酰基辅酶A胆固醇酰基转移酶-1与动脉粥样硬化

酰基辅酶A胆固醇酰基转移酶目前认为是细胞内唯一合成胆固醇酯的酶,维持着细胞以及生物体胆固醇代谢的平衡,而酰基辅酶A胆固醇酰基转移酶-1在动脉粥样硬化的发生、发展过程中发挥着至关重要的作用。自从酰基辅酶A胆固醇酰基转移酶的cDNA在1993年首次被克隆之后,许多学者对酰基辅酶A胆固醇酰基转移酶-1进行了大量的研究,现就近年来酰基辅酶A胆固醇酰基转移酶-1与动脉粥样硬化关系的研究予以综述。     

脂肪分化相关蛋白促进RAW 264.7细胞内酰基辅酶A:胆固醇酰基转移酶1高表达

目的 观察高表达脂肪分化相关蛋白细胞内酰基辅酶A∶胆固醇酰基转移酶1表达的变化,阐明脂肪分化相关蛋白促进细胞内脂质蓄积的机制.方法 构建的pQCXIP-HA-Adi逆转录病毒载体转染PA317包装细胞,获得pQCXIP-HA-Adi逆转录病毒.用病毒感染RAW 264.7细胞, Puromycin筛选后获得稳定高表达脂肪分化相关蛋白的RAW264.7细胞株.应用逆转录聚合酶链反应和免疫印迹法检测经感染后细胞内脂肪分化相关蛋白和酰基辅酶A∶胆固醇酰基转移酶1的表达.并应用阿托伐他汀处理高表达脂肪分化相关蛋白的RAW 264.7细胞,观察酰基辅酶A∶胆固醇酰基转移酶1表达的改变.结果 用pQCXIP-HA-Adi逆转录病毒感染RAW 264.7细胞后,脂肪分化相关蛋白和酰基辅酶A∶胆固醇酰基转移酶1 mRNA和蛋白表达明显升高,而对照组表达无明显变化.加入阿托伐他汀后,即在去除底物对酰基辅酶A∶胆固醇酰基转移酶1表达影响的情况下,高表达脂肪分化相关蛋白细胞内酰基辅酶A∶胆固醇酰基转移酶1表达仍升高.结论 高表达脂肪分化相关蛋白可明显上调RAW 264.7细胞酰基辅酶A∶胆固醇酰基转移酶1的表达.     

肿瘤坏死因子α对乙酰辅酶A:胆固醇酰基转移酶1基因表达的影响

目的研究泡沫细胞形成过程中肿瘤坏死因子-α（TNF-α）对乙酰辅酶A：胆固醇酰基转移酶1（ACAT1）基因的影响。方法体外培养人THP-1单核细胞系,由佛波酯作用使其分化为巨噬细胞,再由乙酰化低密度脂蛋白作用转变为泡沫细胞。TNF-α分别作用于上述三种细胞24h,用Western blot法检测ACAT1蛋白表达,RT-PCR法检测ACAT1 mRNA水平。结果在单核细胞分化为巨噬细胞及转变为泡沫细胞的过程中,ACAT1蛋白及mRNA均增加（P均〈0．05）;TNF-α可上调三种细胞ACAT1蛋白表达及mRNA水平（P均〈0．05）。结论单核细胞分化为巨噬细胞及转变为泡沫细胞的过程中,ACAT1基因表达明显增加,TNF-α可明显促进ACAT1基因表达。     

大鼠乙酰辅酶A∶胆固醇酰基转移酶的C末端片段在大肠杆菌中的融合表达及纯化

为进一步探讨乙酰辅酶Ａ：胆固醇酰基转移酶的结构和功能的关系,经聚合酶链反应扩增得到的乙酰辅酶Ａ∶胆固醇酰基转移酶（ＡＣＡＴ）的Ｃ末端膜外结构域（氨基酸４８０～５４５）用Ｅｃｏｒｌ酶解,插入到表达载体ｐＧＥＸ２ＴＫ中,得到重组表达质粒ｐＧＥＸ２ＴＫ／ＡＣＡＴ（氨基酸４８０～５４５）。阳性重组子在大肠杆菌中经异丙基βＤ硫代半乳糖苷诱导表达谷胱甘肽转移酶－乙酰辅酶Ａ∶胆固醇酰基转移酶（氨基酸４８０～５４５）,重组表达菌裂解上清液经谷胱甘肽ＳｅｐｈａｒｏｓｅＣＬ４Ｂ亲和柱纯化。十二烷基硫酸钠－聚丙烯酰胺凝胶电泳和Ｗｅｓｔｅｒｎｂｌｏｔｉｎｇ分析显示得到了较纯的谷胱甘肽转移酶－乙酰辅酶Ａ∶胆固醇酰基转移酶（氨基酸４８０～５４５）融合蛋白。乙酰辅酶Ａ∶胆固醇酰基转移酶的Ｃ末端膜外结构域（氨基酸４８０～５４５）的分离纯化为抗谷胱甘肽转移酶－乙酰辅酶Ａ∶胆固醇酰基转移酶（Ｃ未端片段）抗体的制备及其乙酰辅酶Ａ∶胆固醇酰基转移酶结构和功能关系的进一步研究打下了基础。     

PPAR-γ对单核/巨噬细胞酰基辅酶A:胆固醇酰基转移酶-1表达效应的研究

目的　研究配体罗格列酮活化的过氧化体增殖物激活型受体 γ(PPAR γ)对单核 /巨噬细胞转分化过程中酰基辅酶A :胆固醇酰基转移酶 1 (Acyl CoA :cholesterolacyltransferases,ACAT 1 )表达效应的影响。 方法　在RPMI1 640培养基中培养人单核细胞 (THP 1 ) ,加入佛波酯(PMA)培养 48h,细胞贴壁呈巨噬细胞样分化。运用免疫细胞化学、逆转录聚合酶链反应、蛋白质免疫印迹等方法 ,观察单核巨噬细胞转分化前后PPAR γ对ACAT 1mRNA和蛋白表达水平的影响。结果　单核巨噬细胞转分化前后ACAT 1表达增加 ,罗格列酮活化的PPAR γ可明显抑制A CAT 1的表达。结论　动脉粥样硬化事件的发生可能与ACAT 1表达增强有关。罗格列酮活化的PPAR γ可能通过抑制ACAT 1表达 ,巨噬细胞摄取脂质降低 ,从而减少泡沫细胞的形成 ,发挥其抗动脉粥样硬化的作用     

卵磷脂胆固醇酰基转移酶与动脉粥样硬化性心血管病

卵磷脂胆固醇酰基转移酶(LCAT)是血浆脂蛋白中催化游离胆固醇酯化的关键酶,在高密度脂蛋白代谢和胆固醇逆转运中起着重要作用。尽管LCAT研究已经进行了半个世纪,其与动脉粥样硬化性心血管病的关系仍存在较大争议。本文就LCAT生物化学特性、在脂代谢中的作用机制、与心血管病的关系及准确测定存在的问题等作一综述。     

葡萄糖对THP-1源性巨噬细胞酰基辅酶A:胆固醇酰基转移酶1表达的影响

研究葡萄糖对人THP-1单核分化巨噬细胞酰基辅酶A：胆固醇酰基转移酶1（ACAT-1）表达的影响。发现高糖作用下，巨噬细胞ACAT-1的mRNA及蛋白表达增加，这可能是糖尿病血管病变机制之一。     

胆固醇诱导未折叠蛋白反应及其调节酰基辅酶A胆固醇酰基转移酶2基因的表达

目的探讨胆固醇对肝细胞和小肠粘膜上皮细胞中未折叠蛋白反应及其调节酰基辅酶A胆固醇酰基转移酶2基因表达的影响。方法以不同浓度(10mg/L和20mg/L)的游离胆固醇和氧化胆固醇温育肝癌细胞系HepG2细胞和小肠粘膜上皮细胞系Caco2细胞,应用半定量逆转录聚合酶链反应法检测两种细胞中X盒结合蛋白1和酰基辅酶A胆固醇酰基转移酶2mRNA的表达水平。结果随着游离胆固醇和氧化胆固醇温育浓度的升高,HepG2细胞和Caco2细胞中的X盒结合蛋白1和酰基辅酶A胆固醇酰基转移酶2mRNA表达均上调,呈现浓度依赖性。结论胆固醇和氧化胆固醇均可诱导未折叠蛋白反应中标记分子X盒结合蛋白1的表达,并上调酰基辅酶A胆固醇酰基转移酶2的表达,提示酰基辅酶A胆固醇酰基转移酶2的表达可能受未折叠蛋白反应的调控;鉴于酰基辅酶A胆固醇酰基转移酶2在胆固醇吸收的酯化过程中具有重要作用,未折叠蛋白反应可能在胆固醇吸收调控中具有重要意义。     

人ACAT1基因P1启动子的克隆及意义

目的克隆人酰基辅酶A:胆固醇酰基转移酶1(ACAT 1)基因P 1启动子。方法应用PCR方法从人单核细胞系THP-1扩增分离出ACAT 1基因P 1启动子全长片段,将PCR产物克隆入T载体,并对所获得的序列进行生物信息学分析。结果经琼脂糖凝胶电泳及直接测序鉴定,克隆的ACAT 1基因P 1启动子片段碱基序列与G enB ank数据库一致,未发现突变。结论人ACAT 1基因P 1启动子克隆成功,此为动脉粥样硬化(A S)过程中ACAT 1基因转录调控机制的研究奠定了基础。     

Conjugated and non-conjugated octadecaenoic acids affect differently intestinal acyl coenzyme A: cholesterol acyltransferase activity

We investigated the relative hypocholesterolemic activity of linoleic acid (LA), conjugated linoleic acid (CLA), -linolenic acid (LN) and conjugated linolenic acid (CLN) in hamsters. Five groups of hamsters (n = 10 each) were fed either the control diet or one of the four fatty acids-supplemented diets for 6 weeks. Results demonstrated that the four octadecaenoic acids decreased plasma cholesterol differently, with CLA being the most effective. Western blotting and RT-PCR analysis demonstrated that the four octadecaenoic acids had no effect on sterol regulatory element binding protein-2 (SREBP-2), liver X receptor (LXR), 3-hydroxy-3-methylglutary-CoA reductase (HMGR), LDL receptor (LDLR), and cholesterol-7-hydroxylase (CYP7A1). However, the four octadecaenoic acids increased the excretion of fecal neutral sterols with CLA being most effective followed by LN, LA and CLN, suggesting they all differentially affect cholesterol absorption. Dietary CLA was associated with the least intestinal acyl coenzyme A: cholesterol acyltransferase (ACAT) activity followed by LN, LA and CLN in a decreasing trend. Since esterification of cholesterol is catalyzed by intestinal ACAT, and is a rate-limiting step in cholesterol absorption, it was concluded that the varying effects of CLA, LN, LA and CLN on blood cholesterol were mediated, at least in part, by their inhibition on intestinal ACAT activity.     

The impact of glycation on apolipoprotein A-I structure and its ability to activate lecithin:cholesterol acyltransferase

Aims/hypothesis Hyperglycaemia, one of the main features of diabetes, results in non-enzymatic glycation of plasma proteins, including apolipoprotein A-I (apoA-I), the most abundant apolipoprotein in HDL. The aim of this study was to determine how glycation affects the structure of apoA-I and its ability to activate lecithin:cholesterol acyltransferase (LCAT), a key enzyme in reverse cholesterol transport. Materials and methods Discoidal reconstituted HDL (rHDL) containing phosphatidylcholine and apoA-I ([A-I]rHDL) were prepared by the cholate dialysis method and glycated by incubation with methylglyoxal. Glycation of apoA-I was quantified as the reduction in detectable arginine, lysine and tryptophan residues. Methylglyoxal-AGE adduct formation in apoA-I was assessed by immunoblotting. (A-I)rHDL size and surface charge were determined by non-denaturing gradient gel electrophoresis and agarose gel electrophoresis, respectively. The kinetics of the LCAT reaction was investigated by incubating varying concentrations of discoidal (A-I)rHDL with a constant amount of purified enzyme. The conformation of apoA-I was assessed by surface plasmon resonance. Results Methylglyoxal-mediated modifications of the arginine, lysine and tryptophan residues in lipid-free and lipid-associated apoA-I were time- and concentration-dependent. These modifications altered the conformation of apoA-I in regions critical for LCAT activation and lipid binding. They also decreased (A-I)rHDL size and surface charge. The rate of LCAT-mediated cholesterol esterification in (A-I)rHDL varied according to the level of apoA-I glycation and progressively decreased as the extent of apoA-I glycation increased. Conclusions/interpretation It is concluded that glycation of apoA-I may adversely affect reverse cholesterol transport in subjects with diabetes.     

Alterations in plasma lecithin:cholesterol acyltransferase and myeloperoxidase in acute myocardial infarction: Implications for cardiac outcome

Background

The cholesterol esterifying enzyme, lecithin:cholesterol acyltransferase (LCAT), plays a key role in HDL maturation and remodeling. Myeloperoxidase (MPO) may compromise LCAT enzymatic activity. We tested the extent to which plasma LCAT activity is altered in acute myocardial infarction (MI) in conjunction with abnormal MPO levels. We also assessed the impact of LCAT and MPO on newly developed major adverse cardiovascular events (MACE).

Methods

Two-hundred one consecutive patients referred for acute chest pain of whom 134 had MI (95 with ST-elevation) participated. Forty-five new MACE were ascertained during 1203 (range 13–1745) days of follow-up among 185 patients. Plasma LCAT activity was measured using an exogenous substrate assay. MPO mass was assayed by chemiluminescent microparticle immunoassay.

Results

Plasma LCAT activity was decreased by 15%, coinciding with 7-fold increased MPO levels in acute MI patients vs. patients with non-cardiac chest pain (p < 0.001 for both; correlation: r = −0.343, p < 0.001). MI at admission was associated independently with both lower plasma LCAT activity and higher MPO (age- and sex-adjusted odds ratio per 1 SD increment: 0.46 (95% CI, 0.31–0.68), p < 0.001 and 7.58 (95% CI, 3.34–17.11), p < 0.001, respectively). In an analysis with LCAT and MPO together these associations were modestly attenuated. MPO mass (hazard ratio: 1.59 (95% CI, 1.15–2.19), p = 0.004), but not LCAT activity (hazard ratio: 0.87 (95% CI, 0.65–1.19), p = 0.39), predicted newly manifest MACE.

Conclusion

In acute MI patients, plasma LCAT activity is decreased coinciding with increased MPO levels. Higher MPO but not lower LCAT activity prospectively predicts adverse cardiac outcome.     

Lecithin:cholesterol acyltransferase activity and high-density lipoprotein subfraction composition in type 1 diabetic patients with improving metabolic control

On initial diagnosis or when metabolic control is poor, subjects with type 1 (insulin-dependent) diabetes mellitus often exhibit decreased high density lipoprotein (HDL) cholesterol levels, which have been associated in numerous studies in non-diabetic subjects with atherosclerosis and coronary artery disease. We measured the activities of plasma lecithin:cholesterol acyltransferase (LCAT), post-heparin lipoprotein lipase, and the composition of the HDL subfractions HDL₂ and HDL₃, in ten poorly controlled type 1 diabetic patients admitted to a metabolic ward (six women and four men, aged 18–37 years). The measurements were repeated after metabolic control had been optimised and again a week after discharge. The results were compared with those of ten healthy normolipidaemic subjects matched for age, sex and body mass. LCAT activity increased significantly (P<0.05) with improved metabolic control in the diabetic patients, and showed positive within — person correlation with HDL₂ cholesterol ester (r=0.67;P<0.01), HDL₂ free cholesterol (r=0.67;P<0.01), phosphatidylcholine (r=0.49;P<0.05), total phospholipids (r=0.50;P<0.01) and apolipoprotein A-I (apo A-I:r=0.72;P<0.01). With improving metabolic control HDL₂ lipid levels increased more than twofold and the compositional changes in HDL₂ were reflected by an increased apo A-I:apo A-II ratio (P<0.05) and a decreased triglyceride:apo A-I ratio (P<0.05). Changes in HDL₃ levels and composition were minor. The results of this study indicate that an increase in LCAT activity increases the concentration and changes the composition of HDL₂ in type 1 diabetic patients with improved metabolic control.     

Determination of hepatic acyl-coenzyme A-cholesterol acyltransferase activity in LPN hamsters: A model for cholesterol gallstone formation

Acyl-coenzyme A-cholesterol acyltransferase (ACAT) catalyses the esterification of cholesterol with long-chain fatty acyl-coenzyme A derivatives and has been implicated in the development of cholesterol gallstones. In this study we have examined several key components of the hepatic ACAT assay in order to develop a reliable and sensitive ACAT assay for LPN hamsters, a breed of golden Syrian hamster which has been characterized recently by this laboratory as a particularly good model for studying the pathogenesis of cholesterol gallstones. The newly developed ACAT assays were subsequently used to examine whether hepatic ACAT activity is altered in this animal model. Important new methodological findings were: (i) ACAT activity displayed two pH optima, one at 7.0 when assayed using endogenous cholesterol as substrate, and the other at about pH 8.5–9.0 when assayed in the presence of exogenous cholesterol; (ii) ACAT activity increased markedly when exogenous cholesterol was delivered to ACAT in Tween 80 (125-fold) or hydroxypropyl-β-cyclodextrin (200-fold) in contrast to the use of cholesterol/phosphatidylcholine liposomes (9-fold); (iii) the addition of dithiothreitol, but not reduced glutathione, to the assay mixture resulted in a marked decrease in ACAT activity. Using the optimal assay conditions (exogenous cholesterol added), hepatic ACAT activity was shown to be significantly reduced in hamsters fed a high sucrose lithogenic diet compared with controls (587 ± 42 vs 737 ± 44 pmol/min per mg; P= 0.025). In contrast, ACAT activity measured using endogenous cholesterol as a substrate was greater in sucrose-fed hamsters compared with controls (22.3 ± 2.5 vs 13.2 ± 2.9 pmol/min per mg; P= 0.030). These results highlight the importance of using an ACAT activity assay which has been well characterized and supports the hypothesis that the pathogenesis of cholesterol gallstones in LPN hamsters is related to an altered hepatic cholesterol metabolism.     

乙酰化低密度脂蛋白促进胆固醇酰基转移酶活性升高的机制研究

目的 :研究乙酰化低密度脂蛋白 (Ac LDL)对人THP 1单核细胞 (MC)分化的巨噬细胞 (MP)酰基辅酶A、胆固醇酰基转移酶 1(ACAT 1)活性的影响及其机制。方法 :体外培养人THP 1单核细胞系 ,由佛波酯 (PMA)作用使其分化为MP ,后者再由乙酰化低密度脂蛋白Ac LDL进行脂质负荷转变为泡沫细胞。该过程中以放射性同位素标记底物法检测ACAT 1酶活性的变化 ,并用Westernblot法检测ACAT 1酶蛋白的表达及RT PCR法检测A CAT 1mRNA的水平。结果 :在MC分化为MP的过程中ACAT 1酶活性升高了 2倍 ,差异有统计学意义 (P <0 .0 5 ) ,酶蛋白及mRNA水平呈现类似变化趋势 ;Ac LDL作用于巨噬细胞 ,使ACAT1酶活性、酶蛋白及mRNA进一步升高 ,差异有统计学意义 (P <0 .0 5 )。结论 :MC分化为MP的过程中ACAT 1表达上调 ,使ACAT 1活性增强 ,而Ac LDL可进一步促进ACAT 1基因表达增加 ,升高ACAT 1活性。     

Diazepam inhibits cholesterol esterification by arterial ACAT and plasma LCAT, in vitro

Benzodiazepine drugs have been reported to have antiatherosclerotic effects in rabbits and roosters and to alter the pattern of circulating lipoproteins in man. The mechanism(s) of these effects has not been elucidated. The studies presented here indicate that diazepam, the most widely used benzodiazepine, is an inhibitor of cholesterol esterification by ACAT in vitro in atheromatous rabbit aortas, in microsomes isolated from atheromatous rabbit aortas, and in normal rat aortas. Diazepam also inhibited LCAT in plasma from man, monkey, rabbit, and rat, in vitro. The ability of diazepam to inhibit these enzyme systems may offer insight into possible in vivo mechanisms of action against atherosclerosis and of lipoprotein modification.