Gut microbiota-derived metabolites regulate fat deposition via N6-methyladenosine (m6A) RNA methylation in germ-free and FMT piglets1

FMT increases serum bile acids (hyodeoxycholic acid, 12-ketodeoxycholic acid, 3b-hydroxy-5-cholenoic acid) and tryptophan metabolites (Indoxyl sulfate, L-kynurenine), correlating with lipid metabolism. 2. Bacteroides , Lactobacillus , Parabacteroides , and Akkermansia drive bile acid and tryptophan metabolism, with Parabacteroides and Akkermansia as key network hubs. 3. Microbial metabolites reduce m 6 A RNA methylation by upregulating FTO in subcutaneous fat and downregulating METTL3 in longissimus dorsi muscle , revealing a microbiota-metabolite-m 6 A axis with potential for improving metabolic health and livestock meat quality. The gut microbiota plays a critical role in regulating host fat deposition, yet the underlying mechanisms remain poorly understood. To investigate this question, this study employed germ-free (GF) and fecal microbiota transplantation (FMT) piglet models to systematically elucidate how microbiota-derived metabolites modulate fat deposition via N 6 -methyladenosine (m 6 A) RNA methylation. Although the FMT group showed no significant change in body weight compared with the GF group, it exhibited markedly increased subcutaneous and intramuscular fat deposition, as evidenced by larger adipocyte size ( P <0.001), elevated serum and longissimus dorsi muscle triglyceride levels ( P <0.05), and upregulated expression of adipogenic genes ( P < 0.05). Non-targeted metabolomics revealed elevated levels of bile acids, including hyodeoxycholic acid (HDCA), 12-ketodeoxycholic acid (12KDCA), and 3b-hydroxy-5-cholenoic acid (3b-h5-CA), as well as tryptophan metabolites indoxyl sulfate (IS) and L-kynurenine (L-Kyn) in the FMT group, and these metabolites were significantly correlated with lipid metabolism parameters. Mechanistic studies showed that these metabolites reduced m 6 A modification levels in mouse stromal vascular fraction (SVF) cells and fibro-adipogenic progenitor (FAPs) cells by upregulating fat mass and obesity-associated protein (FTO) and downregulating methyltransferase-like 3 (METTL3) ( P < 0.05), consistent with the in vivo observations in piglets. Furthermore, Bacteroides , Lactobacillus , Parabacteroides , and Akkermansia were identified as key genera involved in bile acid and tryptophan metabolism. Together, these findings reveal a gut microbiota–metabolite– m 6 A regulatory axis in fat deposition in pigs, providing new insights into host–microbiota interactions and offering potential strategies for improving metabolic health and meat quality in livestock.