Effect of up-regulating individual steps in the reverse cholesterol transport pathway on reverse cholesterol transport in normolipidemic mice

Cholesterol acquired by extrahepatic tissues (from de novo synthesis or lipoproteins) is returned to the liver for excretion in a process called reverse cholesterol transport (RCT). We undertook studies to determine if RCT could be enhanced by up-regulating individual steps in the RCT pathway. Overexpression of 7alpha-hydroxylase, Scavenger receptor B1, lecithin:cholesterol acyltransferase (LCAT), or apoA-I in the liver did not stimulate cholesterol efflux from any extrahepatic tissue. In contrast, infusion of apoA-I.phospholipid complexes (rHDL) that resemble nascent HDL markedly stimulated cholesterol efflux from tissues into plasma. Cholesterol effluxed to rHDL was initially unesterified but by 24 h this cholesterol was largely esterified and had shifted to normal HDL (in mice lacking cholesteryl ester transfer protein) or to apoB containing lipoproteins (in cholesteryl ester transfer protein transgenic mice). Most of the cholesterol effluxed into plasma in response to rHDL came from the liver. However, an even greater proportion of effluxed cholesterol was cleared by the liver resulting in a transient increase in liver cholesterol concentrations. Fecal sterol excretion was not increased by rHDL. Thus, although rHDL stimulated cholesterol efflux from most tissues and increased net cholesterol movement from extrahepatic tissues to the liver, cholesterol flux through the entire RCT pathway was not increased.     

Alcohol consumption stimulates early steps in reverse cholesterol transport.

Alcohol consumption is associated with increased HDL cholesterol levels, which may indicate stimulated reverse cholesterol transport. The mechanism is, however, not known. The aim of this study was to evaluate the effects of alcohol consumption on the first two steps of the reverse cholesterol pathway: cellular cholesterol efflux and plasma cholesterol esterification. Eleven healthy middle-aged men consumed four glasses (40 g of alcohol) of red wine, beer, spirits (Dutch gin), or carbonated mineral water (control) daily with evening dinner, for 3 weeks, according to a 4 x 4 Latin square design. After 3 weeks of alcohol consumption the plasma ex vivo cholesterol efflux capacity, measured with Fu5AH cells, was raised by 6.2% (P < 0.0001) and did not differ between the alcoholic beverages. Plasma cholesterol esterification was increased by 10.8% after alcohol (P = 0.008). Changes were statistically significant after beer and spirits, but not after red wine consumption (P = 0.16). HDL lipids changed after alcohol consumption; HDL total cholesterol, HDL cholesteryl ester, HDL free cholesterol, HDL phospholipids and plasma apolipoprotein A-I all increased (P < 0.01). In conclusion, alcohol consumption stimulates cellular cholesterol efflux and its esterification in plasma. These effects were mostly independent of the kind of alcoholic beverage     

胆固醇逆向转运的分子机制

胆固醇逆向转运是周围细胞胆固醇转运至肝脏转化、清除的重要生理过程,它在维持机体胆固醇代谢平衡和对抗动脉粥样硬化发生及发展中起重要作用。研究证实胆固醇逆向转运直是高密度脂蛋白在多种生物活性分子参与下,由新生前β－ＨＤＬ到成熟α－ＨＤＬ递变的胆固醇转运及代谢过程。     

Is reverse cholesterol transport regulated by active cholesterol?

This review considers the hypothesis that a small portion of plasma membrane cholesterol regulates reverse cholesterol transport in coordination with overall cellular homeostasis. It appears that almost all of the plasma membrane cholesterol is held in stoichiometric complexes with bilayer phospholipids. The minor fraction of cholesterol that exceeds the complexation capacity of the phospholipids is called active cholesterol. It has an elevated chemical activity and circulates among the organelles. It also moves down its chemical activity gradient to plasma HDL, facilitated by the activity of ABCA1, ABCG1, and SR-BI. ABCA1 initiates this process by perturbing the organization of the plasma membrane bilayer, thereby priming its phospholipids for translocation to apoA-I to form nascent HDL. The active excess sterol and that activated by ABCA1 itself follow the phospholipids to the nascent HDL. ABCG1 similarly rearranges the bilayer and sends additional active cholesterol to nascent HDL, while SR-BI simply facilitates the equilibration of the active sterol between plasma membranes and plasma proteins. Active cholesterol also flows downhill to cytoplasmic membranes where it serves both as a feedback signal to homeostatic ER proteins and as the substrate for the synthesis of mitochondrial 27-hydroxycholesterol (27HC). 27HC binds the LXR and promotes the expression of the aforementioned transport proteins. 27HC-LXR also activates ABCA1 by competitively displacing its inhibitor, unliganded LXR. 4 Considerable indirect evidence suggests that active cholesterol serves as both a substrate and a feedback signal for reverse cholesterol transport. Direct tests of this novel hypothesis are proposed.     

胆固醇流出调节蛋白与胆固醇逆向转运

高密度脂蛋白（ＨＤＬ）能从外周细胞摄取我余胆固醇并运输至肝脏排出,防止其在血管壁沉积。外周细胞内胆固醇流向ＨＤＬ的机制一直是人们积极探索但尚未弄清的问题。新近发表在《Ｎａｔｕｒｅ Ｇｅｎｅｔｉｃｓ》上的三篇论文在这个环节上取得了重大突破,他们通过对Ｔａｎｇｉｅｒ病的研究,发现由ＡＴＰ－ｂｉｎｄｉｎｇ－ｃａｓｓｅｔｔｅ ｔｒａｎｓｐｏｒｔｅｒ １基因编码的胆固醇流出调节蛋白（ｃｈｏｌｅｓｔｅｒｏｌ－     

Zymosan-mediated inflammation impairs in vivo reverse cholesterol transport

Inflammation has been proposed to impair HDL function and reverse cholesterol transport (RCT). We investigated the effects of inflammation mediated by zymosan, a yeast glucan, on multiple steps along the RCT pathway in vivo and ex vivo. Acute inflammation with 70 mg/kg zymosan impaired RCT to plasma, liver, and feces similarly by 17-22% (P < 0.05), with no additional block at the liver. Hepatic gene expression further demonstrated no change in ABCG5, ABCB4, and ABCB11 expression but a decline in ABCG8 mRNA (32% P < 0.05). Plasma from zymosan-treated mice had a 21% decrease in cholesterol acceptor ability (P < 0.01) and a 35% decrease in ABCA1-specific efflux capacity (P < 0.01) in vitro. Zymosan treatment also decreased HDL levels and led to HDL remodeling with increased incorporation of serum amyloid A. In addition, cholesterol efflux from cultured macrophages declined with zymosan treatment in a dose dependent manner. Taken together, our results suggest that zymosan impairs in vivo RCT primarily by decreasing macrophage-derived cholesterol entering the plasma, with minimal additional blocks downstream. Our study supports the notion that RCT impairment is one of the mechanisms for the increased atherosclerotic burden observed in inflammatory conditions.     

Study of the components of reverse cholesterol transport in lecithin:cholesterol acyltransferase deficiency

Enzymatic and lipid transfer reactions involved in reverse cholesterol transport were studied in healthy and lecithin:cholesterol acyltransferase (LCAT), deficient subjects. Fasting plasma samples obtained from each individual were labeled with [3H]cholesterol and subsequently fractionated by gel chromatography. The radioactivity patterns obtained corresponded to the elution volumes of the three major ultracentrifugally isolated lipoprotein classes (very low density lipoproteins (VLDL), low density lipoproteins (LDL), and high density lipoproteins (HDL)). In healthy subjects, the LCAT activity was consistently found in association with the higher molecular weight portion of HDL. Similar observations were made when exogenous purified LCAT was added to the LCAT-deficient plasma prior to chromatography. Incubation of the plasma samples at 37 degrees C resulted in significant reduction of unesterified cholesterol (FC) and an increase in esterified cholesterol (CE). Comparison of the data of FC and CE mass measurements of the lipoprotein fractions from normal and LCAT-deficient plasma indicates that: (i) In normal plasma, most of the FC for the LCAT reaction originates from LDL even when large amounts of FC are available from VLDL. (ii) The LCAT reaction takes place on the surface of HDL. (iii) The product of the LCAT reaction (CE) may be transferred to either VLDL or LDL although VLDL appears to be the preferred acceptor when present in sufficient amounts. (iv) CE transfer from HDL to lower density lipoproteins is at least partially impaired in LCAT-deficient patients. Additional studies using triglyceride-rich lipoproteins indicated that neither the capacity to accept CE from HDL nor the lower CE transfer activity were responsible for the decreased amount of CE transferred to VLDL and chylomicrons in LCAT-deficient plasma.     

HDL and reverse cholesterol transport. Role of cholesterol ester transfer protein]

As most of peripheral cells are not able to catabolize cholesterol, the transport of cholesterol excess from peripheral tissues back to the liver, namely "reverse cholesterol transport", is the only way by which cholesterol homeostasis is maintained in vivo. Reverse cholesterol transport pathway can be divided in three major steps: 1) uptake of cellular cholesterol by the high density lipoproteins (HDL), 2) esterification of HDL cholesterol by the lecithin: cholesterol acyltransferase and 3) captation of HDL cholesteryl esters by the liver where cholesterol can be metabolized and excreted in the bile. In several species, including man, cholesteryl esters in HDL can also follow an alternative pathway which consists in their transfer from HDL to very low density (VLDL) and low density (LDL) lipoproteins. The transfer of cholesteryl esters to LDL, catalyzed by the Cholesteryl Ester Transfer Protein (CETP), might affect either favorably or unfavorably the reverse cholesterol transport pathway, depending on whether LDL are finally taken up by the liver or by peripheral tissues, respectively. In order to understand precisely the implication of CETP in reverse cholesterol transport, it is essential to determine its role in HDL metabolism, to know the potential regulation of its activity and to identify the mechanism by which it interacts with lipoprotein substrates. Results from recent studies have demonstrated that CETP can promote the size redistribution of HDL particles. This may be an important process in the reverse cholesterol transport pathway as HDL particles with various sizes have been shown to differ in their ability to promote cholesterol efflux from peripheral cells and to interact with lecithin: cholesterol acyltransferase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anacetrapib promotes reverse cholesterol transport and bulk cholesterol excretion in Syrian golden hamsters

Cholesteryl ester transfer protein (CETP) transfers cholesteryl ester (CE) and triglyceride between HDL and apoB-containing lipoproteins. Anacetrapib (ANA), a reversible inhibitor of CETP, raises HDL cholesterol (HDL-C) and lowers LDL cholesterol in dyslipidemic patients; however, the effects of ANA on cholesterol/lipoprotein metabolism in a dyslipidemic hamster model have not been demonstrated. To test whether ANA (60 mg/kg/day, 2 weeks) promoted reverse cholesterol transport (RCT), 3H-cholesterol-loaded macrophages were injected and (3)H-tracer levels were measured in HDL, liver, and feces. Compared to controls, ANA inhibited CETP (94%) and increased HDL-C (47%). 3H-tracer in HDL increased by 69% in hamsters treated with ANA, suggesting increased cholesterol efflux from macrophages to HDL. 3H-tracer in fecal cholesterol and bile acids increased by 90% and 57%, respectively, indicating increased macrophage-to-feces RCT. Mass spectrometry analysis of HDL from ANA-treated hamsters revealed an increase in free unlabeled cholesterol and CE. Furthermore, bulk cholesterol and cholic acid were increased in feces from ANA-treated hamsters. Using two independent approaches to assess cholesterol metabolism, the current study demonstrates that CETP inhibition with ANA promotes macrophage-to-feces RCT and results in increased fecal cholesterol/bile acid excretion, further supporting its development as a novel lipid therapy for the treatment of dyslipidemia and atherosclerotic vascular disease.     

Abdominal obesity negatively influences key metrics of reverse cholesterol transport

Cardiometabolic risk factors increase the risk of atherosclerotic cardiovascular disease (ASCVD), but whether these metabolic anomalies affect the anti-atherogenic function of reverse cholesterol transport (RCT) is not yet clearly known. The present study aimed to delineate if the function and maturation of high density lipoprotein (HDL) particles cross-sectionally associate with surrogate markers of ASCVD in a population comprising of different degree of cardiometabolic risk.We enrolled 131 subjects and characterized cardiometabolic risk based on the IDF criteria's for metabolic syndrome (MS). In this population, cholesterol efflux capacity (CEC), Lecithin–cholesterol acyltransferase (LCAT) and ApoA-1 glycation was associated with waist circumference, abdominal visceral fat (VFA) and abdominal subcutaneous fat. In multivariate analyses, VFA was identified as a critical contributor for low CEC and LCAT. When stratified into groups based on the presence of cardiometabolic risk factors, we found a prominent reduction in CEC and LCAT as a function of the progressive increase of cardiometabolic risk from 0–2, 0–3 to 0–4/5, whereas an increase in Pre-β-HDL and ApoA-1 glycation was observed between the lowest and highest risk groups.These findings confirm the connection between MS and its predisposing conditions to an impairment of atheroprotective efflux-promoting function of HDLs. Furthermore, we have identified the bona fide pathogenically contribution of abdominal obesity to profound alterations of key metrics of RCT.     

Membrane proteins and phospholipids as effectors of reverse cholesterol transport

The review highlights the membrane aspect of cholesterol efflux from cell membranes to high density lipoproteins (HDL), an initial stage of reverse cholesterol transport to liver. In addition to traditional viewpoints considering cholesterol transport as the step of sequential lipoprotein transformation, which involves blood plasma apoproteins and proteins transporters, employment of proteomic approaches has shown the active role of cell plasma membranes as cholesterol donors and plasma membrane bound proteins in cholesterol transport. These include ATP-binding ABC-A1 transporter and membrane receptor SR-B1. There is experimental and clinical evidence that impairment of genes encoding these proteins cause impairments of reverse cholesterol transport (e.g. Tangier disease and genetic manipulations with experimental animals.) Although precise mechanism involving these membrane proteins remains unknown it is suggested that ABC-AI with free plasma apoA1 facilitates the efflux of membrane phospholipids and formation of their complex with apoAI. This complex accepts membrane cholesterol, with simultaneous formation of a full HDL particle. In certain cells there is correlation between cholesterol efflux into HDL and expression of SR-BI, which reversibly binds to HDL. This receptor protein may influence molecular organization of membrane phospholipids and cholesterol, facilitating cholesterol efflux. The review also deals with properties of ABC-A1 and SR-B1, putative mechanisms of their effects, the role of these proteins in reverse cholesterol transport and their functional coupling to the phospholipid matrix of biomembranes.     

Impact of LDL apheresis on atheroprotective reverse cholesterol transport pathway in familial hypercholesterolemia

In familial hypercholesterolemia (FH), low HDL cholesterol (HDL-C) levels are associated with functional alterations of HDL particles that reduce their capacity to mediate the reverse cholesterol transport (RCT) pathway. The objective of this study was to evaluate the consequences of LDL apheresis on the efficacy of the RCT pathway in FH patients. LDL apheresis markedly reduced abnormal accelerated cholesteryl ester transfer protein (CETP)-mediated cholesteryl ester (CE) transfer from HDL to LDL, thus reducing their CE content. Equally, we observed a major decrease (-53%; P < 0.0001) in pre-β1-HDL levels. The capacity of whole plasma to mediate free cholesterol efflux from human macrophages was reduced (-15%; P < 0.02) following LDL apheresis. Such reduction resulted from a marked decrease in the ABCA1-dependent efflux (-71%; P < 0.0001) in the scavenger receptor class B type I-dependent efflux (-21%; P < 0.0001) and in the ABCG1-dependent pathway (-15%; P < 0.04). However, HDL particles isolated from FH patients before and after LDL apheresis displayed a similar capacity to mediate cellular free cholesterol efflux or to deliver CE to hepatic cells. We demonstrate that rapid removal of circulating lipoprotein particles by LDL apheresis transitorily reduces RCT. However, LDL apheresis is without impact on the intrinsic ability of HDL particles to promote either cellular free cholesterol efflux from macrophages or to deliver CE to hepatic cells.     

急性期应答与胆固醇逆向转运

胆固醇逆向转运(reverse cholesterol transport,RCT)是促进外周胆固醇从细胞内流出,然后转运到肝脏进行代谢的过程,是机体抗动脉粥样硬化相关疾病的重要机制。研究表明,感染、炎症及创伤等诱导的急性期应答(acute phase response,APR)影响高密度脂蛋白的结构和功能,抑制细胞内胆固醇流出、血浆胆固醇转运及肝脏胆固醇代谢和排泌等环节,因此抑制体内RCT。APR短期抑制RCT有利于机体抗感染和组织损伤,然而,APR对RCT的进一步抑制将促进外周组织胆固醇蓄积及代谢紊乱,可能是多种感染免疫性疾病、代谢性疾病与动脉粥样硬化呈正相关的关键因素。本文就APR调节机体RCT的最新研究进展作一综述。     

Hepatic overexpression of cholesteryl ester hydrolase enhances cholesterol elimination and in vivo reverse cholesterol transport

Neutral cholesteryl ester hydrolase (CEH)-mediated hydrolysis of cellular cholesteryl esters (CEs) is required not only to generate free cholesterol (FC) for efflux from macrophages but also to release FC from lipoprotein-delivered CE in the liver for bile acid synthesis or direct secretion into the bile. We hypothesized that hepatic expression of CEH would regulate the hydrolysis of lipoprotein-derived CE and enhance reverse cholesterol transport (RCT). Adenoviral-mediated CEH overexpression led to a significant increase in bile acid output. To assess the role of hepatic CEH in promoting flux of cholesterol from macrophages to feces, cholesterol-loaded and [(3)H]cholesterol-labeled J774 macrophages were injected intraperitoneally into mice and the appearance of [(3)H]cholesterol in gallbladder bile and feces over 48 h was quantified. Mice overexpressing CEH had significantly higher [(3)H]cholesterol radiolabel in bile and feces, and it was associated with bile acids. This CEH-mediated increased movement of [(3)H]cholesterol from macrophages to bile acids and feces was significantly attenuated in SR-BI(-/-) mice. These studies demonstrate that similar to macrophage CEH that rate-limits the first step, hepatic CEH regulates the last step of RCT by promoting the flux of cholesterol entering the liver via SR-BI and increasing hepatic bile acid output.     

Deciphering structural aspects of reverse cholesterol transport: mapping the knowns and unknowns

Atherosclerosis is a major contributor to the onset and progression of cardiovascular disease (CVD). Cholesterol-loaded foam cells play a pivotal role in forming atherosclerotic plaques. Induction of cholesterol efflux from these cells may be a promising approach in treating CVD. The reverse cholesterol transport (RCT) pathway delivers cholesteryl ester (CE) packaged in high-density lipoproteins (HDL) from non-hepatic cells to the liver, thereby minimising cholesterol load of peripheral cells. RCT takes place via a well-organised interplay amongst apolipoprotein A1 (ApoA1), lecithin cholesterol acyltransferase (LCAT), ATP binding cassette transporter A1 (ABCA1), scavenger receptor-B1 (SR-B1), and the amount of free cholesterol. Unfortunately, modulation of RCT for treating atherosclerosis has failed in clinical trials owing to our lack of understanding of the relationship between HDL function and RCT. The fate of non-hepatic CEs in HDL is dependent on their access to proteins involved in remodelling and can be regulated at the structural level. An inadequate understanding of this inhibits the design of rational strategies for therapeutic interventions. Herein we extensively review the structure–function relationships that are essential for RCT. We also focus on genetic mutations that disturb the structural stability of proteins involved in RCT, rendering them partially or completely non-functional. Further studies are necessary for understanding the structural aspects of RCT pathway completely, and this review highlights alternative theories and unanswered questions.