Concentrations Of Chenodeoxycholic Acid Research Articles

Chenodeoxycholic acid through a TGR5-dependent CREB signaling activation enhances Cyclin D1 expression and promotes human endometrial cancer cell proliferation

Endometrial cancer exhibits a strong incidence in western developed countries mainly due to fat-rich diet and obesity. Processing of dietary lipids is triggered by bile acids, amphipathic detergents that are synthesized in the liver and stored in the gallbladder. In addition to their well-recognized role in dietary lipid absorption and cholesterol homeostasis, bile acids can also act as signaling molecules with systemic endocrine functions. In the present study we investigated the biological effects of the primary bile chenodeoxycholic acid (CDCA) on a human endometrial cancer cell line, Ishikawa. Low concentrations of CDCA are able to stimulate Ishikawa cell growth by inducing a significant increase in Cyclin D1 protein and mRNA expression through the activation of the membrane G protein-coupled receptor (TGR5)-dependent pathway. Dissecting the molecular mechanism underlying this effect by mutagenesis, EMSA and ChIP analysis revealed that CDCA-induced Cyclin D1 expression requires the enhanced recruitment of the transcription factor CREB on the cyclic AMP-responsive element motif within the Cyclin D1 gene proximal promoter. Our results suggest a novel molecular mechanism explaining the potential contribution of high-fat diet and obesity to endometrial cancer growth and progression opening the rationale for strategies to prevent the risk of this obesity-related cancer in women.

Gender, but not CYP7A1 or SLCO1B1 Polymorphism, Affects the Fasting Plasma Concentrations of Bile Acids in Human Beings

Cholesterol 7α-hydroxylase (CYP7A1) is the rate-limiting enzyme of bile acid production in human beings, and organic anion-transporting polypeptide 1B1 (OATP1B1) may influence bile acid hepatic uptake and cholesterol and bile acid synthesis rate. Our purpose was to investigate the effects of gender and CYP7A1 and SLCO1B1 polymorphisms on the fasting plasma concentrations of bile acids, bile acid synthesis marker and total cholesterol in a Finnish population. Fasting plasma concentrations of 16 endogenous bile acids, their synthesis marker (7α-hydroxy-4-cholesten-3-one) and total cholesterol were measured in 243 samples from 143 healthy volunteers. The volunteers were genotyped for 6 haplotype-tagging single-nucleotide polymorphisms (SNPs) of CYP7A1 and two functionally relevant SNPs in SLCO1B1. The mean plasma concentrations of chenodeoxycholic acid, glycochenodeoxycholic acid, ursodeoxycholic acid and glycoursodeoxycholic acid were 61-111% higher in men than in women (P ≤ 0.001). Accordingly, the mean concentration of total bile acids was 51% higher in men than in women (P = 0.001). The CYP7A1 rs8192879 and rs1023652 SNPs were associated with deoxycholic acid and hyodeoxycholic acid concentrations, respectively, but the associations were not significant after correction for multiple testing. None of the six CYP7A1 SNPs was associated with the plasma concentrations of cholesterol or 7α-hydroxy-4-cholesten-3-one. SLCO1B1 genotype was associated with total plasma cholesterol concentration only, but the association was not significant after correction for multiple testing. In general, the gender contributes substantially more to variation in fasting plasma bile acid concentrations than CYP7A1 or SLCO1B1 polymorphism do. Common genetic variability in CYP7A1 is unlikely to play a significant role in cholesterol metabolism and bile acid homeostasis under normal physiological conditions.

Helicobacter hepaticus–Induced Liver Tumor Promotion Is Associated with Increased Serum Bile Acid and a Persistent Microbial-Induced Immune Response

Chronic microbial infection influences cancer progression, but the mechanisms that link them remain unclear. Constitutive androstane receptor (CAR) is a nuclear receptor that regulates enzymes involved in endobiotic and xenobiotic metabolism. CAR activation is a mechanism of xenobiotic tumor promotion; however, the effects of chronic microbial infection on tumor promotion have not been studied in the context of CAR function. Here, we report that CAR limits the effects of chronic infection-associated progression of liver cancer. CAR knockout (KO) and wild-type (WT) male mice were treated with or without the tumor initiator diethylnitrosamine (DEN) at 5 weeks of age and then orally inoculated with Helicobacter hepaticus (Hh) or sterile media at 8 weeks of age. At approximately 50 weeks postinoculation, mice were euthanized for histopathologic, microbiological, molecular, and metabolomic analyses. Hh infection induced comparable hepatitis in WT and KO mice with or without DEN that correlated with significant upregulation of Tnfα and toll receptor Tlr2. Notably, DEN-treated Hh-infected KO mice exhibited increased numbers of liver lobes with dysplasia and neoplasia and increased multiplicity of neoplasia, relative to similarly treated WT mice. Enhanced tumor promotion was associated with decreased hepatic expression of P450 enzymes Cyp2b10 and Cyp3a11, increased expression of Camp, and increased serum concentrations of chenodeoxycholic acid. Together, our findings suggest that liver tumor promotion is enhanced by an impaired metabolic detoxification of endobiotics and a persistent microbial-induced immune response.

Fasting plasma chenodeoxycholic acid and cholic acid concentrations are inversely correlated with insulin sensitivity in adults

BackgroundAccumulating data suggest a novel role for bile acids (BAs) in modulating metabolic homeostasis. BA treatment has been shown to improve glucose tolerance and to increase energy expenditure in mice. Here, we investigated the relationship between fasting plasma BAs concentrations and metabolic parameters in humans.FindingsFasting plasma glucose, insulin and lipid profile were measured in 14 healthy volunteers, 20 patients with type 2 diabetes (T2D), and 22 non-diabetic abdominally obese subjects. Insulin sensitivity was also assessed by the determination of the glucose infusion rate (GIR) during a hyperinsulinemic-euglycemic clamp in a subgroup of patients (9 healthy and 16 T2D subjects). Energy expenditure was measured by indirect calorimetry. Plasma cholic acid (CA), chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA) concentrations were analyzed by gas chromatograph-mass spectrometry. In univariable analysis, a positive association was found between HOMA-IR and plasma CDCA (β = 0.09, p = 0.001), CA (β = 0.03, p = 0.09) and DCA concentrations (β = 0.07, p < 0.0001). Spearman analysis retrieved an inverse relationship between plasma CDCA (r = -0.44, p = 0.03), CA (r = -0.65, p = 0.001) and the GIR. HOMA-IR remained positively associated with CDCA (β = 0.11, p = 0.01), CA (β = 0.04, p = 0.01) and DCA (β = 0.06, p = 0.007) in multivariable analysis, after adjustment for age, gender, BMI, HbA1C and plasma lipid parameters. In contrast, HbA1c, energy expenditure and plasma lipid concentrations were not correlated with plasma BAs levels in multivariable analysis.ConclusionsBoth plasma CDCA, CA and DCA concentrations were negatively associated with insulin sensitivity in a wide range of subjects.

T1180 Microbial Liver Tumor Promotion During Nuclear Receptor Deficiency Involves Increased Circulating Chenodeoxycholic Acid and Induction of an Innate Antimicrobial Response

The nuclear receptor constitutive androstane receptor (CAR) regulates the expression of enzymes involved in endobiotic and xenobiotic metabolism. CAR is essential for liver tumor promotion by phenobarbital, a prototype tumor promoter of mice. The mechanism by which chronic microbial infection influences tumor progression is unknown. However, previous studies have suggested a role of endobiotics including certain bile acids in tumor promotion. We investigated the mechanism of liver tumor promotion by Helicobacter hepaticus (Hh), the prototype carcinogenic bacterium of mice. Twenty two CAR-/(KO) and 23 CAR+/+ (wild-type, WT) male mice (C3H/HeN) received a single intraperitoneal injection of the genotoxic carcinogen diethylnitrosamine (DEN) at 5 weeks of age. Another group consisting of 21 KO and 20 WT male mice did not receive DEN. The two groups (with and without DEN) were subsequently stratified into four subgroups (two KO and two WT) and orally inoculated with either Hh or sterile media at 8 weeks of age. Mice were euthanized at 50 weeks postinoculation, complete necropsies were performed, and samples were collected for histopathological, microbiological, molecular, and biochemical analyzes. Hepatic Hh colonization as well as hepatic expression of selected nuclear receptors, cytokines, and innate immune mediators were measured by real-time quantitative PCR. The serum concentrations of selected mono-, di-, and tri-hydroxylated bile acids were measured by liquid chromatography tandem mass spectrometry. Chronic infection with Hh induced hepatitis in WT and KO mice with or without DEN. However, relative to Hh-infected WT mice with DEN, Hhinfected KO mice with DEN exhibited the following statistically significant differences: Increased number of liver lobes with dysplasia/neoplasia (P<0.0001), increased multiplicity of liver tumors (P<0.004), decreased Hh colonization in the liver (P<0.04), increased hepatic expression of genes for vitamin D receptor (Vdr) (P<0.04) and cathelicidin antimicrobial peptide (Camp) (P<0.02), and increased serum concentration of chenodeoxycholic acid (P<0.01). Our findings suggest a mechanism of microbial tumor promotion involving increased systemic bile acid concentration and induction of an innate antimicrobial response against bacterial colonization.

Application of near infrared spectroscopy for rapid analysis of intermediates of Tanreqing injection

A method for rapid quantitative analysis of four kinds of Tanreqing injection intermediates was developed based on Fourier transform near infrared (FT-NIR) spectroscopy and partial least squares (PLS) algorithm. The NIR spectra of 120 samples were collected in transflective mode. The concentrations of chlorogenic acid, caffeic acid, luteoloside, baicalin, ursodesoxycholic acid (UDCA), and chenodeoxycholic acid (CDCA) were determined with the HPLC–DAD/ELSD as reference method. In the PLS calibration, the NIR spectra were pretreated with different methods and the number of PLS factors used in the model calibration was optimized by leave-one-out cross-validation. The performance of the final PLS models was evaluated according to the root mean square error of calibration (RMSEC), root mean square error of cross-validation (RMSECV), root mean square error of prediction (RMSEP), BIAS, standard error of prediction (SEP), and correlation coefficients ( R). The R values in the prediction sets were all higher than 0.93, and the SEPs for the 6 compounds are 1.18, 6.02, 2.71, 155, 126, 30.0 mg/l, respectively. The established models were used for the liquid preparation process analysis of Tanreqing injection in three batches, and a model updating method was proposed for the long-term usage of the established models. This work demonstrated that NIR spectroscopy is more rapid and convenient than the conventional methods to analyze the intermediates of Tanreqing injection, and the presented method is helpful to the implementation of process analytical technology (PAT) in pharmaceutical industry of Chinese Medicines Injections.

Salivary chenodeoxycholic acid and its glycine-conjugate: Their determination method using LC–MS/MS and variation of their concentrations with increased saliva flow rate

Measurement of steroid levels in saliva has been proposed as a new laboratory tool for characterizing steroid metabolism, but it is not known whether the salivary levels of bile acids can be measured with accuracy and if so, whether such measurements provide information that is of clinical value. We developed and validated a sensitive and specific liquid chromatography–electrospray ionization-tandem mass spectrometric (LC–ESI-MS/MS) method for the quantification of chenodeoxycholic acid (CDCA) and glycochenodeoxycholic acid (GCDCA), representative primary non-amidated and glycine-conjugated bile acids, in whole saliva. We also examined whether the salivary bile acid concentrations were dependent on the saliva flow rate, because this is a very important aspect in a discussion of the utility of salivary diagnostics. Saliva was deproteinized with ethanol and purified using a Strata-X cartridge. Bile acids were converted to their hydrazide derivatives using 2-hydrazinopyridine, and subjected to LC–MS/MS. Quantification was based on selected reaction monitoring using characteristic transitions, and deuterated CDCA and GCDCA were used as internal standards. This method allowed the reproducible and accurate quantification of the salivary bile acids using a 200-μl sample and the limits of quantification for CDCA and GCDCA were 25 and 50 pg/ml, respectively. Using this method, the effect of increased saliva flow rate by gum-chewing on the salivary concentrations of CDCA and GCDCA was determined. The salivary level of GCDCA was significantly decreased by gum-chewing, whereas the concentration of CDCA remained constant. These results indicate that there is a good possibility that saliva may be a clinical tool for non-amidated bile acid testing.

Synthesis of 7-ketolithocholic acid via indirect electrooxidation of chenodeoxycholic acid

7-Ketolithocholic acid was synthesized by indirect electrooxidation of chenodeoxycholic acid using the medium of Br−/Br2. Some important factors such as anode material, solvent, initial concentration of chenodeoxycholic acid, current density, electrolysis time have been investigated in detail. By comparing the experiment results, it can be concluded that productivity and current efficiency in the divided electrolytic cell are higher than that in the undivided electrolytic cell. The optimized process conditions have been found to be: the anode material PbO2/Ti, the solvent acetonitrile, and initial concentration of chenodeoxycholic acid 23.8–31.8 mg/mL, current density 95.2–142.9 A/m2. The current efficiency could reach 85% and the productivity of 7-ketolithocholic acid could reach 83%. The melting point, IR spectrum, 1H NMR, 13C NMR and EI-MS were used to characterize the product. The analytical results confirm that the product is 7-ketolithocholic acid.

Plasma Bile Acid Concentrations in Patients with Human Immunodeficiency Virus Infection Receiving Protease Inhibitor Therapy: Possible Implications for Hepatotoxicity

To evaluate whether patients with human immunodeficiency virus (HIV) infection who were receiving protease inhibitor therapy had altered bile acid concentrations compared with noninfected control subjects, and whether bile acid concentrations could predict the onset of hepatotoxicity caused by protease inhibitors. Retrospective sample analysis from a prospectively conducted clinical trial. Academic research center. Eleven adults with advanced HIV disease who were taking protease inhibitor-based antiretroviral therapy, one of whom had developed protease inhibitor-induced hepatotoxicity. Plasma concentrations of cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), ursodeoxycholic acid (UDCA), and taurocholic acid (TC) were analyzed by using a novel high-performance liquid chromatography with tandem mass spectrometry detection method. Comparisons of the relative contribution of each bile acid to the total bile acid pool were made with previously published values and with bile acid concentrations contained in two pooled plasma samples from healthy, non-HIV-infected volunteers analyzed in our laboratory. Each pooled plasma sample used for this analysis contained contributions from three non-HIV-infected volunteers. The LCA and TC concentrations in HIV-infected patients were 3-4-fold higher than those previously reported for non-HIV-infected subjects; concentrations of other bile acids were similar to those of previous reports. The relative contribution of CDCA to the total bile acid pool was 9% in HIV-infected patients compared with 30-50% in noninfected subjects. Total and individual bile acid concentrations in the HIV-infected patient who developed hepatotoxicity were similar to the bile acid concentrations from the other study patients who did not develop hepatotoxicity. These data suggest that bile acid concentrations may be altered by HIV infection and/or protease inhibitor therapy. However, further investigations should be performed to assess whether antiretroviral-associated hepatotoxicity can be predicted by alterations in individual bile acid concentrations.

Effect of SLCO1B1 polymorphism on the plasma concentrations of bile acids and bile acid synthesis marker in humans

Organic anion transporting polypeptide 1B1 (OATP1B1, encoded by SLCO1B1) is a sinusoidal influx transporter of human hepatocytes. Our aim was to characterize the role of OATP1B1 in the hepatic uptake of bile acids in vivo. Fasting blood samples were drawn from 24 healthy volunteers with SLCO1B1 c.388AA-c.521TT (*1A/*1A) genotype, eight with c.388GG-c.521TT (*1B/*1B) genotype, 24 with c.521TC genotype, and nine with c.521CC genotype. Plasma concentrations of 15 endogenous bile acids, their synthesis marker, and cholesterol were determined by liquid chromatography-tandem mass spectrometry. The concentrations of ursodeoxycholic acid, glycoursodeoxycholic acid, chenodeoxycholic acid, and glycochenodeoxycholic acid were approximately 50-240% higher in individuals with the SLCO1B1 c.521CC, c.521TC, or c.388AA-c.521TT genotype than in those with the c.388GG-c.521TT genotype (P<0.05), with the largest differences seen between the c.521CC and c.388GG-c.521TT individuals. The concentration of tauroursodeoxycholic acid was approximately 120% higher in individuals with the c.521TC genotype and that of taurochenodeoxycholic acid 110% higher in individuals with the c.521CC or c.521TC genotype than in those with the c.388GG-c.521TT genotype (P<0.05). The cholic acid concentration was approximately 30% higher in individuals with the c.521CC or c.388AA-c.521TT genotype than in those with the c.388GG-c.521TT genotype (P<0.05), but its conjugates remained unaffected by the genotype. The bile acid synthesis marker 7alpha-hydroxy-4-cholesten-3-one/cholesterol concentration ratio was 62 or 45% higher in the c.388AA-c.521TT participants than in the c.388GG-c.521TT or c.521TC participants, respectively (P<0.05). SLCO1B1 polymorphism considerably affects the disposition of several endogenous bile acids and bile acid synthesis marker, indicating that OATP1B1 plays an important role in the hepatic uptake of bile acids in vivo in humans.

Effect of coadsorbent on the photovoltaic performance of squaraine sensitizednanocrystalline solar cells

The effect of chenodeoxycholic acid as the coadsorbent with a squaraine sensitizer onTiO2 nanocrystalline solar cells was investigated, and it was found that thecoadsorbent prevents the squaraine sensitizer from aggregating on theTiO2 nanoparticles but reduces dye loading leading to an interdependent photovoltaicperformance. Analysis of the absorption spectra, and incident monochromaticphoton-to-current conversion efficiency data showed that the load of squaraine sensitizer aswell as the appearance of H-aggregates is strongly dependent on the molar concentration ofchenodeoxycholic acid coadsorbent. The open circuit voltage of the solar cellswith chenodeoxycholic acid increases due to the enhanced electron lifetime in theTiO2 nanoparticles coupled with the band edge shift ofTiO2 to negative potentials.

Bile acids modulate tight junction structure and barrier function of Caco-2 monolayers via EGFR activation

Intestinal and systemic illnesses have been linked to increased gut permeability. Bile acids, whose luminal profile can be altered in human disease, modulate intestinal paracellular permeability. We investigated the mechanism by which selected bile acids increase gut permeability using a validated in vitro model. Human intestinal Caco-2 cells were grown in monolayers and challenged with a panel of bile acids. Transepithelial electrical resistance and luminal-to-basolateral fluxes of 10-kDa Cascade blue-conjugated dextran were used to monitor paracellular permeability. Immunoprecipitation and immunoblot analyses were employed to investigate the intracellular pathway. Redistribution of tight junction proteins was studied by confocal laser microscopy. Micromolar concentrations of cholic acid, deoxycholic acid (DCA), and chenodeoxycholic acid (CDCA) but not ursodeoxycholic acid decreased transepithelial electrical resistance and increased dextran flux in a reversible fashion. Coincubation of 50 muM CDCA or DCA with EGF, anti-EGF monoclonal antibody, or specific src inhibitor 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP-2) abolished the effect. A concentration of 50 muM of either CDCA or DCA also induced EGF receptor phosphorylation, occludin dephosphorylation, and occludin redistribution at the tight junction level in the same time frame and in a reversible fashion. We conclude that selected bile acids modulate intestinal permeability via EGF receptor autophosphorylation, occludin dephosphorylation, and rearrangement at the tight junction level. The effect is mediated by the src family kinases and is abolished by EGF treatment. These data also support the role of bile acids in the genesis of necrotizing enterocolitis and the protective effect of EGF treatment.

Association between Fecal Bile Acids and Colorectal Cancer: A Meta-analysis of Observational Studies

PurposeTo provide a systematic review with meta-analysis for addressing the relationship between fecal bile acids (FBAs) and colorectal cancer.Materials and MethodsElectronic databases were searched for all observational studies that examined the relationship between FBAs and colorectal cancer or adenoma, and calculated weighted mean difference (WMD) and 95% confidence interval (CI). Publication bias was assessed with funnel plot.ResultsTwenty case-control or cohort studies were identified. All studies were pooled to assess the relationship between total FBAs and cancer/adenoma of the large bowel, however, no association was seen (WMD 0.61 mg/g freeze-dried feces; 95% CI: - 0.35 - 1.57). Significantly increased concentration of chenodeoxycholic acid (CDCA) was seen while pooling to assess the relationship between CDCA and cancer/adenoma of the large bowel (WMD 0.13 mg/g freeze-dried feces; 95% CI: 0.01 - 0.25), especially for colorectal cancer (WMD 0.28 mg/g freeze-dried feces; 95% CI: 0.10 - 0.46). However, no significant differences in deoxycholic acid (DCA), lithocholic acid (LCA), and primary and secondary bile acids, were seen between patients with cancer and patients with matched controls regardless of fixed and random effects models.ConclusionCDCA might play a role in the etiology of colorectal cancer.

Adaptive response to increased bile acids: induction of MDR1 gene expression and P-glycoprotein activity in renal epithelial cells

Cholestatic liver disease and increased serum bile acid concentrations are known to trigger various adaptive responses including the induction of hepatic, intestinal and renal bile acid transport proteins, but renal P-glycoprotein (Pgp, multidrug resistance protein 1, MDR1) remained uninvestigated in this context. We show that treatment of Madin Darby canine kidney (MDCK) cells with pathophysiologically relevant concentrations of chenodeoxycholic acid (CDCA; 100 microM) for 12 h induces MDR1 transcript levels in vitro more than twofold. CDCA and deoxycholic acid pre-treatment for 24-96 h (100 microM) also increased Pgp activity measured as rhodamine efflux, while cholic acid and taurocholic acid were not effective in concentrations up to 600 microM. CDCA pre-treatment (100 microM, 72 h) also resulted in a doubling of rhodamine123 secretion across an epithelium-like monolayer grown on Transwell filters and decreased the sensitivity towards the kidney toxic drugs cyclosporine A and paclitaxel. These findings predict physiologically as well as pharmacologically relevant consequences of liver disease for Pgp substrate transport and toxicity in the kidneys.

Lithocholic acid induction of the FGF19 promoter in intestinal cells is mediated by PXR

To study the effect of the toxic secondary bile acid lithocholic acid (LCA) on the expression of fibroblast growth factor 19 (FGF19) in intestinal cells and to characterize the pregnane-X-receptor (PXR) response of the FGF19 promoter region. The intestinal cell line LS174T was stimulated with various concentrations of chenodeoxy-cholic acid and lithocholic acid for several time points. FGF19 mRNA levels were determined with quantitative realtime RT-PCR. FGF19 deletion promoter constructs were generated and the LCA response was analzyed in reporter assays. Co-transfections with PXR and RXR were carried out to study FGF19 regulation by these factors. LCA and CDCA strongly up-regulate FGF19 mRNA expression in LS174T cells in a time and dose dependent manner. Using reporter gene assays with several deletion constructs we found that the LCA responsive element in the human FGF19 promoter maps to the proximal regulatory region containing two potential binding sites for PXR. Overexpression of PXR and its dimerization partner retinoid X receptor (RXR) and stimulation with LCA or the potent PXR ligand rifampicin leads to a significant induction of FGF19 promoter activity in intestinal cells. LCA induced feedback inhibition of bile acid synthesis in the liver is likely to be regulated by PXR inducing intestinal FGF19 expression.

Disruption of an SP2/KLF6 Repression Complex by SHP Is Required for Farnesoid X Receptor-induced Endothelial Cell Migration

The farnesoid X receptor (FXR) signaling pathway regulates bile acid and cholesterol homeostasis. Here, we demonstrate, using a variety of gain- and loss-of-function approaches, a role of FXR in the process of cell motility, which involves the small heterodimeric partner (SHP)-dependent up-regulation of matrix metalloproteinase-9. We use this observation to reveal a transcriptional regulatory mechanism involving the SP/KLF transcription factors, SP2 and KLF6. Small interference RNA-based silencing studies in combination with promoter, gel shift, and chromatin immunoprecipitation assays indicate that SP2 and KLF6 bind to the matrix metalloproteinase-9 promoter and together function to maintain this gene in a silenced state. However, upon activation of FXR, SHP interacts with SP2 and KLF6, disrupting the SP2/KLF6 repressor complex. Thus, together, these studies identify a mechanism for antagonizing Sp/KLF protein repression function via SHP, with this process regulating endothelial cell motility.

Bile acids stimulate PKCα autophosphorylation and activation: role in the attenuation of prostaglandin E1-induced cAMP production in human dermal fibroblasts

The aim was to identify the specific PKC isoform(s) and their mechanism of activation responsible for the modulation of cAMP production by bile acids in human dermal fibroblasts. Stimulation of fibroblasts with 25-100 microM of chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA) led to YFP-PKCalpha and YFP-PKCdelta translocation in 30-60 min followed by a transient 24- to 48-h downregulation of the total PKCalpha, PKCdelta, and PKCepsilon protein expression by 30-50%, without affecting that of PKCzeta. Increased plasma membrane translocation of PKCalpha was associated with an increased PKCalpha phosphorylation, whereas increased PKCdelta translocation to the perinuclear domain was associated with an increased accumulation of phospho-PKCdelta Thr505 and Tyr311 in the nucleus. The PKCalpha specificity on the attenuation of cAMP production by CDCA was demonstrated with PKC downregulation or inhibition, as well as PKC isoform dominant-negative mutants. Under these same conditions, neither phosphatidylinositol 3-kinase, p38 MAP kinase, p42/44 MAP kinase, nor PKA inhibitors had any significant effect on the CDCA-induced cAMP production attenuation. CDCA concentrations as low as 10 microM stimulated PKCalpha autophosphorylation in vitro. This bile acid effect required phosphatidylserine and was completely abolished by the presence of Gö6976. CDCA at concentrations less than 50 microM enhanced the PKCalpha activation induced by PMA, whereas greater CDCA concentrations reduced the PMA-induced PKCalpha activation. CDCA alone did not affect PKCalpha activity in vitro. In conclusion, although CDCA and UDCA activate different PKC isoforms, PKCalpha plays a major role in the bile acid-induced inhibition of cAMP synthesis in fibroblasts. This study emphasizes potential consequences of increased systemic bile acid concentrations and cellular bile acid accumulation in extrahepatic tissues during cholestatic liver diseases.

Complementary Stimulation of Hepatobiliary Transport and Detoxification Systems by Rifampicin and Ursodeoxycholic Acid in Humans

Rifampicin (RIFA) and ursodeoxycholic acid (UDCA) improve symptoms and biochemical markers of liver injury in cholestatic liver diseases by largely unknown mechanisms. We aimed to study the molecular mechanisms of action of these drugs in humans. Thirty otherwise healthy gallstone patients scheduled for cholestectomy were randomized to RIFA (600 mg/day for 1 week) or UDCA (1 g/day for 3 weeks) or no medication before surgery. Routine biochemistry, lipids, and surrogate markers for P450 activity (4beta-hydroxy cholesterol, 4beta-OH-C) and bile acid synthesis (7alpha-hydroxy-4-cholesten-3-one, C-4) were measured in serum. Bile acids were analyzed in serum, urine, and bile. A wedge liver biopsy specimen was taken to study expression of hepatobiliary ABC transporters as well as detoxification enzymes and regulatory transcription factors. RIFA enhanced bile acid detoxification as well as bilirubin conjugation and excretion as reflected by enhanced expression of CYP3A4, UGT1A1, and MRP2. These molecular effects were paralleled by decreased bilirubin and deoxycholic acid concentrations in serum and decreased lithocholic and deoxycholic acid concentrations in bile. UDCA on the other hand stimulated the expression of BSEP, MDR3, and MRP4. UDCA became the predominant bile acid after UDCA treatment and lowered the biliary cholesterol saturation index. RIFA enhances bile acid detoxification as well as bilirubin conjugation and export systems, whereas UDCA stimulates the expression of transporters for canalicular and basolateral bile acid export as well as the canalicular phospholipid flippase. These independent but complementary effects may justify a combination of both agents for the treatment of cholestatic liver diseases.

Disrupted coordinate regulation of farnesoid X receptor target genes in a patient with cerebrotendinous xanthomatosis

Cerebrotendinous xanthomatosis (CTX), sterol 27-hydroxylase (CYP27A1) deficiency, is associated with markedly reduced chenodeoxycholic acid (CDCA), the most powerful activating ligand for farnesoid X receptor (FXR). We investigated the effects of reduced CDCA on FXR target genes in humans. Liver specimens from an untreated CTX patient and 10 control subjects were studied. In the patient, hepatic CDCA concentration was markedly reduced but the bile alcohol level exceeded CDCA levels in control subjects (73.5 vs. 37.8 +/- 6.2 nmol/g liver). Cholesterol 7alpha-hydroxylase (CYP7A1) and Na+/taurocholate-cotransporting polypeptide (NTCP) were upregulated 84- and 8-fold, respectively. However, small heterodimer partner (SHP) and bile salt export pump were normally expressed. Marked CYP7A1 induction with normal SHP expression was not explained by the regulation of liver X receptor alpha (LXRalpha) or pregnane X receptor. However, another nuclear receptor, hepatocyte nuclear factor 4alpha (HNF4alpha), was induced 2.9-fold in CTX, which was associated with enhanced mRNA levels of HNF4alpha target genes, CYP7A1, 7alpha-hydroxy-4-cholesten-3-one 12alpha-hydroxylase, CYP27A1, and NTCP. In conclusion, the coordinate regulation of FXR target genes was lost in CTX. The mechanism of the disruption may be explained by a normally stimulated FXR pathway attributable to markedly increased bile alcohols with activation of HNF4alpha caused by reduced bile acids in CTX liver.

Regulation of CYP3A4 by the bile acid receptor FXR: evidence for functional binding sites in the CYP3A4 gene.

CYP3A4, the most abundant cytochrome P450 in human liver, is responsible for the metabolism of numerous xenobiotics and endobiotics. CYP3A4 expression is highly variable and is induced by numerous compounds of exogenous and endogenous origin, including elevated concentrations of secondary bile acids via the pregnane X receptor (PXR). We show that physiological concentrations of the primary bile acid chenodeoxycholic acid regulate the expression of CYP3A4 via the bile acid receptor FXR. Experiments performed in vitro in different cell culture systems, gel-mobility shift assays and experiments performed in vivo in transgenic mice lacking FXR or PXR and treated with the synthetic FXR agonist GW4064 were undertaken to study the implication of FXR in the regulation of CYP3A. Our data provide evidence for the presence of two functional FXR recognition sites located in a 345-bp element within the 5'-flanking region of CYP3A4. Mutational analysis of these sites and experiments in transgenic mice lacking FXR or PXR support the relevance of FXR activation for CYP3A regulation. Thus, whereas elevated concentrations of precursors of bile acids and secondary bile acids induce CYP3A via PXR, primary bile acids can modulate the expression of CYP3A via FXR. These findings may explain elevated CYP3A expression in cholestasis and part of the variability of drug responsiveness and toxicity between individuals.