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Introduction: The development of small molecule agonists of the liver X receptors (LXRs) has been an area of interest for over a decade, given the critical role of those receptors in cholesterol metabolism, glucose homeostasis, inflammation, innate immunity and lipogenesis. Many potential indications have been characterized over time including atherosclerosis, diabetes, inflammation, Alzheimer's disease and cancer. However, concerns about the lipogenic effects of full LXRα/β agonists have required extensive efforts aimed at identifying LXRβ agonist with limited activity on the LXRα receptor to increase the safety margins., Areas Covered: This review includes a summary of the LXR agonists that have reached the clinic and summarizes the patent applications for LXR modulators from September 2009 to December 2012 with emphasis on chemical matters, biological data associated with selected analogs and therapeutic indications., Expert Opinion: As LXR agonists have the potential to be useful for many indications, the scientific community, despite setbacks due to on-target side effects, has maintained interest and devised strategies to overcome safety hurdles. While a clinical proof of concept still remains elusive, the recent advancement of compounds into the clinic highlights that acceptable safety margins in preclinical species have been achieved.

The identification of factors that promote β cell proliferation could ultimately move type 1 diabetes treatment away from insulin injection therapy and toward a cure. We have performed high-throughput, cell-based screens using rodent β cell lines to identify molecules that induce proliferation of β cells. Herein we report the discovery and characterization of WS6, a novel small molecule that promotes β cell proliferation in rodent and human primary islets. In the RIP-DTA mouse model of β cell ablation, WS6 normalized blood glucose and induced concomitant increases in β cell proliferation and β cell number. Affinity pulldown and kinase profiling studies implicate Erb3 binding protein-1 and the IκB kinase pathway in the mechanism of action of WS6.

For more than 2 centuries active immunotherapy has been at the forefront of efforts to prevent infectious disease [Waldmann TA (2003) Nat Med 9:269-277]. However, the decreased ability of the immune system to mount a robust immune response to self-antigens has made it more difficult to generate therapeutic vaccines against cancer or chronic degenerative diseases. Recently, we showed that the site-specific incorporation of an immunogenic unnatural amino acid into an autologous protein offers a simple and effective approach to overcome self-tolerance. Here, we characterize the nature and durability of the polyclonal IgG antibody response and begin to establish the generality of p-nitrophenylalanine (pNO(2)Phe)-induced loss of self-tolerance. Mutation of several surface residues of murine tumor necrosis factor-alpha (mTNF-alpha) independently to pNO(2)Phe leads to a T cell-dependent polyclonal and sustainable anti-mTNF-alpha IgG autoantibody response that lasts for at least 40 weeks. The antibodies bind multiple epitopes on mTNF-alpha and protect mice from severe endotoxemia induced by lipopolysaccharide (LPS) challenge. Immunization of mice with a pNO(2)Phe(43) mutant of murine retinol-binding protein (RBP4) also elicited a high titer IgG antibody response, which was cross-reactive with wild-type mRBP4. These findings suggest that this may be a relatively general approach to generate effective immunotherapeutics against cancer-associated or other weakly immunogenic antigens.

A cocrystal structure of T1317 (3) bound to hLXRbeta was utilized in the design of a series of substituted N-phenyl tertiary amines. Profiling in binding and functional assays led to the identification of LXR modulator GSK9772 ( 20) as a high-affinity LXRbeta ligand (IC 50 = 30 nM) that shows separation of anti-inflammatory and lipogenic activities in human macrophage and liver cell lines, respectively. A cocrystal structure of the structurally related analog 19 bound to LXRbeta reveals regions within the receptor that can affect receptor modulation through ligand modification. Mechanistic studies demonstrate that 20 is greater than 10-fold selective for LXR-mediated transrepression of proinflammatory gene expression versus transactivation of lipogenic signaling pathways, thus providing an opportunity for the identification of LXR modulators with improved therapeutic indexes.

The liver X receptors alpha and beta (LXRalpha and LXRbeta) have been shown to play important roles in lipid homeostasis in liver and macrophages, however, their function in adipose tissue is not well defined. Both LXRs are highly expressed in fat, and the expression of LXRalpha increases during adipogenesis. Furthermore, LXRalpha expression is induced by peroxisome proliferator-activated receptor gamma (PPARgamma), the master regulator of fat cell differentiation. Here we investigate the role of LXRs in adipocyte differentiation and gene expression and their potential crosstalk with the PPARgamma pathway. We demonstrate that LXR agonists have no significant effect on the differentiation of 3T3-F442A or 3T3-L1 preadipocytes in vitro and do not alter the expression of differentiation-linked PPARgamma target genes in vivo. Moreover, retroviral expression of LXRalpha in NIH-3T3 cells does not alter the adipogenic potential of these cells and neither augments nor inhibits the action of PPARgamma. However, transcriptional profiling studies reveal that LXRs are important regulators of adipocyte gene expression. We identify the multifunction lipid carrier protein apolipoprotein D and the lipogenic protein Spot 14 as LXR responsive genes both in vitro and in vivo. Thus, although LXRs do not influence adipocyte differentiation per se, these receptors are likely to play an important role in the modulation of lipid metabolism in adipocytes.

Macrophages are an important source of angiogenic activity in wound healing, cancer, and chronic inflammation. Vascular endothelial growth factor (VEGF), a cytokine produced by macrophages, is a primary inducer of angiogenesis and neovascularization in these contexts. VEGF expression by macrophages is known to be stimulated by low oxygen tension as well as by inflammatory signals. In this study, we provide evidence that Vegfa gene expression is also regulated by activation of liver X receptors (LXRs). VEGF mRNA was induced in response to synthetic LXR agonists in murine and human primary macrophages as well as in murine adipose tissue in vivo. The effects of LXR ligands on VEGF expression were independent of hypoxia-inducible factor HIF-1alpha activation and did not require the previously characterized hypoxia response element in the VEGF promoter. Rather, LXR/retinoid X receptor heterodimers bound directly to a conserved hormone response element (LXRE) in the promoter of the murine and human Vegfa genes. Both LXRalpha and LXRbeta transactivated the VEGF promoter in transient transfection assays. Finally, we show that induction of VEGF expression by inflammatory stimuli was independent of LXRs, because these effects were preserved in LXR null macrophages. These observations identify VEGF as an LXR target gene and point to a previously unrecognized role for LXRs in vascular biology.

The control of lipid and glucose metabolism is closely linked. The nuclear receptors liver X receptor (LXR)alpha and LXR beta have been implicated in gene expression linked to lipid homeostasis; however, their role in glucose metabolism is not clear. We demonstrate here that the synthetic LXR agonist GW3965 improves glucose tolerance in a murine model of diet-induced obesity and insulin resistance. Analysis of gene expression in LXR agonist-treated mice reveals coordinate regulation of genes involved in glucose metabolism in liver and adipose tissue. In the liver, activation of LXR led to the suppression of the gluconeogenic program including down-regulation of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1), phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase expression. Inhibition of gluconeogenic genes was accompanied by an induction in expression of glucokinase, which promotes hepatic glucose utilization. In adipose tissue, activation of LXR led to the transcriptional induction of the insulin-sensitive glucose transporter, GLUT4. We show that the GLUT4 promoter is a direct transcriptional target for the LXR/retinoid X receptor heterodimer and that the ability of LXR ligands to induce GLUT4 expression is abolished in LXR null cells and animals. Consistent with their effects on GLUT4 expression, LXR agonists promote glucose uptake in 3T3-L1 adipocytes in vitro. Thus, activation of LXR alters the expression of genes in liver and adipose tissue that collectively would be expected to limit hepatic glucose output and improve peripheral glucose uptake. These results outline a role for LXRs in the coordination of lipid and glucose metabolism.

The liver X receptors (LXRs) are members of the nuclear receptor superfamily that are activated by oxysterols. In response to ligand binding, LXRs regulate a variety of genes involved in the catabolism, transport, and uptake of cholesterol and its metabolites. Here we demonstrate that LXRs also regulate plasma lipoprotein metabolism through control of the phospholipid transfer protein (PLTP) gene. LXR ligands induce the expression of PLTP in cultured HepG2 cells and mouse liver in vivo in a coordinate manner with known LXR target genes. Moreover, plasma phospholipid transfer activity is increased in mice treated with the synthetic LXR ligand GW3965. Unexpectedly, PLTP expression was also highly inducible by LXR in macrophages, a cell type not previously recognized to express this enzyme. The ability of synthetic and oxysterol ligands to regulate PLTP mRNA in macrophages and liver is lost in animals lacking both LXRalpha and LXRbeta, confirming the critical role of these receptors. We further demonstrate that the PLTP promoter contains a high-affinity LXR response element that is bound by LXR/RXR heterodimers in vitro and is activated by LXR/RXR in transient-transfection studies. Finally, immunohistochemistry studies reveal that PLTP is highly expressed by macrophages within human atherosclerotic lesions, suggesting a potential role for this enzyme in lipid-loaded macrophages. These studies outline a novel pathway whereby LXR and its ligands may modulate lipoprotein metabolism.

Macrophages have important roles in both lipid metabolism and inflammation and are central to the pathogenesis of atherosclerosis. The liver X receptors (LXRs) are established mediators of lipid-inducible gene expression, but their role in inflammation and immunity is unknown. We demonstrate here that LXRs and their ligands are negative regulators of macrophage inflammatory gene expression. Transcriptional profiling of lipopolysaccharide (LPS)-induced macrophages reveals reciprocal LXR-dependent regulation of genes involved in lipid metabolism and the innate immune response. In vitro, LXR ligands inhibit the expression of inflammatory mediators such as inducible nitric oxide synthase, cyclooxygenase (COX)-2 and interleukin-6 (IL-6) in response to bacterial infection or LPS stimulation. In vivo, LXR agonists reduce inflammation in a model of contact dermatitis and inhibit inflammatory gene expression in the aortas of atherosclerotic mice. These findings identify LXRs as lipid-dependent regulators of inflammatory gene expression that may serve to link lipid metabolism and immune functions in macrophages.

Recent studies have identified the liver X receptors (LXR alpha and LXR beta) as important regulators of cholesterol metabolism and transport. LXRs control transcription of genes critical to a range of biological functions including regulation of high density lipoprotein cholesterol metabolism, hepatic cholesterol catabolism, and intestinal sterol absorption. Although LXR activity has been proposed to be critical for physiologic lipid metabolism and transport, direct evidence linking LXR signaling pathways to the pathogenesis of cardiovascular disease has yet to be established. In this study bone marrow transplantations were used to selectively eliminate macrophage LXR expression in the context of murine models of atherosclerosis. Our results demonstrate that LXRs are endogenous inhibitors of atherogenesis. Additionally, elimination of LXR activity in bone marrow-derived cells mimics many aspects of Tangier disease, a human high density lipoprotein deficiency, including aberrant regulation of cholesterol transporter expression, lipid accumulation in macrophages, splenomegaly, and increased atherosclerosis. These results identify LXRs as targets for intervention in cardiovascular disease.

Lipid-loaded macrophage "foam cells" accumulate in the subendothelial space during the development of fatty streaks and atherosclerotic lesions. To better understand the consequences of such lipid loading, murine peritoneal macrophages were isolated and incubated with ligands for two nuclear receptors, liver X receptor (LXR) and retinoic acid receptor (RXR). Analysis of the expressed mRNAs using microarray technology led to the identification of four highly induced genes that encode apolipoproteins E, C-I, C-IV, and C-II. Northern blot analysis confirmed that the mRNA levels of these four genes were induced 2-14-fold in response to natural or synthetic ligands for LXR and/or RXR. The induction of all four mRNAs was greatly attenuated in peritoneal macrophages derived from LXRalpha/beta null mice. The two LXR response elements located within the multienhancers ME.1 and ME.2 were shown to be essential for the induction of apoC-II promoter-reporter genes by ligands for LXR and/or RXR. Finally, immunohistochemical studies demonstrate that apoC-II protein co-localizes with macrophages within murine arterial lesions. Taken together, these studies demonstrate that activated LXR induces the expression of the apoE/C-I/C-IV/C-II gene cluster in both human and murine macrophages. These results suggest an alternative mechanism by which lipids are removed from macrophage foam cells.

The nuclear receptors LXRalpha and LXRbeta have been implicated in the control of cholesterol and fatty acid metabolism in multiple cell types. Activation of these receptors stimulates cholesterol efflux in macrophages, promotes bile acid synthesis in liver, and inhibits intestinal cholesterol absorption, actions that would collectively be expected to reduce atherosclerotic risk. However, synthetic LXR ligands have also been shown to induce lipogenesis and hypertriglyceridemia in mice, raising questions as to the net effects of these compounds on the development of cardiovascular disease. We demonstrate here that the nonsteroidal LXR agonist GW3965 has potent antiatherogenic activity in two different murine models. In LDLR(-/-) mice, GW3965 reduced lesion area by 53% in males and 34% in females. A similar reduction of 47% was observed in male apoE(-/-) mice. Long-term (12-week) treatment with LXR agonist had differential effects on plasma lipid profiles in LDLR(-/-) and apoE(-/-) mice. GW3965 induced expression of ATP-binding cassettes A1 and G1 in modified low-density lipoprotein-loaded macrophages in vitro as well as in the aortas of hyperlipidemic mice, suggesting that direct actions of LXR ligands on vascular gene expression are likely to contribute to their antiatherogenic effects. These observations provide direct evidence for an atheroprotective effect of LXR agonists and support their further evaluation as potential modulators of human cardiovascular disease.

Orphan nuclear receptors of the peroxisome proliferator activated receptor (PPAR) and liver X receptor (LXR) subfamilies have been shown to play critical roles in both local and systemic lipid metabolism. The PPARs control fatty acid metabolism in various cell types, including adipocytes, liver, and macrophages. The LXRs have been implicated in the regulation of cholesterol metabolism in the liver, intestines, and macrophages. The importance of these receptors in physiologic lipid metabolism suggests that they may influence the development of metabolic disorders such as obesity, diabetes, and atherosclerosis. Furthermore, the ability of these receptors to be modulated pharmacologically makes them attractive therapeutic targets. This review focuses on the role of PPAR and LXR signaling pathways in macrophage lipid metabolism and the potential of these pathways to modulate the development of atherosclerosis.

The nuclear receptors LXRalpha and LXRbeta have been implicated in the control of lipogenesis and cholesterol homeostasis. Ligand activation of these receptors in vivo induces expression of the LXR target gene SREBP-1c and increases plasma triglyceride levels. Expression of fatty acid synthase (FAS), a central enzyme in de novo lipogenesis and an established target of the SREBP-1 pathway, is also induced by LXR ligands. The effects of LXR ligands on FAS expression have been proposed to be entirely secondary to the induction of SREBP-1c. We demonstrate here that LXRs regulate FAS expression through direct interaction with the FAS promoter as well as through activation of SREBP-1c expression. Induction of FAS expression in HepG2 cells by LXR ligands is reduced, but not abolished, under conditions where SREBP processing is suppressed. Moreover, LXR ligands induce FAS expression in CHO-7 cells without altering expression of SREBP-1. We demonstrate that in addition to tandem SREBP sites, the FAS promoter contains a high affinity binding site for the LXR/RXR heterodimer that is conserved in diverse animal species including birds, rodents, and humans. The LXR and SREBP binding sites independently confer LXR responsiveness on the FAS promoter, and maximal induction requires both transcription factors. Transient elevation of plasma triglyceride levels in mice treated with a synthetic LXR agonist correlates with transient induction of hepatic FAS expression. These results indicate that the LXR signaling pathway modulates FAS expression through distinct but complementary mechanisms and suggest that the FAS gene may be a critical target in the control of lipogenesis by LXRs.

Previous work has implicated the nuclear receptors liver X receptor alpha (LXR alpha) and LXR beta in the regulation of macrophage gene expression in response to oxidized lipids. Macrophage lipid loading leads to ligand activation of LXRs and to induction of a pathway for cholesterol efflux involving the LXR target genes ABCA1 and apoE. We demonstrate here that autoregulation of the LXR alpha gene is an important component of this lipid-inducible efflux pathway in human macrophages. Oxidized low-density lipoprotein, oxysterols, and synthetic LXR ligands induce expression of LXR alpha mRNA in human monocyte-derived macrophages and human macrophage cell lines but not in murine peritoneal macrophages or cell lines. This is in contrast to peroxisome proliferator-activated receptor gamma (PPAR gamma)-specific ligands, which stimulate LXR alpha expression in both human and murine macrophages. We further demonstrate that LXR and PPAR gamma ligands cooperate to induce LXR alpha expression in human but not murine macrophages. Analysis of the human LXR alpha promoter led to the identification of multiple LXR response elements. Interestingly, the previously identified PPAR response element (PPRE) in the murine LXR alpha gene is not conserved in humans; however, a different PPRE is present in the human LXR 5'-flanking region. These results have implications for cholesterol metabolism in human macrophages and its potential to be regulated by synthetic LXR and/or PPAR gamma ligands. The ability of LXR alpha to regulate its own promoter is likely to be an integral part of the macrophage physiologic response to lipid loading.

The farnesoid X-activated receptor (FXR; NR1H4), a member of the nuclear hormone receptor superfamily, induces gene expression in response to several bile acids, including chenodeoxycholic acid. Here we used suppression subtractive hybridization to identify apolipoprotein C-II (apoC-II) as an FXR target gene. Retroviral expression of FXR in HepG2 cells results in induction of the mRNA encoding apoC-II in response to several FXR ligands. EMSAs demonstrate that recombinant FXR and RXR bind to two FXR response elements that are contained within two important distal enhancer elements (hepatic control regions) that lie 11 kb and 22 kb upstream of the transcription start site of the apoC-II gene. A luciferase reporter gene containing the hepatic control region or two copies of the wild-type FXR response element was activated when FXR-containing cells were treated with FXR ligands. In addition, we report that hepatic expression of both apoC-II and phospholipid transfer protein mRNAs increases when mice are fed diets supplemented with cholic acid, an FXR ligand, and this induction is attenuated in FXR null mice. Finally, we observed decreased plasma triglyceride levels in mice fed cholic acid- containing diets. These results identify a mechanism whereby FXR and its ligands lower plasma triglyceride levels. These findings may have important implications in the clinical management of hyperlipidemias.

Apolipoprotein E (apoE) secreted by macrophages in the artery wall exerts an important protective effect against the development of atherosclerosis, presumably through its ability to promote lipid efflux. Previous studies have shown that increases in cellular free cholesterol levels stimulate apoE transcription in macrophages and adipocytes; however, the molecular basis for this regulation is unknown. Recently, Taylor and colleagues [Shih, S. J., Allan, C., Grehan, S., Tse, E., Moran, C. & Taylor, J. M. (2000) J. Biol. Chem. 275, 31567-31572] identified two enhancers from the human apoE gene, termed multienhancer 1 (ME.1) and multienhancer 2 (ME.2), that direct macrophage- and adipose-specific expression in transgenic mice. We demonstrate here that the nuclear receptors LXRalpha and LXRbeta and their oxysterol ligands are key regulators of apoE expression in both macrophages and adipose tissue. We show that LXR/RXR heterodimers regulate apoE transcription directly, through interaction with a conserved LXR response element present in both ME.1 and ME.2. Moreover, we demonstrate that the ability of oxysterols and synthetic ligands to regulate apoE expression in adipose tissue and peritoneal macrophages is reduced in Lxralpha-/- or Lxrbeta-/- mice and abolished in double knockouts. Basal expression of apoE is not compromised in Lxr null mice, however, indicating that LXRs mediate lipid-inducible rather than tissue-specific expression of this gene. Together with our previous work, these findings support a central role for LXR signaling pathways in the control of macrophage cholesterol efflux through the coordinate regulation of apoE, ABCA1, and ABCG1 expression.

Previous work has implicated PPAR gamma in the regulation of CD36 expression and macrophage uptake of oxidized LDL (oxLDL). We provide evidence here that in addition to lipid uptake, PPAR gamma regulates a pathway of cholesterol efflux. PPAR gamma induces ABCA1 expression and cholesterol removal from macrophages through a transcriptional cascade mediated by the nuclear receptor LXR alpha. Ligand activation of PPAR gamma leads to primary induction of LXR alpha and to coupled induction of ABCA1. Transplantation of PPAR gamma null bone marrow into LDLR -/- mice results in a significant increase in atherosclerosis, consistent with the hypothesis that regulation of LXR alpha and ABCA1 expression is protective in vivo. Thus, we propose that PPAR gamma coordinates a complex physiologic response to oxLDL that involves particle uptake, processing, and cholesterol removal through ABCA1.

We obtain sterols from the animal and plant food that we eat. How these plant and animal sterols are absorbed, transported around the body, and excreted has been the subject of much investigation. In a Perspective, Allayee and colleagues discuss a new study (Berge et al.) that implicates two new ABC transporter proteins in the efflux of plant and animal sterols from gut epithelial cells into the gut lumen.

LXR alpha is a nuclear receptor that has previously been shown to regulate the metabolic conversion of cholesterol to bile acids. Here we define a role for this transcription factor in the control of cellular cholesterol efflux. We demonstrate that retroviral expression of LXR alpha in NIH 3T3 fibroblasts or RAW264.7 macrophages and/or treatment of these cells with oxysterol ligands of LXR results in 7- to 30-fold induction of the mRNA encoding the putative cholesterol/phospholipid transporter ATP-binding cassette (ABC)A1. In contrast, induction of ABCA1 mRNA in response to oxysterols is attenuated in cells that constitutively express dominant-negative forms of LXR alpha or LXR beta that lack the AF2 transcriptional activation domain. We further demonstrate that expression of LXR alpha in NIH 3T3 fibroblasts and/or treatment of these cells with oxysterols is sufficient to stimulate cholesterol efflux to extracellular apolipoprotein AI. The ability of oxysterol ligands of LXR to stimulate efflux is dramatically reduced in Tangier fibroblasts, which carry a loss of function mutation in the ABCA1 gene. Taken together, these results indicate that cellular cholesterol efflux is controlled, at least in part, at the level of transcription by a nuclear receptor-signaling pathway. They suggest a model in which activation of LXRs by oxysterols in response to cellular sterol loading leads to induction of the ABCA1 transporter and the stimulation of lipid efflux to extracellular acceptors. These findings have important implications for our understanding of mammalian cholesterol homeostasis and suggest new opportunities for pharmacological regulation of cellular lipid metabolism.

The farnesoid X-activated receptor (FXR; NR1H4) is a member of the nuclear hormone receptor superfamily and functions as a heterodimer with the 9-cis-retinoic acid receptor (RXR). In order to determine the optimal DNA binding sequence for the FXR/RXR heterodimer, we have utilized the selected and amplified binding sequence imprinting technique. This technique identified a number of related sequences that interacted with FXR/RXR in vitro. The consensus sequence contained an inverted repeat of the sequence AGGTCA with a 1-base pair spacing (IR-1). This sequence was shown to be a high affinity binding site for FXR/RXR in vitro and to confer ligand-dependent transcriptional activation by FXR/RXR to a heterologous promoter. Electrophoretic mobility shift assays and transient transfection assays were used to investigate the importance of the core half-site sequences, spacing nucleotide, flanking sequences, and orientation and spacing of the core half-sites on DNA binding and ligand-dependent transcriptional activation by FXR/RXR. These studies demonstrated that the FXR/RXR heterodimer binds to the consensus IR-1 sequence with the highest affinity, although FXR/RXR can bind to and activate through a variety of elements including IR-1 elements with changes in the core half-site sequence, spacing nucleotide, and flanking nucleotides. In addition, FXR/RXR can bind to and transactivate through direct repeats. Three genes were identified that contain IR-1 sequences in their proximal promoters. These elements were shown to bind FXR/RXR in vitro and to confer FXR/RXR-dependent transcriptional activation to a heterologous promoter in response to a bile acid or synthetic retinoid. The endogenous mRNA levels of one of these genes, phospholipid transfer protein, were shown to be induced by FXR and FXR ligands. The identification of the IR-1 and related elements as high affinity binding sites and functional response elements for FXR/RXR and the identification of a target gene for FXR/RXR should assist in the identification of additional genes regulated by FXR/RXR.