Pregnane X receptor knockout mitigates weight gain and hepatic metabolic dysregulation in female C57BL/6 J mice on a long-term high-fat diet

Abstract

Obesity is a significant risk factor for several chronic diseases. However, pre-menopausal females are protected against high-fat diet (HFD)-induced obesity and its adverse effects. The pregnane X receptor (PXR, NR1I2), a xenobiotic-sensing nuclear receptor, promotes short-term obesity-associated liver disease only in male mice but not in females. Therefore, the current study investigated the metabolic and pathophysiological effects of a long-term 52-week HFD in female wild-type (WT) and PXR-KO mice and characterized the PXR-dependent molecular pathways involved. After 52 weeks of HFD ingestion, the body and liver weights and several markers of hepatotoxicity were significantly higher in WT mice than in their PXR-KO counterparts. The HFD-induced liver injury in WT female mice was also associated with upregulation of the hepatic mRNA levels of peroxisome proliferator-activated receptor gamma (Pparg), its target genes, fat-specific protein 27 (Fsp27), and the liver-specific Fsp27b involved in lipid accumulation, apoptosis, and inflammation. Notably, PXR-KO mice displayed elevated hepatic Cyp2a5 (anti-obesity gene), aldo-keto reductase 1b7 (Akr1b7), glutathione-S-transferase M3 (Gstm3) (antioxidant gene), and AMP-activated protein kinase (AMPK) levels, contributing to protection against long-term HFD-induced obesity and inflammation. RNA sequencing analysis revealed a general blunting of the transcriptomic response to HFD in PXR-KO compared to WT mice. Pathway enrichment analysis demonstrated enrichment by HFD for several pathways, including oxidative stress and redox pathway, cholesterol biosynthesis, and glycolysis/gluconeogenesis in WT but not PXR-KO mice. In conclusion, this study provides new insights into the molecular mechanisms by which PXR deficiency protects against long-term HFD-induced severe obesity and its adverse effects in female mice.

Keywords: Female; High-fat diet; Long-term diet; MAFLD; Obesity; Pregnane-X receptor.

Figure 1.. Effects of HFD on Body, liver weight gain, and food intake in WT and PXR-KO female mice

(A) The weekly weight of WT and PXR-KO female control-fed or HFD-fed mice groups over 52 weeks. (B) Weight gain difference in g between the first week (week 0) and the last week (week 52) after control-fed or HFD-fed mice. (C) Liver weight in g. (D) Average weekly food consumption by mice group. (E) 20X objective H and E-stained liver sections in control-fed or HFD-fed WT and PXR-KO female mice. (F-G) Liver steatosis and inflammation score. (H-J) Liver Triglyceride, cholesterol, and NEFA levels. Data represent mean ± SEM (n = 6–10). # represents P<0.05 between control-fed or HFD-fed mice. § represents P<0.05 between mice fed a HFD. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, *** P ≤ 0.0001, compared to indicated groups.

Figure 2.. PXR promotes insulin resistance but not glucose intolerance in female mice.

(A-B) Fasting blood glucose and serum insulin. (C) Oral glucose tolerance test in WT and PXR-KO control-fed and PXR-KO HFD-fed mice. (D) Area under the curve analysis of oral blood glucose in WT and PXR-KO control and HFD-fed mice (E-G) Serum leptin, adiponectin and triglyceride levels (H-I) Liver TBARS levels and serum alanine aminotransferase (ALT) levels Data represent mean ± SEM (n = 6–10). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, *** P ≤ 0.0001, compared to indicated groups.

Figure 3.. HFD increased PXR-targeted lipogenic mRNA

(A-C) Pregnane-X receptor (Pxr), Retinoid X receptor-alpha (RXRa), Constitutive androstane receptor (Car) mRNA levels measured by qPCR. (D-F) Cyp2b10, Cyp2b9, Cyp2b13 mRNA levels measured by qPCR. (G-L) Pparg, Fsp27, Fsp27b, Fsp27a, Cidea, Cideb mRNA levels measured by qPCR. (M-N) Both cd36 mRNA and Cd36 protein levels were measured by qPCR and western blot. (O-P) Both bhmt mRNA and BHMT protein were measured by qPCR and western blot. Data represent mean ± SEM (n = 5–6) for qPCR analysis and (n=3–4) for Western blot analysis. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, *** P ≤ 0.0001, compared to indicated groups.

Figure 4.. PXR inhibits obesity protective genes upon HFD.

(A-D) Akr1b7, Cyp2a5, Pgc1a and HNF4a mRNA levels measured by qPCR. (E-I) Ppara, Cpt1, Acox1 and Sirt1, ER-a mRNA levels were measured by qPCR. (J) p- AMPKa/ AMPKa protein levels measured by western blot analysis (K-L) Foxo1 and Gadd45b mRNA levels measured by qPCR. (M-N) Gstm3 and Sod2 mRNA levels were measured by qPCR. (O-Q) Mmp2, OPN and Colla1, mRNA levels were measured by qPCR. Data represent mean ± SEM (n = 5–6) for qPCR analysis and (n=3–4) for Western blot analysis. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, *** P ≤ 0.0001, compared to indicated groups.

Figure 5.. PXR activated liver fibrotic and cancer-associated genes upon HFD.

(A) PCA plot showing the clustering patterns of WT and PXR-KO mice fed control or HFD (B) Venn diagram illustrating the overlap of the first 100 differentially expressed genes among WT and PXR-KO mice fed control or HFD (C) Volcano plots of WT and PXR-KO, respectively, comparing between control and HFD-diets (D) Volcano plots of control and HFD, respectively, comparing between WT and PXR-KO genotypes-- x-axis represents log2 fold change, y-axis represents log10 p-value, dashed lines represent significance. (E-H) Heatmaps of the top 50 differentially expressed genes with P<0.05 of HFD-fed vs. control-fed WT mice and PXR-KO mice.

Figure 6.. PXR activated multiple inflammatory pathways in HFD-fed mice using IPA database

(A-D) Canonical pathway analysis graph depicting the enriched pathways based on differentially expressed genes in WT and PXR-KO groups fed both control and HFD. The analysis included a differential gene expression dataset of genes with more than 1.5 X up- or down-regulation with a significance of P-value > 0.05 and showed overlap with pathways. The percentage shows overlap with the pathway. Green shows downregulation, red shows upregulation of genes, and white shows no overlap with the pathway.

Figure 6.. PXR activated multiple inflammatory pathways in HFD-fed mice using IPA database

(A-D) Canonical pathway analysis graph depicting the enriched pathways based on differentially expressed genes in WT and PXR-KO groups fed both control and HFD. The analysis included a differential gene expression dataset of genes with more than 1.5 X up- or down-regulation with a significance of P-value > 0.05 and showed overlap with pathways. The percentage shows overlap with the pathway. Green shows downregulation, red shows upregulation of genes, and white shows no overlap with the pathway.

Figure 7:. Wikipathway-based analysis revealed PXR-based lipid metabolism enrichment on HFD.

The top 10 most enriched Wikipathways (by p-value) in genotype and diet comparison are shown as enrichment plots. The magnitude and direction of pathway enrichment (NES) and significance of enrichment (p-value) are plotted for each pathway. Pathway names are indicated at the top of each sub-plot. (A) WT_Control vs HFD diet comparison pathway enrichment (B) PXR-KO_Control vs HFD diet comparison pathway enrichment (C) HFD_WT vs PXR-KO genotype comparison pathway enrichment (D) Control_WT vs PXR-KO genotype comparison pathway enrichment