Farnesoid X Receptor Signaling Research Articles

Luminal bile acids region‐specifically strengthen intestinal epithelial barrier integrity after RYGB through FXR signaling

Roux‐en‐Y gastric bypass surgery (RYGB) provides superior long‐term glycaemic control in severely obese and insulin resistant individuals. The improved peripheral insulin sensitivity at later stages after RYGB has been attributed in part to a reduction in intestinal permeability and associated endotoxemia, but the underlying mechanisms remain unclear. Here, we used diet‐induced obese rat and Caco2 cell monolayer models to show that RYGB region‐specifically strengthens intestinal epithelial barrier (IEB) integrity. This was partly farnesoid X receptor (FXR)‐dependent for the bile‐exposed duodenum and colon but not for the bile‐excluded jejunum. Transfer experiments performed on recipient germ‐free mice additionally demonstrated that jejunal and colonic but duodenal content of RYGB‐operated rats contain microbiota‐produced factors which improve oral glucose tolerance in proportion to suppression in endotoxemia. Collectively, these findings suggest that luminal primary and secondary bile acids acting through FXR in duodenal and colonic epithelial cells respectively, contribute to overall strengthening of IEB integrity and improved glucose homeostasis after RYGB and further support the use of bile acid mimetics for the treatment of metabolic disease.

Mast Cell Signaling Regulates Biliary Farnesoid X Receptor and Apical Sodium Bile Acid Transporter Expression During Cholestatic Liver Injury

BackgroundChronic liver diseases and cholangiopathies (targets cholangiocytes) are diagnosed at advanced stages leading to poor prognosis. Mast cells (MCs) infiltrate the liver during damage, release histamine (HA) and act via H1‐H4 HA receptors (HRs). Cholangiocytes and MCs express apical sodium bile acid transporter (ASBT); farnesoid X receptor (FXR) and H2HR. Introduction of MCs into C57BL/6 (WT) and MC deficient (KitW‐sh) mice increases liver damage, biliary proliferation/liver fibrosis, and total bile acid (TBA) levels. We aimed to determine if loss of MC FXR signaling alters biliary FXR and ASBT, injury and hepatic fibrosis via HA/H2HR signaling.MethodsWT or KitW‐sh mice were subjected to tail vein injection of cultured MCs (ATCC®) pretreated with PBS (PBS‐MC) to induce liver damage or the FXR inhibitor, Z‐guggulsterone (10μM, FXR‐MC) and sacrificed after 3 days. H&E was used to determine pathological changes. Ductular reaction was determined by CK‐19 staining. Biliary proliferation and hepatic senescence were evaluated by Ki‐67 and SA‐b‐galactosidase staining. Hepatic fibrosis was determined by Sirius Red staining. Hepatic stellate cell (HSC) presence was determined by immunofluorescence (IF). Biliary senescence was determined by IF and qPCR. Cholangiocyte FXR and ASBT expression was determined by staining and qPCR in cholangiocytes. Senescence associated secretory phenotypes (SASPs) and HA secretion were measured by EIA in serum and cholangiocyte supernatants. H2HR expression was evaluated in isolated cholangiocytes. TBAs were measured in serum and liver tissues. In vitro, cultured MCs were treated with PBS or Z‐guggulsterone (10μM, 48 hr). Treated MC supernatants were placed on cholangiocytes prior to evaluating biliary ASBT and H2HR expression and secretion of HA, TGF‐b1 and IL‐1b by qPCR and EIA, respectively. In human tissue from patients with PSC or PBC, biliary ASBT, FXR and MC infiltration (marked by tryptase) was measured by IHC.ResultsHepatic damage, ductular reaction, biliary senescence, ASBT expression and liver fibrosis were induced in WT and KitW‐sh mice injected with PBS‐MC; whereas, these parameters were reversed in mice injected with FXR‐MCs. HA secretion and H2HR expression increased in serum and cholangiocytes, respectively in WT and KitW‐sh mice injected with PBS‐MC, but were reduced in mice injected with FXR‐MC. Hepatic and serum TBA increased in WT and KitW‐sh mice injected with PBS‐MC and were reduced in FXR‐MC groups. In vitro, stimulation with MC supernatant increased biliary ASBT, FXR, H2HR, HA, TGF‐b1 and IL‐1b expression. These parameters decreased in cholangiocytes treated with FXR‐MC supernatant. In human tissues, upregulation of MCs was coupled with increased biliary FXR and ASBT expression.ConclusionsReintroduction of MCs recapitulates cholestatic liver injury in WT and KitW‐sh mice. Inhibition of MC FXR signaling decreases biliary ASBT expression, liver injury and hepatic fibrosis via downregulation of HA/H2HR signaling. Manipulation of BA transporters, receptors and MC mediators may be a therapeutic route for patients with cholestatic liver damage.Support or Funding InformationNIH NIDDK R01s, VA Merit, IUSOM

Biological Functions for STARD5 Assessed Using stard5−/− Mice

STARD5 belongs to the StAR‐Related Lipid‐transfer (START) domain protein superfamily and is proposed to contribute to intracellular non‐vesicular cholesterol transport. STARD5 is a soluble sterol‐binding protein that is induced by endoplasmic reticulum (ER) stress, yet testing for a biological function needs to consider the conflicting data on whether the preferred ligand for STARD5 is cholesterol and 25‐hydroxycholesterol or bile acids, including chenodeoxycholic acid (CDCA). Our work has characterized STARD5 expression in kidney and we reported that STARD5 and cholesterol levels are increased in kidneys of the OVE diabetic mouse model compare to wild‐type controls, and that STARD5 is increased in cell culture by the ER stress inducer tunicamycin. Cholesterol accumulation in the ER is known to promote ER stress; however, the significance of the association between elevated renal cholesterol, ER stress, and STARD5 in diabetic kidney remains to be determined. We propose STARD5 indirectly controls cholesterol homeostasis via modulating farnesoid X receptor (FXR) activity. FXR is a nuclear receptor that binds CDCA and activation of FXR contributes to maintaining fatty acid and cholesterol homeostasis in diabetic kidney disease. To test whether STARD5 affects FXR signaling we generated whole body STARD5 knock‐out mice (stard5−/−) using CRISPR‐Cas9 and compared the FXR target gene expression in the knockout and WT kidney. There was no difference in body weight, liver weight/body weight ratio, or kidney weight/body weight ratio between stard5−/− mice compared to wild‐type littermate controls (WT) (male mice n = 5 WT; n= 7 stard5−/−). We will present data from current studies focused on determining whether there are differences in tissue histology, lipid accumulation, and FXR target gene expression between stard5−/− mice and WT littermate controls.Support or Funding InformationBiochemistry & Molecular Genetics Departmental Funds to BJC

Schaftoside alleviates HFD-induced hepatic lipid accumulation in mice via upregulating farnesoid X receptor

Ethnopharmacology relevanceThe farnesoid X receptor (FXR) is a therapeutic target of for the treatment of non-alcoholic fatty liver disease (NAFLD) owing to its regulatory role in lipid homeostasis. Schaftoside (SS) is a bioactive compound of Herba Desmodii Styracifolii, which has traditionally been used to treat hepatitis and cholelithiasis. However, the potential hepatoprotective effect of SS against NAFLD and the underlying mechanisms remain unknown. Aim of the studyWe investigated whether SS could improve NAFLD-induced liver injury by decreasing lipid accumulation via the activation of FXR signalling. Materials and methodsIn vivo, the effects of SS on high-fat diet (HFD)-induced lipid accumulation in the liver of mice were evaluated by serum biochemical parameters and histopathological analysis. In vitro, the intracellular triglyceride (TG) level and Oil Red O staining were used to evaluate the lipid removal ability of SS in Huh-7 cells or FXR knockout mouse primary hepatocytes (MPHs) induced by oleic acid (OA). Moreover, FXR/sterol regulatory element-binding protein 1 (SREBP1) mRNA and protein expression levels were detected. ResultsSS reduced HFD-induced lipid accumulation in the liver, as indicated by decreased aspartate aminotransferase (AST), cholesterol (Ch), and TG levels in serum and TG levels in liver tissue, and subsequently resulting in attenuation of liver histopathological injury. Gene expression profiles demonstrated that SS dose-dependently prevented HFD-induced decrease of FXR expression and inversely inhibited SREBP1 expression in the nucleus. Furthermore, SS significantly suppressed excessive TG accumulation and decreased intracellular TG level in Huh-7 cells or MPHs via the upregulation of FXR and inhibition of SREBP1 expression in the nucleus. ConclusionOur results suggest that SS ameliorates HFD-induced NAFLD by the decrease of lipid accumulation via the control of FXR-SREBP1 signalling.

Farnesoid X receptor contributes to body weight-independent improvements in glycemic control after Roux-en-Y gastric bypass surgery in diet-induced obese mice

ObjectiveRoux-en-Y gastric bypass surgery (RYGB) can achieve long-term remission of type 2 diabetes. However, the specific molecular mechanism through which this occurs has remained largely elusive. Bile acid signaling through the nuclear hormone receptor farnesoid X receptor (FXR) exerts beneficial effects after sleeve gastrectomy (VSG), which has similar effects to RYGB. Therefore, we investigated whether FXR signaling is necessary to mediate glycemic control after RYGB. MethodsRYGB or sham surgery was performed in high-fat diet-induced obese FXR−/− (knockout) and FXR+/+ (wild type) littermates. Sham-operated mice were fed ad libitum (S-AL) or by weight matching (S-WM) to RYGB mice via caloric restriction. Body weight, body composition, food intake, energy expenditure, glucose tolerance tests, insulin tolerance tests, and homeostatic model assessment of insulin resistance were performed. ResultsRYGB surgery decreases body weight and fat mass in WT and FXR-KO mice. RYGB surgery has similar effects on food intake and energy expenditure independent of genotype. In addition, body weight-independent improvements in glucose control were attenuated in FXR −/− relative to FXR +/+ mice after RYGB. Furthermore, pharmacologic blockade of the glucagon-like peptide-1 receptor (GLP-1R) blunts the glucoregulatory effects of RYGB in FXR +/+ but not in FXR −/− mice at 4 weeks after surgery. ConclusionsThese results suggest that FXR signaling is not required for the weight loss up to 16 weeks after RYGB. Although most of the improvements in glucose homeostasis are secondary to RYGB-induced weight loss in wild type mice, FXR signaling contributes to glycemic control after RYGB in a body weight-independent manner, which might be mediated by an FXR-GLP-1 axis during the early postoperative period.

Absence of Bsep/Abcb11 attenuates MCD diet-induced hepatic steatosis but aggravates inflammation in mice.

BackgroundBile acids (BAs) regulate hepatic lipid metabolism and inflammation. Bile salt export pump (BSEP) KO mice are metabolically preconditioned with a hydrophilic BA composition protecting them from cholestasis. We hypothesize that changes in hepatic BA profile and subsequent changes in BA signalling may critically determine the susceptibility to steatohepatitis.MethodsWild‐type (WT) and BSEP KO mice were challenged with methionine choline‐deficient (MCD) diet to induce steatohepatitis. Serum biochemistry, lipid profiling as well as intestinal lipid absorption were assessed. Markers of inflammation, fibrosis, lipid and BA metabolism were analysed. Hepatic and faecal BA profile as well as serum levels of the BA synthesis intermediate 7‐hydroxy‐4‐cholesten‐3‐one (C4) were also investigated.ResultsBile salt export pump KO MCD‐fed mice developed less steatosis but more inflammation than WT mice. Intestinal neutral lipid levels were reduced in BSEP KO mice at baseline and under MCD conditions. Faecal non‐esterified fatty acid concentrations at baseline and under MCD diet were markedly elevated in BSEP KO compared to WT mice. Serum liver enzymes and hepatic expression of inflammatory markers were increased in MCD‐fed BSEP KO animals. PPARα protein levels were reduced in BSEP KO mice. Accordingly, PPARα downstream targets Fabp1 and Fatp5 were repressed, while NFκB subunits were increased in MCD‐fed BSEP KO mice. Farnesoid X receptor (FXR) protein levels were reduced in MCD‐fed BSEP KO vs WT mice. Hepatic BA profile revealed elevated levels of TβMCA, exerting FXR antagonistic action, while concentrations of TCA (FXR agonistic function) were reduced.ConclusionPresence of hydroxylated BAs result in increased faecal FA excretion and reduced hepatic lipid accumulation. This aggravates development of MCD diet‐induced hepatitis potentially by decreasing FXR and PPARα signalling.

Dominant gut Prevotella copri in gastrectomised non-obese diabetic Goto\u2013Kakizaki rats improves glucose homeostasis through enhanced FXR signalling

Aims/hypothesisDrug and surgical-based therapies in type 2 diabetes are associated with altered gut microbiota architecture. Here we investigated the role of the gut microbiome in improved glucose homeostasis following bariatric surgery.MethodsWe carried out gut microbiome analyses in gastrectomised (by vertical sleeve gastrectomy [VSG]) rats of the Goto–Kakizaki (GK) non-obese model of spontaneously occurring type 2 diabetes, followed by physiological studies in the GK rat.ResultsVSG in the GK rat led to permanent improvement of glucose tolerance associated with minor changes in the gut microbiome, mostly characterised by significant enrichment of caecal Prevotella copri. Gut microbiota enrichment with P. copri in GK rats through permissive antibiotic treatment, inoculation of gut microbiota isolated from gastrectomised GK rats, and direct inoculation of P. copri, resulted in significant improvement of glucose tolerance, independent of changes in body weight. Plasma bile acids were increased in GK rats following inoculation with P. copri and P. copri-enriched microbiota from VSG-treated rats; the inoculated GK rats then showed increased liver glycogen and upregulated expression of Fxr (also known as Nr1h4), Srebf1c, Chrebp (also known as Mlxipl) and Il10 and downregulated expression of Cyp7a1.ConclusionsOur data underline the impact of intestinal P. copri on improved glucose homeostasis through enhanced bile acid metabolism and farnesoid X receptor (FXR) signalling, which may represent a promising opportunity for novel type 2 diabetes therapeutics.

Naturally Occurring TPE-CA Maintains Gut Microbiota and Bile Acids Homeostasis via FXR Signaling Modulation of the Liver-Gut Axis.

Antibiotics-induced changes in intestinal flora (dysbiosis) may have various effects on the host. Dysbiosis is associated with numerous metabolites including bile acids, which are produced in the liver from cholesterol and metabolized in the gut by intestinal microbiota. Total phenolic extracts of Citrus aurantium L. (TPE-CA) are rich in dietary flavanones and their glycosyl derivatives, including flavones, flavonols, polymethoxyflavones and coumarins, which exert positive health effects on the microbiota. The aim of this study is to elucidate the interplays between the intestinal microbiota and bile acids metabolism attributed to antibiotics. Mice were exposed to broad-spectrum antibiotics, such as ampicillin, streptomycin and clindamycin, for 14 days. This exposure resulted in reduced bacterial diversity and richness, and destroyed intestinal permeability. The homeostasis of bile acids was also affected. Subsequent TPE-CA administration, counteracted most of the dysbiosis, and reshaped intestinal permeability, these effects occurred via upregulation of zonula occludens 1 and occludin associated proteins and downregulation of serum endotoxin compared to the antibiotics group. TPE-CA maintained the homeostasis of bile acids via modulation of the liver–gut axis related farnesoid X receptor (FXR)/fibroblast growth factor 15 (FGF15) pathway and FXR-targeted protein. Our findings indicated that TPE-CA exerted a protective effect on the restoration of intestinal microbiota composition, reshaped barrier integrity and maintained bile acid homeostasis via the liver–gut axis with antibiotics-induced dysbiosis.

TUDCA Ameliorates Liver Injury Via Activation of SIRT1-FXR Signaling in a Rat Hemorrhagic Shock Model.

The aim of this study was to investigate the changes of bile acids in the liver during hemorrhagic shock (HS) and their potential to attenuate liver injury via activation of SIRT1 (sirtuin 1)-FXR (farnesoid X receptor) signaling. A Sprague-Dawley (SD) rat HS model was established, whereas HepG2 cells were hypoxically cultured to simulate HS in vitro. Liver bile acids (BA) were profiled with ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). FXR expression was detected by western blot and immunohistochemistry. The mRNA levels of SIRT1 and FXR were detected by polymerase chain reaction. Protein expression of SIRT1, FoxM1, NF-κB, acetyl-NF-κB, p53, and acetyl-p53 was analyzed by western blot. Hepatocyte apoptosis and proliferation were measured by TUNEL assay and Ki-67 staining, respectively. Serum and supernatant cytokines were analyzed using ELISA assays. Liver injury was also assessed. To investigate the possible mechanisms, SIRT1 agonist (SRT1720), SIRT1 inhibitor (EX527), and FXR inhibitor (Z-guggulsterone) were used. Tauroursodeoxycholic acid (TUDCA) in the liver decreased significantly after HS. SIRT1 and FXR expression was time-dependently downregulated by HS or hypoxia condition. TUDCA upregulated SIRT1-FXR activity, which inhibited expression and acetylation of NF-κB and p53 and increased FoxM1 expression, leading to decreased inflammatory response and apoptosis and increased proliferative capacity in hepatocytes, and attenuation of liver injury. EX527 pretreatment reversed the protective effect of TUDCA. Moreover, Z-guggulsterone supplementation decreased the protective effect of TUDCA in vitro. TUDCA in the liver decreased during HS. TUDCA supplementation might attenuate HS-induced liver injury by upregulating SIRT1-FXR signaling.

Bile acid receptors FXR and TGR5 signaling in fatty liver diseases and therapy.

Bile acid synthesis is the most significant pathway for catabolism of cholesterol and for maintenance of whole body cholesterol homeostasis. Bile acids are physiological detergents that absorb, distribute, metabolize, and excrete nutrients, drugs, and xenobiotics. Bile acids also are signal molecules and metabolic integrators that activate nuclear farnesoid X receptor (FXR) and membrane Takeda G protein-coupled receptor 5 (TGR5; i.e., G protein-coupled bile acid receptor 1) to regulate glucose, lipid, and energy metabolism. The gut-to-liver axis plays a critical role in the transformation of primary bile acids to secondary bile acids, in the regulation of bile acid synthesis to maintain composition within the bile acid pool, and in the regulation of metabolic homeostasis to prevent hyperglycemia, dyslipidemia, obesity, and diabetes. High-fat and high-calorie diets, dysbiosis, alcohol, drugs, and disruption of sleep and circadian rhythms cause metabolic diseases, including alcoholic and nonalcoholic fatty liver diseases, obesity, diabetes, and cardiovascular disease. Bile acid-based drugs that target bile acid receptors are being developed for the treatment of metabolic diseases of the liver.

SUMOylation inhibitors synergize with FXR agonists in combating liver fibrosis

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.

High-Fat Diet Promotes DSS-Induced Ulcerative Colitis by Downregulated FXR Expression through the TGFB Pathway.

Ulcerative colitis is one of the IBD which cause a chronic intestinal inflammation and dysfunctional of the mucosal barrier. For now, the incident of UC was steadily increased all over the world. It has become a novel independent risk factor of several severe diseases especially colon-rectal cancer. However, the etiology of UC was still obscure. Previous studies show that high-fat diet contributed to the pathogenesis of immune system dysregulation, and farnesoid X receptor (FXR) was also implicated in the pathogenesis of various inflammatory symptoms. Yet, their inner roles in the pathogenesis of UC have not been mentioned. In this study, we aim to investigate the role of FXR in UC. High-fat diet (HFD) promotes the progression of DSS-induced UC, shows an increasing secretion of bile acid in serum, and leads to a downregulation of FXR target genes (FXRα, Shp, and lbabp). Adding FXR agonist FexD rescues the phenotype induced by high-fat diet, whereas TGFBRI inhibitor SB431542 abrogates the restoration by FexD in DSS-induced UC mice. To further verify the relationship between the FXR and TGFB signaling pathway, we made a UC-HFD model in the Caco2 cell line. Results shows the same conclusion that FXR mitigate UC inflammation through a TGFB-dependent pathway. These results expand the role of FXR in ulcerative colitis and suggest that FXR activation may be considered a therapeutic strategy for UC.

Impaired Intestinal Farnesoid X Receptor Signaling in Cystic Fibrosis Mice.

Background & AimsThe bile acid (BA)-activated farnesoid X receptor (FXR) controls hepatic BA synthesis and cell proliferation via the intestinal hormone fibroblast growth factor 19. Because cystic fibrosis (CF) is associated with intestinal dysbiosis, anomalous BA handling, and biliary cirrhosis, we investigated FXR signaling in CF.MethodsIntestinal and hepatic expression of FXR target genes and inflammation markers was assessed in Cftr null mice and controls. Localization of the apical sodium-dependent BA transporter was assessed, and BAs in gastrointestinal tissues were analyzed. The CF microbiota was characterized and FXR signaling was investigated in intestinal tissue and organoids.ResultsIleal murine fibroblast growth factor 19 ortholog (Fgf15) expression was strongly reduced in CF mice, compared with controls. Luminal BA levels and localization of apical sodium-dependent BA transporter was not affected, and BAs induced Fgf15 up to normal levels in CF ileum, ex vivo, and CF organoids. CF mice showed a dysbiosis that was associated with a marked up-regulation of genes involved in host–microbe interactions, including those involved in mucin glycosylation, antimicrobial defense, and Toll-like receptor signaling. Antibiotic treatment reversed the up-regulation of inflammatory markers and restored intestinal FXR signaling in CF mice. Conversely, FXR-dependent gene induction in ileal tissue and organoids was repressed by bacterial lipopolysaccharide and proinflammatory cytokines, respectively. Loss of intestinal FXR activity was associated with a markedly blunted hepatic trophic response to oral BA supplementation, and with impaired repression of Cyp7a1, the gene encoding the rate-limiting enzyme in BA synthesis.ConclusionsIn CF mice, the gut microbiota represses intestinal FXR activity, and, consequently, FXR-dependent hepatic cell proliferation and feedback control of BA synthesis.

Acanthoic acid modulates lipogenesis in nonalcoholic fatty liver disease via FXR/LXRs-dependent manner

Acanthoic acid (AA) is a pimaradiene diterpene isolated from Acanthopanax koreanum Nakai (Araliaceae), with anti-inflammatory and hepatic-protective effects. The present study intended to reveal the effect and mechanism of AA on nonalcoholic fatty liver disease (NAFLD) associated with lipid accumulation by activating Farnesoid X receptor (FXR) and liver X receptors (LXRs) signaling. C57BL/6 mice were received a modified Lieber-DeCarli diet with 71% high-fat (L-D) and treated with AA (20 and 40 mg/kg) or equal volume of saline for 12 weeks. The regulation of AA on lipid accumulation was also detected in pro-steatotic stimulated AML12 cells with palmitic acid (PA). When L-D diet-fed mice were treated with AA, loss in body weight, liver index, and liver lipid droplet were observed along with reduced triglyceride (TG) and serum transaminase. Furthermore, AA decreased sterol regulatory element binding protein 1 (SREBP-1) and target genes expression, regulated PPARα and PPARγ expressions, ameliorated hepatic fibrosis markers, enhanced hepatic FXR and LXR, and regulated AMPK-LKB1 and SIRT1 signaling pathway. Moreover, AA attenuated lipid accumulation via FXR and LXR activation in steatotic AML-12 cells, which was confirmed by guggulsterones (FXR antagonist) or GW3965 (LXR agonist). Activation of FXR and LXR signaling caused by AA might increase AMPK-SIRT1 signaling and then contribute to modulating lipid accumulation and fatty acid synthesis, which suggested that activated FXR-LXR axis by AA represented an effective strategy for relieving NAFLD.

Ursodeoxycholic acid is a GPBAR1 agonist and resets liver/intestinal FXR signaling in a model of diet-induced dysbiosis and NASH

Obeticholic acid (OCA) is a farnesoid-X-receptor (FXR) ligand, shown effective in reducing steatosis and fibrosis in NASH patients. However, OCA causes major side effects including pruritus, while increases the risk for liver decompensation in cirrhotic patients. Ursodeoxycholic acid (UDCA), is a safe and unexpensive bile acid used in the treatment of liver disorders whose mechanism of action is poorly defined. Here we have compared the effects of OCA and UDCA in a mouse model of NASH. In mice exposed to a diet rich in fat/cholesterol and fructose (HFD-F), treatment with OCA or UDCA effectively prevented body weight gain, insulin resistance, as demonstrated by OGTT, and AST plasma levels. After 12 weeks HFD-F mice developed liver microvesicular steatosis, inflammation and mild fibrosis, increased expression of inflammatory (TNFα, IL6, F4/80) and fibrosis (αSma, Col1α1, Tgfβ) markers, reduced liver expression of FXR, dysregulated liver FXR signaling and elevated levels of Tauro-α and β-muricholic acid (T-α and βMCA), two FXR antagonists in mice. Both compounds prevented these changes and improved liver histopathology. OCA reduced primary bile acid synthesis worsening the T-CA/T-βMCA ratio. UDCA effectively transactivated GPBAR1 in vitro. By RNAseq analysis we found that among over 2400 genes modulated by the HFD-F, only 32 and 60 genes were modulated by OCA and UDCA, with only 3 genes (Dbp, Adh7, Osgin1) being modulated by both agents. Both agents partially prevented the intestinal dysbiosis. ConclusionsUDCA is a GPBAR1 ligand and exerts beneficial effects in a rodent model of NASH by activating non-overlapping pathway with OCA.

Ursodeoxycholic acid accelerates bile acid enterohepatic circulation.

Ursodeoxycholic acid (UDCA) is the first-line treatment for primary biliary cholangitis, but its effects on the enterohepatic circulation of bile acid (BA) have been under-investigated. Therefore, we studied the influence of UDCA on BA enterohepatic circulation in vivo and the mechanisms by which UDCA affects the BA kinetics. Mice were treated with UDCA and other BAs to observe changes in BA pool and BA transporters involved in enterohepatic circulation. Isotope dilution techniques and biochemical analyses were applied to study BA kinetics after oral administration of UDCA, and the mechanism involved. Oral administration of UDCA in mice reduced the overall BA pool and produced a unique BA profile with high-abundance conjugated UDCA species, including tauroursodeoxycholic acid (TUDCA) and GUDCA. We found increased expression of several main BA transporters in the ileum and liver. BA kinetic experiment showed that feeding UDCA shortened cycling time of BA and accelerated BA enterohepatic circulation. Additionally, we found evidence that the effect of UDCA administration on accelerating BA enterohepatic circulation was due to the inhibition of farnesoid X receptor (FXR) signalling in the ileum and FGF15/19 in the liver. Oral administration of UDCA produced a unique BA profile with high-abundance TUDCA and GUDCA and significantly accelerated BA enterohepatic circulation through the inhibition of intestinal FXR signalling and reduced level of FGF15/19, which in turn, induced the expression of BA transporters in the liver. These findings highlight a critical role for UDCA in maintaining the homeostasis of BA enterohepatic circulation in vivo.

Yangonin protects against estrogen–induced cholestasis in a farnesoid X receptor-dependent manner

Estrogen–induced cholestasis is a common etiology of hepatic diseases in women with contraceptives administration, pregnancy or hormone replacement therapy. Farnesoid X receptor (FXR) is a member of nuclear receptor super family of ligand-activated transcription factors that is highly expressed in liver. FXR is acknowledged to contribute to the bile acid homeostasis, as well as the pathogenesis and progression of cholestasis. Specific targeting of FXR is an innovative approach for the treatment of cholestasis. The current study aimed to verify the anti-cholestasis effect of yangonin that is a natural product isolated from Kava via FXR signaling pathway in vivo and in vitro. The analyses of FXR gain- or loss-of-function were performed. Yangonin treatment ameliorates estrogen-induced cholestasis through increasing bile flow and biliary bile acid output. The mechanisms were an induction in the hepatic efflux transporters (Bsep and Mrp2) and an inhibition in hepatic uptake transporter (Ntcp) by yangonin. Likewise, yangonin through repressing Cyp7a1, Cyp8b1 and inducing Sult2a1 expression suppressed bile acid synthesis and promoted bile acid metabolism. Furthermore, yangonin improved estrogen–induced inflammatory cell infiltration and the inflammation gene expression. In vitro experiments further consolidated that yangonin alleviated estrogen–caused cholestasis via FXR activation. Noteworthily, the effects of yangonin were enhanced by FXR expression plasmids but abrogated by FXR siRNA. In conclusion, yangonin alleviates estrogen–induced cholestasis, due to FXR-mediated gene regulation.

Trimethylamine N-Oxide Aggravates Liver Steatosis through Modulation of Bile Acid Metabolism and Inhibition of Farnesoid X Receptor Signaling in Nonalcoholic Fatty Liver Disease.

Trimethylamine N-oxide (TMAO), the metabolite of choline generated by gut microbiota, is associated with nonalcoholic fatty liver disease (NAFLD) and could influence bile acid (BA) metabolism. However, whether TMAO aggravates liver steatosis by modulating BA metabolism and the related mechanisms has not been investigated. A case-control study including biopsy-proven NAFLD patients (n=34) and controls (n=14) is conducted to determine the correlation between TMAO and BA metabolism. Serum levels of total BA and the percentage of farnesoid X receptor (FXR)-antagonistic BA species are markedly higher in NAFLD patients than in the controls. Serum levels of TMAO positively correlated with the serum levels of total BA and hepatic mRNA expression of cholesterol 7 alpha hydroxylase (CYP7A1). In a murine model, it is found that 18 weeks administration of TMAO impairs liver function and increases hepatic triglyceride accumulation and lipogenesis in mice fed with a high-fat diet. TMAO increases BA synthesis and shifted hepatic BA composition toward FXR-antagonistic activity. Knockdown of CYP7A1 via small interfering RNA or activation of FXR by GW4064 blocks the effect of TMAO-induced lipogenesis in palmitic acid-treated HepG2 cells. TMAO aggravates liver steatosis by suppressing BA-mediated hepatic FXR signaling.

Enhanced Microbial Bile Acid Deconjugation and Impaired Ileal Uptake in Pregnancy Repress Intestinal Regulation of Bile Acid Synthesis.

Pregnancy is associated with progressive hypercholanemia, hypercholesterolemia, and hypertriglyceridemia, which can result in metabolic disease in susceptible women. Gut signals modify hepatic homeostatic pathways, linking intestinal content to metabolic activity. We sought to identify whether enteric endocrine signals contribute to raised serum bile acids observed in human and murine pregnancies, by measuring fibroblast growth factor (FGF) 19/15 protein and mRNA levels, and 7α‐hydroxy‐4‐cholesten‐3‐one. Terminal ileal farnesoid X receptor (FXR)‐mediated gene expression and apical sodium bile acid transporter (ASBT) protein concentration were measured by qPCR and western blotting. Shotgun whole‐genome sequencing and ultra‐performance liquid chromatography tandem mass spectrometry were used to determine the cecal microbiome and metabonome. Targeted and untargeted pathway analyses were performed to predict the systemic effects of the altered metagenome and metabolite profiles. Dietary CA supplementation was used to determine whether the observed alterations could be overcome by intestinal bile acids functioning as FXR agonists. Human and murine pregnancy were associated with reduced intestinal FXR signaling, with lower FGF19/15 and resultant increased hepatic bile acid synthesis. Terminal ileal ASBT protein was reduced in murine pregnancy. Cecal bile acid conjugation was reduced in pregnancy because of elevated bile salt hydrolase‐producing Bacteroidetes. CA supplementation induced intestinal FXR signaling, which was not abrogated by pregnancy, with strikingly similar changes to the microbiota and metabonome as identified in pregnancy. Conclusion: The altered intestinal microbiota of pregnancy enhance bile acid deconjugation, reducing ileal bile acid uptake and lowering FXR induction in enterocytes. This exacerbates the effects mediated by reduced bile acid uptake transporters in pregnancy. Thus, in pregnant women and mice, there is reduced FGF19/15‐mediated hepatic repression of hepatic bile acid synthesis, resulting in hypercholanemia.

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

Non-alcoholic steatohepatitis (NASH) is a progressive, chronic, liver disease whose prevalence is growing worldwide. Despite several agents being under development for treating NASH, there are no drugs currently approved. The Farnesoid-x-receptor (FXR) and the G-protein coupled bile acid receptor 1 (GPBAR1), two bile acid activated receptors, have been investigated for their potential in treating NASH. Here we report that BAR502, a steroidal dual ligand for FXR/GPBAR1, attenuates development of clinical and liver histopathology features of NASH in mice fed a high fat diet (HFD) and fructose (F). By RNAseq analysis of liver transcriptome we found that BAR502 restores FXR signaling in the liver of mice feed HFD–F, and negatively regulates a cluster of genes including Srebf1 (Srepb1c) and its target genes—fatty acid synthase (Fasn) and Cell death-inducing DFF45-like effector (CIDE) genes, Cidea and Cidec—involved in lipid droplets formation and triglycerides storage in hepatocytes. Additionally, BAR502 increased the intestinal expression of Fgf15 and Glp1 and energy expenditure by white adipose tissues. Finally, exposure to BAR502 reshaped the intestinal microbiota by increasing the amount of Bacteroidaceae. In conclusion, we have shown that dual FXR/GPBAR1 agonism might have utility in treatment of NASH.