The Hidden Players of the Fecal Metabolome: Metabolic Dysregulation Beyond SCFAs Under a High-Fat Diet

Background/Objectives: The interplay between host metabolism and gut microbiota is central to the pathophysiology of metabolic diseases, including metabolic dysfunction-associated steatotic liver disease (MASLD). In this study, we investigated the underexplored fecal host-microbiota co-metabolism profile of male and female Wistar rats after 21 weeks of high-fat diet (HFD), a model previously validated for early MASLD. Methods: Using ¹H-NMR spectroscopy, we detected and quantified metabolites in fecal samples associated with hepatic metabolism beyond short-chain fatty acids (SCFAs), such as energy-related metabolites, amino acid turnover, branched-chain amino acid (BCAA) catabolism, and microbial fermentation. Results: Distinct metabolic signatures were identified according to diet and sex, and statistical analysis was performed. Notably, alterations were observed in bile acids (BAs) such as cholate and glycocholate, suggesting disruptions in enterohepatic circulation. The presence of fucose, a sugar linked to liver pathology, was also elevated. Energy-related metabolites indicated a shift from lactate production to increased acetoacetate and malonate levels, implying redirection of pyruvate metabolism and inhibition of the TCA cycle. BCAA derivatives such as 3-methyl-2-oxovalerate and 3-aminoisobutyrate were altered, supporting earlier findings on disrupted amino acid metabolism under HFD conditions. Furthermore, microbial metabolites including methanol and ethanol showed group-specific differences, suggesting shifts in microbial activity. Conclusions: These findings complement previous longitudinal data and provide a functional interpretation of newly identified metabolites. These metabolites, previously unreported, are now functionally contextualized and linked to hepatic and microbial dysregulation, offering novel biological insights into early MASLD mechanisms.