The Na(+) -taurocholate cotransporting polypeptide knockout mouse: A new tool for study of bile acids and hepatitis B virus biology

The Na+-Taurocholate cotransporting polypeptide (NTCP; gene symbol SLC10A1) was cloned in 1991 and shown to function in a sodium-dependent fashion to efficiently take up all the major glycine and taurine-conjugated bile acids.1 NTCP only weakly transports unconjugated bile acids, and in subsequent years, members of the Organic Anion Transporting Polypeptide (OATP/SLCO) family of facilitative carriers such as OATP1B1 and OATP1B3 in humans and Oatp2b1 in mice were identified as the transporters responsible for hepatic sodium-independent uptake of bile acids, particularly the unconjugated species. NTCP’s properties clearly established it as the major candidate mechanism for hepatic bile acid uptake in multiple species. However definitive in vivo support was lacking until only recently when a pediatric patient was described with a loss-of-function mutation in NTCP and highly elevated serum levels of conjugated bile acids.2 Although the child currently exhibits no jaundice, pruritus, or other signs of liver disease, the finding also highlighted the gaps in our ability to predict the long-term physiologic and metabolic consequences of reduced hepatic bile acid clearance. Following closely on the heels of the first human case of an isolated NTCP defect and partially filling that void, Slijpcevic et al now report the first phenotypic characterization of an Slc10a1 knockout mouse.3 In brief, after confirming the absence of NTCP mRNA and protein in liver, the authors used freshly isolated hepatocytes in sandwich cultures to demonstrate that sodium-dependent taurocholate transport was abolished in the Slc10a1−/− mice. Given that observation, it was expected that the Slc10a1-deficient mice would exhibit markedly elevated serum bile acid levels (hypercholanemia) similar to the recently described NTCP-deficient patient. Surprisingly, serum bile acid levels were in the normal range (1 to 20 μM) for the majority (~70%) of the Slc10a1−/− mice, with the remaining subpopulation exhibiting dramatically elevated levels (> 1000 μM). Slc10a1−/− mice with intermediate levels of serum bile acids were not evident, and the heterogeneity was not explained by gender or by mouse strain genetic background. The remaining studies then proceeded to tell the tale of these two phenotypes. Overall, the Slc10a1−/− mice were grossly similar to their wild type littermates except for reduced body weights, a common observation for knockout mouse models with defects in bile acid biosynthetic enzymes or other transporters important for the enterohepatic circulation of bile acids. The reduced body weight was particularly evident for those mice with highest serum bile acid levels. There was no hepatomegaly or histological evidence of liver injury; serum markers of liver function were unremarkable in the normocholanemic mice, but elevated in hypercholanemic Slc10a1−/− mice. In the normocholanemic Slc10a1−/− mice, the clearance of intravenously administered taurocholate was clearly slowed compared to wild type mice, and the hepatic bile acid clearance defect could be unmasked by feeding a diet containing ursodeoxycholic acid. Bile acid synthesis and enterohepatic cycling appeared to be relatively unperturbed in the normocholanemic Slc10a1−/− mice, with no changes in bile flow, biliary bile acid secretion, or fecal bile acid excretion. In contrast, the hypercholanemic Slc10a1−/− mice exhibited a more substantial defect in taurocholate clearance, as well as decreased bile flow, biliary bile secretion, and fecal bile excretion, and increased urinary bile acid excretion. Overall, the results generally support the concept that NTCP/Slc10a1 is an important hepatic transporter for clearance of conjugated bile acids. However, the phenotypic heterogeneity observed for the Slc10a1−/− mice raises questions regarding alternate hepatic bile acid clearance mechanisms. The sodium-independent bile acid transporters that preferentially function in clearance of unconjugated bile acids may be sufficiently active to compensate and prevent the rise in serum conjugated bile acids in the majority of Slc10a1−/− mice. Indeed, changes were observed in hepatic mRNA and protein levels for several of the Oatps including Oatp1b2. But these changes did not strictly correlate with the presence or absence of hypercholanemia, and additional unidentified modifiers of the phenotype may be involved. Comparison of the Slc10a1−/− mice and index case for human NTCP-deficiency suggests the presence of compensatory mechanisms in mice that are not engaged in humans. This could include higher hepatocyte levels of Oatp1b2 (or another transporter such as other members of the Oatp family), or a greater proportion of the hepatocytes being involved in bile acid clearance (pericentral/zone III as well as periportal/zone I of the liver acinus). Additional studies such as analysis of knockout mice lacking both Slc10a1 and Slco1b2 (Oatp2b1) will be needed to explore those mechanisms. In addition, it is not known if the hypercholanemic phenotype described for NTCP-deficiency is fully penetrant in humans. Only a single NTCP-deficient patient with hypercholanemia has been described, even though non-functional NTCP variants have turned up in population-based sequencing studies.4 As such, the existence of NTCP-deficient normocholanemic human subjects cannot be excluded. In addition to being the bile acid uptake transporter, NTCP was identified as a cell surface receptor for the Hepatitis B virus (HBV) and Hepatitis Delta virus (HDV).5 HBV infection is initiated via low affinity binding of the HBV small surface protein to heparin-sulfate proteoglycans and attachment to the basolateral membrane of hepatocytes. In the next step high-affinity binding occurs between the HBV and the host receptor and allows for viral cellular entry. The identity of the HBV entry receptor(s) had long remained a mystery. An important breakthrough came recently when Yan et al demonstrated that binding of the HBV large surface protein pre-S1 domain myristoylated peptide (amino acids 2–48) to NTCP is essential for infection of human hepatocytes.5 Much of the work to characterize the HBV-NTCP interactions have been performed using the pre-S1 domain-derived myristoylated lipopeptide Myrcludex-B, and the Slc10a1−/− mice provided a new opportunity to further explore the in vivo role of Ntcp in HBV binding. Indeed, in vivo positron emission tomography imaging showed that after administration, radiolabeled Myrcludex B-derived peptides were enriched in liver of wild-type mice but essential absent from liver of the Slc10a1−/− mice. The results further support a key role for NTCP as the HBV entry receptor. It should be noted that mouse Ntcp binds the pre-S1 peptide but does not allow for HBV infections in vitro.6 Nor does introduction of human NTCP confer susceptibility to HBV infection for the mouse hepatoma cell lines.6 As such, the utility of mice to further dissect the role of NTCP in HBV entry is unclear. The dual functions of NCTP in bile acid transport and binding of HBV preS1 peptide raised the question of whether there is mutual interference. In vitro studies showed that binding of preS1 peptide inhibited taurocholate uptake by NTCP, whereas bile acids such as taurocholate blocked/inhibited HBV infection.6,7 In vivo findings with human liver chimeric mice further supported the concept that HBV binding to NTCP limits bile acid uptake and leads to compensatory changes in bile acid metabolism.8 The Slc10a1−/− model may also prove useful as a platform to further dissect the mechanisms responsible for the metabolic changes induced by HBV. Altogether, the first description of the Slc10a1-knockout mouse is supportive of a central role for NTCP in the hepatic uptake of conjugated bile acids and HBV preS1/Myrcludex B binding in vivo. This important work by Slijepcevic et al also provides a valuable new tool to further our understanding of the complex intersection between bile acids, HBV, and hepatic function.