Inhibition of cholesterol efflux by 7-ketocholesterol: comparison between cells, plasma membrane vesicles, and liposomes as cholesterol donors.

Cholesterol removal from lipid-loaded macrophages is an important, potentially antiatherogenic process, and we have previously shown that an oxysterol, 7-ketocholesterol (7K), can impair efflux to lipid-free apoprotein A-1 (apoA-1). This publication investigates whether incorporation of 7K into membranes could account for this impairment of cholesterol efflux. Cholesterol efflux was studied from lipoprotein-loaded THP-1 cells, from plasma membrane vesicles obtained from these cells, and from artificial, protein-free liposomes. Impairment of cholesterol efflux by 7K was observed for all cholesterol donor systems whether measured as decline in cholesterol removal rates or as the percentage mass of total cellular cholesterol exported. 7-Ketocholesterol itself was not removed by apoA-1 from any of the cholesterol donor systems. Increasing membrane cholesterol content increased the rate of cholesterol removal by apoA-1 (as seen with plasma membrane vesicles), the quantity of cholesterol removed at equilibrium (liposomes), or both (whole cells). Although the minimum inhibitory 7K concentrations varied between the cholesterol donor systems, 7K inhibited cholesterol efflux in all systems. It was concluded that 7K induces alteration in membranes which decreased the efficiency of cholesterol efflux and the quantity of removed cholesterol induced by apoA-1. As cell membrane proteins are not essential for cholesterol efflux in these systems, the impairment of such by 7K suggests that its effect on membrane lipid composition and its structure are key regulatory elements in this efflux process.     

Interaction of ApoA-1 and ApoE-3 with triglyceride-phospholipid emulsions containing increasing cholesterol concentrations. Model of triglyceride-rich nascent and remnant lipoproteins

The cholesterol content of triglyceride-rich lipoproteins increases during their catabolism in circulation. We therefore studied the binding of the exchangeable apoprotein apoA-1 and apoE-3 to triolein-rich emulsions with increasing cholesterol content. Five emulsion systems containing 83.1-88.8% (w/w) triolein, 9.3-10.1% egg yolk phosphatidylcholine, and 1.1-7.3% cholesterol were isolated from sonicated lipid mixtures by flotation. Negative stain EM of emulsions containing 1.1 and 7.3% cholesterol showed polydisperse populations of large spherical particles with diameters of 106 +/- 39 and 108 +/- 57 nm. These values are similar to particle diameters calculated from the lipid composition data. No lamellar structures were observed by EM, even after addition of apoA-1 at a molar ratio to lecithin of 10(-2). Apolipoproteins apoA-1 and apoE-3 bound to the particles in a saturable manner without altering particle morphology. We found a dissociation constant Kd = 7.4 x 10(-7) M and a binding capacity N = 3.9 x 10(-3) proteins/lecithin for apoA-1 with particles containing 1.1% cholesterol; the Kd and N values for apoE-3 were very similar. When the emulsion particles were saturated with cholesterol at 7.3%, the protein binding capacity N sharply decreased to 0.6 x 10(-3) (apoA-1) and 0.7 x 10(-3) proteins/lecithin (apoE-3), but the Kd values were virtually unchanged. No change in N occurred when the particle cholesterol content was increased from 1.1 to 3.7%, which spans the normal physiological range. These results suggest that increases in lipoprotein cholesterol content above 3.7% may be responsible for impaired apoprotein redistribution and altered metabolism of remnants such as beta-VLDL.     

β-COP as a Component of Transport Vesicles for HDL Apolipoprotein-Mediated Cholesterol Exocytosis

Objective

HDL and its apolipoproteins protect against atherosclerotic disease partly by removing excess cholesterol from macrophage foam cells. But the underlying mechanisms of cholesterol clearance are still not well defined. We investigated roles of vesicle trafficking of coatomer β-COP in delivering cholesterol to the cell surface during apoA-1 and apoE-mediated lipid efflux from fibroblasts and THP-1 macrophages.

Methods

shRNA knockout, confocal and electron microscopy and biochemical analysis were used to investigate the roles of β-COP in apolipoprotein-mediated cholesterol efflux in fibroblasts and THP-1 macrophages.

Results

We showed that β-COP knockdown by lentiviral shRNA resulted in reduced apoA-1-mediated cholesterol efflux, while increased cholesterol accumulation and formation of larger vesicles were observed in THP-1 macrophages by laser scanning confocal microscopy. Immunogold electron microscopy showed that β-COP appeared on the membrane protrusion complexes and colocalized with apoA-1 or apoE during cholesterol efflux. This was associated with releasing heterogeneous sizes of small particles into the culture media of THP-1 macrophage. Western blotting also showed that apoA-1 promotes β-COP translocation to the cell membrane and secretion into culture media, in which a total of 17 proteins were identified by proteomics. Moreover, β-COP exclusively associated with human plasma HDL fractions.

Conclusion

ApoA-1 and apoE promoted transport vesicles consisting of β-COP and other candidate proteins to exocytose cholesterol, forming the protrusion complexes on cell surface, which were then released from the cell membrane as small particles to media.     

Apolipoprotein A-1 (apoA-1) deposition in, and release from, the enterocyte brush border: a possible role in transintestinal cholesterol efflux (TICE)?

Transintestinal cholesterol efflux (TICE) has been proposed to represent a non-hepatobiliary route of cholesterol secretion directly "from blood to gut" and to play a physiologically significant role in excretion of neutral sterols, but so far little is known about the proteins involved in the process. We have previously observed that apolipoprotein A-1 (apoA-1) synthesized by enterocytes of the small intestine is mainly secreted apically into the gut lumen during fasting where its assembly into chylomicrons and basolateral discharge is at a minimal level. In the present work we showed, both by immunomicroscopy and subcellular fractionation, that a fraction of the apically secreted apoA-1 in porcine small intestine was not released from the cell surface but instead deposited in the brush border. Cholesterol was detected in immunoisolated microvillar apoA-1, and it was partially associated with detergent resistant membranes (DRMs), indicative of localization in lipid raft microdomains. The apolipoprotein was not readily released from microvillar vesicles by high salt or by incubation with phosphatidylcholine-specific phospholipase C or trypsin, indicating a relatively firm attachment to the membrane bilayer. However, whole bile or taurocholate efficiently released apoA-1 at low concentrations that did not solubilize the transmembrane microvillar protein aminopeptidase N. Based on these findings and the well known role played by apoA-1 in extrahepatic cellular cholesterol removal and reverse cholesterol transport (RCT), we propose that brush border-deposited apoA-1 in the small intestine acts in TICE by mediating cholesterol efflux into the gut lumen.     

Kinetic stabilization and fusion of apolipoprotein A-2:DMPC disks: comparison with apoA-1 and apoC-1

Denaturation studies of high-density lipoproteins (HDL) containing human apolipoprotein A-2 (apoA-2) and dimyristoyl phosphatidylcholine indicate kinetic stabilization. Circular dichroism (CD) and light-scattering melting curves show hysteresis and scan rate dependence, indicating thermodynamically irreversible transition with high activation energy E(a). CD and light-scattering data suggest that protein unfolding triggers HDL fusion. Electron microscopy, gel electrophoresis, and differential scanning calorimetry show that such fusion involves lipid vesicle formation and dissociation of monomolecular lipid-poor protein. Arrhenius analysis reveals two kinetic phases, a slower phase with E(a,slow) = 60 kcal/mol and a faster phase with E(a,fast) = 22 kcal/mol. Only the fast phase is observed upon repetitive heating, suggesting that lipid-poor protein and protein-containing vesicles have lower kinetic stability than the disks. Comparison of the unfolding rates and the melting data recorded by differential scanning calorimetry, CD, and light scattering indicates the rank order for the kinetic disk stability, apoA-1 > apoA-2 > apoC-1, that correlates with protein size rather than hydrophobicity. This contrasts with the tighter association of apoA-2 than apoA-1 with mature HDL, suggesting different molecular determinants for stabilization of model discoidal and plasma spherical HDL. Different effects of apoA-2 and apoA-1 on HDL fusion and stability may reflect different metabolic properties of apoA-2 and/or apoA-1-containing HDL.     

Helical domains that mediate lipid solubilization and ABCA1-specific cholesterol efflux in apolipoproteins C-I and A-II

Many of the apolipoproteins in HDL can elicit cholesterol efflux via ABCA1, a critical initial step in HDL formation. Recent work has indicated that omnipresent amphipathic helices play a critical role, and these have been studied intensively in the most common HDL protein, apolipoprotein (apo)A-I. However, little information exists about helical domain arrangement in other apolipoproteins. We studied two of the smallest apolipoproteins known to interact with ABCA1, human apoA-II and apoC-I, in terms of ability to reorganize phospholipid (PL) bilayers and to promote ABCA1-mediated cholesterol. We found that both proteins contained helical domains that were fast and slow with respect to solubilizing PL. ABCA1-medated efflux required a minimum of a bihelical polypeptide comprised of at least one each of a slow and fast lipid reorganizing domain. In both proteins, the fast helix was located at the C terminus preceded by a slow helix. Helical placement in apoC-I was not critical for ABCA1 activity, but helix swaps in apoA-II dramatically disrupted cholesterol efflux, indicating that the tertiary structure of the longer apolipoprotein is important for the pathway. This work has implications for a more complete molecular understanding of apolipoprotein-mediated cholesterol efflux.     

Prdx2 Inhibits Macrophage Lipid Accumulation Through ROS-NF-κB-miR-33a-ABCA1 Pathway

Previous studies have unraveled that peroxiredoxin 2 (Prdx2) inhibits atherogenesis in mice, whereas its role in macrophage lipid accumulation or the underlying mechanisms remain unknown. THP-1 monocyte-derived foam cells were transfected with Prdx2-overexpressing plasmid vectors (pcDNA3.1-Prdx2) or Prdx2 siRNA. The expression of ABCA1, NF-κB p65 and miR-33a were detected by RT-PCR and Western blotting. Percentage of cholesterol efflux was evaluated by liquid scintillation counting. Cellular lipid droplets were assessed using Oil Red O staining. Intracellular cholesterol contents were measured using high performance liquid chromatography (HPLC). Furthermore, cells were pre-treated with NF-κB inhibitor PDTC and/or miR-33a inhibitor, followed by detection of the indices above. The results showed that overexpression of Prdx2 in THP-1 monocyte-derived foam cells significantly increased ABCA1 expression and the percentage of ［³H］-cholesterol efflux to apoA-1 (P＜0.05), whereas NF-κB p65 and miR-33a levels as well as lipid accumulation were decreased (P＜0.05). After pre-treatment with PDTC and/or miR-33a inhibitor, these effects were more obvious (P＜0.05). In contrast, silencing of Prdx2 significantly diminished ABCA1 expression and increased NF-κB p65 and miR-33a levels. At last, we found that Prdx2 overexpression obviously down-regulated the ROS level in THP-1 monocyte-derived foam cells. Altogether, Prdx2 promotes macrophage cholesterol efflux and inhibits intracellular lipid accumulation through the ROS-NF-κB-miR-33a-ABCA1 pathway.     

ABCA1 mediates concurrent cholesterol and phospholipid efflux to apolipoprotein A-I

Prior studies provide data supporting the notion that ATP binding cassette transporter A1 (ABCA1) promotes lipid efflux to extracellular acceptors in a two-step process: first, ABCA1 mediates phospholipid efflux to an apolipoprotein, and second, this apolipoprotein-phospholipid complex accepts free cholesterol in an ABCA1-independent manner. In the current study using RAW264.7 cells, ABCA1-mediated free cholesterol and phospholipid efflux to apolipoprotein A-I (apoA-I) were tightly coupled to each other both temporally and after treatment with ABCA1 inhibitors. The time course and temperature dependence of ABCA1-mediated lipid efflux to apoA-I support a role for endocytosis in this process. Cyclodextrin treatment of RAW264.7 cells partially inhibited 8Br-cAMP-induced efflux of free cholesterol and phospholipid to apoA-I. ABCA1-expressing cells are more sensitive to cell damage by high-dose cyclodextrin and vanadate, leading to increased lactate dehydrogenase leakage and phospholipid release even in the absence of the acceptor apoA-I. Finally, we could not reproduce a two-step effect on lipid efflux using conditioned medium from ABCA1-expressing cells pretreated with cyclodextrin.     

Cholesterol secretion and homeostasis in chondrocytes: a liver X receptor and retinoid X receptor heterodimer mediates apolipoprotein A1 expression.

Cholesterol is required for chondrocyte differentiation and bone formation. Apolipoprotein A1 (apoA-1) plays a major role in lipoprotein clearance and cholesterol redistribution. We report here that apoA-1 is expressed during chondrocyte differentiation in vitro and in vivo. In differentiating chondrocytes, the expression of the liver X receptor (LXR) is modulated and its expression correlates to the expression of apoA-1. The expression of other LXR target genes related to cholesterol homeostasis such as ABCA1 cholesterol transporter and sterol regulatory element-binding protein 1 (SREBP1) is similarly regulated. Small molecule ligands activating either LXR or retinoid X receptor (RXR) lead to a dramatic increase in apoA-1 mRNA and protein expression in cultured chondrocytes. These ligands strongly induce ABCA1 cholesterol transporter expression and effectively mediate cholesterol efflux from hypertrophic chondrocytes. In addition, we report that, in the same cells, the ligands down modulate Serum Amyloid A expression induced by bacterial lipopolysaccharide. Our studies provide evidence that LXR/RXR mediate a fine regulation of cholesterol homeostasis in differentiating chondrocytes.     

Ceramide structural features required to stimulate ABCA1-mediated cholesterol efflux to apolipoprotein A-I

Ceramide is a component of the sphingomyelin cycle and a well-established lipid signaling molecule. We recently reported that ceramide specifically increased ABCA1-mediated cholesterol efflux to apolipoprotein A-I (apoA-I), a critical process that leads to the formation of cardioprotective HDL. In this report, we characterize the structural features of ceramide required for this effect. C2 dihydroceramide, which contains a fully saturated acyl chain and is commonly used as a negative control for ceramide apoptotic signaling, stimulated a 2- to 5-fold increase in ABCA1-mediated cholesterol efflux to apoA-I over a 0-60 muM concentration range without the cell toxicity apparent with native C2 ceramide. Compared with C2 ceramide, C6 and C8 ceramides with medium-length N-acyl chains showed a similar extent of efflux stimulation (a 2- to 5-fold increase) but at a higher onset concentration than the less hydrophobic C2 ceramide. In contrast, the reduced and methylated ceramide analogs, N,N-dimethyl sphingosine and N,N,N-trimethyl sphingosine, failed to stimulate cholesterol efflux. We found that changes in the native spatial orientation at either of two chiral carbon centers (or both) resulted in an approximately 50% decrease compared with native ceramide-stimulated cholesterol efflux. These data show that the overall ceramide shape and the amide bond are critical for the cholesterol efflux effect and suggest that ceramide acts through a protein-mediated pathway to affect ABCA1 activity.     

Efflux of lipid from fibroblasts to apolipoproteins: dependence on elevated levels of cellular unesterified cholesterol.

Earlier work from this laboratory showed that enrichment of cells with free cholesterol enhanced the efflux of phospholipid to lipoprotein acceptors, suggesting that cellular phospholipid may contribute to high density lipoprotein (HDL) structure and the removal of sterol from cells. To test this hypothesis, we examined the efflux of [3H]cholesterol (FC) and [32P]phospholipid (PL) from control and cholesterol-enriched fibroblasts to delipidated apolipoproteins. The percentages of [3H]cholesterol and [32P]phospholipid released from control cells to human apolipoprotein A-I were 2.2 +/- 0.5%/24 h and 0.8 +/- 0.1%/24 h, respectively. When the cellular cholesterol content was doubled, efflux of both lipids increased substantially ([3H]FC efflux = 14.6 +/- 3.6%/24 h and [32P]PL efflux = 4.1 +/- 0.3%/24 h). Phosphatidylcholine accounted for 70% of the radiolabeled phospholipid released from cholesterol-enriched cells. The cholesterol to phospholipid molar ratio of the lipid released from cholesterol-enriched cells was approximately 1. This ratio remained constant throughout an incubation time of 3 to 48 h, suggesting that there was a coordinate release of both lipids. The concentrations of apoA-I, A-II, A-IV, E, and Cs that promoted half-maximal efflux of phospholipid from cholesterol-enriched fibroblasts were 53, 30, 68, 137, and 594 nM, respectively. With apoA-I and A-IV, these values for half-maximal efflux of phospholipid were identical to the concentrations that resulted in half-maximal efflux of cholesterol. Agarose gel electrophoresis of medium containing apoA-I that had been incubated with cholesterol-enriched fibroblasts revealed a particle with alpha to pre-beta mobility. We conclude that the cholesterol content of cellular membranes is an important determinant in the ability of apolipoproteins to promote lipid removal from cells. We speculate that apolipoproteins access cholesterol-phosphatidylcholine domains within the plasma membrane of cholesterol-enriched cells, whereupon HDL is generated in the extracellular compartment. The release of cellular lipid to apolipoproteins may serve as a protective mechanism against the potentially damaging effects of excess membrane cholesterol.     

The establishment of telomerase-immortalized Tangier disease cell lines indicates the existence of an apolipoprotein A-I-inducible but ABCA1-independent cholesterol efflux pathway

Tangier disease (TD) is a human genetic disorder associated with defective apolipoprotein-I-induced lipid efflux and increased atherosclerotic susceptibility. It has been linked to mutations in the ATP-binding cassette protein A1 (ABCA1). Here we describe the establishment of permanent Tangier cell lines using telomerase. Ectopic expression of the catalytic subunit of human telomerase extended the life span of control and TD skin fibroblasts, and (in contrast to immortalization procedures using viral oncogenes) did not impair apolipoprotein A-I-induced lipid efflux. The key characteristics of TD fibroblasts (reduced cholesterol and phospholipid efflux) were observed both in primary and telomerase-immortalized fibroblasts from two unrelated homozygous patients. Surprisingly, the apolipoprotein-inducible cholesterol efflux in TD cells was significantly improved after immortalization (up to 40% of normal values). In contrast to ABCA1-dependent cholesterol efflux, this efflux was not inhibited by brefeldin A, glybenclamide, or intracellular ATP depletion but was inhibited in the presence of cytochalasin D. Apolipoprotein A-I-dependent cholesterol efflux was inversely correlated with the population doubling number in cell culture and was inhibited up to 40% in near-senescent normal diploid fibroblasts. This inhibition was completely reversed by telomerase. Thus ectopic expression of telomerase is a way to circumvent the lack of critical experimental material and represents a major improvement for studying cholesterol efflux pathways in lipid disorders. Our findings indicate the existence of an ABCA1-independent but cytoskeleton-dependent cholesterol removal pathway that may help to prevent early atherosclerosis in Tangier disease but may also be sensitive to aging phenomena ex vivo and possibly in vivo.     

Asymmetry in the lipid affinity of bihelical amphipathic peptides. A structural determinant for the specificity of ABCA1-dependent cholesterol efflux by peptides

ApoA-I contains a tandem array of amphipathic helices with varying lipid affinity, which are critical in its ability to bind and remove lipids from cells by the ABCA1 transporter. In this study, the effect of asymmetry in the lipid affinity of amphipathic helices in a bihelical apoA-I mimetic peptide, 37pA, on lipid efflux by the ABCA1 transporter was examined. Seven peptide variants of 37pA were produced by substituting a varying number of hydrophobic amino acids for alanine on either one or both helices. The 5A peptide with five alanine substitutions in the second helix had decreased helical content compared with 37pA (5A, 12+/-1% helicity; 37pA, 28+/-2% helicity) and showed less self-association but, similar to the parent peptide, was able to readily solubilize phospholipid vesicles. Furthermore, 5A, unlike the parent peptide 37pA, was not hemolytic (37pA, 27+/-2% RBC lysis, 2 h, 18 microm). Finally, the 5A peptide stimulated cholesterol and phospholipid efflux by the ABCA1 transporter with higher specificity (ABCA1-transfected versus untransfected cells) than 37pA (5A, 9.7+/-0.77%, 18 h, 18 microm versus 1.5+/-0.27%, 18 h, 18 microm (p<0.0001); 37pA, 7.4+/-0.85%, 18 h, 18 microm versus 5.8+/-0.20%, 18 h, 18 microm (p=0.03)). In summary, we describe a novel bihelical peptide with asymmetry in the lipid affinity of its helices and properties similar to apoA-I in terms of specificity for cholesterol efflux by the ABCA1 transporter and low cytotoxicity.     

Application of purging biotinylated liposomes from plasma to elucidate influx and efflux processes associated with accumulation of liposomes in solid tumors

Although small, 100-nm liposomes are known to selectively accumulate in solid tumors, the individual contributions of liposome influx and egress rates are not well understood. The aim of this work was to determine influx and efflux kinetics for 100-nm, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/cholesterol (Chol) liposomes by inducing aggregate formation of biotinylated liposomes upon administering avidin. Injecting 50 microg of neutravidin intravenously to mice that had previously been administered 100 mg/kg DPSC/Chol liposomes containing 0.5 mol% biotin-conjugated lipid resulted in >90% elimination of the liposomes from plasma within 1 h. This rapid removal by the reticuloendothelial system (RES) permitted the determination of the tumor efflux kinetics due to negligible tumor influx after neutravidin injection. The tumor efflux rate constant (k(-1)) was determined to be 0.041 h(-1) when neutravidin was injected 4 h after liposome injection. This allowed the determination of the tumor influx rate constant (k(1)), which under these conditions was 0.022 h(-1). Therefore, DSPC/Chol liposomal accumulation, in LS180 solid tumors, is dictated primarily by plasma liposome concentrations and liposome egress is comparable or slightly faster than influx into the tumors. This method is applicable for a wide range of lipid doses, and can be used to characterize influx and efflux parameters at different time points after accumulation. The application, therefore, has the potential to be used to fully characterize the impact of different liposome parameters such as lipid composition, steric stabilization, size and dose on tumor accumulation kinetics.     

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.     

The ATP-binding cassette transporter 1 mediates lipid efflux from Sertoli cells and influences male fertility

The liver X receptor/retinoid X receptor (LXR/RXR)-regulated gene ABCA1 effluxes cellular cholesterol and phospholipid to apolipoprotein A1 (apoA1), which is the rate-limiting step in high-density lipoprotein synthesis. The RXR pathway plays a critical role in testicular lipid trafficking, and RXRbeta-deficient male mice are sterile and accumulate lipids in Sertoli cells. Here, we demonstrate that ABCA1 mRNA and protein are abundant in Sertoli cells, whereas germ cells express little ABCA1. LXR/RXR agonists stimulate ABCA1 expression in cultured Sertoli MSC1 and Leydig TM3 cell lines. However, Sertoli TM4 cells lack ABCA1, and TM4 cells or primary Sertoli cells cultured from ABCA1(-/-) mice both fail to efflux cholesterol to apoA1. Expression of exogenous ABCA1 restores apoA1-dependent cholesterol efflux in Sertoli TM4 cells. In vivo, ABCA1-deficient mice exhibit lipid accumulation in Sertoli cells and depletion of normal lipid droplets from Leydig cells by 2 months of age. By 6 months of age, intratesticular testosterone levels and sperm counts are significantly reduced in ABCA1(-/-) mice compared with wild-type (WT) controls. Finally, a 21% decrease (P = 0.01) in fertility was observed between ABCA1(-/-) males compared with WT controls across their reproductive lifespans. These results show that ABCA1 plays an important role in lipid transport in Sertoli cells and influences male fertility.     

Lateral interactions of pig apolipoprotein A-1 with egg yolk phosphatidylcholine and with cholesterol in mixed monolayers at the triolein-saline interface.

Interfacial tensions of egg yolk phosphatidylcholine (PC) and cholesterol monolayers adsorbed at the triolein-saline interface were measured in the presence and absence of pig apolipoprotein A-1 (apoA-1) in the saline phase. In the absence of apoA-1, the adsorptions of PC and cholesterol at the interface from the triolein phase are cooperative, showing large lateral attractive interactions between the PC molecules and the cholesterol molecules in the monolayer. In the presence of apoA-1, the PC adsorption is anti-cooperative, indicating strong lateral attractive interactions between the PC and the apoA-1 molecules, i.e., apparently, repulsive lateral interactions between the PC molecules. On the other hand, lateral interactions of very low magnitude are observed between the cholesterol and apoA-1 molecules in the monolayer. Values of the lateral interaction energy are evaluated from the adsorption data by the Defay-Prigogine-Flory theory of monolayers. The large difference in lateral interaction energy with apoA-1 between PC and cholesterol in a mixed monolayer is discussed in connection with current problems in lipoprotein catabolism: reverse cholesterol transport, alterations in affinity of lipid particles to apoA-1, and formation of high-density lipoproteins and abnormal lipoproteins.     

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.