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. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/ index.html). (HEPATOLOGY 2006;44:195-204.)

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MRP3 is an ABC transporter localized in the basolateral membrane of epithelial cells such as hepatocytes and enterocytes. In this study, the role of Mrp3 in drug disposition was investigated. Because Mrp3 preferentially transports glucuronide conjugates, we investigated the in vivo disposition of acetaminophen (APAP) and its metabolites. Mrp3 ؉/؉ and Mrp3 ؊/؊ knockout mice received APAP (150 mg/kg), and bile was collected. Basolateral and canalicular excretion of APAP was also assessed in the isolated perfused liver. In separate studies, mice received 400 mg APAP/kg for assessment of hepatotoxicity. No differences were found in the biliary excretion of APAP, APAP-sulfate, and APAPglutathione between Mrp3 ؉/؉ and Mrp3 ؊/؊ mice. However, 20-fold higher accumulation of APAPglucuronide (APAP-GLUC) was found in the liver of Mrp3 ؊/؊ mice. Concomitantly, plasma APAP-GLUC content in Mrp3 ؊/؊ mice was less than 10% of that in Mrp3 ؉/؉ mice. In addition, APAP-GLUC excretion in bile of Mrp3 ؊/؊ mice was tenfold higher than in Mrp3 ؉/؉ mice. In the isolated perfused liver, we also found a strong decrease of APAP-GLUC secretion into the perfusate of Mrp3 ؊/؊ livers. Plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), and histopathology showed that Mrp3 ؊/؊ mice are more resistant to APAP hepatotoxicity than Mrp3 ؉/؉ mice, which is most likely a result of the faster repletion of hepatic GSH. Substrates exported from hepatocytes by MRP1-3 include conjugates of glutathione, glucuronide, and sulfate as well as organic amphiphilic anions. MRP3, which is expressed in liver and gastrointestinal tract, 2,3 prefers glucuronide over glutathione conjugates. 4,5 Mice lacking Mrp3 were recently developed; these mice are viable, fertile, and have no apparent phenotype. 2 Because of the potential role of Mrp3 in basolateral secretion of glucuronidated drugs, the disposition of acetaminophen (APAP) in Mrp3 Ϫ/Ϫ mice was investigated.Acetaminophen is a popular analgesic and antipyretic that can produce acute liver failure with excessive dosing. 6,7 This drug is metabolized in the liver by conjugation with glucuronic acid and sulfate, while a reactive metabolite generated by CYP450 is detoxified by conjugation Abbreviations: MRP, multidrug resistance protein; APAP, acetaminophen; GSH,

Mutations in ATP8B1 cause severe inherited liver disease. The disease is characterized by impaired biliary bile salt excretion (cholestasis), but the mechanism whereby impaired ATP8B1 function results in cholestasis is poorly understood. ATP8B1 is a type 4 P-type ATPase and is a flippase for phosphatidylserine. Atp8b1-deficient mice display a dramatic increase in the biliary extraction of cholesterol from the canalicular (apical) membrane of the hepatocyte. Here we studied the hypothesis that disproportionate cholesterol extraction from the canalicular membrane impairs the activity of the bile salt transporter, ABCB11, and as a consequence causes cholestasis. Using single pass liver perfusions, we show that not only ABCB11-mediated transport but also Abcc2-mediated transport were reduced at least 4-fold in Atp8b1 deficiency. We show that canalicular membranes of cholestatic Atp8b1-deficient mice have a dramatically reduced cholesterol to phospholipid ratio, i.e. 0.75 ؎ 0.24 versus 2.03 ؎ 0.71 for wild type. In vitro depletion of cholesterol from mouse liver plasma membranes using methyl-␤-cyclodextrin demonstrated a near linear relation between cholesterol content of the membranes and ATPdependent taurocholate transport. Abcc2-mediated transport activity was not affected up to 30% of membrane cholesterol depletion but declined to negligible levels at 70% of membrane cholesterol depletion. These effects were reversible as cholesterol repletion of the liver membranes completely restored Abcb11-and Abcc2-mediated transport. Our data demonstrate that membrane cholesterol content is a critical determinant of ABCB11/ABCC2 transport activity, provide an explanation for the etiology of ATP8B1 disease, and suggest a novel mechanism protecting the canalicular membrane against luminal bile salt overload.

Crigler-Najjar (CN) patients have no bilirubin UDP glucuronosyltransferase (UGT1A1) activity and suffer brain damage because of bilirubin toxicity. Vectors based on adeno-associated virus (AAV) serotype 2 transduce liver cells with relatively low efficiency. Recently, AAV serotypes 1, 6, and 8 have been shown to be more efficient for liver cell transduction. We compared AAV serotypes 1, 2, 6, and 8 for correction of UGT1A1 deficiency in the Gunn rat model of CN disease. Adult Gunn rats were injected with CMV-UGT1A1 AAV vectors. Serum bilirubin was decreased over the first year by 64% for AAV1, 16% for AAV2, 25% for AAV6, and 35% for AAV8. Antibodies to UGT1A1 were detected after injection of all AAV serotypes. An AAV1 UGT1A1 vector with the liver-specific albumin promoter corrected serum bilirubin levels but did not induce UGT1A1 antibodies. Two years after injection of AAV vectors all animals had large lipid deposits in the liver. These lipid deposits were not seen in age-matched control animals. AAV1 vectors are promising candidates for CN gene therapy because they can mediate a reduction in serum bilirubin levels in Gunn rats that would be therapeutic in humans.

Cholyl-L-lysyl-fluorescein (CLF) is a fluorescent bile salt derivative that is being developed as an agent for determining in vivo liver function. However, the mechanisms of uptake and excretion by hepatocytes have not been rigorously studied. We have directly assessed the transport capacity of various hepatobiliary transporters for CLF. Uptake experiments were performed in Chinese hamster ovary cells transfected with human NTCP, OATP1B1, OATP1B3, and OATP2B1. Conversely, excretory systems were tested with plasma membrane vesicles from Sf21 insect cells expressing human ABCB11, ABCC2, ABCC3, and ABCG2. In addition, plasma clearance and biliary excretion of CLF were examined in wild-type, Abcc2(Ϫ/Ϫ), and Abcc3(Ϫ/Ϫ) mice. Human Na ϩ -dependent taurocholic-cotransporting polypeptide (NTCP) and ATP-binding cassette B11 (ABCB11) were incapable of transporting CLF. In contrast, high-affinity transport of CLF was observed for organic anion-transporting polypeptide 1B3 (OATP1B3), ABCC2, and ABCC3 with K m values of 4.6 Ϯ 2.7, 3.3 Ϯ 2.0, and 3.7 Ϯ 1.0 M, respectively. In Abcc2(Ϫ/Ϫ) mice biliary excretion of CLF was strongly reduced compared with wild-type mice. This resulted in a much higher hepatic retention of CLF in Abcc2(Ϫ/Ϫ) versus wild-type mice: 64 versus 1% of the administered dose (2 h after administration). In mice intestinal uptake of CLF was negligible compared with that of taurocholate. Our conclusion is that human NTCP and ABCB11 are incapable of transporting CLF, whereas OATP1B3 and ABCC2/Abcc2 most likely mediate hepatic uptake and biliary excretion of CLF, respectively. CLF can be transported back into the blood by ABCC3. Enterohepatic circulation of CLF is minimal. This renders CLF suitable as an agent for assessing in vivo liver function.