Liver X Receptor Regulates Triglyceride Absorption Through Intestinal Down-regulation of Scavenger Receptor Class B, Type 1.

BACKGROUND & AIMS: Reducing postprandial triglyceridemia may be a promising strategy to lower the risk of cardiovascular disorders associated with obesity and type 2 diabetes. In enterocytes, scavenger receptor class B, type 1 (SR-B1, encoded by SCARB1) mediates lipid-micelle sensing to promote assembly and secretion of chylomicrons. The nuclear receptor subfamily 1, group H, members 2 and 3 (also known as liver X receptors [LXRs]) regulate genes involved in cholesterol and fatty acid metabolism. We aimed to determine whether intestinal LXRs regulate triglyceride absorption. METHODS: C57BL/6J mice were either fed a cholesterol-enriched diet or given synthetic LXR agonists (GW3965 or T0901317). We measured the production of chylomicrons and localized SR-B1 by immunohistochemistry. Mechanisms of postprandial triglyceridemia and SR-B1 regulation were studied in Caco-2/TC7 cells incubated with LXR agonists. RESULTS: In mice and in the Caco-2/TC7 cell line, LXR agonists caused localization of intestinal SR-B1 from apical membranes to intracellular organelles and reduced chylomicron secretion. In Caco-2/TC7 cells, LXR agonists reduced SR-B1-dependent lipidic-micelle-induced Erk phosphorylation. LXR agonists also reduced intracellular trafficking of the apical apolipoprotein B pool toward secretory compartments. LXR reduced levels of SR-B1 in Caco-2/TC7 cells via a post-transcriptional mechanism that involves microRNAs. CONCLUSION: In Caco-2/TC7 cells and mice, intestinal activation of LXR reduces the production of chylomicrons by a mechanism dependent on the apical localization of SR-B1.

Liver X receptor activation induces the uptake of cholesteryl esters from high density lipoproteins in primary human macrophages.

OBJECTIVE: Liver X receptors (LXRs) are oxysterol-activated nuclear receptors regulating reverse cholesterol transport, in part by modulating cholesterol efflux from macrophages to apoAI and HDL via the ABCA1 and ABCG1/ABCG4 pathways. Moreover, LXR activation increases intracellular cholesterol trafficking via the induction of NPC1 and NPC2 expression. However, implication of LXRs in the selective uptake of cholesteryl esters from lipoproteins in human macrophages has never been reported. METHODS AND RESULTS: Our results show that (1) selective CE uptake from HDL(3) is highly efficient in human monocyte-derived macrophages; (2) surprisingly, HDL(3)-CE uptake is strongly increased by LXR activation despite antiatherogenic effects of LXRs; (3) HDL(3)-CE uptake increase is not linked to SR-BI expression modulation but it is dependent of proteoglycan interactions; (4) HDL(3)-CE uptake increase is associated with increased expression and secretion of apoE and LPL, two proteins interacting with proteoglycans; (5) HDL(3)-CE uptake increase depends on the integrity of raft domains and is associated with an increased caveolin-1 expression. CONCLUSIONS: Our study identifies a new role for LXRs in the control of cholesterol homeostasis in human macrophages. LXR activation results in enhanced dynamic intracellular cholesterol fluxes through an increased CE uptake from HDL and leads to an increased cholesterol availability to efflux to apoAI and HDL.

P-glycoprotein and cytochrome P450 3A4 involvement in risperidone transport using an in vitro Caco-2/TC7 model and an in vivo model.

The possible involvement of P-glycoprotein (P-gp) and cytochrome P450 (CYP) 3A4 in risperidone transport was investigated using in vitro and in vivo models. Firstly, uptake studies were performed on a Caco-2/TC7 cell monolayer; the effects of 1 microg ml(-1) risperidone on apparent permeability were determined for secretory and absorptive directions, in the presence or absence of various P-gp and CYP3A4 inhibitors (verapamil, ketoconazole, erythromycin), and of an associated multidrug-resistant protein inhibitor (indomethacin). Secondly, on a conscious rat model, risperidone pharmacokinetic parameters, notably absorption parameters, were determined using compartmental and deconvolution methods. Three groups of seven rats received respectively an IV risperidone dose, an oral risperidone dose (PO group) and the same oral risperidone dose after verapamil administration (POV group). No formation of 9-hydroxyrisperidone was observed on Caco-2 cells after risperidone administration; there was no evidence that intestinal CYP3A4 is involved in risperidone metabolising. Risperidone secretory permeation was higher than absorptive permeation. Verapamil increased risperidone absorption permeation and decreased its secretory permeation. Indomethacin did not modify these permeation values. In rats, verapamil led to an increase in both risperidone and 9-hydroxyrisperidone plasmatic concentrations. The fraction absorbed in the verapamil group was 3.18 times higher than in the oral group (65.9% and 20.7% for POV group and PO group). The absorption rate constant was lower in the verapamil group. Our results indicate that P-gp decreases the intestinal absorption of risperidone and that intestinal CYP3A4 is not involved in risperidone metabolism.

Defective VLDL metabolism and severe atherosclerosis in mice expressing human apolipoprotein E isoforms but lacking the LDL receptor.

Differences in affinity of human apolipoprotein E (apoE) isoforms for the low density lipoprotein receptor (LDLR) are thought to result in the differences in lipid metabolism observed in humans with different APOE genotypes. Mice expressing three common human apoE isoforms, E2, E3, and E4, in place of endogenous mouse apoE were used to investigate the relative roles of apoE isoforms in LDLR- and non-LDLR-mediated very low density lipoprotein (VLDL) clearance. While both VLDL particles isolated from mice expressing apoE3 and apoE4 bound to mouse LDLR with affinity and Bmax similar to VLDL containing mouse apoE, VLDL with apoE2 bound with only half the Bmax. In the absence of the LDLR, all lines of mice expressing human apoE showed dramatic increases in VLDL cholesterol and triglycerides (TG) compared to LDLR knockout mice expressing mouse apoE. The mechanism of the hyperlipidemia in mice expressing human apoE isoforms is due to impairment of non-LDL-receptor-mediated VLDL clearance. This results in the severe atherosclerosis observed in mice expressing human apoE but lacking the LDLR, even when fed normal chow diet. Our data show that defects in LDLR independent pathway(s) are a potential factor that trigger hyperlipoproteinemia when the LDLR pathway is perturbed, as in E2/2 mice.

SR-BI does not require raft/caveola localisation for cholesteryl ester selective uptake in the human adrenal cell line NCI-H295R.

Class B type I scavenger receptor (SR-BI) mediates the selective uptake of high-density lipoprotein (HDL)-derived cholesteryl esters (HDL-CE) in steroidogenic cells and hepatocytes. SR-BI is enriched in the caveolae of some cell types, genetically modified or not, and these domains have already been shown to constitute primary acceptors for HDL-CE. Nevertheless, the fate of caveola-free cell types has not yet been discussed.NCI-H295R, a human adrenal cell line, highly active in HDL-CE uptake via SR-BI, does not display any morphologically defined caveolae and expresses caveolin at a very low level. Using two different fractionation protocols, we have shown, in this cell type, that SR-BI is homogeneously distributed along the plasma membrane and consists principally of a non-raft membrane-associated pool. Raft destabilisation and caveolin-1 displacement from plasma membrane did not modify the SR-BI-mediated HDL-CE selective uptake. Moreover, the induction of SR-BI expression that is associated with increased CE selective uptake was not associated with any modification in caveolin-1 expression or any raft-targeting mechanism of SR-BI in NCI-H295R. In conclusion, we provide evidence that SR-BI does not require raft/caveola localisation to be implicated in CE selective uptake either in basal or in induced conditions.

Regulation of the scavenger receptor BI and the LDL receptor by activators of aldosterone production, angiotensin II and PMA, in the human NCI-H295R adrenocortical cell line.

In human adrenal cells, cholesterol for steroidogenesis is derived from both high-density lipoproteins (HDL) via the Scavenger Receptor Class B Type I (SR-BI) and low-density lipoproteins (LDL) via the LDL receptor pathway. We have previously shown that, in the human adrenocortical carcinoma cell line, NCI-H295R, SR-BI and LDL receptor expression and steroidogenesis are coordinately regulated by activators of protein kinase A (PKA) leading to glucocorticoid synthesis. In the present study, we studied whether SR-BI and LDL receptor expression are regulated by activators of the protein kinase C (PKC) signaling pathway, such as angiotensin II, which stimulate mineralocorticoid synthesis. First, it is shown that, in NCI-H295R cells, aldosterone synthesis is stimulated by a phorbol ester (phorbol-12-myristate-13 acetate, PMA), a potent PKC activator. Northern blot analysis indicated that both angiotensin II and PMA stimulated SR-BI expression in a time-dependent manner. LDL receptor expression is slightly stimulated by PMA. The induction of SR-BI gene expression occurs at the transcriptional level, via an activation of the human SR-BI promoter, as shown by transient transfection experiments. Finally, SR-BI protein level was increased in angiotensin II- and PMA-stimulated cells, resulting in higher lipoprotein binding and specific cholesteryl ester (CE) uptake from HDL, as well from LDL after angiotensin II and PMA stimulation.

Human free apolipoprotein A-I and artificial pre-beta-high-density lipoprotein inhibit eNOS activity and NO release.

Little is known about the effects of human free apolipoprotein A-I (Free-Apo A-I) and pre-beta-high density lipoprotein (pre-beta-HDL) on the endothelium function. In this study, we have investigated the effects of Free-Apo A-I and artificial pre-beta-HDL on endothelial NO synthase (eNOS) activity and on NO production by endothelial cells. Free-Apo A-I drastically inhibited NO production in human umbilical cord vein endothelial cells (HUVECs) and eNOS activity in bovine aortic endothelial cells (BAECs). Pre-beta-HDL and serum from human apolipoprotein A-I transgenic rabbits inhibited eNOS activity in BAECs but HDL3 did not. Free-Apo A-I displaced eNOS from BAEC plasma membrane towards intracellular pools without affecting eNOS activity and eNOS mass in BAEC crude homogenates. Free-Apo A-I and HDL3 did not decrease either caveolin bound to BAEC plasma membrane or caveola cholesterol content. As previously described, we showed that HDL3 directly induced endothelium-dependent relaxation of rings from rat aorta. We observed that pre-beta-HDL significantly decreased endothelium-dependent relaxation of rat aortic rings ex vivo.

Early-glycation of apolipoprotein E: effect on its binding to LDL receptor, scavenger receptor A and heparan sulfates.

Glycation is responsible for disruption of lipoprotein functions leading to the development of atherosclerosis in diabetes. The effects of apolipoprotein E (apoE) glycation were investigated with respect to its interaction with receptors. The interaction of apoE with the low density lipoprotein receptor (LDL-R) and scavenger receptor A (SR-A) was measured by competition experiments performed using, respectively, on a human fibroblast cell line 125I-LDL, and on a murine macrophage cell line (J774) 125I-acetylated LDL, and unlabeled apoE/phospholipid complexes. Glycated apoE binding to heparin and heparan sulfates (HS) was assessed by surface plasmon resonance (SPR) technology. Site-directed mutagenesis was then performed on Lys-75, the major glycation site of the protein. The prepared mutant protein proved to be useful as a tool to study the role of Lys-75 in apoE glycation. The findings showed that, although glycation has no effect on apoE binding either to the LDL-R or to SR-A, it impairs its binding to immobilized heparin and HS. The glycation of Lys-75 was found to be proceed rapidly and contributed significantly to total protein glycation. We propose that, in the case of diabetes, glycation may lead to the atherogenicity of apoE-containing lipoproteins disturbing their uptake via the HS proteoglycan pathway.

Apolipoprotein A-II, HDL metabolism and atherosclerosis.

Apolipoprotein (Apo) A-I and apo A-II are the major apolipoproteins of HDL. It is clearly demonstrated that there are inverse relationships between HDL-cholesterol and apo A-I plasma levels and the risk of coronary heart disease (CHD) in the general population. On the other hand, it is still not clearly demonstrated whether apo A-II plasma levels are associated with CHD risk. A recent prospective epidemiological (PRIME) study suggests that Lp A-I (HDL containing apo A-I but not apo A-II) and Lp A-I:A-II (HDL containing apo A-I and apo A-II) were both reduced in survivors of myocardial infarction, suggesting that both particles are risk markers of CHD. Apo A-II and Lp A-I:A-II plasma levels should be rather related to apo A-II production rate than to apo A-II catabolism. Mice transgenic for both human apo A-I and apo A-II are less protected against atherosclerosis development than mice transgenic for human apo A-I only, but the results of the effects of trangenesis of human apo A-II (in the absence of a co-transgenesis of human apo A-I) are controversial. It is highly suggested that HDL reduce CHD risk by promoting the transfer of peripherical free cholesterol to the liver through the so-called 'reverse cholesterol transfer'. Apo A-II modulates different steps of HDL metabolism and therefore probably alters reverse cholesterol transport. Nevertheless, some effects of apo A-II on intermediate HDL metabolism might improve reverse cholesterol transport and might reduce atherosclerosis development while some other effects might be deleterious. In different in vitro models of cell cultures, Lp A-I:A-II induce either a lower or a similar cellular cholesterol efflux (the first step of reverse cholesterol transport) than Lp A-I. Results depend on numerous factors such as cultured cell types and experimental conditions. Furthermore, the effects of apo A-II on HDL metabolism, beyond cellular cholesterol efflux, are also complex and controversial: apo A-II may inhibit lecithin-cholesterol acyltransferase (LCAT) (potential deleterious effect) and cholesteryl-ester-transfer protein (CETP) (potential beneficial effect) activities, but may increase the hepatic lipase (HL) activity (potential beneficial effect). Apo A-II may also inhibit the hepatic cholesteryl uptake from HDL (potential deleterious effect) probably through the SR-BI depending pathway. Therefore, in terms of atherogenesis, apo A-II alters the intermediate HDL metabolism in opposing ways by increasing (LCAT, SR-BI) or decreasing (HL, CETP) the atherogenicity of lipid metabolism. Effects of apo A-II on atherogenesis are controversial in humans and in transgenic animals and probably depend on the complex effects of apo A-II on these different intermediate metabolic steps which are in weak equilibrium with each other and which can be modified by both endogenous and environmental factors. It can be suggested that apo A-II is not a strong determinant of lipid metabolism, but is rather a modulator of reverse cholesterol transport.

Anti-Plasmodium properties of group IA, IB, IIA and III secreted phospholipases A2 are serum-dependent.

Antibacterial, antiparasitidal and antiviral properties have recently been attributed to members of the secreted phospholipases A(2) (sPLA(2)s) superfamily. Seven sPLA(2)s from groups IA, IB, IIA and III, were tested here in different culture conditions for inhibition of the in vitro intraerythrocytic development of Plasmodium falciparum, the causative agent of the most severe form of human malaria. In the presence of human serum, all sPLA(2)s were inhibitory, with three out of seven exhibiting IC(50)<0.1 nM. In all cases, inhibition could be induced by enzymatic pre-treatment of the serum. By contrast, no effect was observed when parasites were grown in a semi-defined medium (AlbuMAX II) devoid of lipoproteins and containing 10 times less phospholipids than the medium with human serum, strongly suggesting that hydrolysis of serum generating toxic lipid by-products, rather than a direct interaction of the sPLA(2) with the infected erythrocyte, is a general feature of the anti-Plasmodium properties of sPLA(2)s. Furthermore, in serum, six out of the seven sPLA(2)s were toxic against both trophozoite and schizont stages of the parasite development, contrasting with the trophozoite-selective bee venom enzyme's toxicity. Deciphering the molecular mechanisms at play in the phenotypic singularity of the bee venom enzyme toxicity might offer new prospects in antimalarial fight.

The human hepatocyte cell lines IHH and HepaRG: models to study glucose, lipid and lipoprotein metabolism.

Metabolic diseases reach epidemic proportions. A better knowledge of the associated alterations in the metabolic pathways in the liver is necessary. These studies need in vitro human cell models. Several human hepatoma models are used, but the response of many metabolic pathways to physiological stimuli is often lost. Here, we characterize two human hepatocyte cell lines, IHH and HepaRG, by analysing the expression and regulation of genes involved in glucose and lipid metabolism. Our results show that the glycolysis pathway is activated by glucose and insulin in both lines. Gluconeogenesis gene expression is induced by forskolin in IHH cells and inhibited by insulin in both cell lines. The lipogenic pathway is regulated by insulin in IHH cells. Finally, both cell lines secrete apolipoprotein B-containing lipoproteins, an effect promoted by increasing glucose concentrations. These two human cell lines are thus interesting models to study the regulation of glucose and lipid metabolism.

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.

Reconstitution of hepatitis C virus envelope glycoproteins into liposomes as a surrogate model to study virus attachment.

The envelope glycoproteins, E1 and E2, of hepatitis C virus (HCV) assemble intracellularly to form a noncovalent heterodimer that is expected to be essential for viral assembly and entry. However, due to the lack of a cell culture system supporting efficient HCV replication, it is very difficult to obtain relevant information on the functions of this glycoprotein oligomer. To get better insights into its biological and biochemical properties, HCV envelope glycoprotein heterodimer expressed by a vaccinia virus recombinant was purified by immunoaffinity. Purified E1E2 heterodimer was recognized by conformation-dependent monoclonal antibodies, showing that the proteins were properly folded. In addition, it interacted with human CD81, a putative HCV receptor, as well as with human low and very low density lipoproteins, which have been shown to be associated with infectious HCV particles isolated from patients. Purified E1E2 heterodimer was also reconstituted into liposomes. E1E2-liposomes were recognized by a conformation-dependent monoclonal antibody as well as by human CD81. Together, these data indicate that E1E2-liposomes are a valuable tool to study the molecular requirements for HCV binding to target cells.

Lipid free apolipoprotein E binds to the class B Type I scavenger receptor I (SR-BI) and enhances cholesteryl ester uptake from lipoproteins.

The Class B type I scavenger receptor I (SR-BI) is a physiologically relevant high density lipoprotein (HDL) receptor that can mediate selective cholesteryl ester (CE) uptake by cells. Direct interaction of apolipoprotein E (apoE) with this receptor has never been demonstrated, and its implication in CE uptake is still controversial. By using a human adrenal cell line (NCI-H295R), we have addressed the role of apoE in binding to SR-BI and in selective CE uptake from lipoproteins to cells. This cell line does not secrete apoE and SR-BI is its major HDL-binding protein. We can now provide evidence that 1) free apoE is a ligand for SR-BI, 2) apoE associated to lipids or in lipoproteins does not modulate binding or CE-selective uptake by the SR-BI pathway, and 3) the direct interaction of free apoE to SR-BI leads to an increase in CE uptake from lipoproteins of both low and high densities. We propose that this direct interaction could modify SR-BI structure in cell membranes and potentiate CE uptake.

Fibrates down-regulate hepatic scavenger receptor class B type I protein expression in mice.

Fibrates are normolipidemic drugs used in atherogenic dyslipidemia because of their ability to raise high density lipoprotein (HDL) and decrease triglyceride levels. They exert multiple effects on lipid metabolism by activating the peroxisome proliferator-activated receptor-alpha (PPAR-alpha), which controls the transcriptional regulation of genes involved in hepatic fatty acid, cholesterol, and lipoprotein metabolism. The hepatic expression of the scavenger receptor class B type I (SR-BI) plays a critical role in lipoprotein metabolism, mainly due to its ability to mediate selective cholesterol uptake. Because fibrates and PPAR-alpha agonists up-regulate SR-BI expression in human and murine macrophages, we tested whether fibrates raised a similar regulatory response on hepatic SR-BI expression in mice. Surprisingly, fibrate treatment suppressed SR-BI protein expression in the liver without changing steady state SR-BI mRNA levels. Decreased hepatic SR-BI protein expression correlated with enlarged HDL particle size. This effect was concomitant with down-regulation of CLAMP, a putative SR-BI-stabilizing protein found in the hepatic plasma membrane, which was also not associated to changes in CLAMP mRNA levels. The post-transcriptional regulatory effect of fibrates over hepatic SR-BI protein levels was dependent on PPAR-alpha expression, because it was absent in PPAR-alpha-deficient mice. Restoring hepatic SR-BI expression in fibrate-treated mice by recombinant adenoviral gene transfer abolished fibrate-mediated HDL particle size enlargement. This study describes a novel effect of fibrates on hepatic SR-BI expression providing an alternative mechanism by which this drug family modulates HDL metabolism in vivo.

Daily melatonin supplementation in mice increases atherosclerosis in proximal aorta.

Considerable evidence supports the hypothesis that LDL oxidation plays an important role in atherosclerosis. Even though high melatonin doses inhibit LDL oxidation in vitro, the effect of melatonin on atherosclerosis has never been studied. We have demonstrated that the feeding of hypercholesterolemic mice with an atherogenic diet supplemented with melatonin highly increases the surface of atherosclerotic lesions in the proximal aorta. These observations occur without detectable lipidic or glucidic phenotype alteration. Melatonin treatment increased highly the sensitivity of atherogenic lipoprotein to Cu(2+) and gamma-radiolysis generated oxyradical ex vivo oxidation during the fasting period. Moreover, these altered lipoproteins were less recognized by the LDL receptor metabolic pathway of murine fibroblasts while they transferred many more cholesteryl esters to murine macrophages. This study suggests that caution should be taken as regards high melatonin dosage in hypercholesterolemic patients.