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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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Correlation of ABCG1-mediated free cholesterol efflux to HDL components in 25 individual human sera. BHK cells expressing ABCG1 were incubated for 4 h with the polyethylene glycol (PEG) supernatants (3.5%) of serum samples obtained from 25 healthy individuals, and the FC efflux was measured. The HDL subfractions of the 25 PEG supernatants were determined using a 2D gel assay (see Materials and Methods). The correlations between the ABCG1-mediated efflux and HDL components were assessed using linear regression. ApoA-I, ▪ (A); apoA-II, • (B); HDL-C, ▴ (C); HDL-PL, ▾ (D); α-2, ♦ (E); preβ, asterisk (F).
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fig5: Correlation of ABCG1-mediated free cholesterol efflux to HDL components in 25 individual human sera. BHK cells expressing ABCG1 were incubated for 4 h with the polyethylene glycol (PEG) supernatants (3.5%) of serum samples obtained from 25 healthy individuals, and the FC efflux was measured. The HDL subfractions of the 25 PEG supernatants were determined using a 2D gel assay (see Materials and Methods). The correlations between the ABCG1-mediated efflux and HDL components were assessed using linear regression. ApoA-I, ▪ (A); apoA-II, • (B); HDL-C, ▴ (C); HDL-PL, ▾ (D); α-2, ♦ (E); preβ, asterisk (F).

Mentions: Although ABCG1 mediates FC efflux to all phospholipid-containing acceptors, HDL is thought to be the main acceptor for ABCG1-mediated efflux (14, 16, 22). We further examined what subfractions and components of HDL correlate with the ABCG1-mediated efflux by using PEG supernatants of 25 individual human sera collected from clinically healthy, normo-lipidemic subjects. Control and ABCG1-upregulated cells were incubated with the PEG supernatants for 4 h, and the ABCG1-mediated efflux was measured. We quantified the HDL components such as cholesterol, phospholipid, and apolipoproteins; and using 2D gel analysis, we characterized and measured the HDL subfractions present in the individual PEG samples. The ABCG1-mediated efflux correlated with the parameters shown in Fig. 5. All HDL components (apoA-I, apoA-II, phospholipids, and cholesterol) were significantly correlated to ABCG1 efflux. Among the HDL subfractions, the α-2 HDL subfraction demonstrated a significant correlation (Fig. 5E). A modest but significant correlation was also observed between ABCG1-mediated efflux and preβ HDL (Fig. 5F).


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)

Correlation of ABCG1-mediated free cholesterol efflux to HDL components in 25 individual human sera. BHK cells expressing ABCG1 were incubated for 4 h with the polyethylene glycol (PEG) supernatants (3.5%) of serum samples obtained from 25 healthy individuals, and the FC efflux was measured. The HDL subfractions of the 25 PEG supernatants were determined using a 2D gel assay (see Materials and Methods). The correlations between the ABCG1-mediated efflux and HDL components were assessed using linear regression. ApoA-I, ▪ (A); apoA-II, • (B); HDL-C, ▴ (C); HDL-PL, ▾ (D); α-2, ♦ (E); preβ, asterisk (F).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5: Correlation of ABCG1-mediated free cholesterol efflux to HDL components in 25 individual human sera. BHK cells expressing ABCG1 were incubated for 4 h with the polyethylene glycol (PEG) supernatants (3.5%) of serum samples obtained from 25 healthy individuals, and the FC efflux was measured. The HDL subfractions of the 25 PEG supernatants were determined using a 2D gel assay (see Materials and Methods). The correlations between the ABCG1-mediated efflux and HDL components were assessed using linear regression. ApoA-I, ▪ (A); apoA-II, • (B); HDL-C, ▴ (C); HDL-PL, ▾ (D); α-2, ♦ (E); preβ, asterisk (F).
Mentions: Although ABCG1 mediates FC efflux to all phospholipid-containing acceptors, HDL is thought to be the main acceptor for ABCG1-mediated efflux (14, 16, 22). We further examined what subfractions and components of HDL correlate with the ABCG1-mediated efflux by using PEG supernatants of 25 individual human sera collected from clinically healthy, normo-lipidemic subjects. Control and ABCG1-upregulated cells were incubated with the PEG supernatants for 4 h, and the ABCG1-mediated efflux was measured. We quantified the HDL components such as cholesterol, phospholipid, and apolipoproteins; and using 2D gel analysis, we characterized and measured the HDL subfractions present in the individual PEG samples. The ABCG1-mediated efflux correlated with the parameters shown in Fig. 5. All HDL components (apoA-I, apoA-II, phospholipids, and cholesterol) were significantly correlated to ABCG1 efflux. Among the HDL subfractions, the α-2 HDL subfraction demonstrated a significant correlation (Fig. 5E). A modest but significant correlation was also observed between ABCG1-mediated efflux and preβ HDL (Fig. 5F).

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