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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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Kinetics of FC efflux from control and ABCG1-upregulated cells to HDL3. After radiolabeling for 24 h with [3H] FC (15 μCi/ml), BHK cells were treated with or without mifepristone for 18 h. The cells were then incubated with HDL3 (100 μg/ml) for 8 h and the medium was collected and radioactivity measured at intervals over this period. The efflux curves for untreated (control) and mifepristone-treated cells were fitted to a mono exponential decay equation as described in Materials and Methods (r2 = 0.997). The data points are mean ± SEM (n = 6) and the error bars are contained within the symbols. Untreated, □; mifepristone, ▴.
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fig8: Kinetics of FC efflux from control and ABCG1-upregulated cells to HDL3. After radiolabeling for 24 h with [3H] FC (15 μCi/ml), BHK cells were treated with or without mifepristone for 18 h. The cells were then incubated with HDL3 (100 μg/ml) for 8 h and the medium was collected and radioactivity measured at intervals over this period. The efflux curves for untreated (control) and mifepristone-treated cells were fitted to a mono exponential decay equation as described in Materials and Methods (r2 = 0.997). The data points are mean ± SEM (n = 6) and the error bars are contained within the symbols. Untreated, □; mifepristone, ▴.

Mentions: We further analyzed the ABCG1-mediated efflux reaction by monitoring the kinetics of FC efflux to HDL3 (Fig. 8). The FC efflux time courses from control and mifepristone-treated cells fitted single-phase exponential decay curves that yielded values for the efflux rate constant (ke) and pool size. The fitting of the efflux values were consistent with one kinetic pool of FC being available for efflux, and the expression of ABCG1 induced a statistically significant (P = 0.01) increase in the size of this FC pool available for efflux from 22.0 ± 1.3% to 28.1 ± 1.4%. The ke for control cells was 0.018 ± 0.001 h−1 (t1/2 = 38 h), while that for mifepristone-treated cells was 0.025 ± 0.002 h−1 (t1/2 = 28 h) (mean ± SEM, n = 6); these values are significantly different from one another (P = 0.01). Thus, the ke and pool size for FC efflux were increased upon expression of 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)

Kinetics of FC efflux from control and ABCG1-upregulated cells to HDL3. After radiolabeling for 24 h with [3H] FC (15 μCi/ml), BHK cells were treated with or without mifepristone for 18 h. The cells were then incubated with HDL3 (100 μg/ml) for 8 h and the medium was collected and radioactivity measured at intervals over this period. The efflux curves for untreated (control) and mifepristone-treated cells were fitted to a mono exponential decay equation as described in Materials and Methods (r2 = 0.997). The data points are mean ± SEM (n = 6) and the error bars are contained within the symbols. Untreated, □; mifepristone, ▴.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig8: Kinetics of FC efflux from control and ABCG1-upregulated cells to HDL3. After radiolabeling for 24 h with [3H] FC (15 μCi/ml), BHK cells were treated with or without mifepristone for 18 h. The cells were then incubated with HDL3 (100 μg/ml) for 8 h and the medium was collected and radioactivity measured at intervals over this period. The efflux curves for untreated (control) and mifepristone-treated cells were fitted to a mono exponential decay equation as described in Materials and Methods (r2 = 0.997). The data points are mean ± SEM (n = 6) and the error bars are contained within the symbols. Untreated, □; mifepristone, ▴.
Mentions: We further analyzed the ABCG1-mediated efflux reaction by monitoring the kinetics of FC efflux to HDL3 (Fig. 8). The FC efflux time courses from control and mifepristone-treated cells fitted single-phase exponential decay curves that yielded values for the efflux rate constant (ke) and pool size. The fitting of the efflux values were consistent with one kinetic pool of FC being available for efflux, and the expression of ABCG1 induced a statistically significant (P = 0.01) increase in the size of this FC pool available for efflux from 22.0 ± 1.3% to 28.1 ± 1.4%. The ke for control cells was 0.018 ± 0.001 h−1 (t1/2 = 38 h), while that for mifepristone-treated cells was 0.025 ± 0.002 h−1 (t1/2 = 28 h) (mean ± SEM, n = 6); these values are significantly different from one another (P = 0.01). Thus, the ke and pool size for FC efflux were increased upon expression of 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