DOI: 10.5061/dryad.95x69p8jg
title: Gut microbiome analysis of high fat diet- and control-fed on PXR-KO mice

description: 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.
description: CEL files were analyzed in R using the oligo package. Genes with
 an average probe intensity &gt;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<sup> </sup>nm absorbance
 ratios, and the integrity of 18S and 28S ribosomal<sup>
 </sup>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,
 <em>BioB</em>, <em>BioC</em>, <em>BioD
 </em>and <em>cre </em>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.
description: 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