Abstract
Although sterol carrier protein-2 (SCP-2) participates in the uptake and intracellular trafficking of cholesterol, its effect on "reverse cholesterol transport" has not been explored. As shown herein, SCP-2 expression inhibited high density lipoprotein (HDL)-mediated efflux of [(3)H]cholesterol and fluorescent 22-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3b-ol (NBD-cholesterol) up to 61 and 157%, respectively. Confocal microscopy of living cells allowed kinetic analysis of two intracellular pools of HDL-mediated NBD-cholesterol efflux: the highly fluorescent lipid droplet pool and the less fluorescent pool outside the lipid droplets, designated the cytoplasmic compartment. Both the whole cell and the cytoplasmic compartment exhibited two similar kinetic pools, the half-times of which were consistent with protein (t(b)(12) near 1 min) and vesicular (t(d)(12) = 10-20 min) mediated sterol transfer. Although SCP-2 expression did not alter cytoplasmic sterol pool sizes, the rapid t(b)(12) decreased 36%, while the slower t(d)(12) increased 113%. Lipid droplets also exhibited two kinetic pools of NBD-cholesterol efflux but with half-times over 200% shorter than those of the cytoplasmic compartment. The lipid droplet slower effluxing pool size and t(d)(12) were increased 48% and 115%, respectively, in SCP-2-expressing cells. Concomitantly, the level of the lipid droplet-specific adipose differentiation-related protein decreased 70%. Overall, HDL-mediated sterol efflux from L-cell fibroblasts reflected that of the cytoplasmic rather than lipid droplet compartment. SCP-2 differentially modulated sterol efflux from the two cytoplasmic pools. However, net efflux was determined primarily by inhibition of the slowly effluxing pool rather than by acceleration of the rapid protein-mediated pool. Finally, SCP-2 expression also inhibited sterol efflux from lipid droplets, an effect related to decreased adipose differentiation-related protein, a lipid droplet surface protein that binds cholesterol with high affinity.
Highlights
The high density lipoprotein (HDL)-mediated1 steps of cholesterol transfer from the cell surface membrane and subsequent fate of choles
The results presented provide fresh insights into the efflux process and for the first time establish new information demonstrating that sterol carrier protein-2 (SCP-2) expression: (i) inhibits HDL-mediated cholesterol efflux and (ii) inhibits cholesterol efflux from a little understood, subcellular compartment, i.e. lipid droplets
HDL (98 g HDL protein/ml) dramatically stimulated efflux of [3H]cholesterol from both mock transfected control (Fig. 1, filled triangles) and SCP-2-overexpressing cells (Fig. 1, open triangles), SCP-2 expression decreased the extent of HDL-mediated [3H]cholesterol efflux by 61% (p Ͻ 0.015, n ϭ 4)
Summary
To co-localize ADRP with Nile red or with NBD-cholesterol, SCP-2 expressors were plated on 8-well Lab-Tek chamber slides (Nunc, Naperville, IL) at subconfluency. The cells were washed with serum-free medium and incubated with 0.005% of either Nile red or NBD-cholesterol in serum-free medium for 30 min, at 37 °C. Data points were fitted to either a biexponential (y ϭ AeϪbt ϩ CeϪdt) or multiparameter (y ϭ (AeϪbT0 ϩ CeϪdT0)(eϪh(tϪT0)) exponential decay equation where y was the fraction of initial cellular NBD-cholesterol remaining in the cells at time t; A and C were the size of the cholesterol pools available for efflux; b, d, and h were the apparent rate constants; and T0 was the time at which depletion of the pools increased to completion. Values with p Ͻ 0.05 were considered statistically significant
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