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1.  Several genetic polymorphisms interact with overweight/obesity to influence serum lipid levels 
Information about the interactions of single nucleotide polymorphisms (SNPs) and overweight/obesity on serum lipid profiles is still scarce. The present study was undertaken to detect ten SNPs and their interactions with overweight/obesity on serum lipid levels.
A total of 978 normal weight and 751 overweight/obese subjects of Bai Ku Yao were randomly selected from our previous stratified randomized cluster samples. Normal weight, overweight and obesity were defined as a body mass index (BMI) < 24, 24–28, and > 28 kg/m2; respectively. Serum total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), apolipoprotein (Apo) A1 and ApoB levels were measured. Genotyping of ATP-binding cassette transporter A1 (ABCA-1) V825I, acyl-CoA:cholesterol acyltransferase-1 (ACAT-1) rs1044925, low density lipoprotein receptor (LDL-R) AvaII, hepatic lipase gene (LIPC) -250G>A, endothelial lipase gene (LIPG) 584C>T, methylenetetrahydrofolate reductase (MTHFR) 677C>T, the E3 ubiquitin ligase myosin regulatory light chain-interacting protein (MYLIP) rs3757354, proprotein convertase subtilisin-like kexin type 9 (PCSK9) E670G, peroxisome proliferator-activated receptor delta (PPARD) +294T>C, and Scavenger receptor class B type 1 (SCARB1) rs5888 was performed by polymerase chain reaction and restriction fragment length polymorphism combined with gel electrophoresis, and then confirmed by direct sequencing. The interactions were detected by factorial design covariance analysis.
The genotypic and allelic frequencies of LIPC and PCSK9 were different between normal weight and overweight/obese subjects, the genotypic frequency of LIPG and allelic frequency of MYLIP were also different between normal weight and overweight/obese subjects (P < 0.05-0.001). The levels of TC, ApoA1 (ABCA-1); TC, LDL-C, ApoA1, ApoB and ApoA1/ApoB (LIPC); TG, HDL-C, and ApoA1 (LIPG); TC, HDL-C, LDL-C, ApoA1 and ApoB (MTHFR); HDL-C and ApoA1 (MYLIP) in normal weight subjects were different among the genotypes (P < 0.01-0.001). The levels of LDL-C, ApoB and ApoA1/ApoB (ABCA-1); HDL-C, ApoA1, ApoB and ApoA1/ApoB (LIPC); TC, HDL-C, ApoA1 and ApoB (LIPG); TC, TG, HDL-C, LDL-C, ApoA1 and ApoB (MTHFR); TC, TG and ApoB (MYLIP); TG (PCSK9); TG, ApoA1 and ApoB (PPARD); and TC, HDL-C, LDL-C, ApoA1 and ApoB (SCARB1) in overweight/obese subjects were different among the genotypes (P < 0.01-0.001). The SNPs of ABCA-1 (LDL-C and ApoA1/ApoB); LIPC (TC, LDL-C, ApoA1 and ApoB); LIPG (ApoB); MTHFR (TC, TG and LDL-C); MYLIP (TC and TG); PCSK9 (TG, HDL-C, ApoB and ApoA1/ApoB); PPARD (TG and ApoA1/ApoB); and SCARB1 (TG, ApoA1 and ApoB) interacted with overweight/obesity to influence serum lipid levels (P < 0.05-0.001).
The differences in serum lipid levels between normal weight and overweight/obese subjects might partly result from different genetic polymorphisms and the interactions between several SNPs and overweight/obesity.
PMCID: PMC3508802  PMID: 23039238
Lipid; Apolipoprotein; Genetic polymorphism; Overweight; Obesity; Interaction
2.  The Interplay between Size, Morphology, Stability, and Functionality of High-Density Lipoprotein Subclasses† 
Biochemistry  2008;47(16):4770-4779.
High-density lipoprotein (HDL) mediates reverse cholesterol transport (RCT), wherein excess cholesterol is conveyed from peripheral tissues to the liver and steroidogenic organs. During this process HDL continually transitions between subclass sizes, each with unique biological activities. For instance, RCT is initiated by the interaction of lipid-free/lipid-poor apolipoprotein A-I (apoA-I) with ABCA1, a membrane-associated lipid transporter, to form nascent HDL. Because nearly all circulating apoA-I is lipid-bound, the source of lipid-free/lipid-poor apoA-I is unclear. Lecithin:cholesterol acyltransferase (LCAT) then drives the conversion of nascent HDL to spherical HDL by catalyzing cholesterol esterification, an essential step in RCT. To investigate the relationship between HDL particle size and events critical to RCT such as LCAT activation and lipid-free apoA-I production for ABCA1 interaction, we reconstituted five subclasses of HDL particles (rHDL of 7.8, 8.4, 9.6, 12.2, and 17.0 nm in diameter, respectively) using various molar ratios of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, free cholesterol, and apoA-I. Kinetic analyses of this comprehensive array of rHDL particles suggest that apoA-I stoichiometry in rHDL is a critical factor governing LCAT activation. Electron microscopy revealed specific morphological differences in the HDL subclasses that may affect functionality. Furthermore, stability measurements demonstrated that the previously uncharacterized 8.4 nm rHDL particles rapidly convert to 7.8 nm particles, concomitant with the dissociation of lipid-free/lipid-poor apoA-I. Thus, lipid-free/lipid-poor apoA-I generated by the remodeling of HDL may be an essential intermediate in RCT and HDL’s in vivo maturation.
PMCID: PMC2902722  PMID: 18366184
3.  Apolipoprotein A-I is a selective target for myeloperoxidase-catalyzed oxidation and functional impairment in subjects with cardiovascular disease 
Journal of Clinical Investigation  2004;114(4):529-541.
In recent studies we demonstrated that systemic levels of protein-bound nitrotyrosine (NO2Tyr) and myeloperoxidase (MPO), a protein that catalyzes generation of nitrating oxidants, serve as independent predictors of atherosclerotic risk, burden, and incident cardiac events. We now show both that apolipoprotein A-I (apoA-I), the primary protein constituent of HDL, is a selective target for MPO-catalyzed nitration and chlorination in vivo and that MPO-catalyzed oxidation of HDL and apoA-I results in selective inhibition in ABCA1-dependent cholesterol efflux from macrophages. Dramatic selective enrichment in NO2Tyr and chlorotyrosine (ClTyr) content within apoA-I recovered from serum and human atherosclerotic lesions is noted, and analysis of serum from sequential subjects demonstrates that the NO2Tyr and ClTyr contents of apoA-I are markedly higher in individuals with cardiovascular disease (CVD). Analysis of circulating HDL further reveals that higher NO2Tyr and ClTyr contents of the lipoprotein are each significantly associated with diminished ABCA1-dependent cholesterol efflux capacity of the lipoprotein. MPO as a likely mechanism for oxidative modification of apoA-I in vivo is apparently facilitated by MPO binding to apoA-I, as revealed by cross-immunoprecipitation studies in plasma, recovery of MPO within HDL-like particles isolated from human atheroma, and identification of a probable contact site between the apoA-I moiety of HDL and MPO. To our knowledge, the present results provide the first direct evidence for apoA-I as a selective target for MPO-catalyzed oxidative modification in human atheroma. They also suggest a potential mechanism for MPO-dependent generation of a proatherogenic dysfunctional form of HDL in vivo.
PMCID: PMC503769  PMID: 15314690
4.  Apolipoprotein A-I glycation by Glucose and Reactive Aldehydes Alters Phospholipid Affinity but Not Cholesterol Export from Lipid-Laden Macrophages 
PLoS ONE  2013;8(5):e65430.
Increased protein glycation in people with diabetes may promote atherosclerosis. This study examined the effects of non-enzymatic glycation on the association of lipid-free apolipoproteinA-I (apoA-I) with phospholipid, and cholesterol efflux from lipid-loaded macrophages to lipid-free and lipid-associated apoA-I. Glycation of lipid-free apoA-I by methylglyoxal and glycolaldehyde resulted in Arg, Lys and Trp loss, advanced glycation end-product formation and protein cross-linking. The association of apoA-I glycated by glucose, methylglyoxal or glycolaldehyde with phospholipid multilamellar vesicles was impaired in a glycating agent dose-dependent manner, with exposure of apoA-I to both 30 mM glucose (42% decrease in kslow) and 3 mM glycolaldehyde (50% decrease in kfast, 60% decrease in kslow) resulting is significantly reduced affinity. Cholesterol efflux to control or glycated lipid-free apoA-I, or discoidal reconstituted HDL containing glycated apoA-I (drHDL), was examined using cholesterol-loaded murine (J774A.1) macrophages treated to increase expression of ATP binding cassette transporters A1 (ABCA1) or G1 (ABCG1). Cholesterol efflux from J774A.1 macrophages to glycated lipid-free apoA-I via ABCA1 or glycated drHDL via an ABCG1-dependent mechanism was unaltered, as was efflux to minimally modified apoA-I from people with Type 1 diabetes, or controls. Changes to protein structure and function were prevented by the reactive carbonyl scavenger aminoguanidine. Overall these studies demonstrate that glycation of lipid-free apoA-I, particularly late glycation, modifies its structure, its capacity to bind phospholipids and but not ABCA1- or ABCG1-dependent cholesterol efflux from macrophages.
PMCID: PMC3669297  PMID: 23741493
5.  Targeted inactivation of hepatic Abca1 causes profound hypoalphalipoproteinemia and kidney hypercatabolism of apoA-I 
Journal of Clinical Investigation  2005;115(5):1333-1342.
Patients with Tangier disease exhibit extremely low plasma HDL concentrations resulting from mutations in the ATP-binding cassette, sub-family A, member 1 (ABCA1) protein. ABCA1 controls the rate-limiting step in HDL particle assembly by mediating efflux of cholesterol and phospholipid from cells to lipid-free apoA-I, which forms nascent HDL particles. ABCA1 is widely expressed; however, the specific tissues involved in HDL biogenesis are unknown. To determine the role of the liver in HDL biogenesis, we generated mice with targeted deletion of the second nucleotide-binding domain of Abca1 in liver only (Abca1–L/–L). Abca1–L/–L mice had total plasma and HDL cholesterol concentrations that were 19% and 17% those of wild-type littermates, respectively. In vivo catabolism of HDL apoA-I from wild-type mice or human lipid-free apoA-I was 2-fold higher in Abca1–L/–L mice compared with controls due to a 2-fold increase in the catabolism of apoA-I by the kidney, with no change in liver catabolism. We conclude that in chow-fed mice, the liver is the single most important source of plasma HDL. Furthermore, hepatic, but not extrahepatic, Abca1 is critical in maintaining the circulation of mature HDL particles by direct lipidation of hepatic lipid-poor apoA-I, slowing its catabolism by the kidney and prolonging its plasma residence time.
PMCID: PMC1074680  PMID: 15841208
6.  Effect of apoA-I Mutations in the Capacity of Reconstituted HDL to Promote ABCG1-Mediated Cholesterol Efflux 
PLoS ONE  2013;8(6):e67993.
ATP binding cassette transporter G1 (ABCG1) mediates the cholesterol transport from cells to high-density lipoprotein (HDL), but the role of apolipoprotein A-I (apoA-I), the main protein constituent of HDL, in this process is not clear. To address this, we measured cholesterol efflux from HEK293 cells or J774 mouse macrophages overexpressing ABCG1 using as acceptors reconstituted HDL (rHDL) containing wild-type or various mutant apoA-I forms. It was found that ABCG1-mediated cholesterol efflux was severely reduced (by 89%) when using rHDL containing the carboxyl-terminal deletion mutant apoA-I[Δ(185–243)]. ABCG1-mediated cholesterol efflux was not affected or moderately decreased by rHDL containing amino-terminal deletion mutants and several mid-region deletion or point apoA-I mutants, and was restored to 69–99% of control by double deletion mutants apoA-I[Δ(1–41)Δ(185–243)] and apoA-I[Δ(1–59)Δ(185–243)]. These findings suggest that the central helices alone of apoA-I associated to rHDL can promote ABCG1-mediated cholesterol efflux. Further analysis showed that rHDL containing the carboxyl-terminal deletion mutant apoA-I[Δ(185–243)] only slightly reduced (by 22%) the ABCG1-mediated efflux of 7-ketocholesterol, indicating that depending on the sterol type, structural changes in rHDL-associated apoA-I affect differently the ABCG1-mediated efflux of cholesterol and 7-ketocholesterol. Overall, our findings demonstrate that rHDL-associated apoA-I structural changes affect the capacity of rHDL to accept cellular cholesterol by an ABCG1-mediated process. The structure-function relationship seen here between rHDL-associated apoA-I mutants and ABCG1-mediated cholesterol efflux closely resembles that seen before in lipid-free apoA-I mutants and ABCA1-dependent cholesterol efflux, suggesting that both processes depend on the same structural determinants of apoA-I.
PMCID: PMC3694867  PMID: 23826352
7.  Why Targeting HDL Should Work as a Therapeutic Tool, but Hasn’t 
Atherosclerosis is one of the most common causes of death and disability in US today despite the availability of statins which reduce hyperlipidemia, a risk factor that predisposes individuals to this disease. Epidemiology of human populations has overwhelmingly demonstrated an inverse correlation between the concentration of plasma HDL cholesterol (HDL-C) and the likelihood of developing cardiovascular disease (CVD). Decades of observations and mechanistic studies suggest that one protective function of HDL is its central role in reverse cholesterol transport (RCT). In this pathway the ATP-binding cassette transporter (ABCA1) releases intracellular cholesterol, which is packaged by apolipoprotein A-I (apoA-I) into nascent HDL (nHDL) particles and released from the plasma membrane. Further lipidation and maturation of HDL occurs in plasma with the eventual uptake by the liver where cholesterol is removed. It is generally accepted that CVD risk can be reduced if plasma HDL-C levels are elevated. Several different pharmacological approaches have been tried, the most popular approach targets the movement of cholesteryl ester from HDL to triglyceride rich particles by cholesteryl ester transfer protein (CETP). Inhibition of CETP increases plasma HDL-C concentration, however, beneficial effects have yet to be demonstrated, likely the result of off-target effects. These revelations have led to a reevaluation of how elevating HDL concentration could decrease risk. A recent, landmark study showed that the inherent cholesterol efflux capacity of an individual’s plasma was a better predictor of CVD status than overall HDL-C concentration. Even more provocative are recent studies showing that apoA-I, the principle protein component of HDL, functions as a modulator of cellular inflammation and oxidation. The following will review all of these potential routes explaining how HDL apoA-I can reduce the risk of CVD.
PMCID: PMC3772973  PMID: 23743767
apolipoprotein A-I; high density lipoprotein; atherosclerosis; lipid raft; inflammation; nascent HDL; cholesterol transport; cardiovascular disease; cholesterol efflux
8.  Hepatic ABCA1 and VLDL triglyceride production 
Biochimica et Biophysica Acta  2011;1821(5):770-777.
Elevated plasma triglyceride (TG) and reduced high density lipoprotein (HDL) concentrations are prominent features of metabolic syndrome (MS) and type 2 diabetes (T2D). Individuals with Tangier disease also have elevated plasma TG concentrations and a near absence of HDL, resulting from mutations in ATP binding cassette transporter A1 (ABCA1), which facilitates the efflux of cellular phospholipid and free cholesterol to assemble with apolipoprotein A-I (apoA-I), forming nascent HDL particles. In this review, we summarize studies focused on the regulation of hepatic very low density lipoprotein (VLDL) TG production, with particular attention on recent evidence connecting hepatic ABCA1 expression to VLDL, LDL, and HDL metabolism. Silencing ABCA1 in McArdle rat hepatoma cells results in diminished assembly of large (>10nm) nascent HDL particles, diminished PI3 kinase activation, and increased secretion of large, TG-enriched VLDL1 particles. Hepatocyte-specific ABCA1 knockout (HSKO) mice have a similar plasma lipid phenotype as Tangier disease subjects, with a twofold elevation of plasma VLDL TG, 50% lower LDL, and 80% reduction in HDL concentrations. This lipid phenotype arises from increased hepatic secretion of VLDL1 particles, increased hepatic uptake of plasma LDL by the LDL receptor, elimination of nascent HDL particle assembly by the liver, and hypercatabolism of apoA-I by the kidney. These studies highlight a novel role for hepatic ABCA1 in the metabolism of all three major classes of plasma lipoproteins and provide a metabolic link between elevated TG and reduced HDL levels that are a common feature of Tangier disease, MS, and T2D.
PMCID: PMC3272310  PMID: 22001232
VLDL overproduction; Tangier disease; liver; metabolic syndrome; type 2 diabetes; nascent HDL; PI3 kinase; triglyceride secretion; VLDL1
9.  Interactions of Apolipoprotein A-I with High-Density Lipoprotein Particles 
Biochemistry  2013;52(11):1963-1972.
Although the partitioning of apolipoprotein A-I (apoA-I) molecules in plasma between high-density lipoprotein (HDL)-bound and -unbound states is an integral part of HDL metabolism, the factors that control binding of apoA-I to HDL particles are poorly understood. To address this gap in knowledge, we investigated how the properties of the apoA-I tertiary structure domains and surface characteristics of spherical HDL particles influence apoA-I binding. The abilities of 14C-labeled human and mouse apoA-I variants to associate with human HDL and lipid emulsion particles were determined using ultracentrifugation to separate free and bound protein. The binding of human apoA-I (243 amino acids) to HDL is largely mediated by its relatively hydrophobic C-terminal domain; the isolated N-terminal helix bundle domain (residues 1–190) binds poorly. Mouse apoA-I, which has a relatively polar C-terminal domain, binds to human HDL to approximately half the level of human apoA-I. The HDL binding abilities of apoA-I variants correlate strongly with their abilities to associate with phospholipid (PL)-stabilized emulsion particles, consistent with apoA-I–PL interactions at the particle surface being important. When equal amounts of HDL2 and HDL3 are present, all of the apoA-I variants partition preferentially to HDL3. Fluorescence polarization measurements using Laurdan-labeled HDL2 and HDL3 indicate that PL molecular packing is looser on the more negatively charged HDL3 particle surface, which promotes apoA-I binding. Overall, it is clear that both apoA-I structural features, especially the hydrophobicity of the C-terminal domain, and HDL surface characteristics such as the availability of free space influence the ability of apoA-I to associate with HDL particles.
PMCID: PMC3603221  PMID: 23425306
10.  A novel compound inhibits rHDL assembly and blocks nascent HDL biogenesis downstream of apoAI binding to ABCA1 expressing cells 
Nascent high-density lipoprotein (HDL) particles form from cellular lipids and extracellular lipid-free apolipoprotein AI (apoAI) in a process mediated by ATP-binding cassette transporter A1 (ABCA1). We have sought out compounds that inhibit nascent HDL biogenesis without affecting ABCA1 activity.
Methods and Results
Reconstituted HDL (rHDL) formation and cellular cholesterol efflux assays were used to show that two compounds that bond via hydrogen with phospholipids inhibit rHDL and nascent HDL production. In rHDL formation assays, the inhibitory effect of compound 1 (methyl 3α-acetoxy-7α,12α-di[(phenylaminocarbonyl)amino]-5β-cholan-24-oate), the more active of the two, depended on its ability to associate with phospholipids. In cell assays, compound 1 suppressed ABCA1-mediated cholesterol efflux to apoAI, the 18A peptide, and taurocholate with high specificity, without affecting ABCA1-independent cellular cholesterol efflux to HDL and endocytosis of acetylated low-density lipoprotein (AcLDL) and transferrin. Furthermore, compound 1 did not affect ABCA1 activity adversely, as ABCA1-mediated shedding of microparticles proceeded unabated and apoAI binding to ABCA1-expressing cells increased in its presence.
The inhibitory effects of compound 1 support a three-step model of nascent HDL biogenesis: plasma membrane remodeling by ABCA1, apoAI binding to ABCA1, and lipoprotein particle assembly. The compound inhibits the final step, causing accumulation of apoAI in ABCA1-expressing cells.
PMCID: PMC3197940  PMID: 21836073
nascent HDL; rHDL; ABCA1; apoAI; reverse cholesterol transport
11.  The Interaction of ApoA-I and ABCA1 Triggers Signal Transduction Pathways to Mediate Efflux of Cellular Lipids 
Molecular Medicine  2011;18(1):149-158.
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.
PMCID: PMC3320140  PMID: 22064972
12.  Adipose tissue ABCA1 contributes to HDL biogenesis in vivo 
Circulation  2011;124(15):1663-1672.
Adipose tissue (AT) is the body’s largest free cholesterol (FC) reservoir and abundantly expresses ATP binding cassette transporter A1 (ABCA1), a key cholesterol transporter for HDL biogenesis. However, the extent to which AT ABCA1 expression contributes to HDL biogenesis in vivo is unknown.
Methods and Results
Adipocyte-specific ABCA1 knockout mice (ABCA1−A/−A) were generated by crossing ABCA1floxed mice with aP2 cre transgenic mice. AT from ABCA1−A/−A mice had <10% of wild type (WT) ABCA1 protein expression, but normal hepatic and intestinal expression. Deletion of adipocyte ABCA1 resulted in a significant decrease in plasma HDL cholesterol (~15%) and apoA-I (~13%) concentrations. AT from ABCA1−A/−A mice had a two-fold increase in FC content, compared to WT mice, and failed to efflux cholesterol to apoA-I. However, cholesterol efflux from AT to plasma HDL was similar for both genotypes of mice. Incubation of WT AT explants with apoA-I resulted in formation of multiple discrete-sized nascent HDL particles ranging in diameter from 7.1–12 nm; similar incubations with ABCA1−A/−A AT explants resulted in nascent HDL <8 nm. Plasma decay and tissue uptake of WT 125I-HDL tracer was similar in both genotypes of recipient mice, suggesting that adipocyte ABCA1 deficiency reduces plasma HDL concentrations solely by reducing nascent HDL particle formation.
We provide in vivo evidence that AT ABCA1-dependent cholesterol efflux and nascent HDL particle formation contribute to systemic HDL biogenesis and that AT ABCA1 expression plays an important role in adipocyte cholesterol homeostasis.
PMCID: PMC3202242  PMID: 21931081
apolipoproteins; cholesterol; lipids; lipoproteins
13.  ABCA1 overexpression leads to hyperalphalipoproteinemia and increased biliary cholesterol excretion in transgenic mice 
Journal of Clinical Investigation  2001;108(2):303-309.
The discovery of the ABCA1 lipid transporter has generated interest in modulating human plasma HDL levels and atherogenic risk by enhancing ABCA1 gene expression. To determine if increased ABCA1 expression modulates HDL metabolism in vivo, we generated transgenic mice that overexpress human ABCA1 (hABCA1-Tg). Hepatic and macrophage expression of hABCA1 enhanced macrophage cholesterol efflux to apoA-I; increased plasma cholesterol, cholesteryl esters (CEs), free cholesterol, phospholipids, HDL cholesterol, and apoA-I and apoB levels; and led to the accumulation of apoE-rich HDL1. ABCA1 transgene expression delayed 125I-apoA-I catabolism in both liver and kidney, leading to increased plasma apoA-I levels, but had no effect on apoB secretion after infusion of Triton WR1339. Although the plasma clearance of HDL-CE was not significantly altered in hABCA1-Tg mice, the net hepatic delivery of exogenous 3H-CEt-HDL, which is dependent on the HDL pool size, was increased 1.5-fold. In addition, the cholesterol and phospholipid concentrations in hABCA1-Tg bile were increased 1.8-fold. These studies show that steady-state overexpression of ABCA1 in vivo (a) raises plasma apoB levels without altering apoB secretion and (b) raises plasma HDL-C and apoA-I levels, facilitating hepatic reverse cholesterol transport and biliary cholesterol excretion. Similar metabolic changes may modify atherogenic risk in humans.
PMCID: PMC203026  PMID: 11457883
14.  High-density lipoprotein and atherosclerosis: Roles of lipid transporters 
World Journal of Cardiology  2014;6(10):1049-1059.
Various previous studies have found a negative correlation between the risk of cardiovascular events and serum high-density lipoprotein (HDL) cholesterol levels. The reverse cholesterol transport, a pathway of cholesterol from peripheral tissue to liver which has several potent antiatherogenic properties. For instance, the particles of HDL mediate to transport cholesterol from cells in arterial tissues, particularly from atherosclerotic plaques, to the liver. Both ATP-binding cassette transporters (ABC) A1 and ABCG1 are membrane cholesterol transporters and have been implicated in mediating cholesterol effluxes from cells in the presence of HDL and apolipoprotein A-I, a major protein constituent of HDL. Previous studies demonstrated that ABCA1 and ABCG1 or the interaction between ABCA1 and ABCG1 exerted antiatherosclerotic effects. As a therapeutic approach for increasing HDL cholesterol levels, much focus has been placed on increasing HDL cholesterol levels as well as enhancing HDL biochemical functions. HDL therapies that use injections of reconstituted HDL, apoA-I mimetics, or full-length apoA-I have shown dramatic effectiveness. In particular, a novel apoA-I mimetic peptide, Fukuoka University ApoA-I Mimetic Peptide, effectively removes cholesterol via specific ABCA1 and other transporters, such as ABCG1, and has an antiatherosclerotic effect by enhancing the biological functions of HDL without changing circulating HDL cholesterol levels. Thus, HDL-targeting therapy has significant atheroprotective potential, as it uses lipid transporter-targeting agents, and may prove to be a therapeutic tool for atherosclerotic cardiovascular diseases.
PMCID: PMC4209431  PMID: 25349649
ATP-binding cassette transporter; ATP-binding cassette A1; ATP-binding cassette G1; Apolipoprotein A-I; High-density lipoprotein; High-density lipoprotein therapy; apoA-I mimetic peptide; Reconstitutedf high-density lipoprotein
15.  Site-specific Oxidation of Apolipoprotein A-I Impairs Cholesterol Export by ABCA1, a Key Cardioprotective Function of HDL 
Biochimica et Biophysica Acta  2011;1821(3):490-501.
The mechanisms that deprive HDL of its cardioprotective properties are poorly understood. One potential pathway involves oxidative damage of HDL proteins by myeloperoxidase (MPO) a heme enzyme secreted by human artery wall macrophages. Mass spectrometric analysis demonstrated that levels of 3-chlorotyrosine and 3-nitrotyrosine—two characteristic products of MPO—are elevated in HDL isolated from patients with established cardiovascular disease. When apolipoprotein A-I (apoA-I), the major HDL protein, is oxidized by MPO, its ability to promote cellular cholesterol efflux by the membrane-associated ATP-binding cassette transporter A1 (ABCA1) pathway is diminished. Biochemical studies revealed that oxidation of specific tyrosine and methionine residues in apoA-I contributes to this loss of ABCA1 activity. Another potential mechanism for generating dysfunctional HDL involves covalent modification of apoA-I by reactive carbonyls, which have been implicated in atherogenesis and diabetic vascular disease. Indeed, modification of apoA-I by malondialdehyde (MDA) or acrolein also markedly impaired the lipoprotein’s ability to promote cellular cholesterol efflux by the ABCA1 pathway. Tandem mass spectrometric analyses revealed that these reactive carbonyls target specific Lys residues in the C-terminus of apoA-I. Importantly, immunochemical analyses showed that levels of MDA-protein adducts are elevated in HDL isolated from human atherosclerotic lesions. Also, apoA-I co-localized with acrolein adducts in such lesions. Thus, lipid peroxidation products might specifically modify HDL in vivo. Our observations support the hypotheses that MPO and reactive carbonyls might generate dysfunctional HDL in humans.
PMCID: PMC3288272  PMID: 22178192
Myeloperoxidase; malondialdehyde; acrolein; dysfunctional HDL; 3-chlorotyrosine; coronary artery disease
16.  The Carboxy-Terminal Region of apoA-I is Required for the ABCA1-Dependent Formation of α-HDL but not preβ-HDL Particles In Vivo 
Biochemistry  2007;46(19):5697-5708.
ABCA1-mediated lipid efflux to lipid poor apoA-I results in the gradual lipidation of apoA-I. This leads to the formation of discoidal HDL which are subsequently converted to spherical HDL by the action of 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[Δ(185-243)] and apoA-I[Δ(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[Δ(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[Δ(185-243)] and apoA-I[Δ(220-243)] mutants formed mainly preβ-HDL particles and few spherical particles enriched in apoE, while WT apoA-I, apoA-I[Δ(232-243)] and apoA-I[E191A/H193A/K195A] formed spherical α-HDL particles. The findings establish that a) deletions that eliminate the 220-231 region of apoA-I prevent the synthesis of α-HDL, but allow the synthesis of preβ-HDL particles in vivo, b) the amino-terminal segment 1-184 of apoA-I can promote synthesis of preβ-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.
PMCID: PMC2528067  PMID: 17447731
17.  A PEST sequence in ABCA1 regulates degradation by calpain protease and stabilization of ABCA1 by apoA-I 
Journal of Clinical Investigation  2003;111(1):99-107.
Cholesterol-loaded macrophage foam cells are a central component of atherosclerotic lesions. ABCA1, the defective molecule in Tangier disease, mediates the efflux of phospholipids and cholesterol from cells to apoA-I, reversing foam cell formation. In ABCA1, we identified a sequence rich in proline, glutamic acid, serine, and threonine (PEST sequence) that enhances the degradation of ABCA1 by calpain protease and thereby controls the cell surface concentration and cholesterol efflux activity of ABCA1. In an apparent positive feedback loop, apoA-I binds ABCA1, promotes lipid efflux, inhibits calpain degradation, and leads to increased levels of ABCA1. ApoA-I infusion also increases ABCA1 in vivo. These studies reveal a novel mode of regulation of ABCA1 by PEST sequence–mediated calpain proteolysis that appears to be reversed by apolipoprotein-mediated phospholipid efflux. Inhibition of ABCA1 degradation by calpain could represent a novel therapeutic approach to increasing macrophage cholesterol efflux and decreasing atherosclerosis.
PMCID: PMC151839  PMID: 12511593
18.  Combined deficiency of ABCA1 and ABCG1 promotes foam cell accumulation and accelerates atherosclerosis in mice 
The Journal of Clinical Investigation  2007;117(12):3900-3908.
HDLs protect against the development of atherosclerosis, but the underlying mechanisms are poorly understood. HDL and its apolipoproteins can promote cholesterol efflux from macrophage foam cells via the ATP-binding cassette transporters ABCA1 and ABCG1. Experiments addressing the individual roles of ABCA1 and ABCG1 in the development of atherosclerosis have produced mixed results, perhaps because of compensatory upregulation in the individual KO models. To clarify the role of transporter-mediated sterol efflux in this disease process, we transplanted BM from Abca1–/–Abcg1–/– mice into LDL receptor–deficient mice and administered a high-cholesterol diet. Compared with control and single-KO BM recipients, Abca1–/–Abcg1–/– BM recipients showed accelerated atherosclerosis and extensive infiltration of the myocardium and spleen with macrophage foam cells. In experiments with isolated macrophages, combined ABCA1 and ABCG1 deficiency resulted in impaired cholesterol efflux to HDL or apoA-1, profoundly decreased apoE secretion, and increased secretion of inflammatory cytokines and chemokines. In addition, these cells showed increased apoptosis when challenged with free cholesterol or oxidized LDL loading. These results suggest that the combined effects of ABCA1 and ABCG1 in mediating macrophage sterol efflux are central to the antiatherogenic properties of HDL.
PMCID: PMC2066200  PMID: 17992262
19.  Adipocyte Modulation of High-Density Lipoprotein Cholesterol 
Circulation  2010;121(11):1347-1355.
Adipose harbors a large depot of free cholesterol. However, a role for adipose in cholesterol lipidation of HDL in vivo is not established. We present the first evidence that adipocytes support transfer of cholesterol to HDL in vivo as well as in vitro and implicate ABCA1 and SR-BI, but not ABCG1, cholesterol transporters in this process.
Methods and Results
Cholesterol efflux from wild-type (WT), ABCA1−/−, SR-BI−/− and ABCG1−/− adipocytes to apoA-I and HDL3 were measured in vitro. 3T3L1-adipocytes, labeled with 3H-cholesterol, were injected intraperitoneally (IP) into WT, apoA-I transgenic and apoA-I−/− mice and tracer movement onto plasma HDL monitored. Identical studies were performed with labeled WT, ABCA1−/− or SR-BI−/− mouse-embryonic-fibroblast (MEF) adipocytes. The effect of TNFα on transporter expression and cholesterol efflux was monitored during adipocyte differentiation. Cholesterol efflux to apoA-I and HDL3 was impaired in ABCA1−/− and SR-BI−/− adipocytes respectively, with no effect observed in ABCG1−/− adipocytes. Injection IP of labeled 3T3L1-adipocytes resulted in increased HDL-associated 3H-cholesterol in apoA-I transgenic mice but reduced levels in apoA-I −/− animals. Injection IP of labeled ABCA1−/− or SR-BI−/− adipocytes reduced plasma counts relative to their respective controls. TNFα reduced both ABCA1 and SR-BI expression and impaired cholesterol efflux from partially-differentiated adipocytes.
These data suggest a novel metabolic function of adipocytes in promoting cholesterol transfer to HDL in vivo and implicate adipocyte SR-BI and ABCA1, but not ABCG1, in this process. Further, adipocyte modulation of HDL may be impaired in adipose inflammatory disease states such as type-2 diabetes.
PMCID: PMC2925122  PMID: 20212278
cholesterol; lipoproteins; adipocytes; atherosclerosis; inflammation
20.  High Density Lipoprotein Structure–Function and Role in Reverse Cholesterol Transport 
Sub-cellular biochemistry  2010;51:183-227.
High density lipoprotein (HDL) possesses important anti-atherogenic properties and this review addresses the molecular mechanisms underlying these functions. The structures and cholesterol transport abilities of HDL particles are determined by the properties of their exchangeable apolipoprotein (apo) components. ApoA-I and apoE, which are the best characterized in structural terms, contain a series of amphipathic α-helical repeats. The helices located in the amino-terminal two-thirds of the molecule adopt a helix bundle structure while the carboxy-terminal segment forms a separately folded, relatively disorganized, domain. The latter domain initiates lipid binding and this interaction induces changes in conformation; the α-helix content increases and the amino-terminal helix bundle can open subsequently. These conformational changes alter the abilities of apoA-I and apoE to function as ligands for their receptors. The apoA-I and apoE molecules possess detergent-like properties and they can solubilize vesicular phospholipid to create discoidal HDL particles with hydrodynamic diameters of ~10 nm. In the case of apoA-I, such a particle is stabilized by two protein molecules arranged in an anti-parallel, double-belt, conformation around the edge of the disc. The abilities of apoA-I and apoE to solubilize phospholipid and stabilize HDL particles enable these proteins to be partners with ABCA1 in mediating efflux of cellular phospholipid and cholesterol, and the biogenesis of HDL particles. ApoA-I-containing nascent HDL particles play a critical role in cholesterol transport in the circulation whereas apoE-containing HDL particles mediate cholesterol transport in the brain. The mechanisms by which HDL particles are remodeled by lipases and lipid transfer proteins, and interact with SR-BI to deliver cholesterol to cells, are reviewed.
PMCID: PMC3215094  PMID: 20213545
HDL; Cholesterol; Lipoprotein; apoA-I; apoE
21.  The roles of C-terminal helices of human apolipoprotein A-I in formation of high-density lipoprotein particles 
Biochimica et biophysica acta  2013;1841(1):10.1016/j.bbalip.2013.10.005.
Apolipoprotein A-I (apoA-I) accepts cholesterol and phospholipids from ATP-binding cassette transporter Al (ABCA1)-expressing cells to form high-density lipoprotein (HDL). Human apoA-I has two tertiary structural domains and the C-terminal domain (approximately amino acids 190–243) plays a key role in lipid binding. Although the high lipid affinity region of the C-terminal domain of apoA-I (residues 223–243) is essential for the HDL formation, the function of low lipid affinity region (residues 191–220) remains unclear. To evaluate the role of residues 191–220, we analyzed the structure, lipid binding properties, and HDL formation activity of Δ191–220 apoA-I, in comparison to wild-type and Δ223–243 apoA-I. Although deletion of residues 191–220 has a slight effect on the tertiary structure of apoA-I, the Δl91–220 variant showed intermediate behavior between wild-type and Δ223–243 regarding the formation of hydrophobic sites and lipid interaction through the C-terminal domain. Physicochemical analysis demonstrated that defective lipid binding of Δl91–220 apoA-I is due to the decreased ability to form α-helix structure which provides the energetic source for lipid binding. In addition, the ability to form HDL particles in vitro and induce cholesterol efflux from ABC Al-expressing cells of Δ191–220 apoA-I was also intermediate between wild-type and Δ223–243 apoA-I. These results suggest that despite possessing low lipid affinity, residues 191–220 play a role in enhancing the ability of apoA-I to bind to and solubilize lipids by forming α-helix upon lipid interaction. Our results demonstrate that the combination of low lipid affinity region and high lipid affinity region of apoA-I is required for efficient ABCA1-dependent HDL formation.
PMCID: PMC3863607  PMID: 24120703
ABCA1; apoA-I; HDL; Cholesterol
22.  The Induction of Atherogenic Dyslipidemia In Poloxamer 407-treated Mice Is Not Mediated Through PPARα 
The copolymer surfactant poloxamer 407 (P-407) has been used to induce a dose-controlled dyslipidemia in both mice and rats. Human macrophages cultured with P-407 exhibit a concentration-dependent reduction in cholesterol efflux to apolipoprotein A1 (apoA1) due to down-regulation of the ATP-binding cassette transporter A1 (ABCA1). Peroxisome proliferator-activated receptor alpha (PPARα) can increase expression of liver X receptor alpha (LXRα) in macrophages and thereby promote the expression of ABCA1, which, in turn, mediates cholesterol efflux to apoA1. The present study investigated point(s) along this signaling pathway at which P-407 might act to inhibit cholesterol efflux from macrophages. A transactivation assay was used to evaluate whether P-407 could either a) activate PPARα, or b) block the activation of PPARα by an established PPARα agonist. P-407 was also evaluated for its potential to alter plasma lipid concentrations following its administration to both normal C57BL/6 and PPARα-deficient mice. P-407 was unable to modulate PPARα activity, as determined in cell-based transactivation assays. Moreover, P-407-induced dyslipidemia occurred at the same rate and to the same extent in PPARα-deficient mice as was observed in C57BL/6 mice, suggesting no role for PPARα in P-407-mediated dyslipidemia. Although PPARs are known to mediate the transcriptional regulation of the two major apolipoproteins associated with HDL (apoA1 and apoA2), P-407 treatment resulted in a similar decrease (∼30%) in the plasma concentration of apoA1 in both control and PPARα-deficient mice. Since our previous work demonstrated that P-407 was unable to abrogate the capacity of a known LXRα agonist to increase cholesterol efflux from macrophages, P-407 is likely to exert its effect, either directly or indirectly, on ABCA1, rather than on LXRα. On the basis of these findings it is concluded that PPARα does not mediate the P-407-dependent reduction in apoA1-facilitated cholesterol efflux from macrophages.
PMCID: PMC2496925  PMID: 18498712
Apolipoprotein; Dyslipidemia; Lipid; Peroxisome proliferator-activated receptor (PPAR); Transactivation assay
23.  Augmented Atherogenesis in LDL Receptor Deficient Mice Lacking Both Macrophage ABCA1 and ApoE 
PLoS ONE  2011;6(10):e26095.
ABCA1 protects against atherosclerosis by facilitating cholesterol efflux from macrophage foam cells in the arterial wall to extracellular apolipoprotein (apo) A-I. In contrast to apoA-I, apoE is secreted by macrophages and can, like apoA-I, induce ABCA1-mediated cholesterol efflux. Yet, the combined effect of macrophage ABCA1 and apoE on lesion development is unexplored.
Methods and Results
LDL receptor knockout (KO) mice were transplanted with bone marrow from ABCA1/apoE double KO (dKO) mice, their respective single KO's, and wild-type (WT) controls and were challenged with a high-fat/high-cholesterol diet for 9 weeks. In vitro cholesterol efflux experiments showed no differences between ABCA1 KO and dKO macrophages. The serum non-HDL/HDL ratio in dKO transplanted mice was 1.7-fold and 2.4-fold (p<0.01) increased compared to WT and ABCA1 KO transplanted mice, respectively. The atherosclerotic lesion area in dKO transplanted animals (650±94×103 µm2), however, was 1.9-fold (p<0.01) and 1.6-fold (p<0.01) increased compared to single knockouts (ABCA1 KO: 341±20×103 µm2; apoE KO: 402±78×103 µm2, respectively) and 3.1-fold increased (p<0.001) compared to WT (211±20×103 µm2). When normalized for serum cholesterol exposure, macrophage ABCA1 and apoE independently protected against atherosclerotic lesion development (p<0.001). Moreover, hepatic expression levels of TNFα and IL-6 were highly induced in dKO transplanted animals (3.0-fold; p<0.05, and 4.3-fold; p<0.001, respectively). In agreement, serum IL-6 levels were also enhanced in ABCA1 KO transplanted mice (p<0.05) and even further enhanced in dKO transplanted animals (3.1-fold as compared to ABCA1 KO transplanted animals; p<0.05).
Combined deletion of macrophage ABCA1 and apoE results in a defect in cholesterol efflux and, compared to ABCA1 KO transplanted mice, elevated serum total cholesterol levels. Importantly, these mice also suffer from enhanced systemic and hepatic inflammation, together resulting in the observed augmented atherosclerotic lesion development.
PMCID: PMC3191178  PMID: 22022523
24.  An In-Silico Model of Lipoprotein Metabolism and Kinetics for the Evaluation of Targets and Biomarkers in the Reverse Cholesterol Transport Pathway 
PLoS Computational Biology  2014;10(3):e1003509.
High-density lipoprotein (HDL) is believed to play an important role in lowering cardiovascular disease (CVD) risk by mediating the process of reverse cholesterol transport (RCT). Via RCT, excess cholesterol from peripheral tissues is carried back to the liver and hence should lead to the reduction of atherosclerotic plaques. The recent failures of HDL-cholesterol (HDL-C) raising therapies have initiated a re-examination of the link between CVD risk and the rate of RCT, and have brought into question whether all target modulations that raise HDL-C would be atheroprotective. To help address these issues, a novel in-silico model has been built to incorporate modern concepts of HDL biology, including: the geometric structure of HDL linking the core radius with the number of ApoA-I molecules on it, and the regeneration of lipid-poor ApoA-I from spherical HDL due to remodeling processes. The ODE model has been calibrated using data from the literature and validated by simulating additional experiments not used in the calibration. Using a virtual population, we show that the model provides possible explanations for a number of well-known relationships in cholesterol metabolism, including the epidemiological relationship between HDL-C and CVD risk and the correlations between some HDL-related lipoprotein markers. In particular, the model has been used to explore two HDL-C raising target modulations, Cholesteryl Ester Transfer Protein (CETP) inhibition and ATP-binding cassette transporter member 1 (ABCA1) up-regulation. It predicts that while CETP inhibition would not result in an increased RCT rate, ABCA1 up-regulation should increase both HDL-C and RCT rate. Furthermore, the model predicts the two target modulations result in distinct changes in the lipoprotein measures. Finally, the model also allows for an evaluation of two candidate biomarkers for in-vivo whole-body ABCA1 activity: the absolute concentration and the % lipid-poor ApoA-I. These findings illustrate the potential utility of the model in drug development.
Author Summary
Epidemiological studies have shown a strong inverse association between HDL-C and cardiovascular risk and led to the formulation of the “HDL cholesterol hypothesis”: under this hypothesis, interventions raising HDL-C should decrease risk. However, the recent failures of HDL-C raising therapies in improving cardiovascular disease risk in outcomes trials have suggested a need to revise the hypothesis to account for the contrary data. An “HDL flux hypothesis” has emerged: it is not HDL-C level per se which forms the basis for reducing risk, but it is the flux rate of reverse cholesterol transport that drives risk reduction. We propose that, the concentration of HDL cholesteryl ester in plasma simply reflects the ratio of input rate of reverse cholesterol transport into the HDL compartments to its clearance rate. A challenge in identifying targets under the new conceptual framework is the feedback process that occurs between the input rate and the clearance rate of HDL-C. To meet this challenge, we have built a systems model which incorporates the main processes of HDL metabolism to elucidate the relationships between target modulations and the reverse cholesterol transport rate.
PMCID: PMC3952822  PMID: 24625468
25.  MicroRNA-144 Regulates Hepatic ABCA1 and Plasma HDL Following Activation of the Nuclear Receptor FXR 
Circulation research  2013;112(12):1602-1612.
The bile acid receptor Farnesoid-X-Receptor (FXR) regulates many aspects of lipid metabolism by various complex and not fully understood molecular mechanisms. We set out to investigate the molecular mechanisms for FXR-dependent regulation of lipid and lipoprotein metabolism.
To identify FXR-regulated microRNAs that were subsequently involved in regulating lipid metabolism.
Methods and Results
ATP binding cassette transporter A1 (ABCA1) is a major determinant of plasma High Density Lipoprotein (HDL)-cholesterol levels. Here we show that activation of the nuclear receptor FXR in vivo increases hepatic levels of miR-144, which in turn lower hepatic ABCA1 and plasma HDL levels. We identified two complementary sequences to miR-144 in the 3′ untranslated region (UTR) of ABCA1 mRNA that are necessary for miR-144-dependent regulation. Overexpression of miR-144 in vitro decreased both cellular ABCA1 protein and cholesterol efflux to lipid-poor apolipoprotein A-I (ApoA-I) protein, whilst overexpression in vivo reduced hepatic ABCA1 protein and plasma HDL-cholesterol. Conversely, silencing miR-144 in mice increased hepatic ABCA1 protein and HDL-cholesterol. In addition, we utilized tissue-specific FXR deficient mice to show that induction of miR-144 and FXR-dependent hypolipidemia requires hepatic, but not intestinal FXR. Finally, we identified functional FXR response elements (FXREs) upstream of the miR-144 locus, consistent with direct FXR regulation.
We have identified a novel pathway involving FXR, miR-144 and ABCA1 that together regulate plasma HDL cholesterol.
PMCID: PMC3995747  PMID: 23519696
FXR; HDL cholesterol; miRNA

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