Dysregulated cysteine metabolism leads to worsened liver pathology in diabetes-tuberculosis comorbid condition.

Diabetes mellitus (DM) is a risk factor for developing active tuberculosis (TB) with a 3-fold increase in susceptibility and a 4-fold higher relapse rate. With increasing DM prevalence in TB endemic regions, understanding pathophysiological changes associated with DM-TB comorbidity is imperative. In this study, streptozotocin (STZ)-induced DM C57BL/6 mice were aerosol infected with low dose (100-120 CFU) Mycobacterium tuberculosis H37Rv. At 3 weeks post infection (w.p.i.), multiple tissue mycobacterial load and metabolites were profiled. The liver proteome of DM-TB and controls were analyzed using quantitative proteomics, and multi-omics data were integrated. DM-TB mice showed dysregulated multi-tissue (lungs, liver, brain, kidney and thigh muscle) metabolism. In contrast, the mycobacterial burden in the lung, spleen and liver was similar at 3 w.p.i. in DM-TB and TB groups. Enrichment analysis of deregulated liver metabolites (n = 20; log2DM-TB/TB>±1.0) showed significant perturbation in cysteine-methionine, glycine-serine, BCAA and fatty acid metabolism. 60 out of 1660 identified liver proteins showed deregulation (log2DM-TB/TB>±1.0) and contributed from perturbed cysteine-methionine metabolism corroborating metabolomics data. In addition, amino acid biosynthesis, retinol metabolism and polyol biosynthetic process were also differentially enriched in the livers of DM-TB groups. Global correlation analysis of liver metabolome and proteome data showed a strong association between aspartic acid, pyruvic acid, leucine and isoleucine with CYP450 enzymes involved in retinol metabolism, while iminodiacetic acid, isoleucine and γ-aminobutyric acid (GABA) strong positive correlation involved in cysteine metabolism. Targeting perturbed cysteine metabolism using micro molecules, like DL-Propargylglycine, might help prevent liver damage in DM-TB comorbid conditions.

Unravelling the Metabolic Underpinnings of Gestational Diabetes Mellitus: A Comprehensive Mendelian Randomisation Analysis Identifying Causal Metabolites and Biological Pathways.

BACKGROUND: Gestational diabetes mellitus (GDM) has a strong genetic predisposition. Integrating metabolomics with Mendelian randomisation (MR) analysis offers a potent method to uncover the metabolic factors causally linked to GDM pathogenesis. OBJECTIVES: This study aims to identify specific metabolites and metabolic pathways causally associated with GDM susceptibility through a comprehensive MR analysis. Additionally, it seeks to explore the potential of these identified metabolites as circulating biomarkers for early GDM detection and risk assessment. Furthermore, it aims to evaluate the implicated metabolic pathways as potential therapeutic targets for preventive or interventional strategies against GDM. METHODS: A two-sample MR study was conducted using summary statistics from a metabolite genome-wide association study (GWAS) of 8299 individuals and a GDM GWAS comprising 13,039 cases and 197,831 controls. Rigorous criteria were applied to select robust genetic instruments for 850 metabolites. RESULTS: MR analysis revealed 47 metabolites exhibiting putative causal associations with GDM risk. Among these, five metabolites demonstrated statistically significant associations after multiple-testing correction: Beta-citrylglutamate, Isobutyrylcarnitine (c4), 1,2-dilinoleoyl-GPC (18:2/18:2), Alliin and Cis-3,4-methyleneheptanoylcarnitine. Importantly, all these metabolites exhibited protective effects against GDM development. Additionally, metabolic pathway enrichment analysis implicated the methionine metabolism and spermidine and spermine biosynthesis pathways in the pathogenesis of GDM. CONCLUSION: This comprehensive MR study has robustly identified specific metabolites and metabolic pathways with causal links to GDM susceptibility. These findings provide novel insights into the metabolic underpinnings of GDM aetiology and offer promising translational implications. The identified metabolites could serve as potential circulating biomarkers for early detection and risk stratification, while the implicated metabolic pathways may represent therapeutic targets for preventive or interventional strategies against GDM.

The role of the mitochondrial trans-sulfuration in cerebro-cardio renal dysfunction during trisomy down syndrome.

One in 700 children is born with the down syndrome (DS). In DS, there is an extra copy of X chromosome 21 (trisomy). Interestingly, the chromosome 21 also contains an extra copy of the cystathionine beta synthase (CBS) gene. The CBS activity is known to contribute in mitochondrial sulfur metabolism via trans-sulfuration pathway. We hypothesize that due to an extra copy of the CBS gene there is hyper trans-sulfuration in DS. We believe that understanding the mechanism of hyper trans-sulfuration during DS will be important in improving the quality of DS patients and towards developing new treatment strategies. We know that folic acid "1-carbon" metabolism (FOCM) cycle transfers the "1-carbon" methyl group to DNA (H3K4) via conversion of s-adenosyl methionine (SAM) to s-adenosyl homocysteine (SAH) by DNMTs (the gene writers). The demethylation reaction is carried out by ten-eleven translocation methylcytosine dioxygenases (TETs; the gene erasers) through epigenetics thus turning the genes off/on and opening the chromatin by altering the acetylation/HDAC ratio. The S-adenosyl homocysteine hydrolase (SAHH) hydrolyzes SAH to homocysteine (Hcy) and adenosine. The Hcy is converted to cystathionine, cysteine and hydrogen sulfide (H2S) via CBS/cystathioneγ lyase (CSE)/3-mercaptopyruvate sulfurtransferase (3MST) pathways. Adenosine by deaminase is converted to inosine and then to uric acid. All these molecules remain high in DS patients. H2S is a potent inhibitor of mitochondrial complexes I-IV, and regulated by UCP1. Therefore, decreased UCP1 levels and ATP production can ensue in DS subjects. Interestingly, children born with DS show elevated levels of CBS/CSE/3MST/Superoxide dismutase (SOD)/cystathionine/cysteine/H2S. We opine that increased levels of epigenetic gene writers (DNMTs) and decreased in gene erasers (TETs) activity cause folic acid exhaustion, leading to an increase in trans-sulphuration by CBS/CSE/3MST/SOD pathways. Thus, it is important to determine whether SIRT3 (inhibitor of HDAC3) can decrease the trans-sulfuration activity in DS patients. Since there is an increase in H3K4 and HDAC3 via epigenetics in DS, we propose that sirtuin-3 (Sirt3) may decrease H3K4 and HDAC3 and hence may be able to decrease the trans-sulfuration in DS. It would be worth to determine whether the lactobacillus, a folic acid producing probiotic, mitigates hyper-trans-sulphuration pathway in DS subjects. Further, as we know that in DS patients the folic acid is exhausted due to increase in CBS, Hcy and re-methylation. In this context, we suggest that folic acid producing probiotics such as lactobacillus might be able to improve re-methylation process and hence may help decrease the trans-sulfuration pathway in the DS patients.

Hepatocyte PPARγ contributes to the progression of non-alcoholic steatohepatitis in male and female obese mice.

Non-alcoholic steatohepatitis (NASH) is associated with obesity and increased expression of hepatic peroxisome proliferator-activated receptor γ (PPARγ). However, the relevance of hepatocyte PPARγ in NASH associated with obesity is still poorly understood. In this study, hepatocyte PPARγ was knocked out (PpargΔHep) in male and female mice after the development of high-fat diet-induced obesity. The diet-induced obese mice were then maintained on their original diet or switched to a high fat, cholesterol, and fructose (HFCF) diet to induce NASH. Hepatic PPARγ expression was mostly derived from hepatocytes and increased by high fat diets. PpargΔHep reduced HFCF-induced NASH progression without altering steatosis, reduced the expression of key genes involved in hepatic fibrosis in HFCF-fed male and female mice, and decreased the area of collagen-stained fibrosis in the liver of HFCF-fed male mice. Moreover, transcriptomic and metabolomic data suggested that HFCF-diet regulated hepatic amino acid metabolism in a hepatocyte PPARγ-dependent manner. PpargΔHep increased betaine-homocysteine s-methyltransferase expression and reduced homocysteine levels in HFCF-fed male mice. In addition, in a cohort of 102 obese patients undergoing bariatric surgery with liver biopsies, 16 cases were scored with NASH and were associated with increased insulin resistance and hepatic PPARγ expression. Our study shows that hepatocyte PPARγ expression is associated with NASH in mice and humans. In male mice, hepatocyte PPARγ negatively regulates methionine metabolism and contributes to the progression of fibrosis.

Mechanistic safety assessment via multi-omic characterisation of systemic pathway perturbations following in vivo MAT2A inhibition.

The tumour suppressor p16/CDKN2A and the metabolic gene, methyl-thio-adenosine phosphorylase (MTAP), are frequently co-deleted in some of the most aggressive and currently untreatable cancers. Cells with MTAP deletion are vulnerable to inhibition of the metabolic enzyme, methionine-adenosyl transferase 2A (MAT2A), and the protein arginine methyl transferase (PRMT5). This synthetic lethality has paved the way for the rapid development of drugs targeting the MAT2A/PRMT5 axis. MAT2A and its liver- and pancreas-specific isoform, MAT1A, generate the universal methyl donor S-adenosylmethionine (SAM) from ATP and methionine. Given the pleiotropic role SAM plays in methylation of diverse substrates, characterising the extent of SAM depletion and downstream perturbations following MAT2A/MAT1A inhibition (MATi) is critical for safety assessment. We have assessed in vivo target engagement and the resultant systemic phenotype using multi-omic tools to characterise response to a MAT2A inhibitor (AZ'9567). We observed significant SAM depletion and extensive methionine accumulation in the plasma, liver, brain and heart of treated rats, providing the first assessment of both global SAM depletion and evidence of hepatic MAT1A target engagement. An integrative analysis of multi-omic data from liver tissue identified broad perturbations in pathways covering one-carbon metabolism, trans-sulfuration and lipid metabolism. We infer that these pathway-wide perturbations represent adaptive responses to SAM depletion and confer a risk of oxidative stress, hepatic steatosis and an associated disturbance in plasma and cellular lipid homeostasis. The alterations also explain the dramatic increase in plasma and tissue methionine, which could be used as a safety and PD biomarker going forward to the clinic.

NAD+ enhances the activity and thermostability of S-adenosyl-L-homocysteine hydrolase from Pyrococcus horikoshii OT3.

S-Adenosyl-L-methionine (SAM) and S-adenosyl-L-homocysteine (SAH) are important biochemical intermediates. SAM is the major methyl donor for diverse methylation reactions in vivo. The SAM to SAH ratio serves as a marker of methylation capacity. Stable isotope-labeled SAM and SAH are used to measure this ratio with high sensitivity. SAH hydrolase (EC 3.13.2.1; SAHH), which reversibly catalyzes the conversion of adenosine and L-homocysteine to SAH, is used to produce labeled SAH. To produce labeled SAH with high efficiency, we focused on the SAHH of Pyrococcus horikoshii OT3, a thermophilic archaeon. We prepared recombinant P. horikoshii SAHH using Escherichia coli and investigated its enzymatic properties. Unexpectedly, the optimum temperature and thermostability of P. horikoshii SAHH were much lower than its optimum growth temperature. However, addition of NAD+ to the reaction mixture shifted the optimum temperature of P. horikoshii SAHH to a higher temperature, suggesting that NAD+ stabilizes the structure of the enzyme.

A mitochondrial STAT3-methionine metabolism axis promotes ILC2-driven allergic lung inflammation.

BACKGROUND: Group 2 innate lymphoid cells (ILC2s), the innate counterpart of TH2 cells, play a critical role in type 2 immune responses. However, the molecular regulatory mechanisms of ILC2s are still unclear. OBJECTIVE: The aim of this study was to explore the importance of signal transducer and activator of transcription 3 (STAT3) to ILC2 function in allergic lung inflammation. METHODS: Acute and chronic asthma models were established by intranasal administration of the protease allergen papain in VavicreStat3fl/fl, Il5tdtomato-creStat3fl/fl, and RorccreStat3fl/fl mice to verify the necessity of functional STAT3 for ILC2 allergic response. The intrinsic role of STAT3 in regulating ILC2 function was examined by generation of bone marrow chimera mice. The underlying mechanism was studied through confocal imaging, metabolomics analysis, and chromatin immunoprecipitation quantitative PCR. RESULTS: STAT3 is essential for ILC2 effector function and promotes ILC2-driven allergic inflammation in the lung. Mechanistically, the alarmin cytokine IL-33 induces a noncanonical STAT3 phosphorylation at serine 727 in ILC2s, leading to translocation of STAT3 into the mitochondria. Mitochondrial STAT3 further facilitates adenosine triphosphate synthesis to fuel the methionine cycle and generation of S-adenosylmethionine, which supports the epigenetic reprogramming of type 2 cytokines in ILC2s. STAT3 deficiency, inhibition of STAT3 mitochondrial translocation, or blockade of methionine metabolism markedly dampened the ILC2 allergic response and ameliorated allergic lung inflammation. CONCLUSION: The mitochondrial STAT3-methionine metabolism pathway is a key regulator that shapes ILC2 effector function through epigenetic regulation, and the related proteins or metabolites represent potential therapeutic targets for allergic lung inflammation.

Comparison of the Metabolomics of Different Dendrobium Species by UPLC-QTOF-MS.

Dendrobium Sw. (family Orchidaceae) is a renowned edible and medicinal plant in China. Although widely cultivated and used, less research has been conducted on differential Dendrobium species. In this study, stems from seven distinct Dendrobium species were subjected to UPLC-QTOF-MS/MS analysis. A total of 242 metabolites were annotated, and multivariate statistical analysis was employed to explore the variance in the extracted metabolites across the various groups. The analysis demonstrated that D. nobile displays conspicuous differences from other species of Dendrobium. Specifically, D. nobile stands out from the remaining six taxa of Dendrobium based on 170 distinct metabolites, mainly terpene and flavonoid components, associated with cysteine and methionine metabolism, flavonoid biosynthesis, and galactose metabolism. It is believed that the variations between D. nobile and other Dendrobium species are mainly attributed to three metabolite synthesis pathways. By comparing the chemical composition of seven species of Dendrobium, this study identified the qualitative components of each species. D. nobile was found to differ significantly from other species, with higher levels of terpenoids, flavonoids, and other compounds that are for the cardiovascular field. By comparing the chemical composition of seven species of Dendrobium, these qualitative components have relevance for establishing quality standards for Dendrobium.

Metabolic and nutritional responses of Nile tilapia juveniles to dietary methionine sources.

Commercial diets for tilapia juveniles contain high levels of plant protein sources. Soybean meal has been utilised due to its high protein content; however, soy-based diets are limited in methionine (Met) and require its supplementation to fulfil fish requirements. dl-Methinone (dl-Met) and Ca bis-methionine hydroxyl analogue (MHA-Ca) are synthetic Met sources supplemented in aquafeeds, which may differ in biological efficiency due to structural differences. The present study evaluated the effect of both methionine sources on metabolism and growth of Nile tilapia. A growth trial was performed using three isonitrogenous and isoenergetic diets, containing plant ingredients as protein sources: DLM and MHA diets were supplemented on equimolar levels of Met, while REF diet was not supplemented. Hepatic free Met and one-carbon metabolites were determined in fish fed for 57 d. Metabolism of dl-Met and MHA was analysed by an in vivo time-course trial using 14C-labelled tracers. Only dl-Met supplementation significantly increased final body weight and improved feed conversion and protein efficiency ratios compared with the REF diet. Our findings indicate that Met in DLM fed fish follows the transsulphuration pathway, while in fish fed MHA and REF diets it is remethylated. The in vivo trial revealed that 14C-dl-Met is absorbed faster and more retained than 14C-MHA, resulting in a greater availability of free Met in the tissues when fish is fed with DLM diet. Our study indicates that dietary dl-Met supplementation improves growth performance and N retention, and that Met absorption and utilisation are influenced by the dietary source in tilapia juveniles.

Ontogeny of hepatic methionine catabolic enzyme activities (Transmethylation and Transsulphuration) and associated physiological amino acids in E10-21 chick embryos and D1-49 broilers.

Developmental changes in hepatic methionine adenosyltransferase, cystathionine ß-synthase, cystathionase, and glycine N-methyltransferase were determined in broiler chick embryos and hatched chicks by using radiometric and spectrometric methods. Hepatic free methionine, S-adenosylmethionine, S-adenosylhomocysteine, homocysteine, cystathionine, and cysteine levels were also investigated. Results showed an increase in hepatic MAT activity from E10 to E21 during embryogenesis, suggesting greater transmethylation rates throughout the rapid embryonic growth and development period. A strong positive correlation between embryo BW and MAT activity also supports this idea. The MAT specific activity continued to increase after hatching, but there was a negative correlation between chick BW and MAT activities from D1 to D49. This may indicate different MAT isozymes exist for chick embryo hepatic tissue compared to hepatic tissue of hatched chick and growing broilers. The developmental pattern of MAT isozymes could be critical for methionine metabolism to cope with the demand imposed on the embryo, chicks, and growing broilers. Additionally, the specific activity of hepatic CBS in chick embryos was determined to be lower compared to that observed in older broilers (35 and 49 days). Since liver CBS specific activity is at the lowest point from D1-7 in young chicks, the ability to convert adequate homocysteine to cysteine through transsulphuration may be limiting for cysteine synthesis at this time. Steady-state hepatic homocysteine levels in chick embryos and chicks may be a function of the rates of homocysteine formation, remethylation, and catabolism via the transsulphuration pathway. The present study indicates young chicks from D1 to D7 may have a limited ability for adequate transsulphuration; therefore, dietary cystine may be needed for optimum performance.

Revisiting the attempts to fortify methionine content in plant seeds.

The sulfur-containing amino acid methionine belongs to the group of essential amino acids, meaning that humans and animals must consume it in their diets. However, plant seeds have low levels of methionine, limiting their nutritional potential. For this reason, efforts have been made over the years to increase methionine levels in seeds. Here, we summarize these efforts and focus particularly on those utilizing diverse genetic and molecular tools. Four main approaches are described: (i) expression of methionine-rich storage proteins in a seed-specific manner to incorporate more soluble methionine into the protein fraction; (ii) reduction of methionine-poor storage proteins inside the seeds to reinforce the accumulation of methionine-rich proteins; (iii) silencing methionine catabolic enzymes; and (iv) up-regulation of key biosynthetic enzymes participating in methionine synthesis. We focus on the biosynthetic genes that operate de novo in seeds and that belong to the sulfur assimilation and aspartate family pathways, as well as genes from the methionine-specific pathway. We also include those enzymes that operate in non-seed tissues that contribute to the accumulation of methionine in seeds, such as S-methylmethionine enzymes. Finally, we discuss the biotechnological potential of these manipulations to increase methionine content in plant seeds and their effect on seed germination.

Tissue nonautonomous effects of fat body methionine metabolism on imaginal disc repair in Drosophila.

Regulatory mechanisms for tissue repair and regeneration within damaged tissue have been extensively studied. However, the systemic regulation of tissue repair remains poorly understood. To elucidate tissue nonautonomous control of repair process, it is essential to induce local damage, independent of genetic manipulations in uninjured parts of the body. Herein, we develop a system in Drosophila for spatiotemporal tissue injury using a temperature-sensitive form of diphtheria toxin A domain driven by the Q system to study factors contributing to imaginal disc repair. Using this technique, we demonstrate that methionine metabolism in the fat body, a counterpart of mammalian liver and adipose tissue, supports the repair processes of wing discs. Local injury to wing discs decreases methionine and S-adenosylmethionine, whereas it increases S-adenosylhomocysteine in the fat body. Fat body-specific genetic manipulation of methionine metabolism results in defective disc repair but does not affect normal wing development. Our data indicate the contribution of tissue interactions to tissue repair in Drosophila, as local damage to wing discs influences fat body metabolism, and proper control of methionine metabolism in the fat body, in turn, affects wing regeneration.

In-Cell Synthesis of Bioorthogonal Alkene Tag S-Allyl-Homocysteine and Its Coupling with Reprogrammed Translation.

In this study, we report our initial results on in situ biosynthesis of S-allyl-l-homocysteine (Sahc) by simple metabolic conversion of allyl mercaptan in Escherichia coli, which served as the host organism endowed with a direct sulfhydration pathway. The intracellular synthesis we describe in this study is coupled with the direct incorporation of Sahc into proteins in response to methionine codons. Together with O-acetyl-homoserine, allyl mercaptan was added to the growth medium, followed by uptake and intracellular reaction to give Sahc. Our protocol efficiently combined the in vivo synthesis of Sahc via metabolic engineering with reprogrammed translation, without the need for a major change in the protein biosynthesis machinery. Although the system needs further optimisation to achieve greater intracellular Sahc production for complete protein labelling, we demonstrated its functional versatility for photo-induced thiol-ene coupling and the recently developed phosphonamidate conjugation reaction. Importantly, deprotection of Sahc leads to homocysteine-containing proteins-a potentially useful approach for the selective labelling of thiols with high relevance in various medical settings.

Alterations of Methionine Metabolism as Potential Targets for the Prevention and Therapy of Hepatocellular Carcinoma.

Several researchers have analyzed the alterations of the methionine cycle associated with liver disease to clarify the pathogenesis of human hepatocellular carcinoma (HCC) and improve the preventive and the therapeutic approaches to this tumor. Different alterations of the methionine cycle leading to a decrease of S-adenosylmethionine (SAM) occur in hepatitis, liver steatosis, liver cirrhosis, and HCC. The reproduction of these changes in MAT1A-KO mice, prone to develop hepatitis and HCC, demonstrates the pathogenetic role of MAT1A gene under-regulation associated with up-regulation of the MAT2A gene (MAT1A:MAT2A switch), encoding the SAM synthesizing enzymes, methyladenosyltransferase I/III (MATI/III) and methyladenosyltransferase II (MATII), respectively. This leads to a rise of MATII, inhibited by the reaction product, with a consequent decrease of SAM synthesis. Attempts to increase the SAM pool by injecting exogenous SAM have beneficial effects in experimental alcoholic and non-alcoholic steatohepatitis and hepatocarcinogenesis. Mechanisms involved in hepatocarcinogenesis inhibition by SAM include: (1) antioxidative effects due to inhibition of nitric oxide (NO•) production, a rise in reduced glutathione (GSH) synthesis, stabilization of the DNA repair protein Apurinic/Apyrimidinic Endonuclease 1 (APEX1); (2) inhibition of c-myc, H-ras, and K-ras expression, prevention of NF-kB activation, and induction of overexpression of the oncosuppressor PP2A gene; (3) an increase in expression of the ERK inhibitor DUSP1; (4) inhibition of PI3K/AKT expression and down-regulation of C/EBPα and UCA1 gene transcripts; (5) blocking LKB1/AMPK activation; (6) DNA and protein methylation. Different clinical trials have documented curative effects of SAM in alcoholic liver disease. Furthermore, SAM enhances the IFN-α antiviral activity and protects against hepatic ischemia-reperfusion injury during hepatectomy in HCC patients with chronic hepatitis B virus (HBV) infection. However, although SAM prevents experimental tumors, it is not curative against already established experimental and human HCCs. The recent observation that the inhibition of MAT2A and MAT2B expression by miRNAs leads to a rise of endogenous SAM and strong inhibition of cancer cell growth could open new perspectives to the treatment of HCC.

Alteration of S-adenosylhomocysteine levels affects lignin biosynthesis in switchgrass.

Methionine (Met) synthesized from aspartate is a fundamental amino acid needed to produce S-adenosylmethionine (SAM) that is an important cofactor for the methylation of monolignols. As a competitive inhibitor of SAM-dependent methylation, the effect of S-adenosylhomocysteine (SAH) on lignin biosynthesis, however, is still largely unknown in plants. Expression levels of Cystathionine γ-synthase (PvCGS) and S-adenosylhomocysteine hydrolase 1 (PvSAHH1) were down-regulated by RNAi technology, respectively, in switchgrass, a dual-purpose forage and biofuel crop. The transgenic switchgrass lines were subjected to studying the impact of SAH on lignin biosynthesis. Our results showed that down-regulation of PvCGS in switchgrass altered the accumulation of aspartate-derived and aromatic amino acids, reduced the content of SAH, enhanced lignin biosynthesis and stunted plant growth. In contrast, down-regulation of PvSAHH1 raised SAH levels in switchgrass, impaired the biosynthesis of both guaiacyl and syringyl lignins and therefore significantly increased saccharification efficiency of cell walls. This work indicates that SAH plays a crucial role in monolignol methylation in switchgrass. Genetic regulation of either PvCGS or PvSAHH1 expression in switchgrass can change intracellular SAH contents and SAM to SAH ratios and therefore affect lignin biosynthesis. Thus, our study suggests that genes involved in Met metabolism are of interest as new valuable targets for cell wall bioengineering in future.

Chronic Alcohol Exposure Differentially Alters One-Carbon Metabolism in Rat Liver and Brain.

BACKGROUND: Epigenetic mechanisms such as DNA methylation play an important role in regulating the pathophysiology of alcoholism. Chronic alcohol exposure leads to behavioral changes as well as decreased expression of genes associated with synaptic plasticity. In the liver, it has been documented that chronic alcohol exposure impairs methionine synthase (Ms) activity leading to a decrease in S-adenosyl methionine/S-adenosyl homocysteine (SAM/SAH) ratio which results in DNA hypomethylation; however, it is not known whether similar alterations of SAM and SAH levels are also produced in brain. METHODS: Male adult Sprague Dawley rats were fed chronically with Lieber-DeCarli ethanol (EtOH) (9% v/v) or control diet. The EtOH-diet-fed rats were withdrawn for 0 and 24 hours. The cerebellum and liver tissues were dissected and used to investigate changes in one-carbon metabolism, SAM, and SAH levels. RESULTS: We found that chronic EtOH exposure decreased SAM levels, SAM/SAH ratio, Ms, methylene tetrahydrofolate reductase, and betaine homocysteine methyltransferase (Bhmt) expression and increased methionine adenosyltransferase-2b (Mat2b) but not Mat2a expression in the liver. In contrast, chronic EtOH exposure decreased SAH levels, increased SAM/SAH ratio and the expression of Mat2a and S-adenosyl homocysteine hydrolase, while the levels of SAM or Bhmt expression in cerebellum remained unaltered. However, in both liver and cerebellum, chronic EtOH exposure decreased the expression of Ms and increased Mat2b expression. All chronic EtOH-induced changes of one-carbon metabolism in cerebellum, but not liver, returned to near-normal levels during EtOH withdrawal. CONCLUSIONS: These results indicate a decreased "methylation index" in liver and an increased "methylation index" in cerebellum. The opposing changes of the "methylation index" suggest altered DNA methylation in liver and cerebellum, thus implicating one-carbon metabolism in the pathophysiology of alcoholism.

Choline and betaine ameliorate liver lipid accumulation induced by vitamin B6 deficiency in rats.

We investigated the efficacy of supplementing the diet with choline or betaine in ameliorating lipid accumulation induced by vitamin B6 (B6) deficiency in rat liver. Male Wistar rats were fed a control, B6-deficient, choline-supplemented (2, 4, or 6 g choline bitartrate/kg diet) B6-deficient diet or betaine-supplemented (1, 2, or 4 g betaine anhydrous/kg diet) B6-deficient diet for 35 d; all diets contained 9 g L-methionine (Met)/kg diet. Choline or betaine supplementation attenuated liver lipid deposition and restored plasma lipid profiles to control levels. These treatments restored the disruptions in Met metabolism and the phosphatidylcholine (PC)/phosphatidylethanolamine (PE) ratio induced by B6 deficiency in liver microsomes. These results suggest that choline and betaine ameliorated liver lipid accumulation induced by B6 deficiency via recovery of Met metabolism and very low-density lipoprotein secretion by restoring the supply of PC derived from PE.

Changes in Methionine Metabolism and Histone H3 Trimethylation Are Linked to Mitochondrial Defects in Multiple Sclerosis.

Mitochondrial changes, including decreased expression of electron transport chain subunit genes and impaired energetic, have been reported in multiple sclerosis (MS), but the mechanisms involved in these changes are not clear. To determine whether epigenetic mechanisms are involved, we measured the concentrations of methionine metabolites by liquid chromatography tandem mass spectrometry, histone H3 methylation patterns, and markers of mitochondrial respiration in gray matter from postmortem MS and control cortical samples. We found decreases in respiratory markers as well as decreased concentrations of the methionine metabolites S-adenosylmethionine, betaine, and cystathionine in MS gray matter. We also found expression of the enzyme betaine homocysteine methyltransferase in cortical neurons. This enzyme catalyzes the remethylation of homocysteine to methionine, with betaine as the methyl donor, and has previously been thought to be restricted to liver and kidney in the adult human. Decreases in the concentration of the methyl donor betaine were correlated with decreases in histone H3 trimethylation (H3K4me3) in NeuN+ neuronal nuclei in MS cortex compared with controls. Mechanistic studies demonstrated that H3K4me3 levels and mitochondrial respiration were reduced in SH-SY5Y cells after exposure to the nitric oxide donor sodium nitroprusside, and betaine was able to rescue H3K4me3 levels and respiratory capacity in these cells. Chromatin immunoprecipitation experiments showed that betaine regulates metabolic genes in human SH-SY5Y neuroblastoma cells. These data suggest that changes to methionine metabolism may be mechanistically linked to changes in neuronal energetics in MS cortex. SIGNIFICANCE STATEMENT: For decades, it has been observed that vitamin B12 deficiency and multiple sclerosis (MS) share certain pathological changes, including conduction disturbances. In the present study, we have found that vitamin B12-dependent methionine metabolism is dysregulated in the MS brain. We found that concentrations of the methyl donor betaine are decreased in MS cortex and are correlated with reduced levels of the histone H3 methyl mark H3K4me3 in neurons. Cell culture and chromatin immunoprecipitation-seq data suggest that these changes may lead to defects in mitochondria and impact neuronal energetics. These data have uncovered a novel pathway linking methionine metabolism with mitochondrial respiration and have important implications for understanding mechanisms involved in neurodegeneration in MS.

The remarkable S. Harvey Mudd - A reminiscence.

Harvey Mudd was the father of methionine metabolic disorders. Beginning with his identification of the enzyme defect in homocystinuria, he co-discovered cobalamin C disorder as the first known human disorder of vitamin B12 metabolism, thereby extending our concept of homocystinuria as a key feature of related disorders rather than a single disease, and identified new disorders that produce hypermethioninemia. He had no equal in our understanding of how critical methionine metabolism is to human homeostasis.

Accumulation of lipid in rat liver was induced by vitamin B6 deficiency and was ameliorated by supplemental phosphatidylcholine in the diet.

We investigated the efficacy of supplementing the diet with pteroylmonoglutamic acid (PGA), choline, or phosphatidylcholine (PC) in ameliorating the lipid accumulation in rat liver that is induced by vitamin B6 (B6) deficiency. In Experiment 1, male Wistar rats were fed a control, B6-deficient, or PGA-, choline-, or PC-supplemented (10 mg, 4 g, and 6.3 g/kg of diet, respectively) B6-deficient diet containing l-methionine at 9 g/kg of diet for 35 days. In Experiment 2, rats were fed a control, B6-deficient, or PC-supplemented (at 3.15, 6.3, or 12.6 g PC/kg of diet) B6-deficient diet for 35 days. Choline or PC supplementation ameliorated liver lipid deposition and returned plasma lipids to normal. Judging from these results, it appeared that B6 deficiency decreased the synthesis of PC in the liver, thereby decreasing the secretion of very low-density lipoproteins, and in consequence producing lipid accumulation in the liver and reductions of plasma lipids.