FXR Activation Research Articles

Western Diet Deregulates Bile Acid Homeostasis, Cell Proliferation, and Tumorigenesis in Colon.

Western-style diets (WD) high in fat and scarce in fiber and vitamin D increase risks of colorectal cancer. Here, we performed a long-term diet study in mice to follow tumorigenesis and characterize structural and metabolic changes in colon mucosa associated with WD and predisposition to colorectal cancer. WD increased colon tumor numbers, and mucosa proteomic analysis indicated severe deregulation of intracellular bile acid (BA) homeostasis and activation of cell proliferation. WD also increased crypt depth and colon cell proliferation. Despite increased luminal BA, colonocytes from WD-fed mice exhibited decreased expression of the BA transporters FABP6, OSTβ, and ASBT and decreased concentrations of secondary BA deoxycholic acid and lithocholic acid, indicating reduced activity of the nuclear BA receptor FXR. Overall, our results suggest that WD increases cancer risk by FXR inactivation, leading to BA deregulation and increased colon cell proliferation. Cancer Res; 77(12); 3352-63. ©2017 AACR.

Abstract 402: Post-transcriptional Regulation of Bile Acid Metabolism by the RNA binding protein ZFP36L1

Bile acids are detergents and important signaling molecules that activate the nuclear receptor FXR to control key metabolic processes, including feedback mechanisms to maintain bile acid homeostasis. Activation of FXR decreases the mRNA levels of several bile acid synthetic genes, including the rate-limiting enzyme Cyp7a1 . Here we show that Cyp7a1 mRNA levels are very rapidly reduced following FXR activation, indicative of a post-transcriptional mechanism. We identify the RNA binding protein Zfp36l1 as an FXR target gene and show that hepatic overexpression of ZFP36L1 in mice decreases Cyp7a1 mRNA levels. In contrast, Zfp36l1 L -KO mice have increased levels of Cyp7a1 mRNA and biliary bile acids as well as reduced plasma cholesterol levels. Zfp36l1 L -KO mice fed a Western diet have reduced diet-induced obesity and steatosis, likely due to impaired lipid absorption, consistent with increased Cyp7a1 levels. Thus, the ZFP36L1-dependent regulation of bile acid metabolism is an important metabolic contributor of dyslipidemia, obesity and hepatosteatosis.

Mo1373 - Evidence of FXR Activation with Obeticholic Acid in an in Vitro Intestinal Model

Mo1373 - Evidence of FXR Activation with Obeticholic Acid in an in Vitro Intestinal Model

Activation of FXR pathway does not alter glial cell function

BackgroundThe nuclear receptor farnesoid-X-receptor (FXR; NR1H4) is expressed not only in the liver, gut, kidney and adipose tissue but also in the immune cells. FXR has been shown to confer protection in several animal models of inflammation, including experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). FXR agonists are currently tested in clinical trials for treatment of human metabolic diseases. The beneficial effect of FXR agonists in EAE suggests that FXR might represent a potential target in inflammatory-demyelinating CNS diseases, such as MS. In MS, oligodendrocytes not only undergo cell death but also contribute to remyelination. This repair mechanism is impaired due to a differentiation block of oligodendroglial progenitor cells. Activation of other nuclear receptors that heterodimerize with FXR promote oligodendroglial differentiation. Therefore, we wanted to address the functional relevance of FXR for glial cells, especially for oligodendroglial differentiation.MethodsWe isolated primary murine oligodendrocytes from FXR-deficient (FXR Ko) and wild-type (WT) mice and determined the effect of FXR deficiency and activation on oligodendroglial differentiation by analysing markers of oligodendroglial progenitor cells (OPCs) and mature oligodendrocytes (OLs) using qRT-PCR and immunocytochemistry. Additionally, we determined whether FXR activation modulates the pro-inflammatory profile of astrocytes or microglia and whether this may subsequently modulate oligodendroglial differentiation. These in vitro studies were complemented by histological analyses of oligodendrocytes in FXR Ko mice.ResultsFXR is expressed by OPCs and mature oligodendrocytes. However, lack of FXR did not affect oligodendroglial differentiation in vitro or in vivo. Furthermore, activation of FXR using the synthetic agonist GW4064 did not affect oligodendroglial differentiation, remyelination in an ex vivo model or the expression of pro-inflammatory molecules in astrocytes or microglia. Concordantly, no effects of supernatants from macrophages cultured in the presence of GW4064 were observed regarding a possible indirect impact on oligodendroglial differentiation.ConclusionsOur data suggest that FXR is dispensable for oligodendroglial differentiation and that FXR agonists, such as GW4064, represent a potential therapeutic approach for MS which specifically targets peripheral immune cells including macrophages but not brain-resident cells, such as oligodendrocytes, astrocytes or microglia.

Role of FXR in Liver Inflammation during Nonalcoholic Steatohepatitis.

About 15-25% of patients with simple steatosis of non-alcoholic fatty liver disease progresses to non-alcoholic steatohepatitis (NASH), and the underlying mechanism for this progression has not been elucidated. NASH ultimately could progress to cirrhosis, an irreversible condition. Farnesoid X receptor (FXR) has been studied for its role in modulating inflammation, and the expression of FXR is down-regulated during NASH development. FXR deficiency has shown to progress and exacerbate NASH development, and FXR activation has been protective against liver inflammation associated with NASH. The expression of factors in both the adaptive and innate immune response in the liver are regulated in a FXR-dependent and -independent manner. Therefore, understanding key signaling pathways of liver inflammation in NASH is important to determine essential components that predispose, progress, or exacerbate NASH. FXR has been identified as a therapeutic target for NASH to prevent liver inflammation.

Farnesoid X Receptor Activation Promotes Hepatic Amino Acid Catabolism and Ammonium Clearance in Mice

The nuclear receptor subfamily 1 group H member 4 (NR1H4 or farnesoid X receptor [FXR]) regulates bile acid synthesis, transport, and catabolism. FXR also regulates postprandial lipid and glucose metabolism. We performed quantitative proteomic analyses of liver tissues from mice to evaluate these functions and investigate whether FXR regulates amino acid metabolism. To study the role of FXR in mouse liver, we used mice with a disruption of Nr1h4 (FXR-knockout mice) and compared them with floxed control mice. Mice were gavaged with the FXR agonist obeticholic acid or vehicle for 11 days. Proteome analyses, as well as targeted metabolomics and chromatin immunoprecipitation, were performed on the livers of these mice. Primary rat hepatocytes were used to validate the role of FXR in amino acid catabolism by gene expression and metabolomics studies. Finally, control mice and mice with liver-specific disruption of Nr1h4 (liver FXR-knockout mice) were re-fed with a high-protein diet after 6 hours fasting and gavaged a 15NH4Cl tracer. Gene expression and the metabolome were studied in the livers and plasma from these mice. In livers of control mice and primary rat hepatocytes, activation of FXR with obeticholic acid increased expression of proteins that regulate amino acid degradation, ureagenesis, and glutamine synthesis. We found FXR to bind to regulatory sites of genes encoding these proteins in control livers. Liver tissues from FXR-knockout mice had reduced expression of urea cycle proteins, and accumulated precursors of ureagenesis, compared with control mice. In liver FXR-knockout mice on a high-protein diet, the plasma concentration of newly formed urea was significantly decreased compared with controls. In addition, liver FXR-knockout mice had reduced hepatic expression of enzymes that regulate ammonium detoxification compared with controls. In contrast, obeticholic acid increased expression of genes encoding enzymes involved in ureagenesis compared with vehicle in C57Bl/6 mice. In livers of mice,FXR regulates amino acid catabolism and detoxification of ammonium via ureagenesis and glutamine synthesis. Failure of the urea cycle and hyperammonemia are common in patients with acute and chronic liver diseases; compounds that activate FXR might promote ammonium clearance in these patients.

Farnesoid X receptor ablation sensitizes mice to hepatitis b virus X protein-induced hepatocarcinogenesis.

We propose that transactivation of FXR by full-length HBx may represent a protective mechanism to inhibit HCC and that this inhibition may be compromised upon the appearance of C-terminally truncated HBx or when the expression and/or activity of FXR is decreased. (Hepatology 2017;65:893-906).

Protective effects of dioscin against doxorubicin-induced nephrotoxicity via adjusting FXR-mediated oxidative stress and inflammation.

Dioscin shows active effects against renal ischemia/reperfusion injury and lipopolysaccharide-induced inflammatory kidney injury, however, little is known concerning the role of it on doxorubicin (Dox)-induced nephrotoxicity. In the present study, in vivo test of Dox-induced nephrotoxicity in rats and in vitro model in NRK-52E cells were developed. The results showed that dioscin significantly attenuated cell injury, obviously reduced ROS level in vitro, and markedly decreased the levels of BUN, Cr, MDA, and notably increased the levels of SOD, GSH and GSH-Px in rats. Mechanistic studies showed that dioscin significantly increased the levels of p-AMPKα, Nrf2, HO-1 and GST by activation of FXR against oxidative stress. In addition, dioscin suppressed the nuclear translocation of NF-κB and HMGB1, and subsequently decreased the mRNA levels of IL-1β, IL-6, and TNF-α against inflammation. These results were further validated by knockdown of FXR using siRNA silencing, and abrogation of FXR using NDB (a FXR inhibitor) in NRK-52E cells, and the results suggested that the protective effect of dioscin against Dox- induced nephrotoxicity via adjusting FXR-mediated signal to suppress oxidative stress and inflammation. In addition, molecular docking assay showed that dioscin directly targeted with FXR through competing with Helix12 (H12) by hydrogen bonding, hydrophobic effect and electrostatic interactions. In a word, our data showed that dioscin is a novel and potent FXR agonist to suppress inflammation and oxidative stress against Dox-induced nephrotoxicity.

SAT-363 - Activation of FXR by steroidal or non-steroidal agonists causes an increase in LDL-cholesterol in mice with humanized chimeric liver

SAT-363 - Activation of FXR by steroidal or non-steroidal agonists causes an increase in LDL-cholesterol in mice with humanized chimeric liver

FXR agonism protects against liver injury in a rat model of intestinal failure-associated liver disease

Intestinal failure-associated liver disease (IFALD) is a clinical challenge. The pathophysiol-ogy is multifactorial and remains poorly understood. Disturbed recirculation of bile salts, e.g. due to loss of bile via an enterocutaneous fistula, is considered a major contributing factor. We hypothesize that impaired signaling via the bile salt receptor FXR underlies the development of IFALD. The aim of this study was to investigate whether activation of FXR improves liver homeostasis during chronic loss of bile in rats. To study consequences of chronic loss of bile, rats underwent external biliary drainage (EBD) or sham surgery for seven days, and the prophylactic potential of the FXR agonist INT-747 was assessed. EBD for 7 days resulted in liver test abnormalities and histological liver damage. Expression of the intestinal FXR target gene Fgf15 was undetectable after EBD, and this was accompanied by an anticipated increase in hepatic Cyp7a1 expression, indicating increased bile salt synthesis. Treatment with INT-747 improved serum biochemistry, reduced loss of bile fluid in drained rats and prevented development of drainage-associated histological liver injury. EBD results in extensive hepatobiliary injury and cholestasis. These data suggest that FXR activation might be a novel therapy in preventing liver dysfunction in patients with intestinal failure. This study demonstrates that chronic loss of bile causes liver injury in rats. Abro-gated recycling of bile salts impairing of enterohepatic bile salt/FXR signaling underlies these pathological changes, as administration of FXR agonist INT747 prevents biliary drainage-induced liver damage. Phar-macological activation of FXR might be a therapeutic strategy to treat disorders accompanied by a per-turbed enterohepatic circulation such as intestinal failure-associated liver disease.

FXR activation by obeticholic acid induces hepatic SR-BI gene expression through a novel mechanism of transcriptional synergy with the nuclear receptor LXR

FXR activation by obeticholic acid induces hepatic SR-BI gene expression through a novel mechanism of transcriptional synergy with the nuclear receptor LXR

SAT-420 - Evidence of FXR activation with obeticholic acid in an in vitro intestinal model

SAT-420 - Evidence of FXR activation with obeticholic acid in an in vitro intestinal model

Geniposide attenuates ANIT-induced cholestasis through regulation of transporters and enzymes involved in bile acids homeostasis in rats

Ethnopharmacological relevanceGeniposide (GE) is one of the major iridoid glycosides isolated from the fruit of Gardenia jasminoides Ellis that has been used to treat hepatic disorders including cholestasis. However, the underlying mechanisms for GE ameliorating the reduction in bile acids accumulation by α-naphthylisothiocyanate (ANIT) remain unclear. Aim of the studyThe purpose of this study is to characterize the efficacy of GE in regulation of bile acids uptake, synthesis, metabolism, and transport in ANIT-induced rats. Materials and methodsSprague–Dawley rats were orally administrated with vehicle, GE (25, 50, and 100mg/kg), and ursodeoxycholic acid (UDCA) (60mg/kg) once daily for seven days. On the fifth day, a single dose of ANIT (75mg/kg) was administrated via oral gavage. Blood biochemical determination, bile flow rate and liver histopathology were measured to evaluate the protective effect of GE. The mRNA expressions and protein levels of transporters and enzymes involved in bile acids homeostasis were determined by quantitative real-time polymerase chain reaction (PCR) and western blot to study the underlying mechanism of GE against ANIT-induced rats. ResultsGE (25, 50, and 100mg/kg, po) dose-dependently prevented ANIT-induced changes in serum markers for liver injury. GE treatment reduced basolateral bile acids uptake via repression of OATP2 (P<0.05). Bile acids biosynthesis was decreased through down-regulation of CYP7A1, CYP8B1, and CYP27A1 (P<0.05). GE significantly increased canalicular bile acids secretion via BSEP (P<0.05), subsequently stimulating bile flow during cholestasis. GE also markedly enhanced mRNA level of basolateral transporter OSTβ (P<0.01). Bile acids transported to the plasma were cleared into the urine, resulting in down-regulation of plasma bile acids. However, GE did not alter the mRNA levels of CYP3A2, UGT1A1 and SULT2A1. Furthermore, the gene and protein expression analysis demonstrated activation of FXR, PXR, and SHP after GE administration. ConclusionGE attenuates ANIT-induced hepatotoxicity and cholestasis in rats, due to regulation enzymes and transporters responsible for bile acids homeostasis.

Calycosin attenuates triglyceride accumulation and hepatic fibrosis in murine model of non-alcoholic steatohepatitis via activating farnesoid X receptor

BackgroundNon-alcoholic steatohepatitis (NASH) represents the more severe end of hepatic steatosis and is associated with progressive liver disease. Calycosin, derived from the root of Radix Astragali, has been demonstrated to have favorable efficacy on acute liver injury. PurposeThe present study was to investigate the hepatoprotective effect of calycosin on attenuating triglyceride accumulation and hepatic fibrosis, as well as explore the potential mechanism in murine model of NASH. Study designThe C57BL/6 male mice were fed with methionine choline deficient (MCD) diet for 4 weeks to induce NASH and treated with or without calycosin by oral gavage for 4 weeks. MethodsThe body weight, liver weight and the liver to body weight ratios were measured. Serum ALT, AST, TG, TC, FFA, MCP-1 and mKC levels were accessed by biochemical methods. H&E staining and Oil red O staining were used to identify the amelioration of liver histopathology. Immunohistochemistry of a-SMA, Masson trichrome staining and Sirius red staining were used to identify the amelioration of hepatic fibrosis. The quantitative real-time-PCR and Western blot were applied to observe the expression changes of key factors involved in triglyceride synthesis, free fatty acid β-oxidation and hepatic fibrosis. ResultsCalycosin significantly inhibited body weight loss induced by MCD diet, decreased the ALT and AST activities, MCP-1 and mKC in a dose-dependent manner. The H&E and Oil red O staining indicated calycosin effectively improved hepatic steatosis, improved the degree of triglyceride accumulation. Masson trichrome and Sirius red staining indicated that calycosin treatment remarkably attenuated the degree of hepatic fibrosis. Immunohistochemistry of a-SMA demonstrated that calycosin attenuated hepatic fibrosis by inhibiting hepatic stellate cell activation. Further, calycosin inhibited the expression of SREBP-1c, FASN, ACC and SCD1 involved in triglyceride synthesis, promoted the expression of PPARa, CPT1, Syndecan-1 and LPL involved in free fatty acid β-oxidation. The above effects of calycosin were attributed to FXR activation. ConclusionCalycosin attenuates triglyceride accumulation and hepatic fibrosis to protect against NASH via FXR activation.

Alisol B 23-acetate protects against non-alcoholic steatohepatitis in mice via farnesoid X receptor activation.

Alisol B 23-acetate (AB23A) is a natural triterpenoid isolated from the traditional Chinese medicine rhizoma alismatis, which exhibits a number of pharmacological activities, including anti-hepatitis virus, anti-cancer and antibacterial effects. In this study we examined whether AB23A protected against non-alcoholic steatohepatitis (NASH) in mice, and the mechanisms underlying the protective effects. NASH was induced in mice fed a methionine and choline-deficient (MCD) diet for 4 weeks. The mice were simultaneously treated with AB23A (15, 30, and 60 mg·kg-1·d-1, ig) for 4 weeks. On the last day, blood samples and livers were collected. Serum liver functional enzymes, inflammatoru markers were assessed. The livers were histologically examined using H&E, Oil Red O, Masson's trichrome and Sirius Red staining. Mouse primary hepatocytes were used for in vitro experiments. The mechanisms underlying AB23A protection were analyzed using siRNA, qRT-PCR, and Western blot assays. AB23A treatment significantly and dose-dependently decreased the elevated levels of serum ALT and AST in MCD diet-fed mice. Furthermore, AB23A treatment significantly reduced hepatic triglyceride accumulation, inflammatory cell infiltration and hepatic fibrosis in the mice. AB23A-induced decreases in serum and hepatic lipids were related to decreased hepatic lipogenesis through decreasing hepatic levels of SREBP-1c, FAS, ACC1 and SCD1 and increased lipid metabolism via inducing PPARα, CPT1α, ACADS and LPL. The reduction in inflammatory cell infiltration corresponded to deceased serum levels of mKC and MCP-1 and decreased hepatic gene expression of MCP-1 and VCAM-1. The reduction in hepatic fibrosis was correlated with decreased hepatic gene expression of fibrosis markers. The protective effects of AB23A were FXR-dependent, because treatment with the FXR agonist CDCA mimicked AB23A-induced hepato-protection in the mice, whereas co-administration of FXR antagonist guggulsterone abrogated AB23A-induced hepato-protection. In mouse primary hepatocytes, FXR gene silencing abrogated AB23A-induced changes in gene expression of Apo C-II, CPT1α, ACADS and LPL. AB23A produces protective effects against NASH in mice via FXR activation.

Discovery of 3α,7α,11β-Trihydroxy-6α-ethyl-5β-cholan-24-oic Acid (TC-100), a Novel Bile Acid as Potent and Highly Selective FXR Agonist for Enterohepatic Disorders.

As a continuation of previous efforts in mapping functional hot spots on the bile acid scaffold, we here demonstrate that the introduction of a hydroxy group at the C11β position affords high selectivity for FXR. In particular, the synthesis and FXR/TGR5 activity of novel bile acids bearing different hydroxylation patterns at the C ring are reported and discussed from a structure-activity standpoint. The results obtained led us to discover the first bile acid derivative endowed with high potency and selectivity at the FXR receptor, 3α,7α,11β-trihydroxy-6α-ethyl-5β-cholan-24-oic acid (TC-100, 7) which also shows a remarkable physicochemical and pharmacological profile. Compound 7 combines the excellent physicochemical properties of hydrophilic bile acids such as ursodeoxycholic acid, with the distinct ability to specifically bind and regulate FXR activity in vivo, thus providing a bona fide novel therapeutic agent to treat enterohepatic disorders such as cholestasis, NASH, and inflammatory bowel disease.

Alterations in Enterohepatic Fgf15 Signaling and Changes in Bile Acid Composition Depend on Localization of Murine Intestinal Inflammation.

Fibroblast growth factor (FGF) 15/19 is part of the gut-liver crosstalk accounting for bile acid (BA) metabolism regulation. Dysregulation of fibroblast growth factor 15/19 signaling is observed in different pathological conditions, for example, in gastrointestinal diseases such as inflammatory bowel disease (IBD). To understand the molecular bases, we analyzed the enterohepatic regulation of Fgf15-mediated pathway in 2 different inflammatory bowel disease mouse models. Target genes of the BA-farnesoid-X-receptor (Fxr)-Ffg15 axis were quantified by RT-PCR or western blotting in gut and liver of dextran sulfate sodium (DSS)-treated and IL10 mice. Serum Fgf15 levels were analyzed by ELISA. Biliary and fecal BA composition was differentiated by HPLC-MS/MS. Dextran sulfate sodium-treated mice with ileum-sparing colitis showed higher Fgf15 serum levels. In contrast, IL10 mice with ileitis had a trend toward decreased Fgf15 serum levels compared with controls and increased expression of Asbt as a negative Fxr-target gene. In hepatic tissue of both models, no histological changes, but higher interleukin 6 (IL-6) mRNA expression and down-regulation of Fxr and Cytochrom P450 7a1 mRNA expression were observed. Fibroblast growth factor receptor 4 up-regulation was in line with higher Fgf15 serum levels in dextran sulfate sodium-treated mice. A distinct fecal BA profile was observed in both models with significantly higher levels of taurine-conjugated BA in particular tauro-β-muricholic acid in IL10 mice. Ileum-sparing colitis is characterized by activation of Fxr-Fgf15 signaling with higher expression of Fxr-target gene Fgf15, whereas ileal inflammation showed no signs of Fxr-Fgf15 activation. Abundance of BA such as T-β-MCA may be important for intestinal Fxr activation in mice.

Restoration of enterohepatic bile acid pathways in pregnant mice following short term activation of Fxr by GW4064

The farnesoid X receptor (Fxr) controls bile acid homeostasis by coordinately regulating the expression of synthesizing enzymes (Cyp7a1, Cyp8b1), conjugating enzymes (Bal, Baat) and transporters in the ileum (Asbt, Ostα/β) and liver (Ntcp, Bsep, Ostβ). Transcriptional regulation by Fxr can be direct, or through the ileal Fgf15/FGF19 and hepatic Shp pathways. Circulating bile acids are increased during pregnancy due to hormone-mediated disruption of Fxr signaling. While this adaptation enhances lipid absorption, elevated bile acids may predispose women to develop maternal cholestasis. The objective of this study was to determine whether short-term treatment of pregnant mice with GW4064 (a potent FXR agonist) restores Fxr signaling to the level observed in virgin mice. Plasma, liver and ilea were collected from virgin and pregnant mice administered vehicle or GW4064 by oral gavage. Treatment of pregnant mice with GW4064 induced ileal Fgf15, Shp and Ostα/β mRNAs, and restored hepatic Shp, Bal, Ntcp, and Bsep back to vehicle-treated virgin levels. Pregnant mice exhibited 2.5-fold increase in Cyp7a1 mRNA compared to virgin controls, which was reduced by GW4064. Similarly treatment of mouse primary hepatocytes with plasma isolated from pregnant mice induced Cyp7a1 mRNA by nearly 3-fold as compared to virgin plasma, which could be attenuated by co-treatment with either GW4064 or recombinant FGF19 protein. Collectively, these data reveal that repressed activity of intestinal and hepatic Fxr in pregnancy, as previously demonstrated, may be restored by pharmacological activation. This study provides the basis for a novel approach to restore bile acid homeostasis in patients with maternal cholestasis.

Cholesterol 7α‐hydroxylase (CYP7A1) plays a critical role in preventing hepatic inflammation and fibrosis

IntroductionCholesterol 7α‐hydroxylase (CYP7A1) is the rate‐limiting enzyme in the bile acid biosynthetic pathway in the liver and controls cholesterol and bile acid homeostasis. Accumulation of toxic bile acids in liver causes cholestasis and inflammatory liver injury. The mechanism of bile acid homeostasis in hepatic inflammation and fibrosis remains unclear.MethodsWe investigated the roles of CYP7A1 in hepatic inflammation and fibrosis using lipopolysaccharides (LPS)‐induced acute liver injury and methionine/choline deficient (MCD) diet‐induced liver injury models. Cyp7a1−/− mice in pure C57BL6 background, bile acid‐activated nuclear receptor farnesoid X receptor deficient (Fxr−/−) mice, and G protein‐coupled bile acid receptor deficient (Tgr5−/−) mice, and adenovirus‐mediated transduction of Cyp7a1 were used to study the roles of CYP7A1 and bile acid receptors in hepatic inflammation and fibrosis. Real‐time PCR, western blot, immunohistochemistry, luciferase report and chromatin immunoprecipitation (ChIP) assays were used in the study.ResultsAdenovirus‐mediated overexpression of CYP7A1 ameliorated LPS‐induced inflammatory cell infiltration and pro‐inflammatory cytokines production in WT and Tgr5−/−, but not Fxr−/− mice suggesting that FXR played a key role in protection against LPS‐induced hepatic inflammation. Luciferase report assay and real‐time PCR showed that FXR agonists, GW4064 or INT‐747 significantly inhibited NF‐κB activation and pro‐inflammatory cytokine mRNA expression in HepG2 cells and human primary hepatocytes. Mammalian two‐hybrid assay, co‐immunoprecipitation and ChIP assays showed that activation of FXR promotes FXR interaction with p65, which in turn blocked p65 binding to NF‐κB response elements on the TNF‐α and IL‐1β gene promoters. Serum AST and ALT levels were significantly increased in the liver of Cyp7a1−/− mice fed with MCD diet compared with WT mice. Pro‐inflammatory cytokine mRNA expression and immunohistochemistry of α‐SMA showed that Cyp7a1−/− mice fed with MCD diet developed more severe hepatic inflammation and fibrosis than WT mice. Cholesterol is aggravated in Cyp7a1−/− mice fed with MCD diet, and may increase reactive oxidizing species and cell death in the liver.ConclusionThese data suggest that increased bile acids ameliorated hepatic inflammation through activation of FXR but not TGR5 signaling. On the other hand, reduced bile acid pool increased susceptibility to MCD diet‐induced hepatic inflammation and fibrosis. Maintaining bile acid homeostasis is important in protection against liver injury and non‐alcoholic fatty liver disease.

Dual activation of bile acid receptors FXR and TGR5 plays a protective role in non‐alcoholic fatty liver disease and atherosclerosis

Nonalcoholic fatty liver disease (NAFLD) is a spectrum of conditions which ranges from fatty liver to non‐alcoholic steatohepatitis (NASH) and liver cirrhosis. The aim of this study is to establish that activation of the bile acid receptors FXR and TGR5 by its semi‐synthetic ligand INT‐767 attenuates the development of NAFLD and atherosclerosis. Administration of the TGR5 specific synthetic ligand INT‐777 led to a significant reduction in liver triglycerides and cholesterol. In addition, treatment with the FXR and TGR5 dual agonist INT‐767 led to a significant reduction in plasma and hepatic triglycerides. In line with this, treatment of high fat diet‐fed C57BL/J6 mice with INT‐767 led to significant decrease in plasma and hepatic triglycerides as well. To further investigate the role of TGR5 and FXR in the development of atherosclerosis, ApoE−/− mice were gavaged with INT‐767 for 60 days. This led to a significant reduction in plasma and liver lipids, and also a marked improvement in the plasma lipoprotein profile. This in turn led to a significant reduction in atherosclerotic plaque formation in INT‐767 treated ApoE−/− mice. These data indicate that dual activation of FXR and TGR5 plays an important role in maintaining hepatic and plasma lipid homeostasis under normal and high fat diet‐fed conditions. These results point to the possibility of targeting both FXR and TGR5 as a potential therapeutic treatment of NAFLD and atherosclerosis.