SUMOylation inhibitors synergize with FXR agonists in combating liver fibrosis.
ABSTRACT: Farnesoid X receptor (FXR) is a promising target for nonalcoholic steatohepatitis (NASH) and fibrosis. Although various FXR agonists have shown anti-fibrotic effects in diverse preclinical animal models, the response rate and efficacies in clinical trials were not optimum. Here we report that prophylactic but not therapeutic administration of obeticholic acid (OCA) prevents hepatic stellate cell (HSC) activation and fibrogenesis. Activated HSCs show limited response to OCA and other FXR agonists due to enhanced FXR SUMOylation. SUMOylation inhibitors rescue FXR signaling and thereby increasing the efficacy of OCA against HSC activation and fibrosis. FXR upregulates Perilipin-1, a direct target gene of FXR, to stabilize lipid droplets and thereby prevent HSC activation. Therapeutic coadministration of OCA and SUMOylation inhibitors drastically impedes liver fibrosis induced by CCl4, bile duct ligation, and more importantly NASH. In conclusion, we propose a promising therapeutic approach by combining SUMOylation inhibitors and FXR agonists for liver fibrosis.
Project description:Accumulating evidence has suggested that farnesoid X receptor (FXR) agonists, such as obeticholic acid (OCA) are therapeutically useful for non-alcoholic steatohepatitis (NASH). However, it is still unclear how FXR agonists protect against NASH and which cell type is the main target of FXR agonists. In this study, we examined the effects of OCA on the development of NASH using melanocortin 4 receptor-deficient (MC4R-KO) mice that progressively developed hepatic steatosis and NASH on Western diet (WD). Treatment with OCA effectively prevented chronic inflammation and liver fibrosis in WD-fed MC4R-KO mice with only marginal effect on body weight and hepatic steatosis. Hepatic crown-like structure (hCLS) is a unique histological structure characteristic of NASH, which triggers hepatocyte death-induced interstitial fibrosis. Intriguingly, treatment with OCA markedly reduced hCLS formation even after MC4R-KO mice developed NASH, thereby inhibiting the progression of liver fibrosis. As its mechanism of action, OCA suppressed metabolic stress-induced p53 activation and cell death in hepatocytes. Our findings in this study highlight the role of FXR in hepatocytes in the pathogenesis of NASH. Collectively, this study demonstrates the anti-fibrotic effect of OCA in a murine model of NASH with obesity and insulin resistance, which suggests the clinical implication for human NASH.
Project description:OBJECTIVE:Nonalcoholic steatohepatitis (NASH) is an unmet need associated with metabolic syndrome. There are no approved therapies for NASH; however, glucagon-like peptide-1 receptor (GLP-1R) and farnesoid-X receptor (FXR) agonists are promising drug targets. We investigated the therapeutic effects of co-administration of a GLP-1R agonist, IP118, with FXR agonist obeticholic acid (OCA) in mice. METHODS:OCA and IP118 alone and in combination were sub-chronically administered to Lepob/Lepob mice with diet-induced NASH or diet-induced obese (DIO) mice. Metabolic (body weight and glucose) and liver (biochemical and histological) endpoints were assessed. NASH severity in Lepob/Lepob mice was graded using a customized integrated scoring system. RESULTS:OCA reduced liver weight and lipid in NASH mice (both by -17%) but had no effect on plasma ALT or AST levels. In contrast, IP118 significantly reduced liver weight (-21%), liver lipid (-15%), ALT (-29%), and AST (-27%). The combination of OCA + IP118 further reduced liver weight (-29%), liver lipid (-22%), ALT (-39%), and AST (-36%). Combination therapy was superior to monotherapies in reducing hepatic steatosis, inflammation, and fibrosis. Hepatic improvements with IP118 and OCA + IP118 were associated with reduced body weight (-4.3% and -3.5% respectively) and improved glycemic control in OCA + IP118-treated mice. In DIO mice, OCA + IP118 co-administration reduced body weight (-25.3%) to a greater degree than IP118 alone (-12.5%) and further improved glucose tolerance and reduced hepatic lipid. CONCLUSION:Our data suggest a complementary or synergistic therapeutic effect of GLP-1R and FXR agonism in mouse models of metabolic disease and NASH.
Project description:Hepatic inflammation drives hepatic stellate cells (HSC), resulting in liver fibrosis. The Farnesoid-X receptor (FXR) antagonizes inflammation through NF-?B inhibition. We investigated preventive and therapeutic effects of FXR agonist obeticholic acid (OCA) on hepatic inflammation and fibrosis in toxic cirrhotic rats. Cirrhosis was induced by thioacetamide (TAA) intoxication. OCA was given during or after intoxication with vehicle-treated rats as controls. At sacrifice, fibrosis, hemodynamic and biochemical parameters were assessed. HSC activation, cell turn-over, hepatic NF-?B activation, pro-inflammatory and pro-fibrotic cytokines were determined. The effect of OCA was further evaluated in isolated HSC, Kupffer cells, hepatocytes and liver sinusoidal endothelial cells (LSEC). OCA decreased hepatic inflammation and fibrogenesis during TAA-administration and reversed fibrosis in established cirrhosis. Portal pressure decreased through reduced intrahepatic vascular resistance. This was paralleled by decreased expression of pro-fibrotic cytokines (transforming growth-factor ?, connective tissue growth factor, platelet-derived growth factor ?-receptor) as well as markers of hepatic cell turn-over, by blunting effects of pro-inflammatory cytokines (e.g. monocyte chemo-attractant protein-1). In vitro, OCA inhibited both LSEC and Kupffer cell activation; while HSC remained unaffected. This related to NF-?B inhibition via up-regulated I?B?. In conclusion, OCA inhibits hepatic inflammation in toxic cirrhotic rats resulting in decreased HSC activation and fibrosis.
Project description:Farnesoid X receptor (FXR) agonism is emerging as an important potential therapeutic mechanism of action for multiple chronic liver diseases. The bile acid-derived FXR agonist obeticholic acid (OCA) has shown promise in a phase 2 study in patients with nonalcoholic steatohepatitis (NASH). Here, we report efficacy of the novel nonbile acid FXR agonist tropifexor (LJN452) in two distinct preclinical models of NASH. The efficacy of tropifexor at <1 mg/kg doses was superior to that of OCA at 25 mg/kg in the liver in both NASH models. In a chemical and dietary model of NASH (Stelic animal model [STAM]), tropifexor reversed established fibrosis and reduced the nonalcoholic fatty liver disease activity score and hepatic triglycerides. In an insulin-resistant obese NASH model (amylin liver NASH model [AMLN]), tropifexor markedly reduced steatohepatitis, fibrosis, and profibrogenic gene expression. Transcriptome analysis of livers from AMLN mice revealed 461 differentially expressed genes following tropifexor treatment that included a combination of signatures associated with reduction of oxidative stress, fibrogenesis, and inflammation. Conclusion: Based on preclinical validation in animal models, tropifexor is a promising investigational therapy that is currently under phase 2 development for NASH.
Project description:Obeticholic acid (OCA) and elafibranor (ELA) are selective and potent agonists for the farnesoid X receptor (FXR) and dual peroxisome proliferator-activated receptor ?/? (PPAR-?/?), respectively. Both agents have demonstrated clinical efficacy in nonalcoholic steatohepatitis (NASH). The present study used OCA and ELA to compare the effects of mono- and combination therapies on metabolic and histological endpoints in Lepob/ob mice with established diet-induced and biopsy-confirmed NASH (ob/ob-NASH). ob/ob-NASH mice were fed the AMLN diet high in trans-fat, fructose and cholesterol for 15 weeks, whereafter they received vehicle, OCA (30?mg/kg, PO, QD), ELA (3, 10?mg/kg, PO, QD), or combinations (OCA?+?ELA) for eight weeks. Within-subject comparisons were performed on histomorphometric changes, including fractional area of liver fat, galectin-3 and Col1a1. OCA and ELA monotherapies improved all quantitative histopathological parameters and OCA?+?ELA combinations exerted additive effects on metabolic and histological endpoints. In agreement with their different molecular mechanisms of action, OCA and ELA monotherapies elicited distinct hepatic gene expression profiles and their combination led to profound transcriptome changes associated with further improvements in lipid handling and insulin signaling, suppression of immune responses and reduced extracellular matrix formation. In conclusion, these findings provide preclinical proof-of-concept for combined FXR and PPAR-?/? agonist-based therapies in NASH.
Project description:Obeticholic acid (OCA), the first FXR-targeting drug, has been claimed effective in the therapy of liver fibrosis. However, recent clinical trials indicated that OCA might not be effective against liver fibrosis, possibly due to the lower dosage to reduce the incidence of the side-effect of pruritus. Here we propose a combinatory therapeutic strategy of OCA and apoptosis inhibitor for combating against liver fibrosis. CCl<sub>4</sub>-injured mice, d-galactosamine/LPS (GalN/LPS)-treated mice and cycloheximide/TNF<i>?</i> (CHX/TNF<i>?</i>)-treated HepG2 cells were employed to assess the effects of OCA, or together with IDN-6556, an apoptosis inhibitor. OCA treatment significantly inhibited hepatic stellate cell (HSC) activation/proliferation and prevented fibrosis. Elevated bile acid (BA) levels and hepatocyte apoptosis triggered the activation and proliferation of HSCs. OCA treatment reduced BA levels but could not inhibit hepatocellular apoptosis. An enhanced anti-fibrotic effect was observed when OCA was co-administrated with IDN-6556. Our study demonstrated that OCA inhibits HSCs activation/proliferation partially by regulating BA homeostasis and thereby inhibiting activation of HSCs. The findings in this study suggest that combined use of apoptosis inhibitor and OCA at lower dosage represents a novel therapeutic strategy for liver fibrosis.
Project description:Bile acids (BAs) activate various dedicated receptors, including the farnesoid X receptor (FXR) and the Takeda G protein-coupled receptor 5 (TGR5). The FXR agonist obeticholic acid (OCA) is licensed for the treatment of primary biliary cholangitis and has shown promising results in NASH patients, whereas TGR5 agonists target inflammation and metabolism. We hypothesized that FXR and/or TGR5 agonists may be therapeutic in early alcoholic liver disease (ALD) in mice, in which hepatic inflammation plays a major role. OCA, INT-777, and INT-767 are BA derivatives with selective agonist properties for FXR, TGR5, or both, respectively. These compounds were tested in two mouse models (3-day binge model and prolonged Lieber DeCarli diet for 12 days) of early ALD. Serum alanine aminotransferase and liver histology were used to assess liver injury, Oil Red O staining of liver sections to assess steatosis, and real-time polymerase chain reaction to assess changes in gene expression. In the ethanol binge model, treatment with OCA and INT-777 decreased hepatic macrovesicular steatosis and protected from ethanol-induced liver injury. After prolonged ethanol administration, mice treated with OCA, INT-767, or INT-777 showed decreased hepatic steatosis, associated with reduced liver fatty acid synthase protein expression, and protection from liver injury. Treatment with BA receptor agonists in both models of ethanol administration modulated lipogenic gene expression, and decreased liver interleukin-1? mRNA expression associated with increased ubiquitination of NLRP3 inflammasome through cyclic adenosine monophosphate-induced activation of protein kinase A. Conclusion: OCA, INT-767, or INT-777 administration is effective in reducing acute and chronic ethanol-induced steatosis and inflammation in mice, with varying degrees of efficacy depending on the duration of ethanol administration, indicating that both FXR and TGR5 activation can protect from liver injury in ALD models.
Project description:Pharmacological treatments for non-alcoholic steatohepatitis (NASH) are still unsatisfactory. Fibrosis is the most significant predictor of mortality and many anti-fibrotic agents are under evaluation. Herein, we assessed in vitro the effects of the FXR agonist obeticholic acid (OCA) and the dual FXR/TGR5 agonist INT-767 in a well-established co-culture NASH model. Co-cultures of human hepatoma and hepatic stellate (HSCs) cells were exposed to free fatty acids (FFAs) alone or in combination with OCA or INT-767. mRNA expression of HSCs activation markers and FXR engagement were evaluated at 24, 96 and 144?hours. Collagen deposition and metalloproteinase 2 and 9 (MMP2-9) activity were compared to tropifexor and selonsertib. FFAs induced collagen deposition and MMP2-9 activity reduction. Co-treatment with OCA or INT-767 did not affect ACTA2 and COL1A1 expression, but significantly reduced FXR and induced SHP expression, as expected. OCA induced a dose-dependent reduction of collagen and induced MMP2-9 activity. Similarly, INT-767 induced collagen reduction at 96?h and a slight increase in MMP2-9. Tropifexor and Selonsertib were also effective in collagen reduction but showed no modulation of MMP2-9. All tested compounds reduced collagen deposition. OCA exerted a more potent and long-lasting effect, mainly related to modulation of collagen turn-over and MMP2-9 activity.
Project description:The main treatments for patients with nonalcoholic fatty liver disease (NAFLD) are currently based on lifestyle changes, including ponderal decrease and dietary management. However, a subgroup of patients with nonalcoholic steatohepatitis (NASH), who are unable to modify their lifestyle successfully, may benefit from pharmaceutical support. Several drugs targeting pathogenic mechanisms of NAFLD have been evaluated in clinical trials for the treatment of NASH. Farnesoid X receptor (FXR) is a nuclear key regulator controlling several processes of the hepatic metabolism. NAFLD has been proven to be associated with abnormal FXR activity. Obeticholic acid (OCA) is a first-in-class selective FXR agonist with anticholestatic and hepato-protective properties. Currently, OCA is registered for the treatment of primary biliary cholangitis. However, promising effects of OCA on NASH and its metabolic features have been reported in several studies.
Project description:microRNAs were recently suggested to contribute to the pathogenesis of nonalcoholic fatty liver disease (NAFLD), a disease lacking specific pharmacological treatments. In that regard, nuclear receptors are arising as key molecular targets for the treatment of nonalcoholic steatohepatitis (NASH). Here we show that, in a typical model of NASH-associated liver damage, microRNA-21 (miR-21) ablation results in a progressive decrease in steatosis, inflammation and lipoapoptosis, with impairment of fibrosis. In a complementary fast food (FF) diet NASH model, mimicking features of the metabolic syndrome, miR-21 levels increase in both liver and muscle, concomitantly with decreased expression of peroxisome proliferator-activated receptor ? (PPAR?), a key miR-21 target. Strikingly, miR-21 knockout mice fed the FF diet supplemented with farnesoid X receptor (FXR) agonist obeticholic acid (OCA) display minimal steatosis, inflammation, oxidative stress and cholesterol accumulation. In addition, lipoprotein metabolism was restored, including decreased fatty acid uptake and polyunsaturation, and liver and muscle insulin sensitivity fully reinstated. Finally, the miR-21/PPAR? axis was found amplified in liver and muscle biopsies, and in serum, of NAFLD patients, co-substantiating its role in the development of the metabolic syndrome. By unveiling that miR-21 abrogation, together with FXR activation by OCA, significantly improves whole body metabolic parameters in NASH, our results highlight the therapeutic potential of nuclear receptor multi-targeting therapies for NAFLD.