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International audience; Background and Aims; Genetically modified mice have been used extensively to study human disease. However, the data gained are not always translatable to humans because of major species differences. Liver‐humanized mice (LHM) are considered a promising model to study human hepatic and systemic metabolism. Therefore, we aimed to further explore their lipoprotein metabolism and to characterize key hepatic species‐related, physiological differences.Approach and Results: Fah−/−, Rag2−/−, and Il2rg−/− knockout mice on the nonobese diabetic (FRGN) background were repopulated with primary human hepatocytes from different donors. Cholesterol lipoprotein profiles of LHM showed a human‐like pattern, characterized by a high ratio of low‐density lipoprotein to high‐density lipoprotein, and dependency on the human donor. This pattern was determined by a higher level of apolipoprotein B100 in circulation, as a result of lower hepatic mRNA editing and low‐density lipoprotein receptor expression, and higher levels of circulating proprotein convertase subtilisin/kexin type 9. As a consequence, LHM lipoproteins bind to human aortic proteoglycans in a pattern similar to human lipoproteins. Unexpectedly, cholesteryl ester transfer protein was not required to determine the human‐like cholesterol lipoprotein profile. Moreover, LHM treated with GW3965 mimicked the negative lipid outcomes of the first human trial of liver X receptor stimulation (i.e., a dramatic increase of cholesterol and triglycerides in circulation). Innovatively, LHM allowed the characterization of these effects at a molecular level.Conclusions: LHM represent an interesting translatable model of human hepatic and lipoprotein metabolism. Because several metabolic parameters displayed donor dependency, LHM may also be used in studies for personalized medicine.

The initiation and progression of autoimmune disorders such as multiple sclerosis (MS) is linked to aberrant cholesterol metabolism and overt inflammation. Liver X receptors (LXR) are nuclear receptors that function at the crossroads of cholesterol metabolism and immunity, and their activation is considered a promising therapeutic strategy to attenuate autoimmunity. However, despite clear functional heterogeneity and cell-specific expression profiles, the impact of the individual LXR isoforms on autoimmunity remains poorly understood. Here, we show that LXR alpha and LXR beta have an opposite impact on immune cell function and disease severity in the experimental autoimmune encephalomyelitis model, an experimental MS model. While Lxr alpha deficiency aggravated disease pathology and severity, absence of Lxr beta was protective. Guided by flow cytometry and by using cell-specific knockout models, reduced disease severity in Lxr beta-deficient mice was primarily attributed to changes in peripheral T cell physiology and occurred independent from alterations in microglia function. Collectively, our findings indicate that LXR isoforms play functionally non-redundant roles in autoimmunity, potentially having broad implications for the development of LXR-based therapeutic strategies aimed at dampening autoimmunity and neuroinflammation.

Summary Non-alcoholic fatty liver disease (NAFLD) is a very common indication for liver transplantation. How fat-rich diets promote progression from fatty liver to more damaging inflammatory and fibrotic stages is poorly understood. Here, we show that disrupting phosphorylation at Ser196 (S196A) in the liver X receptor alpha (LXRα, NR1H3) retards NAFLD progression in mice on a high-fat-high-cholesterol diet. Mechanistically, this is explained by key histone acetylation (H3K27) and transcriptional changes in pro-fibrotic and pro-inflammatory genes. Furthermore, S196A-LXRα expression reveals the regulation of novel diet-specific LXRα-responsive genes, including the induction of Ces1f, implicated in the breakdown of hepatic lipids. This involves induced H3K27 acetylation and altered LXR and TBLR1 cofactor occupancy at the Ces1f gene in S196A fatty livers. Overall, impaired Ser196-LXRα phosphorylation acts as a novel nutritional molecular sensor that profoundly alters the hepatic H3K27 acetylome and transcriptome during NAFLD progression placing LXRα phosphorylation as an alternative anti-inflammatory or anti-fibrotic therapeutic target., Graphical Abstract, Highlights • LXRαS196A induces liver steatosis and prevents cholesterol accumulation • LXRαS196A reduces progression to hepatic inflammation and fibrosis • LXRαS196A modulates hepatic chromatin acetylation • LXRαS196A reveals unique dual LXRα phosphorylation and diet-responsive genes, Progression to inflammatory and fibrotic steatohepatitis is poorly understood. Becares et al. reveal that disrupting LXRα phosphorylation attenuates these processes by promoting a unique diet-induced transcriptome that prevents cholesterol accumulation and reduces hepatic inflammation and fibrosis. Mechanistically, phospho-deficient LXRα promotes chromatin modifications and alters protein-protein interactions in differentially expressed genes.

Liver X receptor (LXR)α and LXRβ play key roles in hepatic de novo lipogenesis through their regulation of lipogenic genes, including sterol regulatory element-binding protein (SREBP)-1c and carbohydrate responsive element-binding protein (ChREBP). LXRs activate lipogenic gene transcription in response to feeding, which is believed to be mediated by insulin. We have previously shown that LXRs are targets for glucose-hexosamine-derived O-linked β-N-acetylglucosamine (O-GlcNAc) modification enhancing their ability to regulate SREBP-1c promoter activity in vitro. To elucidate insulin-independent effects of feeding on LXR-mediated lipogenic gene expression in vivo, we subjected control and streptozotocin-treated LXRα/β(+/+) and LXRα/β(-/-) mice to a fasting-refeeding regime. We show that under hyperglycemic and hypoinsulinemic conditions, LXRs maintain their ability to upregulate the expression of glycolytic and lipogenic enzymes, including glucokinase (GK), SREBP-1c, ChREBPα, and the newly identified shorter isoform ChREBPβ. Furthermore, glucose-dependent increases in LXR/retinoid X receptor-regulated luciferase activity driven by the ChREBPα promoter was mediated, at least in part, by O-GlcNAc transferase (OGT) signaling in Huh7 cells. Moreover, we show that LXR and OGT interact and colocalize in the nucleus and that loss of LXRs profoundly reduced nuclear O-GlcNAc signaling and ChREBPα promoter binding activity in vivo. In summary, our study provides evidence that LXRs act as nutrient and glucose metabolic sensors upstream of ChREBP by modulating GK expression, nuclear O-GlcNAc signaling, and ChREBP expression and activity.

We investigated whether: (1) liver X receptor (LXR)-driven induction of high-density lipoprotein cholesterol (HDL-C) and other LXR-mediated effects on cholesterol metabolism depend on intestinal cholesterol absorption; and (2) combined treatment with the LXR agonist GW3965 and the cholesterol absorption inhibitor ezetimibe results in synergistic effects on cholesterol metabolism that could be beneficial for treatment of atherosclerosis. Mice were fed 0.2 % cholesterol and treated with GW3965+ezetimibe, GW3965 or ezetimibe. GW3965+ezetimibe treatment elevated serum HDL-C and Apolipoprotein (Apo) AI, effectively reduced the intestinal cholesterol absorption and increased the excretion of faecal neutral sterols. No changes in intestinal ATP-binding cassette (ABC) A1 or ABCG5 protein expression were observed, despite increased mRNA expression, while hepatic ABCA1 was slightly reduced. The combined treatment caused a pronounced down-regulation of intestinal Niemann-Pick C1-like 1 (NPC1L1) and reduced hepatic and intestinal cholesterol levels. GW3965 did not affect the intestinal cholesterol absorption, but increased serum HDL-C and ApoAI levels. GW3965 also increased Apoa1 mRNA levels in primary mouse hepatocytes and HEPA1-6 cells. Ezetimibe reduced the intestinal cholesterol absorption, ABCA1 and ABCG5, but did not affect the serum HDL-C or ApoAI levels. Thus, the LXR-driven induction of HDL-C and ApoAI was independent of the intestinal cholesterol absorption and increased expression of intestinal or hepatic ABCA1 was not required. Inhibited influx of cholesterol via NPC1L1 and/or low levels of intracellular cholesterol prevented post-transcriptional expression of intestinal ABCA1 and ABCG5, despite increased mRNA levels. Combined LXR activation and blocked intestinal cholesterol absorption induced effective faecal elimination of cholesterol.

11Beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD-1) converts inactive corticosteroids into biologically active corticosteroids, thereby regulating the local concentration of active glucocorticoids, such as cortisol. 11beta-HSD-1 is particularly expressed in adipocytes and liver and appears to be causally linked to the development of type 2 diabetes and the metabolic syndrome. Liver X receptor (LXR)-alpha and -beta are nuclear oxysterol receptors whose key role in lipid metabolic regulation has recently been established. In this study, we show that treatment of adipocytes derived from 3T3-L1 cells and mouse embryonic fibroblasts in vitro with synthetic or natural LXR agonists decreases mRNA expression of 11beta-HSD-1 by approximately 50%, paralleled by a significant decline in 11beta-HSD-1 enzyme activity. Downregulation of 11beta-HSD-1 mRNA by LXRs started after a lag period of 8 h and required ongoing protein synthesis. Moreover, long-term per os treatment with a synthetic LXR agonist downregulated 11beta-HSD-1 mRNA levels by approximately 50% in brown adipose tissue and liver of wild-type but not of LXRalpha(-/-)beta(-/-) mice and was paralleled by downregulation of hepatic PEPCK expression. In conclusion, LXR ligands could mediate beneficial metabolic effects in insulin resistance syndromes including type 2 diabetes by interfering with peripheral glucocorticoid activation.

Liver X receptors (LXRs) are nuclear receptors with well-known functions in cholesterol transport, fatty acid and glucose metabolism, and modulation of immune responses. Natural and synthetic ligands have been identified and are under development for the treatment of metabolic and inflammatory conditions and diseases. There is mounting evidence pointing to functional roles for LXRs in a variety of malignancies and the potential therapeutic efficacy of their ligands.This review summarizes the discovery and characterization of LXRs and their ligands, surveys their effects and mechanisms of action in cell-based and animal models of cancer, and proposes the future direction of basic and translational studies of LXRs and their ligands in cancer research and therapeutics.Targeting LXRs is a promising strategy for cancer treatment, particularly for those cancers which do not have effective treatment options. Key questions remain, however, regarding the specific mechanisms of action, effects on other target cells within the tumor microenvironment, and receptor status in patient populations. Moreover, LXR ligands optimized for disease-specific functions and cancer-related endpoints are currently not available. These issues represent both challenges and significant opportunities for future research and development efforts.

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.

The organ content of the mevalonate pathway lipids was investigated in liver-X-receptor (LXR) α, β and double knock-out mice. An extensive or moderate increase of total cholesterol in the double KO mice was found in all organs elicited by the increase of the esterified form. In LXRα and double KO mice, coenzyme Q (CoQ) was decreased in liver and increased in spleen, thymus and lung, while dolichol was increased in all organs investigated. This effect was confirmed using LXR- agonist GW 3965. Analysis of CoQ distribution in organelles showed that the modifications are present in all cellular compartments and that the increase of the lipid in mitochondria was the result of a net increase of CoQ without changing the number of mitochondria. It appears that LXR influences not only cellular cholesterol homeostasis but also the metabolism of CoQ and dolichol, in an indirect manner.

The liver X receptors (LXRs) are nuclear receptors that form permissive heterodimers with retinoid X receptor (RXR) and are important regulators of lipid metabolism in the liver. We have recently shown that RXR agonist-induced hypertriglyceridemia and hepatic steatosis in mice are dependent on LXRs and correlate with an LXR-dependent hepatic induction of lipogenic genes. To further investigate the roles of RXR and LXR in the regulation of hepatic gene expression, we have mapped the ligand-regulated genome-wide binding of these factors in mouse liver. We find that the RXR agonist bexarotene primarily increases the genomic binding of RXR, whereas the LXR agonist T0901317 greatly increases both LXR and RXR binding. Functional annotation of putative direct LXR target genes revealed a significant association with classical LXR-regulated pathways as well as peroxisome proliferator-activated receptor (PPAR) signaling pathways, and subsequent chromatin immunoprecipitation-sequencing (ChIP-seq) mapping of PPARα binding demonstrated binding of PPARα to 71 to 88% of the identified LXR-RXR binding sites. The combination of sequence analysis of shared binding regions and sequential ChIP on selected sites indicate that LXR-RXR and PPARα-RXR bind to degenerate response elements in a mutually exclusive manner. Together, our findings suggest extensive and unexpected cross talk between hepatic LXR and PPARα at the level of binding to shared genomic sites.

The acute phase response (APR) classically refers to the rapid reprogramming of gene expression and metabolism in response to inflammatory cytokine signaling. As components of the innate immune system, hepatocyte-derived acute phase proteins (APPs) play a central role in restoring tissue homeostasis. Recently, an intriguing 'metaflammatory' facet of the APR became evident with chronically elevated APP levels being connected to metabolic syndrome disorders. The causality of these connections is unclear but could relate to adverse metabolic and inflammatory disturbances, particularly those affecting lipoprotein properties, cholesterol metabolism and atherogenesis. Here we review these aspects with an emphasis on the emerging importance of lipid-sensing nuclear receptors (LXRs, LRH-1, PPARs), in conjunction with anti-inflammatory transrepression pathways, as physiological and pharmacological relevant modulators of the APR.

The liver X receptors (LXRs) play a key role in cholesterol and bile acid metabolism but are also important regulators of glucose metabolism. Recently, LXRs have been proposed as a glucose sensor affecting LXR-dependent gene expression. We challenged wild-type (WT) and LXRαβ−/−mice with a normal diet (ND) or a high-carbohydrate diet (HCD). Magnetic resonance imaging showed different fat distribution between WT and LXRαβ−/−mice. Surprisingly, gonadal (GL) adipocyte volume decreased on HCD compared with ND in WT mice, whereas it slightly increased in LXRαβ−/−mice. Interestingly, insulin-stimulated lipogenesis of isolated GL fat cells was reduced on HCD compared with ND in LXRαβ−/−mice, whereas no changes were observed in WT mice. Net de novo lipogenesis (DNL) calculated from V̇o2and V̇co2was significantly higher in LXRαβ−/−than in WT mice on HCD. Histology of HCD-fed livers showed hepatic steatosis in WT mice but not in LXRαβ−/−mice. Glucose tolerance was not different between groups, but insulin sensitivity was decreased by the HCD in WT but not in LXRαβ−/−mice. Finally, gene expression analysis of adipose tissue showed induced expression of genes involved in DNL in LXRαβ−/−mice compared with WT animals as opposed to the liver, where expression of DNL genes was repressed in LXRαβ−/−mice. We thus conclude that absence of LXRs stimulates DNL in adipose tissue, but suppresses DNL in the liver, demonstrating opposite roles of LXR in DNL regulation in these two tissues. These results show tissue-specific regulation of LXR activity, a crucial finding for drug development.

Liver X receptors (LXRs) regulate immune cell function and cholesterol metabolism, both factors that are critically involved in Alzheimer's disease (AD). To investigate the therapeutic potential of long-term LXR activation in amyloid-β (Aβ) peptide deposition in an AD model, 13-month-old, amyloid plaque-bearing APP23 mice were treated with the LXR agonist TO901317. Postmortem analysis demonstrated that TO901317 efficiently crossed the blood–brain barrier. Insoluble and soluble Aβ levels in the treated APP23 mice were reduced by 80% and 40%, respectively, compared with untreated animals. Amyloid precursor protein (APP) processing, however, was hardly changed by the compound, suggesting that the observed effects were instead mediated by Aβ disposal. Despite the profound effect on Aβ levels, spatial learning in the Morris water maze was only slightly improved by the treatment. ABCA1 (ATP-binding cassette transporter 1) and apolipoprotein E (ApoE) protein levels were increased and found to be primarily localized in astrocytes. Experiments using primary microglia demonstrated that medium derived from primary astrocytes exposed to TO901317 stimulated phagocytosis of fibrillar Aβ. Conditioned medium from TO901317-treated ApoE−/−or LXRα−/−astrocytes did not increase phagocytosis of Aβ. In APP23 mice, long-term treatment with TO901317 strongly increased the association of microglia and Aβ plaques. Short-term treatment of APP/PS1 mice with TO901317 also increased this association, which was dependent on the presence of LXRα and was accompanied by increased ApoE lipidation. Together, these data suggest that astrocytic LXRα activation and subsequent release of ApoE by astrocytes is critical for the ability of microglia to remove fibrillar Aβ in response to treatment with TO901317.

The orphan receptor LRH-1 and the oxysterol receptors LXRα and LXRβ are established transcriptional regulators of lipid metabolism that appear to control inflammatory processes. Here, we investigate the anti-inflammatory actions of these nuclear receptors in the hepatic acute phase response (APR). We report that selective synthetic agonists induce SUMOylation-dependent recruitment of either LRH-1 or LXR to hepatic APR promoters and prevent the clearance of the N-CoR corepressor complex upon cytokine stimulation. Investigations of the APR in vivo, using LXR knockout mice, indicate that the anti-inflammatory actions of LXR agonists are triggered selectively by the LXRβ subtype. We further find that hepatic APR responses in small ubiquitin-like modifier-1 (SUMO-1) knockout mice are increased, which is due in part to diminished LRH-1 action at APR promoters. Finally, we provide evidence that the metabolically important coregulator GPS2 functions as a hitherto unrecognized transrepression mediator of interactions between SUMOylated nuclear receptors and the N-CoR corepressor complex. Our study extends the knowledge of anti-inflammatory mechanisms and pathways directed by metabolic nuclear receptor–corepressor networks to the control of the hepatic APR, and implies alternative pharmacological strategies for the treatment of human metabolic diseases associated with inflammation.

Transcriptional coregulators, rather than ligand signals, are suspected to confer context and pathway specificity to nuclear receptor signaling, but the identity of such specifying coregulators and the underlying molecular mechanisms remain largely enigmatic. Here we address this issue in metabolic oxysterol receptor LXR pathways and describe the selective requirement of GPS2 for ABCG1 cholesterol transporter gene transcription and cholesterol efflux from macrophages. We implicate GPS2 in facilitating LXR recruitment to an ABCG1-specific promoter/enhancer unit upon ligand activation and identify functional links to histone H3K9 demethylation. We further describe fundamental differences between ABCG1 and ABCA1 with regard to GPS2 in relation to other coregulators, which are likely to apply to additional LXR-regulated genes. Our work identifies a coregulator-dependent epigenetic mechanism governing the access of a nuclear receptor to communicating regulatory regions in the genome. The pathway and coregulator selectivity of this mechanism implies pharmacological possibilities for the development of selective LXR agonists.

RAP250 is a coactivator for nuclear receptors as well as other transcription factors. Recent studies have established RAP250 as an essential coactivator for many important biological processes, but its exact mechanism of action is not fully understood. To identify novel proteins that can associate with RAP250, we used a yeast two-hybrid system to screen cDNA libraries and identified the intracellular mediators of transforming growth factor-beta (TGF-beta) response Smad2 and Smad3 as direct interacting proteins. We show that the interaction between RAP250 and Smad2/3 is dependent upon the second LXXLL interaction motif in RAP250 and the MH2 domain in Smad2 and Smad3. Mouse embryonic fibroblasts lacking RAP250 have reduced expression of the TGF-beta target gene PAI-1 after stimulation by TGF-beta when compared with wild type cells. Furthermore, we demonstrate a cross-talk between TGF-beta and liver X receptors (LXR) signaling pathways and show that stimulation of cells with TGF-beta and LXR agonists have a synergistic effect on the expression of the LXR target gene ABCG1. Our data identify RAP250 as a new coactivator in the TGF-beta signaling pathway that binds Smad2 and Smad3. Our data also suggest that the interaction between RAP250, Smad2, and Smad3 constitutes an important bridging mechanism linking LXR and TGF-beta signaling pathways.

The nuclear receptors LXRalpha (NR1H3) and LXRbeta (NR1H2) are attractive drug targets for the treatment of diabetes and cardiovascular disease due to their established role as regulators of cholesterol and lipid metabolism. A large body of literature has recently indicated their important roles in glucose metabolism and particularly LXRbeta is important for proper insulin production in pancreas. In this study, we report that glucose induces transcription via the LXRB gene promoter. The transcription start site of the human LXRB gene was determined and we identified two highly conserved, and functional, ETS and Elk1 binding sites, respectively, in the LXRB gene promoter. The Elk1 binding site also bound the serum responsive factor (SRF). Mutation of these sites abolished binding. Furthermore, mutation of the binding sites or siRNA knockdown of SRF and Elk1 significantly reduced the promoter activity and impaired the glucose response. Our results indicate that the human LXRB gene is controlled by glucose, thereby providing a novel mechanism by which glucose regulates cellular functions via LXRbeta.

The nuclear hormone receptors liver X receptor α (LXRα) (NR1H3) and LXRβ (NR1H2) are established regulators of cholesterol, lipid, and glucose metabolism and are attractive drug targets for the treatment of diabetes and cardiovascular disease. Adrenal steroid hormones including glucocorticoids and mineralocorticoids are known to interfere with glucose metabolism, insulin signaling, and blood pressure regulation. Here we present genome-wide expression profiles of LXR-responsive genes in both the adrenal and the pituitary gland. LXR activation in cultured adrenal cells inhibited expression of multiple steroidogenic genes and consequently decreased adrenal steroid hormone production. In addition, LXR agonist treatment elevated ACTH mRNA expression and hormone secretion from pituitary cells both in vitro and in vivo. Reduced expression of the glucocortioid-activating enzyme 11β-hydroxysteroid dehydrogenase 1 in pituitary cells upon LXR activation suggests blunting of the negative feedback of glucocorticoids by LXRs. In conclusion, LXRs independently interfere with the hypothalamic-pituitary-adrenal axis regulation at the level of the pituitary and the adrenal gland.

Nuclear hormone receptor liver X receptor-alpha (LXRα) plays a vital role in cholesterol homeostasis and is reported to play a role in adipose function and obesity although this is controversial. Conversely, mesenchymal stem cells (MSCs) are suggested to be a major source of adipocyte generation. Accordingly, we examined the role of LXRα in adipogenesis of MSCs. Adult murine MSCs (mMSCs) were isolated from wild type (WT) and LXR-null mice. Using WT mMSCs, we further generated cell lines stably overexpressing GFP-LXRα (mMSC/LXRα/GFP) or GFP alone (mMSC/GFP) by retroviral infection. Confluent mMSCs were differentiated into adipocytes by the established protocol. Compared with MSCs isolated from WT mice, MSCs from LXR-null mice showed significantly increased adipogenesis, as determined by lipid droplet accumulation and adipogenesis-related gene expression. Moreover, mMSCs stably overexpressing GFP-LXRα (mMSC/LXRα/GFP) exhibited significantly decreased adipogenesis compared with mMSCs overexpressing GFP alone (mMSC/GFP). Since Wnt/beta-catenin signaling is reported to inhibit adipogenesis, we further examined it. The LXR-null group showed significantly decreased Wnt expression accompanied by a decrease of cellular beta-catenin (vs. WT). The mMSC/LXRα/GFP group exhibited significantly increased Wnt expression accompanied by an increase of cellular beta-catenin (vs. mMSC/GFP). These data demonstrate that LXRα has an inhibitory effect on adipogenic differentiation in murine mesenchymal stem cells with Wnt/beta-catenin signaling. These results provide important insights into the pathophysiology of obesity and obesity related consequences such as metabolic syndrome and may identify potential therapeutic targets.

Cholesterol precursors act as activators of the nuclear hormone receptor, liver X receptor (LXR). One of these LXR-activating ligands is meiosis activating sterol (MAS), which also induces resumption of meiosis in oocytes from mice in vitro. Whether LXR participates in the regulation of oocyte maturation and whether the expression of either one of the two paralogues of LXR (alpha and beta) affect fertility of mice has, however, not yet been clarified. Female mice lacking Lxra, Lxrb or both genes (Lxra(-/-), Lxrb(-/-) and Lxrab(-/-), respectively) conceive less frequently and have significantly fewer pups per litter as compared to wild type mice. Both Lxra and Lxrb mRNA were found to be expressed in mouse oocytes. The relative expression of, in particular, Lxrb was almost two orders of magnitude higher than in liver, brain and testis. A water-soluble LXR agonist caused naked oocytes, but not cumulus enclosed oocytes (CEO), from wild type mice to resume meiosis significantly more often than control oocytes. Follicle stimulating hormone (FSH) is a potent stimulator of meiosis in CEO from wild type mice, but was without effect in mice lacking both Lxr genes. Zymosterol, a MAS active substance, induced resumption of meiosis in oocytes from Lxrab(-/-) mice, but significantly less effectively than in oocytes from wild type mice. Taken together, LXRs seem to affect ovarian function, suggesting specific roles of cholesterol precursors in regulation of female reproduction.

The promotion of lipid efflux from macrophages is an important ATP binding cassette A1 (ABCA1)-mediated antiatherosclerotic mechanism that prevents peripheral tissues from foam cell accumulation. Statins exert beneficial antiatherosclerotic effects on cardiovascular disease correlated to the cholesterol-lowering properties and the pleiotropic activities. In this work, we investigated the ability of statins to modulate ABCA1-mediated lipid efflux from macrophages, where the protein expression was differently induced. Pitavastatin (0.1-10 microM) and compactin (10 microM) reduced both cholesterol and phospholipid efflux up to 60% from macrophages expressing ABCA1 upon treatment with 8-(4-chlorophenylthio)-cyclic AMP (cpt-cAMP), and this was secondary to a reduction of ABCA1 mRNA and protein content. Conversely, statins did not affect ABCA1 activity when the protein was up-regulated by 22-hydroxycholesterol/9-cis-retinoic acid or through cholesterol loading. Statin inhibition of lipid efflux induced by cpt-cAMP was reversed in the presence of mevalonate, 22-hydroxycholesterol, and cholesterol but not geranyl geraniol. In macrophages obtained from liver X receptor (LXR)-deficient mice, cpt-cAMP still promoted cholesterol efflux, but pitavastatin did not exert any effect. The present work shows that statins may inhibit ABCA1-mediated lipid efflux in macrophages only when ABCA1 protein expression is induced by cpt-cAMP and provides evidence that cAMP may activate ABCA1 independently of an increase of intracellular sterol synthesis but through at least two pathways: one independent of LXR and one involving an intracellular sterol(s) acting as LXR ligand(s). In addition, the lack of inhibitory effect on lipid efflux in cholesterol-loaded macrophages is likely to exclude a potential negative pleiotropic effect by statins.

The nuclear receptors liver X receptor (LXR)alpha and LXRbeta are sensors of cholesterol metabolism and lipid biosynthesis. They have recently been found to be regulators of inflammatory cytokines, suppressors of hepatic glucose production, and involved in different cell-signaling pathways. LXRalpha is a target gene of the peroxisome proliferator-activated receptor-gamma, a target of drugs used in treating elevated levels of glucose seen in diabetes. Furthermore, insulin induces LXRalpha in hepatocytes, resulting in increased expression of lipogenic enzymes and suppression of key enzymes in gluconeogenesis, including PEPCK. LXR seems to have an important role in the regulation of glucocorticoid action and a role in the overall energy homeostasis suggested by its putative regulatory effect on leptin and uncoupling protein 1. The physiological roles of LXR indicate that it is an interesting potential target for drug treatment of diabetes.

Pancreatic ductal adenocarcinoma (PDAC) is difficult to detect early and is often resistant to standard chemotherapeutic options, contributing to extremely poor disease outcomes. Members of the nuclear receptor superfamily carry out essential biological functions such as hormone signaling and are successfully targeted in the treatment of endocrine-related malignancies. Liver X receptors (LXRs) are nuclear receptors that regulate cholesterol homeostasis, lipid metabolism, and inflammation, and LXR agonists have been developed to regulate LXR function in these processes. Intriguingly, these compounds also exhibit antiproliferative activity in diverse types of cancer cells. In this study, LXR agonist treatments disrupted proliferation, cell-cycle progression, and colony-formation of PDAC cells. At the molecular level, treatments downregulated expression of proteins involved in cell cycle progression and growth factor signaling. Microarray experiments further revealed changes in expression profiles of multiple gene networks involved in biological processes and pathways essential for cell growth and proliferation following LXR activation. These results establish the antiproliferative effects of LXR agonists and potential mechanisms of action in PDAC cells and provide evidence for their potential application in the prevention and treatment of PDAC.

Expression of the LXRalpha nuclear receptor in liver is predicted to affect cholesterol and lipid metabolism. Here we show that a short fragment from the LXRalpha gene promoter spanning the region from -144 to +43 relative to the mRNA initiation site can drive transcription of a reporter gene. Under basal conditions, in vitro DNase I footprinting demonstrated interaction between nuclear proteins and an NF1 recognition site in close vicinity to the transcriptional initiation. Both supershift, mutational analyses in EMSA and transfections provided evidence that the NF1 (nuclear factor I) transcription factor interacts with the LXRalpha promoter. All four members of the NF1 family were found to suppress the transcriptional activity indicating a general inhibitory effect on LXRalpha expression. A similar regulation by NF1 was also observed when using a fragment from the LXRalpha promoter extending up to position -3033 therefore giving the inhibitory effect of NF1 a significant impact on LXRalpha gene expression.

The liver X receptors (LXR)α and LXRβ are transcription factors belonging to the nuclear receptor family, which play a central role in metabolic homeostasis, being master regulators of key target genes in the glucose and lipid pathways. Wild-type (WT), LXRα−/−, and LXRβ−/−mice were fed a chow diet with (treated) or without (control) the synthetic dual LXR agonist GW3965 for 5 wk. GW3965 raised intrahepatic triglyceride (TG) level but, surprisingly, reduced serum TG level through the activation of serum lipase activity. The serum TG reduction was associated with a repression of both catecholamine-stimulated lipolysis and relative glucose incorporation into lipid in isolated adipocytes through activation of LXRβ. We also demonstrated that LXRα is required for basal (nonstimulated) adipocyte metabolism, whereas LXRβ acts as a repressor of lipolysis. On the contrary, in skeletal muscle (SM), the lipogenic and cholesterol transporter LXR target genes were markedly induced in WT and LXRα−/−mice and to a lesser extent in LXRβ−/−mice following treatment with GW3965. Moreover, TG content was reduced in SM of LXRβ−/−mice, associated with increased expression of the main TG-lipase genes Hsl and Atgl. Energy expenditure was increased, and a switch from glucose to lipid oxidation was observed. In conclusion, we provide evidence that LXR might be an essential regulator of the lipid balance between tissues to ensure appropriate control of the flux of fuel. Importantly, we show that, after chronic treatment with GW3965, SM becomes the target tissue for LXR activation, as opposed to liver, in acute treatment.

OR1 is a member of the superfamily of steroid/thyroid hormone nuclear receptors and recognizes DNA as a heterodimer with the 9-cis-retinoic acid receptor RXR (retinoid X receptor). The heterodimeric complex has been shown to be transcriptionally activatable by the RXR ligand as well as certain oxysterols via OR1, but to date uniquely also by heterodimerization itself. Recent studies on other members of the superfamily of nuclear receptors have led to the identification of a number of nuclear receptor-interacting proteins that mediate their regulatory effects on transcription. Here, we address the question of involvement of some of these cofactors in the three modes of activation by the OR1/RXRalpha complex. We show that in vitro the steroid receptor coactivator SRC-1 can be recruited by RXRalpha upon addition of its ligand, and to OR1 upon addition of 22(R)-OH-cholesterol, demonstrating that the latter can act as a direct ligand to OR1. Additionally, heterodimerization is sufficient to recruit SRC-1 to OR1/RXRalpha, indicating SRC-1 as a molecular mediator of dimerization-induced activation. In transfection experiments, coexpression of a nuclear receptor-interacting fragment of SRC-1 abolishes constitutive activation by OR1/RXRalpha, which can be restored by over-expression of full-length SRC-1. This constitutes evidence for an in vivo role of SRC-1 in dimerization-induced activation by OR1/RXRalpha. Additionally, we show that the nuclear receptor-interacting protein RIP140 binds in vitro to OR1 and RXRalpha with requirements distinct from those of SRC-1, and that binding of the two cofactors is competitive. Taken together, our results suggest a complex modulation of differentially induced transactivation by OR1/RXR coregulatory molecules.

Reversing the escalating rate of obesity requires increased knowledge of the molecular mechanisms controlling energy balance. Liver X receptors (LXRs) and thyroid hormone receptors (TRs) are key physiological regulators of energetic metabolism. Analysing interactions between these receptors in the periphery has led to a better understanding of the mechanisms involved in metabolic diseases. However, no data is available on such interactions in the brain. We tested the hypothesis that hypothalamic LXR/TR interactions could co-regulate signalling pathways involved in the central regulation of metabolism. Using in vivo gene transfer we show that LXR activation by its synthetic agonist GW3965 represses the transcriptional activity of two key metabolic genes, Thyrotropin-releasing hormone (Trh) and Melanocortin receptor type 4 (Mc4r) in the hypothalamus of euthyroid mice. Interestingly, this repression did not occur in hypothyroid mice but was restored in the case of Trh by thyroid hormone (TH) treatment, highlighting the role of the triiodothyronine (T3) and TRs in this dialogue. Using shLXR to knock-down LXRs in vivo in euthyroid newborn mice, not only abrogated Trh repression but actually increased Trh transcription, revealing a potential inhibitory effect of LXR on the Hypothalamic-Pituitary-Thyroid axis. In vivo chromatin immunoprecipitation (ChIP) revealed LXR to be present on the Trh promoter region in the presence of T3 and that Retinoid X Receptor (RXR), a heterodimerization partner for both TR and LXR, was never recruited simultaneously with LXR. Interactions between the TR and LXR pathways were confirmed by qPCR experiments. T3 treatment of newborn mice induced hypothalamic expression of certain key LXR target genes implicated in metabolism and inflammation. Taken together the results indicate that the crosstalk between LXR and TR signalling in the hypothalamus centres on metabolic and inflammatory pathways.

Introduction The two oxysterol receptors, 'liver X receptors (LXRs)' LXRα and LXRβ, are amongst the emerging newer drug targets within the nuclear receptor family and targeting LXRs represents novel strategies needed for prevention and treatment of diseases where current therapeutics is inadequate. Areas covered This review discusses the current understanding of LXR biology with an emphasis on the molecular aspects of LXR signalling establishing their potential as drug targets. Recent advances of their transcriptional mechanisms in inflammatory pathways and their physiological roles in inflammation and immunity are described. Expert opinion The new discoveries of LXR-regulated inflammatory pathways have ignited new promises for LXRs as drug targets. The broad physiological roles of LXRs involve a high risk of unwanted side effects. Recent insights into LXR biology of the brain indicate a highly important role in neuronal development and a clinical trial testing an LXR agonist reported adverse neurological side effects. This suggests that drug development must focus on limiting the range of LXR signalling - possibly achieved through subtype, tissue specific, promoter specific or pathway specific activation of LXRs where a successful candidate drug must be carefully studied for its effect in the central nervous system.

Liver X receptors (LXRs) exert key functions in lipid homeostasis and in control of inflammation. In this study we have explored the impact of LXR activation on the macrophage response to the endogenous inflammatory cytokine IFN-γ. Transcriptional profiling studies demonstrate that ∼38% of the IFN-γ–induced transcriptional response is repressed by LXR activation in macrophages. LXRs also mediated inhibitory effects on selected IFN-γ–induced genes in primary microglia and in a model of IFN-γ–induced neuroinflammation in vivo. LXR activation resulted in reduced STAT1 recruitment to the promoters tested in this study without affecting STAT1 phosphorylation. A closer look into the mechanism revealed that SUMOylation of LXRs, but not the presence of nuclear receptor corepressor 1, was required for repression of the NO synthase 2 promoter. We have also analyzed whether IFN-γ signaling exerts reciprocal effects on LXR targets. Treatment with IFN-γ inhibited, in a STAT1-dependent manner, the LXR-dependent upregulation of selective targets, including ATP-binding cassette A1 (ABCA1) and sterol response element binding protein 1c. Downregulation of ABCA1 expression correlated with decreased cholesterol efflux to apolipoprotein A1 in macrophages stimulated with IFN-γ. The inhibitory effects of IFN-γ on LXR signaling did not involve reduced binding of LXR/retinoid X receptor heterodimers to target gene promoters. However, overexpression of the coactivator CREB-binding protein/p300 reduced the inhibitory actions of IFN-γ on the Abca1 promoter, suggesting that competition for CREB-binding protein may contribute to STAT1-dependent downregulation of LXR targets. The results from this study suggest an important level of bidirectional negative cross-talk between IFN-γ/STAT1 and LXRs with implications both in the control of IFN-γ–mediated immune responses and in the regulation of lipid metabolism.

We examined the function of the oxysterol receptors (LXRs) in inflammatory bowel disease (IBD) through studying dextran sodium sulfate (DSS)- and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice and by elucidating molecular mechanisms underlying their anti-inflammatory action. We observed that Lxr-deficient mice are more susceptible to colitis. Clinical indicators of colitis including weight loss, diarrhea and blood in feces appeared earlier and were more severe in Lxr-deficient mice and particularly LXRβ protected against symptoms of colitis. Addition of an LXR agonist led to faster recovery and increased survival. In contrast, Lxr-deficient mice showed slower recovery and decreased survival. In Lxr-deficient mice, inflammatory cytokines and chemokines were increased together with increased infiltration of immune cells in the colon epithelium. Activation of LXRs strongly suppressed expression of inflammatory mediators including TNFα. While LXRα had anti-inflammatory effects in CD11b(+) immune cell populations, LXRβ in addition had anti-inflammatory effects in colon epithelial cells. Lack of LXRβ also induced CD4(+)/CD3(+) immune cell recruitment to the inflamed colon. Expression of both LXRA and LXRB was significantly suppressed in inflamed colon from subjects with IBD compared with non-inflamed colon. Taken together, our observations suggest that the LXRs could provide interesting targets to reduce the inflammatory responses in IBD.

Liver X receptor (LXR) activation stimulates triglyceride (TG) accumulation in the liver. Several lines of evidence indicate that estradiol-17β (E2) reduces TG levels in the liver; however, the molecular mechanism underlying the E2 effect remains unclear. Here, we show that administration of E2 attenuated sterol regulatory element-binding protein (SREBP)-1 expression and TG accumulation induced by LXR activation in mouse liver. In estrogen receptor alpha (ERα) knockout (KO) and liver-specific ERα KO mice, E2 did not affect SREBP-1 expression or TG levels. Molecular analysis revealed that ERα is recruited to the SREBP-1c promoter through direct binding to LXR and inhibits coactivator recruitment to LXR in an E2-dependent manner. Our findings demonstrate the existence of a novel liver-dependent mechanism controlling TG accumulation through the nonclassical ER/LXR pathway. To confirm that a nonclassical ER/LXR pathway regulates ERα-dependent inhibition of LXR activation, we screened ERα ligands that were able to repress LXR activation without enhancing ERα transcriptional activity, and, as a result, we identified the phytoestrogen, phloretin. In mice, phloretin showed no estrogenic activity; however, it did reduce SREBP-1 expression and TG levels in liver of mice fed a high-fat diet to an extent similar to that of E2.We propose that ER ligands reduce TG levels in the liver by inhibiting LXR activation through a nonclassical pathway. Our results also indicate that the effects of ER on TG accumulation can be distinguished from its estrogenic effects by a specific ER ligand.

Tumor-derived oxysterols recruit protumor neutrophils in an LXR-independent, CXCR2-dependent manner, thus favoring tumor growth by promoting neoangiogenesis and immunosuppression., Tumor-infiltrating immune cells can be conditioned by molecules released within the microenvironment to thwart antitumor immune responses, thereby facilitating tumor growth. Among immune cells, neutrophils play an important protumorigenic role by favoring neoangiogenesis and/or by suppressing antitumor immune responses. Tumor-derived oxysterols have recently been shown to favor tumor growth by inhibiting dendritic cell migration toward lymphoid organs. We report that tumor-derived oxysterols recruit protumor neutrophils in a liver X receptor (LXR)–independent, CXCR2-dependent manner, thus favoring tumor growth by promoting neoangiogenesis and immunosuppression. We demonstrate that interfering with the oxysterol–CXCR2 axis delays tumor growth and prolongs the overall survival of tumor-bearing mice. These results identify an unanticipated protumor function of the oxysterol–CXCR2 axis and a possible target for cancer therapy.

Multiple sclerosis is a chronic, inflammatory, demyelinating disease of the central nervous system in which macrophages and microglia play a central role. Foamy macrophages and microglia, containing degenerated myelin, are abundantly found in active multiple sclerosis lesions. Recent studies have described an altered macrophage phenotype after myelin internalization. However, it is unclear by which mechanisms myelin affects the phenotype of macrophages and how this phenotype can influence lesion progression. Here we demonstrate, by using genome wide gene expression analysis, that myelin-phagocytosing macrophages have an enhanced expression of genes involved in migration, phagocytosis and inflammation. Interestingly, myelin internalization also induced the expression of genes involved in liver-X-receptor signalling and cholesterol efflux. In vitro validation shows that myelin-phagocytosing macrophages indeed have an increased capacity to dispose intracellular cholesterol. In addition, myelin suppresses the secretion of the pro-inflammatory mediator IL-6 by macrophages, which was mediated by activation of liver-X-receptor β. Our data show that myelin modulates the phenotype of macrophages by nuclear receptor activation, which may subsequently affect lesion progression in demyelinating diseases such as multiple sclerosis. This work was supported by Agentschap voor Innovatie van Wetenschap en Technology (IWT),Fonds Wetenschappelijk Onderzoek (FWO), transnational University Limburg, Limburg Sterk Merk and Alma-in-Silico (EMR INT4.-1.3.-2008-03/003). This work was partially funded by the Interuniversity Attraction Poles Programme (IAP P6/25 BIOMAGNET), initiated by the Belgian State, Science Policy Office, by the French Community of Belgium (ARC Biomod), and by the European Network of Excellence PASCAL2. VAHT is recipient of a Fonds pour la formation a la Recherche dans l’Industrie et dans l’Agriculture (F.R.I.A.) fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding was received for this study.

Liver X receptors (Lxrα and Lxrβ) are ligand-dependent nuclear receptors critical for ventral midbrain neurogenesis in vivo. However, no endogenous midbrain Lxr ligand has so far been identified. Here we used LC/MS and functional assays to identify cholic acid as a new Lxr ligand. Moreover, 24(S),25-epoxycholesterol (24,25-EC) was found to be the most potent and abundant Lxr ligand in the developing mouse midbrain. Both Lxr ligands promoted neural development in an Lxr-dependent manner in zebrafish in vivo. Notably, each ligand selectively regulated the development of distinct midbrain neuronal populations. Whereas cholic acid increased survival and neurogenesis of Brn3a-positive red nucleus neurons, 24,25-EC promoted dopaminergic neurogenesis. These results identify an entirely new class of highly selective and cell type-specific regulators of neurogenesis and neuronal survival. Moreover, 24,25-EC promoted dopaminergic differentiation of embryonic stem cells, suggesting that Lxr ligands may thus contribute to the development of cell replacement and regenerative therapies for Parkinson's disease.

Liver X receptors (LXRs) are essential for the regulation of intestinal cholesterol absorption. Because two isoforms exist, LXRα and LXRβ, with overlapping but not identical functions, we investigated whether LXRα and LXRβ exert different effects on intestinal cholesterol absorption.Wild-type (WT), LXRα(-/-) and LXRβ(-/-) mice were fed control diet, 0.2% cholesterol-enriched diet or 0.2% cholesterol-enriched diet plus the LXR agonist GW3965.When fed a control diet, all three genotypes showed similar levels of cholesterol absorption. Of interest, a significant increase in cholesterol absorption was found in the LXRα(-/-) mice, but not in the WT or LXRβ(-/-) animals, when fed a diet enriched with 0.2% cholesterol or 0.2% cholesterol + GW3965. Reduced faecal neutral sterol excretion and a hydrophobic bile acid profile were also observed in LXRα(-/-) mice. Greater increases in the apolipoprotein (apo)B-containing lipoproteins in serum were seen in the LXRα(-/-) mice. A 0.2% cholesterol +GW3965 diet suppressed intestinal Npc1l1 protein expression to the same extent for all genotypes, while Abca1 and Abcg5 were elevated to the same degree.In the intestine, LXRα and LXRβ seem to exert similar effects on expression of cholesterol-transporting proteins such as Npc1l1. Selective activation of LXRβ may generate effects such as increased cholesterol absorption and elevated serum levels of apoB-containing lipoproteins, which seem to be counteracted by LXRα. Therefore, an intestinal LXRβ-specific pathway might exist in terms of cholesterol transportation in addition to the main pathway.

Nuclear receptors (NRs) are master regulators of transcriptional programs that integrate the homeostatic control of almost all biological processes. Their direct mode of ligand regulation and genome interaction is at the core of modern pharmacology. The two liver X receptors LXRα and LXRβ are among the emerging newer drug targets within the NR family. LXRs are best known as nuclear oxysterol receptors and physiological regulators of lipid and cholesterol metabolism that also act in an anti-inflammatory way. Because LXRs control diverse pathways in development, reproduction, metabolism, immunity and inflammation, they have potential as therapeutic targets for diseases as diverse as lipid disorders, atherosclerosis, chronic inflammation, autoimmunity, cancer and neurodegenerative diseases. Recent insights into LXR signaling suggest future targeting strategies aiming at increasing LXR subtype and pathway selectivity. This review discusses the current status of our understanding of LXR biology and pharmacology, with an emphasis on the molecular aspects of LXR signaling that constitute the potential of LXRs as drug targets.

Background The liver X receptors (LXRs) are oxysterol sensing nuclear receptors with multiple effects on metabolism and immune cells. However, the complete genome-wide cistrome of LXR in cells of human origin has not yet been provided. Results We performed ChIP-seq in phorbol myristate acetate-differentiated THP-1 cells (macrophage-type) after stimulation with the potent synthetic LXR ligand T0901317 (T09). Microarray gene expression analysis was performed in the same cellular model. We identified 1357 genome-wide LXR locations (FDR < 1%), of which 526 were observed after T09 treatment. De novo analysis of LXR binding sequences identified a DR4-type element as the major motif. On mRNA level T09 up-regulated 1258 genes and repressed 455 genes. Our results show that LXR actions are focused on 112 genomic regions that contain up to 11 T09 target genes per region under the control of highly stringent LXR binding sites with individual constellations for each region. We could confirm that LXR controls lipid metabolism and transport and observed a strong association with apoptosis-related functions. Conclusions This first report on genome-wide binding of LXR in a human cell line provides new insights into the transcriptional network of LXR and its target genes with their link to physiological processes, such as apoptosis. The gene expression microarray and sequence data have been submitted collectively to the NCBI Gene Expression Omnibus http://www.ncbi.nlm.nih.gov/geo under accession number GSE28319.

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.

In amyotrophic lateral sclerosis (ALS), there is selective degeneration of motor neurons that leads to paralysis and death. Although the etiology of ALS is unclear, its heterogeneity suggests that a combination of factors (endogenous and/or environmental) may induce progressive motor neuron stress that results in the activation of different cell death pathways. Alterations of brain cholesterol homeostasis have recently been considered as possible cofactors in many neurodegenerative disorders, including ALS. The liver X receptor beta (LXRbeta) receptor is involved in lipogenesis and cholesterol metabolism, and we previously found that adult-onset motor neuron pathology occurs in LXRbeta mice. Here, we investigated neuromuscular alterations of LXRbeta mice from ages 3 to 24 months. Increased cholesterol levels, gliosis, and inflammation preceded motor neuron loss and clinical disease onset; the mice showed progressivemotor neuron deficits starting from age 7 months. The numbers ofmotor neurons and neuromuscular junctions were decreased in 24-month-old mice, but neither paralysis nor reduced life span was observed. Moreover, other spinal neurons were also lost in these mice. These results suggest that LXRbeta may inhibit neuroinflammation and maintain cholesterol homeostasis, and that LXRbeta mice represent a potential model for investigating the role of cholesterol in ALS and other neurodegenerative disorders.

Sterol metabolism has recently been linked to innate and adaptive immune responses through liver X receptor (LXR) signaling. Whether products of sterol metabolism interfere with antitumor responses is currently unknown. Dendritic cells (DCs) initiate immune responses, including antitumor activity after their CC chemokine receptor-7 (CCR7)-dependent migration to lymphoid organs. Here we report that human and mouse tumors produce LXR ligands that inhibit CCR7 expression on maturing DCs and, therefore, their migration to lymphoid organs. In agreement with this observation, we detected CD83(+)CCR7(-) DCs within human tumors. Mice injected with tumors expressing the LXR ligand-inactivating enzyme sulfotransferase 2B1b (SULT2B1b) successfully controlled tumor growth by regaining DC migration to tumor-draining lymph nodes and by developing overt inflammation within tumors. The control of tumor growth was also observed in chimeric mice transplanted with bone marrow from mice lacking the gene encoding LXR-alpha (Nr1h3(-/-) mice) Thus, we show a new mechanism of tumor immunoescape involving products of cholesterol metabolism. The manipulation of this pathway could restore antitumor immunity in individuals with cancer. Sterol metabolism has recently been linked to innate and adaptive immune responses through liver X receptor (LXR) signaling. Whether products of sterol metabolism interfere with antitumor responses is currently unknown. Dendritic cells (DCs) initiate immune responses, including antitumor activity after their CC chemokine receptor-7 (CCR7)-dependent migration to lymphoid organs. Here we report that human and mouse tumors produce LXR ligands that inhibit CCR7 expression on maturing DCs and, therefore, their migration to lymphoid organs. In agreement with this observation, we detected CD83(+)CCR7(-) DCs within human tumors. Mice injected with tumors expressing the LXR ligand-inactivating enzyme sulfotransferase 2B1b (SULT2B1b) successfully controlled tumor growth by regaining DC migration to tumor-draining lymph nodes and by developing overt inflammation within tumors. The control of tumor growth was also observed in chimeric mice transplanted with bone marrow from mice lacking the gene encoding LXR-alpha (Nr1h3(-/-) mice) Thus, we show a new mechanism of tumor immunoescape involving products of cholesterol metabolism. The manipulation of this pathway could restore antitumor immunity in individuals with cancer.

Udgivelsesdato: 2009-Dec-2 Cold adaptation elicits a paradoxical simultaneous induction of fatty acid synthesis and beta-oxidation in brown adipose tissue. We show here that cold exposure coordinately induced liver X receptor alpha (LXRalpha), adipocyte determination and differentiation-dependent factor 1 (ADD1)/sterol regulatory element-binding protein-1c (SREBP1c) and peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC1alpha) in brown and inguinal white adipose tissues, but not in epididymal white adipose tissue. Using in vitro models of white and brown adipocytes we demonstrate that beta-adrenergic stimulation induced expression of LXRalpha, ADD1/SREBP1c and PGC1alpha in cells with a brown-like adipose phenotype. We demonstrate that ADD1/SREBP1c is a powerful inducer of PGC1alpha expression via a conserved E box in the proximal promoter and that beta-adrenergic stimulation led to recruitment of ADD1/SREBP1c to this E box. The ability of ADD1/SREBP1c to activate the PGC1alpha promoter exhibited a striking cell type dependency, suggesting that additional cell type-restricted factors contribute to ADD1/SREBP1c-mediated activation. In conclusion, our data demonstrate a novel role of ADD1/SREBP1c as a regulator of PGC1alpha expression in brown adipose tissue.

The two liver X receptors (LXRs), LXRα and LXRβ, are transcriptional regulators of cholesterol, lipid, and glucose metabolism and are both activated by oxysterols. Impaired metabolism is linked with obesity, insulin resistance, and type 2-diabetes (T2D). In the present study, we aimed to delineate the specific roles of LXRα and -β in metabolic processes. C57Bl/6 female mice were fed a normal or a high-fat diet (HFD) and metabolic responses in wild-type, LXRα−/−, LXRβ−/−, and LXRαβ−/−mice were analyzed. Whole body fat and intramyocellular lipid contents were measured by nuclear magnetic resonance. Energy expenditure was measured in individual metabolic cages. Glucose, insulin, and pyruvate tolerance tests were performed and gene expression profiles analyzed by qPCR. We found that both LXRβ−/−and LXRαβ−/−mice are resistant to HFD-induced obesity independently of the presence of high cholesterol. Using tolerance tests, we found that, on an HFD, LXRβ−/−mice enhanced their endogenous glucose production and became highly insulin resistant, whereas LXRα−/−and LXRαβ−/−mice remained glucose tolerant and insulin sensitive. Gene expression profiling confirmed that LXRβ is the regulator of lipogenic genes in visceral white adipose tissue (WAT) and muscle tissue and, surprisingly, that Ucp1 and Dio2 are not responsible for the protection against diet-induced obesity observed in LXRβ−/−and LXRαβ−/−mice. LXRα is required for the control of cholesterol metabolism in the liver, while LXRβ appears to be a major regulator of glucose homeostasis and energy utilization and of fat storage in muscle and WAT. We conclude that selective LXRβ agonists would be novel pharmaceuticals in the treatment of T2D.

Liver X receptors (LXRs) are nuclear receptors with established roles in cholesterol, lipid, and carbohydrate metabolism, although their function in adipocytes is not well characterized. Increased adipose tissue mass in obesity is associated with increased adipocyte lipolysis. Fatty acids (FA) generated by lipolysis can be oxidized by mitochondrial β-oxidation, reesterified, or released from the adipocyte. The latter results in higher circulating levels of free FAs, in turn causing obesity-related metabolic complications. However, mitochondrial β-oxidation can at least in part counteract an increased output of FA into circulation. In this study, we provide evidence that activation of LXRs up-regulates mitochondrial β-oxidation in both human and murine white adipocytes. We also show that the expression of a kinase regulating the cellular fuel switch, pyruvate dehydrogenase kinase 4 (PDK4), is up-regulated by the LXR agonist GW3965 in both in vitro differentiated human primary adipocytes and differentiated murine 3T3-L1 cells. Moreover, activation of LXR causes PDK4-dependent phosphorylation of the pyruvate dehydrogenase complex, thereby decreasing its activity and attenuating glucose oxidation. The specificity of the GW3965 effect on oxidation was confirmed by RNA interference targeting LXRs. We propose that LXR has an important role in the regulation of substrate oxidation and the switch between lipids and carbohydrates as cellular fuel in both human and murine white adipocytes.

Modulation of the host inflammatory response to infection may be a key approach to improve the outcome of patients with sepsis and organ injury. We previously reported that pretreatment of rats with the liver X receptor (LXR) agonist GW3965 reduced the liver injury associated with endotoxemia and attenuated the production of TNF-alpha by rat Kupffer cells. Here, we examine the dose-dependent effect of GW3965 on liver injury and cytokine production in a rat model of endotoxemia and explore the mechanisms underlying TNF-alpha attenuation in Kupffer cells. Low doses of GW3965 (0.1 or 0.3 mg/kg) administered 30 min before infusion of LPS and peptidoglycan significantly attenuated the increase in plasma levels of the liver injury markers alanine aminotransferase and bilirubin (6 h) as well as the inflammatory mediators TNF-alpha (1 h) and prostaglandin E2 (6 h) associated with endotoxemia. In contrast, pretreatment with a higher dose of GW3965 (1.0 mg/kg) had no such effect. Studies in primary cultures of rat Kupffer cells demonstrated that LXR agonist treatment attenuated both the secreted and cell-associated levels of TNF-alpha, whereas TNF-alpha mRNA levels were not altered. Phosphorylated p38 mitogen-activated protein kinase, which plays a major role in production of TNF-alpha at the posttranscriptional level, was attenuated by GW3965 treatment in Kupffer cells. Experiments in murine LXR-deficient Kupffer cells demonstrated enhanced production of TNF-alpha in Kupffer cells from LXR-alpha(-/-) mice when challenged with LPS compared with LXR-beta(-/-) and wild-type Kupffer cells. Taken together, these results argue in favor of a novel mechanism for LXR-mediated attenuation of liver injury by interfering with posttranscriptional regulation of TNF-alpha in Kupffer cells.

The oxysterol receptors [liver X receptors (LXRalpha and LXRbeta)] regulate cholesterol and lipid biosynthesis and several studies link dysregulation of these metabolic pathways to aberrant cell growth. Here, we show that activation of LXR significantly reduced proliferation in several human breast cancer cells lines. LXR suppressed messenger RNA and/or protein expression of Skp2, cyclin A2, cyclin D1 and estrogen receptor (ER) alpha, whereas it increased the expression of p53 at the protein level and maintained the retinoblastoma protein in a hypophosphorylated active form. These changes may constitute part of the molecular mechanisms behind the antiproliferative effect of LXR. Furthermore, activation of LXR induced expression of key lipogenic genes including sterol regulatory element-binding protein 1c (SREBP1c), fatty acid synthase and stearoyl-coenzyme A desaturase 1, leading to increased triglyceride production in MCF7 cells. Small interfering RNA knockdown of SREBP1c, a master regulator of the lipid biosynthesis, did not abolish the antiproliferative effect of LXR in these cells. Combined these studies identify LXRs as both antiproliferative and lipogenic factors in breast cancer cells and indicate that the antiproliferative effect of LXRs is independent of lipid biosynthesis.

Udgivelsesdato: 2008-Aug-15 The biological functions of liver X receptors (LXRs) alpha and beta have primarily been linked to pathways involved in fatty acid and cholesterol homeostasis. Here we report a novel role of LXR activation in protecting cells from statin-induced death. When 3T3-L1 preadipocytes were induced to differentiate by standard isobutylmethylxanthine/dexamethasone/insulin treatment in the presence of statins, they failed to differentiate and underwent massive apoptosis. The simultaneous addition of selective LXR agonists prevented the statin-induced apoptosis. By using mouse embryo fibroblasts from wild-type (LXRalpha+/+/LXRbeta+/+), LXRalpha knock-out mice (LXRalpha(-/-)/LXRbeta+/+), LXRbeta knock-out mice (LXRalpha+/-/LXRbeta(-/-)), and LXR double knock-out mice (LXRalpha(-/-)/LXRbeta(-/-)) as well as 3T3-L1 cells transduced with retroviruses expressing either wild-type LXRalpha or a dominant negative version of LXRalpha, we demonstrate that the response to LXR agonists is LXR-dependent. Interestingly, LXR-mediated rescue of statin-induced apoptosis was not related to up-regulation of genes previously shown to be involved in the antiapoptotic action of LXR. Furthermore, forced expression of Bcl-2 did not prevent statin-induced apoptosis; nor did LXR action depend on protein kinase B, whose activation by insulin was impaired in statin-treated cells. Rather, LXR-dependent rescue of statin-induced apoptosis in 3T3-L1 preadipocytes required NF-kappaB activity, since expression of a dominant negative version of IkappaBalpha prevented LXR agonist-dependent rescue of statin-induced apoptosis. Thus, the results presented in this paper provide novel insight into the action of statins on and LXR-dependent inhibition of apoptosis.

Several nuclear hormone receptors have been associated with inflammatory reactions. Particularly, liver X receptors (LXRs) have recently been identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. LXRs are negative regulators of macrophage inflammatory gene expression. Multiple sclerosis (MS), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. Sweden belongs to the countries with a high MS incidence. In Italy, the MS incidence is lower, except on the island of Sardinia where the incidence is even higher than in Sweden. Subjects from Sardinia are ethnically more homogeneous, and differ from Swedes also regarding genetic background and environment. We studied mRNA expression of several nuclear hormone receptors in blood mononuclear cells (MNC) from female patients with untreated relapsing-remitting MS from Sassari, Sardinia, and Stockholm, Sweden. Sex- and age-matched healthy controls (HC) were from both areas. mRNA expression was evaluated by quantitative real-time PCR. We found altered mRNA expression of LXRs, estrogen receptors (ERs), and androgen receptor (AR) in MS. mRNA expression of both LXRalpha and LXRbeta is lower in MS from Stockholm but not from Sassari. In particular, LXRalpha mRNA expression was significantly lower in MS from Stockholm as compared with all groups in the study including MS from Sassari. Low levels of ERalpha mRNA are seen in MS from both Stockholm and Sassari. The splice variant ERbetacx showed significantly higher mRNA expression in MS from Sassari and Stockholm as compared with corresponding HC. In particular, ERbetacx mRNA in MS from Sassari was remarkably higher as compared with all other groups in the study. Higher levels of AR mRNA are present in HC from Sassari. The findings indicate that the expression levels of anti-inflammatory nuclear receptor superfamily genes in MS appear to reflect both ethnic and environmental influences.

Liver X receptor (LXR) paralogues alpha and beta (LXRalpha and LXRbeta) are members of the nuclear hormone receptor family and have oxysterols as endogenous ligands. LXR activation reduces hepatic glucose production in vivo through the inhibition of transcription of the key gluconeogenic enzymes phosphoenolpyruvate carboxykinase and glucose-6-phosphatase (G6Pase). In the present study, we investigated the molecular mechanisms involved in the regulation of G6Pase gene expression by LXR. Both T0901317, a synthetic LXR agonist, and the adenoviral overexpression of either LXRalpha or LXRbeta suppressed G6Pase gene expression in H4IIE hepatoma cells. However, compared to the suppression of G6Pase expression seen by insulin, the decrease of G6Pase mRNA by LXR activation was delayed and was blocked by cycloheximide, an inhibitor of protein synthesis. These observations, together with the absence of a conserved LXR-binding element within the G6Pase promoter, suggest an indirect inhibition of G6Pase gene expression by liver X receptors.

Liver X receptors (LXR) alpha and beta are nuclear oxysterol receptors with established roles in cholesterol, lipid, and carbohydrate metabolism. Although LXRs have been extensively studied in liver and macrophages, the importance for development and metabolism of other tissues and cell types is not as well characterized. We demonstrate here that although LXRalpha and LXRbeta are not required for adipocyte development per se, LXRbeta is required for the increase in adipocyte size that normally occurs with aging and diet-induced obesity. Similar food intake and oxygen consumption in LXRbeta-/- mice suggests that reduced storage of lipid in adipose tissue is not due to altered energy balance. Despite reduced amounts of adipose tissue, LXRbeta-/- mice on a chow diet have insulin sensitivity and levels of adipocyte hormones similar to wild type mice. However, these mice are glucose-intolerant due to impaired glucose-induced insulin secretion. Lipid droplets in pancreatic islets may result from accumulation of cholesterol esters as analysis of islet gene expression reveals that LXRbeta is required for expression of the cholesterol transporters, ABCA1 and ABCG1. Our data establish novel roles for LXRbeta in adipocyte growth, glucose homeostasis, and beta cell function.