Hepatic Expression of the Na+-Taurocholate Cotransporting Polypeptide Is Independent from Genetic Variation.

The hepatic Na+-taurocholate cotransporting polypeptide NTCP/SLC10A1 is important for the uptake of bile salts and selected drugs. Its inhibition results in increased systemic bile salt concentrations. NTCP is also the entry receptor for the hepatitis B/D virus. We investigated interindividual hepatic SLC10A1/NTCP expression using various omics technologies. SLC10A1/NTCP mRNA expression/protein abundance was quantified in well-characterized 143 human livers by real-time PCR and LC-MS/MS-based targeted proteomics. Genome-wide SNP arrays and SLC10A1 next-generation sequencing were used for genomic analyses. SLC10A1 DNA methylation was assessed through MALDI-TOF MS. Transcriptomics and untargeted metabolomics (UHPLC-Q-TOF-MS) were correlated to identify NTCP-related metabolic pathways. SLC10A1 mRNA and NTCP protein levels varied 44-fold and 10.4-fold, respectively. Non-genetic factors (e.g., smoking, alcohol consumption) influenced significantly NTCP expression. Genetic variants in SLC10A1 or other genes do not explain expression variability which was validated in livers (n = 50) from The Cancer Genome Atlas. The identified two missense SLC10A1 variants did not impair transport function in transfectants. Specific CpG sites in SLC10A1 as well as single metabolic alterations and pathways (e.g., peroxisomal and bile acid synthesis) were significantly associated with expression. Inter-individual variability of NTCP expression is multifactorial with the contribution of clinical factors, DNA methylation, transcriptional regulation as well as hepatic metabolism, but not genetic variation.

S-acylation status of bile acid transporter hASBT regulates its function, metabolic stability, membrane expression, and phosphorylation state.

The human apical sodium-dependent bile acid transporter (hASBT, SLC10A2) is the rate-limiting step of intestinal bile acid absorption in the enterohepatic circulation system of bile acids. Therefore, the regulation and stability of hASBT is vital in maintaining bile acid and cholesterol homeostasis and may serve as a potential target for cholesterol-related disorders. We hypothesized that post-translational mechanisms that govern hASBT function and regulation will provide novel insight on intestinal bile acid transport and homeostasis. In this study, we confirm the S-acylation status of hASBT via acyl biotin exchange in COS-1 cells and its impact on hASBT expression, function, kinetics, and protein stability. Using the acylation inhibitor, 2-bromopalmitate, we show that S-acylation is an important modification which modulates the function, surface expression, and maximal transporter flux (Jmax) of hASBT. By means of proteasome inhibitors, S-acylated hASBT was found to be cleared via the proteasome whereas a reduction in the palmitoylation status of hASBT resulted in rapid proteolytic degradation compared to the unmodified transporter. Screening of cysteine mutants in and or near transmembrane domains, some of which are exposed to the cytosol, confirmed Cys314 to be the predominate S-acylated residue. Lastly, we show that S-acylation was reduced in a mutant form of hASBT devoid of cytosolic facing tyrosine residues, suggestive of crosstalk between acylation and phosphorylation post-translational modification mechanisms.

Regulation of renal NaDC1 expression and citrate excretion by NBCe1-A.

Citrate is critical for acid-base homeostasis and to prevent calcium nephrolithiasis. Both metabolic acidosis and hypokalemia decrease citrate excretion and increase expression of Na+-dicarboxylate cotransporter 1 (NaDC1; SLC13A2), the primary protein involved in citrate reabsorption. However, the mechanisms transducing extracellular signals and mediating these responses are incompletely understood. The purpose of the present study was to determine the role of the Na+-coupled electrogenic bicarbonate cotransporter (NBCe1) A variant (NBCe1-A) in citrate metabolism under basal conditions and in response to acid loading and hypokalemia. NBCe1-A deletion increased citrate excretion and decreased NaDC1 expression in the proximal convoluted tubules (PCT) and proximal straight tubules (PST) in the medullary ray (PST-MR) but not in the PST in the outer medulla (PST-OM). Acid loading wild-type (WT) mice decreased citrate excretion. NaDC1 expression increased only in the PCT and PST-MR and not in the PST-MR. In NBCe1-A knockout (KO) mice, the acid loading change in citrate excretion was unaffected, changes in PCT NaDC1 expression were blocked, and there was an adaptive increase in PST-MR. Hypokalemia in WT mice decreased citrate excretion; NaDC1 expression increased only in the PCT and PST-MR. NBCe1-A KO blocked both the citrate and NaDC1 changes. We conclude that 1) adaptive changes in NaDC1 expression in response to metabolic acidosis and hypokalemia occur specifically in the PCT and PST-MR, i.e., in cortical proximal tubule segments; 2) NBCe1-A is necessary for normal basal, metabolic acidosis and hypokalemia-stimulated citrate metabolism and does so by regulating NaDC1 expression in cortical proximal tubule segments; and 3) adaptive increases in PST-OM NaDC1 expression occur in NBCe1-A KO mice in response to acid loading that do not occur in WT mice.

Retrotransposon elements among initial sites of hepatitis B virus integration into human genome in the HepG2-NTCP cell infection model.

Integration of hepatitis B virus (HBV) DNA into host's genome is evident in all stages and models of HBV infection. Investigations of the initial virus-host junctions have been just recently initiated since their nature may promote liver oncogenesis immediately following infection. We examined the time-frame and host sites at which HBV integrates in HepG2 cells overexpressing sodium taurocholate co-transporting polypeptide (NTCP) receptor mediating HBV entry. HepG2-NTCP cells were analyzed from 15 min to 13 days post-infection (p.i.). The results showed that except for 15 min p.i., HBV-host integrations were detected at all time points thereafter. At 30 min p.i., virus junctions with retrotransposon SINE and with neuroblastoma breakpoint family member 1 gene were detected. At one-hour p.i., HBV integration with retrotransposon THE-1B-LTR was identified, while virus insertions into proline-rich protein and protein kinase cGMP-dependent type 1 encoding genes were found at 3 h p.i. Fusion with runt-related transcription factor 1 was detected at 24 h p.i. and merges with 9 different genes at 13 day p.i. The data showed that retrotransposon elements are frequent among first-hit sites of HBV insertion. This may suggest a mechanism by which HBV DNA may spread across host's genome from earliest stages of infection.

Productive HBV infection of well-differentiated, hNTCP-expressing human hepatoma-derived (Huh7) cells.

Feasible and effective cell models for hepatitis B virus (HBV) infection are required for investigating the complete lifecycle of this virus, including the early steps of viral entry. Resistance to dimethyl sulfoxide/polyethylene glycol (DMSO/PEG), hNTCP expression, and a differentiated state are the limiting factors for successful HBV infection models. In the present study, we used a hepatoma cell line (Huh7DhNTCP) to overcome these limiting factors so that it exhibits excellent susceptibility to HBV infection. To achieve this goal, different hepatoma cell lines were tested with 2.5% DMSO / 4% PEG8000, and one resistant cell line (Huh7D) was used to construct a stable hNTCP-expressing cell line (Huh7DhNTCP) using a recombinant lentivirus system. Then, the morphological characteristics and differentiation molecular markers of Huh7DhNTCP cells with or without DMSO treatment were characterized. Finally, the susceptibility of Huh7DhNTCP cells to HBV infection was assessed. Our results showed that Huh7D cells were resistant to 2.5% DMSO / 4% PEG8000, whereas the others were not. Huh7DhNTCP cells were established to express a high level of hNTCP compared to liver extracts, and Huh7DhNTCP cells rapidly transformed into a non-dividing, well-differentiated polarized phenotype under DMSO treatment. Huh7DhNTCP cells fully supported the entire lifecycle of HBV infection. This cell culture system will be useful for the analysis of host-virus interactions, which should facilitate the discovery of antiviral drugs and vaccines.

Hsp90 and hepatobiliary transformation during sea lamprey metamorphosis.

BACKGROUND: Biliary atresia (BA) is a human infant disease with inflammatory fibrous obstructions in the bile ducts and is the most common cause for pediatric liver transplantation. In contrast, the sea lamprey undergoes developmental BA with transient cholestasis and fibrosis during metamorphosis, but emerges as a fecund adult. Therefore, sea lamprey liver metamorphosis may serve as an etiological model for human BA and provide pivotal information for hepatobiliary transformation and possible therapeutics. RESULTS: We hypothesized that liver metamorphosis in sea lamprey is due to transcriptional reprogramming that dictates cellular remodeling during metamorphosis. We determined global gene expressions in liver at several metamorphic landmark stages by integrating mRNA-Seq and gene ontology analyses, and validated the results with real-time quantitative PCR, histological and immunohistochemical staining. These analyses revealed that gene expressions of protein folding chaperones, membrane transporters and extracellular matrices were altered and shifted during liver metamorphosis. HSP90, important in protein folding and invertebrate metamorphosis, was identified as a candidate key factor during liver metamorphosis in sea lamprey. Blocking HSP90 with geldanamycin facilitated liver metamorphosis and decreased the gene expressions of the rate limiting enzyme for cholesterol biosynthesis, HMGCoA reductase (hmgcr), and bile acid biosynthesis, cyp7a1. Injection of hsp90 siRNA for 4 days altered gene expressions of met, hmgcr, cyp27a1, and slc10a1. Bile acid concentrations were increased while bile duct and gall bladder degeneration was facilitated and synchronized after hsp90 siRNA injection. CONCLUSIONS: HSP90 appears to play crucial roles in hepatobiliary transformation during sea lamprey metamorphosis. Sea lamprey is a useful animal model to study postembryonic development and mechanisms for hsp90-induced hepatobiliary transformation.

Hepatitis B virus (HBV) receptors: Deficiency in tumor results in scant HBV infection and overexpression in peritumor leads to higher recurrence risk.

Hepatitis B virus (HBV) infection is a risk factor for hepatocarcinogenesis and recurrence. Here, we sought to characterize intratumoral and peritumoral expression of HBsAg and its specific receptors in HBsAg-positive hepatocellular carcinoma (HCC) patients and further examined their correlation with the recurrence-free survival (RFS). HCC tissue and adjacent normal tissue specimens were acquired from HBsAg-positive patients. The presence of HBsAg and receptors, as well as hepatic progenitor cells (HPCs) were detected by tissue microassay and immunohistochemistry. Necroinflammatory activity was evaluated by HE staining. The mean IOD of HBsAg and HBV DNA in the intratumoral tissues was markedly lower than that in the peritumoral tissues (P < 0.001). Pearson correlation analysis further showed a significant correlation between the expression of HBsAg and NTCP (r = 0.461, P < 0.001) or ASGPR (r = 0.506, P < 0.001) in peritumoral tissues. And the peritumoral HBsAg and receptors presented a positive association with necroinflammatory activity (P < 0.05). Inflammation induced by HBV infection presented a positive association with HPCs activation (P < 0.05). Additionally, due to lack of HBV receptors, HPCs was not preferentially infected with HBV, but activated HPCs had a significant correlation with HBsAg expression in peritumoral tissues, and the peritumoral HPCs activation was associated with RFS of HCC patients, therefore, the overexpression of HBsAg and receptors in peritumor were also with higher recurrence risk (P < 0.05). In conclusion, lack of HBV receptors resulted in scant HBV infection in tumor cells, and overexpression of HBsAg and receptors in peritumor was strongly associated with higher recurrence risk in HCC patients.

The inhibition of hepatic bile acids transporters Ntcp and Bsep is involved in the pathogenesis of isoniazid/rifampicin-induced hepatotoxicity.

Co-treatment of isoniazid (INH) and rifampicin (RFP) is well known for clinically apparent liver injury. However, the mechanism of INH/RFP-induced liver injury is controversial. Emerging evidence shows links between inhibition of bile acids transporters and drug-induced liver injury (DILI). The present study investigates whether sodium taurocholate cotransporting polypeptide (NTCP/Ntcp; SLC10A1) and bile salt export pump (BSEP/Bsep; ABCB11) are involved in the anti-tuberculosis medicines induced liver injury. ICR female mice were intragastrically treated with INH (50 or 100 mg/kg), RFP (100 or 200 mg/kg), or the combination of INH/RFP (50 + 100 mg/kg or 100 + 200 mg/kg) for 14 consecutive days. Liver histopathological examination, serum biochemical and liver malondialdehyde tests were evaluated. Apparent histopathological alterations and hepatic oxidative stress showed in INH (100 mg/kg), RFP (200 mg/kg) and their combination group. The hepatoxic effect was also indicated by increased serum biomarkers, such as aspartate transaminase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), direct bilirubin (DBil), total bilirubin (TBil) and total bile acids (TBA). Both doses of INH/RFP administration significantly down-regulated the expression of Ntcp and Bsep in liver. Furthermore, the combination of INH and RFP displayed stronger effect on the expression of Ntcp compared with the corresponding dose of INH or RFP alone. In conclusion, down-regulated expression of hepatic Ntcp and Bsep might play an important role in the development of INH and RFP induced liver injury.

Effect of Yin-Zhi-Huang on up-regulation of Oatp2, Ntcp, and Mrp2 proteins in estrogen-induced rat cholestasis.

CONTEXT: Yin-Zhi-Huang (YZH), a prescription of traditional Chinese medicine, is widely used to treat neonatal jaundice or cholestasis. OBJECTIVE: This study investigates the regulatory effect of YZH on the localization and expression of organic anion transporting polypeptides 2 (Oatp2), Na(+)-taurocholate co-transporting polypeptide (Ntcp), multidrug-resistance-associated protein 2 (Mrp2), and bile salt export pump (Bsep) in estrogen-induced cholestasis rats. MATERIAL AND METHODS: Cholestasis model rats were induced via subcutaneous injection of estradiol benzoate (EB, 5 mg/kg/d) for 5 d. Other EB-induced rats were treated with saline (2 ml) or YZH (1.5 g/kg, two times a day) for 7, 14, and 21 d. The biochemical and pathologic examinations were performed. Moreover, the localization and expression of Oatp2, Ntcp, Mrp2, and Bsep were determined by immunohistochemisty and Western blotting, respectively. RESULTS: YZH treatment could significantly decrease the serum total bile acids (TBA) (4.9 ± 0.6-2.8 ± 0.8) and direct bilirubin (DBIL) (2.6 ± 0.7-1.0 ± 0.1) levels, improve the histological disorganization, and, respectively, increase the expression of Oatp2 and Ntcp by 46% and 28% compared with saline-treated (p < 0.05) rats at 14 d. The expression of Mrp2 increased by 45% was observed in YZH treated compared with saline-treated (p < 0.05) rats at 7 d. However, there was a little change in the expression of Bsep (p > 0.05) after YZH treatment for 7, 14, and 21 d. DISCUSSION AND CONCLUSION: In conclusion, the therapeutic effect of YZH to cholestasis could be attributed to the regulation of Oatp2, Ntcp, Mrp2, and Bsep.

Prednisolone increases enterohepatic cycling of bile acids by induction of Asbt and promotes reverse cholesterol transport.

BACKGROUND & AIMS: Glucocorticoids, produced by the adrenal gland under control of the hypothalamic-pituitary-adrenal axis, exert their metabolic actions largely via activation of the glucocorticoid receptor (GR). Synthetic glucocorticoids are widely used as anti-inflammatory and immunosuppressive drugs but their application is hampered by adverse metabolic effects. Recently, it has been shown that GR may regulate several genes involved in murine bile acid (BA) and cholesterol metabolism, yet the physiological relevance hereof is controversial. The aim of this study is to provide a mechanistic basis for effects of prednisolone on BA and cholesterol homeostasis in mice. METHODS: Male BALB/c mice were treated with prednisolone (12.5mg/kg/day) for 7days by subcutaneous implantation of slow-release pellets, followed by extensive metabolic profiling. RESULTS: Sustained prednisolone treatment induced the expression of the apical sodium-dependent bile acid transporter (Asbt) in the ileum, which stimulated BA absorption. This resulted in elevated plasma BA levels and enhanced biliary BA secretion. Concomitantly, both biliary cholesterol and phospholipid secretion rates were increased. Enhanced BA reabsorption suppressed hepatic BA synthesis, as evident from hepatic gene expression, reduced plasma C4 levels and reduced fecal BA loss. Plasma HDL cholesterol levels were elevated in prednisolone-treated mice, which likely contributed to the stimulated flux of cholesterol from intraperitoneally injected macrophage foam cells into feces. CONCLUSIONS: Sustained prednisolone treatment increases enterohepatic recycling of BA, leading to elevated plasma levels and reduced synthesis in the absence of cholestasis. Under these conditions, prednisolone promotes macrophage-derived reverse cholesterol transport.

Sodium taurocholate cotransporting polypeptide mediates dual actions of deoxycholic acid in human hepatocellular carcinoma cells: enhanced apoptosis versus growth stimulation.

PURPOSE: The hydrophobic bile acid, deoxycholic acid (DC), can induce apoptosis in hepatocytes. The roles of DC and its transporter are not yet established in hepatocellular carcinoma (HCC) cells. We investigated DC-induced alterations in HCC cell growth, with a particular focus on the effect of the expression of bile acid (BA)-transporting Na(+)-dependent taurocholic cotransporting polypeptides (NTCPs). METHODS: We determined NTCP expression in four human HCC cell lines: Huh-BAT, Huh-7, SNU-761, and SNU-475. NTCP expression and apoptotic signaling cascades were examined by immunoblot analyses. Cell viability was assessed using the 3,4-(5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt assay. Wound healing and invasion assays were performed to evaluate cell migration and invasion abilities. Real-time polymerase chain reaction was performed to measure IL-8 expression levels. Nuclear factor kappa B (NF-κB) activity was evaluated by enzyme-linked immunosorbent assay. RESULTS: The HCC cell lines revealed varying NTCP expression levels, and DC treatment had dual effects, depending on NTCP expression. DC induced apoptosis in NTCP-positive HCC cells, especially under hypoxic conditions. In NTCP-negative HCC cells, simultaneous treatment with DC and cyclooxygenase inhibitor markedly decreased aggressive cellular behaviors via the inhibition of NF-κB/COX-2/IL-8 pathways. CONCLUSION: Hydrophobic bile acid offers therapeutic potential for patients with advanced HCC via different mechanisms depending on NTCP expression levels within the tumor.

Distribution of SLC10A4, a synaptic vesicle protein in the human brain, and the association of this protein with Alzheimer's disease-related neuronal degeneration.

Member 4 of the sodium/bile acid co-transporter family of proteins (SLC10A4) was discovered as a synaptic vesicle protein. The distribution of Slc10a4 protein in the brain has only so far been assessed in adult rats. Here, we assessed the regional distribution of SLC10A4 in aged human brain by immunohistochemistry. The protein was ubiquitously expressed, particularly in the cholinergic and monoaminergic neurons and in the lateral geniculate body. The protein expression was not influenced by the postmortem delay or fixation time. Synaptic alterations are reported to be seen in Alzheimer's disease (AD) and the suggested function of SLC10A4 as a vesicular transporter for cholinergic neurotransmitters proposes a link between this protein and AD. With increased severity of AD-related pathology, depletion of SLC10A4 expression was noted in the transentorhinal cortex. Intriguingly, in the most severely affected cases (Braak V), two patterns were noted, i.e., those with severe depletion of SLC10A4 and those with numerous neurons displaying SLC10A4. In conclusion, assessment of the expression of SLC10A4 by means of immunohistochemistry is feasible. The observed depletion of SLC10A4 with increase in the severity of AD-related neuronal degeneration is interesting and the observation that some subjects in Braak V displayed none and some displayed numerous SLC10A4 immunoreactive neurons is intriguing. Assessment of the SLC10A4 protein in neurodegenerative diseases or diseases affecting lateral geniculate body should be carried out to investigate whether alterations in the expression of SLC10A4 in synaptic vesicles might be used as a marker of transmitter deficits (cholinergic, monoaminorgic) or other synaptic pathology.

Expression and role of the genes involved in the transport of bile acids in the liver and kidneys in mice.

BACKGROUND: Bile acids are synthesized in the liver from cholesterol. METHODS: This study investigated the impact and expression of different carriers of bile acid in the liver and kidneys. Eight-week-old male mice were used, which were fed for 15 days and divided into two groups: 15 mice fed with standard diet (control group) and another 15 mice fed with a rich diet of 5% cholesterol (second group). Bile acid dosage was based on their oxidation by 7α hydroxyl-steroid dehydrogenize. The mRNA expression was quantitatively analyzed by the real time of polymerase chain reaction (RT-PCR), and the expression of the renal carrier bile acid protein was analyzed by Western blot. RESULTS AND CONCLUSIONS: The expression of bile salt export pump involved in the uptake of bile acids in the basolateral membrane of hepatocytes revealed no differences between the two groups of mice. However, the expression of multidrug resistance-associated protein 2 was reduced in mice of the second group. Moreover, the expressions of organic anion transporting polypeptide 4, organic anion transporting polypeptide 1, and sodium taurocholate co-transporting polypeptide (Ntcp) involved in the uptake of bile acids in the apical pole of hepatocytes are suppressed in mice of the second group. The expression of multidrug resistance-associated protein 3 involved in the secretion of bile acids in the apical membrane of hepatocytes revealed no significant differences between the two groups. In mice of the second group, blood concentration of bile acids on the last day was increased. In those mice, the expression of intestinal bile acid transporter was reduced in the kidneys compared with the control mice.

The hepatic bile acid transporters Ntcp and Mrp2 are downregulated in experimental necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency of premature infants and is characterized by an extensive hemorrhagic inflammatory necrosis of the distal ileum and proximal colon. We have previously shown that, during the development of experimental NEC, the liver plays an important role in regulating inflammation in the ileum, and accumulation of ileal bile acids (BA) along with dysregulation of ileal BA transporters contributes to ileal damage. Given these findings, we speculated that hepatic BA transporters would also be altered in experimental NEC. Using both rat and mouse models of NEC, levels of Cyp7a1, Cyp27a1, and the hepatic BA transporters Bsep, Ntcp, Oatp2, Oatp4, Mrp2, and Mrp3 were investigated. In addition, levels of hepatic BA transporters were also determined when the proinflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-18, which are both elevated in NEC, are neutralized during disease development. Ntcp and Mrp2 were decreased in NEC, but elevated ileal BA levels were not responsible for these reductions. However, neutralization of TNF-α normalized Ntcp, whereas removal of IL-18 normalized Mrp2 levels. These data show that the hepatic transporters Ntcp and Mrp2 are downregulated, whereas Cyp27a1 is increased in rodent models of NEC. Furthermore, increased levels of TNF-α and IL-18 in experimental NEC may play a role in the regulation of Ntcp and Mrp2, respectively. These data suggest the gut-liver axis should be considered when therapeutic modalities for NEC are developed.

Hepatoprotection in bile duct ligated mice mediated by darbepoetin-α is not caused by changes in hepatobiliary transporter expression.

AIMS: Darbepoetin-α (DPO), a long-acting erythropoietin analog, has been shown to protect the liver against cholestatic injury, to exert an antifibrotic effect, and to increase the survival time in a model of common bile duct ligation. Here we evaluate whether these tissue-protective effects are caused by DPO induced regulation of hepatobiliary transporters. MAIN METHODS: C57BL/6J mice underwent common bile duct ligation and were treated with either DPO or physiological saline. Time dependent (2, 5, 14, 28 days after bile duct ligation) protein expression of different hepatobiliary transporters which have been established to play an important role in hepatocellular (i) bile acid uptake, (ii) bile acid excretion, and (iii) retrograde bile acid efflux were assessed. mRNA and protein expression of Lhx2, an important negative regulator of hepatic stellate cell activation, was determined. KEY FINDINGS: Saline treated cholestatic mice impress with increased mRNA expression of Lhx2 as a defense mechanism, while there is less need for such an upregulation in mice treated with DPO. Whereas Ntcp (slc10a1) protein expression is suppressed as early as 2 days after bile duct ligation to 40% in untreated animals, DPO treated mice exhibit decreased protein level not before day 5. Similarly, the steady decline of Mrp4 (abcc4) protein level during extrahepatic cholestasis in control treated animals does not occur upon DPO application. SIGNIFICANCE: The collected data show that DPO affects expression of hepatobilliary transporters during obstructive cholestasis but do not provide sufficient evidence to demonstrate a direct correlation between this regulation and hepatoprotection by DPO.

Ostα depletion protects liver from oral bile acid load.

Bile acid homeostasis is tightly maintained through interactions between the liver, intestine, and kidney. During cholestasis, the liver is incapable of properly clearing bile acids from the circulation, and alternative excretory pathways are utilized. In obstructive cholestasis, urinary elimination is often increased, and this pathway is further enhanced after bile duct ligation in mice that are genetically deficient in the heteromeric, basolateral organic solute transporter alpha-beta (Ostα-Ostß). In this study, we examined renal and intestinal function in Ostα-deficient and wild-type mice in a model of bile acid overload. After 1% cholic acid feeding, Ostα-deficient mice had significantly lower serum ALT levels compared with wild-type controls, indicating partial protection from liver injury. Urinary clearance of bile acids, but not clearance of [(3)H]inulin, was significantly higher in cholic acid-fed Ostα-deficient mice compared with wild-type mice but was not sufficient to account for the protection. Fecal excretion of bile acids over the 5 days of cholic acid feeding was responsible for almost all of the bile acid loss in Ostα-deficient mice, suggesting that intestinal losses of bile acids accounted for the protection from liver injury. Thus fecal loss of bile acids after bile acid overload reduced the need for the kidney to filter and excrete the excess bile acids. In conclusion, Ostα-deficient mice efficiently eliminate excess bile acids via the feces. Inhibition of intestinal bile acid absorption might be an effective therapeutic target in early stages of cholestasis when bile acids are still excreted into bile.

Heterogeneous accumulation of fluorescent bile acids in primary rat hepatocytes does not correlate with their homogenous expression of ntcp.

Sodium taurocholate-cotransporting polypeptide (ntcp) is considered to be a major determinant of bile acid uptake into hepatocytes. However, the regulation of ntcp and the degree that it participates in the accumulation of specific substrates are not well understood. We utilized fluorescent bile acid derivatives and direct quantitation of fluorescent microscopy images to examine the regulation of ntcp and its role in the cell-to-cell variability of fluorescent bile acid accumulation. Primary-cultured rat hepatocytes rapidly accumulated the fluorescent bile acids, chenodeoxycholylglycylamidofluorescein (CDCGamF), 7-ß- nitrobenzoxadiazole 3-α hydroxy 5-ß cholan-24-oic acid (NBD-CA), and cholyl-glycylamido-fluorescein (CGamF). However, in stably transfected HeLa cells, ntcp preferred CDCGamF, whereas the organic anion transporter, organic anion transporting polypeptide 1 (oatp1a1), preferred NBD-CA, and neither ntcp nor oatp1a1 showed strong accumulation of CGamF by these methods. Ntcp-mediated transport of CDCGamF was inhibited by taurocholate, cyclosporin, actin depolymerization, and an inhibitor of atypical PKC-ζ. The latter two agents altered the cellular distribution of ntcp as visualized in ntcp-green fluorescent protein-transfected cells. Although fluorescent bile acid accumulation was reproducible by the imaging assays, individual cells showed variable accumulation that was not attributable to changes in membrane permeability or cell viability. In HeLa cells, this was accounted for by variable levels of ntcp, whereas, in hepatocytes, ntcp expression was uniform, and low accumulation was seen in a large portion of cells despite the presence of ntcp. These studies indicate that single-cell imaging can provide insight into previously unrecognized details of anion transport in the complex environment of polarized hepatocytes.

Histone H3K4 trimethylation by MLL3 as part of ASCOM complex is critical for NR activation of bile acid transporter genes and is downregulated in cholestasis.

The nuclear receptor Farnesoid x receptor (FXR) is a critical regulator of multiple genes involved in bile acid homeostasis. The coactivators attracted to promoters of FXR target genes and epigenetic modifications that occur after ligand binding to FXR have not been completely defined, and it is unknown whether these processes are disrupted during cholestasis. Using a microarray, we identified decreased expression of mixed lineage leukemia 3 (MLL3), a histone H3 lysine 4 (H3K4) lysine methyl transferase at 1 and 3 days of post-common bile duct ligation (CBDL) in mice. Chromatin immunoprecipitation analysis (ChIP) analysis revealed that H3K4me3 of transporter promoters by MLL3 as part of activating signal cointegrator-2 -containing complex (ASCOM) is essential for activation of bile salt export pump (BSEP), multidrug resistance associated protein 2 (MRP2), and sodium taurocholate cotransporting polypeptide (NTCP) genes by FXR and glucocorticoid receptor (GR). Knockdown of nuclear receptor coactivator 6 (NCOA6) or MLL3/MLL4 mRNAs by small interfering RNA treatment led to a decrease in BSEP and NTCP mRNA levels in hepatoma cells. Human BSEP promoter transactivation by FXR/RXR was enhanced in a dose-dependent fashion by NCOA6 cDNA coexpression and decreased by AdsiNCOA6 infection in HepG2 cells. GST-pull down assays showed that domain 3 and 5 of NCOA6 (LXXLL motifs) interacted with FXR and that the interaction with domain 5 was enhanced by chenodeoxycholic acid. In vivo ChIP assays in HepG2 cells revealed ligand-dependent recruitment of ASCOM complex to FXR element in BSEP and GR element in NTCP promoters, respectively. ChIP analysis demonstrated significantly diminished recruitment of ASCOM complex components and H3K4me3 to Bsep and Mrp2 promoter FXR elements in mouse livers after CBDL. Taken together, these data show that the "H3K4me3" epigenetic mark is essential to activation of BSEP, NTCP, and MRP2 genes by nuclear receptors and is downregulated in cholestasis.

Enterobacteria modulate intestinal bile acid transport and homeostasis through apical sodium-dependent bile acid transporter (SLC10A2) expression.

In our study, ampicillin (AMP)-mediated decrease of enterobacteria caused increases in hepatic bile acid concentration through (at least in part) elevation of bile acid synthesis in C57BL/6N mice. We investigated the involvement of enterobacteria on intestinal bile acid absorption in AMP-treated mice in the present study. Fecal enterobacterial levels and fecal bile acid excretion rates were markedly decreased in mice treated with AMP (100 mg/kg) for 3 days, whereas bile acid concentrations in portal blood were significantly increased compared with those in mice treated with a vehicle. Ileal apical sodium-dependent bile acid transporter (SLC10A2) mRNA levels and ileal SLC10A2 protein levels in brush-border membranes were significantly increased compared with those in mice treated with the vehicle. In AMP-treated mice, total bile acid levels were increased, whereas levels of enterobacteria-biotransformed bile acid, taurodeoxycholic acid, and cholic acid were decreased in intestinal lumen. These phenomena were also observed in farnesoid X receptor-null mice treated with AMP for 3 days. Discontinuation of AMP administration after 3 days (vehicle administration for 4 days) increased levels of fecal enterobacteria, fecal bile acid excretion, and taurodeoxycholic acid and cholic acid in the intestinal lumen, whereas the discontinuation decreased ileal SLC10A2 expression and bile acid concentrations in the portal blood. Coadministration of taurodeoxycholic acid or cholic acid decreased ileal SLC10A2 expression in mice treated with AMP. These results suggest that enterobacteria-mediated bile acid biotransformation modulates intestinal bile acid transport and homeostasis through down-regulation of ileal SLC10A2 expression.

Up-regulation of the vitamin C transporter SVCT2 upon differentiation and depolarization of myotubes.

In addition to its role as a strong antioxidant, vitamin C regulates the differentiation of several cell lineages. In vertebrate skeletal muscle, the vitamin C transporter SVCT2 is preferentially expressed in slow muscle fibers. To gain insights into the possible involvement of intracellular vitamin C on early myogenesis, we investigated the regulation of SVCT2 expression in cultures of chick fetal myoblasts. SVCT2 expression increases in cultures of both, slow and fast muscle-derived myoblasts, as they fuse to form mainly fast myotubes. Interestingly, we found that SVCT2 could be positively modulated by potassium-induced depolarization of myotubes. These findings suggest that SVCT2-mediated uptake of vitamin C could play diverse roles on skeletal muscle development and physiology.