Age- and sex-dependent alterations of jejunal microbiota in Fischer 344 rats fed with a high-fructose, high-fat diet: depletion of Lactobacillus intestinalis in small bowel contents

Introduction Our previous research demonstrated that a high-fat diet (HFD) induced jejunal inflammation and hepatic steatosis, suggesting that small bowel microbiota contribute to these pathologies. This study investigated age- and sex-specific alterations in jejunal microbiota following a high-fructose, high-fat diet (HFHFD) in F344 rats. Methods Six-week-old and two-year-old rats of both sexes were fed an HFHFD for 8 weeks, after which jejunal contents were collected for metagenomic analysis. Taxonomic profiling and linear discriminant analysis were performed, and Spearman’s rank correlation analysis was used to evaluate associations with jejunal inflammation and hepatic steatosis. Beta-diversity analysis was conducted to assess group separation. In vitro , HIEC-6 human intestinal epithelial cells were used to test the protective effect of Lactobacillus intestinalis under palmitic acid–induced lipotoxic stress. Results HFHFD reduced the Firmicutes/Bacteroidetes ratio in young females and in aged rats of both sexes. Notably, Lactobacillus intestinalis —which supports barrier function—decreased in young males and aged females. In contrast, Akkermansia muciniphila increased across all HFHFD groups, particularly in young females and aged rats. Bacteroides vulgatus increased in aged HFHFD-fed rats of both sexes, while Bacteroides caccae was elevated in females across both age groups. Furthermore, the Lactobacillus reuteri group decreased only in young HFHFD rats. L. intestinalis and L. reuteri groups negatively correlated with jejunal inflammation and hepatic steatosis, whereas B. caccae and A. muciniphila showed positive correlations with both pathogenic phenotypes. Beta-diversity revealed a pronounced diet- and sex-dependent separation in young rats, which was attenuated in aged groups. In HIEC-6 cells, L. intestinalis significantly restored viability under palmitic acid–induced lipotoxic stress, though its conditioned medium did not. Discussion Collectively, HFHFD induces age- and sex-dependent dysbiosis in the jejunum, and L. intestinalis may serve as a potential probiotic for metabolic dysfunction-associated steatotic liver disease.