The role of adiponectin in cholesterol efflux and HDL biogenesis and metabolism.

Cholesterol efflux is the initial step in the reverse cholesterol transport pathway by which excess cholesterol in peripheral cells is exported and subsequently packaged into high-density lipoprotein (HDL) particles. Adiponectin is the most abundantly secreted adipokine that possesses anti-inflammatory and vasculoprotective properties via interaction with transmembrane receptors, AdipoR1 and AdipoR2. Evidence suggests that low levels of adiponectin may be a useful marker for atherosclerotic disease. A proposed anti-atherogenic mechanism of adiponectin involves its ability to promote cholesterol efflux. We performed a systematic review of the role of adiponectin in cholesterol efflux and HDL biogenesis, and of the proteins and receptors believed to be implicated in this process. Nineteen eligible studies (7 clinical, 11 fundamental, 1 clinical + fundamental) were identified through Ovid Medline, Ovid Embase, and Pubmed, that support the notion that adiponectin plays a key role in promoting ABCA1-dependent cholesterol efflux and in modulating HDL biogenesis via activation of the PPAR-γ/LXR-α signalling pathways in macrophages. AdipoR1 and AdipoR2 are suggested to also be implicated in this process, however the data are conflicting/insufficient to establish any firm conclusions. Once the exact mechanisms are unravelled, adiponectin may be critical in defining future treatment strategies directed towards increasing HDL functionality and ultimately reducing atherosclerotic disease.

Apolipoprotein A-1 mimetic peptide 4F promotes endothelial repairing and compromises reendothelialization impaired by oxidized HDL through SR-B1.

Disruption of endothelial monolayer integrity is the primary instigating factor for many cardiovascular diseases. High density lipoprotein (HDL) oxidized by heme enzyme myeloperoxidase (MPO) is dysfunctional in promoting endothelial repair. Apolipoprotein A-1 mimetic 4F with its pleiotropic benefits has been proven effective in many in vivo models. In this study we investigated whether 4F promotes endothelial repair and restores the impaired function of oxidized HDL (Cl/NO2-HDL) in promoting re-endothelialization. We demonstrate that 4F and Cl/NO2-HDL act on scavenger receptor type I (SR-B1) using human aorta endothelial cells (HAEC) and SR-B1 (-/-) mouse aortic endothelial cells. Wound healing, transwell migration, lamellipodia formation and single cell migration assay experiments show that 4F treatment is associated with a recovery of endothelial cell migration and associated with significantly increased endothelial nitric oxide synthase (eNOS) activity, Akt phosphorylation and SR-B1 expression. 4F increases NO generation and diminishes oxidative stress. In vivo, 4F can stimulate cell proliferation and re-endothelialization in the carotid artery after treatment with Cl/NO2-HDL in a carotid artery electric injury model but fails to do so in SR-B1(-/-) mice. These findings demonstrate that 4F promotes endothelial cell migration and has a potential therapeutic benefit against early endothelial injury in cardiovascular diseases.

Desmocollin 1 is abundantly expressed in atherosclerosis and impairs high-density lipoprotein biogenesis.

Aims: The biogenesis of high-density lipoprotein (HDL) particles by cholesterol-laden foam cells in atherosclerotic lesions is crucial for the removal of excess cholesterol from the lesions. Impairment in the HDL biogenic process contributes to the progression of atherosclerosis. The aim of this study is to identify novel cellular factors regulating HDL biogenesis. Methods and results: HDL biogenesis is a process of apolipoprotein (apo)-mediated solubilization of specific plasma membrane (PM) microdomains generated in cholesterol-accumulated cells. We established a new method to isolate PM microdomains interacting with the major HDL protein constituent, apoA-I. Lipidomic and proteomic analyses of an isolated PM microdomain revealed that apoA-I binds to cholesterol-rich and desmocollin 1 (DSC1)-containing microdomains. In this novel apoA-I binding microdomain, DSC1 binds and prevents apoA-I from interacting with another PM microdomain created by adenosine triphosphate-binding cassette transporter A1 (ABCA1) for the formation of HDL. Inhibition of apoA-I-DSC1 binding by silencing DSC1 expression or using DSC1 blocking antibodies increases apoA-I accessibility to ABCA1-created microdomains and thus enhances HDL biogenesis. Importantly, DSC1 is abundantly expressed in macrophages and human atherosclerotic lesions, suggesting that DSC1 may contribute to cholesterol accumulation in atherosclerotic lesions by sequestering apoA-I and impairing HDL biogenesis. Conclusions: The binding of apoA-I to two functionally opposing PM microdomains, ABCA1 and DSC1 domains, suggests that HDL biogenesis and PM cholesterol levels may be regulated by the relative abundance of the two domains and that novel HDL biogenic therapies may be developed by targeting DSC1.

ATP binding cassette A1 (ABCA1) mediates microparticle formation during high-density lipoprotein (HDL) biogenesis.

BACKGROUND AND AIMS: Micro-particles (MP) are secreted by various cells. Their biological roles in health and in disease remain unknown. Here we describe formation of MP in the process of ABCA1-dependent cholesterol efflux in different cell types. METHODS: The ATP-binding cassette transporter, subfamily A, member 1 (ABCA1) is the rate-limiting step in the biogenesis of high-density lipoproteins (HDL). We have found that ABCA1 and apoA-I contribute to the formation of MP. Using cell-based systems with overexpression and selective inactivation of ABCA1, pharmacological blockade and modulation of membrane cholesterol content, we characterized MP release from various cell lines. We studied MP release in BHK cells stably expressing ABCA1 under mifepristone control, human THP-1 macrophages and HepG2 cells without, or with incubation with human apoA-I. RESULTS: ABCA1 mediates the production of MPs containing cholesterol. This was also confirmed in primary human monocyte-derived macrophages (MDMs). Adding apoA-I markedly increases MP release from cells. Inhibition of ABCA1 with probucol or decreasing plasma membrane cholesterol with methyl-ß cyclodextrin (CDX) markedly reduced MP release and nascent HDL formation. MPs do not contain apoA-I, but contain flotilin-2, a marker of plasma membrane, and CD63, an exosome marker. MPs exhibit considerable size heterogeneity (50-250 nm). CONCLUSIONS: We show that MPs are lipoprotein-sized structures created by the ABCA1 transporter, and contribute approximately 30% of ABCA1-and apoA-I mediated cholesterol efflux. In addition, we found that MPs release from cells consists, in part, of exosomes and depends on the same pathway used for HDL biogenesis.

Extracellular lipid-free apolipoprotein E inhibits HCV replication and induces ABCG1-dependent cholesterol efflux.

OBJECTIVE: The HCV life cycle and the lipid metabolism are inextricably intertwined. In the blood, HCV virions are associated with lipoproteins, forming lipoviroparticles (LVPs), which are the most infectious form of the virus. Apolipoprotein E (apoE), a key LVP component, plays an essential role in HCV entry, assembly and egress. ApoE is also a cell host factor involved in lipoprotein homeostasis. Although the majority of apoE is associated with lipoproteins, a lipid-free (LF) form exists in blood. However, the role of LF-apoE in both lipid metabolism and HCV life cycle is poorly understood. DESIGN: In this study, using the cell culture-derived HCV model system in human hepatoma Huh7.5.1 cells and primary human hepatocytes (PHH), we investigated the effect of LF-apoE on the early steps of HCV life cycle and on the lipid metabolism of hepatic cells. RESULTS: A dose-dependent decrease in HCV replication was observed when Huh7.5.1 cells and PHH were treated with increasing amounts of LF-apoE. We showed that LF-apoE acts on HCV replication independently of previously described apoE receptors. We observed that LF-apoE induced a marked hepatic cholesterol efflux via the ATP-binding cassette subfamily G member 1 (ABCG1) protein that in turn inhibits HCV replication. LF-apoE also increases both apolipoprotein AI and high-density lipoprotein production. CONCLUSIONS: Our findings highlight a new mechanism in lipid metabolism regulation and interaction of the lipid metabolism with the HCV life cycle, which may be important for viral pathogenesis and might also be explored for antiviral therapy.

Neo High-Density Lipoprotein Produced by the Streptococcal Serum Opacity Factor Activity against Human High-Density Lipoproteins Is Hepatically Removed via Dual Mechanisms.

Injection of streptococcal serum opacity factor (SOF) into mice reduces the plasma cholesterol level by ∼40%. In vitro, SOF converts high-density lipoproteins (HDLs) into multiple products, including a small HDL, neo HDL. In vitro, neo HDL accounts for ∼60% of the protein mass of the SOF reaction products; in vivo, the accumulated mass of neo HDL is <1% of that observed in vitro. To identify the underlying cause of this difference, we determined the fate of neo HDL in plasma in vitro and in vivo. Following incubation with HDL, neo HDL-PC rapidly transfers to HDL, giving a small remnant, which fuses with HDL. An increased level of SR-B1 expression in Huh7 hepatoma cells and a reduced level of LDLR expression in CHO cells had little effect on neo HDL-[3H]CE uptake. Thus, the dominant receptors for neo HDL uptake are not LDLR or SR-B1. The in vivo metabolic fates of neo HDL-[3H]CE and HDL-[3H]CE were different. Thirty minutes after the injection of neo HDL-[3H]CE and HDL-[3H]CE into mice, plasma [3H]CE counts were 40 and 53%, respectively, of injected counts, with 10 times more [3H]CE appearing in the livers of neo HDL-[3H]CE-injected than in those of HDL-[3H]CE-injected mice. These data support a model of neo HDL-[3H]CE clearance by two parallel pathways. At early post-neo HDL-[3H]CE injection times, some neo HDL is directly removed by the liver; the remainder transfers its PC to HDL, leaving a remnant that fuses with HDL, which is also hepatically removed more slowly. Given that SR-B1 and SOF both remove CE from HDL, this novel mechanism may also underlie the metabolism of remnants released by hepatocytes following selective SR-B1-mediated uptake of HDL-CE.

High-Density Lipoprotein Biogenesis: Defining the Domains Involved in Human Apolipoprotein A-I Lipidation.

The first step in removing cholesterol from a cell is the ATP-binding cassette transporter 1 (ABCA1)-driven transfer of cholesterol to lipid-free or lipid-poor apolipoprotein A-I (apoA-I), which yields cholesterol-rich nascent high-density lipoprotein (nHDL) that then matures in plasma to spherical, cholesteryl ester-rich HDL. However, lipid-free apoA-I has a three-dimensional (3D) conformation that is significantly different from that of lipidated apoA-I on nHDL. By comparing the lipid-free apoA-I 3D conformation of apoA-I to that of 9-14 nm diameter nHDL, we formulated the hypothetical helical domain transitions that might drive particle formation. To test the hypothesis, ten apoA-I mutants were prepared that contained two strategically placed cysteines several of which could form intramolecular disulfide bonds and others that could not form these bonds. Mass spectrometry was used to identify amino acid sequence and intramolecular disulfide bond formation. Recombinant HDL (rHDL) formation was assessed with this group of apoA-I mutants. ABCA1-driven nHDL formation was measured in four mutants and wild-type apoA-I. The mutants contained cysteine substitutions in one of three regions: the N-terminus, amino acids 34 and 55 (E34C to S55C), central domain amino acids 104 and 162 (F104C to H162C), and the C-terminus, amino acids 200 and 233 (L200C to L233C). Mutants were studied in the locked form, with an intramolecular disulfide bond present, or unlocked form, with the cysteine thiol blocked by alkylation. Only small amounts of rHDL or nHDL were formed upon locking the central domain. We conclude that both the N- and C-terminal ends assist in the initial steps in lipid acquisition, but that opening of the central domain was essential for particle formation.

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

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

Biogenesis and cytotoxicity of APOL1 renal risk variant proteins in hepatocytes and hepatoma cells.

Two APOL1 gene variants, which likely evolved to protect individuals from African sleeping sickness, are strongly associated with nondiabetic kidney disease in individuals with recent African ancestry. Consistent with its role in trypanosome killing, the pro-death APOL1 protein is toxic to most cells, but its mechanism of cell death is poorly understood and little is known regarding its intracellular trafficking and secretion. Because the liver appears to be the main source of circulating APOL1, we examined its secretory behavior and mechanism of toxicity in hepatoma cells and primary human hepatocytes. APOL1 is poorly secreted in vitro, even in the presence of chemical chaper-ones; however, it is efficiently secreted in wild-type transgenic mice, suggesting that APOL1 secretion has specialized requirements that cultured cells fail to support. In hepatoma cells, inducible expression of APOL1 and its risk variants promoted cell death, with the G1 variant displaying the highest degree of toxicity. To explore the basis for APOL1-mediated cell toxicity, endoplasmic reticulum stress, pyroptosis, autophagy, and apoptosis were examined. Our results suggest that autophagy represents the predominant mechanism of APOL1-mediated cell death. Overall, these results increase our understanding of the basic biology and trafficking behavior of circulating APOL1 from the liver.

High-density lipoprotein synthesis and metabolism (Review).

High density lipoproteins (HDL) are heterogeneous particles regarding their size and composition. They have vital functions in reverse cholesterol transport (RCT). RCT occurs when lipid-free apolipoprotein AI recruits cholesterol and phospholipid to form nascent HDL particles. Adenosine triphosphate­binding cassette transporters and scavenger receptor class B type I were found to be associated with the synthesis of HDL. Experimental studies have identified several potential anti­atherogenic effects of HDL, including promotion of macrophage cholesterol outflow as well as anti­inflammatory and anti­thrombotic effects. HDL can also transport microRNAs. This review mainly summarizes the present knowledge of HDL synthesis and metabolism.

BH3 domain-independent apolipoprotein L1 toxicity rescued by BCL2 prosurvival proteins.

The potent trypanolytic properties of human apolipoprotein L1 (APOL1) can be neutralized by the trypanosome variant surface antigen gene product known as serum resistance-associated protein. However, two common APOL1 haplotypes present uniquely in individuals of West African ancestry each encode APOL1 variants resistant to serum resistance-associated protein, and each confers substantial resistance to human African sleeping sickness. In contrast to the dominantly inherited anti-trypanosomal activity of APOL1, recessive inheritance of these two trypanoprotective APOL1 alleles predisposes to kidney disease. Proposed mechanisms of APOL1 toxicity have included BH3 domain-dependent autophagy and/or ion channel activity. We probed these potential mechanisms by expressing APOL1 in Xenopus laevis oocytes. APOL1 expression in oocytes increased ion permeability and caused profound morphological deterioration (toxicity). Coexpression of BCL2 family members rescued APOL1-associated oocyte toxicity in the order MCL1 ∼ BCLW > BCLXL ∼ BCL2A1 ≫ BCL2. Deletion of nine nominal core BH3 domain residues abolished APOL1-associated toxicity, but missense substitution of the same residues abolished neither oocyte toxicity nor its rescue by coexpressed MCL1. The APOL1 BH3 domain was similarly dispensable for the ability of APOL1 to rescue intact mice from lethal trypanosome challenge. Replacement of most extracellular Na(+) by K(+) also reduced APOL1-associated oocyte toxicity, allowing demonstration of APOL1-associated increases in Ca(2+) and Cl(-) fluxes and oocyte ion currents, which were similarly reduced by MCL1 coexpression. Thus APOL1 toxicity in Xenopus oocytes is BH3-independent, but can nonetheless be rescued by some BCL2 family proteins.

Cellular Cholesterol Accumulation Facilitates Ubiquitination and Lysosomal Degradation of Cell Surface-Resident ABCA1.

OBJECTIVE: By excreting cellular cholesterol to apolipoprotein A-I, ATP-binding cassette transporter A1 (ABCA1) mediates the biogenesis of high-density lipoprotein in hepatocytes and prevents foam cell formation from macrophages. We recently showed that cell surface-resident ABCA1 (csABCA1) undergoes ubiquitination and later lysosomal degradation through the endosomal sorting complex required for transport system. Herein, we investigated the relevance of this degradation pathway to the turnover of csABCA1 in hypercholesterolemia. APPROACH AND RESULTS: Immunoprecipitation and cell surface-biotinylation studies with HepG2 cells and mouse peritoneal macrophages showed that the ubiquitination level and degradation of csABCA1 were facilitated by treatment with a liver X receptor (LXR) agonist and acetylated low-density lipoprotein. The effects of an LXR agonist and acetylated low-density lipoprotein on the degradation of csABCA1 were repressed completely by treatment with bafilomycin, an inhibitor of lysosomal degradation, and by depletion of tumor susceptibility gene 101, a major component of endosomal sorting complex required for transport-I. RNAi analysis indicated that LXRß inhibited the accelerated lysosomal degradation of csABCA1 by the LXR agonist, regardless of its transcriptional activity. Cell surface coimmunoprecipitation with COS1 cells expressing extracellularly hemagglutinin-tagged ABCA1 showed that LXRß interacted with csABCA1 and inhibited the ubiquitination of csABCA1. Immunoprecipitates with anti-ABCA1 antibodies from the liver plasma membranes showed less LXRß and a higher ubiquitination level of ABCA1 in high-fat diet-fed mice than in normal chow-fed mice. CONCLUSIONS: Under conditions of high cellular cholesterol content, csABCA1 became susceptible to ubiquitination by dissociation of LXRß from csABCA1, which facilitated the lysosomal degradation of csABCA1 through the endosomal sorting complex required for transport system.

apoE3[K146N/R147W] acts as a dominant negative apoE form that prevents remnant clearance and inhibits the biogenesis of HDL.

The K146N/R147W substitutions in apoE3 were described in patients with a dominant form of type III hyperlipoproteinemia. The effects of these mutations on the in vivo functions of apoE were studied by adenovirus-mediated gene transfer in different mouse models. Expression of the apoE3[K146N/R147W] mutant in apoE-deficient (apoE(-/-)) or apoA-I-deficient (apoA-I(-/-))×apoE(-/-) mice exacerbated the hypercholesterolemia and increased plasma apoE and triglyceride levels. In apoE(-/-) mice, the apoE3[K146N/R147W] mutant displaced apoA-I from the VLDL/LDL/HDL region and caused the accumulation of discoidal apoE-containing HDL. The WT apoE3 cleared the cholesterol of apoE(-/-) mice without induction of hypertriglyceridemia and promoted formation of spherical HDL. A unique property of the truncated apoE3[K146N/R147W]202 mutant, compared with similarly truncated apoE forms, is that it did not correct the hypercholesterolemia. The contribution of LPL and LCAT in the induction of the dyslipidemia was studied. Treatment of apoE(-/-) mice with apoE3[K146N/R147W] and LPL corrected the hypertriglyceridemia, but did not prevent the formation of discoidal HDL. Treatment with LCAT corrected hypertriglyceridemia and generated spherical HDL. The combined data indicate that the K146N/R147W substitutions convert the full-length and the truncated apoE3[K146N/R147W] mutant into a dominant negative ligand that prevents receptor-mediated remnant clearance, exacerbates the dyslipidemia, and inhibits the biogenesis of HDL.

Combination BMSC and Niaspan treatment of stroke enhances white matter remodeling and synaptic protein expression in diabetic rats.

OBJECTIVE: White matter remodeling plays an important role in neurological recovery after stroke. Bone marrow stromal cells (BMSCs) and Niaspan, an agent which increases high density lipoprotein (HDL), each induces neurorestorative effects and promotes white matter remodeling after stroke in non-diabetic rats. In this study, we test whether combination of BMSCs with Niaspan induces an enhanced white matter remodeling in the ischemic brain of diabetic rats. RESEARCH DESIGN AND METHODS: Type-1 diabetes (T1DM) rats were subjected to transient middle cerebral artery occlusion (MCAo) and treated with or without BMSCs; Niaspan; and the combination of BMSCs + Niaspan daily for 14 days after MCAo. Immunostaining for white matter remodeling and synaptic protein expression including NG2; CNPase; BS (Bielschowsky silver); LFB (luxol fast blue); Synaptophysin and SMI-31 immunostaining were performed. RESULTS: BMSC monotherapy did not regulate NG2 and CNPase expression compared to T1DM control rats. Both, combination of BMSCs + Niaspan treatment, and Niaspan monotherapy significantly increase NG2 and CNPase expression compared to T1DM control. While combination BMSC+Niaspan, BMSC monotherapy and Niaspan monotherapy groups all increase BS, LFB, synaptophysin, and SMI-31 expression in the ischemic brain compared to T1DM-MCAo control. In addition, the combination treatment significantly enhances LFB, SMI-31, and Synaptophysin expression compared to BMSC monotherapy. CONCLUSIONS: Combination treatment of stroke with BMSCs and Niaspan in T1DM rats increases white matter remodeling and additively increases BMSC monotherapy induced myelination and synaptic plasticity after stroke in T1DM rats.

Significance of the hydrophobic residues 225-230 of apoA-I for the biogenesis of HDL.

We studied the significance of four hydrophobic residues within the 225-230 region of apoA-I on its structure and functions and their contribution to the biogenesis of HDL. Adenovirus-mediated gene transfer of an apoA-I[F225A/V227A/F229A/L230A] mutant in apoA-I⁻/⁻ mice decreased plasma cholesterol, HDL cholesterol, and apoA-I levels. When expressed in apoA-I⁻/⁻ × apoE⁻/⁻ mice, approximately 40% of the mutant apoA-I as well as mouse apoA-IV and apoB-48 appeared in the VLDL/IDL/LDL. In both mouse models, the apoA-I mutant generated small spherical particles of pre-ß- and α4-HDL mobility. Coexpression of the apoA-I mutant and LCAT increased and shifted the-HDL cholesterol peak toward lower densities, created normal αHDL subpopulations, and generated spherical-HDL particles. Biophysical analyses suggested that the apoA-I[225-230] mutations led to a more compact folding that may limit the conformational flexibility of the protein. The mutations also reduced the ability of apoA-I to promote ABCA1-mediated cholesterol efflux and to activate LCAT to 31% and 66%, respectively, of the WT control. Overall, the apoA-I[225-230] mutations inhibited the biogenesis of-HDL and led to the accumulation of immature pre-ß- and α4-HDL particles, a phenotype that could be corrected by administration of LCAT.

Role of the hydrophobic and charged residues in the 218-226 region of apoA-I in the biogenesis of HDL.

We investigated the significance of hydrophobic and charged residues 218-226 on the structure and functions of apoA-I and their contribution to the biogenesis of HDL. Adenovirus-mediated gene transfer of apoA-I[L218A/L219A/V221A/L222A] in apoA-I⁻/⁻ mice decreased plasma cholesterol and apoA-I levels to 15% of wild-type (WT) control mice and generated pre-ß- and α4-HDL particles. In apoA-I⁻/⁻ × apoE⁻/⁻ mice, the same mutant formed few discoidal and pre-ß-HDL particles that could not be converted to mature α-HDL particles by excess LCAT. Expression of the apoA-I[E223A/K226A] mutant in apoA-I⁻/⁻ mice caused lesser but discrete alterations in the HDL phenotype. The apoA-I[218-222] and apoA-I[E223A/K226A] mutants had 20% and normal capacity, respectively, to promote ABCA1-mediated cholesterol efflux. Both mutants had ∼65% of normal capacity to activate LCAT in vitro. Biophysical analyses suggested that both mutants affected in a distinct manner the structural integrity and plasticity of apoA-I that is necessary for normal functions. We conclude that the alteration of the hydrophobic 218-222 residues of apoA-I disrupts apoA-I/ABCA1 interactions and promotes the generation of defective pre-ß particles that fail to mature into α-HDL subpopulations, thus resulting in low plasma apoA-I and HDL. Alterations of the charged 223, 226 residues caused milder but discrete changes in HDL phenotype.

ApoA-IV promotes the biogenesis of apoA-IV-containing HDL particles with the participation of ABCA1 and LCAT.

The objective of this study was to establish the role of apoA-IV, ABCA1, and LCAT in the biogenesis of apoA-IV-containing HDL (HDL-A-IV) using different mouse models. Adenovirus-mediated gene transfer of apoA-IV in apoA-I(-/-) mice did not change plasma lipid levels. ApoA-IV floated in the HDL2/HDL3 region, promoted the formation of spherical HDL particles as determined by electron microscopy, and generated mostly α- and a few pre-ß-like HDL subpopulations. Gene transfer of apoA-IV in apoA-I(-/-) × apoE(-/-) mice increased plasma cholesterol and triglyceride levels, and 80% of the protein was distributed in the VLDL/IDL/LDL region. This treatment likewise generated α- and pre-ß-like HDL subpopulations. Spherical and α-migrating HDL particles were not detectable following gene transfer of apoA-IV in ABCA1(-/-) or LCAT(-/-) mice. Coexpression of apoA-IV and LCAT in apoA-I(-/-) mice restored the formation of HDL-A-IV. Lipid-free apoA-IV and reconstituted HDL-A-IV promoted ABCA1 and scavenger receptor BI (SR-BI)-mediated cholesterol efflux, respectively, as efficiently as apoA-I and apoE. Our findings are consistent with a novel function of apoA-IV in the biogenesis of discrete HDL-A-IV particles with the participation of ABCA1 and LCAT, and may explain previously reported anti-inflammatory and atheroprotective properties of apoA-IV.

Androgen deprivation by flutamide modulates uPAR, MMP-9 expressions, lipid profile, and oxidative stress: amelioration by daidzein.

The growth and development of prostate gland is governed by testosterone. Testosterone helps in maintaining the adipose tissue stores of the body. It is well documented that with advancing age there has been a gradual decline in testosterone levels. Our aim was to study the protective role of daidzein on flutamide-induced androgen deprivation on matrix degrading genes, lipid profile and oxidative stress in Wistar rats. Sub-chronic (60 days) flutamide (30 mg/kg b.wt) administration resulted in marked increase in expressions of matrix degrading genes [matrix metalloproteases 9 and urokinase plasminogen activation receptor]. Additionally, it increased the levels of low density lipoproteins, total cholesterol, triglycerides, and lowered the levels of high density lipoproteins and endogenous antioxidant levels. Oral administration of daidzein (20 and 60 mg/kg b.wt) restituted the levels to normal. Daidzein administration resulted in amelioration of the prostate atrophy, degeneracy and invasiveness induced by flutamide. Our findings suggest that the daidzein may be given as dietary supplement to patients who are on androgen deprivation therapy, to minimize the adverse effects related to it and also retarding susceptibility of patients to cardiovascular diseases.

ABCA1-dependent mobilization of lysosomal cholesterol requires functional Niemann-Pick C2 but not Niemann-Pick C1 protein.

Niemann-Pick disease type C (NPC) is caused by mutations leading to loss of function of NPC1 or NPC2 proteins, resulting in accumulation of unesterified cholesterol in late endosomes and lysosomes. We previously reported that expression of the ATP-binding cassette transporter A1 (ABCA1) is impaired in human NPC1(-/-) fibroblasts, resulting in reduced HDL particle formation and providing a mechanism for the reduced plasma HDL cholesterol seen in the majority of NPC1 patients. We also found that treatment of NPC1(-/-) fibroblasts with an agonist of liver X-receptor corrects ABCA1 expression and HDL formation and reduces lysosomal cholesterol accumulation. We have confirmed that ABCA1 expression is also reduced in NPC2(-/-) cells, and found that α-HDL particle formation is impaired in these cells. To determine whether selective up-regulation of ABCA1 can correct lysosomal cholesterol accumulation in NPC disease cells and HDL particle formation, we produced and infected NPC1(-/-) and NPC2(-/-) fibroblasts with an adenovirus expressing full-length ABCA1 and enhanced green fluorescent protein (AdABCA1-EGFP). ABCA1-EGFP expression in NPC1(-/-) fibroblasts resulted in normalization of cholesterol efflux to apolipoprotein A-I (apoA-I) and α-HDL particle formation, plus a marked reduction in filipin staining of unesterified cholesterol in late endosomes/lysosomes. In contrast, AdABCA1-EGFP treatment of NPC2(-/-) fibroblasts to normalize ABCA1 expression had no effect on cholesterol efflux to apoA-I or accumulation of excess cholesterol in lysosomes, and only partially corrected α-HDL formation by these cells. These results suggest that correction of ABCA1 expression can bypass the mutation of NPC1 but not NPC2 to mobilize excess cholesterol from late endosomes and lysosomes in NPC disease cells. Expression of ABCA1-EGFP in NPC1(-/-) cells increased cholesterol available for esterification and reduced levels of HMG-CoA reductase protein, effects that were abrogated by co-incubation with apoA-I. A model can be generated in which ABCA1 is able to mobilize cholesterol, to join the intracellular regulatory pool or to be effluxed for HDL particle formation, either directly or indirectly from the lysosomal membrane, but not from the lysosomal lumen. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).

ApoA-I enhances generation of HDL-like lipoproteins through interaction between ABCA1 and phospholipase Cγ in rat astrocytes.

In the previous paper, we reported that apolipoprotein (apo) A-I enhances generation of HDL-like lipoproteins in rat astrocytes to be accompanied with both increase in tyrosine phosphorylation of phospholipase Cγ (PL-Cγ) and PL-Cγ translocation to cytosolic lipid-protein particles (CLPP) fraction. In this paper, we studied the interaction between apoA-I and ATP-binding cassette transporter A1 (ABCA1) to relate with PL-Cγ function for generation of HDL-like lipoproteins in the apoA-I-stimulated astrocytes. ABCA1 co-migrated with exogenous apoA-I with apparent molecular weight over 260kDa on SDS-PAGE when rat astrocytes were treated with apoA-I and then with a cross-linker, BS3. The solubilized ABCA1 of rat astrocytes was associated with the apoA-I-immobilized Affi-Gel 15. An LXR agonist, To901317, increased the cellular level of ABCA1, association of apoA-I with ABCA1 and apoA-I-mediated lipid release in rat astrocytoma GA-1/Mock cells where ABCA1 expression at baseline is very low. PL-Cγ was co-isolated by apoA-I-immobilized Affi-Gel 15 and co-immunoprecipitated by anti-ABCA1 antibody along with ABCA1 from the solubilized membrane fraction of rat astrocytes. The SiRNA of ABCA1 suppressed not only the PL-Cγ binding to ABCA1 but also the tyrosine phosphorylation of PL-Cγ. A PL-C inhibitor, U73122, prevented generation of apoA-I-mediated HDL-like lipoproteins in rat astrocytes. To901317 increased the association of PL-Cγ with ABCA1 in GA-1/Mock cells dependently on the increase of cellular level of ABCA1 without changing that of PL-Cγ. These findings suggest that the exogenous apoA-I augments the interaction between PL-Cγ and ABCA1 to stimulate tyrosine phosphorylation and activation of PL-Cγ for generation of HDL-like lipoproteins in astrocytes.