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Effects of acceptor composition and mechanism of ABCG1-mediated cellular free cholesterol efflux.

Sankaranarayanan S, Oram JF, Asztalos BF, Vaughan AM, Lund-Katz S, Adorni MP, Phillips MC, Rothblat GH - J. Lipid Res. (2008)

Bottom Line: ABCG1 did not enhance influx of FC or cholesteryl oleyl ether (COE) when cells were incubated with radiolabeled HDL(3).ABCG1 expression did not increase the association of HDL(3) with cells.In conclusion, composition and particle size determine the acceptor efficiency for ABCG1-mediated efflux.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Children's Hospital of Philadelphia, Philadelphia, PA, USA.

ABSTRACT
Among the known mechanisms of reverse cholesterol transport (RCT), ATP binding cassette transporter G1 (ABCG1)-mediated free cholesterol (FC) transport is the most recent and least studied. Here, we have characterized the efficiencies of different acceptors using baby hamster kidney (BHK) cells transfected with human ABCG1 cDNA, which is inducible upon treatment with mifepristone. When normalized on particle number and particle surface area, the acceptor efficiency for FC efflux was as follows: small unilamellar vesicles (SUV)>LDL>reconstituted HDL>HDL(2) = HDL(3). Based on phospholipid content, the order was reversed. ABCG1 also mediated phospholipid efflux to human serum and HDL(3). ABCG1-mediated FC efflux correlated significantly with a number of HDL subfractions and components in serum collected from 25 normolipidemic individuals: apolipoprotein A-II (apoA-II) (r(2) = 0.7), apolipoprotein A-I (apoA-I) (r(2) = 0.5), HDL-C (r(2) = 0.4), HDL-PL (r(2) = 0.4), alpha-2 HDL (r(2) = 0.4), and prebeta HDL (r(2) = 0.2). ABCG1 did not enhance influx of FC or cholesteryl oleyl ether (COE) when cells were incubated with radiolabeled HDL(3). ABCG1 expression did not increase the association of HDL(3) with cells. Compared with control cells, ABCG1 expression significantly increased the FC pool available for efflux and the rate constant for efflux. In conclusion, composition and particle size determine the acceptor efficiency for ABCG1-mediated efflux. ABCG1 increases cell membrane FC pools and changes its rate of desorption into the aqueous phase without enhancing the association with the acceptor.

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ATP binding cassette GI (ABCG1)-mediated free cholesterol efflux to different acceptors. Baby hamster kidney (BHK) cells transfected with ABCG1 were radiolabeled with [3H] free cholesterol (FC) for 24 h and were treated with or without mifepristone for 18 h. The cells were incubated with various acceptors at the indicated concentrations for 4 h, and % of efflux was measured. The different acceptors were normalized based on their protein (A), phospholipid (B), particle number concentration calculated from particle compositions and molecular weights (C; (39, 56), and a function of particle size (D; (38). The ABCG1-mediated FC efflux data (i.e., the difference in efflux between mifepristone-treated and control cells) are fitted to the Michaelis-Menten equation. The efflux values are mean ± SD of triplicate measurements. Reconstituted HDL disc (rHDL disc), ♦; HDL3, ▴; HDL2, ▾; LDL, ▪; SUV, •.
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fig1: ATP binding cassette GI (ABCG1)-mediated free cholesterol efflux to different acceptors. Baby hamster kidney (BHK) cells transfected with ABCG1 were radiolabeled with [3H] free cholesterol (FC) for 24 h and were treated with or without mifepristone for 18 h. The cells were incubated with various acceptors at the indicated concentrations for 4 h, and % of efflux was measured. The different acceptors were normalized based on their protein (A), phospholipid (B), particle number concentration calculated from particle compositions and molecular weights (C; (39, 56), and a function of particle size (D; (38). The ABCG1-mediated FC efflux data (i.e., the difference in efflux between mifepristone-treated and control cells) are fitted to the Michaelis-Menten equation. The efflux values are mean ± SD of triplicate measurements. Reconstituted HDL disc (rHDL disc), ♦; HDL3, ▴; HDL2, ▾; LDL, ▪; SUV, •.

Mentions: Using the ABCG1-expressing BHK cell system (22, 23), we have tested a variety of acceptors for their efficiencies in mediating cell cholesterol efflux. The different acceptors included HDL2, HDL3, LDL, rHDL discs, and egg PC SUVs at a range of concentrations (Fig. 1). Because these acceptors differ in composition, efflux data are normalized on the basis of their protein content (Fig. 1A), phospholipid content (Fig. 1B), particle number concentration (Fig. 1C), and particle size (Fig. 1D). All of these particles, with the exception of lipid-free apoA-I (data not shown) act as cholesterol acceptors. The most efficient acceptor when normalized on the basis of protein was rHDL, and HDL2 and HDL3 were equally effective (Fig. 1A), while HDL3 proved to be the best acceptor when normalized on phospholipid concentration (Fig. 1B). The Km values for HDL3 and HDL2 were 47 ± 8 μg/ml and 52 ± 12 μg/ml, respectively, which were generally similar to the Km value for HDL (31 μg/ml) obtained by Vaughan and Oram (22). At a given protein or phospholipid concentration, the smaller acceptor particlesm such as rHDL disc, HDL3, and HDL2, were present in higher numbers and therefore gave higher ABCG1-mediated efflux compared with large particles such as LDL and SUV. However, at a similar particle number (Fig. 1C), and when particle size was taken into account (Fig. 1D), the larger SUV and LDL particles proved to be the most effective.


Effects of acceptor composition and mechanism of ABCG1-mediated cellular free cholesterol efflux.

Sankaranarayanan S, Oram JF, Asztalos BF, Vaughan AM, Lund-Katz S, Adorni MP, Phillips MC, Rothblat GH - J. Lipid Res. (2008)

ATP binding cassette GI (ABCG1)-mediated free cholesterol efflux to different acceptors. Baby hamster kidney (BHK) cells transfected with ABCG1 were radiolabeled with [3H] free cholesterol (FC) for 24 h and were treated with or without mifepristone for 18 h. The cells were incubated with various acceptors at the indicated concentrations for 4 h, and % of efflux was measured. The different acceptors were normalized based on their protein (A), phospholipid (B), particle number concentration calculated from particle compositions and molecular weights (C; (39, 56), and a function of particle size (D; (38). The ABCG1-mediated FC efflux data (i.e., the difference in efflux between mifepristone-treated and control cells) are fitted to the Michaelis-Menten equation. The efflux values are mean ± SD of triplicate measurements. Reconstituted HDL disc (rHDL disc), ♦; HDL3, ▴; HDL2, ▾; LDL, ▪; SUV, •.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2636919&req=5

fig1: ATP binding cassette GI (ABCG1)-mediated free cholesterol efflux to different acceptors. Baby hamster kidney (BHK) cells transfected with ABCG1 were radiolabeled with [3H] free cholesterol (FC) for 24 h and were treated with or without mifepristone for 18 h. The cells were incubated with various acceptors at the indicated concentrations for 4 h, and % of efflux was measured. The different acceptors were normalized based on their protein (A), phospholipid (B), particle number concentration calculated from particle compositions and molecular weights (C; (39, 56), and a function of particle size (D; (38). The ABCG1-mediated FC efflux data (i.e., the difference in efflux between mifepristone-treated and control cells) are fitted to the Michaelis-Menten equation. The efflux values are mean ± SD of triplicate measurements. Reconstituted HDL disc (rHDL disc), ♦; HDL3, ▴; HDL2, ▾; LDL, ▪; SUV, •.
Mentions: Using the ABCG1-expressing BHK cell system (22, 23), we have tested a variety of acceptors for their efficiencies in mediating cell cholesterol efflux. The different acceptors included HDL2, HDL3, LDL, rHDL discs, and egg PC SUVs at a range of concentrations (Fig. 1). Because these acceptors differ in composition, efflux data are normalized on the basis of their protein content (Fig. 1A), phospholipid content (Fig. 1B), particle number concentration (Fig. 1C), and particle size (Fig. 1D). All of these particles, with the exception of lipid-free apoA-I (data not shown) act as cholesterol acceptors. The most efficient acceptor when normalized on the basis of protein was rHDL, and HDL2 and HDL3 were equally effective (Fig. 1A), while HDL3 proved to be the best acceptor when normalized on phospholipid concentration (Fig. 1B). The Km values for HDL3 and HDL2 were 47 ± 8 μg/ml and 52 ± 12 μg/ml, respectively, which were generally similar to the Km value for HDL (31 μg/ml) obtained by Vaughan and Oram (22). At a given protein or phospholipid concentration, the smaller acceptor particlesm such as rHDL disc, HDL3, and HDL2, were present in higher numbers and therefore gave higher ABCG1-mediated efflux compared with large particles such as LDL and SUV. However, at a similar particle number (Fig. 1C), and when particle size was taken into account (Fig. 1D), the larger SUV and LDL particles proved to be the most effective.

Bottom Line: ABCG1 did not enhance influx of FC or cholesteryl oleyl ether (COE) when cells were incubated with radiolabeled HDL(3).ABCG1 expression did not increase the association of HDL(3) with cells.In conclusion, composition and particle size determine the acceptor efficiency for ABCG1-mediated efflux.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Children's Hospital of Philadelphia, Philadelphia, PA, USA.

ABSTRACT
Among the known mechanisms of reverse cholesterol transport (RCT), ATP binding cassette transporter G1 (ABCG1)-mediated free cholesterol (FC) transport is the most recent and least studied. Here, we have characterized the efficiencies of different acceptors using baby hamster kidney (BHK) cells transfected with human ABCG1 cDNA, which is inducible upon treatment with mifepristone. When normalized on particle number and particle surface area, the acceptor efficiency for FC efflux was as follows: small unilamellar vesicles (SUV)>LDL>reconstituted HDL>HDL(2) = HDL(3). Based on phospholipid content, the order was reversed. ABCG1 also mediated phospholipid efflux to human serum and HDL(3). ABCG1-mediated FC efflux correlated significantly with a number of HDL subfractions and components in serum collected from 25 normolipidemic individuals: apolipoprotein A-II (apoA-II) (r(2) = 0.7), apolipoprotein A-I (apoA-I) (r(2) = 0.5), HDL-C (r(2) = 0.4), HDL-PL (r(2) = 0.4), alpha-2 HDL (r(2) = 0.4), and prebeta HDL (r(2) = 0.2). ABCG1 did not enhance influx of FC or cholesteryl oleyl ether (COE) when cells were incubated with radiolabeled HDL(3). ABCG1 expression did not increase the association of HDL(3) with cells. Compared with control cells, ABCG1 expression significantly increased the FC pool available for efflux and the rate constant for efflux. In conclusion, composition and particle size determine the acceptor efficiency for ABCG1-mediated efflux. ABCG1 increases cell membrane FC pools and changes its rate of desorption into the aqueous phase without enhancing the association with the acceptor.

Show MeSH
Related in: MedlinePlus