EFFECT OF HIGH FAT DIET ON OBESITY PHENOTYPES
Adult male and female WT and PXR-KO mice were fed either a standard rodent chow diet or a HFD diet for 16 weeks. The depletion of PXR in PXR-KO was confirmed by RT-qPCR in livers of these mice (Fig. S1). Daily food consumption by weight was higher in HFD-fed mice group than control diet-fed group of the same genotypes and sexes (Fig. S2). In addition, HFD-fed female PXR-KO mice consumed less food than female WT mice (Fig. S2).
In both male and female WT mice, HFD markedly increased the body weight starting from Week 2 to the end of the HFD exposure at week 16 (Fig. 1A). Interestingly, although HFD also increased the body weight of both male and female PXR-KO mice, the onset of weight gain occurred much later than in HFD-fed WT mice, which was Week 9 in male PXR-KO and week 7 in female PXR-KO mice. In addition, the percentage of weight gain in both male and female PXR-KO mice was markedly attenuated at all time points, as compared to the HFD-fed WT mice of the same sex.
In male mice, HFD increased both the absolute liver weight and liver to-body-weight ratio in a PXR-dependent manner (Table 1). In female mice, HFD did not alter the absolute liver weight or liver to-body-weight ratio in WT mice, whereas it decreased the liver/body weight ratio in PXR-KO mice. In male mice, HFD did not alter the mass of the epididymal white adipose tissue (WAT) in either genotype; however, in females, HFD increased the WAT mass in both genotypes, and the presence of PXR resulted in a greater WAT mass gain. HFD increased mesenteric WAT mass in all groups except for female PXR-KO mice where no change was observed (Table 1). HFD increased brown adipose tissue (BAT) mass in a PXR-dependent manner. HFD did not alter the lean mass in any groups; however, HFD increased the fat mass of both males and females in a PXR-dependent manner. Together, these observations suggest that the presence of PXR promotes obesity related metabolic parameters and male mice are more susceptible to HFD-induced liver and fat mass gain than females.
Regarding the serum metabolic parameters, as shown in Table 1, in males, HFD increased serum alanine aminotransferase (ALT), which is a well-established biomarker for liver injury (36), increased serum total bile acids (BAs), which is an indicator of cholestatic liver injury (37) and increased fibroblast growth factor 21 (FGF21), which is a liver-derived hormone that promotes hepatic fatty acid oxidation, ketogenesis, and insulin sensitivity (38), all in a PXR-dependent manner. Interestingly, in HFD-fed female mice, serum ALT, BAs, or Fgf21 were unchanged in either genotype. In male mice, HFD increased serum insulin in both WT and PXR-KO mice; whereas in females, HFD did not have any effect in either WT or PXR-KO conditions. HFD increased fasting glucose in male PXR-KO mice, but did not have any effect in other groups. Serum leptin was increased by HFD in both sexes and genotypes, whereas serum adiponectin was not altered in any group. These observations suggest that both the presence of PXR and the male sex render the mice more susceptible to HFD-induced injuries than females. However, whereas HFD-induced liver injury and lipid disorders were promoted by PXR, PXR may be beneficial in maintaining insulin sensitivity (as evidenced by increased fasting glucose in male PXR-KO mice).
H&E staining was performed in control and HFD-fed mice to determine the effects of PXR and sex on lipid accumulation and inflammation. The presence of PXR promoted HFD-induced substantial increase in lipid accumulation (observed mostly as macrovesicular with some microvesicular steatosis) as well as greater inflammatory cell infiltration in both male and female WT mice, whereas such phenotype was much attenuated in the absence of PXR in both sexes (Fig.1B-1D). In addition, HFD-fed male WT mice had even more lipid accumulation and inflammation than HFD-fed female WT mice. Col1a1 mRNA was increased by HFD in male mice in a PXR-dependent manner; COL1A1 is a well-known early biomarker for liver fibrosis and hepatocellular carcinoma (39). Mild necrosis was also observed in livers of HFD-fed male WT mice with no noteworthy signs of apoptosis, fibrosis, and/or biliary hyperplasia by HFD exposure in either sexes or genotypes (Data not shown). HFD increased the levels of hepatic triglyceride in PXR-dependent manner in both sexes (Fig. S3A). In summary, these results indicate that PXR in both male and female mice promotes HFD-induced body weight gain, steatosis, inflammation and hepatic triglyceride accumulation.
EFFECT OF HIGH FAT DIET ON GLUCOSE TOLERANCE
Impaired glucose tolerance is the earliest identifiable metabolic abnormality in the pathogenesis of type II diabetes. To establish whether PXR or sex has any influence on insulin signaling and glucose clearance, a glucose tolerance test was performed in control- or HFD-fed male and female WT and PXR-KO mice (Fig. 1E). Notably, in male mice, deficiency of PXR resulted in a higher area under the curve (AUC) of serum glucose in both control diet and HFD groups. This observation aligns with the finding that PXR-KO males had elevated increased fasting glucose (Table 1), and suggests that PXR is beneficial in maintaining insulin signaling in males. However, in female mice, PXR worsens the ability to clear glucose, because HFD-fed female WT mice had higher AUC than in HFD-fed female PXR-KO mice (Fig. S3B). Together these observations suggest that although the presence of PXR worsens HFD-induced lipid accumulation and inflammation, its role in insulin signaling is divergent and sex-specific.
HFD-MEDIATED FUNCTIONAL CHANGES PREDICTED FROM HEPATIC TRANSCRIPTOMIC RESPONSE
To examine the role of PXR in the HFD-mediated hepatic transcriptomic response in liver, we employed microarray and predicted functional changes using Gene ontology (GO) enrichment analysis. HFD produced a distinct separation in the liver transcriptomes in both genotypes and sexes (Fig. S4). Interestingly, the presence of PXR was associated with the HFD-mediated hepatic up-regulation in multiple pro-inflammatory signaling pathways and microbial responses in male mice (Fig. 2A), suggesting the presence of a leaky gut. These observations suggest the importance of an indirect role for PXR in regulating the gut microbiome community to modify hepatic transcriptome along the gut-liver axis. In contrast, in livers of male PXR-KO mice, neither pro-inflammation nor microbial response pathways were enriched (FDR < 0.01 cut-off, Fig. 2B).
In male mice, the presence of PXR did not enrich down-regulated hepatic pathways following HFD-exposure (Fig. 2C); whereas the absence of PXR potentiated HFD-mediated down-regulation of several pathways related to immune cell mobilization/infiltration (Fig. 2D)and positive regulation of leukocyte and cell activation. Other down-regulated functions related to immune response include antimicrobial humoral immune response mediated by antimicrobial peptide and acute inflammatory response. In addition, the down-regulation of superoxide anion generation and sensory responses was potentiated by the absence of PXR.
In HFD-fed female mice, the absence of PXR potentiated the up-regulation of pathways involved in pro-inflammation and microbial response (Fig. S5A-S5B), suggesting that PXR in females may render protection against HFD-induced injuries (Fig. S5). The presence of PXR drives the HFD-mediated down-regulation of several lipid and nucleotide metabolism pathways, whereas the absence of PXR potentiates the hepatic down-regulation of pathways involved in hormonal response and thermogenesis in females (Fig. S5C-S5D).
Together these observations suggested PXR-dependent and sex-divergent regulation of the hepatic transcriptome following HFD exposure. Observations in HFD-fed male mice suggest that PXR contributes to HFD-induced liver injury in male mice by promoting pro-inflammatory cascade and microbial response and preventing the down-regulation of immune cell infiltration and oxidative stress. Observations in HFD-fed female mice suggest that PXR offers protection against HFD-induced liver injury by preventing a pro-inflammatory cascade and microbial response.
HFD-INDUCED PXR-DEPENDENT AND -INDEPENDENT GENE EXPRESSION IN LIVER
To determine the importance of PXR in HFD-mediated changes in intermediary metabolism, microarray analysis was done for differentially regulated genes involved in the following pathways: lipid metabolism, carbohydrate metabolism, cholesterol and BA metabolism, as well as nuclear receptor signaling (Fig. 3A). In WT male livers, HFD increased the expression of lipid metabolism-related genes Abcd2, Apoa4, and Echs1, but decreased the expression of cholesterol metabolism-related genes Abca1, Cyp4a12a, and Cyp4a12b and the BA synthetic enzyme Cyp7a1, all in a PXR-dependent manner (Fig. 3A). In WT female livers, HFD increased the expression of lipid metabolism-related genes Acadm, Apoc2, Elovl3, and Mtor and most of the genes related to cholesterol and BA metabolism (Slc10a2, Abcb4, and Srebf2 except for Mbtps1), whereas HFD decreased the expression of lipid metabolism-related genes Fabp5 and Slc25a17, most of the genes related to glucose and glycogen metabolism (Gpd2, Gpi1, and Pygl except for Entpd5), and nuclear receptor genes Nr2c1 and Nr3c1 in a PXR-dependent manner (Fig. 3B).
In male PXR-KO mice livers, HFD increased the expression of some genes related to lipid metabolism (Acaa1a, Apoa1, Hadha, and Phyh) and glucose metabolism (Gpd1 and Pdha1), but decreased the expression of other genes related to lipid metabolism (Npc2 and Ppargc1a) and cholesterol metabolism (Sqle) (Fig. 3C). In female PXR-KO mice livers, HFD increased the expression of lipid metabolism-related genes Decr2 and Fads1 and the glucose metabolism-related gene Gck (Fig. 3D).
To examine the potential therapeutic significance of hepatic transcriptomic changes following HFD treatment, we compared PXR-dependent gene expression signatures with the Library of Integrated Network-Based Cellular Signatures (LINCS) L1000 database (Fig. 3E). In Fig. 3E (top left), the drugs and endogenous ligands that most closely match the up-regulated signatures for male PXR-dependent changes by HFD are all cancer-related (details described in Table S2-3). In females, PXR dependent HFD-mediated gene expression signatures match the LINCS L1000 database related to innate immunity, self-regulation, and cancer treatment (Table S4-5). In summary, the gene expression signature resembled the ones in LINCS database of inflammation, self-regulation, and cancer treatments. These therapeutic options may be beneficial to mitigate PXR-dependent liver toxicities.
EFFECT OF HFD ON GUT MICROBIOTA DIVERSITY
.Alpha diversity, which is a measure of the richness of the gut microbiome, was determined using the Chao 1 index (QIIME). Only males showed significant differences in species richness of the gut microbiome (alpha diversity) (Fig. S7A). Regarding beta diversity, there was a distinct separation between control and HFD-fed groups in WT males; however, there was minimal separation between control and HFD-fed groups for WT females, also for both sexes of PXR-KO mice (Fig. S7C-F). The top 14 most abundant taxa are shown in Fig. S8A-B. Most of these taxa were altered by diet and genotype, which mostly belong to the Firmicutes phylum.
Notably, in males, PXR was associated with a more stringent separation in the gut microbiome composition between control and HFD treated groups (Fig. 4A), whereas a more diffusive and overlapping pattern was observed between control and HFD fed male PXR-KO mice and female mice of both genotypes. Interestingly, there was a PXR-dependent and male-specific down-regulation of the anti-inflammatory Bifidobacterium genus and in the pro-inflammatory Lactobacillus genus (Fig. 4B), indicating a pro-inflammatory microbial signature that is associated with increased liver inflammation and liver injuries. The anti-obesity marker Allobaculum (35) was down-regulated by HFD in a male-specific PXR-dependent manner, whereas the two pro-inflammatory taxa Ruminococcus gnavus and Peptococcaceae were down-regulated in a PXR-dependent manner in females.
UNIQUE MICROBIAL BIOMARKERS AND EFFECT OF HFD ON FIRMICUTES/BACTEROIDETES (F/B) RATIO
The most prominent microbial biomarkers in each treatment group varied by sex, genotype and diet as follow in Fig. 5A. The F/B ratio, an indicator of obesity (36), was up-regulated by HFD in male (and to a much lesser extent female) WT mice in a PXR-dependent manner (Fig. 5B). Moreover, the F/B ratio remained unchanged by HFD in female PXR-KO mice. Thus, it is possible that PXR exacerbates diet-induced obesity partly through modulation of the gut microbiome.
CORRELATIONS AMONG DIFFERENTIALLY REGULATED INTESTINAL BACTERIA, BONA FIDE PXR-TARGET GENES, AND LIVER BAs FOLLOWING HFD TREATMENT
Pearson’s correlation was performed between differentially regulated intestinal bacteria and PXR-target genes that are differentially regulated by HFD in a PXR-dependent manner (i.e. bona fide PXR-target genes), as well as between differentially regulated bacteria and hepatic BAs (Fig. 6A-D). For bona fide PXR-target genes, we focused on 3 categories, xenobiotic biotransformation (including BA-metabolizing enzymes), energy metabolism, and inflammation.
In general, there were more intestinal bacteria and PXR-target genes associations in male mice than female mice. In male mice, 3 taxa in the Bifidobacterium genus were negatively associated with drug-processing genes and inflammation genes, whereas, 3 taxa in the Lactobacillus genus were positively associated with drug-processing and inflammation genes (Fig. 6A-B).
In male livers, there was a positive association between Allobaculum, S24-7, and Bifidobacterium genus in intestine and most of the liver BAs, except for T-αMCA and T- ßMCA, which were negatively associated (Fig. 6C). Conversely, there was a negative association between Lactobacillus genus and Bacteroides caccae and most of the liver BAs except for LCA which was positively associated (Fig. 6C). In female livers, there was a positive association between Desulfovibrio with most of the liver BAs except for T-αMCA, T- ßMCA, T-LCA, DCA, and LCA, which had negative association (Fig. 6D). However, there was a negative association between Allobaculum with most of the liver BAs except for T-LCA, which had positive association and T-HDCA. Furthermore, for Faecalibacterium prausnitzii, Jeotgalicoccus, Paenibacillus, Parabacteroides distasonis, and Collinsella aerofaciens, negative associations were shown in most of the liver BAs except for T-LCA, which was positively associated (Fig. 6D).