Nonalcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease due to the current epidemics of obesity and diabetes. The pregnane X receptor (PXR) is a xenobiotic-sensing nuclear receptor known for trans-activating liver genes involved in drug metabolism and transport, and more recently implicated in energy metabolism. The gut microbiota can modulate the host xenobiotic biotransformation and contribute to the development of obesity. While the male sex confers a higher risk for NAFLD than women before menopause, the mechanism remains unknown. We hypothesized that the presence of PXR promotes obesity by modifying the gut-liver axis in a sex-specific manner. Male and female C57BL/6 (wild-type/WT) and PXR-knockout (PXR-KO) mice were fed control or high fat diet (HFD) for 16-weeks. Serum parameters, liver histopathology, transcriptomic profiling, 16S-rDNA sequencing, and bile acid (BA) metabolomics were performed. PXR enhanced HFD-induced weight gain, hepatic steatosis and inflammation especially in males, accompanied by PXR-dependent up-regulation in hepatic genes involved in microbial response, inflammation, oxidative stress, and cancer; PXR-dependent increase in intestinal Firmicutes/Bacteroides ratio (hallmark of obesity) and the pro-inflammatory Lactobacillus, as well as a decrease in the anti-obese Allobaculum and the anti-inflammatory Bifidobacterum, with a PXR-dependent reduction of beneficial BAs in liver. The resistance to NAFLD in females may be explained by PXR-dependent decrease in pro-inflammatory bacteria (Ruminococcus gnavus and Peptococcaceae). In conclusion, PXR exacerbates hepatic steatosis and inflammation accompanied by obesity- and inflammation-prone gut microbiome signature, suggesting that gut microbiome may contribute to PXR-mediated exacerbation of NAFLD.

CEL files were analyzed in R using the oligo package. Genes with an average probe intensity >100 were considered to be significantly expressed. Probe IDs were annotated using the clariomsmousehttranscriptcluster.db package. Upper quartile normalization and differential expression between vehicle and HFD-exposed groups of the same genotype and sex was determined using EdgeR.  The RUVSeq package was used to remove unwanted variations as described previously, and genes with a false discovery rate below 0.05 were considered statistically significant.  Volcano plots and heatmaps were plotted for the differentially regulated genes with at least 50% up- or down-regulation using prcomp and ComplexHeatmap, respectively.  Pathway analysis was performed using the R package TopGo. Bar plots of representative differentially regulated genes were generated using SigmaPlot (Systat Software, Inc).  Total RNA were isolated with Trizol from liver and intestinal tissues of male and female WT and PXR-KO mice fed control or a HFD for 16 weeks and purified using RNeasy spin columns (Germantown, MD), according to the supplier’s instructions. The quality of the RNA was evaluated by measuring the 260:280 nm absorbance ratios, and the integrity of 18S and 28S ribosomal RNA bands assessed by electrophoresis. Total RNA (250 ng) was used to synthesize fragmented and labeled sense-strand cDNA and hybridize onto Affymetrix arrays. The Affymetrix GeneChip® WT PLUS Reagent Kit Manual were followed to prepare the samples. Briefly, the GeneChip® WT PLUS Reagent Kit (Affymetrix) was used to generate sense-strand cDNA from total RNA. Following synthesis of sense-strand cDNA, the cDNA was fragmented and labeled with the Affymetrix GeneChip Terminal Labeling Kit. Fragmented and labeled cDNA were then added to a hybridization cocktail (25 µg/µl fragmented cDNA, 50 pM control oligonucleotide B2, BioB, BioC, BioD and cre hybridization controls, 7 % DMSO, 100 mM MES, 1 M [Na+], 20 mM EDTA, 0.01% Tween 20).  Affymetrix arrays (Affymetrix, Santa Clara, CA) were hybridized for 16 hours at 45°C in the GeneChip Hybridization Oven 645 (Affymetrix). The arrays were washed and stained with R-phycoerythrin streptavidin in the GeneChip Fluidics Station 450 (Affymetrix). The arrays were scanned with the GeneChip Scanner 3000 7G Plus with autoloader. GeneChip Command Console Software (AGCC) was used for washing, staining and scanning control of the instrumentation.

Kim_et_al_2021_microbiome_applications

This file contains description CEL files for WT and PXR-KO extracted from large intestinal content to produce various gut microbiome analyses reported in the associated paper. 

Metadata_Kim_et_al_HFDPXRKO.csv