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The activation of nuclear receptors, peroxisome proliferator-activated receptor gamma (PPARγ) and liver X receptor alpha (LXRα), has been shown to inhibit the growth of prostate cancer cells. This study examined whether the anti-proliferative effect of lycopene on androgen-dependent human prostate cancer (LNCaP) cells involves the up-regulation of the expression of PPARγ and LXRα. As expected, lycopene treatment (2.5-10 μM) significantly inhibited the proliferation of LNCaP cells during incubation for 96 h. Lycopene significantly increased the protein and mRNA expression of PPARγ and LXRα at 24 and 48 h, while the increased in the expression of ATP-binding cassette transporter 1 (ABCA1) was only evident 96 h. In addition, lycopene significantly decreased cellular total cholesterol levels and increased apoA1 protein expression at 96 h. Incubation of LNCaP cells with lycopene (10 μM) in the presence (20 μM) of a specific antagonist of PPARγ (GW9662) and LXRα (GGPP) restored the proliferation of LNCaP cells to the control levels and significantly suppressed protein expression of PPARγ and LXRα as well as increased cellular total cholesterol levels. LXRα knockdown by siRNA against LXRα significantly enhanced the proliferation of LNCaP cells, whereas si-LXRα knockdown followed by incubation with lycopene (10 μM) restored the proliferation to the control level. The present study is the first to demonstrate that the anti-proliferative effect of lycopene on LNCaP cells involves the activation of the PPARγ-LXRα-ABCA1 pathway, leading to reduced cellular total cholesterol levels. Show less

The liver plays a pivotal role in the physiological adaptation to fasting and a better understanding of the metabolic adaptive responses may give hints on new therapeutic strategies to control the metabolic diseases. The liver X receptors (LXRs) are well-established regulators of lipid and glucose metabolism. More recently fibroblast growth factor 21 (FGF21) has emerged as an important regulator of energy homeostasis. We hypothesized that the LXR transcription factors could influence Fgf21 expression, which is induced in response to fasting. Wild-type, LXRα(-/-), and LXRβ(-/-) mice were treated for 3 d with vehicle or the LXR agonist GW3965 and fasted for 12 h prior to the killing of the animals. Interestingly, serum FGF21 levels were induced after fasting, but this increase was blunted when the mice were treated with GW3965 independently of genotypes. Compared with wild-type mice, GW3965-treated LXRα(-/-) and LXRβ(-/-) mice showed improved insulin sensitivity and enhanced ketogenic response at fasting. Of note is that during fasting, GW3965 treatment tended to reduce liver triglycerides as opposed to the effect of the agonist in the fed state. The LXR-dependent repression of Fgf21 seems to be mainly mediated by the recruitment of LXRβ onto the Fgf21 promoter upon GW3965 treatment. This repression by LXRβ occurs through the recruitment and stabilization of the repressor complex composed of retinoid-related orphan receptor-α/Rev-Erbα/histone deacetylase 3 onto the Fgf21 promoter. Our data clearly demonstrate that there is a cross talk between the LXR and FGF21 signaling pathways in the adaptive response to fasting. Show less

The gradually increasing changes in a human hyperlipidemic diet along with chronic stress might play an important role in the increased numbers of fatty liver. This study investigated the effects of Ilex asprella root decoction on related genes of lipid metabolism in chronic stress in hyperlipidemic fatty liver in rats. Forty-eight male Wistar rats were randomly divided into four groups: normal control group, model control group, simvastatin group, and Ilex asprella root group. To establish chronic stress and hyperlipidemic fatty liver models in rats, the levels of serum lipids, glucose, liver index, insulin (INS), insulin resistant (IR) index, adiponectin, superoxide dismutase (SOD), glutathione peroxidase (GSH-pX), glutathione (GSH), liver X receptor (LXR), and sterol responsive element binding protein (SREBP)-1c in rats were measured. When compared to the normal control group, the levels of serum lipids, glucose, liver index, INS, IR index, and GSH in the model control group significantly increased (P < 0.01). The protein levels of LXRα and SREBP-1c increased (P < 0.05), and the serum adiponectin and the SOD and GSH-pX decreased significantly (P < 0.01). When compared to the model control group, the levels of serum lipids, glucose, liver index, INS, IR index, SOD, and GSH-pX in the simvastatin group and Ilex asprella root group increased in varying degrees (P < 0.01 or 0.05); the serum adiponectin and GSH decreased (P < 0.05), while the protein levels of LXRα and SREBP-1c decreased in varying degrees (P < 0.01 or 0.05). When compared to the simvastatin group, the IR index and protein levels of LXRα in the Ilex asprella root group decreased (P < 0.05), and the serum adiponectin and SOD increased (P < 0.05). The Ilex asprella root decoction has some protective effects on regulating the related genes of lipid metabolism caused by chronic stress and hyperlipidemic fatty liver in rats. Show less

CYP1A1 and CYP1A2 are involved in both detoxification and metabolic activation of xenobiotics. Human CYP1A1 (hCYP1A1) and hCYP1A2 exist in a head-to-head orientation in chromosome 15 with the overlapping 5'-flanking region. We have recently reported that nuclear receptor constitutive androstane receptor (CAR), in addition to aryl hydrocarbon receptor, bidirectionally transactivates these genes through common motifs. In this study, we have investigated a role of liver X receptor α (LXRα), another liver-enriched nuclear receptor, in the expression hCYP1A1 and hCYP1A2. In reporter assays with dual-reporter constructs containing their promoter region between two different reporter genes, LXRα simultaneously transactivated hCYP1A1 and hCYP1A2 through two regions, independent of aryl hydrocarbon receptor. In electrophoretic mobility shift assays, LXRα/retinoid X receptor α heterodimer bound to two ER8-type motifs found at around -520 and -460 of hCYP1A1. The former corresponds to the CAR-binding motif previously identified. Reporter assays using mutated constructs confirmed the critical roles of these motifs in the LXRα-mediated simultaneous transcription of hCYP1A1 and hCYP1A2. hCYP1A1 and hCYP1A2 mRNA levels were increased in human hepatoma HuH-7 cells and human primary hepatocytes, respectively, after treatment with the LXRα ligand GW3965. Our results suggest that LXRα transactivates the expression of hCYP1A1 and hCYP1A2 through common two cis-elements. Show less

Endothelin-1 (ET-1), predominantly produced by vascular endothelial cells (VECs), plays an important role in the pathogenesis of inflammatory diseases. Liver X receptor (LXR), a typical nuclear receptor, is known for inhibiting expression of inflammatory molecules. However, it remains unclear whether LXR suppresses ET-1 expression. In the present study, we showed that pretreatment with GW3965, a specific ligand of LXR, significantly attenuated lipopolysaccharide (LPS)-induced ET-1 in mice plasma. The in vitro experiments showed that both LXRα and β were expressed in human VECs, and they are functional as demonstrated by induction of the target gene ABCA1 after treatment with GW3965. Moreover, activation of LXR with GW3965 in human VECs dramatically attenuated the basal and LPS-stimulated ET-1 production at both transcriptional and translational levels. Luciferase reporter assays indicated that LXR activation suppressed the transcriptional activity of the human ET-1 gene promoter, and repressed the activity of a heterologous promoter driven by the response elements of activator-1 (AP-1) or nuclear factor-κB (NF-κB). Electrophoretic mobility shift and chromatin immunoprecipitation assays showed that activation of LXR reduced the binding of the transcriptional factors AP-1 and NF-κB to the ET-1 gene promoter region. In conclusion, activation of LXR represses ET-1 expression in vivo and in vitro, which may be involved in the negatively interfering with AP-1/NF-κB signaling. These results suggest that LXRs may serve as a novel molecular target for modulating ET-1 expression in VECs, and even for the treatment of ET-1-associated inflammatory diseases. Show less

A novel liver X receptor (LXR) modulator, iristectorigenin B isolated from Belamcanda chinensis, stimulated the transcriptional activity of both LXR-α and LXR-β. In macrophages, iristectorigenin B suppressed cholesterol accumulation in a dose-dependent manner and induced the transcriptional activation of LXR-α/-β-responsive genes, ATP-binding cassette transporters A1 and G1. It did not induce hepatic lipid accumulation nor the expression of the lipogenesis genes sterol regulatory element-binding protein-1c, fatty acid synthase, and stearoyl-CoA desaturase-1. Iristectorigenin B thus is a dual-LXR agonist that regulates the expression of key genes in cholesterol homeostasis in macrophage cells without inducing hepatic lipid accumulation. Show less

The purpose of this study was to examine selective macrophage differentiation occurring in areas of intraplaque hemorrhage in human atherosclerosis. Macrophage subsets are recognized in atherosclerosis, but the stimulus for and importance of differentiation programs remain unknown. We used freshly isolated human monocytes, a rabbit model, and human atherosclerotic plaques to analyze macrophage differentiation in response to hemorrhage. Macrophages characterized by high expression of both mannose and CD163 receptors preferentially exist in atherosclerotic lesions at sites of intraplaque hemorrhage. These hemoglobin (Hb)-stimulated macrophages, M(Hb), are devoid of neutral lipids typical of foam cells. In vivo modeling of hemorrhage in the rabbit model demonstrated that sponges exposed to red cells showed an increase in mannose receptor-positive macrophages only when these cells contained Hb. Cultured human monocytes exposed to Hb:haptoglobin complexes, but not interleukin-4, expressed the M(Hb) phenotype and were characterized by their resistance to cholesterol loading and up-regulation of ATP-binding cassette (ABC) transporters. M(Hb) demonstrated increased ferroportin expression, reduced intracellular iron, and reactive oxygen species (ROS). Degradation of ferroportin using hepcidin increased ROS and inhibited ABCA1 expression and cholesterol efflux to apolipoprotein A-I, suggesting reduced ROS triggers these effects. Knockdown of liver X receptor alpha (LXRα) inhibited ABC transporter expression in M(Hb) and macrophages differentiated in the antioxidant superoxide dismutase. Last, LXRα luciferase reporter activity was increased in M(Hb) and significantly reduced by overnight treatment with hepcidin. Collectively, these data suggest that reduced ROS triggers LXRα activation and macrophage reverse cholesterol transport. Hb is a stimulus for macrophage differentiation in human atherosclerotic plaques. A decrease in macrophage intracellular iron plays an important role in this nonfoam cell phenotype by reducing ROS, which drives transcription of ABC transporters through activation of LXRα. Reduction of macrophage intracellular iron may be a promising avenue to increase macrophage reverse cholesterol transport. Show less

α-Eleostearic acid and punicic acid, two typical conjugated linolenic acid (CLnA) isomers present in bitter gourd and snake gourd oil respectively, exhibit contrasting cis-trans configuration which made them biologically important. Rats were divided into six groups. Group 1 was control and group 2 was treated control. Rats in the groups 3 and 4 were treated with mixture of α-eleostearic acid and punicic acid (1:1) (0.5% and 1.0% respectively) while rats in the groups 5 and 6 were treated with 0.5% of α-eleostearic acid and 0.5% of punicic acid respectively along with sodium arsenite by oral gavage once per day. Results showed that increase in nitric oxide synthase (NOS) activity, inflammatory markers expression, platelet aggregation, lipid peroxidation, protein oxidation, DNA damage and altered expression of liver X receptor-α (LXR-α) after arsenite treatment were restored with the supplementation of oils containing CLnA isomers. Altered activities of different antioxidant enzymes such as superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and ferric reducing ability of plasma (FRAP) also restored after oil supplementation. Altered morphology and fluidity of erythrocyte membrane studied by atomic force and scanning electron microscopy, after stress induction were significantly improved due to amelioration in cholesterol/phospholipid ratio and fatty acid profile of membrane. Oils treatment also improved morphology of liver and fatty acid composition of hepatic lipid. Overall two isomers showed synergistic antioxidant and anti-inflammatory effect against induced perturbations and membrane disintegrity. Synergistic antioxidant and anti-inflammatory role of these CLnA isomers were established by this study. Show less

Psychological stress is associated with an increased risk of cardiovascular diseases. However, the connecting mechanisms of the stress-inducing activation of the hypothalamic-pituitary-adrenal axis with atherosclerosis are not well-understood. To study the effect of acute psychological stress on reverse cholesterol transport (RCT), which transfers peripheral cholesterol to the liver for its ultimate fecal excretion. C57Bl/6J mice were exposed to restraint stress for 3 hours to induce acute psychological stress. RCT in vivo was quantified by measuring the transfer of [(3)H]cholesterol from intraperitoneally injected mouse macrophages to the lumen of the small intestine within the stress period. Surprisingly, stress markedly increased the contents of macrophage-derived [(3)H]cholesterol in the intestinal lumen. In the stressed mice, intestinal absorption of [(14)C]cholesterol was significantly impaired, the intestinal mRNA expression level of peroxisome proliferator-activated receptor-α increased, and that of the sterol influx transporter Niemann-Pick C1-like 1 decreased. The stress-dependent effects on RCT rate and peroxisome proliferator-activated receptor-α gene expression were fully mimicked by administration of the stress hormone corticosterone (CORT) to nonstressed mice, and they were blocked by the inhibition of CORT synthesis in stressed mice. Moreover, the intestinal expression of Niemann-Pick C1-like 1 protein decreased when circulating levels of CORT increased. Of note, when either peroxisome proliferator-activated receptor α or liver X receptor α knockout mice were exposed to stress, the RCT rate remained unchanged, although plasma CORT increased. This indicates that activities of both transcription factors were required for the RCT-accelerating effect of stress. Acute psychological stress accelerated RCT by compromising intestinal cholesterol absorption. The present results uncover a novel functional connection between the hypothalamic-pituitary-adrenal axis and RCT that can be triggered by a stress-induced increase in circulating CORT. Show less

Conjugated linoleic acid (CLA) reduces adiposity in vivo. However, mechanisms mediating these changes are unclear. Therefore, we treated cultures of human adipocytes with trans-10, cis-12 (10,12) CLA, cis-9, trans-11 (9,11) CLA or other trans fatty acids (FA), and measured indices of lipid metabolism. The lipid-lowering effects of 10,12 CLA were unique, as other trans FA did not reduce TG content to the same extent. Using low levels of [(14)C]-CLA isomers, it was shown that both isomers were readily incorporated into acylglycerols and phospholipids, albeit at lower levels than [(14)C]-oleic or [(14)C]-linoleic acids. When using [(14)C]-acetic acid and [(14)C]-pyruvic acid as substrates, 30 μM 10,12 CLA, but not 9,11 CLA, decreased de novo synthesis of triglyceride, free FA, diacylglycerol, cholesterol esters, cardiolipin, phospholipids and ceramides within 3-24 h. Treatment with 30 μM 10,12 CLA, but not 9,11 CLA, decreased total cellular lipids within 3 days and the ratio of monounsaturated FA (MUFA) to saturated FA, and increased C18:0 acyl-CoA levels within 24 h. Consistent with these data, stearoyl-CoA desaturase (SCD)-1 mRNA and protein levels were down-regulated by 10,12 CLA within 7-12 h, respectively. The mRNA levels of liver X receptor (LXR)α and sterol regulatory element binding protein (SREBP)-1c, transcription factors that regulate SCD-1, were decreased by 10,12 CLA within 5 h. These data suggest that the isomer-specific decrease in de novo lipid synthesis by 10,12 CLA is due, in part, to the rapid repression of lipogenic transcription factors that regulate MUFA synthesis, suggesting an anti-obesity mechanism unique to this trans FA. Show less

Following the widespread use of genome-wide association studies (GWAS), focus is turning towards identification of causal variants rather than simply genetic markers of diseases and traits. As a step towards a high-throughput method to identify genome-wide, non-coding, functional regulatory variants, we describe the technique of allele-specific FAIRE, utilising large-scale genotyping technology (FAIRE-gen) to determine allelic effects on chromatin accessibility and regulatory potential. FAIRE-gen was explored using lymphoblastoid cells and the 50,000 SNP Illumina CVD BeadChip. The technique identified an allele-specific regulatory polymorphism within NR1H3 (coding for LXR-α), rs7120118, coinciding with a previously GWAS-identified SNP for HDL-C levels. This finding was confirmed using FAIRE-gen with the 200,000 SNP Illumina Metabochip and verified with the established method of TaqMan allelic discrimination. Examination of this SNP in two prospective Caucasian cohorts comprising 15,000 individuals confirmed the association with HDL-C levels (combined beta = 0.016; p = 0.0006), and analysis of gene expression identified an allelic association with LXR-α expression in heart tissue. Using increasingly comprehensive genotyping chips and distinct tissues for examination, FAIRE-gen has the potential to aid the identification of many causal SNPs associated with disease from GWAS. Show less

The present report describes our efforts to convert an existing LXR agonist into an LXR antagonist using a structure-based approach. A series of benzenesulfonamides was synthesized based on structural modification of a known LXR agonist and was determined to be potent dual liver X receptor (LXR α/β) ligands. Herein we report the identification of compound 54 as the first reported LXR antagonist that is suitable for pharmacological in vivo evaluation in rodents. Show less

We previously reported that grape skin ethanol extract (GSE) decreases adipogenic transcription factor gene expression, inhibiting triglyceride accumulation in 3T3-L1 adipocytes. In this study, we hypothesized that GSE may induce differential expression profiles in adipocytes, thus providing protection against obesity. Thirty-five genes involved in the peroxisome proliferator-activated receptor-γ (PPARγ) signaling pathway, lipid metabolism, or adipogenesis were identified through microarray analysis of adipocytes treated with GSE. Expression of the genes involved in PPARγ signaling, Adipoq, Scd1, Nr1h3, Fabp5, Scd2, and Pparg decreased with GSE treatment, whereas expression of Ppargc1a increased. Lipid metabolism-associated genes Mlxp1, Stat5a, Hsl, Plin1, and Vdr were down-regulated. Interestingly, GSE also affected expression of genes related to the mitogen-activated protein kinases pathway. GSE extract treatment decreased expression of aP2, Fas, and Tnfa, known markers of adipogenesis, as measured by real-time polymerase reaction. These findings demonstrate the antiadipogenic effects of GSE on 3T3-L1 adipocytes at the genetic level, primarily on the PPARγ signaling pathway. Show less

Cholesterol synthesis and transport in oligodendrocytes are essential for optimal myelination and remyelination in pathological conditions such as multiple sclerosis. However, little is known about cholesterol homeostasis in the myelin-forming oligodendrocytes. Liver X receptors (LXRs) are nuclear oxysterol receptors that regulate genes involved in cholesterol homeostasis and may therefore play an important role in de- and remyelination. We investigated whether LXRs regulate cholesterol homeostasis in oligodendrocytes. mRNA expression of genes encoding LXR-α and LXR-β and their target genes (ABCA1, ABCG1, ABCG4, apoE, and LDLR) was detected in oligodendrocytes derived from both neonatal and adult rats using quantitative real-time PCR. The expression of LXR-β and several target genes was increased during oligodendrocyte differentiation. We further demonstrated that treatment of primary neonatal rat oligodendrocytes with the synthetic LXR agonist T0901317 induced the expression of several established LXR target genes, including ABCA1, ABCG1, apoE, and LDLR. Treatment of oligodendrocytes with T0901317 resulted in an enhanced cholesterol efflux in the presence of apolipoprotein A-I or high-density lipoprotein particles. These data show that LXRs are involved in regulating cholesterol homeostasis in oligodendrocytes. Show less

Leptin, a key hormone in regulating energy homeostasis, is mainly produced by adipocytes. Cogent evidence indicates a unique role of leptin in the promotion of liver fibrosis. Hepatic stellate cell (HSC) activation is a pivotal step in the process of liver fibrosis. Sterol regulatory element binding protein (SREBP)-1c, a critical transcription factor for lipid synthesis and adipocyte differentiation, functions as a key transcription factor in inhibition of HSC activation. SREBP-1c is highly expressed in quiescent HSCs and downregulated upon HSC activation. The aim of this study is to examine the effect of leptin on SREBP-1c gene expression in HSCs in vitro and in vivo and elucidate the underlying mechanisms. The results of the present study demonstrated that leptin strongly inhibited SREBP-1c expression in HSCs in vivo and in vitro. p38 MAPK was involved in leptin regulation of SREBP-1c expression in cultured HSCs. Leptin-induced activation of p38 MAPK led to the decreases in liver X receptor (LXR)-α protein level, activity and its binding to the SREBP-1c promoter, which caused the downregulation of SREBP-1c expression. Moreover, leptin inhibition of SREBP-1c expression via p38 MAPK increased the expression of alpha1(I) collagen in HSCs. Our results might provide new insights into the mechanisms of the unique role of leptin in the development of liver fibrosis and might have potential implications for clarifying the molecular mechanisms underlying liver fibrosis in diseases in which circulating leptin levels are elevated such as nonalcoholic steatohepatitis, type 2 diabetes mellitus and alcoholic cirrhosis. Show less

Endothelial progenitor cells (EPCs), critical for mediating vascular repair, are dysfunctional in a hyperglycemic and/or hypercholesterolemic environment. Their dysfunction contributes to the progression of diabetic macro- and microvascular complications. Activation of "cholesterol-sensing" nuclear receptors, the liver X receptors (LXRα/LXRβ), protects against atherosclerosis by transcriptional regulation of genes important in promoting cholesterol efflux and inhibiting inflammation. We hypothesized that LXR activation with a synthetic ligand would correct diabetes-induced EPC dysfunction and improve diabetic retinopathy. Studies were performed in streptozotocin (STZ)-injected DBA/2J mice fed a high-fat Western diet (DBA/STZ/WD) and treated with the LXR agonist GW3965 and in LXRα(-/-), LXRβ(-/-), and LXRα/β(-/-) mice. Retinas were evaluated for number of acellular capillaries and glial fibrillary acidic protein (GFAP) immunoreactivity. Bone marrow EPCs were analyzed for migratory function and gene expression. Compared with vehicle-treated DBA/STZ/WD mice, GW3965 treated mice showed fewer acellular capillaries and reduced GFAP expression. These mice also exhibited enhanced EPC migration and restoration of inflammatory and oxidative stress genes toward nondiabetic levels. LXRα(-/-), LXRβ(-/-), and LXRα/β(-/-) mice developed acellular capillaries and EPC dysfunction similar to the DBA/STZ/WD mice. These studies support a key role for LXR in retinal and bone marrow progenitor dysfunction associated with type 1 diabetes. LXR agonists may represent promising pharmacologic targets for correcting retinopathy and EPC dysfunction. Show less

Migration and homing of DCs to lymphoid organs is pivotal for inducing adaptive immunity and tolerance. DC homing depends on the chemokine receptor CCR7. However, expression of CCR7 alone is not sufficient for effective DC migration. A second signal, mediated by prostaglandin E(2) (PGE(2)), is critical for the development of a migratory DC phenotype. PGE(2) is important for inducing efficient immune responses, but, if deregulated, contributes to chronic inflammation, autoimmune diseases through Th17-cell development and tumorigenesis. In contrast, activation of liver X receptor (LXR)α has recently been shown to interfere with CCR7 expression and migration of DCs resulting in a reduced immune response. Here, we demonstrate that PGE(2) downregulates LXRα expression in human monocyte derived as well as ex vivo DCs. Moreover, PGE(2) stimulation dampens LXR activation, auto-regulation and LXR-mediated gene transcription. Consequently, we show that PGE(2) enhances CCR7 expression and migration of LXR-activated DCs. Furthermore, we provide evidence that PGE(2) signaling and LXR activation specifically elicit converse effects on CCR7 expression and DC migration. In contrast, production of MMP9, CCL4, COX-2, and IL-23 is solely regulated by PGE(2) , but not by LXR activation, offering new perspectives for therapeutic interventions. Show less

To establish if the juice of Moro, an anthocyanin-rich orange, may improve liver damage in mice with diet-induced obesity. Eight-week-old mice were fed a high-fat diet (HFD) and were administrated water or Moro juice for 12 wk. Liver morphology, gene expression of lipid transcription factors, and metabolic enzymes were assessed. Mice fed HFD displayed increased body weight, insulin resistance and dyslipidemia. Moro juice administration limited body weight gain, enhanced insulin sensitivity, and decreased serum triglycerides and total cholesterol. Mice fed HFD showed liver steatosis associated with ballooning. Dietary Moro juice markedly improved liver steatosis by inducing the expression of peroxisome proliferator-activated receptor-α and its target gene acylCoA-oxidase, a key enzyme of lipid oxidation. Consistently, Moro juice consumption suppressed the expression of liver X receptor-α and its target gene fatty acid synthase, and restored liver glycerol-3-phosphate acyltransferase 1 activity. Moro juice counteracts liver steatogenesis in mice with diet-induced obesity and thus may represent a promising dietary option for the prevention of fatty liver. Show less

To obtain novel transrepression-selective liver X receptor (LXR) ligands, we adopted a strategy of reducing the transactivational agonistic activity of the 5,11-dihydro-5-methyl-11-methylene-6H-dibenz[b,e]azepin-6-one derivative 10, which exhibits LXR-mediated transrepressional and transactivational activity. Structural modification of 10 based on the reported X-ray crystal structure of the LXR ligand-binding domain led to a series of compounds, of which almost all exhibited transrepressional activity at 1 or 10 μM but showed no transactivational activity even at 30 μM. Among the compounds obtained, 18 and 22 were confirmed to have LXR-dependent transrepressional activity by using peritoneal macrophages from wild-type and LXR-null mice. A newly developed fluorescence polarization assay indicated that they bind directly to LXRα. Next, further structural modification was performed with the guidance of docking simulations with LXRα, focusing on enhancing the binding of the ligands with LXRα through the introduction of substituents or heteroatom(s). Among the compounds synthesized, compound 48, bearing a hydroxyl group, showed potent, selective, and dose-dependent transrepressional activity. Show less

Recent studies have underlined the role of nuclear receptors in the involvement of prostate cancer signalling pathways. A total of 84 benign prostate hyperplasia (BPH), 84 low risk prostate cancer (LPC) and 64 advanced disease (APC) cases were sampled on a tissue microarray (TMA) and stained for retinoic acid receptor (RAR)-α, retionoid X receptor (RXR)-α, liver X receptor (LXR)-α, farnesoid X receptor (FXR) and proliferate-activated receptor gamma (PPAR)-γ and the (pro)-inflammatory molecules cyclooxygenase 2 (COX2), tumor necrosis factor (TNF)-α and inducible Nitric oxide synthase (iNOS) immunohistochemically. PPAR-γ expression in APC tissues was found to be significantly higher than that in LPC and BPH specimens (p<0.001). In contrast, RXR-a expression was significantly lower (p<0.001). COX2 staining demonstrated a trend towards overexpression in APC (p=0.025). No significant differences were found for RAR-α, iNOS and TNF-α expression. Staining of FXR and LXR was seen diffusely in the cytoplasm as well as in the nucleus, preventing sufficient evaluation by definition. This study provides the basis for applying PPAR-γ ligands clinically in treatment of APC. Show less

Liver X receptor (LXR) α and β are nuclear receptors that are crucial for the regulation of carbohydrate and lipid metabolism. Activation of LXRs in the brain facilitates cholesterol clearance and improves cognitive deficits, thus they are considered as promising drug targets to treat diseases such as atherosclerosis and Alzheimer's disease. Nevertheless, little is known about the function and localization of LXRs in the brain. Here, we studied the expression of LXR in the brains of rats that received free access to 10% (w/v) fructose group (FG) in their beverages or water control drinks (control group (CG)). After 6 weeks rats in the FG presented with hypertriglyceridemia, hyperinsulinemia, and became glucose intolerant, suggesting a progression toward type 2 diabetes. We found that hypothalamic LXR expression was altered in fructose-fed rats. Rats in the FG presented with a decrease in LXRβ levels while showing an increase in LXRα expression in the hypothalamus but not in the hippocampus, cerebellum, or neocortex. Moreover, both LXRα and β expression correlated negatively with insulin and triglyceride levels. Interestingly, LXRβ showed a negative correlation with the area under the curve during the glucose tolerance test in the CG and a positive correlation in the FG. Immunocytochemistry revealed that the paraventricular and ventromedial nuclei express mainly LXRα whereas the arcuate nucleus expresses LXRβ. Both LXR immunosignals were found in the median preoptic area. This is the first study showing a relationship between glucose and lipid homeostasis and the expression of LXRs in the hypothalamus, suggesting that LXRs may trigger neurochemical and neurophysiological responses for the control of food intake and energy expenditure through these receptors. Show less

Secretion of cholesterol into bile is important for the elimination of cholesterol from the body. Thyroid hormone (TH) increases biliary cholesterol secretion and hepatic gene expression of adenosine triphosphate (ATP)-binding cassette, subfamily G (WHITE), member 5 (ABCG5) and ATP-binding cassette, subfamily G (WHITE), member 8 (ABCG8), two half-transporters that act as a heterodimeric complex promoting sterol secretion. In addition, nuclear liver x receptor-alpha (LXRa), also regulated by TH, induces gene expression of ABCG5/G8. We here investigated if the TH-induced stimulation of biliary cholesterol secretion is mediated by the ABCG5/G8 complex in vivo, and if so, whether LXRa is involved. Mice homozygous for disruption of Abcg5 (Abcg5(-/-) ) or Lxra (Lxra(-/-) ) and their wild-type counterparts were treated with triiodothyronine (T3) for 14 days and compared to untreated mice of corresponding genetic backgrounds. Bile was collected by gallbladder cannulation, and liver samples were analyzed for gene expression levels. Basal biliary cholesterol secretion in Abcg5(-/-) mice was 72% lower than in Abcg5(+/+) mice. T3 treatment increased cholesterol secretion 3.1-fold in Abcg5(+/+) mice, whereas this response was severely blunted in Abcg5(-/-) mice. In contrast, biliary cholesterol secretion in T3-treated Lxra(+/+) and Lxra(-/-) mice was increased 3.5- and 2.6-fold, respectively, and did not differ significantly. TH-induced secretion of cholesterol into bile is largely dependent on an intact ABCG5/G8 transporter complex, whereas LXRa is not critical for this effect. Show less

Liver X receptor (LXR) α and LXRβ function as physiological sensors of cholesterol metabolites (oxysterols), regulating key genes involved in cholesterol and lipid metabolism. LXRs have been extensively studied in both human and rodent cell systems, revealing their potential therapeutic value in the contexts of atherosclerosis and inflammatory diseases. The LXR genome landscape has been investigated in murine macrophages but not in human THP-1 cells, which represent one of the frequently used monocyte/macrophage cell systems to study immune responses. We used a whole-genome screen to detect direct LXR target genes in THP-1 cells treated with two widely used LXR ligands [N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)-ethyl]phenyl]-benzenesulfonamide (T0901317) and 3-[3-[N-(2-chloro-3-trifluoromethylbenzyl)-(2,2-diphenylethyl)amino]propyloxy] phenylacetic acid hydrochloride (GW3965)]. This screen identified the sphingomyelin phosphodiesterase acid-like 3A (SMPDL3A) gene as a novel LXR-regulated gene, with an LXR response element within its promoter. We investigated the regulation of SMPDL3A gene expression by LXRs across several human and mouse cell types. These studies indicate that the induction of SMPDL3A is LXR-dependent and is restricted to human blood cells with no induction observed in mouse cellular systems. Show less

To observe the effects of Guanxinkang (GXK) decoction, a compound traditional Chinese herbal medicine, on expressions of peroxisome proliferator-activated receptor γ (PPARγ), liver X receptor α (LXRα) and ATP-binding cassette transporter A1 (ABCA1) in apolipoprotein E (ApoE)-knockout mice with atherosclerosis. Fourteen 6-week-old C57BL/6 J mice were used as normal control group. Seventy 6-week-old ApoE-knockout mice receiving a high-cholesterol diet to induce atherosclerosis were randomly divided into untreated group, simvastatin group and low-dose (concentration of crude drugs at 0.864 g/mL), medium-dose (crude drugs at 1.728 g/mL) and high-dose (crude drugs at 3.456 g/mL) GXK groups. After treated with the drugs for eight weeks continuously, the livers and aortas of mice were separated. The expressions of PPARγ, LXRα and ABCA1 were measured by real-time quantitative polymerase chain reaction and Western blotting respectively. Compared with the normal control group, mRNAs and proteins of PPARγ, LXRα and ABCA1 over-expressed in the untreated group (P<0.05). After the treatment, GXK decoction and simvastatin decreased the expressions of PPARγ, LXRα and ABCA1 (P<0.05). High-dose GXK decoction had more marked effects than low- and medium-dose GXK and simvastatin. The PPARγ-LXRα-ABCA1 pathway is involved in lipid regulation and inflammation activities. Over-expression of the genes has complicated effects on atherosclerosis in ApoE-knockout mice with high-cholesterol diet. GXK decoction has anti-inflammatory and anti-matrix metalloproteinase activities by regulating PPARγ, LXRα and ABCA1 interactions in the ApoE-knockout mice. Show less

ATP-binding cassette transporters (ABC) A1 and G1 are key molecules in cholesterol efflux from macrophages, which is an initial step of reverse cholesterol transport (RCT), a major anti-atherogenic property of high-density lipoprotein (HDL). Astaxanthin is one of the naturally occurring carotenoids responsible for the pink-red pigmentation in a variety of living organisms. Although astaxanthin is known to be a strong antioxidant, it remains unclear through what mechanism of action it affects cholesterol homeostasis in macrophages. We therefore investigated the effects of astaxanthin on cholesterol efflux and ABCA1/G1 expressions in macrophages. Astaxanthin enhanced both apolipoprotein (apo) A-I- and HDL-mediated cholesterol efflux from RAW264.7 cells. In supporting these enhanced cholesterol efflux mechanisms, astaxanthin promoted ABCA1/G1 expression in various macrophages. In contrast, peroxisome proliferator-activated receptor γ, liver X receptor (LXR) α and LXRβ levels remained unchanged by astaxanthin. An experiment using actinomycin D demonstrated that astaxanthin transcriptionally induced ABCA1/G1 expression, and oxysterol depletion caused by overexpression of cholesterol sulfotransferase further revealed that these inductions in ABCA1/G1 were independent of LXR-mediated pathways. Finally, we performed luciferase assays using human ABCA1/G1 promoter-reporter constructs to reveal that astaxanthin activated both promoters irrespective of the presence or absence of LXR-responsive elements, indicating LXR-independence of these activations. In conclusion, astaxanthin increased ABCA1/G1 expression, thereby enhancing apoA-I/HDL-mediated cholesterol efflux from the macrophages in an LXR-independent manner. In addition to the anti-oxidative properties, the potential cardioprotective properties of astaxanthin might therefore be associated with an enhanced anti-atherogenic function of HDL. Show less

Menopause is associated with changes in lipid levels resulting in increased risk of atherosclerosis and cardiovascular events. Hormone therapy (HT) and atorvastatin have been used to improve lipid profile in postmenopausal women. Effects of HT, atorvastatin and APOE polymorphisms on serum lipids and APOE and LXRA expression were evaluated in 87 hypercholesterolemic postmenopausal women, randomly selected for treatment with atorvastatin (AT, n=17), estrogen or estrogen plus progestagen (HT, n=34) and estrogen or estrogen plus progestagen associated with atorvastatin (HT+AT, n=36). RNA was extracted from peripheral blood mononuclear cells (PBMC) and mRNA expression was measured by TaqMan(®) PCR. APOE ɛ2/ɛ3/ɛ4 genotyping was performed using PCR-RFLP. Total cholesterol (TC), LDL-c and apoB were reduced after each treatment (p<0.001). Triglycerides, VLDL-c and apoAI were reduced only after atorvastatin (p<0.05), whereas triglycerides and VLDL-c were increased after HT (p=0.01). HT women had lower reduction on TC, LDL-c and apoB than AT and HT+AT groups (p<0.05). APOE mRNA expression was reduced after atorvastatin treatment (p=0.03). Although LXRA gene expression was not modified by atorvastatin, it was correlated with APOE mRNA before and after treatments. Basal APOE mRNA expression was not influenced by gene polymorphisms, however the reduction on APOE expression was more pronounced in ɛ3ɛ3 than in ɛ3ɛ4 carriers. Atorvastatin down-regulates APOE mRNA expression and it is modified by APOE genotypes in PBMC from postmenopausal women. Show less

Oxidized sterols are toxic to endothelial cells and play a central role in promoting atherogenesis. In this study, we evaluated the impact of anthocyanin, a class of flavonoid compounds, on oxysterol efflux from endothelial cells and the underlying mechanism. The human aortic ECs (HAECs) were incubated with anthocyanin cyanidin-3-O-β-glucoside (C3G) for different times. C3G treatment upregulates ABCG1 and ABCA1 expression in a dose-dependent manner in HAECs. Moreover, C3G promotes the efflux of cholesterol mainly 7-ketocholesterol (7-KC) from HAECs in an ABCG1-dependent manner. As a result, C3G abrogated the 7-KC-mediated increase of reactive oxygen species (ROS) and apoptosis in HAECs. Furthermore, C3G treatment reverses the inhibition of endothelial nitric oxide synthase (eNOS) activity by 7-KC, leading to the preservation of nitric oxide (NO) bioavailability. The induction of ABCG1 and its mediated 7-KC efflux from HAECs by C3G resulted from liver X receptor α (LXRα) activation, which was confirmed by its blockage of ABCG1 expression after pharmacological or small interfering RNA inhibition of LXRα. These data uncover a novel mechanism by which C3G ameliorates oxysterol-induced oxidative damage on endothelial cells. Show less

Liver X receptor (LXR)α regulates the genes involved in cholesterol, fatty acid and glucose metabolism. Soy protein (SP) consumption reduces the hepatic accumulation of cholesterol and triacylglycerol, and improves insulin sensitivity. However, it is not known whether these effects are mediated via LXRα. We therefore investigated whether the consumption of SP regulates metabolic changes in cholesterol metabolism and insulin sensitivity via LXRα. Wild-type (WT) and Lxrα(-/-) (Lxrα, also known as Nr1h3) mice were fed an SP diet with or without cholesterol for 28 days. The expression of LXRα target genes was measured in liver and intestine, as were hepatic lipid content and faecal bile acid concentration. Oral glucose and insulin tolerance tests were also performed. Hepatocytes were used to study the effect of isoflavones on LXR activity. The livers of WT and Lxrα(-/-) mice fed an SP high-cholesterol diet showed less steatosis than those fed casein. The SP diet increased the expression of the ATP-binding cassette (ABC) sub-family genes Abca1, Abcg5 and Abcg8 in the liver and intestine, as well as increasing total faecal bile acid excretion and insulin sensitivity in WT mice compared with mice fed a casein diet. However, these effects of SP were not observed in Lxrα(-/-) mice. The SP isoflavone, genistein, repressed the activation of LXRα target genes by T0901317, whereas it stimulated the activation of LXRβ target genes. The AMP-activated protein kinase inhibitor, compound C, had the opposite effects to those of genistein. Our results suggest that SP isoflavones stimulate the phosphorylation of LXRα or LXRβ, resulting in different biological effects for each LXR isoform. Show less

Liver X receptors (LXRα, LXRβ) are master regulators of cholesterol homeostasis. In the endothelium, perturbations of cell cholesterol have an impact on fundamental processes. We, therefore, assessed the effects of LXR activation on endothelial functions related to angiogenesis in vitro and in vivo. LXR agonists (T0901317, GW3965) blunted migration, tubulogenesis, and proliferation of human umbilical vein endothelial cells. By affecting endothelial cholesterol homeostasis, LXR activation impaired the compartmentation of vascular endothelial growth factor receptor-2 in lipid rafts/caveolae and led to defective phosphorylation and downstream signaling of vascular endothelial growth factor receptor-2 upon vascular endothelial growth factor-A stimulation. Consistently, the antiangiogenic actions of LXR agonists could be prevented by coadministration of exogenous cholesterol. LXR agonists reduced endothelial sprouting from wild-type but not from LXRα(-/-)/LXRβ(-/-) knockout aortas and blunted the vascularization of implanted angioreactors in vivo. Furthermore, T0901317 reduced the growth of Lewis lung carcinoma grafts in mice by impairing angiogenesis. Pharmacological activation of endothelial LXRs reduces angiogenesis by restraining cholesterol-dependent vascular endothelial growth factor receptor-2 compartmentation and signaling. Thus, administration of LXR agonists could exert therapeutic effects in pathological conditions characterized by uncontrolled angiogenesis. Show less

ATP-binding cassette transporter A1 (ABCA1) plays a crucial role in exporting cholesterol from macrophages, a function relevant to its involvement in the prevention of atherosclerosis. Quercetin, one of flavonoids, has been described to reduce atherosclerotic lesion formation. This study is aimed to investigate the effect of quercetin on regulation of ABCA1 expression and to explore its underlying mechanisms in macrophages. The results show that quercetin markedly enhanced cholesterol efflux from macrophages in a concentration-dependent manner, which was associated with an increase in ABCA1 mRNA and protein expression. Remarkably, quercetin is able to stimulate the phosphorylation of p38 by up to 234-fold at 6 h via an activation of the transforming growth factor β-activated kinase 1 (TAK1) and mitogen-activated kinase kinase 3/6 (MKK3/6). Inhibition of p38 with a pharmacological inhibitor or small hairpin RNA (shRNA) suppressed the stimulatory effects of quercetin on ABCA1 expression and cholesterol efflux. Moreover, knockdown of p38 reduced quercetin-enhanced ABCA1 promoter activity and the binding of specificity protein 1 (Sp1) and liver X receptor α (LXRα) to the ABCA1 promoter using chromatin immunoprecipitation assays. These findings provide evidence that p38 signaling is essential for the regulation of quercetin-induced ABCA1 expression and cholesterol efflux in macrophages. Show less