Metabolic dysfunction-associated steatotic liver disease exhibits sex-specific microbial heterogeneity within intestinal compartments

Abstract

Background/aims: Evidence suggests that the gastrointestinal microbiome plays a significant role in the biology of metabolic dysfunction-associated steatotic liver disease (MASLD). However, it remains unclear whether disparities in the gut microbiome across intestinal tissular compartments between the sexes lead to MASLD pathogenesis.

Methods: Sex-specific analyses of microbiome composition in two anatomically distinct regions of the gut, the small intestine and colon, were performed using an experimental model of MASLD. The study involved male and female spontaneously hypertensive rats and the Wistar-Kyoto control rat strain, which were fed either a standard chow diet or a high-fat diet for 12 weeks to induce MASLD (12 rats per group). High-throughput 16S sequencing was used for microbiome analysis.

Results: There were significant differences in the overall microbiome composition of male and female rats with MASLD, including variations in topographical gut regions. The beta diversity of the jejunal and colon microbiomes was higher in female rats than in male rats (PERMANOVA p-value=0.001). Sex-specific analysis and discriminant features using LEfSe showed considerable variation in bacterial abundance, along with distinct functional properties, in the jejunum and colon of animals with MASLD. Significantly elevated levels of lipopolysaccharide and protein expression of Toll-like receptor 4 were observed in the livers of male rats with MASLD compared with their female counterparts.

Conclusion: This study uncovered sexual dimorphism in the gut microbiome of MASLD and identified microbial heterogeneity within intestinal compartments. Insights into sex-specific variations in gut microbiome composition could facilitate customised treatment strategies.

Keywords: Gut microbiome; MASLD; Metabolic syndrome; NAFLD; Sexual dimorphism.

Figure 1.

Hierarchical taxonomic structure by disease group. A heat phylogenetic tree was constructed to demonstrate the hierarchical structure of taxonomic features in MASLD and control samples in the jejunum (A) and the descending colon (B). The heat phylogenetic tree analysis used the hierarchical structure of taxonomic classifications to show abundance profiles between animals with MASLD and the control group in a quantitative (using the median abundance) and statistical manner (using the nonparametric Wilcoxon Rank Sum test). The figure illustrates taxonomic features at the genus level. In this taxonomic tree, each circle represents a taxon and the lines show the hierarchical relationships between them. MASLD, metabolic dysfunction-associated steatotic liver disease.

Figure 2.

Taxonomic diversity profiling by sex. The plots illustrate variations in the community profiles of females and males in the small and descending intestines. Alpha diversity was assessed in samples from the jejunum (A) and descending colon (B) using the Chao1 nonparametric method to estimate the community species richness. Chao1 was used as an estimator of abundance-based bacterial richness. We used the Wilcoxon rank-sum test to assess the differences in diversity between males and females. The Bray-Curtis algorithm was used to calculate beta diversity, which measures the differences in community composition between samples from the jejunum (C) and colon (D). To test how much of the inter-individual microbial variation (Bray-Curtis and Jaccard distances) could be explained by sex, we performed permutational multivariate analysis of variance (PERMANOVA) using the adonis function. The P-value was determined using 1,000 permutations, and differences were considered significant at P<0.05. To aid in pattern identification and to gain biological insight, samples displayed on the PCoA were color-coded based on metadata (pink for female animals and light blue for male animals).

Figure 3.

Hierarchical taxonomic structure by sex. A heat phylogenetic tree was constructed to demonstrate the hierarchical structure of taxonomic features in female and male animals in the jejunum (A) and the descending colon (B). The heat phylogenetic tree analysis uses the hierarchical structure of taxonomic classifications to show abundance profiles in a quantitative (using the median abundance) and statistical manner (using the nonparametric Wilcoxon rank-sum test). The figure illustrates taxonomic features at the genus level. In this taxonomic tree, each circle represents a taxon and the lines show the hierarchical relationships between them.

Figure 4.

Dot plot representing significantly differential genera of the gut microbiome in MASLD stratified by sex. A dot plot is presented, showing the genera of the gut microbiome that were significantly different in MASLD stratified by sex. The LEfSe procedure was used for linear discriminant analysis (LDA) at an adjusted false discovery rate (FDR/q-value < 0.05). Significant taxa were ranked in decreasing order based on their LDA scores (x-axis). The mini-heat map on the right side of the plot indicates whether the number of taxa was higher (red/brown) or lower (blue/violet) in each group. (A) jejunum samples; (B) descending colon. MASLD, metabolic dysfunction-associated steatotic liver disease.

Figure 5.

Liver lipopolysaccharides (LPS) and Tlr4 staining. LPS (A–F) and toll-like receptor 4 (Tlr4) (G–L) protein expression in liver tissue samples from male and female animals with MASLD and controls were detected using immunohistochemistry. Representative samples are shown at the original magnification of ×400. MASLD, metabolic dysfunction-associated steatotic liver disease; LD, lipid droplet; BH, ballooned hepatocytes; CV, central vein; H, hepatocytes.

Figure 6.

Predicted Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in proximal small intestine (jejunum) by sex. The KEGG pathways were significantly different between the two groups (female and male animals). The bars on the left show the abundance (mean proportion, %) of different functional classifications in the two groups. The dots on the right side show the differences in the mean proportions (%) within the 95% confidence interval between the two groups. The rightmost values indicate Benjamini-Hochberg-adjusted P values. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States 2 (PICRUSt2) analysis resulted in KEGG. Statistical analysis was performed using the STAMP software. The figure was divided into two panels for readability.

Figure 7.

Predicted Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in the descending colon by sex. The KEGG pathways were significantly different between the two groups (female and male animals). The bars on the left show the abundance (mean proportion, %) of different functional classifications in the two groups. The dots in the right show the differences in the mean proportions (%) within the 95% confidence interval between the two groups. The rightmost values indicate Benjamini-Hochberg-adjusted P-values. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States 2 (PICRUSt2) analysis resulted in KEGG. Statistical analysis was performed using the STAMP software.