ß-elemene blocks lipid-induced inflammatory pathways via PPARß activation in heart failure.

This study aims to investigate the effects of ß-elemene on a mouse model of heart failure (HF) and to elucidate the underlying mechanisms in vitro approaches. In this study, left anterior descending (LAD)-induced HF mouse model and oxygen-glucose deprivation/recovery (OGD/R)-induced H9C2 model were leveraged to assess the therapeutic effects of ß-elemene. Histological examination, western blot and quantitative real-time PCR analysis (RT-qPCR) and immunofluorescence staining was utilized to elucidate mechanism of ß-elemene in lipid-induced inflammation. Results showed that ß-elemene improved heart function in HF mice evidenced by the increase of cardiac ejection fraction (EF) and fractional shortening (FS) values. Furthermore, ß-elemene administration rescued ventricular dilation, lipid accumulation, and inflammatory infiltration in arginal areas of mice myocardial infarction. At transcription level, ß-elemene augmented the mRNA expression of fatty acid oxidation-associated genes, such as peroxisome proliferator-activated receptor-ß (PPARß). In vitro, treatment of ß-elemene increased carnitine palmitoyltransferase 1A (CPT1A) and sirtuin 3 (SIRT3). Hallmarks of inflammation including the nuclear translocation of nuclear factor κB (NF-κB) and the degradation of inhibitory κBα (IκBα) were significantly suppressed. Consistently, we observed down-regulation of interleukin-6 (IL-6) and pro-inflammatory cytokines (such as TNFα) in ß-elemene treated H9C2 cells. Finally, molecular docking model predicted an interaction between ß-elemene and PPARß protein. Furthermore, ß-elemene increased the expression of PPARß, which was validated by antagonist of PPARß and siRNA for PPARß.

Co-Incubation with PPARß/δ Agonists and Antagonists Modeled Using Computational Chemistry: Effect on LPS Induced Inflammatory Markers in Pulmonary Artery.

Peroxisome proliferator activated receptor beta/delta (PPARß/δ) is a nuclear receptor ubiquitously expressed in cells, whose signaling controls inflammation. There are large discrepancies in understanding the complex role of PPARß/δ in disease, having both anti- and pro-effects on inflammation. After ligand activation, PPARß/δ regulates genes by two different mechanisms; induction and transrepression, the effects of which are difficult to differentiate directly. We studied the PPARß/δ-regulation of lipopolysaccharide (LPS) induced inflammation (indicated by release of nitrite and IL-6) of rat pulmonary artery, using different combinations of agonists (GW0742 or L-165402) and antagonists (GSK3787 or GSK0660). LPS induced release of NO and IL-6 is not significantly reduced by incubation with PPARß/δ ligands (either agonist or antagonist), however, co-incubation with an agonist and antagonist significantly reduces LPS-induced nitrite production and Nos2 mRNA expression. In contrast, incubation with LPS and PPARß/δ agonists leads to a significant increase in Pdk-4 and Angptl-4 mRNA expression, which is significantly decreased in the presence of PPARß/δ antagonists. Docking using computational chemistry methods indicates that PPARß/δ agonists form polar bonds with His287, His413 and Tyr437, while antagonists are more promiscuous about which amino acids they bind to, although they are very prone to bind Thr252 and Asn307. Dual binding in the PPARß/δ binding pocket indicates the ligands retain similar binding energies, which suggests that co-incubation with both agonist and antagonist does not prevent the specific binding of each other to the large PPARß/δ binding pocket. To our knowledge, this is the first time that the possibility of binding two ligands simultaneously into the PPARß/δ binding pocket has been explored. Agonist binding followed by antagonist simultaneously switches the PPARß/δ mode of action from induction to transrepression, which is linked with an increase in Nos2 mRNA expression and nitrite production.

The Endocannabinoid System and PPARs: Focus on Their Signalling Crosstalk, Action and Transcriptional Regulation.

Peroxisome proliferator-activated receptors (PPARs) are a family of nuclear receptors including PPARα, PPARγ, and PPARß/δ, acting as transcription factors to regulate the expression of a plethora of target genes involved in metabolism, immune reaction, cell differentiation, and a variety of other cellular changes and adaptive responses. PPARs are activated by a large number of both endogenous and exogenous lipid molecules, including phyto- and endo-cannabinoids, as well as endocannabinoid-like compounds. In this view, they can be considered an extension of the endocannabinoid system. Besides being directly activated by cannabinoids, PPARs are also indirectly modulated by receptors and enzymes regulating the activity and metabolism of endocannabinoids, and, vice versa, the expression of these receptors and enzymes may be regulated by PPARs. In this review, we provide an overview of the crosstalk between cannabinoids and PPARs, and the importance of their reciprocal regulation and modulation by common ligands, including those belonging to the extended endocannabinoid system (or "endocannabinoidome") in the control of major physiological and pathophysiological functions.

Investigation of binding and activity of perfluoroalkyl substances to the human peroxisome proliferator-activated receptor ß/δ.

Previously, perfluoroalkyl substances (PFASs) have been found to be associated with many adverse effects mediated by the peroxisome proliferator-activated receptor α (PPARα) and PPARγ. Here, we found another subtype of the peroxisome proliferator-activated receptors (PPARs); the PPARß/δ mediated pathway might also be a potential adverse outcome pathway for PFASs. We investigated the direct binding and transcriptional activity of PFASs toward human PPARß/δ, and further revealed the structure-binding and structure-activity relationship between PFASs and PPARß/δ. The receptor binding experiment showed that their binding potency was dependent on the carbon chain length and the terminal functional group. For twelve perfluoroalkyl carboxylic acids (PFCAs), an inverted U-shaped relationship existed between the PPARß/δ binding potency and the carbon chain length, with perfluorododecanoc acid (C12) showing the highest binding potency. The three perfluoroalkane sulfonic acids (PFSAs) exhibited a stronger binding potency than their PFCA counterparts. The two fluorotelomer alcohols (FTOHs) showed no binding potency. In receptor transcriptional activity assays, they enhanced the PPARß/δ transcriptional activity. Their transcriptional activity was also related to the carbon chain length and the terminal functional group. Molecular docking analysis showed the PFASs fitted into the ligand binding pocket of PPARß/δ with a binding geometry similar to a fatty acid.

Mechanism of Action of Magnesium Lithospermate B against Aging and Obesity-Induced ER Stress, Insulin Resistance, and Inflammsome Formation in the Liver.

Magnesium lithospermate B (MLB) is the biologically active compound of the water-soluble fraction of Salvia miltiorrhiza. Magnesium lithospermate B exhibits various biological functions, including antidiabetic, neuroprotective, and antioxidant effects. However, its beneficial effects on insulin sensitivity and related signaling pathways in the liver need to be elucidated. Our previous study reported that MLB is a PPARß/δ agonist in fibroblasts. Because insulin-sensitizing and anti-inflammatory effects of PPARß/δ has been reported in the liver, we investigated whether MLB has a beneficial effect on insulin-, ER stress- and inflammasome-related signaling in the livers of aging and obese animal models. Western blotting and protein-ligand docking simulation showed that MLB activated PPARß/δ and improved glucose tolerance in the livers of aging and obese animal models. MLB supplementation ameliorated aging or obesity-induced disruption of insulin signaling in the liver. Consistently, aging and obesity-induced increase in the protein levels of a gluconeogenic phosphoenolpyruvate carboxykinase was decreased by MLB. When molecular signaling pathways related to insulin signaling were examined in the liver, MLB supplementation suppressed ER stress- and inflammasome-related signaling molecules induced by aging and obesity. These results suggest that MLB may improve insulin resistance in the liver at least partially by suppressing ER stress and inflammasome formation in aging and obese animal models.

Exposure to 2,4-dichlorophenoxyacetic acid induced PPARß-dependent disruption of glucose metabolism in HepG2 cells.

2,4-Dichlorophenoxyacetic acid is one of the most widely used herbicides. Its impact on health is increasingly attracting great attentions. This study aimed to investigate the effect of 2,4-dichlorophenoxyacetic acid on glucose metabolism in HepG2 cells and the underlying mechanism. After 24 h exposure to 2,4-dichlorophenoxyacetic acid, glycogen was measured by PAS staining and glucose by ELISA in HepG2 cells. The expression of genes involved in glucose metabolism was measured by real-time PCR, Western blotting, and immunofluorescence. HepG2 cells presented more extracellular glucose consumption and glycogen content after exposed to 2,4-dichlorophenoxyacetic acid. Expression of gluconeogenesis-related genes, FoxO1, and CREB is significantly elevated. Moreover, PPARß was up-regulated dose-dependently. SiRNA knockdown of PPARß completely rescued the increase of glycogen accumulation and glucose uptake, and the up-regulation of FOXO1 and CREB expression. Our findings propose novel mechanisms that 2,4-dichlorophenoxyacetic acid causes glucose metabolism dysfunction through PPARß in HepG2 cells.

Monitoring Solution Structures of Peroxisome Proliferator-Activated Receptor ß/δ upon Ligand Binding.

Peroxisome proliferator-activated receptors (PPARs) have been intensively studied as drug targets to treat type 2 diabetes, lipid disorders, and metabolic syndrome. This study is part of our ongoing efforts to map conformational changes in PPARs in solution by a combination of chemical cross-linking and mass spectrometry (MS). To our best knowledge, we performed the first studies addressing solution structures of full-length PPAR-ß/δ. We monitored the conformations of the ligand-binding domain (LBD) as well as full-length PPAR-ß/δ upon binding of two agonists. (Photo-) cross-linking relied on (i) a variety of externally introduced amine- and carboxyl-reactive linkers and (ii) the incorporation of the photo-reactive amino acid p-benzoylphenylalanine (Bpa) into PPAR-ß/δ by genetic engineering. The distances derived from cross-linking experiments allowed us to monitor conformational changes in PPAR-ß/δ upon ligand binding. The cross-linking/MS approach proved highly advantageous to study nuclear receptors, such as PPARs, and revealed the interplay between DBD (DNA-binding domain) and LDB in PPAR-ß/δ. Our results indicate the stabilization of a specific conformation through ligand binding in PPAR-ß/δ LBD as well as full-length PPAR-ß/δ. Moreover, our results suggest a close distance between the N- and C-terminal regions of full-length PPAR-ß/δ in the presence of GW1516. Chemical cross-linking/MS allowed us gaining detailed insights into conformational changes that are induced in PPARs when activating ligands are present. Thus, cross-linking/MS should be added to the arsenal of structural methods available for studying nuclear receptors.

PPARß/δ and lipid metabolism in the heart.

Cardiac lipid metabolism is the focus of attention due to its involvement in the development of cardiac disorders. Both a reduction and an increase in fatty acid utilization make the heart more prone to the development of lipotoxic cardiac dysfunction. The ligand-activated transcription factor peroxisome proliferator-activated receptor (PPAR)ß/δ modulates different aspects of cardiac fatty acid metabolism, and targeting this nuclear receptor can improve heart diseases caused by altered fatty acid metabolism. In addition, PPARß/δ regulates glucose metabolism, the cardiac levels of endogenous antioxidants, mitochondrial biogenesis, cardiomyocyte apoptosis, the insulin signaling pathway and lipid-induced myocardial inflammatory responses. As a result, PPARß/δ ligands can improve cardiac function and ameliorate the pathological progression of cardiac hypertrophy, heart failure, cardiac oxidative damage, ischemia-reperfusion injury, lipotoxic cardiac dysfunction and lipid-induced cardiac inflammation. Most of these findings have been observed in preclinical studies and it remains to be established to what extent these intriguing observations can be translated into clinical practice. This article is part of a Special Issue entitled: Heart Lipid Metabolism edited by G.D. Lopaschuk.

Identification and structural characterization of two peroxisome proliferator activated receptors and their transcriptional changes at different developmental stages and after feeding with different fatty acids.

Peroxisome proliferator activated receptors beta1 (PPARß1) and beta2 (PPARß2) were investigated in loach (Misgurnus anguillicaudatus). The PPARß1 and PPARß2 were widely distributed in loach tissues. Multiple alignments of deduced amino acid sequences revealed homologous characteristics of the two subtypes of PPARß with 88% identity. PPARß1 was markedly expressed in the liver, about 100-fold higher than liver PPARß2. The two subtypes in unfertilized ovum (UO) showed the highest transcriptions in early life stages, and there were great divergences in expression between unfertilized and fertilized stages. The regulation of PPARß1 and PPARß2 in response to dietary fatty acids was studied in liver of loach fed with diets containing fish oil (FO, rich in n-3 highly unsaturated fatty acid) or soybean oil (SO, rich in 18:2n-6) for 75days. Results showed that hepatic transcription of PPARß1 in the SO group was higher than in the FO group. However, PPARß2 expression was similar. The differences of molecular characterization, tissue expressions in early life stages, and transcriptional regulation by lipid resources indicated that PPARß1 and PPARß2 were functionally different. This is the first report of differential expression of PPARß1 and PPARß2 in various tissues and early life stages of loach are regulated by lipid resources. These results will stimulate further studies to better understand the functional characterization of PPARß1 and PPARß2.

Integrative and systemic approaches for evaluating PPARß/δ (PPARD) function.

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, ß/δ and γ) have been identified, among which PPARß/δ is the most difficult to functionally examine due to its tissue-specific diversity in cell fate determination, energy metabolism and housekeeping activities. PPARß/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 PPARß/δ-interacting corepressor or coactivator complexes and PPARß/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 PPARß/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 PPARß/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.

Activation of PPARß/δ protects pancreatic ß cells from palmitate-induced apoptosis by upregulating the expression of GLP-1 receptor.

We previously showed that activated peroxisome proliferator-activated receptor (PPAR)ß/δ can protect pancreatic ß cells against lipotoxic apoptosis. However, the molecular mechanism remained unclear. Glucagon-like peptide-1 receptor (GLP-1R) has been reported to exhibit a protective effect against lipotoxic apoptosis in pancreatic ß cells. In the present study, we aimed to investigate the underlying molecular mechanisms that PPARß/δ activation suppressed apoptosis and improved ß cell function impaired by fatty acids, focusing on contribution of GLP-1R. Isolated rat islets and rat insulin-secreting INS-1 cells were treated with the PPARß/δ agonist GW501516 (GW) in the presence or absence of palmitate (PA) and transfected with siRNA for PPARß/δ or treated with the PPARß/δ antagonist GSK0660. Apoptosis was assessed by DNA fragmentation, Hoechst 33342 staining and flow cytometry. GLP-1R expression in INS-1 cells and islets was assayed by immunoblotting, quantitative PCR (qPCR) and immunofluorescence staining. SREBP-1c, Caveolin-1, Akt, Bcl-2, Bcl-xl and caspase-3 expression was measured using immunoblotting and qPCR. Our results showed that PPARß/δ activation decreased apoptosis in ß cells and robustly stimulated GLP-1R expression under lipotoxic conditions. GW enhanced glucose-stimulated insulin secretion (GSIS) impaired by PA through stimulation of GLP-1R expression in ß cells. Moreover, SREBP-1c/Caveolin-1 signaling was involved in PPARß/δ-regulated GLP-1R expression. Finally, GW exerted anti-apoptotic effects via interfering with GLP-1R-dependent Akt/Bcl-2 and Bcl-xl/caspase-3 signaling pathways. Our study suggested that the anti-apoptotic action of GW may involve its transcriptional regulation of GLP-1R, and PPARß/δ activation may represent a new therapeutic method for protecting pancreatic ß cells from lipotoxicity.

Harnessing the benefits of PPARß/δ agonists.

Lipid mediators have complex effects on the cell; one of the key transcriptional factors that moderate proliferation and inflammatory effects is PPARß/δ. Following highly successful clinical trials using the PPARß/δ agonists GW501516 for treatment of diabetes, GSK announced that any further research would be discontinued due to preclinical trials in rodents which linked this drug to wide spread tumour development. In this review we outline the dual molecular functions of PPARß/δ and connect these to the diverse results from in vitro studies, and draw parallels with the outcomes of animal and human studies. The PPARß/δ agonists have a great potential in terms of therapy, and we hope to provide some insight into the reasons why such contrasting results have been published. The discussion presented here is important to the future development of PPARß/δ agonists for the clinic, and for a fuller understanding for their complex regulatory roles in the cell.

Ombuin-3-O-ß-D-glucopyranoside from Gynostemma pentaphyllum is a dual agonistic ligand of peroxisome proliferator-activated receptors α and δ/ß.

We demonstrated that ombuin-3-O-ß-D-glucopyranoside (ombuine), a flavonoid from Gynostemma pentaphyllum, is a dual agonist for peroxisome proliferator-activated receptors (PPARs) α and δ/ß. Using surface plasmon resonance (SPR), time-resolved fluorescence resonance energy transfer (FRET) analyses, and reporter gene assays, we showed that ombuine bound directly to PPARα and δ/ß but not to PPARγ or liver X receptors (LXRs). Cultured HepG2 hepatocytes stimulated with ombuine significantly reduced intracellular concentrations of triglyceride and cholesterol and downregulated the expression of lipogenic genes, including sterol regulatory element binding protein-1c (SREBP1c) and stearoyl-CoA desaturase-1 (SCD-1), with activation of PPARα and δ/ß. Activation of LXRs by ombuine was confirmed by reporter gene assays, however, SPR and cell-based FRET assays showed no direct binding of ombuine to either of the LXRs suggesting LXR activation by ombuine may be operated via PPARα stimulation. Ombuine-stimulated macrophages showed significantly induced transcription of ATP binding cassette cholesterol transporter A1 (ABCA1) and G1 (ABCG1), the key genes in reverse cholesterol transport, which led to reduced cellular cholesterol concentrations. These results suggest that ombuine is a dual PPAR ligand for PPARα and δ/ß with the ability to decrease lipid concentrations by reducing lipogenic gene expression in hepatocytes and inducing genes involved in cholesterol efflux in macrophages.

Scaffold-based pan-agonist design for the PPARα, PPARß and PPARγ receptors.

As important members of nuclear receptor superfamily, Peroxisome proliferator-activated receptors (PPAR) play essential roles in regulating cellular differentiation, development, metabolism, and tumorigenesis of higher organisms. The PPAR receptors have 3 identified subtypes: PPARα, PPARß and PPARγ, all of which have been treated as attractive targets for developing drugs to treat type 2 diabetes. Due to the undesirable side-effects, many PPAR agonists including PPARα/γ and PPARß/γ dual agonists are stopped by US FDA in the clinical trials. An alternative strategy is to design novel pan-agonist that can simultaneously activate PPARα, PPARß and PPARγ. Under such an idea, in the current study we adopted the core hopping algorithm and glide docking procedure to generate 7 novel compounds based on a typical PPAR pan-agonist LY465608. It was observed by the docking procedures and molecular dynamics simulations that the compounds generated by the core hopping and glide docking not only possessed the similar functions as the original LY465608 compound to activate PPARα, PPARß and PPARγ receptors, but also had more favorable conformation for binding to the PPAR receptors. The additional absorption, distribution, metabolism and excretion (ADME) predictions showed that the 7 compounds (especially Cpd#1) hold high potential to be novel lead compounds for the PPAR pan-agonist. Our findings can provide a new strategy or useful insights for designing the effective pan-agonists against the type 2 diabetes.

Deficiency of PPARbeta/delta in the epidermis results in defective cutaneous permeability barrier homeostasis and increased inflammation.

In cultured human keratinocytes or murine epidermis, peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) (NR1C2) activators (1) stimulate keratinocyte differentiation; (2) decrease keratinocyte proliferation; (3) accelerate permeability barrier repair; (4) increase epidermal lipid synthesis; and (5) reduce cutaneous inflammation. Since these results suggest that PPARbeta/delta could play an important role in cutaneous homeostasis, we assessed here the skin phenotype of mice deficient in PPARbeta/delta. Gross cutaneous abnormalities were not evident, and both stratum corneum (SC) skin hydration and surface pH were normal. However, the epidermis was thickened and proliferating cell nuclear antigen (PCNA) staining was increased, indicating increased cell proliferation. No change in apoptosis was observed but the expression of differentiation markers, such as filaggrin, involucrin, and loricrin, was slightly increased in PPARbeta/delta(-/-) mice. Although basal permeability barrier function was normal, PPARbeta/delta knockout (KO) mice show a significant delay in barrier recovery rates following acute barrier disruption by either acetone treatment or tape-stripping. Delayed barrier recovery correlated with decreased production and secretion of lamellar bodies (LBs), and with reduced numbers of extracellular lamellar membranes in the SC. Finally, PPARbeta/delta KO mice displayed increased inflammation in response to 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. Together, these results further demonstrate that PPARbeta/delta in the epidermis: (1) is required for permeability barrier homeostasis; (2) regulates keratinocyte proliferation; and (3) modulates cutaneous inflammation.

The oxidative stress mediator 4-hydroxynonenal is an intracellular agonist of the nuclear receptor peroxisome proliferator-activated receptor-beta/delta (PPARbeta/delta).

Liver insufficiency and damage are major causes of death and disease worldwide and may result from exposure to environmental toxicants, specific combinations or dosages of pharmaceuticals, and microbial metabolites. The generation of reactive intermediates, in particular 4-hydroxynonenal (4-HNE), is a common event in liver damage caused by a variety of hepatotoxic drugs and solvents. The peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that are involved in the transcriptional regulation of lipid metabolism as well as other biological functions. Importantly, we have observed that the PPARbeta/delta-/- mouse is more susceptible to chemically induced hepatotoxicity than its wild-type counterpart, and our objective in this study was to elucidate the mechanism(s) by which PPARbeta/delta confers protection to hepatocytes. We hypothesized that PPARbeta/delta plays a protective role by responding to toxic lipids and altering gene expression accordingly. In support, oxidized-VLDL and constituents including 13-S-hydroxyoctadecadienoic acid (13-S-HODE) and 4-HNE are PPARbeta/delta ligands. A structure-activity relationship was established where 4-HNE and 4-hydroperoxynonenal (4-HpNE) enhanced the activity of the PPARbeta/delta subtype while 4-hyroxyhexenal (4-HHE), 4-oxo-2-Nonenal (4-ONE), and trans-4,5-epoxy-2(E)-decenal did not activate this receptor. Increasing PPARbeta/delta activity with a synthetic agonist decreased sensitivity of hepatocytes to 4-HNE and other toxic agents, whereas inhibition of this receptor had the opposite result. Gene expression microarray analysis identified several important PPARbeta/delta-regulated detoxification enzymes involved in 4-HNE metabolism that are regulated at the transcript level. This research established 4-HNE as an endogenous modulator of PPARbeta/delta activity and raises the possibility that agonists of this nuclear receptor may be utilized to prevent or treat liver disease associated with oxidative damage.

Studies towards the conception of new selective PPARbeta/delta ligands.

In order to define new PPARbeta/delta ligands, SAR study on the selective PPARbeta/delta activator L-165,041 led to the identification of one key functional group for selective PPARbeta/delta activation. Furthermore, taking advantage of SAR studies done elsewhere on the most selective PPARbeta/delta ligand GW501516, the conception of new ligands showing good affinity for PPARbeta/delta is reported. Finally, synthesis and biological evaluation of pyridine analogues have shown the benefical effect of the pyridine ring on the PPARbeta/delta subtype selectivity.

Recombinant human PPAR-beta/delta ligand-binding domain is locked in an activated conformation by endogenous fatty acids.

High-resolution crystallographic structures of recombinant human peroxisome proliferator-activated receptor ligand-binding domain (isotype beta/delta) reveal a fatty acid in the binding site. Mass spectrometry confirmed the presence of C16:0, C16:1, C18:0 and C18:1 in a ratio of approximately 3:2:1:4 with 11, Z-octadecenoic acid (cis-vaccenic acid) identified as the predominant species. These are endogenous fatty acids acquired from the bacterial expression system, and serve to lock the ligand-binding domain into the activated conformation. A requirement for crystal growth, the additive n-heptyl-beta-d-glucopyranoside, binds near the activation function helix where recognition of co-activator proteins occurs. Our observations suggest potential physiological ligands for human PPAR-beta/delta and highlight that reported binding studies must be treated with caution unless endogenous fatty acids have been removed from the sample prior to analysis.