Bile acid receptor agonist GW4064 regulates PPARγ coactivator-1α expression through estrogen receptor-related receptor α

Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) is induced in energy-starved conditions and is a key regulator of energy homeostasis. This makes PGC-1α an attractive therapeutic target for metabolic syndrome and diabetes. In our effort to identify new regulators of PGC-1α expression, we found that GW4064, a widely used synthetic agonist for the nuclear bile acid receptor [farnesoid X receptor (FXR)] strongly enhances PGC-1α promoter reporter activity, mRNA, and protein expression. This induction in PGC-1α concomitantly enhances mitochondrial mass and expression of several PGC-1α target genes involved in mitochondrial function. Using FXR-rich or FXR-nonexpressing cell lines and tissues, we found that this effect of GW4064 is not mediated directly by FXR but occurs via activation of estrogen receptor-related receptor α (ERRα). Cell-based, biochemical and biophysical assays indicate GW4064 as an agonist of ERR proteins. Interestingly, FXR disruption alters GW4064 induction of PGC-1α mRNA in a tissue-dependent manner. Using FXR-null [FXR knockout (FXRKO)] mice, we determined that GW4064 induction of PGC-1α expression is not affected in oxidative soleus muscles of FXRKO mice but is compromised in the FXRKO liver. Mechanistic studies to explain these differences revealed that FXR physically interacts with ERR and protects them from repression by the atypical corepressor, small heterodimer partner in liver. Together, this interplay between ERRα-FXR-PGC-1α and small heterodimer partner offers new insights into the biological functions of ERRα and FXR, thus providing a knowledge base for therapeutics in energy balance-related pathophysiology.     

Farnesoid X receptor protects liver cells from apoptosis induced by serum deprivation in vitro and fasting in vivo

The farnesoid X receptor (FXR) is a key metabolic regulator in the liver by maintaining the homeostasis of liver metabolites. Recent findings suggest that FXR may have a much broader function in liver physiology and pathology. In the present work, we identify a novel role of FXR in protecting liver cell from apoptosis induced by nutritional withdrawal including serum deprivation in vitro or starvation in vivo. Two FXR ligands, chenodeoxycholic acid (CDCA) and GW4064, rescued HepG2 cells from serum deprivation-induced apoptosis in a dose-dependent manner. This effect of FXR on apoptotic suppression was compromised when FXR was knocked down by short interfering RNA. Similarly, the effects of both CDCA and GW4064 were abolished after inhibition of the MAPK pathway by a specific inhibitor of MAPK kinase 1/2. Immunoblotting results indicated that FXR activation by CDCA and GW4064 induced ERK1/2 phosphorylation, which was attenuated by serum deprivation. In vivo, FXR(-/-) mice exhibited an exacerbated liver apoptosis and lower levels of phosphorylated-ERK1/2 compared to wild-type mice after starvation. In conclusion, our results suggest a novel role of FXR in modulating liver cell apoptosis.     

Inhibition of endothelin-1-mediated contraction of hepatic stellate cells by FXR ligand

Activation of hepatic stellate cells (HSCs) plays an important role in the development of cirrhosis through the increased production of collagen and the enhanced contractile response to vasoactive mediators such as endothelin-1 (ET-1). The farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily that is highly expressed in liver, kidneys, adrenals, and intestine. FXR is also expressed in HSCs and activation of FXR in HSCs is associated with significant decreases in collagen production. However, little is known about the roles of FXR in the regulation of contraction of HSCs. We report in this study that treatment of quiescent HSCs with GW4064, a synthetic FXR agonist, significantly inhibited the HSC transdifferentiation, which was associated with an inhibition of the upregulation of ET-1 expression. These GW4064-treated cells also showed reduced contractile response to ET-1 in comparison to HSCs without GW4064 treatment. We have further shown that GW4064 treatment inhibited the ET-1-mediated contraction in fully activated HSCs. To elucidate the potential mechanism we showed that GW4064 inhibited ET-1-mediated activation of Rho/ROCK pathway in activated HSCs. Our studies unveiled a new mechanism that might contribute to the anti-cirrhotic effects of FXR ligands.     

The farnesoid X receptor modulates adiposity and peripheral insulin sensitivity in mice

The farnesoid X receptor (FXR) is a bile acid (BA)-activated nuclear receptor that plays a major role in the regulation of BA and lipid metabolism. Recently, several studies have suggested a potential role of FXR in the control of hepatic carbohydrate metabolism, but its contribution to the maintenance of peripheral glucose homeostasis remains to be established. FXR-deficient mice display decreased adipose tissue mass, lower serum leptin concentrations, and elevated plasma free fatty acid levels. Glucose and insulin tolerance tests revealed that FXR deficiency is associated with impaired glucose tolerance and insulin resistance. Moreover, whole-body glucose disposal during a hyperinsulinemic euglycemic clamp is decreased in FXR-deficient mice. In parallel, FXR deficiency alters distal insulin signaling, as reflected by decreased insulin-dependent Akt phosphorylation in both white adipose tissue and skeletal muscle. Whereas FXR is not expressed in skeletal muscle, it was detected at a low level in white adipose tissue in vivo and induced during adipocyte differentiation in vitro. Moreover, mouse embryonic fibroblasts derived from FXR-deficient mice displayed impaired adipocyte differentiation, identifying a direct role for FXR in adipocyte function. Treatment of differentiated 3T3-L1 adipocytes with the FXR-specific synthetic agonist GW4064 enhanced insulin signaling and insulin-stimulated glucose uptake. Finally, treatment with GW4064 improved insulin resistance in genetically obese ob/ob mice in vivo. Although the underlying molecular mechanisms remain to be unraveled, these results clearly identify a novel role of FXR in the regulation of peripheral insulin sensitivity and adipocyte function. This unexpected function of FXR opens new perspectives for the treatment of type 2 diabetes.     

激活FXR抑制结肠癌细胞浸润转移及MMP-7的表达

目的:探讨法尼酯X受体(FXR)特异性激动剂GW4064抑制结肠癌细胞浸润转移的机制。方法:在体外培养人结肠癌细胞HT-29,应用GW4064作用于结肠癌细胞,以四唑氮蓝还原法(MTT)检测细胞活性的变化。用transwell小室研究结肠癌细胞的迁移及浸润。用RT-PCR检测FXRm RNA及MMP-7mRNA表达的变化,用western blot检测FXR及MMP-7蛋白表达的变化。结果:MTT结果显示GW4064作用于人结直肠HT-29细胞的生长抑制率呈浓度依赖性;transwell小室结果显示GW4064抑制结肠癌细胞的浸润转移,与对照组相比,差异具有统计学意义(P0.05),RT-PCR及Western blot显示GW4064促进FXR m RNA及蛋白表达,抑制MMP-7mRNA及蛋白的表达,与对照组相比差异有统计学意义(P0.05)。结论:GW4064抑制结肠癌细胞的生长及转移,上调HT-29细胞FXR m RNA及蛋白的表达,下调HT-29细胞MMP-7 m RNA及蛋白的表达。FXR被激活后抑制结肠癌细胞转移,MMP-7可能是其作用通路之一。     

Src-Mediated Cross-Talk between Farnesoid X and Epidermal Growth Factor Receptors Inhibits Human Intestinal Cell Proliferation and Tumorigenesis

Besides its essential role in controlling bile acid and lipid metabolism, the farnesoid X receptor (FXR) protects against intestinal tumorigenesis by promoting apoptosis and inhibiting cell proliferation. However, the mechanisms underlying these anti-proliferative actions of FXR remain to be elucidated. In the present study, we examined the effects of FXR activation (FXR overexpression and treatment with an FXR agonist GW4064) and inactivation (treatment with FXR siRNA and an FXR antagonist guggulsterone) on colon cancer cell proliferation in vitro using human colon cancer cell lines (H508, SNU-C4 and HT-29) and in vivo using xenografts in nude mice. Blocking FXR activity with guggulsterone stimulated time- and dose-dependent EGFR (Tyr845) phosphorylation and ERK activation. In contrast, FXR overexpression and activation with GW4064 attenuated cell proliferation by down-regulating EGFR (Tyr845) phosphorylation and ERK activation. Treatment with guggulsterone and GW4064 also caused dose-dependent changes in Src (Tyr416) phosphorylation. In stably-transfected human colon cancer cells, overexpression of FXR reduced EGFR, ERK, Src phosphorylation and cell proliferation, and in nude mice attenuated the growth of human colon cancer xenografts (64% reduction in tumor volume; 47% reduction in tumor weight; both P<0.01). Moreover, guggulsterone-induced EGFR and ERK phosphorylation and cell proliferation were abolished by inhibiting activation of Src, EGFR and MEK. Collectively these data support the novel conclusion that in human colon cancer cells Src-mediated cross-talk between FXR and EGFR modulates ERK phosphorylation, thereby regulating intestinal cell proliferation and tumorigenesis.     

In vivo imaging of farnesoid X receptor activity reveals the ileum as the primary bile acid signaling tissue

We generated and characterized a firefly luciferase reporter mouse for the nuclear receptor farnesoid X receptor (FXR). This FXR reporter mouse has basal luciferase expression in the terminal ileum, an organ with well-characterized FXRalpha signaling. In vivo luciferase activity reflected the diurnal activity pattern of the mouse, and is regulated by both natural (bile acids, chenodeoxycholic acid) and synthetic (GW4064) FXRalpha ligands. Moreover, in vivo and in vitro analysis showed luciferase activity after GW4064 administration in the liver, kidney, and adrenal gland, indicating that FXRalpha signaling is functional in these tissues. Hepatic luciferase activity was robustly induced in cholestatic mice, showing that FXRalpha signaling pathways are activated in this disease. In conclusion, we have developed an FXR reporter mouse that is useful to monitor FXRalpha signaling in vivo in health and disease. The use of this animal could facilitate the development of new therapeutic compounds that target FXRalpha in a tissue-specific manner.     

Bile acid signaling through FXR induces intracellular adhesion molecule-1 expression in mouse liver and human hepatocytes

Previous studies have demonstrated a dramatic induction of inflammatory gene expression in livers from mice fed a high-fat, high-cholesterol diet containing cholate after 3-5 wk. To determine the contribution of cholate in mediating these inductions, C57BL/6 mice were fed a chow diet supplemented with increasing concentrations of cholic acid (CA) for 5 days. A dose-dependent induction in the hepatic levels of TNF-alpha, VCAM-1, ICAM-1, and SAA-2 mRNA were observed. As positive controls, a dose-dependent repression of cholesterol 7alpha-hydroxylase and a dose-dependent induction of small heterodimer partner (SHP) expression were also observed, suggesting that farnesoid X receptor (FXR) was activated. In addition, ICAM-1 and SHP mRNA levels were also induced in primary human hepatocytes when treated with chenodeoxycholic acid or GW4064, a FXR-selective agonist. The involvement of FXR in CA-induced inflammatory gene expression was further investigated in the human hepatic cell line HepG2. Both ICAM-1 and SHP expression were induced in a dose- and time-dependent manner by treatment with the FXR-selective agonist GW4064. Moreover, the induction of ICAM-1 by GW4064 was inhibited by the FXR antagonist guggulsterone or with transfection of FXR siRNA. Finally, the activity of FXR was mapped to a retinoic acid response element (RARE) site containing an imbedded farnesoid X response element (FXRE) on the human ICAM-1 promoter and FXR and retinoid X receptor were demonstrated to bind to this site. Finally, FXR-mediated activation of ICAM-1 could be further enhanced by TNF-alpha cotreatment in hepatocytes, suggesting a potential cooperation between cytokine and bile acid-signaling pathways during hepatic inflammatory events.     

A potential role of GW4064 to inhibit gut bacterial overgrowth by activating FXR in suppression of ethanol-induced liver injury

Hepatic parenchymal and nonparenchymal cells are highly susceptible to ethanol and its metabolites, and excessive alcohol consumption results in damage to the liver. Ethanol induces an increased prevalence for bacterial overgrowth in the small intestine and translocation of endotoxin into the portal blood. Some studies have pointed to a role for activation of Kupffer cells by gut bacteria-derived endotoxin as a primary event in mechanisms of alcoholic liver injury (ALD). GW4064, a potent farnesoid X receptor (FXR) agonist, has been developed as a hepatoprotective agent, and has been used in animal models of a variety of liver diseases. At the same time, previous experimental results showed that BAs and GW4064 inhibit bacterial overgrowth in the small intestine. It is logical to postulate that GW4064 may control or alleviate the ethanol-induced liver injury through inhibiting gut bacterial overgrowth. GW4064 activates FXR, which induces the expression of several genes with potential functions in mucosal defense to prevent intestinal bacteria overgrowth and translocation into the circulation induced by ethanol, and then will alleviate ethanol-induced liver injury. The hypothesis will provide the brand-new direction that we may prevent and treat ALD by using GW4064 through activating FXR to control gut bacteria overgrowth.     

Secondary (iso)BAs cooperate with endogenous ligands to activate FXR under physiological and pathological conditions

IsoBAs, stereoisomers of primary and secondary BAs, are found in feces and plasma of human individuals. BA signaling via the nuclear receptor FXR is crucial for regulation of hepatic and intestinal physiology/pathophysiology. Aim: Investigate the ability of BA-stereoisomers to bind and modulate FXR under physiological/pathological conditions. Methods: Expression-profiling, luciferase-assays, fluorescence-based coactivator-association assays, administration of (iso)-BAs to WT and cholestatic mice. Results: Compared to CDCA/isoCDCA, administration of DCA/isoDCA, UDCA/isoUDCA only slightly increased mRNA expression of FXR target genes; the induction was more evident looking at pre-mRNAs. Notably, almost 50% of isoBAs were metabolized to 3-oxo-BAs within 4 h in cell-based assays, making it difficult to study their actions. FRET-based real-time monitoring of FXR activity revealed that isoCDCA>CDCA stimulated FXR, and isoDCA and isoUDCA allowed fully activated FXR to be re-stimulated by a second dose of GW4064. In vivo co-administration of a single dose of isoBAs followed by GW4064 cooperatively activated FXR, as did feeding of UDCA in a background of endogenous FXR ligands. However, in animals with biliary obstruction and concomitant loss of intestinal BAs, UDCA was unable to increase intestinal Fgf15. In contrast, mice with an impaired enterohepatic circulation of BAs (Asbt?/?, Ostα?/?), administration of UDCA was still able to induce ileal Fgf15 and repress hepatic BA-synthesis, arguing that UDCA is only effective in the presence of endogenous FXR ligands. Conclusion: Secondary (iso)BAs cooperatively activate FXR in the presence of endogenous BAs, which is important to consider in diseases linked to disturbances in BA enterohepatic cycling.     

Lithocholic acid decreases expression of bile salt export pump through farnesoid X receptor antagonist activity

Bile salt export pump (BSEP) is a major bile acid transporter in the liver. Mutations in BSEP result in progressive intrahepatic cholestasis, a severe liver disease that impairs bile flow and causes irreversible liver damage. BSEP is a target for inhibition and down-regulation by drugs and abnormal bile salt metabolites, and such inhibition and down-regulation may result in bile acid retention and intrahepatic cholestasis. In this study, we quantitatively analyzed the regulation of BSEP expression by FXR ligands in primary human hepatocytes and HepG2 cells. We demonstrate that BSEP expression is dramatically regulated by ligands of the nuclear receptor farnesoid X receptor (FXR). Both the endogenous FXR agonist chenodeoxycholate (CDCA) and synthetic FXR ligand GW4064 effectively increased BSEP mRNA in both cell types. This up-regulation was readily detectable at as early as 3 h, and the ligand potency for BSEP regulation correlates with the intrinsic activity on FXR. These results suggest BSEP as a direct target of FXR and support the recent report that the BSEP promoter is transactivated by FXR. In contrast to CDCA and GW4064, lithocholate (LCA), a hydrophobic bile acid and a potent inducer of cholestasis, strongly decreased BSEP expression. Previous studies did not identify LCA as an FXR antagonist ligand in cells, but we show here that LCA is an FXR antagonist with partial agonist activity in cells. In an in vitro co-activator association assay, LCA decreased CDCA- and GW4064-induced FXR activation with an IC(50) of 1 microm. In HepG2 cells, LCA also effectively antagonized GW4064-enhanced FXR transactivation. These data suggest that the toxic and cholestatic effect of LCA in animals may result from its down-regulation of BSEP through FXR. Taken together, these observations indicate that FXR plays an important role in BSEP gene expression and that FXR ligands may be potential therapeutic drugs for intrahepatic cholestasis.     

Farnesoid X receptor activates transcription of the phospholipid pump MDR3

The human multidrug resistance gene MDR3 encodes a P-glycoprotein that belongs to the ATP-binding cassette transporter family (ABCB4). MDR3 is a critical trans-locator for phospholipids across canalicular membranes of hepatocytes, evidenced by the fact that human MDR3 deficiencies result in progressive familial intrahepatic cholestasis type III. It has been reported previously that MDR3 expression is modulated by hormones, cellular stress, and xenobiotics. Here we show that the MDR3 gene is trans-activated by the farnesoid X receptor (FXR) via a direct binding of FXR/retinoid X receptor alpha heterodimers to a highly conserved inverted repeat element (a FXR response element) at the distal promoter (-1970 to -1958). In FXR trans-activation assays, both the endogenous FXR agonist chenodeoxycholate and the synthetic agonist GW4064 activated the MDR3 promoter. Deletion or mutation of this inverted repeat element abolished FXR-mediated MDR3 promoter activation. Consistent with these data, MDR3 mRNA was significantly induced by both chenodeoxycholate and GW4064 in primary human hepatocytes in time- and dose-dependent fashions. In conclusion, we demonstrate that MDR3 expression is directly up-regulated by FXR. These results, together with the previous report that the bile salt export pump is a direct FXR target, suggest that FXR coordinately controls secretion of bile salts and phospholipids. Results of this study further support the notion that FXR is a master regulator of lipid metabolism.     

FXR regulates organic solute transporters alpha and beta in the adrenal gland, kidney, and intestine

Expression of the farnesoid X receptor (FXR; NR1H4) is limited to the liver, intestine, kidney, and adrenal gland. However, the role of FXR in the latter two organs is unknown. In the current study, we performed microarray analysis using RNA from H295R cells infected with constitutively active FXR. Several putative FXR target genes were identified, including the organic solute transporters alpha and beta (OSTalpha and OSTbeta). Electromobility shift assays and promoter-reporter studies identified functional farnesoid X receptor response elements (FXREs) in the promoters of both human genes. These FXREs are conserved in both mouse genes. Treatment of wild-type mice with 3-(2,6-dichlorophenyl)-4-(3'-carboxy-2-chloro-stilben-4-yl)-oxymethyl-5-isopropyl-isoxazole (GW4064), a synthetic FXR agonist, induced OSTalpha and OSTbeta mRNAs in the intestine and kidney. Both mRNAs were also induced when wild-type, but not FXR-deficient (FXR-/-), adrenals were cultured in the presence of GW4064. OSTalpha and OSTbeta mRNA levels were also induced in the adrenals and kidneys of wild-type, but not FXR-/-, mice after the increase of plasma bile acids in response to the hepatotoxin alpha-naphthylisothiocyanate. Finally, overexpression of human OSTalpha and OSTbeta facilitated the uptake of conjugated chenodeoxycholate and the activation of FXR target genes. These results demonstrate that OSTalpha and OSTbeta are novel FXR target genes that are expressed in the adrenal gland, kidney, and intestine.     

Human kininogen gene is transactivated by the farnesoid X receptor

Human kininogen belongs to the plasma kallikreinkinin system. High molecular weight kininogen is the precursor for two-chain kinin-free kininogen and bradykinin. It has been shown that the two-chain kinin-free kininogen has the properties of anti-adhesion, anti-platelet aggregation, and anti-thrombosis, whereas bradykinin is a potent vasodilator and mediator of inflammation. In this study we show that the human kininogen gene is strongly up-regulated by agonists of the farnesoid X receptor (FXR), a nuclear receptor for bile acids. In primary human hepatocytes, both the endogenous FXR agonist chenodeoxycholate and synthetic FXR agonist GW4064 increased kininogen mRNA with a maximum induction of 8-10-fold. A more robust induction of kininogen expression was observed in HepG2 cells, where kininogen mRNA was increased by chenodeoxycholate or GW4064 up to 130-140-fold as shown by real time PCR. Northern blot analysis confirmed the up-regulation of kininogen expression by FXR agonists. To determine whether kininogen is a direct target of FXR, we examined the sequence of the kininogen promoter and identified a highly conserved FXR response element (inverted repeat, IR-1) in the proximity of the kininogen promoter (-66/-54). FXR/RXRalpha heterodimers specifically bind to this IR-1. A construct of a minimal promoter with the luciferase reporter containing this IR-1 was transactivated by FXR. Deletion or mutation of this IR-1 abolished FXR-mediated promoter activation, indicating that this IR-1 element is responsible for the promoter transactivation by FXR. We conclude that kininogen is a novel and direct target of FXR, and bile acids may play a role in the vasodilation and anti-coagulation processes.     

Inhibitory effects of bile acids and synthetic farnesoid X receptor agonists on rotavirus replication

Rotaviruses (group A rotaviruses) are the most important cause of severe gastroenteritis in infants and children worldwide. Currently, an antiviral drug is not available and information on therapeutic targets for antiviral development is limited for rotavirus infection. Previously, it was shown that lipid homeostasis is important in rotavirus replication. Since farnesoid X receptor (FXR) and its natural ligands bile acids (such as chenodeoxycholic acid [CDCA]) play major roles in cholesterol and lipid homeostasis, we examined the effects of bile acids and synthetic FXR agonists on rotavirus replication in association with cellular lipid levels. In a mouse model of rotavirus infection, effects of oral administration of CDCA on fecal rotavirus shedding were investigated. The results demonstrate the following. First, the intracellular contents of triglycerides were significantly increased by rotavirus infection. Second, CDCA, deoxycholic acid (DCA), and other synthetic FXR agonists, such as GW4064, significantly reduced rotavirus replication in cell culture in a dose-dependent manner. The reduction of virus replication correlated positively with activation of the FXR pathway and reduction of cellular triglyceride contents (r(2) = 0.95). Third, oral administration of CDCA significantly reduced fecal virus shedding in mice (P < 0.05). We conclude that bile acids and FXR agonists play important roles in the suppression of rotavirus replication. The inhibition mechanism is proposed to be the downregulation of lipid synthesis induced by rotavirus infection.     

Bile Acid Binding Resin Improves Metabolic Control through the Induction of Energy Expenditure

Background

Besides well-established roles of bile acids (BA) in dietary lipid absorption and cholesterol homeostasis, it has recently become clear that BA is also a biological signaling molecule. We have shown that strategies aimed at activating TGR5 by increasing the BA pool size with BA administration may constitute a significant therapeutic advance to combat the metabolic syndrome and suggest that such strategies are worth testing in a clinical setting. Bile acid binding resin (BABR) is known not only to reduce serum cholesterol levels but also to improve glucose tolerance and insulin resistance in animal models and humans. However, the mechanisms by which BABR affects glucose homeostasis have not been established. We investigated how BABR affects glycemic control in diet-induced obesity models.

Methods and Findings

We evaluated the metabolic effect of BABR by administrating colestimide to animal models for the metabolic syndrome. Administration of BABR increased energy expenditure, translating into significant weight reduction and insulin sensitization. The metabolic effects of BABR coincide with activation of cholesterol and BA synthesis in liver and thermogenesis in brown adipose tissue. Interestingly, these effects of BABR occur despite normal food intake and triglyceride absorption. Administration of BABR and BA had similar effects on BA composition and thermogenesis, suggesting that they both are mediated via TGR5 activation.

Conclusion

Our data hence suggest that BABR could be useful for the management of the impaired glucose tolerance of the metabolic syndrome, since they not only lower cholesterol levels, but also reduce obesity and improve insulin resistance.