Integrative and systemic approaches for evaluating PPAR β/δ (PPARD) function Greta MP Giordano Attianese and Béatrice Desvergne Center for Integrative Genomics, University of Lausanne, Switzerland Footnotes: Corresponding author, BD: [email protected] Competing interests: The authors declare no competing financial interests

Author contributions: Both authors have been involved in drafting the manuscript and revising it critically.

Received December 5, 2014; Accepted March 9, 2015; Published April 27, 2015

Copyright © 2015 Giordano Attianese and Desvergne. This is an open-access article distributed under the terms of the Creative Commons Non-Commercial Attribution License, which permits unrestricted non-commercial use distribution and reproduction in any medium, provided the original work is properly cited.

Abbreviations: αMyHC, α-Myosin Heavy Chain; BCL6, B-cell lymphoma 6 protein; BAT, Brown adipose tissue; ChIP, Chromatin Immunoprecipitation; CHD, Coronary heart disease; DBD, DNA-binding domain; FAO, Fatty Acid Oxidation; FA, Fatty Acid; GSIS, Glucose-stimulated insulin secretion; HSC, Hematopoietic Stem cells; H&E, Hematoxylin and Eosin; HDAC1, Histone deacetylase 1; LBD, Ligand binding domain; MCP1, Monocyte chemotactic protein 1; NFkB, Nuclear factor kappa-light-chain- enhancer of activated B cells; NR, Nuclear Receptor; NCoR1, Nuclear receptor co-repressor 1; PPARs, Peroxisome proliferator- activated receptors; PPRE, PPAR-responsive element; RER, Respiratory Exchange Ratio; RA, Retinoic Acid; RXR, Retinoid X receptor; SMRT, Silencing mediator of retinoic acid and thyroid hormone receptors; SNPs, Single Nucleotide Polymorphisms; SUMO, Small Ubiquitin-like Modifier; TZDs, Thiazolidinediones; TR, Thyroid hormone receptor; TG, Triglycerides; VLDL, Very large density lipoprotein; WOSCOPS, West of Scotland Coronary Prevention Study; WAT, White adipose tissue.

Citation: Giordano Attianese G and Desvergne B (2015) Integrative and systemic approaches for evaluating PPAR β/δ (PPARD) function. Nucl Recept Signal 13, e001. doi:10.1621/nrs.13001

Keywords: PPAR β/δ, Systems Biology, nuclear receptor, ChIP

The peroxisome proliferator-activated receptors (PPARs) are a group of nuclear receptors that function as transcription factors regulating the expression of genes involved in cellular differentiation, development, metabolism and also tumorigenesis. Three PPAR isotypes ( α, β/δ and γ) have been identified, among which PPAR β/δ (PPARD) is the most difficult to functionally examine due to its tissue-specific diversity in cell fate determination, energy metabolism and housekeeping activities. PPAR β/δ acts both in a ligand-dependent and -independent manner. The specific type of regulation, activation or repression, is determined by many factors, among which the type of ligand, the presence/absence of PPAR β/δ-interacting corepressor or coactivator complexes and PPAR β/δ protein post- translational modifications play major roles. Recently, new global approaches to the study of nuclear receptors have made it possible to evaluate their molecular activity in a more systemic fashion, rather than deeply digging into a single pathway/function. This systemic approach is ideally suited for studying PPAR β/δ, due to its ubiquitous expression in various organs and its overlapping and tissue-specific transcriptomic signatures. The aim of the present review is to present in detail the diversity of PPAR β/δ function, focusing on the different information gained at the systemic level, and describing the global and unbiased approaches that combine a systems view with molecular understanding.

Introduction Several qualities make PPAR β/δ an interesting Systems biology aims at deciphering complex molecule to explore using system approaches: its biological units by aggregating biological information ubiquitous expression, the rather broad and poorly of various natures (gene, RNA, protein) in order to specific nature of its ligands (mainly unsaturated free gain the most comprehensive view of the events that fatty acids) and the fundamental functions that shape life. Another way of saying this is that systems PPAR β/δ controls, such as energy metabolism or cell biology aims to understand how a biological system, survival. Herein, we will start with a chapter made up of multiple interacting linear or non-linear concerning the gene, its structure and evolutionary pathways, behaves, and explains the global pattern history, and the main classical properties of PPAR β/δ observable at the level of the organism. The present as a transcription factor. We will then proceed with the review compiles a diversity of details regarding the systemic and integrative views that mouse models activity of peroxisome proliferator-activated receptor carrying alterations of PPAR β/δ activities provide. The β/δ (PPAR β/δ), viewed at the system level. third part will describe the global and unbiased

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approaches that bridge a system view and molecular [Yoshikawa et al., 1996], and Ppard has been mechanisms. These approaches encompass assigned to chromosome 17 in mouse. The six exons microarray and genome-wide Chromatin in the 3’ part encode the full PPAR β/δ protein. Immunoprecipitation (ChIP). This review will end with the description of polymorphisms in the human PPAR β/δ protein structure PPARD gene that are associated with various pathologies. Like most NRs, the PPAR protein structure consists of four main domains: the unstructured N-terminal A/B- Molecular properties of PPAR β/δ domain, the C-domain folded in two zinc fingers which corresponds to the DNA-binding domain (DBD), the PPAR β/δ ( PPARD ) gene D-domain or hinge region, and finally the E-domain forming a bundle of helices and stranded beta-sheet, Shortly after the characterization of a new nuclear which accommodates a ligand binding pocket. While receptor (NR) involved in the peroxisome proliferation the A/B-and D-domains are only poorly conserved response to some chemicals in mice, which was between the PPAR isotypes, the C- and E-domains accordingly referred to as peroxisome-proliferator share a high degree of sequence and structural activated receptor (PPAR) [Issemann and Green, homology (reviewed in [Escher and Wahli, 2000]). 1990], three PPAR genes, α ( PPARA ), β ( PPARD ) and γ ( PPARG ), were identified in Xenopus laevi Whether and by which mechanism the N-terminal [Krey et al., 1993]. When characterized in mouse, rat activator domain (AF1) of PPARs may regulate or human, the mammalian PPAR α and PPAR γ genes transcriptional activity remains debated. This domain were easily identified, while the third gene was less appears to be a determinant for PPAR subtype- clearly homologous to the Xenopus PPAR β and was specific activity [Bugge et al., 2009; Castillo et al., alternatively called NUC-1 in human [Schmidt et al., 1999; Hummasti and Tontonoz, 2006], also restricting 1992], fatty acid activated receptor (FAAR) [Amri et the number of genes transcriptionally regulated by al., 1995] in mice, and lastly PPAR δ [Evans et al., each of the PPAR subtypes [Hummasti and Tontonoz, 2004]. It now appears clear that they are indeed 2006]. homologous to the Xenopus PPAR β [Germain et al., 2006; Laudet, 1997]. These laborious identification The ligand binding domain (LBD) in all three PPARs efforts explain the different names for PPAR β/δ in the is a very large Y-shaped cavity (about 1400 cubic literature. angstroms) as compared to other NRs, and this characteristic allows the PPARs to interact with PPARs belong to a subfamily of the NR superfamily, numerous structurally-distinct ligands [Nolte et al., together with the thyroid hormone receptors, retinoic 1998]. However, a novel aspect of the PPAR β/δ acid (RA), vitamin D, ecdysone, and the orphan pocket is the narrowness of one of the Y arms, which receptors Rev-ErbA α (5ear1;NR1D1) and E75 cannot accommodate bulky polar heads [Xu et al., (NR1D3, from Drosophila). The latter two represent 2001; Zoete et al., 2007] such as Thiazolidinediones the closest relatives of the PPARs [Laudet et al., (TZDs) and L-tyrosine-based agonists. When the 1992]. The ancestral genes in this subfamily human PPAR β/δ LBD was first crystallized in absence appeared more than 500 million years ago [Knoll, of ligand, the crystals indicated the presence of 1992], followed by further duplication. The ancestral vaccenic acid, of bacterial origin, in the ligand pocket thyroid hormone receptor (TR) gene duplicated into [Fyffe et al., 2006]. Crystallization in the presence of two genes, TR α (NR1A1) and TR β (NR1A2), and the the potent and selective PPAR β/δ ligand GW0742 ancestral retinoic acid receptor (RAR) gene was later performed [Batista et al., 2012] and both duplicated into three genes, RAR α (NR1B1), RAR β studies suggest that two residues in the hormone (NR1B2), and RAR γ (NR1B3). Similarly, the three binding pocket (Val312 and Ile328) are important for PPAR loci, α, β, and γ, appeared during this second ligand-selective binding to PPAR β/δ. Finally, in the period [Laudet et al., 1992], with the duplication true absence of ligand, the structure of PPAR β/δ events likely being contemporaneous with the seems to correspond to an equilibrium of different appearance of the early vertebrates [Keese and conformations, among which are those favoring Gibbs, 1992]. Interestingly, PPAR genes have coregulator recruitment. evolved 2–3 times faster than the RAR and TR genes according to the amino acid sequence differences PPARs bind to DNA as obligate heterodimers with observed between the Xenopus and mammalian members of the retinoid X receptor (RXR) subfamily homologs. Among the PPAR subtypes, PPAR β/δ of NRs. The PPAR:RXR complex behaves as a exhibits an even higher rate of evolution. This permissive heterodimer since it can regulate gene relatively rapid evolution emphasizes the need for expression upon activation by either RXR or PPAR careful evaluation when studying PPAR activities in ligands. DNA binding properties of PPAR β/δ are various species. PPARD has been assigned to similar to those of PPAR α and PPAR γ, with a chromosome 6, at position 6p21.1-p21.2 in human common consensus sequence (PPAR-responsive

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element; PPRE), which consists of a direct repeat of a Takahashi et al., 2006]. In the reproductive organs, close derivative of the AGGTCA consensus hexamer, the nucleus of both spermatid/spermatocytes in the separated by one base-pair, the PPAR moiety testis and follicular epithelial cells in the ovary are occupying the 5’ half-site [IJpenberg et al., 1997]. The positively stained for PPAR β/δ [Higashiyama et al., only difference that contributes to the strength and 2007]. Finally, PPAR β/δ has been also found in specificity of PPAR isotype binding lies in the 5’ cartilage and bone compartment [Scholtysek et al., flanking region, though it seems mainly important for 2013]. PPAR α and not for PPAR β/δ [Juge-Aubry et al., 1997]. However, there are some divergent aspects of studies PPAR β/δ expression patterns that cannot be listed β δ Patterns of PPAR / expression herein. These relate to experimental conditions,

mouse strain and environment at the time of PPAR β/δ gene expression is truly ubiquitous measurement, as PPAR β/δ expression level is [Braissant et al., 1996; Kliewer et al., 1994], dictated by both exogenous and endogenous signals. suggesting the importance of this receptor in both systemic activity and/or for basic cellular functions. The use of different approaches (in situ hybridization, Different methods such as in situ hybridization, qPCR qPCR for RNA levels, various antibodies and and tissue microarray-based immunochemistry have methods for protein levels) to evaluate PPAR β/δ been used to provide a large survey of PPAR β/δ- expression also likely contributes to some of the expressing cells in the body, the most exhaustive one divergencies found in the literature. Of particular note, being that of Higashiyama et al. [Higashiyama et al., questions remain regarding how truly specific 2007]. This study confirmed the wide distribution of commercial antibodies are, suggesting that off-target PPAR β/δ protein in mouse tissues. A brief summary activity may contribute to false positive signals. with key references is given below. Despite these difficulties, the ubiquitous expression of PPAR β/δ - with the basal highest expression in PPAR β/δ is expressed in organs/cells highly gastrointestinal tract and skeletal muscle [Braissant et associated with fatty acid catabolism such as al., 1996] - likely reflects its role in multiple biological hepatocytes in the liver [Sanderson et al., 2010; functions. Sanderson et al., 2009; Shan et al., 2008], adipocytes in the brown and white adipose tissue (BAT and Transcriptional activity of PPAR β/δ WAT) [Leibowitz et al., 2000; Mottillo et al., 2012; Pan et al., 2009; Reilly and Lee, 2008; Roberts et al., The classical view of the prototypic activity of 2011; Wang et al., 2003] and skeletal muscle cells PPAR β/δ, as for other NRs, is to activate transcription [Giordano et al., 2009]. in a ligand-dependent manner, via binding to specific response element in the promoter of target genes and PPAR β/δ is also widely observed in the nucleus of recruitment of coactivator complexes. The somewhat epithelial lineage cells from keratinocytes [Kim et al., dogmatic concept is that agonist ligands induce a 2006; Schmuth et al., 2004; Tan et al., 2001; conformational change in PPAR β/δ:RXR that favors Westergaard et al., 2001] to enterocytes [Girroir et al., the dissociation of corepressors and the association 2008; Gupta et al., 2000; Gupta et al., 2004; He et al., 1999; Park et al., 2001]. In the nervous system, with coactivators [Yu and Reddy, 2007]. A large PPAR β/δ is found in both axons and dendrites of number of coactivator complexes interacting with neurons residing in different brain areas and in transcription factors have been identified and it is microglia cells [Higashiyama et al., 2007; Xiao et al., hypothesized that combinatorial usage of these 2010] of the central nervous system, as well as in complexes provides the basis for cell type-specific, astrocytes [Chistyakov et al., 2014] and in the gene-specific, and signal-specific transcriptional neurofibers of the peripheral nerves and spinal cord responses. Where and with which complexes active [Jana et al., 2012; Peters et al., 2000]. PPAR β/δ interacts is, however, not yet clear [Khozoie et al., 2012; Ricote and Glass, 2007]. In the immune system, PPAR β/δ expression has been particularly characterized in macrophages [Bouhlel et One intriguing question concerns ligand-dependent al., 2009; Chawla, 2010; Desvergne, 2008; Lee et al., repression of gene expression, particularly with 2003]. In the cardiovascular system, PPAR β/δ respect to PPAR β/δ, as it may represent an important immunostaining is present in the nucleus of activity of this receptor. The best-documented cardiomyocytes and vascular smooth muscle cells in mechanism was shown in macrophages where, in the aorta [Cheng et al., 2004a; Cheng et al., 2004b; absence of ligand stimulation, PPAR β/δ sequesters Finck, 2007] and other vascular districts [Zhang et al., the transcriptional repressor B-cell lymphoma 6 2002]. In the endocrine system, PPAR β/δ is seen in protein (BCL6). Upon ligand stimulation, PPAR β/δ the delta cells of the Langerhans islet and in the releases BCL6, thereby triggering BCL6-dependent secretory cells of the adrenal cortex [Lee et al., 2006; repression of NF-kB target genes such as Monocyte

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chemotactic protein 1 (MCP1) [Lee et al., 2003]. region (K185) [Wadosky and Willis, 2012], no Other mechanisms are likely involved, depending on experimental data to date has confirmed a role for cellular context, but still remain to be elucidated. SUMOylation in regulating PPAR β/δ activity.

In absence of ligand, PPAR β/δ may also exert PPAR β/δ ligands transcriptional repression. This was proposed to explain the regulation of a set of genes whose Natural ligands expression increased upon deletion of PPAR β/δ [Adhikary et al., 2011; Khozoie et al., 2012]. Previous As for PPAR α and PPAR γ, many unsaturated fatty studies had shown that by occupying PPREs while acids can bind to PPAR β/δ, in a pattern closely interacting in a ligand-independent manner with resembling that of binding to PPAR α [Desvergne and silencing mediator of retinoic acid and thyroid Wahli, 1999]. One proposed model of fatty acids hormone receptors (SMRT) and Histone deacetylase delivery to PPAR β/δ is via the very large density 1 HDAC1, PPAR β/δ could down-regulate PPAR α- lipoprotein (VLDL), through release of their triglycerides (TG), as shown in macrophages [Chawla and PPAR γ-dependent transactivation [Shi et al., et al., 2003; Ziouzenkova and Plutzky, 2004]. 2002]. Corepressor interactions with PPAR β/δ are indeed particularly strong and might even impair its However, it is still unresolved which particular fatty classical ligand-dependent positive transcriptional acids act as endogenous ligand specific for PPAR β/δ. activity when either Nuclear receptor corepressor Arachidonic acid derivatives were the first to be (NCoR) or SMRT is overexpressed [Krogsdam et al., pointed at, starting from those obtained upon 2002], thereby contributing to the complexity of the cyclooxygenase 2 (COX2) activation, such as system. Since these studies, little if any progress has prostacyclin (PGI2) and other prostaglandins [Yu et been made with regard to assessing the mechanism al., 1995], but also metabolite derivatives obtained by which PPAR β/δ could act as a bona fide repressor. through the 12/15 lipoxygenase activity, such as 9- HODE,13-HODE, 12-HETE and 15-HETE. While they In addition to LBD conformational changes upon are known as low affinity PPAR γ activators [Nagy et ligand binding, there are some important post- al., 1998], these metabolites activate PPAR β/δ with translational modifications that may also alter the some intriguing results since 13-s HODE is reported activity of PPAR β/δ in the absence/presence of to be an inhibitor of PPAR β/δ in colon epithelial cells ligand. Phosphorylation is one of the most common [Shureiqi et al., 2003], but an agonist in pre- modifications and several consensus phosphorylation adipocytes [Coleman et al., 2007]. Such observations sites can be mapped along the PPAR β/δ sequence. point to the concept of tissue-specific response, due Along these lines, both cAMP and PKA increased the for example to presence or absence of other basal and ligand-dependent activity of PPAR β/δ coregulators. Along these lines, a report suggested [Burns and Vanden Heuvel, 2007], while Wortmannin, that retinoic acid (RA) could be a ligand for either an inhibitor of the PI3K, could inhibit the positive PPAR β/δ or RARs, depending on the relative transcriptional activity of PPAR β/δ on prostaglandin expression of CRABPII (delivering RA to RARs) and E2 receptor EP4 gene expression [Han et al., 2005]. FABP5 (delivering RA to PPAR β/δ) [Schug et al., 2007]. Further studies from the same group However, at the molecular level, the phosphorylation highlighted some of the biological outcomes of this events of the PPAR β/δ protein and their functional crosstalk between RA and PPAR β/δ [Schug et al., consequences remain to be directly explored. 2008], albeit discrepancies concerning the effectiveness of RA stimulation on PPAR β/δ have Ligand-induced proteolysis through the ubiquitin- been reported [Rieck et al., 2008]. proteasome system is a common mechanism to end transcriptional activity promoted by the ligand- Exogenous fatty acids are also studied, such as activated receptors. However, this mechanism seems ombuin-3-O-beta-D-glucopyranoside (ombuine), a to be reversed for PPAR β/δ, as in the absence of flavonoid from Gynostemma pentaphyllum, activating ligand, PPAR β/δ has a very short half-life due to both PPAR β/δ and PPAR α [Malek et al., 2013]. This efficient ubiquitination and proteosomal degradation. example underscores the fact that most of the natural Ligand binding inhibits this ubiquitination thereby ligands known to interact with PPAR β/δ also interact increasing its half-life [Genini and Catapano, 2007; with the other PPAR subtypes. It is an interesting Rieck et al., 2007; Wadosky and Willis, 2012], albeit feature when searching for dual agonists, albeit it may this phenomenon may depend on the level of also lead to off-target effects. PPAR β/δ expression at the time of ligand stimulation [Rieck et al., 2007]. Interestingly, ubiquitin-C is itself a Synthetic ligands target gene of PPAR β/δ [Kim et al., 2004]. Finally, even though PPAR β/δ presents a potential Small Tremendous effort has been devoted towards Ubiquitin-like Modifier (SUMO)ylation site in its D optimizing synthetic ligand binding selectivity to

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PPAR β/δ, thereby avoiding off-target effects. Recently target genes [Shearer et al., 2008] and increasing the a high-throughput screening (HTS) assay [Xia et al., recruitment of corepressors [Palkar et al., 2010]. 2013] has been established in order to select PPAR More recently two novel compounds have been agonists with low toxicity and high efficiency. Below described in the class of PPAR β/δ antagonists: are a few, and by no means exhaustive, illustrations DG172 and PT-S58. DG172 exhibits high binding of synthetic PPAR β/δ agonists and antagonists that affinity and potent inverse agonistic properties, have been used or are presently being tested in in enhancing transcriptional corepressor recruitment and vivo studies. down-regulating transcription of PPAR β/δ target genes [Lieber et al., 2012]. Moreover, DG172 is Agonists bioavailable after oral treatment in mice. PT-S58, a cell-permeable diaryl-sulfonamide, acts as a pure The very first synthetic compound, called L165041 competitive PPAR β/δ subtype-specific antagonist [Berger et al., 1999], was an established leukotriene targeting the ligand binding site of PPAR β/δ while not antagonist, which activates human PPAR β/δ, but also allowing coregulator interactions [Levi et al., 2013; triggers PPAR γ activation at high doses [Forman et Naruhn et al., 2011]. al., 1997]. The compound GW501516 [Sznaidman et Altogether, these molecules represent important tools al., 2003], which was more potent and more specific, to study the systemic impact of PPAR β/δ activities in subsequently took over in most published animal models, together with the generation of experimental work and became the reference genetically-modified mouse models. compound as a PPAR β/δ agonist [Pelton, 2006]. However, while being investigated for its potential Integrative physiology of PPAR β/δ activity in metabolic disorders, as reviewed in Lamers et al. [Lamers et al., 2012], its uncontrolled use in β δ human as a doping substance and subsequent high In the context of this review on PPAR / and systems risks of abuse contributed to stop all further biology, the present section focuses on knowledge development of the molecule. Today, GW0742, which gained with a systemic view from animal models, was developed together with GW501516, is a highly either through gene modification or through animal selective PPAR β/δ agonist that is commercially treatment with agonist or antagonist ligands. Although available for non-human research purposes these reported studies have deeply contributed in [Sznaidman et al., 2003]. The most recent agonists shaping our present understanding of PPAR β/δ, they developed to target PPAR β/δ in clinical settings were also present several caveats. It indeed remains MBX-8025/RWJ800025 (MBX-8025 presently in difficult when using transgenesis approaches (total phase II trials, generated by Metabolex) [Billin, 2008] body or tissue-specific) activating or deleting and KD-3010 developed by Kalypsys [Iwaisako et al., PPAR β/δ, to disentangle the truly specific effects from 2012]. the secondary and compensatory effects. The use of constitutively active PPAR β/δ via its fusion with trans- Antagonists and Inverse Agonists activator domains, such as VP16, can alter the secondary/tertiary structure of PPAR β/δ, leading to The irreversible PPAR γ antagonist GW9662 different interaction surfaces and in turn diverse [Leesnitzer et al., 2002] was initially used since it also transactivation functions. Agonist and antagonist exerted a potent PPAR β/δ antagonistic activity compounds are also not exempt from off-target [Seimandi et al., 2005]. The first selective PPAR β/δ activities. These few examples underline the need to antagonist was GSK0660, which was identified and cautiously interpret observations made in such characterized in 2008 [Shearer et al., 2008], although models. its poor bioavailability has impaired its use in in vivo studies. The same was true for SR13904, which in Generation of PPAR β/δ null mice addition to PPAR β/δ also antagonizes PPAR γ transactivation, albeit with much weaker potency Four models of PPAR β/δ total body Knock Out (KO) [Zaveri et al., 2009]. Finally, GSK3787 is a potent have been reported. In the first published model PPAR β/δ antagonist with good pharmacokinetic [Peters et al., 2000], Ppard was disrupted via insertion properties. It has a good bioavailability and can be of phosphoribosyltransferase II expression cassette in used in animal studies [Palkar et al., 2010; Shearer et the last exon, encoding the ligand binding domain. al., 2010]. However, this compound is also an Mice for this modification presented significantly lower irreversible antagonist, which covalently binds survival rate in mixed background but this phenotype PPAR β/δ and it is not as selective as hoped. These was back to normal in C57BL/6N background, in antagonists can also be referred to as inverse striking contrast with the three other models, agonists, as they bind PPAR β/δ as an agonist, but described below. It must be noted that the strategy of induce an opposite pharmacological response, the gene alteration has likely driven the generation of decreasing the basal expression level of PPAR β/δ a hypomorph allele rather than a null allele, since

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theoretically, PPAR β/δ obtained from this gene could without interference from the PPAR β/δ systemic be expressed, albeit truncated of its C-terminus 60 deletion (see Table 1). amino-acids, but left with intact N terminus and DNA binding domains. Another strategy consisted of PPAR β/δ as a potent regulator of metabolism and framing the exon 4 encoding the first moiety of the inflammation DNA-binding domain of PPAR β/δ with two LoxP sites, allowing the generation of either a conditional knock- Energy metabolism in muscle out allele, or a constitutive deletion of the exon 4, with a subsequent frame shift of the encoding sequence While initially overshadowed by PPAR α and PPAR γ, [Barak et al., 2002]. Using the latter, a PPAR β/δ null interest in PPAR β/δ rose significantly when its role in mouse model was generated, although this gene muscle energy metabolism was identified. The first alteration resulted in severe placental defects and indications that PPAR β/δ might play a role in muscle frequent mid-gestation lethality. Notable changes in metabolism came from the observations that the structure of the placenta of PPAR β/δ KO mice PPAR β/δ expression in skeletal muscle is increased include reduced size of the entire tissue with upon fasting [Holst et al., 2003] and upon exercise loosening of its attachment to decidua and [Russell et al., 2005], suggesting a role for PPAR β/δ hemorrhages at various locations due to flooding of in the adaptive response of skeletal muscle to maternal blood. Replacing exon 4 with the β- increased demand for fatty acid oxidation. Skeletal galactosidase gene resulted in placental defects and muscle indeed represents an important consumer of lethality similar to those previously reported [Barak et fatty acids. In a transgenic mouse model, the al., 2008]. Using a similar strategy, Nadra et al. overexpression of PPAR β/δ in skeletal muscle [Nadra et al., 2006] replaced exon 4 and part of exon provokes a shift towards more oxidative fibers and 5 with a PGK-neo cassette deleting the two zinc promotes a general decrease of body fat content [Luquet et al., 2003]. Similar results were obtained by fingers of the DNA-binding domain. Here again, expressing overactive PPAR β/δ fusion protein deletion of PPAR β/δ severely affected placental (PPAR β/δ-VP16) under the control of the alpha-actin development, leading to embryonic lethality at promoter [Wang et al., 2004], which resulted in an embryonic day 9.5 (E9.5) to E10.5 of most, but not all, average 2-fold increase in type I muscle fibers, and a PPAR β/δ-null mutant embryos. The observed subsequent increase in muscle oxidative capacity, placental alterations mainly reside in the Giant cell leading to a remarkable increase in running distance layer. In fact, both molecular and cellular studies and time on a treadmill [Wang et al., 2004]. Because β δ highlighted the role of PPAR / in the differentiation of this phenotype, the latter model was also called the pathway of these particular cells through activation of “marathon mice”. Akt signaling and inhibition of Id-2 [Nadra et al., 2006]. Conversely, when PPAR β/δ is specifically deleted in skeletal muscle, using the CRE-recombinase system Further analyses demonstrated that the lethality under the control of alpha-actin promoter, the muscle observed is 100% in pure background (C57BL/6 and fibers exhibited lower oxidative activity, while the body SV129, B. Yaacov and B. Desvergne personal fat mass increased and led to insulin resistance communication, respectively), whereas it is partial on [Schuler et al., 2006]. Altogether, these observations mixed backgrounds (C57Bl6/FVB or C57Bl6/SV129, suggest PPAR β/δ activation plays a role in skeletal Barak Yaacov and B. Desvergne personal muscle adaptation to physical stress, although the communication, respectively). The viability and molecular mechanisms underlying this effect are not maintenance of PPAR β/δ null mice is achieved by the yet clearly understood. interbreeding of survivors on mixed genetic backgrounds. The fact that mixed background To further study the PPAR-dependent energy increases chances to obtain null mutant pups, in substrate usage in muscle and to characterize it at the addition to a “founder” effect rather than a specific molecular level, Gan et al. [Gan et al., 2011] genetic background, underline the complexity of the generated transgenic mouse lines expressing either genetic network in which PPAR β/δ is involved with PPAR β/δ or PPAR α under the control of skeletal respect to placenta development. muscle-specific promoter (MCK-PPAR β/δ or MCK- PPAR α constructs). Comparing these two mouse The development of the surviving mice was grossly models, the authors demonstrated that PPAR β/δ normal and numerous studies could benefit from this interacts with the exercise-inducible kinase AMP- model, as seen in the next section. In parallel, tissue- activated protein kinase (AMPK), which in turn specific knockout mice and transgenic mice promotes glucose uptake, fatty acid oxidation, expressing normal, mutated or strongly-active forms mitochondrial biogenesis, and insulin sensitivity. In of PPAR β/δ have been generated in order to study addition, PPAR β/δ-mediated activation of AMPK tissue-specific functions exerted by this receptor leads to a synergistic activation of lactate

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Table 1. PPAR β/δ mutant phenotypes. The main PPAR β/δ-based mouse models are herein listed according to the type of mutation and tissue/cell specificity. The main observations reported from the mutant are also briefly commented.

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dehydrogenase b (Ldhb) gene transcription, Mirroring the PPAR β/δ deletion phenotype, these increasing the ratio Ldhb/Ldha and diminishing the mice presented increased myocardial glucose accumulation of lactate. The high glycogen stores and utilization, with upregulated expression of Glut4 and increased levels of GLUT4 in mouse muscles with glycolytic genes, and were resistant to diet-induced MCK-driven PPAR β/δ expression further suggested a lipid accumulation in cardiomyocytes and subsequent broad reprogramming of glucose utilization pathways lipotoxic cardiomyopathy [Burkart et al., 2007]. More mediated by PPAR β/δ [Gan et al., 2011]. In parallel, recently, a fourth mouse model overexpressing slow-twitch genes are increased in MCK-PPAR β/δ PPAR β/δ specifically in cardiac muscle has been soleus muscle, via an indirect mechanism involving a generated. The constitutively active form of PPAR β/δ- complex NR/microRNA circuitry [Gan et al., 2013]. VP16 was placed under the control of the promoter αMyHC for cardiac-muscle-restricted expression, and Altogether, these results clearly confirm that PPARβ/δ was constructed to be inducible by Tamoxifen in skeletal muscle controls different phases of the administration. This report mainly describes how this adaptive response to training (i.e., the number of methodology may be used to efficiently generate oxidative myofibers), and metabolic switch (i.e., the transgenic mouse models expressing a constitutively increase in fat burning capability), by reprogramming active form of PPAR β/δ upon Tamoxifen the expression of glucose utilization genes. We might administration in a tissue-specific manner [Kim et al., also ask whether these observations could be of any 2013]. However, the overexpression of such use for improving structural muscular disorders. Miura constitutively activated PPAR β/δ cannot take into et al.,[Miura et al., 2009] found that GW501516 account subtle activities depending on expression systemic treatment stimulated utrophin A mRNA levels in muscle cells derived from the Duchenne levels and ligand availability, and this may lead to muscular dystrophy mdx mouse model. Over a 4- overestimates of the importance of certain regulatory week trial period, the agonist treatment augmented activities. the percentage of muscle fibers expressing slower myosin heavy chain isoforms and stimulated PPAR β/δ in adipose tissue transcription of utrophin A and its expression in the sarcolemma. The mdx sarcolemma integrity was The role of PPAR β/δ in fatty acid catabolism is also improved, together with a limitation of the eccentric effective in white and brown adipose tissue (WAT and contraction-induced damage of mdx skeletal muscles BAT, respectively). Transgenic mice expressing, in [Miura et al., 2009]. More generally, a better the WAT, a hyper-active form of PPAR β/δ (PPAR β/δ- knowledge of the metabolic role of PPAR β/δ in VP16) are resistant to both high-fat diet-induced and skeletal muscle, where it contributes to enhanced genetically predisposed obesity, together with muscle endurance, might be useful in designing decreased lipid accumulation in adipose tissue and therapeutic strategies for muscular degenerative diminished lipidemia, in line with an increased fatty diseases such as muscular dystrophy. acid consumption [Wang et al., 2003]. The effect of PPAR β/δ over-activation was even more dramatic in Adult cardiomyocytes express a high level of metabolically-active BAT, with an increased PPAR β/δ, and also utilize fatty acid as their main expression of genes involved in fatty acid hydrolysis, energy source. With respect to PPAR β/δ functional oxidation, and uncoupling of oxidative delineation in cardiac muscle, three different mouse phosphorylation [Wang et al., 2003]. This over- models have been generated. The cre-loxP-mediated activation may, however, mask more subtle regulatory deletion of PPAR β/δ restricted to cardiomyocytes activity, such as the PPAR β/δ-mediated induction of presented the most dramatic phenotype due to the Twist1, which acts as a negative regulator of energy loss of this receptor, with congestive heart failure and dissipation, in part via a decrease of UCP1 [Pan et al., reduced mouse survival [Cheng et al., 2004a]. This 2009]. was associated with down-regulation of the constitutive expression of key fatty acid oxidation genes, and a consequent decrease of basal Lessons learned from PPAR β/δ deletion in mice are myocardial fatty acid oxidation with lipid accumulation. painting a more complex picture. Total body PPAR β/δ In a second model, PPAR β/δ cardiac-specific deletion null mice exhibit a paradoxically leaner phenotype was induced at 2 months of age. This “adult” model with a significant reduction of both WAT and BAT underscored the overall alteration of energy substrate mass, an effect possibly resulting from an alteration of usage, with decreases of both fatty acid and glucose fatty acid transport [Barak et al., 2002; Peters et al., oxidation rates concomitant with a reduction in the 2000]. However, PPAR β/δ null mice fed with high fat expression of genes in both pathways [Wang et al., diet displayed an increased susceptibility to weight 2010]. It also suggests that the severity of the cardiac gain, coupled with blunted BAT UCP1 expression failure in the first model might be due to a role of [Wang et al., 2003]. Finally, mice carrying an aP2- PPAR β/δ in cardiac development. In the third model, driven adipose-specific deletion of PPAR β/δ did not mice overexpressed PPAR β/δ under the control of the show any altered fat mass content [Barak et al., 2002] alpha myosin heavy chain ( αMyHC) promoter. (reviewed in [Christodoulides and Vidal-Puig, 2010]).

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These results suggest that most effects on adipose questions regarding their respective roles. Microarray tissue may be the consequence of systemic and analyses comparing the liver transcriptome of PPAR α muscle metabolism rather than reflecting a direct null mice versus PPAR β/δ null mice revealed only activity of PPAR β/δ in the adipose tissue. minor overlap between PPAR α- and PPAR β/δ- dependent gene regulation, and further reinforced the PPAR β/δ-specific roles in liver metabolism observations that PPAR β/δ governs glucose utilization and lipoprotein metabolism and has an Liver is one of the most important organs in important anti-inflammatory role in liver [Sanderson et controlling energy homeostasis, via regulation of al., 2010]. energy availability and storage. PPAR β/δ is expressed in all the main cell types present in the PPAR β/δ-specific roles in the pancreas liver: hepatocytes, Kupffer cells and hepatic stellate cells. The exploration of PPAR β/δ-mediated response Pancreas-specific deletion of PPAR β/δ, obtained in the liver suggested the dual implication of PPARβ/δ through PDX1-mediated PPAR β/δ gene deletion, not only in fatty acid metabolism, but also directly in caused an increased number of islets and, more glucose metabolism. In particular, Lee et al. [Lee et importantly, enhanced insulin secretion, leading to al., 2006] demonstrated that PPAR β/δ enhances hyperinsulinemia and lower glycemia in mutant mice. glucose utilization in the liver, via activation of the This was due to alterations in the machinery of pentose-phosphate pathway, and promotes liver exocytosis, from Golgi functions to routing of granules lipogenesis, recapitulating an insulin-sensitizing and vesicles to the cell periphery, rather than an effect. Conversely, PPAR β/δ null mice suffer from altered metabolic response [Iglesias et al., 2012]. In glucose intolerance [Lee et al., 2006]. contrast, systemic treatment with PPAR β/δ agonists Deciphering what is due to a direct activity of resulted in increasing glucose-stimulated insulin PPAR β/δ in the liver from a combined effect on liver, secretion (GSIS) and normalizing pancreatic islet muscle, and adipose tissue remains difficult. In an hypertrophy in ob/ob mice [Tanaka et al., 2003] and effort to address this question, Liu et al. [Liu et al., db/db mice [Winzell et al., 2010]. Treatment with the 2011] achieved a liver-specific PPAR β/δ activation by PPAR β/δ agonist GW501516 also restored the employing an adenoviral-mediated gene delivery impaired GSIS observed in mice carrying a pancreas- system and used, as a control, liver-specific PPAR β/δ selective deletion of Desnutrin (also called null mice. The comparison between mice ATGL/PNPLA2) [Tang et al., 2013]. This study also overexpressing PPAR β/δ in liver with those bearing a suggested that in homeostatic conditions, the ATGL- liver-specific deletion confirmed its role as an insulin mediated lipolysis would provide ligand for PPAR β/δ, sensitizer. In fact, overexpressing PPAR β/δ in liver whose activity on mitochondrial oxidation would causes a genetic reprogramming that leads to an contribute to islet β cell GSIS. increased glucose utilization and increased lipogenesis. However, no specific alterations have Finally, and more indirectly, PPAR β/δ agonists been pointed out regarding the liver-specific deletion increase the production of glucagon-like peptide-1 of PPAR β/δ, suggesting a moderate activity under (GLP-1), an intestinal incretin that can preserve the normal conditions. morphology and function of pancreatic β-cells [Daoudi et al., 2011]. This mechanism might in part explain the These effects of PPAR β/δ in controlling hepatic lipid apparent contradiction between the observations metabolism may also have consequences on its role made in the model of pancreas-specific deletion of in the resident macrophages in liver, the Kupffer cells, PPAR β/δ and those made with systemic PPAR β/δ that have also been implicated in fatty liver disease agonist treatment. However, and as mentioned and insulin resistance [Lanthier et al., 2010]. More above, we cannot ignore the caveats that each model specifically, PPAR β/δ is a key regulator of the carries (compensatory effects and off-target activities alternative activation of Kupffer cells towards anti- for example). inflammatory activity (macrophage M2 subtype) in the presence of IL4 and IL13 stimulation [Odegaard et al., Systemic metabolic effects of PPAR β/δ 2008]. An imbalance of inflammatory mediators in liver can also affect hepatic stellate cells where, The tissue-specific deletions of PPAR β/δ allowed the regardless of their activation status, PPAR β/δ is evaluation of its tissue-specific activity. Systemic expressed at high levels [Hellemans et al., 2003]. treatments of mice with specific PPAR β/δ agonists However, no in vivo mouse studies using PPAR β/δ allowed an integrated analysis of these responses, activation or inactivation mouse models have yet mainly affecting the overall metabolic homeostasis. pushed these cellular studies forward. For example, the treatment of obese animals with specific PPAR β/δ agonists results in normalizing the These observations confirmed a key role of PPAR β/δ metabolic parameters with decrease of circulating in tissues and circumstances where PPAR α is also a triglycerides and reduction of adiposity [Tanaka et al., key regulator of fatty acid oxidation, thereby raising 2003; Wang et al., 2003; Wang et al., 2004].

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Circulating insulin levels also declined, whereas the of PPAR β/δ generates some concerns regarding the improvement in glucose tolerance and insulin possible onset of adverse side-effects, due to the sensitivity, as determined by the glucose and insulin activation of PPAR β/δ in tissues not related to the tolerance tests (GTT and ITT), was moderate [Tanaka therapeutic effects. et al., 2003]. This PPAR β/δ-mediated systemic improvement in lipid homeostasis also led to Inflammation decreased high-fat-diet-induced liver steatosis [Wu et al., 2011]. Such PPAR β/δ-driven treatment Macrophages are key players in inflammation. The recapitulates the improved carbohydrate catabolism in first report on PPAR β/δ acting on inflammation in the liver, the promotion of fatty acid oxidation in the macrophages described the association and muscle and the inhibition of free fatty acid release dissociation of PPAR β/δ with the transcriptional from adipocytes, thereby explaining the overall corepressor BCL6, in absence and presence of enhanced insulin sensitivity [Lee et al., 2006]. Similar ligand, respectively. This results in ligand-dependent observations were made in OLETF rats, an animal PPAR β/δ-mediated inhibition of NFkB target genes, model of type II diabetes with obesity, whose thereby limiting the inflammation [Lee et al., 2003]. treatment with the highly PPAR β/δ-specific agonist Macrophages, however, differentiate from tissue GW0742 attenuates hepatic fat accumulation and resident monocytes either toward a pro-inflammatory improves insulin signaling [Lee et al., 2012]. (M1 or classically activated macrophage, e.g. by IFN γ, TNF α or bacterial LPS) or an anti-inflammatory PPAR β/δ also participates in regulating lipid systemic (M2 or alternatively activated, e.g. by IL-4 or IL-13) transport through lipoproteins. Treating obese rhesus phenotype according to the specific stimuli of the monkeys, an animal model for human obesity and environment. Specific deletion of PPAR β/δ in associated metabolic disorders, as well as diabetic macrophages (Lys-Cre-driven specific deletion; [Kang db/db mice, with a selective PPAR β/δ agonist caused et al., 2008]) and irradiated wild-type mice subjected a beneficial increase in serum HDL cholesterol and a to PPAR β/δ-/- bone marrow transplantation decrease in small-dense LDL [Leibowitz et al., 2000; [Odegaard et al., 2008] both demonstrated that Oliver et al., 2001], together with a reduced alternative activation of resident macrophages in liver cholesterol absorption in the intestine, associated with and adipose tissue depends highly on PPAR β/δ a decrease in Niemannn-Pick C1 like 1 protein [van activity, which contributes to the M2a-specific gene der Veen et al., 2005]. Conversely, PPAR β/δ deficient expression program. Of interest is the fact that M2a mice exhibit LDL hypertriglyceridemia, due to macrophages not only limit inflammation but also increased hepatic production of VLDL and decreased affect metabolic regulation, in a variable manner LPL-mediated catabolism [Akiyama et al., 2004]. depending on the animal model used [Desvergne, 2008]. Finally, PPAR β/δ also acts as a transcriptional Altogether, these observations emphasize the sensor of dying cells, facilitating the engulfment of intertissular, reciprocal, metabolic regulation that apoptotic cells by macrophages. Mice bearing a PPAR β/δ coordinates. It explains the difficulties, macrophage-specific deletion of PPAR β/δ exhibit a particularly in the adipose tissue, in discriminating decreased expression of opsonins, impairing the tissue-specific from systemic regulation. It also capability of phagocytes to recognize apoptotic bodies permits insistence on the potential therapeutic and to clear them from the environment. This leads to activities of PPAR β/δ in obesity and/or in type 2 a progressive formation of autoantibodies that diabetes. Indeed, the implication of PPAR β/δ in predispose PPAR β/δ KO mice to develop energy consumption rendered this receptor an autoimmune disease [Mukundan et al., 2009]. attractive therapeutic target, especially for its anti- obesity activity. The first human clinical trial using a The vascular compartment and its endothelial cells PPAR β/δ agonist has been concluded by Sprecher et also play an important role in inflammation, which is al.[Sprecher et al., 2007], in which GW501516 associated with increased local vasodilation, vascular treatment has been tested in a small cohort of healthy permeability, and leukocyte recruitment due to volunteers. Several successive clinical trials have augmented expression of adhesion molecules on been conducted, or have recently been completed, as endothelial cells. Selective PPAR β/δ ligand reviewed in [Lamers et al., 2012] to treat GW501516 treatment inhibits the expression of major hyperlipidemia, insulin resistance and obesity. endothelial cell inflammatory responses (V-CAM-1, E- However, despite its promising potential as a selectine, ICAM-1) involved in leukocyte recruitment treatment for obesity and dyslipidemia, the use of in vivo [Piqueras et al., 2009]. PPAR β/δ also protects PPAR β/δ agonist, in particular GW501516, in clinical endothelial cells from tissue damaging oxidative trials was limited, due to its possible use as a doping stress, which is produced by immune cell activity and substance (http://www.independent.co.uk/life- may contribute to increased inflammatory response style/health-and-wellbeing/health-news/warning-to- [Fan et al., 2008; Liou et al., 2006] [Jiang et al., beijing-olympics-over-pills-that-mimic-exercise- 2009a] [d'Uscio et al., 2012]. Other observations 882608.html). In addition, the ubiquitous expression include the anti-inflammatory effect of PPAR β/δ ligand

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administration, which limited the development of tetradecanoylphorbol acetate topical application, hair atherosclerosis in models using LDL receptor plucking, or skin wound healing, with a demonstrated knockout mice [Barish et al., 2008; Graham et al., role in the timing and efficiency of keratinocyte 2005; Lee et al., 2003; Takata et al., 2008]. differentiation [Michalik et al., 2001].

These studies likely explained the positive effects of The role in keratinocyte differentiation and the high PPAR β/δ agonist treatment in a variety of models of constitutive expression of PPAR β/δ in the epithelial induced inflammation in mice, such as LPS-induced compartment of the gastrointestinal tract raised the pulmonary inflammation [Haskova et al., 2008], question as to whether PPAR β/δ would similarly affect chronic inflammation in the adipose tissue homeostatic regulation of intestinal cell [Rodriguez-Calvo et al., 2008], experimental colitis proliferation/differentiation. Indeed, in PPAR β/δ null [Hollingshead et al., 2007], systemic septic shock mice, Varnat et al. demonstrated that PPAR β/δ [Kapoor et al., 2010], TPA-induced hyperplasia and contributes to Paneth cell terminal differentiation inflammation in the skin [Peters et al., 2000] and [Varnat et al., 2006], via a PPAR β/δ-dependent down- hepatotoxicity [Shan et al., 2008] (reviewed in regulation of Hedgehog signaling, suggesting that [Bishop-Bailey and Bystrom, 2009]). The anti- PPAR β/δ is downstream of the Wnt-β-catenin/TCF4 inflammatory activity of PPAR β/δ might also be pathway. beneficial in the brain. For instance, activation of PPAR β/δ with GW0742 in a murine model of In the brain, PPAR β/δ is highly expressed during the experimental autoimmune encephalomyelitis limits the development of the central nervous system [Braissant appearance of cortical lesions and decreases IL-1β and Wahli, 1998; Cullingford et al., 1998; Moreno et expression, and reduces the incidence of clinical al., 2004; Woods et al., 2003]. PPAR β/δ null mice symptoms [Polak et al., 2005]. PPAR β/δ may also exhibit myelination defects [Peters et al., 2000] contribute to the anti-inflammatory activity of suggesting a role in oligodendrocyte differentiation Palmitoylethanolamide (PEA) in spinal cord injury and maturation, further confirmed in studies using [Paterniti et al., 2013]. As can be seen from this cells in culture [Saluja et al., 2001; Vittoria Simonini et paragraph, the diverse importance of the mechanisms al., 2010]. proposed is difficult to establish when studying the integrated model represented by the mouse. In PPAR β/δ importantly contributes to the bone particular, the reciprocal link between inflammation differentiation/remodeling cell program in mice and metabolism, along with the attendant question of [Scholtysek et al., 2013]. First of all, PPAR β/δ is the which starts first, is very intriguing and is clearly most abundantly expressed PPAR isotype in bone. Its highlighted when phenotyping the activity of PPAR β/δ activation decreases osteoclastogenesis and in the full organism. reinforces osteoblast differentiation via increased WNT signaling to mesenchymal cells. A mirror image PPAR β/δ in cell fate is observed in PPAR β/δ null mice that present a higher number of osteoclasts and progressive PPAR β/δ in cell differentiation osteopenia, particularly aggravated upon ovariectomy in females [Scholtysek et al., 2013]. Mouse observations have identified a certain number of cell types sensitive to PPAR β/δ activation in their PPAR β/δ is the predominant PPAR isotype expressed differentiation process, as we described in the in hematopoietic stem cells (HSCs) [Ito et al., 2012]. preceding section concerning placental Giant Bone marrow transplantation using PPAR β/δ-deleted Trophoblastic cells. KitposScaposLinneg cells revealed that PPAR β/δ loss did not affect homing of HSCs but profoundly In the skin, PPAR β/δ promotes keratinocyte impacted long-term repopulating capacity, whereas differentiation [Kim et al., 2005; Schmuth et al., 2004; treatment with PPAR β/δ agonists improved HSC Tan et al., 2001]. Particularly, in the context of wound maintenance in vivo [Ito et al., 2012]. and inflammation, which triggers increased PPAR β expression and promotes the synthesis of a ligand so Finally, as discussed above, the role of PPAR β/δ in far unidentified [Tan et al., 2001]. The end of the adipose tissue is highly complex and intimately linked wound healing process is marked by increased with the systemic metabolic consequences of activity of the TGF-β/Smad pathway that terminates PPAR β/δ activation, and it seems that PPAR β/δ has PPAR β/δ-induced expression and activation [Tan et little or no specific activity on adipocyte differentiation. al., 2004]. This is consistent with the high expression However, while total body KO mice have reduced of PPAR β/δ in the mouse epidermis during fetal adipose stores, mice with selective adipocyte development, which progressively disappears from PPAR β/δ KO have normal adipose tissue. the interfollicular epithelium after birth, but is re- Considering that the aP2 promoter which drives activated in the presence of various stimuli such as PPAR β/δ deletion is activated at a late stage of

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adipocyte differentiation, we cannot exclude a PPAR β/δ agonist L-165041 protects the testis from possible role at an earlier stage, as suggested by ischemia and reperfusion damage, reducing TNF α, IL- studies using cell culture [Bastie et al., 2000; Hansen 6 along with tissue injury via inhibition of extracellular- et al., 2001; Matsusue et al., 2004]. regulated kinase 1/2 phosphorylation [Minutoli et al., 2009]. In conclusion, these in vivo observations are quite heterogenous. PPAR β/δ seems to act in many PPAR β/δ in cell proliferation and tumor biology different cell types and in different tissue contexts to modulate cell fate. Is there a unifying mechanism for The role of PPAR β/δ in cell proliferation is more all these observations? It is indeed tempting to ambiguous. Different groups observed that PPAR β/δ hypothesize that these activities come from a has a negative role towards proliferation of PPAR β/δ role in cellular basal metabolism. However, keratinocytes [Tan et al., 2001] [Burdick et al., 2007; this remains to be demonstrated. Burdick et al., 2006; Kim et al., 2006; Kim et al., 2005], at least in part through down-regulation of PPAR β/δ in cell survival cyclin A [Tan et al., 2001], through reduced ubiquitination of PKC α [Kim et al., 2005], or via The study of Di Poi et al. [Di-Poi et al., 2002], Cdkn1c gene encoding the cell cycle inhibitor discussed in the previous section, highlighted another p57(Kip2) [Muller-Brusselbach et al., 2007]. However, important PPAR β/δ-mediated response to the other studies have shown that PPAR β/δ can promote inflammatory insult: cell survival. This occurs via keratinocyte proliferation [Di-Poi et al., 2002; Michalik PPAR β/δ-dependent up-regulation of integrin-linked et al., 2001; Romanowska et al., 2010]. For example, kinase (ILK) and 3-phosphoinositide-dependent overexpression of PPAR β/δ in the skin, driven by the kinase-1 (PDK1), which activate Akt1 via human CYP1A1 promoter, provoked hyper- phosphorylation, and through down-regulation of proliferation of keratinocytes, dendritic cell PTEN [Di-Poi et al., 2002]. This is in turn accumulation, and endothelial activation, closely accompanied by an increased response to mimicking a psoriasis lesion [Romanowska et al., chemotactic signals, in particular through cytoskeleton 2010]. Moreover, under UV stimulation, PPAR β/δ remodeling [Tan et al., 2007]. activates Src protein, that in turn promotes the Epithelial to Mesenchymal Transition. This complex Accordingly, different models of ischemia-reperfusion process leads to a progressive de-differentiation of have been studied to evaluate the importance of this keratinocytes, thereby contributing to a higher activity in various tissues. Ischemia-reperfusion is proliferation rate and, in turn, to tumor progression [Montagner et al., 2014]. A potential and likely only characterized by a first step of adaptation to the lack partial explanation for these discrepancies may of nutrients and oxygen creating a cellular stress with involve in vivo an intricate combination of PPAR β/δ acidosis, swelling, changes in the expression of activation not only in keratinocytes but also in the adhesion molecules and other modifications due to adjacent fibroblasts, regulating the expression of adaptation of the cell to a forced anaerobic condition. various cytokines, a hypothesis verified at least in a Reperfusion in this context results in an oxidative system of organotypic culture [Chong et al., 2009]. insult with induction of oxidative stress in cells rather than restoration of normal function. Such a model is At first glance, due to its role in cell differentiation and for example used for mimicking ischemic acute renal the limited evidence supporting its pro-proliferative failure, where the major cellular damage is acute activity to date, PPAR β/δ should not be thought of as tubular necrosis. In such a mouse model, a pro-tumorigenic molecule. However, two main preconditioning with PPAR β/δ agonist dramatically features link PPAR β/δ to tumorigenesis in the protected against the damage, via PPAR β/δ- intestine: i) PPAR β/δ is a Wnt target gene in this dependent increased Akt activity, whereas PPAR β/δ tissue [He et al., 1999], a feature often associated heterozygous and PPAR β/δ null mice were very with pro-tumorigenic activity, and ii) PPAR β/δ has a sensitive to kidney failure [Letavernier et al., 2005]. strong anti-apoptotic activity, which is particularly These results, reproduced in a diabetic rat model remarkable under conditions of stress (see above). [Collino et al., 2011] and in endothelial cells in culture Moreover, the analyses of PPAR β/δ expression, [Jiang et al., 2009b], point to PPAR β/δ as a performed in various contexts, underscore its high remarkable target for preconditioning strategies. The expression in cancer cells. same protective properties are found in the heart, where in vivo activation of PPAR β/δ via agonist The discussion below will only present examples of treatment preserves the heart from contradictory results obtained in in vivo studies, as ischemia/reperfusion injury in Zucker fatty rats [Yue et numerous exhaustive reviews have been written on al., 2008], as well as in brain undergoing ischemia the subject [Michalik and Wahli, 2008; Muller- [Iwashita et al., 2007]. Finally, treatment with the Brusselbach et al., 2007; Muller et al., 2008; Peters et

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Table 2. PPAR β/δ microarray datasets. Compilation of the microarrays data available in the GEO (NCBI) and EBI array-express databases. Data are listed according to tissue/cell specificity. The main observations gained along each data analysis are reported. Data on PPARb microarray can be found in: GEO data sets: http://www.ncbi.nlm.nih.gov/gds/?term=ppar+beta; EBI ArrayExpress: http://www.ebi.ac.uk/arrayexpress/ +keyword

al., 2011]. Most of the in vivo studies evaluate the role size of polyps in intestine [Gupta et al., 2004] [Wang of PPAR β/δ in intestinal cancers and can be divided et al., 2006], whereas GW0742 administered to in two principal conditions: PPAR β/δ deficiency and PPAR β/δ activation. ApcMin/+ mice did not affect size or number of polyps [Marin et al., 2006]. In contrast, treatment with high In the context of ApcMin/+ genetic background, doses of GW0742 agonist in the AOM-induced model PPAR β/δ deletion enhances the number of colon of carcinogenesis leads to a significant decrease in polyps formed in the intestine [Harman et al., 2004; tumor incidence [Hollingshead et al., 2008] [Marin et Reed et al., 2004]. In the same manner, the treatment al., 2006]. of PPAR β/δ-deficient mice with a potent carcinogen (azoxymethane, AOM) causes an increased number A partial explanation for the controversial findings of polyps formed within this tissue [Hollingshead et reported in the in vivo studies may come from al., 2008; Marin et al., 2006], suggesting a protective PPAR β/δ activity in the tumor microenvironment. For effect of PPAR β/δ in tumorigenesis. However, other example, PPAR β/δ up-regulates VEGF expression in studies showed that PPAR β/δ disruption (total body epithelial cells [Piqueras et al., 2007; Wang et al., KO or intestinal-specific PPAR β/δ deletion), either 2006], a critical pro-angiogenic factor, also affecting combined with ApcMin/+ genetic background [Wang vascular permeability, and immune cell activity and et al., 2006] or with AOM treatment [Zuo et al., 2009], their response to tumor. An elegant demonstration led to reduction of polyp formation and to a marked comes from the study of Muller-Brusselbach et al. resistance against carcinogen action. Finally, two [Muller-Brusselbach et al., 2007], who showed that other reports stated that total body deletion of growth of syngeneic PPAR β/δ wild-type tumors is PPAR β/δ in ApcMin/+ genetic background or impaired in PPAR β/δ null mice, due to a deficit in intestinal-specific PPAR β/δ deletion combined with microvessel formation and maturation. This is also carcinogenic treatment did not result in any changes consistent with the correlation found in human tumors in colon tumor formation [Barak et al., 2002; Monk et between high levels of PPAR β/δ and cyclooxygenase al., 2012]. 2 expression on the one hand, and the microvascular

density and venous vessel tumor invasion on the Some contradictions were shown in the case of other hand [Yoshinaga et al., 2009], thereby PPAR β/δ activation via treatment of mice with confirming its role in affecting the tumor PPAR β/δ agonist. GW501516 administration in ApcMin/+ mice leads to an increased number and microenvironment.

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This brief description is by no means exhaustive, as et al., 2010], serving to confirm the role of PPAR β/δ numerous papers continue to feed the controversy, as a key regulator of oxidative metabolism. The global but fail to provide definitive reasons for such gene expression pattern in PPAR β/δ-depleted discrepancies. The controversies are not over [Peters pancreatic cells also highlighted its positive regulatory et al., 2008; Xu et al., 2013], and further studies need activity on a combination of genes involved in insulin to be done in order to establish a role for PPAR β/δ in secretion and its repressive role on genes involved in cancer, and in doing so to ensure the safety of pancreatic cell proliferation [Iglesias et al., 2012]. PPAR β/δ activation through synthetic agonists. Genetic reprogramming of oxidative metabolism genes is also involved in PPAR β/δ regulation of Genome-wide analyses of PPAR β/δ muscle response to physical stress and muscle transcriptional activities endurance. Activation of PPAR β/δ by ligand treatment or transgenic expression leads to a general Mouse models are crucial for gaining a systemic view upregulation of genes involved in fatty acid transport, of PPAR β/δ functions relevant to the full organism. To beta-oxidation, and mitochondrial respiration [Tanaka gain insights into the mechanisms by which the et al., 2003], which further increases when coupled transcription factor PPAR β/δ may act, whole genome with exercise [Narkar et al., 2008]. Microarray also studies allow a broad and unbiased view of the identified Lactate b dehydrogenase and AMPK as two primary targets it affects. of the main enzymes involved in the endurance program [Gan et al., 2011]. Moreover, the classical Microarray studies switch toward fiber type I observed in PPAR β/δ- activated genetic reprogramming goes through a Due to the fact that PPAR β/δ is ubiquitously distinct muscle microRNA (miRNA) network and the expressed and controls many different biological signaling of estrogen-related receptor gamma pathways, the compilation of microarray data, in (ERRgamma) [Gan et al., 2013]. different contexts, could be helpful in order to highlight some “core” functions and tissue- or cell-specific Another aspect of PPAR β/δ-mediated gene regulation activity on gene expression from a wider point of view. relates to this receptor’s role as a potent anti- Table 2 recapitulates the microarrays available in the inflammatory player [Iwaisako et al., 2012; Kaddatz et GEO (NCBI) and EBI array-express databases at the al., 2010; Mukundan et al., 2009; Stockert et al., following links: http://www.ncbi.nlm.nih.gov/gds and 2011; Stockert et al., 2013]. In fact, PPAR β/δ deletion http://www.ebi.ac.uk/arrayexpress/+keyword. The triggered a reduced expression of anti-inflammatory keywords PPAR beta, PPAR β/δ, PPARdelta and cytokine and opsonins in macrophages [Mukundan et PPAR beta/delta enable you to find all the available al., 2009]. As a mirror image, PPAR β/δ activated by microarray data. This search is likely not complete, ligand stimulation is able in fibroblasts to counteract but it provides a good handle on conducting a deeper TGF β-activated genes and to activate TGF β- search. Doing a rigorous meta-analysis across all repressed genes [Kaddatz et al., 2010; Stockert et al., data obtained in microarrays remains very 2011]. Importantly, different agonists can exert challenging, due to different initial conditions, different different activation of genetic regulation by PPAR β/δ. platforms used, and most importantly, to the For example in liver, KD3010 but not GW501516, experimental design aimed at answering a different attenuates the gene expression signature consistent and specific question for each study. The summary with inflammation and hepatocyte cell death, presented below only aims at presenting the main stimulates ROS protection pathways and down- observations and leads that these microarray studies regulates pro-fibrotic factor expression [Iwaisako et highlighted, albeit many of them have been previously al., 2012]. stressed (see section on Integrative Physiology). Genome-wide regulation of gene expression Firstly, microarray represents a powerful technique for mediated by PPAR β/δ in tumor development also reflects the controversies discussed above. In assessment of gene expression regulation in mammary epithelium, PPAR β/δ acts as an oncogene understanding the impact of PPAR β/δ affecting cell invasion, motility, but also inflammation- deletion/activation on lipid and glucose metabolism. related pathways [Yuan et al., 2013]. Actin The main observations were achieved studying liver, cytoskeleton and cell migration genes were adipose tissue, and pancreas comparing both deregulated after PPAR β/δ agonist treatment and PPAR β/δ wild type and KO mice, or agonist-treated ANGPLT4 has been highlighted as a central step in versus vehicle control mice expression profiles. In tumor cell invasion [Adhikary et al., 2012]. In contrast, particular, PPAR β/δ deletion caused suppression of activation of PPAR β/δ in keratinocytes seems to be the expression of several genes involved in glucose protective against tumor formation by controlling the and lipid catabolism [Mandard et al., 2007; Sanderson expression of members of the ubiquitin-proteasome

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degradation pathway [Kim et al., 2004] or by the relatively poor efficiency and specificity of NR repressing E2F target genes through the interaction monoclonal or polyclonal antibodies available with p130/p107 [Zhu et al., 2012]. These commercially. However, we present below first a discrepancies may be due to the fact that the analysis series of papers demonstrating PPAR β/δ interaction has been performed in isolated tissues/cells, thereby on known gene sequences, and second two papers losing the contribution of the paracrine activity exerted analyzing genome-wide binding sites of PPAR β/δ. by other cell types residing in the tumor environment (tumor stroma). ChIP has successfully elucidated PPAR β/δ interaction with the promoter of proteolipid protein (PLP) when Particular attention should be given to two microarray cells are treated with the fibrate Gemfibrosil [Jana et studies, performed in myofibroblasts [Adhikary et al., al., 2012]. PPRE sequences binding to PPAR β/δ were 2011] and in keratinocytes [Khozoie et al., 2012], also identified by ChIP in the promoter region of the where all the collected data comparing the absence calreticulin gene [Riahi et al., 2010]. Animal studies and presence of a PPAR β/δ activator have been demonstrated that oral administration of GSK3787 filtered by PPAR β/δ silenced or PPAR β/δ KO derived antagonizes the GW0742-induced PPAR β/δ promoter gene expression profiles, in cells treated with the occupancy of Angptl4 and ADRP genes. This same experimental conditions in order to exclude correlates with a reduced ADRP and Angptl4 mRNA non-specific signals. By combining microarray profile expression in WT but not in PPAR β/δ null mice colon, with ChIP-seq data, the authors described different which is consistent with the ChIP results [Palkar et al., types of PPAR β/δ-mediated gene regulation 2010]. The PPAR β/δ antibody used in this study was (summarized in the next section “ChIP and ChIP-seq developed by Girroir et al. [Girroir et al., 2008]. with PPAR β/δ antibody”), from repression in presence of ligand to activation in absence of ligand. ChIP experiments were also very useful in suggesting Interestingly, genes belonging to a given regulatory indirect mechanisms of PPAR β/δ-mediated pattern were directly associated with particular transcriptional regulation. Indeed, the enhanced functional pathways, thereby confirming the biological expression of the SIRT1 gene after PPAR β/δ ligand relevance of these findings. activation is not associated with PPAR β/δ binding to its 5’ flanking region, but is mediated instead by a In conclusion, microarray analysis of PPAR β/δ canonical Sp1 binding site. Consistently, this potent activities contributed to a better knowledge of the trans-activating effect of PPAR β/δ/GW501506 was molecular properties and biological functions of this completely abolished in the presence of Mithramycin, ubiquitously-expressed receptor, highlighting both an inhibitor of Sp1, suggesting that Sp1 could act as pan-tissue and tissue-specific genetic regulation. an ancillary factor for PPAR β/δ [Okazaki et al., 2010]. Nonetheless, a rigorous meta-analysis across all data Finally, in HCT116 colorectal carcinoma cells, VEGFA obtained in microarrays remains to be done. transcription is regulated by PPAR β/δ via β-catenin- mediated chromatin loops. The authors show that ChIP and ChIP-seq with PPAR β/δ antibodies chromatin loops around VEGFA are released upon PPAR β/δ activation. The model predicts that β- The analysis of the binding landscape of a given catenin mediates repressive looping and that transcription factor is an important approach currently PPAR β/δ-specific ligands release the loops, creating used to identify its possible target genes in the an active transcription unit [Hwang et al., 2012]. genome. However, binding of a TF in the vicinity of a gene is not sufficient, but only increases the In concert, this limited number of examples (this probability that this gene is regulated by this TF. presentation is not exhaustive) demonstrate that ChIP Therefore, combining the binding analysis above with of PPAR β/δ can be obtained in certain contexts. a gene expression analysis greatly facilitates Other contexts were not so successful and required, interpretation of the data. Irrespective of for example, the use of a V5- PPAR β/δ tagged protein transcriptional regulation, the whole genome binding [Yamamoto et al., 2011]. Recently, however, two landscape may also give some insights into general groups have succeeded in performing genome-wide DNA-binding properties. analyses of PPAR β/δ binding. Adhikary et al.,[Adhikary et al., 2011] performed genome-wide Chromatin Immunoprecipitation (ChIP) is a technique analyses of human myofibroblasts (WPMY-1 cell line), used to study the interaction between DNA and treated with or without agonist (GW501516), in control proteins within the cellular context, rather than using a cells and in PPAR β/δ-depleted cells, to identify synthetic piece of DNA in in vitro experiments such as PPAR β/δ-mediated gene regulation. ChIP-Seq the gel electro-mobility shift assay. It is clear that the analyses of PPAR β/δ, RNA POL II, and H3K4me3 quality (specificity and affinity) of the antibody is a have been performed, using for PPAR β/δ a Santa crucial determinant of the effectiveness of such an Cruz antibody. The sequencing data identified a total experiment. This is indeed a critical issue for of 4542 enriched peaks for PPAR β/δ, most of them PPAR β/δ, as well as for many other NRs, because of inside transcribed genomic regions or less than 25Kb

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Review PPAR β/δ: integrative and systemic approaches

upstream. Among the 4542 enriched peaks for depending on the type of regulation. More particularly, PPAR β/δ, the high confidence peak set defined as ATF4 is required for ligand-dependent induction of having a false discovery rate (FDR) <0.05 led to the PPAR β/δ target genes for some of the types of gene identification of 443 peaks. This high confidence peak regulation identified in the study [Khozoie et al., set was then filtered for H3K4m3 and RNA POL II 2012]. enrichment as markers for active and inducible proximal promoter. The identified enriched sites were In conclusion, these studies offer an important subsequently combined with transcriptional profiling glimpse into the complexity of PPAR β/δ-mediated derived from both agonist GW501516-treated or transcriptional regulation. Not only ligand stimulation, PPAR β/δ-silenced myofibroblasts. This additional but also the DNA binding of other transcription factors analysis enabled the definition of 112 bona fide influences the transcriptional activity of PPAR β/δ itself PPAR β/δ target genes classified according to three [Khozoie et al., 2012]. However, it would be of major distinct types of transcriptional response: ligand- interest to complete the picture with genome-wide independent repression by PPAR β/δ; ligand-induced analyses in different cellular and treatment contexts. activation and/or derepression by PPAR β/δ; and This would require having access to a fully reliable ligand-independent activation by PPAR β/δ. The antibody. Alternatively, developing a flag-tagged majority of enriched pathways were associated with knock-in PPAR β/δ mouse model might help to carbohydrate and lipid metabolism. However, the overcome the lack of a highly specific and efficient analysis highlighted PPAR β/δ involvement in non- PPAR β/δ antibody. metabolic functions including hematopoiesis and muscle/heart development. No analysis for the Human PPAR β/δ ( PPARD ) gene polymorphisms presence of other transcription factor sites coupled to PPAR β/δ genome-specific peaks was proposed. The search for human polymorphisms and their Sequencing data were deposited at EBI ArrayExpress consequences on human physiology might reveal (E-MTAB-371) [Adhikary et al., 2011]. important activities of PPAR β/δ in human. 90 single nucleotide polymorphisms have been identified and In 2012, a second study coupling the expression listed in the NCBI reference assembly, among which profiles with ChIP-seq allowed examination of 21 SNPs have been cited in the literature. We briefly PPAR β/δ-dependent regulation of gene expression in describe below a few polymorphisms that have been primary culture of mouse keratinocytes, [Khozoie et associated with a phenotype (see Table 3). It must be al., 2012]. The study was carried out on primary noted that none of the polymorphisms identified to keratinocytes derived from both PPAR β/δ wild type date change the amino acids sequence of the protein, and PPAR β/δ KO mice, in the presence and absence being either in untranslated regions, promoter of the selective agonist GW0742. An initial microarray sequences, intron sequences, or giving rise to a analysis identified a total of 612 target genes for synonymous codon. Some studies evaluated the PPAR β/δ. These target genes were then classified impact of such variations on PPAR β/δ expression according to the type of transcriptional regulation, levels, as mentioned in the table (Table 3), albeit no resulting in a total of eight different types of regulation further mechanistic studies have been done so far. ranging from repression to activation with or without However, we believe that at this present time of major ligand stimulation, plus combinations of these effects. expansion of individual genome analyses, GWAS analyses will be significantly empowered. ChIP-seq was subsequently performed to examine the molecular mechanism by which PPAR β/δ Skogsberg et al. [Skogsberg et al., 2003a] were the differentially regulates these target genes. The first to screen the proximal promoter and the 5'-UTR authors used the polyclonal antibody anti-PPAR β/δ for functional polymorphisms in the human gene for described by Girroir et al. [Girroir et al., 2008]. ChIP- use as tools in association studies. Four seq data revealed 6839 sites occupied by PPAR β/δ in polymorphisms were detected: -409C/T in the chromatin prepared from control cells, while more promoter region, +73C/T in exon 1, +255A/G in exon than twice as many sites were identified in chromatin 3, and T+294C (rs2016520) in exon 4. The authors prepared from agonist-treated cells, the specificity of found that only the rs2016520 showed significant which was controlled by comparing with chromatin association with a metabolic trait. Homozygotes for prepared from PPAR β/δ null mice. Only 203 out of the the rare C allele had a higher plasma LDL–cholesterol 612 PPAR β/δ-regulated genes displayed in the concentration than homozygotes for the common T microarray have been found to be occupied by allele. This association was verified in an independent PPAR β/δ at the chromatin level within 10Kb of the cohort of 282 healthy men. Transfection studies transcriptional start site. The search for DNA binding showed that the rare C allele is more efficiently motifs proximal to PPAR β/δ binding regions identified transcribed than the common T allele. Electrophoretic two main phylogenic families of transcription factors, mobility shift assays demonstrated that the T+294C the ETS and the CREB/ATF/AP1, associated with the polymorphism influenced binding of Sp-1. Using the PPAR β/δ binding site in various combinations West of Scotland Coronary Prevention Study

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Review PPAR β/δ: integrative and systemic approaches

(WOSCOPS), the authors found that individuals interaction between the three PPAR isotypes, Luo et carrying the rare PPAR β/δ T+294C allele had al. [Luo et al., 2012] found that in the group of 820 significantly lower high-density lipoprotein cholesterol Chinese subjects (non obese and obese) rs2016520 (HDL-C) concentration than subjects homozygous for (T+294C) is associated with lower obesity risk, in the common T-allele. Homozygous carriers of the C- contrast to the observations of Skogsberg et al. allele also showed a tendency towards higher risk of [Skogsberg et al., 2003a]. However, the authors Coronary Heart Disease (CHD) compared with pointed out that interactions among rs2016520, homozygous carriers of the T-allele. In addition, a rs9794 on PPAR β/δ and rs10865170 (PPAR γ) are gene–gene interaction involving T+294C associated with higher obesity risk. polymorphism and the PPAR α L162V polymorphism may influence the plasma LDL-C concentration Because of the link between cholesterol metabolism [Skogsberg et al., 2003b]. Finally, a recent study on and Alzheimer's disease (AD) pathogenesis, another PPAR β/δ polymorphism, the rs9794, Holzapfel et al. [Holzapfel et al., 2006] investigated suggested its association with hypertriglyceridemia three frequent polymorphisms located in exons 4 [Gu et al., 2013]. (rs2016520) and 9 (rs3734254 and rs9794) of PPARD for their putative influence on the risk of Alzheimer's In 2004, in an effort to identify polymorphic markers in disease and on cholesterol, 24S-hydroxycholesterol candidate genes for type 2 diabetes and associated and 27-hydroxycholesterol plasma levels. The study phenotypes, Shin et al. [Shin et al., 2004] identified population consisted of 167 AD patients and 194 nine polymorphisms in PPAR β/δ: four in the intron, controls. No single PPAR β/δ haplotypes influenced one in the 5' untranslated region (UTR), and four in the risk of AD. However, a two-marker haplotype the 3' UTR. Among identified polymorphisms, five consisting of rs2016520 and rs9794 in AD patients were common sites, namely c.-13454G>T, c.-87T>C, showed a significant effect on the relative plasma c.2022-12G>A, c.2629T>C and c.2806C>G. While the levels of 24S-hydroxycholesterol and 27- authors did not find significant associations with the hydroxycholesterol, but not in non-demented controls. risk of type 2 diabetes, several positive associations Their results suggest that PPAR β/δ haplotypes might of PPAR β/δ polymorphisms with fasting plasma influence levels of cholesterol metabolites in AD glucose and BMI were detected in non-diabetic patients, but not act as risk factors for AD. Indeed, a control subjects [Shin et al., 2004]. Intriguingly, in a Finnish study showed no association between study encompassing more than 11000 individuals, PPAR β/δ polymorphism and AD [Helisalmi et al., association of PPAR β/δ polymorphisms with BMI, HDL, leptin and TNF α was positive only in a gender- 2008]. dependent manner, for example significant association of rs2016520 and increased HDL As described above, PPAR β/δ plays an important role cholesterol in males, whereas HDL cholesterol tended in energy metabolism, particularly in muscle. Studies to be decreased in females [Burch et al., 2010]. were therefore designed to investigate whether SNPs Increased risk of diabetes was also observed in in the PPARD gene can modulate the effect of female but not male carriers of the C allele of exercise training on aerobic physical fitness and rs6902123 in a study including 769 subjects insulin sensitivity. Stefan et al., genotyped and participating in the STOP-NIDDM trial [Andrulionyte et described in detail SNPs rs1053049, rs6902123 and al., 2006]. Along this line, but independent of gender, rs2267668 in PPARD [Stefan et al., 2007]. After 9 the C allele of the T+294C polymorphism was months of lifestyle intervention (Tuebingen Lifestyle associated with higher fasting plasma glucose Intervention Program), the minor G allele of concentrations in both normoglycemic and diabetic rs2267668 in PPAR β/δ and the gly482-to-ser subjects in a study of 663 Shangaï men and women polymorphism in PGC1 α (PPAR gamma co-activator [Hu et al., 2006]. The same polymorphism (genotype 1alpha) were independently associated with less CC) is associated with increased risk of coronary increase in aerobic physical fitness, with a possible artery disease [Aberle et al., 2006; Nikitin et al., additive effect of these two SNPs. The interpretation 2010]. In other studies, association of the T+294C is that the minor alleles of the tested PPAR β/δ SNPs allele with the metabolic syndrome or with Type 2 would allow a lesser benefit from exercise and weight diabetes was at best suggested [Robitaille et al., loss than the major alleles, thereby providing a 2007] or denied [Villegas et al., 2011]. mechanistic explanation for the reduced aerobic physical fitness and insulin sensitivity of these A series of studies revealed the interaction between substances [Stefan et al., 2007]. These results are in polymorphisms. One in particular, is the interaction of line with those of Thamer et al. [Thamer et al., 2008], PPAR β/δ polymorphisms with the gly482-to-ser showing that SNPs rs1053049, rs6902123 and polymorphism in PGC1 α, shown to increase the risk rs2267668 in PPAR β/δ after lifestyle intervention for diabetes [Andrulionyte et al., 2006], or be induced changes in overall adiposity, hepatic fat associated with polycystic ovary syndrome [San- storage, and relative muscle mass. In contrast, no Millan and Escobar-Morreale, 2010]. Searching for association of PPAR β/δ polymorphism with

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Table 3. PPAR β/δ single nucleotide polymorphisms associated with disease. PPAR β/δ SNPs associated with diseases are listed according to SNPs Reference, Human Genome Variation Society (HGVS) nomenclature, chromosome and functional position. The target sequences are also reported together with the main observations on associated disease phenotype.

SNPs HGVS Names Chr 6 position Functional Sequence Associated manifestations References Reference position

rs1053049 *1762C>T 35395618(+) 3’ UTR CCCTA T/CGGGCG ↓ Loss of fat mass and hepac lipid aer Life [Thamer et al., Intervention 2008] Heterozygous minor allele carrier present [Ordelheide et ↑ mRNA expression of PPARβ/δ, associated al., 2011] with ↑ADIPOR1 expression. rs6902123 -102+15494T>C 35330421(+) Intron CCTGG T/CGCTGT ↑ Insulin Resistance and risk of diabetes; no [Andrulionyte et change in mRNA expression levels. al., 2006] ↑ Risk of diabetes in women [Cresci, 2008] ↑ Risk of diabetes in elderly Chinese [Lu et al., 2012] ↓ Loss of fat mass and hepac lipid aer Life [Thamer et al., Intervention 2008] rs2267668 -101-842G>A 35377922(+) Intron TATCT A/GAGAGT ↑ aerobic physical fitness and insulin [Stefan et al., sensitivity 2007] ↓ Loss of fat mass aer Life Intervenon [Thamer et al., 2008] ↑ mRNA expression of PPARβ/δ [Ordelheide et al., 2011] Not associated with higher longevity [Santiago et al., 2013] rs2016520 -87C>T or T+294C 35378778(-) 5’UTR ACAGG A/GTGGTT ↑ plasma LDL cholesterol ; the rare variant [Skogsberg et al., Exon 4 5’UTR confers higher PPARβ/δ expression 2003] Exonic splicing ↓ plasma cholesterol level in Alzheimer's [Holzapfel et al., enhancer disease but not in controls 2006] ↓ plasma HDL in women; ↑ Coronary heart [Aberle et al., disease 2006; Nikitin et al., 2010] ↑ plasma cholesterol [Gallicchio et al., 2008] ↑ and ↓ body mass index in women and men, [Burch et al., respectively. ↓ and ↑ HDL cholesterol in 2010] women and men, respectively. ↓ obesity risk in Chinese Han populaon [Luo et al., 2013] ↑ fasng insulin ↓insulin sensivity in [San-Millan& Polycystic ovary syndrome Escobar- Morreale, 2010] ↑ fasng plasma glucose in HGT or diabec [Hu et al., 2006] Chinese subject/ impaired insulin sensitivity ↓ stature adults & children; [Burch et al., 2009] Associated with elite-level endurance athletes [Eynon et al., 2009]

↑ risk of chronic kidney disease [Hishida et al., 2013] rs3734254 *1154C>T 35395010(+) 3’UTR CTGCC T/CCTCCA ↑ risk of diabetes; no change in mRNA levels [Andrulionyte et al., 2006] No significant association with plasma [Holzapfel et al., cholesterol levels in Alzheimer patients 2006] rs9794 *1939G>A or 35395795(+) 3’UTR GCCCC A/C/GACCCG ↓ plasma cholesterol in Alzheimer's disease [Holzapfel et al., *1939G>C Exon 9 patients 2006] Hypertriglyceridemia in Chinese Han [Gu et al., 2013] Population ↓ Obesity risk in Chinese Han Populaon [Luo et al., 2013] rs2267665 -101-9273A>G 35369491(+) Intron GTGCC G/ACACCT Significant association with susceptibility to [Zandi et al., Bipolar disorder using a family-based study 2008]

rs9462082 131-1863A>G 35386041(+) Intron CAGGC A/GTCCAC Significant association with susceptibility to [Zandi et al., Bipolar disorder using a family-based study 2008]

rs2076167 c.489C>T 35391787(-) Exon 6 CCCTC A/GTTTGC ↑obesity risk [Saez et al., -substitution 2008] N [Asn] ⇒ N [Asn] -Coding synonimous rs2076168 424+264G>A or 35389999(-) Intron GAATG A/C/TGTAAA ↓ PPARβ/δ mRNA expression of rare allele. [Nilsson et al., 424+264G>T PPARβ/δ basal level of expression correlates 2007] with slow and fast twitch markers expression.

rs2076169 285+421A>G 35388479(-) Intron AGATT T/CTGATG ↑ insulin sensivity [Grarup et al., 2007]

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performance of marathon athletes was found [Tsianos National de Recherche Suisse, the Etat de Vaud, and et al., 2010], while the interaction of rs2016520 and the European Initial Training Networks NR-NET. Gly482-to-Ser polymorphism in PGC1 α is suggested in elite level endurance athletes [Eynon et al., 2009]. References

In conclusion, some but not all studies indicate a Aberle, J., Hopfer, I., Beil, F. U.and Seedorf, U. significant association with either propensity for (2006). Association of the T+294C polymorphism in diabetes or obesity. These difficulties in pointing to a PPAR delta with low HDL cholesterol and coronary specific phenotype underline the role of PPAR β/δ as a heart disease risk in women. International journal of subtle regulator rather than a master key in metabolic medical sciences 3, 108-111. regulation. It is likely that the galloping development of the new generation sequencing technology will Adhikary, T., Brandt, D. T., Kaddatz, K., Stockert, J., provide a wealth of difficult to handle but extremely Naruhn, S., Meissner, W., Finkernagel, F., Obert, J., rich information, particularly in the context of gene- Lieber, S., Scharfe, M., et al. (2012). Inverse gene interaction studies. PPARbeta/delta agonists suppress oncogenic signaling to the ANGPTL4 gene and inhibit cancer cell Conclusion and future perspectives invasion. Oncogene, 32, 5241-52.

In the present review we have summarized different Adhikary, T., Kaddatz, K., Finkernagel, F., issues surrounding PPAR β/δ trying to sum up the Schonbauer, A., Meissner, W., Scharfe, M., Jarek, M., present knowledge in a systems biology-oriented Blocker, H., Muller-Brusselbach, S.and Muller, R. view. The different mouse models plus the ligand (2011). Genomewide analyses define different modes treatment strategies, microarray, and ChIP-seq of transcriptional regulation by peroxisome highlighted that PPAR β/δ plays its role in three main proliferator-activated receptor-beta/delta fields: i) regulation of energy metabolism, ii) cell (PPARbeta/delta). PloS one 6, e16344. proliferation and differentiation and iii) protection in stress conditions such as oxidative stress and Akiyama, T. E., Lambert, G., Nicol, C. J., Matsusue, inflammation. Whereas interest in developing new K., Peters, J. M., Brewer, H. B., Jr.and Gonzalez, F. J. PPAR β/δ-based therapeutics for obesity remain, this (2004). Peroxisome proliferator-activated receptor is impaired by its possible use as a doping substance. beta/delta regulates very low density lipoprotein In addition, a design for tissue-specific delivery of production and catabolism in mice on a Western diet. PPAR β/δ agonists would limit the systemic effects The Journal of biological chemistry 279, 20874- 20881. due to improper activation of PPAR β/δ in other compartments. A better understanding and Amri, E. Z., Bonino, F., Ailhaud, G., Abumrad, N. characterization of coregulators (coactivators and/or A.and Grimaldi, P. A. (1995). Cloning of a protein that corepressors) interacting with PPAR β/δ in different mediates transcriptional effects of fatty acids in cell types will be fundamental in this regard. However, preadipocytes. Homology to peroxisome proliferator- our knowledge about the PPAR β/δ interactome activated receptors. The Journal of biological remains poor because of the difficulties inherent in chemistry 270, 2367-2371. generating suitable tools, such as high performance antibodies or modified (e.g. epitope-tagged) Andrulionyte, L., Peltola, P., Chiasson, J. L.and PPAR β/δ. Laakso, M. (2006). Single nucleotide polymorphisms of PPARD in combination with the Gly482Ser In conclusion, while significant strides have been substitution of PGC-1A and the Pro12Ala substitution made in understanding PPAR β/δ function, a number of PPARG2 predict the conversion from impaired of questions still remain open. Further studies are glucose tolerance to type 2 diabetes: the STOP- needed in order to better characterize this receptor in NIDDM trial. Diabetes 55, 2148-2152. a more systemic manner, to strengthen the possibility that PPAR β/δ might be used as a therapeutic target in Barak, Y., Liao, D., He, W., Ong, E. S., Nelson, M. C., metabolic disorders, muscle endurance improvement Olefsky, J. M., Boland, R.and Evans, R. M. (2002). and inflammation control. Effects of peroxisome proliferator-activated receptor delta on placentation, adiposity, and colorectal Acknowledgements cancer. Proceedings of the National Academy of Sciences of the United States of America 99, 303- The authors are grateful to Yaakov Barak for his in- 308. depth reading and suggestions. We would also like to thank Federica Gilardi and all members of the BD Barak, Y., Sadovsky, Y.and Shalom-Barak, T. (2008). Laboratory for their critical reading. The laboratory of PPAR Signaling in Placental Development and Béatrice Desvergne is supported by the Fond Function. PPAR research 2008, 142082.

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Burdick, A. D., Bility, M. T., Girroir, E. E., Billin, A. N., Berger, J., Leibowitz, M. D., Doebber, T. W., Elbrecht, Willson, T. M., Gonzalez, F. J.and Peters, J. M. A., Zhang, B., Zhou, G., Biswas, C., Cullinan, C. A., (2007). Ligand activation of peroxisome proliferator- Hayes, N. S., Li, Y., et al. (1999). Novel peroxisome activated receptor-beta/delta(PPARbeta/delta) inhibits proliferator-activated receptor (PPAR) gamma and cell growth of human N/TERT-1 keratinocytes. PPARdelta ligands produce distinct biological effects. Cellular signalling 19, 1163-1171. The Journal of biological chemistry 274, 6718-6725.

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Roberts, L. D., Murray, A. J., Menassa, D., Ashmore, Schmidt, A., Endo, N., Rutledge, S. J., Vogel, R., T., Nicholls, A. W.and Griffin, J. L. (2011). The Shinar, D.and Rodan, G. A. (1992). Identification of a contrasting roles of PPARdelta and PPARgamma in new member of the steroid hormone receptor regulating the metabolic switch between oxidation and superfamily that is activated by a peroxisome storage of fats in white adipose tissue. Genome proliferator and fatty acids. Molecular endocrinology biology 12, R75. 6, 1634-1641.

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Review PPAR β/δ: integrative and systemic approaches

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