The Na⁺-taurocholate cotransporting polypeptide (NTCP, encoded by SLC10A1) is the major hepatic uptake transporter of conjugated bile salts in basolateral membrane of hepatocytes. In addition to being an important component of enterohepatic circulation of bile acids, NTCP also serves as a functional receptor of hepatitis B virus (HBV) and hepatitis D virus (HDV). Bile acids are important physiological agents for intestinal nutrient absorption and also are signaling molecules in regualting hepatic lipid, glucose, and energy homeostasis. However, the consequence of NTCP dysfunction and potential use of NTCP inhibitor in metabolic diseases largely remain unexplored. Here, we studied the pathophysiological changes of NTCP dysfuction in mice and humans and we also developed a new NTCP inhibitor and evaluated its potentials in mice models with metabolic diseases.

We combined multidisciplinary approaches from molecular biology, biochemistry, and pathology to RNA-Seq, CyTOF and high-end UPLC-MS/MS analysis and we studied NTCP knockout (Slc10a1^-/-) mice as well as samples from more than 30 individuals with NTCP p.Ser267Phe (NTCP S267F) loss-of-function variant. Our experimental results demonstrated consequences of NTCP dysfunction, including lifelong changes of total bile acids (TBA) level, reduced risk of liver cancer and increased risk of developing gallbladder abnormalities. A new developed NTCP inhibitor also showed some potentials in metabolic diseases.

We first characterized the spatiotemporal expression of NTCP during liver development in mice using a newly developed monoclonal NTCP antibody. Membrane expression of NTCP was detectable as early as embryonic day 19.5. In adult mice, heterogeneous expression pattern of NTCP was also observed in hepatocytes which had higher levels of membranous NTCP expression through the sinosoids around the portal vein to a lower level in hepatocytes around the central vein in adulthood liver. NTCP deficiency in mice and human constantly resulted in hypercholanemia. However, we found that TBA levels tended to decrease gradually with age increasing in both Slc10a1^-/- mice and NTCP S267F individuals. And enhanced bile acids sulfation and excretion are likely to be responsible for the re-normalization of TBA level as age increasing under NTCP deficiency.

We reported that Slc10a1^-/- mice with long-term hypercholanemia do not develop spontaneous hepatocellular carcinoma (HCC). Using a diethylnitrosamine (DEN)-induced mouse model of HCC, we found that Slc10a1^-/- mice exhibited lower risk of developing HCC and fewer liver tumors, Interestingly, a transgenic mouse constitutively overexpressing human NTCP in hepatocytes (hNTCP-Tg) also showed significantly inhibition of HCC development. Slc10a1^-/- mice with hypercholanemia had increased number of Kupffer cells in liver. Under hypercholanemia, conjugated bile acids can induce the expression of heme oxygenase-1 (HO-1) in hepatic macrophages.

Guided by the finding of gallbladder abnormalities in Slc10a1^-/- mic, we found that NTCP S267F individuals also had increased risk of developing gallbladder abnormalities, such as gallstones in gallbladder and gallbladder polyps at different ages.

  To evaluate the (possible) metabolic benefits of pharmacologically targeting NTCP, we developed a novel non-immunosuppressive derivative of Cyclosporin A, CsA055, as a NTCP inhibitor. Treatment with CsA055 significantly increased TBA level in mice. And the bile acid profiles in these mice also mimiced that in Slc10a1^-/- mice. CsA055 lowerd the serum cholesterol levels in hypercholesterolemic Apoe^?/? mice either fed with chow diet or high-fat diet. For the diabetic ob/ob mice, CsA055 averted the glucose intolerance and partially detered the liver steatosis. Oral CsA055 treatment also ameliorated hepatic inflammation and liver damage in methionine-choline-deficient (MCD) diet induced mouse model of nonalcoholic steatohepatitis (NASH).

  In conclusion, these studies shed new lights on NTCP deficiency. Meanwhile, our results also indicated that NTCP is a druggable target that has potential to be used for developing therapies against HBV/HDV infection and metabolic-related diseases.