S-adenosylmethionine Concentrations Research Articles

Modulation of mercaptopurine intestinal toxicity and pharmacokinetics by gut microbiota

The interaction between the gut microbiota and mercaptopurine (6-MP), a crucial drug used in pediatric acute lymphoblastic leukemia (ALL) treatment, has not been extensively studied. Here we reveal the significant perturbation of gut microbiota after 2-week 6-MP treatment in beagles and mice followed by the functional prediction that showed impairment of SCFAs production and altered amino acid synthesis. And the targeted metabolomics in plasma also showed changes in amino acids. Additionally, targeted metabolomics analysis of feces showed changes in amino acids and SCFAs. Furthermore, ablating the intestinal microbiota by broad-spectrum antibiotics exacerbated the imbalance of amino acids, particularly leading to a significant decrease in the concentration of S-adenosylmethionine (SAM). Importantly, the depletion of gut microbiota worsened the damage of small intestine caused by 6-MP, resulting in increased intestinal permeability. Considering the relationship between toxicity and 6-MP metabolites, we conducted a pharmacokinetic study in pseudo germ-free rats to confirm that gut microbiota depletion altered the methylation metabolites of 6-MP. Specifically, the concentration of MeTINs, a secondary methylation metabolite, showed a negative correlation with SAM, the pivotal methyl donor. Additionally, we observed a strong correlation between Alistipes and SAM levels in both feces and plasma. In conclusion, our study demonstrates that 6-MP disrupts the gut microbiota, and depleting the gut microbiota exacerbates 6-MP-induced intestinal toxicity. Moreover, SAM derived from microbiota plays a crucial role in influencing plasma SAM and the methylation of 6-MP. These findings underscore the importance of comprehending the role of the gut microbiota in 6-MP metabolism and toxicity.

SMG5 Inhibition Restrains Hepatocellular Carcinoma Growth and Enhances Sorafenib Sensitivity.

Hepatocellular carcinoma (HCC) has a pathogenesis that remains elusive with restricted therapeutic strategies and efficacy. This study aimed to investigate the role of SMG5, a crucial component in nonsense-mediated mRNA decay (NMD) that degrades mRNA containing a premature termination codon, in HCC pathogenesis and therapeutic resistance. We demonstrated an elevated expression of SMG5 in HCC and scrutinized its potential as a therapeutic target. Our findings revealed that SMG5 knockdown not only inhibited the migration, invasion, and proliferation of HCC cells but also influenced sorafenib resistance. Differential gene expression analysis between the control and SMG5 knockdown groups showed an upregulation of methionine adenosyltransferase 1A in the latter. High expression of methionine adenosyltransferase 1A, a catalyst for S-adenosylmethionine (SAM) production, as suggested by The Cancer Genome Atlas data, was indicative of a better prognosis for HCC. Further, an ELISA showed a higher concentration of SAM in SMG5 knockdown cell supernatants. Furthermore, we found that exogenous SAM supplementation enhanced the sensitivity of HCC cells to sorafenib alongside changes in the expression of Bax and Bcl-2, apoptosis-related proteins. Our findings underscore the important role of SMG5 in HCC development and its involvement in sorafenib resistance, highlighting it as a potential target for HCC treatment.

Longitudinal Analysis of One-Carbon Metabolism-Related Metabolites in Maternal and Cord Blood of Japanese Pregnant Women.

One-carbon metabolism (OCM) is a complex and interconnected network that undergoes drastic changes during pregnancy. In this study, we investigated the longitudinal distribution of OCM-related metabolites in maternal and cord blood and explored their relationships. Additionally, we conducted cross-sectional analyses to examine the interrelationships among these metabolites. This study included 146 healthy pregnant women who participated in the Chiba Study of Mother and Child Health. Maternal blood samples were collected during early pregnancy, late pregnancy, and delivery, along with cord blood samples. We analyzed 18 OCM-related metabolites in serum using stable isotope dilution liquid chromatography/tandem mass spectrometry. We found that serum S-adenosylmethionine (SAM) concentrations in maternal blood remained stable throughout pregnancy. Conversely, S-adenosylhomocysteine (SAH) concentrations increased, and the total homocysteine/total cysteine ratio significantly increased with advancing gestational age. The betaine/dimethylglycine ratio was negatively correlated with total homocysteine in maternal blood for all sampling periods, and this correlation strengthened with advances in gestational age. Most OCM-related metabolites measured in this study showed significant positive correlations between maternal blood at delivery and cord blood. These findings suggest that maternal OCM status may impact fetal development and indicate the need for comprehensive and longitudinal evaluations of OCM during pregnancy.

Development and validation of a generic methyltransferase enzymatic assay based on an SAH riboswitch

Methylation of proteins and nucleic acids plays a fundamental role in epigenetic regulation, and discovery of methyltransferase (MT) inhibitors is an area of intense activity. Because of the diversity of MTs and their products, assay methods that detect S-adenosylhomocysteine (SAH) – the invariant product of S-adenosylmethionine (SAM)-dependent methylation reactions - offer some advantages over methods that detect specific methylation events. However, direct, homogenous detection of SAH requires a reagent capable of discriminating between SAH and SAM, which differ by a single methyl group. Moreover, MTs are slow enzymes and many have submicromolar affinities for SAM; these properties translate to a need for detection of SAH at low nanomolar concentrations in the presence of excess SAM. To meet these needs, we leveraged the exquisite molecular recognition properties of a naturally occurring SAH-sensing RNA aptamer, or riboswitch. By splitting the riboswitch into two fragments, such that SAH binding induces assembly of a trimeric complex, we engineered sensors that transduce binding of SAH into positive fluorescence polarization (FP) and time resolved Förster resonance energy transfer (TR-FRET) signals. The split riboswitch configuration, called the AptaFluor™ SAH Methyltransferase Assay, allows robust detection of SAH (Z’ > 0.7) at concentrations below 10 nM, with overnight signal stability in the presence of typical MT assay components. The AptaFluor assay tolerates diverse MT substrates, including histones, nucleosomes, DNA and RNA, and we demonstrated its utility as a robust, enzymatic assay method for several methyltransferases with SAM Km values < 1 µM. The assay was validated for HTS by performing a pilot screen of 1,280 compounds against the SARS-CoV-2 RNA capping enzyme, nsp14. By enabling direct, homogenous detection of SAH at low nanomolar concentrations, the AptaFluor assay provides a universal platform for screening and profiling MTs at physiologically relevant SAM concentrations.

Abstract PO5-08-06: Metabolic shift to serine pathway induced by lipids confers oncogenic properties in non-transformed breast cells

Abstract Introduction. Oncogenic factors that are local/in-breast are of great interest as they may be more specifically targetable for breast cancer prevention than systemic factors. We have identified a lipid metabolism gene signature that is enriched in breast tissue at risk for estrogen negative breast cancer (ER- BC). Utilizing the medium chain fatty acid Octanoic acid (OA) to probe lipid metabolism in non-transformed breast epithelial cells, we observed increased flux through several metabolic reactions and altered histone methylation with consequent changes in gene expression (e.g. neural genes). Neuronal signaling and regulatory circuits are observed in cancer cells of multiple origins, not just ones with ontological relationships to neurons. We hypothesize that the first and rate limiting step in the de novo serine pathway, which is catalyzed by Phosphoglycerate Dehydrogenase (PHGDH) is key to these observations. In the forward direction, PHGDH participates in the serine, one-carbon, glycine (SOG) and methionine pathways that produce the methyl donor S-adenosylmethionine (SAM) and in the reverse direction produces the oncometabolite 2-Hydroxyglycerate (2-HG). Methods. Non-transformed MCF-10A cells exposed to OA were utilized for U13C-glucose tracing. SAM and 2-HG concentrations following treatment with OA ± PHGDH inhibitor were measured by liquid chromatography. CUT&amp;RUN for H3K4me3 was performed and genes affected by OA (PMID: 28263391) were compared with OA-responsive peaks. Single cell RNA-sequencing was carried out using breast microstructures derived from reduction mammoplasty tissue exposed to vehicle or OA. Microstructures were dissociated into single cells and sequenced using the 10x Genomics platform. The digital expression matrix file containing UMIs were analyzed with Seurat. Alkaline comet assay was performed to detect DNA breaks. Results. U13C-glucose tracing in presence of OA revealed that one-carbon-THF was redirected to the methionine cycle increasing flux to methylation. Concentrations of SAM and 2-HG increased after 15- and 30-min OA exposure, respectively; PHGDH inhibitor blocked these increases. H3K4me3 CUT&amp;RUN revealed 661 differential peaks (FDR &amp;lt; 0.05) comparing OA to control. 73% of H3K4me3 OA-associated peaks were in regulatory regions of OA-induced genes (FDR &amp;lt; 0.01), these genes are involved in neural pathways, EMT and ER- BC. Motif analysis revealed an overrepresentation of binding sites for transcription factors ATF3/4 (p &amp;lt; 0.05), which are regulators of the serine pathway. Single cell RNA-sequencing revealed OA not only affected the distribution of cell subpopulations but also modulated the expression of many genes within each subcluster. The percentage of luminal progenitor subcluster 3 increased upon OA from less than 1% to about 13%. Within basal subcluster 3, OA drives the expression of ATF3, along with two of the enzymes in the de novo serine pathway: PHGDH and PSAT1. Alkaline comet assay showed DNA breaks in OA- and control 2-HG- treated cells. Conclusions. Metabolism of OA in preference to glucose and glutamine results in a metabolic shift toward the serine pathway increasing the production of SAM and 2-HG, with implications for oncogenesis: 1. SAM production results in epigenetic fostered plasticity leading to reprogramming/selecting cells that express genes consistent with a neural/neural crest-like state. These co-opted neuronal regulatory mechanisms can make critical contributions to the acquired functional capabilities that drive cancer development. 2. 2-HG exposure results in appearance of DNA breaks, which are likely consequent to the inhibition of the alpha-ketoglutarate-dependent dioxygenases KDM 4A/B by 2-HG. Their catalytic activity is required for homologous recombination repair; inhibition results in metabolic “BRCAness”. Citation Format: Mariana Bustamante Eduardo, Gannon Cottone, Shiyu Liu, Maria Paula Zappia, Elizaveta V. Benevolenskaya, Abul Bashar Mir Md. Khademul Islam, Maxim V. Frolov, Seema Khan, Susan Clare. Metabolic shift to serine pathway induced by lipids confers oncogenic properties in non-transformed breast cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-08-06.

Abstract 444: Lipid exposure re-wires cellular metabolism away from glycolysis toward the serine pathway conferring oncogenic properties to non-transformed breast cells

Abstract Introduction. A lipid metabolism gene signature is associated with the risk of estrogen negative breast cancer (ER-BC). In vitro, lipid exposure alters histone methylation affecting gene expression and increases flux through various metabolic reactions including those involved in serine, one-carbon, glycine (SOG) and methionine. We hypothesized that the metabolism of lipids in preference to glucose and glutamine results in a metabolic shift toward the serine pathway increasing S-adenosylmethionine (SAM) leading to histone methylation increases and changes in gene expression. Methods. MCF-10A cells exposed to octanoic acid (OA) were utilized for U13C-glucose tracing. SAM, glutathione (GSH) and 2-hydroxyglutarate (2-HG) concentrations were measured following treatment with OA ± the PHGDH inhibitor CBR-5884. ROS-induced redox changes were monitored live in cells transduced with ORP1-roGFP2 vectors. CUT&amp;RUN was performed for H3K4me3. Expression of OA-induced genes was measured by rt-qPCR upon exposure to OA ± CBR-5884 or OA ± the histone methyltransferase (HMT) inhibitor Piribedil. Alkaline comet assay was done to detect DNA breaks. Single-cell RNA-seq (scRNAseq) was performed on tissue-derived breast microstructures exposed to ± OA. Results. Upon OA treatment, the Cancer index increased and 1C-THF was redirected to the methionine cycle increasing flux to methylation. OA significantly increased the production of SAM, GSH and 2-HG after 15 min. Blocking the first enzyme in the serine pathway, PHGDH, prevented these increases. After 5 min OA exposure, mitochondrial and nuclear ROS increased significantly (p &amp;lt; 0.0001), peaking at 15 min. H3K4me3 CUT&amp;RUN revealed 661 differential peaks (FDR &amp;lt; 0.05) comparing OA to control. 73% of H3K4me3 OA-associated peaks were in regulatory regions of OA-induced genes (FDR &amp;lt; 0.01) including neural, EMT and ER- BC related genes. Motif analysis revealed an overrepresentation of binding sites for serine pathway transcriptional activators ATF3/4 (p &amp;lt; 0.05). Blocking PHGDH or HMT prevented OA-induced gene expression changes. Alkaline comet assay showed that OA significantly increased comet tails. scRNAseq revealed OA increased the numbers of cells within all cell clusters expressing ATF3 and PHGDH. Also observed was an increase in the percentage of basal BSL1, luminal progenitor LP3 and hormone sensing HS1 cells. Conclusions. Metabolism of OA results in a metabolic shift toward the SOG and methionine increasing the production of SAM, GSH and 2-HG which have implications for oncogenesis. The increased SAM fosters epigenetic phenotypic plasticity via altered histone methylation. The increased proportion of specific cell types likely reflects the survival of cells able to mitigate oxidative stress. Increased 2-HG may produce metabolic BRCAness by inhibiting 2-oxoglutarate-dependent dioxygenases. Citation Format: Mariana Bustamante Eduardo, Gannon Cottone, Shiyu Liu, Flavio R. Palma, Maria Paula Zappia, Abul B.M.M.K. Islam, Elizaveta V. Benevolenskaya, Maxim V. Frolov, Jason Locasale, Marcelo G. Bonini, Navdeep S. Chandel, Seema Khan, Susan Clare. Lipid exposure re-wires cellular metabolism away from glycolysis toward the serine pathway conferring oncogenic properties to non-transformed breast cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 444.

MetJ-Based Mutually Interfering SAM-ON/SAM-OFF Biosensors.

SAM (S-adenosylmethionine) is an important metabolite that operates as a major donor of methyl groups and is a controller of various physiological processes. Its availability is also believed to be a major bottleneck in the biological production of numerous high-value metabolites. Here, we constructed SAM-sensing systems using MetJ, an SAM-dependent transcriptional regulator, as a core component. SAM is a corepressor of MetJ, which suppresses the MetJ promoter with an increasing cellular concentration of SAM (SAM-OFF sensor). The application of transcriptional interference and evolutionary tuning effectively inverted its response, yielding a SAM-ON sensor (signal increases with increasing SAM concentration). By linking two genes encoding fluorescent protein reporters in such a way that their transcription events interfere with each other's and by placing one of them under the control of MetJ, we could increase the effective signal-to-noise ratio of the SAM sensor while decreasing the batch-to-batch deviation in signal output, likely by canceling out the growth-associated fluctuation in translational resources. By taking the ratio of SAM-ON/SAM-OFF signals and by resetting the default pool size of SAM, we could rapidly identify SAM synthetase (MetK) mutants with increased cellular activity from a random library. The strategy described herein should be widely applicable for identifying activity mutants, which would be otherwise overlooked because of the strong homeostasis of metabolic networks.

Depletion of SAM leading to loss of heterochromatin drives muscle stem cell ageing.

The global loss of heterochromatin during ageing has been observed in eukaryotes from yeast to humans, and this has been proposed as one of the causes of ageing. However, the cause of this age-associated loss of heterochromatin has remained enigmatic. Here we show that heterochromatin markers, including histone H3K9 di/tri-methylation and HP1, decrease with age in muscle stem cells (MuSCs) as a consequence of the depletion of the methyl donor S-adenosylmethionine (SAM). We find that restoration of intracellular SAM in aged MuSCs restores heterochromatin content to youthful levels and rejuvenates age-associated features, including DNA damage accumulation, increased cell death, and defective muscle regeneration. SAM is not only a methyl group donor for transmethylation, but it is also an aminopropyl donor for polyamine synthesis. Excessive consumption of SAM in polyamine synthesis may reduce its availability for transmethylation. Consistent with this premise, we observe that perturbation of increased polyamine synthesis by inhibiting spermidine synthase restores intracellular SAM content and heterochromatin formation, leading to improvements in aged MuSC function and regenerative capacity in male and female mice. Together, our studies demonstrate a direct causal link between polyamine metabolism and epigenetic dysregulation during murine MuSC ageing.

Selective utilization of organic carbon molecules promotes arsenic methylation by increasing methyltransferase activity in arsM-harboring microbes of paddy soils

The composition of organic carbon molecules (OCMs) impacts microbial diversity and in turn regulates the microbial transformation of elements in soils. However, the potential association of OCMs characteristics with arsenic methylation mediated by arsenite S-adenosylmethionine (SAM) methyltransferase gene (arsM)-harboring microbes in paddy soils remains unclear. Here, we investigated the impacts of OCMs on microbial methylators and arsenic methylation by identifying OCMs with Fourier transform ion cyclotron resonance mass spectrometry. The results showed that arsenic methylation efficiency increased by 57.4–260.1% in pig manure- or rice straw-treated paddy soils compared to the control. Molecules containing carbon, hydrogen, oxygen, and nitrogen (CHONs) with the atomic oxygen to carbon ratio (O/C) ranging from 0.5 to 0.8 had a more significant correlation (p < 0.01) with the abundance of the arsM gene than with that of 16S rRNA. This was confirmed by a significant positive correlation (p < 0.001) between consumed CHONs with O/C of 0.5–0.8 and increased methylated arsenic. CHONs (with O/C of 0.5–0.8) increased the intracellular SAM concentration and methyltransferase activity, stimulating arsenic methylation in vivo. The specialization of arsM-harboring taxa in CHONs with different O/C were identified. These observations provide a basis for predicting the risk of straight-head disease in paddy soils.

Dietary methionine supplementation during the estrous cycle improves follicular development and estrogen synthesis in rats.

The follicle is an important unit for the synthesis of steroid hormones and the oocyte development and maturation in mammals. However, the effect of methionine supply on follicle development and its regulatory mechanism are still unclear. In the present study, we found that dietary methionine supplementation during the estrous cycle significantly increased the number of embryo implantation sites, as well as serum contents of a variety of amino acids and methionine metabolic enzymes in rats. Additionally, methionine supplementation markedly enhanced the expression of rat ovarian neutral amino acid transporters, DNA methyltransferases (DNMTs), and cystathionine gamma-lyase (CSE); meanwhile, it significantly increased the ovarian concentrations of the metabolite S-adenosylmethionine (SAM) and glutathione (GSH). In vitro data showed that methionine supply promotes rat follicle development through enhancing the expression of critical gene growth differentiation factor 9 and bone morphogenetic protein 15. Furthermore, methionine enhanced the relative protein and mRNA expression of critical genes related to estrogen synthesis, ultimately increasing estrogen synthesis in primary ovarian granulosa cells. Taken together, our results suggested that methionine promoted follicular growth and estrogen synthesis in rats during the estrus cycle, which improved embryo implantation during early pregnancy. These findings provided a potential nutritional strategy to improve the reproductive performance of animals.

S-Adenosylmethionine Inhibits the Proliferation of Retinoblastoma Cell Y79, Induces Apoptosis and Cell Cycle Arrest of Y79 Cells by Inhibiting the Wnt2/β-Catenin Pathway.

Retinoblastoma is one of the most common primary intraocular malignancies in young children. Traditional treatment methods such as chemotherapy often come with significant adverse effects, such as hearing loss, cognitive impairment, and vision loss. Therefore, there is an urgent need to explore a novel therapeutic drug that is both effective and safe. S-adenosylmethionine (SAM) is a natural compound known to exhibit anti-proliferative effects in various cancer cell lines. However, to date, no studies investigated the effects of SAM on retinoblastoma cells and its potential mechanisms of action. Therefore, this study aims to investigate the impact of SAM on retinoblastoma cells and explore its possible mechanisms of action, with the hope of providing new insights into the treatment of this disease. The optimal concentration of SAM was determined using the Cell Counting Kit-8 assay. The effect of SAM on retinoblastoma proliferation was assessed using the 5-ethynyl-2'-deoxyuridine cell proliferation assay. Y79 cells were subjected to hematoxylin and eosin stain and electron microscopy to observe any morphological changes induced by SAM. The stages of SAM's action on the retinoblastoma cell cycle and its apoptotic effects were measured using flow cytometry. The apoptotic effect of SAM on retinoblastoma was further confirmed using the TUNEL assay. Differential expression of related genes was detected through RT-PCR. In vivo subcutaneous tumor formation in nude mice and immunohistochemistry were employed to validate the effect of SAM on retinoblastoma-related phenotypes. Western blotting was conducted to investigate whether SAM modulated retinoblastoma-related phenotypes via the Wnt2/β-catenin pathway. SAM arrested the cell cycle of retinoblastoma at the G1 phase, induced apoptosis of retinoblastoma cells through the Wnt2/β-catenin pathway, and affected their morphology and even ultrastructure. In addition, in vitro and in vivo experiments demonstrated that SAM had an oncogenic effect on retinoblastoma. In this study, we verify in vitro and in vivo whether SAM inhibits the proliferation of retinoblastoma cell Y7, induces apoptosis and cell cycle arrest of Y79 cells by inhibiting the Wnt2/β-catenin pathway, and affects the morphology and structure of retinoblastoma cell Y79.

DIPG-36. H3K27M HISTONE MUTANT GLIOMAS ARE SENSITIVE TO METHIONINE LOSS AND MAT2A INHIBITION THROUGH A NOVEL FEEDBACK MECHANISM BETWEEN AMD1 AND METTL16

Abstract Diffuse Midline gliomas (DMGs) are grade IV tumors by the World Health Organization. They are inoperable and resistant to chemo/radiotherapies resulting in a median survival of 8-11 months and a 5-year survival of &amp;lt;2%. DMG is an epigenetic disease characterized by mutations on histone H3.3 K27M resulting in global transcriptional reprogramming. This disease lacks appropriate models to predict disease biology and response to treatment. Therefore, we developed a novel syngeneic H3K27M mouse model using clinically relevant co-alterations in Olig2+ neural progenitor cells (NPCs). Using an unbiased systems biology approach, we identified a reliance of H3K27M but not isogenic controls to the amino acid methionine, and the enzymes methionine adenosyltransferase 2A (MAT2A), and adenosylmethionine decarboxylase 1 (AMD1). MAT2A is a master regulator of methionine metabolism that converts methionine into the universal methyl donor S-adenosylmethionine (SAM) which is later converted into decarboxylated SAM (dcSAM) by AMD1 for polyamine metabolism. We postulated that targeting methionine regulator MAT2A through genetic/pharmacological abrogation would selectively alter DMG viability by disrupting the methylome. We discovered a novel mechanism demonstrating H3K27M cells are sensitive to MAT2A loss independent of methylthioadenosine phosphorylase (MTAP) deletions but rather through AMD1 overexpression. The current paradigm shows that MAT2A protein expression is inversely correlated with cellular SAM concentrations as sensed by splicing complex and m6A reader methyltransferase-like protein 16 (METTL16). To investigate the molecular mechanism by which H3K27M represses MAT2A, we postulated that dcSAM, the resultant metabolite of AMD1, promote(s) high turnover of METTL16–MAT2A transcript interactions like SAM, thereby diminishing MAT2A transcript and protein expression. We found that exogenous dcSAM promoted MAT2A intron retention and lower mature transcript levels. Our findings demonstrate that H3K27M leads to increased AMD1 protein expression resulting in diminished MAT2A expression. Combinatorial treatments inhibiting MAT2A and AMD1 may presents exploitable therapeutic vulnerabilities in these gliomas.

Revisiting One-Carbon Metabolites in Human Breast Milk: Focus on S-Adenosylmethionine.

Breastfeeding is the gold standard for early nutrition. Metabolites from the one-carbon metabolism pool are crucial for infant development. The aim of this study is to compare the breast-milk one-carbon metabolic profile to other biofluids where these metabolites are present, including cord and adult blood plasma as well as cerebrospinal fluid. Breast milk (n = 142), cord blood plasma (n = 23), maternal plasma (n = 28), aging adult plasma (n = 91), cerebrospinal fluid (n = 92), and infant milk formula (n = 11) samples were analyzed by LC-MS/MS to quantify choline, betaine, methionine, S-adenosylmethionine, S-adenosylhomocysteine, total homocysteine, and cystathionine. Differences between groups were visualized by principal component analysis and analyzed by Kruskal-Wallis test. Correlation analysis was performed between one-carbon metabolites in human breast milk. Principal component analysis based on these metabolites separated breast milk samples from other biofluids. The S-adenosylmethionine (SAM) concentration was significantly higher in breast milk compared to the other biofluids and was absent in infant milk formulas. Despite many significant correlations between metabolites in one-carbon metabolism, there were no significant correlations between SAM and methionine or total homocysteine. Together, our data indicate a high concentration of SAM in breast milk, which may suggest a strong demand for this metabolite during infant early growth while its absence in infant milk formulas may indicate the inadequacy of this vital metabolic nutrient.

식이제한이 마우스 신장의 글루타치온 대사과정에 미치는 영향

Various benefits for the maintenance of metabolic homeostasis can be obtained through dietary restriction. Short-term fasting of mice may induce systemic metabolic remodeling, which may have a significant effect on the level of endogenous metabolites. This study was conducted to examine metabolic changes of sulfur-containing substances in the kidneys of fasted mice over 48 h. No change in the concentration of renal methionine was observed over 48 h; however, significant decreases in the concentrations of S-adenosylmethionine and S-adenosylhomocysteine were observed at both 24 and 48 h after fasting. A significant decrease in the concentration of renal cysteine was observed from 24 h, and this was maintained at 48 h. A significant decrease in the concentration of glutathione (GSH) was observed in the kidney at 48 h, whereas increased concentrations of taurine were observed 48 h after fasting. Although significantly increased expression levels of BHMT were observed, markedly decreased expression levels of CβS were observed at 48 h after fasting, indicating activated generation of methionine, but downregulation of cysteine synthesis. The expression level of γ-GCL was not altered by fasting for 48 h; however, a significant increase of cysteine sulfinate decarboxylase mediating the taurine synthesis was observed. Significantly decreased protein expression levels of solute carrier family 7 member 11, which imports extracellular cystine, were observed at 48 h. These results suggest that the renal methionine concentration was maintained during short-term fasting through an increase in the re-methylation of homocysteine. Moreover, a reduction in the concentration of cysteine was the result of an increase in the synthesis of taurine and a decrease in the uptake of extracellular cystine, which may result in a decreased concentration of renal GSH.

Integration of Transcriptome and Metabolome Analyses Reveals the Mechanistic Basis for Cadmium Accumulation in Maize.

Cadmium (Cd) pollution in soil has become a major environmental issue worldwide. However, the underlying molecular mechanism of low grain-Cd accumulation (GCA) in maize is still largely unknown. Herein, we report the mechanistic basis for low GCA in maize by a multiomics approach. The low GCA genotype L63 showed normal vacuolar formation and a lower capacity of xylem loading of Cd than the high-accumulator L42 under Cd stress. Transcriptomic sequencing identified 84 low-GCA-associated genes which are mainly involved in the S-adenosylmethionine (SAM) cycle, metal transport, and vacuolar sequestration. A metabolome analysis revealed that L63 plants had a more active SAM cycle and a greater capacity for terpenoid synthesis and phenylalanine metabolism than L42. Combining the analysis of transcriptome and metabolome characterized several genes as key genes involved in the determination of Cd accumulation. Our study identifies a mechanistic basis for low Cd accumulation in maize grains and provides candidate genes for genetic improvement of crops.

Association among urinary polycyclic aromatic hydrocarbons metabolites, SAHH activity and H19 expression in coke oven workers

Objective: To investigate the relationship of polycyclic aromatic hydrocarbons (PAHs) exposure, S-adenosylhomocysteine hydrolase (SAHH) activity and long noncoding RNA H19 gene expression in the urine of coke oven workers. Methods: In September 2019, in a coking plant in Taiyuan City, 146 male workers who had worked in coke oven operations for one year were selected through a completely random sampling method, and their basic personal information was collected by questionnaire survey, and blood and urine samples were collected. The levels of 4 PAHs metabolites 2-hydroxfluorene (2-FLU), 2- hydroxynaphthalene (2-NAP), 9-hydroxyphenanthren (9-PHE), and 1-hydroxypyrene (1-OHP) in urine were detected by high performance liquid chromatography (HPLC) -fluorescence detection method. HPLC-UV detection method was used to detect the content of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) in plasma, and the SAHH activity value was obtained by calculating the ratio. Reverse transcription PCR method was used to determine the H19 gene expression level. Urine levels of 2-FLU, 2-NAP, 9-PHE, and 1-OHP were divided into Q(1), Q(2), Q(3), and Q(4) groups according to quartiles (P(25), P(50), P(75)). Regression, trend test and restricted cubic splines were used to analyze the relationship among PAHs metabolites, SAHH activity, H19 gene expression and their dose-response. Results: The median age of coke oven workers was 39.60 years old, the median length of service was 20.38 years, and the urinary levels of 2-FLU, 2-NAP, 9- PHE, and 1-OHP were 0.29, 0.74, 0.09, and 0.06 μg/mmol Cr, respectively. The levels of 2-FLU, 2-NAP and 9-PHE in the urine of workers were significantly different between groups with different 1-OHP levels (P<0.05). After adjusting for age, length of service, smoking, drinking, and levels of 2-FLU, 2-NAP and 9-PHE, SAHH activity decreased with the increase of urinary 1-OHP level (OR=0.63, 95%CI: 0.41-0.98, P=0.038), showing a nonlinear relationship (P(nonlinear)= 0.030). H19 gene expression increased with the increase of urinary 1- OHP level (OR=1.51, 95%CI: 1.03-2.19, P=0.033), there was a linear relationship (P(trend)= 0.058). The relationship between the other three metabolites in urine and SAHH activity and H19 gene expression was not statistically significant (P>0.05) . Conclusion: Urinary 1-OHP level may be a risk factor for decreased SAHH activity and increased H19 gene expression in coke oven workers.

Metformin Treatment Abrogates the Competitive Advantage of Dnmt3a R878H HSPCs By Enhancing DNA Methylation Activity

Clonal hematopoiesis of indeterminate potential (CHIP) refers to the clonal expansion of hematopoietic stem and progenitor cells (HSPCs) carrying leukemogenic mutations in individuals without evidence of a blood disorder. CHIP carriers are at an increased risk of blood cancers and cardiovascular diseases. Targeting the clonal expansion of mutant HSPCs has the potential to mitigate CHIP-related illnesses. The most commonly mutated gene in CHIP is DNMT3A, which encodes an enzyme that catalyzes the methyl transfer from S-adenosylmethionine (SAM) to cytosines in DNA. The heterozygous DNMT3A hotspot mutation at R882 exerts a dominant negative effect on the wild-type (WT) allele, resulting in decreased DNA methylation activity. Prior studies have shown that murine HSPCs carrying mutant Dnmt3a R878H (equivalent to R882 in humans) have an advantage over WT cells in competition assays. Here, we aimed to first identify differences in the functional properties between Dnmt3a-mutated and WT HSPCs. Given the crosstalk between mitochondrial metabolism and DNA methylation, we investigated potential differences in mitochondrial activity and found that the level of mitochondrial respiration was higher in Dnmt3aR878H/+ HSPCs than in WT cells. To determine if this translated to differential dependencies, we silenced the expression of critical subunits in the each of five electron transport chain (ETC) complexes. Knockdown of NDUFV1 in Complex I was the most effective in reducing the competitive advantage of Dnmt3aR878H/+ cells in vitro. To explore translational relevance of this finding, we tested the impact of metformin, a pharmacologic inhibitor of Complex I, on Dnmt3a-mutated HSPCs. Treatment with metformin at clinically relevant concentrations suppressed the competitive advantage of mutant cells in vitro. Importantly, this effect was rescued by expression of NDI1, a metformin-resistant yeast analog of Complex I. To extend these findings, we conducted a competitive repopulation assay between CD45.2+ Dnmt3aR878H/+ and CD45.1+ Dnmt3a+/+ HSPCs. Metformin treatment abrogated the in vivo competitive advantage of mutant cells over an 8-month period based on peripheral blood chimerism. To decipher the mechanism of action, we performed transcriptome and metabolome profiling of mutant HSPCs treated with or without metformin for 1 month. GSEA analysis of the transcriptome data revealed a downregulation of genes associated with stemness in metformin-treated cells. Intriguingly, we found that metformin increased the expression of genes involved in one-carbon metabolism, which generates SAM. Consistent with these findings, metabolomics analysis showed that the levels of several key metabolites in one-carbon metabolism, including SAM, were higher in metformin-treated cells compared with controls. Genetic and pharmacologic inhibition of serine hydroxymethyltransferase 2 (SHMT2), a key enzyme in one-carbon metabolism, as well as the addition of exogeneous S-adenosylhomocysteine (SAH), an inhibitor of SAM-dependent methyltransferases, effectively rescued the effect of metformin on mutant cells in vitro. Based on the above findings, we hypothesized that metformin treatment might enhance the residual DNA methylation activity found in Dnmt3aR878H/+ mutated cells by increasing SAM concentration. In support of this hypothesis, we found that exogeneous SAM reproduced the effect of metformin, and HSPCs with homozygous Dnmt3aR878H mutations were insensitive to metformin. Furthermore, we performed DNA methylome profiling of untreated and metformin-treated mutant and WT HSPCs using RRBS. This analysis revealed that the hypomethylated regions (HMRs) in untreated Dnmt3aR878H/+ HSPCs relative to WT controls were enriched for regions of repressed chromatin with H3K27 methylation. Importantly, metformin treatment increased the level of DNA methylation at these regions in mutated HSPCs. In summary, our findings demonstrate that Dnmt3aR878H/+ HSPCs depend on ETC activity and particularly complex I activity to maintain their competitive advantage over WT cells. We propose a model in which inhibition of complex I activity with metformin increases one-carbon metabolism and SAM level, thereby restoring DNA methylation at HMRs and the differentiation of mutant HSPCs. Our findings have important implications for the development of preventive interventions against DNMT3A-mutated CHIP.

Efficacy and pharmacokinetics of betaine in CBS and cblC deficiencies: a cross-over randomized controlled trial

BackgroundBetaine is an “alternate” methyl donor for homocysteine remethylation catalyzed by betaine homocysteine methyltransferase (BHMT), an enzyme mainly expressed in the liver and kidney. Betaine has been used for more than 30 years in pyridoxine non-responsive cystathionine beta-synthase (pnrCBS) and cobalamin C (cblC) deficiencies to lower the hyperhomocysteinemia, although little is known about the optimal therapeutic dosage and its pharmacokinetic in these patients.AimsWe compared 2 betaine doses (100 mg/kg/day vs. 250 mg/kg/day) in children affected by pnrCBS or cblC deficiencies. We also measured the pharmacokinetics parameters after a single dose of betaine (100 or 250 mg/kg) in these patients.MethodsWe conducted a prospective, randomized, crossover clinical trial with blinded evaluation. The primary outcome was the equivalence of total plasma homocysteine (tHcy) concentrations upon one-month oral treatment with betaine at 100 versus 250 mg/kg/day.ResultsEleven patients completed the study (5 pnrCBS and 6 cblC). tHcy concentrations were equivalent after a one-month treatment period for the two betaine dosages. Multivariate analysis showed a significant effect of betaine dose on methionine (Met) (p = 0.01) and S-adenosylmethionine (SAM) concentrations (p = 0.006).ConclusionsOur analysis shows that there is no overt benefit to increasing betaine dosage higher than 100 mg/kg/day to lower tHcy concentrations in pnrCBS and cblC deficiencies. However, increasing betaine up to 250 mg/kg/d could benefit cblC patients through the increase of methionine and SAM concentrations, as low Met and SAM concentrations are involved in the pathophysiology of this disease. In contrast, in pnrCBS deficiency, betaine doses higher than 100 mg/kg/day could be harmful to these patients with pre-existing hypermethioninemia.Trial registration: Clinical Trials, NCT02404337. Registered 23 May 2015—prospectively registered, https://clinicaltrials.gov.

Lipopolysaccharide affects energy metabolism and elevates nicotinamide N-methyltransferase level in human aortic endothelial cells (HAEC).

This study aimed to investigate the putative role of nicotinamide N-methyltransferase in the metabolic response of human aortic endothelial cells. This enzyme catalyses S-adenosylmethionine-mediated methylation of nicotinamide to methylnicotinamide. This reaction is accompanied by the reduction of the intracellular nicotinamide and S-adenosylmethionine content. This may affect NAD+ synthesis and various processes of methylation, including epigenetic modifications of chromatin. Particularly high activity of nicotinamide N-methyltransferase is detected in liver, many neoplasms as well as in various cells in stressful conditions. The elevated nicotinamide N-methyltransferase content was also found in endothelial cells treated with statins. Although the exogenous methylnicotinamide has been postulated to induce a vasodilatory response, the specific metabolic role of nicotinamide N-methyltransferase in vascular endothelium is still unclear. Treatment of endothelial cells with bacterial lipopolysaccharide evokes several metabolic and functional consequences which built a multifaceted physiological response of endothelium to bacterial infection. Among the spectrum of biochemical changes substantially elevated protein level of nicotinamide N-methyltransferase was particularly intriguing. Here it has been shown that silencing of the nicotinamide N-methyltransferase gene influences several changes which are observed in cells treated with lipopolysaccharide. They include altered energy metabolism and rearrangement of the mitochondrial network. A complete explanation of the mechanisms behind the protective consequences of the nicotinamide N-methyltransferase deficiency in cells treated with lipopolysaccharide needs further investigation.

As3MT-mediated SAM consumption, which inhibits the methylation of histones and LINE1, is involved in arsenic-induced male reproductive damage

Studies have demonstrated that arsenic (As) induces male reproductive injury, however, the mechanism remains unknown. The high levels of arsenic (3) methyltransferase (As3MT) promote As-induced male reproductive toxicity. For As-exposed mice, the germ cells in seminiferous tubules and sperm quality were reduced. Exposure to As caused lower S-adenosylmethionine (SAM) and 5-methylcytosine (5 mC) levels, histone and DNA hypomethylation, upregulation of long interspersed element class 1 (LINE1, or L1), defective repair of double-strand breaks (DSBs), and the arrest of meiosis, resulting in apoptosis of germ cells and lower litter size. For GC-2spd (GC-2) cells, As induced apoptosis, which was prevented by adding SAM or by reducing the expression of As3MT. The levels of LINE1, affected by SAM content, were involved in As-induced apoptosis. Furthermore, folic acid (FA) and vitamin B12 (VB12) supplements restored SAM, 5 mC, and LINE1 levels and blocked impairment of spermatogenesis and testes and lower litter size. Exposed to As, mice with As3MT knockdown showed less impairment of spermatogenesis and testes and greater litter size compared to As-exposed wild-type (WT) mice. Thus, the high As3MT levels induced by As consume SAM and block histone and LINE1 DNA methylation, elevating LINE1 expression and evoking impairment of spermatogenesis, which causes male reproductive damage. Overall, we have found a mechanism for As-induced male reproductive damage, which provides biological insights into the alleviation of reproductive injury induced by environmental factors.