Front Microbiol. 2026;17:1779112.

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.

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제1저자 하성찬 · 교신저자 김나영

PMID 41847199 PMC12989582 DOI 10.3389/fmicb.2026.1779112 IF 5.8 소화기내과

소속 (4)

Department of Health Science and Technology, Graduate School of Convergence Science and Technology, Seoul National University, Seoul, Republic of Korea.
Department of Internal Medicine, Seoul National University Bundang Hospital, Seongnam, Republic of Korea.
Research Center for Sex- and Gender-Specific Medicine, Seoul National University Bundang Hospital, Seongnam, Republic of Korea.
Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea.

Abstract

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.

Keywords

agehigh-fructose high-fat dietmicrobiotanon-alcoholic fatty liver diseasesexsmall bowel

Figures

**Figure 1** Taxonomic composition and Firmicutes/Bacteroidetes (F/B) ratio, and their correlations with hepatic steatosis and jejunal inflammation in the jejunal microbiota of rats fed different diets. Stacked bar plots show the relative taxonomic composition at the phylum level **(A,B)**. Scatter plots represent the relative abundance of **(C)** Firmicutes and **(D)** Bacteroidetes. **(E)** Comparison of F/B ratio according to age, sex, and diet. **(F)** Comparison of F/B regardless of sex. **(G)** Correlation between jejunal inflammation and F/B ratio. **(H)** Correlation between hepatic steatosis and F/B ratio. Scatter plot data are expressed as mean ± SEM. Statistical significance was determined using the Kruskal–Wallis test (p-values shown above the graphs) followed by the Mann–Whitney U-test (*p < 0.05, **p < 0.01, ***p < 0.001). Correlation was assessed using Spearman’s rank correlation (n = 6 per group, except for 2-year-old male CON, n = 5); 6w, 6 weeks; 2 yr., 2 years; CON, control; HFHFD, high-fructose high-fat diet.

**Figure 2** Alterations in the abundance ratio at the genus **(A–C)** and species **(D–H)** levels, along with major species, following exposure to a high-fat diet. Scatter plots show relative taxonomic abundance, with data expressed as mean ± SEM. **(A)** *Lactobacillus*; **(B)** *Bacteroides*; **(C)** *Akkermansia*; **(D)** *Lactobacillus intestinalis*; **(E)** *Bacteroides caccae*; **(F)** *Akkermansia muciniphila*; **(G)** *Lactobacillus reuteri group*; **(H)** *Bacteroides vulgatus*. In this dataset, *Akkermansia muciniphila* accounted for nearly all reads assigned to the genus *Akkermansia*, resulting in highly similar genus- and species-level abundance patterns. Stacked bar plots show the relative taxonomic composition at the genus and species level **(I,J)**. Statistical significance was determined using the Kruskal–Wallis test (p-values shown above the graphs) followed by the Mann–Whitney U-test (*p < 0.05, **p < 0.01, ***p < 0.001) (n = 6 per group, except for 2-year-old male CON, n = 5); 6w, 6 weeks; 2 yr., 2 years; CON, control; HFHFD, high-fructose high-fat diet.

**Figure 3** Correlations between histological parameters and bacterial abundance ratios in the jejunal microbiota of rats fed different diets. Scatter plots show correlations between histological features and the relative abundance of specific bacterial species. **(A)** Jejunal inflammation vs *Lactobacillus intestinalis*, **(B)** hepatic steatosis vs *L. intestinalis*, **(C)** jejunal inflammation vs *Bacteroides caccae*, **(D)** hepatic steatosis vs *B.caccae*, **(E)** jejunal inflammation vs *Akkermansia muciniphila*, **(F)** hepatic steatosis vs *A. muciniphila*, **(G)** jejunal inflammation vs *Lactobacillus reuteri* group, **(H)** hepatic steatosis vs *L. reuteri* group, **(I)** hepatic steatosis vs *Bacteroides vulgatus*. Statistical correlations were analyzed using Spearman’s rank correlation, and r- and p-values are indicated on the graphs (n = 6 per group, except for 2-year-old male CON, n = 5); 6w, 6 weeks; 2yr, 2 years.

**Figure 4** Distribution of jejunal microbiota according to diet, sex, and age in rats. Principal coordinate analysis (PCoA) plots illustrate the distribution of gut microbial communities according to dietary condition (CON vs HFHFD) and sex (male and female) in **(A)** 6-week-old and **(B)** 2-year-old rats. Each ellipse represents a 95% confidence interval for each group. Pairwise comparisons of microbial community composition between groups were performed using the PERMANOVA method on the EzBioCloud platform, and corresponding p- and q-values are summarized in the tables below each plot (all pairwise comparisons showed p = 0.001, as shown above the graphs). PC1 explains 40.464% of the variance, PC2 explains 18.869% of the variance for **(A)** 6-week-old rats and PC1 explains 33.106% of the variance, PC2 explains 10.732% of the variance for **(B)** 2-year-old rats (n = 6 per group, except for 2-year-old male CON, n = 5); CON, control; HFHFD, high-fructose high-fat diet.

**Figure 5** Differentially abundant bacterial species identified by LEfSe analysis in the jejunal microbiota of rats fed a control diet or HFHFD. LEfSe (Linear Discriminant Analysis Effect Size) was performed at the species level to identify significantly enriched bacterial taxa between the two dietary groups. Bar plots indicate bacterial species with LDA scores exceeding the threshold (LDA score > 2.0), representing those differentially abundant in each group. **(A)** 6-week-old males, **(B)** 6-week-old females, **(C)** 2-year-old males, **(D)** 2-year-old females. The length of each bar represents the effect size (LDA score) (n = 6 per group, except for 2-year-old male CON, n = 5); 6w, 6 weeks; 2 yr., 2 years; CON, control; HFHFD, high-fructose high-fat diet.

**Figure 6** Differentially abundant bacterial species identified by LEfSe analysis in the jejunal microbiota of male and female rats within each diet and age group. LEfSe (Linear Discriminant Analysis Effect Size) was performed at the species level to identify sex-dependent differences in bacterial abundance under each dietary condition. Bar plots show bacterial species with LDA scores exceeding the threshold (LDA score > 2.0), representing those differentially abundant between males and females. **(A)** 6-week-old CON, **(B)** 6-week-old HFHFD, **(C)** 2-year-old CON, **(D)** 2-year-old HFHFD. The length of each bar represents the effect size (LDA score) (n = 6 per group, except for 2-year-old male CON, n = 5); 6w, 6 weeks; 2 yr., 2 years; CON, control; HFHFD, high-fructose high-fat diet.

**Figure 7** Differentially abundant bacterial species identified by LEfSe analysis in the jejunal microbiota of rats of different ages within each diet and sex group. LEfSe (Linear Discriminant Analysis Effect Size) was performed at the species level to identify age-related differences in bacterial abundance between 6-week-old and 2-year-old rats under each dietary condition. Bar plots show bacterial species with LDA scores exceeding the threshold (LDA score > 2.0), representing those differentially abundant between age groups. **(A)** Male CON, **(B)** male HFHFD, **(C)** female CON, **(D)** female HFHFD. The length of each bar represents the effect size (LDA score) (n = 6 per group, except for 2-year-old male CON, n = 5); 6w, 6 weeks; 2 yr., 2 years; CON, control; HFHFD, high-fructose high-fat diet.

**Figure 8** Functional biomarkers predicted by LEfSe analysis based on KEGG pathway classification in the jejunal microbiota of rats. LEfSe (Linear Discriminant Analysis Effect Size) was used to identify significantly different functional biomarkers inferred from KEGG pathway analysis. Heatmaps display pathways with significantly different LDA scores between the indicated groups (*p < 0.05, **p < 0.01). **(A–D)** Comparison between CON and HFHFD groups in **(A)** 6-week-old males, **(B)** 6-week-old females, **(C)** 2-year-old males, and **(D)** 2-year-old females. **(E,F)** Comparison between males and females within each dietary condition: **(E)** 6-week CON and **(F)** 6-week HFHFD. **(G–J)** Comparison between 6-week-old and 2-year-old rats within each sex and dietary group: **(G)** male CON, **(H)** male HFHFD, **(I)** female CON, and **(J)** female HFHFD. Color intensity represents the relative LDA score of each KEGG pathway category (n = 6 per group, except for 2-year-old male CON, n = 5); 6w, 6 weeks; 2 yr., 2 years; CON, control; HFHFD, high-fructose high-fat diet.

**Figure 9** *In vitro* study design and cell viability analysis from three repeated experiments. Experimental design schematic for *in vitro* treatment with *L. intestinalis* and PA under HFD conditions **(A)**. Cell viability measured by CCK-8 assay following exposure to PA, with and without *L. intestinalis* treatment **(B)**. Data expressed as mean ± SEM of three independent experiments and 12 wells per group. Statistical significance was determined using the Kruskal–Wallis test (p-values shown above the graphs) followed by the Mann–Whitney U-test; PA, Palmitic acid; CM, conditioned media.

그림 출처: PubMed Central 공개 논문 (PMC12989582)

Conflict of interest statement

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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