Abstract
An important mechanism contributing to cell cholesterol efflux is aqueous transfer in which cholesterol diffuses from cells into the aqueous phase and becomes incorporated into an acceptor particle. Some compounds can enhance diffusion by acting as shuttles transferring cholesterol to cholesterol acceptors, which act as cholesterol sinks. We have examined whether particles in serum can enhance cholesterol efflux by acting as shuttles. This task was accomplished by incubating radiolabeled J774 cells with increasing concentrations of lipoprotein-depleted sera (LPDS) or components present in serum as shuttles and a constant amount of LDL, small unilamellar vesicles, or red blood cells (RBC) as sinks. Synergistic efflux was measured as the difference in fractional efflux in excess of that predicted by the addition of the individual efflux values of sink and shuttle alone. Synergistic efflux was obtained when LPDS was incubated with cells and LDL. When different components of LPDS were used as shuttles, albumin produced synergistic efflux, while apoA-I did not. A synergistic effect was also obtained when RBC was used as the sink and albumin as shuttle. The previously observed negative association of albumin with coronary artery disease might be linked to reduced cholesterol shuttling that would occur when serum albumin levels are low.
Highlights
An important mechanism contributing to cell cholesterol efflux is aqueous transfer in which cholesterol diffuses from cells into the aqueous phase and becomes incorporated into an acceptor particle
The earliest cholesterol flux mechanism that was identified was aqueous transfer, in which there is a desorption of free cholesterol (FC) molecules from a donor, such as a cell membrane, into the aqueous phase followed by diffusion of the molecules and their subsequent collision with and incorporation into an acceptor particle [4]
The earliest mechanism demonstrated to play a role in the movement of FC molecules between cells and lipoproteins was termed aqueous diffusion
Summary
Materials Tissue culture plasticware was purchased from Thermo Fisher (Rochester, NY) and Corning (Corning, NY). After labeling plus or minus cholesterol enrichment, the cells were washed 2× with MEM HEPES and equilibrated with RPMI medium containing 0.2% BSA and cAMP (0.3 mmol/l) for 18 h After this equilibration period, the cells were washed 2× with MEM-HEPES buffer and incubated with MEM-HEPES media containing exogenous acceptors alone or in combination (shuttle and sink) for 4 h, or as indicated in the figures, to measure the labeled FC efflux. Protein and cholesterol mass determination At the end of the efflux experiment, the cell monolayers were washed with DPBS, and cell lipid was extracted using 0.5 ml of 2-propanol containing 5 μg/ml of cholesteryl methyl ether (CME; Sigma, St. Louis, MO) as an internal standard for gas liquid chromatography (GLC) analysis.
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