Acyl-coenzyme A: cholesterol acyltransferase family

The enzymes of the acyl-coenzyme A: cholesterol acyltransferase (ACAT) family are responsible for the in vivo synthesis of neutral lipids. They are potential drug targets for the intervention of atherosclerosis, hyperlipidemia, obesity, type II diabetes and even Alzheimer’s disease. ACAT family enzymes are integral endoplasmic reticulum (ER) membrane proteins and can be divided into ACAT branch and acyl-coenzyme A: diacylglycerol acyltransferase 1 (DGAT1) branch according to their substrate specificity. The ACAT branch catalyzes synthesis of cholesteryl esters using long-chain fatty acyl-coenzyme A and cholesterol as substrates, while the DGAT1 branch catalyzes synthesis of triacylglycerols using fatty acylcoenzyme A and diacylglycerol as substrates. In this review, we mainly focus on the recent progress in the structural research of ACAT family enzymes, including their disulfide linkage, membrane topology, subunit interaction and catalysis mechanism.     

Acyl-coenzyme A:cholesterol acyltransferase family

The enzymes of the acyl-coenzyme A:cholesterol acyltransferase (ACAT) family are responsible for the in vivo synthesis of neutral lipids.They are potential drug targets for the intervention of atherosclerosis,hyperlipidemia,obesity,type Ⅱ diabetes and even Alzheimer's disease.ACAT family enzymes are integral endoplasmic reticulum (ER) membrane proteins and can be divided into ACAT branch and acyl-coenzyme A:diacylglycerol acyltransferase 1 (DGATI) branch according to their substrate specificity.The ACAT branch catalyzes synthesis of cholesteryl esters using long-chain fatty acyl-coenzyme A and cholesterol as substrates,while the DGAT1 branch catalyzes synthesis of triacylglycerols using fatty acylcoenzyme A and diacylglycerol as substrates.In this review,we mainly focus on the recent progress in the structural research of ACAT family enzymes,including their disulfide linkage,membrane topology,subunit interaction and catalysis mechanism.     

Decreased activity of lecithin:cholesterol acyltransferase and hepatic lipase in chronic hypothyroid rats: Implications for reverse cholesterol transport

Chronic hypothyroidism is frequently associated with atherosclerosis due to increased cholesterol plasma levels; nevertheless, the contribution of impaired reverse cholesterol transport (RCT) in this process has not been completely elucidated. The aim of this study was to evaluate the effect of thyroidectomy (Htx) upon the main stages of RCT in rats. Plasma lipid alterations induced by thyroidectomy showed a slight, but significant, reduction of total plasma triglycerides, a 300% increase of LDL-cholesterol and a 25% decrease in HDL-cholesterol compared to control rats. We evaluated the first stage of RCT determining ₃H-cholesterol efflux in Fu5AH cells. The capacity of HDL obtained from Htx rats to promote cholesterol efflux was similar to that of controls. Lecithin:cholesterol acyltransferase (LCAT) activity, the second stage and the driving force of RCT was 30% lower in Htx animals compared to controls, as determined by reconstituted HDL used as an external substrate. Lipoproteins are remodeled by hepatic lipase; the mean activity of this enzyme in postheparin plasma of Htx animals was reduced by 30% compared to controls, thus suggesting an impaired HDL remodeling by this enzyme in the hypothyroid status. In contrast, lipoprotein lipase activity in the Htx group was unchanged. In summary, this study demonstrates that chronic hypothyroidism in the rat induced an impaired RCT mainly at the cholesterol esterification, and HDL remodeling mediated by hepatic lipase. The latter probably results in an abnormal HDL structure, i.e. phospholipid enrichment, which contributes to decrease HDL-apo AI fractional catabolic rates.     

Effects of high-density lipoprotein2 on cholesterol transport and acyl-coenzyme A:cholesterol acyltransferase activity in P388D1 macrophages

High-density lipoproteins are the putative vehicles for cholesterol removal from monocyte-derived macrophages, which are an important cell type in all stages of atherosclerosis. The role of HDL₂, an HDL subclass that accounts for most variation in plasma HDL-cholesterol concentration, in cholesterol metabolism in monocyte-derived macrophages is not known. In this study, the dose-dependent effects of HDL₂ on cellular cholesterol mass, efflux, and esterification, and on cellular cholesteryl ester (CE) hydrolysis using the mouse macrophage P388D1 cell line was investigated. HDL₂ at low concentrations (40 μg protein/ml) decreased CE content without affecting cellular free cholesterol content (FC), CE hydrolysis, or cholesterol biosynthesis. In addition, HDL₂ at low concentrations reduced cellular acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity and increased FC efflux from macrophages. Thus, HDL₂ has two potential roles in reverse cholesterol transport. In one, HDL₂ is an acceptor of macrophage FC. In the other, more novel role, HDL₂ increases the availability of macrophage FC through the inhibition of ACAT. Elucidation of the mechanism by which HDL₂ inhibits ACAT could identify new therapeutic targets that enhance the transfer of cholesterol from macrophages to the liver.     

Plasma lecithin:cholesterol acyltransferase activity modifies the inverse relationship of C-reactive protein with HDL cholesterol in nondiabetic men

Lecithin:cholesterol acyltransferase (LCAT) is instrumental in high-density lipoprotein (HDL) maturation, but high LCAT levels do not predict low cardiovascular risk. LCAT may affect antioxidative or anti-inflammatory properties of HDL. We determined the relationship of plasma high-sensitivity C-reactive protein (CRP) with LCAT activity and evaluated whether LCAT activity modifies the decreasing effect of HDL cholesterol (HDL-C) on CRP, as an estimate of its anti-inflammatory properties. Plasma HDL-C, apolipoprotein (apo) A-I and LCAT activity (exogenous substrate method) were measured in 260 nondiabetic men without cardiovascular disease. CRP was correlated inversely with HDL-C and apo A-I, and positively with LCAT activity (P < 0.01 to 0.001). Multivariate regression analysis demonstrated that age- and smoking-adjusted plasma CRP levels were associated negatively with HDL-C (β = − 0.224, P < 0.001) and positively with LCAT activity (β = 0.119, P = 0.034), as well as with the interaction between HDL-C and LCAT activity (β = 0.123, P = 0.026). There was also an interaction between apo A-I and LCAT activity on CRP (β = 0.159, P = 0.005). These relationships remained similar after adjustment for apo B-containing lipoproteins. In conclusion, the inverse relationship of HDL-C with CRP is attenuated by LCAT activity at higher HDL-C levels. It is hypothesized that LCAT could mitigate HDL's anti-inflammatory or antioxidative properties at higher HDL-C concentrations.     

Hypocholesterolemic effect of rice protein is due to regulating hepatic cholesterol metabolism in adult rats

Aging is one of major risk factors for developing hypercholesterolemia. To elucidate the cholesterol-lowering mechanism exerted by rice protein (RP), the effects on hepatic cholesterol outputs and cholesterol metabolism related enzymes were investigated in adult rats, which were fed by casein (CAS) and RP without cholesterol in diets. After 2 weeks of feeding, the significant cholesterol-lowering effect was observed in adult rats fed by RP compared to CAS. The hepatic total- and VLDL-cholesterol secretions into circulation were significantly depressed in RP group, whereas biliary outputs of bile acids and cholesterol were effectively stimulated by RP-feeding, causing an increase in fecal sterol excretion compared to CAS. As a result, the apparent cholesterol absorption was significantly inhibited by RP. RP-feeding significantly increased the activity and gene expression of cholesterol 7α-hydroxylase, whereas acyl-CoA:cholesterol acyltransferase-2 activity and gene expression were significantly decreased by RP as compared with CAS. Neither activity nor gene expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase of RP did differ from CAS in the liver. The present study demonstrates that rice protein can prevent hypercholesterolemia through modifying hepatic cholesterol metabolism under cholesterol-free dietary condition. The findings suggest that hypocholesterolemic action induced by rice protein is attributed in part to the inhibition of cholesterol absorption during the adult period.     

Continuous monitoring of cholesterol oleate hydrolysis by hormone-sensitive lipase and other cholesterol esterases

Hormone-sensitive lipase (HSL) contributes importantly to the hydrolysis of cholesteryl ester in steroidogenic tissues, releasing the cholesterol required for adrenal steroidogenesis. HSL has broad substrate specificity, because it hydrolyzes triacylglycerols (TAGs), diacylglycerols, monoacylglycerols, and cholesteryl esters. In this study, we developed a specific cholesterol esterase assay using cholesterol oleate (CO) dispersed in phosphatidylcholine and gum arabic by sonication. To continuously monitor the hydrolysis of CO by HSL, we used the pH-stat technique. For the sake of comparison, the hydrolysis of CO dispersion was also tested using other cholesteryl ester-hydrolyzing enzymes. The specific activities measured on CO were found to be 18, 100, 27, and 3 micromol/min/mg for HSL, cholesterol esterase from Pseudomonas species, Candida rugosa lipase-3, and cholesterol esterase from bovine pancreas, respectively. The activity of HSL on CO is approximately 4- to 5-fold higher than on long-chain TAGs. In contrast, with all other enzymes tested, the rates of TAG hydrolysis were higher than those of CO hydrolysis. The relatively higher turnover of HSL on CO observed in vitro adds further molecular insight on the physiological importance of HSL in cholesteryl ester catabolism in vivo. Thus, HSL could be considered more as a cholesteryl ester hydrolase than as a TAG lipase.     

HIV-1 Nef mobilizes lipid rafts in macrophages through a pathway that competes with ABCA1-dependent cholesterol efflux

HIV infection, through the actions of viral accessory protein Nef, impairs activity of cholesterol transporter ABCA1, inhibiting cholesterol efflux from macrophages and elevating the risk of atherosclerosis. Nef also induces lipid raft formation. In this study, we demonstrate that these activities are tightly linked and affect macrophage function and HIV replication. Nef stimulated lipid raft formation in macrophage cell line RAW 264.7, and lipid rafts were also mobilized in HIV-1-infected human monocyte-derived macrophages. Nef-mediated transfer of cholesterol to lipid rafts competed with the ABCA1-dependent pathway of cholesterol efflux, and pharmacological inhibition of ABCA1 functionality or suppression of ABCA1 expression by RNAi increased Nef-dependent delivery of cholesterol to lipid rafts. Nef reduced cell-surface accessibility of ABCA1 and induced ABCA1 catabolism via the lysosomal pathway. Despite increasing the abundance of lipid rafts, expression of Nef impaired phagocytic functions of macrophages. The infectivity of the virus produced in natural target cells of HIV-1 negatively correlated with the level of ABCA1. These findings demonstrate that Nef-dependent inhibition of ABCA1 is an essential component of the viral replication strategy and underscore the role of ABCA1 as an innate anti-HIV factor.     

Changes in cholesterol absorption and cholesterol synthesis caused by ezetimibe and/or simvastatin in men

This study evaluates changes in cholesterol balance in hypercholesterolemic subjects following treatment with an inhibitor of cholesterol absorption or cholesterol synthesis or coadministration of both agents. This was a randomized, double blind, placebo-controlled, four-period crossover study to evaluate the effects of coadministering 10 mg ezetimibe with 20 mg simvastatin (ezetimibe/simvastatin) on cholesterol absorption and synthesis relative to either drug alone or placebo in 41 subjects. Each treatment period lasted 7 weeks. Ezetimibe and ezetimibe/simvastatin decreased fractional cholesterol absorption by 65% and 59%, respectively (P < 0.001 for both relative to placebo). Simvastatin did not significantly affect cholesterol absorption. Ezetimibe and ezetimibe/simvastatin increased fecal sterol excretion (corrected for dietary cholesterol), which also represents net steady state cholesterol synthesis, by 109% and 79%, respectively (P < 0.001). Ezetimibe, simvastatin, and ezetimibe/simvastatin decreased plasma LDL-cholesterol by 20, 38, and 55%, respectively. The coadministered therapy was well tolerated. The decreases in net cholesterol synthesis and increased fecal sterol excretion yielded nearly additive reductions in LDL-cholesterol for the coadministration of ezetimibe and simvastatin.     

Cellular cholesterol homeostasis and Alzheimer's disease: Thematic Review Series: ApoE and Lipid Homeostasis in Alzheimer's Disease

Alzheimer's disease (AD) is the most common form of dementia in older adults. Currently, there is no cure for AD. The hallmark of AD is the accumulation of extracellular amyloid plaques composed of amyloid-β (Aβ) peptides (especially Aβ1-42) and neurofibrillary tangles, composed of hyperphosphorylated tau and accompanied by chronic neuroinflammation. Aβ peptides are derived from the amyloid precursor protein (APP). The oligomeric form of Aβ peptides is probably the most neurotoxic species; its accumulation eventually forms the insoluble and aggregated amyloid plaques. ApoE is the major apolipoprotein of the lipoprotein(s) present in the CNS. ApoE has three alleles, of which the Apoe4 allele constitutes the major risk factor for late-onset AD. Here we describe the complex relationship between ApoE4, oligomeric Aβ peptides, and cholesterol homeostasis. The review consists of four parts: 1) key elements involved in cellular cholesterol metabolism and regulation; 2) key elements involved in intracellular cholesterol trafficking; 3) links between ApoE4, Aβ peptides, and disturbance of cholesterol homeostasis in the CNS; 4) potential lipid-based therapeutic targets to treat AD. At the end, we recommend several research topics that we believe would help in better understanding the connection between cholesterol and AD for further investigations.     

The exchangeability of human erythrocyte membrane cholesterol

A new method has been used to determine what fraction of human erythrocyte cholesterol is available for exchange with plasma unesterified cholesterol. Erythrocytes labeled with ³H-cholesterol by this exchange process were incubated with sonicated phosphatidylcholine vesicles, giving rise to a net movement of cholesterol out of the cells. The specific activity of cholesterol taken up by the vesicles depended on the length of time of incubation. Initially the specific activity in the vesicles was greater than that in the cells, but after approximately 10% of cell cholesterol had been removed, the specific activity of subsequently removed cholesterol was equal to that of the remaining erythrocyte cholesterol. We conclude from these data that (a) all of the cholesterol in the erythrocyte is exchangeable with plasma, and (b) approximately 10% of erythrocyte cholesterol is in a more rapidly exchangeable pool than the remainder.     

类植物乳杆菌的耐酸、耐胆盐及降胆固醇特性

摘要：【目的】研究了类植物乳杆菌Ⅱ32的酸、胆盐耐受性和胆固醇去除能力,并通过菌株生长和胆固醇去除的关系探讨可能的机理。【方法】菌株Ⅱ32生长在高胆固醇MRS培养基中,胆固醇的检测通过气相色谱法。检测菌株在不同生长阶段对胆固醇的去除情况。【结果】菌株Ⅱ32具有酸耐受性、胆盐耐受性和一定的胆固醇清除能力。菌株在pH2.0培养2 h后仍能达到104 cfu/mL;加胆盐（0.3%～0.4%）对菌株生长量达到OD值0.6的时间延迟在0.5 h以内;热杀死的和休眠的细胞能去除很少的胆固醇,分别是5.64、5.90 mg/g细胞干重,而生长的细胞去除的胆固醇达到16.98 mg/g细胞干重。另外,研究表明胆固醇去除与菌体的生长有一定的相关性。【结论】菌株Ⅱ32去除胆固醇可能的机理是菌体对胆固醇的吸附及菌体在生长过程中对胆固醇的吸收利用,为此该菌株具有添加到食品中来降低血液胆固醇的潜能。     

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.     

Production of testosterone from cholesterol using a single-step microbial transformation of Mycobacterium sp

A novel single-step microbial transformation process for the production of testosterone (TS) from cholesterol by Mycobacterium sp was investigated. It was found that the supply of reducing power, NADH, from the metabolism of glucose was necessary for the reduction of androst-4-en-3,17-dione (AD) to TS. The cultivation time for the maximum accumulation of TS and the residual glucose increased in parallel with the amount of glucose supplemented in fermentation cultures. After the glucose in the fermentation culture was completely consumed, most of the TS was oxidized to AD. Adding a larger amount of glucose could prevent oxidation of TS to AD. Under optimal fermentation conditions, the maximum molar conversion rate of TS from cholesterol was 51% in a 5-L surface-aerated fermentor after 120 h cultivation. Received 27 June 1997/ Accepted in revised form 11 August 1997