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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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Influence of ABCG1 expression on HDL3 association with cells. BHK cells were treated with or without mifepristone for 18 h after which they were incubated with increasing concentrations of HDL3 labeled with [125I] for 2 h at 37°C. The cell monolayers were solubilized in 0.1 M NaOH, after being washed with MEM-HEPES containing 1% BSA (3×) followed by washing with PBS (2×). Radioactivity and protein concentrations were then measured. ABCG1-specific association with [125I] HDL3 was calculated by subtracting the values for untreated from mifepristone-treated cells. The values are mean ± SD of triplicate measurements. Data were fitted by linear regression (P ≤ 0.05). Untreated, □; mifepristone, ▴; ABCG1-specific, •.
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fig7: Influence of ABCG1 expression on HDL3 association with cells. BHK cells were treated with or without mifepristone for 18 h after which they were incubated with increasing concentrations of HDL3 labeled with [125I] for 2 h at 37°C. The cell monolayers were solubilized in 0.1 M NaOH, after being washed with MEM-HEPES containing 1% BSA (3×) followed by washing with PBS (2×). Radioactivity and protein concentrations were then measured. ABCG1-specific association with [125I] HDL3 was calculated by subtracting the values for untreated from mifepristone-treated cells. The values are mean ± SD of triplicate measurements. Data were fitted by linear regression (P ≤ 0.05). Untreated, □; mifepristone, ▴; ABCG1-specific, •.

Mentions: The mechanism of ABCG1-mediated FC and phospholipid efflux to HDL is unknown. To explore the role of HDL association to the transporter, BHK control and upregulated cells were incubated at 37°C for 2 h with increasing concentrations of HDL3 labeled with [125I]. As shown in Fig. 7, the amount of cell-associated [125I] HDL radioactivity was the same for control and mifepristone-treated cells. Thus, the upregulation of ABCG1 had no impact on HDL-cell association, consistent with a lack of specific binding of HDL to ABCG1.


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)

Influence of ABCG1 expression on HDL3 association with cells. BHK cells were treated with or without mifepristone for 18 h after which they were incubated with increasing concentrations of HDL3 labeled with [125I] for 2 h at 37°C. The cell monolayers were solubilized in 0.1 M NaOH, after being washed with MEM-HEPES containing 1% BSA (3×) followed by washing with PBS (2×). Radioactivity and protein concentrations were then measured. ABCG1-specific association with [125I] HDL3 was calculated by subtracting the values for untreated from mifepristone-treated cells. The values are mean ± SD of triplicate measurements. Data were fitted by linear regression (P ≤ 0.05). Untreated, □; mifepristone, ▴; ABCG1-specific, •.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig7: Influence of ABCG1 expression on HDL3 association with cells. BHK cells were treated with or without mifepristone for 18 h after which they were incubated with increasing concentrations of HDL3 labeled with [125I] for 2 h at 37°C. The cell monolayers were solubilized in 0.1 M NaOH, after being washed with MEM-HEPES containing 1% BSA (3×) followed by washing with PBS (2×). Radioactivity and protein concentrations were then measured. ABCG1-specific association with [125I] HDL3 was calculated by subtracting the values for untreated from mifepristone-treated cells. The values are mean ± SD of triplicate measurements. Data were fitted by linear regression (P ≤ 0.05). Untreated, □; mifepristone, ▴; ABCG1-specific, •.
Mentions: The mechanism of ABCG1-mediated FC and phospholipid efflux to HDL is unknown. To explore the role of HDL association to the transporter, BHK control and upregulated cells were incubated at 37°C for 2 h with increasing concentrations of HDL3 labeled with [125I]. As shown in Fig. 7, the amount of cell-associated [125I] HDL radioactivity was the same for control and mifepristone-treated cells. Thus, the upregulation of ABCG1 had no impact on HDL-cell association, consistent with a lack of specific binding of HDL to ABCG1.

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