Synthetic LXR ligand inhibits the development of atherosclerosis in mice

The nuclear receptors LXRalpha and LXRbeta have been implicated in the control of cholesterol and fatty acid metabolism in multiple cell types. Activation of these receptors stimulates cholesterol efflux in macrophages, promotes bile acid synthesis in liver, and inhibits intestinal cholesterol absorption, actions that would collectively be expected to reduce atherosclerotic risk. However, synthetic LXR ligands have also been shown to induce lipogenesis and hypertriglyceridemia in mice, raising questions as to the net effects of these compounds on the development of cardiovascular disease. We demonstrate here that the nonsteroidal LXR agonist GW3965 has potent antiatherogenic activity in two different murine models. In LDLR(-/-) mice, GW3965 reduced lesion area by 53% in males and 34% in females. A similar reduction of 47% was observed in male apoE(-/-) mice. Long-term (12-week) treatment with LXR agonist had differential effects on plasma lipid profiles in LDLR(-/-) and apoE(-/-) mice. GW3965 induced expression of ATP-binding cassettes A1 and G1 in modified low-density lipoprotein-loaded macrophages in vitro as well as in the aortas of hyperlipidemic mice, suggesting that direct actions of LXR ligands on vascular gene expression are likely to contribute to their antiatherogenic effects. These observations provide direct evidence for an atheroprotective effect of LXR agonists and support their further evaluation as potential modulators of human cardiovascular disease.     

LXRβ activation increases intestinal cholesterol absorption,leading to an atherogenic lipoprotein profile

Abstract. Hu X, Steffensen KR, Jiang Z‐Y, Parini P, Gustafsson J‐Å, Gåfvels M, Eggertsen G (Karolinska Institutet, Huddinge, Stockholm, Sweden; Karolinska Institutet, Huddinge, Stockholm, Sweden; Shanghai Institute of Digestive Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China; and University of Houston, Houston, TX, USA). LXRβ activation increases intestinal cholesterol absorption, leading to an atherogenic lipoprotein profile. J Intern Med 2012; 272: 452–464. Objectives. Liver X receptors (LXRs) are essential for the regulation of intestinal cholesterol absorption. Because two isoforms exist, LXRα and LXRβ, with overlapping but not identical functions, we investigated whether LXRα and LXRβ exert different effects on intestinal cholesterol absorption. Design. Wild‐type (WT), LXRα^?/? and LXRβ^?/? mice were fed control diet, 0.2% cholesterol‐enriched diet or 0.2% cholesterol‐enriched diet plus the LXR agonist GW3965. Results. When fed a control diet, all three genotypes showed similar levels of cholesterol absorption. Of interest, a significant increase in cholesterol absorption was found in the LXRα^?/? mice, but not in the WT or LXRβ^?/? animals, when fed a diet enriched with 0.2% cholesterol or 0.2% cholesterol + GW3965. Reduced faecal neutral sterol excretion and a hydrophobic bile acid profile were also observed in LXRα^?/? mice. Greater increases in the apolipoprotein (apo)B‐containing lipoproteins in serum were seen in the LXRα^?/? mice. A 0.2% cholesterol + GW3965 diet suppressed intestinal Npc1l1 protein expression to the same extent for all genotypes, while Abca1 and Abcg5 were elevated to the same degree. Conclusions. In the intestine, LXRα and LXRβ seem to exert similar effects on expression of cholesterol‐transporting proteins such as Npc1l1. Selective activation of LXRβ may generate effects such as increased cholesterol absorption and elevated serum levels of apoB‐containing lipoproteins, which seem to be counteracted by LXRα. Therefore, an intestinal LXRβ‐specific pathway might exist in terms of cholesterol transportation in addition to the main pathway.     

Increased fecal neutral sterol loss upon liver X receptor activation is independent of biliary sterol secretion in mice

BACKGROUND & AIMS: Reverse cholesterol transport (RCT) is defined as high-density lipoprotein (HDL)-mediated flux of excess cholesterol from peripheral cells to liver, followed by secretion into bile and disposal via the feces. Various steps of this pathway are controlled by the liver X receptor (LXR). We addressed the role of the intestine in LXR-dependent stimulation of fecal cholesterol excretion. METHODS: To segregate biliary from intestine-derived cholesterol, wild-type and Mdr2 P-glycoprotein-deficient mice ( Mdr2 -/- ), which are unable to secrete cholesterol into bile, were treated with the LXR agonist GW3965. RESULTS: Treatment with GW3965 increased biliary cholesterol secretion by 74% in wild-type mice but had no effect in Mdr2 -/- mice. LXR activation increased fecal neutral sterol excretion 2.1-fold in wild-type mice. Surprisingly, an identical increase was observed in Mdr2 -/- mice. Fractional cholesterol absorption was reduced on LXR activation in both strains but was more pronounced in Mdr2 -/- mice, coinciding with reduced Npc111 expression. Intestinal gene expression of ATP-binding cassette transporters (Abc) Abca1 , Abcg1 , Abcg5 , and Abcg8 was strongly induced upon LXR activation in both strains, whereas expression of HMGCoA reductase , controlling cholesterol synthesis, remained unaffected. Additionally, LXR activation stimulated the excretion of plasma-derived [ 3 H]cholesterol into the fecal neutral sterol fraction in Mdr2 -/- mice. CONCLUSIONS: Increased fecal cholesterol loss upon LXR activation is independent of biliary cholesterol secretion in mice. An important part of excess cholesterol is excreted directly via the intestine, supporting the existence of an alternative, quantitatively important route for cholesterol disposal.     

Study of ABCA1 function in transgenic mice

The ATP-binding cassette transporter A1 (ABCA1), identified in 1999 as the gene defective in Tangier disease, promotes efflux of cellular cholesterol from macrophages and other peripheral tissues to apolipoprotein acceptors. These ABCA1-mediated processes are anticipated to have antiatherogenic properties, prompting the development of pharmacological agents that increase ABCA1 gene expression as well as the establishment of ABCA1-transgenic mouse lines. Preliminary studies of ABCA1-Tg mice seem to validate the selection of this transporter as a therapeutic target for the treatment of low HDL syndromes and cardiovascular disease but have also raised new questions regarding the function of ABCA1. In particular, the relative contribution of hepatic and peripheral ABCA1 to plasma HDL levels and to reverse cholesterol transport, as well as the potential role of ABCA1 in modulating the plasma concentrations of the apolipoprotein B-containing lipoproteins and protecting against atherosclerosis, seem to be promising areas of investigation. The present review summarizes the most recent studies and discusses insights provided by these transgenic mouse models.     

肝脏X受体α对肝细胞HepG2胆固醇代谢基因表达的调控

目的:研究肝细胞X受体α(liver X receptorα,LXRα)基因对HepG2肝细胞胆固醇代谢相关基因表达的调控作用。方法:将特异性LXRα小片段干扰RNA(siRNA)经脂质体Lipofectamine 2000介导转染人HepG2肝细胞,同时行HepG2肝细胞LXRα的激动(T0901317)实验,采用实时定量PCR方法分别测定干扰和激动前后的LXRα以及胆固醇代谢相关基因ATP结合盒(ABC)G5、ABCG8、ABCA1 mRNA的表达。结果:LXRαsiRNA干扰24h后,HepG2实验组LXRα mRNA相对表达量显著低于阴性对照组(P0.05)。HepG2加入激动剂(T0901317)48h后,实验组ABCG8和ABCA1基因表达均显著升高(P0.05)。结论:RNA干扰使HepG2肝细胞LXRα表达受抑制,继而有使靶基因ABCG5、ABCG8、ABCA1表达降低趋势;人工合成配体T0901317与LXRα结合,使其活性增加,上调了ABCG8、ABCA1和ABCG5 mRNA的表达。提示LXRα可调控HepG2肝细胞的胆固醇代谢基因。     

ATP结合盒转运蛋白A1研究进展

动脉粥样硬化是心脑血管疾病的重要基础病理改变,而胆固醇在巨噬细胞内聚集和泡沫细胞形成是动脉粥样硬化的始动环节。ATP结合盒转运蛋白Al（ABCAl）是一种重要的胆固醇流出调节蛋白,能介导细胞内胆固醇逆向转运到细胞外,使之与载脂蛋白A-Ⅰ结合并包装形成高密度脂蛋白（HDL）的膜转运蛋白。因此,ABCAl是调节血浆HDL及细胞内胆固醇水平的重要膜蛋白,ABCAl的表达水平与动脉粥样硬化的发生关系密切。同时,细胞内脂质、核受体PPAR、LXR、RXR和细胞因子等对ABCAl蛋白表达具有凋控作用。本文将ABCAl研究进展作一综述。     

Pharmacological LXR activation reduces presence of SR-B1 in liver membranes contributing to LXR-mediated induction of HDL-cholesterol

ObjectivePharmacological LXR activation has anti-atherosclerotic actions in animal models. Part of these beneficial effects may be explained by accelerated reverse cholesterol transport since both plasma high density lipoprotein (HDL) cholesterol and fecal neutral sterol secretion are higher upon LXR activation. Mechanisms underlying these LXR-mediated effects have not been fully elucidated.MethodsWe investigated the roles of the isoforms LXRα and LXRβ and the HDL cholesterol uptake receptor SR-B1 in modulation of cholesterol metabolism upon treatment of mice with the LXR ligand T0901317.ResultsHDL cholesterol was maximally 60% increased in a time-dependent fashion due to appearance of more and larger HDL particles. Fecal neutral sterol secretion was maximally induced after 1 week treatment. T0901317 treatment induced fecal neutral sterol secretion by ～300% in wild-type but not in Lxrα deficient mice. Surprisingly, LXR activation reduced SR-B1 protein amount in hepatic membranes, suggesting that this might contribute to elevated HDL cholesterol. However, T0901317 still elevated plasma HDL cholesterol in Sr-b1 deficient mice, suggesting that SR-B1 is not the only step involved in LXR-mediated induction of plasma HDL cholesterol. In addition, SR-B1 is not essential for LXR-induced cholesterol removal from the body.ConclusionInduction of fecal neutral sterol secretion by T0901317 critically depends on LXRα but not on LXRβ. LXR activation reduces SR-B1 in hepatic membranes, probably partly contributing to elevated HDL cholesterol. SR-B1 is not required to enhance fecal neutral sterol secretion.     

Plasma levels of 27-hydroxycholesterol in humans and mice with monogenic disturbances of high density lipoprotein metabolism

Secretion of 27-hydroxycholesterol (27OHC) from macrophages is considered as an alternative to HDL-mediated reverse transport of excess cholesterol. We investigated 27OHC-concentrations in plasma of humans and mice with monogenic disorders of HDL metabolism. As compared to family controls mutations in the genes for apolipoprotein A-I, ATP binding cassette transporter (ABC) A1 and lecithin:cholesterol acylstransferase (LCAT) were associated with reduced concentrations of both HDL-cholesterol and HDL-27OHC whereas mutations in the genes for cholesterylester transfer protein (CETP), scavenger receptor type BI and hepatic lipase were associated with elevated HDL concentrations of either sterol. Compared to family controls and relative to the concentrations of total 27OHC and cholesterol, lower 27OHC-ester but normal cholesterylester levels were found in HDL of heterozygous LCAT mutation carriers and nonHDL of heterozygous CETP mutation carriers. In family controls, LCAT activity and CETP mass were more strongly correlated with 27OHC-ester than cholesterylester concentrations in HDL and nonHDL, respectively. These findings suggest that the formation and transfer of 27OHC-esters are more sensitive to reduced activities of LCAT and CETP, respectively, than the formation and transfer of cholesterylesters. 27OHC plasma levels were also decreased in apoA-I-, ABCA1- or LCAT-knockout mice but increased in SR-BI-knockout mice. Transplantation of ABCA1- and/or ABCG1-deficient bone marrow into LDL receptor deficient mice decreased plasma levels of 27OHC. In conclusion, mutations or absence of HDL genes lead to distinct alterations in the quantity, esterification or lipoprotein distribution of 27OHC. These findings argue against the earlier suggestion that 27OHC-metabolism in plasma occurs independently of HDL.     

Quantifying anomalous intestinal sterol uptake, lymphatic transport, and biliary secretion in Abcg8(-/-) mice

Sitosterolemia is caused by mutations in either ABCG5 or ABCG8, but simultaneous mutations of these genes have never been observed. To explore whether ABCG8, the sterol efflux (hemi-)transporter, plays a major role in determining intestinal absorption efficiency and hepatic secretion rates of cholesterol and sitostanol, we performed direct measurements of the absorption and lymphatic transport of these sterols in mice with chronic biliary and lymphatic fistulae, as well as the transport rates of radiolabeled cholesterol and sitostanol from plasma high-density lipoprotein (HDL) into bile in male Abcg8(-/-) and wild-type mice. We observed that the absorption and lymphatic transport rates of radiolabeled cholesterol and sitostanol were increased by approximately 40% and approximately 500%, respectively, in Abcg8(-/-) mice in the setting of constant intraduodenal infusion of micellar taurocholate and lecithin. Both strains displayed identical intestinal Npc1l1 expression levels and small intestinal transit rates. After 45 minutes of intraduodenal infusion, acute intestinal uptake rates of trace [(14)C]cholesterol and [(3)H]sitostanol were essentially similar in both groups of mice with intact biliary secretion. Furthermore, in wild-type mice, mass transport rate of [(3)H]sitostanol from plasma HDL into bile was significantly faster than that of [(14)C]cholesterol; however, no [(3)H]sitostanol and only traces of [(14)C]cholesterol were detected in bile of Abcg8(-/-) mice. CONCLUSION: Deletion of the Abcg8 gene alone significantly increases the mass of intestinal cholesterol and sitostanol absorption and reduces but does not eliminate hepatic secretion of cholesterol. Moreover, the mutation has no influence on acute uptake of cholesterol and sitostanol by the enterocyte nor small intestinal transit time.     

A novel liver X receptor agonist establishes species differences in the regulation of cholesterol 7alpha-hydroxylase (CYP7a)

The liver X receptors, LXRalpha and LXRbeta, are members of the nuclear receptor superfamily. Originally identified as orphans, both receptor subtypes have since been shown to be activated by naturally occurring oxysterols. LXRalpha knockout mice fail to regulate cyp7a mRNA levels upon cholesterol feeding, implicating the role of this receptor in cholesterol homeostasis. LXR activation also induces the expression of the lipid pump involved in cholesterol efflux, the gene encoding ATP binding cassette protein A1 (ABCA1). Therefore, LXR is believed to be a sensor of cholesterol levels and a potential therapeutic target for atherosclerosis. Here we describe a synthetic molecule named F(3)MethylAA [3-chloro-4-(3-(7-propyl-3-trifluoromethyl-6-(4,5)-isoxazolyl)propylthio)-phenyl acetic acid] that is more potent than 22(R)-hydroxycholesterol in LXR in vitro assays. F(3)MethylAA is capable not only of inducing ABCA1 mRNA levels, but also increasing cholesterol efflux from THP-1 macrophages. In rat hepatocytes, F(3)MethylAA induced cyp7a mRNA, confirming conclusions from the knockout mouse studies. Furthermore, in rat in vivo studies, F(3)MethylAA induced liver cyp7a mRNA and enzyme activity. A critical species difference is also reported in that neither F(3)MethylAA nor 22(R)-hydroxycholesterol induced cyp7a in human primary hepatocytes. However, other LXR target genes, ABCA1, ABCG1, and SREBP1, were regulated.     

Effects of hepatic expression of the high-density lipoprotein receptor SR-BI on lipoprotein metabolism and female fertility

The etiology of human female infertility is often uncertain. The sterility of high-density lipoprotein (HDL) receptor-negative (SR-BI(-/-)) female mice suggests a link between female infertility and abnormal lipoprotein metabolism. SR-BI(-/-) mice exhibit elevated plasma total cholesterol [with normal-sized and abnormally large HDL and high unesterified to total plasma cholesterol (UC:TC) ratio]. We explored the influence of hepatic SR-BI on female fertility by inducing hepatic SR-BI expression in SR-BI(-/-) animals by adenovirus transduction or stable transgenesis. For transgenes, we used both wild-type SR-BI and a double-point mutant, Q402R/Q418R (SR-BI-RR), which is unable to bind to and mediate lipid transfer from wild-type HDL normally, but retains virtually normal lipid transport activities with low-density lipoprotein. Essentially wild-type levels of hepatic SR-BI expression in SR-BI(-/-) mice restored to nearly normal the HDL size distribution and plasma UC:TC ratio, whereas approximately 7- to 40-fold overexpression dramatically lowered plasma TC and increased biliary cholesterol secretion. In contrast, SR-BI-RR overexpression had little effect on SR-BI(+/+) mice, but in SR-BI(-/-) mice, it substantially reduced levels of abnormally large HDL and normalized the UC:TC ratio. In all cases, hepatic transgenic expression restored female fertility. Overexpression in SR-BI(-/-) mice of lecithin:cholesterol acyl transferase, which esterifies plasma HDL cholesterol, did not normalize the UC:TC ratio, probably because the abnormal HDL was a poor substrate, and did not restore fertility. Thus, hepatic SR-BI-mediated lipoprotein metabolism influences murine female fertility, raising the possibility that dyslipidemia might contribute to human female infertility and that targeting lipoprotein metabolism might complement current assisted reproductive technologies.     

ABCA1基因调控与动脉粥样硬化的关系

ATP结合盒转运体A1(ABCA1)足一种整合膜蛋白,它以ATP为能源,转运细胞内的游离胆固醇、磷脂至细胞外并与载脂蛋白A1结合,形成高密度脂蛋白.ABCA1基因是胆固醇逆转运和高密度脂蛋白(HDL)生成起始步骤的关键基因.ABCA1的表达在转录和转录后水平受肝X受体/视黄醇X受体(LXR/RXR)等多种因子调控.ABCA1功能障碍将导致动脉粥样硬化的发生、发展,与心脑血管疾病密切相关.