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Interleukin (IL)-10 is a prototypical anti-inflammatory cytokine that has been shown to attenuate atherosclerosis development. In addition to its anti-inflammatory properties, the anti-atherogenic effect of IL-10 has recently also been suggested to reflect a complex effect of IL-10 on lipid metabolism in macrophages. In the present study we examined the effects of IL-10 on cholesterol efflux mechanism in lipid-loaded THP-1 macrophages. Our main findings were: (i) IL-10 significantly enhanced cholesterol efflux induced by fetal-calf serum, high-density lipoprotein (HDL)2 and apolipoprotein A-1. (ii) The IL-10-mediated effects on cholesterol efflux were accompanied by an increased IL-10-mediated expression of the ATP-binding cassette transporters ABCA1 and ABCG1, that was further enhanced when the cells were co-activated with the liver X receptor (LXR)α agonist (22R)-hydroxycholesterol. (iii) The effect of LXRα activation on the IL-10-mediated effects on the ATP-binding cassette transporters seems to include enhancing effects on the IL-10 receptor 1 (IL10R1) expression and interaction with STAT-3 signaling. (iv) These enhancing effects on ABCA1 and ABCG1 was not seen when the cells were stimulated with the IL-10 family members IL-22 and IL-24. This study suggests that the anti-atherogenic properties of IL-10 may include enhancing effects on cholesterol efflux mechanism that involves cross-talk with LXRα activation. Show less

Preeclampsia is a serious pregnancy complication. Soluble endoglin (sEng) is released from the placenta and contributes to the maternal endothelial dysfunction seen in preeclampsia. Recently oxysterols, which activate the Liver X Receptor (LXR), have been implicated in producing sEng, by upregulating matrix metalloproteinase-14 (MMP14; cleaves endoglin to produce sEng) and down-regulating tissue inhibitor of metalloproteinase-3 (TIMP-3; inhibitor of MMP14). The functional experiments in that study were performed on JAR cells (human choriocarcinoma cell line) and placental explants. We characterized LXR in severe preeclamptic placentas, and assessed whether oxysterols increase release of sEng from primary human umbilical vein endothelial cells (HUVECs), primary trophoblasts and placental explants. Given pravastatin is thought to block oxysterol production and inhibit the LXR, we examined whether pravastatin reduces sEng release. LXRα and β were localized to the syncytiotrophoblast and villous tips and were significantly up-regulated in preeclamptic placenta. Oxysterols upregulated sEng production in HUVECs and placental explants although the increases were far more modest than that recently reported. Oxysterols did not upregulate sEng in primary trophoblasts. Furthermore, mRNA expression of MMP14 and TIMP-3 were not altered by oxysterols in any tissue. Surprisingly, pravastatin did not decrease oxysterol-induced upregulation of sEng. LXR is up-regulated in preeclamptic placenta. Oxysterols upregulate sEng production from human tissues, but the increase is modest, suggesting this may not be the main mechanism for the very significant elevations in sEng seen in preeclampsia. Pravastatin does not decrease sEng production. Oxysterols modestly up-regulate sEng production which is not quenched by pravastatin. Show less

To investigate whether RNA interference (RNAi) of LXRα gene in donor rats with fatty liver improves liver graft function after transplantation. Fifty donor SD rats were fed a high-fat diet and 56% alcohol to induce macrovesicular steatosis exceeding 60% in the liver. The donor rats were injected via the portal veins with 7 × 10⁷ TU LXRα-RNAi-LV mixture (n=25) or negative control-LV (NC-LV) vector (n=25) 72 h before orthotopic liver transplantation. At 2, 24, and 72 h after the transplantation, the recipient rats were sacrificed to examine liver transaminases, liver graft histology, immunostaining (TUNEL), and protein and mRNA levels of LXRα. Lentivirus-LXRα RNAi inhibited LXRα gene expression at both the mRNA and protein levels in the liver graft and reduced the expressions of SREBP-1c and CD36 as compared with the controls, resulting also in reduced fatty acid accumulation in the hepatocytes. The recipient rats receiving RNAi-treated grafts showed more obvious reduction in serum ALT, AST, IL-1β and TNF-α levels, and exhibited milder hepatic pathologies than the control rats after the transplantation. TUNEL assay demonstrated a significant reduction in cell apoptosis in LXRα-RNAi-LV-treated liver grafts, and the rats receiving treated liver grafts had a prolonged mean overall survival time. LXRα-RNAi-LV treatment of the donor rats with fatty liver can significantly down-regulate LXRα gene expression in the liver graft and improve the graft function and recipient rat survival after liver transplantation. Show less

Liver X receptor-α (LXR-α) which belongs to the nuclear receptor superfamily, is a ligand-activated transcription factor. Best known for its ability to regulate lipid metabolism and transport, LXRs have recently also been implicated in regulation of inflammatory response. The aim of this study was to investigate the preventive effects of synthetic LXR-α agonist T0901317 on LPS-induced mastitis in mice. The mouse model of mastitis was induced by injection of LPS through the duct of mammary gland. T0901317 was injected 1h before and 12h after induction of LPS intraperitoneally. The results showed that T0901317 significantly attenuated the infiltration of neutrophilic granulocytes, and the activation of myeloperoxidase (MPO); down-regulated the level of pro-inflammatory mediators including TNF-α, IL-1β, IL-6, COX-2 and PEG2; inhibited the phosphorylation of IκB-α and NF-κB p65, caused by LPS. Moreover, we report for the first time that LXR-α activation impaired LPS-induced mastitis. Taken together, these data indicated that T0901317 had protective effect on mastitis and the anti-inflammatory mechanism of T0901317 on LPS induced mastitis in mice may be due to its ability to inhibit NF-κB signaling pathway. LXR-α activation can be used as a therapeutic approach to treat mastitis. Show less

Hepatocyte-like cells, differentiated from different stem cell sources, are considered to have a range of possible therapeutic applications, including drug discovery, metabolic disease modelling, and cell transplantation. However, little is known about how stem cells differentiate into mature and functional hepatocytes. Using transcriptomic screening, a transcription factor, liver X receptor α (NR1H3), was identified as increased during HepaRG cell hepatogenesis; this protein was also upregulated during embryonic stem cell and induced pluripotent stem cell differentiation. Overexpressing NR1H3 in human HepaRG cells promoted hepatic maturation; the hepatocyte-like cells exhibited various functions associated with mature hepatocytes, including cytochrome P450 (CYP) enzyme activity, secretion of urea and albumin, upregulation of hepatic-specific transcripts and an increase in glycogen storage. Importantly, the NR1H3-derived hepatocyte-like cells were able to rescue lethal fulminant hepatic failure using a non-obese diabetic/severe combined immunodeficient mouse model. In this study, we found that NR1H3 accelerates hepatic differentiation through an HNF4α-dependent reciprocal network. This contributes to hepatogenesis and is therapeutically beneficial to liver disease. Show less

Cholesterol is the template for steroid hormone biosynthesis. Cholesterol homeostasis is regulated by Cyt-P450 oxygenated cholesterols acting as ligands on LXR-α and LXR-β transcription factors that are now emerging as drug targets. Heterodimerization of LXRs with retinoic acid receptor is considered a prerequisite for target gene activation. Dietary plant oxysterol 28-homobrassinolide (28-HB) is a proven antihyperglycemic and a pro-steroidogenic agent in the rat. Whether 28-HB has a role in LXR gene expression was therefore investigated using oral gavage (15 days) of 28-HB (333 µg/kg b w) to normal and diabetic rat. PCR amplified LXR-α and β mRNA transcripts from treated rat liver and testis exhibited quantitative differences in their expression. Conformational differences in 28-HB docking to LXR-α and β binding domains were also noted through in silico studies, LXR-β adopting lesser specificity. We report that 28-HB transactivates LXR genes in the rat tissues. Show less

Liver X receptor (LXR) is an oxysterol-activated nuclear receptor involved in the control of major metabolic pathways for cholesterol homeostasis and lipogenesis. Although the role of LXR in hepatic steatosis is well known, its correlation with intrahepatic inflammation and fibrosis has not been thoroughly studied. We investigated the association between LXRα, hepatic inflammation, and fibrosis, as well as its correlation with other intrahepatic lipid transporters in patients with nonalcoholic fatty liver disease (NAFLD). We evaluated clinical characteristics including sex, age, body mass index, and laboratory findings from 40 NAFLD and 16 control patients. Immunohistochemical staining was carried out on liver biopsy samples from all patients. The positive rate of LXRα expression was 30 % in the control group, 50 % in the NAFLD group, and 97 % in NASH groups. LXRα expression was positively correlated with not only the amount of intrahepatic fat, but also with intrahepatic inflammation and hepatic fibrosis. LXRα expression showed positive correlation with intrahepatic expression of ABCG5/8, CD36, and SREBP-1c. The expression of ABCA1, ABCG5/8, SREBP-1c, and CD36 was higher in NAFLD than in controls and there was no further increase in the NASH group. NPC1L1 was abundant in human liver. Expression of NPC1L1 was negatively correlated with intrahepatic inflammation and LXRα intensity. LXR expression correlated with the degree of hepatic fat deposition, as well as with hepatic inflammation and fibrosis in NAFLD patients. Our research suggests that LXR is an attractive target for treatment and regulation of hepatic inflammation and fibrosis. Show less

In nonruminants, the alternative splicing of peroxisome proliferator-activated receptor γ (PPARG) generates PPARG1 and PPARG2 isoforms. Although transcriptional control differences between isoforms have been reported in human adipose tissue, their roles in ruminant mammary cells are not well known. To assess which of these isoforms is more closely associated with the regulation of mammary lipogenic pathways, their tissue distribution was analyzed and the expression of key genes regulating lipogenic gene networks was measured after overexpression of the 2 isoforms in goat mammary epithelial cells (GMEC). The expression of PPARG2 was markedly greater in adipose tissue, whereas PPARG1 is the main isoform in goat mammary tissue (ratio of PPARG1:PPARG2 was close to 37:1). As was reported in previous work, PPARG1 upregulated the transcription regulators SREBF1 and PPARG and the lipogenic genes FASN, ACACA, and SCD. Along with a tendency for greater expression of AGPAT6, DGAT1, and PLIN2, these data suggest that PPARG1 is the isoform controlling lipogenesis in mammary cells. Addition of the PPARG ligand rosiglitazone (ROSI) to GMEC overexpressing both isoforms upregulated the expression of LPL and CD36, which help control uptake of long-chain fatty acids into mammary cells. Other responses to ROSI addition to GMEC overexpressing PPARG1 and PPARG2 included upregulation of AGPAT6, DGAT1, INSIG1, SREBF1, and NR1H3. Although the data suggest that both PPARG1 and PPARG2 could affect mammary lipogenesis via control of gene expression when stimulated (e.g., by ROSI), the fact that PPARG1 is more abundant in mammary tissue and that its overexpression alone upregulated key lipogenic gene networks suggest that it is the more important isoform in goat mammary cells. Show less

We evaluated in vitro anti-diabetic activities of 497 native plants of Jeju Island (South Korea) by measuring the induction of adipocyte differentiation. Among the plants, Daphniphyllum macropodum fruit extract (DME) had the highest peroxisome proliferator-activated receptor γ (PPARγ) agonist activity and was therefore selected as a potential source of anti-diabetic agents. To elucidate the active components of DME, constituent compounds were purified and their effects on the adipocyte differentiation were studied. Using activity-guided fractionation, four compounds were isolated from DME and their adipogenic effects were evaluated. Among the compounds isolated, 5,7-dihydroxychromone potently induced the differentiation of mouse 3T3-L1 preadipocytes. DME and 5,7-dihydroxychromone increased PPARγ and liver X receptor α (LXRα) mRNA expression levels. To determine whether the adipogenic effects we observed might affect serum glucose levels, we undertook in vivo experiment using streptozotocin-/high-fat diet-induced type 2 diabetes mouse model. DME supplementation reduced serum glucose, total cholesterol, and triacylglycerol levels in diabetes mice. These results suggest that DME may be useful for the prevention and treatment of type 2 diabetes mellitus. Moreover, it was proposed that 5,7-dihydroxychromone isolated from DME is one of the active compounds that may contribute to regulate blood glucose levels. Show less

Chlorogenic acid (CGA) is one of the most abundant polyphenols in the human diet and is suggested to be a potential antiatherosclerotic agent due to its proposed hypolipidemic, anti-inflammatory and antioxidative properties. The aim of this study was to evaluate the effect of CGA on atherosclerosis development in ApoE(-/-) mice and its potential mechanism. ApoE(-/-) mice were fed a cholesterol-rich diet without (control) or with CGA (200 and 400 mg/kg) or atorvastatin (4 mg/kg) for 12 weeks. During the study plasma lipid and inflammatory parameters were determined. Treatment with CGA (400 mg/kg) reduced atherosclerotic lesion area and vascular dilatation in the aortic root, comparable to atorvastatin. CGA (400 mg/kg) also significantly decreased plasma levels of total cholesterol, triglycerides and low-density lipoprotein-cholesterol as well as inflammatory markers. Supplementation with CGA or CGA metabolites-containing serum suppressed oxidized low-density lipoprotein (oxLDL)-induced lipid accumulation and stimulated cholesterol efflux from RAW264.7 cells. CGA significantly increased the mRNA levels of PPARγ, LXRα, ABCA1 and ABCG1 as well as the transcriptional activity of PPARγ. Cholesterol efflux assay showed that three major metabolites, caffeic, ferulic and gallic acids, significantly stimulated cholesterol efflux from RAW264.7 cells. These results suggest that CGA potently reduces atherosclerosis development in ApoE(-/-) mice and promotes cholesterol efflux from RAW264.7 macrophages. Caffeic, ferulic and gallic acids may be the potential active compounds accounting for the in vivo effect of CGA. Show less

Adenosine triphosphate-binding cassette transporter A1 (ABCA1) and ABCG1 play crucial roles in reverse cholesterol transport, and have anti-atherosclerosis effects, and liver X receptor alpha (LXRα) can stimulate cholesterol efflux through these transporters. Angiotensin (Ang)-(1-7) can protect endothelial cells, inhibit smooth muscle cell growth, ameliorate inflammation and exert anti-atherosclerotic effects. In the present study, we attempted to clarify the effect of Ang-(1-7) on expression of ABCA1 and ABCG1, and explored the role of LXRα in the regulation of ABCA1 and ABCG1 in THP-1 macrophages that had been incubated with angiotensin-II (AngII). Ang-(1-7) increased ABCA1 and ABCG1 expression in a concentration-dependent manner at both the mRNA and protein levels, promoted cholesterol efflux, and decreased cholesterol content in THP-1 macrophages treated with AngII. Furthermore, Ang-(1-7) upregulated the expression of LXRα in a concentration-dependent manner in these cells. LXRα small interfering RNA, as well as the Mas receptor antagonist A-779, completely abolished these effects of Ang-(1-7). In summary, Ang-(1-7) upregulates ABCA1 and ABCG1 expression in THP-1 macrophages treated with AngII through the Mas receptor, via the LXRα pathway. This novel insight into the molecular mechanism underlying Ang-(1-7) and AngII interaction could prove useful for developing new strategies for treatment of cardiovascular diseases. Show less

Collaborative regulation of liver X receptor (LXR) and sterol regulatory element binding protein (SREBP)-1 are main determinants in hepatic steatosis, as shown in both animal models and human patients. Recent studies indicate that selective intervention of overly functional LXRα in the liver shows promise in treatment of fatty liver disease. In the present study, we evaluated the effects of meso-dihydroguaiaretic acid (MDGA) on LXRα activation and its ability to attenuate fatty liver in mice. MDGA inhibited activation of the LXRα ligand-binding domain by competitively binding to the pocket for agonist T0901317 and decreased the luciferase activity in LXRE-tk-Luc-transfected cells. MDGA significantly attenuated hepatic neutral lipid accumulation in T0901317- and high fat diet (HFD)-induced fatty liver. The effect of MDGA was so potent that treatment with 1mg/kg for 2 weeks completely reversed the lipid accumulation induced by HFD feeding. MDGA reduced the expression of LXRα co-activator protein RIP140 and LXRα target gene products associated with lipogenesis in HFD-fed mice. These results demonstrate that MDGA has the potential to attenuate nonalcoholic steatosis mediated by selective inhibition of LXRα in the liver in mice. Show less

This study was designed to investigate the direct effects of fatty acids (FAs) on the cell viability and the expression levels of genes involved in lipid metabolism in LO2 human liver cells. Palmitate (PA), oleate (OA) and docosahaexenoic acid (DHA) were used to represent saturated, mono-unsaturated and polyunsaturated FAs, respectively. At concentrations of ≤3.2 µg/ml, treatment with single FAs increased the viability of the LO2 cells. At FA concentrations of >3.2 µg/ml, cell viability following OA treatment was increased, but PA or DHA treatment at these concentrations reduced cell viability. Administration of mixtures of these FAs in three ratios (PA:OA:DHA = 1:2:1, 1:1:1 and 1:1:2, respectively) increased the cell viability compared with the control group. The intracellular triglyceride (TG) levels following all types of treatment were significantly increased and the accumulation of TGs was markedly increased with high doses of DHA. In addition, peroxisome proliferator-activated receptor-γ was significantly upregulated in all groups, with the exception of the 1:1:1 group at 3.2 µg/ml and the 1:1:2 group at 12.8 µg/ml. The expression levels of sterol regulatory-element binding protein‑1c, liver X receptor α and apolipoprotein C‑I were significantly reduced in all groups with the exception of the DHA‑treated group and the 1:2:1 groups at 3.2 and 12.8 µg/ml. In conclusion, these results indicate that the type, concentration and mixture ratios of FAs are all important in determining the cell viability and lipid metabolism-related gene expression in LO2 hepatocytes. Show less

Inflammation marks all stages of atherogenesis. DNA hypermethylation in the whole genome or specific genes is associated with inflammation and cardiovascular diseases. Therefore, we aimed to study whether inhibiting DNA methylation by DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) ameliorates atherosclerosis in low-density lipoprotein receptor knockout (Ldlr(-/-)) mice. Ldlr(-/-) mice were fed an atherogenic diet and adminisered saline or 5-aza-dC (0.25 mg/kg) for up to 30 weeks. 5-aza-dC treatment markedly decreased atherosclerosis development in Ldlr(-/-) mice without changes in body weight, plasma lipid profile, macrophage cholesterol levels and plaque lipid content. Instead, this effect was associated with decreased macrophage inflammation. Macrophages with 5-aza-dC treatment had downregulated expression of genes involved in inflammation (TNF-α, IL-6, IL-1β, and inducible nitric oxidase) and chemotaxis (CD62/L-selectin, chemokine [C-C motif] ligand 2/MCP-1 [CCL2/MCP-1], CCL5, CCL9, and CCL2 receptor CCR2). This resulted in attenuated macrophage migration and adhesion to endothelial cells and reduced macrophage infiltration into atherosclerotic plaques. 5-aza-dC also suppressed macrophage endoplasmic reticulum stress, a key upstream signal that activates macrophage inflammation and apoptotic pathways. Finally, 5-aza-dC demethylated liver X receptor α (LXRα) and peroxisome proliferator-activated receptor γ1 (PPARγ1) promoters, which are both enriched with CpG sites. This led to overexpression of LXRα and PPARγ, which may be responsible for 5-aza-dC's anti-inflammatory and atheroprotective effect. Our findings provide strong evidence that DNA methylation may play a significant role in cardiovascular diseases and serve as a therapeutic target for prevention and treatment of atherosclerosis. Show less

Stearoyl-CoA desaturase 1 (SCD1) is the rate limiting enzyme in unsaturated fatty acid biosynthesis. This enzyme has an important role in the regulation of hepatic lipogenesis and lipid oxidation, and alterations in these pathways may lead to several diseases. We examined, in HepG2 cell cultures, the mechanism of SCD1 regulation considering the involvement of two transcription factors: liver X receptor alpha (LXRα) and sterol regulatory element-binding protein-1 (SREBP-1), also investigating the effect of dietary polyunsaturated fatty acids (PUFAs) on this process. The analysis of SCD1 promoter allowed to identify a functional SREBP-1 binding site (SRE 1). LXRα activation increased SCD1 protein level through upregulation of SREBP-1 and its consequent binding to SRE 1 sequence. Polyunsaturated docosahexaenoic acid (DHA, C22:6), eicosapentaenoic acid (EPA, C20:5) and arachidonic acid (AA, C20:4) were able to reduce SREBP-1 binding to SCD1 promoter, while saturated stearic acid (SA, C18:0) did not give any effect. Surface plasmon resonance analysis showed a direct binding of DHA, EPA and AA to LXRα. These data indicate a direct inhibitory interaction of PUFAs with LXRα, a consequent reduction of SREBP-1 and of its binding to SCD1 promoter. This information provides a mechanism to explain the regulation of lipogenic pathways induced by PUFAs. Show less

Arsenic exposure has been linked to an increased incidence of atherosclerosis. Previously, we have shown in vitro and in vivo that arsenic inhibits transcriptional activation of the liver X receptors (LXRs), key regulators of lipid homeostasis. Therefore, we evaluated the role of LXRα in arsenic-induced atherosclerosis using the apoE(-/-) mouse model. Indeed, deletion of LXRα protected apoE(-/-) mice against the proatherogenic effects of arsenic. We have previously shown that arsenic changes the plaque composition in apoE(-/-) mice. Arsenic decreased collagen content in the apoE(-/-) model, and we have observed the same diminution in LXRα(-/-)apoE(-/-) mice. However, the collagen-producing smooth muscle cells (SMCs) were decreased in apoE(-/-), but increased in LXRα(-/-)apoE(-/-). Although transcriptional activation of collagen remained the same in SMC from both genotypes, arsenic-exposed LXRα(-/-)apoE(-/-) plaques had increased matrix metalloproteinase activity compared with both control LXRα(-/-)apoE(-/-) and apoE(-/-), which could be responsible for both the decrease in plaque collagen and the SMC invasion. In addition, arsenic increased plaque lipid accumulation in both genotypes. However, macrophages, the cells known to retain lipid within the plaque, were unchanged in arsenic-exposed apoE(-/-) mice, but decreased in LXRα(-/-)apoE(-/-). We confirmed in vitro that these cells retained more lipid following arsenic exposure and are more sensitive to apoptosis than apoE(-/-). Mice lacking LXRα are resistant to arsenic-enhanced atherosclerosis, but arsenic-exposed LXRα(-/-)apoE(-/-) mice still present a different plaque composition pattern than the arsenic-exposed apoE(-/-) mice. Show less

Liver-X-receptors, LXRα (NR1H3) and LXRβ (NR1H2), encode 2 different but highly homologous isoforms of transcription factors belonging to the nuclear receptor superfamily. Whether LXRα and LXRβ subtypes have discrete roles in the regulation of cardiac physiology/pathology is unknown. We determine the role of each LXR subtype in myocardial ischemia/reperfusion (MI/R) injury. Mice (wild type; those genetically depleted of LXRα, LXRβ, or both; and those overexpressing LXRα or LXRβ by in vivo intramyocardial adenoviral vector) were subjected to MI/R injury. Both LXRα and LXRβ were detected in wild-type mouse heart. LXRα, but not LXRβ, was significantly upregulated after MI/R. Dual activation of LXRα and LXRβ by natural and synthetic agonists reduced myocardial infarction and improved contractile function after MI/R. Mechanistically, LXR activation inhibited MI/R-induced oxidative stress and nitrative stress, attenuated endoplasmic reticulum stress and mitochondrial dysfunction, and reduced cardiomyocyte apoptosis in ischemic/reperfused myocardium. The aforementioned cardioprotective effects of LXR agonists were impaired in the setting of cardiac-specific gene silencing of LXRα, but not LXRβ subtype. Moreover, LXRα/β double-knockout and LXRα-knockout mice, but not LXRβ-knockout mice, increased MI/R injury, exacerbated MI/R-induced oxidative/nitrative stress, and aggravated endoplasmic reticulum stress and mitochondrial dysfunction. Furthermore, cardiac LXRα, not LXRβ, overexpression via adenoviral transfection suppressed MI/R injury. Our study provides the first direct evidence that the LXRα, but not LXRβ, subtype is a novel endogenous cardiac protective receptor against MI/R injury. Drug development strategies specifically targeting LXRα may be beneficial in treating ischemic heart disease. Show less

Recent studies have revealed critical roles that nuclear receptors like LXR-α (Liver X Receptor- alpha) plays as a class of post-transcriptional gene regulator in skin development and diseases. Keeping in view the fact that LXR-α plays crucial role in keratinocyte proliferation and differentiation, it becomes imperative to dissect the pathways and role of LXR-α genomics in the pathogenesis of psoriasis with ultimate aim to explore novel preventive/therapeutic strategies as treatment options. To explore the effects of agonists and activators of LXR-α on its own gene expression and the putative targets in psoriatic keratinocytes. Identification of promoter sequences for (vitamin D receptor) VDR and Catalase were done using in silico analysis followed by β-galactosidase (β-gal) reporter plasmid assay in keratinocytes from clinically heathy subjects. Determination of relative levels of LXR-α,VDR and catalase in control versus treated cells upon activation of LXR-α with Atorvastatin + 22R hydroxycholestrol and Ascorbic acid + 22R hydroxycholestrol was done by PCR and Cell Proliferation Assay. The cells transfected with the reporter plasmid element for VDR and catalase showed more than 5 and 4 fold increase respectively in the β-gal activity compared to the control. An increase of 55% in LXR-α gene expression at RNA level was observed in Atorvastatin + 22-R hydroxycholestrol compared to 24% in Ascorbic acid + 22-ROH cholesterol. The expression of the VDR and Catalase was significantly increased in both treated keratinocytes compared to its normal counterpart. Show less

We aimed to elucidate the effect of bilirubin on dyslipidemia and nephropathy in a diabetes mellitus (DM) type I animal model. Sprague-Dawley rats were separated into control, DM, and bilirubin-treated DM (Bil) groups. The Bil group was injected intraperitoneally with 60 mg/kg bilirubin 3 times per week and hepatoma cells were cultured with bilirubin at a concentration of 0.3 mg/dL. The Bil group showed lower serum creatinine levels 5 weeks after diabetes onset. Bilirubin treatment also decreased the amount of mesangial matrix, lowered the expression of renal collagen IV and transforming growth factor (TGF)-β1, and reduced the level of apoptosis in the kidney, compared to the DM group. These changes were accompanied by decreased tissue levels of hydrogen superoxide and NADPH oxidase subunit proteins. Bilirubin decreased serum total cholesterol, high-density lipoprotein cholesterol (HDL-C), free fatty acids, and triglycerides (TGs), as well as the TG content in the liver tissues. Bilirubin suppressed protein expression of LXRα, SREBP-1, SCD-1, and FAS, factors involved in TG synthesis that were elevated in the livers of DM rats and hepatoma cells under high-glucose conditions. In conclusion, bilirubin attenuates renal dysfunction and dyslipidemia in diabetes by suppressing LXRα and SREBP-1 expression and oxidative stress. Show less

To determine whether the transcription factors liver X receptors (LXRs) and their downstream genes, which are involved in the regulation of several testicular functions in mouse models, are differentially expressed in testes of men with nonobstructive azoospermia (NOA) or obstructive azoospermia (OA). Prospective study. University hospital. Patients with various types of NOA (n=22) and with OA (n=5). Human testicular biopsies. Transcript levels were measured in testicular biopsies with the use of quantitative polymerase chain reaction. Correlations of LXR mRNA levels with the number of germ cells, the expression of proliferation and apoptosis markers, and the amount of intratesticular lipids and testosterone were evaluated. The localization of LXRα was analyzed by immunofluorescence. LXR mRNA levels were decreased by 49%-98% in NOA specimens and positively correlated with germ cell number. Accumulations of IDOL and SREBP1c (LXR targets involved in lipid homeostasis) were 1.8-2.1 times lower in NOA samples and mRNA levels of the SREBP1c target gene ELOVL6 were increased 1.9-2.4-fold. Interestingly, the amount of triglycerides and free fatty acids were higher in NOA testes (3.4-12.2-fold). LXRα was present in Leydig cells. Accumulations of LXR downstream genes encoding the steroidogenic proteins StAR and 3βHSD2 were higher in NOA testes (5.9-12.8-fold). Knowledge of changes in the transcript levels of LXRs and some of their downstream genes during altered spermatogenesis may help us to better understand the physiopathology of testicular failure in azoospermic patients. Show less

Lipid deposition in artery walls is implied in the pathogenesis of atherosclerosis and imbalance between uptake and efflux of cholesterol favors the deposition. We investigated the effect of vitamin E with the same dose and duration on the different stages of atherosclerosis in Apolipoprotein E knockout (ApoE KO) mice and explored the potential mechanisms. The results showed that the ApoE KO mouse spontaneously develops atherosclerosis in an age-dependent manner from 14 to 46 weeks on the regular chow. Vitamin E (100 mg/kg) supplementation to ApoE KO mice at 6, 14, and 22 weeks for 8 weeks significantly reduced the atherosclerotic lesion area by 41, 29 and 19% respectively compared to the age-matched control mice; however had no significant effect on the lesion when given at 30 and 38 weeks. In addition, vitamin E supplemented at the ages from 6 to 30 weeks decreased the contents of serum oxLDL and TBARS without affecting the TC and TAG contents in serum and liver. Furthermore, vitamin E supplemented at 6, 14 and 22 weeks down-regulated vasculature mRNA expressions of scavenger receptor CD36 and up-regulated mRNA expressions of PPARγ, LXRα and ABCA1 which are involved in reverse cholesterol transportation; however had no significant effects on these genes when given at 30 and 38 weeks. In conclusion, vitamin E with same dose and duration inhibits the early but not advanced atherosclerotic lesion in ApoE KO mice by anti-oxidation and regulation of mRNA expression of genes involved in cholesterol uptake and efflux, which favors the improvement of atherosclerosis. Show less

Ezetimibe is a potent inhibitor of Niemann-Pick type C1-Like 1 and has been approved for the treatment of hypercholesterolemia. Our preliminary study showed that ezetimibe promotes cholesterol efflux from vascular smooth muscle cells (VSMCs). Our aim was to investigate the cellular mechanisms underlying the ezetimibe actions. Rat VSMCs were converted to foam cells by incubation with cholesterol:methyl-β-cyclodextrin. The intracellular free cholesterol, total cholesterol, and the ratio of cholesteryl ester to total cholesterol were decreased after the incubation of VSMCs with different concentrations of ezetimibe (3, 10, 30, and 30 μmol/l) or treated with 30 μmol/l of ezetimibe for different time periods (6, 12, 24, and 48 h). Our results also showed that the expression of caveolin-1, liver X receptor α, and ATP-binding cassette transporter ABCA1 was enhanced, but the expression of nSREBP-1c was decreased in a concentration- and time-dependent manner. RNA interference was used to determine the roles of caveolin-1 and SREBP-1 in the lipid-lowering effect of ezetimibe. The results showed that caveolin-1 was involved in the regulation of intracellular cholesterol content, and the expression of caveolin-1 was repressed by SREBP-1. The present study indicates that ezetimibe protects VSMCs from cholesterol accumulation by regulating the expression of lipid metabolism-related genes. Show less

Liver X receptor α (LXRα) plays an important role in the cholesterol metabolism process, and LXRα activation can reduce atherosclerosis. In the present study, using an LXRα-GAL4 luciferase reporter screening, we discovered IMB-170, a structural analog of quinazolinone, which showed potent LXRα agonistic activity. IMB-170 significantly activated LXRα, with an EC50 value of 0.27μM. Interestingly, IMB-170 not only increased the expression of ATP-binding cassette transporter A1 (ABCA1) and G1 (ABCG1), which are related to the reverse cholesterol transport (RCT) process, but also influenced the expression levels of other genes involved in the cholesterol metabolism pathway in many cell lines. Moreover, IMB-170 significantly reduced cellular lipid accumulation and increased cholesterol efflux from RAW264.7 and THP-1 macrophages. Interestingly, compared with TO901317, IMB-170 only slightly increased protein expression levels of lipogenesis-related genes in HepG2 cells, indicating that IMB-170 may have a lower lipogenesis side effect in vivo. These results suggest that IMB-170 showed the selective agonistic activity for LXRα. Moreover, compared with full LXR-agonists, IMB-170 possesses a differential ability to recruit coregulators. This suggests that IMB-170 has distinct interactions with the active sites in the LXRα ligand-binding domain. In summary, IMB-170 is a novel partial LXRα agonist without the classical lipogenesis side effects, which could be used as a potential anti-atherosclerotic leading compound in the future. Show less

Gestational diabetes (GD) alters normal fetal development and is related to a diabetogenic effect in the progeny. Liver X receptors (LXRs) are considered to be potential drug targets for the regulation, treatment, or prevention of diabetes. The aim of this study was to evaluate early and late changes of LXR in the hippocampus and hypothalamus of the male and female offspring of control (CO) and diabetic (DO) mothers. We used an experimental model of streptozotocin-induced GD to assess the protein expression of LXRα (NR1H3) and LXRβ (NR1H2) by western blotting. The tissues were obtained from CO and DO animals at postnatal day 1 (1D), day 10 (10D), and day 35 (35D) and 9 months (9M). In CO, the LXR expression showed significant differences among the groups, which were tissue- and receptor-specific (P<0.05). Sex differences in CO were found only in the hypothalamus for LXRβ expression at 35D and 9M (P<0.05). When CO and DO were compared, differences between them were observed in the majority of the studied groups at 1D (male hippocampus, LXRα 31% and LXRβ 161%; female hippocampus, LXRβ 165%; male hypothalamus, LXRβ 182%; and female hypothalamus, LXRα 85%; P<0.05). However, these differences disappeared later with the exception of LXRβ expression in the male hypothalamus (P<0.05). The area under the curve during the glucose tolerance test correlated negatively with LXRβ in CO but not in DO animals. Moreover, in a male DO subpopulation this correlation was positive as it occurs in intolerant animals. These results indicate that GD affects hypothalamic LXR expression differently in male and female offspring. Show less

The objective of this study was to determine the effects of pentraxin3 (PTX3) on human oxidized low density lipoprotein (oxLDL) uptake and cholesterol efflux from human macrophage foam cells, which may play a critical role in atherogenesis. The effects of PTX3 on oxLDL uptake and cholesterol efflux were determined after transfection of human THP-1 macrophages with pSG5hPTX3 or PTX3siRNA plasmids. To evaluate the role of specific signaling pathways, human THP-1 cells were pre-treated with inhibitors of the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), phosphatidylinositide 3-kinases (PI3-K), and p38 mitogen-activated protein kinase (MAPK) pathways (PD98059, LY294002, and SB203580, respectively), and then exposed to oxLDL for the uptake assay or oxLDL and [(3)H]-cholesterol and apolipoprotein A-I (apoA-I) for the cholesterol efflux assay. PTX3 overexpression not only promoted oxLDL uptake but also significantly reduced cholesterol efflux to apoA-I; it also significantly decreased the expression of peroxisome proliferator-activated receptor-γ (PPARγ), liver X receptor alpha (LXRα) and ATP-binding membrane cassette transporter A-1 (ABCA1), which was increased with PTX3 silencing. Furthermore, PTX3 significantly increased p-ERK1/2 levels in THP-1-derived foam cells, and inhibition of ERK1/2 by PD98059 significantly reduced the oxLDL uptake and promoted the cholesterol efflux induced by PTX3 overexpression. Here, we demonstrate that PTX3 affects lipid accumulation in human macrophages, increasing oxLDL uptake and inhibiting cholesterol efflux. That is the underlying possible mechanisms of PTX3 contribution to the progression of atherosclerosis. Show less

To examine the cholesterol efflux and the expressions of ATP-binding cassette transporter G1 (ABCG1) in macrophages of diabetic patients and the roles of liver-X receptor (LXR) in regulation of ABCG1 expressions. Blood was collected from patients with type 2 diabetes mellitus and healthy controls. The peripheral blood monocytes were differentiated into macrophages with macrophage colony stimulating factor (M-CSF). The cells were radio labeled with [(3)H] cholesterol and were performed with cholesterol efflux assays. Quantitative real-time PCR (qRT PCR) and Western blot were performed to measure the mRNA and protein expressions of ABCA1 and ABCG1. To test the effects of LXR on ABCG1 expressions, inhibition of LXRα and LXRβ by siRNA were performed. The DNA-protein complex of LXR and LXR element (LXRE) located in the promoter region of ABCG1 gene were detected with electrophery mobility supershift assay (EMSA). Macrophage ABCG1 expressions and high-density lipoprotein (HDL) induced cholesterol efflux were significantly reduced (19.0%±1.2% vs. 12.8%±3.6%, t=2.532, P=0.016) in the diabetic subjects whereas ABCA1 expressions and apolipoprotein A1 (ApoA1) induced cholesterol efflux were comparable (12.0%±1.2% vs. 10.2%±2.3%, t=1.771, P=0.109) between the diabetic patients and healthy subjects. The mRNA expressions of LXRα and LXRβ had no changes between the diabetes subjects and healthy controls (t=1.025, P=0.315; t=-0.531, P=0.600). The LXR-LXRE DNA-protein complex detected by EMSA were also similar between the diabetes subjects and healthy controls (t=1.483, P=0.164). Moreover, ABCG1 expressions were not altered by inhibition of LXRα/β siRNA (t=2.143, P=0.061). Our data indicated that expression of ABCG1 and HDL induced cholesterol efflux were reduced in type 2 diabetic patients. However, the LXR mRNA expression and binding complex of LXR and ABCG1 promoter were not changed. The impairment of cholesterol efflux and ABCG1 gene expressions might be regulated via an LXR-independent pathway. Show less

Cholesterol efflux from macrophages is a critical mechanism to prevent the development of atherosclerosis. Although PPARγ is known to be a potent sterol sensor that play a fundamental role in cholesterol metabolism, the potential effects of PPARγ responsive miRNA still need to be revealed. In this study, we found that miR-613 is inversely correlated with LXRα and ABCA1 in PPARγ activated THP-1 cells. PPARγ negatively regulates the expression of miR-613 at transcriptional level, and miR-613 suppressed LXRα and ABCA1 by targeting the 3'-UTR of their mRNAs. Furthermore, downregulation of LXRα and ABCA1 by miR-613 inhibited cholesterol efflux from PPARγ activated THP-1 macrophages. These results revealed an alternative mechanism for PPARγ regulation and provided a potential target for the treatment of cholesterol metabolic diseases. Show less

Liver X receptors (LXRs)-mediated signals in acanthoic acid (AA) ameliorating liver fibrosis were examined in carbon tetrachloride (CCl4)-induced mice and TGF-β stimulated hepatic stellate cells (HSCs). AA was isolated from the root of Acanthopanax koreanum Nakai (Araliaceae). CCl4-treated mice were intraperitoneally injected with 10% CCl4 in olive oil (2 mL/kg for 8 weeks). In AA treated groups, mice were intragastrically administrated with AA (20 mg/kg or 50 mg/kg) 3 times per week for 8 weeks. Administration of AA reduced serum aminotransferase and tissue necrosis factor-α (TNF-α) levels evoked by CCl4, and the reverse of liver damage was further confirmed by histopathological staining. Administration of AA reduced the expression of fibrosis markers and regulated the ratio of MMP-13/TIMP-1, further reversed the development of liver fibrosis. TGF-β (5 ng/ml) was added to activate HSC-T6 cells for 2 h, and then treated with AA (1, 3, or 10 μmol/l) for 24 h before analysis. Cells were collected and proteins were extracted to detect the expressions of LXRs. AA could inhibit the expression of α-SMA stimulated by TGF-β and increase the expression of LXRβ. In vivo and in vitro experiments, AA could modulate liver fibrosis induced by CCl4-treatment via activation of LXRα and LXRβ, while inhibit HSCs activation only via activation of LXRβ. Acanthoic acid might ameliorate liver fibrosis induced by CCl4 via LXRs signals. Show less

To investigate associations of single nucleotide polymorphisms (SNPs) rs2228314 of sterol regulatory element-binding protein-2 (SREBP-2) or rs11039155 of liver X receptor α (LXRα) with susceptibility to polycystic ovary syndrome (PCOS) in a Chinese Han population. SREBP-2 rs2228314 and LXRα rs11039155 polymorphisms were genotyped in patients with PCOS and age- and sex-matched PCOS-free controls from a Chinese Han population. A total of 605 patients with PCOS and 615 controls were recruited in this study. We found that GC and CC genotypes of rs2228314, and variant C, were associated with a significantly increased risk of PCOS. In addition, GA and AA genotypes of rs11039155, as well as variant A, were also associated with a significantly increased risk of PCOS. Our results showed that SREBP-2 rs2228314 G to C change and variant C genotype as well as LXRα rs11039155 G to A change and variant A may contribute to PCOS in Chinese Han population. Show less

The Liver X receptors (LXRs), Liver X receptor A (LXRA) and Liver X receptor B (LXRB), regulate lipid metabolism and antimicrobial response. LXRs have a crucial role in the control of Mycobacterium tuberculosis (M.tb). Lacking LXRs mice is more susceptibility to infection M.tb, developing higher bacterial burdens and an increase in the size and number of granulomatous lesions. We aimed to assess the associations between single nucleotide polymorphisms (SNPs) in LXRs and risk of tuberculosis. We sequenced the LXRs genes to detect SNPs and to examine genotypic frequencies in 600 patients and 620 healthy controls to investigate for associations with tuberculosis (TB) in the Chinese Han population. DNA re-sequencing revealed eight common variants in the LXRs genes. The G allele of rs1449627 and the T allele of rs1405655 demonstrated an increased risk of developing TB (p<0.001, p = 0.002), and the T allele of rs3758673, the T allele of rs2279238, and the C allele of rs1449626 in LXRA and the C allele of rs17373080, the G allele of rs2248949, and the C allele of rs1052677 in LXRB were protective against TB patients compared to healthy controls (p = 0.0002, p = 0.006, p<0.001, p = 0.004, p = 0.008, p = 0.003, respectively). All SNP genotypes were significantly associated with TB. An estimation of the frequencies of haplotypes revealed two potential risk haplotypes,GGCG in LXRB (p = 0.004,) and TTCG in LXRA (p<0.001, p = 0.004). Moreover, three protective haplotypes, TTAT and CCAT in LXRA and CATC in LXRB, were significantly "protective" (p = 0.008, p<0.001, p = 0.031) for TB. Furthermore, we determined that the LXRs SNPs were nominally associated with the clinical pattern of disease. Our study data supported that LXRs play a fundamental role in the genetic susceptibility to TB and to different clinical patterns of disease. Thus, further investigation is required in larger populations and in additional areas. Show less