Transcriptome Analysis of Dual FXR and GPBAR1 Agonism in Rodent Model of NASH Reveals Modulation of Lipid Droplets Formation

Figures

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  Figure 1. BAR502 reduces body weight gain, ameliorated insulin resistance and biochemical profile in mice fed a high fat diet and fructose (HFD–F). BAR502, 20 mg/kg/day, was administered by gavage in C57BL/6J male mice starting on day 10 of HFD and fructose for an additional 11 weeks. The data shown are: (A) % of body weight; (B) Food intake (g/day); (C) areas under curve (AUCs) of body weight expressed in arbitrary units; Anthropometrical parameters (D) body mass index (BMI) and (E) Lee Index, measured at the end of the study; (F) Glucose plasma levels response to oral glucose tolerance test (OGTT) and (G) AUCs of glucose plasma levels expressed in arbitrary units; (H) Plasma levels of aspartate transaminase (AST), alanine transaminase (ALT), Triglycerides, Cholesterol, and HDL and measured at the end of the study. The data shown are mean ± SE of 7–10 mice/group. In each panel # denotes p < 0.05 versus Naïve mice, * denotes p < 0.05 versus HFD–F mice.

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  Figure 2. BAR502 attenuated non-alcoholic steatohepatitis (NASH) like features and redirect lipid partition in mice fed HFD–F. BAR502, 20 mg/kg/day, was administered by gavage in C57BL/6J male mice starting on day 10 of high fat diet (HFD) and fructose for additional 11 weeks. (A) Hematoxylin and eosin (H&E) staining on mice liver tissues showing severe steatosis and ballooning of hepatocytes in mice feed a HFD–F for 12 weeks, magnification 10×; (B) NAS activity score assessed in at least 5 different fields per liver in a blind manner; (C) Liver weight expressed as ratio Liver weight to Body weight. Panels D–F: Analysis of hepatic gene expression conducted with RT-PCR. Liver expression of (D) markers of inflammation (Il-6, Tnfα, Il-1β, F4/80), (E) genes involved in bile acids metabolism, (F) genes involved in lipid synthesis. The RT-PCR values were normalized against β2-Microglobulin and Gapdh, and the relative mRNA is expressed according the Ct method as described in Materials and Methods. The data shown are mean ± SEM of 7–10 mice/group. In each panel # denotes p < 0.05 versus Naïve mice, * denotes p < 0.05 versus HFD–F mice.

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  Figure 3. BAR502 ameliorated Fibrosis caused by HFD–F administration. BAR502, 20 mg/kg/day, was administered by gavage in C57BL/6J male mice starting on day 10 of HFD and fructose for an additional 11 weeks. (A) Sirius Red staining of liver sections. The images shown are representative of at least 10 others, each one obtained from an individual mouse, showing a similar pattern of regulation. Magnification 10×. (B) % of Collagen Deposition (Liver Fibrosis Score) was measured as described in Material and Methods by Image J analysis. (C) Hepatic expression of pro-fibrotic genes Tgfβ, Col1α1, and αSma. The RT-PCR values were normalized against β2-Microglobulin and Gapdh, and the relative mRNA is expressed according the Ct method as described in Materials and Methods. The data shown are mean ± SEM of 7–10 mice/group. In each panel # denotes p < 0.05 versus Naïve mice, * denotes p < 0.05 versus HFD–F mice.

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  Figure 4. RNA-seq of BAR502 treated mice. BAR502, 20 mg/kg/day, was administered by gavage in C57BL/6J male mice starting on day 10 of high fat diet (HFD) and fructose for an additional 11 weeks. (A) Venn diagram of differentially expressed genes showing the overlapping regions (identified as ABC, AC, AB, and BC sets) between the three experimental groups of mice (Fold Change <−2 or >2, p value < 0.05). (B) Table showing the fold change of expression of genes included in region C, AC, and ABC of the corresponding Venn Diagram. Panels C,D. Analysis of genes involved in triglycerides synthesis and lipid storage into the hepatocytes, that were found differentially expressed both in HFD–F mice compared to Naïve mice, both in HFD–F + BAR502 and HFD–F + UDCA compared to HFD–F mice. In each panel # denotes p < 0.05 versus Naïve mice, * denotes p < 0.05 versus HFD–F mice.

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  Figure 5. Effects of HFD–F on adipose tissue and ileum. BAR502, 20 mg/kg/day, was administered by gavage in C57BL/6J male mice starting on day 10 of HFD and fructose for additional 11 weeks. Panels A,B. Analysis of epididymal White Adipose Tissue (eWAT). (A) Ratio of eWAT weight to body weight. (B) Changes in mRNA expression of genes involved in: lipid synthesis and metabolism (Srebf1, Fasn), brite/beige trans-differentiation and β-oxidation (Prdm16, Ucp1, Pparγ, Pgc1α). Panels C–E. Analysis of Brown Adipose Tissue (BAT). (C) Ratio of BAT weight to body weight. (D) Thermogenic activity of BAT as measured by infrared spectroscopy. (E) Physical activity of mice expressed as Global activity and distance. The data showed in Panels D–E are recorded after 10 weeks of HFD–F. (F) Change in transcript levels of genes involved in regulation of intestinal metabolism (Shp, Fgf15, Glp1). The RT-PCR values were normalized against β2-Microglobulin and Gapdh, and the relative mRNA is expressed according the Ct method as described in Materials and Methods. The data shown are mean ± SEM of 7–10 mice/group. In each panel # denotes p < 0.05 versus Naïve mice, * denotes p < 0.05 versus HFD–F mice.

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  Figure 6. Analysis of gallbladder and fecal BAs composition. BAR502, 20 mg/kg/day, was administered by gavage in C57BL/6J male mice starting on day 10 of HFD and fructose for an additional 11 weeks. (A) Total concentrations of gallbladder bile acids. (B) Composition % of gallbladder bile acids pool in NT, HFD–F, and HFD–F + BAR502 mice. (C) Ratio tauro- cholic acid (T-CA)/tauro-beta muricholic acid (T-βMCA 0in gallbladder samples. (D) Composition of fecal bile acids pool. Data are the mean ± SEM of 7–10 mice per group. In each panel # denotes p < 0.05 versus Naïve mice, * denotes p < 0.05 versus HFD–F mice.

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  Figure 7. Analysis of fecal microbiota composition BAR502, 20 mg/kg/day, was administered by gavage in C57BL/6J male mice starting on day 10 of HFD and fructose for an additional 11 weeks. (A) Relative abundance of Phyla, calculated as percent of Mapped Reads. (B) Relative abundance of bacterial Families, calculated as percent of Mapped Reads. (C) Quantitative β analysis of PCoA conducted by family (Bray-Curtis analysis), that showed a major dissimilarity between untreated mice and mice under HFD–F.

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  Figure 8. Schematic representation of the transcriptional regulation and role of CIDE pathway in controlling hepatic lipid metabolism (red and green arrows indicate respectively up- and down- regulated genes) Under HFD–F treatment the gene expression of protein involved in triglycerides synthesis and storage, resulted increased. Particularly the HFD–F via Srebf1 activation, promoted the transcription of lipidogenic genes, such as Fasn and Cidea. BAR502 activates FXR thus suppressing a cluster of genes downstream to Srebf1 including fatty acid synthase (Fasn) and Cell death-inducing DFF45-like effector (CIDE) genes, Cidea and Cidec, involved in lipid droplets formation and triglycerides storage.