Glycochenodeoxycholate and glycodeoxycholate 3-O-glucuronides, but not hexadecanedioate and tetradecanedioate, detected weak inhibition of OATP1B caused by GDC-0810 in humans.

Endogenous biomarkers of drug transporters are promising tools to evaluate in vivo transporter function and potential alterations in the pharmacokinetics of their substrates. We have previously reported that coproporphyrin I/III captured the weak inhibition of OATP1B transporters by GDC-0810. In this study, we measured plasma concentrations of additional biomarkers, namely fatty acids, bile acids and their sulphate or glucuronide conjugates in the presence and absence of GDC-0810. Concentrations of hexadecanedioate and tetradecanedioate did not increase in the presence of GDC-0810. Among bile acids and their conjugates, glycochenodeoxycholate and glycodeoxycholate 3-O-glucuronides (GCDCA-3G and GDCA-3G) showed Cmax increases with geometric mean ratio (95% confidence interval) of 1.58 (1.13-2.22) and 1.49 (1.21-1.83), consistent with previous reports from low-dose rifampin co-administration and pharmacogenetic studies. These results suggest that GCDCA-3G and GDCA-3G are two more promising biomarkers that may capture weak OATP1B inhibition in addition to coproporphyrin I/III.

Bile acids and ceramide overcome the entry restriction for GII.3 human norovirus replication in human intestinal enteroids.

Human noroviruses (HuNoVs) cause sporadic and epidemic outbreaks of gastroenteritis in all age groups worldwide. We previously reported that stem cell-derived human intestinal enteroid (HIE) cultures support replication of multiple HuNoV strains and that some strains (e.g., GII.3) replicate only in the presence of bile. Heat- and trypsin-treatment of bile did not reduce GII.3 replication, indicating a nonproteinaceous component in bile functions as an active factor. Here we show that bile acids (BAs) are critical for GII.3 replication and replication correlates with BA hydrophobicity. Using the highly effective BA, glycochenodeoxycholic acid (GCDCA), we show BAs act during the early stage of infection, BA-dependent replication in HIEs is not mediated by detergent effects or classic farnesoid X receptor or Takeda G protein-coupled receptor 5 signaling but involves another G protein-coupled receptor, sphingosine-1-phosphate receptor 2, and BA treatment of HIEs increases particle uptake. We also demonstrate that GCDCA induces multiple cellular responses that promote GII.3 replication in HIEs, including enhancement of 1) endosomal uptake, 2) endosomal acidification and subsequent activity of endosomal/lysosomal enzyme acid sphingomyelinase (ASM), and 3) ceramide levels on the apical membrane. Inhibitors of endosomal acidification or ASM reduce GII.3 infection and exogenous addition of ceramide alone permits infection. Furthermore, inhibition of lysosomal exocytosis of ASM, which is required for ceramide production at the apical surface, decreases GII.3 infection. Together, our results support a model where GII.3 exploits rapid BA-mediated cellular endolysosomal dynamic changes and cellular ceramide to enter and replicate in jejunal HIEs.

Scopoletin and umbelliferone protect hepatocytes against palmitate- and bile acid-induced cell death by reducing endoplasmic reticulum stress and oxidative stress.

BACKGROUND: The number of patients with non-alcoholic fatty liver disease (NAFLD) is rapidly increasing due to the growing epidemic of obesity. Non-alcoholic steatohepatitis (NASH), the inflammatory stage of NAFLD, is characterized by lipid accumulation in hepatocytes, chronic inflammation and hepatocyte cell death. Scopoletin and umbelliferone are coumarin-like molecules and have antioxidant, anti-cancer and anti-inflammatory effects. Cytoprotective effects of these compounds have not been described in hepatocytes and the mechanisms of the beneficial effects of scopoletin and umbelliferone are unknown. AIM: To investigate whether scopoletin and/or umbelliferone protect hepatocytes against palmitate-induced cell death. For comparison, we also tested the cytoprotective effect of scopoletin and umbelliferone against bile acid-induced cell death. METHODS: Primary rat hepatocytes were exposed to palmitate (1 mmol/L) or the hydrophobic bile acid glycochenodeoxycholic acid (GCDCA; 50 µmol/L). Apoptosis was assessed by caspase-3 activity assay, necrosis by Sytox green assay, mRNA levels by qPCR, protein levels by Western blot and production of reactive oxygen species (ROS) by fluorescence assay. RESULTS: Both scopoletin and umbelliferone protected against palmitate and GCDCA-induced cell death. Both palmitate and GCDCA induced the expression of ER stress markers. Scopoletin and umbelliferone decreased palmitate- and GCDCA-induced expression of ER stress markers, phosphorylation of the cell death signaling intermediate JNK as well as ROS production. CONCLUSION: Scopoletin and umbelliferone protect against palmitate and bile acid-induced cell death of hepatocytes by inhibition of ER stress and ROS generation and decreasing phosphorylation of JNK. Scopoletin and umbelliferone may hold promise as a therapeutic modality for the treatment of NAFLD.

Multiple Timescale Dynamic Analysis of Functionally-Impairing Mutations in Human Ileal Bile Acid-Binding Protein.

Human ileal bile acid-binding protein (hI-BABP) has a key role in the enterohepatic circulation of bile salts. Its two internal binding sites exhibit positive cooperativity accompanied by a site-selectivity of glycocholate (GCA) and glycochenodeoxycholate (GCDA), the two most abundant bile salts in humans. To improve our understanding of the role of dynamics in ligand binding, we introduced functionally impairing single-residue mutations at two key regions of the protein and subjected the mutants to NMR relaxation analysis and MD simulations. According to our results, mutation in both the vicinity of the C/D (Q51A) and the G/H (Q99A) turns results in a redistribution of motional freedom in apo hI-BABP. Mutation Q51A, deteriorating the site-selectivity of GCA and GCDA, results in the channeling of ms fluctuations into faster motions in the binding pocket hampering the realization of key side chain interactions. Mutation Q99A, abolishing positive binding cooperativity for GCDA, leaves ms motions in the C-terminal half unchanged but by decoupling ßD from a dynamic cluster of the N-terminal half displays an increased flexibility in the vicinity of site 1. MD simulations of the variants indicate structural differences in the portal region and mutation-induced changes in dynamics, which depend on the protonation state of histidines. A dynamic coupling between the EFGH portal, the C/D-region, and the helical cap is evidenced highlighting the interplay of structural and dynamic effects in bile salt recognition in hI-BABP.

Short communication: Chemical structure, concentration, and pH are key factors influencing antimicrobial activity of conjugated bile acids against lactobacilli.

Effects of chemical structure, concentration, and pH on antimicrobial activity of conjugated bile acids were investigated in 4 strains of lactobacilli. Considerable differences were observed in the antimicrobial activity between the 6 human conjugated bile acids, including glycocholic acid, taurocholic acid, glycodeoxycholic acid, taurodeoxycholic acid, glycochenodeoxycholic acid, and taurochenodeoxycholic acid. Glycodeoxycholic acid and glycochenodeoxycholic acid generally showed significantly higher antimicrobial activity against the lactobacilli, but glycocholic acid and taurocholic acid exhibited the significantly lower antimicrobial activity. Glycochenodeoxycholic acid was selected for further analysis, and the results showed its antimicrobial activity was concentration-dependent, and there was a significantly negative linear correlation (R2 > 0.98) between bile-antimicrobial index and logarithmic concentration of the bile acid for each strain of lactobacilli. Additionally, the antimicrobial activity of glycochenodeoxycholic acid was also observed to be pH-dependent, and it was significantly enhanced with the decreasing pH, with the result that all the strains of lactobacilli were unable to grow at pH 5.0. In conclusion, chemical structure, concentration, and pH are key factors influencing antimicrobial activity of conjugated bile acids against lactobacilli. This study provides theoretical guidance and technology support for developing a scientific method for evaluating the bile tolerance ability of potentially probiotic strains of lactobacilli.

Glycochenodeoxycholate promotes the metastasis of gallbladder cancer cells by inducing epithelial to mesenchymal transition via activation of SOCS3/JAK2/STAT3 signaling pathway.

The incidence of gallbladder cancer (GBC) is relatively rare but a high degree of malignancy. The migration and invasion potential of GBC severely affects the prognosis of patients with GBC. Glycochenodeoxycholate (GCDC) is one of the most important components in GBC-associated microenvironment. However, the role of GCDC in the metastatic feature of GBC cells is not fully understood. First, the results of this study found that GCDC could effectively enhance the metastasis of GBC cells. Furthermore, GCDC could lead to the enhancement of epithelial to mesenchymal transition (EMT) phenotype in GBC cells, which is concerned to be an important mechanism of tumor metastasis. Further studies showed that GCDC treatment induced the upregulation of matrix metalloproteinase-3 (MMP3), MMP9, and SOCS3/JAK2/p-STAT3 signal pathway in GBC cells, which could regulate the level of EMT. Beside that, we also found the positive expression of farnesoid X receptor (FXR) in GBC cells and inhibition of FXR could significantly block the effect of GCDC on the metastasis of GBC cells. These results indicated that GCDC promoted GBC cells metastasis by enhancing the level of EMT and inhibition of FXR could significantly block the effect of GCDC. On one hand, FXR might be an indicator for predicting the metastasis of patient with GBC. On the other hand, FXR might serve as a potential antimetastasis target in GBC therapy.

Glycochenodeoxycholate induces cell survival and chemoresistance via phosphorylation of STAT3 at Ser727 site in HCC.

Glycochenodeoxycholate (GCDA) is closely associated with carcinogenesis and chemoresistance of hepatocellular carcinoma (HCC). Signal transducer and activator of transcription 3 (STAT3), a transcription factor, is involved in various human tumors. Whether GCDA induces chemoresistance through STAT3 and the mechanism of action remains elusive. In this study, we firstly found that the expression level of STAT3 has a positive correlation with chemoresistance of HCC cells. Moreover, GCDA can upregulate the expression of STAT3 protein. Hence, we suspect that GCDA may induce chemoresistance of HCC cells via STAT3. Mechanistically, GCDA stimulates phosphorylation of STAT3 at Ser727 site and mediates pSer727-STAT3 protein to translocate and aggregate in the nucleus, which is important for cell survival. When Ser727 of STAT3 mutated to Asp, the capacity of STAT3 to accumulate in the nucleus was attenuated, STAT3-induced cell survival was impaired and GCDA-induced chemoresistance was abolished. In addition, while activation of extracellular signal-regulated kinase 1/2 (ERK1/2) was inhibited by PD98059, phosphorylation of STAT3 at Ser727 induced by GCDA was suppressed. Taken together, these data demonstrate that GCDA-enhanced survival of liver cancer cells may occur through the activation of STAT3 by phosphorylation at Ser727 site via mitogen-activated protein kinase/ERK1/2 pathway, which may contribute to the progression of human liver cancer and chemoresistance.

Bile salt (glycochenodeoxycholate acid) induces cell survival and chemoresistance in hepatocellular carcinoma.

OBJECTIVE: Glycochenodeoxycholate acid (GCDA) is a toxic component in bile salts. It plays an important role in the development and progression of liver cancer. In this study, we investigated the underlying mechanism of GCDA in hepatocarcinogenesis and chemotherapy resistance. MATERIALS AND METHODS: Cell proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and clonality by Ki-67 and colony-formation assay. Apoptosis was examined by flow cytometry. Real-time polymerase chain reaction (PCR) and western blot analysis were used to measure messenger RNA and protein levels, respectively. Short hairpin RNA was used to silence signal transducer and activator of transcription 3 (Stat3) expression. RESULTS: Bile salts (GCDA) promoted the proliferation of hepatocellular carcinoma (HCC) cells (HepG2 and QGY-7703), and GCDA treatment reduced the chemosensitivity of 5-fluorouracil (5FU) in HepG2 and QGY-7703 cells. GCDA upregulated the expression of antiapoptosis proteins Mcl-1/Survivin/Bcl-2. GCDA had no discernible effect on basal protein level or subcellular localization of phosphorylated Stat3. 5FU increased the apoptosis of HepG2 cells with silenced Stat3 expression, but GCDA-induced chemoresistance was not reversed. CONCLUSIONS: GCDA-reduced HCC cell chemosensitivity may occur by upregulating antiapoptosis proteins Mcl-1/Survivin/Bcl-2. Stat3 may be a target for enhancing the chemosensitivity of hepatocellular carcinoma cells, but GCDA-induced chemoresistance is independent of Stat3.

Regulation of Plasma Membrane Localization of the Na⁺-Taurocholate Co-Transporting Polypeptide by Glycochenodeoxycholate and Tauroursodeoxycholate.

BACKGROUND/AIMS: Hydrophobic bile salts, such as glycochenodeoxycholate (GCDC) can trigger hepatocyte apoptosis, which is prevented by tauroursodesoxycholate (TUDC), but the effects of GCDC and TUDC on sinusoidal bile salt uptake via the Na⁺-taurocholate transporting polypeptide (Ntcp) are unclear. METHODS: The effects of GCDC and TUDC on the plasma membrane localization of Ntcp were studied in perfused rat liver by means of immunofluorescence analysis and super-resolution microscopy. The underlying signaling events were investigated by Western blotting and inhibitor studies. RESULTS: GCDC (20 µmol/l) induced within 60 min a retrieval of Ntcp from the basolateral membrane into the cytosol, which was accompanied by an activating phosphorylation of the Src kinases Fyn and Yes. Both, Fyn activation and the GCDC-induced Ntcp retrieval from the plasma membrane were sensitive to the NADPH oxidase inhibitor apocynin, the antioxidant N-acetylcysteine and the Src family kinase inhibitors SU6656 and PP-2, whereas PP-2 did not inhibit GCDC-induced Yes activation. Internalization of Ntcp by GCDC was also prevented by the protein kinase C (PKC) inhibitor Gö6850. TUDC (20 µmol/l) reversed the GCDC-induced retrieval of Ntcp from the plasma membrane and prevented the activation of Fyn and Yes in GCDC-perfused rat livers. Reinsertion of Ntcp into the basolateral membrane in GCDC-perfused livers by TUDC was sensitive to the protein kinase A (PKA) inhibitor H89 and the integrin-inhibitory peptide GRGDSP, whereas the control peptide GRADSP was ineffective. Ex posure of cultured rat hepatocytes to GCDC (50 µmol/l, 15min) increased the fluorescence intensity of the reactive oxygen fluorescent indicator DCF to about 1.6-fold of untreated controls in a TUDC (50 µmol/l)-sensitive way. GCDC caused a TUDC-sensitive canalicular dilatation without evidence for Bsep retrieval from the canalicular membrane. CONCLUSION: The present study suggests that GCDC triggers the retrieval of Ntcp from the basolateral membrane into the cytosol through an oxidative stress-dependent activation of Fyn. TUDC prevents the GCDC-induced Fyn activation and Ntcp retrieval through integrin-dependent activation of PKA.

Tauroursodeoxycholate protects from glycochenodeoxycholate-induced gene expression changes in perfused rat liver.

Tauroursodeoxycholate (TUDC) is well known to protect against glycochenodeoxycholate (GCDC)-induced apoptosis in rat hepatocytes. In the present study, we analyzed whether TUDC also exerts protective effects by modulating GCDC-induced gene expression changes. For this, gene array-based transcriptome analysis and quantitative polymerase chain reaction (qPCR) were performed on RNA isolated from rat livers perfused with GCDC, TUDC or a combination of both (each 20 µm for 2 h). GCDC led to a significant increase of lactate dehydrogenase (LDH) into the effluent perfusate, which was prevented by TUDC. GCDC, TUDC and co-perfusion induced distinct gene expression changes. While GCDC upregulated the expression of several pro-inflammatory genes, co-perfusion with TUDC increased the expression of pro-proliferative and anti-apoptotic p53 target genes. In line with this, levels of serine20-phosphorylated p53 and of its target gene p21 were elevated by GCDC in a TUDC-sensitive way. GCDC upregulated the oxidative stress surrogate marker 8OH(d)G and the pro-apoptotic microRNAs miR-15b/16 and these effects were prevented by TUDC. The upregulation of miR-15b and miR-16 in GCDC-perfused livers was accompanied by a downregulation of several potential miR-15b and miR-16 target genes. The present study identified changes in the transcriptome of the rat liver which suggest, that TUDC is hepatoprotective by counteracting GCDC-induced gene expression changes.

Organic solute transporter OSTα/ß is overexpressed in nonalcoholic steatohepatitis and modulated by drugs associated with liver injury.

The heteromeric steroid transporter organic solute transporter α/ß (OSTα/ß, SLC51A/B) was discovered over a decade ago, but its physiological significance in the liver remains uncertain. A major challenge has been the lack of suitable models expressing OSTα/ß. Based on observations first reported here that hepatic OSTα/ß is upregulated in nonalcoholic steatohepatitis, the aim of this research was to develop an in vitro model to evaluate OSTα/ß function and interaction with drugs and bile acids. OSTα/ß expression in human liver tissue was analyzed by quantitative RT-PCR, Western blotting, and immunofluorescence. Radiolabeled compounds were used to determine OSTα/ß-mediated transport in the established in vitro model. The effect of bile acids and drugs, including those associated with cholestatic drug-induced liver injury, on OSTα/ß-mediated transport was evaluated. Expression of OSTα/ß was elevated in the liver of patients with nonalcoholic steatohepatitis and primary biliary cholangitis, whereas hepatocyte expression of OSTα/ß was low in control liver tissue. Studies in the novel cell-based system showed rapid and linear OSTα/ß-mediated transport for all tested compounds: dehydroepiandrosterone sulfate, digoxin, estrone sulfate, and taurocholate. The interaction study with 26 compounds revealed novel OSTα/ß inhibitors: a biomarker for cholestasis, glycochenodeoxycholic acid; the major metabolite of troglitazone, troglitazone sulfate; and a macrocyclic antibiotic, fidaxomicin. Additionally, some drugs (e.g., digoxin) consistently stimulated taurocholate uptake in OSTα/ß-overexpressing cells. Our findings demonstrate that OSTα/ß is an important transporter in liver disease and imply a role for this transporter in bile acid-bile acid and drug-bile acid interactions, as well as cholestatic drug-induced liver injury. NEW & NOTEWORTHY The organic solute transporter OSTα/ß is highly expressed in hepatocytes of liver tissue obtained from patients with nonalcoholic steatohepatitis and primary biliary cholangitis. OSTα/ß substrates exhibit rapid, linear, and concentration-driven transport in an OSTα/ß-overexpressing cell line. Drugs associated with hepatotoxicity modulate OSTα/ß-mediated taurocholate transport. These data suggest that hepatic OSTα/ß plays an essential role in patients with cholestasis and may have important clinical implications for bile acid and drug disposition.

Mesenchymal stem cells enhance autophagy of human intrahepatic biliary epithelial cells in vitro.

Dysfunctional autophagy in intrahepatic biliary epithelial cells (IBECs) is the main mechanism underlying the pathogenesis of bile duct lesions in primary biliary cholangitis. Autophagy may be a key pathogenesis for aetiology of primary biliary cholangitis. Immunoblotting and immunofluorescence analyses were used for the evaluation of autophagy in human intrahepatic biliary epithelial cells (HiBECs) at various time points. Glycochenodeoxycholate (GCDC) induced autophagy in HiBECs; the ratio of microtubule-associated protein light chain 3-II/microtubule-associated protein light chain 3-I (LC3-II/LC3-I) expression markedly increased at 48 hours, and then declined. However, compared with cells treated with GCDC alone, the expression of LC3-II increased and the clearance of autophagosome enhanced in GCDC-treated cells cocultured with mesenchymal stem cells (MSCs). Furthermore, the level of phosphorylation of signal transducer and activator of transcription 3 (pSTAT3) decreased in HiBECs cocultured with MSCs relative to those cultured without MSCs. Following STAT3 silencing, decreased expression of phosphorylated eukaryotic initiation factor 2α was consistently observed. The present data suggest that mesenchymal stem cells may enhance autophagic flux of HiBECs through the inhibition of STAT3 activity. SIGNIFICANCE PARAGRAPH: The present findings constitute the first report that human umbilical cord-derived MSCs enhance autophagic flux in HiBECs through a STAT3-dependent way: MSCs enhance the autophagic flux by increasing the formation of autophagosome and autolysosome in GCDC-treated HiBECs. MSCs decrease the STAT3 activity and the expression of eIF2α in GCDC-treated HiBECs; in addition, MSCs increase the expression of PKR. With STAT3 silencing, MSCs enhance neither the levels of LC3II nor the expression of PKR in GCDC-treated HiBECs.

Barrett's metaplasia develops from cellular reprograming of esophageal squamous epithelium due to gastroesophageal reflux.

Gastroesophageal reflux disease (GERD) clinically predisposes to columnar Barrett's metaplasia (BM) in the distal esophagus. We demonstrate evidence supporting the cellular origin of BM from reprograming or transcommitment of resident normal esophageal squamous (NES) epithelial cells in response to acid and bile (A + B) exposure using an in vitro cell culture model. The hTERT-immortalized NES cell line NES-B10T was exposed 5 min/day to an A + B mixture for 30 wk. Morphological changes, mRNA, and protein expression levels for the inflammatory marker cyclooxygenase-2; the lineage-determining transcription factors TAp63 (squamous), CDX2, and SOX9 (both columnar); and the columnar lineage markers Villin, Muc-2, CK8, and mAb Das-1 (incomplete phenotype of intestinal metaplasia) were assessed every 10 wk. Markers of columnar lineage and inflammation increased progressively, while squamous lineage-determining transcriptional factors were significantly decreased both at the mRNA and/or protein level in the NES-B10T cells at/after A + B treatment for 30 wk. Distinct modifications in morphological features were only observed at/after 30 wk of A + B exposure. These changes acquired by the NES-B10T 30-wk cells were retained even after cessation of A + B exposure for at least 3 wk. This study provides evidence that chronic exposure to the physiological components of gastric refluxate leads to repression of the discernable squamous transcriptional factors and activation of latent columnar transcriptional factors. This reflects the alteration in lineage commitment of the precursor-like biphenotypic, NES-B10T cells in response to A + B exposure as the possible origin of BM from the resident NES cells.NEW & NOTEWORTHY This study provides evidence of the origins of Barrett's metaplasia from lineage transcommitment of resident esophageal cells after chronic exposure to gastroesophageal refluxate. The preterminal progenitor-like squamous cells alter their differentiation and develop biphenotypic characteristics, expressing markers of incomplete-type columnar metaplasia. Development of these biphenotypic precursors in vitro is a unique model to study pathogenesis of Barrett's metaplasia and esophageal adenocarcinoma.

Mcl-1 suppresses abasic site repair following bile acid-induced hepatic cellular DNA damage.

In cholestasis, increases in bile acid levels result in the generation of reactive oxygen species and the induction of DNA damage and mutation. It is believed that bile acid accumulation is associated with liver tumorigenesis. However, the mechanism that underpins this phenomenon remains to be elucidated. Mcl-1, which is overexpressed in hepatic cells, is a pro-survival member of the Bcl-2 family. In this study, we observed that Mcl-1 potently suppresses the repair of bile acid-induced abasic (apurinic/apyrimidinic) sites in DNA lesions. Upon exposure of hepatic cells to glycochenodeoxycholate, one of the major conjugated human bile acids, we observed an increase in AP site accumulation along with induction of poly(ADP-ribose) polymerase and XRCC1 ( X-Ray Repair Cross Complementing 1). In addition, accumulation of Mcl-1 was observed in the nuclei of QGY-7703 cells in response to glycochenodeoxycholate stimulation. Knockdown of endogenous Mcl-1 by RNA interference significantly accelerated the repair of DNA lesions in glycochenodeoxycholate-treated cells. However, unlike XRCC1, poly(ADP-ribose) polymerase was induced following Mcl-1 knockdown. Conversely, poly(ADP-ribose) polymerase suppression was observed following glycochenodeoxycholate treatment of cells overexpressing Mcl-1. Moreover, AP-site counting analyses revealed that DNA repair activity was enhanced in cells overexpressing poly(ADP-ribose) polymerase under glycochenodeoxycholate stress conditions. It is well known that poly(ADP-ribose) polymerase plays a crucial role in the base excision repair pathway. Thus, our findings suggest that Mcl-1 suppresses base excision repair by inhibiting poly(ADP-ribose) polymerase induction following glycochenodeoxycholate-induced DNA damage. These results potentially explain how bile acid accumulation results in genetic instability and carcinogenesis.

Investigation of Glycochenodeoxycholate Sulfate and Chenodeoxycholate Glucuronide as Surrogate Endogenous Probes for Drug Interaction Studies of OATP1B1 and OATP1B3 in Healthy Japanese Volunteers.

PURPOSE: To assess the use of glycochenodeoxycholate-3-sulfate (GCDCA-S) and chenodeoxycholate 3- or 24-glucuronide (CDCA-3G or -24G) as surrogate endogenous substrates in the investigation of drug interactions involving OATP1B1 and OATP1B3. METHODS: Uptake of GCDCA-S and CDCA-24G was examined in HEK293 cells transfected with cDNA for OATP1B1, OATP1B3, and NTCP and in cryopreserved human hepatocytes. Plasma concentrations of bile acids and their metabolites (GCDCA-S, CDCA-3G, and CDCA-24G) were determined by LC-MS/MS in eight healthy volunteers with or without administration of rifampicin (600 mg, po). RESULTS: GCDCA-S and CDCA-24G were substrates for OATP1B1, OATP1B3, and NTCP. The uptake of [3H]atorvastatin, GCDCA-S, and CDCA-24G by human hepatocytes was significantly inhibited by both rifampicin and pioglitazone, whereas that of taurocholate was inhibited only by pioglitazone. Rifampicin elevated plasma concentrations of GCDCA-S more than those of other bile acids. The area under the plasma concentration-time curve for GCDCA-S was 20.3 times higher in rifampicin-treated samples. CDCA-24G could be detected only in plasma from the rifampicin-treatment phase, and CDCA-3G was undetectable in both phases. CONCLUSIONS: We identified GCDCA-S and CDCA-24G as substrates of NTCP, OATP1B1, and OATP1B3. GCDCA-S is a surrogate endogenous probe for the assessment of drug interactions involving hepatic OATP1B1 and OATP1B3.

Hydrophobic bile acid apoptosis is regulated by sphingosine-1-phosphate receptor 2 in rat hepatocytes and human hepatocellular carcinoma cells.

The hepatotoxic bile acid glycochenodeoxycholate (GCDC) modulates hepatocyte cell death through activation of JNK, Akt, and Erk. The nonhepatotoxic bile acid taurocholate activates Akt and Erk through the sphingosine-1-phosphate receptor 2 (S1PR2). The role of the S1PR2 in GCDC-mediated apoptosis and kinase activation is unknown. Studies were done in rat hepatocytes, HUH7 cells, and HUH7 cells stably transfected with rat Ntcp (HUH7-Ntcp). Cells were treated with GCDC and apoptosis was monitored morphologically by Hoechst staining and biochemically by immunoblotting for the active cleaved fragment of caspase 3. Kinase activation was determined by immunoblotting with phospho-specific antibodies. JTE-013, an inhibitor of S1PR2, significantly attenuated morphological evidence of GCDC-induced apoptosis and prevented caspase 3 cleavage in rat hepatocytes and HUH7-Ntcp cells. In hepatocytes, JTE-013 mildly suppressed, augmented, and had no effect on GCDC-induced JNK, Akt, and Erk phosphorylation, respectively. Similar results were seen in HUH7-Ntcp cells except for mild suppression of JNK and Erk phosphorylation. Knockdown of S1PR2 in HUH7-Ntcp augmented Akt, inhibited JNK, and had no effect on Erk phosphorylation. GCDC failed to induce apoptosis or kinase activation in HUH7 cells. In conclusion, SIPR2 inhibition attenuates GCDC-induced apoptosis and inhibits and augments GCDC-induced JNK and Akt phosphorylation, respectively. In addition, GCDC must enter hepatocytes to mediate cell death or activate kinases. These results suggest that SIPR2 activation is proapoptotic in GCDC-induced cell death but that this effect is not due to direct ligation of the S1PR2 by the bile acid.

Binding Isotherms and Time Courses Readily from Magnetic Resonance.

Evidence is presented that binding isotherms, simple or biphasic, can be extracted directly from noninterpreted, complex 2D NMR spectra using principal component analysis (PCA) to reveal the largest trend(s) across the series. This approach renders peak picking unnecessary for tracking population changes. In 1:1 binding, the first principal component captures the binding isotherm from NMR-detected titrations in fast, slow, and even intermediate and mixed exchange regimes, as illustrated for phospholigand associations with proteins. Although the sigmoidal shifts and line broadening of intermediate exchange distorts binding isotherms constructed conventionally, applying PCA directly to these spectra along with Pareto scaling overcomes the distortion. Applying PCA to time-domain NMR data also yields binding isotherms from titrations in fast or slow exchange. The algorithm readily extracts from magnetic resonance imaging movie time courses such as breathing and heart rate in chest imaging. Similarly, two-step binding processes detected by NMR are easily captured by principal components 1 and 2. PCA obviates the customary focus on specific peaks or regions of images. Applying it directly to a series of complex data will easily delineate binding isotherms, equilibrium shifts, and time courses of reactions or fluctuations.

Bile Acid-Induced Suicidal Erythrocyte Death.

BACKGROUND/AIMS: In nucleated cells, bile acids may activate cation channels subsequently leading to entry of Ca2+. In erythrocytes, increase of cytosolic Ca2+ activity triggers eryptosis, the suicidal death of erythrocytes characterized by phosphatidylserine exposure at the cell surface and cell shrinkage. Eryptosis is triggered by bile duct ligation, an effect partially attributed to conjugated bilirubin. The present study explored, whether bile acids may stimulate eryptosis. METHODS: Phosphatidylserine exposing erythrocytes have been identified utilizing annexin V binding, cell volume estimated from forward scatter, cytosolic Ca2+ activity determined using Fluo-3 fluorescence, and ceramide abundance at the erythrocyte surface utilizing specific antibodies. RESULTS: The exposure of human erythrocytes to glycochenodesoxycholic (GCDC) and taurochenodesoxycholic (TCDC) acid was followed by a significant decrease of forward scatter and significant increase of Fluo-3 fluorescence, ceramide abundance as well as annexin V binding. The effect on annexin V binding was significantly blunted, but not abolished by removal of extracellular Ca2+. CONCLUSION: Bile acids stimulate suicidal cell death, an effect paralleled by and in part due to Ca2+ entry and ceramide. The bile acid induced eryptosis may in turn lead to accelerated clearance of circulating erythrocytes and, thus, may contribute to anemia in cholestatic patients.

Decreasing mitochondrial fission prevents cholestatic liver injury.

Mitochondria frequently change their shape through fission and fusion in response to physiological stimuli as well as pathological insults. Disrupted mitochondrial morphology has been observed in cholestatic liver disease. However, the role of mitochondrial shape change in cholestasis is not defined. In this study, using in vitro and in vivo models of bile acid-induced liver injury, we investigated the contribution of mitochondrial morphology to the pathogenesis of cholestatic liver disease. We found that the toxic bile salt glycochenodeoxycholate (GCDC) rapidly fragmented mitochondria, both in primary mouse hepatocytes and in the bile transporter-expressing hepatic cell line McNtcp.24, leading to a significant increase in cell death. GCDC-induced mitochondrial fragmentation was associated with an increase in reactive oxygen species (ROS) levels. We found that preventing mitochondrial fragmentation in GCDC by inhibiting mitochondrial fission significantly decreased not only ROS levels but also cell death. We also induced cholestasis in mouse livers via common bile duct ligation. Using a transgenic mouse model inducibly expressing a dominant-negative fission mutant specifically in the liver, we demonstrated that decreasing mitochondrial fission substantially diminished ROS levels, liver injury, and fibrosis under cholestatic conditions. Taken together, our results provide new evidence that controlling mitochondrial fission is an effective strategy for ameliorating cholestatic liver injury.

Tauroursodeoxycholate Protects Rat Hepatocytes from Bile Acid-Induced Apoptosis via ß1-Integrin- and Protein Kinase A-Dependent Mechanisms.

BACKGROUND/AIMS: Ursodeoxycholic acid, which in vivo is rapidly converted into its taurine conjugate, is frequently used for the treatment of cholestatic liver disease. Apart from its choleretic effects, tauroursodeoxycholate (TUDC) can protect hepatocytes from bile acid-induced apoptosis, but the mechanisms underlying its anti-apoptotic effects are poorly understood. METHODS: These mechanisms were investigated in perfused rat liver and isolated rat hepatocytes. RESULTS: It was found that TUDC inhibited the glycochenodeoxycholate (GCDC)-induced activation of the CD95 death receptor at the level of association between CD95 and the epidermal growth factor receptor. This was due to a rapid TUDC-induced ß1-integrin-dependent cyclic AMP (cAMP) signal with induction of the dual specificity mitogen-activated protein (MAP) kinase phosphatase 1 (MKP-1), which prevented GCDC-induced phosphorylation of mitogen-activated protein kinase kinase 4 (MKK4) and c-jun-NH2-terminal kinase (JNK) activation. Furthermore, TUDC induced a protein kinase A (PKA)-mediated serine/threonine phosphorylation of the CD95, which was recently identified as an internalization signal for CD95. Furthermore, TUDC inhibited GCDC-induced CD95 targeting to the plasma membrane in a ß1-integrin-and PKA-dependent manner. In line with this, the ß1-integrin siRNA knockdown in sodium taurocholate cotransporting polypeptide (Ntcp)-transfected HepG2 cells abolished the protective effect of TUDC against GCDC-induced apoptosis. CONCLUSION: TUDC exerts its anti-apoptotic effect via a ß1-integrin-mediated formation of cAMP, which prevents CD95 activation by hydrophobic bile acids at the levels of JNK activation and CD95 serine/threonine phosphorylation.