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Background/aim Methionine addiction is a general and fundamental hallmark of cancer due to the excess use of methionine for transmethylation reactions, termed the "Hoffman Effect". Methionine addiction has been shown to be a highly-effective target for cancer therapy by methionine restriction with oral recombinant methioninase (o-rMETase) in preclinical studies, including patient- derived orthotopic xenograft (PDOX) mouse models of cancer. A clinical study of o-rMETase as a supplement showed a 70% reduction of PSA levels in a patient with bone-metastatic prostate cancer. Materials and methods In the present study, two advanced prostate-cancer patients took o-rMETase as a supplement for approximately one month. Results One of the patients taking o-rMETase showed a 38% reduction of PSA levels and the second patient showed a 20% PSA reduction. Conclusion o-rMETase shows promise for treating patients with advanced prostate cancer.

The present review explores the nutritional and performance boosting functions of betaine in poultry and pig ration as a feed additive. Betaine, a trimethyl derivative of glycine is a normal metabolite in many plant and animal tissues. In all the animals, betaine is produced by oxidation of choline or supplied through feed. Its characteristics chemical structure makes betaine a dipolar zwitterion, conferring it osmoprotective and methyl donating properties. Over the past decades, numerous studies have investigated and published the favorable effects of betaine on performance in different animal species. Betaine is involved in transmethylation reactions for the synthesis of several metabolically active substances like creatine, carnitine etc. Also, it has shown evidence to increasing nutrient utilization, digestibility and methionine availability. Boosting immune status and reducing oxidative or heat stress are also some of the important functions of betaine. Most of the researches have specifically studied the growth promoting, carcass modulating, immune boosting or stress reducing properties of betaine in different species. Literature covering on different benefits of betaine as an essential feed supplement is not yet abundant. As both poultry and pig are important species of domesticated animals, betaine can be a better and cheaper, alternative feed supplement for enhancing the nutrient utilization and performance of poultry and pig. Therefore, an attempts have been made to delineate the functional effects of supplementary betaine in poultry and pig.

Developmental changes in hepatic methionine adenosyltransferase, cystathionine β-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.

Absztrakt: Bevezetés: A zsírmáj kialakulhat betegségek, műtétek, gyógyszerek, éhezés vagy túlzott mértékű alkoholfogyasztás következtében, de a helytelen táplálkozást, a zsírbő és rostszegény étkezést tartják az elsődleges oknak. A nem alkoholos zsírmájbetegség a lakosság 20–30%-át érintheti. A táplálkozási eredetű zsírmájat korai stádiumban általában szövődménymentes májkárosodásnak írják le. Célkitűzés: Kutatásaink célja a zsírdús táplálkozás bél–máj tengelyre gyakorolt hatásának a feltérképezése volt. Módszerek: Rutinlaboratóriumi, analitikai és szövettani módszerekkel patkányokban vizsgáltuk a bél és a máj redoxparamétereit, valamint a máj zsírsavösszetételét és elemtartalmát. Eredmények: A máj elzsírosodása során a vérszérumban mérhető májenzim- és metabolitértékek eltérései mellett a redox-homeosztázis mutatóiban is szignifikáns változások igazolódtak. Változás volt kimutatható a májban, a vérben és a belekben. A máj elzsírosodásának folyamata együtt jár a transzmetiláló képesség csökkenésével. Megváltozott a zsírsavösszetétel és a fémion-homeosztázis is a májban. Szövettani vizsgálatoknál a májlobulusok centrális részén a hepatocyták megduzzadtak, bennük zsírcseppek láthatók, piknotikus sejtmagok alakultak ki, ami a zsíros elfajulásra jellemző. A vékony- és vastagbél enterocytái sérültek, helyenként lelökődtek a felszínről, és gyulladásos reakciók figyelhetők meg a nyálkahártyában. Következtetés: Eredményeink alapján leszögezhető, hogy a zsírdús diéta okozta steatosis már korai stádiumban sem tekinthető egyszerűen reverzibilis változásnak, mert a változások kedvezőtlenül befolyásolják az egész szervezet redox-homeosztázisát, és egyben alapját képezhetik súlyos szív- és metabolikus betegségeknek, valamint siettethetik a gastrointestinalis tumorok kialakulását. Orv Hetil. 2020; 161(35): 1456–1465.

Background: Elevated maternal homocysteine concentration has been described as a consequence of disruptions in methionine and/or folate metabolism which is associated with adverse outcomes and several obstetric complications inclusive of neural tube defects in offspring such as spina bifida, preeclampsia and spontaneous abortion. Aims and Objectives: The current study investigated the influence of maternal homocysteine metabolism in singleton pregnant women on the prevalence of Spina bifida in Osun State. Materials and Methods: Sixty (60) subjects attending Ladoke Akintola University of Technology Teaching Hospital and State Specialist Hospital in Osogbo, Osun State were randomly recruited for the study which spanned from January 2018 through April 2018 involving 30 apparently healthy singleton pregnant women classified according to gestational age and 30 apparently healthy age- and socio-economic status-matched non-pregnant women as controls. Haematocrit, haemoglobin concentration, total white cell count as well as the levels of homocysteine concentration were assessed. Results: Haematocrit and haemoglobin concentration in all three trimesters were significantly (p< 0.05) decreased when compared with their non-pregnant counterparts. There was however a significant (p< 0.05) increase in the expression of circulating homocysteine in all three trimesters compared to the control group, and an observed steady decline in the second trimester through the third trimester. Conclusion: These findings therefore imply that gestational perturbation of homocysteine metabolism may contribute to reduced risk of spina bifida in Osun State, Nigeria.

To date, it has been established that hyperhomocysteinemia plays a significant role in the development and progression of many diseases. The accumulation of homocysteine occurs due to a violation of the relationship between its production and excretion from the body. The liver plays an important role in the metabolism of homocysteine, because it undergoes most of the reactions of its transmethylation, and, therefore, it is the first to be adversely affected. The aim of the study is to identify the features of electron microscopic changes in the liver structure of young rats with hyperhomocysteinemia. The experimental study was performed on 22 white nonlinear young (1-2 months) male rats, which were divided into a control group and an experimental group. A model of persistent hyperhomocysteinemia was created by administering to rats the experimental group of thiolactone homocysteine at a dose of 200 mg/kg body weight intragastrically for 60 days. The study of ultrastructural changes in the liver of rats was performed using an electron microscope PEM-125K. It was found that the introduction of thiolactone homocysteine at a dose of 200 mg/kg in rats led to the development of degenerative changes in hepatocytes. Changes in the structure of liver cells manifested themselves in the form of edema of the cytoplasm and mitochondria, destruction of mitochondrial cristae, dilation of the tubules of the granular endoplasmic reticulum and tanks of the Golgi complex. The activity of fat-accumulating liver cells and stellate macrophages is characteristically. In the lumens of the sinusoidal capillaries found sweeter shaped blood elements, the cytoplasm of endothelial cells had signs of edema. Thus, in experimental hyperhomocysteinemia revealed changes at the ultrastructural level in all structural components of the liver of young rats. The identified changes are compensatory-adaptive in nature and are reversible.

Methionine adenosyltransferase (MAT) catalyzes the adenosine 5'-triphosphate (ATP) and l-methionine (l-Met) dependent formation of S-adenosyl-l-methionine (SAM), the principal methyl donor of most biological transmethylation reactions. We carried out in-depth kinetic studies to further understand its mechanism and interaction with a potential regulator, Mat2B. The initial velocity pattern and results of product inhibition by SAM, phosphate, and pyrophosphate, and dead-end inhibition by the l-Met analog cycloleucine (l-cLeu) suggest that Mat2A follows a strictly ordered kinetic mechanism where ATP binds before l-Met and with SAM released prior to random release of phosphate and pyrophosphate. Isothermal titration calorimetry (ITC) showed binding of ATP to Mat2A with a Kd of 80 ± 30 μM, which is close to the Km(ATP) of 50 ± 10 μM. In contrast, l-Met or l-cLeu showed no binding to Mat2A in the absence of ATP; however, binding to l-cLeu was observed in the presence of ATP. The ITC results are fully consistent with the product and dead-inhibition results obtained. We also carried out kinetic studies in the presence of the physiological regulator Mat2B. Under conditions where all Mat2A is found in complex with Mat2B, no significant change in the kinetic parameters was observed despite confirmation of a very high binding affinity of Mat2A to Mat2B (Kd of 6 ± 1 nM). Finally, we found that while Mat2A is unstable at low concentrations (

Hyperhomocysteneinemia (HHcy) is common in the general population and is a risk factor for atherosclerosis by mechanisms that are still elusive. A hypomethylated status of epigenetically relevant targets may contribute to the vascular toxicity associated with HHcy. Ketogenic diets (KD) are diets with a severely restricted amount of carbohydrates that are being widely used, mainly for weight-loss purposes. However, studies associating nutritional ketosis and HHcy are lacking. This pilot study investigates the effects of mild HHcy induced by nutritional manipulation of the methionine metabolism in the absence of dietary carbohydrates on disease progression and specific epigenetic changes in the apolipoprotein-E deficient (apoE–/–) mouse model. ApoE–/– mice were either fed a KD, a diet with the same macronutrient composition but low in methyl donors (low methyl KD, LMKD), or control diet. After 4, 8 or 12 weeks plasma was collected for the quantification of: (1) nutritional ketosis, (i.e., the ketone body beta-hydroxybutyrate using a colorimetric assay), (2) homocysteine by HPLC, (3) the methylating potential S-adenosylmethionine to S-adenosylhomocysteine ratio (AdoHcy/AdoMet) by LC-MS/MS, and (4) the inflammatory cytokine monocyte chemoattractant protein 1 (MCP1) by ELISA. After 12 weeks, aortas were collected to assess: (1) the vascular AdoHcy/AdoMet ratio, (2) the volume of atherosclerotic lesions by high-field magnetic resonance imaging (14T-MRI), and (3) the content of specific epigenetic tags (H3K27me3 and H3K27ac) by immunofluorescence. The results confirmed the presence of nutritional ketosis in KD and LMKD mice but not in the control mice. As expected, mild HHcy was only detected in the LMKD-fed mice. Significantly decreased MCP1 plasma levels and plaque burden were observed in control mice versus the other two groups, together with an increased content of one of the investigated epigenetic tags (H3K27me3) but not of the other (H3K27ac). Moreover, we are unable to detect any significant differences at the p &lt, 0.05 level for MCP1 plasma levels, vascular AdoMet:AdoHcy ratio levels, plaque burden, and specific epigenetic content between the latter two groups. Nevertheless, the systemic methylating index was significantly decreased in LMKD mice versus the other two groups, reinforcing the possibility that the levels of accumulated homocysteine were insufficient to affect vascular transmethylation reactions. Further studies addressing nutritional ketosis in the presence of mild HHcy should use a higher number of animals and are warranted to confirm these preliminary observations.

Objective. S-Adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) are indicators of global transmethylation and may play an important role as markers of severity of COVID-19. Methods. The levels of plasma SAM and SAH were determined in patients admitted with COVID-19 ( n = 56 , mean age = 61 ). Lung injury was identified by computed tomography (CT) in accordance with the CT0-4 classification. Results. SAM was found to be a potential marker of lung damage risk in COVID-19 patients ( SAM > 80 nM ; CT3,4 vs. CT 0-2: relative ratio (RR) was 3.0; p = 0.0029 ). SAM / SAH > 6.0 was also found to be a marker of lung injury (CT2-4 vs. CT0,1: RR = 3.47 , p = 0.0004 ). There was a negative association between SAM and glutathione level ( ρ = − 0.343 , p = 0.011 ). Interleukin-6 (IL-6) levels were associated with SAM ( ρ = 0.44 , p = 0.01 ) and SAH ( ρ = 0.534 , p = 0.001 ) levels. Conclusions. A high SAM level and high methylation index are associated with the risk of lung injury in patients with COVID-19. The association of SAM with IL-6 and glutathione indicates an important role of transmethylation in the development of cytokine imbalance and oxidative stress in patients with COVID-19.

Methionine is an aliphatic, sulfur-containing, essential amino acid that has been demonstrated to have crucial roles in metabolism, innate immunity, and activation of endogenous antioxidant enzymes, including methionine sulfoxide reductase A/B and the biosynthesis of glutathione to counteract oxidative stress. Still, methionine restriction avoids altered methionine/transmethylation metabolism, thus reducing DNA damage and possibly avoiding neurodegenerative processes. In this study, we wanted to study the preventive effects of methionine in counteracting 6-hydroxydopamine (6-OHDA)-induced injury. In particular, we analyzed the protective effects of the amino acid L-methionine in an in vitro model of Parkinson’s disease and dissected the underlying mechanisms compared to the known antioxidant taurine to gain insights into the potential of methionine treatment in slowing the progression of the disease by maintaining mitochondrial functionality. In addition, to ascribe the effects of methionine on mitochondria and oxidative stress, methionine sulfoxide was used in place of methionine. The data obtained suggested that an L-methionine-enriched diet could be beneficial during aging to protect neurons from oxidative imbalance and mitochondrial dysfunction, thus preventing the progression of neurodegenerative processes.

The level of homoarginine (hArg) in terms of prognostic significance may exceed the natriuretic peptides and other well-known markers according to the latest data about the progression of cardiovascular diseases. The lack of data on the association of hArg levels with levels of other metabolites makes it difficult to understand its role in the pathogenesis of cardiovascular diseases. Relationships of hArg and other amino acids, including methionine (Met) and total homocysteine (tHcy), and their ratio in patients with ischemic heart disease were evaluated. The study included 74 patients with coronary heart disease (57 men and 17 women) aged 62 (57 - 67) years before coronary artery bypass surgery and 27 healthy people of similar age. In patients, the level of hArg was almost 2 times lower (p

Objective The loss of liver glycine N-methyltransferase (GNMT) promotes liver steatosis and the transition to hepatocellular carcinoma (HCC). Previous work showed endogenous glucose production is reduced in GNMT-null mice with gluconeogenic precursors being used in alternative biosynthetic pathways that utilize methyl donors and are linked to tumorigenesis. This metabolic programming occurs before the appearance of HCC in GNMT-null mice. The metabolic physiology that sustains liver tumor formation in GNMT-null mice is unknown. The studies presented here tested the hypothesis that nutrient flux pivots from glucose production to pathways that incorporate and metabolize methyl groups in GNMT-null mice with HCC. Methods 2H/13C metabolic flux analysis was performed in conscious, unrestrained mice lacking GNMT to quantify glucose formation and associated nutrient fluxes. Molecular analyses of livers from mice lacking GNMT including metabolomic, immunoblotting, and immunochemistry were completed to fully interpret the nutrient fluxes. Results GNMT knockout (KO) mice showed lower blood glucose that was accompanied by a reduction in liver glycogenolysis and gluconeogenesis. NAD+ was lower and the NAD(P)H-to-NAD(P)+ ratio was higher in livers of KO mice. Indices of NAD+ synthesis and catabolism, pentose phosphate pathway flux, and glutathione synthesis were dysregulated in KO mice. Conclusion Glucose precursor flux away from glucose formation towards pathways that regulate redox status increase in the liver. Moreover, synthesis and scavenging of NAD+ are both impaired resulting in reduced concentrations. This metabolic program blunts an increase in methyl donor availability, however, biosynthetic pathways underlying HCC are activated., Highlights • Loss of glycine N-methyltransferase results in hepatocellular carcinoma. • Metabolic reprogramming ensues to attenuate the increased S-adenosylmethionine. • The metabolic changes include dysregulated liver NAD+ homeostasis and redox state. • Liver glucose formation is reduced and precursors directed to biosynthetic pathways.

Gemcitabine (GEM) has been widely used for pancreatic cancer (PC) treatment but limited by the development of drug resistance. The agents that reverse its resistance and improve the chemo-sensitivity are urgently needed. S-Adenosylmethionine (SAM) is a precursor for polyamine biosynthesis in mammalian cells and plays a key role in biological transmethylation events. It is reported that SAM could be used as a therapeutic reagent for cancer treatments. In this study, we investigated the chemo-sensitization of SAM to potentiate the antitumor effect of GEM in PC. After treating PC cells with GEM and/or SAM, different subsequent experiments were performed. Results showed that SAM plus GEM could significantly inhibit the growth and proliferation of PC cells, and SAM acts synergistically with GEM. The combinative treatment could induce cell apoptosis and inhibit invasion and migration through JAK2/STAT3 inactivation. Inhibition of JAK2/STAT3 pathway significantly enhanced the pro-apoptotic effect of SAM, suggesting the key roles of JAK2/STAT3 in the process. Moreover, co-treatment with GEM and SAM exhibited more efficient inhibition of tumor weight and volume on PANC-1 xenograft mouse model compared to GEM or SAM alone and has no significant effect on the function of the liver and kidney. In general, this study indicated that SAM synergistically enhanced the antitumor effect of GEM against PC through suppressed JAK2/STAT3 pathway, and SAM is applicable as a promising agent to improve the sensitivity of PC to GEM.

The objective of this study was to determine the dose-response relationship between dietary choline intake and one-carbon cycle activity in overweight cats at maintenance energy requirements. This study was approved by the University of Guelph Animal Care Committee (AUP#4118). Overweight (body condition score:≥6/9) adult male neutered cats (n = 14) received each of five choline chloride doses individually for three weeks in a 5x5 latin square design. Doses were based on body weight (BW) and the daily recommended allowance (RA) for choline for adult cats (63 mg/kg BW0.67). Doses were: 2xRA (126 mg/kg BW0.67), 4xRA (252 mg/kg BW0.67), 6xRA (378 mg/kg BW0.67), 8xRA (504 mg/kg BW0.67), and control (no additional choline). Choline was administered onto the commercial extruded cat food (3620 mg choline/kg diet), given once a day at maintenance energy requirements (130 kcal/kg BW0.4). Fasted serum samples were analyzed for metabolomics through quantitative nuclear magnetic resonance (NMR) spectroscopy and direct infusion mass spectrometry (DI-MS) at the end of each period. Data was analyzed using proc GLIMMIX in SAS, with group and period as the random effects, and dose as the fixed effect. Statistical significance was considered at P < 0.05. Tukey’s post-hoc test was performed to assess multiple comparisons. Choline at 6x and 8x RA appears to increase transmethylation through the one-carbon cycle, as suggested by increased serum betaine, dimethylglycine (DMG) and methionine (P < 0.05). Carnitine, methylated by s-adenosylmethionine (SAM), increased (P < 0.05). Serine and threonine, precursors for the biosynthesis of homocysteine, appear to have been spared (P < 0.05). Supplemental dietary choline (6x and 8xRA) may be beneficial for overweight cats, as it may increase activity of the one-carbon cycle and methyl status; which are necessary for hepatic function. To better understand the impact of choline on methylation reactions, the quantification of specific proteins, gene expression and/or conversion rates is required.

Patients with active lupus have altered T cells characterized by low DNA methyltransferase levels. We hypothesized that low DNA methyltransferase levels synergize with low methionine levels to cause greater overexpression of genes normally suppressed by DNA methylation. CD4+ T cells from lupus patients and controls were stimulated with PHA then cultured in custom media with normal or low methionine levels. Oxidative stress was induced by treating the normal CD4+ T cells with peroxynitrite prior to culture. Methylation sensitive gene expression was measured by flow cytometry. Results showed low methionine levels caused greater overexpression of methylation sensitive genes in peroxynitrite treated T cells relative to untreated T cells, and in T cells from lupus patients relative to T cells from healthy controls. In conclusion, low dietary transmethylation micronutrient levels and low DNA methyltransferase levels caused either by oxidative stress or lupus, have additive effects on methylation sensitive T cell gene expression.

Homocysteine (Hcy) is a sulfur-containing amino acid generated during methionine metabolism, accumulation of which may be caused by genetic defects or the deficit of vitamin B12 and folate. A serum level greater than 15 micro-mols/L is defined as hyperhomocysteinemia (HHcy). Hcy has many roles, the most important being the active participation in the transmethylation reactions, fundamental for the brain. Many studies focused on the role of homocysteine accumulation in vascular or degenerative neurological diseases, but the results are still undefined. More is known in cardiovascular disease. HHcy is a determinant for the development and progression of inflammation, atherosclerotic plaque formation, endothelium, arteriolar damage, smooth muscle cell proliferation, and altered-oxidative stress response. Conversely, few studies focused on the relationship between HHcy and small vessel disease (SVD), despite the evidence that mice with HHcy showed a significant end-feet disruption of astrocytes with a diffuse SVD. A severe reduction of vascular aquaporin-4-water channels, lower levels of high-functioning potassium channels, and higher metalloproteinases are also observed. HHcy modulates the N-homocysteinylation process, promoting a pro-coagulative state and damage of the cellular protein integrity. This altered process could be directly involved in the altered endothelium activation, typical of SVD and protein quality, inhibiting the ubiquitin-proteasome system control. HHcy also promotes a constant enhancement of microglia activation, inducing the sustained pro-inflammatory status observed in SVD. This review article addresses the possible role of HHcy in small-vessel disease and understands its pathogenic impact.

Methionine addiction is a fundamental and general hallmark of cancer discovered by us almost a half-century ago [Proc Natl Acad Sci U S A 73 (1976) 1523-1527]. Methionine addiction is defined as the requirement, specific for cancer cells of all types, for exogenous methionine despite the normal ability to synthesize methionine from homocysteine. The methionine addiction of cancer is termed the Hoffman-effect, analogous to the Warburg-effect of the high glucose requirement of cancer cells. Methionine addiction is due to excess transmethylation reactions resulting in high methionine flux in cancer cells, which causes them to selectively arrest under methionine restriction due to depletion of free methionine and S-adenosyl methionine. Recently we have shown methionine-addicted cancer cells over-methylate histone H3 lysine marks which are not over-methylated in normal cells or in low-malignancy methionine-independent revertants derived from methionine-addicted cancer cells. In the present report, we show that in patient-derived xenograft (PDX) mouse models of the most common soft tissue sarcomas: myxofibrosarcoma, undifferentiated pleomorphic sarcoma (UPS) and liposarcoma, histone H3K4me3 and H3K9me3 are super over-methylated compared to normal muscle tissue. This new result is discussed along with our previous reports, regarding the potential of histone H3 over-methylation as a basis of malignancy.

Homocysteine is a sulfur-containing amino acid obtained from the metabolism of methionine. Dietary methionine is converted to S-adenosylmethionine and is demethylated to S-adenosylhomocysteine and homocysteine. Under conditions of protein deficient diet, intracellularly homocysteine is metabolized by one of two pathways: re-methylation and trans-sulfuration where vitamin B12 and folic acid participate as cofactors respectively. In the folate cycle, the enzyme methionine synthase and vitamin B12 as well as the enzyme 5,10-methylenetetrahydrofolate reductase are required. Folate enters the re-methylation cycle and is converted to 5-methyltetrahydrofolate, an important methyl donor in the conversion of homocysteine to methionine. Remethylation reactions also occur in the liver in which the enzyme betaine homocysteine methyltransferase, transfers a methyl group to homocysteine via the demethylation of betaine to dimethylglycine [1]. Therefore, disruption of S-adenosylmethionine -dependent transmethylation reactions leads to high concentrations of homocysteine in the blood and has been associated with deleterious effects. The normal level of homocysteine ranges between 5–15 μM, however in diseased conditions it can range from 50 μM in mild cases to 500 μM in severe cases [2]. The etiology of cancer is multifactorial. Studies have established a close association between cancer and hyperhomocystinemia [3]. Genetic & environmental factors play an important role in its pathogenesis. High levels of homocysteine are found in epigenetic regulation of DNA silencing and posttranslational modification of histones [3]. Specifically, the carcinogenic potential of hyper homocysteinemia is dependent on the detoxification pathways in homocysteine metabolism, venous thromboembolism, deficiency of folate and/or polymorphisms. In this chapter, the role of homocysteine on atherosclerosis, cardiovascular disease and cancer is discussed.

The mechanistic target of rapamycin complex 1 (mTORC1) signaling plays pivotal roles in cell growth and diseases. However, it remains mechanistically unclear about how to maintain mTORC1 activity during mammary glands development. Here we showed that mammary glands suffered from aggravated oxidative stress as pregnancy advanced and was accompanied by an increase in H2O2 levels, while the consumption for methionine and S-adenosylmethionine (SAM) rather than S-adenosylhomocysteine (SAH) were promoted in vivo. Likewise, H2O2 promoted SAM synthesis and reduced SAM utilization for methylation depending on H2O2 levels and treatment time in vitro. H2O2 inhibited phosphorylation of S6 kinase Thr 389 (p-S6K1 (T389)), 4E-BP1 Thr 37/46 and ULK1 Ser 757, the downstream of mTORC1, in mammary epithelial cells. However, methionine and SAM were shown to activate mTORC1 under H2O2-exposed condition. Moreover, this effect was not disabled by SGI-1027 which inhibits SAM transmethylation. In conclusion, methionine appeared to protect mammary cells against oxidative stress through producing SAM to maintain mTORC1 signaling activity.

Scope Vitamin B12 deficiency presents various neurological manifestations, such as cognitive dysfunction, mental retardation, or memory impairment. However, the involved molecular mechanisms remain to date unclear. Vitamin B12 is essential for synthesizing S-adenosyl methionine (SAM), the methyl group donor used for almost all transmethylation reactions. Here, we investigated the m6A methylation of mRNAs and their related gene expression in models of vitamin B12 deficiency. Methods and results We studied two cellular models deficient in vitamin B12 and hippocampi of mice knock-out for the CD320 receptor. The decrease in SAM levels resulting from vitamin B12 deficiency was associated with m6 A reduced levels in mRNAs. This was also potentially mediated by the overexpression of the eraser FTO. We further investigated mRNA methylation of some genes involved in neurological functions targeted by the m6A reader YTH proteins. We notably observed a m6A hypermethylation of Prkca mRNA and a consistently increased expression of PKCα, a kinase involved in brain development and neuroplasticity, in the two cellular models. Conclusion Our data show that m6A methylation in mRNA could be one of the contributing mechanisms that underlie the neurological manifestations produced by vitamin B12 deficiency. This article is protected by copyright. All rights reserved.

Obesity is increasing worldwide in prepubertal children, reducing the age of onset of associated comorbidities, including type 2 diabetes. Sulfur-containing amino acids, methionine, cysteine, and their derivatives play important roles in the transmethylation and transsulfuration pathways. Dysregulation of these pathways leads to alterations in the cellular methylation patterns and an imbalanced redox state. Therefore, we tested the hypothesis that one-carbon metabolism is already dysregulated in prepubertal children with obesity. Peripheral blood was collected from 64 children, and the plasma metabolites from transmethylation and transsulfuration pathways were quantified by HPLC. The cohort was stratified by BMI z-scores and HOMA-IR indices into healthy lean (HL), healthy obese (HO), and unhealthy obese (UHO). Fasting insulin levels were higher in the HO group compared to the HL, while the UHO had the highest. All groups presented normal fasting glycemia. Furthermore, high-density lipoprotein (HDL) was lower while triglycerides and lactate levels were higher in the UHO compared to HO subjects. S-adenosylhomocysteine (SAH) and total homocysteine levels were increased in the HO group compared to HL. Additionally, glutathione metabolism was also altered. Free cystine and oxidized glutathione (GSSG) were increased in the HO as compared to HL subjects. Importantly, the adipocyte secretory function was already compromised at this young age. Elevated circulating leptin and decreased adiponectin levels were observed in the UHO as compared to the HO subjects. Some of these alterations were concomitant with alterations in the DNA methylation patterns in the obese group, independent of the impaired insulin levels. In conclusion, our study informs on novel and important metabolic alterations in the transmethylation and the transsulfuration pathways in the early stages of obesity. Moreover, the altered secretory function of the adipocyte very early in life may be relevant in identifying early metabolic markers of disease that may inform on the increased risk for specific future comorbidities in this population.

Background/aim Methionine addiction, a fundamental and general hallmark of cancer, is due to the excess use of methionine for transmethylation, and is described as the Hoffman-effect. Methionine-addicted cancer cells can revert at low frequency to methionine independence when selected under methionine-restriction. We report here that highly-malignant methionine-addicted H460 human lung-cancer cells, when selected for methionine independence, have greatly-reduced tumorigenic potential. Materials and methods Methionine-addicted H460 parental cancer cells and methionine-independent revertant H460-R1 cells were injected in nude mice subcutaneously. Results When the parental H460 methionine-addicted cells were injected in nude mice at 2.5×105, 1×105 and 5×104, the cells could form tumors. In contrast, the H460-R1 methionine-independent revertant cells could not form tumors when the above-listed cell numbers were injected in nude mice. Conclusion There is a tight linkage between methionine addiction and malignancy.

Methionine addiction is a fundamental and general hallmark of cancer and is an area of current intense interest. Methionine addiction results from the overuse of methionine by cancer cells for excess transmethylation reactions. In order to identify excess transmethylation reactions in cancer and further understand the basis of methionine addiction, we compared the histone H3 lysine methylation status between methionine-addicted cancer cells, normal cells and rare revertants of methionine-addicted cancer cells which regained methionine independence and lost malignancy. The levels of H3K4me3, H3K9me3 and H3K36me3 were strongly elevated in methionine-addicted cancer cells in vitro compared to methionine-independent revertants isolated from the cancer cells. Tumorigenicity and experimental metastatic potential in nude mice were highly reduced in the methionine-independent revertants compared to the parental cells. Our previous studies showed that methionine restriction (MR) selectively arrests methionine-addicted cancer cells due to loss of histone H3 lysine methylation which was stable in normal cells under MR. Our previous and present results suggest that overmethylation of histone H3 lysine is linked with methionine addiction of cancer, required for the growth of cancer cells in vitro and in vivo, and necessary for malignancy as the histone lysine overmethylation disappears when the methionine-addicted cells revert to methionine independence and reduced malignancy. Methionine addiction has revealed fundamental molecular changes necessary for malignancy and presents great potential as a pan-cancer therapeutic target.

Organophosphates (OPs) are used worldwide as pesticides. However, acute and chronic exposure to OPs can cause serious adverse health effects. The mechanism of delayed OP toxicity is thought to involve off-target inhibition of serine proteases, although the precise molecular details remain unclear owing to the lack of an analytical method for global detection of protein targets of OPs. Here, we report the development of a mass spectrometry method to identify OP-adducted proteins from complex mixtures in a nontargeted manner. Human plasma was incubated with the OP dichlorvos that was 50% isotopically labeled and 50% unlabeled. Proteins and protein adducts were extracted, digested, and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect "twin ions" of peptides that were covalently modified by a chemical reaction with dichlorvos. The LC-MS/MS data were processed by a blended data analytics software (Xenophile) to detect the amino acid residue sites of proteins that were covalently modified by exposure to OPs. We discovered that OPs can transmethylate the N, S, and O side chains of His, Cys, Glu, Asp, and Lys residues. For model systems, such transmethylation reactions were confirmed by LC-MS, nuclear magnetic resonance (NMR), and rationalized using electronic structure calculations. Methylation of the ubiquitous antioxidant glutathione by dichlorvos can decrease the reducing/oxidizing equilibrium of glutathione in liver extracts, which has been implicated in diseases and pathological conditions associated with delayed OP toxicity.

[This corrects the article DOI: 10.1371/journal.pmed.1003012.].

The molecular mechanism of Alzheimer-like cognitive impairment induced by manganese (Mn) exposure has not yet been fully clarified, and there are currently no effective interventions to treat neurodegenerative lesions related to manganism. Protein phosphatase 2 A (PP2A) is a major tau phosphatase and was recently identified as a potential therapeutic target molecule for neurodegenerative diseases; its activity is directed by the methylation status of the catalytic C subunit. Methionine is an essential amino acid, and its downstream metabolite S-adenosylmethionine (SAM) participates in transmethylation pathways as a methyl donor. In this study, the neurotoxic mechanism of Mn and the protective effect of methionine were evaluated in Mn-exposed cell and rat models. We show that Mn-induced neurotoxicity is characterized by PP2Ac demethylation accompanied by abnormally decreased LCMT-1 and increased PME-1, which are associated with tau hyperphosphorylation and spatial learning and memory deficits, and that the poor availability of SAM in the hippocampus is likely to determine the loss of PP2Ac methylation. Importantly, maintenance of local SAM levels through continuous supplementation with exogenous methionine, or through specific inhibition of PP2Ac demethylation by ABL127 administration in vitro, can effectively prevent tau hyperphosphorylation to reduce cellular oxidative stress, apoptosis, damage to cell viability, and rat memory deficits in cell or animal Mn exposure models. In conclusion, our data suggest that SAM and PP2Ac methylation may be novel targets for the treatment of Mn poisoning and neurotoxic mechanism-related tauopathies. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13311-020-00930-6) contains supplementary material, which is available to authorized users.

Background/aim Cancers are selectively sensitive to methionine (MET) restriction (MR) due to their addiction to MET which is overused for elevated methylation reactions. MET addiction of cancer was discovered by us 45 years ago. MR of cancer results in depletion of S-adenosylmethionine (SAM) for transmethylation reactions, resulting in selective cancer-growth arrest in the late S/G2-phase of the cell cycle. The aim of the present study was to determine if blockade of the MET-methylation axis is a highly-effective strategy for cancer chemotherapy. Materials and methods In the present study, we demonstrated the efficacy of MET-methylation-axis blockade using MR by oral-recombinant methioninase (o-rMETase) combined with decitabine (DAC), an inhibitor of DNA methylation, and an inhibitor of SAM synthesis, cycloleucine (CL). We determined a proof-of-concept of the efficacy of the MET-methylation-axis blockade on a recalcitrant undifferentiated/unclassified soft-tissue sarcoma (USTS) patient-derived orthotopic xenograft (PDOX) mouse model. Results The o-rMETase-CL-DAC combination regressed the USTS PDOX with extensive cancer necrosis. Conclusion The new concept of combination MET-methylation-axis blockade is effective and can now be tested on many types of recalcitrant cancer.

The remethylation of homocysteine to methionine is important for chick embryos to sustain the S-adenosylmethionine transmethylation reactions, which are essential for the rapid proliferation of cells. Developmental changes in hepatic 5-methyltetrahydrofolate-homocysteine methyltransferase (MFMT), betaine-homocysteine methyltransferase (BHMT) and hepatic serine hydroxymethyltransferase (SHMT) were determined in E10-21 Cobb 500 broiler chick embryos and hatched chicks from D1-49. Hepatic levels of free serine, glycine, putrescine, spermidine and spermine levels were also determined. Analyses showed hepatic MFMT-specific activity doubled from E10 to E12, with remaining embryo development experiencing small fluctuations in activity through E21. Hepatic MFMT doubled immediately after hatch, with peak activity occurring at D3. Afterwards, hepatic MFMT-specific activity steadily declined from D7-49. Hepatic BHMT activity was higher from E10 to E16 of embryogenesis, decreased rapidly at E17 and remained lower through E21 (p < .05). Hepatic BHMT-specific activity was also lower in chicks, with the exception of a peak in specific activity on D7. BHMT activity returned to lower levels by D21. Throughout embryogenesis, hepatic SHMT activity in chick embryos remained relatively constant except for a decrease at 13E, followed by an increase at 14E. Maximal activity of SHMT was found the first day post-hatch. Additionally, SHMT activity was significantly lower in growing chicks than that in embryos. Hepatic-free serine and glycine levels were negatively correlated with SHMT in hatched chicks. Hepatic polyamine, putrescine and spermidine shared a similar development pattern: peak level in the middle of incubation, low at late embryogenesis and lowest during the post-hatch period except an increase within one week after hatch. The sharp increase in hepatic concentrations of glycine, serine and putrescine, along with increased specific activities of MHMT, BHMT and SHMT from D1-7, suggests that methionine conservation (remethylation from homocysteine) and glycine/serine is critical for young chicks for organ growth, maturation, and development.

Background/aims Methodological challenges have been associated with the dynamic measurement of muscle protein breakdown (MPB), as have the measurement of both muscle protein synthesis (MPS) and MPB within the same experiment. Our aim was to use the transmethylation properties of methionine as proof-of-concept to measure rates of MPB via its methylation of histidine within skeletal muscle myofibrillar proteins, whilst simultaneously utilising methionine incorporation into bound protein to measure MPS. Results During the synthesis measurement period, incorporation of methyl[D3]-13C-methionine into cellular protein in C2C12 myotubes was observed (representative of MPS), alongside an increase in the appearance of methyl[D3]-methylhistidine into the media following methylation of histidine (representative of MPB). For further validation of this approach, fractional synthetic rates (FSR) of muscle protein were increased following treatment of the cells with the anabolic factors insulin-like growth factor-1 (IGF-1) and insulin, while dexamethasone expectedly reduced MPS. Conversely, rates of MPB were reduced with IGF-1 and insulin treatments, whereas dexamethasone accelerated MPB. Conclusions This is a novel stable isotope tracer approach that permits the dual assessment of muscle cellular protein synthesis and breakdown rates, through the provision of a single methionine amino acid tracer that could be utilised in a wide range of biological settings., Highlights • Measuring protein synthesis and breakdown simultaneously is methodologically challenging. • The stable isotope tracer methyl[D3]-13C-methionine was able to measure protein synthesis in vitro. • The same tracer could also measure breakdown after its conversion to methyl[D3]-methylhistidine. • This method could be widely used by researchers within the field of musculoskeletal research.

Background There is growing evidence that Alzheimer disease (AD) is a pervasive metabolic disorder with dysregulation in multiple biochemical pathways underlying its pathogenesis. Understanding how perturbations in metabolism are related to AD is critical to identifying novel targets for disease-modifying therapies. In this study, we test whether AD pathogenesis is associated with dysregulation in brain transmethylation and polyamine pathways. Methods and findings We first performed targeted and quantitative metabolomics assays using capillary electrophoresis-mass spectrometry (CE-MS) on brain samples from three groups in the Baltimore Longitudinal Study of Aging (BLSA) (AD: n = 17; Asymptomatic AD [ASY]: n = 13; Control [CN]: n = 13) (overall 37.2% female; mean age at death 86.118 ± 9.842 years) in regions both vulnerable and resistant to AD pathology. Using linear mixed-effects models within two primary brain regions (inferior temporal gyrus [ITG] and middle frontal gyrus [MFG]), we tested associations between brain tissue concentrations of 26 metabolites and the following primary outcomes: group differences, Consortium to Establish a Registry for Alzheimer’s Disease (CERAD) (neuritic plaque burden), and Braak (neurofibrillary pathology) scores. We found significant alterations in concentrations of metabolites in AD relative to CN samples, as well as associations with severity of both CERAD and Braak, mainly in the ITG. These metabolites represented biochemical reactions in the (1) methionine cycle (choline: lower in AD, p = 0.003; S-adenosyl methionine: higher in AD, p = 0.005); (2) transsulfuration and glutathione synthesis (cysteine: higher in AD, p < 0.001; reduced glutathione [GSH]: higher in AD, p < 0.001); (3) polyamine synthesis/catabolism (spermidine: higher in AD, p = 0.004); (4) urea cycle (N-acetyl glutamate: lower in AD, p < 0.001); (5) glutamate-aspartate metabolism (N-acetyl aspartate: lower in AD, p = 0.002); and (6) neurotransmitter metabolism (gamma-amino-butyric acid: lower in AD, p < 0.001). Utilizing three Gene Expression Omnibus (GEO) datasets, we then examined mRNA expression levels of 71 genes encoding enzymes regulating key reactions within these pathways in the entorhinal cortex (ERC; AD: n = 25; CN: n = 52) and hippocampus (AD: n = 29; CN: n = 56). Complementing our metabolomics results, our transcriptomics analyses also revealed significant alterations in gene expression levels of key enzymatic regulators of biochemical reactions linked to transmethylation and polyamine metabolism. Our study has limitations: our metabolomics assays measured only a small proportion of all metabolites participating in the pathways we examined. Our study is also cross-sectional, limiting our ability to directly test how AD progression may impact changes in metabolite concentrations or differential-gene expression. Additionally, the relatively small number of brain tissue samples may have limited our power to detect alterations in all pathway-specific metabolites and their genetic regulators. Conclusions In this study, we observed broad dysregulation of transmethylation and polyamine synthesis/catabolism, including abnormalities in neurotransmitter signaling, urea cycle, aspartate-glutamate metabolism, and glutathione synthesis. Our results implicate alterations in cellular methylation potential and increased flux in the transmethylation pathways, increased demand on antioxidant defense mechanisms, perturbations in intermediate metabolism in the urea cycle and aspartate-glutamate pathways disrupting mitochondrial bioenergetics, increased polyamine biosynthesis and breakdown, as well as abnormalities in neurotransmitter metabolism that are related to AD., Uma Mahajan and coworkers study metabolic perturbations in specific brain regions in Alzheimer disease., Author summary Why was this study done? A growing body of evidence suggests that Alzheimer disease (AD) may be associated with dysregulation of multiple metabolic pathways, and identifying novel molecular targets underlying AD pathogenesis is essential for developing effective AD treatments. Past studies have shown that abnormalities in choline-related biochemical pathways may be associated with AD pathogenesis, specifically the transmethylation, polyamine synthesis/catabolism and related pathways. Our study tested the hypothesis that dysregulation of choline-related biochemical pathways in the brain is associated with AD pathogenesis; we examined metabolites within biochemical reactions linked to transmethylation and polyamine synthesis/catabolism. What did the researchers do and find? We performed quantitative and targeted metabolomics on brain tissue samples (AD: n = 17; Asymptomatic AD [ASY]: n = 13; Control [CN]: n = 13) and transcriptomics from Gene Expression Omnibus data (entorhinal cortex [AD: n = 25; CN: n = 52] and hippocampus [AD: n = 29; CN: n = 56]) to identify aberrations across 6 biochemical reactions linked to the transmethylation and polyamine pathways: methionine cycle, transsulfuration and glutathione synthesis, polyamine synthesis and catabolism, urea cycle, glutamate-aspartate metabolism, and neurotransmitter metabolism. We found significant metabolite alterations associated with AD mainly in the inferior temporal gyrus (ITG) across all pathways tested, as well as associations between metabolite concentrations and severity of AD pathology. Complementing our metabolomics results, our transcriptomics analyses also revealed significant alterations in gene expression of key enzymatic regulators of biochemical reactions linked to transmethylation and polyamine metabolism. What do these findings mean? Our results implicate alterations in cellular methylation potential and increased flux in the transmethylation pathways, increased demand on antioxidant defense mechanisms, perturbations in intermediate metabolism in the urea cycle and aspartate-glutamate pathways disrupting mitochondrial bioenergetics, increased polyamine biosynthesis and breakdown, as well as abnormalities in neurotransmitter metabolism that are related to severity of AD pathology and the expression of clinical symptoms. This study adds to a comprehensive understanding of the metabolic basis of AD pathogenesis and provides insights into novel targets for disease-modifying therapies. The cross-sectional nature of the study limits our ability to directly test how AD progression may impact changes in metabolite concentrations or differential-gene expression.

In recent years, numerous studies have confirmed the vital role and therapeutic potential of quaternary ammonium compounds and endogenous formaldehyde in the prevention and treatment of diseases. These compounds participate in the transmethylation processes and play a role in the metabolism and in the regulation of cellular processes. The present research indicates that sour cherry fruit contains large quantities of quaternary ammonium compounds in the early developmental phase (burgeoning). The quantity of methyl-donor compounds (choline, carnitine) and easily mobilizable methyl groups were measured in various fruit parts (stalk, fruit flesh, seed kernel) of five sour cherry cultivars using OPLC technology, and the cultivar dependence of the detected compounds was examined. The results of comparative examinations established the presence of choline in the stalk and seed and of a significant amount of carnitine in the fruit flesh. The clear, significant differences between the genotypes will facilitate the selection of cultivars containing the largest proportion of components beneficial for human health.

Metabolism of excess methionine (Met) to homocysteine (Hcy) by transmethylation is facilitated by the expression of methionine adenosyltransferase (MAT) I/III and glycine N-methyltransferase (GNMT) in liver, and a lack of either enzyme results in hypermethioninemia despite normal concentrations of MATII and methyltransferases other than GNMT. The further metabolism of Hcy by the transsulfuration pathway is facilitated by activation of cystathionine β-synthase (CBS) by S-adenosylmethionine (SAM) as well as the relatively high KM of CBS for Hcy. Transmethylation plus transsulfuration effects catabolism of the Met molecule along with transfer of the sulfur atom of Met to serine to synthesize cysteine (Cys). Oxidation and excretion of Met sulfur depend upon Cys catabolism and sulfur oxidation pathways. Excess Cys is oxidized by cysteine dioxygenase 1 (CDO1) and further metabolized to taurine or sulfate. Some Cys is normally metabolized by desulfhydration pathways, and the hydrogen sulfide (H2S) produced is further oxidized to sulfate. If Cys or Hcy concentrations are elevated, Cys or Hcy desulfhydration can result in excess H2S and thiosulfate production. Excess Cys or Met may also promote their limited metabolism by transamination pathways.

Metabolic analysis of ADA2-deficient cells shows that extracellular purine nucleosides trigger cellular innate immunity., Mechanisms linking immune sensing of DNA danger signals in the extracellular environment to innate pathways in the cytosol are poorly understood. Here, we identify a previously unidentified immune-metabolic axis by which cells respond to purine nucleosides and trigger a type I interferon-β (IFN-β) response. We find that depletion of ADA2, an ectoenzyme that catabolizes extracellular dAdo to dIno, or supplementation of dAdo or dIno stimulates IFN-β. Under conditions of reduced ADA2 enzyme activity, dAdo is transported into cells and undergoes catabolysis by the cytosolic isoenzyme ADA1, driving intracellular accumulation of dIno. dIno is a functional immunometabolite that interferes with the cellular methionine cycle by inhibiting SAM synthetase activity. Inhibition of SAM-dependent transmethylation drives epigenomic hypomethylation and overexpression of immune-stimulatory endogenous retroviral elements that engage cytosolic dsRNA sensors and induce IFN-β. We uncovered a previously unknown cellular signaling pathway that responds to extracellular DNA–derived metabolites, coupling nucleoside catabolism by adenosine deaminases to cellular IFN-β production.

Carnitine is demonstrated as an effective methyl donor in the formation of the plant growth regulator N, N-dimethylpiperidinium (mepiquat), encompassing either N-methylation/decarboxylation of pipecolic acid, or Maillard pathways followed by transmethylation reactions. The formation of mepiquat and the intermediate compounds was monitored (180-300 °C, up to 180 min) using HPLC-MS/MS in different binary or ternary model systems composed of (i) lysine/fructose/carnitine, (ii) lysine/glucose/carnitine, or (iii) pipecolic acid (PipAc)/carnitine. The highest yield of mepiquat was 2.4% after 120 min incubation at 290 °C (PipAc/carnitine model system). The highest yield was recorded in fructose and glucose (Maillard) systems after 180 min at 230 °C. The full-scan mode was used to monitor the formation of the corresponding intermediates (piperidine and N-methylpiperidine, the demethylated intermediates of carnitine). The new pathways of mepiquat formation indicate that the occurrence of low levels of this thermally induced compound is potentially more widespread in some selected cooked foodstuffs. For the first time, mepiquat was detected in oven-cooked beef, reaching up to 82.5 μg/kg. These amounts are not expected to significantly contribute to the overall exposure via different foodstuffs, as reported in previous studies.

Tuberous sclerosis complex (TSC) is an autosomal dominant neurodevelopmental disorder and the quintessential disorder of mechanistic Target of Rapamycin Complex 1 (mTORC1) dysregulation. Loss of either causative gene, TSC1 or TSC2, leads to constitutive mTORC1 kinase activation and a pathologically anabolic state of macromolecular biosynthesis. Little is known about the organ-specific metabolic reprogramming that occurs in TSC-affected organs. Using a mouse model of TSC in which Tsc2 is disrupted in radial glial precursors and their neuronal and glial descendants, we performed an unbiased metabolomic analysis of hippocampi to identify Tsc2-dependent metabolic changes. Significant metabolic reprogramming was found in well-established pathways associated with mTORC1 activation, including redox homeostasis, glutamine/tricarboxylic acid cycle, pentose and nucleotide metabolism. Changes in two novel pathways were identified: transmethylation and polyamine metabolism. Changes in transmethylation included reduced methionine, cystathionine, S-adenosylmethionine (SAM—the major methyl donor), reduced SAM/S-adenosylhomocysteine ratio (cellular methylation potential), and elevated betaine, an alternative methyl donor. These changes were associated with alterations in SAM-dependent methylation pathways and expression of the enzymes methionine adenosyltransferase 2A and cystathionine beta synthase. We also found increased levels of the polyamine putrescine due to increased activity of ornithine decarboxylase, the rate-determining enzyme in polyamine synthesis. Treatment of Tsc2(+/−) mice with the ornithine decarboxylase inhibitor α-difluoromethylornithine, to reduce putrescine synthesis dose-dependently reduced hippocampal astrogliosis. These data establish roles for SAM-dependent methylation reactions and polyamine metabolism in TSC neuropathology. Importantly, both pathways are amenable to nutritional or pharmacologic therapy.

Diet-related bioactive compounds are intensively examined in vitro, although the in vivo and in vitro effects are different, and the mode of actions in in vivo is not yet known in detail. Metals are also important both in the free radical formation and in the antioxidant defence as well as in signal transduction. It is a proven fact that methylation agents have an important role in preventing cancer and improve redox homeostasis. The root of Beta vulgaris L. ssp . esculenta var. rubra (table beet) as a functional food has been used for centuries as a traditional and popular food in many national dishes. Since several bioactive agents e.g. betaine, betanins, betacyanins, betaxanthins, vulgaxanthine, polyphenols, flavonoids, vitamins (thiamine, riboflavine, piridoxine, ascorbic acid, biotin and folic acid) as well as soluble fibre, pectin and different metal elements (e.g. Al, B, Ba, Ca, Cu, Fe, K, Mg, Mn, Na, Zn) can be found in large quantities in vegetable table beet, therefore a relative simple methodological repertoire to interpret the impact of variety of active substances on the vital function of the metastatic cancerous patients is needed to be developed. It is also required to examine in addition to routine laboratory parameters, values of HbA1c, 9 cytokines and levels of 3 growth factors, the global parameter of redox-homeostasis, few metal elements and levels of free and Zn-protoporphyrins as well as transmethylation, since low transmethylation ability, high free protoporphyrin and Zn-protoporphyrin concentrations and high induced free radical levels of erythrocytes are very important indexes in cancer.

Gemcitabine (GEM) has been widely used for pancreatic cancer (PC) treatment but limited by the development of drug resistance. The agents that reverse its resistance and improve the chemo-sensitivity are urgently needed. S-Adenosylmethionine (SAM) is a precursor for polyamine biosynthesis in mammalian cells and plays a key role in biological transmethylation events. It is reported that SAM could be used as a therapeutic reagent for cancer treatments. In this study, we investigated the chemo-sensitization of SAM to potentiate the antitumor effect of GEM in PC. After treating PC cells with GEM and/or SAM, different subsequent experiments were performed. Results showed that SAM plus GEM could significantly inhibit the growth and proliferation of PC cells, and SAM acts synergistically with GEM. The combinative treatment could induce cell apoptosis and inhibit invasion and migration through JAK2/STAT3 inactivation. Inhibition of JAK2/STAT3 pathway significantly enhanced the pro-apoptotic effect of SAM, suggesting the key roles of JAK2/STAT3 in the process. Moreover, co-treatment with GEM and SAM exhibited more efficient inhibition of tumor weight and volume on PANC-1 xenograft mouse model compared to GEM or SAM alone and has no significant effect on the function of the liver and kidney. In general, this study indicated that SAM synergistically enhanced the antitumor effect of GEM against PC through suppressed JAK2/STAT3 pathway, and SAM is applicable as a promising agent to improve the sensitivity of PC to GEM.

Context Homocysteine is an independent cardiovascular risk factor and is elevated in essential hypertension. Insulin stimulates homocysteine catabolism in healthy individuals. However, the mechanisms of hyperhomocysteinemia and its relationship with insulin resistance in essential hypertension are unknown. Objective To investigate whole body methionine and homocysteine kinetics and the effects of insulin in essential hypertension. Design and Setting Eight hypertensive male subjects and six male normotensive controls were infused with l-[methyl-2H3,1-13C]methionine for 6 hours. In the last 3 hours a euglycemic, hyperinsulinemic clamp was performed. Steady-state methionine and homocysteine kinetics were determined in postabsorptive and hyperinsulinemic conditions. Results Postabsorptive hypertensive subjects had elevated homocysteine concentrations (+30%, P = 0.035) and slightly (by 15% to 20%) but insignificantly lower methionine rates of appearance (Ras) (P = 0.07 to P = 0.05) and utilization for protein synthesis (P = 0.06) than postabsorptive normotensive controls. Hyperinsulinemia suppressed methionine Ra and protein synthesis, whereas it increased homocysteine trans-sulfuration, clearance, and methionine transmethylation (the latter only in the normotensive subjects). However, in the hypertensive subjects trans-sulfuration was significantly lower (P < 0.05) and increased ~50% less [by +1.59 ± 0.34 vs +3.45 ± 0.52 µmol/kg lean body mass (LBM) per hour, P < 0.005] than in normotensive controls. Homocysteine clearance through trans-sulfuration was ~50% lower in hypertensive than in normotensive subjects (P < 0.005). In the hypertensive subjects, insulin-mediated glucose disposal was ~45% lower (460 ± 44 vs 792 ± 67 mg/kg LBM per hour, P < 0.0005) than in normotensive controls and was positively correlated with the increase of trans-sulfuration (P < 0.0015). Conclusions In subjects with essential hypertension, hyperhomocysteinemia is associated with decreased homocysteine trans-sulfuration and probably represents a feature of insulin resistance.

Methionine is an aliphatic, sulfur-containing, essential amino acid, and a precursor of succinyl-CoA, homocysteine, cysteine, creatine, and carnitine. Recent research has demonstrated that methionine can regulate metabolic processes, the innate immune system, and digestive functioning in mammals. It also intervenes in lipid metabolism, activation of endogenous antioxidant enzymes such as methionine sulfoxide reductase A, and the biosynthesis of glutathione to counteract oxidative stress. In addition, methionine restriction prevents altered methionine/transmethylation metabolism, thereby decreasing DNA damage and carcinogenic processes and possibly preventing arterial, neuropsychiatric, and neurodegenerative diseases. This review focuses on the role of methionine in metabolism, oxidative stress, and related diseases.

Methionine cycle plays an essential role in regulating many cellular events, especially transmethylation reactions, incorporating the methyl donor S-adenosylmethionine (SAM). The transmethylations and substances involved in the cycle have shown complicated effects and mechanisms on immunocytes developments and activations, and exert crucial impacts on the pathological processes in immune disorders. Areas covered: Methionine cycle has been considered as an effective means of drug developments. This review discussed the role of methionine cycle in immune responses and summarized the potential therapeutic strategies based on the cycle, including SAM analogs, methyltransferase inhibitors, S-adenosylhomocysteine hydrolase (SAHH) inhibitors, adenosine receptors specific agonists or antagonists and homocysteine (Hcy)-lowering reagents, in treating human immunodeficiency virus (HIV) infections, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), multiple sclerosis (MS), systemic sclerosis (SSc) and other immune disorders. Expert opinion: New targets and biomarkers grown out of methionine cycle have developed rapidly in the past decades. However, impacts of epigenetic regulations on immune disorders are unclear and whether the substances in methionine cycle can be clarified as biomarkers remains controversial. Therefore, further elucidation on the role of epigenetic regulations and substances in methionine cycle may contribute to exploring the cycle-derived biomarkers and drugs in immune disorders.

Nicotinamide N-methyltransferase (NNMT) transfers the methyl from S-adenosyl-L-methionine (SAM) to nicotinamide (NA) to produce S-adenosyl-L-homocysteine (SAH) and 1-methylnicotinamide (MeN). NNMT has been implicated in a variety of diseases; however, the role of NNMT in drug addiction is largely unknown. Here, we found that the expression of Nnmt was significantly upregulated in the dorsal striatum (DS) of cocaine-conditioned mice. Cocaine significantly decreased SAM/SAH ratio levels in the DS, which was accompanied with the decreased activities of Rac1 and RhoA. Lentivirus-mediated knockdown of Nnmt in the dorsomedial striatum (DMS) attenuated cocaine conditioned place preference (CPP) reward, but increased striatal SAM/SAH ratio levels as well as Rac1 and RhoA activities. In addition, pharmacological inhibition of NNMT through intra-DMS infusion of MeN attenuated cocaine CPP and the activities of Rac1 and RhoA, but increased SAM/SAH ratio. These results suggest that NNMT-dependent transmethylation is involved in the activation of Rac1 and RhoA, which utilize SAM as a methyl donor cofactor. Co-immunoprecipitation assay using a RhoGDIα antibody indirectly captured Rac1 or RhoA that were bound to RhoGDIα. The results showed that cocaine increased the association of RhoGDIα with Rac1 or RhoA, whereas such effect was inhibited by Nnmt knockdown. Collectively, our findings show that NNMT regulates cocaine CPP through SAM-mediated modification of Rac1 and RhoA.

Background: Low-quality dietary protein intake and vitamin B-12 deficiency could interact to decrease methionine transmethylation and remethylation rates during pregnancy and may affect epigenetic modifications of the fetal genome.Objective: The objective of this randomized, partially open-labeled intervention trial was to examine the effect of supplemental high-quality protein and vitamin B-12 on third-trimester methionine kinetics in pregnant Indian women with a low vitamin B-12 status.Methods: Pregnant women with low serum vitamin B-12 concentrations ( 0.1). Placental mRNA expression, LINE-1, and VEGF promoter methylation did not differ between groups.Conclusions: Combined vitamin B-12 and balanced protein-energy supplementation increased the homocysteine remethylation rate in late pregnancy. Thus, vitamin B-12 along with balanced protein-energy supplementation is critical for optimal functioning of the methionine cycle in the third trimester of pregnancy in Indian women with low serum vitamin B-12 in early pregnancy. This trial was registered at clinicaltrials.gov as CTRI/2016/01/006578.

We propose here a hypothesis of the cause of cancer that brings together fundamental changes in methyl-group metabolism resulting in methionine dependence and global DNA hypomethylation which destabilizes the genome leading to aneuploid karyotypes which evolve and stabilize into autonomous cancer. Experimental support for this hypothesis is that methioine dependence is a general metabolic defect in caner. Methionine dependence is due to excess use of methionene for aberrant transmethylation reactions that apparently divert methyl groups from DNA. The resulting global DNA hypomethylation is also a general phenomena in cancer. Global hypomethylation leads to an unstable genomes and aneuploid karyotypes, another general phenomena in cancer. The excessive and aberrant use of methionine in cancer is strongly observed in [11C]methionine PET imaging, where high uptake of [11C]methionine results in a very strong and selective tumor signal compared with normal tissue background. [11C]methionine is superior to [18C] fluorodeoxyglucose (FDG)-PET for PET imaging, suggesting methionine dependence is more tumor-specific than glucose dependence.

Methionine dependence is due to the overuse of methionine for aberrant transmethylation reactions in cancer. Methionine dependence may be the only general metabolic defect in cancer. In order to exploit methionine dependence for therapy, our laboratory previously cloned L-methionine α-deamino-γ-mercaptomethane lyase [EC 4.4.1.11]). The cloned methioninase, termed recombinant methioninase, or rMETase, has been tested in mouse models of human cancer cell lines. Ewing's sarcoma is recalcitrant disease even though development of multimodal therapy has improved patients'outcome. Here we report efficacy of rMETase against Ewing's sarcoma in a patient-derived orthotopic xenograft (PDOX) model. The Ewing's sarcoma was implanted in the right chest wall of nude mice to establish a PDOX model. Eight Ewing's sarcoma PDOX mice were randomized into untreated control group (n = 4) and rMETase treatment group (n = 4). rMETase (100 units) was injected intraperitoneally (i.p.) every 24 hours for 14 consecutive days. All mice were sacrificed on day-15, 24 hours after the last rMETase administration. rMETase effectively reduced tumor growth compared to untreated control. The methionine level both of plasma and supernatants derived from sonicated tumors was lower in the rMETase group. Body weight did not significantly differ at any time points between the 2 groups. The present study is the first demonstrating rMETase efficacy in a PDOX model, suggesting potential clinical development, especially in recalcitrant cancers such as Ewing's sarcoma.

BACKGROUND The neonatal methionine requirement must consider not only the high demand for rapid tissue protein expansion but also the demands as the precursor for a suite of critical transmethylation reactions. However, methionine metabolism is inherently complex because upon transferring its methyl group during transmethylation, methionine can be reformed by the dietary methyl donors choline (via betaine) and folate. OBJECTIVE We sought to determine whether dietary methyl donors contribute to methionine availability for protein synthesis in neonatal piglets. METHODS Yucatan miniature piglets aged 4-8 d were fed a diet that provided 38 μg folate/(kg·d), 60 mg choline/(kg·d), and 238 mg betaine/(kg·d) [methyl-sufficient (MS); n = 8] or a diet devoid of these methyl precursors [methyl-deficient (MD); n = 8]. After 5 d, dietary methionine was reduced from 0.30 to 0.20 g/(kg·d) in both groups. On day 6, piglets received a constant [1-13C]phenylalanine infusion to measure whole-body protein kinetics, and on day 8 they received a constant [3H-methyl]methionine infusion to measure tissue-specific protein synthesis in skeletal muscle, the liver, and the jejunum. RESULTS Whole-body phenylalanine flux, protein synthesis, and protein breakdown were 13%, 12%, and 22% lower, respectively, in the MD group than in the MS group (P < 0.05). Reduced whole-body protein synthesis in the MD piglets was attributed to 50% lower protein synthesis in skeletal muscle and the jejunum than in the MS piglets (P < 0.05). Furthermore, methionine availability in skeletal muscle was halved in piglets fed the MD diet (P < 0.05), and the specific radioactivity of methionine was doubled in the jejunum of MD piglets (P < 0.05), suggesting lower intestinal remethylation. Liver protein synthesis did not significantly differ between the groups, but secreted proteins were not measured. CONCLUSIONS Dietary methyl donors can affect whole-body and tissue-specific protein synthesis in neonatal piglets and should be considered when determining the methionine requirement.

Cystic fibrosis (CF) is an autosomal recessive disorder, caused by genetic mutations in CF transmembrane conductance regulator protein. Several reports have indicated the presence of specific fatty acid alterations in CF patients, most notably decreased levels of plasmatic and tissue docosahexaenoic acid (DHA), the precursor of specialized pro-resolving mediators. We hypothesized that DHA supplementation could restore the production of DHA-derived products and possibly contribute to a better control of the chronic pulmonary inflammation observed in CF subjects. Sputum samples from 15 CF and 10 chronic obstructive pulmonary disease (COPD) subjects were collected and analyzed by LC/MS/MS, and blood fatty acid were profiled by gas chromatography upon lipid extraction and transmethylation. Interestingly, CF subjects showed increased concentrations of leukotriene B4 (LTB4), prostaglandin E2 (PGE2), and 15-hydroxyeicosatetraenoic acid (15-HETE), when compared with COPD patients, whereas the concentrations of DHA metabolites did not differ between the two groups. After DHA supplementation, not only DHA/arachidonic acid (AA) ratio and highly unsaturated fatty acid index were significantly increased in the subjects completing the study (p < 0.05) but also a reduction in LTB4 and 15-HETE was observed, together with a tendency for a decrease in PGE2, and an increase in 17-hydroxy-docosahexaenoic acid (17OH-DHA) levels. At the end of the washout period, LTB4, PGE2, 15-HETE, and 17OH-DHA showed a trend to return to baseline values. In addition, 15-HETE/17OH-DHA ratio in the same sample significantly decreased after DHA supplementation (p < 0.01) when compared with baseline. In conclusion, our results show here that in CF patients, an impairment in fatty acid metabolism, characterized by increased AA-derived metabolites and decreased DHA-derived metabolites, could be partially corrected by DHA supplementation.

The general cancer-specific metabolic defect of methionine (MET) dependence is due to MET overuse for aberrant transmethylation reactions. The excess use of MET for aberrant transmethylation reactions apparently diverts methyl groups from DNA. The resulting global DNA hypomethylation is also a general phenomenon in cancer and leads to unstable genomes and aneuploid karyotypes. The excessive and aberrant use of MET in cancer is readily observed in [11C]-MET-PET imaging, where high uptake of [11C]-MET results in a very strong and selective tumor signal compared to normal tissue background for brain cancer and possibly other cancers. [11C]-MET is superior to [18C]-fluorodeoxyglucose (FDG) for PET imaging, suggesting that MET overuse in cancer ("Hoffman effect") is greater than glucose overuse in cancer ("Warburg effect").

Methionine (MET) dependence is a cancer-specific metabolic abnormality that is due to MET overuse for aberrant transmethylation reactions. [11C]-MET is very useful for positron-emission tomography (PET) due to MET overuse in malignant tumors. Many benefits of MET-PET have been demonstrated. MET-PET can differentiate recurrent glioma and necrosis. [11C]-MET-PET can also predict prognosis in gliomas better than [18F]-FDG PET. [11C]-MET-PET is better than MRI for predicting survival in low-grade glioma (LGG). MET-PET has greater specificity for detecting residual tumor after surgery than MRI.

Many different types of cancer cells have been shown to be methionine (MET) dependent. Cancer cells, unlike normal cells, grow poorly or not at all when MET is restricted. Cancer cells have an elevated requirement for exogenous MET for growth, despite high levels of endogenous synthesis. This requirement reflects increased utilization of MET by cancer cells, analogous to increased utilization glucose by cancer cells (Warburg effect). To answer the critical question of whether MET-dependent cancer cells synthesize normal amounts of MET, we determined the levels of MET, S-adenosylmethionine (AdoMET), and S-adenosylhomocysteine (AdoHCY) that were synthesized by MET-dependent cancer cells under conditions of MET restriction. We demonstrated that MET-dependent cells synthesize a normal amount of endogenously synthesized MET but are still deficient in AdoMET. In contrast, exogenously supplied MET results in normal AdoMET levels. The ratio of AdoMET to AdoHCY is low in MET-dependent cells growing in MET-restricted medium but is normal when MET is supplied. Under conditions of MET restriction, the low AdoMET/AdoHCY ratio probably limits proliferation of MET-dependent cancer cells. The amount of free MET is also low in MET-dependent cancer cells under MET restriction. The elevated MET requirement for cancer cells may be due to enhanced overall rates of transmethylation compared to normal human cells. Thus, MET-dependent cancer cells have low levels of free MET, low levels of AdoMET, and elevated levels of AdoHCY under conditions of MET restriction probably due to overuse of MET for transmethylation reactions ("Hoffman effect"), thereby blocking cellular proliferation.