FXR, a Key Regulator of Lipid Metabolism, Is Inhibited by ER Stress-Mediated Activation of JNK and p38 MAPK in Large Yellow Croakers (Larimichthys crocea) Fed High Fat Diets

Abstract

High-fat diets induced abnormal lipid accumulation in the liver of cultured fish that caused body damage and diseases. The purpose of this research was to investigate the role and mechanism of farnesoid X receptor (FXR) in regulating lipid metabolism and to determine how high-fat diets affect FXR expression in large yellow croakers. The results showed that ligand-meditated FXR-activation could prevent abnormal lipid accumulation in the liver and hepatocytes of large yellow croakers. FXR activation increased the expression of lipid catabolism-related genes while decreasing the expression of lipogenesis-related genes. Further investigation found that the promoter activity of proliferator-activated receptor α (PPARα) could be increased by croaker FXR. Through the influence of SHP on LXR, FXR indirectly decreased the promoter activity of sterol regulatory element binding protein 1 (SREBP1) in large yellow croakers. Furthermore, the findings revealed that endoplasmic reticulum (ER)-stress-induced-activation of JNK and P38 MAPK participated in the reduction of FXR induced by high-fat diets. Then, hepatocyte nuclear factor 1α (HNF1α) was confirmed to be an FXR regulator in large yellow croaker, and it was reduced by high-fat diets and ER stress. In addition, co-expression of c-Jun with HNF1α inhibited the effect of HNF1α on FXR promoter, and suppression of P38 MAPK could relieve the HNF1α expression reduction caused by ER stress activation. In summary, the present study showed that FXR mediated lipid metabolism can prevent abnormal lipid accumulation through regulating PPARα and SREBP1 in large yellow croakers, while high-fat diets can suppress FXR expression by ER stress mediated-activation of JNK and P38 MAPK pathways. This research could benefit the study of FXR functions in vertebrate evolution and the development of therapy or preventative methods for nutrition-related disorders.

Keywords: ER stress; FXR; MAPK; high-fat diets; lipid metabolism.

Figure 1

Effects of HFD and supplementation of CDCA on: (A) Food intake; (B–D) lipid content of whole body, muscle, and liver; (E) hepatosomatic index; (F,G) TG and NEFA of liver; (H,I) ALT and AST of serum in large yellow croakers. The data are provided as means ± SEMs (n = 3) and were analyzed using Duncan’s multiple range test. The letters a and b indicate that there is a significant difference between means, p < 0.05.

Figure 2

Effect of HFD and CDCA supplementation on expression of lipid metabolism related genes. Expression of genes involved in: (A) Lipolysis and β-oxidation; (B) lipogenesis; (C) transport. (D) Expression of fxr and target genes (shp and bsep). The data are provided as means ± SEMs (n = 3) and were analyzed using Duncan’s multiple range test. The letters a–c indicate that there is a significant difference between means, p < 0.05.

Figure 3

Effects of FXR overexpression or ligand-mediated FXR activation on the TG content of LYCL cells and primary hepatocytes of large yellow croakers: (A–C) Fluorescence analysis, immunoblots, and qRT-PCR assays for FXR in LYCL cells infected by croaker fxr adenovirus; (D–F) TG content of LYCL cells with overexpression of FXR or after treatment with CDCA and GW4064. Data are shown as means ± SEMs (n = 3) and were analyzed using Duncan’s multiple range test and Student’s t-test. The letters a–c indicate that there is a significant difference between means, * p < 0.05.

Figure 4

FXR affects genes involved in lipolysis and β-oxidation of large yellow croakers: (A,B) qRT-PCR assays or immunoblots for PPARα, CPT1, and ATGL in LYCL cells after FXR overexpression and treatment with FA; (C,D) qRT-PCR assays or immunoblots for PPARα, CPT1, and ATGL in LYCL cells after FXR knockdown and treatment with FA; (E–G) gene expression of pparα, cpt1, and atgl in primary hepatocytes after GW4064 or CDFA treatment or FXR knockdown; (H) promoter activity of croaker PPARα in HEK 293T cells after FXR overexpression and treatment with FXR agonists; (I) ChIP analysis and identification of the FXR binding site in the croaker PPARα promoter region; (J,K) promoter activity of croaker ATGL in HEK 293T cells after FXR or PPARα overexpression. Data are shown as means ± SEMs (n = 3) and were analyzed using Duncan’s multiple range test and Student’s t-test. The letters a–d indicate that there is a significant difference between means, * p < 0.05.

Figure 5

Effects of FXR on genes involved in lipogenesis of large yellow croakers: (A,B) Gene expression of lxrα, srebp1, fas, and scd1 in LYCL cells after FXR overexpression or knockdown and treatment with FA; (C–E) gene expression of lxrα, srebp1, fas, and scd1 in primary hepatocytes after GW4064 or CDFA treatment or FXR knockdown; (F) promoter activity of croaker SREBP1, SCD1, and FAS in HEK 293T cells after FXR overexpression; (G) promoter activity of croaker SREBP1 after LXRα and SHP overexpression; (H) analysis of protein interactions between LXRα and SHP of large yellow croaker. Data are shown as means ± SEMs (n = 3) and were analyzed using Duncan’s multiple range test and Student’s t-test. The letters a–d indicate that there is a significant difference between means, * p < 0.05.

Figure 6

Effects of HFD and ER stress on the expression of FXR in large yellow croaker: (A,B) Immunoblots or qRT-PCR assays for FXR and ER stress markers in the liver of large yellow croakers fed HFD diet; (C–F) immunoblots or qRT-PCR assays for FXR and ER stress markers in LYCL cells after PA, TG, or TM treatment; (G) immunoblots for FXR in LYCL cells after TUDCA treatment. Data are shown as means ± SEMs (n = 3) and were analyzed using Student’s t-test, * p < 0.05.

Figure 7

Effects of MAPK signaling pathway on the expression of FXR: (A) Immunoblots for MAPK signaling pathway in liver of large yellow croakers fed HFD diet and LYCL cells after PA, TM, or TG treatment; (B) immunoblots for FXR in LYCL cells after TG treatment alone or with MAPK inhibitors. Data are shown as means ± SEMs (n = 3) and were analyzed using Duncan’s multiple range test and Student’s t-test. The letters a–c indicate that there is a significant difference between means, p < 0.05.

Figure 8

HNF1α is involved in er stress effect on FXR. (A) Promoter activity of croaker FXR in HEK 293T cells after HNF1α overexpression. (B) qRT-PCR assays for HNF1α in the liver of large yellow croakers fed HFD diet. (C) Immunoblots for HNF1α and c-Jun in LYCL cells after TG treatment. (D) Promoter activity of croaker FXR in HEK 293T cells after HNF1 overexpression alone or co-transfected with c-Jun. (E,F) Immunoblots or qRT-PCR assays for HNF1α in LYCL cells after TG treatment alone or with MAPK inhibitors. Data are shown as means ± SEMs (n = 3) and were analyzed using Duncan’s multiple range test and Student’s t-test. The letters a–c indicate that there is a significant differences between the means, * p < 0.05.