ILA mitigates HFD-induced metabolic dysfunction via lipid-immune crosstalk and gut microbiota modulation in zebrafish.

High-fat-induced metabolic syndrome poses many challenges to fish farming. In our previous study, we found that probiotic Akkermansia muciniphila can increase the tryptophan microbiota metabolite indole-3-lactic acid (ILA). Therefore, this study further investigates the effect of ILA on resisting HFD-induced metabolic disorders and preliminarily explores its mechanism in zebrafish. We found that supplementation with 100 μg/g ILA significantly attenuated HFD-induced weight gain, improved feed conversion ratio, and had no effect on survival rate. Additionally, ILA alleviated hepatic steatosis, significantly reducing triglyceride accumulation by downregulating lipogenic genes (DGAT2, SREBP-1c; p < 0.01, p < 0.05), while enhancing the expression of lipolytic‌ gene, such as UCP2 (p < 0.01). Concurrently, ILA preserved intestinal barrier integrity by attenuating intestinal villi damage and restoring tight junction ultrastructure. qRT-PCR results showed that ILA supplementation enhanced both intestinal mechanical and chemical barrier integrity by upregulating tight junction proteins (Muc2, TJP-1α, Cldn12) and complement C3b. Additionally, HFD increased serum LPS and hepatic LBP, which were reversed by ILA. And it could also ameliorate HFD-induced low-grade inflammation, as evidenced by suppressing pro-inflammatory cytokines (TNF-α, IL-6; p < 0.05) while elevating anti-inflammatory IL-10 (p < 0.05). Gut microbiota analysis revealed ILA intake exerted no significant effects on alpha diversity, yet profoundly restructured microbial composition by reducing Bacillota, Bacteroidota abundance and enriching Fusobacteriota, Verrucomicrobiota compared to the HFD group, accompanied by reduced abundance of pathogenic genera (Shewanella, Acinetobacter, Malacoplasma, Culicoidibacter, and Pseudomonas). Transcriptome sequencing analysis identified 3594 differentially expressed genes (DEGs) following ILA supplementation. These DEGs were significantly enriched in lipid metabolism and immune-related pathways, including drug metabolism-cytochrome P450, steroid hormone biosynthesis, fatty acid degradation, glycerolipid metabolism, as well as the PPAR/FoxO/insulin signaling pathways. It was further verified that the effects of ILA could be mediated via AhR2 receptor activation, followed by the modulation of Cyp1a1, a downstream gene involved in drug metabolism-cytochrome P450. Multi-omics analyses revealed significant correlation between gut microbial genera abundance and pathway-specific gene expression. These findings collectively demonstrated that ILA ameliorates HFD-induced metabolic dysfunction through dual regulation of lipid homeostasis, intestinal barrier function, and inflammation, providing insights into its potential mechanisms.