PPARß/δ-ANGPTL4 axis mediates the promotion of mono-2-ethylhexyl phthalic acid on MYCN-amplified neuroblastoma development.

Di-2-ethylhexyl phthalic acid (DEHP) is one of the most widely used plasticizers in the industry, which can improve the flexibility and durability of plastics. It is prone to migrate from various daily plastic products through wear and leaching into the surrounding environment and decompose into the more toxic metabolite mono-2-ethylhexyl phthalic acid (MEHP) after entering the human body. However, the impacts and mechanisms of MEHP on neuroblastoma are unclear. We exposed MYCN-amplified neuroblastoma SK-N-BE(2)C cells to an environmentally related concentration of MEHP and found that MEHP increased the proliferation and migration ability of tumor cells. The peroxisome proliferator-activated receptor (PPAR) ß/δ pathway was identified as a pivotal signaling pathway in neuroblastoma, mediating the effects of MEHP through transcriptional sequencing analysis. Because MEHP can bind to the PPARß/δ protein and initiate the expression of the downstream gene angiopoietin-like 4 (ANGPTL4), the PPARß/δ-specific agonist GW501516 and antagonist GSK3787, the recombinant human ANGPTL4 protein, and the knockdown of gene expression confirmed the regulation of the PPARß/δ-ANGPTL4 axis on the malignant phenotype of neuroblastoma. Based on the critical role of PPARß/δ and ANGPTL4 in the metabolic process, a non-targeted metabolomics analysis revealed that MEHP altered multiple metabolic pathways, particularly lipid metabolites involving fatty acyls, glycerophospholipids, and sterol lipids, which may also be potential factors promoting tumor progression. We have demonstrated for the first time that MEHP can target binding to PPARß/δ and affect the progression of neuroblastoma by activating the PPARß/δ-ANGPTL4 axis. This mechanism confirms the health risks of plasticizers as tumor promoters and provides new data support for targeted prevention and treatment of neuroblastoma.

Elafibranor upregulates the EMT-inducer S100A4 via PPARß/δ.

Elafibranor is a dual peroxisome proliferator-activated receptor (PPAR)α and ß/δ agonist that has reached a phase III clinical trial for the treatment of metabolic dysfunction-associated steatotic liver disease (MASLD). Here, we examined the effects of elafibranor in mice fed a choline-deficient high-fat diet (CD-HFD), a model of metabolic dysfunction-associated steatohepatitis (MASH) that presents obesity and insulin resistance. Our findings revealed that elafibranor treatment ameliorated steatosis, inflammation, and fibrogenesis in the livers of CD-HFD-fed mice. Unexpectedly, elafibranor also increased the levels of the epithelial-mesenchymal transition (EMT)-promoting protein S100A4 via PPARß/δ activation. The increase in S100A4 protein levels caused by elafibranor was accompanied by changes in the levels of markers associated with the EMT program. The S100A4 induction caused by elafibranor was confirmed in the BRL-3A rat liver cells and a mouse primary hepatocyte culture. Furthermore, elafibranor reduced the levels of ASB2, a protein that promotes S100A4 degradation, while ASB2 overexpression prevented the stimulating effect of elafibranor on S100A4. Collectively, these findings reveal an unexpected hepatic effect of elafibranor on increasing S100A4 and promoting the EMT program.

A novel PPARß/FFA1 dual agonist Y8 promotes diabetic wound healing.

BACKGROUND: Diabetes ulcer is one of the leading causes of disability and death in diabetics. Y8 [(2-(2-fluoro-4-((4-methyl-2-(4-(trifluoromethyl)phenyl)thiazol-5-yl)methoxy) phenoxy)acetic acid)], a dual agonist of peroxisome proliferation activated receptorß (PPARß) and free fatty acid receptor 1 (FFA1/FFAR1/GPR40), a new compound molecule with the potential for diabetes ulcer treatment. OBJECTIVE: To research the effect of the dual target agonist Y8 and its mechanism of action in the treatment of diabetic ulcers. METHODS: We have established a wound model in diabetic mice. After treatment with Y8, wound healing was evaluated by tissue pathology, reactive oxygen species (ROS) levels, and gene expression testing. Under high sugar conditions, the mechanism of Y8 affecting fibroblasts' proliferation and keratinocytes' migration is further studied. RESULTS: We found that Y8 accelerated wound healing and shortened healing time in diabetic mice. Granulation tissue generation and extracellular matrix (ECM) deposition were significantly increased in Y8-treated mice. Mechanistically, Y8 promotes keratinocyte proliferation by activating PPARß and migration of keratinocytes by triggering FFA1 in vitro. In addition, Y8 also decreased ROS levels in fibroblasts in vitro and in vivo by activating PPARß, reducing their release of superoxide anions. CONCLUSION: Our results suggest that PPARß/FFA1 dual agonist Y8 has the effect of promoting the healing of diabetic ulcer wounds in vivo and in vitro, and its therapeutic effect is better than that of single-target agonists.

Pharmacological activation of PPARß/δ preserves mitochondrial respiratory function in ischemia/reperfusion via stimulation of fatty acid oxidation-linked respiration and PGC-1α/NRF-1 signaling.

Myocardial ischemia/reperfusion (I/R) injury leads to significant impairment of cardiac function and remains the leading cause of morbidity and mortality worldwide. Activation of peroxisome proliferator-activated receptor ß/δ (PPARß/δ) confers cardioprotection via pleiotropic effects including antioxidant and anti-inflammatory actions; however, the underlying mechanisms are not yet fully elucidated. The aim of this study was to investigate the effect of PPARß/δ activation on myocardial mitochondrial respiratory function and link this effect with cardioprotection after ischemia/reperfusion (I/R). For this purpose, rats were treated with the PPARß/δ agonist GW0742 and/or antagonist GSK0660 in vivo. Mitochondrial respiration and ROS production rates were determined using high-resolution fluororespirometry. Activation of PPARß/δ did not alter mitochondrial respiratory function in the healthy heart, however, inhibition of PPARß/δ reduced fatty acid oxidation (FAO) and complex II-linked mitochondrial respiration and shifted the substrate dependence away from succinate-related energy production and towards NADH. Activation of PPARß/δ reduced mitochondrial stress during in vitro anoxia/reoxygenation. Furthermore, it preserved FAO-dependent mitochondrial respiration and lowered ROS production at oxidative phosphorylation (OXPHOS)-dependent state during ex vivo I/R. PPARß/δ activation was also followed by increased mRNA expression of components of FAO -linked respiration and of transcription factors governing mitochondrial homeostasis (carnitine palmitoyl transferase 1b and 2-CPT-1b and CPT-2, electron transfer flavoprotein dehydrogenase -ETFDH, peroxisome proliferator-activated receptor gamma co-activator 1 alpha- PGC-1α and nuclear respiratory factor 1-NRF-1). In conclusion, activation of PPARß/δ stimulated both FAO-linked respiration and PGC-1α/NRF -1 signaling and preserved mitochondrial respiratory function during I/R. These effects are associated with reduced infarct size.

PPARß/δ priming enhances the anti-apoptotic and therapeutic properties of mesenchymal stromal cells in myocardial ischemia-reperfusion injury.

BACKGROUND: Mesenchymal Stromal Cells (MSC) have been widely used for their therapeutic properties in many clinical applications including myocardial infarction. Despite promising preclinical results and evidences of safety and efficacy in phases I/ II, inconsistencies in phase III trials have been reported. In a previous study, we have shown using MSC derived from the bone marrow of PPARß/δ (Peroxisome proliferator-activated receptors ß/δ) knockout mice that the acute cardioprotective properties of MSC during the first hour of reperfusion are PPARß/δ-dependent but not related to the anti-inflammatory effect of MSC. However, the role of the modulation of PPARß/δ expression on MSC cardioprotective and anti-apoptotic properties has never been investigated. OBJECTIVES: The aim of this study was to investigate the role of PPARß/δ modulation (inhibition or activation) in MSC therapeutic properties in vitro and ex vivo in an experimental model of myocardial infarction. METHODS AND RESULTS: Naïve MSC and MSC pharmacologically activated or inhibited for PPARß/δ were challenged with H2O2. Through specific DNA fragmentation quantification and qRT-PCR experiments, we evidenced in vitro an increased resistance to oxidative stress in MSC pre-treated by the PPARß/δ agonist GW0742 versus naïve MSC. In addition, PPARß/δ-priming allowed to reveal the anti-apoptotic effect of MSC on cardiomyocytes and endothelial cells in vitro. When injected during reperfusion, in an ex vivo heart model of myocardial infarction, 3.75 × 105 PPARß/δ-primed MSC/heart provided the same cardioprotective efficiency than 7.5 × 105 naïve MSC, identified as the optimal dose in our experimental model. This enhanced short-term cardioprotective effect was associated with an increase in both anti-apoptotic effects and the number of MSC detected in the left ventricular wall at 1 h of reperfusion. By contrast, PPARß/δ inhibition in MSC before their administration in post-ischemic hearts during reperfusion decreased their cardioprotective effects. CONCLUSION: Altogether these results revealed that PPARß/δ-primed MSC exhibit an increased resistance to oxidative stress and enhanced anti-apoptotic properties on cardiac cells in vitro. PPARß/δ-priming appears as an innovative strategy to enhance the cardioprotective effects of MSC and to decrease the therapeutic injected doses. These results could be of major interest to improve MSC efficacy for the cardioprotection of injured myocardium in AMI patients.

Gene Doping with Peroxisome-Proliferator-Activated Receptor Beta/Delta Agonists Alters Immunity but Exercise Training Mitigates the Detection of Effects in Blood Samples.

Synthetic ligands of peroxisome-proliferator-activated receptor beta/delta (PPARß/δ) are being used as performance-enhancing drugs by athletes. Since we previously showed that PPARß/δ activation affects T cell biology, we wanted to investigate whether a specific blood T cell signature could be employed as a method to detect the use of PPARß/δ agonists. We analyzed in primary human T cells the in vitro effect of PPARß/δ activation on fatty acid oxidation (FAO) and on their differentiation into regulatory T cells (Tregs). Furthermore, we conducted studies in mice assigned to groups according to an 8-week exercise training program and/or a 6-week treatment with 3 mg/kg/day of GW0742, a PPARß/δ agonist, in order to (1) determine the immune impact of the treatment on secondary lymphoid organs and to (2) validate a blood signature. Our results show that PPARß/δ activation increases FAO potential in human and mouse T cells and mouse secondary lymphoid organs. This was accompanied by increased Treg polarization of human primary T cells. Moreover, Treg prevalence in mouse lymph nodes was increased when PPARß/δ activation was combined with exercise training. Lastly, PPARß/δ activation increased FAO potential in mouse blood T cells. Unfortunately, this signature was masked by training in mice. In conclusion, beyond the fact that it is unlikely that this signature could be used as a doping-control strategy, our results suggest that the use of PPARß/δ agonists could have potential detrimental immune effects that may not be detectable in blood samples.

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.

Cardioprotective Effects of PPARß/δ Activation against Ischemia/Reperfusion Injury in Rat Heart Are Associated with ALDH2 Upregulation, Amelioration of Oxidative Stress and Preservation of Mitochondrial Energy Production.

Accumulating evidence support the cardioprotective properties of the nuclear receptor peroxisome proliferator activated receptor ß/δ (PPARß/δ); however, the underlying mechanisms are not yet fully elucidated. The aim of the study was to further investigate the mechanisms underlying PPARß/δ-mediated cardioprotection in the setting of myocardial ischemia/reperfusion (I/R). For this purpose, rats were treated with PPARß/δ agonist GW0742 and/or antagonist GSK0660 in vivo and hearts were subjected to ex vivo global ischemia followed by reperfusion. PPARß/δ activation improved left ventricular developed pressure recovery, reduced infarct size (IS) and incidence of reperfusion-induced ventricular arrhythmias while it also up-regulated superoxide dismutase 2, catalase and uncoupling protein 3 resulting in attenuation of oxidative stress as evidenced by the reduction in 4-hydroxy-2-nonenal protein adducts and protein carbonyl formation. PPARß/δ activation also increased both mRNA expression and enzymatic activity of aldehyde dehydrogenase 2 (ALDH2); inhibition of ALDH2 abrogated the IS limiting effect of PPARß/δ activation. Furthermore, upregulation of PGC-1α and isocitrate dehydrogenase 2 mRNA expression, increased citrate synthase activity as well as mitochondrial ATP content indicated improvement in mitochondrial content and energy production. These data provide new mechanistic insight into the cardioprotective properties of PPARß/δ in I/R pointing to ALDH2 as a direct downstream target and suggesting that PPARß/δ activation alleviates myocardial I/R injury through coordinated stimulation of the antioxidant defense of the heart and preservation of mitochondrial function.

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.

Recent Insights on the Role of PPAR-ß/δ in Neuroinflammation and Neurodegeneration, and Its Potential Target for Therapy.

Peroxisome proliferator-activated receptor (PPAR) ß/δ belongs to the family of hormone and lipid-activated nuclear receptors, which are involved in metabolism of long-chain fatty acids, cholesterol, and sphingolipids. Similar to PPAR-α and PPAR-γ, PPAR-ß/δ also acts as a transcription factor activated by dietary lipids and endogenous ligands, such as long-chain saturated and polyunsaturated fatty acids, and selected lipid metabolic products, such as eicosanoids, leukotrienes, lipoxins, and hydroxyeicosatetraenoic acids. Together with other PPARs, PPAR-ß/δ displays transcriptional activity through interaction with retinoid X receptor (RXR). In general, PPARs have been shown to regulate cell differentiation, proliferation, and development and significantly modulate glucose, lipid metabolism, mitochondrial function, and biogenesis. PPAR-ß/δ appears to play a special role in inflammatory processes and due to its proangiogenic and anti-/pro-carcinogenic properties, this receptor has been considered as a therapeutic target for treating metabolic syndrome, dyslipidemia, carcinogenesis, and diabetes. Until now, most studies were carried out in the peripheral organs, and despite of its presence in brain cells and in different brain regions, its role in neurodegeneration and neuroinflammation remains poorly understood. This review is intended to describe recent insights on the impact of PPAR-ß/δ and its novel agonists on neuroinflammation and neurodegenerative disorders, including Alzheimer's and Parkinson's, Huntington's diseases, multiple sclerosis, stroke, and traumatic injury. An important goal is to obtain new insights to better understand the dietary and pharmacological regulations of PPAR-ß/δ and to find promising therapeutic strategies that could mitigate these neurological disorders.

Comparison of PPAR Ligands as Modulators of Resolution of Inflammation, via Their Influence on Cytokines and Oxylipins Release in Astrocytes.

Neuroinflammation is a key process of many neurodegenerative diseases and other brain disturbances, and astrocytes play an essential role in neuroinflammation. Therefore, the regulation of astrocyte responses for inflammatory stimuli, using small molecules, is a potential therapeutic strategy. We investigated the potency of peroxisome proliferator-activated receptor (PPAR) ligands to modulate the stimulating effect of lipopolysaccharide (LPS) in the primary rat astrocytes on (1) polyunsaturated fatty acid (PUFAs) derivative (oxylipins) synthesis; (2) cytokines TNFα and interleukin-10 (IL-10) release; (3) p38, JNK, ERK mitogen-activated protein kinase (MAPKs) phosphorylation. Astrocytes were exposed to LPS alone or in combination with the PPAR ligands: PPARα (fenofibrate, GW6471); PPARß (GW501516, GSK0660); PPARγ (rosiglitazone, GW9662). We detected 28 oxylipins with mass spectrometry (UPLC-MS/MS), classified according to their metabolic pathways: cyclooxygenase (COX), cytochrome P450 monooxygenases (CYP), lipoxygenase (LOX) and PUFAs: arachidonic (AA), docosahexaenoic (DHA), eicosapentaenoic (EPA). All tested PPAR ligands decrease COX-derived oxylipins; both PPARß ligands possessed the strongest effect. The PPARß agonist, GW501516 is a strong inducer of pro-resolution substances, derivatives of DHA: 4-HDoHE, 11-HDoHE, 17-HDoHE. All tested PPAR ligands decreased the release of the proinflammatory cytokine, TNFα. The PPARß agonist GW501516 and the PPARγ agonist, rosiglitazone induced the IL-10 release of the anti-inflammatory cytokine, IL-10; the cytokine index, (IL-10/TNFα) was more for GW501516. The PPARß ligands, GW501516 and GSK0660, are also the strongest inhibitors of LPS-induced phosphorylation of p38, JNK, ERK MAPKs. Overall, our data revealed that the PPARß ligands are a potential pro-resolution and anti-inflammatory drug for targeting glia-mediated neuroinflammation.

MHY2013 Modulates Age-related Inflammation and Insulin Resistance by Suppressing the Akt/FOXO1/IL-1ß Axis and MAPK-mediated NF-κB Signaling in Aged Rat Liver.

Chronic inflammation is a major risk factor underlying aging and age-associated diseases. It impairs normal lipid accumulation, adipose tissue function, and mitochondrial function, which eventually lead to insulin resistance. Peroxisome proliferator-activated receptors (PPARs) critically regulate gluconeogenesis, lipid metabolism, and the lipid absorption and breakdown process, and PPAR activity decreases in the liver during aging. In the present study, we investigated the ability of 2-(4-(5,6-methylenedioxybenzo[d]thiazol-2-yl)-2-methylphenoxy)-2-methylpropanoic acid (MHY2013), synthesized PPARα/PPARß/PPARγ pan agonist, to suppress the inflammatory response and attenuate insulin resistance in aged rat liver. Six- and 20-month-old rats were divided into 4 groups: young and old rats fed ad libitum; and old rats fed ad libitum supplemented with MHY2013 (1 mg and 5 mg/kg/d for 4 wk). We found that MHY2013 supplementation efficiently downregulated the activity of nuclear factor-κB through JNK/ERK/p38 mitogen-activated protein kinase signaling in the liver of aged rats. In addition, MHY2013 treatment increased hepatic insulin signaling, and the downstream signaling activity of FOXO1, which is negatively regulated by Akt. Downregulation of Akt increases expression of FOXO1, which acts as a transcription factor and increases transcription of interleukin-1ß, leading to hepatic inflammation. The major finding of this study is that MHY2013 acts as a therapeutic agent against age-related inflammation associated with insulin resistance by activating PPARα, PPARß, and PPARγ. Thus, the study provides evidence for the anti-inflammatory properties of MHY2013, and the role it plays in the regulation of age-related alterations in signal transduction pathways.

Peroxisome Proliferator-Activated Receptor Beta/Delta Agonist Suppresses Inflammation and Promotes Neovascularization.

The effects of peroxisome proliferator-activated receptor (PPAR)ß/δ ophthalmic solution were investigated in a rat corneal alkali burn model. After alkali injury, GW501516 (PPARß/δ agonist) or vehicle ophthalmic solution was topically instilled onto the rat's cornea twice a day until day 7. Pathological findings were evaluated, and real-time reverse transcription polymerase chain reaction was performed. GW501516 strongly suppressed infiltration of neutrophils and pan-macrophages, and reduced the mRNA expression of interleukin-6, interleukin-1ß, tumor necrosis factor alpha, and nuclear factor-kappa B. On the other hand, GW501516 promoted infiltration of M2 macrophages, infiltration of vascular endothelial cells associated with neovascularization in the wounded area, and expression of vascular endothelial growth factor A mRNA. However, 7-day administration of GW501516 did not promote neovascularization in uninjured normal corneas. Thus, the PPARß/δ ligand suppressed inflammation and promoted neovascularization in the corneal wound healing process. These results will help to elucidate the role of PPARß/δ in the field of ophthalmology.

Molecular and nanoscale evaluation of N-cadherin expression in invasive bladder cancer cells under control conditions or GW501516 exposure.

N-cadherin is a transmembrane glycoprotein expressed by mesenchymal origin cells and is located at the adherens junctions. It regulates also cell motility and contributes to cell signaling. In previous studies, we identified that its anomalous expression in bladder carcinoma was a tumor progression marker. A pharmacological approach to inhibit N-cadherin expression or to block its function could be relevant to prevent disease progression and metastasis development. The morphological exploration of T24 invasive bladder cancer cells by atomic force microscopy (AFM) revealed a spindle-like shape with fibrous structures. By engaging force spectroscopy with AFM tip functionalized with anti-E or anti-N-cadherin antibodies, results showed that T24 cells expressed only N-cadherin as also demonstrated by Western blotting and confocal microscopy. For the first time, we demonstrated by RTqPCR and Western blotting analyses that the peroxisome proliferator-activated receptor ß/δ (PPARß/δ) agonist GW501516 significantly decreased N-cadherin expression in T24 cells. Moreover, high non-cytotoxic doses of GW501516 inhibited confluent T24 cell wound healing closure. By using AFM, a more sensitive nanoanalytical method, we showed that the treatment modified the cellular morphology and diminished N-cadherin cell surface coverage through the decreasing of these adhesion molecule-mediated interaction forces. We observed a greater decrease of N-cadherin upon GW501516 exposure with AFM than that detected with molecular biology techniques. AFM was a complementary tool to biochemical techniques to perform measurements on living cells at the nanometer resolution level. Taken together, our data suggest that GW501516 could be an interesting therapeutic strategy to avoid bladder cancer cell spreading through N-cadherin decrease.

Human Endometrial Stromal Cell Differentiation is Stimulated by PPARß/δ Activation: New Targets for Infertility?

CONTEXT: Implantation is a reproductive bottleneck in women, regulated by fluctuations in ovarian steroid hormone concentrations. However, other nuclear receptor ligands are modifiers of endometrial differentiation leading to successful pregnancy. In the present study we analyzed the effects of peroxisome-proliferator-activated receptor ß/δ (PPARß/δ) activation on established cellular biomarkers of human endometrial differentiation (decidualization). OBJECTIVE: The objective of this work is to test the effects of PPARß/δ ligation on human endometrial cell differentiation. DESIGN: Isolated primary human endometrial stromal cells (ESCs) were treated with synthetic (GW0742) or natural (all trans-retinoic acid, RA) ligands of PPARß/δ, and also with receptor antagonists (GSK0660, PT-S58, and ST247) in the absence or presence of decidualizing hormones (10 nM estradiol, 100 nM progesterone, and 0.5 mM dibutyryl cAMP [3',5'-cyclic adenosine 5'-monophosphate]). In some cases interleukin (IL)-1ß was used as an inflammatory stimulus. Time course and dose-response relationships were evaluated to determine effects on panels of well characterized in vitro biomarkers of decidualization. RESULTS: PPARß/δ, along with estrogen receptor α (ERα) and PR-A and PR-B, were expressed in human endometrial tissue and isolated ESCs. GW0742 treatment enhanced hormone-mediated ESC decidualization in vitro as manifested by upregulation of prolactin, insulin-like growth factor-binding protein 1, IL-11, and vascular endothelial growth factor (VEGF) secretion and also increased expression of ERα, PR-A and PR-B, and connexin 43 (Cx43). RA treatment also increased VEGF, ERα, PR-A, and PR-B and an active, nonphosphorylated isoform of Cx43. IL-1ß and PPARß/δ antagonists inhibited biomarkers of endometrial differentiation. CONCLUSION: Ligands that activate PPARß/δ augment the in vitro expression of biomarkers of ESC decidualization. By contrast, PPARß/δ antagonists impaired decidualization markers. Drugs activating these receptors may have therapeutic benefits for embryonic implantation.

Context-dependent regulation of endothelial cell metabolism: differential effects of the PPARß/δ agonist GW0742 and VEGF-A.

Peroxisome proliferator activated receptor ß/δ (PPARß/δ) has pro-angiogenic functions, but whether PPARß/δ modulates endothelial cell metabolism to support the dynamic phenotype remains to be established. This study characterised the metabolic response of HUVEC to the PPARß/δ agonist, GW0742, and compared these effects with those induced by VEGF-A. In HUVEC monolayers, flux analysis revealed that VEGF-A promoted glycolysis at the expense of fatty acid oxidation (FAO), whereas GW0742 reduced both glycolysis and FAO. Only VEGF-A stimulated HUVEC migration and proliferation whereas both GW0742 and VEGF-A promoted tubulogenesis. Studies using inhibitors of PPARß/δ or sirtuin-1 showed that the tubulogenic effect of GW0742, but not VEGF-A, was PPARß/δ- and sirtuin-1-dependent. HUVEC were reliant on glycolysis and FAO, and inhibition of either pathway disrupted cell growth and proliferation. VEGF-A was a potent inducer of glycolysis in tubulogenic HUVEC, while FAO was maintained. In contrast, GW0742-induced tubulogenesis was associated with enhanced FAO and a modest increase in glycolysis. These novel data reveal a context-dependent regulation of endothelial metabolism by GW0742, where metabolic activity is reduced in monolayers but enhanced during tubulogenesis. These findings expand our understanding of PPARß/δ in the endothelium and support the targeting of PPARß/δ in regulating EC behaviour and boosting tissue maintenance and repair.

Peroxisome proliferator-activated receptor ß/δ and γ agonists differentially affect prostaglandin E2 and cytokine synthesis and nutrient transporter expression in porcine trophoblast cells during implantation.

Peroxisome proliferator-activated receptors (PPARs) belong to the nuclear receptor family of ligand-dependent transcription factors. PPARs have been shown to be important regulators of female reproductive functions, including conceptus development and placenta formation. This study examines the effect of PPARß/δ and PPARγ agonists and antagonists on (1) the synthesis of prostaglandin (PG) E2, interleukin (IL) 6, interferon (IFN) γ, and tumor necrosis factor (TNF) α and (2) the mRNA expression of genes encoding nutrient transporters and/or binding proteins in Day 15 conceptus trophoblast cells. The study also examines whether PPAR agonist-modulated IL6, IFNγ, and TNFα secretion is mediated via mitogen-activated protein kinase (MAPK) pathways. Trophoblast cells were exposed to L-165,041 (a PPARß/δ agonist) or rosiglitazone (a PPARγ agonist) in the presence or absence of GSK3787 (a PPARß/δ antagonist) or GW9662 (a PPARγ antagonist) or in the presence or absence of U0126 (a MAPK inhibitor). Rosiglitazone stimulated PGE synthase and IFNG mRNA expression in trophoblast cells and enhanced PGE2 concentrations in the incubation medium. Moreover, cells treated with rosiglitazone exhibited increased abundance of the solute carrier organic anion transporter family member 2A1 (SLCO2A1, a PG transporter) and of fatty acid binding protein (FABP) 5 transcripts. All these effects were abolished by the addition of GW9662, which indicates that the action of rosiglitazone is PPARγ-dependent in the studied cells. L-165,041 inhibited TNFα synthesis and decreased the mRNA expression of FABP3 and IL6 in trophoblast cells. However, this effect was not abolished by the addition of GSK3787 into the incubation medium, suggesting that L-165,041 action is independent of PPARß/δ. The inhibitory effect of L-165,041 on TNFα concentration and the stimulatory effect of rosiglitazone on IFNγ accumulation in the medium were not observed in the presence of the MAPK inhibitor, suggesting that the action of both agonists may be mediated by MAPKs. In conclusion, PPARß/δ and PPARγ agonists are differentially involved in the trophoblast expression of genes related to conceptus development and implantation in pigs. Furthermore, L-165,041 and rosiglitazone may have PPAR-dependent and -independent effects in conceptus trophoblast cells.

PPARß/δ Agonism Upregulates Forkhead Box A2 to Reduce Inflammation in C2C12 Myoblasts and in Skeletal Muscle.

Daily activities expose muscles to innumerable impacts, causing accumulated tissue damage and inflammation that impairs muscle recovery and function, yet the mechanism modulating the inflammatory response in muscles remains unclear. Our study suggests that Forkhead box A2 (FoxA2), a pioneer transcription factor, has a predominant role in the inflammatory response during skeletal muscle injury. FoxA2 expression in skeletal muscle is upregulated by fatty acids and peroxisome proliferator-activated receptors (PPARs) but is refractory to insulin and glucocorticoids. Using PPARß/δ agonist GW501516 upregulates FoxA2, which in turn, attenuates the production of proinflammatory cytokines and reduces the infiltration of CD45+ immune cells in two mouse models of muscle inflammation, systemic LPS and intramuscular injection of carrageenan, which mimic localized exercise-induced inflammation. This reduced local inflammatory response limits tissue damage and restores muscle tetanic contraction. In line with these results, a deficiency in either PPARß/δ or FoxA2 diminishes the action of the PPARß/δ agonist GW501516 to suppress an aggravated inflammatory response. Our study suggests that FoxA2 in skeletal muscle helps maintain homeostasis, acting as a gatekeeper to maintain key inflammation parameters at the desired level upon injury. Therefore, it is conceivable that certain myositis disorders or other forms of painful musculoskeletal diseases may benefit from approaches that increase FoxA2 activity in skeletal muscle.

GQ-130, a novel analogue of thiazolidinedione, improves obesity-induced metabolic alterations in rats: Evidence for the involvement of PPARß/δ pathway.

The present investigation aimed to characterize the effect of a short-time treatment with a new thiazolidinedione (TZD) derivative, GQ-130, on metabolic alterations in rats fed a high-fat diet (HFD). We investigated whether metabolic alterations induced by GQ-130 were mediated though a mechanism that involves PPARß/δ transactivation. Potential binding and transactivation of PPARα, PPARß/δ or PPARγ by GQ-130 were examined through cell transactivation, 8-anilino-1-naphthalenesulfonic acid (ANS) fluorescence quenching assays and thermal shift assay. For in vivo experiments, male 8-week-old Wistar rats were divided into three groups fed for 6 weeks with: (a) a standard rat chow (14% fat) (control group), (b) a HFD (57.8% fat) alone (HFD group), or (c) a HFD associated with an oral treatment with GQ-130 (10 mg/kg/d) during the last week (HFD-GQ group). In 293T cells, unlike rosiglitazone, GQ-130 did not cause significant transactivation of PPARγ but was able to activate PPARß/δ by 153.9 folds in comparison with control values (DMSO). Surprisingly, ANS fluorescence quenching assay reveals that GQ-130 does not bind directly to PPARß/δ binding site, a finding that was further corroborated by thermal shift assay which evaluates the thermal stability of PPARß/δ in the presence of GQ-130. Compared to the control group, rats of the HFD group showed obesity, increased systolic blood pressure (SBP), insulin resistance, impaired glucose intolerance, hyperglycaemia, and dyslipidaemia. GQ-130 treatment abolished the increased SBP and improved all metabolic dysfunctions observed in the HFD group. Oral treatment with GQ-130 was effective in improving HFD-induced metabolic alterations probably through a mechanism that involves PPARß/δ activation.

PPARα/ß Activation Alleviates Age-Associated Renal Fibrosis in Sprague Dawley Rats.

Age-associated renal fibrosis is commonly observed, with a decline in renal function during aging. Although peroxisome proliferator-activated receptors α/ß (PPARα/ß) activation has been shown to exert beneficial effects on age-associated renal changes, its effects on age-associated renal fibrosis have not been investigated yet. Here, we show that the PPARα/ß activator, MHY2013, can significantly alter lipid metabolism in renal tubule epithelial cells and attenuate renal fibrosis in aged Sprague Dawley (SD) rats. We found that MHY2013 significantly increased nuclear translocation and activity of PPARα/ß in NRK52E renal epithelial cells. Moreover, the enhanced PPARα/ß activity increased the expression of fatty acid oxidation-associated PPARα/ß target genes. In addition, transforming growth factor-ß (TGF-ß)- and oleic acid-induced lipid accumulation and fibrosis-associated gene expression were decreased in NRK52E cells by MHY2013 pretreatment. To evaluate the effects of MHY2013 on age-associated renal fibrosis, aged SD rates were orally administered MHY2013 (1 and 5 mg/kg) daily for 1 month. MHY2013 efficiently increased PPARα/ß activation and reduced renal lipid accumulation in aged SD rat kidneys. Furthermore, renal fibrosis was significantly decreased by MHY2013, indicating the importance of renal lipid metabolism in age-associated renal fibrosis. Taken together, our results suggest that activation of PPARα/ß signaling during aging prevents age-associated renal fibrosis.