Search Results (265)

Betaine-homocysteine methyltransferase (BHMT) is an enzyme involved in one-carbon metabolism and plays a crucial role in maintaining liver health. In this study, we investigated the impact of liver-specific deletion of BHMT on liver dysfunction using a mouse model. We generated BHMT floxed mice and bred them with albumin Cre to generate liver-specific BHMT knockout (BHMT LKO) mice. Liver tissues harvested from six-month-old chow-fed BHMT floxed and LKO mice were characterized through histological, biochemical, and molecular analyses. BHMT LKO mice displayed a complete loss of hepatic expression of BHMT mRNA, protein and enzyme activity. Histopathological analysis revealed the development of hepatic steatosis in BHMT LKO mice compared to the floxed mice. These morphological changes were supported by biochemical analysis showing elevated levels of hepatic triglycerides in conjunction with a profound decrease in the methylation potential (i.e., reduced S-adenosylmethionine (SAM): S-adenosylhomocysteine (SAH) ratio), which was mainly driven by a six- to sevenfold increase in SAH levels. BHMT LKO mice also exhibited increased lipid peroxidation and lysosomal dysfunction compared to floxed mice. Early signs of inflammation were seen in the livers of BHMT LKO mice of both sexes, as evident from significant increase in CD68-positive cells and interleukin 1β levels. Additionally, there was a moderate increase in fibrosis, as evidenced by the upregulated expression of α-smooth muscle actin and collagen II levels and the histological assessment of picrosirius red-stained liver sections of BHMT LKO mice of both sexes compared to their respective counterparts. These findings demonstrate that hepatic BHMT deficiency promotes lipid accumulation, lysosomal/proteasomal dysfunction, and early inflammatory and fibrotic changes in the liver by reducing the methylation potential. Collectively, our results underscore BHMT as a critical regulator of liver homeostasis and a potential therapeutic target in liver-related disorders. Full article

Polyamines, putrescine, spermidine and spermine, are essential polycationic metabolites present in all eukaryotic cells, where they regulate fundamental processes including nucleic acid stabilization, translation, and stress responses. Spermidine synthase (SPDS), a member of the aminopropyltransferase (APT) family, catalyzes the transfer of an aminopropyl group from decarboxylated S-adenosylmethionine (dc-SAM) to putrescine to form spermidine. Although genomic analyses predict the presence of SPDS homologs in multiple fungal species, polyamine biosynthesis has not been experimentally characterized in the multidrug-resistant fungal pathogen Candidozyma auris. Here, we report the biochemical and functional characterization of the C. auris spermidine synthase, CauSpe3. The CauSPE3 gene complemented a Saccharomyces cerevisiae spe3Δ mutant demonstrating conserved function in vivo. Recombinant CauSpe3 was expressed in Escherichia coli, purified and analyzed using the fluorescence-based DAB-APT assay, which uses 1,2-diacetylbenzene (DAB) for polyamine detection. CauSpe3 catalyzed efficient conversion of putrescine to spermidine in the presence of dc-SAM, with K_half values of 65.5 ± 7.11 µM for putrescine and 66.9 ± 2.09 µM for dc-SAM, and V_max values of 7.1 ± 0.57 and 7.9 ± 0.12 nmol·µg⁻¹·min⁻¹, respectively. A catalytic-site mutant and heat-inactivated enzyme showed no detectable activity, and product formation was confirmed by means of thin-layer chromatography and mass spectrometry. These findings establish CauSpe3 as a functional spermidine synthase. Full article

Cystathionine β-synthase (CBS) deficiency causes classical homocystinuria with severe hyperhomocysteinemia (HHcy) that is inadequately controlled by current therapies. We tested whether liver-targeted CBS gene therapy provides durable biochemical and phenotypic rescue. Using a Cre-inducible adult mouse model of whole-body CBS loss, a single intravenous dose of AAV-DJ-hCBS (3 × 10¹² or 3 × 10¹³ vg/kg) was administered, and the animals were followed for 12 months. Vector biodistribution showed ~100-fold hepatic enrichment over the kidney and spleen. Both doses rapidly normalized plasma homocysteine (<8 µM), maintaining correction throughout the study while preventing alopecia, weight loss, and loss of adiposity. Liver histology showed resolution of inflammation, and only 2 of 19 mice developed anti-hCBS antibodies. Liver proteomics (3998 proteins quantified) revealed CBS deficiency-associated suppression of tRNA aminoacylation and dysregulation of lipid and carbon metabolism with an HNF4A transcriptional signature, all normalized by therapy. Liver metabolomics demonstrated accumulation of S-adenosylmethionine and S-adenosylhomocysteine and disruption of phosphatidylcholine synthesis, also corrected by treatment. Plasma metabolomics revealed systemic disturbances fully normalized by hepatic CBS restoration. These findings identify the liver as the central metabolic control point in CBS deficiency and support liver-targeted gene therapy as a durable corrective strategy. Full article

Background/Objectives: Folic acid (FA) intake impacts one-carbon metabolism (OCM), which is crucial for fetal development and epigenetic regulation. While FA supplementation is known to lower homocysteine levels, its comprehensive effects on OCM-related metabolites in maternal and cord blood remain unclear. This study aimed to investigate the association between FA use and serum OCM-related metabolite profiles in Japanese pregnant women. Methods: We analyzed 146 mother-infant pairs from the Chiba study of Mother and Child Health (C-MACH) cohort. Blood samples were collected in early pregnancy, late pregnancy, and at delivery (maternal and cord blood). FA use was assessed via self-administered questionnaires. Serum concentrations of 18 OCM-related metabolites, including 5-methyltetrahydrofolate (5-MTHF) and homocysteine, were measured using LC-MS/MS. Results: FA users exhibited significantly higher 5-MTHF and lower total homocysteine concentrations in maternal blood at all time points and in cord blood compared to non-users. Compared to non-users, FA users exhibited a lower serine/glycine ratio in early pregnancy, a higher betaine/DMG ratio in maternal blood at delivery, and higher S-adenosylmethionine and total cysteine concentrations in maternal blood during late pregnancy. In cord blood, unmetabolized folic acid concentrations did not differ significantly between FA users and non-users. Furthermore, the cord-to-maternal 5-MTHF ratio was significantly lower in FA users. Conclusions: Our findings suggest that FA use during pregnancy may contribute to the optimization of OCM in both the mother and fetus. Full article

Ziziphus jujuba Mill. is a characteristic resource with both medicinal and edible values. At present, its lactic acid bacteria-fermented products are plagued by ambiguous variety selection and low added value. To clarify the variety-specific regulatory effects of Z. jujuba cultivars on metabolic profiles during lactic acid bacteria fermentation, this study analyzed the metabolic characteristics of fermented broths of Tan jujube, Jun jujube, and Ban jujube under a unified fermentation system using LC-MS untargeted metabolomics technology. Significantly differential metabolites were screened with the criteria of p < 0.05 and VIP > 1, and the metabolic regulatory mechanisms were further elucidated, combined with KEGG pathway enrichment analysis. The results showed that a total of 570 metabolites were identified in the three fermented broths. Tan jujube was enriched in linolenic acid, Ban jujube was rich in D-xylitol and dethiobiotin, and Jun jujube had prominent contents of S-adenosylmethionine and pyridoxine. All the aforementioned metabolites are involved in important physiological processes such as anti-inflammation and intestinal homeostasis maintenance. The differential metabolites were mainly enriched in 6 key pathways, including central carbon metabolism, ABC transporters, and phenylpropanoid biosynthesis, among which central carbon metabolism and ABC transporters were the core regulatory pathways. This study constructed an association network of Z. jujuba variety–differential metabolite–key pathway, systematically elucidated the metabolic differentiation mechanisms of fermented broths from different Z. jujuba cultivars, and provided a scientific basis for the precise selection of Z. jujuba varieties dedicated to fermentation and the targeted development of high-value-added functional fermented foods. Full article

S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) are essential intermediates in one-carbon metabolism and key regulators of cellular methylation capacity. Their concentrations and the SAM/SAH ratio are increasingly studied as biomarkers across metabolic, cardiovascular, neurological, and cancer-related diseases. This review outlines validated analytical methods for quantifying SAM and SAH, focusing primarily on liquid chromatography–tandem mass spectrometry (LC–MS/MS), which is considered the gold standard in both clinical and research settings. A comprehensive literature search identified studies on method development, validation, and clinical use of SAM and SAH measurements. Special attention is given to analytical challenges arising from their high polarity, structural similarity, endogenous presence, and limited stability. The review also discusses preanalytical variables, including biological matrix selection, sample handling, and storage conditions. LC–MS/MS methods are compared with alternative techniques, such as immunoassays, with respect to sensitivity, specificity, matrix effects, and clinical relevance. Additionally, the review summarizes the concentration ranges of SAM and SAH, and their ratio, in healthy and patient populations, noting current standardization limitations. Overall, the review highlights the importance of harmonized analytical protocols and matrix-specific validation to enable reliable clinical interpretation of SAM and SAH as methylation biomarkers. Full article

Proteins can be methylated at either of the two N atoms of the imidazole ring of histidine, yielding 1-methylhistidine (or pi-methylhistidine) or 3-methylhistidine (tau-methylhistidine). While protein histidine methylation in mammals was discovered more than 50 years ago, the first histidine methyltransferases were identified only recently. So far, four different human protein histidine methyltransferases have been uncovered, and one of these is METTL9, which is responsible for introducing 1-methylhistidine in a number of proteins. The minimal sequence motif that is required, though not always sufficient, for METTL9-mediated methylation is His-X-His (HxH), where X is preferentially a small uncharged residue. Many METTL9 substrates are methylated at stretches of alternating histidines, i.e., several adjoining HxH motifs, such as HxHxH. Histidines are frequently involved in binding metal ions, such as zinc. Accordingly, it has been shown for several sequences targeted by METTL9, for example, in the immunomodulatory and antibacterial protein S100A9 and the zinc transporter SLC39A7, that histidine methylation diminishes zinc binding and thereby modulates protein function. In this review, we present a detailed account of METTL9-mediated histidine methylation, regarding its discovery, biochemical mechanism, structural features, and biological significance. Full article

The SARS-CoV-2 papain-like protease (PL^pro) is an essential viral enzyme that promotes viral polyprotein processing while simultaneously suppressing the host innate immune response, which makes it a primary target for developing antiviral drugs. The present study employs a comprehensive approach integrating untargeted metabolomic profiling, in silico molecular docking and dynamics simulations, Molecular Mechanics Generalized Born Surface Area (MM-GBSA) energetic assessments, and biochemical enzyme assays. This integrated method aims to discover natural PL^pro inhibitors from two ethnomedicinal plants, Lippia javanica and Acorus calamus, which have long been utilized in African traditional medicine to treat respiratory diseases. Comprehensive metabolite profiling using untargeted Ultra-Performance Liquid Chromatography–Tandem Mass Spectrometry (UPLC-MS/MS) and Global Natural Products Social (GNPS) molecular networking revealed flavonoid glucuronides and phenylpropanoid derivatives as the major constituents in both plant species. In situ histochemical staining further offered spatial validation of phenolic- and lignin-associated tissues, supporting the phenolic-dominated molecular families detected by GNPS molecular networking. In silico evaluation of six selected compounds demonstrated spontaneous and thermodynamically favorable binding to PL^pro, with ΔG_bind values ranging from −5.63 to −6.43 kcal/mol. Catechin-7-glucoside emerged as the lead compound, establishing multiple hydrogen bond networks with Asp164, Gln269, Tyr264, and Asn267, supplemented by hydrophobic engagement with Pro247 and Pro248, and π-π stacking with the blocking loop 2 (BL2 loop). Molecular dynamics simulations confirmed the stability of the protein–ligand complexes. Biochemical enzyme assays confirmed concentration-dependent inhibition of PL^pro proteolytic and deubiquitinating activity by both crude plant extracts and isolated bioactive compounds. However, S-adenosyl-methionine showed comparatively high PL^pro proteolytic activity (IC₅₀ 5.872 µM) compared to catechin-7-glucoside, with an IC₅₀ of 7.493 µM, exhibiting efficacy similar to the reference inhibitor GRL0617. Both the extracts of L. javanica and A. calamus have shown significant inhibitory activity while maintaining cell viability in Human embryonic kidney 293T cell (HEK293T) culture models, indicating a favorable safety profile of the tested concentrations. Based on these results, catechin-based polyphenols and phenylpropanoid derivatives appear as promising lead compounds for the development of PL^pro inhibitors. To progress toward therapeutic use, further work is necessary in pharmacokinetics, structural optimization, and antiviral validation in cell models. Full article

Glycine N-methyltransferase (GNMT), a S-adenosylmethionine (SAM)-dependent methyltransferase, is primarily expressed in the liver and plays a key role in regulating liver metabolism and protecting against liver injury. Several studies have shown that deficiency or downregulation of GNMT is strongly associated with the pathogenesis of hepatocellular carcinoma (HCC), highlighting its critical role as a tumor suppressor. Other studies have shown that GNMT is also strongly correlated with the pathogenesis of metabolic dysfunction-associated fatty liver disease (MAFLD). Although many factors regulate GNMT expression, recent studies have identified microRNAs (miRNAs), such as miR-873-5p and miR-224, as key post-transcriptional regulators that directly target GNMT mRNA and suppress its expression in HCC and MAFLD. This review provides an overview of GNMT’s role in liver physiology and how its dysregulation contributes to the progression of HCC and MAFLD, with a focus on the regulation of GNMT by miR-873-5p and miR-224. We also highlight the potential of these two miRNAs as biomarkers and therapeutic targets for HCC and MAFLD, discussing emerging strategies such as antisense-based inhibition, gene therapy, and small-molecule inducers aimed at restoring GNMT expression. Full article

Background/Objectives: The ketogenic diet (KD) induces profound metabolic shifts, yet the sex-specific long-term effects on skeletal muscle metabolism and sterol homeostasis across tissues remain insufficiently characterized. This study tested the hypothesis that a prolonged KD would elicit distinct, sex-dependent metabolic and sterol adaptations in mice. Methods: We examined how a 12-week KD, compared with a standard diet, affected body mass, the skeletal muscle metabolome, hepatic lipid and collagen content, and sterol profiles in the skeletal muscle, liver, spleen, and serum in male and female C57BL/6J mice. Three-month-old mice of both sexes were randomized to a KD or standard diet and evaluated using the histological quantification of hepatic steatosis and collagen deposition, matrix-assisted laser desorption/ionization time-of-flight imaging mass spectrometry (MALDI-TOF IMS) of skeletal muscle, and LC-MS/MS-based sterol profiling. Results: The KD induced rapid body mass gain in males and delayed weight gain in females, promoted hepatic steatosis in both sexes, and generated clearly segregated, sex- and diet-specific skeletal muscle metabolomic signatures. These signatures included reduced tricarboxylic acid cycle precursors and a marked decrease in S-adenosylmethionine in KD-fed females. Across tissues, the KD consistently suppressed precursor sterols, including 7-dehydrocholesterol and desmosterol in the skeletal muscle, liver, and spleen, while elevating serum cholesterol and desmosterol (male-biased), with changes generally more pronounced in males. Conclusions: Collectively, these findings demonstrate that a long-term KD drives sex- and organ-specific metabolic remodeling, with evidence of greater metabolic flexibility but a shared risk of hepatic steatosis in females. These results underscore the importance of personalized, sex-stratified approaches when considering long-term ketogenic interventions. Full article

Age-related osteoporosis is driven in part by senescence-associated rewiring of bone marrow mesenchymal stem cells (MSCs) from osteogenic toward adipogenic fates. Accumulating evidence indicates that epigenetic drift and reduced autophagy are not isolated lesions but are mechanistically coupled through a bidirectional DNA methylation and autophagy axis. Here, we summarize how promoter hypermethylation of genes involved in autophagy and osteogenesis suppresses autophagic flux and osteoblast lineage transcriptional programs. Conversely, autophagy insufficiency reshapes the methylome by limiting methyl donor availability, most notably S-adenosylmethionine (SAM), and by reducing the turnover of key epigenetic regulators, including DNA methyltransferases (DNMTs), ten-eleven translocation (TET) dioxygenases, and histone deacetylases (HDACs). This self-reinforcing circuitry exacerbates mitochondrial dysfunction, oxidative stress, and inflammation driven by the senescence-associated secretory phenotype (SASP), thereby stabilizing adipogenic bias and progressively impairing marrow niche homeostasis and bone remodeling. We further discuss therapeutic strategies to restore balance within this axis, including selective modulation of epigenetic enzymes; activation of AMP-activated protein kinase (AMPK) and mechanistic target of rapamycin (mTOR) signaling with downstream engagement of Unc-51-like autophagy-activating kinase 1 (ULK1) and transcription factor EB (TFEB); targeting sirtuin pathways; mitochondria- and autophagy-supportive natural compounds; and bone-targeted delivery approaches or rational combination regimens. Full article

Neuroblastoma (NB), a pediatric cancer of sympatho-adrenal (SA) lineage, is marked by disrupted differentiation and cellular heterogeneity. INSM1, a zinc-finger transcription factor, is highly expressed in NB and developing SA tissues, where it regulates neuroendocrine differentiation, especially in chromaffin cells. We investigated INSM1’s role in maintaining an undifferentiated, progenitor-like state in NB and its regulation via metabolic and epigenetic mechanisms. Transcriptomic profiling, promoter assays, and metabolic flux analysis revealed that INSM1 expression correlates with methionine cycle activity, particularly the S-adenosylmethionine (SAM)/S-adenosylhomocysteine (SAH) ratio. Disruption of SAM/SAH balance altered INSM1 promoter activity and histone methylation, implicating epigenetic control in NB cell fate. Retinoic acid-induced differentiation downregulated INSM1 and N-Myc, linking INSM1 to tumor cell immaturity. INSM1 overexpression in SH-SY-5Y cells upregulated neuroendocrine and thyroid hormone-related genes (CHGA, CHGB, DDC, NCAM1, DIO3, TH), while suppressing genes involved in cell cycle (RRM, CDC25A), methionine metabolism (AHCY, MAT2A), transcriptional regulation (MYBL2, EZH2), and oncogenic signaling (ALK, LINC011667). These findings suggest that INSM1 promotes NB aggressiveness by sustaining a neuroendocrine progenitor-like phenotype through metabolic-epigenetic coupling. Full article

Alcohol-associated liver disease (ALD) is a leading cause of liver-related morbidity, mortality, and premature death worldwide. Its pathogenesis is complex and incompletely understood, with disrupted methionine metabolism as a key contributor. This pathway converts methionine into S-adenosylmethionine (SAM or SAMe), the principal methyl donor, a precursor of glutathione (GSH), and a critical regulator of hepatocellular function. Alterations in methionine metabolism are primarily driven by downregulation of methionine adenosyltransferase 1A (MAT1A), the liver-specific gene encoding the MATα1 subunit responsible for SAMe biosynthesis. Reduced MAT1A expression and activity lead to hepatic SAMe and GSH deficiency, resulting in global hypomethylation, mitochondrial dysfunction, impaired lipid metabolism, and progressive liver injury, hallmarks of ALD. Recent studies show that MATα1 also localizes to hepatocyte mitochondria, where its selective depletion contributes to mitochondrial dysfunction in ALD. Experimental models demonstrate that SAMe supplementation restores methylation capacity, replenishes GSH, reduces oxidative stress, and improves mitochondrial function and liver histology. Preservation of mitochondrial MATα1 also protects against ALD, underscoring its importance in hepatocellular health. Clinical exploration of SAMe in early-stage ALD suggests potential benefit and motivates continued investigation into treatment strategies that build on and extend beyond supplementation. This review summarizes current knowledge on the role of the MAT1A/SAMe axis in ALD pathophysiology, emphasizing molecular functions and critically evaluating preclinical and clinical evidence for potential therapy. Full article

Inhibition of respiratory chain complex I (NADH dehydrogenase) is a widely encountered biochemical consequence of drug intoxication and a primary consequence of mtDNA mutations and other mitochondrial defects. In an organ-selective form, it is also deployed as antidiabetic pharmacological treatment. Complex I inhibition evokes a pronounced metabolic reprogramming of uncertain purposefulness, as in several cases, anabolism appears to be fostered in a state of bioenergetic shortage. A hallmark of complex I inhibition is the enhanced biosynthesis of serine, usually accompanied by an induction of folate-converting enzymes. Here, we have revisited the differential transcriptional induction of these metabolic pathways in three published models of selective complex I inhibition: MPP-treated neuronal cells, methionine-restricted rats, and patient fibroblasts harboring an NDUFS2 mutation. We find that in a coupled fashion, serinogenesis and circular folate cycling provide an unrecognized alternative pathway of complete glucose oxidation that is mostly dependent on NADP instead of the canonic NAD cofactor (NADP:NAD ≈ 2:1) and thus evades the shortage of oxidized NAD produced by complex I inhibition. In contrast, serine utilization for anabolic purposes and C₁-folate provision for S-adenosyl-methionine production and transsulfuration cannot explain the observed transcriptional patterns, while C₁-folate provision for purine biosynthesis did occur in some models, albeit not universally. We conclude that catabolic glucose oxidation to CO₂, linked with NADPH production for indirect downstream respiration through fatty acid cycling, is the general purpose of the remarkably strong induction of serinogenesis after complex I inhibition. Full article

Background: Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide, increasingly arising in patients with metabolic dysfunction-associated steatotic liver disease (MASLD). Epigenetic dysregulation, particularly DNA methylation, has been implicated in MASLD-HCC development, yet the roles that the principal DNA methylation precursor metabolites, S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH), play in this association are unclear. Objective: We investigated associations of circulating SAM, SAH, the SAM/SAH ratio, with MASLD-HCC. Methods: In a multi-center pilot case–control study, we evaluated 69 MASLD-HCC cases and 136 cancer-free MASLD controls. Plasma SAM and SAH levels were quantified by liquid chromatography–tandem mass spectrometry. Metabolite levels were categorized as greater than or less than the median based on distribution in controls. Logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs), adjusting for age, sex, body mass index, smoking status, and type 2 diabetes. Results: MASLD-HCC cases had significantly higher plasma SAM levels (mean 121 vs. 96 nmol/L; p = 0.001) and SAM/SAH ratios (2.09 vs. 1.48; p = 6.42 × 10⁻⁷) than MASLD controls. In multivariable-adjusted models, elevated SAM levels (OR_{≥median vs. <median} = 2.76; 95% CI: 1.38–5.72) and higher SAM/SAH ratio (OR_{≥median vs. <median} = 2.30; 95% CI: 1.15–4.73) were associated with higher odds of MASLD-HCC. SAH alone was associated with MASLD-HCC. Conclusions: Higher plasma SAM levels and SAM/SAH ratios are independently linked to MASLD-HCC development. These metabolites might serve as noninvasive markers for HCC risk stratification in patients with MASLD and improve early detection efforts for MASLD-HCC. Full article