BAR502, a dual FXR and GPBAR1 agonist, promotes browning of white adipose tissue and reverses liver steatosis and fibrosis - PubMed (original) (raw)
BAR502, a dual FXR and GPBAR1 agonist, promotes browning of white adipose tissue and reverses liver steatosis and fibrosis
Adriana Carino et al. Sci Rep. 2017.
Abstract
Non-alcoholic steatohepatitis (NASH) is a highly prevalent chronic liver disease. Here, we have investigated whether BAR502, a non-bile acid, steroidal dual ligand for FXR and GPBAR1, reverses steato-hepatitis in mice fed a high fat diet (HFD) and fructose. After 9 week, mice on HFD gained ≈30% of b.w (P < 0.01 versus naïve) and were insulin resistant. These overweighting and insulin resistant mice were randomized to receive HFD or HFD in combination with BAR502. After 18 weeks, HFD mice developed NASH like features with severe steato-hepatitis and fibrosis, increased hepatic content of triacylglycerol and cholesterol and expression of SREPB1c, FAS, ApoC2, PPARα and γ, α-SMA, α1 collagen and MCP1 mRNAs. Treatment with BAR502 caused a ≈10% reduction of b.w., increased insulin sensitivity and circulating levels of HDL, while reduced steatosis, inflammatory and fibrosis scores and liver expression of SREPB1c, FAS, PPARγ, CD36 and CYP7A1 mRNA. BAR502 increased the expression of SHP and ABCG5 in the liver and SHP, FGF15 and GLP1 in intestine. BAR502 promoted the browning of epWAT and reduced liver fibrosis induced by CCl4. In summary, BAR502, a dual FXR and GPBAR1 agonist, protects against liver damage caused by HFD by promoting the browning of adipose tissue.
Figures
Figure 1. BAR502 reduced body weight gain and reduced hepatic lipid partition in mice fed HFD.
Mice were fed a HFD and fructose for 18 weeks. BAR502 was administered at the dose of 15 mg/kg/day starting on day 63 (week 9). The data shown are (A) body weight (% delta weight); (B) glycemic response to oral glucose tolerance test (OGTT) and to insulin-tolerance test (ITT) (both after 9 weeks of HFD). (C) plasma levels of AST, cholesterol, HDL and triacylglycerols measured at the end of the study. The data shown in Panels (A–C), are mean ± SE of 9 mice. (D) Hematoxylin and eosin (H&E) staining on mice liver tissues showing severe steatosis and ballooning of hepatocytes in mice feed a HFD for 19 weeks. These changes were significantly attenuated by treating the HFD mice with BAR502. The data are mean ± SE of 9 mice. (E) Food intake through the study expressed as mg/week/mouse. The data shown in Panels A–C, are mean ± SE of 9 mice. The data show that while HFD mice had reduced food intake in comparison to mice on standard chow diet, BAR502 did not reduced food intake. Panels F–L. Impact of BAR502 on (F) liver weight, (G) steatosis (Steatosis score) and liver TG and Cholesterol content. (H) Sirius red staining of liver sections; (I) fibrosis score and hepatic expression of αSMA and COL1α1 mRNA; (L) inflammation score and hepatic expression of pro-inflammatory genes. F4/80 is a markers for macrophages. Results are the mean ± SE of 5–9 mice per group. *p < 0.05 versus naive mice, #p < 0.05 versus HFD mice. Values are normalized to B2M and ACTβ, the relative mRNA expression is expressed as 2(−ΔΔCt).
Figure 2. BAR502 represses fatty acid synthesis, activates export of lipids from hepatocytes and activates FXR target genes in the liver.
Relative hepatic mRNA expression of genes involved in (A) Triacylglycerols and fatty acid metabolism, (B) Cholesterol metabolism, and (C) Nuclear receptors. Results are the mean ± SE of 5 mice per group. *p < 0.05 versus naive mice, #p < 0.05 versus HFD mice. Values are normalized to B2M and ACTβ, the relative mRNA expression is expressed as 2(−ΔΔCt).
Figure 3. BAR502 improved morphometry and promotes the browning of epWAT of C57BL6 fed HFD mice.
(A) H&E staining on mice epWAT tissues. BAR502 reduced the size of adipocytes and reduced the extent of inflammatory infiltration; Magnification in 20X; (B) Immunohistochemistry analysis of the expression of uncoupling protein 1 (UCP1) in epWAT of naïve mice and mice on HFD with or without BAR502. Magnification is 20x. Inset: enlargement of the area enclosed by square showing staining of UCP1. Magnification is 40X (C) Effect of BAR502 on epWAT weight. Changes in mRNA expression of epWAT genes. Data shown are relative expression of genes involved in: (D) adipogenesis and fatty acid transport and metabolism; (E) brite/beige trans-differentiation and (F) nuclear receptors. (G) The expression of UCP1 in brown adipose tissue of C57BL6 mice was assayed by immunohistochemistry and used as positive control. (H) Effect of BAR502 on BAT weight. (I) Changes in mRNA expression of BAT marker genes and nuclear receptors. Results are the mean ± SE of 5 mice per group. *p < 0.05 versus naive mice, #p < 0.05 versus HFD mice. Values are normalized to B2M and ACTβ, the relative mRNA expression is expressed as 2(−ΔΔCt).
Figure 4. Effects of BAR502 on muscle and intestine.
Change in transcript levels of genes involved in regulating: (A) muscular energy metabolism, and (B) enteric metabolism. Results are the mean ± SE of 5 mice per group. *p < 0.05 versus naive mice, #p < 0.05 versus HFD mice. Values are normalized to B2M and ACTβ, the relative mRNA expression is expressed as 2(−ΔΔCt).
Figure 5. Effects of BAR502 on glucose and insulin sensitivity.
The data shown are the glycemic response to (A) oral glucose tolerance test (OGTT) and to insulin-tolerance test (ITT) (after 13 weeks of HFD). Mice were fasted by were assuming fructose in drinking water. (B) Insulin plasma levels measure after 18 weeks of HFD. (C). Liver expression of genes involved in glucose metabolism. Results are the mean ± SE of 5 mice per group. *p < 0.05 versus naive mice, #p < 0.05 versus HFD mice. Values are normalized to B2M and ACTβ, the relative mRNA expression is expressed as 2(−ΔΔCt).
Figure 6. BAR502 activates FXR and reduces the synthesis of primary bile acids.
Bile acid pool was assayed by measuring fecal levels of (A) unconjugated (CA, HCA, CDCA, UDCA, CDCA, DCA, LCA) and (B) conjugated (TβMCA, TCA, THCA, TCDCA, TDCA) bile acids. Results are the mean ± SE of 5 mice per group. *p < 0.05 versus naive mice, #p < 0.05 versus HFD mice.
Figure 7. Exposure of 3T3-L1 pre-adipocytes to BAR502 increases Ucp1 expression.
(A) 3T3-L1 pre-adipocytes were differentiated into mature white adipocytes for 4 days and then stimulated 24 hours with 10 μM BAR502. (B) Total RNA was extracted from cells and used to evaluated the relative mRNA expression of adipogenic marker genes, genes involved in brite differentiation and autophagic genes by RT-PCR. Results are the mean ± SE of 2 experiments. *p < 0.05 versus undifferentiated cells (Day 0); #p < 0.05 versus differentiated cells (Day 4). Values are normalized to GAPDH, the relative mRNA expression is expressed as 2(−ΔΔCt). (C) Expression of UCP1 wa also investigated by western blot analysis. Then Western blot shows that exposure of 3T3 –L1 cells to BAR502 increases the expression of UCP1 protein. The blot shown is representative of two others showing the same pattern.
Figure 8. BAR502 protects against liver fibrosis induced by CCl4.
Liver fibrosis was induced by carbon tetrachloride (CCl4) administration (i.p. 500 μL/Kg body weight, twice a week for 8 weeks). CCl4 mice were randomized to receive BAR502 (15 mg/Kg daily by gavage) or vehicle (distilled water). Data shown are (A) Portal pressure, (B) plasmatic levels of AST, Bilirubin and Albumin. (C) Hematoxylin and eosin (H&E) staining and (D) Sirius red staining on mice liver tissues; (E) Image J quantification of Sirius red staining. Total RNA extracted from liver was used to evaluate by RT-PCR the relative mRNA expression of (F) marker genes of fibrosis, (G) genes involved in endothelial function and (H) Nuclear Receptor genes. Results are the mean ± SE of 6–12 mice per group. *p < 0.05 versus naive mice. #p < 0.05 versus CCl4 alone.
Figure 9. BAR502 modulates bile acid pool in CCl4 mice.
(A,B) Gallbladder levels of unconjugated (CA, HCA, CDCA, UDCA, CDCA, DCA, LCA) and conjugated (TβMCA, TCA, THCA, TCDCA, TDCA) bile acids are shown. Results are the mean ± SE of 6–12 mice per group. *p < 0.05 versus naive mice. #p < 0.05 versus CCl4 alone.
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