Cloning and sequencing of human cholesteryl ester transfer protein cDNA

The transfer of insoluble cholesteryl esters among lipoprotein particles is a vital step in normal cholesterol homeostasis and may be involved in the development of atherosclerosis. Extrahepatic tissues lack the enzymes required for the degradation of sterols to the excretable form of bile acids. Cholesterol synthesized in these tissues in excess of that needed for the synthesis of cell membranes or steroid hormones must accordingly be returned through the plasma to the liver for catabolism. The series of reactions involved has been termed reverse cholesterol transport. Catalysed steps of this pathway are believed to include an efflux from peripheral cells, which generates a diffusion gradient between these membranes and extracellular fluid; esterification of this cholesterol by lecithin-cholesterol acyltransferase (LCAT) (phosphatidylcholine-sterol acyltransferase) acting on species of high-density lipoproteins; transfer of the cholesteryl esters formed (largely to low- and very low-density lipoproteins) (LDL and VLDL) by a cholesteryl ester transfer protein (CETP); and removal of these lipoproteins, together with their cholesteryl ester content, by the liver through receptor-mediated and nonspecific endocytosis. Of these steps, the CETP reaction is the least characterized. Several laboratories have reported the purification from human plasma of proteins active on cholesteryl ester transfer between lipoprotein particles and possibly between cells and plasma. However, the reported relative molecular mass (Mr), abundance and specificity of the purified activities have differed considerably. We have recently described the preparation of a highly active CETP of Mr 74,000 purified about 100,000-fold from human plasma, which may represent the functional component of earlier preparations. Using a partial amino-acid sequence from this purified protein, CETP complementary DNA derived from human liver DNA has been cloned and sequenced and the cloned DNA used to detect CETP messenger RNA in a number of human tissues.     

Molecular basis of lipid transfer protein deficiency in a family with increased high-density lipoproteins

Plasma high density lipoproteins (HDL) are a negative risk factor for atherosclerosis. Increased HDL is sometimes clustered in families, but a genetic basis has never been clearly documented. The plasma cholesteryl ester transfer protein (CETP) catalyses the transfer of cholesteryl ester from HDL to other lipoproteins and therefore might influence HDL levels. Using monoclonal antibodies, we show that CETP is absent in two Japanese siblings who have markedly increased and enlarged HDL. Furthermore, they are homozygous for a point mutation in the 5'-splice donor site of intron 14 of the gene for CETP, a change that is incompatible with normal splicing of pre-messenger RNA. The results indicate that the family has an inherited deficiency of CETP due to a gene splicing defect, and illustrate the key role that CETP has in human HDL metabolism.     

Direct evidence that reverse cholesterol transport is mediated by high-density lipoprotein in rabbit

Mammalian cells obtain cholesterol for membrane synthesis mostly via the receptor-mediated endocytosis of low-density lipoprotein (LDL). Macrophages and vascular endothelium additionally have receptors that recognize certain modified forms of LDL (for example, acetyl-LDL). The process by which cholesterol returns from peripheral cells to hepatocytes (reverse cholesterol transport) has not been established; although tissue culture studies have favoured high-density lipoprotein (HDL) as the principal vehicle, the in vivo evidence for this is meagre. When cholesterol-loaded macrophages are incubated in medium containing plasma, cholesterol moves from the cells to HDL and is then esterified by lecithin/cholesterol acyltransferase. The accumulation of cholesteryl esters in the particles increases their size and decreases their density; enrichment with apoprotein E (apo E) also occurs, producing a decrease in electrophoretic mobility. We now report that similar changes occur in the circulating HDL of rabbits, when their peripheral tissues are loaded with cholesterol by intravenous (i.v.) injection of acetylated or native human LDL. This result suggests that HDL is involved in reverse cholesterol transport in vivo.     

Ectopic beta-chain of ATP synthase is an apolipoprotein A-I receptor in hepatic HDL endocytosis

The effect of high-density lipoprotein (HDL) in protecting against atherosclerosis is usually attributed to its role in 'reverse cholesterol transport'. In this process, HDL particles mediate the efflux and the transport of cholesterol from peripheral cells to the liver for further metabolism and bile excretion. Thus, cell-surface receptors for HDL on hepatocytes are chief partners in the regulation of cholesterol homeostasis. A high-affinity HDL receptor for apolipoprotein A-I (apoA-I) was previously identified on the surface of hepatocytes. Here we show that this receptor is identical to the beta-chain of ATP synthase, a principal protein complex of the mitochondrial inner membrane. Different experimental approaches confirm this ectopic localization of components of the ATP synthase complex and the presence of ATP hydrolase activity at the hepatocyte cell surface. Receptor stimulation by apoA-I triggers the endocytosis of holo-HDL particles (protein plus lipid) by a mechanism that depends strictly on the generation of ADP. We confirm this effect on endocytosis in perfused rat liver ex vivo by using a specific inhibitor of ATP synthase. Thus, membrane-bound ATP synthase has a previously unsuspected role in modulating the concentrations of extracellular ADP and is regulated by a principal plasma apolipoprotein.     

Inhibition of early atherogenesis in transgenic mice by human apolipoprotein AI

Epidemiological surveys have identified a strong inverse relationship between the amount in the plasma of high density lipoproteins (HDL), apolipoprotein AI (ApoA-I), the major protein component of HDL, and the risk for atherosclerosis in humans. It is not known if this relationship arises from a direct antiatherogenic effect of these plasma components or if it is the result of other factors also associated with increases in ApoA-I and HDL levels. Because some strains of mice are susceptible to diet-induced formation of preatherosclerotic fatty streak lesions, and because of available techniques for the genetic manipulation of this organism, the murine system offers a unique setting in which to investigate the process of early atherogenesis. To test the hypothesis that induction of a high plasma concentration of ApoA-I and HDL would inhibit this process, we studied the effects of atherogenic diets on transgenic mice expressing high amounts of human ApoA-I. We report that transgenic mice with high plasma ApoA-I and HDL levels were significantly protected from the development of fatty streak lesions.     

Complete protein sequence and identification of structural domains of human apolipoprotein B

Epidemiological, pathological and genetic studies show a strong positive correlation between elevated plasma concentrations of low-density lipoprotein (LDL) cholesterol and the risk of premature coronary heart disease. Apolipoprotein (apo) B-100 is the sole protein component of LDL and is the ligand responsible for the receptor-mediated uptake and clearance of LDL from the circulation. Apo B-100 is made by the liver and is essential for the assembly of triglyceride-rich very low-density lipoproteins (VLDL) in the cisternae of the endoplasmic reticulum and for their secretion into the plasma. VLDL transports triglyceride to peripheral muscle and adipose tissue, where the triglyceride is hydrolysed by lipoprotein lipase. The resultant particle, relatively enriched in cholesteryl ester, constitutes LDL. LDL delivers cholesterol to peripheral tissues where it is used for membrane and steroid hormone biosynthesis and to the liver, the only organ which can catabolize and excrete cholesterol. Plasma LDL levels are therefore determined by the balance between their rate of production from VLDL and clearance by the hepatic LDL (apo B/E) receptor pathway. Here we report the complete 4,563-amino-acid sequence of apo B-100 precursor (relative molecular mass (Mr) 514,000 (514K] determined from complementary DNA clones. Numerous lipid-binding structures are distributed throughout the extraordinary length of apo B-100 and must underlie its special functions as a nucleus for lipoprotein assembly and maintenance of plasma lipoprotein integrity. A domain enriched in basic amino-acid residues has been identified as important for the cellular uptake of cholesterol by the LDL receptor pathway.     

决明子提取物降脂作用的研究

探讨决明子溶剂分部提取物实验性高脂血症大鼠血脂的影响。将大鼠制成高脂血症模型，以多烯康为阳性对照，测定决明子粉及其不同溶剂的提取物给药后，对高脂血症大鼠的血清总胆固醇（TC）、甘油三酯（TG）、高密度脂蛋白（HDL）和低密度脂蛋白（LDL）含量的影响。结果显示决明子粉、乙酸乙酯提取物、正丁醇提取物、乙醇提取物和水提取物均可使高脂血症大鼠的TC明显降低（P＜0.05或P＜0.01)；且能显著升高HDL水平（P＜0.05或P＜0.01)。表明决明子溶剂分部提取物的降脂作用随提取溶剂极性的增大而增强。推测决明子中能有效降低血脂的主要成分可能是甙类、蛋白质、多糖等成分。     

以极低密度脂蛋白为指标选育低胆固醇蛋鸡品系

以血浆极低密度脂蛋白(VLDL)为间接选择指标,对蛋鸡血浆VLDL浓度进行双向选择.经过两个世代的选择,在测定第一和第二世代母鸡血浆VLDL浓度及各相关性状值的基础上,估计了部分性状间的相关系数.结果为：血浆高VLDL浓度组母鸡的腹脂重、腹脂含量、蛋黄重、蛋黄比例、全蛋胆固醇总量和全蛋胆固醇含量等虽略高于血浆低VLDL浓度组,但不同处理之间的差异未达到显著水平（p>0.05）.而血浆低VLDL浓度组母鸡的体重、蛋白重和蛋重则相应提高,其中蛋黄比例的差异在第一、第二世代分别达到显著（p<0.05）和极显著水平（p<0.01）.血浆VLDL浓度与蛋重、蛋白重呈负遗传相关,而与体重、腹脂重、蛋黄重、蛋黄比例、全蛋胆固醇总量和全蛋胆固醇含量呈正遗传相关.实验结果表明,血浆VLDL浓度作为间接性状对全蛋胆固醇含量进行选择,来培育生产低胆固醇蛋的蛋鸡品系具有可行性.     

瑞舒伐他汀钙片联合盐酸吡格列酮片对Ⅱ型糖尿病患者血脂及颈动脉粥样硬化的影响

目的观察瑞舒伐他汀钙片联合盐酸吡格列酮片对Ⅱ型糖尿病患者血脂及颈动脉粥样硬化的影响.方法96例Ⅱ型糖尿病患者随机分成对照组与治疗组,对照组单独服用瑞舒伐他汀钙片,治疗组同时服用瑞舒伐他汀钙片与盐酸吡格列酮片.治疗20个月后观察患者空腹血糖(FPG)、甘油三酯(TG)、总胆固醇(TC)、高密度脂蛋白(HDL)、低密度脂蛋白(LDL)、颈动脉内中膜厚度(IMT)及颈动脉斑块Crouse积分等变化.结果治疗前后各组组内比较,对照组TC及LDL明显下降,具有统计学意义(P0.01);IMT、颈动脉斑块Crouse积分下降,具有统计学意义(P0.05);HDL呈升高趋势,但无统计学意义(P0.05).治疗组TC,LDL,TG及FPG明显下降,具有统计学意义(P0.01);IMT、颈动脉斑块Crouse积分均下降,具有统计学意义(P0.05);HDL升高,具有统计学意义(P0.05).治疗后各组组间比较HDL明显升高,具有统计学意义(P0.01);FPG,TG,IMT,颈动脉斑块Crouse积分下降,具有统计学意义(P0.05).结论与单独应用瑞舒伐他汀钙片比较,联合应用瑞舒伐他汀钙片与盐酸吡格列酮片能进一步降低TG,IMT及颈动脉斑块Crouse积分,显著升高HDL.     

载脂蛋白E-基因敲除鼠VLDL和IDL在动脉硬化中的作用

探讨载脂蛋白Ｅ（ａｐｏＥ）－基因敲除鼠极低密度脂蛋白（ＶＬＤＬ）和中间密度脂蛋白（ＩＤＬ）组分（ａｐｏＥｋｏ－ＶＬＤＬ／ＩＤＬ）致动脉硬化作用．采用超离法从ａｐｏＥ－基因敲除鼠血浆中分离ＶＬＤＬ和ＩＤＬ组分,与鼠腹腔巨噬细胞共育,观察ａｐｏＥｋｏ－ＶＬＤＬ／ＩＤＬ与巨噬细胞的相互作用．ＡｐｏＥｋｏ－ＶＬＤＬ／ＩＤＬ导致细胞胆固醇酯含量显著增加．其诱导的细胞［３Ｈ］胆固醇油酸脂量高达１５．１ｎｍｏｌ／ｍｇ细胞蛋白,是天然低密度脂蛋白的８．４倍．形态学观察显示,经ａｐｏＥｋｏ－ＶＬＤＬ／ＩＤＬ处理的巨噬细胞与苏丹黑Ｂ呈阳性染色．细胞结合实验表明,１２５Ｉ－ａｐｏＥｋｏ－ＶＬＤＬ／ＩＤＬ与巨噬细胞的总结合可被非标记配基取代８０％以上,特异结合呈一饱和图形．同时细胞缔合和细胞内降解实验也显示相似结果．非修饰的ａｐｏＥｋｏ－ＶＬＤＬ／ＩＤＬ可通过一特异而不依赖于ａｐｏＥ的途径导致巨噬细胞胆固醇酯的显著蓄积     

Vascular respiratory uncoupling increases blood pressure and atherosclerosis

The observations that atherosclerosis often occurs in non-smokers without elevated levels of low-density lipoprotein cholesterol, and that most atherosclerosis loci so far identified in mice do not affect systemic risk factors associated with atherosclerosis, suggest that as-yet-unidentified mechanisms must contribute to vascular disease. Arterial walls undergo regional disturbances of metabolism that include the uncoupling of respiration and oxidative phosphorylation, a process that occurs to some extent in all cells and may be characteristic of blood vessels being predisposed to the development of atherosclerosis. To test the hypothesis that inefficient metabolism in blood vessels promotes vascular disease, we generated mice with doxycycline-inducible expression of uncoupling protein-1 (UCP1) in the artery wall. Here we show that UCP1 expression in aortic smooth muscle cells causes hypertension and increases dietary atherosclerosis without affecting cholesterol levels. UCP1 expression also increases superoxide production and decreases the availability of nitric oxide, evidence of oxidative stress. These results provide proof of principle that inefficient metabolism in blood vessels can cause vascular disease.     

穿山龙水提取物对大鼠血液生化指标作用的研究

目的研究穿山龙水提取物对大鼠血液生化指标的影响。方法大鼠游泳训练力竭后测定其血液中总胆固醇（TG）、甘油三酯（TG）、高密度脂蛋白（HDL）、低密度脂蛋白（LDL）和极低密度脂蛋白（VLDL）含量、超氧化物歧化酶（SOD）、谷草转氨酶（GOT）、谷丙转氨酶（GPT）活性及血糖（BS）、丙二醛（MDA）和全血血红蛋白（Hb）含量。结果穿山龙水提取物能使力竭运动训练大鼠TC的含量显著下降;HDL的含量显著升高;LDL和VLDL的含量显著下降;SOD活性、BS、Hb含量显著升高;MDA含量、GOT活性显著下降。结论穿山龙水提取物对运动训练大鼠可显著降低血脂、清除运动产生的脂质过氧化物和提高BS、Hb含量。     

枸杞降血脂作用的实验研究

以大鼠为实验动物,研究了枸杞对大鼠血脂水平的影响,结果表明,枸杞可有效降低血清中甘油三脂（TG）,胆固醇（TC）,低密度脂蛋白（LDL－C）含量及LDL－C／HDL－C（高密度脂蛋白）比值,具有明显的降血脂作用。     

Lipoprotein(a) modulation of endothelial cell surface fibrinolysis and its potential role in atherosclerosis

Endothelial cells play a critical role in thromboregulation by virtue of a surface-connected fibrinolytic system. Cultured endothelial cells synthesize and secrete tissue-type plasminogen activator (t-PA) which can bind to at least two discrete sites on the cell surface. These binding sites preserve the catalytic activity of t-PA and protect it from its physiological inhibitor (PAI-1). N-terminal glutamic acid plasminogen (Glu-PLG), the main circulating fibrinolytic zymogen, also interacts specifically with the endothelial cell surface. Binding is associated with a 12-fold increase in catalytic efficiency of plasmin generation by t-PA which may reflect conversion of Glu-PLG to its plasmin-modified form, N-terminal lysine plasminogen (Lys-PLG). Lipoprotein(a) is an atherogenic lipoprotein particle which contains the plasminogen-like apolipoprotein(a) bound to low density lipoprotein. We report here that lipoprotein(a) interferes with endothelial cell fibrinolysis by inhibiting plasminogen binding and hence plasmin generation. In addition, we demonstrate lipoprotein(a) accumulation in atherosclerotic lesions. These findings may provide a link between impaired cell surface fibrinolysis and progressive atherosclerosis.     

黄米醇溶蛋白对小鼠胆固醇代谢的调节作用

比较了分别饲喂酪蛋白、大豆分离蛋白、黄米醇溶蛋白小鼠的血清总胆固醇、高密度脂蛋白胆固醇、低密度脂蛋白胆固醇,甘油三酯浓度和动脉硬化指数,旨在评价黄米醇溶蛋白对小鼠胆固醇代谢的特定作用。结果显示:黄米醇溶蛋白(添加1.82%Lys和0.23%Trp)能显著提高小鼠血清高密度脂蛋白胆固醇浓度(P<0.05),降低动脉硬化指数;但体外实验表明:黄米醇溶蛋白不能抑制羟甲基戊二酸单酰辅酶A(HMG-CoA)还原酶的活性,即不能抑制胆固醇的合成。线性回归分析得出黄米醇溶蛋白对小鼠血清胆固醇代谢的调节作用与其蛋氧酸与甘氨酸含量之比、疏水性氨基酸、含硫氨基酸含量密切相关。     

高脂血症食蟹猴模型的建立及分析

目的 建立高脂血症食蟹猴模型。方法 采用高脂膳食饲料诱导食蟹猴,分别在4、8、12、18个月检测血清总胆固醇(TC)、高密度脂蛋白(HDL)、低密度脂蛋白(LDL)、甘油三脂(TG)指标,对其血脂指标的变化趋势进行分析。结果 模型组在饲喂高脂膳食饲料4个月后,TC、HDL、LDL与实验前相比差异极显著(P<0.01);TG在饲喂高脂膳食饲料8个月后明显升高,与实验前相比差异极显著(P<0.01);造模后,高脂饲料组与正常对照组比较,TC、HDL、LDL、TG均高于对照组,差异极显著(P<0.01)。结论 在本实验中,模型组TC、HDL、LDL、TG均呈现递增趋势,尤其是TC、LDL明显高于食蟹猴正常参考值水平,说明模型组食蟹猴具有高胆固醇和高、低密度脂蛋白血症的特征,初步建立了食蟹猴高脂血症动物模型。     

A DNA insertion in the apolipoprotein A-I gene of patients with premature atherosclerosis

Apolipoprotein A-I (apo A-I) is the major protein constituent of high-density lipoprotein (HDL). The study of the apo A-I gene is of interest because plasma levels of HDL have been inversely correlated with the development of coronary artery disease and because polymorphisms related to this gene have been associated with hypertriglyceridaemia and premature atherosclerosis. We have recently isolated and characterized the human apo A-I gene and have shown that apo A-I and apolipoprotein C-III (apo C-III) genes are physically linked and that a polymorphism (of unknown frequency in the general population) of the apo A-I gene is inherited as a mendelian trait linked to premature atherosclerosis in an affected family (not the same polymorphism as has previously been reported to be associated with hypertriglyceridaemia). Here we report that this polymorphism is due an at least 6.5-kilobase (kb) DNA insertion in the coding region of the apo A-I gene and that there is no detectable alteration (as determined by limited genomic blotting analysis) of the apo C-III gene of these patients.     

血脂检测预测冠状动脉病学的临床意义

目的通过检测血脂在冠状动脉病变中的变化,了解血脂分析预测冠状动脉病变的临 床意义。方法设置病变组和对照组,检测胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白(RDL)、载脂 蛋白AI(APOAI)、载脂蛋白B(APOB)、脂蛋白a[Lp(a)]。结果病变组APOAI、APOB与对照组比较 具有高度显著性差异(P《0．001);TC、TG、HDL、Lp(a)次之(P<0．01);各项指标检测异常比例以 APOAI、APOB、LP(a)最高(69．5％、72.O％、65．9％)。结论血脂6项指标对预测冠状动脉病变均有 临床意义,建议这6项指标均应成为冠状动脉病变预测的常规检测项目。     

Subendothelial retention of atherogenic lipoproteins in early atherosclerosis

Complications of atherosclerosis are the most common cause of death in Western societies. Among the many risk factors identified by epidemiological studies, only elevated levels of lipoproteins containing apolipoprotein (apo) B can drive the development of atherosclerosis in humans and experimental animals even in the absence of other risk factors. However, the mechanisms that lead to atherosclerosis are still poorly understood. We tested the hypothesis that the subendothelial retention of atherogenic apoB-containing lipoproteins is the initiating event in atherogenesis. The extracellular matrix of the subendothelium, particularly proteoglycans, is thought to play a major role in the retention of atherogenic lipoproteins. The interaction between atherogenic lipoproteins and proteoglycans involves an ionic interaction between basic amino acids in apoB100 and negatively charged sulphate groups on the proteoglycans. Here we present direct experimental evidence that the atherogenicity of apoB-containing low-density lipoproteins (LDL) is linked to their affinity for artery wall proteoglycans. Mice expressing proteoglycan-binding-defective LDL developed significantly less atherosclerosis than mice expressing wild-type control LDL. We conclude that subendothelial retention of apoB100-containing lipoprotein is an early step in atherogenesis.