ATP11C targets basolateral bile salt transporter proteins in mouse central hepatocytes

Abstract

ATP11C is a homolog of ATP8B1, both of which catalyze the transport of phospholipids in biological membranes. Mutations in ATP8B1 cause progressive familial intrahepatic cholestasis type1 in humans, which is characterized by a canalicular cholestasis. Mice deficient in ATP11C are characterized by a conjugated hyperbilirubinemia and an unconjugated hypercholanemia. Here, we have studied the hypothesis that ATP11C deficiency interferes with basolateral uptake of unconjugated bile salts, a process mediated by organic anion-transporting polypeptide (OATP) 1B2. ATP11C localized to the basolateral membrane of central hepatocytes in the liver lobule of control mice. In ATP11C-deficient mice, plasma total bilirubin levels were 6-fold increased, compared to control, of which ~65% was conjugated and ~35% unconjugated. Plasma total bile salts were 10-fold increased and were mostly present as unconjugated species. Functional studies in ATP11C-deficient mice indicated that hepatic uptake of unconjugated bile salts was strongly impaired whereas uptake of conjugated bile salts was unaffected. Western blotting and immunofluorescence analysis demonstrated near absence of basolateral bile salt uptake transporters OATP1B2, OATP1A1, OATP1A4, and Na+-taurocholate-cotransporting polypeptide only in central hepatocytes of ATP11C-deficient liver. In vivo application of the proteasome inhibitor, bortezomib, partially restored expression of these proteins, but not their localization. Furthermore, we observed post-translational down-regulation of ATP11C protein in livers from cholestatic mice, which coincided with reduced OATP1B2 levels. Conclusions ATP11C is essential for basolateral membrane localization of multiple bile salt transport proteins in central hepatocytes and may act as a gatekeeper to prevent hepatic bile salt overload. Conjugated hyperbilirubinemia and unconjugated hypercholanemia and loss of OATP expression in ATP11C-deficient liver strongly resemble the characteristics of Rotor syndrome, suggesting that mutations in ATP11C can predispose to Rotor syndrome.

Blocking Sodium‐Taurocholate Cotransporting Polypeptide Stimulates Biliary Cholesterol and Phospholipid Secretion in Mice

“…Atp11c ‐deficient mice are a model with a zonation difference in bile salt uptake given that basolateral bile salt uptake transporters are absent in the pericentral region restricting bile salt uptake to periportal regions, where NTCP expression is maintained in the periportal hepatocyte (Fig. E) . In this experiment, the relation between biliary cholesterol and bile salt levels was not significantly different between Atp11c‐ deficient mice and WT littermates under conditions where the periportal region predominates uptake (before TC infusion).…”

“…To assess whether altered zonated hepatic bile salt uptake can contribute to an increase in biliary lipid secretion, we analyzed the relation between biliary cholesterol and bile salt secretion in two models with zonation‐related phenotypes. First, we reassessed a TC‐infusion experiment, which was previously performed in WT and Atp11c ‐deficient mice . Atp11c ‐deficient mice are a model with a zonation difference in bile salt uptake given that basolateral bile salt uptake transporters are absent in the pericentral region restricting bile salt uptake to periportal regions, where NTCP expression is maintained in the periportal hepatocyte (Fig.…”

“…First, we reassessed a TC-infusion experiment, which was previously performed in WT and Atp11c-deficient mice. (12) Atp11cdeficient mice are a model with a zonation difference in bile salt uptake given that basolateral bile salt uptake transporters are absent in the pericentral region restricting bile salt uptake to periportal regions, where NTCP expression is maintained in the periportal hepatocyte ( Fig. 4E).…”

“…Bile was collected in aliquots every 10 minutes for a total of 60 minutes ( Sr‐bI −/− mice, WT C57BL/6J mice, and Abcg8 −/− mice). Taurocholate (TC) infusion and bile collection in Atp11c ‐deficient mice was performed as described . Bile flow was determined gravimetrically assuming a density of 1 g/mL for bile.…”

“…Taurocholate (TC) infusion and bile collection in Atp11c-deficient mice was performed as described. (12) Bile flow was determined gravimetrically assuming a density of 1 g/mL for bile. A heating pad maintained body temperature at 37°C.…”