Peroxisome proliferator-activated receptor delta activation leads to increased transintestinal cholesterol efflux

Peroxisome proliferator-activated receptor delta (PPARδ) is involved in regulation of energy homeostasis. Activation of PPARδ markedly increases fecal neutral sterol secretion, the last step in reverse cholesterol transport. This phenomenon can neither be explained by increased hepatobiliary cholesterol secretion, nor by reduced cholesterol absorption. To test the hypothesis that PPARδ activation leads to stimulation of transintestinal cholesterol efflux (TICE), we quantified it by intestine perfusions in FVB mice treated with PPARδ agonist GW610742. To exclude the effects on cholesterol absorption, mice were also treated with cholesterol absorption inhibitor ezetimibe or ezetimibe/GW610742. {"type":"entrez-nucleotide","attrs":{"text":"GW601742","term_id":"299511820","term_text":"GW601742"}}GW601742 treatment had little effect on plasma lipid levels but stimulated both fecal neutral sterol excretion (∼200%) and TICE (∼100%). GW610742 decreased intestinal Npc1l1 expression but had no effect on Abcg5/Abcg8. Interestingly, expression of Rab9 and LIMPII, encoding proteins involved in intracellular cholesterol trafficking, was increased upon PPARδ activation. Although treatment with ezetimibe alone had no effect on TICE, it reduced the effect of GW610742 on TICE. These data show that activation of PPARδ stimulates fecal cholesterol excretion in mice, primarily by the two-fold increase in TICE, indicating that this pathway provides an interesting target for the development of drugs aiming at the prevention of atherosclerosis.     

Serum, but not monocyte macrophage foam cells derived from low HDL-C subjects, displays reduced cholesterol efflux capacity

The main antiatherogenic function of HDL is to promote the efflux of cholesterol from peripheral cells and transport it to the liver for excretion in a process termed reverse cholesterol transport. The aim of this study was to evaluate the cholesterol efflux capacity in low- and high-HDL subjects by utilizing monocytes and serum from 18 low-HDL and 15 high-HDL subjects. Low and high HDL levels were defined, respectively, as HDL < or =10(th) and HDL > or =90(th) Finnish age/sex-specific percentile. Cholesterol efflux from [(3)H]cholesterol-oleate-acetyl-LDL-loaded monocyte-derived macrophages to standard apolipoprotein A-I (apoA-I), HDL(2), and serum was measured. In addition, cholesterol efflux from acetyl-LDL-loaded human THP-1 macrophages to individual sera (0.5%) derived from the study subjects was evaluated. Cholesterol efflux to apoA-I, HDL(2), and serum from macrophage foam cells derived from low- and high-HDL subjects was similar. The relative ABCA1 and ABCG1 mRNA expression levels in unloaded macrophages, as well as their protein levels in loaded macrophage foam cells, were similar in the two study groups. Cholesterol efflux from THP-1 foam cells to serum recovered from high-HDL subjects was slightly higher than that to serum from low-HDL subjects (P = 0.046). Cholesterol efflux from THP-1 macrophages to serum from study subjects correlated with serum apoB (P = 0.033), apoA-I (P = 0.004), apoA-II (P < 0.0001), and the percentage of apoA-I present in the form of prebeta-HDL (P = 0.0001). Our data reveal that macrophages isolated from either low- or high-HDL subjects display similar cholesterol efflux capacity to exogenous acceptors. However, sera from low-HDL subjects have poorer cholesterol acceptor ability as compared with sera from high-HDL subjects.     

Enhancing apolipoprotein A-I-dependent cholesterol efflux elevates cholesterol export from macrophages in vivo

Eight proteins potentially involved in cholesterol efflux [ABCA1, ABCG1, CYP27A1, phospholipid transfer protein (PLTP), scavenger receptor type BI (SR-BI), caveolin-1, cholesteryl ester transfer protein, and apolipoprotein A-I (apoA-I)] were overexpressed alone or in combination in RAW 264.7 macrophages. When apoA-I was used as an acceptor, overexpression of the combination of ABCA1, CYP27A1, PLTP, and SR-BI (Combination I) enhanced the efflux by 4.3-fold. It was established that the stimulation of efflux was due to increased abundance of ABCA1 and increased apoA-I binding to non-ABCA1 sites on macrophages. This combination caused only a small increase of the efflux to isolated HDL. When HDL was used as an acceptor, overexpression of caveolin-1 or a combination of caveolin-1 and SR-BI (Combination II) was the most active, doubling the efflux to HDL, without affecting the efflux to apoA-I. When tested in the in vivo mouse model of cholesterol efflux, overexpression of ABCA1 and Combination I elevated cholesterol export from macrophages to plasma, liver, and feces, whereas overexpression of caveolin-1 or Combination II did not have an effect. We conclude that pathways of cholesterol efflux using apoA-I as an acceptor make a predominant contribution to cholesterol export from macrophages in vivo.     

In vivo tissue cholesterol efflux is reduced in carriers of a mutation in APOA1

Atheroprotection by high density lipoprotein (HDL) is considered to be mediated through reverse cholesterol transport (RCT) from peripheral tissues. We investigated in vivo cholesterol fluxes through the RCT pathway in patients with low plasma high density lipoprotein cholesterol (HDL-c) due to mutations in APOA1. Seven carriers of the L202P mutation in APOA1 (mean HDL-c: 20 ± 19 mg/dl) and seven unaffected controls (mean HDL-c: 54 ± 11 mg/dl, P < 0.0001) received a 20 h infusion of ¹³C₂-cholesterol (¹³C-C). Enrichment of plasma and erythrocyte free cholesterol and plasma cholesterol esters was measured. With a three-compartment SAAM-II model, tissue cholesterol efflux (TCE) was calculated. TCE was reduced by 19% in carriers (4.6 ± 0.8 mg/kg/h versus 5.7 ± 0.7 mg/kg/h in controls, P = 0.02). Fecal ¹³C recovery and sterol excretion 7 days postinfusion did not differ significantly between carriers and controls: 21.3 ± 20% versus 13.3 ± 6.3% (P = 0.33), and 2,015 ± 1,431 mg/day versus 1456 ± 404 mg/day (P = 0.43), respectively. TCE is reduced in carriers of mutations in APOA1, suggesting that HDL contributes to efflux of tissue cholesterol in humans. The residual TCE and unaffected fecal sterol excretion in our severely affected carriers suggest, however, that non-HDL pathways contribute to RCT significantly.     

Recycling of apoprotein E is associated with cholesterol efflux and high density lipoprotein internalization

After receptor-mediated endocytosis of triglyceride-rich lipoproteins (TRL) into the liver, TRL particles are immediately disintegrated in peripheral endosomal compartments. Whereas core lipids and apoprotein B are delivered for degradation into lysosomes, TRL-derived apoE is efficiently recycled back to the plasma membrane. This is followed by apoE re-secretion and association of apoE with high density lipoproteins (HDL). Because HDL and apoE can independently promote cholesterol efflux, we investigated whether recycling of TRL-derived apoE in human hepatoma cells and fibroblasts could be linked to intracellular cholesterol transport. In this study we demonstrate that HDL(3) does not only act as an extracellular acceptor for recycled apoE but also stimulates the recycling of internalized TRL-derived apoE. Furthermore, radioactive pulse-chase experiments indicate that apoE recycling is accompanied by cholesterol efflux. Confocal imaging reveals co-localization of apoE and cholesterol in early endosome antigen 1-positive endosomes. During apoE re-secretion, HDL(3)-derived apoA-I is found in these early endosome antigen 1, cholesterol-containing endosomes. As shown by time-lapse fluorescence microscopy, apoE recycling involves the intracellular trafficking of apoA-I to pre-existing and TRL-derived apoE/cholesterol-containing endosomes in the periphery. Thus, these studies provide evidence for a new intracellular link between TRL-derived apoE, cellular cholesterol transport, and HDL metabolism.     

Age-related impairment of HDL-mediated cholesterol efflux

Our aim in this study was to investigate the effect of aging on the capacity of HDLs to promote reverse cholesterol transport. HDLs were isolated from plasma of young (Y-HDL) and elderly (E-HDL) subjects. HDL-mediated cholesterol efflux was studied using THP-1 and J774 macrophages. Our results show that E-HDLs present a lower capacity to promote cholesterol efflux than Y-HDLs (41.7 +/- 1.4% vs. 49.0 +/- 2.2%, respectively; P = 0.013). Reduction in the HDL-mediated cholesterol efflux capacity with aging was more significant with HDL(3) than HDL(2) (Y-HDL(3), 57.3 +/- 1% vs. E-HDL(3), 50.9 +/- 2%; P = 0.012). Moreover, our results show that ABCA1-mediated cholesterol efflux is the more affected pathway in terms of cholesterol-removing capacity. Interestingly, the composition and structure of HDL revealed a reduction in the phosphatidylcholine-sphingomyelin ratio (E-HDL, 32.7 +/- 2.7 vs. Y-HDL, 40.0 +/- 1.9; P = 0.029) and in the phospholipidic layer membrane fluidity in E-HDL compared with Y-HDL as well as an alteration in the apolipoprotein A-I structure and charge. In conclusion, our results shown that E-HDLs present a reduced capacity to promote cholesterol efflux, principally through the ABCA1 pathway, and this may explain the increase of the incidence of cardiovascular diseases observed during aging.     

Role of LCAT in HDL remodeling: investigation of LCAT deficiency states

To better understand the role of LCAT in HDL metabolism, we compared HDL subpopulations in subjects with homozygous (n = 11) and heterozygous (n = 11) LCAT deficiency with controls (n = 22). Distribution and concentrations of apolipoprotein A-I (apoA-I)-, apoA-II-, apoA-IV-, apoC-I-, apoC-III-, and apoE-containing HDL subpopulations were assessed. Compared with controls, homozygotes and heterozygotes had lower LCAT masses (-77% and -13%), and LCAT activities (-99% and -39%), respectively. In homozygotes, the majority of apoA-I was found in small, disc-shaped, poorly lipidated prebeta-1 and alpha-4 HDL particles, and some apoA-I was found in larger, lipid-poor, discoidal HDL particles with alpha-mobility. No apoC-I-containing HDL was noted, and all apoA-II and apoC-III was detected in lipid-poor, prebeta-mobility particles. ApoE-containing particles were more disperse than normal. ApoA-IV-containing particles were normal. Heterozygotes had profiles similar to controls, except that apoC-III was found only in small HDL with prebeta-mobility. Our data are consistent with the concepts that LCAT activity: 1) is essential for developing large, spherical, apoA-I-containing HDL and for the formation of normal-sized apoC-I and apoC-III HDL; and 2) has little affect on the conversion of prebeta-1 into alpha-4 HDL, only slight effects on apoE HDL, and no effect on apoA-IV HDL particles.     

Robust passive and active efflux of cellular cholesterol to a designer functional mimic of high density lipoprotein

The ability of HDL to support macrophage cholesterol efflux is an integral part of its atheroprotective action. Augmenting this ability, especially when HDL cholesterol efflux capacity from macrophages is poor, represents a promising therapeutic strategy. One approach to enhancing macrophage cholesterol efflux is infusing blood with HDL mimics. Previously, we reported the synthesis of a functional mimic of HDL (fmHDL) that consists of a gold nanoparticle template, a phospholipid bilayer, and apo A-I. In this work, we characterize the ability of fmHDL to support the well-established pathways of cellular cholesterol efflux from model cell lines and primary macrophages. fmHDL received cell cholesterol by unmediated (aqueous) and ABCG1- and scavenger receptor class B type I (SR-BI)-mediated diffusion. Furthermore, the fmHDL holoparticle accepted cholesterol and phospholipid by the ABCA1 pathway. These results demonstrate that fmHDL supports all the cholesterol efflux pathways available to native HDL and thus, represents a promising infusible therapeutic for enhancing macrophage cholesterol efflux. fmHDL accepts cholesterol from cells by all known pathways of cholesterol efflux: unmediated, ABCG1- and SR-BI-mediated diffusion, and through ABCA1.     

对氧磷降低RAW264.7巨噬细胞源性泡沫细胞ABCA1表达和胆固醇流出

三磷酸腺苷结合盒转运体A1(ABCA1)是体内胆固醇逆向转运的关键环节.对氧磷是广泛使用的有机磷农药的活性代谢产物.研究发现,对氧磷能增加巨噬细胞中胆固醇的堆积,但具体机制还不清楚.以RAW264.7巨噬细胞源性泡沫细胞为研究对象,观察对氧磷对RAW264.7巨噬细胞源性泡沫细胞ABCA1表达和胆固醇流出的影响并探讨其机制.结果显示,对氧磷以时间和剂量依赖的方式增加RAW264.7巨噬细胞源性泡沫细胞中总胆固醇、游离胆固醇和胆固醇酯水平,降低ABCA1表达和胆固醇流出,同时对氧磷降低细胞中环磷酸腺苷(cAMP)的水平及腺苷酸环化酶(AC)的活性和增加磷酸二酯酶(PDE)的活性,而cAMP的类似物双丁酰环腺苷酸(dBcAMP)能够阻断对氧磷降低ABCA1表达和部分阻断对氧磷降低胆固醇流出,对氧磷导致的cAMP水平的降低也可被AC激动剂福斯高林(Forskolin)和PDE抑制剂3-异丁基-1-甲基黄嘌呤(IBMX)所阻断.以上结果表明,对氧磷通过cAMP信号通路下调RAW264.7巨噬细胞源性泡沫细胞ABCA1的表达,降低细胞内胆固醇流出和增加细胞内胆固醇堆积.     

Plasma cholesterol efflux capacity from human THP-1 macrophages is reduced in HIV-infected patients: impact of HAART

The capacity of HDL to remove cholesterol from macrophages is inversely associated with the severity of angiographic coronary artery disease. The effect of human immunodeficiency virus (HIV) infection or its treatment on the ability of HDL particles to stimulate cholesterol efflux from human macrophages has never been studied. We evaluated the capacity of whole plasma and isolated HDL particles from HIV-infected subjects (n = 231) and uninfected controls (n = 200), as well as in a subset of 41 HIV subjects receiving highly active antiretroviral therapy (HAART) to mediate cholesterol efflux from human macrophages. Plasma cholesterol efflux capacity was reduced (−12%; P = 0.001) in HIV patients as compared with controls. HIV infection reduced by 27% (P < 0.05) the capacity of HDL subfractions to promote cholesterol efflux from macrophages. We observed a reduced ABCA1-dependent efflux capacity of plasma (−27%; P < 0.0001) from HIV-infected subjects as a result of a reduction in the efflux capacity of HDL3 particles. HAART administration restored the capacity of plasma from HIV patients to stimulate cholesterol efflux from human macrophages (9.4%; P = 0.04). During HIV infection, the capacity of whole plasma to remove cholesterol from macrophages is reduced, thus potentially contributing to the increased coronary heart disease in the HIV population. HAART administration restored the removal of cholesterol from macrophages by increasing HDL functionality.     

Incorporation of cholesterol into serum high density lipoprotein apoprotein and recombinants

The uptake of cholesterol (CHL) by serum high density lipoprotein (HDL) delipidated apoproteins and phospholipid-apoprotein recombinants has been studied with two methods; by incubation with Celite-dispersed cholesterol or with cholesterol crystals. The apoproteins bind very small amounts of cholesterol with a maximum of about 6 micrograms/mg apoprotein. Recombinants with dimyristoyl phosphatidylcholine (DMPC) or egg phosphatidylcholine (EPC) as phospholipid component gave similar values for cholesterol uptake. The initial rate for uptake from Celite-cholesterol by recombinants was high (0.1 mol cholesterol/mol phospholipid/h) and somewhat higher than that for phospholipid vesicles. The maximal uptake was by gel filtration shown to depend on the size of the complexes with values about 0.95 mol cholesterol per phospholipid for vesicular complexes, 0.75 for discoidal complexes and between 0.5 and 0.2 for small 'protein-rich' complexes. During the incubation of recombinants with cholesterol there was considerable decomposition of discoidal complexes and formation of larger ones. The results show that phospholipid-apoprotein complexes are efficient acceptors for cholesterol but also that about 25% of the phospholipid in the discoidal complexes is excluded from interaction with cholesterol by interaction with apoprotein.     

ATP binding cassette G1-dependent cholesterol efflux during inflammation

ATP binding cassette transporter G1 (ABCG1) mediates the transport of cellular cholesterol to HDL, and it plays a key role in maintaining macrophage cholesterol homeostasis. During inflammation, HDL undergoes substantial remodeling, acquiring lipid changes and serum amyloid A (SAA) as a major apolipoprotein. In the current study, we investigated whether remodeling of HDL that occurs during acute inflammation impacts ABCG1-dependent efflux. Our data indicate that lipid free SAA acts similarly to apolipoprotein A-I (apoA-I) in mediating sequential efflux from ABCA1 and ABCG1. Compared with normal mouse HDL, acute phase (AP) mouse HDL containing SAA exhibited a modest but significant 17% increase in ABCG1-dependent efflux. Interestingly, AP HDL isolated from mice lacking SAA (SAAKO mice) was even more effective in promoting ABCG1 efflux. Hydrolysis with Group IIA secretory phospholipase A(2) (sPLA(2)-IIA) significantly reduced the ability of AP HDL from SAAKO mice to serve as a substrate for ABCG1-mediated cholesterol transfer, indicating that phospholipid (PL) enrichment, and not the presence of SAA, is responsible for alterations in efflux. AP human HDL, which is not PL-enriched, was somewhat less effective in mediating ABCG1-dependent efflux compared with normal human HDL. Our data indicate that inflammatory remodeling of HDL impacts ABCG1-dependent efflux independent of SAA.