ABCA1 Research Articles

Cholesterol efflux mechanism revealed by structural analysis of human ABCA1 conformational states.

ATP-binding cassette transporter A1 (ABCA1) utilizes energy derived from ATP hydrolysis to export cholesterol and phospholipids from macrophages. ABCA1 plays a central role in the biosynthesis of high-density lipoprotein (HDL), which mediates reverse cholesterol transport and prevents detrimental lipid deposition. Mutations in ABCA1 cause Tangier disease characterized by a remarkable reduction in the amount of HDL in blood. Here we present cryo-electron microscopy structures of human ABCA1 in ATP-bound and nucleotide-free states. Structural comparison reveals that ATP molecules pull the nucleotide-binding domains together, inducing movements of transmembrane helices 1, 2, 7 and 8 through a series of salt-bridge interactions. Subsequently, extracellular domains (ECDs) undergo a rotation and introduce conformational changes in the ECD-transmembrane interface. In addition, while we observe a sterol-like molecule in ECDs, no such density was observed in the structure of an HDL-deficiency mutant ABCA1Y482C, demonstrating the physiological importance of ECDs and a putative interaction mode between ABCA1 and its lipid acceptors. Thus, these structures, along with cholesterol efflux assays, advance the understanding ABCA1-mediated reverse cholesterol transport.

ABCA7 rs3764650 Polymorphism is Associated with Delayed Neurocognitive Recovery.

BackgroundSeveral studies have shown that ATP-binding cassette transporter A7 (ABCA7) gene variation is associated with cognitive impairment. This study was aimed to investigate the relationship between ABCA7 rs3764650 polymorphism and perioperative neurocognitive disorder (pNCD).MethodsA total of 132 elderly patients aged 65 and over who underwent elective non-cardiac surgery were enrolled in the study, while 28 healthy volunteers matching age and sex were recruited as the control group. A battery of neuropsychological tests was conducted 1 day before, 7 days, and 3 months after surgeries. Delayed neurocognitive recovery (dNCR) and postoperative mild or major neurocognitive disorder (POCD) were determined using the Z value method. The venous blood sample of the surgical patients was taken before the operation. Genotyping of rs3764650 was performed using polymerase chain reaction amplification and restriction fragment length polymorphism analysis.ResultsThe incidences of dNCR and POCD were 29.7% and 16.8% at 7 days and 3 months after surgery, respectively. The G allele frequency and GG frequency of dNCR patients were significantly higher than that of non-dNCR patients (43.3% vs 28.2%, P=0.035; 23.3% vs 4.2%, P=0.013, respectively) at 7 days following surgery. No significant differences in ABCA7 alleles between POCD and non-POCD patients were observed 3 months postoperatively.ConclusionABCA7 rs3764650 gene polymorphism is associated with dNCR and GG genotype might be a predisposing factor for postoperative cognitive impairment in Chinese Han elderly populations.

The role of HDL- and non-HDL-related parameters in cell-cholesterol efflux capacity

Background and aimsThe regulation of cell-cholesterol efflux is not completely understood. Our aim was to assess the role of HDL- and non-HDL-related parameters in ATP-binding cassette transporter-A1 (ABCA1) and scavenger receptor class B-type-I (SRBI) cell-cholesterol efflux capacity (CEC) in coronary heart disease (CHD) cases and controls. MethodsLipids and apoA-I-containing HDL particles (by 2D gel-electrophoresis and immunodetection) were measured in 534 statin-treated CHD patients and in 1076 age-, gender-, and BMI-matched controls. ABCA1-CEC and SRBI-CEC were measured in apoB-depleted serum of 100 cases and 100 controls. ResultsCases had significantly higher concentrations of preβ-1 particles (88%) and ABCA1-CEC (34%) compared to controls. ABCA1-CEC was positively correlated with the concentrations of preβ-1 particles, triglycerides, small-dense (sd) LDL-C, and LDL-C in both cases and controls. Moreover, both the concentration and the functionality of preβ-1 particles (ABCA1-CEC/mg preβ-1) were positively associated with the concentrations of sdLDL-C and triglycerides. Cases had 27% lower levels of large HDL particles but similar SRBI-CEC compared to controls. SRBI-CEC was correlated positively with HDL-C, apoA-I, and large-HDL particle levels. However, the functionality of large-HDL particles (SRBI-CEC/mg large particles) was significantly and positively correlated with the preβ-1/α-1 ratio, sdLDL-C, and triglycerides. ConclusionsCHD patients have significantly higher concentration, but less functional preβ-1 particles in term of cholesterol efflux capacity compared to controls. Triglyceride-rich lipoproteins have significant influence on either the concentration or the functionality or both of HDL particles and consequently HDL-CEC.

Perivascular adipose-derived exosomes reduce macrophage foam cell formation through miR-382-5p and the BMP4-PPARγ-ABCA1/ABCG1 pathways

BackgroundPerivascular adipose tissue (PVAT) releases exosomes (EXOs) to regulate vascular homeostasis. PVAT-derived EXOs reduce macrophage foam cell formation, but the underlying molecular mechanism has yet to be fully elucidated. We hypothesize that PVAT release miRNA through EXOs and regulate the expression of cholesterol transporter of macrophages, thereby reducing foam cell formation.Methods and resultsThrough RT-qPCR, we identified that miR-382-5p, which was expressed at lower levels in PVAT-EXOs from coronary atherosclerotic heart disease patients than healthy individuals, was expressed at higher levels in wild-type C57BL/6 J mouse aortic PVAT-EXOs than in subcutaneous adipose tissue-derived EXOs. We explored macrophage lipid accumulation through oil red O staining, assessed cholesterol uptake and efflux, and verified cholesterol transporter expression. We found that transfection with a miR-382-5p inhibitor offset PVAT-EXO-related reductions in macrophage foam cell formation and increases in cholesterol efflux mediated by ATP-binding cassette transporter A1 (ABCA1) and ATP-binding cassette transporter G1 (ABCG1). In addition, bone morphogenetic protein 4 (BMP4) pretreatment and si-peroxisome proliferator-activated receptor γ (PPARγ) transfection showed that BMP4-PPARγ participated in PVAT-EXO-mediated upregulation of the cholesterol efflux transporters ABCA1 and ABCG1.ConclusionsPVAT-EXOs reduce macrophage foam cell formation through miR-382-5p- and BMP4-PPARγ-mediated upregulation of the cholesterol efflux transporters ABCA1 and ABCG1. This finding suggests a promising strategy for the prevention and treatment of atherosclerosis.

POS-209 OXYSTEROL-BINDING PROTEIN LIKE 7 IN CHRONIC KIDNEY DISEASE

ATP-binding cassette transporter A1 (ABCA1)-mediated, cholesterol ester-induced podocyte injury plays a major role in the progression of glomerular disease. Overexpression of ABCA1 or pharmacological induction of ABCA1 (ABCA1i) was sufficient to partially rescue glomerular injury in proteinuric mice. Interestingly, these ABCA1i’s were developed to compete specifically with oxysterol binding to oxysterol-binding protein (OSBP) like 7 (OSBPL7), a member of a group of lipid-binding proteins involved in the movement of lipids between membranes.

Effects of pre-eclampsia on HDL-mediated cholesterol efflux capacity after pregnancy

Background and aimsPreeclampsia (PE) is associated with life-long increased risk of cardiovascular disease. One of the main protective functions of high-density lipoprotein (HDL) is its role in reverse cholesterol transport. HDL-mediated cholesterol efflux capacity (CEC) is decreased during pregnancy in women with PE. Whether this persists postpartum is unknown. MethodsBasal and transporter-specific CEC were determined 6 months postpartum in women who had a normotensive (n = 44) or a PE (n = 42) pregnancy. CEC was also measured in 23 normotensive and 20 PE women for whom samples were collected 24 months postpartum. Basal, ATP-binding cassette transporter-A1 (ABCA1)- and -G1 (ABCG1)-specific CEC were primarily determined using Chinese hamster ovary cells stably expressing human ABCA1 or ABCG1, and were also assessed using a J774 mouse macrophage cell line. ResultsABCA1-specific CEC was significantly lower in women who had PE 6 months postpartum (0.57 ± 0.1 vs 0.53 ± 0.08; p < 0.05), whilst basal and ABCG1-specific efflux were not significantly different. cAMP-specific CEC in J774 cells was also lower 6 months after PE (0.85 ± 0.21 vs 0.75 ± 0.25, p < 0.05). Although apoA-I, apoE, plasminogen and PON-1 levels were not significantly different in women who had PE compared with controls, ABCA1 efflux did correlate with apoA-l, HDL-C and apoE levels after a normal, and with apoA-l and HDL-C levels after a PE pregnancy. ABCA1-specific efflux decreased in all women between 6 and 24 months postpartum, by 11 ± 1.6% in women who had a normotensive pregnancy and 9 ± 1.3% in women who had PE. After adjustment for apoA-I levels, there was no significant difference in ABCA1-specific efflux between the groups at 6 months postpartum and in normotensive women over time, but remained significantly different between 6 and 24 months in women who had PE. ConclusionsABCA1-mediated CEC is impaired 6 months postpartum after a PE pregnancy and decreases thereafter in both normotensive and PE pregnancies. ABCA1-mediated efflux is dynamic after pregnancy but is unlikely to explain the long-term increased CVD risk in women with PE.

Multifaceted Protective Effects of Hesperidin by Aromatic Hydrocarbon Receptor in Endothelial Cell Injury Induced by Benzo[a]Pyrene.

Benzo[a]pyrene (BaP) causes atherosclerosis by activating the aromatic hydrocarbon receptor (AHR) signaling pathway to trigger lipid peroxidation and inflammation, thereby promoting the development of atherosclerosis. Hesperidin (Hsd), one of the 60 flavonoids of citrus, exhibits therapeutic effects on atherosclerosis. However, its antagonistic function for BaP remains unclear. In this study, the EA.hy926 cell model was used to systematically examine the antagonistic effect of Hsd with BaP, especially in low-density lipoprotein (LDL) oxidation and transport. Results showed that Hsd could reduce BaP-induced AHR activation in mRNA and protein expression level, and reduce LDL accumulation by decreasing the BaP-induced expression of advanced glycation end products and enhancing the BaP-inhibited Adenosine Triphosphate-binding cassette transporter A1 (ABCA1) protein and mRNA expression in EA.hy926 cells. In addition, Hsd could antagonize BaP-induced interaction of reactive oxygen species and the subsequent generation of oxidized LDL and malondialdehyde. Finally, Hsd could alleviate BaP-induced inflammatory response by decreasing IL-1β and TNF-α expression. All these results suggest that Hsd suppresses LDL accumulation, oxidation, and inflammatory response, and thus strongly impedes the AHR pathway activated by BaP.

Elucidating the Structural Features of ABCA1 in its Heterogeneous Membrane Environment.

ATP Binding Cassette Transporter A1 (ABCA1) plays an integral part in Reverse Cholesterol Transport (RCT) and is critical for maintaining lipid homeostasis. One theory of lipid efflux by the transporter (alternating access) proposes that ABCA1 harbours two different conformations that provide alternating access for lipid binding and release. This is followed by sequestration via a direct interaction between ABCA1 and its partner, ApoA1. The other theory (lateral access) proposes that ABCA1 obtains lipids laterally from the membrane to form a temporary extracellular “reservoir”. This reservoir contains an isolated lipid monolayer due to the net accumulation of lipids in the exofacial leaflet. Recently, a full-length Cryo-EM structure of this 2,261-residue transmembrane protein showed its discreetly folded domains and have detected the presence of a tunnel enclosed within the extracellular domains (ECDs) but not in the TMDs, giving it an outward-facing conformation. This structure was hypothesized to substantiate the lateral access theory. Utilizing long time-scale multiple replica atomistic molecular dynamics simulations (MDS), we simulated the structure in a large heterogeneous lipid environment and found that the protein undergoes several large conformational changes in its extremities. We observed that the cavity enclosed within ATP unbound form of ABCA1 is narrow at the distal ends of TMD as well as the ECD region substantiating the “lateral access” theory. We have also characterized ABCA1 and the lipid dynamics along with the protein-lipid interactions in the heterogeneous environment, providing novel insights into understanding ABCA1 conformation at an atomistic level.

Beyond Thermal Sensation: Roles of Transient Receptor Potential Vanilloid Subfamily Member 1 and Spicy Food in Cardiometabolic Diseases

Beyond Thermal Sensation: Roles of Transient Receptor Potential Vanilloid Subfamily Member 1 and Spicy Food in Cardiometabolic Diseases

Korelasi Kadar Adiponektin dengan Kadar Kolesterol High Density Lipoprotein pada Penyandang Obes

Adiponectin and HDL cholesterol have a protective cardiovascular effect. Adiponectin levels decrease in obese people. This causes ATP-binding cassette transporter A1 expression and apolipoprotein A-I synthesis in hepatocytes to decrease, resulting in impaired HDL cholesterol synthesis. Objectives: To determined the correlation between adiponectin levels with HDL cholesterol levels in obese. Methods: This was the analytic study with a cross-sectional design of 59 obese people who met inclusion and exclusion criteria at RSUP dr. M. Djamil Padang, from January to August 2019. Research subjects were grouped into two groups based on WHO Asia Pacific body mass index (BMI) classification. First-degree obesity group (25.0 £ BMI &lt;30.0 kg/m2) and second-degree obesity group (BMI ≥ 30.0kg/ m2). Adiponectin levels were examined by the enzyme-linked immunosorbent assay (ELISA) method and HDL cholesterol levels by the enzymatic colorimetric method. Data analyzed by Pearson correlation test, significant if p &lt;0.05. Results: Research subjects 59 people (17 men, 42 women), average age 35 (8.04) years. Adiponectin levels in the first-degree obesity group were higher than second-degree obesity group, with a mean difference between groups 1.03 μg/mL (CI: 95%: 0.14 - 1.92, p = 0.02). Results showed adiponectin levels positively correlated with HDL cholesterol levels of moderate strength (r = 0.45) and statistically significant (p = &lt;0.001). The results of the population subanalysis show relatively similar patterns. First-degree obesity population (r = 0.55; p = 0.002), second-degree obesity population (r = 0.41; p = 0.026). Conclusions: Adiponectin levels correlate with HDL cholesterol levels.Keywords: adiponectin, HDL cholesterol, obesity

A high-fat/high-cholesterol diet, but not high-cholesterol alone, increases free cholesterol and apoE-rich HDL serum levels in rats and upregulates hepatic ABCA1 expression

A high-fat/high-cholesterol (HFC) diet, but not a high-cholesterol (HC) diet, is known to induce elevated serum apolipoprotein E (apoE)-rich high-density lipoprotein (HDL) levels in animal models. However, the exact mechanisms by which the combination of dietary fat and cholesterol induces apoE-rich HDL production is not well understood. Here, we investigated the effects of dietary fat and cholesterol on serum lipoprotein profiles and hepatic mRNA expression that are associated with HDL production, cholesterol transport, and bile acid metabolism. Male Sprague-Dawley rats were fed HFC, HC, high-fat, or control diets and then evaluated. The HFC diet induced significant increases in hepatic free-cholesterol accumulation (1.4-fold, p < 0.01) and serum apoE-rich HDL cholesterol (8.7-fold, p < 0.001) levels compared with the HC diet. The apoE-rich HDL induced by the HFC diet was remarkably rich in free cholesterol. Liver gene-expression was mostly similar between the HC and HFC diet groups. However, there was a significant increase of ATP-binding cassette transporter A1 (ABCA1) levels in the HFC group compared to the HC group for both mRNA (1.9-fold, p < 0.001) and protein (6.6-fold, p < 0.01). These results suggest that an increase in apoE-rich HDL induced by dietary fat and cholesterol may be involved in cholesterol efflux from the liver through increased ABCA1-mediated free-cholesterol efflux.

Acid-Sensing Ion Channel 1/Calpain1 Activation Impedes Macrophage ATP-Binding Cassette Protein A1-Mediated Cholesterol Efflux Induced by Extracellular Acidification.

BackgroundExtracellular acidification is a common feature of atherosclerotic lesions, and such an acidic microenvironment impedes ATP-binding cassette transporter A1 (ABCA1)-mediated cholesterol efflux and promotes atherogenesis. However, the underlying mechanism is still unclear. Acid-sensing ion channel 1 (ASIC1) is a critical H+ receptor, which is responsible for the perception and transduction of extracellular acidification signals.AimIn this study, we explored whether or how ASIC1 influences extracellular acidification-induced ABCA1-mediated cholesterol efflux from macrophage-derived foam cells.MethodsRAW 264.7 macrophages were cultured in an acidic medium (pH 6.5) to generate foam cells. Then the intracellular lipid deposition, cholesterol efflux, and ASIC1/calpain1/ABCA1 expressions were evaluated.ResultsWe showed that extracellular acidification enhanced ASIC1 expression and translocation, promoted calpain1 expression and lipid accumulation, and decreased ABCA1 protein expression as well as ABCA1-mediated cholesterol efflux. Of note, inhibiting ASIC1 activation with amiloride or Psalmotoxin 1 (PcTx-1) not only lowered calpain1 protein level and lipid accumulation but also enhanced ABCA1 protein levels and ABCA1-mediated cholesterol efflux of macrophages under extracellular acidification conditions. Furthermore, similar results were observed in macrophages treated with calpain1 inhibitor PD150606.ConclusionExtracellular acidification declines cholesterol efflux via activating ASIC1 to promote calpain1-mediated ABCA1 degradation. Thus, ASIC1 may be a novel therapeutic target for atherosclerosis.

SREBP2 promotes the viability, proliferation, and migration and inhibits apoptosis in TGF-β1-induced airway smooth muscle cells by regulating TLR2/NF-κB/NFATc1/ABCA1 regulatory network

ABSTRACT Asthma is a respiratory disease with complex pathogenesis. Sterol-responsive element-binding proteins 2 (SREBP2) was found to bind to promoter sequences of ABCA1 to suppress ABCA1 promoter activity. This study aimed to explore the expression level of SREBP2 and ATP-binding cassette transporter A1 (ABCA1), and their effects on the development of airway smooth muscle cells (ASMCs) in asthma. ASMCs were treated with different concentrations of TGF-β1 (0, 0.5, 1, 5 and 10 ng/mL). Short hairpin SREBP2 (shSREBP2), SREBP2, shABCA1 or ABCA1 were transfected into ASMCs. Cell viability, proliferation, apoptosis, migration, and the expression of SREBP2, ABCA1 and related pathway proteins were detected by MTT assay, Brdu staining, flow cytometer, Transwell assay, qRT-PCR, and Western blotting, respectively. The results showed that TGF-β1 increased the viability, proliferation, migration and inhibited apoptosis in ASMCs. Moreover, TGF-β1 also decreased the expression of ABCA1, cleaved caspase-3, cleaved PARP, E-cadherin, and increased the expression of vimentin, TLR2, p-p65 and NFATc1. SREBP2 knockdown alleviated these TGF-β1-induced changes. SREBP2 overexpression inhibited ABCA1 expression and apoptosis, and promoted cell migration and the expression of TLR2, p-p65, NFATc1 in ASMCs. ABCA1 overexpression alleviated these SREBP2-induced promoting and inhibition effects. In conclusion, SREBP2 activates TLR2/NF-κB/NFATc1 regulatory network and promotes TGF-β1-induced cell movement through inhibiting ABCA1 expression.

Conformational flexibility of apolipoprotein A-I amino- and carboxy-termini is necessary for lipid binding but not cholesterol efflux

Because of its critical role in HDL formation, significant efforts have been devoted to studying apolipoprotein A-I (APOA1) structural transitions in response to lipid binding. To assess the requirements for the conformational freedom of its termini during HDL particle formation, we generated three dimeric APOA1 molecules with their termini covalently joined in different combinations. The dimeric (d)-APOA1C-N mutant coupled the C-terminus of one APOA1 molecule to the N-terminus of a second with a short alanine linker, whereas the d-APOA1C-C and d-APOA1N-N mutants coupled the C-termini and the N-termini of two APOA1 molecules, respectively, using introduced cysteine residues to form disulfide linkages. We then tested the ability of these constructs to generate reconstituted HDL by detergent-assisted and spontaneous phospholipid microsolubilization methods. Using cholate dialysis, we demonstrate WT and all APOA1 mutants generated reconstituted HDL particles of similar sizes, morphologies, compositions, and abilities to activate lecithin:cholesterol acyltransferase. Unlike WT, however, the mutants were incapable of spontaneously solubilizing short chain phospholipids into discoidal particles. We found lipid-free d-APOA1C-N and d-APOA1N-N retained most of WT APOA1’s ability to promote cholesterol efflux via the ATP binding cassette transporter A1, whereas d-APOA1C-C exhibited impaired cholesterol efflux. Our data support the double belt model for a lipid-bound APOA1 structure in nascent HDL particles and refute other postulated arrangements like the “double super helix.” Furthermore, we conclude the conformational freedom of both the N- and C-termini of APOA1 is important in spontaneous microsolubilization of bulk phospholipid but is not critical for ABCA1-mediated cholesterol efflux.

Effects of 2-Methoxyestradiol, a Main Metabolite of Estradiol on Hepatic ABCA1 Expression in HepG2 Cells.

ATP-binding cassette transporter A1 (ABCA1) is a key regulator of lipid efflux, and the absence of ABCA1 induces hepatic lipid accumulation, which is one of the major causes of fatty liver. 2-Methoxyestradiol (2-ME2) has been demonstrated to protect against fatty liver. In this study, we investigated the effects of 2-ME2 on the hepatic lipid content and ABCA1 expression. We found that 2-ME2 dose-dependently increased ABCA1 expression, and therefore, the lipid content was significantly decreased in HepG2 cells. 2-ME2 enhanced the ABCA1 promoter activity; however, this effect was reduced after the inhibition of the PI3K pathway. The overexpression of Akt or p110 induced ABCA1 promoter activity, while dominant-negative Akt diminished the ability of 2-ME2 on ABCA1 promoter activity. Further, 2-ME2 stimulated the rapid phosphorylation of Akt and FoxO1 and reduced the nuclear accumulation of FoxO1. Chromatin immunoprecipitation confirmed that FoxO1 bonded to the ABCA1 promoter region. The binding was reduced by 2-ME2, which facilitated ABCA1 gene transcription. Furthermore, mutating FoxO1-binding sites in the ABCA1 promoter region or treatment with FoxO1-specific siRNA disrupted the effect of 2-ME2 on ABCA1 expression. All of our results demonstrated that 2-ME2 might upregulate ABCA1 expression via the PI3K/Akt/FoxO1 pathway, which thus reduces the lipid content in hepatocytes.

Apolipoprotein A1-Related Proteins and Reverse Cholesterol Transport in Antiatherosclerosis Therapy: Recent Progress and Future Perspectives.

Hyperlipidemia characterized by abnormal deposition of cholesterol in arteries can cause atherosclerosis and coronary artery occlusion, leading to atherosclerotic coronary heart disease. The body prevents atherosclerosis by reverse cholesterol transport to mobilize and excrete cholesterol and other lipids. Apolipoprotein A1, the major component of high-density lipoprotein, plays a key role in reverse cholesterol transport. Here, we reviewed the role of apolipoprotein A1-targeting molecules in antiatherosclerosis therapy, in particular ATP-binding cassette transporter A1, lecithin-cholesterol acyltransferase, and scavenger receptor class B type 1.

Apolipoprotein A-I Inhibits Transendothelial Transport of Apolipoprotein B-Carrying Lipoproteins and Enhances Its Associated High-Density Lipoprotein Formation.

Caveola-located scavenger receptor type B class I (SR-BI) and activin receptor-like kinase-1 (ALK1) are involved in transendothelial transport of apolipoprotein B-carrying lipoproteins (apoB-LPs). Transport of apoB-LPs though mouse aortic endothelial cells (MAECs) is associated with apoE-carrying high-density lipoprotein (HDL)-like particle formation and apoAI induces raft-located proteins to shift to non-raft membranes by upregulation of ATP-binding cassette transporter A1 (ABCA1). To investigate apoAI's effect on transendothelial transport of apoB-LPs, MAECs and human coronary artery endothelial cells (HCAECs) were treated with apoB-LPs ± apoAI. Our data demonstrated that apoAI neither altered SR-BI and ALK1 expression nor affected apoB-LP binding to MAECs. ApoAI inhibited MAEC uptake, transcellular transport, and intracellular accumulation of apoB-LPs and accelerated their resecretion in MAECs. ApoAI enhanced transendothelial apoB-LP transport-associated HDL-like particle formation, upregulated ABCA1 expression, shifted SR-BI and ALK1 to the non-raft membrane in MAECs, inhibited transcellular transport of apoB-LPs, and enhanced associated HDL-like particle formation in HCAECs. ABCA1 knockdown attenuated apoAI-induced membrane SR-BI and ALK1 relocation and diminished apoAI's effect on transendothelial apoB-LP transport and HDL-like particle formation in MAECs. This suggests that upregulation of ABCA1 expression is a mechanism, whereby apoAI provokes caveola-located receptor relocation, inhibits transendothelial apoB-LP transport, and promotes associated HDL-like particle formation.

A Fluorescence-Based In Vitro Method to Assess Cholesterol Efflux.

Cholesterol efflux (ChE) capacity is associated with the incidence of cardiovascular events and has been proposed as an emerging cardiovascular risk factor. ChE has been traditionally assessed by in vitro radioactive methods but these are not appropriate when assessing a large number of samples. Therefore, alternative, reproducible nonradioactive methods have been developed. This chapter describes a robust nonradioactive method using a fluorescent tracer to assess ChE in vitro.The measurement of ChE in vitro requires three main components: a cholesterol-loaded donor cell, a cholesterol tracer, and a cholesterol acceptor. This method involves labeling of murine macrophage J774A.1 cells using the fluorescent sterol dipyrromethene boron difluoride (BODIPY)-cholesterol. The cholesterol acceptors from humans or animals include lipid-free apolipoprotein (ApoA)-1, high-density lipoprotein (HDL), HDL2 and HDL3 subfractions, serum, plasma or ApoB-depleted serum or plasma. While lipid-free ApoA-1 mediates ChE via only ATP-binding cassette (ABC)A1 transporter, the remaining acceptors mediate ChE via ABCA1 , ABCG1 and scavenger receptor class B type 1 (SRB1) transporters. The reproducibility of this BODIPY-ChE assay is excellent as the intra-assay coefficients of variation (CVs) were <10% (30 replicates on the same day) and the interassay CVs were <14% (10 experiments performed on different days, with 3 replicates each). The fluorescent method therefore represents a reproducible, safe and useful tool to evaluate ChE as an emerging cardiovascular risk factor.

Curcumin ameliorates lipid metabolic disorder and cognitive dysfunction via the ABCA1 transmembrane transport system in APP/PS1 double transgenic mice.

The disorder of lipid metabolism, especially cholesterol metabolism, can promote Alzheimer's Disease. Curcumin can ameliorate lipid metabolic disorder in the brain of Alzheimer's Disease patients, while the mechanism is not clear. APP/PS1 (APPswe/PSEN1dE9) double transgenic mice were divided into dementia, low-dose, and high-dose groups and then fed for six months with different dietary concentrations of curcumin. Morris water maze was used to evaluate the transgenic mice's special cognitive and memory ability in each group. In contrast, the cholesterol oxidase-colorimetric method was used to measure total serum cholesterol and high-density lipoprotein levels. Immunohistochemistry was used to evaluate the expression of liver X receptor-β, ATP binding cassette A1 and apolipoprotein A1 of the hippocampus and Aβ42 in the brains of transgenic mice. The mRNA and protein expression levels of liver X receptor-β, retinoid X receptor-α and ATP binding cassette A1 were evaluated using qRT-PCR and Western blotting, respectively. Curcumin improved the special cognitive and memory ability of transgenic Alzheimer's Disease Mice. The total serum cholesterol decreased in Alzheimer's Disease mice fed the curcumin diet, while the high-density lipoprotein increased. The curcumin diet was associated with reduced expression of Aβ and increased expression of liver X receptor-β, ATP binding cassette A1, and apolipoprotein A1 in the CA1 region of the hippocampus. The mRNA and protein levels of retinoid X receptor-α, liver X receptor-β, and ATP binding cassette A1 were higher in the brains of Alzheimer's Disease mice fed the curcumin diet. Our results point to the mechanism by which curcumin improves lipid metabolic disorders in Alzheimer's Disease via the ATP binding cassette A1 transmembrane transport system.

Apolipoprotein A-I carboxy-terminal domain residues 187–243 are required for adiponectin-induced cholesterol efflux

Adiponectin exerts its atheroprotection by stimulating adenosine triphosphate binding cassette transporter A1 (ABCA1)-mediated cholesterol efflux to apolipoprotein A-I (apoA-I). However, involvement of the apoA-I residues in this process have not been studied. In Tamm-Horsfall 1 (THP-1) macrophages and baby hamster kidney (BHK) cells we assessed adiponectin's potential to restore cholesterol efflux in the presence of apoA-I and ABCA1 mutants, respectively. Adiponectin was unable to restore efflux from THP-1 macrophages in the presence of apoA-I carboxy-terminal domain (CTD) successive mutants from residues 187–243 versus apoA-I mutants alone. Furthermore, adiponectin did not significantly influence cholesterol efflux to apoA-I from BHK-ABCA1 mutant cells. Adiponectin appears to require functional apoA-I CTD residues 187–243 and wild-type ABCA1 to mediate efficient cholesterol efflux from THP-1 macrophages and BHK cells, respectively. Therefore, adiponectin cannot rescue defective cholesterol efflux in apoA-I- or ABCA1-mutant conditions, but rather increases cholesterol efflux in wild-type apoA-I conditions compared to apoA-I exposure alone.