Hyperhomocysteinemia (HHcy), characterized by elevated plasma homocysteine (>15 µmol/L), is a risk factor for cardiovascular diseases and glucose intolerance. A high-methionine (HM) diet is a common model for inducing HHcy in animals. While the liver is considered the primary site of methionine and homocysteine metabolism, recent studies suggest that the gastrointestinal tract (GIT) contributes to net homocysteine production. The gut microbiota, which is significantly influenced by diet, plays a crucial role in host metabolism, including amino acid metabolism. However, the involvement of gut microbiota in homocysteine metabolism remains unclear. This study aimed to investigate whether gut microbiota ablation could alleviate HM diet-induced HHcy and glucose intolerance and elucidate the underlying mechanisms.

Numerous studies have established HHcy as an independent risk factor for various cardiovascular diseases. Furthermore, HHcy has been linked to glucose intolerance, insulin resistance, and hepatic steatosis. Plasma homocysteine levels are influenced by several factors, including genetics, diet, and vitamin deficiencies. The liver is widely recognized as the primary site of methionine and homocysteine metabolism. However, emerging evidence indicates that the GIT contributes significantly to homocysteine production. Prior research highlights the gut microbiota's influence on various aspects of host metabolism, particularly amino acid metabolism, with studies demonstrating the microbiota's impact on tryptophan metabolism. However, the specific role of gut microbiota in homocysteine metabolism remained largely unexplored before this study.

Male C57BL/6J mice (8 weeks old) were fed either a chow diet or an HM diet (2%, 20 g/kg L-methionine) for 4 weeks. One group within each diet group received nonabsorbable broad-spectrum antibiotics in their drinking water to deplete the gut microbiota. Body weight and food intake were monitored. Plasma homocysteine, methionine, insulin, triglyceride, and total cholesterol levels were measured. Glucose tolerance tests (GTT) and insulin tolerance tests (ITT) were performed. Liver gene expression (CBS, BHMT, MetRS, MAT1A, MAT2A, MTHFR) and protein levels (CBS, BHMT) and enzyme activity (CBS) were assessed. Fecal samples were collected for metagenomic shotgun sequencing to analyze gut microbiota composition and functional shifts (KEGG pathway analysis). The correlation between microbiota composition and plasma homocysteine levels was analyzed. Homocysteine production by *Dubosiella newyorkensis* was examined via LC-MS analysis of cultured supernatants. Intestinal epithelial homocysteine and methionine levels were also measured via LC-MS. Statistical analysis included unpaired two-tailed Student’s t-test, unpaired one-way ANOVA with Tukey’s post hoc test, and Mann-Whitney test where appropriate.

The HM diet significantly increased plasma homocysteine levels compared to the chow diet (23.04 vs. 10.04 µM, P < 0.0001). Antibiotic treatment significantly reduced plasma homocysteine levels in HM diet-fed mice (13.67 vs. 23.04 µM, P < 0.0001). The HM diet also increased intestinal epithelial homocysteine levels, which were reduced by antibiotic treatment. The HM diet-induced glucose intolerance, as demonstrated by GTT, was also alleviated by antibiotic treatment. Antibiotic treatment did not significantly affect the gene expression, protein levels, or enzyme activity of homocysteine metabolic enzymes in the liver. Metagenomic sequencing revealed that the HM diet altered the gut microbiota composition, increasing the abundance of *Faecalibaculum* and *Dubosiella* and decreasing the abundance of *Alistipes*. These changes were positively correlated with plasma homocysteine levels. KEGG pathway analysis showed an upregulation of the cysteine and methionine metabolism pathway, specifically two homocysteine biosynthesis-related KOs (K01739 and K14155). LC-MS analysis confirmed homocysteine production by *Dubosiella newyorkensis* in vitro.

This study provides strong evidence that the gut microbiota plays a critical role in HM diet-induced HHcy and glucose intolerance. The finding that antibiotic treatment reduced plasma homocysteine and improved glucose tolerance without affecting liver homocysteine metabolism points to the gut microbiota as the primary source of the observed effects. The identification of *Faecalibaculum* and *Dubosiella* as potential homocysteine producers, coupled with the observed positive correlation between their abundance and plasma homocysteine levels, strengthens this conclusion. The upregulation of homocysteine biosynthesis-related pathways within the gut microbiota further supports this hypothesis. These findings highlight a novel mechanism linking diet, gut microbiota, and HHcy pathogenesis.

This study demonstrates that gut microbiota ablation alleviates HM diet-induced HHcy and glucose intolerance in mice. *Faecalibaculum* and *Dubosiella* were identified as potential producers of homocysteine under the HM diet. Targeting the gut microbiota may offer a novel therapeutic strategy for HHcy-related diseases. Future research should focus on exploring the precise mechanisms of homocysteine production by these bacteria and investigating the translational potential of targeting the gut microbiota in clinical settings.

This study utilized a mouse model and antibiotic treatment, which may not fully reflect the complexities of human gut microbiota and its interaction with homocysteine metabolism. Further studies are needed to confirm these findings in humans and to explore the long-term effects of microbiota modulation on HHcy and related metabolic disorders. The observed colonic structural changes in the HM + ABX group warrant further investigation to determine their contribution to the observed effects.