Gpbar1 agonism promotes a Pgc-1α-dependent browning of white adipose tissue and energy expenditure and reverses diet-induced steatohepatitis in mice - PubMed (original) (raw)
Gpbar1 agonism promotes a Pgc-1α-dependent browning of white adipose tissue and energy expenditure and reverses diet-induced steatohepatitis in mice
Adriana Carino et al. Sci Rep. 2017.
Abstract
Gpbar1 is a bile acid activated receptor for secondary bile acids. Here we have investigated the mechanistic role of Gpbar1 in the regulation of adipose tissues functionality in a murine model of steatohepatitis (NASH). Feeding wild type and Gpbar1-/- mice with a high fat diet-fructose (HFD-F) lead to development of NASH-like features. Treating HFD-F mice with 6β-ethyl-3a,7b-dihydroxy-5b-cholan-24-ol (BAR501), a selective Gpbar1-ligand, reversed insulin resistance and histologic features of NASH, increased the weight of epWAT and BAT functionality and promoted energy expenditure and the browning of epWAT as assessed by measuring expression of Ucp1 and Pgc-1α. The beneficial effects of BAR501 were lost in Gpbar1-/- mice. In vitro, BAR501 promoted the browning of 3T3-L1 cells a pre-adipocyte cell line and recruitment of CREB to the promoter of Pgc-1α. In conclusion, Gpbar1 agonism ameliorates liver histology in a rodent model of NASH and promotes the browning of white adipose tissue.
Conflict of interest statement
The study was partially supported by a research contribution by BAR Pharmaceuticals (Italy) to the Department of Surgical and Biomedical Sciences, University of Perugia, and Department of Pharmacy, University of Naples.
Figures
Figure 1
Gpbar1+/+ and Gpbar1−/− mice feed HFD-F for 18 weeks develop a similar metabolic phenotype, insulin resistance and NASH-like phenotype. Wild type and Gpbar1−/− mice were fed a HFD-F for 18 weeks. The data shown are: (A) Body weight (% delta weight) and glucose plasma levels response to oral glucose tolerance test (OGTT) carried out after 18 weeks of HFD-F; (B) Motor activity (Global activity and Total distance); (C) Plasma levels of AST, cholesterol, HDL, triacylglycerols and serum Bile Acids measured at the end of the study. (D–F) Liver histopathology obtained after feeding mice a HFD-F diet for 18 weeks. Liver sections where stained with Hematoxylin and eosin (H and E) staining (D), or (E) Sirius red. (F) Steatosis score and Inflammation score; (G) Fibrosis score and liver mRNA expression of pro-fibrotic genes. Values are normalized to β2-Microglobulin and Actin-β, the relative mRNA expression is expressed as 2(−ΔΔCt) as described in Materials and Methods. Results are the mean ± SE of 5–9 mice per group. *p < 0.05 versus Gpbar1+/+ naïve mice, #p < 0.05 versus Gpbar1−/− naïve mice.
Figure 2
Feeding Gpbar1+/+ and Gpbar1−/− mice with a HFD-F diet for 18 weeks results in a similar liver metabolic phenotype. Hepatic expression of genes involved in: (A) Triglycerides and fatty acids metabolism; (B) Cholesterol metabolism; (C) Nuclear receptors; (D) Glucose metabolism. Data are the mean ± SE of 5 mice per group. *p < 0.05 versus Gpbar1+/+ naïve mice, #p < 0.05 versus Gpbar1−/− naïve mice. Values are normalized to β2-Microglobulin and Actin-β, the relative mRNA expression is expressed as 2(−ΔΔCt) as described in Materials and Methods.
Figure 3
Feeding a HFD-F differentially modulates adipose tissues, intestinal and muscle metabolism in Gpbar1+/+ and Gpbar1−/− mice. Panels A-C: mRNA expression of epWAT genes. Data shown are relative mRNA expression of genes involved in: (A) Adipogenesis and fatty acids transport and metabolism; (B) Brite/beige transdifferentiation; and (C) Nuclear receptors. Panels D–F: Effects of HFD administration on mRNA expression in the: (D) BAT, (E) Muscle (gastrocnemius), and (F) Terminal ileum. Data are the mean ± SE of 5 mice per group. *p < 0.05 versus Gpbar1+/+ naïve mice, #p < 0.05 versus Gpbar1−/− naïve mice. Values are normalized to β2-Microglobulin and Actin-β, the relative mRNA expression is expressed as 2(−ΔΔCt) as described in Materials and Methods.
Figure 4
Treating wild type mice with a Gpbar1 ligand, BAR501, reverses NASH like features and redirect lipid partition in mice fed HFD-F. BAR501, 15 mg/kg/day, was administered by gavage starting on day 63 (week 9) for additional 9 weeks. The data shown are: (A) body weight (% weight gain and grams); (B) Glucose plasma levels response to oral glucose tolerance test (OGTT) and to insulin-tolerance test (ITT) at the 18th week of HFD-F. (C) Plasma levels of AST, cholesterol, HDL and triacylglycerols measured at the end of the study; liver body weight ratio. The data shown in Panels A–C, are mean ± SE of 9 mice. (D) Hematoxylin and eosin (H and E) staining and Sirius Red staining of liver tissues obtained at the end of the study (19 weeks of HFD-F). The data are mean ± SE of 9 mice. Panels E–I. Effects of BAR501 on: (E) Steatosis (steatosis score); (F) inflammation (inflammation score) and (G) hepatic expression of pro-inflammatory genes; (H) Fibrosis score and (I) hepatic expression of αSMA and Col1α1 mRNA. Data are the mean ± SE of 5–9 mice per group. *p < 0.05 versus Gpbar1+/+ naïve mice, #p < 0.05 versus Gpbar1+/+ HFD-F mice. Values are normalized to β2-Microglobulin and Actin-β, the relative mRNA expression is expressed as 2(−ΔΔCt) as described in Materials and Methods.
Figure 5
Effects of BAR501 on liver, muscle and intestinal metabolism. Change in transcript levels of genes involved in: (A) Liver triacylglycerols and fatty acids metabolism, (B) Liver cholesterol metabolism, (C) Liver nuclear receptors, (D) Liver glucose metabolism and (E) Liver Bile Acids transport and metabolism. Change in transcript levels of genes regulating: (F) Intestinal metabolism; and (G) Muscular metabolism. Data are the mean ± SE of 5 mice per group. *p < 0.05 versus Gpbar1+/+ naïve mice, #p < 0.05 versus Gpbar1+/+ HFD-F mice. Values are normalized to β2-Microglobulin and Actin-β, the relative mRNA expression is expressed as 2(−ΔΔCt) as described in Materials and Methods.
Figure 6
Gpbar1 agonism by BAR501 promotes the browning of epWAT. (A) epWAT weight (mg) and epWAT/Body weight ratio (%) obtained from experimental groups after 19th weeks of feeding with a HFD-F alone or in combination with BAR501, 15 mg/kg/day. *p < 0.05 versus Gpbar1+/+ naïve mice, # p < 0.05 versus Gpbar1+/+HFD-F, n = 6–11 mice. (B) H&E staining on epWAT. Magnification is 20X; (C) Immunohistochemistry analysis of UCP1 protein in epWAT. Magnification is 20x. (D) Effects of BAR501 on mRNA expression of epWAT genes: (D) Adipogenesis and fatty acids transport and metabolism; (E) Brite/beige transdifferentiation; and (F) Nuclear receptors. Results are the mean ± SE of 5 mice per group. *p < 0.05 versus Gpbar1+/+ naïve mice, #p < 0.05 versus Gpbar1+/+HFD-F mice. Values are normalized to β2-Microglobulin and Actin-β, the relative mRNA expression is expressed as 2(−ΔΔCt) as described in Materials and Methods.
Figure 7
Gpbar1 agonism by BAR501 increases the weight and improves the functionality of BAT. (A) BAT weight (mg) and BAT/Body weight ratio (%) obtained from experimental groups after 19 weeks of feeding with a HFD-F alone or in combination with BAR501, 15 mg/kg/day. # < 0.05 versus Gpbar1+/+HFD-F, n = 6–11 mice. (B and C) BAT temperature measured by infrared images of interscapular area after 8 weeks treatment with BAR501. Data are mean ± SE of 6–11 mice per group. *p < 0.05 versus Gpbar1+/+ naïve mice, #p < 0.05 versus Gpbar1+/+HFD-F mice. (D) Effects of BAR501 on mRNA expression of BAT genes. Results are the mean ± SE of 5 mice per group. *p < 0.05 versus Gpbar1+/+ naïve mice, #p < 0.05 versus Gpbar1+/+ HFD-F mice. Values are normalized to β2-Microglobulin and Actin-β, the relative mRNA expression is expressed as 2(−ΔΔCt) as described in Materials and Methods.
Figure 8
Effect of BAR501 on gallbladder volume and gallbladder bile acids composition in mice feed HFD-F. (A) Gallbladder weight and (B) bile acid species. Data are the mean ± SE of 6 mice per group. *p < 0.05 versus Gpbar1+/+ naïve mice, #p < 0.05 versus Gpbar1+/+HFD-F mice.
Figure 9
Exposure of 3T3-L1 cells to BAR501 induces the brite transdifferentiation. (A) 3T3-L1 were differentiated for 8 days and then exposed for 24 hours to 10 μM (Supplementary Figure 2) or 50 µM BAR501. Total RNA was extracted from cells and used to evaluate the relative mRNA expression of adipogenic marker genes and genes involved in brite differentiation. Results are the mean ± SE of 2 experiments carried out in triplicate. *p < 0.05 versus undifferentiated cells (Day 0); #p < 0.05 versus differentiated cells (Day 8). Values are normalized to Gapdh, the relative mRNA expression is expressed as 2(−ΔΔCt). Brite transdifferentiation was detected by a UCP1 staining on 3T3-L1 cells. (B) Immunofluorescence revealed that cells treated with BAR501 (50 µM), showed an enhanced Ucp1 staining compared both with undifferentiated and differentiated cells. Magnification 63x. (C) Chromatin immunoprecipitation with anti-pCREB antibody on 3T3-L1 cells differentiated 8 days and stimulated with 50 µM BAR501 for 18 h. Real-Time PCR was performed on Pgc-1α murine promoter (proximal, distal and control regions) to demonstrate the recruitment of the transcription factor pCREB. Precipitation with an unrelated antibody (anti-IgG) was used as negative control. Values are normalized with Fold Enrichment Method and expressed as 2(−ΔΔCt). *p < 0.05 versus Negative Control (anti-IgG).
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