Hepatic SR-BI-mediated cholesteryl ester selective uptake occurs with unaltered efficiency in the absence of cellular energy

Abstract

Scavenger receptor class B type I (SR-BI) plays a critical role in the delivery of HDL cholesterol and cholesteryl esters (CEs) to liver and steroidogenic tissues by a selective process that does not result in significant degradation of HDL protein. Recently, SR-BI-mediated endocytosis and recycling of HDL have been demonstrated. However, it remains unclear whether efficient SR-BI-mediated selective uptake occurs strictly at the plasma membrane or at additional sites along its endocytic itinerary. To examine the requirement for SR-BI endocytosis in HDL selective uptake, we determined the effects of energy depletion on the levels of cell-associated HDL protein and CE in primary mouse hepatocytes. Compared with CHO cells, we observed a much larger energy-dependent effect on CE uptake in primary mouse hepatocytes. Although varying the levels of caveolin-1 and carboxyl ester lipase altered the efficiency of selective uptake, neither was able to account for the energydependent component of HDL-CE uptake. Finally, we demonstrate that the hepatocyte-specific, energy-dependent effects on HDL-apolipoprotein A-I and -CE uptake are independent of SR-BI and are not required to achieve efficient SR-BI-mediated selective uptake of CE. Together, these data support the conclusion that neither the intracellular trafficking of HDL nor any energy-dependent cellular process affects the ability of the cell to maximally acquire CE through SR-BI-mediated selective uptake from HDL.-Harder, C. J., G. HDL has a functional role in the protection against atherosclerosis, and its plasma concentration is inversely correlated with the risk of cardiovascular disease. One of the protective actions of HDL involves cholesterol removal from peripheral cells for transport to the liver for biliary secretion (reviewed in 1). This process, termed ''reverse cholesterol transport,'' is mediated, in part, by the well-established HDL receptor, scavenger receptor class B type I (SR-BI) (2). In contrast to the holoparticle uptake of the LDL pathway (3), SR-BI mediates cholesterol, cholesteryl ester (CE), and phospholipid uptake via a pathway that does not involve significant degradation of the HDL particle, a process known as ''selective uptake.'' This process was originally described to consist of two phases. First, the lipoprotein binds to SR-BI on the cell surface, and second, the lipids in the lipoprotein are transferred to a membrane (reviewed in 1).Despite intense interest in SR-BI, the cellular mechanism by which SR-BI contributes to the selective uptake and intracellular trafficking of HDL-derived CE is still not fully understood. Early kinetic studies demonstrated that the rate of CE transfer is proportional to the amount of CE in HDL, suggesting that SR-BI forms a hydrophobic channel that facilitates the movement of sterol down a concentration gradient into the plasma membrane (4). Experiments involving reconstitution of SR-BI into liposomes showed that SR-BI mediates CE selective uptake from HDL into multilamellar vesicles independent of...

Scavenger Receptor Class B Type I Mediates Biliary Cholesterol Secretion Independent of ATP-Binding Cassette Transporter g5/g8 in Mice†

“…Although some in vitro studies showed that SR-BI-mediated selective uptake does not require endocytosis 30,31 and SR-BI is able to mediate selective uptake into phospholipid vesicles in the absence of a cellular environment, 32 other data suggested that SR-BI is an endocytic receptor. 33 Vesicular trafficking of SR-BI together with HDL through the early endosome system to subapical compartments, where cholesteryl ester hydrolysis likely takes place, has been demonstrated.…”

Scavenger receptor class B type I localizes to a late endosomal compartment

“…Although it is established that SR-BI can effectively mediate selective uptake without the concomitant internalization of the HDL particle, there is evidence that a fraction of HDL is internalized together with SR-BI (13,14,16). The functional significance of HDL internalization is not understood.…”

“…Cells were cultured in DMEM supplemented with 10% (v/v) fetal calf serum, 100 U/ml penicillin, 100 mg/ml streptomycin, and 2 mM L-glutamine. Primary hepatocytes were isolated from retired breeders of the C57BL6 (wild-type) mouse strain purchased from Charles River (Wilmington, MA) and SR-BI 2/2 mice (18) as previously described (16). Other reagents and suppliers included: filipin (Sigma Mississauga, ON, Canada), human plasma HDL (Calbiochem, San Diego, CA), anti-LAMP-2 and anti-EEA1 Abs (Transduction Laboratories, Mississauga, ON, Canada), anti-SR-BI Ab (NB400-101) (Novus Biologicals, Little-ton, CO), anti-Rab7 Ab (Sigma), and Lipofectamine 2000 (Invitrogen, Burlington, ON, Canada).…”

“…These data suggest that in addition to its well-described role in selective uptake at the plasma membrane, a pool of SR-BI that resides in intracellular structures may have an independent role in intracellular lipid movement. The functional significance of SR-BI trafficking is not known; however, we and others have reported that endocytosis and recycling of SR-BI occur in but are not required for efficient selective uptake (16,17).…”

Phosphatidylinositol-3-Kinase Regulates Scavenger Receptor Class B Type I Subcellular Localization and Selective Lipid Uptake in Hepatocytes

“…8 Other studies, however, have provided strong evidence that selective lipid uptake does not depend on HDL internalization by SR-BI and that the bulk of selective lipid uptake occurs at the plasma membrane. 11,12 The importance of SR-BI in the regulation of HDL levels, as well as in biliary cholesterol secretion, has been well demonstrated in studies of SR-BI genetically altered mice. 3,13 In addition to functioning in the liver as an HDL receptor, SR-BI also contributes to the metabolism of chylomicron remnants.…”