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Liver X receptors (LXRα and LXRβ) are important regulators of cholesterol and lipid metabolism, and their activation has been shown to inhibit cardiovascular disease and reduce atherosclerosis in animal models. Small molecule agonists of LXR activity are therefore of great therapeutic interest. However, the finding that such agonists also promote hepatic lipogenesis has led to the idea that hepatic LXR activity is undesirable from a therapeutic perspective. To investigate whether this might be true, we performed gene targeting to selectively delete LXRα in hepatocytes. Liver-specific deletion of LXRα in mice substantially decreased reverse cholesterol transport, cholesterol catabolism, and cholesterol excretion, revealing the essential importance of hepatic LXRα for whole body cholesterol homeostasis. Additionally, in a pro-atherogenic background, liver-specific deletion of LXRα increased atherosclerosis, uncovering an important function for hepatic LXR activity in limiting cardiovascular disease. Nevertheless, synthetic LXR agonists still elicited anti-atherogenic activity in the absence of hepatic LXRα, indicating that the ability of agonists to reduce cardiovascular disease did not require an increase in cholesterol excretion. Furthermore, when non-atherogenic mice were treated with synthetic LXR agonists, liver-specific deletion of LXRα eliminated the detrimental effect of increased plasma triglycerides, while the beneficial effect of increased plasma HDL was unaltered. In sum, these observations suggest that therapeutic strategies that bypass the liver or limit the activation of hepatic LXRs should still be beneficial for the treatment of cardiovascular disease. Show less

Recent studies have revealed that PPARγ's transactivation function is regulated by extracellular signals. In particular, cytokines and Wnt family proteins suppress the ligand-inducible transactivation function of PPARγ and attenuate adipogenesis/osteoblastogenesis switching in mesenchymal stem cells (MSCs). For example, Wnt5a suppresses PPARγ transcriptional activity through the NLK/SETDB1/CHD7 pathway. Among these factors, BMP2 strongly induces bone formation, but the effect of BMP2 on PPARγ function remains unclear. We examined the effect of BMP2 and PPARγ in ST2 cells and found that PPARγ activation affected BMP2's signaling pathway through epigenetic regulation. Although BMP2 did not interfere with PPARγ-mediated adipogenesis, BMP2 increased mRNA expression levels of PPARγ target genes (such as Fabp4 and Nr1h3) when cells were first treated with troglitazone (TRO). Moreover, PPARγ activation affected BMP2 through enhancement of histone activation markers (acetylated histone H3 and trimethylated Lys4 of histone H3) on the Runx2 promoter. After TRO treatment for three hours, BMP2 enhanced the levels of active histone marks on the promoter of a PPARγ target gene. These results suggest that the order of treatment with BMP2 and a PPARγ ligand is critical for adipogenesis and osteoblastogenesis switching in MSCs. Show less

The present study demonstrates a key role for the oxysterol receptor liver X receptor β (LXRβ) in the etiology of diabetes insipidus (DI). Given free access to water, LXRβ(-/-) but not LXRα(-/-) mice exhibited polyuria (abnormal daily excretion of highly diluted urine) and polydipsia (increased water intake), both features of diabetes insipidus. LXRβ(-/-) mice responded to 24-h dehydration with a decreased urine volume and increased urine osmolality. To determine whether the DI was of central or nephrogenic origin, we examined the responsiveness of the kidney to arginine vasopressin (AVP). An i.p. injection of AVP to LXRβ(-/-) mice revealed a partial kidney response: There was no effect on urine volume, but there was a significant increase of urine osmolality, suggesting that DI may be caused by a defect in central production of AVP. In the brain of WT mice LXRβ was expressed in the nuclei of magnocellular neurons in the supraoptic and paraventricular nuclei of the hypothalamus. In LXRβ(-/-) mice the expression of AVP was markedly decreased in the magnocellular neurons as well as in urine collected over a 24-h period. The persistent high urine volume after AVP administration was traced to a reduction in aquaporin-1 expression in the kidney of LXRβ(-/-) mice. The LXR agonist (GW3965) in WT mice elicited an increase in urine osmolality, suggesting that LXRβ is a key receptor in controlling water balance with targets in both the brain and kidney, and it could be a therapeutic target in disorders of water balance. Show less

The polycystic ovary syndrome (PCOS) is a complex endocrine-metabolic disorder consisting of reproductive disturbances associated with all aspects of the metabolic syndrome and genetic components in the pathology of this complex disease is very likely. Accordingly, variations in single genes might affect specific features of PCOS and thereby help to define different subgroups. SREBP-1 or LXRα have been shown to be genetically linked to lipid metabolism or insulin sensitivity. As these are two major aspects of the PCOS phenotype, we evaluated both genes in a cohort of 153 PCOS patients. Analyses of both genes revealed in SREBF-1, i.e. SREBP-1a and SREBP-1c, not any variation and in the LXRα gene no novel sequence variations. Common variants of LXRα (rs2279238:G; all:0.8658; PCOS:0.8627; controls: 0.8686 or A: all:0.13412; PCOS:0.1373; controls:0.1314; (OR (95% CI) 0.9508 (0.4226-2.1385); rs11039155: G: all:0.8767; PCOS:0.8663; controls:0.8857 and A all:0.1233; PCOS:0.1337; controls:0.1143; (OR (95% CI) 0.8383 (0.3618-1.9371)) were also not directly associated to PCOS. Combined analyses of both polymorphism revealed that there was no difference of distribution between the groups. In contrast, analyses of the impact of these polymorphisms on metabolic parameters of the syndrome indicated significant differences related to genotypes. The data indicated that rs11039155 increases metabolic risk, whereas rs2279238 has a protective effect on the overall metabolic risk. The investigation of the PCOS group presented indicates that the combined analyses of variations in putative candidate genes allowed a genotype-phenotype correlation for metabolic features. Show less

The ATP-binding cassette transporter A1 (ABCA1) is a membrane transporter that directly contributes to high-density lipoprotein (HDL) biogenesis by regulating the cellular efflux of cholesterol. Since ABCA1 plays a pivotal role in cholesterol homeostasis and HDL metabolism, identification of a novel substance that is capable of increasing its expression would be beneficial for the prevention and therapy of atherosclerosis. In the present study, we studied the effects of ethanolic extracts of Brazilian red propolis (EERP) on ABCA1 expression and cholesterol efflux in THP-1 macrophages. EERP enhanced PPARγ and liver X receptor (LXR) transcriptional activity at 5-15μg/ml, which was associated with upregulation of PPARγ and LXRα expression. It was also found that EERP increase the activity of the ABCA1 promoter, which is positively regulated by LXR. Consistent with these findings, treatment with EERP increased both mRNA and protein expression of ABCA1. Finally, EERP upregulated ApoA-I-mediated cholesterol efflux. Our results showed that EERP promote ApoA-I-mediated cholesterol efflux from macrophages by increasing ABCA1 expression via induction of PPARγ/LXR. Show less

To investigate the regulation of cholesterol transporters, including ATP-binding cassette transporter A1 (ABCA1), ABCG1 and scavenger receptor class B, type I (SR-BI), by inflammatory stimuli in macrophages. MATERIALS AND TREATMENTS: RAW 264.7 macrophages and mouse peritoneal macrophages were treated with inflammatory stimuli with or without rosiglitazone, a peroxisome proliferator activated receptor γ (PPARγ) agonist, or T0901317, a liver X receptor (LXR) agonist. Real-time PCR and Western blotting for cholesterol transporters as well as cellular cholesterol efflux to high-density lipoprotein 2 (HDL(2)) were determined. In RAW 264.7 macrophages, lipopolysaccharide (LPS) significantly reduced ABCG1 and PPARγ as well as cholesterol efflux to HDL(2). Rosiglitazone and T0901317 induced ABCA1 and ABCG1 several-fold, but LPS reduced only ABCG1. ABCG1 and SR-BI proteins, but not ABCA1, were decreased by LPS. In mouse peritoneal macrophages, LPS, tumor necrosis factor α and interleukin-1β decreased ABCG1, SR-BI, LXRα and PPARγ mRNA. The agonists increased ABC transporter expression but LPS reduced mRNA of T0901317-induced ABCA1 as well as basal and agonists-induced ABCG1. SR-BI protein was increased by rosiglitazone but LPS decreased the levels. The data suggest that inflammatory insults repress ABCG1 and SR-BI expression partly dependent on PPARγ with a minimal effect on ABCA1 expression. Show less

Cytosolic sulfotransferase (SULT2B1b) catalyzes oxysterol sulfation. 5-Cholesten-3β-25-diol-3-sulfate (25HC3S), one product of this reaction, decreases intracellular lipids in vitro by suppressing liver X receptor/sterol regulatory element binding protein (SREBP)-1c signaling, with regulatory properties opposite to those of its precursor 25-hydroxycholesterol. Upregulation of SULT2B1b may be an effective strategy to treat hyperlipidemia and hepatic steatosis. The objective of the study was to explore the effect and mechanism of oxysterol sulfation by SULT2B1b on lipid metabolism in vivo. C57BL/6 and LDLR(-/-) mice were fed with high-cholesterol diet or high-fat diet for 10 weeks and infected with adenovirus encoding SULT2B1b. SULT2B1b expressions in different tissues were determined by immunohistochemistry and Western blot. Sulfated oxysterols in liver were analyzed by high-pressure liquid chromatography. Serum and hepatic lipid levels were determined by kit reagents and hematoxylin and eosin staining. Gene expressions were determined by real-time reverse transcriptase polymerase chain reaction and Western Blot. Following infection, SULT2B1b was successfully overexpressed in the liver, aorta, and lung tissues, but not in the heart or kidney. SULT2B1b overexpression, combined with administration of 25-hydroxycholesterol, significantly increased the formation of 25HC3S in liver tissue and significantly decreased serum and hepatic lipid levels, including triglycerides, total cholesterol, free cholesterol, and free fatty acids, as compared with controls in both C57BL/6 and LDLR(-/-) mice. Gene expression analysis showed that increases in SULT2B1b expression were accompanied by reduction in key regulators and enzymes involved in lipid metabolism, including liver X receptor α, SREBP-1, SREBP-2, acetyl-CoA carboxylase-1, and fatty acid synthase. These findings support the hypothesis that 25HC3S is an important endogenous regulator of lipid biosynthesis. Show less

Organotins, including tri-butyltin chloride (TBTC), are widely used in agricultural and chemical industries and cause persistent and widespread pollution. TBTC has been shown to activate nuclear receptor retinoid X receptor (RXR)/PPARγ signaling by interacting with RXR to modulate adipogenesis. However, whether TBTC affects liver X receptor (LXR)/RXR activity and subsequently the expression of cholesterol mobilizing genes is not known. In this study, we evaluated the ability of TBTC to activate LXR/RXR and ABC transporter A1 (ABCA1) expression. ABCA1 plays a critical role in HDL generation, maintaining cholesterol homeostasis, and cholesterol accumulation-induced diseases, such as atherosclerosis and pancreatic islet dysfunction. In a reporter gene assay, TBTC activated LXRα/RXR but not LXRβ/RXR. In mouse macrophage RAW264 cells, TBTC activated the ABCA1 promoter in an LXR-responsive element dependent manner and increased ABCA1 mRNA expression. TBTC augmented ABCA1 protein levels and apolipoprotein A-I-dependent cellular cholesterol efflux (HDL generation). The LXR-target fatty acid synthase and Spα mRNA levels were also increased by TBTC exposure. We conclude that TBTC has the ability to activate permissive LXRα/RXR signaling and thereby modulate cellular cholesterol efflux. Show less

To investigate the effect of Hepatitis B Virus X Protein (HBx) on the expression of lipid metabolism-related genes and its role in pathogenesis of hepatocyte fatty degeneration. Hepatitis B Virus X gene eukaryon expression vector pIRES2-eGFP-HBx was transfected into HepG2 cells to establish HepG2/HBx cell model for HBx expression. HepG2 cells transfected with pIRES2-eGFP (HepG2/pIRES2 cell) and non-transfected were used as controls. At 24, 48 and 72 hours after transfection, the expression of green fluorescent protein (GFP) was observed by fluorescence microscope and the triglyceride(TG) content was detected. RT-PCR and Western blot were applied to detect the levels of sterol regulatory element binding protein-1 (SREBP-1), liver x receptor alpha (LXRalpha) mRNA and the levels of HBx, LXRalpha and fatty acid synthase (FAS) protein. At 24, 48 and 72 hours after transfection, the expression of GFP was found in HepG2/HBx and HepG2/pIRES2 cells, and increased gradually. The expression of HBx was detected only in HepG2/HBx cells, and was increased with time after transfection (F = 32.21, P less than 0.01). These suggested successful obtaining of HepG2-HBx cell model for HBx expression. At 24h, 48h and 72h after transfection, the expression levels of LXRalpha mRNA (0.386+/-0.055, 0.505+/-0.071, 0.649+/-0.058 ) and SREBP-1 mRNA (0.395+/-0.055, 0.548+/-0.047, 0.795+/-0.058), as well as the levels of LXRalpha protein(0.178+/-0.036, 0.263+/-0.047, 0.347+/-0.058) and FAS protein(0.436+/-0.055, 0.608+/-0.053, 0.827+/-0.046) in HepG2-HBx group were dramatically higher than those in the controls at the same time points (all P less than 0.05/0.01), and were gradually increased with time (all P less than 0.05/0.01). A positive correlationship was observed between HBX protein level and the LXRalpha, SREbP-1 mRNA and LXRalpha, FAS protein levels. The difference of TG content between HepG2/HBx group and control groups was not statistically significant (P more than 0.05). HBx-LXRalpha-SREBP-1/FAS pathway suggested regulating transcription and expression of lipid metabolism-related genes, which might be one of the important molecular mechanism causing hepatocyte fatty degeneration. Show less

To investigate the relationship of NOR-1 with the inhibition of inflammatory reaction in mice Kupffer cells (KCs) induced by lipopolysaccharide (LPS) via liver X receptor alpha (LXR alpha). KCs from male KM mice were isolated by density gradient centrifugation, incubated and then randomly assigned to three groups: control group, LPS treated group and LPS+T0901317 treated group. The mRNA and protein expressions of LXR alpha and NOR-1 in each group were determined by RT-PCR, immunofluorescent assay and western blot, respectively. The densities of TNF alpha and IL-10 in supernatants were evaluated by enzyme linked immunosorbent assay (ELISA). The mRNA and protein expression levels of LXR alpha in LPS + T0901317 group were the highest as compared to the other two groups (0.748+/-0.072 and 1.217+/-0.133 respectively), The mRNA and protein expression levels of NOR-1 in LPS+ T0901317 group were the highest as compared to the other two groups (2.726+/-0.065 and 0.842+/-0.058 respectively). The densities of supernatant TNF alpha in LPS group and IL-10 in LPS+T0901317 group were the highest [(450.89+/-78.52) ng/L and (537.41+/-36.41) ng/L respectively]. Promoting the expression of LXR alpha in KCs can elevate the NOR-1 expression and then inhibit inflammatory reaction. Show less

Synthetic activators of peroxisome proliferator-activated receptors (PPARs) stimulate cholesterol removal from macrophages through PPAR-dependent up-regulation of liver × receptor α (LXRα) and subsequent induction of cholesterol exporters such as ATP-binding cassette transporter A1 (ABCA1) and scavenger receptor class B type 1 (SR-BI). The present study aimed to test the hypothesis that the hydroxylated derivative of linoleic acid (LA), 13-HODE, which is a natural PPAR agonist, has similar effects in RAW264.7 macrophages. RAW264.7 macrophages were treated without (control) or with LA or 13-HODE in the presence and absence of PPARα or PPARγ antagonists and determined protein levels of LXRα, ABCA1, ABCG1, SR-BI, PPARα and PPARγ and apolipoprotein A-I mediated lipid efflux. Treatment of RAW264.7 cells with 13-HODE increased PPAR-transactivation activity and protein concentrations of LXRα, ABCA1, ABCG1 and SR-BI when compared to control treatment (P < 0.05). In addition, 13-HODE enhanced cholesterol concentration in the medium but decreased cellular cholesterol concentration during incubation of cells with the extracellular lipid acceptor apolipoprotein A-I (P < 0.05). Pre-treatment of cells with a selective PPARα or PPARγ antagonist completely abolished the effects of 13-HODE on cholesterol efflux and protein levels of genes investigated. In contrast to 13-HODE, LA had no effect on either of these parameters compared to control cells. 13-HODE induces cholesterol efflux from macrophages via the PPAR-LXRα-ABCA1/SR-BI-pathway. Show less

Atherosclerosis is a major cause of cardiovascular disease caused by high cholesterol. Stains are widely prescribed to lower cholesterol levels, but natural dietary compounds may also be effective. Therefore, we studied the effect of the natural dietary compound curcumin on atherosclerosis and its underlying mechanisms based on plasma and hepatic lipid metabolism. LDLR(-/-) mice were fed a high-cholesterol diet and treated with curcumin, lovastatin or control (n=10 per group) for 18 wk. Aortic arch sections revealed curcumin ameliorated early atherosclerotic lesions, lipid infiltration, ICAM-1 and VCAM-1 localization, similar to lovastatin treatment. Furthermore, curcumin lowered plasma cholesterol, triglycerides, LDL cholesterol and Apo B levels as well as CETP activity, while curcumin increased plasma HDL cholesterol and liver Apo A-I expression, similar to lovastatin treatment. Curcumin caused transcriptional inhibition of HMG-CoA reductase, independent of ACAT1 and ACAT2 expression. Hepatic PPARα and LXRα expression was upregulated by curcumin treatment. Hepatic complement factor D (Cfd) and systemic CRP levels, markers of immune complement pathway activation, were significantly reduced by curcumin treatment. Long-term curcumin treatment lowers plasma and hepatic cholesterol and suppresses early atherosclerotic lesions comparable to the protective effects of lovastatin. The anti-atherogenic effect of curcumin is mediated via multiple mechanisms including altered lipid, cholesterol and immune gene expression. Show less

Preeclampsia is a frequent complication of pregnancy and a leading cause of perinatal mortality. Both genetic and environmental risk factors have been identified. Lipid metabolism, particularly cholesterol metabolism, is associated with this disease. Liver X receptors alpha (NR1H3, also known as LXRalpha) and beta (NR1H2, also known as LXRbeta) play a key role in lipid metabolism. They belong to the nuclear receptor superfamily and are activated by cholesterol derivatives. They have been implicated in preeclampsia because they modulate trophoblast invasion and regulate the expression of the endoglin (CD105) gene, a marker of preeclampsia. The aim of this study was to investigate associations between the NR1H3 and NR1H2 genes and preeclampsia. We assessed associations between single nucleotide polymorphisms of NR1H3 (rs2279238 and rs7120118) and NR1H2 (rs35463555 and rs2695121) and the disease in 155 individuals with preeclampsia and 305 controls. Genotypes were determined by high-resolution melting analysis. We then used a logistic regression model to analyze the different alleles and genotypes for those polymorphisms as a function of case/control status. We found no association between NR1H3 SNPs and the disease, but the NR1H2 polymorphism rs2695121 was found to be strongly associated with preeclampsia (genotype C/C: adjusted odds ratio, 2.05; 95% CI, 1.04-4.05; p = 0.039 and genotype T/C: adjusted odds ratio, 1.85; 95% CI, 1.01-3.42; p = 0.049). This study provides the first evidence of an association between the NR1H2 gene and preeclampsia, adding to our understanding of the links between cholesterol metabolism and this disease. Show less

ATP-binding cassette transporter A1 (ABCA1) promotes cholesterol and phospholipid efflux from cells to lipid-poor apolipoprotein A-I and plays an important role in atherosclerosis. In a previous study, we developed a high-throughput screening method using an ABCA1p-LUC HepG2 cell line to find upregulators of ABCA1. Using this method in the present study, we found that mycophenolic acid (MPA) upregulated ABCA1 expression (EC50=0.09 μM). MPA upregulation of ABCA1 expression was confirmed by real-time quantitative reverse transcription-PCR and Western blot analysis in HepG2 cells. Previous work has indicated that MPA is a potent agonist of peroxisome proliferator-activated receptor gamma (PPARγ; EC50=5.2-9.3 μM). Liver X receptor α (LXRα) is a target gene of PPARγ and may directly regulate ABCA1 expression. Western blot analysis showed that MPA induced LXRα protein expression in HepG2 cells. Addition of PPARγ antagonist GW9662 markedly inhibited MPA-induced ABCA1 and LXRα protein expression. These data suggest that MPA increased ABCA1 expression mainly through activation of PPARγ. Thus, the effects of MPA on upregulation of ABCA1 expression were due mainly to activation of the PPARγ-LXRα-ABCA1 signaling pathway. This is the first report that the antiatherosclerosis activity of MPA is due to this mechanism. Show less

Liver X receptor α (LXRα) and sterol regulatory element binding protein-1c (SREBP-1c) were studied in rats with non-alcoholic steatohepatitis (NASH) induced by a high-fat diet. Forty 5-week-old rats were fed either a high-fat diet (n = 30) or a normal diet (n = 10) for 9, 13 or 17 weeks. The mRNA and protein levels for LXRα and SREBP-1c were measured at each time point, as was fatty acid synthase (FAS) activity and the serum levels of free fatty acid (FFA) and triglyceride (TG). The mRNA and protein levels for LXRα and SREBP-1c, FAS activity and serum levels of FFA and TG all significantly increased from week 9 in the high-fat diet rats versus controls. In conclusion, a high-fat diet upregulates LXRα which, in turn, upregulates SREBP-1c, increasing the activity of FAS and FFA and accumulation of TG in hepatocytes. Thus, LXRα and SREBP-1c contribute to the development of NASH. Show less

PLAC1 expression, first characterized as restricted to developing placenta among normal tissues, is also found in a wide range of tumors and transformed cell lines. To understand the basis for its unusual expression profile, we have analyzed the gene structure and its mode of transcription. We find that the gene has a hitherto unique feature, with two promoters, P1 and P2, separated by 105 kb. P2 has been described before. Here we define P1 and show that it and P2 are activated by RXRα in conjunction with LXRα or LXRβ. In placenta, P2 is the preferred promoter, whereas various tumor cell lines tend to express predominantly either one or the other promoter. Furthermore, when each promoter is fused to a luciferase reporter gene and transfected into cancer cell lines, the promoter corresponding to the more active endogenous promoter is preferentially transcribed. Joint expression of activating nuclear receptors can partially account for the restricted expression of PLAC1 in placenta, and may be co-opted for preferential P1 or P2 PLAC1 expression in various tumor cells. Show less

Our previous study using interleukin-1α/β-knockout (IL-1-KO) and wild-type (WT) mice demonstrated that IL-1 acts as a positive factor for constitutive gene expression of hepatic cytochrome P4507a1 (Cyp7a1). In this study, to clarify the role of IL-1 in the expression of the hepatic Cyp7a1 gene, we focused on Cyp7a1 transcriptional regulators such as α-fetoprotein transcription factor (FTF), liver X receptor α (LXRα), hepatocyte nuclear factor 4α (HNF4α) and small heterodimer partner (SHP) and examined the effects of IL-1 on their gene expression by real-time reverse-transcription polymerase chain reaction using IL-1-KO and WT mice. We observed no significant differences between sex-matched IL-1-KO and WT mice with regard to gene expression levels of FTF, LXRα, and HNF4α, all of which are positive transcriptional regulators for the Cyp7a1 gene. However, interindividual differences in hepatic FTF and LXRα expression were closely dependent on the gene expression level(s) of hepatic IL-1 and tumor necrosis factor-α (TNF-α), while interindividual differences in hepatic HNF4α were clearly correlated with the expression of IL-1, but not TNF-α. In contrast, the gene expression level of SHP, which is a negative transcriptional regulator of the Cyp7a1 gene through inhibition of FTF function, was higher in IL-1-KO mice than in sex-matched WT mice. These findings demonstrate that, like TNF-α, IL-1 positively controls the gene expression of Cyp7a1 transcriptional upregulators but, in contrast to the previously reported action of TNF-α, IL-1 also acts to downregulate SHP gene expression. Show less

Atherosclerosis is a chronic and progressive inflammatory disease of the arteries that is characterized by subendothelial accumulation of lipid-rich macrophages, called foam cells. We sought to identify the molecular details of cross-talk between liver X receptor α (LXRα) and hypoxia-inducible factor 1α (HIF-1α) for the formation of triglyceride-rich foam cells under hypoxic conditions. We first observed that expression of LXRα and its target lipogenic genes was time-dependently induced in human primary macrophages and RAW 264.7 cells under hypoxia. Similarly, TO901317, an activator of LXRα, enhanced the expression level and the transcriptional activity of HIF-1α. Second, we demonstrated that LXRα increased HIF-1α protein stability through a physical interaction between the ligand binding domain of LXRα and the oxygen-dependent degradation domain of HIF-1α. Third, we found that the activation of HIF-1α or LXRα synergistically induced triglyceride accumulation in macrophages. Finally, we showed that LXRα and HIF-1α were codistributed in the macrophages of atherosclerotic lesions of patients. These results suggest that the positive feed-forward regulation of transcriptional activity and protein stability of LXRα and HIF-1α has an important impact in foam cell formation. Show less

Foam cell formation is the hallmark of early atherosclerosis. Lipid uptake by scavenger receptors (SR) in macrophages initiates chronic proinflammatory cascades linked to atherosclerosis. It has been reported that the upregulation of cholesterol efflux may be protective in the development of atherosclerosis. Ellagic acid, a polyphenolic compound mostly found in berries, walnuts, and pomegranates, possesses antioxidative, growth-inhibiting and apoptosis-promoting activities in cancer cells. However, the antiatherogenic actions of ellagic acid are not well defined. The current study elucidated oxidized LDL handling of ellagic acid in J774A1 murine macrophages. Noncytotoxic ellagic acid suppressed SR-B1 induction and foam cell formation within 6 h after the stimulation of macrophages with oxidized LDL, confirmed by Oil red O staining of macrophages. Ellagic acid at ≤5 μmol/L upregulated PPARγ and ATP binding cassette transporter-1 in lipid-laden macrophages, all responsible for cholesterol efflux. In addition, 5 μmol/L ellagic acid accelerated expression and transcription of the nuclear receptor of liver X receptor-α highly implicated in the PPAR signaling. Furthermore, ellagic acid promoted cholesterol efflux in oxidized LDL-induced foam cells. These results provide new information that ellagic acid downregulated macrophage lipid uptake to block foam cell formation of macrophages and boosted cholesterol efflux in lipid-laden foam cells. Therefore, dietary and pharmacological interventions with berries rich in ellagic acid may be promising treatment strategies to interrupt the development of atherosclerosis. Show less

To investigate the potential role of synthetic liver X receptors (LXRs) agonists T0901317 in lung of rats with acute lung injury induced by lipopolysaccharide (LPS). Rats infused with LPS served as acute lung injury (ALI) models. Specific mRNA was quantified by semi-quantitative reverse transcription polymerase (RT-PCR) and protein expression by western blotting. Inflammatory cytokine and MPO activity assays were studied by ELISA. Histopathology analysis was evaluated by hematoxylin and eosin. The expressions of LXRα and LXRβ were gradually decreased after LPS challenge. T0901317 pretreatment efficiently reduced the production of TNF-α, IL-1β, and IL-6, while elevated the level of IL-10 in BALF of rats with ALI. T0901317 also decreased the number of inflammatory cells and the concentration of total proteins in the BALF. Compared with the LPS group, rats with ALI which were pretreated with T0901317 had lower pulmonary tissue MPO activity and lightened histopathologic changes of lung. Furthermore, the expressions of NF-κB and ICAM-1 were markedly reduced after T0901317 administration. The expressions of LXRs were significantly decreased and synthetic agonist T0901317 suppresses lung inflammatory responses and lightened histopathologic changes of lung in rats with ALI. The mechanisms of this action for T0901317 may associate with the inhibition of NF-κB activation and downregulation of adhesion molecules ICAM-1 gene. Show less

To study the effects of Jiangzhi Granule (JZG), a compound traditional Chinese herbal medicine, in regulating liver X receptor α (LXRα) and sterol regulatory element-binding protein-1c (SREBP-1c) expressions in a rat model of non-alcoholic fatty liver disease (NAFLD). Forty specific pathogen-free Wistar male rats were randomly divided into normal group, untreated group, pioglitazone (PIO) group and JZG group. All rats were fed with high-fat diet (88% normal chow plus 10% lard plus 2% cholesterol) for 4 weeks except for the normal group. After the NAFLD model was established, PIO and JZG were fed to rats in the corresponding groups respectively for another 4 weeks. At the end of the 8th week, liver steatosis level was observed under a light microscope with hematoxylin and eosin (HE) staining; serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities and triacylglycerol (TAG) and free fatty acid (FFA) contents in liver tissues were measured. LXRα and SREBP-1c expressions in liver tissues were determined by real-time polymerase chain reaction and Western blot methods. Compared with the normal group, there were physiological changes for hepatic steatosis in liver tissues in the untreated group as observed by HE staining. JZG improved serum ALT and AST levels which were significantly increased in the untreated group. Both JZG and PIO improved FFA and TAG levels in liver tissues which were significantly increased in the untreated group. mRNA and protein levels of LXRα and SREBP-1c in the untreated group were higher than those in the normal group, while the treatment of JZG and PIO lowered their expressions. JZG may regulate fatty acid metabolic disorder by decreasing the levels of LXRα and SREBP-1c. Show less

Although animal studies indicate that liver X receptor alpha (LXRα) might influence risk of atherosclerosis, data in humans remain scarce. We tested the hypothesis that genetic variation in LXRα associates with risk of ischemic vascular disease and/or plasma lipid and lipoprotein levels in the general population. We studied 10,281 white persons of Danish ancestry from a general population cohort, including 1,986 in whom ischemic heart disease (IHD) developed, and 989 in whom ischemic cerebrovascular disease developed. We examined another 51,429 white persons of Danish ancestry from a general population study, including 3,789 with IHD. We genotyped 10 genetic variants identified by resequencing LXRα. Homozygosity for -840AA/-115AA(=2.7%) predicted hazard ratios of 1.3 (95% confidence interval, 1.0-1.7) for IHD, 1.6 (1.2-2.2) for myocardial infarction, and 1.7 (1.3-2.4) for ischemic cerebrovascular disease. The corresponding odds ratios in the second cohort were 1.1 (0.9-1.4) for IHD and 1.5 (1.1-2.0) for myocardial infarction. In the combined studies, odds ratios were 1.2 (1.0-1.4) for IHD and 1.5 (1.2-1.9) for myocardial infarction. Homozygosity for -840AA/-115AA did not associate with lipid or lipoprotein levels. LXRα -1830T>C (tagging the haplotype -1830C/-840A/-115A, all r(2)≥0.97) associated with 91% increased transcriptional activity. This study suggests that functional genetic variation in LXRα predicts risk of ischemic vascular disease in the general population. Show less

The nuclear receptors Liver X receptors, LXRα and LXRβ, regulate cholesterol and triglyceride metabolism. We and others have previously reported that synthetic LXR agonists reduced atherosclerosis in models of mouse with no detectable plasma cholesteryl ester transfer protein (CETP) activity, which plays an important role in reverse cholesterol transport. In the present study, we investigated the effect of LXR activation in rabbits to elucidate the influence of CETP activity. First, we cloned rabbit LXRs cDNA. The data indicated that rabbit LXRα was mostly highly expressed in the liver, whereas LXRβ expression was ubiquitous. Next, we investigated the effect of LXR agonist on lipid levels. Treatment with LXR agonist T0901317 increased plasma CETP activity and consequently elevated LDL, but no change in HDL. High cholesterol (HC) diet-feeding, which is thought to provide oxysterols as the natural agonists, could also increase expression of CETP and other LXR target genes. Finally, we tested T0901317 in the atherosclerosis intervention study. Chronic administration of T0901317 significantly reduced atherosclerosis in HC diet-fed rabbits despite less favorable lipid profiles, i.e. increases of plasma triglycerides and no change of HDL. T0901317 induced ATP-binding cassette transporters ABCA1 and ABCG1 and suppressed inflammatory genes expression in the aorta, suggesting that direct actions of LXR agonist on vascular gene expression are likely to contribute to the antiatherogenic effect. The present work strongly supports the idea that LXR agonists could be beneficial as therapeutic agents for treatment of atherosclerosis. Show less

Pioglitazone, a peroxisome proliferator-activated receptor γ (PPARγ) agonist, reportedly reduces cardiovascular events in diabetic patients. ATP cassette binding transporters (ABC) A1 and G1 are pivotal molecules for cholesterol efflux (ChE) from macrophages and high density-lipoprotein biogenesis, and the A1 transporter is regulated by a PPARγ-liver receptor X (LXR) pathway. Also, pioglitazone induces ABCG1 expression, though the exact mechanism remains unclear. We therefore investigated the effects of pioglitazone on ABCA1/G1 expression in vitro and ex vivo. The effects of pioglitazone on ChE and ABCA1/G1 expressions in macrophages were assessed. Then, mRNA was quantified in macrophages when PPARγ/LXR inhibition by siRNA or overexpression of oxysterol sulfotransferase was performed. ABCA1/G1 promoter activity with mutated LXR-responsive elements was also measured. As an ex vivo study, 15 type 2 diabetic patients were administered pioglitazone or placebo, and ChE assays and protein expressions were determined using macrophages cultured with the corresponding sera. Pioglitazone increased LXRα/ABCA1/G1 expressions, which enhanced ChE from macrophages. Inhibition of PPARγ/LXR pathways revealed that LXR was primarily involved in pioglitazone's transactivation of ABCA1 but only partially involved for ABCG1. Promoter assays showed that ABCG1 was regulated more by the promoter in intron 4 than that upstream of exon 1 but both promoters were responsive to LXR activation. Sera obtained after pioglitazone treatment promoted ChE and ABCA1/G1 expressions in macrophages. Pioglitazone enhanced ChE from macrophages by increasing ABCA1/G1 in LXR-dependent and -independent manners. Our comparable in vitro and ex vivo results shed new light on pioglitazone's novel anti-atherogenic property. Show less

It has previously been shown that dual activation of the Liver X Receptors (LXRα and LXRβ) by the agonist, GW3965, enhances pathology in a murine model of collagen-induced arthritis. To determine whether LXRα or LXRβ have discrete roles in driving articular inflammation. Arthritis was induced in male C57BL/6 wild-type (WT), LXRα-/-, LXRβ-/- and LXRα/β double KO mice by injection with type II collagen and treated with 30 mg/kg of the LXR agonist GW3965 or vehicle control. The mice were monitored for articular inflammation and cartilage degradation by scoring for clinical signs of arthritis and by histological examination of the joints. Administration of 30 mg/kg GW3965 significantly increases the severity of arthritis in WT but not LXRα-/-, LXRβ-/- or LXRα/β KO mice as assessed by an increase in the clinical score, paw thickness and articular histological analysis. The proinflammatory effects associated with the administration of GW3965 are mediated specifically through LXRs. The absence of increased disease severity in the LXRα-/- and LXRβ-/- GW3965-treated groups shows for the first time that agonism of both LXRα and LXRβ is required to drive proinflammatory pathways in vivo. Show less

Increasing plasma glucose levels are associated with increasing risk of vascular disease. We tested the hypothesis that there is a glycaemia-mediated impairment of reverse cholesterol transport (RCT). We studied the influence of plasma glucose on expression and function of a key mediator in RCT, the ATP binding cassette transporter-A1 (ABCA1) and expression of its regulators, liver X receptor-α (LXRα) and peroxisome proliferator-activated receptor-γ (PPARγ). Leukocyte ABCA1, LXRα and PPARγ expression was measured by polymerase chain reaction in 63 men with varying degrees of glucose homeostasis. ABCA1 protein concentrations were measured in leukocytes. In a sub-group of 25 men, ABCA1 function was quantified as apolipoprotein-A1-mediated cholesterol efflux from 2-3 week cultured skin fibroblasts. Leukocyte ABCA1 expression correlated negatively with circulating HbA1c and glucose (rho = -0.41, p<0.001; rho = -0.34, p = 0.006 respectively) and was reduced in Type 2 diabetes (T2DM) (p = 0.03). Leukocyte ABCA1 protein was lower in T2DM (p = 0.03) and positively associated with plasma HDL cholesterol (HDL-C) (rho = 0.34, p = 0.02). Apolipoprotein-A1-mediated cholesterol efflux correlated negatively with fasting glucose (rho = -0.50, p = 0.01) and positively with HDL-C (rho = 0.41, p = 0.02). It was reduced in T2DM compared with controls (p = 0.04). These relationships were independent of LXRα and PPARγ expression. ABCA1 expression and protein concentrations in leukocytes, as well as function in cultured skin fibroblasts, are reduced in T2DM. ABCA1 protein concentration and function are associated with HDL-C levels. These findings indicate a glycaemia-related, persistent disruption of a key component of RCT. Show less

The physiological regulation of hepatic apoE gene has not been clarified, although the expression of apoE in adipocytes and macrophages has been known to be regulated by LXR. We investigated the effect of TO901317, a LXR agonist, on hepatic apoE production utilizing HepG2 cells cultured in spheroid form, known to be more differentiated than HepG2 cells in monolayer culture. Spheroid HepG2 cells were prepared in alginate-beads. The secretions of albumin, apoE and apoA-I from spheroid HepG2 cells were significantly increased compared to those from monolayer HepG2 cells, and these increases were accompanied by increased mRNA levels of apoE and apoA-I. Several nuclear receptors including LXRα also became abundant in nuclear fractions in spheroid HepG2 cells. Treatment with TO901317 significantly increased apoE protein secretion from spheroid HepG2 cells, which was also associated with the increased expression of apoE mRNA. Separation of the media with FPLC revealed that the production of apoE-rich large HDL particles were enhanced even at low concentration of TO901317, and at higher concentration of TO901317, production of VLDL particles increased as well. LXR activation enhanced the expression of hepatic apoE, together with the alteration of lipoprotein particles produced from the differentiated hepatocyte-derived cells. HepG2 spheroids might serve as a good model of well-differentiated human hepatocytes for future investigations of hepatic lipid metabolism. Show less

Liver X receptor (LXR) is a transcription factor of the nuclear receptor family, regulating genes involved in metabolism, inflammation, and apoptosis. In the present investigation, we examined the role of LXR in organ injury and systemic inflammation in rodent models of polymicrobial peritonitis caused by cecal ligation and puncture (CLP). Rats were subjected to CLP sepsis or a sham operation. Some were treated with the synthetic LXR agonist GW3965 0.3 mg/kg 30 min prior to the CLP procedure, and organs and plasma were harvested at 3, 10, 18, or 24 h. Organs were analyzed for RNA expression by quantitative polymerase chain reaction or for morphologic differences by histologic review. Organ injury and inflammatory markers were measured in plasma. Expression of the LXRα gene was decreased in the livers of CLP rats compared with sham-operated rats. Administration of a synthetic agonist of LXR (GW3965) reduced biochemical indices of liver injury in the blood of CLP rats. We also demonstrated that liver injury associated with CLP is aggravated in LXRα- and LXRαβ-deficient mice compared with wild-type and LXRβ-deficient mice, indicating a role for LXRα in protecting the liver. The enhanced liver injury in LXR-deficient mice was associated with elevated plasma concentrations of high mobility group box 1, a late mediator of inflammation and a known factor in the pathology of this model. Collectively, these results argue in favor of a role for LXRα in protection against liver injury in experimental sepsis induced by CLP. Show less

Liver X receptor (LXR) α and LXRβ belong to the nuclear receptor superfamily. For many years, they have been called orphan receptors, as no natural ligand was identified. In the last decade, the LXR natural ligands have been shown to be oxysterols, molecules derived from cholesterol. While these nuclear receptors have been abundantly studied for their roles in the regulation of lipid metabolism, it appears that they also present crucial activities in reproductive organs such as testis and epididymis, as well as prostate. Phenotypic analyses of mice lacking LXRs (lxr-/-) pointed out their physiological activities in the various cells and organs regulating reproductive functions. This review summarizes the impact of LXR-deficiency in male reproduction, highlighting the novel information coming from the phenotypic analyses of lxrα-/-, lxrβ-/- and lxrα;β-/- mice. This article is part of a Special Issue entitled: Translating nuclear receptor from health to disease. Show less

In the previous experiment, we found that there was a different response between E3 rats and DA.1U rats to high-fat-diet-induced metabolic syndrome (HFD-MetS). The aim of this study was to explore the cause and molecular mechanism of the genetic difference in susceptibility to metabolic syndrome in E3 rats as compared with DA.1U rats. Firstly, a 12-week HFD-MetS model in E3 and DA.1U rats was carried out and assessed. Then, the expression of key insulin signaling molecules, metabolic nuclear receptors, metabolic key enzymes and histone deacetylases (Hdacs) was determined by different methods. Finally, the effects of overexpression and disruption of Hdac3 on metabolic nuclear receptors were analyzed in CBRH-7919 cells and primarily-hepatic cells from DA.1U and E3 rats. We found that E3 rats were susceptible, while DA.1U rats were resisted to HFD-MetS. The expression of liver X receptor α,β (LXR-α,β), farnesoid X receptor (FXR), peroxisome proliferator activated receptor γ (PPAR-γ) and cholesterol 7α-hydroxylase (CYP7A1) increased markedly in DA.1U rat liver, whereas they decreased significantly in E3 rats. The expression of Hdac3 increased by HFD treatment in both E3 and DA.1U rat livers, but the constitutive Hdac3 expression was lower in DA.IU rat liver than in E3 rat liver. Importantly, overexpression of Hdac3 could downregulate the expression of LXR-α, PPAR-γ and CYP7A1 in both CBRH-7919 cells and primarily cultured hepatic cells from DA.IU rats. On the contrary, disruption of Hdac3 by shRNA upregulated the expression of LXR-α, PPAR-γ and CYP7A1 in both CBRH-7919 cells and primarily cultured hepatic cells from E3 rats. The results suggested that a high constitutive expression of Hdac3 inhibiting the expression of PPAR-γ, LXR-α and CYP7A1 in liver contributes to HFD-MetS in E3 rats. Show less