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.

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.

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.

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.

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.

Vascular PPARß/δ Promotes Tumor Angiogenesis and Progression.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors, which function as transcription factors. Among them, PPARß/δ is highly expressed in endothelial cells. Pharmacological activation with PPARß/δ agonists had been shown to increase their angiogenic properties. PPARß/δ has been suggested to be involved in the regulation of the angiogenic switch in tumor progression. However, until now, it is not clear to what extent the expression of PPARß/δ in tumor endothelium influences tumor progression and metastasis formation. We addressed this question using transgenic mice with an inducible conditional vascular-specific overexpression of PPARß/δ. Following specific over-expression of PPARß/δ in endothelial cells, we induced syngenic tumors. We observed an enhanced tumor growth, a higher vessel density, and enhanced metastasis formation in the tumors of animals with vessel-specific overexpression of PPARß/δ. In order to identify molecular downstream targets of PPARß/δ in the tumor endothelium, we sorted endothelial cells from the tumors and performed RNA sequencing. We identified platelet-derived growth factor receptor beta (Pdgfrb), platelet-derived growth factor subunit B (Pdgfb), and the tyrosinkinase KIT (c-Kit) as new PPARß/δ -dependent molecules. We show here that PPARß/δ activation, regardless of its action on different cancer cell types, leads to a higher tumor vascularization which favors tumor growth and metastasis formation.

PPARß/δ agonist GW501516 inhibits TNFα-induced repression of adiponectin and insulin receptor in 3T3-L1 adipocytes.

Previous reports have shown that PPARß/δ agonists ameliorate insulin resistance associated with type 2 diabetes mellitus (T2DM). To determine the role of PPARß/δ in tumor necrosis factor α (TNFα)-mediated insulin resistance, we investigated expression levels of adiponectin and insulin receptor (IR) in response to treatment with the PPARß/δ agonist GW501516 with or without TNFα, a proinflammatory cytokine, in differentiated 3T3-L1 adipocytes. GW501516 induced adipocyte differentiation and the expression of adiponectin in a dose-dependent manner in differentiated adipocytes. TNFα treatment reduced adiponectin expression at the end of differentiation. This effect was reversed by GW501516 co-treatment with TNFα. TNFα treatment decreased adipogenic marker genes such as PPARγ, aP2, resistin, and GLUT4, and GW501516 reversed the effects of TNFα. GW501516 treatment increased the expression of insulin receptor and inhibited TNFα-mediated repression of insulin receptor. Our results showed that GW501516 abrogated TNFα-induced insulin resistance. In summary, our study demonstrated that the PPARß/δ agonist, GW501516 reversed TNFα-induced decreases in adipocyte differentiation and adiponectin expression, and improved insulin sensitivity by increasing the expression of insulin receptor. Therefore, PPARδ may be a promising therapeutic target for treatment of insulin resistance in patients with T2DM.

Activation of Peroxisome Proliferator-Activated Receptors α and δ Synergizes with Inflammatory Signals to Enhance Adoptive Cell Therapy.

Memory CD8+ T cells (Tmem) are superior mediators of adoptive cell therapy (ACT) compared with effector CD8+ T cells (Teff) due to increased persistence in vivo. Underpinning Tmem survival is a shift in cellular metabolism away from aerobic glycolysis towards fatty acid oxidation (FAO). Here we investigated the impact of the peroxisome proliferator-activated receptor (PPAR) agonist GW501516 (GW), an agent known to boost FAO in other tissues, on CD8+ T-cell metabolism, function, and efficacy in a murine ACT model. Via activation of both PPARα and PPARδ/ß, GW treatment increased expression of carnitine palmitoyl transferase 1a, the rate-limiting enzyme of FAO, in activated CD8+ T cells. Using a metabolomics approach, we demonstrated that GW increased the abundance of multiple different acylcarnitines, consistent with enhanced FAO. T cells activated in the presence of GW and inflammatory signals, either mature dendritic cells or IL12, also demonstrated enhanced production of IFNγ and expression of T-bet. Despite high expression of T-bet, a characteristic of short-lived effector cells, GW-treated cells demonstrated enhanced persistence in vivo and superior efficacy in a model of ACT. Collectively, these data identify combined PPARα and PPARδ/ß agonists as attractive candidates for further studies and rapid translation into clinical trials of ACT. SIGNIFICANCE: Dual activation of peroxisome proliferator-activated receptors α and δ improves the efficacy of adoptive cell therapy by reprogramming T-cell metabolism and cytokine expression.

Retinoic Acid Increases Fatty Acid Oxidation and Irisin Expression in Skeletal Muscle Cells and Impacts Irisin In Vivo.

BACKGROUND/AIMS: All-trans retinoic acid (ATRA) has protective effects against obesity and metabolic syndrome. We here aimed to gain further insight into the interaction of ATRA with skeletal muscle metabolism and secretory activity as important players in metabolic health. METHODS: Cultured murine C2C12 myocytes were used to study direct effects of ATRA on cellular fatty acid oxidation (FAO) rate (using radioactively-labelled palmitate), glucose uptake (using radioactively-labelled 2-deoxy-D-glucose), triacylglycerol levels (by an enzymatic method), and the expression of genes related to FAO and glucose utilization (by RT-real time PCR). We also studied selected myokine production (using ELISA and immunohistochemistry) in ATRA-treated myocytes and intact mice. RESULTS: Exposure of C2C12 myocytes to ATRA led to increased fatty acid consumption and decreased cellular triacylglycerol levels without affecting glucose uptake, and induced the expression of the myokine irisin at the mRNA and secreted protein level in a dose-response manner. ATRA stimulatory effects on FAO-related genes and the Fndc5 gene (encoding irisin) were reproduced by agonists of peroxisome proliferator-activated receptor ß/δ and retinoid X receptors, but not of retinoic acid receptors, and were partially blocked by an AMP-dependent protein kinase inhibitor. Circulating irisin levels were increased by 5-fold in ATRA-treated mice, linked to increased Fndc5 transcription in liver and adipose tissues, rather than skeletal muscle. Immunohistochemistry analysis of FNDC5 suggested that ATRA treatment enhances the release of FNDC5/irisin from skeletal muscle and the liver and its accumulation in interscapular brown and inguinal white adipose depots. CONCLUSION: These results provide new mechanistic insights on how ATRA globally stimulates FAO and enhances irisin secretion, thereby contributing to leaning effects and improved metabolic status.

Baicalin Attenuates Cardiac Dysfunction and Myocardial Remodeling in a Chronic Pressure-Overload Mice Model.

BACKGROUND/AIMS: Baicalin has been shown to be effective for various animal models of cardiovascular diseases, such as pulmonary hypertension, atherosclerosis and myocardial ischaemic injury. However, whether baicalin plays a role in cardiac hypertrophy remains unknown. Here we investigated the protective effects of baicalin on cardiac hypertrophy induced by pressure overload and explored the potential mechanisms involved. METHODS: C57BL/6J-mice were treated with baicalin or vehicle following transverse aortic constriction or Sham surgery for up to 8 weeks, and at different time points, cardiac function and heart size measurement and histological and biochemical examination were performed. RESULTS: Mice under pressure overload exhibited cardiac dysfunction, high mortality, myocardial hypertrophy, increased apoptosis and fibrosis markers, and suppressed cardiac expression of PPARα and PPARß/δ. However, oral administration of baicalin improved cardiac dysfunction, decreased mortality, and attenuated histological and biochemical changes described above. These protective effects of baicalin were associated with reduced heart and cardiomyocyte size, lower fetal genes expression, attenuated cardiac fibrosis, lower expression of profibrotic markers, and decreased apoptosis signals in heart tissue. Moreover, we found that baicalin induced PPARα and PPARß/δ expression in vivo and in vitro. Subsequent experiments demonstrated that long-term baicalin treatment presented no obvious cardiac lipotoxicity. CONCLUSIONS: The present results demonstrated that baicalin attenuates pressure overload induced cardiac dysfunction and ventricular remodeling, which would be due to suppressed cardiac hypertrophy, fibrosis, apoptosis and metabolic abnormality.

Effect of PPAR-ß/δ agonist GW0742 treatment in the acute phase response and blood-brain barrier permeability following brain injury.

The systemic response to ischemic stroke is associated with the hepatic acute phase response (APR) that modulates leukocytes recruitment to the injured brain. The inappropriate recruitment of leukocytes to the brain parenchyma can result in blood-brain barrier (BBB) breakdown. Emerging data suggest that peroxisome proliferator-activated receptor beta/delta (PPAR-ß/δ) activation has a potential neuroprotective role in ischemic stroke. However, mechanisms of PPAR-ß/δ mediated protection in ischemic insults remain unclear. In the present study, we determined for the first time, the effects of GW0742, a PPAR-ß/δ agonist on the APR following brain injury and assessed the effects on BBB permeability and tight junction integrity via claudin-5, occludin, and zona occludens-1 expression. C57/BL6 mice were exposed to 1 hour of ischemia and received 10 minutes before reperfusion either a vehicle solution or GW0742. Hepatic expression of chemokines (C-X-C motif ligand: CXCL1, CXCL2, and CXCL10), serum amyloid A-1, tumor necrosis factor alpha, interleukin-1ß, and interleukin-6 was measured, and the extent of brain and hepatic neutrophil infiltration was determined. The results showed that GW0742 treatment decreased infarct volume and edema, reactant production and neutrophil recruitment to the brain and liver, which is a hallmark of the APR. GW0742 significantly reduced BBB leakage and metalloproteinase 9 expression and upregulated the expression of tight junction proteins. These findings may help to guide the experimental and clinical therapeutic use of PPAR-ß/δ agonists against brain injury.

Apoptotic effect of the selective PPARß/δ agonist GW501516 in invasive bladder cancer cells.

GW501516 is a selective and high-affinity synthetic agonist of peroxisome proliferator-activated receptor ß/δ (PPARß/δ). This molecule promoted the inhibition of proliferation and apoptosis in few cancer cell lines, but its anticancer action has never been investigated in bladder tumor cells. Thus, this study was undertaken to determine whether GW501516 had antiproliferative and/or apoptotic effects on RT4 and T24 urothelial cancer cells and to explore the molecular mechanisms involved. Our results indicated that, in RT4 cells (derived from a low-grade papillary tumor), GW501516 did not induce cell death. On the other hand, in T24 cells (derived from an undifferentiated high-grade carcinoma), this PPARß/δ agonist induced cytotoxic effects including cell morphological changes, a decrease of cell viability, a G2/M cell cycle arrest, and the cell death as evidenced by the increase of the sub-G1 cell population. Furthermore, GW501516 triggered T24 cell apoptosis in a caspase-dependent manner including both extrinsic and intrinsic apoptotic pathways through Bid cleavage. In addition, the drug led to an increase of the Bax/Bcl-2 ratio, a mitochondrial dysfunction associated with the dissipation of ΔΨm, and the release of cytochrome c from the mitochondria to the cytosol. GW501516 induced also ROS generation which was not responsible for T24 cell death since NAC did not rescue cells upon PPARß/δ agonist exposure. For the first time, our data highlight the capacity of GW501516 to induce apoptosis in invasive bladder cancer cells. This molecule could be relevant as a therapeutic drug for high-grade urothelial cancers.

Role of UCP2 in the protective effects of PPARß/δ activation on lipopolysaccharide-induced endothelial dysfunction.

Bacterial endotoxin lipopolysaccharide (LPS) activates inflammatory pathways, induces cytokine expression in the endothelium, augments reactive oxygen species (ROS) production in the vascular wall, and induces endothelial dysfunction. The aim of the present study was to analyze the effects of peroxisome proliferator-activated receptor (PPAR)ß/δ activation on LPS-induced inflammation, oxidative stress and endothelial dysfunction and to determine whether uncoupling protein-2 (UCP2) plays a role in these effects. In vivo, the PPARß/δ agonist GW0742 treatment prevented the LPS-induced reduction in aortic relaxation, the increase in vascular ROS production, the upregulation of NOX1, NOX2, p47(phox), and p22(phox) mRNA levels, and the endoplasmic reticulum (ER) stress markers in mice. We show that in mouse aortic endothelial cells (MAECs), GW0742 prevented the decreased A23187-stimulated nitric oxide (NO) production, and the increased intracellular ROS levels caused by exposure to LPS in vitro. The PPARß/δ antagonist GSK0660 abolished all these in vivo and in vitro protective effects induced by GW0742. This agonist also restored the reduced expression of UCP2 and mitofusin-2 induced by LPS. The effects of GW0742 on NO and ROS production in MAEC exposed to LPS were abolished by the UCP2 inhibitor genipin or by siRNA targeting UCP-2. Genipin also suppressed the expressional changes on NADPH oxidase and ER stress markers induced by GW0742. In conclusion, PPARß/δ activation restored the LPS-induced endothelial dysfunction by upregulation of UCP2, with the subsequent alleviation of ER stress and NADPH oxidase activity, thus reducing intracellular ROS production and increasing NO bioavailability.

Picrasidine N Is a Subtype-Selective PPARß/δ Agonist.

Recently, growing evidence of the pivotal roles of peroxisome proliferator-activated receptor (PPAR) ß/δ in various physiological functions, including lipid homeostasis, cancer, and inflammation, has raised interest in this receptor. In this study, the naturally occurring dimeric alkaloid picrasidine N (1) from Picrasma quassioides was identified as a novel PPARß/δ agonist from a library consisting of plant extracts and natural compounds using a mammalian one-hybrid assay, and this compound was characterized. Compound 1 activated PPARß/δ but did not activate or slightly activated PPARα and PPARγ. Furthermore, a peroxisome proliferator response element-driven luciferase reporter gene assay demonstrated that 1 enhanced PPARß/δ transcriptional activity. Moreover, 1 selectively induced mRNA expression of ANGPTL4, which is a PPAR target gene. This observation is quite different from previously identified synthetic PPARß/δ agonists, which can induce the expression of not only ANGPTL4 but also other PPAR target genes, such as ADRP, PDK4, and CPT-1. These results demonstrate that 1 is a potent subtype-selective and gene-selective PPARß/δ agonist, suggesting its potential as a lead compound for further drug development. This compound would also be a useful chemical tool for elucidating the mechanism of PPARß/δ-regulated specific gene expression and the biological significance of PPARß/δ.

PPAR-pan activation induces hepatic oxidative stress and lipidomic remodelling.

The peroxisome proliferator-activated receptors (PPARs) are ligand activated nuclear receptors that regulate cellular homoeostasis and metabolism. PPARs control the expression of genes involved in fatty-acid and lipid metabolism. Despite evidence showing beneficial effects of their activation in the treatment of metabolic diseases, particularly dyslipidaemias and type 2 diabetes, PPAR agonists have also been associated with a variety of side effects and adverse pathological changes. Agonists have been developed that simultaneously activate the three PPAR receptors (PPARα, γ and δ) in the hope that the beneficial effects can be harnessed while avoiding some of the negative side effects. In this study, the hepatic effects of a discontinued PPAR-pan agonist (a triple agonist of PPAR-α, -γ, and -δ), was investigated after dietary treatment of male Sprague-Dawley (SD) rats. The agonist induced liver enlargement in conjunction with metabolomic and lipidomic remodelling. Increased concentrations of several metabolites related to processes of oxidation, such as oxo-methionine, methyl-cytosine and adenosyl-methionine indicated increased stress and immune status. These changes are reflected in lipidomic changes, and increased energy demands as determined by free fatty acid (decreased 18:3 n-3, 20:5 n-3 and increased ratios of n-6/n-3 fatty acids) triacylglycerol, phospholipid (decreased and increased bulk changes respectively) and eicosanoid content (increases in PGB2 and 15-deoxy PGJ2). We conclude that the investigated PPAR agonist, GW625019, induces liver enlargement, accompanied by lipidomic remodelling, oxidative stress and increases in several pro-inflammatory eicosanoids. This suggests that such pathways should be monitored in the drug development process and also outline how PPAR agonists induce liver proliferation.