Chenodeoxycholic Acid

Kinase experiment:

Briefly, transfected HEK-293 cells, incubated in charcoal-treated Dulbecco's modified Eagle's medium for 24 h, are washed once with Hanks' solution and resuspended in a buffer containing 100 mM NaCl, 1 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 250 mMsucrose, 20 mM Tris-HCl, pH 7.4. Cells are lysed by freezing in liquid nitrogen. Dehydrogenase activity is measured in a final volume of 20 μL containing the appropriate concentration of bile acid, 30 nCi of [3H]cortisol, and unlabeled cortisol to a final concentrations of 50 nM. The reaction is started by mixing cell lysate with the reaction mixture. Alternatively, endoplasmic reticulum microsomes are prepared from transfected HEK-293 cells and incubated with reaction mixture containing various concentrations of cortisol and CDCA. Incubation proceeded for 20 min, and the conversion of cortisol to cortisone is determined by thin layer chromatography (TLC). Because of the inaccuracy of the TLC method at low conversion rates and the end-product inhibition of 11βHSD2 at conversion rates higher than 60-70%, only conversion rates between 10 and 60% are considered for calculation. The inhibitory constant IC50 is evaluated using the curve-fitting program. Results are expressed as means±S.E. and consist of at least four independent measurements.

Cell experiment:

The cell viability is analyzed by incubating transfected HEK-293 cells and CHO cells for 1 h with the corresponding concentration of bile acid and staining with trypan blue. The toxicity of bile acids is analyzed using the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) according to the cell proliferation kit I. No significant differences between control and bile acid-treated cells are obtained in both tests.

References:

[1]. Stauffer AT, et al. Chenodeoxycholic acid and deoxycholic acid inhibit 11 beta-hydroxysteroid dehydrogenase type 2 and cause cortisol-induced transcriptional activation of the mineralocorticoid receptor. J Biol Chem. 2002 Jul 19;277(29):26286-92
[2]. Casaburi I, et al. Chenodeoxycholic acid through a TGR5-dependent CREB signaling activation enhances cyclin D1 expression and promotes human endometrial cancer cell proliferation. Cell Cycle. 2012 Jul 15;11(14):2699-710
[3]. Kawabe Y, et al. The molecular mechanism of the induction of the low density lipoprotein receptor by chenodeoxycholic acid in cultured human cells. Biochem Biophys Res Commun. 1995 Mar 8;208(1):405-11.
[4]. Ao M, et al. Chenodeoxycholic acid stimulates Cl(-) secretion via cAMP signaling and increases cystic fibrosis transmembrane conductance regulator phosphorylation in T84 cells. Am J Physiol Cell Physiol. 2013 Aug 15;305(4):C447-56
[5]. Noh K, et al. Farnesoid X receptor activation by chenodeoxycholic acid induces detoxifying enzymes through AMP-activated protein kinase and extracellular signal-regulated kinase 1/2-mediated phosphorylation of CCAAT/enhancer binding protein β. Drug Metab