Search Results (71)

Carbon dioxide (CO₂) is a cost-effective, abundant, and renewable carbon source, but its utilization technologies face several issues. The reductive glycine pathway (RGP) is recognized as one of the most efficient one-carbon (C1) assimilation routes in nature, with its core component—the glycine cleavage system (GCS: GcvP, GcvH, GcvT, and GcvL)—playing an essential role in C1 metabolism. To develop efficient CO₂ conversion and utilization pathways, we identified NhFtfL and AmFchA-MtdA with high catalytic efficiency through gene mining and constructed a four-plasmid co-expression system in E. coli BL21(DE3) using Gibson Assembly. This system integrated GcvP-GcvH, GcvT-GcvL, NhFtfL-AmFchA-MtdA, and RsPPK2, thereby reconstituting the complete RGP while enhancing ATP supply. The engineered strain functioned as an efficient whole-cell biocatalyst, achieving a glycine space–time productivity of 0.125 mmol/L/h via one-pot conversion of formate. Furthermore, we expanded the application scope by developing a whole-cell electrocatalysis system that directly synthesized glycine from CO₂ and NH₄Cl, achieving a glycine space–time productivity of 0.135 mmol/L/h. This study demonstrates the potential of the engineered RGP system for upgrading C1 resources and supports the transition toward carbon neutrality. Full article

Background/Objectives: Vitamin B₁₂ deficiency is increasingly recognized as a contributor in both vascular and neurodegenerative aging-related disorders. Its deficiency disrupts one-carbon metabolism, leading to impaired homocysteine (Hcy) cycling. Elevated Hcy is a well-established risk factor for vascular dysfunction. Previously, we established that elevated Hcy contributes to aging retinal diseases and plays a central role in blood retinal barrier (BRB) dysfunction. Building on this foundation, the present study examines how B-vitamin deficiency disrupts one-carbon metabolism and whether restoring these vitamins can serve as a preventive or therapeutic strategy. Since B-vitamins (B₆, B₉, and B₁₂) are crucial cofactors in the metabolism of Hcy, we investigated how dietary changes in these vitamins affect serum Hcy levels and retinal vascular integrity in mice. Methods: C57BL/6- Wild-type (WT) and cbs^+/− mice (Cystathionine Beta-Synthase heterozygotes, common mouse model for elevated Hcy) were fed specially formulated diets, which contained different levels of B-vitamins (normal, deficient (B-Vit (−)) or enriched (B-Vit (+)). Initially, two groups of mice were placed on either a normal or a deficient diet. After 12–16 weeks, the success of the diet regimes was confirmed by observing serum B₁₂ deficiency in the B-Vit (−) group, along with elevated Hcy levels. Subsequently, a subgroup of the B-Vit (−) mice was switched to an enriched diet. The BRB integrity was evaluated in living mice using fluorescein angiography (FA), optical coherence tomography (OCT), and in the perfused mice retinas with Western blot analysis of leaked retinal albumin and tight junction proteins (occludin and ZO-1) levels. Results: The B-vitamin deficiency caused significant drop in serum vitamin B₁₂ and an increase in plasma Hcy, leading to vascular leakage, altered retinal thickness, choroidal neovascular changes, increased retinal albumin leak, and decreased tight junction protein expression, indicating BRB disruption, which was restored with B-vitamin supplementation. Conclusions: a long-term deficiency of vitamins B₆, B₉, and B₁₂ can lead to disruptions in the BRB. However, supplementation with these B-vitamins has the potential to reverse these effects and help maintain the integrity of BRB. This under-score the significance of one-carbon metabolism for retinal health and suggests that ensuring adequate levels of B-vitamins may aid in preventing aging retinal diseases with BRB disruption such as diabetic retinopathy and age-related macular degeneration. Full article

Background an Objectives: The Mediterranean sea urchins Paracentrotus lividus and Arbacia lixula co-occur on shallow rocky reefs but display contrasting ecological and physiological traits. We compared their gonadal metabolomes to identify species-specific metabolic strategies. Methods: High-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR) spectroscopy to intact gonadal tissues, combining multivariate chemometric modelling with targeted integration, boxplot-based univariate analysis and pathway analysis. Results:A. lixula showed an osmolyte- and redox-oriented phenotype with elevated betaine, taurine, sarcosine, trimethylamine (TMA), trimethylamine N-oxide (TMAO), carnitine, creatine, malonate, methylmalonate, uridine and xanthine. In contrast, P. lividus exhibited an amino-acid-enriched anabolic profile dominated by lysine, glycine and glutamine, together with higher levels of formaldehyde, methanol and 3-carboxypropyl-trimethylammonium. Pathway analysis indicated that A. lixula metabolites mapped onto glycine/serine–threonine metabolism and the folate-linked one-carbon pool, whereas P. lividus metabolites were enriched in glyoxylate/dicarboxylate, nitrogen and amino-acid pathways. These contrasting osmolyte–C₁ versus nitrogen–amino-acid strategies are compatible with species-specific host–microbiota metabolic interactions inferred from published microbiome data. Conclusions: Overall, our results support a framework in which A. lixula adopts a resilience-oriented osmolyte strategy and P. lividus an efficiency-oriented anabolic strategy, highlighting HR-MAS NMR metabolomics as a powerful approach to investigate adaptive biochemical diversity in marine invertebrates. Full article

Background/Objectives: Numerous studies have demonstrated that dietary plant-derived polyphenols suppress signaling and metabolic pathways in various malignancies, including neuroblastoma. In the present study, we compared the inhibitory activities of selected polyphenols and their combinations on key metabolic and signaling pathways in two human neuroblastoma cell lines and two noncancerous cell lines—mesenchymal stem cells (MSCs). Methods: The influence of polyphenols on neuroblastoma cells and MSCs were studied via an MTT-assay, cell cycle analysis, and an apoptosis assay (flow cytometry). Chou-Talalay algorithms were used to quantify drug interactions. SeaHorse energy profiling was applied to study energy metabolism. The influence of the compounds on metabolic enzymes and signaling pathways was examined using immunoblotting. Total protein biosynthesis was assessed using o-propargyl-puromycin labeling (flow cytometry). Results: While most of the studied polyphenols displayed a more significant inhibitory effect on neuroblastoma cells than on mesenchymal stem cells (MSCs), we found that the combinations of curcumin and quercetin (CQ) and curcumin, quercetin, and resveratrol (CQR) were significantly superior to the individual compounds. These combinations displayed synergistic effects and inhibited the cell cycle while inducing apoptosis. The CQ and CQR combinations effectively suppressed metabolic reprogramming by downregulating key enzymes of glycolysis, respiration, one-carbon metabolism, glutaminolysis, and fatty acid biosynthesis, as well as N-Myc and c-Myc, which are master regulators of metabolic processes. Furthermore, CQ and CQR inhibited AKT/mTOR, MAPK/ERK, and WNT/β-catenin signaling pathways and total protein biosynthesis and sensitized malignant cells to doxorubicin. Conclusions: Polyphenol combinations exert multifaceted inhibitory effects on metabolic rewiring and signaling networks in neuroblastoma cells. Full article

Vascular health relies on the proper function of endothelial cells, which regulate vascular tone, blood fluidity, and barrier integrity. Endothelial dysfunction, often aggravated by inadequate vitamin absorption, contributes to a spectrum of clinical disorders, including cardiovascular disease, cerebrovascular disease, peripheral artery disease, age-related macular degeneration, lymphedema, and chronic venous insufficiency. B-group vitamins (especially folate, or vitamin B9), along with vitamins B12, B6, C, D, and E, are essential in maintaining endothelial function, supporting DNA synthesis, regulating methylation, enhancing cellular repair, mitigating oxidative stress and inflammatory signaling, and curtailing vascular damage. Folate is noted for its central function in one-carbon metabolism and in converting homocysteine to methionine, thereby reducing vascular toxicity. We cover natural dietary sources of folate, synthetic folic acid, and the biologically active forms 5-methyl-(6S)-tetrahydrofolate (L-5-MTHF, L-methylfolate) and 5-formyl-(6S)-tetrahydrofolate (levoleucovorin). Therapeutic strategies to address vascular health and prevent hyperhomocysteinemia in order to preclude follow-on disorders include targeted vitamin supplementation, dietary improvements to ensure a sufficient intake of bioavailable nutrient forms, and, in certain clinical contexts, the use of active L-methylfolate or levoleucovorin (a drug product) to bypass metabolic conversion issues. These evidence-based interventions aim to restore endothelial homeostasis, slow disease progression, and improve patient outcomes across a variety of disorders linked to poor vascular health. Full article

Background/Objectives: This study examined the effects of 2 weeks of betaine versus placebo supplementation (3 g/d) on 60 km cycling performance, gut permeability, and shifts in plasma metabolites. Methods: Participants included 21 male and female non-elite cyclists. A randomized, placebo-controlled, double-blind, crossover design was used with two 2-week supplementation periods and a 2-week washout period. Supplementation periods were followed by a 60 km cycling time trial. Six blood samples were collected before and after supplementation (overnight fasted state), and at 0 h, 1.5 h, 3 h, and 24 h post-exercise. Five-hour urine samples were collected pre-supplementation and post-60 km cycling after ingesting a sugar solution containing lactulose 5 g, ¹³C mannitol 100 mg, and ¹²C mannitol 1.9 g in 450 mL water. Other outcome measures included plasma intestinal fatty acid binding protein-1 (I-FABP), muscle damage biomarkers (serum creatine kinase, myoglobin), serum cortisol, complete blood cell counts, and shifts in plasma metabolites using untargeted metabolomics. Results: The time to complete the 60 km cycling bout differed significantly between the betaine and placebo trials (mean ± SE, 112.8 ± 2.3, 114.2 ± 2.6 min, respectively, (−1.41 ± 0.7 min) (effect size = 0.475, p = 0.042). No trial differences were found for I-FABP (interaction effect, p = 0.076), L:¹³CM (p = 0.559), the neutrophil/lymphocyte ratio (p = 0.171), serum cortisol (p = 0.982), serum myoglobin (p = 0.942), or serum creatine kinase (p = 0.694). Untargeted metabolomics showed that 214 metabolites exhibited significant trial treatment effects and 130 significant trial x time interaction effects. Betaine versus placebo supplementation was linked to significant increases in plasma betaine, dimethylglycine (DMG), sarcosine, methionine, S-adenosylhomocysteine (SAH), alpha-ketoglutaramate, and 5′methylthioadensone (MTA), and decreases in plasma carnitine and numerous acylcarnitines. Conclusions: Betaine supplementation modestly improved 60 km cycling performance but had no effect on gut permeability. The metabolomics data supported a strong influence of 2-week intake of betaine on the one-carbon metabolism pathway during the 24 h recovery period. Full article

Methyl groups can be obtained either from the diet (labile methyl groups) or produced endogenously (methylneogenesis) via one-carbon (C1-) metabolism as S-adenosylmethionine (SAM). The essential nutrients folate and choline (through betaine) are metabolically entwined to feed their methyl groups into C1-metabolism. A choline-deficient diet in rats produces a 31–40% reduction in liver folate content, 50% lower hepatic SAM levels, and a doubling of plasma homocysteine. Similarly, folate deficiency results in decreased total hepatic choline. Thus, sufficient intakes of both folate and choline (or betaine) contribute to safeguarding the methyl balance in the body. A significant amount of choline (as phosphatidylcholine) is produced in the liver via the SAM-dependent phosphatidylethanolamine methyltransferase. Experimental studies using diets deficient in several methyl donors have shown that supplemental betaine was able to rescue not only plasma betaine but also plasma folate. Fasting plasma homocysteine concentrations are mainly determined by folate intake or status, while the effect of choline or betaine on fasting plasma homocysteine is minor. This appears to contradict the finding that approximately 50% of cellular SAM is provided via the betaine-homocysteine methyltransferase (BHMT) pathway, which uses dietary choline (after oxidation to betaine) or betaine to convert homocysteine to methionine and then to SAM. However, it has been shown that the relative contribution of choline and betaine to cellular methylation is better reflected by measuring plasma homocysteine after a methionine load test. Choline or betaine supplementation significantly lowers post-methionine load homocysteine, whereas folate supplementation has a minor effect on post-methionine load homocysteine concentrations. This review highlights the interactions between folate and choline and the essentiality of choline as a key player in C1-metabolism. We further address some areas of interest for future work. Full article

Epigenetic modifications act as crucial regulators of gene activity and are influenced by both internal and external environmental factors, with diet being the most impactful external factor. On the other hand, cellular metabolism encompasses a complex network of biochemical reactions essential for maintaining cellular function, and it impacts every cellular process. Many metabolic cofactors are critical for the activity of chromatin-modifying enzymes, influencing methylation and the global acetylation status of the epigenome. For instance, dietary nutrients, particularly those involved in one-carbon metabolism (e.g., folate, vitamins B12 and B6, riboflavin, methionine, choline, and betaine), take part in the generation of S-adenosylmethionine (SAM), which represents the main methyl donor for DNA and histone methylation; α-ketoglutarate and ascorbic acid (vitamin C) act, respectively, as a co-substrate and cofactor for Ten-eleven Translocation (TET), which is responsible for DNA demethylation; and metabolites such as Acetyl-CoA directly impact histone acetylation, linking metabolism of the TCA cycle to epigenetic regulation. Further, bioactive compounds, such as polyphenols, modulate epigenetic patterns by affecting methylation processes or targeting epigenetic enzymes. Since diet and nutrition play a critical role in shaping epigenome functions and supporting human health, this review offers a comprehensive update on recent advancements in metabolism, epigenetics, and nutrition, providing insights into how nutrients contribute to metabolic balance, epigenome integrity maintenance and, consequently, disease prevention. Full article

Helicobacter pylori is a gastric pathogen implicated in peptic ulcer disease and gastric cancer. The increasing prevalence of antibiotic-resistant strains underscores the urgent need for alternative therapeutic strategies. In this study, we investigated the chemical composition and antibacterial activity of an aqueous extract from Caulerpa lentillifera (sea grape), a farm-cultivated edible green seaweed collected from Krabi Province, Thailand. Ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) revealed that the extract was enriched in bioactive nucleosides and phenolic compounds. In vitro assays demonstrated dose-dependent inhibition of H. pylori growth following exposure to sea grape extract. Furthermore, untargeted intracellular metabolomic profiling of H. pylori cells treated with the extract uncovered significant perturbations in central carbon and nitrogen metabolism, including pathways associated with the tricarboxylic acid (TCA) cycle, one-carbon metabolism, and alanine, aspartate, and glutamate metabolism. Pyrimidine biosynthesis was selectively upregulated, indicating a potential stress-induced shift toward nucleotide salvage and DNA repair. Of particular note, succinate levels were markedly reduced despite accumulation of other TCA intermediates, suggesting disruption of electron transport-linked respiration. These findings suggest that bioactive metabolites from C. lentillifera impair essential metabolic processes in H. pylori, highlighting its potential as a natural source of antimicrobial agents targeting bacterial physiology. Full article

Reactive oxygen species (ROS) generated by cold atmospheric plasma (CAP) cause irreversible damage to cancer cell DNA, RNA, mitochondria, and antioxidant defense systems, leading to apoptosis. Plasma-induced disruption of the antioxidant defense system of cancer cells by cystine uptake via xC⁻ antiporter has been widely studied, while folate uptake by cancer cells via high expression of hSLC19A1, which generates Nicotinamide Adenine Dinucleotide Phosphate (NADPH) via one-carbon metabolism, is also an important component of the antioxidant defense mechanism of cancer cells. Disrupting folate transport in cancer cells is an important potential pathway for synergizing with pemetrexed (PMX) to induce apoptosis in cancer cells, which is of great research value. In this paper, classical molecular dynamics simulations were employed to study the effect of plasma oxidation of hSLC19A1 on the uptake of 5-Methyltetrahydrofolate (5-MTHF), which is the predominant dietary and circulatory folate, and the antifolate chemotherapeutic agent PMX by cancer cells. The results showed that the channel radius of hSLC19A1 for transporting 5MTHF after oxidation became narrower and the conformation tended to be closed, which was unfavorable for the transport of 5-MTHF; hydrogen bonding and hydrophobic interactions between hSLC19A1 and 5-MTHF decreased, the predicted docking affinity decreased, and the binding energy decreased from −28.023 kcal/mol to −16.866 kcal/mol, while that with PMX was stable around −28 kcal/mol, suggesting that the oxidative modification reduced the binding capacity of hSLC19A1 and 5-MTHF while barely affecting the transport of PMX, which contributed to weakening the antioxidant defense system of cancer cells and synergizing with PMX to induce apoptosis in cancer cells. Our simulations provide theoretical insights for CAP-induced apoptosis in cancer cells at the microscopic level and help promote the further development of cold atmospheric plasma in the field of cancer therapy. Full article

Background/Objectives: Melanoma is an aggressive skin cancer with intratumor metabolic heterogeneity, which drives its progression and therapy resistance. Natural anthraquinones, such as emodin and aloe-emodin, exhibit anti-cancer properties, but their effects on metabolic plasticity remain unclear. This study evaluated their impact on proliferation and metabolic pathways in heterogenous melanoma human cell lines. Methods: COLO 800, COLO 794, and A375 melanoma cell lines representing distinct metabolic phenotypes were analyzed. Targeted and untargeted metabolomics analyses integrated with Seahorse assays were performed to assess the effects of emodin and aloe-emodin on cell proliferation, mitochondrial function, and redox homeostasis. Glucose tracing using [U-¹³C₆] glucose and metabolic flux analysis (MFA) were carried out to evaluate the glycolysis and TCA cycle dynamics. Results: Emodin and aloe-emodin inhibited proliferation by disrupting glycolysis, oxidative phosphorylation, and energy production across all cell lines. Both compounds impaired glucose metabolism, reduced TCA cycle intermediates, and induced mitochondrial ROS accumulation, causing oxidative stress and redox imbalance. Despite intrinsic metabolic differences, COLO 800 and COLO 794 upregulated antioxidant defenses; A375 enhanced one-carbon metabolism and amino acid pathways to maintain redox balance and nucleotide biosynthesis. Conclusions: Emodin and aloe-emodin can disrupt the metabolic plasticity of melanoma cells by impairing glycolysis, mitochondrial function, and redox homeostasis. Their ability to target metabolic vulnerabilities across diverse phenotypes highlights their therapeutic potential for overcoming resistance mechanisms and advancing melanoma treatment strategies. Full article

Background: Understanding the metabolic adaptations of wild bighorn sheep (Ovis c. canadensis) to nutritional stress is crucial for their conservation. Methods: This study employed ¹H nuclear magnetic resonance (NMR) metabolomics to investigate the biochemical responses of these animals to varying sub-maintenance nutritional states. Serum samples from 388 wild bighorn sheep collected between 2014 and 2017 from December (early sub-maintenance) through March (severe sub-maintenance) across Wyoming and Montana were analyzed. Multivariate statistics and machine learning analyses were employed to identify characteristic metabolic patterns and metabolic interactions between early and severe sub-maintenance nutritional states. Results: Significant differences were observed in the levels of 15 of the 49 quantified metabolites, including formate, thymine, glucose, choline, and others, pointing to disruptions in one-carbon, amino acid, and central carbon metabolic pathways. These metabolites may serve as indicators of critical physiological processes such as nutritional intake, immune function, energy metabolism, and protein catabolism, which are essential for understanding how wild bighorn sheep adapt to nutritional stress. Conclusions: This study has generated valuable insights into molecular networks underlying the metabolic resilience of wild bighorn sheep, highlighting the potential for using specific biochemical markers to evaluate nutritional and energetic states in free-ranging ungulates. These insights may help wildlife managers and ecologists compare populations across different times in seasonal cycles, providing information to assess the adequacy of seasonal ranges and support conservation efforts. This research strengthens our understanding of metabolic adaptations to environmental stressors in wild ruminants, offering a foundation for improving management practices to maintain healthy bighorn sheep populations. Full article

Background: Patients suffering from chronic kidney disease (CKD) have a high risk of premature cardiovascular morbidity and mortality. It has been suggested that elevated homocysteine (Hcy) or disturbances in the transmethylation pathway may contribute to this high cardiovascular risk burden due to epigenetic mechanisms. The objective of this study was to explore the prognostic value of Hcy, S-adenosylhomocysteine (SAH) and S-adenosylmethionine (SAM) (one-carbon (C1)-metabolites) among patients with CKD. Methods: Plasma concentrations of Hcy, SAM and SAH were measured among 297 participants with CKD (KDIGO GFR category G2–G5). The predefined endpoint was the occurrence of major cardiovascular events (MACE), defined as carotid, coronary and peripheral arterial revascularization, stroke, acute myocardial infarction, major amputation, cardiovascular death and all-cause mortality during a median (IQR) follow-up period of 4.0 [3.2; 4.3] years. Results: Among all participants, the median (IQR) of plasma Hcy, SAH, and SAM levels were 16.6 [13.5; 21.2] µmol/L, 41.5 [26.6; 63.9] nmol/L, 183.4 [151.1; 223.5] nmol/L, respectively. Estimated glomerular filtration rate (eGFR) correlated more strongly with plasma SAH (r = −0.588) than with SAM (r = −0.497) and Hcy (r = −0.424). During the follow-up period, 55 participants experienced MACE. In a univariate Kaplan Meier analysis, all three C1-metabolites were significantly associated with the occurrence of the primary outcome. In a Cox-regression analysis, the association between Hcy and MACE was not significant after adjustment for age and sex (hazard ratio (HR) and 95% confidence intervals (95% CI) for the 3rd vs. 1st tertile = 1.804 (0.868–3.974)). Both SAH and SAM were not associated with MACE after adjustment for age, sex and additionally for renal function markers (SAH: HR 3rd vs. 1st tertile 1.645 95% (0.654–4.411); SAM: HR 3rd vs. 1st tertile 1.920 95% CI (0.764–5.138)). Conclusions: In people with CKD, plasma Hcy, SAH and SAM were not independent predictors of MACE after adjustment for age, sex and renal function. Disturbed renal function may explain elevated C1-metabolites and disturbed transmethylation, while this pathway is not likely to be an appropriate access point to modify the risk of cardiovascular events in CKD patients. Full article

Medulloblastoma (MB) is the most common malignant brain tumor in children, typically arising during infancy and childhood. Despite multimodal therapies achieving a response rate of 70% in children older than 3 years, treatment remains challenging. Ferroptosis, a form of regulated cell death, can be induced in medulloblastoma cells in vitro using erastin or RSL3. Using two independent medulloblastoma RNA-sequencing cohorts (MB-PBTA and MTAB-10767), we investigated the expression of ferroptosis-related molecules through multiple approaches, including Weighted Gene Co-Expression Network Analysis (WGCNA), molecular subtype stratification, protein–protein interaction (PPI) networks, and univariable and multivariable overall survival analyses. A prognostic expression score was computed based on a cross-validated ferroptosis signature. In training and validation cohorts, the regulation of the ferroptosis transcriptional program distinguished the four molecular subtypes of medulloblastoma. WGCNA identified nine gene modules in the MB tumor transcriptome; five correlated with molecular subtypes, implicating pathways related to oxidative stress, hypoxia, and trans-synaptic signaling. One module, associated with disease recurrence, included epigenetic regulators and nucleosome organizers. Univariable survival analyses identified a 45-gene ferroptosis prognostic signature associated with nutrient sensing, cysteine and methionine metabolism, and trans-sulfuration within a one-carbon metabolism. The top ten unfavorable ferroptosis genes included CCT3, SNX5, SQOR, G3BP1, CARS1, SLC39A14, FAM98A, FXR1, TFAP2C, and ATF4. Patients with a high ferroptosis score showed a worse prognosis, particularly in the G3 and SHH subtypes. The PPI network highlighted IL6 and CBS as unfavorable hub genes. In a multivariable overall survival model, which included gender, age, and the molecular subtype classification, the ferroptosis expression score was validated as an independent adverse prognostic marker (hazard ratio: 5.8; p-value = 1.04 × 10⁻⁹). This study demonstrates that the regulation of the ferroptosis transcriptional program is linked to medulloblastoma molecular subtypes and patient prognosis. A cross-validated ferroptosis signature was identified in two independent RNA-sequencing cohorts, and the ferroptosis score was confirmed as an independent and adverse prognostic factor in medulloblastoma. Full article

Background/Objectives: The MTHFR^677C>T gene variant results in a thermolabile MTHFR enzyme associated with elevated plasma homocysteine in TT individuals. Health risks associated with the TT genotype may be modified by dietary and supplemental folate intake. Supplementation with methyltetrahydrofolate (methylTHF) may be preferable to folic acid because it is the MTHFR product, and does not require reduction by DHFR to enter one-carbon folate metabolism. In the Mthfr^677C>T mouse model for this variant, female 677TT (TT) mice have an increased incidence of hepatic steatosis. The objective of this study was to compare the effects of methylTHF and folic acid supplementation on hepatic steatosis and one-carbon metabolism in this model. Methods: Male and female C57BL/6J 677CC (CC) and TT mice were fed control (CD), 5xmethylTHF-supplemented (MFSD), or 5xfolic-acid-supplemented (FASD) diets for 4 months. Liver sections were assessed for steatosis by Oil Red O staining. One-carbon metabolites were measured in the liver and plasma. MTHFR protein expression was evaluated in the liver. Results: MFSD had no significant effect on plasma homocysteine, liver SAM/SAH ratios, or hepatic steatosis in males or females as compared to CD. MTHFR protein increased in MFSD TT female liver, but remained <50% of the CC. FASD had no effect on plasma homocysteine but it decreased the liver MTHFR protein and SAM/SAH ratios, and increased hepatic steatosis in CC females. Conclusions: MethylTHF and folic acid supplementation had limited benefits for TT mice, while folic acid supplementation had negative effects on CC females. Further investigation is required to determine if these effects are relevant in humans. Full article