调控胆固醇吸收的分子通路

机体内的胆固醇失衡会引发多种疾病,如高胆固醇血症、心脑血管疾病等,而其平衡由胆固醇的合成、吸收、代谢和循环共同维持,其中胆固醇的吸收至关重要。胆固醇的吸收主要发生在小肠和近段空肠,受众多蛋白的调控。尼曼-匹克C1样蛋白1(NPC1L1)负责胆固醇的摄取;ATP结合盒转运蛋白(ABCG5/ABCG8)则抑制胆固醇的吸收,酰基辅酶A-胆固醇酰基转移酶(ACAT)催化胆固醇脂化提高胆固醇吸收;ATP结合盒转运蛋白A1(ABCA1)负责外周组织胆固醇的转运,而这些蛋白又受到其他调控因子的影响。解析胆固醇吸收的分子通路对胆固醇失衡相关疾病的预防及治疗具有重大指导意义。因此,本文就调控胆固醇吸收的分子通路进行综述。     

胆固醇的内源合成与小肠吸收

以乙酰辅酶A为原料的从头合成和小肠从食物中吸收是人体获得胆固醇的主要来源。胆固醇的内源合成在转录水平上受SREBP通路调控,在转录后水平上主要受胆固醇合成途径限速酶HMGCR和SM的降解调控。小肠对胆固醇吸收是一个涉及胆汁乳化、转运及酯化等多个步骤的复杂过程。定位于小肠上皮刷状缘膜上的Niemann-Pick C1 Like 1(NPC1L1)是介导胆固醇吸收的关键蛋白,负责跨膜运输胆固醇进入小肠吸收细胞。现主要总结胆固醇合成途径的调控机制、NPC1L1蛋白介导胆固醇吸收的分子途径,以及讨论小肠胆固醇吸收与人体血液胆固醇水平之间的相关性。     

饮食胆固醇吸收的研究进展

膜蛋白尼曼-匹克C1型类似蛋白1(Niemann-Pick C1 Like 1,NPC1L1)是介导肝脏和小肠细胞从胆汁或食物中吸收胆固醇的关键蛋白质。本文综述了NPC1L1蛋白的结构、功能及其介导肝肠细胞吸收外源胆固醇的分子机制。NPC1L1蛋白与脂筏蛋白Flotillin-1或Flotillin-2结合,在细胞质膜上形成富含胆固醇的膜微结构域,通过clathrin/AP2介导的囊泡内吞机制,将该NPC1L1-Flotillin-Cholesterol膜微结构域内吞运输至细胞内的内吞循环体上;内吞循环体上的胆固醇浓度下降后,NPC1L1-Flotillin复合物则由Cdc42和Myosin Vb.Rab11a.Rab11-FIP2蛋白运输至质膜,以执行下一轮的胆固醇吸收功能。NPC1L1蛋白的N端结构域可特异性结合胆固醇,是NPC1L1-Flotillin-Cholesterol膜微结构域形成所必需的,同时决定了胆固醇吸收的特异性。人群中NPC1L1基因的多态性与胆固醇吸收异常相关。本文还对未来的研究方向进行了探讨。     

NPC1L1:固醇脂质吸收的关键蛋白质

NPC1L1是最近发现的一种与NPC1同源的蛋白质。在体内的分布有物种差异性,其亚细胞定位存在很大争议。近些年发现NPC1L1在固醇类脂质代谢途径中起着重要作用,是肠道吸收固醇类脂质尤其是胆固醇的关键蛋白质,这项新发现使得人们对固醇类脂质的吸收机制有了了解。高胆固醇血症是心血管系统疾病的一个高危因子,因此,对NPC1L1的研究具有重大的实际意义,正逐渐成为研究的热点。     

缺血后处理降低高胆固醇血症大鼠心肌对缺血/再灌注损伤的易感性及其机制

目的:探讨缺血后处理对高胆固醇血症基础上发生的心肌缺血/再灌注损伤的影响及其可能的机制。方法:建立食源性高胆固醇血症大鼠模型,运用TTC染色、酶活性检测等方法测定缺血/再灌注所致的心肌损伤,用实时定量RT-PCR方法检测心肌组织中低氧诱导因子-1α(HIF-1α)mRNA水平,用Western blot方法检测HIF-1α蛋白水平。结果:高胆固醇血症加重了缺血/再灌注造成的心肌损伤,而缺血后处理显著缩小了高胆固醇血症大鼠缺血/再灌注所致的心梗面积,降低了血清肌酸激酶(CK)的活性,减少了心肌细胞凋亡。同时,缺血后处理提高了高胆固醇血症大鼠缺血心肌组织中HIF-1α的蛋白水平。结论:缺血后处理可以降低高胆固醇血症大鼠心肌对缺血/再灌注损伤的敏感性,其效应与心肌组织中HIF-1α的蛋白水平存在着相关性。     

三磷酸腺苷结合盒转运体和细胞胆固醇外排

胆固醇是细胞质膜的重要组成成分。然而,过多的胆固醇累积可导致细胞中毒。异常的胆固醇胞内迁移与蓄积是造成许多心血管疾病如动脉粥样硬化的分子基础。细胞内胆固醇稳态由胆固醇的吸收、合成及外排等一系列过程调控。在哺乳动物细胞中,调节胆固醇合成、吸收和外排是维持体内胆固醇平衡的必要生理过程。本综述着重概述了三磷酸腺苷结合盒转运体(ABC)家族,如ABCA1、ABCG1、ABCG5和ABCG8的细胞功能及生理作用,以及这些转运体在调控胆固醇胞外转运中的分子机制。     

高胆固醇血症血液流变性的研究

对实验性高胆固醇血症家兔血液流变性、红细胞流变性、红细胞膜脂分析以及与血清胆固醇浓度之间关系的研究指出随血清胆固醇浓度升高,全血表观粘度和血浆粘度升高,红细胞(?)积和(?)降低.进一步分析表明,随血清胆固醇浓度升高,红细胞膜中胆固醇Ch含量增加,引起膜胆固醇和磷脂(P1)的克分子比(Ch,P1)升高,膜的荧光偏振度(P)增大,表示红细胞流动性降低,而对照组各项指标在实验过程中保持稳定.     

痛风与单纯性高尿酸血症人群高脂血症的对比分析

目的:对比分析痛风与单纯高尿酸血症合并高脂血症的情况。方法:收集青岛大学附属医院痛风专病门诊2009年5月至2016年1月收治的痛风患者7207例(男性6759例,女性448例),单纯高尿酸血症患者2095例(男性1852例,女性243例)。测量受试者身高、体重、腰围、臀围、血压、空腹血糖(FPG)、血甘油三酯(TG)、血胆固醇(TC)及血尿酸(UA),计算并比较两组高甘油三脂血症、高胆固醇血症的患病率,并分析其在痛风发生中的独立作用。结果:痛风组高甘油三酯血症和高胆固醇血症的患病率分别为57.8%、47.5%;单纯高尿酸血症组为51.8%、52.9%;两组率相比的比值比,高甘油三脂血症1.274[95%CI(1.155,1.404)],胆固醇血症0.805[95%CI(0.730,0.887)]。按性别分层分析,男性痛风组高甘油三脂血症、高胆固醇血症患病率分别为56.2%,46.8%;单纯高尿酸血症组分别为52.3%,52.6%。两组率相比的比值比,高甘油三脂血症1.25[95CI%(1.13,1.39)],高胆固醇血症0.80[95CI%(0.72,0.89)]。女性中痛风组高甘油三脂血症、高胆固醇血症患病率分别为52.2%,58.90%;单纯高尿酸血症组分别为46.6%,58.0%;两组差异无统计学意义。高甘油三脂血症与痛风的发生正相关OR=1.29,95%CI(1.12,1.48),高胆固醇血症与痛风的发生负相关OR=0.80,95%CI(0.73,0.89)。结论:痛风与单纯高尿酸血症患者存在不同的脂代谢状态,高甘油三酯血症可能是单纯高尿酸血症发展为痛风的危险因素。     

pcsk9基因突变与胆固醇血症

  常染色体显性高胆固醇血症（ADH）是家族早发性动脉粥样硬化的最主要的危险因素.在与ADH有关的基因突变中,LDL-R、apoB100基因突变导致ADH的机制已经比较明确,而pcsk9基因突变与ADH相关是最近发现的.pcsk9基因编码神经凋亡调节转化酶即NARC-1, 它通过在蛋白水平降低肝细胞上LDL受体的数量,使血液中LDL不能被清除,从而与高胆固醇血症相关联.研究pcsk9与LDL之间的关系,探索pcsk9在高胆固醇血症以及动脉粥样硬化发生中的作用及机制,将有助于高胆固醇血症和动脉粥样硬化发病机制的研究,也能为防治高胆固醇血症和动脉粥样硬化提供新思路.     

胆汁酸的合成调控及其在生理与病理中的功能机制

胆汁酸是一类胆固醇的代谢物,在机体胆固醇与能量代谢平衡和小肠营养物质吸收等方面起着重要作用。肝脏是合成胆汁酸的主要场所。饥饿条件下,胆汁酸从肝脏分泌进入胆管并被储存到胆囊;进食后胆囊收缩,贮存的胆汁酸被排出进入小肠。在小肠中,95%的胆汁酸会被小肠重新吸收,通过肝门静脉返回肝脏,这一过程被称为胆汁酸的肝肠循环。胆汁酸一方面作为乳化剂促进小肠中脂类等物质的吸收及转运,同时也作为重要的信号分子与多种受体结合,包括核受体法呢醇X受体(farnesoidXreceptor,FXR)、维生素D受体(vitaminD receptor,VDR)、孕烷X受体(pregnaneXreceptor,PXR)以及细胞膜表面受体G蛋白偶联受体(cellmembrane surface receptor-G protein coupled receptor, TGR5)等,在调节体内胆汁酸的代谢平衡、糖脂代谢与能量代谢平衡等方面发挥重要作用。肝细胞生长因子(hepatocyte growth factor, HGF)、白介素1-(interleukin-1, IL-1)及肿瘤坏死因子(tumor necrosis factor, TNF-)等协同作用构成了胆汁酸合成的精密调控网络。本文主要综述了胆汁酸的合成调控及其功能方面的最新研究进展,旨在为胆汁酸代谢相关研究提供参考。     

Niemann–Pick C1 Like 1 (NPC1L1) an intestinal sterol transporter

Niemann–Pick C1 Like 1 (NPC1L1) has been identified and characterized as an essential protein in the intestinal cholesterol absorption process. NPC1L1 localizes to the brush border membrane of absorptive enterocytes in the small intestine. Intestinal expression of NPC1L1 is down regulated by diets containing high levels of cholesterol. While otherwise phenotypically normal, Npc1l1 null mice exhibit a significant reduction in the intestinal uptake and absorption of cholesterol and phytosterols. Characterization of the NPC1L1 pathway revealed that cholesterol absorption inhibitor ezetimibe specifically binds to an extracellular loop of NPC1L1 and inhibits its sterol transport function. Npc1l1 null mice are resistant to diet-induced hypercholesterolemia, and when crossed with apo E null mice, are completely resistant to the development of atherosclerosis. Intestinal gene expression studies in Npc1l1 null mice indicated that no exogenous cholesterol was entering enterocytes lacking NPC1L1, which resulted in an upregulation of intestinal and hepatic LDL receptor and cholesterol biosynthetic gene expression. Polymorphisms in the human NPC1L1 gene have been found to influence cholesterol absorption and plasma low density lipoprotein levels. Therefore, NPC1L1 is a critical intestinal sterol uptake transporter which influences whole body cholesterol homeostasis.     

Niemann-Pick C1 Like 1 (NPC1L1) is the intestinal phytosterol and cholesterol transporter and a key modulator of whole-body cholesterol homeostasis

Niemann-Pick C1 Like 1 (NPC1L1) is a protein localized in jejunal enterocytes that is critical for intestinal cholesterol absorption. The uptake of intestinal phytosterols and cholesterol into absorptive enterocytes in the intestine is not fully defined on a molecular level, and the role of NPC1L1 in maintaining whole body cholesterol homeostasis is not known. NPC1L1 null mice had substantially reduced intestinal uptake of cholesterol and sitosterol, with dramatically reduced plasma phytosterol levels. The NPC1L1 null mice were completely resistant to diet-induced hypercholesterolemia, with plasma lipoprotein and hepatic cholesterol profiles similar to those of wild type mice treated with the cholesterol absorption inhibitor ezetimibe. Cholesterol/cholate feeding resulted in down-regulation of intestinal NPC1L1 mRNA expression in wild type mice. NPC1L1 deficiency resulted in up-regulation of intestinal hydroxymethylglutaryl-CoA synthase mRNA and an increase in intestinal cholesterol synthesis, down-regulation of ABCA1 mRNA, and no change in ABCG5 and ABCG8 mRNA expression. NPC1L1 is required for intestinal uptake of both cholesterol and phytosterols and plays a major role in cholesterol homeostasis. Thus, NPC1L1 may be a useful drug target for the treatment of hypercholesterolemia and sitosterolemia.     

Transgenic expression of CYP7A1 in LDL receptor-deficient mice blocks diet-induced hypercholesterolemia

Constitutive expression of a cholesterol-7alpha-hydroxylase (CYP7A1) transgene in LDL receptor-deficient mice blocked the ability of a cholesterol-enriched diet to increase plasma levels of apolipoprotein B-containing lipoproteins. LDL receptor-deficient mice expressing the CYP7A1 transgene exhibited complete resistance to diet-induced hypercholesterolemia and to the accumulation of cholesterol in the liver. Hepatic mRNA expression of liver X receptor-inducible ABCG5 and ABCG8 was decreased in CYP7A1 transgenic, LDL receptor-deficient mice fed a cholesterol-enriched diet. Thus, increased biliary cholesterol excretion could not account for the maintenance of cholesterol homeostasis. CYP7A1 transgenic, LDL receptor-deficient mice fed the cholesterol-enriched diet exhibited decreased jejunal Niemann-Pick C1-Like 1 protein (NPC1L1) mRNA expression, an important mediator of intestinal cholesterol absorption. A taurocholate-enriched diet also decreased NPC1L1 mRNA expression in a farnesoid X receptor-independent manner. Reduced expression of NPC1L1 mRNA was associated with decreased cholesterol absorption ( approximately 20%; P < 0.05) exhibited by CYP7A1 transgenic LDL receptor-deficient mice fed the cholesterol-enriched diet. The combined data show that enhanced expression of CYP7A1 is an effective means to prevent the accumulation of cholesterol in the liver and of atherogenic apolipoprotein B-containing lipoproteins in plasma.     

Niemann-Pick C1 like 1 gene expression is down-regulated by LXR activators in the intestine

Niemann-Pick C1 like 1 (NPC1L1) is a protein critical for intestinal cholesterol absorption. The nuclear receptors peroxisome proliferator-activated receptor alpha (PPARalpha) and liver X receptors (LXRalpha and LXRbeta) are major regulators of cholesterol homeostasis and their activation results in a reduced absorption of intestinal cholesterol. The goal of this study was to define the role of PPARalpha and LXR nuclear receptors in the regulation of NPC1L1 gene expression. We show that LXR activators down-regulate NPC1L1 mRNA levels in the human enterocyte cell line Caco-2/TC7, whereas PPARalpha ligands have no effect. Furthermore, NPC1L1 mRNA levels are decreased in vivo, in duodenum of mice treated with the LXR agonist T0901317. In conclusion, the present study identifies NPC1L1 as a novel LXR target gene further supporting a crucial role of LXR in intestinal cholesterol homeostasis.     

Ezetimibe blocks the internalization of NPC1L1 and cholesterol in mouse small intestine

The multiple transmembrane protein Niemann-Pick C1 like1 (NPC1L1) is essential for intestinal cholesterol absorption. Ezetimibe binds to NPC1L1 and is a clinically used cholesterol absorption inhibitor. Recent studies in cultured cells have shown that NPC1L1 mediates cholesterol uptake through vesicular endocytosis that can be blocked by ezetimibe. However, how NPC1L1 and ezetimibe work in the small intestine is unknown. In this study, we found that NPC1L1 distributed in enterocytes of villi and transit-amplifying cells of crypts. Acyl-CoA cholesterol acyltransferase 2 (ACAT2), another important protein for cholesterol absorption by providing cholesteryl esters to chylomicrons, was mainly presented in the apical cytoplasm of enterocytes. NPC1L1 and ACAT2 were highly expressed in jejunum and ileum. ACAT1 presented in the Paneth cells of crypts and mesenchymal cells of villi. In the absence of cholesterol, NPC1L1 was localized on the brush border of enterocytes. Dietary cholesterol induced the internalization of NPC1L1 to the subapical layer beneath the brush border and became partially colocalized with the endosome marker Rab11. Ezetimibe blocked the internalization of NPC1L1 and cholesterol and caused their retention in the plasma membrane. This study demonstrates that NPC1L1 mediates cholesterol entering enterocytes through vesicular endocytosis and that ezetimibe blocks this step in vivo.     

Interaction of cholesterol with sphingosine: physicochemical characterization and impact on intestinal absorption

Molecular associations between sphingomyelin and cholesterol provide a molecular basis for the colocalization of these lipids in plasma membrane microdomains (lipid rafts) and for the inhibitory effect of sphingomyelin on the intestinal absorption of cholesterol. Using surface pressure measurements at the air-water interface, we showed that sphingosine, the common sphingoid backbone of most sphingolipids, formed condensed lipid complexes with cholesterol. Structure-activity relationship studies with long-chain analogs of sphingosine, together with molecular mechanics simulations, were consistent with a specific interaction between sphingosine and the alpha face of cholesterol. The uptake of micellar cholesterol and the effect of sphingosine on cholesterol absorption were studied with two human model intestinal epithelial cell lines, Caco-2 and HT-29-D4. Real-time PCR quantifications of the putative cholesterol transporter Niemann-Pick C1 like 1 (NPC1L1) mRNA revealed that, in these cell lines, the activity of cholesterol transport correlated with the level of NPC1L1 expression. In both cell lines, sphingosine induced a dose-dependent decrease of cholesterol absorption. Yet the effect of sphingosine was more dramatic in Caco-2 cells, which also displayed the highest expression of NPC1L1 mRNA. Altogether, these data suggested that sphingosine interacts specifically with cholesterol and inhibits the intestinal NPC1L1-dependent transport of micellar cholesterol.     

Luteolin and Quercetin Affect the Cholesterol Absorption Mediated by Epithelial Cholesterol Transporter Niemann–Pick C1-Like 1 in Caco-2 Cells and Rats

Niemann–Pick C1-Like 1 (NPC1L1) mediates cholesterol absorption, and ezetimibe is a potent NPC1L1 inhibitor applicable for medication of hypercholesterolemia. Epidemiological studies demonstrated that consumption of polyphenols correlates with a decreased risk for atherosclerosis due to their antioxidant effect. This activity can hardly be attributable to the antioxidant activity only, and we hypothesized that polyphenols inhibit intestinal transport of cholesterol. We elucidated the kinetic parameters of intestinal cholesterol absorption, screened several polyphenols for their ability to specifically inhibit intestinal cholesterol absorption, and determined the inhibitory effects of selected flavonoids in vitro and in vivo. The concentration-dependent uptake of cholesterol by Caco-2 cells obeyed a monophasic saturation process. This indicates the involvement of an active-passive transport, i.e., NPC1L1. Parameters of cholesterol uptake by Caco-2 cells were as follows: J _max, K _t, and K _d were 6.89±2.96 19.03±11.58 µM, and 0.11±0.02 pmol/min/mg protein, respectively. Luteolin and quercetin inhibited cholesterol absorption by Caco-2 cells and human embryonic kidney 293T cells expressing NPC1L1. When preincubated Caco-2 cells with luteolin and quercetin before the assay, cholesterol uptake significantly decreased. The inhibitory effects of these flavonoids were maintained for up to 120 min. The level of inhibition and irreversible effects were similar to that of ezetimibe. Serum cholesterol levels significantly decreased more in rats fed both cholesterol and luteolin (or quercetin), than in those observed in the cholesterol feeding group. As quercetin induced a significant decrease in the levels of NPC1L1 mRNA in Caco-2 cells, the in vivo inhibitory effect may be due to the expression of NPC1L1. These results suggest that luteolin and quercetin reduce high blood cholesterol levels by specifically inhibiting intestinal cholesterol absorption mediated by NPC1L1.     

Ezetimibe interferes with cholesterol trafficking from the plasma membrane to the endoplasmic reticulum in CaCo-2 cells

Niemann-Pick C1-like 1 protein (NPC1L1) is the putative intestinal sterol transporter and the molecular target of ezetimibe, a potent inhibitor of cholesterol absorption. To address the role of NPC1L1 in cholesterol trafficking in intestine, the regulation of cholesterol trafficking by ezetimibe was studied in the human intestinal cell line, CaCo-2. Ezetimibe caused only a modest decrease in the uptake of micellar cholesterol, but markedly prevented its esterification. Cholesterol trafficking from the plasma membrane to the endoplasmic reticulum was profoundly disrupted by ezetimibe without altering the trafficking of cholesterol from the endoplasmic reticulum to the plasma membrane. Cholesterol oxidase-accessible cholesterol at the apical membrane was increased by ezetimibe. Cholesterol synthesis was modestly increased. Although the amount of cholesteryl esters secreted at the basolateral membrane was markedly decreased by ezetimibe, the transport of lipids and the number of lipoprotein particles secreted were not altered. NPC1L1 gene and protein expression were decreased by sterol influx, whereas cholesterol depletion enhanced NPC1L1 gene and protein expression. These results suggest that NPC1L1 plays a role in cholesterol uptake and cholesterol trafficking from the plasma membrane to the endoplasmic reticulum. Interfering with its function will profoundly decrease the amount of cholesterol transported into lymph.     

Sequence variation in NPC1L1 and association with improved LDL-cholesterol lowering in response to ezetimibe treatment

Niemann-Pick C1-like 1 (NPC1L1) is an intestinal cholesterol transporter and the molecular target of ezetimibe, a cholesterol absorption inhibitor demonstrated to reduce LDL-cholesterol (LDL-C) both as monotherapy and when co-administered with 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins). Interestingly, significant interindividual variability has been observed for rates of intestinal cholesterol absorption and LDL-C reductions at both baseline and post ezetimibe treatment. To test the hypothesis that genetic variation in NPC1L1 could influence the LDL-C response to ezetimibe, we performed extensive resequencing of the gene in 375 apparently healthy individuals and genotyped hypercholesterolemic patients from clinical trial cohorts. No association was observed between NPC1L1 single-nucleotide polymorphism and baseline cholesterol. However, significant associations to LDL-C response to treatment with ezetimibe were observed in patients treated with ezetimibe in two large clinical trials. Our data demonstrate that DNA sequence variants in NPC1L1 are associated with an improvement in response to ezetimibe pharmacotherapy and suggest that detailed analysis of genetic variability in clinical trial cohorts can lead to improved understanding of factors contributing to variable drug response.