Postprandial lipemia enhances the capacity of large HDL2 particles to mediate free cholesterol efflux via SR-BI and ABCG1 pathways in type IIB hyperlipidemia

Lipid and cholesterol metabolism in the postprandial phase is associated with both quantitative and qualitative remodeling of HDL particle subspecies that may influence their anti-atherogenic functions in the reverse cholesterol transport pathway. We evaluated the capacity of whole plasma or isolated HDL particles to mediate cellular free cholesterol (FC) efflux, cholesteryl ester transfer protein (CETP)-mediated cholesteryl ester (CE) transfer, and selective hepatic CE uptake during the postprandial phase in subjects displaying type IIB hyperlipidemia (n = 16). Postprandial, large HDL2 displayed an enhanced capacity to mediate FC efflux via both scavenger receptor class B type I (SR-BI)-dependent (+12%; P < 0.02) and ATP binding cassette transporter G1 (ABCG1)-dependent (+31%; P < 0.008) pathways in in vitro cell systems. In addition, the capacity of whole postprandial plasma (4 h and 8 h postprandially) to mediate cellular FC efflux via the ABCA1-dependent pathway was significantly increased (+19%; P < 0.0003). Concomitantly, postprandial lipemia was associated with elevated endogenous CE transfer rates from HDL2 to apoB lipoproteins and with attenuated capacity (−17%; P < 0.02) of total HDL to deliver CE to hepatic cells. Postprandial lipemia enhanced SR-BI and ABCG1-dependent efflux to large HDL2 particles. However, postprandial lipemia is equally associated with deleterious features by enhancing formation of CE-enriched, triglyceride-rich lipoprotein particles through the action of CETP and by reducing the direct return of HDL-CE to the liver.     

Cellular SR-BI and ABCA1-mediated cholesterol efflux are gender-specific in healthy subjects

We evaluated the impact of gender differences in both the quantitative and qualitative features of HDL subspecies on cellular free cholesterol efflux through the scavenger receptor class B type I (SR-BI), ABCA1, and ABCG1 pathways. For that purpose, healthy subjects (30 men and 26 women) matched for age, body mass index, triglyceride, apolipoprotein A-I, and high density lipoprotein-cholesterol (HDL-C) levels were recruited. We observed a significant increase (+14%; P < 0.03) in the capacity of whole sera from women to mediate cellular free cholesterol efflux via the SR-BI-dependent pathway compared with sera from men. Such enhanced efflux capacity resulted from a significant increase in plasma levels of large cholesteryl ester-rich HDL2 particles (+20%; P < 0.04) as well as from an enhanced capacity (+14%; P < 0.03) of these particles to mediate cellular free cholesterol efflux via SR-BI. By contrast, plasma from men displayed an enhanced free cholesterol efflux capacity (+31%; P < 0.001) via the ABCA1 transporter pathway compared with that from women, which resulted from a 2.4-fold increase in the plasma level of prebeta particles (P < 0.008). Moreover, in women, SR-BI-mediated cellular free cholesterol efflux was significantly correlated with plasma HDL-C (r = 0.72, P < 0.0001), whereas this relationship was not observed in men. In conclusion, HDL-C level may not represent the absolute indicator of the efficiency of the initial step of the reverse cholesterol transport.     

SR-BI inhibits ABCG1-stimulated net cholesterol efflux from cells to plasma HDL

This study compares the roles of ABCG1 and scavenger receptor class B type I (SR-BI) singly or together in promoting net cellular cholesterol efflux to plasma HDL containing active LCAT. In transfected cells, SR-BI promoted free cholesterol efflux to HDL, but this was offset by an increased uptake of HDL cholesteryl ester (CE) into cells, resulting in no net efflux. Coexpression of SR-BI with ABCG1 inhibited the ABCG1-mediated net cholesterol efflux to HDL, apparently by promoting the reuptake of CE from medium. However, ABCG1-mediated cholesterol efflux was not altered in cholesterol-loaded, SR-BI-deficient (SR-BI(-/-)) macrophages. Briefly cultured macrophages collected from SR-BI(-/-) mice loaded with acetylated LDL in the peritoneal cavity did exhibit reduced efflux to HDL. However, this was attributable to reduced expression of ABCG1 and ABCA1, likely reflecting increased macrophage cholesterol efflux to apolipoprotein E-enriched HDL during loading in SR-BI(-/-) mice. In conclusion, cellular SR-BI does not promote net cholesterol efflux from cells to plasma HDL containing active LCAT as a result of the reuptake of HDL-CE into cells. Previous findings of increased atherosclerosis in mice transplanted with SR-BI(-/-) bone marrow probably cannot be explained by a defect in macrophage cholesterol efflux.     

ABCA1 increases extracellular ATP to mediate cholesterol efflux to ApoA-I

ATP-binding cassette protein A1 (ABCA1) is a key plasma membrane protein required for the efflux of cellular cholesterol to extracellular acceptors, particularly to apolipoprotein A-I (apoA-I). This process is essential to maintain cholesterol homeostasis in the body. The detailed molecular mechanisms, however, are still insufficiently understood. Also, the molecular identity of ABCA1, i.e., channel, pump, or flippase, remains unknown. In this study we analyzed extracellular ATP levels in the medium of ABCA1-expressing BHK cells and RAW macrophages and compared them to the medium of nonexpressing cells. We found that extracellular ATP concentrations are significantly elevated when cells express ABCA1. Importantly, a dysfunctional ABCA1 mutant (A937V), when expressed similarly as wild-type ABCA1, is unable to raise extracellular ATP concentration, which suggests a casual relationship between functional ABCA1 and elevated extracellular ATP. To explore the physiological role of extracellular ATP, we analyzed ABCA1-mediated cholesterol efflux under conditions where extracellular ATP levels were modulated. We found that increasing extracellular ATP within the physiological range, i.e., <μM, promotes cholesterol efflux to apoA-I. On the other hand, removing extracellular ATP, either by adding apyrase to the medium or by expressing a plasma membrane-bound ectonucleotidase, CD39, abolishes cholesterol efflux to apoA-I. On the basis of these results, we conclude that, through direct or indirect mechanisms, ABCA1 functions to raise ATP levels in the medium. This elevated extracellular ATP is required for ABCA1-mediated cholesterol efflux to apoA-I.     

Discrete subspecies of human low density lipoproteins are heterogeneous in their interaction with the cellular LDL receptor.

The low density lipoproteins (LDL) of human plasma consist of a series of discrete particle subspecies of distinct physicochemical, immunological, and hydrodynamic properties. Such structural differences are intimately linked to the metabolic heterogeneity of circulating LDL in vivo. The current studies were designed to evaluate and compare the interaction of discrete LDL subspecies from normolipidemic subjects with the LDL receptor. Plasma LDL of d 1.019-1.063 g/ml from healthy males were fractionated into 15 subspecies of defined physicochemical characteristics by isopycnic density gradient ultracentrifugation as described earlier (Chapman et al., J. Lipid Res. 1988. 29: 442-458). The major LDL subspecies, LDL-5 to LDL-10, exhibited an overall range in density from 1.0244 to 1.0435 g/ml; individual subspecies increased in density by increments of 0.027 (LDL-5), 0.026 (LDL-6), 0.030 (LDL-7), 0.031 (LDL-8), 0.035 (LDL-9), and 0.042 g/ml (LDL-10), respectively. Taken together, these subspecies accounted for approximately 70% of the total mass of LDL of d 1.019-1.063 g/ml; their cholesterol: protein ratios decreased from 1.70 to 1.12 and particle size from 275 to 260 A with increase in density. ApoB-100 was the unique protein component in subspecies 5-8, with trace amounts (less than 0.2% of apoLDL) of both apoA-I and apoE in subspecies 9 and 10. The interaction of individual LDL subspecies with the LDL receptor on cultured human U-937 monocyte-like cells was compared by determining receptor binding affinities at 4 degrees C. Scatchard analysis of specific binding curves demonstrated a single class of binding site for each subspecies. The lowest dissociation constants were displayed by LDL subspecies 6 (Kd 5.71 nM), 7 (Kd 5.24 nM) and 8 (Kd 4.67 nM), while subspecies 5, 9, and 10 displayed significantly higher Kd values (8.35, 7.20, and 6.87 nM, respectively). Competitive displacement studies at 4 degrees C, in which unlabeled subspecies from the same gradient series competed for binding with 125I-labeled LDL subspecies, confirmed the relative binding affinities of these subspecies. As the hydrophobic lipid core of LDL undergoes a thermotropic transition at approximately 37 degrees C, which may in turn influence the surface structure of the particle, internalization and degradation studies were performed at 37 degrees C. No effect of temperature was detectable; again, LDL subspecies at each extreme of the density distribution (LDL-5 and LDL-10) displayed significantly lower binding affinities for the LDL receptor than that from the peak region (LDL-7).(ABSTRACT TRUNCATED AT 400 WORDS)     

Scavenger receptor BI (SR-BI) mediates free cholesterol flux independently of HDL tethering to the cell surface

In addition to its effect on high density lipoprotein (HDL) cholesteryl ester (CE) uptake, scavenger receptor BI (SR-BI) was recently reported to stimulate free cholesterol (FC) flux from Chinese hamster ovary (CHO) cells stably expressing mouse SR-BI, a novel function of SR-BI that may play a role in cholesterol removal from the vessel wall where the receptor can be found. It is possible that SR-BI stimulates flux simply by tethering acceptor HDL particles in close apposition to the cell surface thereby facilitating the movement of cholesterol between the plasma membrane and HDL. To test this, we used transiently transfected cells and compared the closely related class B scavenger receptors mouse SR-BI and rat CD36 for their ability to stimulate cholesterol efflux as both receptors bind HDL with high affinity. The results showed that, although acceptor binding to SR-BI may contribute to efflux to a modest extent, the major stimulation of FC efflux occurs independently of acceptor binding to cell surface receptors. Instead our data indicate that SR-BI mediates alterations to membrane FC domains which provoke enhanced bidirectional FC flux between cells and extracellular acceptors.     

Oxidized LDL increase free cholesterol and fail to stimulate cholesterol esterification in murine macrophages

Oxidatively modified low density lipoproteins (Ox-LDL) may be involved in determining the formation of foam cells by inducing cellular cholesteryl ester accumulation. We studied the effect of copper oxidized LDL (Ox-LDL) on cholesterol accumulation and esterification in murine macrophages. Ox-LDL (44 micrograms/ml of lipoprotein cholesterol) increased the total cholesterol content of the cells from 29 to 69 micrograms/mg cell protein. Free cholesterol accounted for 85% of this increase. Acetyl LDL (Ac-LDL) (38 micrograms/ml of lipoprotein cholesterol), raised total cellular cholesterol content to a similar extent (76 micrograms/mg cell protein), however only 25% of the accumulated cholesterol was unesterified. When ACAT activity was determined after incubation of J774 cell with Ox- or Ac-LDL, Ox-LDL were 12 times less effective than Ac-LDL in stimulating cholesteryl ester formation. This was not due to an inhibition of ACAT by Ox-LDL since these lipoproteins failed to inhibit pre activated enzyme in cholesteryl ester-loaded macrophages. The uptake of 125I-Ox-LDL: was 175% that of 125I-Ac-LDL, while degradation was only 20%. All together these data suggest an altered intracellular processing of Ox-LDL, which may be responsible for free cholesterol accumulation.     

Glycosphingolipid accumulation inhibits cholesterol efflux via the ABCA1/apolipoprotein A-I pathway: 1-phenyl-2-decanoylamino-3-morpholino-1-propanol is a novel cholesterol efflux accelerator

Cellular glycosphingolipid (GSL) storage is known to promote cholesterol accumulation. Although physical interactions between GSLs and cholesterol are thought to cause intracellular cholesterol "trapping," it is not known whether cholesterol homeostatic mechanisms are also impaired under these conditions. ApoA-I-mediated cholesterol efflux via ABCA1 (ATP-binding cassette transporter A1) is a key regulator of cellular cholesterol balance. Here, we show that apoA-I-mediated cholesterol efflux was inhibited (by up to 53% over 8 h) when fibroblasts were treated with lactosylceramide or the glucocerebrosidase inhibitor conduritol B epoxide. Furthermore, apoA-I-mediated cholesterol efflux from fibroblasts derived from patients with genetic GSL storage diseases (Fabry disease, Sandhoff disease, and GM1 gangliosidosis) was impaired compared with control cells. Conversely, apoA-I-mediated cholesterol efflux from fibroblasts and cholesterol-loaded macrophage foam cells was dose-dependently stimulated (by up to 6-fold over 8 h) by the GSL synthesis inhibitor 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP). Unexpectedly, a structurally unrelated GSL synthesis inhibitor, N-butyldeoxynojirimycin, was unable to stimulate apoA-I-mediated cholesterol efflux despite achieving similar GSL depletion. PDMP was found to up-regulate ABCA1 mRNA and protein expression, thereby identifying a contributing mechanism for the observed acceleration of cholesterol efflux to apoA-I. This study reveals a novel defect in cellular cholesterol homeostasis induced by GSL storage and identifies PDMP as a new agent for enhancing cholesterol efflux via the ABCA1/apoA-I pathway.     

Proteomic analysis of HDL from inbred mouse strains implicates APOE associated with HDL in reduced cholesterol efflux capacity via the ABCA1 pathway

Cholesterol efflux capacity associates strongly and negatively with the incidence and prevalence of human CVD. We investigated the relationships of HDL’s size and protein cargo with its cholesterol efflux capacity using APOB-depleted serum and HDLs isolated from five inbred mouse strains with different susceptibilities to atherosclerosis. Like humans, mouse HDL carried >70 proteins linked to lipid metabolism, the acute-phase response, proteinase inhibition, and the immune system. HDL’s content of specific proteins strongly correlated with its size and cholesterol efflux capacity, suggesting that its protein cargo regulates its function. Cholesterol efflux capacity with macrophages strongly and positively correlated with retinol binding protein 4 (RBP4) and PLTP, but not APOA1. In contrast, ABCA1-specific cholesterol efflux correlated strongly with HDL’s content of APOA1, APOC3, and APOD, but not RBP4 and PLTP. Unexpectedly, APOE had a strong negative correlation with ABCA1-specific cholesterol efflux capacity. Moreover, the ABCA1-specific cholesterol efflux capacity of HDL isolated from APOE-deficient mice was significantly greater than that of HDL from wild-type mice. Our observations demonstrate that the HDL-associated APOE regulates HDL’s ABCA1-specific cholesterol efflux capacity. These findings may be clinically relevant because HDL’s APOE content associates with CVD risk and ABCA1 deficiency promotes unregulated cholesterol accumulation in human macrophages.     

Macrophage cholesterol efflux to free apoprotein A-I in C3H and C57BL/6 mice

Cholesterol efflux from peritoneal macrophages of mice C57BL/6 susceptible and C3H resistant to atherosclerosis was compared, using apoprotein A-I as acceptor. The elicited macrophages were labeled with 3H-cholesterol and cholesterol enriched by incubation for 24 h with acetylated LDL. After incubation for 6 or 24 h, 3H-cholesterol efflux to free apoA-I (10 microg/ml) was significantly higher with macrophages derived from C3H mice compared to C57BL/6 mice. The cells were also pretreated with 0.3-0.45 mM cyclic AMP, 10 microM 9-cis-retinoic acid or 10 microM 22(R)-hydroxycholesterol, RXR and LXR ligands. Treatment with cyclic AMP, RXR, or LXR ligands, resulted in enhancement of 3H-cholesterol efflux in both strains. Under all conditions, 3H-cholesterol efflux was significantly higher in C3H compared to C57BL/6 macrophages. In conclusion, the higher cholesterol efflux from C3H macrophages could contribute toward the resistance of this strain to diet-induced atherosclerosis despite hypercholesterolemia.     

Membrane lipid domains distinct from cholesterol/sphingomyelin-rich rafts are involved in the ABCA1-mediated lipid secretory pathway

Efflux of excess cellular cholesterol mediated by lipid-poor apolipoproteins occurs by an active mechanism distinct from passive diffusion and is controlled by the ATP-binding cassette transporter ABCA1. Here we examined whether ABCA1-mediated lipid efflux involves the selective removal of lipids associated with membrane rafts, plasma membrane domains enriched in cholesterol and sphingomyelin. ABCA1 was not associated with cholesterol and sphingolipid-rich membrane raft domains based on detergent solubility and lack of colocalization with marker proteins associated with raft domains. Lipid efflux to apoA-I was accounted for by decreases in cellular lipids not associated with cholesterol/sphingomyelin-rich membranes. Treating cells with filipin, to disrupt raft structure, or with sphingomyelinase, to digest plasma membrane sphingomyelin, did not impair apoA-I-mediated cholesterol or phosphatidylcholine efflux. In contrast, efflux of cholesterol to high density lipoproteins (HDL) or plasma was partially accounted for by depletion of cholesterol from membrane rafts. Additionally, HDL-mediated cholesterol efflux was partially inhibited by filipin and sphingomyelinase treatment. Apo-A-I-mediated cholesterol efflux was absent from fibroblasts with nonfunctional ABCA1 (Tangier disease cells), despite near normal amounts of cholesterol associated with raft domains and normal abilities of plasma and HDL to deplete cholesterol from these domains. Thus, the involvement of membrane rafts in cholesterol efflux applies to lipidated HDL particles but not to lipid-free apoA-I. We conclude that cholesterol and sphingomyelin-rich membrane rafts do not provide lipid for efflux promoted by apolipoproteins through the ABCA1-mediated lipid secretory pathway and that ABCA1 is not associated with these domains.     

Increased maternal and fetal cholesterol efflux capacity and placental CYP27A1 expression in preeclampsia

Preeclampsia is a pregnancy-specific condition that leads to increased cardiovascular risk in later life. A decrease in cholesterol efflux capacity is linked to CVD. We hypothesized that in preeclampsia there would be a disruption of maternal/fetal plasma to efflux cholesterol, as well as differences in the concentrations of both placental sterol 27-hydroxylase (CYP27A1) and apoA1 binding protein (AIBP). Total, HDL-, and ABCA1-mediated cholesterol effluxes were performed with maternal and fetal plasma from women with preeclampsia and normotensive controls (both n = 17). apoA1 and apoE were quantified by chemiluminescence, and 27-hydroxycholesterol (27-OHC) by GC-MS. Immunohistochemistry was used to determine placental expression/localization of CYP27A1, AIBP, apoA1, apoE, and SRB1. Maternal and fetal total and HDL-mediated cholesterol efflux capacities were increased in preeclampsia (by 10–20%), but ABCA1-mediated efflux was decreased (by 20–35%; P < 0.05). Maternal and fetal apoE concentrations were higher in preeclampsia. Fetal plasma 27-OHC levels were decreased in preeclamptic samples (P< 0.05). Placental protein expression of both CYP27A1 and AIBP were localized around fetal vessels and significantly increased in preeclampsia (P = 0.04). Placental 27-OHC concentrations were also raised in preeclampsia (P < 0.05). Increased HDL-mediated cholesterol efflux capacity and placental CYP27A1/27-OHC could be a rescue mechanism in preeclampsia, to remove cholesterol from cells to limit lipid peroxidation and increase placental angiogenesis.     

Elevated triglyceride content diminishes the capacity of high density lipoprotein to deliver cholesteryl esters via the scavenger receptor class B type I (SR-BI)

The selective uptake of high density lipoprotein (HDL) cholesteryl ester (CE) by the scavenger receptor class B type I (SR-BI) is well documented. However, the effect of altered HDL composition, such as occurs in hyperlipidemia, on this important process is not known. This study investigated the impact of variable CE and triglyceride (TG) content on selective uptake. CE selective uptake by Y1 and HepG2 cells was strongly affected by modification of either the CE or TG content of HDL. Importantly, TG, like CE, was selectively taken up by a dose-dependent, saturable process in these cells. As shown by ACTH up-regulation and receptor overexpression experiments, SR-BI mediated the selective uptake of both CE and TG. With in vitro modified HDLs of varying CE and TG composition, the selective uptake of CE and TG was dependent on the abundance of each lipid within the HDL particle. Furthermore, total selective uptake (CE + TG) remained constant, indicating that these lipids competed for cellular uptake. These data support a novel mechanism whereby SR-BI binds HDL and mediates the incorporation of a nonspecific portion of the HDL lipid core. In this way, TG directly affects the ability of HDL to donate CE to cells. Processes that raise the TG/CE ratio of HDL will impair the delivery of CE to cells via this receptor and may compromise the efficiency of sterol balancing pathways such as reverse cholesterol transport.     

Hormone-sensitive lipase deficiency affects the expression of SR-BI,LDLr, and ABCA1 receptors/transporters involved in cellular cholesterol uptake and efflux and disturbs fertility in mouse testis

Hormone-sensitive lipase (HSL) hydrolyse acylglycerols, cholesteryl and retinyl esters. HSL is a key lipase in mice testis, as HSL deficiency results in male sterility. The present work study the effects of the deficiency and lack of HSL on the localization and expression of SR-BI, LDLr, and ABCA1 receptors/transporters involved in uptake and efflux of cholesterol in mice testis, to determine the impact of HSL gene dosage on testis morphology, lipid homeostasis and fertility. The results of this work show that the lack of HSL in mice alters testis morphology and spermatogenesis, decreasing sperm counts, sperm motility and increasing the amount of Leydig cells and lipid droplets. They also show that there are differences in the localization of HSL, SR-BI, LDLr and ABCA1 in HSL^+/+, HSL^+/− and HSL^−/− mice. The deficiency or lack of HSL has effects on protein and mRNA expression of genes involved in lipid metabolisms in mouse testis. HSL^−/− testis have augmented expression of SR-BI, LDLr, ABCA1 and LXRβ, a critical sterol sensor that regulate multiple genes involved in lipid metabolism; whereas LDLr expression decreased in HSL^+/− mice. Plin2, Abca1 and Ldlr mRNA levels increased; and LXRα (Nr1h3) and LXRβ (Nr1h2) decreased in testis from HSL^−/− compared with HSL^+/+; with no differences in Scarb1. Together these data suggest that HSL deficiency or lack in mice testis induces lipid homeostasis alterations that affect the cellular localization and expression of key receptors/transporter involved in cellular cholesterol uptake and efflux (SR-BI, LDRr, ABCA1); alters normal cellular function and impact fertility.     

NARC-1/PCSK9 and its natural mutants: zymogen cleavage and effects on the low density lipoprotein (LDL) receptor and LDL cholesterol

The discovery of autosomal dominant hypercholesterolemic patients with mutations in the PCSK9 gene, encoding the proprotein convertase NARC-1, resulting in the missense mutations suggested a role in low density lipoprotein (LDL) metabolism. We show that the endoplasmic reticulum-localized proNARC-1 to NARC-1 zymogen conversion is Ca2+-independent and that within the zymogen autocatalytic processing site SSVFAQ [downward arrow]SIP Val at P4 and Pro at P3' are critical. The S127R and D374Y mutations result in approximately 50-60% and > or =98% decrease in zymogen processing, respectively. In contrast, the double [D374Y + N157K], F216L, and R218S natural mutants resulted in normal zymogen processing. The cell surface LDL receptor (LDLR) levels are reduced by 35% in lymphoblasts of S127R patients. The LDLR levels are also reduced in stable HepG2 cells overexpressing NARC-1 or its natural mutant S127R, and this reduction is abrogated in the presence of 5 mm ammonium chloride, suggesting that overexpression of NARC-1 increases the turnover rate of the LDLR. Adenoviral expression of wild type human NARC-1 in mice resulted in a maximal approximately 9-fold increase in circulating LDL cholesterol, while in LDLR-/- mice a delayed approximately 2-fold increase in LDL cholesterol was observed. In conclusion, NARC-1 seems to affect both the level of LDLR and that of circulating apoB-containing lipoproteins in an LDLR-dependent and -independent fashion.     

Analysis of chimeric receptors shows that multiple distinct functional activities of scavenger receptor, class B, type I (SR-BI), are localized to the extracellular receptor domain

Scavenger receptor BI (SR-BI) mediates the selective uptake of high-density lipoprotein (HDL) cholesteryl ester (CE), a process by which HDL CE is taken into the cell without degradation of the HDL particle. In addition, SR-BI stimulates the bi-directional flux of free cholesterol (FC) between cells and lipoproteins, an activity that may be responsible for net cholesterol efflux from peripheral cells as well as the rapid hepatic clearance of FC from plasma HDL. SR-BI also increases cellular cholesterol mass and alters cholesterol distribution in plasma membrane domains as judged by the enhanced sensitivity of membrane cholesterol to extracellular cholesterol oxidase. In contrast, CD36, a closely related class B scavenger receptor, has none of these activities despite binding HDL with high affinity. In the present study, analyses of chimeric SR-BI/CD36 receptors and domain-deleted SR-BI have been used to test the various domains of SR-BI for functional activities related to HDL CE selective uptake, bi-directional FC flux, and the alteration of membrane cholesterol mass and distribution. The results show that each of these activities localizes to the extracellular domain of SR-BI. The N-terminal cytoplasmic tail and transmembrane domains appear to play no role in these activities other than targeting the receptor to the plasma membrane. The C-terminal tail of SR-BI is dispensable for activity as well for targeting to the plasma membrane. Thus, multiple distinct functional activities are localized to the SR-BI extracellular domain.     

The molecular structure of apolipoprotein A-II modulates the capacity of HDL to promote cell cholesterol efflux

The influence of apolipoprotein A-II (apoA-II) molecular structure on the capacity of high density lipoproteins (HDL) to promote cellular cholesterol efflux was investigated in cultured mouse peritoneal macrophages (MPM). Conversion by reduction and carboxamidomethylation of the naturally occurring dimeric apoA-II to its monomeric form in both native or reconstituted HDL did not change apolipoprotein secondary structure and lipoprotein size/composition. All particles containing monomeric apoA-II, i.e., native HDL₃ or reconstituted HDL with or without apoA-I, showed a higher ability to promote cholesterol efflux originating from plasma membrane and intracellular stores, compared to particles containing dimeric apoA-II. These findings indicate that apolipoprotein molecular structure is a major determinant of HDL capacity to promote cholesterol efflux from cells.     

The role of apolipoprotein A-I helix 10 in apolipoprotein-mediated cholesterol efflux via the ATP-binding cassette transporter ABCA1

Recent studies of Tangier disease have shown that the ATP-binding cassette transporter A1 (ABCA1)/apolipoprotein A-I (apoA-I) interaction is critical for high density lipoprotein particle formation, apoA-I integrity, and proper reverse cholesterol transport. However, the specifics of this interaction are unknown. It has been suggested that amphipathic helices of apoA-I bind to a lipid domain created by the ABCA1 transporter. Alternatively, apoA-I may bind directly to ABCA1 itself. To better understand this interaction, we created several truncation mutants of apoA-I and then followed up with more specific point mutants and helix translocation mutants to identify and characterize the locations of apoA-I required for ABCA1-mediated cholesterol efflux. We found that deletion of residues 221-243 (helix 10) abolished ABCA1-mediated cholesterol efflux from cultured RAW mouse macrophages treated with 8-bromo-cAMP. Point mutations in helix 10 that affected the helical charge distribution reduced ABCA1-mediated cholesterol efflux versus the wild type. We noted a strong positive correlation between cholesterol efflux and the lipid binding characteristics of apoA-I when mutations were made in helix 10. However, there was no such correlation for helix translocations in other areas of the protein as long as helix 10 remained intact at the C terminus. From these observations, we propose an alternative model for apolipoprotein-mediated efflux.     

Human apolipoprotein A-IV binds to apolipoprotein A-I/A-II receptor sites and promotes cholesterol efflux from adipose cells

Cholesterol efflux was studied in cultured mouse adipose cells after preloading with low density lipoprotein cholesterol. Exposure to complexes containing human apolipoprotein A-IV and L-alpha-dimyristoylphosphatidylcholine (DMPC) as well as to human lipoprotein particles containing apolipoprotein A-IV but not apolipoprotein A-I and particles containing apolipoproteins A-IV and A-I showed that both artificial and native apolipoprotein A-IV-containing particles were able to promote cholesterol efflux at 37 degrees C as a function of time and concentration. The half-maximal concentration was found to be 0.3 X 10(-6) M for apolipoprotein A-IV.DMPC complexes. Binding experiments performed in intact cells at 4 degrees C with labeled apolipoprotein A-IV.DMPC complexes showed the existence of specific binding sites, with a Kd value of 0.32 x 10(-6) M and a maximal binding capacity of 223,000 sites/cell. By cross-competition experiments with labeled and unlabeled complexes containing apolipoprotein A-IV, A-I, or A-II, it appeared that all three apolipoproteins bind to the same cell-surface recognition sites. It is suggested that apolipoprotein A-IV, which is present in the interstitial fluid surrounding adipose cells in vivo at concentrations similar to those required in vitro for the promotion of cholesterol efflux, plays a critical role in cholesterol removal from peripheral cells.     

Plasma cholesterol efflux capacity from human THP-1 macrophages is reduced in HIV-infected patients: impact of HAART

The capacity of HDL to remove cholesterol from macrophages is inversely associated with the severity of angiographic coronary artery disease. The effect of human immunodeficiency virus (HIV) infection or its treatment on the ability of HDL particles to stimulate cholesterol efflux from human macrophages has never been studied. We evaluated the capacity of whole plasma and isolated HDL particles from HIV-infected subjects (n = 231) and uninfected controls (n = 200), as well as in a subset of 41 HIV subjects receiving highly active antiretroviral therapy (HAART) to mediate cholesterol efflux from human macrophages. Plasma cholesterol efflux capacity was reduced (−12%; P = 0.001) in HIV patients as compared with controls. HIV infection reduced by 27% (P < 0.05) the capacity of HDL subfractions to promote cholesterol efflux from macrophages. We observed a reduced ABCA1-dependent efflux capacity of plasma (−27%; P < 0.0001) from HIV-infected subjects as a result of a reduction in the efflux capacity of HDL3 particles. HAART administration restored the capacity of plasma from HIV patients to stimulate cholesterol efflux from human macrophages (9.4%; P = 0.04). During HIV infection, the capacity of whole plasma to remove cholesterol from macrophages is reduced, thus potentially contributing to the increased coronary heart disease in the HIV population. HAART administration restored the removal of cholesterol from macrophages by increasing HDL functionality.