The differentiation of a preadipocyte into a mature adipocyte is a highly regulated process that requires a scripted program of transcriptional events leading to changes in gene expression. Several genes are associated with adipogenesis, including the CAAT/enhancer-binding protein (C/EBPs) and peroxisome proliferator-activated receptor (PPAR) families of transcription factors. In this study, we have investigated the role of the farnesoid X receptor (FXR), a bile acid-activated nuclear receptor, in regulating adipogenesis in a preadipocyte cell line (3T3-L1 cells). Our results show that FXR is expressed in the white adipose tissue of adult mice and in differentiated 3T3-L1 cells but not in undifferentiated preadipocytes. Exposure of 3T3-L1 cells to INT-747 (6-ethyl cheno-deoxycholic acid), a potent and selective FXR ligand, increases preadipocyte differentiation induced by a differentiating mixture containing insulin. Augmentation of differentiating mixture-induced differentiation of 3T3-L1 cells by INT-747 associated with induction of aP2, C/EBPalpha, and PPARgamma2 mRNAs along with other adipocyte-related genes. This effect was reversed by guggulsterone, an FXR antagonist, and partially reverted by GW9662 (2-chloro-5-nitro-N-phenylbenzamide), a selective PPARgamma antagonist, indicating that FXR modulates adipocyte-related genes by PPARgamma-dependent and -independent pathways. Regulation of adipocyte-related genes by INT-747 was lost in FXR-/- mice, indicating that modulation of these genes by INT-747 requires an intact FXR. In addition, INT-747 enhances both insulin-induced serine phosphorylation of Akt and glucose uptake by 3T3-L1 cells. Taken together, these results suggest that activation of FXR plays a critical role in regulating adipogenesis and insulin signaling.

Several studies have argued that G-protein-coupled receptors (GPCR) have the capacity to promote activation of receptor tyrosine kinases. The current studies were performed to examine the regulation of the extracellular regulated kinase (ERK)1/2 and AKT pathways by conjugated and unconjugated bile acids in primary hepatocytes. Deoxycholic acid (DCA), chenodeoxycholic acid (CDCA), taurodeoxycholic acid (TDCA), glycodeoxycholic acid (GDCA), taurochenodeoxycholic acid (TCDCA), glycochenodeoxycholic acid (GCDCA), taurocholic acid (TCA), glycocholic acid (GCA), and tauroursodeoxycholic acid (TUDCA) all activated ERK1/2 in primary rat hepatocytes that was abolished by inhibition of ERBB1, and significantly reduced by ROS quenching agents. Bile acid-induced AKT activation was blunted by preventing ERBB1 activation and ROS generation. Treatment of rat hepatocytes with pertussis toxin (PTX) did not alter ERK1/2 and AKT activation induced by DCA or CDCA but abolished pathway activations by conjugated bile acids. Similar data to those with PTX were obtained when a dominant negative form of G(i1alpha) was overexpressed. Treatment of rat hepatocytes with TDCA and TCA promoted guanosine triphosphate (GTP) loading of G(i1alpha), G(i2alpha), and G(i3alpha) in vitro. Treatment of rat hepatocytes with PTX abolished TDCA-induced tyrosine phosphorylation of ERBB1. Similar findings to those in rat hepatocytes were also obtained in primary mouse and human hepatocytes, but not in established rodent or human hepatoma cell lines. In conclusion, collectively our findings demonstrate that unconjugated bile acids activate hepatocyte receptor tyrosine kinases and intracellular signaling pathways in a ROS-dependent manner. In contrast, conjugated bile acids primarily activate receptor tyrosine kinases and intracellular signaling pathways in a GPCR (G(ialpha))-dependent and ROS-dependent manner.

The promoting effect of dietary corn oil (CO), safflower oil (SO), olive oil (OO), coconut oil (CC), and medium-chain triglycerides (MCT) on azoxymethane (AOM)-induced colon tumors was studied in female F344 rats. The animals were fed low-fat diets containing 5% CO, 5% SO, or 5% OO 2 weeks before, during, and 1 week after sc injection of 20 mg AOM/kg body weight. One week after the AOM treatment, groups of animals were transferred to high-fat diets containing 23.52% CO, 23.52% SO, 23.52% OO, and 23.52% CC, or 5.88% CO + 17.64% MCT; the remaining animals were continued on 5% fat diets. All animals were fed these diets until the termination of the experiment. Body weights and intakes of calories, protein, and micronutrients were comparable among the various dietary groups. The incidence of colon tumors was increased in rats fed diets containing high-CO and high-SO compared to those fed low-CO and low-SO diets, whereas the diets containing high OO, CC, or MCT had no promoting effect on colon tumor incidence. There was a significant increase in the excretion of fecal deoxycholic acid, lithocholic acid, and 12-ketolithocholic acid in animals fed the high-CO and high-SO diets and no difference in these secondary bile acids excretion in animals fed the high-OO and high-CC diets compared to those animals fed their respective 5% fat diets. This study thus indicates that not only the amount of dietary fat but also the fatty acid composition (type) of fat are important factors in the determination of the promoting effect in colon carcinogenesis.

Colorectal cancer is often lethal when invasion and/or metastasis occur. Tumor progression to the metastatic phenotype is mainly dependent on tumor cell invasiveness. Secondary bile acids, particularly deoxycholic acid (DCA), are implicated in promoting colon cancer growth and progression. Whether DCA modulates beta-catenin and promotes colon cancer cell growth and invasiveness remains unknown. Because beta-catenin and its target genes urokinase-type plasminogen activator receptor (uPAR) and cyclin D1 are overexpressed in colon cancers, and are linked to cancer growth, invasion, and metastasis, we investigated whether DCA activates beta-catenin signaling and promotes colon cancer cell growth and invasiveness. Our results show that low concentrations of DCA (5 and 50 microM) significantly increase tyrosine phosphorylation of beta-catenin, induce urokinase-type plasminogen activator, uPAR, and cyclin D1 expression and enhance colon cancer cell proliferation and invasiveness. These events are associated with a substantial loss of E-cadherin binding to beta-catenin. Inhibition of beta-catenin with small interfering RNA significantly reduced DCA-induced uPAR and cyclin D1 expression. Blocking uPAR with a neutralizing antibody significantly suppressed DCA-induced colon cancer cell proliferation and invasiveness. These findings provide evidence for a novel mechanism underlying the oncogenic effects of secondary bile acids.

The orphan nuclear receptor pregnane X receptor (PXR) plays an important role in the detoxification of foreign and endogenous chemicals, including bile acids. PXR promotes bile acid elimination by activating bile acid-detoxifying enzymes and transporters. Certain bile acids are known to promote colonic carcinogenesis by inducing colon cancer cell apoptosis. However, whether and how PXR plays a role in colon cancer apoptosis has not been reported. In this study, we showed that activation of PXR by genetic (using a constitutively activated PXR) or pharmacological (using PXR agonist rifampicin) means protected the PXR-overexpressing colon cancer HCT116 cells from deoxycholic acid-induced apoptosis. Interestingly, activation of PXR also protected HCT116 cells from adriamycin-induced cell death, suggesting that the antiapoptotic effect of PXR was not bile acid specific. Moreover, the antiapoptotic effect of PXR in HCT116 cells appeared to be independent of xenobiotic enzyme regulation, because these cells had little basal and inducible expression of bile acid-detoxifying enzymes. Instead, SuperArray analysis showed that PXR-mediated deoxycholic acid resistance was associated with up-regulation of multiple antiapoptotic genes, including BAG3, BIRC2, and MCL-1, and down-regulation of proapoptotic genes, such as BAK1 and TP53/p53. Treatment with rifampicin in colon cancer LS180 cells, a cell line known to express endogenous PXR, also inhibited apoptosis. Activation of PXR in transgenic mice inhibited bile acid-induced colonic epithelial apoptosis and sensitized mice to dimethylhydrazine-induced colonic carcinogenesis, suggesting that the antiapoptotic effect of PXR is conserved in normal colon epithelium. In summary, our results have established the antiapoptotic role of PXR in both human colon cancer cells and normal mouse colon epithelium.

Obesity increases the risk of cancers, including hepatocellular carcinomas (HCC). However, the precise molecular mechanisms through which obesity promotes HCC development are still unclear. Recent studies have shown that gut microbiota may influence liver diseases by transferring its metabolites and components. Here, we show that the hepatic translocation of obesity-induced lipoteichoic acid (LTA), a Gram-positive gut microbial component, promotes HCC development by creating a tumor-promoting microenvironment. LTA enhances the senescence-associated secretory phenotype (SASP) of hepatic stellate cells (HSC) collaboratively with an obesity-induced gut microbial metabolite, deoxycholic acid, to upregulate the expression of SASP factors and COX2 through Toll-like receptor 2. Interestingly, COX2-mediated prostaglandin E2 (PGE2) production suppresses the antitumor immunity through a PTGER4 receptor, thereby contributing to HCC progression. Moreover, COX2 overexpression and excess PGE2 production were detected in HSCs in human HCCs with noncirrhotic, nonalcoholic steatohepatitis (NASH), indicating that a similar mechanism could function in humans.Significance: We showed the importance of the gut-liver axis in obesity-associated HCC. The gut microbiota-driven COX2 pathway produced the lipid mediator PGE2 in senescent HSCs in the tumor microenvironment, which plays a pivotal role in suppressing antitumor immunity, suggesting that PGE2 and its receptor may be novel therapeutic targets for noncirrhotic NASH-associated HCC. Cancer Discov; 7(5); 522-38. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 443.

This investigation was undertaken in order to (a) characterize the postprandial inflow of individual bile acids to the liver and (b) determine if peripheral venous bile acid levels always adequately reflect the portal venous concentration, or if saturation of hepatic bile acid uptake can occur under physiological conditions. In five patients with uncomplicated cholesterol gallstone disease, the umbilical cord was cannulated during cholecystectomy, and a catheter was left in the left portal branch for 5 to 7 d. The serum concentrations of cholic acid, chenodeoxycholic acid, and deoxycholic acid in portal venous and systemic circulation were then determined at intervals of 15 to 30 min before and after a standardized meal. A highly accurate and specific gas chromatographic/mass spectrometric technique was used. The sum of the fasting concentrations of the three bile acids averaged 14.04+/-4.13 mumol/liter in portal venous serum, and 2.44+/-0.31 mumol/liter in peripheral venous serum. The estimated hepatic fractional uptake of cholic acid was approximately 90%, and those of chenodeoxycholic acid and deoxycholic acid were 70-80%. This resulted in an enrichment of systemic bile acids in the dihydroxy bile acid species. In response to a standardized meal, portal venous bile acid concentrations increased two- to sixfold, with a peak seen 15-60 min after the meal. The maximum postprandial portal venous bile acid concentration averaged 43.04+/-6.12 mumol/liter, and the corresponding concentration in peripheral serum was 5.22+/-0.74 mumol/liter. The estimated fractional uptakes of the individual bile acids were not affected by the increased inflow to the liver. The peripheral venous concentrations of individual as well as total bile acids were well correlated with those in portal venous serum. The results (a) give a quantitation of postprandial bile acid inflow to the liver and (b) indicate that the hepatic uptake system for bile acids in healthy man cannot be saturated during maximal inflow of endogenous bile acids. Measurement of peripheral serum bile acids can thus give important information on the status of the enterohepatic circulation.

Epidemiological studies have suggested that the concentration and composition of fecal bile acids are important determining factors in the etiology of colon cancer. However, the mechanism by which these compounds influence tumor development is not understood. To begin to elucidate their mechanism of action, four bile acids, cholic acid, chenodeoxycholic acid, deoxycholic acid (DCA), and ursodeoxycholic acid, were examined for their effects on the growth of several different tumor cell lines. We found that incubating cells with chenodeoxycholic acid or DCA caused morphological changes, seen by electron and light microscopy, that were characteristic of apoptosis, whereas incubating cells with ursodeoxycholic acid inhibited cell proliferation but did not induce apoptosis. Cholic acid had no discernible effect on cells. Notably, the apoptosis induced by DCA could be suppressed by inhibiting protein kinase C activity with calphostin C. These results indicate that different bile acids exhibit distinct biological activities and suggest that the cytotoxicity reported for DCA may be due to its capacity to induce apoptosis via a protein kinase C-dependent signaling pathway.

The farnesoid X receptor (FXR), an endogenous sensor for bile acids, regulates a program of genes involved in bile acid biosynthesis, conjugation, and transport. Cholestatic liver diseases are a group of immunologically and genetically mediated disorders in which accumulation of endogenous bile acids plays a role in the disease progression and symptoms. Here, we describe the effect of 6-ethyl chenodeoxycholic acid (6-ECDCA or INT-747), a semisynthetic bile acid derivative and potent FXR ligand, in a model of cholestasis induced by 5-day administration of 17alpha-ethynylestradiol (E(2)17alpha) to rats. The exposure of rat hepatocytes to 1 microM 6-ECDCA caused a 3- to 5-fold induction of small heterodimer partner (Shp) and bile salt export pump (bsep) mRNA and 70 to 80% reduction of cholesterol 7alpha-hydroxylase (cyp7a1), oxysterol 12beta-hydroxylase (cyp8b1), and Na(+)/taurocholate cotransporting peptide (ntcp). In vivo administration of 6-ECDCA protects against cholestasis induced by E(2)17alpha. Thus, 6-ECDCA reverted bile flow impairment induced by E(2)17alpha, reduced secretion of cholic acid and deoxycholic acid, but increased muricholic acid and chenodeoxycholic acid secretion. In vivo administration of 6-ECDCA increased liver expression of Shp, bsep, multidrug resistance-associated protein-2, and multidrug resistance protein-2, whereas it reduced cyp7a1 and cyp8b1 and ntcp mRNA. These changes were reproduced by GW4064, a synthetic FXR ligand. In conclusion, by demonstrating that 6-ECDCA protects against E(2)17alpha cholestasis, our data support the notion that development of potent FXR ligands might represent a new approach for the treatment of cholestatic disorders.

The recently identified type VI secretion system (T6SS) of proteobacteria has been shown to promote pathogenicity, competitive advantage over competing microorganisms, and adaptation to environmental perturbation. By detailed phenotypic characterization of loss-of-function mutants, in silico, in vitro and in vivo analyses, we provide evidence that the enteric pathogen, Campylobacter jejuni, possesses a functional T6SS and that the secretion system exerts pleiotropic effects on two crucial processes--survival in a bile salt, deoxycholic acid (DCA), and host cell adherence and invasion. The expression of T6SS during initial exposure to the upper range of physiological levels of DCA (0.075%-0.2%) was detrimental to C. jejuni proliferation, whereas down-regulation or inactivation of T6SS enabled C. jejuni to resist this effect. The C. jejuni multidrug efflux transporter gene, cmeA, was significantly up-regulated during the initial exposure to DCA in the wild type C. jejuni relative to the T6SS-deficient strains, suggesting that inhibition of proliferation is the consequence of T6SS-mediated DCA influx. A sequential modulation of the efflux transporter activity and the T6SS represents, in part, an adaptive mechanism for C. jejuni to overcome this inhibitory effect, thereby ensuring its survival. C. jejuni T6SS plays important roles in host cell adhesion and invasion as T6SS inactivation resulted in a reduction of adherence to and invasion of in vitro cell lines, while over-expression of a hemolysin co-regulated protein, which encodes a secreted T6SS component, greatly enhanced these processes. When inoculated into B6.129P2-IL-10(tm1Cgn) mice, the T6SS-deficient C. jejuni strains did not effectively establish persistent colonization, indicating that T6SS contributes to colonization in vivo. Taken together, our data demonstrate the importance of bacterial T6SS in host cell adhesion, invasion, colonization and, for the first time to our knowledge, adaptation to DCA, providing new insights into the role of T6SS in C. jejuni pathogenesis.

There is considerable evidence that the level of deoxycholic acid in the bile influences biliary cholesterol saturation. Deoxycholic acid is formed in the colon and absorbed slowly. Hence changes in colonic transit rate might influence biliary deoxycholic acid and the cholesterol saturation of bile. When 14 constipated subjects took standardised senna tablets for six weeks in a dose sufficient to lower mean whole gut transit time from 134 to 54 hours, deoxycholic acid as a proportion of biliary bile acids fell from 25.9 +/- 8.6 to 17.2 +/- 8.3% (p less than 0.0001) and deoxycholic acid pool measured by isotope dilution fell from 0.64 +/- 0.34 to 0.45 +/- 0.29 g (p less than 0.0001). In those subjects (n = 8) whose bile was initially supersaturated with cholesterol, the saturation index fell from 1.40 +/- 0.22 to 1.20 +/- 0.19 (p = 0.02). Conversely, when 12 normal volunteers took loperamide capsules sufficient to cause symptomatic constipation and to prolong mean transit-time from 48 to 103 hours, the deoxycholic acid pool increased from 0.40 +/- 0.24 to 0.57 +/- 0.17 g (p = 0.008). The percentage deoxycholic acid did not alter significantly, because the estimated total bile acid pool expanded (from 1.98 +/- 0.61 to 2.81 +/- 0.48 g; p less than 0.001), presumably because of loperamide slowing down small bowel transit. Despite this expansion of the bile acid pool, loperamide increased the cholesterol saturation index from 1.10 +/- 0.31 to 1.20 +/- 0.32 (p = 0.01). Changes in colonic transit rate alter the size of the deoxycholic acid pool and bile cholesterol saturation. These findings suggest that constipation or slow colonic transit might increase the chance of supersaturated bile and hence of gall stones.

Bile acids have been reported to activate several different cell signaling cascades in rat hepatocytes. However, the mechanism(s) of activation of these pathways have not been determined. This study aims to determine which bile acids activate the Raf-1/MEK/ERK cascade and the mechanism of activation of this pathway. Taurodeoxycholic acid (TDCA) stimulated (+235%) the phosphorylation of p(74) Raf-1 in a time (5 to 20 minutes) and concentration-dependent (10 to 100 micromol/L) manner. Raf-1 and ERK activities were both significantly increased by most bile acids tested. Deoxycholic acid (DCA) was the best activator of ERK (3.6-fold). A dominant negative Ras (N17) construct expressed in primary hepatocytes prevented the activation of ERK by DCA. The epidermal growth factor receptor (EGFR)-specific inhibitor (AG1478) significantly inhibited (approximately 81%) the activation of ERK by DCA. DCA rapidly (30 to 60 seconds) increased phosphorylation of the EGFR (approximately 2-fold) and Shc (approximately 4-fold). A dominant negative mutant of the EGFR (CD533) blocked the ability of DCA to activate ERK. In conclusion, these results show that DCA activates the Raf-1/MEK/ERK signaling cascade in primary hepatocytes primarily via an EGFR/Ras-dependent mechanism.

Previous studies have demonstrated in hepatocytes that deoxycholic acid (DCA) promotes inactivation of protein tyrosine phosphatases (PTPases) and activation of ERBB1 and the extracellular-regulated kinase (ERK) 1/2 pathway. The present studies have determined the biochemical mechanism(s) through which these events occur. DCA and taurodeoxycholic acid (TDCA) (100 micromol/L) caused activation of ERBB1, insulin receptor, and the ERK1/2 and AKT pathways in primary rodent hepatocytes. DCA- and TDCA-induced receptor and signaling pathway activations were blocked by the reactive oxygen species (ROS) scavengers N-acetyl cysteine (NAC) and Trolox (TX), as well as by cyclosporin A (CsA) and bongkrekic acid (BKA). DCA activated the ERK1/2 pathway in HuH7 human hepatoma cells that was blocked by the incubation of cells with an ERBB1 inhibitor, NAC, TX, CsA, or BKA. DCA did not activate the ERK1/2 pathway in mitochondria-defective HuH7 Rho 0 cells. In HuH7 cells and primary hepatocytes, DCA enhanced the production of ROS, an effect that was abolished in Rho 0 cells and by prior incubation of cells with CsA or BKA. In hepatocytes and HuH7 cells, DCA inhibited PTPase activity. Incubation of hepatocytes with either CsA or BKA prevented DCA-induced inhibition of PTPase activity. Loss of mitochondrial function in Rho 0 cells also abolished the inhibitory effects of DCA on PTPase activity. In conclusion, DCA and TDCA cause ROS generation in hepatocytes that is dependent on metabolically active mitochondria. The generation of ROS is essential for PTPase inactivation, receptor tyrosine kinase activation, and enhanced signaling down the ERK1/2 and AKT pathways.

Bile acids up-regulate death receptor 5 (DR5)/TRAIL-receptor 2 (TRAIL-R2) expression thereby sensitizing hepatocytes to TRAIL-mediated apoptosis. However, the precise mechanism by which bile acids enhance DR5/TRAIL-R2 expression is unknown. Although several bile acids enhanced DR5/TRAIL-R2 expression, deoxycholic acid (DCA) was the most potent. DCA stimulated JNK activation and the JNK inhibitor SP600125 blocked DCA-induced DR5/TRAIL-R2 mRNA and protein expression. Reporter gene analysis identified a 5'-flanking region containing two Sp1 binding sites within the DR5/TRAIL-R2 promoter as bile acid responsive. Sp1 binding to one of the two sites was enhanced by DCA treatment as evaluated by electrophoretic mobility shift assays and chromatin immunoprecipitation studies. JNK inhibition with SP600125 also blocked binding of Sp1 to the DR5/TRAIL-R2 promoter. Finally, point mutations of the Sp1 binding site attenuated promoter activity. In conclusion, Sp1 is a bile acid-responsive transcription factor that mediates DR5/TRAIL-R2 gene expression downstream of JNK.

The hydrophilic bile salt ursodeoxycholic acid (UDCA) is a potent inhibitor of apoptosis. In this paper, we further characterize the mechanism by which UDCA inhibits apoptosis induced by deoxycholic acid, okadaic acid and transforming growth factor beta1 in primary rat hepatocytes. Our data indicate that coincubation of cells with UDCA and each of the apoptosis-inducing agents was associated with an approximately 80% inhibition of nuclear fragmentation (P<0.001). Moreover, UDCA prevented mitochondrial release of cytochrome c into the cytoplasm by 70 - 75% (P<0.001), thereby, inhibiting subsequent activation of DEVD-specific caspases and cleavage of poly(ADP-ribose) polymerase. Each of the apoptosis-inducing agents decreased mitochondrial transmembrane potential and increased mitochondrial-associated Bax protein levels. Coincubation with UDCA was associated with significant inhibition of these mitochondrial membrane alterations. The results suggest that the mechanism by which UDCA inhibits apoptosis involves an interplay of events in which both depolarization and channel-forming activity of the mitochondrial membrane are inhibited.

Impairment of gut barrier is associated with a fat-rich diet, but mechanisms are unknown. We have earlier shown that dietary fat modifies fecal bile acids in mice, decreasing the proportion of ursodeoxycholic acid (UDCA) vs. deoxycholic acid (DCA). To clarify the potential role of bile acids in fat-induced barrier dysfunction, we here investigated how physiological concentrations of DCA and UDCA affect barrier function in mouse intestinal tissue. Bile acid experiments were conducted in vitro in Ussing chambers using 4- and 20-kDa FITC-labeled dextrans. Epithelial integrity and inflammation were assayed by histology and Western blot analysis for cyclooxygenase-2. LPS was studied in DCA-induced barrier dysfunction. Finally, we investigated in a 10-wk in vivo feeding trial in mice the barrier-disrupting effect of a diet containing 0.1% DCA. DCA disrupted epithelial integrity dose dependently at 1-3 mM, which correspond to physiological concentrations on a high-fat diet. Low-fat diet-related concentrations of DCA had no effect. In vivo, the DCA-containing diet increased intestinal permeability 1.5-fold compared with control (P = 0.016). Hematoxylin-eosin staining showed a clear disruption of the epithelial barrier by 3 mM DCA in vitro. A short-term treatment by DCA did not increase cyclooxygenase-2 content in colon preparations. UDCA did not affect barrier function itself, but it ameliorated DCA-induced barrier disruption at a 0.6 mM concentration. LPS had no significant effect on barrier function at 0.5-4.5 μg/ml concentrations. We suggest a novel mechanism for barrier dysfunction on a high-fat diet involving the effect of hydrophobic luminal bile acids.

Ursodeoxycholic acid (UDCA), a hydrophilic bile acid, is known as a cytoprotective agent. UDCA prevents apoptosis induced by a variety of stress stimuli including cytotoxic bile acids such as deoxycholic acid (DCA). Here we examined the molecular mechanism by which UDCA can antagonize DCA-induced apoptosis in human colon cancer cells. UDCA pretreatment decreases the number of apoptotic cells caused by exposure to DCA and UDCA. Further studies of the signaling pathway showed that UDCA pretreatment suppressed DNA binding activity of activator protein-1 and this was accompanied by downregulation of both extracellular signal-regulated kinase (ERK) and Raf-1 kinase activities stimulated by exposure to DCA. DCA was also found to activate epidermal growth factor receptor (EGFR) activity and UDCA inhibited this. Collectively, these findings suggest that the inhibitory effect of UDCA in DCA-induced apoptosis is partly mediated by modulation of EGFR/Raf-1/ERK signaling.

Six normal men were fed formula diets containing either highly saturated fat (cocoa butter, iodine value 32) or polyunsaturated fat (corn oil, iodine value 125). The sterol balance technique was used to compare the changes in serum cholesterol concentration with the excretion of fecal steroids. The method used for the analysis of fecal steroids was chemical, with a final identification and quantification by gas-liquid chromatography. It was confirmed that the chemical method for fecal steroid analysis was accurate and reproducible. The three dietary periods were each 3 wk in length. In sequence, cocoa butter (period I), corn oil, and cocoa butter (period III) were fed at 40% of the total calories. All diets were cholesterol free, contained similar amounts of plant sterols, and were identical in other nutrients. Corn oil had a hypocholesterolemic effect. Mean serum cholesterol concentrations were 222 mg/100 ml (cocoa butter, period I), 177 during corn oil, and 225 after the return to cocoa butter. Individual fecal steroids were determined from stools pooled for 7 days. Both neutral steroids and bile acids were altered significantly by dietary polyunsaturated fat. The change in bile acid excretion was considerably greater than the change in neutral steroids. Corn oil caused a greater fecal excretion of both deoxycholic and lithocholic acids. The total mean excretion (milligrams per day) of fecal steroids was 709 for cocoa butter (period I), 915 for corn oil, and 629 for the second cocoa butter period. The enhanced total fecal steroid excretion by the polyunsaturated fat of corn oil created a negative cholesterol balance vis-à-vis the saturated fat of cocoa butter. The hypocholesterolemic effect of polyunsaturated fat was associated with total fecal sterol excretion twice greater than the amount of cholesterol calculated to leave the plasma. This finding suggested possible loss of cholesterol from the tissues as well.

Hypertransaminasemia is a frequent side effect during chenodeoxycholic administration for gallstone dissolution. Evidence suggests that this effect is not mediated by lithocholic acid, the intestinal metabolite of chenodeoxycholic acid, but that toxicity is due to the chenodeoxycholic acid itself. In vitro cytotoxicity of bile salts is positively proportional to their detergent effect, which is, on the other hand, related to their hydrophobic-hydrophilic balance. We hypothesize that in vivo also liver injury can occur when the liver is perfused by an high proportion of strongly detergent bile salts. The more detergent bile salts are unconjugated or glycine conjugated, while the lesser are taurine conjugated and sulfated. Within each class the following order of decreasing detergent power can be indicated: lithocholic greater than deoxycholic greater than chenodeoxycholic greater than cholic greater than ursodeoxycholic acid. Besides chronic exogenous administration of chenodeoxycholic or deoxycholic acids, conditions in which the liver is perfused by an high mass of highly detergent bile salts are those characterized by an enhanced intestinal biodegradation of bile salts. These conditions, which are common features of some chronic inflammatory bowel diseases, are frequently associated with liver damage. On the other hand, a normally detergent bile salt pool can become hepatotoxic for liver cells which have already been injured. In this respect, as already reported for increased sulfation, the increased proportion of taurine conjugates and the reduced formation of deoxycholic acid in liver cirrhosis can be regarded as protective mechanisms. Liver toxicity induced by bile salts' detergent action can be prevented by favouring tauroconjugation or reducing the intestinal degradation of bile salts or by administering poorly detergent bile salts.

Barrett's oesophagus patients accumulate chromosomal defects during the histological progression to cancer, one of the most prominent of which is the amplification of the whole of chromosome 4. We aimed to study the role that the transcription factor NF-kappaB, a candidate cancer- promoting gene, present on chromosome 4, plays in Barrett's oesophagus, using OE33 cells as a model. Specifically, we wanted to determine if NF-kappaB was activated by exposure to bile acid (deoxycholic acid) in oesophageal cells. We employed pathway specific cDNA microarrays and real-time PCR, to first identify bile acid induced genes and specifically to investigate the role of NF-kappaB. An NF-kappaB reporter system was used, as well as an inhibitor of NF-kappaB (pyrrolidine dithiocarbamate) to confirm the activation of NF-kappaB by bile. We show that physiological levels of DCA (100-300 microM) were capable of activating NF-kappaB in OE33 cells and inducing NF-kappaB target gene expression (particularly IkappaB and IL-8). Other gene expression abnormalities were also shown to be induced by DCA. Importantly, preliminary experiments showed that NF-kappaB activation by bile occurred at neutral pH, but not at acid pH. Acidic bile did however cause over-expression of the c-myc oncogene, as reported previously. Hence, we present data showing that NF-kappaB may be a key mediator of carcinogenesis in bile exposed Barrett's tissues. In addition, neutral bile acids appear to play a significant part in reflux induced gene expression changes. We postulate that the activation of the survival factor NF-kappaB by bile may be linked to the previous cytogenetic data from our laboratory showing the amplification of NF-kappaB's chromosome (chromosome 4), during Barrett's cancer progression. Hence chromosome 4 amplification may provide a survival mechanism for bile exposed oesophageal tissues via NF-kappaB.

A targeted intracellular delivery system of paclitaxel (PTX) was successfully developed based on redox-sensitive hyaluronic acid-deoxycholic acid (HA-ss-DOCA) conjugates. The conjugates self-assembled into nano-size micelles in aqueous media and exhibited excellent drug-loading capacities (34.1%) and entrapment efficiency (93.2%) for PTX. HA-ss-DOCA micelles were sufficiently stable at simulated normal physiologic condition but fast disassembled in the presence of 20 mm reducing agent, glutathione. In vitro drug release studies showed that the PTX-loaded HA-ss-DOCA micelles accomplished rapid drug release under reducing condition. Intracellular release of fluorescent probe nile red indicated that HA-ss-DOCA micelles provide an effective approach for rapid transport of cargo into the cytoplasm. Enhanced cytotoxicity of PTX-loaded HA-ss-DOCA micelles further confirmed that the sensitive micelles are more potent for intracellular drug delivery as compared to the insensitive control. Based on flow cytometry and confocal microscopic analyses, observations revealed that HA-ss-DOCA micelles were taken up to human breast adenocarcinoma cells (MDA-MB-231) via HA-receptor mediated endocytosis. In vivo investigation of micelles in tumor-bearing mice confirmed that HA-ss-DOCA micelles possessed much higher tumor targeting capacity than the insensitive control. These results suggest that redox-sensitive HA-ss-DOCA micelles hold great potential as targeted intracellular delivery carriers of lipophilic anticancer drugs.

Bile acids (BAs) play important roles not only in lipid metabolism, but also in signal transduction. TGR5, a transmembrane receptor of BAs, is an immunomodulative factor, but its detailed mechanism remains unclear. Here, we aimed to delineate how BAs operate in immunological responses via the TGR5 pathway in human mononuclear cell lineages. We examined TGR5 expression in human peripheral blood monocytes, several types of in vitro differentiated macrophages (Mϕs) and dendritic cells. Mϕs differentiated with macrophage colony-stimulating factor and interferon-γ (Mγ-Mϕs), which are similar to the human intestinal lamina propria CD14(+) Mϕs that contribute to Crohn's disease (CD) pathogenesis by production of pro-inflammatory cytokines, highly expressed TGR5 compared with any other type of differentiated Mϕ and dendritic cells. We also showed that a TGR5 agonist and two types of BAs, deoxycholic acid and lithocholic acid, could inhibit tumour necrosis factor-α production in Mγ-Mϕs stimulated by commensal bacterial antigen or lipopolysaccharide. This inhibitory effect was mediated by the TGR5-cAMP pathway to induce phosphorylation of c-Fos that regulated nuclear factor-κB p65 activation. Next, we analysed TGR5 levels in lamina propria mononuclear cells (LPMCs) obtained from the intestinal mucosa of patients with CD. Compared with non-inflammatory bowel disease, inflamed CD LPMCs contained more TGR5 transcripts. Among LPMCs, isolated CD14(+) intestinal Mϕs from patients with CD expressed TGR5. In isolated intestinal CD14(+) Mϕs, a TGR5 agonist could inhibit tumour necrosis factor-α production. These results indicate that TGR5 signalling may have the potential to modulate immune responses in inflammatory bowel disease.

OBJECTIVE

There is an unclear relationship among bowel symptoms, excretion of unconjugated fecal bile acid (UBA), and colonic transit in irritable bowel syndrome (IBS). We measured total and main individual UBA in fecal samples of patients with IBS and assessed relationships among stool frequency or consistency, fecal UBA (total and individual), and colonic transit.

METHODS

In this study 30 healthy volunteers (controls), 31 subjects with IBS with diarrhea (IBS-D), and 30 with IBS with constipation (IBS-C) were placed on 4-day diets containing 100 g fat; we measured stool characteristics, total fecal UBA and fat levels, and overall colonic transit. We assessed univariate associations of total and individual levels of fecal UBA with phenotype (controls, IBS-D, IBS-C) by using the Kruskal-Wallis test; associations between end points were assessed by using Spearman correlations. With response surface regression models, we assessed relationships between stool, colonic transit, and fecal total and secretory UBA.

RESULTS

There was a significant association between total fecal UBA and phenotype (P = .029); the association was greater for IBS-D than IBS-C, compared with controls. Fecal levels of primary UBAs (cholic and chenodeoxycholic acids) were higher in subjects with IBS-D, compared with controls (both P < .01). Levels of fecal secretory UBAs (chenodeoxycholic acid, P = .019; deoxycholic acid, P = .025) were lower in subjects with IBS-C compared with controls, whereas levels of the nonsecretory UBA, lithocholic acid, were higher (P = .020). There were significant univariate associations between stool number and form and total fecal UBA (including percentages of lithocholic acid, chenodeoxycholic acid and cholic acid), fecal fat, and colonic transit at 24 and 48 hours after eating. In the regression models, the relative contribution of colonic transit was consistently greater and largely independent of the contribution of bile acids.

CONCLUSIONS

Measurements of individual UBAs identify changes associated with stool characteristics in patients with IBS; these effects are independent of the effects of colonic transit.

Male Sprague-Dawley rats were randomly divided into five groups in which group 1 received a sham operation (controls), groups 2-5 underwent common bile duct ligation and transection 14 days before the experiments. Two days prior to the studies, animals in groups 1 and 2 received saline orally, while groups 3-5 received an oral administration of either cholic acid, deoxycholic acid or whole bile. Specimens were taken for bacterial culture, and blood was collected for endotoxin assay. The rate of positive bacterial cultures from mesenteric lymph nodes in jaundiced saline-treated animals was significantly higher (p < 0.05) as compared with both controls and the other jaundiced animals treated with either bile or bile acids. Assays were positive for endotoxin in the jaundiced saline-treated group, whereas they were negative in both controls and bile- or bile-acid-treated animals. We conclude that oral administration of cholic acid, deoxycholic acid or whole bile inhibited bacterial translocation and endotoxin absorption in obstructive jaundice in the rat.