Bile Salt Transport Research Articles

Expression of ATP-binding cassette multidrug transporters in the giant liver fluke Fasciola gigantica and their possible involvement in the transport of bile salts and anthelmintics

ATP-binding cassette (ABC) transporters belong to one of the largest protein families that either import or export a wide spectrum of different substrates. Certain members of this superfamily have been implicated in multidrug resistance in various types of cancer as well as in pathogenic microorganisms. The role of ABC proteins in parasitic multidrug resistance becomes increasingly evident. However, studies on ABC transporters in helminths have been limited to MDR1 and MRP orthologues. In the present study, we reported, for the first time, the expression and localization of ABC proteins including orthologues of MDR1, MRP1, BCRP, and BSEP in the giant liver fluke Fasciola gigantica. Furthermore, the functional activities of these ABC transporters were characterized in isolated fluke cells using a fluorescent substrate, rhodamine. The results revealed the inhibition of rhodamine efflux by cyclosporin A, a potent inhibitor of ABC transporters. Interestingly, our data suggested that these proteins might play a role in the export of bile salts, in particular, taurocholate. Although, we did not observe any substantial changes in rhodamine transport in the presence of anthelmintics under experimental conditions, however, our findings altogether shed light on the possible involvement of several members of ABC proteins in the mechanism of drug resistance as well as detoxification process in helminths to survive inside their hosts.

Bile acids: chemistry, pathochemistry, biology, pathobiology, and therapeutics.

Bile acids and bile alcohols in the form of their conjugates are amphipathic end products of cholesterol metabolism with multiple physiological functions. The great variety of bile acids and bile alcohols that are present in vertebrates are tabulated. Bile salts have an enterohepatic circulation resulting from efficient vectorial transport of bile salts through the hepatocyte and the ileal enterocyte; such transport leads to the accumulation of a pool of bile salts that cycles between the liver and intestine. Bile salt anions promote lipid absorption, enhance tryptic cleavage of dietary proteins, and have antimicrobial effects. Bile salts are signaling molecules, activating nuclear receptors in the hepatocyte and ileal enterocyte, as well as an increasing number of G-protein coupled receptors. Bile acids are used therapeutically to correct deficiency states, to decrease the cholesterol saturation of bile, or to decrease the cytotoxicity of retained bile acids in cholestatic liver disease.

Dexamethasone Exposure of Neonatal Rats Modulates Biliary Lipid Secretion and Hepatic Expression of Genes Controlling Bile Acid Metabolism in Adulthood Without Interfering With Primary Bile Acid Kinetics

Literature suggests that glucocorticoid (GC) exposure during early life may have long-term consequences into adult life. GCs are known to influence hepatic bile acid synthesis and their transport within the enterohepatic circulation. This study addresses effects of early postnatal exposure to GC on hepatic expression of key genes in bile acid metabolism and bile acid kinetics in adult rats. Male rats were treated with either dexamethasone (DEX) or saline at days 1-3 d after birth. Liver tissue and blood were collected from 2 d to 50 wk of age. Bile acid kinetics were determined at week 8. DEX acutely induced hepatic mRNA levels of cholesterol 7alpha-hydroxylase (Cyp7a1), cholesterol 27-hydroxylase (Cyp27), and in particular sterol 12alpha-hydroxylase (Cyp8b1), whereas expression of the bile acid transporters bile salt export pump (Bsep) and sodium taurocholate cotransporting polypeptide (Ntcp) was moderately affected. Neonatal DEX administration led to increased bilary lipid secretion, decreased Cyp8B1 mRNA expression and a 3-fold higher Cyp7a1/Cyp8b1 mRNA ratio in rats at week 8 compared with age-matched controls without alterations in bile acid kinetics. Therefore, neonatal DEX administration causes altered gene expressions later in life that are not translated into quantitative changes in bile acid kinetics.

Multidrug resistance–associated proteins are crucial for the viability of activated rat hepatic stellate cells†‡

Hepatic stellate cells (HSCs) survive and proliferate in the chronically injured liver. ATP-binding cassette (ABC) transporters play a crucial role in cell viability by transporting toxic metabolites or xenobiotics out of the cell. ABC transporter expression in HSCs and its relevance to cell viability and/or activation have not been reported so far. The aim of this study was to investigate the expression, regulation, and function of multidrug resistance-associated protein (Mrp)-type and multidrug resistance protein (Mdr)-type ABC transporters in activated rat HSCs. Rat HSCs were exposed to cytokines or oxidative stress. ABC transporter expression was determined by quantitative polymerase chain reaction and immunohistochemistry. HSCs were exposed to the Mdr inhibitors verapamil and PSC-833 and the Mrp inhibitor MK571. Mdr and Mrp transporter function was evaluated with flow cytometry. Apoptosis was determined by activated caspase-3 and acridine orange staining, and necrosis was determined by Sytox green nuclear staining. An in vivo model of carbon tetrachloride (CCl(4))-induced liver fibrosis was used. With respect to hepatocytes, activated HSCs expressed high levels of Mrp1 and comparable levels of Mrp3, Mrp4, Mdr1a, and Mdr1b but not the hepatocyte-specific transporters bile salt export pump, Mrp2, and Mrp6. Mrp1 protein staining correlated with desmin staining in livers from CCl(4)-treated rats. Mrp1 expression increased upon activation of HSCs. Cytokines induced Mdr1b expression only. Oxidative stress was not a major regulator of Mdr and Mrp transporter expression. Activated HSCs became necrotic when exposed to the Mrp inhibitors. Activated HSCs contain relatively high levels of Mrp1. Mrp-type transporters are required for the viability of activated HSCs. Mrp-dependent export of endogenous metabolites is important for the survival of activated HSCs in chronic liver diseases.

Localization of the Sodium-Taurocholate cotransporting polypeptide in membrane rafts and modulation of its activity by cholesterol in vitro

Background The relevance of discrete localization of hepatobiliary transporters in specific membrane microdomains is not well known. Aim To determine whether the Na +/taurocholate cotransporting polypeptide (Ntcp), the main hepatic sinusoidal bile salt transporter, is localized in specific membrane microdomains. Methods Presence of Ntcp in membrane rafts obtained from mouse liver was studied by immunoblotting and immunofluorescence. HEK-293 cells stably transfected with rat Ntcp were used for in vitro studies. Expression, localization and function of Ntcp in these cells were assessed by immunoblotting, immunofluorescence and biotinylation studies and Na +-dependent taurocholate uptake assays, respectively. The effect of cholesterol depletion/repletion assays on Ntcp function was also investigated. Results Ntcp localized primarily to membrane rafts in in vivo studies and localized partially in membrane rafts in transfected HEK-293 cells. In these cells, membrane cholesterol depletion resulted in a shift of Ntcp localization into non-membrane rafts, which correlated with a 2.5-fold increase in taurocholate transport. Cholesterol repletion shifted back part of Ntcp into membrane rafts, and normalized taurocholate transport to values similar to control cells. Conclusion Ntcp localizes in membrane rafts and its localization and function are regulated by membrane cholesterol content. This may serve as a novel regulatory mechanism of bile salt transport in liver.

Mechanisms of human hepatobiliary transporter gene regulation in the hepatocyte-like cell line HepaRG

Background&Aim: Functional studies on human hepatic gene regulation are limited due to the lack of suitable models. Currently used human hepatoma cell lines such as HepG2 or Huh7 are deficient in several transporters such as NTCP or BSEP whereas primary human hepatocytes and freshly prepared liver slices are limited in availability. The highly differentiated hepatic cell line HepaRG, which exhibits a hepatocyte-like equipment with various CYP P450s, nuclear receptors and a broad range of hepatobiliary transporters, represents an ideal model system to study human transporter gene regulation (LeVee et al. 2006, Aninat et al. 2006). The aim of the present study was to functionally characterize inflammatory and bile salt-induced mechanisms of human transporter gene regulation in the unique HepaRG cell system (kindly provided by Biopredic). Methods: Highly differentiated HepaRG cells maintained in DMSO containing medium were treated with either 100ng/ml LPS or 10ng/ml IL–1s or 10µM taurocholate (exemplary for inflammatory conditions or bile salt overload) for 16 hours. RNA and protein were prepared and analyzed by real-time RT-PCR and Western blotting. Results: Upon LPS-treatment NTCP mRNA expression decreased to 13% whereas OATP-C and BSEP expression showed a moderate reduction of 56% and 39%, respectively, compared to untreated controls. Effects of IL–1s treatment were even more pronounced (NTCP 4%, OATP-C 10% and BSEP 18%). MRP2 is down-regulated by inflammatory signals to 40–60% at the protein level whereas mRNA was 65–77% for IL–1s and LPS stimulation, respectively. MRP4 mRNA is increased around 2-fold while MRP3 expression remained stable under these conditions. Taurocholate treatment increased BSEP mRNA to 258%, slightly enhanced NTCP as well as MRP4 expression while OATP-C, MRP3 and MRP2 were unaffected. Conclusions: Human bile salt transporters NTCP and BSEP are suppressed by similar inflammatory mechanisms as their rat counterparts. Inflammation-induced MRP2 down-regulation occurs by posttranscriptional mechanisms. During bile salt accumulation MRP4 appears to be the primary human rescue system whereas other transporters are rather moderately suppressed or unaffected. Our data allow a comprehensive insight into cholestatic human transporter gene regulation and enable further mechanistic studies in the future.

Review article: the function and regulation of proteins involved in bile salt biosynthesis and transport

Bile salts are produced and secreted by the liver and are required for intestinal absorption of fatty food components and excretion of endobiotics and xenobiotics. They are reabsorbed in the terminal ileum and transported back to the liver via the portal tract. Dedicated bile salt transporters in hepatocytes and enterocytes are responsible for the unidirectional transport of bile salts in the enterohepatic cycle. To give an overview of the function and regulations of proteins involved in bile salt synthesis and transport. Data presented are obtained from PubMed-accessible literature combined with our own recent research. Hepatocytes and enterocytes contain unique bile salt importers (sodium-taurocholate cotransporting polypeptide and apical sodium-dependent bile acid transporter, respectively) and exporters (bile salt export pump and organic solute transporter alpha-beta, respectively). Enzymes involved in bile salt biosynthesis reside in different subcellular locations, including the endoplasmic reticulum, mitochondria, cytosol and peroxisomes. Defective expression or function of the transporters or enzymes may lead to cholastasis. The bile salt-activated transcription factor Farnesoid X receptor controls expression of genes involved in bile salt biosynthesis and transport. Detailed knowledge is available about the enzymes and transporters involved in bile salt homeostasis and how their defective function is associated with cholestasis. In contrast, the process of intracellular bile salt transport is largely unexplored.

Gene expression profiling in livers of mice after acute inhibition of β-oxidation

Inborn errors of mitochondrial β-oxidation cause ectopic fat accumulation, particularly in the liver. Fatty liver is associated with insulin resistance and predisposes to hepatic fibrosis. The factors underlying the pathophysiological consequences of hepatic fat accumulation have remained poorly defined. Gene expression profiling in a model of acute fatty liver disease induced by blocking long-chain fatty acid β-oxidation was performed to study the early effects of steatosis on the transcriptome. Tetradecylglycidic acid (TDGA) was used to irreversibly inhibit carnitine palmitoyltransferase 1, a key enzyme in the control of mitochondrial β-oxidation. TDGA treatment induced massive microvesicular hepatic steatosis within a 12-h time frame in male C57BL6/J mice. Increased hepatic long-chain acyl-CoA content, particularly of C16:0, C16:1 and C18:1, was associated with profound effects on the transcriptome as revealed by unbiased gene expression profiling and quantitative real-time PCR. The results indicate drastic changes in the expression of genes encoding proteins involved in lipid, carbohydrate, and amino acid metabolism. Pathway analysis identified transcription factors and coregulators such as hepatocyte nuclear factor 4 (HNF4), peroxisome proliferator-activated receptor-α (PPAR-α), and PPAR gamma coactivator 1α (PGC-1α ) as key players in these metabolic adaptations. Apoptotic and profibrotic responses were also affected. Surprisingly, a strong reduction in the expression of genes involved in hepatic bile salt metabolism and transport was observed. Therefore, this transcriptome analysis opens new avenues for research.

Levels of plasma membrane expression in progressive and benign mutations of the bile salt export pump (Bsep/Abcb11) correlate with severity of cholestatic diseases

Human BSEP (ABCB11) mutations are the molecular basis for at least three clinical forms of liver disease, progressive familial intrahepatic cholestasis type 2 (PFIC2), benign recurrent intrahepatic cholestasis type 2 (BRIC2), and intrahepatic cholestasis of pregnancy (ICP). To better understand the pathobiology of these disease phenotypes, we hypothesized that different mutations may cause significant differences in protein defects. Therefore we compared the effect of two PFIC2 mutations (D482G, E297G) with two BRIC2 mutations (A570T and R1050C) and one ICP mutation (N591S) with regard to the subcellular localization, maturation, and function of the rat Bsep protein. Bile salt transport was retained in all but the E297G mutant. Mutant proteins were expressed at reduced levels on the plasma membrane of transfected HEK293 cells compared with wild-type (WT) Bsep in the following order: WT > N591S > R1050C approximately A570T approximately E297G >> D482G. Total cell protein and surface protein expression were reduced to the same extent, suggesting that trafficking of these mutants to the plasma membrane is not impaired. All Bsep mutants accumulate in perinuclear aggresome-like structures in the presence of the proteasome inhibitor MG-132, suggesting that mutations are associated with protein instability and ubiquitin-dependent degradation. Reduced temperature, sodium butyrate, and sodium 4-phenylbutyrate enhanced the expression of the mature and cell surface D482G protein in HEK293 cells. These results suggest that the clinical phenotypes of PFIC2, BRIC2, and ICP may directly correlate with the amount of mature protein that is expressed at the cell surface and that strategies to stabilize cell surface mutant protein may be therapeutic.

Obstructive cholestasis down-regulates Oatp1 by TNFα signalling without affecting Oatp2 and Oatp4 expression and Sodium-independent bile salt uptake in rat liver

Background & Aims: Hepatic uptake of bile salts is mediated by sodium-dependent and sodium-independent transport systems. During extrahepatic cholestasis both function and expression of the Na+/taurocholate cotransporting polypeptide (Ntcp) are down-regulated. To test whether hepatic sodium-independent organic anion transporting polypeptides (Oatp1/Oatp1a1, Oatp2/Oatp1a4, Oatp4/Oatp1b2) are also affected by extrahepatic cholestasis, function and expression of all three Oatps have been determined in rats subjected to common bile duct ligation (CBDL; n=4) for up to 7 days with and without anti-cytokine pretreatment with etanercept and anakinra. Results: Protein expression of Oatp1 protein mass as determined by Western blotting remained unchanged after CBDL for 1 day, but then declined by 75±7% and 90±17%, respectively, after 3 and 7 days. In contrast Oatp2 and 4 protein expression was not affected by CBDL as compared to both untreated and sham-operated controls. After CBDL, Oatp1 steady-state mRNA as determined by Northern blotting was rapidly down-regulated by 68±21% of untreated controls (P<0.05) within 24 hours, and remained at similar levels at 3 and 7 days. Cytokine-inactivation studies with etanercept pretreatment demonstrated that TNFα dependent signals mediated the down-regulation of this transporter gene at both protein and mRNA level during obstructive cholestasis. Sodium-independent uptake of taurocholate and cholate into freshly isolated hepatocyte suspensions showed neither significant differences in Km nor Vmax values. Conclusions: These results indicate that sodium-independent transport of bile salts may be mediated by Oatp2 and 4 during biliary obstruction, since its expression remains unaffected and may compensate for loss of Oatp1 expression and function in cholestatic hepatocytes. However, continous bile salt uptake through maintained Oatp2 and 4 expression under these conditions could contribute to the progressive nature of obstructive liver damage.

Extrahepatic cholestasis downregulates Oatp1 by TNF‐α signalling without affecting Oatp2 and Oatp4 expression and sodium‐independent bile salt uptake in rat liver

Hepatic uptake of bile salts is mediated by sodium-dependent and sodium-independent transport systems. During extrahepatic cholestasis, both the function and the expression of the Na(+)/taurocholate cotransporting polypeptide (Ntcp) are downregulated. To test whether sodium-independent organic anion-transporting polypeptides are also affected by extrahepatic cholestasis, the function and expression of all three Oatps have been determined in common bile duct-ligated (CBDL) rats. Oatp1/Oatp1a1 protein mass remained unchanged after CBDL for 1 day, but then declined by 75+/-7% and 90+/-17%, respectively, after 3 and 7 days. In contrast, Oatp2/Oatp1a4 and Oatp4/Oatp1b2 protein expression was not affected by CBDL as compared with controls. After CBDL, Oatp1 mRNA was rapidly downregulated by 68+/-21% of untreated controls (P<0.05) within 24 h, and remained at similar levels at 3 and 7 days. Cytokine-inactivation studies with etanercept pretreatment demonstrated that TNF-alpha-dependent signals mediated the down-regulation of this transporter gene at both protein and mRNA levels during obstructive cholestasis. Sodium-independent uptake of taurocholate and cholate into freshly isolated hepatocyte suspensions showed neither significant differences in K(m) nor V(max) values. These results indicate that sodium-independent transport of bile salts may be mediated by Oatp2 and 4 during biliary obstruction, because its expression remains unaffected and may compensate for loss of Oatp1 expression and function in cholestatic hepatocytes.

Intestinal bile salt absorption in Atp8b1 deficient mice

Mutations in the ATP8B1 gene can cause Progressive Familial Intrahepatic Cholestasis type 1. We have previously reported that Atp8b1(G308V/G308V) mice, a model for PFIC1, have slightly, but significantly, higher baseline serum bile salt (BS) concentrations compared to wt mice. Upon BS feeding, serum BS concentrations strongly increased in Atp8b1-deficient mice. Despite these findings, we observed only mildly impaired canalicular BS transport. In the present report we tested the hypothesis that Atp8b1(G308V/G308V) mice hyperabsorb BS in the intestine during BS feeding. Intestinal BS absorption was measured in intestinal perfusion and in intestinal explants. In addition, we measured BS concentrations in portal blood. Ileal expression of the Fxr-targets Asbt, Ilbp and Shp was assessed. In wt and Atp8b1(G308V/G308V) mice, intestinal taurocholate absorption is primarily mediated by the ileal bile salt transporter Asbt. Neither of the experimental systems revealed enhanced absorption of BS in Atp8b1(G308V/G308V) mice compared to wt mice. In line with these observations, we found no difference in the ileal protein expression of Asbt. Induction of Shp expression during BS feeding also demonstrated that Fxr signalling is intact in Atp8b1(G308V/G308V) mice. The accumulation of BS in plasma of Atp8b1(G308V/G308V) mice during BS feeding is not caused by increased intestinal BS absorption.

Human and rat bile acid–CoA:amino acid N -acyltransferase are liver-specific peroxisomal enzymes: Implications for intracellular bile salt transport

Bile acid-coenzyme A:amino acid N-acyltransferase (BAAT) is the sole enzyme responsible for conjugation of primary and secondary bile acids to taurine and glycine. Previous studies indicate a peroxisomal location of BAAT in peroxisomes with variable amounts up to 95% detected in cytosolic fractions. The absence or presence of a cytosolic pool of BAAT has important implications for the intracellular transport of unconjugated/deconjugated bile salts. We used immunofluorescence microscopy and digitonin permeabilization assays to determine the subcellular location of endogenous BAAT in primary human and rat hepatocytes. In addition, green fluorescent protein (GFP)-tagged rat Baat (rBaat) and human BAAT (hBAAT) were transiently expressed in primary rat hepatocytes and human fibroblasts. Catalase and recombinant GFP-SKL and DsRed-SKL were used as peroxisomal markers. Endogenous hBAAT and rBaat were found to specifically localize to peroxisomes in human and rat hepatocytes, respectively. No significant cytosolic fraction was detected for either protein. GFP-tagged hBAAT and rBaat were efficiently sorted to peroxisomes of primary rat hepatocytes. Significant amounts of GFP-tagged hBAAT or rBaat were detected in the cytosol only when coexpressed with DsRed-SKL, suggesting that hBAAT/rBaat and DsRed-SKL compete for the same peroxisomal import machinery. When expressed in fibroblasts, GFP-tagged hBAAT localized to the cytosol, confirming earlier observations. hBAAT and rBaat are peroxisomal enzymes present in undetectable amounts in the cytosol. Unconjugated or deconjugated bile salts returning to the liver need to shuttle through the peroxisome before reentering the enterohepatic circulation.

Novel cationic and neutral glycocholic acid and polyamine conjugates able to inhibit transporters involved in hepatic and intestinal bile acid uptake

To obtain novel drugs able to inhibit transporters involved in bile acid uptake, three compounds were synthesized by conjugating N-(3-aminopropyl)-1,3-propanediamine (PA) with one (BAPA-3), two (BAPA-6), or three (BAPA-8) moieties of glycocholic acid (GC) through their carboxylic group. The expected net charge in aqueous solutions was 2+ (BAPA-3), 1+ (BAPA-6), and 0 (BAPA-8). They were purified by liquid chromatography and their purity checked by HPLC before being chemically characterized by elemental analysis, NMR, and FAB-MS. Using brush-border membranes isolated from rat ileum; their ability to inhibit [ 14C]-GC transport (BAPA-3 > BAPA-6 > BAPA-8) was suggested. This was further investigated 48 h after injecting Xenopus laevis oocytes with the mRNA of rat sodium/taurocholate (TC)-cotransporting polypeptide (Ntcp), rat apical sodium-dependent bile salt transporter (Asbt), or the human isoforms OATP-C/1B1 and OATP8/1B3 of organic anion-transporting polypeptides, when maximal functional expression was detected. BAPA-8, BAPA-6, and BAPA-3 induced no inhibition of OATP8/1B3-mediated [ 3H]-TC uptake, but dramatically reduced [ 3H]-TC uptake by OATP-C/1B1. In the cases of Ntcp- and Asbt-mediated [ 3H]-TC uptake, these were sodium-dependent and were inhibited by BAPA-6 > BAPA-8 > BAPA-3 and BAPA-8 > BAPA-6 > BAPA-3, respectively. In conclusion, our results suggest that these compounds are potentially interesting research tools for the selective modulation of liver and intestinal uptake of bile acids and other cholephilic compounds. Moreover, they may be of pharmacological usefulness to prevent the acute toxicity of compounds reaching liver cells through specific transporters or to enhance both fecal elimination of bile acids and hence cholesterol consumption for the ‘de novo’ synthesis of bile acids.

Mutations and polymorphisms in the bile salt export pump and the multidrug resistance protein 3 associated with drug-induced liver injury

Increasing evidence suggests that a genetically determined functional impairment of the hepatocellular efflux transporters bile salt export pump (BSEP, ABCB11) and multidrug resistance protein 3 (MDR3, ABCB4) play a pathophysiological role in the development of drug-induced liver injury. The aim of this study was therefore to describe the extent of genetic variability in ABCB11 and ABCB4 in patients with drug-induced liver injury and to in vitro functionally characterize newly detected ABCB11 mutations and polymorphisms. ABCB11 and ABCB4 were sequenced in 23 patients with drug-induced cholestasis and 13 patients with drug-induced hepatocellular injury. Ninety-five healthy Caucasians served as the control group. Reference and mutant BSEP were expressed in Sf9 cells and ATP-dependent transport of [H]-taurocholate was measured in a rapid filtration assay. Four highly conserved nonsynonymous mutations were specific for drug-induced liver injury [ABCB11: D676Y (drug-induced cholestasis) and G855R (drug-induced cholestasis); ABCB4: I764L (drug-induced cholestasis) and L1082Q (drug-induced hepatocellular injury)]. Furthermore, a polymorphism in exon 13 of ABCB11 (V444A), which is associated with decreased hepatic BSEP expression was significantly more frequent in drug-induced cholestasis patients than in drug-induced hepatocellular injury patients and healthy controls (76 versus 50 and 59% in drug-induced cholestasis patients, drug-induced hepatocellular injury patients and healthy controls, respectively; P<0.05). The in-vitro transport activity of the V444A and the D676Y BSEP constructs was similar, whereas the G855R mutation was nonfunctional. In summary, our data support a role of ABCB11 and ABCB4 mutations and polymorphisms in drug-induced cholestasis. Genotyping of selected patients with acquired cholestasis might help to identify individuals with a genetic predisposition.

Differentiation of Non-Adherent Hematopoietic Stem Cells from Umbilical Cord Blood Cells into Adherent Hepatocytic Lineage.

The liver consists of a variety of different cell types, the vast majority of which are hepatocytes. It has been shown that adult, as well as umbilical cord blood (UCB) mesenchymal stem cells (MSC) are capable of differentiation into mesenchymal and non-mesenchymal tissues including hepatocytes. Only a limited number of UCB units (20%) such as frozen, unfractionated and concentrate leukocytes induced by hydroxyethyl starch volume reduced UCB units can generate MSC. In light of this, we have developed the technology to generate potent MSC from all frozen UCB samples tested (3/3). In the present study, the incubation of thawed out UCB mononuclear cells (MNC) and non-selected CD34+ cells in the presence of SCF (25ng/ml), FLt-3 (25ng/ml), MGDF (10ng/ml) & IL-6 (20ng/ml) and 10% human serum in stroma-free liquid culture not only generated long term expansion of transplantable UCB hematopoietic stem cells (non-adherent) (Peters et al., 2004), but also long-term expansion of MSC (adherent cells). Adherent cells were enriched by medium changes followed by trypsinization and subsequent culture and passing on fibronectin in the presence of 10% human serum and 30ng/ml bFGF. The stroma-free liquid cultures were tested for the production of hematopoietic stem cells (HSC), as well as MSC over a period of 15 weeks; MSC increased throughout expansion of HSC. We used the CFU-Fibroblast assay to demonstrate MSC activity in stromal cell formation in vitro. Expanded MSC between passages 3–5 were plated using MesenCult™ (Stem Cell Technologies). After 14 days incubation at 37oC, 5% CO2, very large and small CFU-fibroblast colonies generated from 105 MNC/ml (initiated on Day 0 in stroma-free liquid culture) were continuously detected throughout expansion. In order to test the capacity of MSC to differentiate into hepatocytes, cells were cultured in DMEM supplemented with dexamethasone and 25ng/ml HGF, the latter of which was replaced with 50ng/ml oncostatin M after incubation day 14. The cells were able to expand to cell lines and took on the common hexagonal shape characteristic of hepatocytes; this parameter was used as a primary screening criterion. In addition, the hepatocyte-like cells were further assessed by reverse transcription-polymerase chain reaction and found to express mRNA for albumin, as well as two hepatocyte-specific bile salt transport proteins, basolateral bile transport system NTCP and the canalicular bile salt export pump BSEP. These data demonstrate the presence of pluripotent stem cells in UCB with the capacity to differentiate into a hepatocytic lineage.The research in this area has great potential, both with respect to future application in patients with end-stage liver disease, as well as for learning more about the role of MSC in new clinical cellular therapies.

Increased Serum Concentrations of Secondary Bile Salts during Cholate Feeding Are Due to Coprophagy. A Study with Wild-Type and Atp8b1-Deficient Mice

Coprophagy (i.e., consumption of feces) is a behavior seen in rodents and other animal species. This behavior can substantially influence the enterohepatic cycling of compounds, including bile salts. Since many studies involve the feeding of rodents with bile salt supplemented diets, it is of importance to know the influence of coprophagy on bile salt composition in such studies. We compared the peripheral and portal bile salt composition of mice in conventional and metabolic cages when fed a control diet or a diet containing 0.5% cholate. We also performed these experiments with Atp8b1-deficient mice as it has been suggested that in the absence of this transporter bile salt absorption in the intestine would be increased. In mice on a control diet there is little difference in bile salt composition between conventional housing and metabolic housing. Metabolic housing led to a near complete disappearance of the low levels of dihydroxy bile salts (i.e., deoxycholate + chenodeoxycholate) in peripheral serum. In mice fed a control diet, the portal blood concentration of unconjugated dihydroxy bile salts was extremely low (<2%), but these rose to about 10% when mice were fed a cholate-supplemented diet. In metabolic cages the portal blood content of these unconjugated dihydroxy bile salts was reduced to undetectable levels. Whether housed in conventional cages or in metabolic cages, wild-type and Atp8b1-deficient mice had similar concentrations in portal blood, suggesting that intestinal bile salt absorption is not altered in Atp8b1-deficient mice.

Atp8b1 deficiency in mice reduces resistance of the canalicular membrane to hydrophobic bile salts and impairs bile salt transport

Progressive familial intrahepatic cholestasis type 1 (PFIC1, Byler disease, OMIM 211600) is a severe inherited liver disease caused by mutations in ATP8B1. ATP8B1 is a member of the type 4 subfamily of P-type ATPases, which are phospholipid flippases. PFIC1 patients generally develop end-stage liver disease before the second decade of life. The disease is characterized by impaired biliary bile salt excretion, but the mechanism whereby impaired ATP8B1 function results in cholestasis is unclear. In a mouse model for PFIC1, we observed decreased resistance of the hepatocanalicular membrane to hydrophobic bile salts as evidenced by enhanced biliary recovery of phosphatidylserine, cholesterol, and ectoenzymes. In liver specimens from PFIC1 patients, but not in those from control subjects, ectoenzyme expression at the canalicular membrane was markedly deficient. In isolated mouse livers Atp8b1 deficiency impaired the transport of hydrophobic bile salts into bile. In conclusion, our study shows that Atp8b1 deficiency causes loss of canalicular phospholipid membrane asymmetry that in turn renders the canalicular membrane less resistant toward hydrophobic bile salts. The loss of phospholipid asymmetry may subsequently impair bile salt transport and cause cholestasis.

315 Fasting-induced upregulation of basolateral hepatic bile salt transporters in rats is mediated by the PGC-1α/HNF-4α pathway

315 Fasting-induced upregulation of basolateral hepatic bile salt transporters in rats is mediated by the PGC-1α/HNF-4α pathway

Relation of bile salt transporters BSEP, MRP2 and NTCP to cholelithiasis

目的 探讨不同的胆盐转运子(BSEP、MRP2、NTCP)与胆囊结石形成的关系.方法胆囊结石病人的肝组织标本20例,正常肝组织标本10例.应用逆转录聚合酶链反应(RT-PCR)技术和Western blot技术检测BSEP、MRP2、NTCPmRNA和蛋白的表达.结果(1)胆囊结石病人的肝组织中的BSEP、MRP2和NTCP mRNA的表达较正常肝组织减少,光密度平均值分别为0.47±0.18、1.12±0.39和0.56±0.31,而正常肝的组织中相应的光密度值分别为0.90±0.42、2.48±0.89和0.76±0.28.统计分析结果显示,BSEP、MRP2在两组间均存在显著性差异(P＜0.01),而NTCP在两组间无统计学差异(P＞0.05).(2)所有组织中BSEP、MRP2蛋白均有表达,但胆囊结石病人的肝组织中BSEP、MRP2蛋白的表达要显著低于正常的肝组织(P＜0.01).结论胆囊结石病人肝组织中BSEP、MRP2的mRNA和蛋白的表达均下降,BSEP、MRP2的表达降低可能与胆囊结石的形成有关.NTCP可能与结石的形成无关。