Systemic Deficiency of GHR in Pigs leads to Hepatic Steatosis via Negative Regulation of AHR Signaling

Abstract

Laron syndrome (LS) is an autosomal recessive genetic disease mainly caused by mutations in the human growth hormone receptor (GHR) gene. Previous studies have focused on Ghr mutant mice, but compared with LS patients, Ghr knockout (KO) mice exhibit differential lipid metabolism. To elucidate the relationship between GHR mutation and lipid metabolism, the role of GHR in lipid metabolism was examined in GHR KO pigs and hepatocytes transfected with siGHR. We observed high levels of free fatty acids and hepatic steatosis in GHR KO pigs, which recapitulates the abnormal lipid metabolism in LS patients. RNAseq analysis revealed that genes related to the fatty acid oxidation pathway were significantly altered in GHR KO pigs. AHR, a transcription factor related to lipid metabolism, was significantly downregulated in GHR KO pigs and siGHR-treated human hepatocytes. We found that AHR directly regulated fatty acid oxidation by directly binding to the promoters of ACOX1 and CPT1A and activating their expression. These data indicate that loss of GHR disturbs the ERK-AHR-ACOX1/CPT1A pathway and consequently leads to hepatic steatosis. Our results established AHR as a modulator of hepatic steatosis, thereby providing a therapeutic target for lipid metabolism disorder.

Keywords: AHR; GHR; Laron syndrome; hepatic steatosis.

Figure 1

Phenotype of GHR KO pigs. (A) Representative photographs of pigs of the two genotypes. (B-C) Body weight of male and female GHR KO pigs and their WT littermates. (D-F) Hepatic GHR mRNA and protein levels in GHR KO pigs. (D) Hepatic GHR mRNA levels in GHR KO pigs. (E-F) Hepatic GHR protein levels in GHR KO pigs by Western blotting, and quantification of the content by ImageJ. (G-H) Hepatic GHR protein levels in GHR KO pigs by IHC and quantification of the content by ImageJ. (I) Representative photographs of organs from GHR KO pigs and their WT littermates. (J) Absolute weight and relative weight of the organs. Scale bar: 100 µm. n = 3 pigs per group. The data are presented as the mean ± SD values. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Annotation: relative weight, the ratio of organ weight to body weight.

Figure 2

GHR deficiency disrupted lipid homeostasis in pigs. (A-E) Serum TG, TC, HDL, LDL and FFA levels in WT and GHR KO pigs. (F-G) Liver function markers, including ALT and AST, in WT and GHR KO pigs. n = 3 pigs per group. The data are presented as the mean ± SD values. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 3

GHR KO pigs developed hepatic steatosis. (A) Representative image of H&E staining. (B) Hepatic TG levels in WT and GHR KO pigs. (C-D) Representative image of Oil red O staining and relative Oil red O-stained area. (E) mRNA levels of key genes in fatty acid oxidation and VLDL secretion. (F-G) Protein levels of key genes in fatty acid oxidation and quantification of the content by ImageJ. Scale bar: 100 µm. n = 3 pigs per group. The data are presented as the mean ± SD values. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 4

GHR depletion caused intracellular lipid accumulation in cultured human hepatocytes. (A-E) Hepatocytes were treated with NC or GHR siRNA (siGHR). (A-B) The GHR protein expression levels in human hepatocytes, and quantification of the content by ImageJ. (C) TG levels in human hepatocytes. (D) Nile red staining of human hepatocytes with NC or siGHR. (E) The neutral lipid content was quantified with ImageJ and normalized to the number of nuclei. Scale bar: 100 µm. n = 3 per group. The data are presented as the mean ± SD values. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 5

Transcriptome analysis of hepatic gene expression profiles in GHR KO pigs. (A) The five enriched biological processes contributing to GHR function were determined by GO analysis based on the DEGs. (B) KEGG pathway enrichment analysis of the six identified lipid metabolism-related processes. (C-E) mRNA and protein levels of AHR in the livers of WT and GHR KO pigs. (F-H) mRNA and protein levels of AHR in NC and siGhr mouse hepatocytes. (I-M) mRNA and protein levels of AHR in NC and siGHR human hepatocytes. (I) mRNA levels of AHR in NC and siGHR human hepatocytes. (J-K) Protein levels of AHR in NC and siGHR human hepatocytes by Western blotting and quantification of the content by ImageJ. (L-M) Protein levels of AHR in NC and siGHR human hepatocytes by immunofluorescence and quantification of the content by ImageJ. Scale bar: 100 µm. n = 3 per group. The data are presented as the mean ± SD values. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 6

GHR regulates AHR expression through the MAPK signaling pathway. (A-B) IF staining of p-ERK1/2 in pigs and hepatocytes, and quantification of the content by ImageJ. (C-D) Protein levels of p-ERK1/2 and ERK1/2 in pigs and hepatocytes by Western blotting and quantification of the content by ImageJ. (E-G) Protein levels of AHR, p-ERK1/2 and ERK1/2 in L02 cells treated with GDC-0994 (an ERK inhibitor), and quantification of the content by ImageJ. Scale bar = 50 µm. n = 3 per group. The data are presented as the mean ± SD values. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 7

AHR directly activated fatty acid oxidation gene expression. (A-D) Protein levels of CPT1A, ACOX1 and AHR in cells treated with Tapinarof (an AHR ligand) and/or AHR siRNA (siAHR), and quantification of the content by ImageJ. (E) Effect of Tapinarof and/or siAHR treatment on ACOX1 luciferase reporter activity. (F) Effect of Tapinarof and/or siAHR treatment on CPT1A luciferase reporter activity. (G) ChIP assays were performed with the CYP1A1, CPT1A and ACOX1 promoters in L02 cells with or without Tapinarof (10 µM) treatment for 12 h. n = 3 per group. The data are presented as the mean ± SD values. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with the first group, ^#, P < 0.05; ^##, P < 0.01; ^###, P < 0.001 compared with the second group.

Figure 8

Overexpression of AHR alleviated lipid deposition induced by GHR deletion. (A) Nile red staining and (C) TG content in NC and siGHR cells with or without pCMV-Myc-AHR transfection. (B) The neutral lipid content was quantified with ImageJ and normalized to the number of nuclei. The data are presented as the mean ± SD values. Scale bar: 100 µm. n = 3 per group. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with the first group; ^#, P < 0.05; ^##, P < 0.01; ^###, P < 0.001 compared with the second group.

Figure 9

Schematic illustration of the proposed role of GHR in hepatic steatosis. Normal liver (left): GH binds to GHR and promotes the expression of AHR by activating ERK1/2 via phosphorylation. Activated AHR directly binds to the promoter regions of ACOX1 and CPT1A to transcriptionally activate their expression and allow normal fatty acid oxidation function. Hepatic steatosis (right): Loss of GHR reduces the expression of AHR by reducing the phosphorylation level of ERK1/2. AHR downregulation directly reduces fatty acid oxidation, leading to hepatic steatosis.