High Concentrations Of Bile Acids Research Articles

Suppression of Autophagic Flux by Bile Acids in Hepatocytes

Retention of bile acids (BAs) in the liver during cholestasis plays an important role in the development of cholestatic liver injury. Several studies have reported that high concentrations of certain BAs induce cell death and inflammatory response in the liver, and BAs may promote liver tumorigenesis. Macroautophagy (hereafter referred to as autophagy) is a lysosomal degradation process that regulates organelle and protein homeostasis and serves as a cell survival mechanism under a variety of stress conditions. However, it is not known if BAs modulate autophagy in hepatocytes. In the present study, we determined autophagic flux in livers of farnesoid X receptor (FXR) knockout (KO) mice that have increased concentrations of hepatic BAs and in primary cultured mouse hepatocytes treated with BAs. The results showed that autophagic flux was impaired in livers of FXR KO mice and in BA-treated primary mouse hepatocytes. Mechanistically, BAs did not affect the activities of cathepsin or the proteasome, but impaired autophagosomal-lysosomal fusion likely due to reduction of Rab7 protein expression and targeting to autophagosomes. In conclusion, BAs suppress autophagic flux in hepatocytes by impairing autophagosomal-lysosomal fusion, which may be implicated in bile acid-induced liver tumor promotion observed in FXR KO mice.

Oral bioavailability of ospemifene improves with food intake

To assess the effect of concomitant food intake on the relative bioavailability of ospemifene and its main metabolite, 4-hydroxyospemifene, after single oral dosing. This was an open-label, randomized, balanced, two-treatment (fed vs. fasted), two-period, two-sequence cross-over study in 24 healthy male subjects. Single 60-mg doses of ospemifene were administered without food or with a high-fat, high-energy breakfast (860 kcal). In an extension study, a single 60-mg dose of ospemifene was given to 12 subjects with a low-fat, light breakfast (300 kcal). Additional information was acquired by determining tablet dissolution profiles in media which reflected fasted and fed intestinal conditions. The AUC0-72 h and Cmax of ospemifene were 2.8- and 3.6-fold higher after a high-fat breakfast and 1.9- and 2.3-fold higher after a low-fat breakfast when compared with an overnight fast. The variability in both primary pharmacokinetic parameters was considerably reduced (by up to 50%) with a meal, indicating more consistent absorption of ospemifene with concomitant food intake. Dissolution in conditions simulating fed intestinal fluid (high bile acid concentration) was increased 3-fold compared with dissolution in simulated fasted intestinal fluid. wood markedly enhanced the extent and predictability of ospemifene absorption. The increase in bioavailability was not linearly related with the fat content of the meal. In vitro dissolution results were consistent with these clinical observations. Administration with food enhances and standardizes the oral bioavailability of ospemifene. Thus, it is recommended that ospemifene tablets should be taken with food.

Bile acids induce arrhythmias in human atrial myocardium—implications for altered serum bile acid composition in patients with atrial fibrillation

ObjectiveHigh bile acid serum concentrations have been implicated in cardiac disease, particularly in arrhythmias. Most data originate from in vitro studies and animal models. We tested the hypotheses that (1)...

Why more attentions to fetus in cases of intrahepatic cholestasis of pregnancy?

Intrahepatic cholestasis of pregnancy (ICP) is a peculiar disease in middle-late pregnancy with the pathological characteristics of hepatic capillary bile duct silts and is accompanied by clinical presentations of pruritus and bile acid (BA) elevation in serum. Maternal outcomes for patients diagnosed with ICP are usually good. However, fetal outcomes can be devastating with high frequencies of perinatal complications. Patients with ICP generally have an early delivery due to fetal complications. The current hypothesis is that ICP has higher frequencies of fetal complications due to high concentrations of BA which has toxic cellular effects to many organs. In lungs, it destroys the AT-II cells, decreasing phospholipids synthesis leading to the alveolar capillary permeability to increase and pulmonary surfactant to decrease. In heart, cholate can cross into the fetal compartment and causing fetal arrhythmias and decreased contractility. In the nervous system, high BAs can cause nerve cell denaturation and necrosis, mitochondria edema and membrane dissolve. In the placenta, high BA concentration can cause edema of the villous, decrease number of villous, intervillous thickening and balloon formation. EDITORIAL

Screening and Identification of Functional Lactobacillus Specific for Vegetable Fermentation

In this study, functional lactic acid bacteria (LAB) specific for vegetable fermentation were screened from Chinese sauerkraut brine by designing purpose media. The selected LAB strains showed excellent tolerance to high concentrations of bile salt and acids. They can also survive passage through the simulated gastric fluid and may be able to reach to the intestine. Through the performance measurements of vegetable fermentation, one of these strains, NCU116 was found to have the best fermentation ability, and further subjected to a series of analyses including identification of physiological and biochemical characteristics, 16S rDNA and pheS sequencing. Taken together, the strain NCU116 was identified as Lactobacillus plantarum subsp. plantarum. These results laid a good foundation for the fermentation research in the future.

NUCLEAR RECEPTORS

NUCLEAR RECEPTORS

Deoxycholic and chenodeoxycholic bile acids induce apoptosis via oxidative stress in human colon adenocarcinoma cells

The continuous exposure of the colonic epithelium to high concentrations of bile acids may exert cytotoxic effects and has been related to pathogenesis of colon cancer. A better knowledge of the mechanisms by which bile acids induce toxicity is still required and may be useful for the development of new therapeutic strategies. We have studied the effect of deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA) treatments in BCS-TC2 human colon adenocarcinoma cells. Both bile acids promote cell death, being this effect higher for CDCA. Apoptosis is detected after 30 min-2 h of treatment, as observed by cell detachment, loss of membrane asymmetry, internucleosomal DNA degradation, appearance of mitochondrial transition permeability (MPT), and caspase and Bax activation. At longer treatment times, apoptosis is followed in vitro by secondary necrosis due to impaired mitochondrial activity and ATP depletion. Bile acid-induced apoptosis is a result of oxidative stress with increased ROS generation mainly by activation of plasma membrane enzymes, such as NAD(P)H oxidases and, to a lower extent, PLA2. These effects lead to a loss of mitochondrial potential and release of pro-apoptotic factors to the cytosol, which is confirmed by activation of caspase-9 and -3, but not caspase-8. This initial apoptotic steps promote cleavage of Bcl-2, allowing Bax activation and formation of additional pores in the mitochondrial membrane that amplify the apoptotic signal.

A Study of the Prevalence of Genetic Polymorphisms in Bile Acid Diarrhea Patients

Introduction Bile acid diarrhoea (BAD) is a common cause of chronic diarrhoea and results from high concentrations of bile acids in the colon. It has recently been suggested that impaired production of fibroblast growth factor 19 (FGF19) in the ileum can result in excessive hepatic bile acid synthesis through reduced negative feedback regulation, and this, rather than malabsorption, is the cause of the diarrhoea. The reason for the reduced ileal FGF19 production remains unclear but it has been established that ileal FGF19 synthesis is markedly stimulated by bile acids binding to the farnesoid X receptor (FXR). In the liver, FGF19 acts through the FGFR4 receptor and its co-receptor Klotho β. Organic solute transporter α (OSTα) is the ileal basolateral bile acid transporter. The aim of this study was to determine the prevalence of 6 common single nucleotide polymorphisms (SNPs) in four genes involved in bile acid metabolism and FGF19 action, comparing the prevalence in patients with primary BAD, a similar number of controls with diarrhoea, and a larger numbers of healthy controls. Methods Two common SNPs (rs948992 and rs1789170) in the FGF19 gene were analysed as well as 2 SNPs (M173T, rs61755050 and −1g>t, rs56163822) in the FXR gene. Mutations in FXR are implicated in patients with obstetric cholestasis and have been shown to reduce function and translation efficiency of FXR. A SNP in Klotho β (rs17618244) has been associated with colonic transit in IBS-D patients. A further SNP in the OSTα gene (rs939885) was also studied. Genomic DNA was extracted from 92 primary BAD patients diagnosed on the basis of an abnormal ( Results Polymorphisms in all the genes studied were detectable in controls and in cases. No significant differences in the genotypes and allele frequencies were found in the Caucasian cohorts in the control and primary BAD groups (p > 0.1). Further analysis of patients of different ethnic origin again did not show significant differences in the allele frequencies. Conclusion Major differences in the frequency of genetic polymorphisms in four candidate genes involved with FGF19 and bile acid metabolism are unlikely to be associated with primary BAD. However, the SNPs identified may contribute small effects, with a combination of SNPs in several genes producing a cumulative effect. Larger numbers of cases are required to define specific interactions or minor effects. At present the cause of the reduced FGF19 action in BAD remains uncertain.

* A study of the prevalence of genetic polymorphisms in bile acid diarrhoea patients

IntroductionBile acid diarrhoea (BAD) is a common cause of chronic diarrhoea and results from high concentrations of bile acids in the colon. It has recently been suggested that impaired production...

290 CD8+ T CELLS WITH DOUBLE STRAND DNA BREAKS ACCUMULATE WITH INCREASED FIBROSIS STAGE IN CHRONIC HCV INFECTION AND FAIL TO RESPOND TO IFN-α

Background and Aims: Innate immune recognition of microbial components is dependent on pattern recognition receptors (PRRs), which detect components of foreign pathogens referred to as pathogen associated molecular patterns (PAMPs). One important group of PRRs consists of the so called Toll like receptors (TLRs), which activate a network of signal pathways leading to activation of the transcription factors IRF3, NF-uB and AP-1 and subsequent expression of pro-inflammatory cytokines and type I interferons (IFN). Methods: Peripheral blood mononuclear cells (PBMC) were isolated from human blood (healthy volunteers, man) by Ficoll Paque centrifugation. CD14 positive cells were separated by magnetically labelled beads and cultured for 9 days in the presence of M-CSF to differentiate them into macrophages. Thereafter cells were stimulated with different TLR ligands in presence or absence of hydrophobic bile acids like taurolithocholic acid (TLC), glycochenodeoxycholic acid (GCDC) and taurochenodeoxycholic acid (TCDC). Cells were lysed and mRNA and protein expression were measured by realtime PCR and immunoblot respectively. Results: Hydrophobic bile acids changed cytokine expression pattern of TLR ligand stimulated macrophages. LPS-induced TNF-a, IL-6 and IFN-bmRNA expression and expression of antiviral proteins like MxA and PKR were significantly downregulated in response to hydrophobic bile acids, however LPS-induced expression of the strong inhibitory anti-inflammatory interleukin-10 was not modulated. These effects could be mimicked by cAMP. TLC also inhibited TNF-a and IFN-b mRNA expression induced by other TLR ligands like TLR 2, 3, 7 and 9. Phosphorylation of TLR4 dependent signaling elements like p-IRF3 (Ser) and p-p65 (Ser) were not effected by bile acids. However, the inhibitory effect of bile acids on cytokine expression was accompanied by elevation of cAMP and could be reverted by blocking proteinkinase A. This suggested an involvement of the bile acid receptor TGR5 in inhibition of TLR-responses, which we detected in human macrophages by immunostaining. Conclusions: These data show that bile acids mediate immunosuppressive effects by blocking TLR-induced activation of innate immunity responses in human macrophages, thereby probably changing differentiation and activation of macrophages. This may contribute to the known clinical observations that cholestatic patients with high serum bile acid concentrations show reduced cell mediated immunity.

289 INHIBITION OF TOLL-LIKE RECEPTOR SIGNALING IN HUMAN MACROPHAGES BY BILE ACIDS

Background and Aims: Innate immune recognition of microbial components is dependent on pattern recognition receptors (PRRs), which detect components of foreign pathogens referred to as pathogen associated molecular patterns (PAMPs). One important group of PRRs consists of the so called Toll like receptors (TLRs), which activate a network of signal pathways leading to activation of the transcription factors IRF3, NF-uB and AP-1 and subsequent expression of pro-inflammatory cytokines and type I interferons (IFN). Methods: Peripheral blood mononuclear cells (PBMC) were isolated from human blood (healthy volunteers, man) by Ficoll Paque centrifugation. CD14 positive cells were separated by magnetically labelled beads and cultured for 9 days in the presence of M-CSF to differentiate them into macrophages. Thereafter cells were stimulated with different TLR ligands in presence or absence of hydrophobic bile acids like taurolithocholic acid (TLC), glycochenodeoxycholic acid (GCDC) and taurochenodeoxycholic acid (TCDC). Cells were lysed and mRNA and protein expression were measured by realtime PCR and immunoblot respectively. Results: Hydrophobic bile acids changed cytokine expression pattern of TLR ligand stimulated macrophages. LPS-induced TNF-a, IL-6 and IFN-bmRNA expression and expression of antiviral proteins like MxA and PKR were significantly downregulated in response to hydrophobic bile acids, however LPS-induced expression of the strong inhibitory anti-inflammatory interleukin-10 was not modulated. These effects could be mimicked by cAMP. TLC also inhibited TNF-a and IFN-b mRNA expression induced by other TLR ligands like TLR 2, 3, 7 and 9. Phosphorylation of TLR4 dependent signaling elements like p-IRF3 (Ser) and p-p65 (Ser) were not effected by bile acids. However, the inhibitory effect of bile acids on cytokine expression was accompanied by elevation of cAMP and could be reverted by blocking proteinkinase A. This suggested an involvement of the bile acid receptor TGR5 in inhibition of TLR-responses, which we detected in human macrophages by immunostaining. Conclusions: These data show that bile acids mediate immunosuppressive effects by blocking TLR-induced activation of innate immunity responses in human macrophages, thereby probably changing differentiation and activation of macrophages. This may contribute to the known clinical observations that cholestatic patients with high serum bile acid concentrations show reduced cell mediated immunity.

Bile acids decrease intracellular bilirubin levels in the cholestatic liver: implications for bile acid-mediated oxidative stress

High plasma concentrations of bile acids (BA) and bilirubin are hallmarks of cholestasis. BA are implicated in the pathogenesis of cholestatic liver damage through mechanisms involving oxidative stress, whereas bilirubin is a strong antioxidant. We evaluated the roles of bilirubin and BA on mediating oxidative stress in rats following bile duct ligation (BDL). Adult female Wistar and Gunn rats intraperitoneally anaesthetized with ketamine and xylazine underwent BDL or sham operation. Cholestatic markers, antioxidant capacity, lipid peroxidation and heme oxygenase (HO) activity were determined in plasma and/or liver tissue 5 days after surgery. HepG2-rNtcp cells were used for in vitro experiments. Plasma bilirubin levels in control and BDL animals positively correlated with plasma antioxidant capacity. Peroxyl radical scavenging capacity was significantly higher in the plasma of BDL Wistar rats (210 ± 12%, P < 0.0001) compared to controls, but not in the liver tissues. Furthermore after BDL, lipid peroxidation in the livers increased (179 ± 37%, P < 0.01), whereas liver HO activity significantly decreased to 61% of control levels (P < 0.001). Addition of taurocholic acid (TCA, ≥50 μmol/l) to liver homogenates increased lipid peroxidation (P < 0.01) in Wistar, but not in Gunn rats or after the addition of bilirubin. In HepG2-rNtcp cells, TCA decreased both HO activity and intracellular bilirubin levels. We conclude that even though plasma bilirubin is a marker of cholestasis and hepatocyte dysfunction, it is also an endogenous antioxidant, which may counteract the pro-oxidative effects of BA in circulation. However, in an animal model of obstructive cholestasis, we found that BA compromise intracellular bilirubin levels making hepatocytes more susceptible to oxidative damage.

Hydrophobicity and haemolytic potential of oxo derivatives of cholic, deoxycholic and chenodeoxycholic acids

The objective of this work was to study the effect of structure of bile acids on their membranolytic potential and extent of overlapping of the information about the membranolytic potential of bile acids and their physico-chemical parameters, namely: retention index RM0 (as a measure of bile acid hydrophobicity, reversed-phase thin-layer chromatography (RPTLC)), lecithin solubilisation (measure of the interaction of bile acids with phospholipids) and critical micellar concentration (CMC).It was found that bile acid concentrations at 100% lysis of erythrocyte membranes is described best by their CMC values, whereas at 50% lysis the parameter used is lecithin solubilisation. This indicates that different mixed micelles are formed in the membrane lysis at lower and higher concentrations of bile acids. Replacement of the hydroxyl (OH) group in the bile acid molecule with an oxo group yields derivatives with lowered hydrophobicity, power of lecithin solubilisation, tendency for self-aggregation as well as the membranolytic activity.

Inflammation Mediated Down-Regulation of Hepatobiliary Transporters Contributes to Intrahepatic Cholestasis and Liver Damage in Murine Biliary Atresia

To investigate the hypothesis that during the development of biliary atresia, early changes in hepatobiliary transport are mainly related to the inflammatory process and lead to intrahepatic cholestasis and subsequent liver injury, livers from mice with rhesus rotavirus-induced biliary atresia were analyzed for mRNA expression of hepatobiliary transporters, nuclear receptors, and inflammatory cytokines. Seven days after inoculation, despite high bile acid concentrations in the liver, gene expression of canalicular and basolateral hepatobiliary transporters and their regulatory nuclear receptors was down-regulated with concomitant increase in gene expression of inflammatory cytokines and rise in serum unconjugated bilirubin. At 14 d, hepatobiliary transporters and nuclear receptors remained down-regulated although the inflammatory response subsided. The percentage of conjugated bilirubin started to increase as extrahepatic biliary obstruction occurred. At 18 d, expression of hepatobiliary transporters remained low, expression of nuclear receptors returned to normal, while expression of inflammatory cytokines decreased further. Moreover, histology demonstrated progressive inflammation, bile duct damage, ductular proliferation, and hepatocyte necrosis. In conclusion, intrahepatic cholestasis due to inflammation-related down-regulation of basolateral and canalicular hepatobiliary transporters is an early event in the development of biliary atresia. Intrahepatic cholestasis contributes to the development of jaundice and liver injury.

Physiological concentrations of bile acids down‐regulate agonist induced secretion in colonic epithelial cells

In patients with bile acid malabsorption, high concentrations of bile acids enter the colon and stimulate Cl(-) and fluid secretion, thereby causing diarrhoea. However, deoxycholic acid (DCA), the predominant colonic bile acid, is normally present at lower concentrations where its role in regulating transport is unclear. Thus, the current study set out to investigate the effects of physiologically relevant DCA concentrations on colonic epithelial secretory function. Cl(-) secretion was measured as changes in short-circuit current across voltage-clamped T(84) cell monolayers. At high concentrations (0.5-1 mM), DCA acutely stimulated Cl(-) secretion but this effect was associated with cell injury, as evidenced by decreased transepithelial resistance (TER) and increased lactate dehydrogenase (LDH) release. In contrast, chronic (24 hrs) exposure to lower DCA concentrations (10-200 microM) inhibited responses to Ca(2+) and cAMP-dependent secretagogues without altering TER, LDH release, or secretagogue-induced increases in intracellular second messengers. Other bile acids - taurodeoxycholic acid, chenodeoxycholic acid and cholic acid - had similar antisecretory effects. DCA (50 microM) rapidly stimulated phosphorylation of the epidermal growth factor receptor (EGFr) and both ERK and p38 MAPKs (mitogen-activated protein kinases). The EGFr inhibitor, AG1478, and the protein synthesis inhibitor, cycloheximide, reversed the antisecretory effects of DCA, while the MAPK inhibitors, PD98059 and SB203580, did not. In summary, our studies suggest that, in contrast to its acute prosecretory effects at pathophysiological concentrations, lower, physiologically relevant, levels of DCA chronically down-regulate colonic epithelial secretory function. On the basis of these data, we propose a novel role for bile acids as physiological regulators of colonic secretory capacity.

T1920 Amplification of HER2/Neu Oncogene in Reflux-Barrett's Metaplasia-Dysplasia-Adenocarcinoma Sequence in Correlation to H. pylori Infection

foveolar-type cells positive for GOS, which were similar to those in cardiac-type mucosa in SSBE. A fewCDX2-positive cells were detected in themetaplastic glands. However, specialized intestinal metaplasia, dysplasia, and carcinoma were not observed. Discussion: Induction of cardiac-type mucosa similar to that observed in SSBE was successfully achieved by using a novel rat reflux model. It has been reported that CDX2 plays important roles in the induction and maintenance of BE, and that the bile acids induce ectopic expression of CDX2 in esophageal epithelium. Barrett's dysplasia and esophageal adenocarcinoma were not detected in the present model, probably because of the dilution of the bile acids by gastric juice in reflux contents. Therefore, high concentrations of bile acids appear to play an important role in the development of specialized intestinal metaplasia leading to adenocarcinoma.

Bile acids cause secretory phospholipase A2 activity enhancement, revertible by exogenous surfactant administration

Bile acids have been implicated in some forms of acute lung injury, including meconium aspiration and bile acid pneumonia in neonates, or aspiration related ARDS in adults. Secretory phospholipase A2 (sPLA2) is now known as a key enzyme in the lung injury pathways and is supposed to be responsible for surfactant dysfunction. Our aim was to investigate the interaction between bile acids and sPLA2 in an extracellular environment representing an in vitro model of aspiration. In vitro study using broncho-alveolar lavage (BAL) of 23 neonates/infants (<6 m) with healthy lungs. BAL supernatants were assayed for sPLA2 activity in basal condition and after addition of randomly assigned concentrations of bile acids (BA) or normal saline. Samples coming from neonates were then challenged with poractant-alfa up to a phospholipid concentration equal to that found in babies after the surfactant treatment for respiratory distress syndrome. sPLA2 activity was again measured, being corrected for serum/supernatant urea ratio and for confounding factors. High concentrations of BA (5 micromol/l) significantly increased (P = 0.012) sPLA2 activity, leading to increased surfactant catabolism. This finding was not observed with lower BA concentration and this is consistent with available literature data and may indicate an anionic activation of the enzyme by bile acids. Increased activity was significantly reverted by the addition of exogenous surfactant (P = 0.004) which was able to reduce sPLA2 activity almost to the baseline level. BA are likely to contribute to lung injury, causing surfactant inactivation through the increased sPLA2 activity. Other mechanisms cannot be excluded and require further studies to be clarified.

Ischemia and Cholestasis: More Than (Just) the Bile Ducts!

Ischemia and Cholestasis: More Than (Just) the Bile Ducts!

Nuclear Receptors

Nuclear Receptors

Effects of bile acids on pancreatic ductal bicarbonate secretion in guinea pig

Background and aims:Acute pancreatitis is associated with significant morbidity and mortality. Bile reflux into the pancreas is a common cause of acute pancreatitis and, although the bile can reach both...