The lipidation status of acute-phase protein serum amyloid A determines cholesterol mobilization via scavenger receptor class B, type I

During the acute-phase reaction, SAA (serum amyloid A) replaces apoA-I (apolipoprotein A-I) as the major HDL (high-density lipoprotein)-associated apolipoprotein. A remarkable portion of SAA exists in a lipid-free/lipid-poor form and promotes ABCA1 (ATP-binding cassette transporter A1)-dependent cellular cholesterol efflux. In contrast with lipid-free apoA-I and apoE, lipid-free SAA was recently reported to mobilize SR-BI (scavenger receptor class B, type I)-dependent cellular cholesterol efflux [Van der Westhuyzen, Cai, de Beer and de Beer (2005) J. Biol. Chem. 280, 35890-35895]. This unique property could strongly affect cellular cholesterol mobilization during inflammation. However, in the present study, we show that overexpression of SR-BI in HEK-293 cells (human embryonic kidney cells) (devoid of ABCA1) failed to mobilize cholesterol to lipid-free or lipid-poor SAA. Only reconstituted vesicles containing phospholipids and SAA promoted SR-BI-mediated cholesterol efflux. Cholesterol efflux from HEK-293 and HEK-293[SR-BI] cells to lipid-free and lipid-poor SAA was minimal, while efficient efflux was observed from fibroblasts and CHO cells (Chinese-hamster ovary cells) both expressing functional ABCA1. Overexpression of SR-BI in CHO cells strongly attenuated cholesterol efflux to lipid-free SAA even in the presence of an SR-BI-blocking IgG. This implies that SR-BI attenuates ABCA1-mediated cholesterol efflux in a way that is not dependent on SR-BI-mediated re-uptake of cholesterol. The present in vitro experiments demonstrate that the lipidation status of SAA is a critical factor governing cholesterol acceptor properties of this amphipathic apolipoprotein. In addition, we demonstrate that SAA mediates cellular cholesterol efflux via the ABCA1 and/or SR-BI pathway in a similar way to apoA-I.     

Serum amyloid A is a ligand for scavenger receptor class B type I and inhibits high density lipoprotein binding and selective lipid uptake

Serum amyloid A is an acute phase protein that is carried in the plasma largely as an apolipoprotein of high density lipoprotein (HDL). In this study we investigated whether SAA is a ligand for the HDL receptor, scavenger receptor class B type I (SR-BI), and how SAA may influence SR-BI-mediated HDL binding and selective cholesteryl ester uptake. Studies using Chinese hamster ovary cells expressing SR-BI showed that (125)I-labeled SAA, both in lipid-free form and in reconstituted HDL particles, functions as a high affinity ligand for SR-BI. SAA also bound with high affinity to the hepatocyte cell line, HepG2. Alexa-labeled SAA was shown by fluorescence confocal microscopy to be internalized by cells in a SR-BI-dependent manner. To assess how SAA association with HDL influences HDL interaction with SR-BI, SAA-containing HDL was isolated from mice overexpressing SAA through adenoviral gene transfer. SAA presence on HDL had little effect on HDL binding to SR-BI but decreased (30-50%) selective cholesteryl ester uptake. Lipid-free SAA, unlike lipid-free apoA-I, was an effective inhibitor of both SR-BI-dependent binding and selective cholesteryl ester uptake of HDL. We have concluded that SR-BI plays a key role in SAA metabolism through its ability to interact with and internalize SAA and, further, that SAA influences HDL cholesterol metabolism through its inhibitory effects on SR-BI-mediated selective lipid uptake.     

ABCA1 and scavenger receptor class B,type I,are modulators of reverse sterol transport at an in vitro blood-brain barrier constituted of porcine brain capillary endothelial cells

The objective of the present study was to investigate the involvement of key players in reverse cholesterol/24(S)OH-cholesterol transport in primary porcine brain capillary endothelial cells (pBCEC) that constitute the BBB. We identified that, in addition to scavenger receptor class B, type I (SR-BI), pBCEC express ABCA1 and apolipoprotein A-I (apoA-I) mRNA and protein. Studies on the regulation of ABCA1 by the liver X receptor agonist 24(S)OH-cholesterol revealed increased ABCA1 expression and apoA-I-dependent [3H]cholesterol efflux from pBCEC. In unpolarized pBCEC, high density lipoprotein, subclass 3 (HDL3)-dependent [3H]cholesterol efflux, was unaffected by 24(S)OH-cholesterol treatment but was enhanced 5-fold in SR-BI overexpressing pBCEC. Efflux of cellular 24(S)-[3H]OH-cholesterol was highly efficient, independent of ABCA1, and correlated with SR-BI expression. Polarized pBCEC were cultured on porous membrane filters that allow separate access to the apical and the basolateral compartment. Addition of cholesterol acceptors to the apical compartment resulted in preferential [3H]cholesterol efflux to the basolateral compartment. HDL3 was a better promoter of basolateral [3H]cholesterol efflux than lipid-free apoA-I. Basolateral pretreatment with 24(S)OH-cholesterol enhanced apoA-I-dependent basolateral cholesterol efflux up to 2-fold along with the induction of ABCA1 at the basolateral membrane. Secretion of apoA-I also occurred preferentially to the basolateral compartment, where the majority of apoA-I was recovered in an HDL-like density range. In contrast, 24(S)-[3H]OH-cholesterol was mobilized efficiently to the apical compartment of the in vitro BBB by HDL3, low density lipoprotein, and serum. These results suggest the existence of an autoregulatory mechanism for removal of potentially neurotoxic 24(S)OH-cholesterol. In conclusion, the apoA-I/ABCA1- and HDL/SR-BI-dependent pathways modulate polarized sterol mobilization at the BBB.     

Serum amyloid A promotes ABCA1-dependent and ABCA1-independent lipid efflux from cells

Serum amyloid A (SAA) is an acute phase protein that associates with HDL. In order to examine the role of SAA in reverse-cholesterol transport, lipid efflux was tested to SAA from HeLa cells before and after transfection with the ABCA1 transporter. ABCA1 expression increased efflux of cholesterol and phospholipid to SAA by 3-fold and 2-fold, respectively. In contrast to apoA-I, SAA also removed lipid without ABCA1; cholesterol efflux from control cells to SAA was 10-fold higher than for apoA-I. Furthermore, SAA effluxed cholesterol from Tangier disease fibroblasts and from cells after inhibition of ABCA1 by fixation with paraformaldehyde. In summary, SAA can act as a lipid acceptor for ABCA1, but unlike apoA-I, it can also efflux lipid without ABCA1, by most likely a detergent-like extraction process. These results suggest that SAA may play a unique role as an auxiliary lipid acceptor in the removal of lipid from sites of inflammation.     

Influence of HDL particles on cell-cholesterol efflux under various pathological conditions

It has been reported that low cell-cholesterol efflux capacity (CEC) of HDL is an independent risk factor for CVD. To better understand CEC regulation, we measured ABCA1- and scavenger receptor class B type I (SR-BI)-dependent cell-cholesterol efflux, HDL anti-oxidative capacity, HDL particles, lipids, and inflammatory- and oxidative-stress markers in 122 subjects with elevated plasma levels of triglyceride (TG), serum amyloid A (SAA), fibrinogen, myeloperoxidase (MPO), or β-sitosterol and in 146 controls. In controls, there were strong positive correlations between ABCA1-dependent cholesterol efflux and small preβ-1 concentrations (R² = 0.317) and SR-BI-dependent cholesterol efflux and large (α-1 + α-2) HDL particle concentrations (R² = 0.774). In high-TG patients, both the concentration and the functionality (preβ-1 concentration-normalized ABCA1 efflux) of preβ-1 particles were significantly elevated compared with controls; however, though the concentration of large particles was significantly decreased, their functionality (large HDL concentration-normalized SR-BI efflux) was significantly elevated. High levels of SAA or MPO were not associated with decreased functionality of either the small (preβ-1) or the large (α-1 + α-2) HDL particles. HDL anti-oxidative capacity was negatively influenced by high plasma β-sitosterol levels, but not by the concentrations of HDL particles, TG, SAA, fibrinogen, or MPO. Our data demonstrate that under certain conditions CEC is influenced not only by quantitative (concentration), but also by qualitative (functional) properties of HDL particles.     

Nascent HDL formation in hepatocytes and role of ABCA1, ABCG1, and SR-BI

To study the mechanisms of hepatic HDL formation, we investigated the roles of ABCA1, ABCG1, and SR-BI in nascent HDL formation in primary hepatocytes isolated from mice deficient in ABCA1, ABCG1, or SR-BI and from wild-type (WT) mice. Under basal conditions, in WT hepatocytes, cholesterol efflux to exogenous apoA-I was accompanied by conversion of apoA-I to HDL-sized particles. LXR activation by T0901317 markedly enhanced the formation of larger HDL-sized particles as well as cellular cholesterol efflux to apoA-I. Glyburide treatment completely abolished the formation of 7.4 nm diameter and greater particles but led to the formation of novel 7.2 nm-sized particles. However, cells lacking ABCA1 failed to form such particles. ABCG1-deficient cells showed similar capacity to efflux cholesterol to apoA-I and to form nascent HDL particles compared with WT cells. Cholesterol efflux to apoA-I and nascent HDL formation were slightly but significantly enhanced in SR-BI-deficient cells compared with WT cells under basal but not LXR activated conditions. As in WT but not in ABCA1-deficient hepatocytes, 7.2 nm-sized particles generated by glyburide treatment were also detected in ABCG1-deficient and SR-BI-deficient hepatocytes. Our data indicate that hepatic nascent HDL formation is highly dependent on ABCA1 but not on ABCG1 or SR-BI.     

Testosterone up-regulates scavenger receptor BI and stimulates cholesterol efflux from macrophages

By lowering high density lipoprotein (HDL) cholesterol, testosterone contributes to the gender difference in HDL cholesterol and has been accused to be pro-atherogenic. The mechanism by which testosterone influences HDL cholesterol is little understood. We therefore investigated the effect of testosterone on the gene expression of apolipoprotein A-I (apoA-I), hepatic lipase (HL), scavenger receptor B1 (SR-BI), and the ATP binding cassette transporter A1 (ABCA1), all of which are important regulators of HDL metabolism. In both cultivated HepG2 hepatocytes and primary human monocyte-derived macrophages, testosterone led to a dose-dependent up-regulation of SR-BI, which was assessed on both the mRNA and the protein levels. As a functional consequence, we observed an increased HDL(3)-induced cholesterol efflux from macrophages. At supraphysiological dosages, testosterone also increased the expression of HL in HepG2 cells. Testosterone had no effect on the expression of apoA-I in HepG2 cells and ABCA1 in either HepG2 cells or macrophages. These data suggest that testosterone, despite lowering HDL cholesterol, intensifies reverse cholesterol transport and thereby exerts an anti-atherogenic rather than a pro-atherogenic effect.     

ATP binding cassette G1-dependent cholesterol efflux during inflammation

ATP binding cassette transporter G1 (ABCG1) mediates the transport of cellular cholesterol to HDL, and it plays a key role in maintaining macrophage cholesterol homeostasis. During inflammation, HDL undergoes substantial remodeling, acquiring lipid changes and serum amyloid A (SAA) as a major apolipoprotein. In the current study, we investigated whether remodeling of HDL that occurs during acute inflammation impacts ABCG1-dependent efflux. Our data indicate that lipid free SAA acts similarly to apolipoprotein A-I (apoA-I) in mediating sequential efflux from ABCA1 and ABCG1. Compared with normal mouse HDL, acute phase (AP) mouse HDL containing SAA exhibited a modest but significant 17% increase in ABCG1-dependent efflux. Interestingly, AP HDL isolated from mice lacking SAA (SAAKO mice) was even more effective in promoting ABCG1 efflux. Hydrolysis with Group IIA secretory phospholipase A(2) (sPLA(2)-IIA) significantly reduced the ability of AP HDL from SAAKO mice to serve as a substrate for ABCG1-mediated cholesterol transfer, indicating that phospholipid (PL) enrichment, and not the presence of SAA, is responsible for alterations in efflux. AP human HDL, which is not PL-enriched, was somewhat less effective in mediating ABCG1-dependent efflux compared with normal human HDL. Our data indicate that inflammatory remodeling of HDL impacts ABCG1-dependent efflux independent of SAA.     

Depletion of pre-beta-high density lipoprotein by human chymase impairs ATP-binding cassette transporter A1- but not scavenger receptor class B type I-mediated lipid efflux to high density lipoprotein

The ATP-binding cassette transporter A1 (ABCA1) mediates the efflux of cellular unesterified cholesterol and phospholipid to lipid-poor apolipoprotein A-I. Chymase, a protease secreted by mast cells, selectively cleaves pre-beta-migrating particles from high density lipoprotein (HDL)(3) and reduces the efflux of cholesterol from macrophages. To evaluate whether this effect is the result of reduction of ABCA1-dependent or -independent pathways of cholesterol efflux, in this study we examined the efflux of cholesterol to preparations of chymase-treated HDL(3) in two types of cell: 1) in J774 murine macrophages endogenously expressing low levels of scavenger receptor class B, type I (SR-BI), and high levels of ABCA1 upon treatment with cAMP; and 2) in Fu5AH rat hepatoma cells endogenously expressing high levels of the SR-BI and low levels of ABCA1. Treatment of HDL(3) with the human chymase resulted in rapid depletion of pre-beta-HDL and a concomitant decrease in the efflux of cholesterol and phospholipid (2-fold and 3-fold, respectively) from the ABCA1-expressing J774 cells. In contrast, efflux of free cholesterol from Fu5AH to chymase-treated and to untreated HDL(3) was similar. Incubation of HDL(3) with phospholipid transfer protein led to an increase in pre-beta-HDL contents as well as in ABCA1-mediated cholesterol efflux. A decreased cholesterol efflux to untreated HDL(3) but not to chymase-treated HDL(3) was observed in ABCA1-expressing J774 with probucol, an inhibitor of cholesterol efflux to lipid-poor apoA-I. Similar results were obtained using brefeldin and gliburide, two inhibitors of ABCA1-mediated efflux. These results indicate that chymase treatment of HDL(3) specifically impairs the ABCA1-dependent pathway without influencing either aqueous or SR-BI-facilitated diffusion and that this effect is caused by depletion of lipid-poor pre-beta-migrating particles in HDL(3). Our results are compatible with the view that HDL(3) promotes ABCA1-mediated lipid efflux entirely through its lipid-poor fraction with pre-beta mobility.     

Lipidation of apolipoprotein A-I by ATP-binding cassette transporter (ABC) A1 generates an interaction partner for ABCG1 but not for scavenger receptor BI

The ATP-binding cassette transporters ABCA1 and ABCG1 as well as scavenger receptor BI (SR-BI) mediate the efflux of lipids from macrophages to apolipoprotein A-I (apoA-I) and high density lipoproteins (HDL). We used RNA interference in RAW264.7 macrophages to study the interactions of ABCA1, ABCG1, and SR-BI with lipid-free apoA-I, native and reconstituted HDL with apoA-I:phosphatidylcholine ratios of either 1:40 (rHDL(1:40)) or 1:100 (rHDL(1:100)). Knock-down of ABCA1 inhibits the cellular binding at 4 degrees C of lipid-free apoA-I but not of HDL whereas suppression of ABCG1 or SR-BI reduces the binding of HDL but not lipid-free apoA-I. The degree of lipidation influences the interactions of rHDL with ABCG1 and SR-BI. Knock-down of ABCG1 inhibits more effectively the binding and cholesterol efflux capacities of lipid-poorer rHDL(1:40) whereas knock-down of SR-BI has a more profound effect on the binding and cholesterol efflux capacities of lipid-richer rHDL(1:100). Moreover, knock-down of ABCG1 but not SR-BI interferes with the association of lipid-free apoA-I during prolonged incubation at 37 degrees C. Finally, knock-down of ABCG1 inhibits the binding of initially lipid-free apoA-I which has been preconditioned by cells with high ABCA1 activity. The gained ability of initially lipid-free apoA-I to interact with ABCG1 is accompanied by its shift from electrophoretic pre-beta- to alpha-mobility. Taken together, these data suggest that the interaction of lipid-free apoA-I with ABCA1 generates a particle that immediately interacts with ABCG1 but not with SR-BI. Furthermore, the degree of lipidation influences the interaction of HDL with ABCG1 or SR-BI.     

Serum amyloid A generates high density lipoprotein with cellular lipid in an ABCA1- or ABCA7-dependent manner

Serum amyloid A (SAA) is an amphiphilic helical protein that is found associated with plasma HDL in various pathological conditions, such as acute or chronic inflammation. Cellular lipid release and generation of HDL by this protein were investigated, in comparison with the reactions by apolipoprotein A-I (apoA-I) and several types of cells that appear with various specific profiles of cholesterol and phospholipid release. SAA mediated cellular lipid release from these cells with the same profile as apoA-I. Upregulation of cellular ABCA1 protein by liver X receptor/retinoid X receptor agonists resulted in an increase of cellular lipid release by apoA-I and SAA. SAA reacted with the HEK293-derived clones that stably express human ABCA1 (293/2c) or ABCA7 (293/6c) to generate cholesterol-containing HDL in a similar manner to apoA-I. Dibutyryl cyclic AMP and phorbol 12-myristate 13-acetate, which differentiate apoA-I-mediated cellular lipid release between 293/2c and 293/6c, also exhibited the same differential effects on the SAA-mediated reactions. No evidence was found for the ABCA1/ABCA7-independent lipid release by SAA. Characterization of physicochemical properties of the HDL revealed that SAA-generated HDL particles had higher density, larger diameter, and slower electrophoretic mobility than those generated by apoA-I. These results demonstrate that SAA generates cholesterol-containing HDL directly with cellular lipid and that the reaction is mediated by ABCA1 and ABCA7.     

In vivo modulation of HDL phospholipid has opposing effects on SR-BI- and ABCA1-mediated cholesterol efflux

The effects of in vivo modulation of HDL phospholipid (PL) on scavenger receptor class BI (SR-BI)- and ATP binding cassette transporter 1 (ABCA1)-mediated efflux were examined by overexpressing either endothelial lipase (EL) or phosphatidylserine phospholipase (PS-PLA1) in human apolipoprotein A-I (apoA-I) transgenic mice. Overexpression of EL led to large reductions in the serum PL/apoA-I ratio (-60%), total cholesterol (TC; -89%), and HDL cholesterol (-91%). Relative to the serum before overexpression of EL, the efflux potential of the serum via SR-BI decreased by 90% and ABCA1-mediated efflux increased by 63%. In contrast to overexpression of EL, overexpression of PS-PLA1 led to increases in the PL/apoA-I ratio (88%), TC (78%), HDL cholesterol (57%), and HDL size. The efflux potential of the serum increased by 60% via SR-BI and decreased by 57% via ABCA1. There were significant positive correlations between SR-BI-mediated efflux and a number of serum parameters, including PL/apoA-I ratio, PL, TC, free cholesterol (FC), and HDL cholesterol. In striking contrast, the same correlations were seen with ABCA1-mediated efflux, but the relationships were inverse. In summary, in vivo modulation of HDL PL content affects ABCA1- and SR-BI-mediated efflux in a reciprocal manner. These findings indicate that the type of lipase acting on HDL in vivo will determine which FC efflux pathway the HDL serves. Additionally, the extent of lipolysis will determine the efficiency of FC removal via this pathway.     

ATP-binding cassette transporter A1 mediates cellular secretion of alpha-tocopherol

Alpha-tocopherol (alpha-TOH) is associated with plasma lipoproteins and accumulates in cell membranes throughout the body, suggesting that lipoproteins play a role in transporting alpha-TOH between tissues. Here we show that secretion of alpha-TOH from cultured cells is mediated in part by ABCA1, an ATP-binding cassette protein that transports cellular cholesterol and phospholipids to lipid-poor high density lipoprotein (HDL) apolipoproteins such as apoA-I. Treatment of human fibroblasts and murine RAW264 macrophages with cholesterol and/or 8-bromo-cyclic AMP, which induces ABCA1 expression, enhanced apoA-I-mediated alpha-TOH efflux. ApoA-I lacked the ability to remove alpha-TOH from Tangier disease fibroblasts that have a nonfunctional ABCA1. BHK cells that lack an active ABCA1 pathway markedly increased secretion of alpha-TOH to apoA-I when forced to express ABCA1. ABCA1 also mediated a fraction of the alpha-TOH efflux promoted by lipid-containing HDL particles, indicating that HDL promotes alpha-TOH efflux by both ABCA1-dependent and -independent processes. Exposing apoA-I to ABCA1-expressing cells did not enhance its ability to remove alpha-TOH from cells lacking ABCA1, consistent with this transporter participating directly in the translocation of alpha-TOH to apolipoproteins. These studies provide evidence that ABCA1 mediates secretion of cellular alpha-TOH into the HDL metabolic pathway, a process that may facilitate vitamin transport between tissues and influence lipid oxidation.     

Enhancing apolipoprotein A-I-dependent cholesterol efflux elevates cholesterol export from macrophages in vivo

Eight proteins potentially involved in cholesterol efflux [ABCA1, ABCG1, CYP27A1, phospholipid transfer protein (PLTP), scavenger receptor type BI (SR-BI), caveolin-1, cholesteryl ester transfer protein, and apolipoprotein A-I (apoA-I)] were overexpressed alone or in combination in RAW 264.7 macrophages. When apoA-I was used as an acceptor, overexpression of the combination of ABCA1, CYP27A1, PLTP, and SR-BI (Combination I) enhanced the efflux by 4.3-fold. It was established that the stimulation of efflux was due to increased abundance of ABCA1 and increased apoA-I binding to non-ABCA1 sites on macrophages. This combination caused only a small increase of the efflux to isolated HDL. When HDL was used as an acceptor, overexpression of caveolin-1 or a combination of caveolin-1 and SR-BI (Combination II) was the most active, doubling the efflux to HDL, without affecting the efflux to apoA-I. When tested in the in vivo mouse model of cholesterol efflux, overexpression of ABCA1 and Combination I elevated cholesterol export from macrophages to plasma, liver, and feces, whereas overexpression of caveolin-1 or Combination II did not have an effect. We conclude that pathways of cholesterol efflux using apoA-I as an acceptor make a predominant contribution to cholesterol export from macrophages in vivo.     

Regulatory effects of synthetic liver X receptor- and peroxisome-proliferator activated receptor agonists on sterol transport pathways in polarized cerebrovascular endothelial cells

The blood-brain barrier contributes to maintain brain cholesterol metabolism and protects this uniquely balanced system from exchange with plasma lipoprotein cholesterol. Brain capillary endothelial cells, representing a physiological barrier to the central nervous system, express apolipoprotein A-I (apoA-I, the major high-density lipoprotein (HDL)-associated apolipoprotein), ATP-binding cassette transporter A1 (ABCA1), and scavenger receptor, class B, type I (SR-BI), proteins that promote cellular cholesterol mobilization. Liver X receptors (LXRs) and peroxisome-proliferator activated receptors (PPARs) are regulators of cholesterol transport, and activation of LXRs and PPARs has potential therapeutic implications for lipid-related neurodegenerative diseases. To clarify the functional impact of LXR/PPAR activation, sterol transport along the: (i) ABCA1/apoA-I and (ii) SR-BI/HDL pathway was investigated in primary, polarized brain capillary endothelial cells, an in vitro model of the blood-brain barrier. Activation of LXR (24(S)OH-cholesterol, TO901317), PPARalpha (bezafibrate, fenofibrate), and PPARgamma (troglitazone, pioglitazone) modulated expression of apoA-I, ABCA1, and SR-BI on mRNA and/or protein levels without compromising transendothelial electrical resistance or tight junction protein expression. LXR-agonists and troglitazone enhanced basolateral-to-apical cholesterol mobilization in the absence of exogenous sterol acceptors. Along with the induction of cell surface-located ABCA1, several agonists enhanced cholesterol mobilization in the presence of exogenous apoA-I, while efflux of 24(S)OH-cholesterol (the major brain cholesterol metabolite) in the presence of exogenous HDL remained unaffected. Summarizing, in cerebrovascular endothelial cells apoA-I, ABCA1, and SR-BI represent drug targets for LXR and PPAR-agonists to interfere with cholesterol homeostasis at the periphery of the central nervous system.     

Cross-inhibition of SR-BI- and ABCA1-mediated cholesterol transport by the small molecules BLT-4 and glyburide

Scavenger receptor class B type I (SR-BI) and ABCA1 are structurally dissimilar cell surface proteins that play key roles in HDL metabolism. SR-BI is a receptor that binds HDL with high affinity and mediates both the selective lipid uptake of cholesteryl esters from lipid-rich HDL to cells and the efflux of unesterified cholesterol from cells to HDL. ABCA1 mediates the efflux of unesterified cholesterol and phospholipids from cells to lipid-poor apolipoprotein A-I (apoA-I). The activities of ABCA1 and other ATP binding cassette superfamily members are inhibited by the drug glyburide, and SR-BI-mediated lipid transport is blocked by small molecule inhibitors called BLTs. Here, we show that one BLT, [1-(2-methoxy-phenyl)-3-naphthalen-2-yl-urea] (BLT-4), blocked ABCA1-mediated cholesterol efflux to lipid-poor apoA-I at a potency similar to that for its inhibition of SR-BI (IC(50) approximately 55-60 microM). Reciprocally, glyburide blocked SR-BI-mediated selective lipid uptake and efflux at a potency similar to that for its inhibition of ABCA1 (IC(50) approximately 275-300 microM). As is the case with BLTs, glyburide increased the apparent affinity of HDL binding to SR-BI. The reciprocal inhibition of SR-BI and ABCA1 by BLT-4 and glyburide raises the possibility that these proteins may share similar or common steps in their mechanisms of lipid transport.     

Differential effects of HDL subpopulations on cellular ABCA1- and SR-BI-mediated cholesterol efflux

Our objective was to evaluate the associations of individual apolipoprotein A-I (apoA-I)-containing HDL subpopulation levels with ABCA1- and scavenger receptor class B type I (SR-BI)-mediated cellular cholesterol efflux. HDL subpopulations were measured by nondenaturing two-dimensional gel electrophoresis from 105 male subjects selected with various levels of apoA-I in pre-beta-1, alpha-1, and alpha-3 HDL particles. ApoB-containing lipoprotein-depleted serum was incubated with [(3)H]cholesterol-labeled cells to measure efflux. The difference in efflux between control and ABCA1-upregulated J774 macrophages was taken as a measure of ABCA1-mediated efflux. SR-BI-mediated efflux was determined using cholesterol-labeled Fu5AH hepatoma cells. Fractional efflux values obtained from these two cell systems were correlated with the levels of individual HDL subpopulations. A multivariate analysis showed that two HDL subspecies correlated significantly with ABCA1-mediated efflux: small, lipid-poor pre-beta-1 particles (P=0.0022) and intermediate-sized alpha-2 particles (P=0.0477). With regard to SR-BI-mediated efflux, multivariate analysis revealed significant correlations with alpha-2 (P=0.0004), alpha-1 (P=0.0030), pre-beta-1 (P=0.0056), and alpha-3 (P=0.0127) HDL particles. These data demonstrate that the small, lipid-poor pre-beta-1 HDL has the strongest association with ABCA1-mediated cholesterol even in the presence of all other HDL subpopulations. Cholesterol efflux via the SR-BI pathway is associated with several HDL subpopulations with different apolipoprotein composition, lipid content, and size.     

Dual effects on HDL metabolism by cholesteryl ester transfer protein inhibition in HepG2 cells

Cholesteryl ester transfer protein (CETP) promotes reverse cholesterol transport via exchange of cholesteryl ester and triglyceride among lipoproteins. Here, we focused on HDL metabolism during inhibition of CETP expression by using CETP antisense oligodeoxynucleotides (ODNs) in HepG2 cells. CETP secretion was decreased by 70% in mRNA levels and by 52% in mass 20 h after ODNs against CETP were delivered to HepG2 cells. Furthermore, as a consequence of the downregulation of CETP, the expression of scavenger receptor class B type I (SR-BI), an HDL receptor, was also reduced by approximately 50% in mRNA and protein levels, whereas the apolipoprotein A-I (apoA-I) expression and secretion were increased by 30 and 92%, respectively. In a functional study, the selective uptake of (125)I-[(14)C]cholesteryl oleate-labeled HDL(3) was decreased. Cholesterol efflux to apoA-I and HDL(3) was significantly increased by 88 and 37%, respectively. Moreover, the CE levels in cells after antisense treatment were elevated by 20%, which was related to the about twofold increase of cholesterol esterification and increased acyl-CoA:cholesterol acyltransferase 1 mRNA levels. Taken together, these findings suggest that although acute suppression of CETP expression leads to an elevation in cellular cholesterol stores, apoA-I secretion, and cellular cholesterol efflux to apoA-I, the return of HDL-CE to hepatocytes via an SR-BI pathway was inhibited in vitro. Thus antisense inhibition of hepatic CETP expression manifests dual effects: namely, increased formation of HDL and suppression of catabolism of HDL-CE, probably via the SR-BI pathway.