The Therapeutic Potential of Nuclear Receptor Modulators for Treatment of Metabolic Disorders: PPARγ, RORs, and Rev-erbs

Cell Metabolism

Review The Therapeutic Potential of Nuclear Receptor Modulators for Treatment of Metabolic Disorders: PPARg, RORs, and Rev-erbs David P. Marciano,1 Mi Ra Chang,1 Cesar A. Corzo,1 Devrishi Goswami,1 Vinh Q. Lam,1 Bruce D. Pascal,1 and Patrick R. Griffin1,* 1Department of Molecular Therapeutics, The Scripps Research Institute, Scripps Florida, Jupiter, FL 33458, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.cmet.2013.12.009

Nuclear receptors (NRs) play central roles in metabolic syndrome, making them attractive drug targets despite the challenge of achieving functional selectivity. For instance, members of the thiazolidinedione class of insulin sensitizers offer robust efficacy but have been limited due to adverse effects linked to activation of genes not involved in insulin sensitization. Studies reviewed here provide strategies for targeting subsets of PPARg target genes, enabling development of next-generation modulators with improved therapeutic index. Additionally, emerging evidence suggests that targeting the NRs ROR and Rev-erb holds promise for treating metabolic syndrome based on their involvement in circadian rhythm and metabolism. Metabolic Disorders The percentage of the global population categorized as obese has skyrocketed over the last two decades. This trend is predicted to continue as developing nations increasingly adopt more sedentary lifestyles and gain easier access to high-calorie diets. As recently as 10 years ago in the United States, obese adults (defined as BMI >30) made up less than 12% of the population. Now more than 20% of the adult population meets the CDC criteria for obesity, and greater than 40% are considered overweight. Metabolic syndrome is characterized as a clustering of factors associated with an increased risk of cardiovascular disease and stroke, and is becoming more common (Moller and Kaufman, 2005). The essential risk factors that constitute metabolic syndrome are age, atherogenic dyslipidemia (high triglycerides and low HDL-C), hypertension, elevated plasma glucose, a prothrombotic state, and a proinflammatory state. According to current views, there are two major underlying causes of metabolic syndrome: obesity and type 2 diabetes mellitus (T2DM) (Grundy et al., 2005). Obesity is conceptually defined as an excess of body lipid of sufficient magnitude to impair health and longevity. T2DM is a chronic metabolic disorder that results partly in the inability of the body to respond adequately to circulating insulin, a condition termed insulin resistance. The comorbidities of metabolic syndrome will continue to strain global health care systems and require the development of alternative strategies to combat this epidemic. The current standard of care for treating metabolic syndrome includes pharmacologic intervention and lifestyle modification such as nutritional consultation and modification of diet, rigorous weight control, and increased regular exercise. Modest weight loss can result in an improvement in metabolic parameters (Grundy et al., 2004). However, weight loss and exercise often are not sufficient due to poor compliance and perhaps confounding genetic factors (Bouchard, 1988; Moller et al., 1996). Pharmacologic approaches have focused on the use of combination therapy for diabetes treatment in an effort to postpone the inevitable need for insulin therapy (Gavin,

2006). Therapeutic strategies to directly combat obesity have demonstrated successful outcomes in the clinic by suppressing either appetite or absorption of calories (Berlie and Hurren, 2013; Carter et al., 2012; Hung et al., 2010); however, adverse effects and other challenges persist with these approaches (Bloom et al., 2008; Heal et al., 2009; Jain et al., 2011). Recent efforts to target obesity have expanded to therapeutic approaches that include restoring dysregulated circadian rhythms that are associated with obesity (Maury et al., 2010), and induction of brown adipose tissue (BAT) or browning of white adipose tissue (WAT) resulting in dissipation of energy as heat (Seale and Lazar, 2009). Many of these strategies evolved from our increased understanding of nuclear receptor (NR) function, and they represent opportunities for therapeutic development. Nuclear Receptors as Therapeutic Targets for Metabolic Syndrome NRs are a unique superfamily of ligand-dependent transcription factors that control a diverse set of biological activities by translating dietary and endocrine signals into changes in expression of gene networks. NRs are attractive therapeutic targets for the treatment of metabolic syndrome because their dysfunction due to naturally occurring mutations can result in metabolic disorders (Gurnell, 2003; Hegele et al., 2002; Savage et al., 2003), and their activity can be robustly modulated by small lipophilic molecules that can be substituted by exogenous synthetic small molecules. NRs are the molecular target of approximately 10%–15% of drugs currently approved by the FDA, highlighting their tractability for therapeutic intervention (Overington et al., 2006). There are 48 NRs in the human genome that are all thought to share a common evolutionary origin as evidenced by the significant sequence homology and conserved cellular function across the superfamily (Figure 1). NRs are characterized by a multidomain architecture comprised of an N-terminal ligandindependent Activating Function 1 (AF1) domain, DNA-binding Cell Metabolism 19, February 4, 2014 ª2014 Elsevier Inc. 193

Cell Metabolism

Review

Figure 1. Sequence Alignment of Nuclear Receptor Superfamily Alignment of all 48 human NR receptors demonstrates significant conservation of the DBD and LBD across the superfamily, with significant divergence of the A/B, Hinge, and F domains. All receptors are aligned by their DNA binding domains and are drawn to scale based on length of amino acid sequence.

domain (DBD), hinge, and ligand-binding domain (LBD) containing the ligand-dependent AF2 (Evans, 1988). The AF1 and hinge are the most divergent in sequence and length across the superfamily and are considered intrinsically disordered (Krasowski et al., 2008), and their function and significance have been reviewed previously (Clinckemalie et al., 2012; Moore et al., 2006; Tremblay et al., 1999; Wa¨rnmark et al., 2003; Zwart et al., 2010). The DBD is the most highly conserved sequence among NRs and contains two zinc-finger motifs to bind distinct DNA response elements. NR response elements are commonly arranged as either direct or inverted repeats of a consensus half-site (RGGTCA; R = purine). NRs can bind DNA as monomers, homodimers, or heterodimers with a member of the retinoid X receptor (RXR) subfamily. The LBD is often the focus of drug-discovery efforts and is structurally conserved across the superfamily and is described as having three stacked a-helical sheets that create an internal hydrophobic cavity to which small-molecule ligands can bind (Moore et al., 2006). The ligand-dependent AF2 structural element contained in the LBD is the surface of the receptor directly involved in interactions with coactivator and corepressor proteins that either have intrinsic chromatin remodeling activity or tether in such enzymes. Coactivator proteins contain a common LXXLL motif known as ‘‘NR boxes,’’ which interact at AF2 when the receptor is in an active conformation (Heery et al., 1997). Coactivators like steroid receptor coactivator 1 (SRC-1) can act locally to acetylate histones, unraveling DNA and allow194 Cell Metabolism 19, February 4, 2014 ª2014 Elsevier Inc.

ing recruitment of the basal transcription complex to the initiation site of NR target genes (Spencer et al., 1997). In contrast, corepressor proteins like the NR corepressor (NCoR) and silencing mediators of retinoid and thyroid (SMRT) contain an L/IxxI/VI motif referred to as ‘‘CoRNR boxes,’’ which interact with high affinity at AF2 when the receptor is in the inactive conformation (Hu and Lazar, 1999). SMRT and NCoR recruit histone deacetylase 3 (HDAC3), which keeps chromatin compact leading to repression of basal transcriptional activity (Privalsky, 2004). NR Ligand Binding NRs are classified as ligand-dependent transcription factors, as changes in their conformational dynamics induced by binding ligand ultimately drive downstream transcriptional events. In the apo state, NRs typically have high affinity for corepressor leading to transcriptional repression. Activation of NRs by agonist ligands occurs primarily through conformational changes in AF2 that displace corepressor and recruit coactivators. Alternatively, ligands that repress NR transcriptional activity below basal level through recruitment of corepressor are termed inverse agonists (Germain et al., 2006). The Rev-erb subfamily of ‘‘constitutive repressors’’ is an exception, as repressive ligands that induce increased affinity for NCoR are considered agonists (Woo et al., 2007). Over the past two decades it has become clear that there is significantly more complexity to the functional regulation of

Cell Metabolism

Review

Figure 2. Mechanisms of PPARg Functional Control Ligand-mediated control of PPARg can be achieved through classical AF2 activation, modulation of PTM status, RXRa, cofactor, and DNA affinity.

NRs than simply a ligand-induced on/off switch. Ligands not only differentially affect the conformational dynamics of AF2 leading to various degrees of activation but can also impact NR affinity for DNA (Thuillier et al., 1998; Zhang et al., 2011), receptor degradation (Hauser et al., 2000), interaction with cofactor proteins (Ohno et al., 2012), and modulation of posttranslational modification (PTM) status (Choi et al., 2010; Choi et al., 2011), a central focus of this review (Figure 2). NR ligands can also demonstrate tissue-specific effects as a result of differing cofactor populations, NR subtypes, and isoforms (Stossi et al., 2004; Tee et al., 2004). This exceptional biological complexity in response to ligand binding is the primary challenge of translating readily identifiable, high-affinity NR ligands into clinical therapeutics. Understanding this complexity may afford the opportunity to develop functional selective modulators that translate into improved drugs. Here we review the evolution of PPARg targeted ligands for the treatment of T2DM from blunt instruments (super agonists) that carry significant adverse effects, to next-generation ligand classes that selectively modulate receptor function with improved therapeutic index. Similar strategies will likely apply to therapeutic development efforts for ROR and Rev-erb targeted ligands, for which recent studies reviewed here demonstrate promise for the treatment of metabolic syndrome. The PPAR Subfamily of NRs The peroxisome proliferator-activated receptor (PPAR) NR subfamily was identified with the discovery that a diverse class of rodent hepatocarcinogens induced proliferation of peroxisomes (Issemann and Green, 1990). Three PPAR subtypes have been identified—PPARa (NR1C1), PPARb/d (NR1C2), and PPARg (NR1C3)—each with unique expression profiles, ligand specificity, and functional roles (Berger and Moller, 2002). These receptors regulate transcriptional activity by forming obligate heterodimers with RXR and binding to specific DNA response elements (PPREs) that consist of two repeats of the core AGGTCA sequence separated by one nucleotide (DR-1) in the promoter or enhancer regions of their target genes (Kliewer et al., 1992; Mangelsdorf and Evans, 1995).

PPARa is a major regulator of lipid metabolism in the liver and is activated under conditions of energy deprivation and prolonged fasting to initiate ketogenesis (Kersten et al., 1999). Activation of PPARa by synthetic ligands, including the fibrate drugs used to treat hyperlipidemia, promote uptake, utilization, and catabolism of fatty acids via increased expression of classic target genes PDK4, ACOX1, and CPT1 (Rakhshandehroo et al., 2010; Staels et al., 1998). The therapeutic potential of PPARa for the treatment of metabolic syndrome has been reviewed previously (Cheng and Leiter, 2008; Hiukka et al., 2010; Staels and Fruchart, 2005). PPARd (also called PPARb) is highly expressed in intestinal epithelium, liver, and keratinocytes, consistent with its significant biological role in these tissues, and is also detected at lower levels in the brain, skin, kidney, lung, and testes (Girroir et al., 2008). It has been reported that forced PPARd activation in transgenic models protects against obesity and induces an exerciselike metabolic status, spurring interest in the development of PPARd modulators for the treatment of metabolic syndrome (Wang et al., 2004). Studies in animals and humans demonstrate that PPARd activation with synthetic ligands like GW501516 exerts desirable effects that include reduced weight gain, increased skeletal muscle metabolic rate and endurance, improved insulin sensitivity, and reduced atherogenic inflammation (Rise´rus et al., 2008; Tanaka et al., 2003). The potential of PPARd as a therapeutic target for the treatment of metabolic syndrome has been reviewed previously (Reilly and Lee, 2008). PPARg is the most well-characterized member of the PPAR subfamily, as its central role in glucose metabolism and fatty acid storage has made it a desirable pharmacological target. There are two PPARg isoforms, PPARg1, which is expressed ubiquitously except for muscle, and PPARg2, which is expressed primarily in WAT under normal metabolic conditions (Fajas et al., 1997). PPARg2 is often called the ‘‘master regulator of adipogenesis’’ because it is required to drive the terminal differentiation of preadipocytes toward maturation (Chandra et al., 2008). Fat-specific PPARg knockout mice fail to generate adipose tissue in response to high-fat diet, demonstrating the receptors’ critical role in regulating fatty acid storage and glucose metabolism (Jones et al., 2005). The proper function of PPARg is critical to metabolic homeostasis, as genetic variants in humans can lead to familial partial lipodystrophy, an extreme monogenic form of metabolic syndrome (Jeninga et al., 2009). Interestingly, no general defects are observed in transgenic heterozygote PPARg knockout mice that demonstrate protection from high-fat-diet-induced obesity, fatty liver, and insulin resistance as a result of reduced triglyceride content in WAT, skeletal muscle, and liver (Akune et al., 2004; Kubota et al., 1999; Yamauchi et al., 2001). Activation of PPARg by endogenous ligands which may include polyunsaturated fatty acids (Kliewer et al., 1997; Krey et al., 1997), prostanoids like 15-deoxyD12,14 prostaglandin J2 (15-dPGJ2) (Forman et al., 1995; Kliewer et al., 1995), and oxidized fatty acids like 9-HODE and 13-HODE (Nagy et al., 1998) leads to increased expression of many target genes including aP2, GLUT4, PEPCK, C/EBP, Adipsin, and adiponectin (Perera et al., 2006; Tontonoz et al., 1994a, 1994b, 1995). The promiscuity of PPARg activation is a major challenge of avoiding adverse effects when pharmacologically targeting the receptor, and therapeutic development Cell Metabolism 19, February 4, 2014 ª2014 Elsevier Inc. 195

Cell Metabolism

Review efforts now aim to selectively modulate subsets of PPARg target genes by harnessing tissue-specific effects (Festuccia et al., 2009; Montague, 2002; Zı´dek et al., 2013) and nonclassical AF2 mechanisms of receptor control (Ahmadian et al., 2013; Cariou et al., 2012; Rosenson et al., 2012).

retain comparable antidiabetic efficacy to full agonist TZDs? And to the broader point, why is synthetic activation of PPARg so effective at insulin sensitization, when many obese and diabetic patients that respond to TZDs have no defects in the receptor?

PPARg Is the Pharmacological Target of the TZDs PPARg is the pharmacological target of the antidiabetic thiazolidinediones (TZDs) class of drugs that include rosiglitazone (Avandia) and pioglitazone (Actos) (Lehmann et al., 1995). The precursor lead to the TZDs, 2-chloro-3-phenylpropanoic acid ethyl ester (AL-294), was discovered through in vivo screening efforts against insulin-resistant rodent models (Ikeda et al., 1981; Iwatsuka et al., 1970; Kawamatsu et al., 1980). As such, the molecular target of the TZDs and their mechanism of action were unknown until several years after their discovery (Lehmann et al., 1995). TZDs have proven robust as insulin sensitizers and work synergistically with other T2DM therapeutic approaches (Elte and Blickle´, 2007; Garber et al., 2007; Hanefeld, 2007; Zinman et al., 2007, 2009). The mechanism by which TZDs derive efficacy is not fully understood but is thought to be through a combination of induction of insulin-sensitizing adipokines, antiinflammatory effects, and fatty acid partitioning from muscle to adipocytes (Girard, 2001; Martin et al., 1998; Saraf et al., 2012). These drugs have also been shown to have positive effects on cardiovascular disease (Hamblin et al., 2009; Plutzky, 2011), Alzheimer’s disease (Heneka et al., 2011), Parkinson’s disease (Carta et al., 2011), cancer (Blanquicett et al., 2008), and the browning of fat (Ohno et al., 2012) through various PPARg-mediated mechanisms. Unfortunately, concerns over adverse side effects including weight gain (Fonseca, 2003), edema (Nesto et al., 2003), plasma volume expansion (PVE) (Staels, 2005), increased risk of congestive heart failure (Nesto et al., 2003), and bone fracture (Aubert et al., 2010) have limited their utility. In particular, rosiglitazone has been restricted or withdrawn from most markets due to the increased incidence of myocardial infarction, likely a result of increased backpressure on the heart due to edema and PVE (Hsiao et al., 2009; Lipscombe et al., 2007; Loke et al., 2011). Pioglitazone, a comparatively less potent PPARg agonist with modest PPARa activity, has demonstrated improved cardiovascular outcomes, providing a potential mechanistic explanation for this improved therapeutic index (Juurlink et al., 2009; Winkelmayer et al., 2008). Based on these findings, efforts have focused on the pharmacological development of a class of ‘‘selective PPARg modulators’ (SPPARMs).

Posttranslational Modification of PPARg Some aspects of the PPARg paradox were addressed by the discovery that inflammatory cytokines associated with obesity activate the protein kinase cyclin-dependent kinase 5 (Cdk5), resulting in phosphorylation of PPARg at S273 (pS273) (Choi et al., 2010). This modification of PPARg correlated with repression of a subset of target genes shown to be dysregulated in obesity, including repression of the insulin-sensitizing adipokine, adiponectin. The efficacy of antidiabetic PPARg ligands such as rosiglitazone and the partial agonist MRL24 were shown to correlate with their ability to block pS273, independent of classical receptor transcriptional agonism (Choi et al., 2010; Rocchi et al., 2001). Based on these findings, an alternative class of high-affinity PPARg-targeting insulin sensitizers was developed that effectively block pS273 while avoiding classical AF2-driven receptor activation, termed functional selective PPARg modulators (FSPPARMs). SR1664 is a representative compound from this class that, in terms of insulin sensitization, was shown to be equally efficacious as rosiglitazone, without causing weight gain or hemodilution in vivo (Choi et al., 2011). SR1664 was derived from the potent partial agonist SPPARM GSK538 (Lamotte et al., 2010) and was designed to actively antagonize the receptor through an AF2 clash to ablate classical agonism as previously described for the estrogen receptor (Brzozowski et al., 1997; Shiau et al., 1998). Thus, this class of compounds is functionally selective in that they are devoid of classical AF2 agonism (do not drive expression of adipogenic genes) yet potently modulate a specific PTM to control expression of a unique subset of PPARg target genes. The picture that emerges from these findings is that the design of compounds targeting PPARg must move beyond the traditional paradigm of agonist/antagonist and include a focus on the modulation of PTMs. Recent work from the Manglesdorf and Kliewer labs suggesting a path toward more selective modulators has demonstrated that both beneficial and adverse effects of TZDs are a product of the induced expression of Fibroblast Growth Factor-21 (FGF21) and its ability to modulate receptor activity through SUMOylation of K107 (Dutchak et al., 2012; Qiang and Accili, 2012). A thorough review of PPARg PTMs to AF1 has previously been published (van Beekum et al., 2009). Here we focus on PTMs to the ligand binding domain that have been demonstrated to be modulated by synthetic ligand binding as strategies for targeting metabolic syndrome.

SPPARMs and the PPARg Paradox SPPARMs display reduced transcriptional activity in reporter assays (partial agonists), exhibit potent insulin sensitization on the same order as TZDs, and are anitiadipogenic with reduced adverse effects in animal models (Rangwala and Lazar, 2002). Importantly, SPPARMs demonstrate that the insulin-sensitizing effects of PPARg-targeted ligands can be separated from the adverse adipogenic effects. Several SPPARMs have advanced into clinical trials and may hold therapeutic promise (Gregoire et al., 2009; Motani et al., 2009); however, mechanistically they create a paradox. If PPARg-targeted ligands derive antidiabetic efficacy by activating the receptor, why do these poor agonists 196 Cell Metabolism 19, February 4, 2014 ª2014 Elsevier Inc.

SUMOylation of the PPARg LBD Leads to Repression of NFkB Inflammatory Genes Efforts to elucidate the mechanism by which PPARg agonists are capable of simultaneously activating PPARg and repressing NFkB target genes led to the discovery of a SUMOylation site at lysine 365 on the ligand binding domain (Pascual et al., 2005). Rosiglitazone and the SPPARM GW00072 were reported to promote SUMOylation of the receptor, targeting PPARg to the

Cell Metabolism

Review NCoR/HDAC3 complex on NFkB inflammatory gene promoters. This association prevents ubiquitylation and proteosomal degradation of the repressor complex that would typically lead to target gene activation, instead maintaining a repressed state (Pascual et al., 2005). Ligand-induced repression of these NFkB inflammatory target genes is associated with the desirable antidiabetic and antiatherogenic efficacy of rosiglitazone. This finding highlights the multiple roles of PPARg, which acts not only as a transcription factor to activate its own target genes but also as a cofactor capable of regulating the activity of other transcription factors. Acetylation of the PPARg LBD Controls Induction of Brown Adipose Tissue Adipose tissue in mammals can be distinguished as either WAT used to store excess energy or BAT that acts to dissipate energy in the form of heat. The strategy to promote the development of BAT pharmacologically is conceptually appealing as a defense against obesity and has been demonstrated viable in animal models (Enerba¨ck, 2010; Fukui et al., 2000; Sell et al., 2004; Wilson-Fritch et al., 2003) and in humans (Bogacka et al., 2005). PPARg ligands have been shown to drive the conversion of WAT to BAT by modulating association with PRDM16, a factor that controls the development of classical brown fat (Ohno et al., 2012). Despite the observation that full agonist TZDs are most efficacious at driving the ‘‘browning’’ effect, efforts to develop functional selective ligands with improved therapeutic index are buoyed by the finding that the association of PPARg:PRDM16 is regulated by recently identified PTMs (Qiang et al., 2012). Two acetylation sites on the PPARg ligand binding domain (lysine 268 and lysine 293) were recently identified in a report addressing the mechanism by which NAD-dependent deacetylase SirT1 gain of function mimics the insulin-sensitizing and ‘‘browning’’-of-WAT effects associated with TZDs (Qiang et al., 2012). The acetylation state of these sites was found to be associated with physiological cues such as low temperature, which triggers reduced acetylation and increased browning of WAT, while high-fat diet led to increased acetylation, reduced SirT1 association, and decreased insulin sensitivity. The TZDs rosiglitazone and troglitazone were both reported to increase association of PPARg:SirT1, reducing acetylation of both K268 and K293. Site-directed mutagenesis studies revealed that deacetylation of K293 was requisite for association of PPARg with PRDM16, while K268 PTM status had no effect. However, acetylation of both lysines was required to associate with NCoR, while deacetylation of either site was sufficient to displace. These findings provide a potential strategy for developing functional SPPARMs that drive the conversion of WAT to BAT by modulating acetylation of the receptor while avoiding AF2 activation that would lead to induction of adipogenesis and the associated adverse effects of TZDs. Development of PPARg Structure Activity Relationship Significant efforts have been made to structurally characterize the interaction between PPARg and synthetic ligands to develop optimized therapeutics and better characterize the functional differences between TZDs, SPPARMs, and FSPPARMs. Several cocrystal structures of isolated PPARg LBD bound to represen-

tative synthetic ligands have been reported, leading to the observation that full agonist TZDs and non-TZDs like MRL20 make a conserved hydrogen bond with Y473 on helix 12, while partial agonist SPPARMs do not (Bruning et al., 2007; Einstein et al., 2008; Nolte et al., 1998). These crystal structures have been valuable in identifying differences between TZDs and SPPARMs; however, no large-scale changes in the global protein fold of PPARg have been observed to explain the differential effects on corepressor and coactivator affinities (Bruning et al., 2007). The full-length crystal structure of PPARg in complex with RXRa and DNA was a significant achievement and provided the first insight into the interplay between various receptor domains, DNA, and ligands (Chandra et al., 2008). However, solution structures of the same full-length PPARg complex obtained using SAXS, SANS, and FRET demonstrate an extended asymmetric shape highlighting the dynamic nature of these interactions and the importance of utilizing complimentary structural approaches when developing structure activity relationships (SAR) (Rochel et al., 2011). It is important to note that none of these structures have been solved for the various PTMs of the receptor mentioned above. In efforts to better understand the complexity of functional response induced by ligand binding, hydrogen/deuterium exchange mass spectrometry (HDX-MS) has been applied to characterize changes in protein dynamics (Bruning et al., 2007; Dai et al., 2008, 2009; Wright et al., 2011; Zhang et al., 2010, 2011). Some of these studies have demonstrated that the degree to which ligand binding stabilizes the AF2 domain correlates with receptor activation (Bruning et al., 2007; Choi et al., 2011), while other modulators stabilize different regions of the LBD. This approach has been used to demonstrate that at least some partial-agonist SPPARMs activate PPARg using a helix 12independent mechanism (Bruning et al., 2007), a finding now supported by other reports (Klein et al., 2005; Puigserver et al., 1998; Waku et al., 2010). NMR studies have also demonstrated that ligand and receptor dynamics on a subsecond timescale affect the graded transcriptional output of PPARg modulators (Hughes et al., 2012). The application of complimentary structural approaches will continue to be critical in understanding the complex functional control of PPARg and other NRs by endogenous and synthetic ligands. Rev-erbs and RORs: Regulators of Circadian Rhythm, Lipid Homeostasis, and Metabolism Circadian rhythm is a biological process that displays a pattern of daily oscillation, is controlled by endogenous factors, and is entrainable. Circadian oscillations are necessary for an organism’s survival, as they allow the anticipation and adaptation to predictable changes in light/dark cycles, activity/rest cycles, and feeding times. The central circadian clock is located in the suprachiasmatic nucleus within the hypothalamus and directly receives environmental cues from ganglion cells in the retina (Green et al., 2008). Disturbances of the circadian clock have been associated with increased incidence of obesity, diabetes, and other disorders (Gachon et al., 2004; Sahar and SassoneCorsi, 2009; Weldemichael and Grossberg, 2010). Hormones and adipokines are expressed and secreted in a circadian rhythmic fashion; however, their expression patterns in obese animals and humans are distorted (Ando et al., 2005; Yildiz et al., 2004). Cell Metabolism 19, February 4, 2014 ª2014 Elsevier Inc. 197

Cell Metabolism

Review Figure 3. Nuclear Receptors and Circadian Rhythm Members of the PPAR, ROR, and Rev-erb NR subfamilies regulate circadian rhythms and expression of core clock genes. Therapeutic targeting of these NRs to normalize dysregulated circadian rhythm may afford a treatment strategy for metabolic syndrome.

The observation that shift workers, who typically have irregular sleep and eating times, disproportionately display impaired insulin sensitivity, higher body mass, and hypertension highlights the correlation between proper circadian clock oscillation and metabolic homeostasis (Gangwisch et al., 2005; Rudic et al., 2004). Metabolic control of the circadian rhythm starts with the oligomerization and binding of core clock activators brain and muscle aryl hydrocarbon receptor nuclear translocator (ARNT)like protein 1 (BMAL1), circadian locomotor output cycles kaput (CLOCK), and neuronal PAS domain protein 2 (NPAS2) to E box (50 -CACGTC-30 ) enhancer elements (Church et al., 1985; DeBruyne et al., 2007; Gachon et al., 2004; Hao et al., 1997). This event drives the activation of several clock genes, including Per and Cry, which heterodimerize, and at critical concentrations can translocate into the nucleus repressing BMAL1/CLOCK, completing the negative feedback loop in a circadian pattern (Ko and Takahashi, 2006). Environmental cues such as light, temperature, and food can induce the rapid activation of cyclic AMP production, Ca2+ signaling, and MAPK pathway, leading to induction of Per and Cry genes and demonstrating the complex interplay of circadian factors that control energy balance, feeding behavior, and metabolic processes (Hirota and Fukada, 2004; Panda et al., 2002; Storch et al., 2002). The RORs and Rev-erbs are considered core clock machinery because they regulate the cyclic expression of BMAL1 and CLOCK, providing an essential link between the positive and negative loops of the circadian clock (Figure 3) (Charoensuksai and Xu, 2010; Solt et al., 2011a). The PPARs and core clock genes have been demonstrated to crossregulate transcriptional outputs in peripheral tissues (Canaple et al., 2006; Gatfield et al., 2009; Nakamura et al., 2008; Oishi et al., 2005; Wang et al., 2008), and CLOCK mutant mice have been shown to disrupt the circadian expression of PPARa in the liver (Lemberger et al., 1996; Oishi et al., 2005). Rev-erbs (a and b isoforms) and RORs (a and g isoforms) are coexpressed in adipose tissue, liver, skeletal muscle, and brain (Bonnelye et al., 1994; Dumas et al., 1994; Forman et al., 1994; Moore et al., 2006). As monomers, both Rev-erbs and RORs occupy the same RORE ‘‘half-site’’ with a 50 AT-rich region preceding the response element (Harding and Lazar, 1993, 1995; Moraitis and Gigue`re, 1999). The occupancy of the RORE is synergized between Rev-erbs acting as 198 Cell Metabolism 19, February 4, 2014 ª2014 Elsevier Inc.

constitutive repressors and RORs as constitutive activators to fine-tune the expression of BMAL1, CLOCK, and several other oscillating gene networks involved in glucose and lipid metabolism (Cho et al., 2012; Guillaumond et al., 2005; Jetten, 2009; Sato et al., 2004; Solt et al., 2010). Here we review the current understanding of the ROR and Reverb NR subfamilies’ role in metabolic disorder, and the most recent efforts to develop synthetic ligand modulators to serve as chemical probes and therapeutics. Therapeutic Targeting of RORs for Metabolic Syndrome The first member of the ROR subfamily of receptors (RORa) was identified based on sequence similarities to the retinoic acid receptor (RAR) and the RXR, yielding the name ‘‘retinoic acid receptor-related orphan receptor’’ (Gigue`re et al., 1994). Two other members of this NR subfamily, RORb and RORg, were subsequently identified (Hirose et al., 1994). The RORs display distinct patterns of tissue expression and are involved in the regulation of various physiological processes. RORa is predominantly expressed in lung, muscle, brain, heart, peripheral blood leukocytes, spleen, liver, and ovary, whereas expression of RORb is limited to the central nervous system (Andre´ et al., 1998a, 1998b). Two isoforms of RORg are found in both humans and mice (RORg1 and RORg2), with RORg2 commonly referred to as RORgt, as it was originally identified in the thymus (Jetten et al., 2001), although its expression is not limited to T cells. RORgt, specifically RORgt2, is highly expressed in immune tissue including the thymus, but there is also significant expression of RORg in the liver, skeletal muscle, adipose tissue, and kidney (Hirose et al., 1994; Jetten, 2009). Given the specific tissue distribution of each ROR receptor and their potential role in pathophysiological conditions, there is considerable interest in developing synthetic ligand modulators as therapeutics. RORa has been shown to regulate the expression of several components of the circadian clock system. These include genes encoding enzymes and transporters involved in nutrient transport and metabolism, cellular cholesterol homeostasis, xenobiotic detoxification, and energy balance (Panda et al., 2002). Furthermore, RORa regulates the expression of apolipoprotein CIII, a component of HDL and very-low-density lipoprotein that plays a role in regulation of triglyceride levels and lipoprotein lipase (LPL) activity (Raspe´ et al., 2001). Genetic, molecular, and biochemical studies have demonstrated that RORadeficient mice develop severe atherosclerosis and have low HDL-C, hypo-a-lipoproteinemia, muscular atrophy, and heightened inflammatory response (Jarvis et al., 2002; Lau et al., 1999; Steinmayr et al., 1998). More recently, RORa has been

Cell Metabolism

Review shown to control the expression of genes involved in lipid absorption, b-oxidation, cholesterol efflux, and energy expenditure in skeletal muscle cells (C2C12) (Lau et al., 2004). RORa has also been shown to regulate the expression of FGF21, a potential pharmacological target gene for treatment of metabolic disease (Wang et al., 2010). Observations in the RORasg/sg stagger mouse provide insight into the role of the receptor in metabolic homeostasis. These mice, which express nonfunctional RORa, are resistant to dietinduced obesity (Lau et al., 2008). Analysis of these mice showed a significant reduction in the levels of expression of SREBP1c and FAS. SREBP1 is a critical transcriptional regulator that controls the expression of genes involved in fatty acid biogenesis, and directly regulates lipogenic enzymes such as FAS (Shimano et al., 1999). Interestingly, genes related to the thermogenic program such as b2-AR, UCP-1, NOR-1, and PGC1(a/b) were increased in BAT in these mice (Bertin et al., 1990; Crunkhorn et al., 2007; Lau et al., 2008). These findings suggest that the development of synthetic RORa agonist/inverse agonist ligands may hold therapeutic potential for the treatment of metabolic disorders and in parallel will serve as useful chemical probes to better understand the receptors’ functional role. RORg, along with RORa, is expressed in skeletal muscle, a tissue that accounts for approximately 40% of total body mass and 50% of energy expenditure and is a major site of fatty acid and glucose oxidation (Rasmussen and Wolfe, 1999). It has been shown that RORg controls expression of genes that regulate muscle activity, fat mass, and lipid homeostasis (FABP4, CD36, and LPL) and plays a role in the regulation of reactive oxygen species (ROS) (Raichur et al., 2007). Microarray analyses of liver tissue from RORasg/sg, RORg/, and RORsg/sg RORg/ double knockout mice (Kang et al., 2007) revealed that RORa and RORg are critical regulators of hepatic genes encoding several phase I and phase II metabolic enzymes, including 3b-hydroxysteroid dehydrogenases, cytochrome P450 enzymes, and sulfotransferases. Mice deficient in RORg also exhibit reduced blood glucose levels (Kang et al., 2007). In double knockout mice, a similar reduction in cholesterol, triglyceride, and blood glucose levels was observed as compared to single gene knockout (Kang et al., 2007; Meissburger et al., 2011). As muscle and liver are critical mediators of insulin sensitivity, lipid metabolism, and energy balance (Lau et al., 2004; Ramakrishnan et al., 2005), the pharmacological targeting of RORa and RORg holds promise for the treatment of metabolic syndrome and associated diseases. Ligand Modulation of the RORs RORa, RORb, and RORg have long been considered orphan receptors, as endogenous ligands have yet to be unanimously agreed upon. Regardless, crystal structures of these receptors bound to naturally occurring ligands have provided insight into molecules that may be functionally relevant. The first ROR LBD subtype to be crystallized was RORb in complex with stearic acid, a prevalent saturated fatty acid (Stehlin et al., 2001). Subsequently, all-trans retinoic acid (ATRA) and a synthetic analog (ALRT 1550) were identified as functional ligands (Stehlin-Gaon et al., 2003). RORa LBD crystal structures identified cholesterol and cholesterol sulfate as potential ligands and served as early indicators that RORa played a central role in lipid metabolism

(Kallen et al., 2002, 2004). It is interesting to note that in spite of the high sequence similarity and similar-sized ligand binding pockets (722 A˚3 and 766 A˚3, respectively) of the RORa and RORb LBDs, cholesterol has no effect on RORb activity. More recently, the crystal structures of RORg in complex with 20a-, 22R-, and 25- hydroxycholesterol were reported (Jin et al., 2010). These oxysterols were shown to have agonist activity on the receptor driving an active AF2 confirmation that facilitates recruitment of coactivator to the conserved charge clamp comprised of helices 3, 4, 5, and 12 of the LBD. This finding served as a critical first step toward designing synthetic RORg modulators and further suggests the receptors’ role as a central mediator of metabolism and lipid homeostasis. In contrast, the crystal structure of RORg in complex with antagonist digoxin, an extract from the foxglove plant (Digitalis lanata), highlights the molecular interactions critical for antagonizing coactivator interaction (Fujita-Sato et al., 2011). This structure reveals that digoxin protrudes between helices 3 and 11 of the LBD and prevents positioning of helix 12 into an active conformation. These observations provide a template by which to design synthetic small-molecule modulators of RORg. Due to the emerging role of RORs in several human diseases, considerable efforts have been made to develop synthetic exogenous ligands with improved potency, specificity, and pharmacokinetics. The synthetic agonist of the liver X receptor (LXR) T0901317 was the first modest affinity synthetic small molecule to be identified as a dual RORa/g antagonist/inverse agonist (Figure 4) (Houck et al., 2004; Kumar et al., 2010b; Li et al., 2006; Mitro et al., 2007). Removal of the sulfonamide alkyl group from the T0901317 scaffold resulted in RORa/g antagonists/ inverse agonist devoid of LXR activity (Kumar et al., 2010a; Solt et al., 2011b). One such example, SR1001, repressed both RORa-Gal4 and RORg-Gal4 transcriptional activity with EC50’s 4–6 mm (Solt et al., 2011b). SR1001 inhibited the development of murine TH17 cells, as demonstrated by inhibition of interleukin-17A gene expression and protein production. Furthermore, SR1001 inhibited the expression of cytokines (IL17a, IL17f, IL21, and IL22) when added to differentiated murine or human TH17 cells. Finally, SR1001 effectively delayed the onset and clinical severity of autoimmune disease in a MOGinduced mouse model (EAE) of multiple sclerosis. HDX experiments have provided the first structural insight into the mechanism of transcriptional repression, demonstrating that SR1001 disrupts interaction with the receptor interacting domain (RID) of coactivator SRC2 (Solt et al., 2011b). Further optimization of the T091317 scaffold led to the identification of SR3335, a RORa selective antagonist/inverse agonist (Kumar et al., 2010a; Solt et al., 2012a). SR3335 significantly inhibited the constitutive activity of RORa in a cell-based transactivation assay (IC50 = 480 nM) but had no effect on the activity of LXRa, RORb, and RORg. Diet-induced obese (DIO) mice were treated with SR3335 for 6 days, and a pyruvate tolerance test revealed reduced plasma glucose levels compared with vehicle-treated cohorts. Importantly, mice treated with SR3335 displayed no difference in body weight or food intake after 7 days of treatment, indicating the effects on glucose homeostasis are not secondary to weight loss and represent a metabolic response. These data suggest SR3335 is a useful chemical probe for evaluating the in vitro and in vivo functions of RORa Cell Metabolism 19, February 4, 2014 ª2014 Elsevier Inc. 199

Cell Metabolism

Review Figure 4. Evolution of Synthetic RORa/g Inverse Agonists Next-generation selective RORa/g duel agonist and subtype-specific inverse agonists from T0901317, a synthetic dual RORa/g inverse agonist with LXR agonist activity. T0901317 RORa IC50 0.132 mM, RORg 0.051 mM; SR1078 IC50 RORa 1–3 mM, RORg 1–3 mM; SR1001 IC50 RORa 0.172 mM, RORg 0.111 mM; SR3335 IC50 RORa 0.480 mM; SR2211 IC50 RORg 0.320 mM; SR1555 IC50 RORg 1.5 mM.

and suggest that compounds like SR3335 may hold utility in the treatment of type 2 diabetes. Optimization of synthetic ligands for the RORs has recently been reviewed (Kamenecka et al., 2013). It is particularly interesting that subtle changes to the same chemical scaffold can yield ROR subtype selective ligands and alter their pharmacology from antagonists/inverse agonists to agonists. Therapeutic Targeting of the Rev-Erbs for Metabolic Syndrome The Rev-erb subfamily of NRs consists of two members, Reverba (NR1D1) (Lazar et al., 1989) and Rev-erbb (NR1D2) (Dumas et al., 1994), similar in protein structure and function. The Reverbs are unique NRs in that they have a truncated C terminus (devoid of helix 12 of AF2) which prevents association with coactivator proteins (Woo et al., 2007). Importantly, the Rev-erbs retain the ability to complex with corepressor proteins like NCoR (Zamir et al., 1996), forming a potent transcriptional repressor complex that is critical for proper metabolic function. 200 Cell Metabolism 19, February 4, 2014 ª2014 Elsevier Inc.

Rev-erb proteins are expressed in multiple tissues including adipose, liver, brain, and skeletal muscle. The orchestrated expression pattern of Rev-erba in adipose tissue has been demonstrated to be a critical regulator of adipogenesis (Chawla and Lazar, 1993; Laitinen et al., 2005; Wang and Lazar, 2008) and can be modulated by TZD-induced activation of PPARg (Fontaine et al., 2003). The Reverbs’ role in lipid metabolism and glucose homeostasis and involvement in cholesterol trafficking are also a result of their ability to modulate key enzymes in each pathway. Rev-erbs have been reported to regulate the expression of LPL, an enzyme involved in the uptake and accumulation of lipids in multiple organs and cell types (Babaev et al., 1999; Kern, 1997; Ong et al., 1994; Pulinilkunnil and Rodrigues, 2006). Rev-erba deficiency in mice elevates LPL levels in peripheral tissues (liver, muscle, and adipose tissue) correlating with increases in body weight and overall adiposity (Delezie et al., 2012). Further, the sterol regulatory elementbinding protein 1c (Srebp-1c) and its target gene fatty acid synthase (FAS), key regulators of lipogenesis, were reduced upon silencing of Rev-erba (Le Martelot et al., 2009). Rev-erba deficiency in skeletal muscle leads to reduced mitochondrial content and oxidative function resulting in compromised exercise capacity, while overexpression and pharmacological activation of the receptor led to an improvement (Woldt et al., 2013). Cholesterol trafficking is partly regulated by Rev-erb proteins through transcriptional regulation of various lipoproteins. APOC3, a component of HDL and VLDL that regulates triglyceride levels, is present in high levels in Rev-erba null mice (Coste and Rodrı´guez, 2002). These same mice also suffer from abnormally high VLDL and triglyceride levels. ApoA1, a significant component of HDL necessary for the transport of cholesterol to the liver for excretion into the bile, is repressed by fibrates (Lefebvre et al., 2009). Fibrates, a class of hypolipidemic drugs and known PPARa activators, increased Rev-erb mRNA levels 10-fold in rat livers, likely mediated through PPARa transactivation (Gervois et al., 1999). Rev-erbs also can modulate bile acid

Cell Metabolism

Review

Figure 5. Evolution of Synthetic Rev-Erb Modulators Generation of synthetic Rev-erb ligands started from the discovery of heme as an endogenous ligand. To date, various synthetic modulators with altered function have been developed. GSK4112 EC50 Rev-erba 0.40 mM; SR9009 IC50 Rev-erba 0.67 mM, Rev-erbb 0.80 mM; SR9011 IC50 Rev-erba 0.79 mM, Rev-erbb 0.56 mM; SR8278 EC50 Rev-erba 0.47 mM.

synthesis through regulation of cholesterol 7a-hydroxylase (Cyp7A1), the rate-limiting enzyme in the classic pathway of bile acid synthesis from cholesterol in the liver (Duez et al., 2008). Mice lacking Rev-erba have asynchronous circadian rhythms, with a predisposition to diet-induced obesity, impaired glucose, and lipid utilization leading to increased susceptibility to diabetes (Delezie et al., 2012; Preitner et al., 2002). Depletion of both Rev-erb a/b isoforms in mice results in elevated hepatic triglyceride levels triggering hepatosteatosis (Bugge et al., 2012). Rev-erbs have been shown to regulate hepatic glucose production, with increased heme binding demonstrated to repress the gluconeogenic gene PepCK in HepG2 cells (Yin et al., 2007). Synthetic modulators of Rev-erbs have been shown to alter expression of G6Pase, the enzyme controlling the final step of gluconeogenesis (Grant et al., 2010; Kojetin et al., 2011). These findings support the role of Rev-erbs in both lipid and glucose homeostasis, making these receptors attractive targets of therapeutic intervention for metabolic disorders. Rev-Erb Ligands Rev-erba and Rev-erbb were identified as orphan nuclear receptors based on their conserved NR domain structure and homology with other NRs (Bonnelye et al., 1994; Dumas et al., 1994; Lazar et al., 1989). The identification of the porphorin heme as

a physiological ligand capable of regulating receptor function provided further molecular and mechanistic insights into the role of Rev-erbs (Burris, 2008; Raghuram et al., 2007; Yin et al., 2007). Heme binding modulates the repressive activity of Rev-erb by increasing recruitment of the corepressor NCoR and HDAC3 to DNA response elements in promoter regions of Rev-erb target genes (Guillaumond et al., 2005; Kumar et al., 2010c; Raspe´ et al., 2002; Yin et al., 2007). Heme has also been shown to promote proteasomal degradation of the receptor, a critical event for adipogenesis (Kumar et al., 2010c; Wang and Lazar, 2008). The cocrystal structure of Rev-erbb in complex with heme shows that a single molecule of the porphorin is bound, and it has been demonstrated that the receptor can adopt multiple conformational states depending on the iron oxidation state (Marvin et al., 2009; Pardee et al., 2009). Despite these findings, the utility of heme as a chemical probe to study the functional effects of Rev-erb ligand binding is limited due to its cell toxicity at high concentrations and the lack of selectivity for Rev-erb (Alayash, 2004; Kim et al., 2004). To address these shortcomings, efforts to develop synthetic Rev-erb ligands were initiated with the development of a fluorescence resonance energy transfer (FRET) biochemical assay monitoring the interaction between the Rev-erb LBD and a peptide representing the first interaction domain of NCoR (NCoR 1D1) (Grant et al., 2010). Using this assay to screen targeted chemical libraries, the first synthetic agonist GSK4112 was identified, and it was shown that this compound enhanced the interaction of Rev-erbs with NCoR (Figure 5) (Grant et al., 2010; Kumar et al., 2010c; Meng et al., 2008). Importantly, GSK4112 was compatible with cellular studies and was shown to inhibit the expression of the circadian target gene bmal1, to repress expression of gluconeogenic genes in liver cells, and to reduce glucose output in primary hepatocytes (Grant et al., 2010). GSK4112 was also reported to induce adipogenesis in 3T3-L1 cells, leading to increased lipid accumulation and increased expression of adipogenic genes including aP2, NR1C3, and AdipoQ (Kumar et al., 2010c). Interestingly, GSK4112 was also shown to synergize with rosiglitazone in promoting lipid accumulation (Kumar et al., 2010c). More recently, the Rev-erb agonists SR9009 and SR9011 were described, representing chemical tools with improved pharmacokinetics over GSK4112 that were sufficient to enable in vivo studies (Solt et al., 2012b). Treatment of obese mice with SR9009 resulted in weight loss, improved dyslipidemia, and improved hyperglycemia. SR9009 and SR9011 were reported to alter gene expression in several tissues including liver, skeletal muscle, and WAT. In skeletal muscle the Rev-erb agonists induced expression of genes involved in fatty acid oxidation and glycolysis such as Cpt1b, Ucp3, Ppargc1b, Pkm2, and Hk1. These Rev-erb agonists also decreased expression of the lipogenic enzymes Fasn and Scd1 and the cholesterologenic regulatory proteins Hmgcr and Srebf2, consistent with the decreased triglyceride and cholesterol synthesis observed in liver and WAT from treated animals. Taken together, these observations suggest Rev-erb agonists increase whole-body energy expenditure, offering an approach for treatment of metabolic syndrome. Recently, the first synthetic Rev-erba antagonist (SR8278) was described to oppose the action of heme and drive Cell Metabolism 19, February 4, 2014 ª2014 Elsevier Inc. 201

Cell Metabolism

Review increased expression of Rev-erba target genes (Kojetin et al., 2011). SR8278 is structurally similar to the synthetic agonist GSK4112; however, it too has poor pharmacokinetic properties. Despite this shortcoming, SR8278 represents a chemical probe to characterize Rev-erba in cell-based models. Future Directions and Perspective The TZD class of PPARg-targeted insulin sensitizers for the treatment of T2DM had an unfettered decade-long run of commercial and clinical success. However, the long-term safety concerns associated with this class of drugs have significantly increased the scrutiny which all next-generation PPARg-targeted therapeutics must face. While the TZD pioglitazone is still widely used clinically for the treatment of T2DM, its future is somewhat uncertain as reports of bladder cancer incidence and bone fractures continue to persist (Stephenson, 2011). Nevertheless, PPARg’s role as the ‘‘master regulator of adipogenesis’’ and the growing need for safe and effective drugs to treat metabolic syndrome will ensure it remains a target of interest for pharmacological development. It will be critical for future development efforts to better understand the complexity of functional response induced by ligand binding. These can include the effects of ligand binding on PTM status, oligomeric state, and cofactor and DNA affinity, in addition to classical AF2-dependent agonism. Monitoring, in an unbiased fashion, the expression profile of PPARg target genes in response to a wide range of pharmacologically distinct ligands will be critical in delineating function in this increasingly complex, multivariable system. The development of functional SPPARMs like SR1664 demonstrates how improved understanding of the complex NR regulatory networks can lead to new approaches to modulate old targets. The central role that has emerged for the RORs and Rev-erbs in regulating the circadian clock and metabolic gene networks that control glucose and lipid metabolism strongly suggests their potential for therapeutic targeting for the treatment of metabolic syndrome. Pharmacological development efforts are still in their infancy, but the increasing interest in these targets promises that a wave of synthetic ligands will emerge covering the spectrum of functional outputs and with optimized pharmacokinetics. If the lessons learned from PPARg are predictive, it is likely that functional selective ROR and Rev-erb modulators that affect receptor oligomeric state, cofactor and DNA affinities, and PTMs will be critical for formulating pharmacological strategies and limiting adverse effects. Functionally relevant PTMs for both Rev-erba (Yin et al., 2006) and RORa (Ermisch et al., 2011; Hwang et al., 2009; Lechtken et al., 2007; Lee et al., 2010) have been reported, and understanding the effect of ligand binding on modulating these sites will be critical. It has been demonstrated that inhibition of GSK3b with lithium can destabilize Rev-erba and affect the circadian clock, potentially leading to its efficacy in circadian diseases such as bipolar disorder (Yin et al., 2006). Targeting the NR substrate as opposed to the modifying enzymes, as demonstrated with PPARg, may prove to be a desirable strategy with reduced off-target effects. As the complexity of NR biology comes more into focus, the opportunities to develop mechanism-based, functional selective ligands that translate into therapeutics targeting metabolic syndrome and a variety of other diseases will continue to emerge. 202 Cell Metabolism 19, February 4, 2014 ª2014 Elsevier Inc.

REFERENCES Ahmadian, M., Suh, J.M., Hah, N., Liddle, C., Atkins, A.R., Downes, M., and Evans, R.M. (2013). PPARg signaling and metabolism: the good, the bad and the future. Nat. Med. 19, 557–566. Akune, T., Ohba, S., Kamekura, S., Yamaguchi, M., Chung, U.I., Kubota, N., Terauchi, Y., Harada, Y., Azuma, Y., Nakamura, K., et al. (2004). PPARgamma insufficiency enhances osteogenesis through osteoblast formation from bone marrow progenitors. J. Clin. Invest. 113, 846–855. Alayash, A.I. (2004). Oxygen therapeutics: can we tame haemoglobin? Nat. Rev. Drug Discov. 3, 152–159. Ando, H., Yanagihara, H., Hayashi, Y., Obi, Y., Tsuruoka, S., Takamura, T., Kaneko, S., and Fujimura, A. (2005). Rhythmic messenger ribonucleic acid expression of clock genes and adipocytokines in mouse visceral adipose tissue. Endocrinology 146, 5631–5636. Andre´, E., Conquet, F., Steinmayr, M., Stratton, S.C., Porciatti, V., and BeckerAndre´, M. (1998a). Disruption of retinoid-related orphan receptor beta changes circadian behavior, causes retinal degeneration and leads to vacillans phenotype in mice. EMBO J. 17, 3867–3877. Andre´, E., Gawlas, K., and Becker-Andre´, M. (1998b). A novel isoform of the orphan nuclear receptor RORbeta is specifically expressed in pineal gland and retina. Gene 216, 277–283. Aubert, R.E., Herrera, V., Chen, W., Haffner, S.M., and Pendergrass, M. (2010). Rosiglitazone and pioglitazone increase fracture risk in women and men with type 2 diabetes. Diabetes Obes. Metab. 12, 716–721. Babaev, V.R., Fazio, S., Gleaves, L.A., Carter, K.J., Semenkovich, C.F., and Linton, M.F. (1999). Macrophage lipoprotein lipase promotes foam cell formation and atherosclerosis in vivo. J. Clin. Invest. 103, 1697–1705. Berger, J., and Moller, D.E. (2002). The mechanisms of action of PPARs. Annu. Rev. Med. 53, 409–435. Berlie, H.D., and Hurren, K.M. (2013). Evaluation of lorcaserin for the treatment of obesity. Expert Opin. Drug Metab. Toxicol. 9, 1053–1059. Bertin, R., Guastavino, J.M., and Portet, R. (1990). Effects of cold acclimation on the energetic metabolism of the staggerer mutant mouse. Physiol. Behav. 47, 377–380. Blanquicett, C., Roman, J., and Hart, C.M. (2008). Thiazolidinediones as anti-cancer agents. Cancer Ther. 6 (A), 25–34. Bloom, S.R., Kuhajda, F.P., Laher, I., Pi-Sunyer, X., Ronnett, G.V., Tan, T.M., and Weigle, D.S. (2008). The obesity epidemic: pharmacological challenges. Mol. Interv. 8, 82–98. Bogacka, I., Xie, H., Bray, G.A., and Smith, S.R. (2005). Pioglitazone induces mitochondrial biogenesis in human subcutaneous adipose tissue in vivo. Diabetes 54, 1392–1399. Bonnelye, E., Vanacker, J.M., Desbiens, X., Begue, A., Stehelin, D., and Laudet, V. (1994). Rev-erb beta, a new member of the nuclear receptor superfamily, is expressed in the nervous system during chicken development. Cell Growth Differ. 5, 1357–1365. Bouchard, C. (1988). Genetic factors in the regulation of adipose tissue distribution. Acta Med. Scand. Suppl. 723, 135–141. Bruning, J.B., Chalmers, M.J., Prasad, S., Busby, S.A., Kamenecka, T.M., He, Y., Nettles, K.W., and Griffin, P.R. (2007). Partial agonists activate PPARgamma using a helix 12 independent mechanism. Structure 15, 1258– 1271. Brzozowski, A.M., Pike, A.C., Dauter, Z., Hubbard, R.E., Bonn, T., Engstro¨m, O., Ohman, L., Greene, G.L., Gustafsson, J.A., and Carlquist, M. (1997). Molecular basis of agonism and antagonism in the oestrogen receptor. Nature 389, 753–758. Bugge, A., Feng, D., Everett, L.J., Briggs, E.R., Mullican, S.E., Wang, F., Jager, J., and Lazar, M.A. (2012). Rev-erba and Rev-erbb coordinately protect the circadian clock and normal metabolic function. Genes Dev. 26, 657–667. Burris, T.P. (2008). Nuclear hormone receptors for heme: REV-ERBalpha and REV-ERBbeta are ligand-regulated components of the mammalian clock. Mol. Endocrinol. 22, 1509–1520.

Cell Metabolism

Review Canaple, L., Rambaud, J., Dkhissi-Benyahya, O., Rayet, B., Tan, N.S., Michalik, L., Delaunay, F., Wahli, W., and Laudet, V. (2006). Reciprocal regulation of brain and muscle Arnt-like protein 1 and peroxisome proliferator-activated receptor alpha defines a novel positive feedback loop in the rodent liver circadian clock. Mol. Endocrinol. 20, 1715–1727. Cariou, B., Charbonnel, B., and Staels, B. (2012). Thiazolidinediones and PPARg agonists: time for a reassessment. Trends Endocrinol. Metab. 23, 205–215. Carta, A.R., Pisanu, A., and Carboni, E. (2011). Do PPAR-gamma agonists have a future in Parkinson’s disease therapy? Parkinsons Dis. 2011, 689181. Carter, R., Mouralidarane, A., Ray, S., Soeda, J., and Oben, J. (2012). Recent advancements in drug treatment of obesity. Clin. Med. 12, 456–460. Chandra, V., Huang, P., Hamuro, Y., Raghuram, S., Wang, Y., Burris, T.P., and Rastinejad, F. (2008). Structure of the intact PPAR-gamma-RXR- nuclear receptor complex on DNA. Nature 456, 350–356. Charoensuksai, P., and Xu, W. (2010). PPARs in rhythmic metabolic regulation and implications in health and disease. PPAR Res. 2010, 2010, http://dx.doi. org/10.1155/2010/243643.

Duez, H., van der Veen, J.N., Duhem, C., Pourcet, B., Touvier, T., Fontaine, C., Derudas, B., Bauge´, E., Havinga, R., Bloks, V.W., et al. (2008). Regulation of bile acid synthesis by the nuclear receptor Rev-erbalpha. Gastroenterology 135, 689–698. Dumas, B., Harding, H.P., Choi, H.S., Lehmann, K.A., Chung, M., Lazar, M.A., and Moore, D.D. (1994). A new orphan member of the nuclear hormone receptor superfamily closely related to Rev-Erb. Mol. Endocrinol. 8, 996–1005. Dutchak, P.A., Katafuchi, T., Bookout, A.L., Choi, J.H., Yu, R.T., Mangelsdorf, D.J., and Kliewer, S.A. (2012). Fibroblast growth factor-21 regulates PPARg activity and the antidiabetic actions of thiazolidinediones. Cell 148, 556–567. Einstein, M., Akiyama, T.E., Castriota, G.A., Wang, C.F., McKeever, B., Mosley, R.T., Becker, J.W., Moller, D.E., Meinke, P.T., Wood, H.B., and Berger, J.P. (2008). The differential interactions of peroxisome proliferatoractivated receptor gamma ligands with Tyr473 is a physical basis for their unique biological activities. Mol. Pharmacol. 73, 62–74. Elte, J.W., and Blickle´, J.F. (2007). Thiazolidinediones for the treatment of type 2 diabetes. Eur. J. Intern. Med. 18, 18–25. Enerba¨ck, S. (2010). Human brown adipose tissue. Cell Metab. 11, 248–252.

Chawla, A., and Lazar, M.A. (1993). Induction of Rev-ErbA alpha, an orphan receptor encoded on the opposite strand of the alpha-thyroid hormone receptor gene, during adipocyte differentiation. J. Biol. Chem. 268, 16265–16269.

Ermisch, M., Firla, B., and Steinhilber, D. (2011). Protein kinase A activates and phosphorylates RORa4 in vitro and takes part in RORa activation by CaMK-IV. Biochem. Biophys. Res. Commun. 408, 442–446.

Cheng, A.Y., and Leiter, L.A. (2008). PPAR-alpha: therapeutic role in diabetesrelated cardiovascular disease. Diabetes Obes. Metab. 10, 691–698.

Evans, R.M. (1988). The steroid and thyroid hormone receptor superfamily. Science 240, 889–895.

Cho, H., Zhao, X., Hatori, M., Yu, R.T., Barish, G.D., Lam, M.T., Chong, L.W., DiTacchio, L., Atkins, A.R., Glass, C.K., et al. (2012). Regulation of circadian behaviour and metabolism by REV-ERB-a and REV-ERB-b. Nature 485, 123–127.

Fajas, L., Auboeuf, D., Raspe´, E., Schoonjans, K., Lefebvre, A.M., Saladin, R., Najib, J., Laville, M., Fruchart, J.C., Deeb, S., et al. (1997). The organization, promoter analysis, and expression of the human PPARgamma gene. J. Biol. Chem. 272, 18779–18789.

Choi, J.H., Banks, A.S., Estall, J.L., Kajimura, S., Bostro¨m, P., Laznik, D., Ruas, J.L., Chalmers, M.J., Kamenecka, T.M., Blu¨her, M., et al. (2010). Anti-diabetic drugs inhibit obesity-linked phosphorylation of PPARgamma by Cdk5. Nature 466, 451–456. Choi, J.H., Banks, A.S., Kamenecka, T.M., Busby, S.A., Chalmers, M.J., Kumar, N., Kuruvilla, D.S., Shin, Y., He, Y., Bruning, J.B., et al. (2011). Antidiabetic actions of a non-agonist PPARg ligand blocking Cdk5-mediated phosphorylation. Nature 477, 477–481. Church, G.M., Ephrussi, A., Gilbert, W., and Tonegawa, S. (1985). Cell-typespecific contacts to immunoglobulin enhancers in nuclei. Nature 313, 798–801. Clinckemalie, L., Vanderschueren, D., Boonen, S., and Claessens, F. (2012). The hinge region in androgen receptor control. Mol. Cell. Endocrinol. 358, 1–8. Coste, H., and Rodrı´guez, J.C. (2002). Orphan nuclear hormone receptor Reverbalpha regulates the human apolipoprotein CIII promoter. J. Biol. Chem. 277, 27120–27129. Crunkhorn, S., Dearie, F., Mantzoros, C., Gami, H., da Silva, W.S., Espinoza, D., Faucette, R., Barry, K., Bianco, A.C., and Patti, M.E. (2007). Peroxisome proliferator activator receptor gamma coactivator-1 expression is reduced in obesity: potential pathogenic role of saturated fatty acids and p38 mitogenactivated protein kinase activation. J. Biol. Chem. 282, 15439–15450. Dai, S.Y., Chalmers, M.J., Bruning, J., Bramlett, K.S., Osborne, H.E., Montrose-Rafizadeh, C., Barr, R.J., Wang, Y., Wang, M., Burris, T.P., et al. (2008). Prediction of the tissue-specificity of selective estrogen receptor modulators by using a single biochemical method. Proc. Natl. Acad. Sci. USA 105, 7171–7176.

Festuccia, W.T., Blanchard, P.G., Turcotte, V., Laplante, M., Sariahmetoglu, M., Brindley, D.N., and Deshaies, Y. (2009). Depot-specific effects of the PPARgamma agonist rosiglitazone on adipose tissue glucose uptake and metabolism. J. Lipid Res. 50, 1185–1194. Fonseca, V. (2003). Effect of thiazolidinediones on body weight in patients with diabetes mellitus. Am. J. Med. 115 (Suppl 8A ), 42S–48S. Fontaine, C., Dubois, G., Duguay, Y., Helledie, T., Vu-Dac, N., Gervois, P., Soncin, F., Mandrup, S., Fruchart, J.C., Fruchart-Najib, J., and Staels, B. (2003). The orphan nuclear receptor Rev-Erbalpha is a peroxisome proliferator-activated receptor (PPAR) gamma target gene and promotes PPARgamma-induced adipocyte differentiation. J. Biol. Chem. 278, 37672– 37680. Forman, B.M., Chen, J., Blumberg, B., Kliewer, S.A., Henshaw, R., Ong, E.S., and Evans, R.M. (1994). Cross-talk among ROR alpha 1 and the Rev-erb family of orphan nuclear receptors. Mol. Endocrinol. 8, 1253–1261. Forman, B.M., Tontonoz, P., Chen, J., Brun, R.P., Spiegelman, B.M., and Evans, R.M. (1995). 15-deoxy-delta 12, 14-prostaglandin J2 is a ligand for the adipocyte determination factor PPAR gamma. Cell 83, 803–812. Fujita-Sato, S., Ito, S., Isobe, T., Ohyama, T., Wakabayashi, K., Morishita, K., Ando, O., and Isono, F. (2011). Structural basis of digoxin that antagonizes RORgamma t receptor activity and suppresses Th17 cell differentiation and interleukin (IL)-17 production. J. Biol. Chem. 286, 31409–31417. Fukui, Y., Masui, S., Osada, S., Umesono, K., and Motojima, K. (2000). A new thiazolidinedione, NC-2100, which is a weak PPAR-gamma activator, exhibits potent antidiabetic effects and induces uncoupling protein 1 in white adipose tissue of KKAy obese mice. Diabetes 49, 759–767.

Dai, S.Y., Burris, T.P., Dodge, J.A., Montrose-Rafizadeh, C., Wang, Y., Pascal, B.D., Chalmers, M.J., and Griffin, P.R. (2009). Unique ligand binding patterns between estrogen receptor alpha and beta revealed by hydrogen-deuterium exchange. Biochemistry 48, 9668–9676.

Gachon, F., Nagoshi, E., Brown, S.A., Ripperger, J., and Schibler, U. (2004). The mammalian circadian timing system: from gene expression to physiology. Chromosoma 113, 103–112.

DeBruyne, J.P., Weaver, D.R., and Reppert, S.M. (2007). CLOCK and NPAS2 have overlapping roles in the suprachiasmatic circadian clock. Nat. Neurosci. 10, 543–545.

Gangwisch, J.E., Malaspina, D., Boden-Albala, B., and Heymsfield, S.B. (2005). Inadequate sleep as a risk factor for obesity: analyses of the NHANES I. Sleep 28, 1289–1296.

Delezie, J., Dumont, S., Dardente, H., Oudart, H., Gre´chez-Cassiau, A., Klosen, P., Teboul, M., Delaunay, F., Pe´vet, P., and Challet, E. (2012). The nuclear receptor REV-ERBa is required for the daily balance of carbohydrate and lipid metabolism. FASEB J. 26, 3321–3335.

Garber, A.J., Schweizer, A., Baron, M.A., Rochotte, E., and Dejager, S. (2007). Vildagliptin in combination with pioglitazone improves glycaemic control in patients with type 2 diabetes failing thiazolidinedione monotherapy: a randomized, placebo-controlled study. Diabetes Obes. Metab. 9, 166–174.

Cell Metabolism 19, February 4, 2014 ª2014 Elsevier Inc. 203

Cell Metabolism

Review Gatfield, D., Le Martelot, G., Vejnar, C.E., Gerlach, D., Schaad, O., FleuryOlela, F., Ruskeepa¨a¨, A.L., Oresic, M., Esau, C.C., Zdobnov, E.M., and Schibler, U. (2009). Integration of microRNA miR-122 in hepatic circadian gene expression. Genes Dev. 23, 1313–1326.

Hauser, S., Adelmant, G., Sarraf, P., Wright, H.M., Mueller, E., and Spiegelman, B.M. (2000). Degradation of the peroxisome proliferator-activated receptor gamma is linked to ligand-dependent activation. J. Biol. Chem. 275, 18527–18533.

Gavin, J.R., 3rd. (2006). How can we implement current therapies and interventions to achieve glycemic control? Endocr. Pract. 12 (Suppl 1 ), 93–97.

Heal, D.J., Gosden, J., and Smith, S.L. (2009). Regulatory challenges for new drugs to treat obesity and comorbid metabolic disorders. Br. J. Clin. Pharmacol. 68, 861–874.

Germain, P., Staels, B., Dacquet, C., Spedding, M., and Laudet, V. (2006). Overview of nomenclature of nuclear receptors. Pharmacol. Rev. 58, 685–704. Gervois, P., Chopin-Delannoy, S., Fadel, A., Dubois, G., Kosykh, V., Fruchart, J.C., Najı¨b, J., Laudet, V., and Staels, B. (1999). Fibrates increase human REV-ERBalpha expression in liver via a novel peroxisome proliferator-activated receptor response element. Mol. Endocrinol. 13, 400–409.

Heery, D.M., Kalkhoven, E., Hoare, S., and Parker, M.G. (1997). A signature motif in transcriptional co-activators mediates binding to nuclear receptors. Nature 387, 733–736. Hegele, R.A., Cao, H., Frankowski, C., Mathews, S.T., and Leff, T. (2002). PPARG F388L, a transactivation-deficient mutant, in familial partial lipodystrophy. Diabetes 51, 3586–3590.

Gigue`re, V., Tini, M., Flock, G., Ong, E., Evans, R.M., and Otulakowski, G. (1994). Isoform-specific amino-terminal domains dictate DNA-binding properties of ROR alpha, a novel family of orphan hormone nuclear receptors. Genes Dev. 8, 538–553.

Heneka, M.T., Reyes-Irisarri, E., Hu¨ll, M., and Kummer, M.P. (2011). Impact and therapeutic potential of PPARs in Alzheimer’s disease. Curr. Neuropharmacol. 9, 643–650.

Girard, J. (2001). [Mechanisms of action of thiazolidinediones]. Diabetes Metab. 27, 271–278.

Hirose, T., Smith, R.J., and Jetten, A.M. (1994). ROR gamma: the third member of ROR/RZR orphan receptor subfamily that is highly expressed in skeletal muscle. Biochem. Biophys. Res. Commun. 205, 1976–1983.

Girroir, E.E., Hollingshead, H.E., He, P., Zhu, B., Perdew, G.H., and Peters, J.M. (2008). Quantitative expression patterns of peroxisome proliferatoractivated receptor-beta/delta (PPARbeta/delta) protein in mice. Biochem. Biophys. Res. Commun. 371, 456–461.

Hirota, T., and Fukada, Y. (2004). Resetting mechanism of central and peripheral circadian clocks in mammals. Zoolog. Sci. 21, 359–368.

Grant, D., Yin, L., Collins, J.L., Parks, D.J., Orband-Miller, L.A., Wisely, G.B., Joshi, S., Lazar, M.A., Willson, T.M., and Zuercher, W.J. (2010). GSK4112, a small molecule chemical probe for the cell biology of the nuclear heme receptor Rev-erba. ACS Chem. Biol. 5, 925–932. Green, C.B., Takahashi, J.S., and Bass, J. (2008). The meter of metabolism. Cell 134, 728–742. Gregoire, F.M., Zhang, F., Clarke, H.J., Gustafson, T.A., Sears, D.D., Favelyukis, S., Lenhard, J., Rentzeperis, D., Clemens, L.E., Mu, Y., and Lavan, B.E. (2009). MBX-102/JNJ39659100, a novel peroxisome proliferator-activated receptor-ligand with weak transactivation activity retains antidiabetic properties in the absence of weight gain and edema. Mol. Endocrinol. 23, 975–988. Grundy, S.M., Hansen, B., Smith, S.C., Jr., Cleeman, J.I., and Kahn, R.A.; American Heart Association; National Heart, Lung, and Blood Institute; American Diabetes Association (2004). Clinical management of metabolic syndrome: report of the American Heart Association/National Heart, Lung, and Blood Institute/American Diabetes Association conference on scientific issues related to management. Circulation 109, 551–556. Grundy, S.M., Cleeman, J.I., Daniels, S.R., Donato, K.A., Eckel, R.H., Franklin, B.A., Gordon, D.J., Krauss, R.M., Savage, P.J., Smith, S.C., Jr., et al.; American Heart Association; National Heart, Lung, and Blood Institute (2005). Diagnosis and management of the metabolic syndrome: an American Heart Association/National Heart, Lung, and Blood Institute Scientific Statement. Circulation 112, 2735–2752. Guillaumond, F., Dardente, H., Gigue`re, V., and Cermakian, N. (2005). Differential control of Bmal1 circadian transcription by REV-ERB and ROR nuclear receptors. J. Biol. Rhythms 20, 391–403. Gurnell, M. (2003). PPARgamma and metabolism: insights from the study of human genetic variants. Clin. Endocrinol. (Oxf.) 59, 267–277. Hamblin, M., Chang, L., Fan, Y., Zhang, J., and Chen, Y.E. (2009). PPARs and the cardiovascular system. Antioxid. Redox Signal. 11, 1415–1452. Hanefeld, M. (2007). Pioglitazone and sulfonylureas: effectively treating type 2 diabetes. Int. J. Clin. Pract. Suppl. 2007, 20–27. Hao, H., Allen, D.L., and Hardin, P.E. (1997). A circadian enhancer mediates PER-dependent mRNA cycling in Drosophila melanogaster. Mol. Cell. Biol. 17, 3687–3693.

Hiukka, A., Maranghi, M., Matikainen, N., and Taskinen, M.R. (2010). PPARalpha: an emerging therapeutic target in diabetic microvascular damage. Nat. Rev. Endocrinol. 6, 454–463. Houck, K.A., Borchert, K.M., Hepler, C.D., Thomas, J.S., Bramlett, K.S., Michael, L.F., and Burris, T.P. (2004). T0901317 is a dual LXR/FXR agonist. Mol. Genet. Metab. 83, 184–187. Hsiao, F.Y., Huang, W.F., Wen, Y.W., Chen, P.F., Kuo, K.N., and Tsai, Y.W. (2009). Thiazolidinediones and cardiovascular events in patients with type 2 diabetes mellitus: a retrospective cohort study of over 473,000 patients using the National Health Insurance database in Taiwan. Drug Saf. 32, 675–690. Hu, X., and Lazar, M.A. (1999). The CoRNR motif controls the recruitment of corepressors by nuclear hormone receptors. Nature 402, 93–96. Hughes, T.S., Chalmers, M.J., Novick, S., Kuruvilla, D.S., Chang, M.R., Kamenecka, T.M., Rance, M., Johnson, B.A., Burris, T.P., Griffin, P.R., and Kojetin, D.J. (2012). Ligand and receptor dynamics contribute to the mechanism of graded PPARg agonism. Structure 20, 139–150. Hung, M.S., Chang, C.P., Li, T.C., Yeh, T.K., Song, J.S., Lin, Y., Wu, C.H., Kuo, P.C., Amancha, P.K., Wong, Y.C., et al. (2010). Discovery of 1-(2,4dichlorophenyl)-4-ethyl-5-(5-(2-(4-(trifluoromethyl)phenyl)ethynyl)thiophen-2yl)-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide as a potential peripheral cannabinoid-1 receptor inverse agonist. ChemMedChem 5, 1439–1443. Hwang, E.J., Lee, J.M., Jeong, J., Park, J.H., Yang, Y., Lim, J.S., Kim, J.H., Baek, S.H., and Kim, K.I. (2009). SUMOylation of RORalpha potentiates transcriptional activation function. Biochem. Biophys. Res. Commun. 378, 513–517. Ikeda, H., Shino, A., Matsuo, T., Iwatsuka, H., and Suzuoki, Z. (1981). A new genetically obese-hyperglycemic rat (Wistar fatty). Diabetes 30, 1045–1050. Issemann, I., and Green, S. (1990). Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators. Nature 347, 645–650. Iwatsuka, H., Shino, A., and Suzuoki, Z. (1970). General survey of diabetic features of yellow KK mice. Endocrinol. Jpn. 17, 23–35. Jain, R., Chung, S.M., Jain, L., Khurana, M., Lau, S.W., Lee, J.E., Vaidyanathan, J., Zadezensky, I., Choe, S., and Sahajwalla, C.G. (2011). Implications of obesity for drug therapy: limitations and challenges. Clin. Pharmacol. Ther. 90, 77–89.

Harding, H.P., and Lazar, M.A. (1993). The orphan receptor Rev-ErbA alpha activates transcription via a novel response element. Mol. Cell. Biol. 13, 3113–3121.

Jarvis, C.I., Staels, B., Brugg, B., Lemaigre-Dubreuil, Y., Tedgui, A., and Mariani, J. (2002). Age-related phenotypes in the staggerer mouse expand the RORalpha nuclear receptor’s role beyond the cerebellum. Mol. Cell. Endocrinol. 186, 1–5.

Harding, H.P., and Lazar, M.A. (1995). The monomer-binding orphan receptor Rev-Erb represses transcription as a dimer on a novel direct repeat. Mol. Cell. Biol. 15, 4791–4802.

Jeninga, E.H., Gurnell, M., and Kalkhoven, E. (2009). Functional implications of genetic variation in human PPARgamma. Trends Endocrinol. Metab. 20, 380–387.

204 Cell Metabolism 19, February 4, 2014 ª2014 Elsevier Inc.

Cell Metabolism

Review Jetten, A.M. (2009). Retinoid-related orphan receptors (RORs): critical roles in development, immunity, circadian rhythm, and cellular metabolism. Nucl. Recept. Signal. 7, e003.

Kojetin, D., Wang, Y., Kamenecka, T.M., and Burris, T.P. (2011). Identification of SR8278, a synthetic antagonist of the nuclear heme receptor REV-ERB. ACS Chem. Biol. 6, 131–134.

Jetten, A.M., Kurebayashi, S., and Ueda, E. (2001). The ROR nuclear orphan receptor subfamily: critical regulators of multiple biological processes. Prog. Nucleic Acid Res. Mol. Biol. 69, 205–247.

Krasowski, M.D., Reschly, E.J., and Ekins, S. (2008). Intrinsic disorder in nuclear hormone receptors. J. Proteome Res. 7, 4359–4372.

Jin, L., Martynowski, D., Zheng, S., Wada, T., Xie, W., and Li, Y. (2010). Structural basis for hydroxycholesterols as natural ligands of orphan nuclear receptor RORgamma. Mol. Endocrinol. 24, 923–929. Jones, J.R., Barrick, C., Kim, K.A., Lindner, J., Blondeau, B., Fujimoto, Y., Shiota, M., Kesterson, R.A., Kahn, B.B., and Magnuson, M.A. (2005). Deletion of PPARgamma in adipose tissues of mice protects against high fat dietinduced obesity and insulin resistance. Proc. Natl. Acad. Sci. USA 102, 6207–6212. Juurlink, D.N., Gomes, T., Lipscombe, L.L., Austin, P.C., Hux, J.E., and Mamdani, M.M. (2009). Adverse cardiovascular events during treatment with pioglitazone and rosiglitazone: population based cohort study. BMJ 339, b2942. Kallen, J.A., Schlaeppi, J.M., Bitsch, F., Geisse, S., Geiser, M., Delhon, I., and Fournier, B. (2002). X-ray structure of the hRORalpha LBD at 1.63 A: structural and functional data that cholesterol or a cholesterol derivative is the natural ligand of RORalpha. Structure 10, 1697–1707. Kallen, J., Schlaeppi, J.M., Bitsch, F., Delhon, I., and Fournier, B. (2004). Crystal structure of the human RORalpha ligand binding domain in complex with cholesterol sulfate at 2.2 A˚. J. Biol. Chem. 279, 14033–14038. Kamenecka, T.M., Lyda, B., Chang, M.R., and Griffin, P.R. (2013). Synthetic modulators of the retinoic acid receptor-related orphan receptors. Medchemcomm. 4, 764–776. Kang, H.S., Angers, M., Beak, J.Y., Wu, X., Gimble, J.M., Wada, T., Xie, W., Collins, J.B., Grissom, S.F., and Jetten, A.M. (2007). Gene expression profiling reveals a regulatory role for ROR alpha and ROR gamma in phase I and phase II metabolism. Physiol. Genomics 31, 281–294. Kawamatsu, Y., Asakawa, H., Saraie, T., Mizuno, K., Imamiya, E., Nishikawa, K., and Hamuro, Y. (1980). Studies on antihyperlipidemic agents. III. Synthesis and biological activities of 2-chloro-3-arylpropionic acids containing a quarternary carbon atom. Arzneimittelforschung 30, 751–758. Kern, P.A. (1997). Potential role of TNFalpha and lipoprotein lipase as candidate genes for obesity. J. Nutr. 127, 1917S–1922S. Kersten, S., Seydoux, J., Peters, J.M., Gonzalez, F.J., Desvergne, B., and Wahli, W. (1999). Peroxisome proliferator-activated receptor alpha mediates the adaptive response to fasting. J. Clin. Invest. 103, 1489–1498. Kim, E.Y., Kim, J.S., Kim, M.Y., Koh, W.S., Guengerich, F.P., and Yun, C.H. (2004). Non-specific inhibition of human cytochrome P450-catalyzed reactions by hemin. Toxicol. Lett. 153, 239–246. Klein, F.A., Atkinson, R.A., Potier, N., Moras, D., and Cavarelli, J. (2005). Biochemical and NMR mapping of the interface between CREB-binding protein and ligand binding domains of nuclear receptor: beyond the LXXLL motif. J. Biol. Chem. 280, 5682–5692. Kliewer, S.A., Umesono, K., Noonan, D.J., Heyman, R.A., and Evans, R.M. (1992). Convergence of 9-cis retinoic acid and peroxisome proliferator signalling pathways through heterodimer formation of their receptors. Nature 358, 771–774. Kliewer, S.A., Lenhard, J.M., Willson, T.M., Patel, I., Morris, D.C., and Lehmann, J.M. (1995). A prostaglandin J2 metabolite binds peroxisome proliferator-activated receptor gamma and promotes adipocyte differentiation. Cell 83, 813–819. Kliewer, S.A., Sundseth, S.S., Jones, S.A., Brown, P.J., Wisely, G.B., Koble, C.S., Devchand, P., Wahli, W., Willson, T.M., Lenhard, J.M., and Lehmann, J.M. (1997). Fatty acids and eicosanoids regulate gene expression through direct interactions with peroxisome proliferator-activated receptors alpha and gamma. Proc. Natl. Acad. Sci. USA 94, 4318–4323. Ko, C.H., and Takahashi, J.S. (2006). Molecular components of the mammalian circadian clock. Hum. Mol. Genet. 15 (Spec No 2), R271–R277.

Krey, G., Braissant, O., L’Horset, F., Kalkhoven, E., Perroud, M., Parker, M.G., and Wahli, W. (1997). Fatty acids, eicosanoids, and hypolipidemic agents identified as ligands of peroxisome proliferator-activated receptors by coactivatordependent receptor ligand assay. Mol. Endocrinol. 11, 779–791. Kubota, N., Terauchi, Y., Miki, H., Tamemoto, H., Yamauchi, T., Komeda, K., Satoh, S., Nakano, R., Ishii, C., Sugiyama, T., et al. (1999). PPAR gamma mediates high-fat diet-induced adipocyte hypertrophy and insulin resistance. Mol. Cell 4, 597–609. Kumar, N., Solt, L.A., Conkright, J., Wang, Y., Istrate, M.A., Busby, S.A., Garcia-Ordonez, R.D., Nuhant, P., Burris, T., Mercer, B.A., et al. (2010a). Campaign to identify novel modulators of the Retinoic acid receptor-related Orphan Receptors (ROR). In Probe Reports from the NIH Molecular Libraries Program (Bethesda, MD: National Center for Biotechnology Information). Kumar, N., Solt, L.A., Conkright, J.J., Wang, Y., Istrate, M.A., Busby, S.A., Garcia-Ordonez, R.D., Burris, T.P., and Griffin, P.R. (2010b). The benzenesulfoamide T0901317 [N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-trifluoro-1-hydroxy-1(trifluoromethyl)ethyl]phenyl]-benzenesulfonamide] is a novel retinoic acid receptor-related orphan receptor-alpha/gamma inverse agonist. Mol. Pharmacol. 77, 228–236. Kumar, N., Solt, L.A., Wang, Y., Rogers, P.M., Bhattacharyya, G., Kamenecka, T.M., Stayrook, K.R., Crumbley, C., Floyd, Z.E., Gimble, J.M., et al. (2010c). Regulation of adipogenesis by natural and synthetic REV-ERB ligands. Endocrinology 151, 3015–3025. Laitinen, S., Fontaine, C., Fruchart, J.C., and Staels, B. (2005). The role of the orphan nuclear receptor Rev-Erb alpha in adipocyte differentiation and function. Biochimie 87, 21–25. Lamotte, Y., Martres, P., Faucher, N., Laroze, A., Grillot, D., Ancellin, N., Saintillan, Y., Beneton, V., and Gampe, R.T., Jr. (2010). Synthesis and biological activities of novel indole derivatives as potent and selective PPARgamma modulators. Bioorg. Med. Chem. Lett. 20, 1399–1404. Lau, P., Bailey, P., Dowhan, D.H., and Muscat, G.E. (1999). Exogenous expression of a dominant negative RORalpha1 vector in muscle cells impairs differentiation: RORalpha1 directly interacts with p300 and myoD. Nucleic Acids Res. 27, 411–420. Lau, P., Nixon, S.J., Parton, R.G., and Muscat, G.E. (2004). RORalpha regulates the expression of genes involved in lipid homeostasis in skeletal muscle cells: caveolin-3 and CPT-1 are direct targets of ROR. J. Biol. Chem. 279, 36828–36840. Lau, P., Fitzsimmons, R.L., Raichur, S., Wang, S.C., Lechtken, A., and Muscat, G.E. (2008). The orphan nuclear receptor, RORalpha, regulates gene expression that controls lipid metabolism: staggerer (SG/SG) mice are resistant to diet-induced obesity. J. Biol. Chem. 283, 18411–18421. Lazar, M.A., Hodin, R.A., Darling, D.S., and Chin, W.W. (1989). A novel member of the thyroid/steroid hormone receptor family is encoded by the opposite strand of the rat c-erbA alpha transcriptional unit. Mol. Cell. Biol. 9, 1128–1136. Le Martelot, G., Claudel, T., Gatfield, D., Schaad, O., Kornmann, B., Lo Sasso, G., Moschetta, A., and Schibler, U. (2009). REV-ERBalpha participates in circadian SREBP signaling and bile acid homeostasis. PLoS Biol. 7, e1000181. Lechtken, A., Ho¨rnig, M., Werz, O., Corvey, N., Zu¨ndorf, I., Dingermann, T., Brandes, R., and Steinhilber, D. (2007). Extracellular signal-regulated kinase2 phosphorylates RORalpha4 in vitro. Biochem. Biophys. Res. Commun. 358, 890–896. Lee, J.M., Kim, I.S., Kim, H., Lee, J.S., Kim, K., Yim, H.Y., Jeong, J., Kim, J.H., Kim, J.Y., Lee, H., et al. (2010). RORalpha attenuates Wnt/beta-catenin signaling by PKCalpha-dependent phosphorylation in colon cancer. Mol. Cell 37, 183–195. Lefebvre, P., Cariou, B., Lien, F., Kuipers, F., and Staels, B. (2009). Role of bile acids and bile acid receptors in metabolic regulation. Physiol. Rev. 89, 147–191. Lehmann, J.M., Moore, L.B., Smith-Oliver, T.A., Wilkison, W.O., Willson, T.M., and Kliewer, S.A. (1995). An antidiabetic thiazolidinedione is a high affinity

Cell Metabolism 19, February 4, 2014 ª2014 Elsevier Inc. 205

Cell Metabolism

Review ligand for peroxisome proliferator-activated receptor gamma (PPAR gamma). J. Biol. Chem. 270, 12953–12956. Lemberger, T., Saladin, R., Va´zquez, M., Assimacopoulos, F., Staels, B., Desvergne, B., Wahli, W., and Auwerx, J. (1996). Expression of the peroxisome proliferator-activated receptor alpha gene is stimulated by stress and follows a diurnal rhythm. J. Biol. Chem. 271, 1764–1769. Li, L., Liu, J., Zhu, L., Cutler, S., Hasegawa, H., Shan, B., and Medina, J.C. (2006). Discovery and optimization of a novel series of liver X receptor-alpha agonists. Bioorg. Med. Chem. Lett. 16, 1638–1642. Lipscombe, L.L., Gomes, T., Le´vesque, L.E., Hux, J.E., Juurlink, D.N., and Alter, D.A. (2007). Thiazolidinediones and cardiovascular outcomes in older patients with diabetes. JAMA 298, 2634–2643. Loke, Y.K., Kwok, C.S., and Singh, S. (2011). Comparative cardiovascular effects of thiazolidinediones: systematic review and meta-analysis of observational studies. BMJ 342, d1309. Mangelsdorf, D.J., and Evans, R.M. (1995). The RXR heterodimers and orphan receptors. Cell 83, 841–850. Martin, G., Schoonjans, K., Staels, B., and Auwerx, J. (1998). PPARgamma activators improve glucose homeostasis by stimulating fatty acid uptake in the adipocytes. Atherosclerosis Suppl. 137, S75–S80. Marvin, K.A., Reinking, J.L., Lee, A.J., Pardee, K., Krause, H.M., and Burstyn, J.N. (2009). Nuclear receptors homo sapiens Rev-erbbeta and Drosophila melanogaster E75 are thiolate-ligated heme proteins which undergo redoxmediated ligand switching and bind CO and NO. Biochemistry 48, 7056–7071. Maury, E., Ramsey, K.M., and Bass, J. (2010). Circadian rhythms and metabolic syndrome: from experimental genetics to human disease. Circ. Res. 106, 447–462. Meissburger, B., Ukropec, J., Roeder, E., Beaton, N., Geiger, M., Teupser, D., Civan, B., Langhans, W., Nawroth, P.P., Gasperikova, D., et al. (2011). Adipogenesis and insulin sensitivity in obesity are regulated by retinoid-related orphan receptor gamma. EMBO Mol. Med. 3, 637–651. Meng, Q.J., McMaster, A., Beesley, S., Lu, W.Q., Gibbs, J., Parks, D., Collins, J., Farrow, S., Donn, R., Ray, D., and Loudon, A. (2008). Ligand modulation of REV-ERBalpha function resets the peripheral circadian clock in a phasic manner. J. Cell Sci. 121, 3629–3635. Mitro, N., Vargas, L., Romeo, R., Koder, A., and Saez, E. (2007). T0901317 is a potent PXR ligand: implications for the biology ascribed to LXR. FEBS Lett. 581, 1721–1726. Moller, D.E., and Kaufman, K.D. (2005). Metabolic syndrome: a clinical and molecular perspective. Annu. Rev. Med. 56, 45–62. Moller, D.E., Bjørbaek, C., and Vidal-Puig, A. (1996). Candidate genes for insulin resistance. Diabetes Care 19, 396–400. Montague, C.T. (2002). Adipose depot-specific effects of PPAR gamma agonists: a consequence of differential expression of PPAR gamma in adipose tissue depots? Diabetes Obes. Metab. 4, 356–361. Moore, J.T., Collins, J.L., and Pearce, K.H. (2006). The nuclear receptor superfamily and drug discovery. ChemMedChem 1, 504–523. Moraitis, A.N., and Gigue`re, V. (1999). Transition from monomeric to homodimeric DNA binding by nuclear receptors: identification of RevErbAalpha determinants required for RORalpha homodimer complex formation. Mol. Endocrinol. 13, 431–439. Motani, A., Wang, Z., Weiszmann, J., McGee, L.R., Lee, G., Liu, Q., Staunton, J., Fang, Z., Fuentes, H., Lindstrom, M., et al. (2009). INT131: a selective modulator of PPAR gamma. J. Mol. Biol. 386, 1301–1311. Nagy, L., Tontonoz, P., Alvarez, J.G., Chen, H., and Evans, R.M. (1998). Oxidized LDL regulates macrophage gene expression through ligand activation of PPARgamma. Cell 93, 229–240. Nakamura, K., Inoue, I., Takahashi, S., Komoda, T., and Katayama, S. (2008). Cryptochrome and period proteins are regulated by the CLOCK/BMAL1 gene: crosstalk between the PPARs/RXRalpha-regulated and CLOCK/BMAL1-regulated systems. PPAR Res. 2008, 348610. Nesto, R.W., Bell, D., Bonow, R.O., Fonseca, V., Grundy, S.M., Horton, E.S., Le Winter, M., Porte, D., Semenkovich, C.F., Smith, S., et al.; American Heart

206 Cell Metabolism 19, February 4, 2014 ª2014 Elsevier Inc.

Association; American Diabetes Association (2003). Thiazolidinedione use, fluid retention, and congestive heart failure: a consensus statement from the American Heart Association and American Diabetes Association. October 7, 2003. Circulation 108, 2941–2948. Nolte, R.T., Wisely, G.B., Westin, S., Cobb, J.E., Lambert, M.H., Kurokawa, R., Rosenfeld, M.G., Willson, T.M., Glass, C.K., and Milburn, M.V. (1998). Ligand binding and co-activator assembly of the peroxisome proliferator-activated receptor-gamma. Nature 395, 137–143. Ohno, H., Shinoda, K., Spiegelman, B.M., and Kajimura, S. (2012). PPARg agonists induce a white-to-brown fat conversion through stabilization of PRDM16 protein. Cell Metab. 15, 395–404. Oishi, K., Shirai, H., and Ishida, N. (2005). CLOCK is involved in the circadian transactivation of peroxisome-proliferator-activated receptor alpha (PPARalpha) in mice. Biochem. J. 386, 575–581. Ong, J.M., Simsolo, R.B., Saghizadeh, M., Pauer, A., and Kern, P.A. (1994). Expression of lipoprotein lipase in rat muscle: regulation by feeding and hypothyroidism. J. Lipid Res. 35, 1542–1551. Overington, J.P., Al-Lazikani, B., and Hopkins, A.L. (2006). How many drug targets are there? Nat. Rev. Drug Discov. 5, 993–996. Panda, S., Antoch, M.P., Miller, B.H., Su, A.I., Schook, A.B., Straume, M., Schultz, P.G., Kay, S.A., Takahashi, J.S., and Hogenesch, J.B. (2002). Coordinated transcription of key pathways in the mouse by the circadian clock. Cell 109, 307–320. Pardee, K.I., Xu, X., Reinking, J., Schuetz, A., Dong, A., Liu, S., Zhang, R., Tiefenbach, J., Lajoie, G., Plotnikov, A.N., et al. (2009). The structural basis of gas-responsive transcription by the human nuclear hormone receptor REV-ERBbeta. PLoS Biol. 7, e43. Pascual, G., Fong, A.L., Ogawa, S., Gamliel, A., Li, A.C., Perissi, V., Rose, D.W., Willson, T.M., Rosenfeld, M.G., and Glass, C.K. (2005). A SUMOylation-dependent pathway mediates transrepression of inflammatory response genes by PPAR-gamma. Nature 437, 759–763. Perera, R.J., Marcusson, E.G., Koo, S., Kang, X., Kim, Y., White, N., and Dean, N.M. (2006). Identification of novel PPARgamma target genes in primary human adipocytes. Gene 369, 90–99. Plutzky, J. (2011). The PPAR-RXR transcriptional complex in the vasculature: energy in the balance. Circ. Res. 108, 1002–1016. Preitner, N., Damiola, F., Lopez-Molina, L., Zakany, J., Duboule, D., Albrecht, U., and Schibler, U. (2002). The orphan nuclear receptor REV-ERBalpha controls circadian transcription within the positive limb of the mammalian circadian oscillator. Cell 110, 251–260. Privalsky, M.L. (2004). The role of corepressors in transcriptional regulation by nuclear hormone receptors. Annu. Rev. Physiol. 66, 315–360. Puigserver, P., Wu, Z., Park, C.W., Graves, R., Wright, M., and Spiegelman, B.M. (1998). A cold-inducible coactivator of nuclear receptors linked to adaptive thermogenesis. Cell 92, 829–839. Pulinilkunnil, T., and Rodrigues, B. (2006). Cardiac lipoprotein lipase: metabolic basis for diabetic heart disease. Cardiovasc. Res. 69, 329–340. Qiang, L., and Accili, D. (2012). FGF21 and the second coming of PPARg. Cell 148, 397–398. Qiang, L., Wang, L., Kon, N., Zhao, W., Lee, S., Zhang, Y., Rosenbaum, M., Zhao, Y., Gu, W., Farmer, S.R., and Accili, D. (2012). Brown remodeling of white adipose tissue by SirT1-dependent deacetylation of Pparg. Cell 150, 620–632. Raghuram, S., Stayrook, K.R., Huang, P., Rogers, P.M., Nosie, A.K., McClure, D.B., Burris, L.L., Khorasanizadeh, S., Burris, T.P., and Rastinejad, F. (2007). Identification of heme as the ligand for the orphan nuclear receptors REVERBalpha and REV-ERBbeta. Nat. Struct. Mol. Biol. 14, 1207–1213. Raichur, S., Lau, P., Staels, B., and Muscat, G.E. (2007). Retinoid-related orphan receptor gamma regulates several genes that control metabolism in skeletal muscle cells: links to modulation of reactive oxygen species production. J. Mol. Endocrinol. 39, 29–44. Rakhshandehroo, M., Knoch, B., Mu¨ller, M., and Kersten, S. (2010). Peroxisome proliferator-activated receptor alpha target genes. PPAR Res. 2010, 612089.

Cell Metabolism

Review Ramakrishnan, S.N., Lau, P., Burke, L.J., and Muscat, G.E. (2005). Reverbbeta regulates the expression of genes involved in lipid absorption in skeletal muscle cells: evidence for cross-talk between orphan nuclear receptors and myokines. J. Biol. Chem. 280, 8651–8659. Rangwala, S.M., and Lazar, M.A. (2002). The dawn of the SPPARMs? Sci. STKE 2002, pe9. Rasmussen, B.B., and Wolfe, R.R. (1999). Regulation of fatty acid oxidation in skeletal muscle. Annu. Rev. Nutr. 19, 463–484. Raspe´, E., Duez, H., Gervois, P., Fie´vet, C., Fruchart, J.C., Besnard, S., Mariani, J., Tedgui, A., and Staels, B. (2001). Transcriptional regulation of apolipoprotein C-III gene expression by the orphan nuclear receptor RORalpha. J. Biol. Chem. 276, 2865–2871. Raspe´, E., Duez, H., Manse´n, A., Fontaine, C., Fie´vet, C., Fruchart, J.C., Vennstro¨m, B., and Staels, B. (2002). Identification of Rev-erbalpha as a physiological repressor of apoC-III gene transcription. J. Lipid Res. 43, 2172–2179. Reilly, S.M., and Lee, C.H. (2008). PPAR delta as a therapeutic target in metabolic disease. FEBS Lett. 582, 26–31. Rise´rus, U., Sprecher, D., Johnson, T., Olson, E., Hirschberg, S., Liu, A., Fang, Z., Hegde, P., Richards, D., Sarov-Blat, L., et al. (2008). Activation of peroxisome proliferator-activated receptor (PPAR)delta promotes reversal of multiple metabolic abnormalities, reduces oxidative stress, and increases fatty acid oxidation in moderately obese men. Diabetes 57, 332–339. Rocchi, S., Picard, F., Vamecq, J., Gelman, L., Potier, N., Zeyer, D., Dubuquoy, L., Bac, P., Champy, M.F., Plunket, K.D., et al. (2001). A unique PPARgamma ligand with potent insulin-sensitizing yet weak adipogenic activity. Mol. Cell 8, 737–747. Rochel, N., Ciesielski, F., Godet, J., Moman, E., Roessle, M., Peluso-Iltis, C., Moulin, M., Haertlein, M., Callow, P., Me´ly, Y., et al. (2011). Common architecture of nuclear receptor heterodimers on DNA direct repeat elements with different spacings. Nat. Struct. Mol. Biol. 18, 564–570. Rosenson, R.S., Wright, R.S., Farkouh, M., and Plutzky, J. (2012). Modulating peroxisome proliferator-activated receptors for therapeutic benefit? Biology, clinical experience, and future prospects. Am. Heart J. 164, 672–680. Rudic, R.D., McNamara, P., Curtis, A.M., Boston, R.C., Panda, S., Hogenesch, J.B., and Fitzgerald, G.A. (2004). BMAL1 and CLOCK, two essential components of the circadian clock, are involved in glucose homeostasis. PLoS Biol. 2, e377.

Solt, L.A., Griffin, P.R., and Burris, T.P. (2010). Ligand regulation of retinoic acid receptor-related orphan receptors: implications for development of novel therapeutics. Curr. Opin. Lipidol. 21, 204–211. Solt, L.A., Kojetin, D.J., and Burris, T.P. (2011a). The REV-ERBs and RORs: molecular links between circadian rhythms and lipid homeostasis. Future Med. Chem. 3, 623–638. Solt, L.A., Kumar, N., Nuhant, P., Wang, Y., Lauer, J.L., Liu, J., Istrate, M.A., , D., et al. (2011b). Suppression of Kamenecka, T.M., Roush, W.R., Vidovic TH17 differentiation and autoimmunity by a synthetic ROR ligand. Nature 472, 491–494. Solt, L.A., Kumar, N., He, Y., Kamenecka, T.M., Griffin, P.R., and Burris, T.P. (2012a). Identification of a selective RORg ligand that suppresses T(H)17 cells and stimulates T regulatory cells. ACS Chem. Biol. 7, 1515–1519. Solt, L.A., Wang, Y., Banerjee, S., Hughes, T., Kojetin, D.J., Lundasen, T., Shin, Y., Liu, J., Cameron, M.D., Noel, R., et al. (2012b). Regulation of circadian behaviour and metabolism by synthetic REV-ERB agonists. Nature 485, 62–68. Spencer, T.E., Jenster, G., Burcin, M.M., Allis, C.D., Zhou, J., Mizzen, C.A., McKenna, N.J., Onate, S.A., Tsai, S.Y., Tsai, M.J., and O’Malley, B.W. (1997). Steroid receptor coactivator-1 is a histone acetyltransferase. Nature 389, 194–198. Staels, B. (2005). Fluid retention mediated by renal PPARgamma. Cell Metab. 2, 77–78. Staels, B., and Fruchart, J.C. (2005). Therapeutic roles of peroxisome proliferator-activated receptor agonists. Diabetes 54, 2460–2470. Staels, B., Dallongeville, J., Auwerx, J., Schoonjans, K., Leitersdorf, E., and Fruchart, J.C. (1998). Mechanism of action of fibrates on lipid and lipoprotein metabolism. Circulation 98, 2088–2093. Stehlin, C., Wurtz, J.M., Steinmetz, A., Greiner, E., Schu¨le, R., Moras, D., and Renaud, J.P. (2001). X-ray structure of the orphan nuclear receptor RORbeta ligand-binding domain in the active conformation. EMBO J. 20, 5822–5831. Stehlin-Gaon, C., Willmann, D., Zeyer, D., Sanglier, S., Van Dorsselaer, A., Renaud, J.P., Moras, D., and Schu¨le, R. (2003). All-trans retinoic acid is a ligand for the orphan nuclear receptor ROR beta. Nat. Struct. Biol. 10, 820–825.

Sahar, S., and Sassone-Corsi, P. (2009). Metabolism and cancer: the circadian clock connection. Nat. Rev. Cancer 9, 886–896.

Steinmayr, M., Andre´, E., Conquet, F., Rondi-Reig, L., Delhaye-Bouchaud, N., Auclair, N., Daniel, H., Cre´pel, F., Mariani, J., Sotelo, C., and Becker-Andre´, M. (1998). staggerer phenotype in retinoid-related orphan receptor alphadeficient mice. Proc. Natl. Acad. Sci. USA 95, 3960–3965.

Saraf, N., Sharma, P.K., Mondal, S.C., Garg, V.K., and Singh, A.K. (2012). Role of PPARg2 transcription factor in thiazolidinedione-induced insulin sensitization. J. Pharm. Pharmacol. 64, 161–171.

Stephenson, J. (2011). Diabetes drug may be associated with increase in risk of bladder cancer. JAMA 306, 143.

Sato, T.K., Panda, S., Miraglia, L.J., Reyes, T.M., Rudic, R.D., McNamara, P., Naik, K.A., FitzGerald, G.A., Kay, S.A., and Hogenesch, J.B. (2004). A functional genomics strategy reveals Rora as a component of the mammalian circadian clock. Neuron 43, 527–537. Savage, D.B., Tan, G.D., Acerini, C.L., Jebb, S.A., Agostini, M., Gurnell, M., Williams, R.L., Umpleby, A.M., Thomas, E.L., Bell, J.D., et al. (2003). Human metabolic syndrome resulting from dominant-negative mutations in the nuclear receptor peroxisome proliferator-activated receptor-gamma. Diabetes 52, 910–917. Seale, P., and Lazar, M.A. (2009). Brown fat in humans: turning up the heat on obesity. Diabetes 58, 1482–1484. Sell, H., Berger, J.P., Samson, P., Castriota, G., Lalonde, J., Deshaies, Y., and Richard, D. (2004). Peroxisome proliferator-activated receptor gamma agonism increases the capacity for sympathetically mediated thermogenesis in lean and ob/ob mice. Endocrinology 145, 3925–3934. Shiau, A.K., Barstad, D., Loria, P.M., Cheng, L., Kushner, P.J., Agard, D.A., and Greene, G.L. (1998). The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen. Cell 95, 927–937. Shimano, H., Yahagi, N., Amemiya-Kudo, M., Hasty, A.H., Osuga, J., Tamura, Y., Shionoiri, F., Iizuka, Y., Ohashi, K., Harada, K., et al. (1999). Sterol regulatory element-binding protein-1 as a key transcription factor for nutritional induction of lipogenic enzyme genes. J. Biol. Chem. 274, 35832–35839.

Storch, K.F., Lipan, O., Leykin, I., Viswanathan, N., Davis, F.C., Wong, W.H., and Weitz, C.J. (2002). Extensive and divergent circadian gene expression in liver and heart. Nature 417, 78–83. Stossi, F., Barnett, D.H., Frasor, J., Komm, B., Lyttle, C.R., and Katzenellenbogen, B.S. (2004). Transcriptional profiling of estrogen-regulated gene expression via estrogen receptor (ER) alpha or ERbeta in human osteosarcoma cells: distinct and common target genes for these receptors. Endocrinology 145, 3473–3486. Tanaka, T., Yamamoto, J., Iwasaki, S., Asaba, H., Hamura, H., Ikeda, Y., Watanabe, M., Magoori, K., Ioka, R.X., Tachibana, K., et al. (2003). Activation of peroxisome proliferator-activated receptor delta induces fatty acid betaoxidation in skeletal muscle and attenuates metabolic syndrome. Proc. Natl. Acad. Sci. USA 100, 15924–15929. Tee, M.K., Rogatsky, I., Tzagarakis-Foster, C., Cvoro, A., An, J., Christy, R.J., Yamamoto, K.R., and Leitman, D.C. (2004). Estradiol and selective estrogen receptor modulators differentially regulate target genes with estrogen receptors alpha and beta. Mol. Biol. Cell 15, 1262–1272. Thuillier, P., Baillie, R., Sha, X., and Clarke, S.D. (1998). Cytosolic and nuclear distribution of PPARgamma2 in differentiating 3T3-L1 preadipocytes. J. Lipid Res. 39, 2329–2338. Tontonoz, P., Hu, E., Graves, R.A., Budavari, A.I., and Spiegelman, B.M. (1994a). mPPAR gamma 2: tissue-specific regulator of an adipocyte enhancer. Genes Dev. 8, 1224–1234.

Cell Metabolism 19, February 4, 2014 ª2014 Elsevier Inc. 207

Cell Metabolism

Review Tontonoz, P., Hu, E., and Spiegelman, B.M. (1994b). Stimulation of adipogenesis in fibroblasts by PPAR gamma 2, a lipid-activated transcription factor. Cell 79, 1147–1156.

Wright, E., Busby, S.A., Wisecarver, S., Vincent, J., Griffin, P.R., and Fernandez, E.J. (2011). Helix 11 dynamics is critical for constitutive androstane receptor activity. Structure 19, 37–44.

Tontonoz, P., Hu, E., Devine, J., Beale, E.G., and Spiegelman, B.M. (1995). PPAR gamma 2 regulates adipose expression of the phosphoenolpyruvate carboxykinase gene. Mol. Cell. Biol. 15, 351–357.

Yamauchi, T., Kamon, J., Waki, H., Murakami, K., Motojima, K., Komeda, K., Ide, T., Kubota, N., Terauchi, Y., Tobe, K., et al. (2001). The mechanisms by which both heterozygous peroxisome proliferator-activated receptor gamma (PPARgamma) deficiency and PPARgamma agonist improve insulin resistance. J. Biol. Chem. 276, 41245–41254.

Tremblay, G.B., Tremblay, A., Labrie, F., and Gigue`re, V. (1999). Dominant activity of activation function 1 (AF-1) and differential stoichiometric requirements for AF-1 and -2 in the estrogen receptor alpha-beta heterodimeric complex. Mol. Cell. Biol. 19, 1919–1927. van Beekum, O., Fleskens, V., and Kalkhoven, E. (2009). Posttranslational modifications of PPAR-gamma: fine-tuning the metabolic master regulator. Obesity (Silver Spring) 17, 213–219. Waku, T., Shiraki, T., Oyama, T., Maebara, K., Nakamori, R., and Morikawa, K. (2010). The nuclear receptor PPARg individually responds to serotonin- and fatty acid-metabolites. EMBO J. 29, 3395–3407. Wang, J., and Lazar, M.A. (2008). Bifunctional role of Rev-erbalpha in adipocyte differentiation. Mol. Cell. Biol. 28, 2213–2220. Wang, Y.X., Zhang, C.L., Yu, R.T., Cho, H.K., Nelson, M.C., Bayuga-Ocampo, C.R., Ham, J., Kang, H., and Evans, R.M. (2004). Regulation of muscle fiber type and running endurance by PPARdelta. PLoS Biol. 2, e294. Wang, N., Yang, G., Jia, Z., Zhang, H., Aoyagi, T., Soodvilai, S., Symons, J.D., Schnermann, J.B., Gonzalez, F.J., Litwin, S.E., and Yang, T. (2008). Vascular PPARgamma controls circadian variation in blood pressure and heart rate through Bmal1. Cell Metab. 8, 482–491. Wang, Y., Solt, L.A., and Burris, T.P. (2010). Regulation of FGF21 expression and secretion by retinoic acid receptor-related orphan receptor alpha. J. Biol. Chem. 285, 15668–15673. Wa¨rnmark, A., Treuter, E., Wright, A.P., and Gustafsson, J.A. (2003). Activation functions 1 and 2 of nuclear receptors: molecular strategies for transcriptional activation. Mol. Endocrinol. 17, 1901–1909. Weldemichael, D.A., and Grossberg, G.T. (2010). Circadian rhythm disturbances in patients with Alzheimer’s disease: a review. Int. J. Alzheimers Dis. 2010, 716453. Wilson-Fritch, L., Burkart, A., Bell, G., Mendelson, K., Leszyk, J., Nicoloro, S., Czech, M., and Corvera, S. (2003). Mitochondrial biogenesis and remodeling during adipogenesis and in response to the insulin sensitizer rosiglitazone. Mol. Cell. Biol. 23, 1085–1094. Winkelmayer, W.C., Setoguchi, S., Levin, R., and Solomon, D.H. (2008). Comparison of cardiovascular outcomes in elderly patients with diabetes who initiated rosiglitazone vs pioglitazone therapy. Arch. Intern. Med. 168, 2368– 2375. Woldt, E., Sebti, Y., Solt, L.A., Duhem, C., Lancel, S., Eeckhoute, J., Hesselink, M.K., Paquet, C., Delhaye, S., Shin, Y., et al. (2013). Rev-erb-a modulates skeletal muscle oxidative capacity by regulating mitochondrial biogenesis and autophagy. Nat. Med. 19, 1039–1046. Woo, E.J., Jeong, D.G., Lim, M.Y., Jun Kim, S., Kim, K.J., Yoon, S.M., Park, B.C., and Ryu, S.E. (2007). Structural insight into the constitutive repression function of the nuclear receptor Rev-erbbeta. J. Mol. Biol. 373, 735–744.

208 Cell Metabolism 19, February 4, 2014 ª2014 Elsevier Inc.

Yildiz, B.O., Suchard, M.A., Wong, M.L., McCann, S.M., and Licinio, J. (2004). Alterations in the dynamics of circulating ghrelin, adiponectin, and leptin in human obesity. Proc. Natl. Acad. Sci. USA 101, 10434–10439. Yin, L., Wang, J., Klein, P.S., and Lazar, M.A. (2006). Nuclear receptor Reverbalpha is a critical lithium-sensitive component of the circadian clock. Science 311, 1002–1005. Yin, L., Wu, N., Curtin, J.C., Qatanani, M., Szwergold, N.R., Reid, R.A., Waitt, G.M., Parks, D.J., Pearce, K.H., Wisely, G.B., and Lazar, M.A. (2007). Reverbalpha, a heme sensor that coordinates metabolic and circadian pathways. Science 318, 1786–1789. Zamir, I., Harding, H.P., Atkins, G.B., Ho¨rlein, A., Glass, C.K., Rosenfeld, M.G., and Lazar, M.A. (1996). A nuclear hormone receptor corepressor mediates transcriptional silencing by receptors with distinct repression domains. Mol. Cell. Biol. 16, 5458–5465. Zhang, J., Chalmers, M.J., Stayrook, K.R., Burris, L.L., Garcia-Ordonez, R.D., Pascal, B.D., Burris, T.P., Dodge, J.A., and Griffin, P.R. (2010). Hydrogen/ deuterium exchange reveals distinct agonist/partial agonist receptor dynamics within vitamin D receptor/retinoid X receptor heterodimer. Structure 18, 1332–1341. Zhang, J., Chalmers, M.J., Stayrook, K.R., Burris, L.L., Wang, Y., Busby, S.A., Pascal, B.D., Garcia-Ordonez, R.D., Bruning, J.B., Istrate, M.A., et al. (2011). DNA binding alters coactivator interaction surfaces of the intact VDR-RXR complex. Nat. Struct. Mol. Biol. 18, 556–563. Zı´dek, V., Mlejnek, P., Sima´kova´, M., Silhavy, J., Landa, V., Kazdova´, L., Pravenec, M., and Kurtz, T.W. (2013). Tissue-specific peroxisome proliferator activated receptor gamma expression and metabolic effects of telmisartan. Am. J. Hypertens. 26, 829–835. Zinman, B., Hoogwerf, B.J., Dura´n Garcı´a, S., Milton, D.R., Giaconia, J.M., Kim, D.D., Trautmann, M.E., and Brodows, R.G. (2007). The effect of adding exenatide to a thiazolidinedione in suboptimally controlled type 2 diabetes: a randomized trial. Ann. Intern. Med. 146, 477–485. Zinman, B., Gerich, J., Buse, J.B., Lewin, A., Schwartz, S., Raskin, P., Hale, P.M., Zdravkovic, M., and Blonde, L.; LEAD-4 Study Investigators (2009). Efficacy and safety of the human glucagon-like peptide-1 analog liraglutide in combination with metformin and thiazolidinedione in patients with type 2 diabetes (LEAD-4 Met+TZD). Diabetes Care 32, 1224–1230. Zwart, W., de Leeuw, R., Rondaij, M., Neefjes, J., Mancini, M.A., and Michalides, R. (2010). The hinge region of the human estrogen receptor determines functional synergy between AF-1 and AF-2 in the quantitative response to estradiol and tamoxifen. J. Cell Sci. 123, 1253–1261.