La carence n’induit pas de modification de l’expression des certaines proteines impliquees dans le voies AKT/PKB. Conclusion Chez le raton carence les modifications du BNP temoignent d’un stress de la paroi myocardique. Puisque les acides gras sont la principale source d’energie du myocarde, un deficit en acylcarnitine suggere un probleme de production d’energie qui pourrait expliquer l’hypertrophie par un mecanisme adaptatif.