The lipidation by hepatocytes of human apolipoprotein A-I occurs by both ABCA1-dependent and -independent pathways

The pathways of hepatic intra- and peri-cellular lipidation of apolipoprotein A-I (apoA-I) were studied by infecting primary mouse hepatocytes from either apoA-I-deficient or ABCA1-deficient mice with a recombinant adenovirus expressing the human apoA-I (hapoA-I) cDNA (endo apoA-I) or incubating the hepatocytes with exogenously added hapoA-I (exo apoA-I) and examining the hapoA-I-containing lipoproteins formed. The cells, maintained in serum-free medium, were labeled with [(3)H]choline, and the cell medium was separated by fast protein liquid chromatography or immunoprecipitated to quantify labeled choline phospholipids specifically associated with hapoA-I. With the apoA-I-deficient hepatocytes, the high density lipoprotein fraction formed with endo apoA-I contained proportionally more phospholipids than that formed with exo apoA-I. However, the lipoprotein size and electrophoretic mobility and phospholipid profiles were similar for exo apoA-I and endo apoA-I. Taken together, these data demonstrate that a significant proportion of hapoA-I is secreted from hepatocytes in a phospholipidated state but that hapoA-I is also phospholipidated peri-cellularly. With primary hepatocytes from ABCA1-deficient mice, the expression and net secretion of adenoviral-generated endogenous apoA-I was unchanged compared with control mice, but (3)H-phospholipids associated with endo apoA-I and exo apoA-I decreased by 63 and 25%, respectively. The lipoprotein size and electrophoretic migration and their phospholipid profiles remained unchanged. In conclusion, we demonstrated that intracellular and peri-cellular lipidation of apoA-I represent distinct and additive pathways that may be regulated independently. Hepatocyte expression of ABCA1 is central to the lipidation of newly synthesized apoA-I but also contributes to the lipidation of exogenous apoA-I. However, a significant basal level of phospholipidation occurs in the absence of ABCA1.     

Intracellular lipidation of newly synthesized apolipoprotein A-I in primary murine hepatocytes

Hepatocytes, which are the main site of apolipoprotein (apo)A-I and ATP-binding cassette transporter A1 (ABCA1) expression, are also the main source of circulating high density lipoprotein. Here we have characterized the intracellular lipidation of newly synthesized apoA-I, in primary hepatocytes cultured with [3H]choline to label choline-phospholipids, low density lipoprotein-[3H]cholesterol to label the cell surface, or [3H]mevalonate to label de novo synthesized cholesterol. Phospholipidation of apoA-I is significant and most evident in endoplasmic reticulum (ER) and medial Golgi, both in the lumen and on the membrane fractions of the ER and medial Golgi. In the presence of cycloheximide, endogenous apoA-I is substantially phospholipidated intracellularly but acquires some additional lipid after export out of the cell. In cells labeled with low density lipoprotein-[3H]cholesterol, intracellular cholesterol lipidation of apoA-I is entirely absent, but the secreted apoA-I rapidly accumulates cholesterol after secretion from the cell in the media. On the other hand, de novo synthesized cholesterol can lipidate apoA-I intracellularly. We also showed the interaction between apoA-I and ABCA1 in ER and Golgi fractions. In hepatocytes lacking ABCA1, lipidation by low density lipoprotein-cholesterol was significantly reduced at the plasma membrane, phospholipidation and lipidation by de novo synthesized sterols were both reduced in Golgi compartments, whereas ER lipidation remained mostly unchanged. Therefore, the early lipidation in ER is ABCA1 independent, but in contrast, the lipidation of apoA-I in Golgi and at the plasma membrane requires ABCA1. Thus, we demonstrated that apoA-I phospholipidation starts early in the ER and is partially dependent on ABCA1, with the bulk of lipidation by phospholipids and cholesterol occurring in the Golgi and at the plasma membrane, respectively. Finally, we showed that the previously reported association of newly synthesized apoA-I and apoB (Zheng, H., Kiss, R. S., Franklin, V., Wang, M. D., Haidar, B., and Marcel, Y. L. (2005) J. Biol. Chem. 280, 21612-21621) occurs after secretion at the cell surface.     

ABCA1-dependent lipid efflux to apolipoprotein A-I mediates HDL particle formation and decreases VLDL secretion from murine hepatocytes

High levels of expression of the ATP binding cassette transporter A1 (ABCA1) in the liver and the need to over- or underexpress hepatic ABCA1 to impact plasma HDL levels in mice suggest a major role of the liver in HDL formation and in determining circulating HDL levels. Cultured murine hepatocytes were used to examine the role of hepatic ABCA1 in mediating the lipidation of apolipoprotein A-I (apoA-I) for HDL particle formation. Exogenous apoA-I stimulated cholesterol efflux to the medium from wild-type hepatocytes, but not from ABCA1-deficient (abca1(-/-)) hepatocytes. ApoA-I induced the formation of new HDL particles and enhanced the lipidation of endogenously secreted murine apoA-I in ABCA1-expressing but not abca1(-/-) hepatocytes. ABCA1-dependent cholesterol mobilization to apoA-I increased new cholesterol synthesis, indicating depletion of the regulatory pool of hepatocyte cholesterol during HDL formation. Secretion of triacylglycerol and apoB was decreased following apoA-I incubation with ABCA1-expressing but not abca1(-/-) hepatocytes. These results support a major role for hepatocyte ABCA1 in generating a critical pool of HDL precursor particles that enhance further HDL generation and passive cholesterol mobilization in the periphery. The results also suggest that diversion of hepatocyte cholesterol into the "reverse" cholesterol transport pathway diminishes cholesterol availability for apoB-containing lipoprotein secretion by the liver.     

ABCA1 is required for normal central nervous system ApoE levels and for lipidation of astrocyte-secreted apoE

ABCA1 is an ATP-binding cassette protein that transports cellular cholesterol and phospholipids onto high density lipoproteins (HDL) in plasma. Lack of ABCA1 in humans and mice causes abnormal lipidation and increased catabolism of HDL, resulting in very low plasma apoA-I, apoA-II, and HDL. Herein, we have used Abca1-/- mice to ask whether ABCA1 is involved in lipidation of HDL in the central nervous system (CNS). ApoE is the most abundant CNS apolipoprotein and is present in HDL-like lipoproteins in CSF. We found that Abca1-/- mice have greatly decreased apoE levels in both the cortex (80% reduction) and the CSF (98% reduction). CSF from Abca1-/- mice had significantly reduced cholesterol as well as small apoE-containing lipoproteins, suggesting abnormal lipidation of apoE. Astrocytes, the primary producer of CNS apoE, were cultured from Abca1+/+, +/-, and -/- mice, and nascent lipoprotein particles were collected. Abca1-/- astrocytes secreted lipoprotein particles that had markedly decreased cholesterol and apoE and had smaller apoE-containing particles than particles from Abca1+/+ astrocytes. These findings demonstrate that ABCA1 plays a critical role in CNS apoE metabolism. Since apoE isoforms and levels strongly influence Alzheimer's disease pathology and risk, these data suggest that ABCA1 may be a novel therapeutic target.     

Detergent-mediated phospholipidation of plasma lipoproteins increases HDL cholesterophilicity and cholesterol efflux via SR-BI

Cellular cholesterol efflux is an early, obligatory step in reverse cholesterol transport, the putative antiatherogenic mechanism by which human plasma high-density lipoproteins (HDL) transport cholesterol from peripheral tissue to the liver for recycling or disposal. HDL-phospholipid content is the essential cholesterol-binding component of lipoproteins and therefore a major determinant of cholesterol efflux. Thus, increased phospholipidation of lipoproteins, particularly HDL, is one strategy for increasing cholesterol efflux. This study validates a simple, new detergent perturbation method for the phospholipidation of plasma lipoproteins; we have quantified the cholesterophilicity of human plasma lipoproteins and the effects of lipoprotein phospholipidation on cholesterophilicity and cellular cholesterol efflux mediated by the class B type I scavenger receptor (SR-BI). We determined that low-density lipoproteins (LDL) are more cholesterophilic than HDL and that LDL has a higher affinity for phospholipids than HDL whereas HDL has a higher phospholipid capacity than LDL. Phospholipidation of total human plasma lipoproteins enhances cholesterol efflux, an effect that occurs largely through the preferential phospholipidation of HDL. We conclude that increasing HDL phospholipid increases its cholesterophilicity, thereby making it a better acceptor of cellular cholesterol efflux. Phospholipidation of lipoproteins by detergent perturbation is a simple way to increase HDL cholesterophilicity and cholesterol efflux in a way that may be clinically useful.     

Carboxyl terminus of apolipoprotein A-I (ApoA-I) is necessary for the transport of lipid-free ApoA-I but not prelipidated ApoA-I particles through aortic endothelial cells

High density lipoproteins (HDL) and apolipoprotein A-I (apoA-I) must leave the circulation and pass the endothelium to exert their atheroprotective actions in the arterial wall. We previously demonstrated that the transendothelial transport of apoA-I involves ATP-binding cassette transporter (ABC) A1 and re-secretion of lipidated particles. Transendothelial transport of HDL is modulated by ABCG1 and the scavenger receptor BI (SR-BI). We hypothesize that apoA-I transport is started by the ABCA1-mediated generation of a lipidated particle which is then transported by ABCA1-independent pathways. To test this hypothesis we analyzed the endothelial binding and transport properties of initially lipid-free as well as prelipidated apoA-I mutants. Lipid-free apoA-I mutants with a defective carboxyl-terminal domain showed an 80% decreased specific binding and 90% decreased specific transport by aortic endothelial cells. After prior cell-free lipidation of the mutants, the resulting HDL-like particles were transported through endothelial cells by an ABCG1- and SR-BI-dependent process. ApoA-I mutants with deletions of either the amino terminus or both the amino and carboxyl termini showed dramatic increases in nonspecific binding but no specific binding or transport. Prior cell-free lipidation did not rescue these anomalies. Our findings of stringent structure-function relationships underline the specificity of transendothelial apoA-I transport and suggest that lipidation of initially lipid-free apoA-I is necessary but not sufficient for specific transendothelial transport. Our data also support the model of a two-step process for the transendothelial transport of apoA-I in which apoA-I is initially lipidated by ABCA1 and then further processed by ABCA1-independent mechanisms.     

Pitavastatin increases ABCA1-mediated lipid efflux from Fu5AH rat hepatoma cells

ATP binding cassette A1 (ABCA1) is responsible in vivo for the formation of HDL by promoting the lipidation of apoprotein A-I (apoA-I) via cholesterol and phospholipid efflux from the liver. Treatment of patients with statins produces an increase in HDL plasma level, but the underlying mechanism is not completely understood. In this work we investigated the ability of pitavastatin to modulate ABCA1-mediated efflux from Fu5AH rat hepatoma cells, that here we demonstrate to express functional ABCA1 upon treatment with 22OH/cRA. In both basal and ABCA1 expressing cells pitavastatin 0.1-50microM induced a dose-dependent increase in cholesterol efflux to apoA-I; this effect was reversed by mevalonate or geranyl geraniol. A stimulatory effect was also observed on phospholipid efflux. Similar results were obtained with compactin, suggesting a class-related effect of statins. These results indicate a potential mechanism for the improvement in HDL plasma profile observed in patients treated with statins.     

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.     

Preferential ATP-binding cassette transporter A1-mediated cholesterol efflux from late endosomes/lysosomes

Recently, ATP-binding cassette transporter A1 (ABCA1), the defective molecule in Tangier disease, has been shown to stimulate phospholipid and cholesterol efflux to apolipoprotein A-I (apoA-I); however, little is known concerning the cellular cholesterol pools that act as the source of cholesterol for ABCA1-mediated efflux. We observed a higher level of isotopic and mass cholesterol efflux from mouse peritoneal macrophages labeled with [(3)H]cholesterol/acetyl low density lipoprotein (where cholesterol accumulates in late endosomes and lysosomes) compared with cells labeled with [(3)H]cholesterol with 10% fetal bovine serum, suggesting that late endosomes/lysosomes act as a preferential source of cholesterol for ABCA1-mediated efflux. Consistent with this idea, macrophages from Niemann-Pick C1 mice that have an inability to exit cholesterol from late endosomes/lysosomes showed a profound defect in cholesterol efflux to apoA-I. In contrast, phospholipid efflux to apoA-I was normal in Niemann-Pick C1 macrophages, as was cholesterol efflux following plasma membrane cholesterol labeling. These results suggest that cholesterol deposited in late endosomes/lysosomes preferentially acts as a source of cholesterol for ABCA1-mediated cholesterol efflux.     

The carboxy-terminal region of apoA-I is required for the ABCA1-dependent formation of alpha-HDL but not prebeta-HDL particles in vivo

ATP-binding cassette transporter A-1 (ABCA1)-mediated lipid efflux to lipid-poor apolipoprotein A-I (apoA-I) results in the gradual lipidation of apoA-I. This leads to the formation of discoidal high-density lipoproteins (HDL), which are subsequently converted to spherical HDL by the action of lecithin:cholesterol acyltransferase (LCAT). We have investigated the effect of point mutations and deletions in the carboxy-terminal region of apoA-I on the biogenesis of HDL using adenovirus-mediated gene transfer in apoA-I-deficient mice. It was found that the plasma HDL levels were greatly reduced in mice expressing the carboxy-terminal deletion mutants apoA-I[Delta(185-243)] and apoA-I[Delta(220-243)], shown previously to diminish the ABCA1-mediated lipid efflux. The HDL levels were normal in mice expressing the WT apoA-I, the apoA-I[Delta(232-243)] deletion mutant, or the apoA-I[E191A/H193A/K195A] point mutant, which promote normal ABCA1-mediated lipid efflux. Electron microscopy and two-dimensional gel electrophoresis showed that the apoA-I[Delta(185-243)] and apoA-I[Delta(220-243)] mutants formed mainly prebeta-HDL particles and few spherical particles enriched in apoE, while WT apoA-I, apoA-I[Delta(232-243)], and apoA-I[E191A/H193A/K195A] formed spherical alpha-HDL particles. The findings establish that (a) deletions that eliminate the 220-231 region of apoA-I prevent the synthesis of alpha-HDL but allow the synthesis of prebeta-HDL particles in vivo, (b) the amino-terminal segment 1-184 of apoA-I can promote synthesis of prebeta-HDL-type particles in an ABCA1-independent process, and (c) the charged residues in the 191-195 region of apoA-I do not influence the biogenesis of HDL.     

Structure-function relationships in reconstituted HDL: Focus on antioxidative activity and cholesterol efflux capacity

AimsHigh-density lipoprotein (HDL) contains multiple components that endow it with biological activities. Apolipoprotein A-I (apoA-I) and surface phospholipids contribute to these activities; however, structure-function relationships in HDL particles remain incompletely characterised.MethodsReconstituted HDLs (rHDLs) were prepared from apoA-I and soy phosphatidylcholine (PC) at molar ratios of 1:50, 1:100 and 1:150. Oxidative status of apoA-I was varied using controlled oxidation of Met112 residue. HDL-mediated inactivation of PC hydroperoxides (PCOOH) derived from mildly pre-oxidized low-density lipoprotein (LDL) was evaluated by HPLC with chemiluminescent detection in HDL + LDL mixtures and re-isolated LDL. Cellular cholesterol efflux was characterised in RAW264.7 macrophages.ResultsrHDL inactivated LDL-derived PCOOH in a dose- and time-dependent manner. The capacity of rHDL to both inactivate PCOOH and efflux cholesterol via ATP-binding cassette transporter A1 (ABCA1) increased with increasing apoA-I/PC ratio proportionally to the apoA-I content in rHDL. Controlled oxidation of apoA-I Met112 gradually decreased PCOOH-inactivating capacity of rHDL but increased ABCA1-mediated cellular cholesterol efflux.ConclusionsIncreasing apoA-I content in rHDL enhanced its antioxidative activity towards oxidized LDL and cholesterol efflux capacity via ABCA1, whereas oxidation of apoA-I Met112 decreased the antioxidative activity but increased the cholesterol efflux. These findings provide important considerations in the design of future HDL therapeutics.Non-standard abbreviations and acronyms: AAPH, 2,2′-azobis(-amidinopropane) dihydrochloride; ABCA1, ATP-binding cassette transporter A1; apoA-I, apolipoprotein A-I; BHT, butylated hydroxytoluene; CV, cardiovascular; EDTA, ethylenediaminetetraacetic acid; HDL-C, high-density lipoprotein cholesterol; LOOH, lipid hydroperoxides; Met(O), methionine sulfoxide; Met112, methionine 112 residue; Met86, methionine 86 residue; oxLDL, oxidized low-density lipoprotein; PBS, phosphate-buffered saline; PC, phosphatidylcholine; PL, phospholipid; PCOOH, phosphatidylcholine hydroperoxide; PLOOH, phospholipid hydroperoxide.     

On the hepatic mechanism of HDL assembly by the ABCA1/apoA-I pathway

The mechanism for the assembly of HDL with cellular lipid by ABCA1 and helical apolipoprotein was investigated in hepatocytes. Both HepG2 cells and mouse primary culture hepatocytes produced HDL with apolipoprotein A-I (apoA-I) whether endogenously synthesized or exogenously provided. Probucol, an ABCA1 inactivator, inhibited these reactions, as well as the reversible binding of apoA-I to HepG2. Primary cultured hepatocytes of ABCA1-deficient mice also lacked HDL production regardless of the presence of exogenous apoA-I. HepG2 cells secreted apoA-I into the medium even when ABCA1 was inactivated by probucol, but it was all in a free form as HDL production was inhibited. When a lipid-free apoA-I-specific monoclonal antibody, 725-1E2, was present in the culture medium, production of HDL was suppressed, whether with endogenous or exogenously added apoA-I, and the antibody did not influence HDL already produced by HepG2 cells. We conclude that the main mechanism for HDL assembly by endogenous apoA-I in HepG2 cells is an autocrine-like reaction in which apoA-I is secreted and then interacts with cellular ABCA1 to generate HDL.     

The interaction of ApoA-I and ABCA1 triggers signal transduction pathways to mediate efflux of cellular lipids

Reverse cholesterol transport (RCT) has been characterized as a crucial step for antiatherosclerosis, which is initiated by ATP-binding cassette A1 (ABCA1) to mediate the efflux of cellular phospholipids and cholesterol to lipid-free apolipoprotein A-I (apoA-I). However, the mechanisms underlying apoA-I/ABCA1 interaction to lead to the lipidation of apoA-I are poorly understood. There are several models proposed for the interaction of apoA-I with ABCA1 as well as the lipidation of apoA-I mediated by ABCA1. ApoA-I increases the levels of ABCA1 protein markedly. In turn, ABCA1 can stabilize apoA-I. The interaction of apoA-I with ABCA1 could activate signaling molecules that modulate posttranslational ABCA1 activity or lipid transport activity. The key signaling molecules in these processes include protein kinase A (PKA), protein kinase C (PKC), Janus kinase 2 (JAK2), Rho GTPases and Ca²⁺, and many factors also could influence the interaction of apoA-I with ABCA1. This review will summarize these mechanisms for the apoA-I interaction with ABCA1 as well as the signal transduction pathways involved in these processes.     

Biogenesis of HDL by SAA is dependent on ABCA1 in the liver in vivo

Serum amyloid A (SAA) was markedly increased in the plasma and in the liver upon acute inflammation induced by intraperitoneal injection of lipopolysaccharide (LPS) in mice, and SAA in the plasma was exclusively associated with HDL. In contrast, no HDL was present in the plasma and only a small amount of SAA was found in the VLDL/LDL fraction (d < 1.063 g/ml) after the induction of inflammation in ABCA1-knockout (KO) mice, although SAA increased in the liver. Primary hepatocytes isolated from LPS-treated wild-type (WT) and ABCA1-KO mice both secreted SAA into the medium. SAA secreted from WT hepatocytes was associated with HDL, whereas SAA from ABCA1-KO hepatocytes was recovered in the fraction that was >1.21 g/ml. The behavior of apolipoprotein A-I (apoA-I) was the same as that of SAA in HDL biogenesis by WT and ABCA1-KO mouse hepatocytes. Lipid-free SAA and apoA-I both stabilized ABCA1 and caused cellular lipid release in WT mouse-derived fibroblasts, but not in ABCA1-KO mouse-derived fibroblasts, in vitro when added exogenously. We conclude that both SAA and apoA-I generate HDL largely in hepatocytes only in the presence of ABCA1, likely being secreted in a lipid-free form to interact with cellular ABCA1. In the absence of ABCA1, nonlipidated SAA is seemingly removed rapidly from the extracellular space.     

Membrane microdomains modulate oligomeric ABCA1 function: impact on apoAI-mediated lipid removal and phosphatidylcholine biosynthesis

Recent studies have identified an ABCA1-dependent, phosphatidylcholine-rich microdomain, called the “high-capacity binding site” (HCBS), that binds apoA-I and plays a pivotal role in apoA-I lipidation. Here, using sucrose gradient fractionation, we obtained evidence that both ABCA1 and [¹²⁵I]apoA-I associated with the HCBS were found localized to nonraft microdomains. Interestingly, phosphatidylcholine (PtdCho) was selectively removed from nonraft domains by apoA-I, whereas sphingomyelin and cholesterol were desorbed from both detergent-resistant membranes and nonraft domains. The modulatory role of cholesterol on apoA-I binding to ABCA1/HCBS was also examined. Loading cells with cholesterol resulted in a drastic reduction in apoA-I binding. Conversely, depletion of membrane cholesterol by methyl-β-cyclodextrin treatment resulted in a significant increase in apoA-I binding. Finally, we obtained evidence that apoA-I interaction with ABCA1 promoted the activation and gene expression of key enzymes in the PtdCho biosynthesis pathway. Taken together, these results provide strong evidence that the partitioning of ABCA1/HCBS to nonraft domains plays a pivotal role in the selective desorption of PtdCho molecules by apoA-I, allowing an optimal environment for cholesterol release and regeneration of the PtdCho-containing HCBS. This process may have important implications in preventing and treating atherosclerotic cardiovascular disease.     

Structural modification of plasma HDL by phospholipids promotes efficient ABCA1-mediated cholesterol release

It has been suggested that ABCA1 interacts preferentially with lipid-poor apolipoprotein A-I (apoA-I). Here, we show that treatment of plasma with dimyristoyl phosphatidylcholine (DMPC) multilamellar vesicles generates prebeta(1)-apoA-I-containing lipoproteins (LpA-I)-like particles similar to those of native plasma. Isolated prebeta(1)-LpA-I-like particles inhibited the binding of (125)I-apoA-I to ABCA1 more efficiently than HDL(3) (IC(50) = 2.20 +/- 0.35 vs. 37.60 +/- 4.78 microg/ml). We next investigated the ability of DMPC-treated plasma to promote phospholipid and unesterified (free) cholesterol efflux from J774 macrophages stimulated or not with cAMP. At 2 mg DMPC/ml plasma, both phospholipid and free cholesterol efflux were increased ( approximately 50% and 40%, respectively) in cAMP-stimulated cells compared with unstimulated cells. Similarly, both phospholipid and free cholesterol efflux to either isolated native prebeta(1)-LpA-I and prebeta(1)-LpA-I-like particles were increased significantly in stimulated cells. Furthermore, glyburide significantly inhibited phospholipid and free cholesterol efflux to DMPC-treated plasma. Removal of apoA-I-containing lipoproteins from normolipidemic plasma drastically reduced free cholesterol efflux mediated by DMPC-treated plasma. Finally, treatment of Tangier disease plasma with DMPC affected the amount of neither prebeta(1)-LpA-I nor free cholesterol efflux. These results indicate that DMPC enrichment of normal plasma resulted in the redistribution of apoA-I from alpha-HDL to prebeta-HDL, allowing for more efficient ABCA1-mediated cellular lipid release. Increasing the plasma prebeta(1)-LpA-I level by either pharmacological agents or direct infusions might prevent foam cell formation and reduce atherosclerotic vascular disease.     

Akt Inhibition Promotes ABCA1-Mediated Cholesterol Efflux to ApoA-I through Suppressing mTORC1

ATP-binding cassette transporter A1 (ABCA1) plays an essential role in mediating cholesterol efflux to apolipoprotein A-I (apoA-I), a major housekeeping mechanism for cellular cholesterol homeostasis. After initial engagement with ABCA1, apoA-I directly interacts with the plasma membrane to acquire cholesterol. This apoA-I lipidation process is also known to require cellular signaling processes, presumably to support cholesterol trafficking to the plasma membrane. We report here that one of major signaling pathways in mammalian cells, Akt, is also involved. In several cell models that express ABCA1 including macrophages, pancreatic beta cells and hepatocytes, inhibition of Akt increases cholesterol efflux to apoA-I. Importantly, Akt inhibition has little effect on cells expressing non-functional mutant of ABCA1, implicating a specific role of Akt in ABCA1 function. Furthermore, we provide evidence that mTORC1, a major downstream target of Akt, is also a negative regulator of cholesterol efflux. In cells where mTORC1 is constitutively activated due to tuberous sclerosis complex 2 deletion, cholesterol efflux to apoA-I is no longer sensitive to Akt activity. This suggests that Akt suppresses cholesterol efflux through mTORC1 activation. Indeed, inhibition of mTORC1 by rapamycin or Torin-1 promotes cholesterol efflux. On the other hand, autophagy, one of the major pathways of cholesterol trafficking, is increased upon Akt inhibition. Furthermore, Akt inhibition disrupts lipid rafts, which is known to promote cholesterol efflux to apoA-I. We therefore conclude that Akt, through its downstream targets, mTORC1 and hence autophagy, negatively regulates cholesterol efflux to apoA-I.