Mathematical Analysis of Hepatic High Density Lipoprotein Transport Based on Quantitative Imaging Data

Abstract

Hepatocytes internalize high density lipoprotein (HDL) at the basolateral membrane. Most HDL is recycled while some is shuttled to the canalicular membrane by transcytosis. Here, transport of HDL was analyzed by mathematical modeling based on measurements in polarized hepatic HepG2 cells. Recycling of HDL from basolateral sorting endosomes was modeled by applying the rapid equilibrium approach. Analytical expressions were derived, which describe in one model the transport of HDL to the subapical compartment/apical recycling compartment, the biliary canaliculus (BC), and to late endosomes and lysosomes (LE/LYS). Apical endocytosis of HDL predicted by the model was confirmed for rhodamine-dextran and fluorescent asialoorosomucoid, markers for LE/LYS in living HepG2 cells. Budding of endocytic vesicles from the BC was directly observed by time lapse imaging of a fluorescent lipid probe. Based on fitted kinetic parameters and their covariance matrix a Monte Carlo simulation of HDL transport in hepatocytes was performed. The model was used to quantitatively assess release of HDL-associated free cholesterol by scavenger receptor BI. It is shown that only 6% of HDLassociated sterol reaches the BC as a constituent of the HDL particles, whereas the remaining sterol is rapidly released from HDL and shuttled to the BC by non-vesicular transport.Hepatic transport of high density lipoprotein (HDL) 1 and processing of its associated lipids including sterols is a key step during reverse cholesterol transport. Free cholesterol and cholesteryl esters (CE) are transferred from the HDL particle to the hepatocyte (1-3). Biliary secretion of cholesterol along with bile salts and phosphatidylcholine (PC) is the only pathway of sterol clearance from the body (4). Uptake of sterols from HDL in the hepatocyte is partially independent of the processing of the HDL protein moiety. This so-called selective uptake is mediated largely by the scavenger receptor BI (SR-BI) at the basolateral membrane of hepatic cells (3,5). It has been proposed that endocytosis and recycling of the whole HDL particle occurs in hepatocytes and contributes to selective CE uptake (6 -8). Recently, we demonstrated that the natural cholesterol analog dehydroergosterol (DHE) incorporated into fluorescent HDL is rapidly released from the lipoprotein at the basolateral membrane of polarized HepG2 cells (9). This process required SR-BI but was not dependent on metabolic energy in contrast to uptake of the HDL particles. DHE was rapidly shuttled to the apical canalicular membrane of HepG2 cells by ATP-independent non-vesicular transport (9, 10). The canalicular membrane of HepG2 cells forms apical closed vacuoles that resemble the biliary canaliculi (BC) of hepatocytes in many respects (11). We found evidence for internalization of DHE and fluorescent PC from the BC of HepG2 cells (10, 12). We demonstrated that fluorescent PC and DHE become delivered to a subapical compartment or apical recycling compartment (SAC/ ARC) in vesicles in 15-30 min. The identity of this SAC/A...

High Density Lipoprotein Endocytosis by Scavenger Receptor SR-BII Is Clathrin-dependent and Requires a Carboxyl-terminal Dileucine Motif

“…We interpret these results to suggest that localization of the receptor to the cell surface is optimal for selective uptake. SR-BI-dependent endocytosis of HDL has been reported by various groups (6,7,8,13), but increasing evidence points against a role for HDL endocytosis in selective uptake. As recently shown by Nieland et al (13), for example, blocking of SR-BI-dependent HDL endocytosis appeared not to inhibit selective uptake, and blocking of selective uptake by specific inhibitors did not affect endocytosis of HDL.…”

Hepatic SR-BI, not endothelial lipase, expression determines biliary cholesterol secretion in mice

“…Thus far studies tracing the cellular fate of HDL cholesterol taken up via SR-BI have largely focused on HDL-associated free sterols. These studies established that in polarized cells, uptake of free sterols via SR-BI and their respective transport to the bile canaliculus occurs in a rapid, nonvesicular, and largely energy-independent manner ( 42,43 ). However, the bulk of HDL cholesterol enters the cell via SR-BI as cholesteryl ester, and it has been acknowledged as a limitation to the above-mentioned studies that there might be fundamental The effect of EL overexpression on bile salt-independent biliary cholesterol secretion (chol/BA ratio) dependent on the hepatic SR-BI expression level.…”

“…These data indicate an important role for hepatocyte SR-BI in directing cholesterol toward biliary secretion and stress the relevance of SR-BI in RCT. differences in the intracellular routing of cholesteryl esters ( 42 ). Further research will be required to delineate these pathways.…”

Quantitative analysis of SR-BI-dependent HDL retroendocytosis in hepatocytes and fibroblasts

“…Mathematical modeling of the kinetic data derived from this study predicted a transport pathway whereby internalized HDL is targeted from the basolateral membrane to sorting endosomes from which rapid recycling occurs. Some HDL escaping this recycling pathway is transcytosed to bile canaliculi via the subapical compartment/apical recycling compartment or, alternatively, delivered to the late endosome/ lysosome for degradation (62). In another recently published study, it was estimated that z40% of 125 I-HDL 3 associated with HepG2 cells was resecreted during a 2 h, 378C chase (35).…”