Lipid composition of hepatocyte canalicular membrane : relevance to bile formation

The work presented in this thesis aims to investigate the role of the hepatocyte canalicular membrane lipid composition in the molecular mechanism of bile formation. Coordinate hepatic secretion of bile salts, phospholipids and cholesterol into bile forms the basis for elimination of excess cholesterol and a wide variety of xenobiotics from the body. As the canalicular liver plasma membrane (cLPM) is the primary site for bile formation, biliary lipid secretion ultimately will be dictated by the physical properties of the cLPM and the component lipids in the membrane. Phosphatidylcholine (PC) and sphingomyelin (SM) are the major phospholipids on the outer leaflet of the cLPM (facing the bile canaliculus). However, under the influence of bile salts, PC is preferentially secreted into bile (>95% of total biliary phospholipids), whereas SM is nearly absent. To understand the physico-chemical basis for preferential biliary PC secretion, we studied the interactions of the bile salt taurocholate (TC) with different natural PCs and SMs in various in vitro model systems. Different structural arrangements of PC/TC micelles compared to SM/TC micelles were observed. In presence of cholesterol, PC preferentially distributed in micelles (mimicking lipid structures present in bile) and SM in vesicles (mimicking lipid membranes). Since SM has a high affinity for cholesterol, we have defined the SM molecular species present in cLPM and hepatic bile of rats. Bile was highly enriched with more hydrophilic SM species as compared to cLPM, suggesting that more hydrophilic species in bile-destined lipid microdomains are laterally separated from more hydrophobic species in microdomains resistant to solubilization into bile. We further investigated the role of cLPM lipid composition in biliary lipid secretion using a diosgenin-fed rat model. Diosgenin induced hypersecretion of biliary cholesterol without influencing the phospholipid and bile salt secretion. Hepatic bile was significantly enriched with more hydrophilic PC species as compared to canalicular PCs. However, no other differences in bile or membrane lipid composition and species were observed between control- and diosgenin-fed rats, suggesting that biliary cholesterol hypersecretion in diosgenin-fed rats was independent of the cLPM lipid composition. An alternative, possibly protein-mediated, mechanism might be responsible for biliary cholesterol hypersecretion. Since prolonged diosgenin feeding induced significantly higher biliary cholesterol outputs in mice as compared to rats, this model appeared more suitable to identify specific genes that are associated with biliary cholesterol hypersecretion. Cholesterol contents of liver, blood and plasma membrane subfractions were unchanged, but hepatic expression levels of the genes encoding HMG-CoA reductase, Oatp2 and the transcription factor Egr-1 were significantly increased and levels for Abcg8 were decreased. Levels of other known genes involved in cholesterol homeostasis remained unaltered by diosgenin feeding. This suggests that during biliary cholesterol hypersecretion the increased loss of cholesterol into bile is compensated for by increased de novo synthesis, without significant contribution of other known genes associated with cholesterol homeostasis. However, since biliary cholesterol secretion does not seem to be regulated by the cLPM lipid composition, it could be possible that as yet unidentified proteins are involved in regulation of biliary cholesterol secretion