Endogenous n-3 PUFAs Improve Non-Alcoholic Fatty Liver Disease through FFAR4-Mediated Gut-Liver Crosstalk

Abstract

The gut-liver axis plays a key role in the development and progression of non-alcoholic fatty liver disease (NAFLD). Due to the complexity and incomplete understanding of the cross-talk between the gut and liver, effective therapeutic targets are largely unknown. Free fatty acid receptors (FFARs) may bridge the cross-talk between the gut and liver. FFAR4 has received considerable attention due to its important role in lipid metabolism. However, the role of FFAR4 in this cross talk in NAFLD remains unclear. In this study, mice with high endogenous n-3 PUFAs but FFAR4 deficiency were generated by crossbreeding Fat-1 and FFAR4 knockout mice. FFAR4 deficiency blocked the protective effects of high endogenous n-3 PUFAs on intestinal barrier dysfunction and hepatic steatosis. In addition, FFAR4 deficiency decreased gut microbiota diversity and enriched Rikenella, Anaerotruncus, and Enterococcus, and reduced Dubosiella, Ruminococcaceae UCG-010, Ruminococcaceae UCG-014, Coriobacteriaceae UCG-002, Faecalibaculum, Ruminococcaceae UCG-009, and Akkermansia. Notably, FFAR4 deficiency co-regulated pantothenic acid and CoA biosynthesis, β-alanine metabolism, and sphingolipid metabolism pathways in the gut and liver, potentially associated with the aggravation of NAFLD. Together, the beneficial effects of n-3 PUFAs on the gut and liver were mediated by FFAR4, providing insights on the role of FFAR4 in the treatment of NAFLD through the gut-liver axis.

Keywords: free fatty acid receptor 4; gut–liver axis; n-3 polyunsaturated fatty acids; non-alcoholic fatty liver disease.

Figure 1

Endogenous n-3 PUFAs improve HFD-induced intestinal barrier dysfunction and hepatic steatosis. (A) Final body weight and liver weight at 16 weeks. (B) Liver pathology of different groups (200× magnification; scale bar = 50 μm). (C) Hepatic TC, TG, ALT, and AST content. (D) Representative images of H&E-stained colon sections (100× magnification; scale bar = 100 μm). Villus length quantified using ImageJ software (right). (E) qPCR analysis of genes involved in the intestinal barrier function. Results are presented as mean ± SD (n = 8 for each group). * p < 0.05, ** p < 0.01, and *** p < 0.001 by one-way ANOVA and Student’s t-test.

Figure 2

FFAR4 deficiency blocks the beneficial effects of endogenous n-3 PUFA. (A) Final body weight and liver weight at 16 weeks. (B) Liver pathology of different groups (200× magnification; scale bar = 50 μm). (C) Hepatic TC, TG, ALT, and AST content. (D) Representative images of H&E-stained colon sections (100× magnification; scale bar = 100 μm). Villus length quantified using ImageJ software (right). (E) Intestinal barrier function genes analyzed with qPCR. Results are presented as mean ± SD (n = 7, per group). * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 3

Diversity analysis of gut microbiota among different groups. Alpha diversity of the gut microbiome shown as evenness (A), observed OTUs (B), and Shannon index (C) among the WT-ND, Fat-1-ND, WT-HFD, and Fat-1-HFD groups. (D) PCoa plot of beta diversity in four groups (WT-ND, Fat-1-ND, WT-HFD, and Fat-1-HFD group). Alpha diversity of gut microbiome shown as evenness (E), observed OTUs (F), and Shannon index (G) between the Fat-1-HFD and FFAR4^−/−/Fat-1-HFD groups. (H) PCoa of beta diversity in two groups (Fat-1-HFD and FFAR4^−/−/Fat-1-HFD). Results are shown as mean ± SD (n = 5–6 for each group). * p < 0.05, ** p < 0.01.

Figure 4

Gut microbial composition and microbial functional pathways. (A) Linear Discriminant Analysis (LDA) effect size (LEfSe) analysis between the Fat-1-HFD and FFAR4^−/−/Fat-1-HFD groups (LDA score > 2 and p < 0.05). (B) Taxonomic cladogram was performed to identify the differentially abundant taxa. (C) Heat map of the differentially genus level (Top 10) between two groups. (D) Differential pathways between the Fat-1-HFD and FFAR4^−/−/Fat-1-HFD groups (p < 0.05). Results are presented as mean ± SD (n = 6 for each group).

Figure 5

Liver metabolomic analysis. (A) PCA score plot. (B) OPLS-DA score plot. (C) Heat maps of differential metabolites between the Fat-1-HFD and FFAR4^−/−/Fat-1-HFD groups, as determined by VIP > 1 and p < 0.05. (D) Overview of the metabolite set enrichment analysis. Results are shown as mean ± SD (n = 7, per group).

Figure 6

Key liver metabolites and gut microbiota affected by FFAR4. (A) Venn diagram analysis. (B) Correlation analysis of differential bacterial genera and metabolites. * p < 0.05 and ** p < 0.01.

Figure 7

Endogenous n-3 PUFAs improved NAFLD through FFAR4-mediated gut–liver axis.