FXR (IC50 = 40 μM)

In Ntcp-HepG2 cells, GCDCA markedly increased apoptosis after 4h. Co-incubation with TβMCA reduced apoptosis to 49% (p<0.01 vs. GCDCA, each; n=6). While GCDCA (100μmol/L) reduced the MMP to 34% after 6h, combination treatment with TβMCA restored the MMP to control levels at all time points (n=4). TβMCA also restored breakdown of the MMP induced by palmitate. GCDCA induced a translocation of Bax from the cytosol to mitochondria that was inhibited by simultaneous treatment with TβMCA in eqimolar concentrations.
Conclusions: TβMCA restricts hepatocellular apoptosis induced by low micromolar concentrations of GCDCA or palmitate via inhibition of Bax translocation to mitochondria and preservation of the MMP. Thus, further studies are warranted to evaluate a potential use of TβMCA in ameliorating liver injury in cholestasis. [1]

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T-MCAs Are FXR Antagonists [2]
It is known that CAs but not MCAs are FXR agonists (Reschly et al., 2008), and thus the similar levels of CAs observed in small intestines of GF and CONV-R mice (Figures 2 and S2) are hard to reconcile with the higher expression of FXR-dependent genes in the ileum of CONV-R mice (Figures 4A and S4). However, MCA levels were significantly higher in the ileum of GF mice (Figure S2). MCAs are much less hydrophobic than primary human bile acids CDCA and CA, which have affinities to FXR directly related to their hydrophobicity (Reschly et al., 2008). Docking of TβMCA into the ligand binding pocket of FXR with the FXR 6E-CDCA (OCA, obeticholic acid; INT-747) cocrystal structure suggested that, in contrast to the 6α ethyl group of 6E-CDCA, the 6β hydroxyl group of TβMCA does not occupy the pocket near Tyr358, Phe363, and Tyr366 (Figure 6A). This pocket is known to be critical for the activation of FXR (Mi et al., 2003), and led us to hypothesize that TβMCA may act as an FXR antagonist.

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To test our hypothesis, we performed a coactivator recruitment assay and showed that both TαMCA and TβMCA were FXR antagonists with IC50 values of 28 μM and 40 μM, respectively (Figure 6B). Taurine conjugation was essential for antagonistic activity and, as expected, neither TαMCA nor TβMCA activated FXR in this assay (data not shown). To exclude the possibility that the observed antagonistic activity was due to nonspecific detergent effects at high bile acid concentrations, we showed that the concentration-response curve for the selective FXR agonist GW4064 was shifted to the right in the presence of 100 μM or 400 μM TβMCA (Figure 6C). We next investigated whether TβMCA could also function as an FXR antagonist in the small intestine, which has been shown to have the highest Fgf15 expression of all tissues (Larsson et al., 2012). We treated ileal explants from CONV-R mice with TCA to induce Fgf15 expression and found that TβMCA prevented this induction in a concentration-dependent fashion (Figure 6D). In addition, we induced ileal Fgf15 and Shp expression in GF mice in vivo by treatment with TCA and showed that simultaneous treatment with TβMCA significantly reduced this induction (Figures 6E and 6F). We could not repeat this experiment in CONV-R mice since the gut microbiota rapidly deconjugates TβMCA to βMCA. Taken together, these data indicate that TβMCA is a competitive and reversible antagonist for ligand-activated FXR [2].

Antibiotic Treatment of CONV-R Mice Modifies Bile Acid Composition and Expression of Fgf15 and Cyp7a1 [2]
To investigate whether microbiota-induced changes in bile acid composition and Fgf15 and Cyp7a1 expression are reversible in colonized mice, we treated CONV-R mice with an antibiotic cocktail consisting of bacitracin, neomycin, and streptomycin, which are nonabsorbable from the intestine. We showed that antibiotic treatment increased the levels of TCA and TβMCA in the gallbladder of wild-type mice and reduced levels of secondary bile acids in serum (Figures 5A and 5B). These results are in agreement with a recent study showing increased levels of TCA and TβMCA in the intestinal lumen of ampicillin-treated mice (Kuribayashi et al., 2012). Antibiotic treatment also promoted a dramatic suppression of Fgf15 expression and a corresponding increase of Cyp7a1 expression (Figure 5C). Thus, antibiotic treatment results in a phenotype similar to that observed in GF mice, namely modified bile acid composition together with reduced Fgf15 and increased Cyp7a1 expression.

FXR Coactivator Recruitment Assay [2]
αMCA and βMCA and their respective taurine conjugates were tested for direct FXR activity in a coactivator recruitment assay as previously described (Solaas et al., 2004). The ligand binding domain of human FXR (amino acids 222–472) was expressed in Eschericha coli as an NH2-terminal His-tagged protein with the pET28a vector and the protein was purified by affinity chromatography. The activity of recombinant human FXR was determined in a FXR coactivator recruitment assay in white 384-well plates. Eu3+-coupled anti-His antibody (anti-His-Eu3+) and allophycocyanin-coupled streptavidin were used. An N-terminally biotinylated peptide (NH2-HSSLTERHKILHRLLQEGSPS-COOH) derived from steroid receptor coactivator 1 was used as coactivator. A 20 μl reaction volume contained 20 mM Tris (pH 7.5), 0.125% CHAPS, 2 mM dithiotreitol, 0.05% bovine serum albumin, 0.14 μg/ml anti-His-Eu3+, 2.9 μg/ml allophycocyanin-coupled streptavidin, 75 nM human FXR-ligand binding domain, 150 nM biotinylated steroid receptor coactivator 1 peptide, and the appropriate ligand. Antagonist activity was measured in the presence of 80 μM CDCA in the reaction mixture. After addition of all reagents, plates were incubated for 1 hr at room temperature and time-resolved fluorescence was measured in a Pherastar platereader. Excitation was at 340 nm, and fluorescence was measured at 615 nm and 665 nm. Specific signals were calculated by dividing the 665 nm signal by the 615 nm signal and multiplying the fraction by 10,000. TαMCA and TβMCA and GW4064 was obtained from XXX and was used at concentrations ranging from 5.1 nM to 100 μM. XLFit was used to fit the experimental data points to curves and to calculate IC50 values.

Transfection and culture of human HepG2 hepatoblastoma cells [1]
Similar as described previously, rat Ntcp was inserted in a pcDNA3.1 vector and stably transfected into the human hepatoma cell line HepG2 with Fugene transfection reagent). Cells were cultured in minimal essential medium (MEM) from PAA supplemented with 10% fetal calf serum, 2 mmol/L l-glutamine, 1 mmol/L Na-pyruvate, non-essential amino acids (1% of a 100× stock solution), 100 U/mL penicillin, 0.1 g/L streptomycin, and 1 g/L G418 sulfate. After 24 h of culture Ntcp-HepG2 cells were incubated for another 4 h with the bile acids GCDCA, TUDCA, or TβMCA alone, or with combinations of GCDCA + TUDCA or GCDCA + TβMCA, at concentrations of 25 μmol/L each, or with the solvent DMSO (0.1%) as control. In addition, cells were incubated with the free fatty acid palmitate 200 μmol/L) alone or in the combination with TβMCA.

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Mitochondrial membrane potential [1]
Ntcp-HepG2 cells were cultured for 24 h in poly-l-lysine-coated 96-well dishes (4 × 104 cells/well) and incubated for 2, 4, and 6 h with 25, 50, and 100 μmol/L GCDCA, TβMCA, or equimolar combinations of GCDCA and TβMCA. In addition, cells were treated with palmitate in combination with TβMCA. Cells were stained with 2 μmol/L 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyaniniodide (JC-1) from Molecular Probes in cell culture media for 30 min and washed twice with HBSS after staining. Fluorescence was determined with a Cyto-Fluor 4000 plate reader at wavelengths of 485 nm (excitation) and 530 and 580 nm (emission). The ratio of green and red fluorescence signals serve as a parameter for the mitochondrial membrane potential ΔΨm independent of the mitochondrial mass.

GF male Swiss Webster and C57BL/6 mice were maintained under a strict 12 hr light cycle in flexible plastic film isolators. Fxr−/− mice backcrossed four generations on to a C57BL/6 background were obtained from the Jackson Laboratory through a MTA with Frank Gonzalez and backcrossed four more generations onto C57BL/6 (backcrossed eight generations in total). The Fxr−/− mice were rederived as GF through embryo transfer and maintained under a strict 12 hr light cycle in flexible plastic film isolators.
All mice were fed an autoclaved chow diet ad libitum. GF isolators were routinely tested for sterility by culturing and PCR analysis of feces amplifying the 16S rRNA gene. All mice were aged 9–16 weeks at the time of analysis and were age matched (±2 weeks) for the individual experiments. Blood was collected from the inferior vena cava under deep isoflurane-induced anesthesia after a 4 hr fasting period. Liver, gallbladder, small intestine, cecum, and colon were harvested, and the small intestine was divided into three equal segments numbered in ascending order. A small biopsy from the most distal part of the small intestine was taken for RNA analysis of ileum. All tissues for bile acid and RNA analysis were snap frozen in liquid nitrogen and stored in −80°C until further processed. Fecal droppings from each cage were collected.

TCA or a mixture of TCA and TβMCA (400 mg/kg body weight of each compound dissolved in sterile water at a concentration of 40 mg/ml) or vehicle (sterile water) was administered to GF C57BL/6 mice by intragastric gavage on two occasions (2 and 14 hr before the mice were killed).

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Ex Vivo Experiments [2]
CONV-R mice were killed by cervical dislocation, and approximately 1 cm section of distal ileum was collected. Intestinal contents from the section were removed through flushing with cold PBS, and the section was divided into four equal longitudinal parts. Each part was placed in cell culture plates containing growth medium (Dulbecco’s modified Eagle’s medium with glutamine and pyruvate, 4.5 g/l glucose, 10% fetal calf serum, 100 U/ml penicillin, and 100 μg/ml streptomycin) with different concentrations of the bile acids TCA and TβMCA. The plates were then incubated overnight at 37°C in 5% CO2. The mixture containing the tissue was transferred to Eppendorf tubes and centrifuged at 14,000 rpm for 5 min. The tissue was collected after removal of the supernatant and used for analysis of mRNA expression.

[1]. Tauro-β-muricholic acid restricts bile acid-induced hepatocellular apoptosis by preserving the mitochondrial membrane potential. Biochem Biophys Res Commun. 2012 Aug 10;424(4):758-64.

[2]. Gut microbiota regulates bile acid metabolism by reducing the levels of tauro-beta-muricholic acid, a naturally occurring FXR antagonist. Cell Metab. 2013 Feb 5;17(2):225-35.

Tauro-beta-muricholic acid is a bile acid taurine conjugate derived from beta-muricholic acid. It has a role as a human metabolite and a rat metabolite. It is a bile acid taurine conjugate, a monocarboxylic acid amide, a 3alpha-hydroxy steroid, a 6beta-hydroxy steroid and a 7beta-hydroxy steroid. It is functionally related to a beta-muricholic acid. It is a conjugate acid of a tauro-beta-muricholate.

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Purpose: β-Muricholic acid (βMCA) is a trihydroxylated bile acid that constitutes the major bile acid in rat and mouse. βMCA is more hydrophilic than ursodeoxycholic acid and has been evaluated for dissolution of cholesterol gallstones. Since it is unknown if βMCA has beneficial effects on hepatocyte cell death we determined the effect of tauro-βMCA (TβMCA) on apoptosis in vitro. Methods: Human Ntcp-transfected HepG2 cells and primary hepatocytes from rat and mouse were incubated with the proapoptotic glycochenodeoxycholic acid (GCDCA) as well as the free fatty acid palmitate in the absence and presence of TβMCA. Apoptosis was quantified using caspase 3/7-assays and after Hoechst 33342 staining. The mitochondrial membrane potential (MMP) was measured fluorometrically using JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazol-carbocyaniniodide). Immunoblotting was performed against the proapoptotic Bcl-2-protein Bax. Results: In Ntcp-HepG2 cells, GCDCA markedly increased apoptosis after 4h. Co-incubation with TβMCA reduced apoptosis to 49% (p<0.01 vs. GCDCA, each; n=6). While GCDCA (100μmol/L) reduced the MMP to 34% after 6h, combination treatment with TβMCA restored the MMP to control levels at all time points (n=4). TβMCA also restored breakdown of the MMP induced by palmitate. GCDCA induced a translocation of Bax from the cytosol to mitochondria that was inhibited by simultaneous treatment with TβMCA in eqimolar concentrations. Conclusions: TβMCA restricts hepatocellular apoptosis induced by low micromolar concentrations of GCDCA or palmitate via inhibition of Bax translocation to mitochondria and preservation of the MMP. Thus, further studies are warranted to evaluate a potential use of TβMCA in ameliorating liver injury in cholestasis. [1]

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It has been shown that elevated serum free fatty acids are features of non-alcoholic fatty liver disease. The saturated C16 free fatty acid palmitate can induce hepatocyte lipoapoptosis in a JNK-dependent manner by activating Bax thereby triggering the mitochondrial apoptotic pathway. TβMCA also prevented palmitate-induced breakdown of the mitochondrial potential. Thus, the beneficial effect of TβMCA appears not to be restricted to bile acid-induced liver damage. In conclusion, we were able to demonstrate substantial antiapoptotic effects of TβMCA on bile acid-induced apoptosis in cell models from three different species, possibly via inhibition of Bax translocation to mitochondria and preservation of the mitochondrial membrane potential. [1]

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Bile acids are synthesized from cholesterol in the liver and further metabolized by the gut microbiota into secondary bile acids. Bile acid synthesis is under negative feedback control through activation of the nuclear receptor farnesoid X receptor (FXR) in the ileum and liver. Here we profiled the bile acid composition throughout the enterohepatic system in germ-free (GF) and conventionally raised (CONV-R) mice. We confirmed a dramatic reduction in muricholic acid, but not cholic acid, levels in CONV-R mice. Rederivation of Fxr-deficient mice as GF demonstrated that the gut microbiota regulated expression of fibroblast growth factor 15 in the ileum and cholesterol 7α-hydroxylase (CYP7A1) in the liver by FXR-dependent mechanisms. Importantly, we identified tauro-conjugated beta- and alpha-muricholic acids as FXR antagonists. These studies suggest that the gut microbiota not only regulates secondary bile acid metabolism but also inhibits bile acid synthesis in the liver by alleviating FXR inhibition in the ileum. [2]

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In summary, we have demonstrated that the gut microbiota has a profound systemic effect on bile acid metabolism. Not only does the gut microbiota exert its effects within the gut, but also in other parts of the enterohepatic system, such as regulating bile acid synthesis in the liver. We demonstrate that the microbial suppression of biosynthetic genes in the liver is consistent with increased FXR-dependent activation of Fgf15 in the ileum due to reduced levels of TβMCA.[2]

C26H45NO7S

515.292

C, 60.56; H, 8.80; N, 2.72; O, 21.72; S, 6.22

25696-60-0

145022-92-0 (sodium)

168408

Typically exists as solid at room temperature

3.839

5

7

7

35

891

8

OS(CCNC(CCC(C1CCC2C3C(O)C(O)C4CC(CCC4(C)C3CCC12C)O)C)=O)(=O)=O

XSOLDPYUICCHJX-OEYGYFRSSA-N

InChI=1S/C26H45NO7S/c1-15(4-7-21(29)27-12-13-35(32,33)34)17-5-6-18-22-19(9-11-25(17,18)2)26(3)10-8-16(28)14-20(26)23(30)24(22)31/h15-20,22-24,28,30-31H,4-14H2,1-3H3,(H,27,29)(H,32,33,34)/t15-,16-,17-,18?,19?,20+,22?,23+,24-,25-,26-/m1/s1

2-[[(4R)-4-[(3R,5R,6S,7R,10R,13R,17R)-3,6,7-trihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoyl]amino]ethanesulfonic acid

tauro-beta-muricholic acid; tauro-b-muricholic acid; Tauro-b-muricholate; T-beta-MCA; beta-tauromuricholic acid; CHEBI:133057; N-(3alpha,6beta,7beta-trihydroxy-5beta-cholan-24-oyl)-taurine; 25696-60-0; Tauromuricholic acid, T-alpha-MC, Tauro-alpha-muricholate, Tauro-alpha-muricholic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)