The relationship between bile acid concentration,glucagon-like-peptide 1, fibroblast growth factor 15 and bile acid receptors in rats during progression of glucose intolerance

Background

Recent studies show that bile acids are involved in glucose and energy homeostasis through activation of G protein coupled membrane receptor (TGR5) and farnesoid X receptor (FXR). A few researches have explored changes of TGR5 and FXR in animals with impaired glucose regulation. This study aimed to observe changes of plasma total bile acids (TBA), glucagon-like-peptide 1 (GLP-1), fibroblast growth factor 15 (FGF15), intestinal expressions of TGR5 and FXR, and correlations between them in rats with glucose intolerance.

Methods

Besides plasma fasting glucose, lipid, TBAs, alanine transaminase (ALT), active GLP-1(GLP-1A) and FGF15, a postprandial meal test was used to compare responses in glucose, insulin and GLP-1A among groups. The expressions of TGR5 and FXR in distal ileum and ascending colon were quantified by real-time PCR and western blot.

Results

TGR5 expression was significantly decreased in distal ileum in DM group compared to other groups, and TGR5 and FXR expressions in ascending colon were also decreased in DM group compared to other groups. Correlation analysis showed correlations between TBA and GLP-1A or FGF15. GLP-1A was correlated with TGR5 mRNA expression in colon, and FGF15 was correlated with FXR mRNA expression in colon.

Conclusions

These results indicates that bile acid-TGR5/FXR axis contributes to glucose homeostasis.

     

Identification of a lead pharmacophore for the development of potent nuclear receptor modulators as anticancer and X syndrome disease therapeutic agents

A series of tetrahydroisoquinoline-N-phenylamide derivatives were designed, synthesized, and tested for their relative binding affinity and antagonistic activity against androgen receptor (AR). Compound 1b (relative binding affinity, RBA = 6.4) and 1h (RBA = 12.6) showed higher binding affinity than flutamide (RBA = 1), a potent AR antagonist. These two compounds also exerted optimal antagonistic activity against AR in reporter assays. The derivatives were also tested for their activities against another nuclear receptor, farnesoid x receptor (FXR), with most compounds acting as weak antagonists, however, compound 1h behaved as a FXR agonist with activity slightly less than that of chenodeoxycholic acid (CDCA), a natural FXR agonist.     

Polyunsaturated fatty acids are FXR ligands and differentially regulate expression of FXR targets

Polyunsaturated fatty acids (PUFAs) have been previously reported as agonists of peroxisome proliferatoractivated receptor and antagonists of the liver X receptor. The activities on these two nuclear receptors have been attributed to their beneficial effects such as improvement of dyslipidemia and insulin sensitivity and decrease of hepatic lipogenesis. Here we report that PUFAs are ligands of farnesoid X receptor (FXR), a nuclear receptor for bile acids. In a conventional FXR binding assay, arachidonic acid (AA, 20:4), docosahexaenoic acid (DA, 22:6), and linolenic acid (LA, 18:3) had an affinity of 2.6, 1.5, and 3.5 microM, respectively. In a cell-free coactivator association assay, AA, DA, and LA decreased FXR agonist-induced FXR activation with IC(50)s ranging from 0.9 to 4.7 microM. In HepG2 cells, PUFAs regulated the expression of two FXR targets, BSEP and kininogen, in an opposite fashion, although both genes were transactivated by FXR. All three PUFAs dose-dependently enhanced FXR agonist-induced BSEP expression but decreased FXR agonist-induced human kininogen mRNA. Saturated fatty acids such as stearic acid (SA, 18:0) and palmitic acid (PA, 16:0) did not bind to FXR and did not change BSEP or kininogen expression. The pattern of BSEP and kininogen regulation by PUFAs is closely similar to that of the guggulsterone, previously reported as a selective bile acid receptor modulator. Our results suggest that PUFAs may belong to the same class of FXR ligands as guggulsterone, and that the selective regulation of FXR targets may contribute to the beneficial effects of PUFAs in lipid metabolism.     

The p300 acetylase is critical for ligand-activated farnesoid X receptor (FXR) induction of SHP

The primary bile acid receptor farnesoid X receptor (FXR) maintains lipid and glucose homeostasis by regulating expression of numerous bile acid-responsive genes, including an orphan nuclear receptor and metabolic regulator SHP. Using SHP as a model gene, we studied how FXR activity is regulated by p300 acetylase. FXR interaction with p300 and their recruitment to the SHP promoter and acetylated histone levels at the promoter were increased by FXR agonists in mouse liver and HepG2 cells. In contrast, p300 recruitment and acetylated histones at the promoter were not detected in FXR-null mice. p300 directly interacted with and acetylated FXR in vitro. Overexpression of p300 wild type increased, whereas a catalytically inactive p300 mutant decreased, acetylated FXR levels and FXR transactivation in cells. While similar results were observed with a related acetylase, CBP, GCN5 did not enhance FXR transactivation, and its recruitment to the promoter was not increased by FXR agonists, suggesting functional specificity of acetylases in FXR signaling. Down-regulation of p300 by siRNA decreased acetylated FXR and acetylated histone levels, and occupancy of FXR at the promoter, resulting in substantial inhibition of SHP expression. These results indicate that p300 acts as a critical coactivator of FXR induction of SHP by acetylating histones at the promoter and FXR itself. Surprisingly, p300 down-regulation altered expression of other metabolic FXR target genes involved in lipoprotein and glucose metabolism, such that beneficial lipid and glucose profiles would be expected. These unexpected findings suggest that inhibition of hepatic p300 activity may be beneficial for treating metabolic diseases.     

Structural optimization and in vitro profiling of N-phenylbenzamide-based farnesoid X receptor antagonists

Activation of the nuclear farnesoid X receptor (FXR) which acts as cellular bile acid sensor has been validated as therapeutic strategy to counter liver disorders such as non-alcoholic steatohepatitis by the clinical efficacy of obeticholic acid. FXR antagonism, in contrast, is less well studied and potent small molecule FXR antagonists are rare. Here we report the systematic optimization of a novel class of FXR antagonists towards low nanomolar potency. The most optimized compound antagonizes baseline and agonist induced FXR activity in a full length FXR reporter gene assay and represses intrinsic expression of FXR regulated genes in hepatoma cells. With this activity and a favorable toxicity-, stability- and selectivity-profile it appears suitable to further study FXR antagonism in vitro and in vivo.     

Histone H3K4 trimethylation by MLL3 as part of ASCOM complex is critical for NR activation of bile acid transporter genes and is downregulated in cholestasis

The nuclear receptor Farnesoid x receptor (FXR) is a critical regulator of multiple genes involved in bile acid homeostasis. The coactivators attracted to promoters of FXR target genes and epigenetic modifications that occur after ligand binding to FXR have not been completely defined, and it is unknown whether these processes are disrupted during cholestasis. Using a microarray, we identified decreased expression of mixed lineage leukemia 3 (MLL3), a histone H3 lysine 4 (H3K4) lysine methyl transferase at 1 and 3 days of post-common bile duct ligation (CBDL) in mice. Chromatin immunoprecipitation analysis (ChIP) analysis revealed that H3K4me3 of transporter promoters by MLL3 as part of activating signal cointegrator-2 -containing complex (ASCOM) is essential for activation of bile salt export pump (BSEP), multidrug resistance associated protein 2 (MRP2), and sodium taurocholate cotransporting polypeptide (NTCP) genes by FXR and glucocorticoid receptor (GR). Knockdown of nuclear receptor coactivator 6 (NCOA6) or MLL3/MLL4 mRNAs by small interfering RNA treatment led to a decrease in BSEP and NTCP mRNA levels in hepatoma cells. Human BSEP promoter transactivation by FXR/RXR was enhanced in a dose-dependent fashion by NCOA6 cDNA coexpression and decreased by AdsiNCOA6 infection in HepG2 cells. GST-pull down assays showed that domain 3 and 5 of NCOA6 (LXXLL motifs) interacted with FXR and that the interaction with domain 5 was enhanced by chenodeoxycholic acid. In vivo ChIP assays in HepG2 cells revealed ligand-dependent recruitment of ASCOM complex to FXR element in BSEP and GR element in NTCP promoters, respectively. ChIP analysis demonstrated significantly diminished recruitment of ASCOM complex components and H3K4me3 to Bsep and Mrp2 promoter FXR elements in mouse livers after CBDL. Taken together, these data show that the "H3K4me3" epigenetic mark is essential to activation of BSEP, NTCP, and MRP2 genes by nuclear receptors and is downregulated in cholestasis.     

Endogenous bile acids are ligands for the nuclear receptor FXR/BAR.

The major metabolic pathway for elimination of cholesterol is via conversion to bile acids. In addition to this metabolic function, bile acids also act as signaling molecules that negatively regulate their own biosynthesis. However, the precise nature of this signaling pathway has been elusive. We have isolated an endogenous biliary component (chenodeoxycholic acid) that selectively activates the orphan nuclear receptor, FXR. Structure-activity analysis defined a subset of related bile acid ligands that activate FXR and promote coactivator recruitment. Finally, we show that ligand-occupied FXR inhibits transactivation from the oxysterol receptor LXR alpha, a positive regulator of cholesterol degradation. We suggest that FXR (BAR) is the endogenous bile acid sensor and thus an important regulator of cholesterol homeostasis.     

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.     

Pharmacological Activation of the Bile Acid Nuclear Farnesoid X Receptor Is Feasible in Patients with Quiescent Crohn's Colitis

Background

The bile acid-activated nuclear receptor Farnesoid X Receptor (FXR) is critical in maintaining intestinal barrier integrity and preventing bacterial overgrowth. Patients with Crohn''s colitis (CC) exhibit reduced ileal FXR target gene expression. FXR agonists have been shown to ameliorate inflammation in murine colitis models. We here explore the feasibility of pharmacological FXR activation in CC.

Methods

Nine patients with quiescent CC and 12 disease controls were treated with the FXR ligand chenodeoxycholic acid (CDCA; 15 mg/kg/day) for 8 days. Ileal FXR activation was assessed in the fasting state during 6 hrs after the first CDCA dose and on day 8, by quantification of serum levels of fibroblast growth factor (FGF) 19. Since FGF19 induces gallbladder (GB) refilling in murine models, we also determined concurrent GB volumes by ultrasound. On day 8 ileal and cecal biopsies were obtained and FXR target gene expression was determined.

Results

At baseline, FGF19 levels were not different between CC and disease controls. After the first CDCA dose, there were progressive increases of FGF19 levels and GB volumes during the next 6 hours in CC patients and disease controls (FGF19: 576 resp. 537% of basal; GB volumes: 190 resp. 178% of basal) without differences between both groups, and a further increase at day 8. In comparison with a separate untreated control group, CDCA affected FXR target gene expression in both CC and disease controls, without differences between both groups.

Conclusions

Pharmacological activation of FXR is feasible in patients with CC. These data provide a rationale to explore the anti-inflammatory properties of pharmacological activation of FXR in these patients.

Trial Registration

TrialRegister.nl NTR2009     

Identification of small molecule antagonists of the human mas-related gene-X1 receptor

The recently identified mas-related-gene (MRG) family of receptors, located primarily in sensory neurons of the dorsal root ganglion, has been implicated in the perception of pain. Thus, antagonists of this class of receptors have been postulated to be useful analgesics. Toward this end, we developed a cell-based beta-lactamase (BLA) reporter gene assay to identify small molecule antagonists of the human MRG-X1 receptor from a library of compounds. Single-cell clones expressing functional receptors were selected using the BLA reporter gene technology. The EC50 for the MRG agonist peptide, BAM15, appeared to be comparable between the BLA assay and the intracellular Ca2+ transient assays in these cells. Ultra high-throughput screening of approximately 1 million compounds in a 1.8-microl cell-based BLA reporter gene assay was conducted in a 3456-well plate format. Compounds exhibiting potential antagonist profile in the BLA assay were confirmed in the second messenger Ca2+ transient assay. A cell-based receptor trafficking assay was used to further validate the mechanism of action of these compounds. Several classes of compounds, particularly the 2,3-disubstituted azabicyclo-octanes, appear to be relatively potent antagonists at the human MRG-X1 receptors, as confirmed by the receptor trafficking assay and radioligand binding studies. Furthermore, the structure-activity relationship reveals that within this class of compounds, the diphenylmethyl moiety is constant at the 2-substituent, whereas the 3-substituent is directly correlated with the antagonist activity of the compound.     

Pyrazolidine-3,5-dione derivatives as potent non-steroidal agonists of farnesoid X receptor: virtual screening, synthesis, and biological evaluation

The identification of a novel pyrazolidine-3,5-dione based scaffold hit compound as Farnesoid X receptor (FXR) partial or full agonist has been accomplished by means of virtual screening techniques. A series of pyrazolidine-3,5-dione derivatives (1a-u and 7) was designed, synthesized, and evaluated by a cell-based luciferase transactivation assay for their agonistic activities against FXR. Most of them showed agonistic potencies and 10 of them (1a, 1b, 1d-f, 1j, 1n, 1t, 5b, and 7) exhibited lower EC(50) values than the reference drug CDCA. Molecular modeling studies for the representative compounds 1a, 1d, 1f, 1j, 1n, 1u, 5b, and 7 were also presented. The novel structural scaffold has provided a new direction for finding potent and selective FXR partial and full agonists (referred to as 'selective bile acid receptor modulators', SBARMs).     

The cholesterol-raising factor from coffee beans, cafestol, as an agonist ligand for the farnesoid and pregnane X receptors

Cafestol, a diterpene present in unfiltered coffee brews such as Scandinavian boiled, Turkish, and cafetière coffee, is the most potent cholesterol-elevating compound known in the human diet. Several genes involved in cholesterol homeostasis have previously been shown to be targets of cafestol, including cholesterol 7alpha-hydroxylase (CYP7A1), the rate-limiting enzyme in bile acid biosynthesis. We have examined the mechanism by which cafestol elevates serum lipid levels. Changes in several lipid parameters were observed in cafestol-treated APOE3Leiden mice, including a significant increase in serum triglyceride levels. Microarray analysis of these mice identified alterations in hepatic expression of genes involved in lipid metabolism and detoxification, many of which are regulated by the nuclear hormone receptors farnesoid X receptor (FXR) and pregnane X receptor (PXR). Further studies demonstrate that cafestol is an agonist ligand for FXR and PXR, and that cafestol down-regulates expression of the bile acid homeostatic genes CYP7A1, sterol 12alpha-hydroxylase, and Na(+)-taurocholate cotransporting polypeptide in the liver of wild-type but not FXR null mice. Cafestol did not affect genes known to be up-regulated by FXR in the liver of wild-type mice, but did increase expression of the positive FXR-target genes intestinal bile acid-binding protein and fibroblast growth factor 15 (FGF15) in the intestine. Because FGF15 has recently been shown to function in an enterohepatic regulatory pathway to repress liver expression of bile acid homeostatic genes, its direct induction in the gut may account for indirect effects of cafestol on liver gene expression. PXR-dependent gene regulation of cytochrome P450 3A11 and other targets by cafestol was also only seen in the intestine. Using a double FXR/PXR knockout mouse model, we found that both receptors contribute to the cafestol-dependent induction of intestinal FGF15 gene expression. In conclusion, cafestol acts as an agonist ligand for both FXR and PXR, and this may contribute to its impact on cholesterol homeostasis.     

5beta-Cholane activators of the farnesol X receptor

The farnesoid X receptor (FXR) is activated by bile acids, natural agonists for this nuclear receptor. FXR-target genes play important roles in cholesterol and lipid metabolism. We have found that a series of 5beta-cholanic acid derivatives, even though without a hydroxyl group or any other substituent on the steroidal rings, can activate FXR more potently than hydroxylated bile acids in a reporter gene assay. The most potent compound among these derivatives, N-methyl-5beta-glycocholanic acid (NMGCA), induces the formation of receptor/coactivator complex in a gel-shift assay and also increases the expression of FXR target genes in human hepatoma HepG2 cells. Furthermore, in rats, NMGCA causes hypolipidemic effects as well as induction of the FXR target genes in liver. Our results suggest that NMGCA and its derivatives are important FXR activators in the study of the physiological functions of FXR and are potentially useful as pharmaceutical agents for treatment of cholesterol and lipid-related diseases.     

FXR, a target for different diseases

Great progress has been made in the understanding of the physiological roles of the nuclear receptor farnesoid X receptor (FXR) during the last several years. Roles for FXR were initially identified in the regulation of bile acid, cholesterol, triglyceride, and glucose metabolism. More recently, our group has identified additional functional roles of FXR. Specifically, we have shown that FXR regulates normal liver regeneration and plays a protective role in liver carcinogenesis. These exciting findings suggest that FXR has a broader role than previously thought, and also highlight potential new opportunities for using FXR as a drug target for different diseases. Here we summarize the latest results from studies on FXR response elements, target genes and functions in different diseases.     

Bile Acid-Induced Arrhythmia Is Mediated by Muscarinic M2 Receptors in Neonatal Rat Cardiomyocytes

Background

Intrahepatic cholestasis of pregnancy (ICP) is a common disease affecting up to 5% of pregnancies and which can cause fetal arrhythmia and sudden intrauterine death. We previously demonstrated that bile acid taurocholate (TC), which is raised in the bloodstream of ICP, can acutely alter the rate and rhythm of contraction and induce abnormal calcium destabilization in cultured neonatal rat cardiomyocytes (NRCM). Apart from their hepatic functions bile acids are ubiquitous signalling molecules with diverse systemic effects mediated by either the nuclear receptor FXR or by a recently discovered G-protein coupled receptor TGR5. We aim to investigate the mechanism of bile-acid induced arrhythmogenic effects in an in-vitro model of the fetal heart.

Methods and Results

Levels of bile acid transporters and nuclear receptor FXR were studied by quantitative real time PCR, western blot and immunostaining, which showed low levels of expression. We did not observe functional involvement of the canonical receptors FXR and TGR5. Instead, we found that TC binds to the muscarinic M₂ receptor in NRCM and serves as a partial agonist of this receptor in terms of inhibitory effect on intracellular cAMP and negative chronotropic response. Pharmacological inhibition and siRNA-knockdown of the M₂ receptor completely abolished the negative effect of TC on contraction, calcium transient amplitude and synchronisation in NRCM clusters.

Conclusion

We conclude that in NRCM the TC-induced arrhythmia is mediated by the partial agonism at the M₂ receptor. This mechanism might serve as a promising new therapeutic target for fetal arrhythmia.