The bile salt export pump (BSEP) encoded by ABCB11 is located in the canalicular membrane of hepatocytes and mediates the secretion of numerous conjugated bile salts into the bile canaliculus. In this study, 28 ABCB11 exons (including non-coding exon 1) and their flanking introns were comprehensively screened for genetic variations in 120 Japanese subjects. Fifty-nine genetic variations, including 19 novel ones, were found: 14 in the coding exons (6 nonsynonymous and 8 synonymous variations), 4 in the 3'-UTR, and 41 in the introns. Three novel nonsynonymous variations, 361C>A (Gln121Lys), 667C>T (Arg223Cys), and 1460G>T (Arg487Leu), were found as heterozygotes and at 0.004 allele frequencies. These data provide fundamental and useful information for genotyping ABCB11 in the Japanese and probably other Asian populations.

Transcriptional coregulators are important components of nuclear receptor (NR) signaling machinery and provide additional mechanisms for modulation of NR activity. Expression of a mutated nuclear corepressor 1 (NCoR1) that lacks 2 NR interacting domains (NCoRΔID) in the liver leads to elevated expression of genes regulated by thyroid hormone receptor (TR) and liver X receptor (LXR), both of which control hepatic cholesterol metabolism. Here, we demonstrate that expression of NCoRΔID in mouse liver improves dietary cholesterol tolerance in an LXRα-independent manner. NCoRΔID-associated cholesterol tolerance was primarily due to diminished intestinal cholesterol absorption as the result of changes in the composition and hydrophobicity of the bile salt pool. Alterations of the bile salt pool were mediated by increased expression of genes encoding the bile acid metabolism enzymes CYP27A1 and CYP3A11 as well as canalicular bile salt pump ABCB11. We have determined that these genes are regulated by thyroid hormone and that TRβ1 is recruited to their regulatory regions. Together, these data indicate that interactions between NCoR1 and TR control a specific pathway involved in regulation of cholesterol metabolism and clearance.

OBJECTIVE

The aim of the study was to elucidate the role and characteristics of ATP8B1 gene mutations in mainland Chinese children with progressive intrahepatic cholestasis and low gamma-glutamyltransferase (GGT).

METHODS

Twenty-four children who presented with progressive intrahepatic cholestasis and low GGT were admitted to a tertiary pediatric hospital in eastern China from January 2004 to July 2007. Five children with homozygous or compound heterozygous ABCB11 gene mutations were excluded from the study. All encoding exons and their flanking areas of ATP8B1 gene were sequenced in the remaining 19 patients, in whom only 1 or no mutation of ABCB11 was found. Clinical features and liver histology obtained by reviewing the medical records were compared among patients with different genotypes.

RESULTS

Nine mutations of ATP8B1 gene were found in 9 patients. All of them were novel except for mutations I694N and R952X. A linked P209T and IVS6+5G>T mutation was found in 4 of 9 patients, including 2 homozygotes and 2 heterozygotes. Giant cell transformation of hepatocytes was demonstrated in 1 of 6 patients with ATP8B1 mutations and 4 of 5 patients with ABCB11 mutations.

CONCLUSIONS

ATP8B1 gene mutations play an important role in Chinese patients with progressive intrahepatic cholestasis and low GGT. The linked mutation P209T and IVS6+5G>T is a hot mutation in the Chinese population. Histological examination may be helpful in differentiating familial intrahepatic cholestasis type 1 from bile salt export pump-related disease.

The cytokine oncostatin M (OSM) is a member of the interleukin (IL)-6 family, known to down-regulate expression of drug metabolizing cytochromes P-450 in human hepatocytes. The present study was designed to determine whether OSM may also impair expression of sinusoidal and canalicular drug transporters, which constitute important determinants of drug hepatic clearance. Exposure of primary human hepatocytes to OSM down-regulated mRNA levels of major sinusoidal solute carrier (SLC) influx transporters, including sodium-taurocholate co-transporting polypeptide (NTCP), organic anion transporting polypeptide (OATP) 1B1, OATP1B3, OATP2B1, organic cation transporter 1 and organic anion transporter 2. OSM also repressed mRNA expressions of ATP binding cassette (ABC) efflux transporters such as multidrug resistance protein (MRP) 2/ABCC2 and breast cancer resistance protein/ABCG2, without however impairing those of multidrug resistance gene 1/P-glycoprotein/ABCB1, MRP3/ABCC3, MRP4/ABCC4 and bile salt export pump/ABCB11. The cytokine concomitantly reduced NTCP, OATP1B1, OATP2B1 and ABCG2 protein expression and NTCP and OATP transport activities. OSM effects towards transporters were found to be dose-dependent and highly correlated with those of IL-6, but not with those of other inflammatory cytokines such as tumor necrosis factor-α or interferon-γ. In addition, OSM-mediated repression of some transporters such as NTCP, OATP1B1 and OATP2B1, was counteracted by knocking-down expression of the type II OSM receptor subunits through siRNA transfection. This OSM-mediated down-regulation of drug SLC transporters and ABCG2 in human hepatocytes may contribute to alterations of pharmacokinetics in patients suffering from diseases associated with increased production of OSM.

Identifying rare genetic forms of infantile cholestasis is challenging due to their similar clinical presentation and their diverse etiology. After exclusion of common non-genetic causes a huge list of rare differential diagnosis remains to be solved. More than 90 genes are associated with monogenic forms of infantile cholestasis, thus preventing routine genetic workup by Sanger sequencing. Here we demonstrate a next generation sequencing approach to discover the underlying cause in clinically well characterized patients in whom common causes of infantile cholestasis have been excluded. After validation of the analytical sensitivity massive parallel sequencing was performed for 93 genes in six prospectively studied patients. Six novel mutations (PKHD1: p.Thr777Met, p.Tyr2260Cys; ABCB11: p.Val1112Phe, c.611+1G>> A, p.Gly628Trpfs*3 and NPC1: p.Glu391Lys) and two known pathogenic mutations were detected proving our multi gene panel for infantile cholestasis to be a sensitive and specific method overcoming the complexity of the phenotype-based, candidate gene approach. Three exemplary clinical cases of infants with cholestasis are presented and discussed in the context of their genetic and histopathological findings (autosomal recessive polycystic kidney disease, atypical PFIC and Niemann-Pick syndrome type C1). These case reports highlight the critical impact of integrating clinical, histopathological and genetic data during the process of multi gene panel testing to ultimately pinpoint rare genetic diagnoses.

The bile salt export pump (BSEP, ABCB11), multidrug resistance protein 3 (MDR3, ABCB4) and the ATPase familial intrahepatic cholestasis 1 (FIC1, ATP8B1) mediate bile formation. This study aimed to determine the contribution of mutations and common variants in the FIC1, BSEP and MDR3 genes to cholestatic disorders of differing disease onset and severity.

Coding exons with flanking intron regions of ATP8B1, ABCB11, and ABCB4 were sequenced in cholestatic patients with assumed genetic cause. The effects of new variants were evaluated by bioinformatic tools and 3D protein modeling.

In 427 patients with suspected inherited cholestasis, 149 patients carried at least one disease-causing mutation in FIC1, BSEP or MDR3, respectively. Overall, 154 different mutations were identified, of which 25 were novel. All 13 novel missense mutations were disease-causing according to bioinformatics analyses and homology modeling. Eighty-two percent of patients with at least one disease-causing mutation in either of the three genes were children. One or more common polymorphism(s) were found in FIC1 in 35.3%, BSEP in 64.3% and MDR3 in 72.6% of patients without disease-causing mutations in the respective gene. Minor allele frequencies of common polymorphisms in BSEP and MDR3 varied in our cohort compared to the general population, as described by gnomAD. However, differences in ethnic background may contribute to this effect.

In a large cohort of patients, 154 different variants were detected in FIC1, BSEP, and MDR3, 25 of which were novel. In our cohort, frequencies for risk alleles of BSEP (p.V444A) and MDR3 (p.I237I) polymorphisms were significantly overrepresented in patients without disease-causing mutation in the respective gene, indicating that these common variants can contribute to a cholestatic phenotype.

FIC1, BSEP, and MDR3 represent hepatobiliary transport proteins essential for bile formation. Genetic variants in these transporters underlie a broad spectrum of cholestatic liver diseases. To confirm a genetic contribution to the patients' phenotypes, gene sequencing of these three major cholestasis-related genes was performed in 427 patients and revealed 154 different variants of which 25 have not been previously reported in a database. In patients without a disease-causing mutation, common genetic variants were detected in a high number of cases, indicating that these common variants may contribute to cholestasis development.

The safety and effectiveness of drugs used to treat chronic diseases critically depend on their propensity to interact with co-administered drugs. Induction of enzymes and drug transporters involved in the clearance and distribution of drugs may critically reduce exposure with their substrates and thus lead to nonresponse. We therefore investigated the impact of the endothelin-1 receptor antagonists bosentan and ambrisentan on the expression of relevant human efflux and uptake transporters and on phase 1 and phase 2 enzymes. LS180 adenocarcinoma cells were treated for four days with bosentan or ambrisentan (1-50 μM), the positive control rifampicin, or medium only (negative control). For evaluation of bosentan also HuH-7 human hepatoma cells were used and treated similarly. Gene expression was quantified at the mRNA level by real-time reverse transcription polymerase chain reaction and for some genes also at the protein level by western blot analysis. Comparable to rifampicin, bosentan was a moderate to strong inductor for all cytochrome P450 isozymes and ATP-binding cassette transporters tested, and it also induced organic anion transporting polypeptides. 50 μM bosentan up-regulated e.g. CYP3A4 8.5-fold, ABCB1 5.1-fold, and ABCB11 1.9-fold at the mRNA level in LS180 cells. In HuH-7 cells induction was much less pronounced (e.g. CYP3A4 1.9-fold for bosentan). In contrast, ambrisentan only weakly induced some of the genes investigated in LS180 cells. These findings corroborate the in vivo finding that bosentan is much more prone to drug interactions than ambrisentan.

Bile salts are predominantly taken up by hepatocytes via the basolateral Na(+)-taurocholate cotransporting polypeptide (NTCP/SLC10A1) and secreted into the bile by the bile salt export pump (BSEP/ABCB11). In the present study, we transfected rat Ntcp and rat Bsep into polarized Madin-Darby canine kidney cells and characterized the transport properties of these cells for eight bile salts. Immunohistochemical staining demonstrated that Ntcp was expressed at the basolateral domains, whereas Bsep was expressed at the apical domains. Basal-to-apical transport of taurocholate across the monolayer expressing only Ntcp and that coexpressing Ntcp/Bsep was observed, whereas the flux across the monolayer of control and Bsep-expressing cells was symmetrical. Basal-to-apical transport of taurocholate across Ntcp/Bsep-coexpressing monolayers was significantly higher than that across monolayers expressing only Ntcp. Kinetic analysis of this vectorial transport of taurocholate gave an apparent K(m) value of 13.9 +/- 4.7 microM for cells expressing Ntcp alone, which is comparable with 22.2 +/- 4.5 microM for cells expressing both Ntcp and Bsep and V(max) values of 15.8 +/- 4.2 and 60.8 +/- 9.0 pmol.min(-1).mg protein(-1) for Ntcp alone and Ntcp and Bsep-coexpressing cells, respectively. Transcellular transport of cholate, glycocholate, taurochenodeoxycholate, chenodeoxycholate, glycochenodeoxycholate, tauroursodeoxycholate, ursodeoxycholate, and glycoursodeoxycholate, but not that of lithocholate was also observed across the double transfectant. This double-expressing system can be used as a model to clarify vectorial transport of bile salts across hepatocytes under physiological conditions.

Adenosine triphosphate-binding cassette, subfamily B, member 4 (ABCB4) gene alterations can cause two distinct clinical entities: progressive familial intrahepatic cholestasis type 3 (PFIC3) and low phospholipid-associated cholelithiasis (LPAC). Based on the findings in two siblings and a review of the literature, we aimed to identify determinants of disease phenotypic traits associated with ABCB4 gene alterations. Two siblings presented, before the age of 30 years, recurrent symptomatic cholelithiasis and extensive biliary fibrosis that progressed towards portal hypertension and liver failure necessitating liver transplantation. We analysed the sequence of the ABCB4 gene and immunolocalization of the protein in the liver. Sequence analysis of ABCB11, potentially involved in similar symptoms, was also performed. Two heterozygous non-synonymous variants of ABCB4 were found in both siblings. One of them (c.959C>T; p.Ser320Phe) was previously implicated in LPAC and the second one (c.2858C>A; p.Ala953Asp) in PFIC3. Both patients were also heterozygous for the ABCB11 variant Val444Ala, which predisposes to cholestatic disorders. ABCB4 was normally detected at the canalicular membrane of hepatocytes. The review of ABCB4 gene variants reported so far shows that the vast majority of variants causing PFIC3 and LPAC are distinct. Also as a general rule, homozygous variants cause PFIC3 while heterozygous variants lead to LPAC. Combined PFIC3 and LPAC phenotype is a rare clinical event, which may be determined by the coexistence of ABCB4 variants related to both phenotypes and also potentially to the ABCB11 variant. Thus, most of the patients presenting with LPAC are not at a particular risk of developing PFIC3 features in adulthood.

OBJECTIVE

Generation of bile is an important function of the liver. Its impairment can be caused by inherited mutations or by acquired factors and leads to cholestasis. Bile salts are an important constituent of bile and are secreted by the bile salt export pump (BSEP) from hepatocytes.

RESULTS

Significant progress was made in the understanding of mechanisms and consequences of malfunctioning BSEP. This information was gained from extensive characterization of patients with inherited BSEP deficiency and the subsequent characterization of the identified mutations in heterologous expression systems. Furthermore and importantly, clinical evidence shows that patients with severe BSEP deficiency are at risk to develop hepatocellular carcinoma. Bile salts are now recognized to be important in the modulation of whole body energy homeostasis. Because BSEP is the rate-limiting step in hepatocellular bile salt transport, it controls the spill over of bile salts into the systemic circulation. Therefore, an indirect role of BSEP in energy homeostasis becomes more and more likely.

CONCLUSIONS

In summary, knowledge on the physiologic and pathophysiologic role of BSEP is rapidly progressing. It can be anticipated that the next major step in better understanding BSEP should come from information on structure-function relationship. However, given the difficulty in structure determination of mammalian transporters, this will require major efforts.

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.

BACKGROUND

Cholesterol gallstone disease is a complex genetic trait and induced by multiple but as yet unknown genes. A major Lith gene, Lith1 was first identified on chromosome 2 in gallstone-susceptible C57L mice compared with resistant AKR mice. Abcb11, encoding the canalicular bile salt export pump in the hepatocyte, co-localizes with the Lith1 QTL region and its hepatic expression is significantly higher in C57L mice than in AKR mice.

METHODS

To investigate whether Abcb11 influences cholesterol gallstone formation, we created an Abcb11 transgenic strain on the AKR genetic background and fed these mice with a lithogenic diet for 56 days.

RESULTS

We excluded functionally relevant polymorphisms of the Abcb11 gene and its promoter region between C57L and AKR mice. Overexpression of Abcb11 significantly promoted biliary bile salt secretion and increased circulating bile salt pool size and bile salt-dependent bile flow rate. However, biliary cholesterol and phospholipid secretion, as well as gallbladder size and contractility were comparable in transgenic and wild-type mice. At 56 days on the lithogenic diet, cholesterol saturation indexes of gallbladder biles and gallstone prevalence rates were essentially similar in these two groups of mice.

CONCLUSIONS

Overexpression of Abcb11 augments biliary bile salt secretion, but does not affect cholelithogenesis in mice.

OBJECTIVE

Rodent obesity models have been shown to display impaired bile secretory functions. We have shown that glucagon-like peptide 1 (GLP-1) attenuates hepatic lipogenesis, and in the present study we investigated whether GLP-1 also improves high fat diet-associated cholestatic injury.

METHODS

Wild type (WT) and dipeptidyl peptidase 4-deficient rats (DPP4-) with chronic elevated serum levels of active GLP-1 were fed regular chow and a Western diet for 2 months. Primary hepatocytes were used to assess GLP-1 effects on mRNA expression and transcription of genes encoding bile acid synthesis enzymes and transporters.

RESULTS

DPP4- exhibited attenuated liver injury as expressed by lower serum AST and ALT after 2 months of a Western diet. In addition, DPP4- had better insulin sensitivity, lower serum triglycerides, cholesterol and bile acids. Hepatic expression of cyp7A1, the rate limiting enzyme in conversion of cholesterol into bile acids, was strongly attenuated in DPP4- fed with a Western diet. Moreover, hepatic expression of bile transporter, ABCB11, was increased, facilitating a higher rate of bile secretion. Mechanistically, we showed that GLP-1 directly reduced basal and LXR-induced cyp7A1 mRNA expression and suppressed cyp7A1 transcription in transient transfection assays in primary hepatocytes. However, GLP-1 and its analog exendin 4 also induced mRNA expression of bile acid transporter ABCC3 in primary rat hepatocyte cultures.

CONCLUSIONS

Our data suggest that GLP-1 analogs may serve as a novel therapeutic drug to alleviate obesity-induced liver injury by reducing bile acid synthesis and improving liver bile secretory function.

More than 1·4 billion individuals are overweight or obese worldwide. While complications often require therapeutic intervention, data regarding the impact of obesity on drug disposition are scarce. As the influence of diet-induced obesity on drug transport and metabolic pathways is currently unclear, the objective of the present study was to investigate the effect of high fat feeding for 13 weeks in female Sprague-Dawley rats on the hepatic expression of the nuclear receptors pregnane X receptor (PXR), constitutive androstane receptor (CAR), liver X receptor (LXR) and farnesoid X receptor (FXR) and several of their target genes. We hypothesised that high fat feeding would alter the gene expression of major hepatic transporters through a dysregulation of the expression of the nuclear receptors. The results demonstrated that, along with a significant increase in body fat and weight, a high-fat diet (HFD) induced a significant 2-fold increase in the expression of PXR as well as a 2-, 5- and 2·5-fold increase in the hepatic expression of the PXR target genes Abcc2, Abcb1a and Cyp3a2, respectively (P< 0·05). The expression levels of FXR were significantly increased in rats fed a HFD in addition to the increase in the expression levels of FXR target genes Abcb11 and Abcb4. The expression levels of both LXRα and LXRβ were slightly but significantly increased in rats fed a HFD, and the expression levels of their target genes Abca1 and Abcg5, but not Abcg8, were significantly increased. The expression of the nuclear receptor CAR was not significantly altered between the groups. This suggests that a HFD may induce changes in the hepatobiliary transport and metabolism of endogenous and exogenous compounds.

The outcome of hepatitis C virus (HCV) infection and the likelihood of a sustained virological response (SVR) to antiviral therapy depends on both viral and host characteristics. In vitro studies demonstrated that bile acids (BA) interfere with antiviral interferon effects. We investigate the influence of plasma BA concentrations and an ABCB11 polymorphism associated with lower transporter expression on viral load and SVR. Four hundred and fifty-one Caucasian HCV-patients treated with PEG-interferon and ribavirin were included in the study. ABCB11 1331T>C was genotyped, and plasma BA levels were determined. The 1331C allele was slightly overrepresented in HCV-patients compared to controls. In HCV-patients, a significant difference between patients achieving SVR vs non-SVR was observed for HCV-2/3 (5 vs 9 μm; P=0.0001), while median BA levels in HCV-1 were marginally elevated. Normal BA levels <8 μm were significantly associated with SVR (58.3%vs 36.3%; OR 2.48; P=0.0001). This difference was significant for HCV-2/3 (90.7%vs 67.6%; P=0.002) but marginal in HCV-1 (38.7%vs 27.8%; P=0.058). SVR rates were equivalent between ABCB11 genotypes for HCV-1, but increased for HCV-2/3 (TT 100%vs CC 78%; OR 2.01; P=0.043). IL28B genotype had no influence on these associations. No correlation between BA levels and HCV RNA was detected for any HCV genotype. The higher allelic frequency of ABCB11 1331C in HCV-patients compared to controls may indirectly link increased BA to HCV chronicity. Our data support a role for BA as host factor affecting therapy response in HCV-2/3 patients, whereas a weaker association was found for HCV-1.

OBJECTIVE

Progressive familial intrahepatic cholestasis (PFIC) and to a lesser extent, Alagille syndrome, often lead to end-stage liver disease during childhood. We report our experience of DNA-based prenatal diagnosis of PFIC1-3 and Alagille syndrome.

METHODS

Four molecular antenatal diagnoses were performed in 3 PFIC families and 17 in 11 Alagille syndrome families. DNA was isolated from chorionic villus or cultured amniocyte samples from women, without pregnancy complications.

RESULTS

All four foetuses with a family history of PFIC1, 2, or 3 were heterozygous for an ATP8B1, ABCB11, or ABCB4 mutation and pregnancies were continued. Three of the infants were healthy after birth, and 1 premature infant, who had an ABCB4 mutation, experienced transient neonatal cholestasis. Among the families with a history of de novo JAG1 mutation, none of the foetuses was mutated, versus 40% of those with a history of familial mutation. Of 4 pregnant women with a JAG1-mutated foetus, 3 cut short their pregnancy and 1 gave birth to a child with overt Alagille syndrome.

CONCLUSIONS

Molecular antenatal diagnosis of PFIC1-3 and Alagille syndrome is reliable because clinical outcome after birth corresponded to molecular foetal data.

Bile formation is a key function of the liver and is driven by active secretion of bile salts and other organic compounds into the biliary tree. Bile salts represent the major organic constituent of bile. They are released with bile into the small intestine, where they are almost quantitatively reabsorbed and transported via the portal circulation back to the liver. In the liver, they are taken up into hepatocytes and secreted into bile. This cycling between the liver and the small intestine is called enterohepatic circulation of bile salts. Bile salts are secreted from hepatocytes into the bile by the bile salt export pump BSEP. This step constitutes the rate-limiting step of handling of bile salts in the liver and is the major driving force of the enterohepatic circulation of bile salts. Improper functioning of BSEP leads to an accumulation of bile salts within hepatocytes, where bile salts become cytotoxic. If persistent, accumulation of bile salts in hepatocytes will lead to liver disease. This review summarizes the essential concepts of bile formation and the current knowledge of mechanisms known to impair BSEP function. Finally, it sets the current therapeutic approaches for cholestatic liver disease into perspective to the pathophysiologic mechanisms of impaired BSEP function.

Genetic polymorphisms of human ABC-transporter genes have been suggested to modulate breast cancer risk in the general population. In particular ABCC11 (MRP8), which is highly expressed in breast cancer tissue and involved in the efflux of conjugated estrogen metabolites such as estrone-3-sulfate and estradiol-17beta-glucuronide, has recently been proposed as a potential risk factor for female breast cancer. The wet earwax-associated G-allele of the c.538G>A polymorphism was associated with an increased risk for breast cancer in Japanese women. In contrast, no evidence for such an association could be observed in Caucasian women. We aimed to confirm/refute the association of the c.538G>A variant in ABCC11 with breast cancer risk and/or histo-pathological tumor characteristics in an independent population-based breast cancer case-control study from Germany comprising 1021 cases and 1015 age-matched controls. No association for allele and genotype frequencies of the 538G>A variant in ABCB11 with breast cancer risk was found. Our data suggest that the c.538G>A variation in ABCC11 does not contribute to breast carcinogenesis in women of European descent.

Severe cholestasis may result in end-stage liver disease with the need of liver transplantation (LTX). In children, about 10 % of LTX are necessary because of cholestatic liver diseases. Apart from bile duct atresia, three types of progressive familial intrahepatic cholestasis (PFIC) are common causes of severe cholestasis in children. The three subtypes of PFIC are defined by the involved genes: PFIC-1, PFIC-2, and PFIC-3 are due to mutations of P-type ATPase ATP8B1 (familial intrahepatic cholestasis 1, FIC1), the ATP binding cassette transporter ABCB11 (bile salt export pump, BSEP), or ABCB4 (multidrug resistance protein 3, MDR3), respectively. All transporters are localized in the canalicular membrane of hepatocytes and together mediate bile salt and phospholipid transport. In some patients with PFIC-2 disease, recurrence has been observed after LTX, which mimics a PFIC phenotype. It could be shown by several groups that inhibitory anti-BSEP antibodies emerge, which most likely cause disease recurrence. The prevalence of severe BSEP mutations (e.g., splice site and premature stop codon mutations) is very high in this group of patients. These mutations often result in the complete absence of BSEP, which likely accounts for an insufficient auto-tolerance against BSEP. Although many aspects of this "new" disease are not fully elucidated, the possibility of anti-BSEP antibody formation has implications for the pre- and posttransplant management of PFIC-2 patients. This review will summarize the current knowledge including diagnosis, pathomechanisms, and management of "autoimmune BSEP disease."

Hepatocytes are used widely as a cell model for investigation of xenobiotic metabolism and the toxic mechanism of drugs. Simvastatin is the first statin drug used extensively in clinical practice for control of elevated cholesterol or hypercholesterolemia. However, it has also been reported to cause adverse effects in liver due to cellular damage. In this study, for proteomic and transcriptomic analysis, rat primary hepatocytes were exposed to simvastatin at IC20 concentration for 24 h. Among a total of 607 differentially expressed proteins, 61 upregulated and 29 downregulated proteins have been identified in the simvastatin-treated group. At the mRNA level, results of transcriptomic analysis revealed 206 upregulated and 41 downregulated genes in the simvastatin-treated group. Based on results of transcriptomic and proteomic analysis, NRF2-mediated oxidative stress response, xenobiotics by metabolism of cytochrome P450, fatty acid metabolism, bile metabolism, and urea cycle and inflammation metabolism pathways were focused using IPA software. Genes (FASN, UGT2B, ALDH1A1, CYP1A2, GSTA2, HAP90, IL-6, IL-1, FABP4, and ABC11) and proteins (FASN, CYP2D1, UG2TB, ALDH1A1, GSTA2, HSP90, FABP4, and ABCB11) related to several important pathways were confirmed by real-time PCR andWestern blot analysis, respectively. This study will provide new insight into the potential toxic pathways induced by simvastatin.

BACKGROUND

Divergently selected Lean and Fat mouse lines represent unique models for a polygenic form of resistance and susceptibility to obesity development. Previous research on these lines focused mainly on obesity-susceptible factors in the Fat line. This study aimed to examine the molecular basis of obesity-resistant mechanisms in the Lean line by analyzing various fat depots and organs, the liver transcriptome of selected metabolic pathways, plasma and lipid homeostasis and expression of selected skeletal muscle genes.

RESULTS

Expression profiling using our custom Steroltalk v2 microarray demonstrated that Lean mice exhibit a higher hepatic expression of cholesterol biosynthesis genes compared to the Fat line, although this was not reflected in elevation of total plasma or liver cholesterol. However, FPLC analysis showed that protective HDL cholesterol was elevated in Lean mice. A significant difference between the strains was also found in bile acid metabolism. Lean mice had a higher expression of Cyp8b1, a regulatory enzyme of bile acid synthesis, and the Abcb11 bile acid transporter gene responsible for export of acids to the bile. Additionally, a higher content of blood circulating bile acids was observed in Lean mice. Elevated HDL and upregulation of some bile acids synthesis and transport genes suggests enhanced reverse cholesterol transport in the Lean line--the flux of cholesterol out of the body is higher which is compensated by upregulation of endogenous cholesterol biosynthesis. Increased skeletal muscle Il6 and Dio2 mRNA levels as well as increased activity of muscle succinic acid dehydrogenase (SDH) in the Lean mice demonstrates for the first time that changes in muscle energy metabolism play important role in the Lean line phenotype determination and corroborate our previous findings of increased physical activity and thermogenesis in this line. Finally, differential expression of Abcb11 and Dio2 identifies novel strong positional candidate genes as they map within the quantitative trait loci (QTL) regions detected previously in crosses between the Lean and Fat mice.

CONCLUSIONS

We identified novel candidate molecular targets and metabolic changes which can at least in part explain resistance to obesity development in the Lean line. The major difference between the Lean and Fat mice was in increased liver cholesterol biosynthesis gene mRNA expression, bile acid metabolism and changes in selected muscle genes' expression in the Lean line. The liver Abcb11 and muscle Dio2 were identified as novel positional candidate genes to explain part of the phenotypic difference between the Lean and Fat lines.

To overcome the pathological phenomena caused by altered function of ABC (ATP Binding Cassette) proteins, their mechanisms of action are extensively investigated, often involving the design of mutant constructs for experiments. Designing mutagenetic constructs, interpreting the result of mutagenetic experiments, and finding individual genetic variants require an extensive knowledge of previously published mutations. To aid the recapitulation of mutations described in the literature, we set up a database of ABC protein mutations (ABCMdb) extracted from full-text papers using an automatic mining approach. We have also developed a Web application interface to compare mutations in different ABC proteins using sequence alignments and to interactively map the mutations to 3D structural models. Currently our database contains protein mutations published for ABCB1, ABCB11, ABCC1, ABCC6, ABCC7, and the proteins of the ABCG subfamily. The database will be extended to include other members and subfamilies, and to provide information on whether or not a mutation is disease causing, represents a high-incidence polymorphism, or was generated only in vitro. The ABCMdb database should already help to compare the effects of mutations at homologous positions in different ABC proteins, and its interactive tools aim to advance the design of experiments for a wider range of proteins.

Various disease models have been shown to alter hepatic drug-metabolizing enzyme (DME) and transporter expression and to induce cholestasis through altered enzyme and transporter expression. Previously, we detailed the regulation of hepatic DMEs during infectious colitis caused by Citrobacter rodentium infection. We hypothesized that this infection would also modulate hepatic drug transporter expression and key genes of bile acid (BA) synthesis and transport. Mice lacking Toll-like receptor 4 (TLR4), interleukin-6 (IL-6), or interferon-gamma (IFNγ) and appropriate wild-type animals were orally infected with C. rodentium and sacrificed 7 days later. In two wild-type strains, drug transporter mRNA expression was significantly decreased by infection for Slc22a4, Slco1a1, Slco1a4, Slco2b1, and Abcc6, whereas the downregulation of Abcc2, Abcc3, and Abcc4 were strain-dependent. In contrast, mRNA expressions of Slco3a1 and Abcb1b were increased in a strain-dependent manner. Expression of Abcb11, Slc10a1, the two major hepatic BA transporters, and Cyp7a1, the rate-limiting enzyme of BA synthesis, was also significantly decreased in infected animals. None of the above effects were caused by bacterial lipopolysaccharide, since they still occurred in the absence of functional TLR4. The downregulation of Slc22a4 and Cyp7a1 was absent in IFNγ-null mice, and the downregulation of Slco1a1 was abrogated in IL-6-null mice, indicating in vivo roles for these cytokines in transporter regulation. These data indicate that C. rodentium infection modulates hepatic drug processing through alteration of transporter expression as well as DMEs. Furthermore, this infection downregulates important genes of BA synthesis and transport and may increase the risk for cholestasis.

Progressive familial intrahepatic cholestasis type 2 (PFIC-2) is caused by mutations in ABCB11, encoding the bile salt export pump (BSEP). In 2009, we described a child with PFIC-2 who developed PFIC-like symptoms after orthotopic liver transplantation (OLT). BSEP-reactive antibodies were demonstrated to account for disease recurrence. Here, we characterize the nature of this antibody response in 7 more patients with antibody-induced BSEP deficiency (AIBD). Gene sequencing and immunostaining of native liver biopsies indicated absent or strongly reduced BSEP expression in all 7 PFIC-2 patients who suffered from phenotypic disease recurrence post-OLT. Immunofluorescence, western blotting analysis, and transepithelial transport assays demonstrated immunoglobulin (Ig) G-class BSEP-reactive antibodies in these patients. In all cases, the N-terminal half of BSEP was recognized, with reaction against its first extracellular loop (ECL1) in six sera. In five, antibodies reactive against the C-terminal half also were found. Only the sera recognizing ECL1 showed inhibition of transepithelial taurocholate transport. In a vesicle-based functional assay, transport inhibition by anti-BSEP antibodies binding from the cytosolic side was functionally proven as well. Within 2 hours of perfusion with antibodies purified from 1 patient, rat liver showed canalicular IgG staining that was absent after perfusion with control IgG.

CONCLUSIONS

PFIC-2 patients carrying severe BSEP mutations are at risk of developing BSEP antibodies post-OLT. The antibody response is polyclonal, targeting both extra- and intracellular BSEP domains. ECL1, a unique domain of BSEP, likely is a critical target involved in transport inhibition as demonstrated in several patients with AIBD manifest as cholestasis.