Dimethyl itaconate attenuates palmitate-induced insulin resistance in skeletal muscle cells through the AMPK/FGF21/PPARδ-mediated suppression of inflammation.

AIM: Itaconate (ITA), a derivative of the tricarboxylic acid cycle, has been documented to have a direct antimicrobial effect by inhibiting isocitrate lyase and suppressing proinflammatory cytokines in LPS-treated macrophages. However, the effects of dimethyl ITA (DITA), a membrane-permeable derivative of ITA, on insulin signaling and inflammation in skeletal muscle in an obese state remain to be elucidated. Thus, this study was designed to investigate the effects of DITA on the impairment of insulin signaling and inflammation in palmitate-treated C2C12 myocytes. MATERIALS AND METHODS: Western blotting was used to determine the expression of insulin signaling associated genes, inflammatory markers, fibroblast growth factor 21 (FGF21), and PPARδ expression, as well as AMPK phosphorylation in mouse skeletal muscle cells. Secreted proinflammatory cytokine levels were detected by enzyme-linked immunosorbent assay. Insulin signaling was assessed by glucose uptake assay. KEY FINDINGS: Treating C2C12 myocytes with DITA attenuated palmitate-induced aggravation of insulin signaling markers, such as insulin receptor substrate-1 (IRS-1) and Akt phosphorylation and inflammatory markers, such as NFκB and IκB phosphorylation. AMPK phosphorylation, as well as PPARδ and myokine FGF21 expression, were enhanced in C2C12 myocytes by DITA treatment. siRNA-mediated suppression of AMPK or FGF21 expression abolished the effects of DITA on insulin resistance and inflammation in palmitate-treated C2C12 myocytes. SIGNIFICANCE: In sum, DITA suppresses inflammation through the AMPK/FGF21/PPARδ signaling, thereby alleviating insulin resistance in palmitate-treated C2C12 myocytes. The current study appears to be an essential basis for performing animal experiments to develop insulin resistance therapeutics.

Exploration of novel candidate genes involved in epidermal keratinocyte differentiation and skin barrier repair in man.

A proper skin barrier function requires constant formation of stratum corneum, i.e. the outermost layer of epidermis composed of terminally differentiated keratinocytes. The complex process of converting proliferative basal keratinocytes into corneocytes relies on programmed changes in the activity of many well-established genes. Much remains however to be investigated about this process, e.g. in conjunction with epidermal barrier defects due to genetic errors as in ichthyosis. To this end, we re-analyzed two sets of microarray-data comparing altered gene expression in differentiated vs. proliferating keratinocytes and in the skin of patients with autosomal recessive congenital ichthyosis (ARCI) vs. healthy controls, respectively. We thus identified 24 genes to be upregulated in both sets of array and not previously associated with keratinocyte differentiation. For 10 of these genes (AKR1B10, BLNK, ENDOU, GCNT4, GLTP, RHCG, SLC15A1, TMEM45B, TMEM86A and VSNL1), qPCR analysis confirmed the array results and subsequent immunostainings of normal epidermis showed superficial expression of several of the proteins. Furthermore, induction of keratinocyte differentiation using phorbol esters (PMA) resulted in increased expression of eight of the genes, whereas siRNA silencing of PPARδ, a transcription factor supporting differentiation, had the opposite effect. In summary, our results identify ten new candidate genes seemingly involved in human epidermal keratinocyte differentiation and possibly important for epidermal repair in a genetic skin disease characterized by barrier failure.

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 nutrient sensing pathways FoxO1/3 and mTOR in the heart are coordinately regulated by central leptin through PPARß/δ. Implications in cardiac remodeling.

BACKGROUND: Cardiovascular disease in obese individuals with type 2 diabetes is often associated with hyperleptinemia and leptin resistance, while other studies support that leptin has cardioprotective effects. Besides, the role of leptin in regulating cardiac atrophy or hypertrophy remains to be clearly defined. In fact, in rats with normal leptin sensitivity, the molecular underpinnings of the effects of central leptin regulating cardiac structural pathways remain poorly understood. OBJECTIVE: Hence, we assessed the effects of intracerebroventricular (icv) leptin infusion on cardiac remodeling analyzing FOXO1/3 and mTORC1 pathways, focusing special attention to PPARß/δ as mediator of central leptin's effects on cardiac metabolism. METHODS: Male 3-months-old Wistar rats, infused with icv leptin (0.2 µg/day) for 7 days, were daily co-treated intraperitoneally with the specific PPARß/δ antagonist GSK0660, at 1 mg/kg per day along leptin treatment. RESULTS: Central leptin regulated dynamically, in an opposite manner, the network between FOXOs and mTORC1 and induced an atrophy-related gene program in cardiac tissue. Leptin activated the anti-hypertrophic kinase GSK3ß and increased the protein levels of muscle-specific ubiquitin ligases, muscle RING finger 1 (MuRF1) and muscle atrophy F-box (MAFbx)/Atrogin-1 involved in limiting cardiac hypertrophy. FOXO1 activity and the expression of their target genes, Sod2 and Lpl, were also increased in the heart upon central leptin infusion. Besides, Beclin-1 and LC3B-II, gene products of the autophagic pathway response, were upregulated, while the content and expression levels of phenotypic markers of cardiac hypertrophy as ANP and ß-myosin heavy chain, gene product of Myh7 were significantly decreased. On the other hand, mTORC1 activity and OXPHOS protein levels were decreased suggesting a key role of central leptin preventing cardiac oxidative stress. In fact, the content of carbonylated proteins, TBARS and ROS/RSN were not increased in cardiac tissue in response to central leptin infusion. Finally, the pharmacological inhibition of PPARß/δ, via in vivo administration of the selective antagonist GSK0660, blunted the induction of FOXO1/3, Atrogin-1, MuRF1 and GSK3ß in the heart mediated by icv leptin infusion. CONCLUSIONS: Our results demonstrate that, in lean rats with normal leptin sensitivity, central leptin regulates nutrient sensing pathways in heart contributing to balance cardiac remodeling through the anti- and pro-hypertrophic programs, and in this process is involved PPARß/δ.

Network pharmacology of bioactives from Sorghum bicolor with targets related to diabetes mellitus.

BACKGROUND: Sorghum bicolor (SB) is rich in protective phytoconstituents with health benefits and regarded as a promising source of natural anti-diabetic substance. However, its comprehensive bioactive compound(s) and mechanism(s) against type-2 diabetes mellitus (T2DM) have not been exposed. Hence, we implemented network pharmacology to identify its key compounds and mechanism(s) against T2DM. METHODS: Compounds in SB were explored through GC-MS and screened by Lipinski's rule. Genes associated with the selected compounds or T2DM were extracted from public databases, and the overlapping genes between SB-compound related genes and T2DM target genes were identified using Venn diagram. Then, the networking between selected compounds and overlapping genes was constructed, visualized, and analyzed by RStudio. Finally, affinity between compounds and genes was evaluated via molecular docking. RESULTS: GC-MS analysis of SB detected a total of 20 compounds which were accepted by the Lipinski's rule. A total number of 16 compounds-related genes and T2DM-related genes (4,763) were identified, and 81 overlapping genes between them were selected. Gene set enrichment analysis exhibited that the mechanisms of SB against T2DM were associated with 12 signaling pathways, and the key mechanism might be to control blood glucose level by activating PPAR signaling pathway. Furthermore, the highest affinities were noted between four main compounds and six genes (FABP3-Propyleneglyco monoleate, FABP4-25-Oxo-27-norcholesterol, NR1H3-Campesterol, PPARA-ß-sitosterol, PPARD-ß-sitosterol, and PPARG-ß-sitosterol). CONCLUSION: Our study overall suggests that the four key compounds detected in SB might ameliorate T2DM severity by activating the PPAR signaling pathway.

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.

Targeting Peroxisome Proliferator-Activated Receptor Delta (PPARδ): A Medicinal Chemistry Perspective.

One of the three subtypes of the peroxisome proliferator-activated receptor (PPAR) functioning as a transcription factor is the PPARß or PPARδ. PPARδ is crucial to pathophysiological processes, including metabolic disorders, liver diseases, and cardiovascular diseases. In the past, the clinical development of PPARδ-selective agonist drugs has been stalled due to potential safety-related issues. Despite the elusiveness of such a drug, efforts continue in developing drugs that target PPARδ due to advances in the knowledge of the PPARδ receptor's structure and functions. While several preclinical and clinical studies are reported on PPARδ agonists, there is limited data with no clinical evidence available for PPARδ-selective antagonists. In this review, we mainly focus on the challenges of PPARδ selectivity and the medicinal chemistry of most active agonists discovered by different pharmaceutical companies and institutes. With this in mind, we also provide an update on the development status of PPARδ agonists that are undergoing clinical trials and their therapeutic promise for the treatment of various diseases.

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.

Treating the Disease, Not the Symptom: Beyond NSAIDs.

Mitigating inflammation is clearly important in cancer prevention and control. Traditionally, pharmaceuticals have taken the lead in this problem. In an attempt to 'head them off at the pass', this Forum takes a hard look at the concept of 'better living through chemicals' and limiting proinflammatory chemicals entering the body.

Pharmacological Blockade of PPAR Isoforms Increases Conditioned Fear Responding in the Presence of Nociceptive Tone.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors with three isoforms (PPARα, PPARß/δ, PPARγ) and can regulate pain, anxiety, and cognition. However, their role in conditioned fear and pain-fear interactions has not yet been investigated. Here, we investigated the effects of systemically administered PPAR antagonists on formalin-evoked nociceptive behaviour, fear-conditioned analgesia (FCA), and conditioned fear in the presence of nociceptive tone in rats. Twenty-three and a half hours following fear conditioning to context, male Sprague-Dawley rats received an intraplantar injection of formalin and intraperitoneal administration of vehicle, PPARα (GW6471), PPARß/δ (GSK0660) or PPARγ (GW9662) antagonists, and 30 min later were re-exposed to the conditioning arena for 15 min. The PPAR antagonists did not alter nociceptive behaviour or fear-conditioned analgesia. The PPARα and PPARß/δ antagonists prolonged context-induced freezing in the presence of nociceptive tone without affecting its initial expression. The PPARγ antagonist potentiated freezing over the entire trial. In conclusion, pharmacological blockade of PPARα and PPARß/δ in the presence of formalin-evoked nociceptive tone, impaired short-term, within-trial fear-extinction in rats without affecting pain response, while blockade of PPARγ potentiated conditioned fear responding. These results suggest that endogenous signalling through these three PPAR isoforms may reduce the expression of conditioned fear in the presence of nociceptive tone.

Peroxisome proliferator-activated receptor agonists and antagonists: a patent review (2014-present).

Introduction: Peroxisome proliferator-activated receptors (PPARs), PPARα, PPARδ, and PPARγ, play an important role in the regulation of various physiological processes, specifically lipid and energy metabolism and immunity. PPARα agonists (fibrates) and PPARγ agonists (thiazolidinediones) are used for the treatment of hypertriglyceridemia and type 2 diabetes, respectively. PPARδ activation enhances mitochondrial and energy metabolism but PPARδ-acting drugs are not yet available. Many synthetic ligands for PPARs have been developed to expand their therapeutic applications.Areas covered: The authors searched recent patent activity regarding PPAR ligands. Novel PPARα agonists, PPARδ agonists, PPARγ agonists, PPARα/γ dual agonists, and PPARγ antagonists have been claimed for the treatment of metabolic disease and inflammatory disease. Methods for the combination of PPAR ligands with other drugs and expanded application of PPAR agonists for bone and neurological disease have been also claimed.Expert opinion: Novel PPAR ligands and the combination of PPAR ligands with other drugs have been claimed for the treatment of mitochondrial disease, inflammatory/autoimmune disease, neurological disease, and cancer in addition to metabolic diseases including dyslipidemia and type 2 diabetes. Selective therapeutic actions of PPAR ligands should be exploited to avoid adverse effects. More basic studies are needed to elucidate the molecular mechanisms of selective actions.

The peroxisome proliferator-activated receptor-ß/δ antagonist GSK0660 mitigates retinal cell inflammation and leukostasis.

Diabetic retinopathy (DR) is triggered by retinal cell damage stimulated by the diabetic milieu, including increased levels of intraocular free fatty acids. Free fatty acids may serve as an initiator of inflammatory cytokine release from Müller cells, and the resulting cytokines are potent stimulators of retinal endothelial pathology, such as leukostasis, vascular permeability, and basement membrane thickening. Our previous studies have elucidated a role for peroxisome proliferator-activated receptor-ß/δ (PPARß/δ) in promoting several steps in the pathologic cascade in DR, including angiogenesis and expression of inflammatory mediators. Furthermore, PPARß/δ is a known target of lipid signaling, suggesting a potential role for this transcription factor in fatty acid-induced retinal inflammation. Therefore, we hypothesized that PPARß/δ stimulates both the induction of inflammatory mediators by Müller cells as well the paracrine induction of leukostasis in endothelial cells (EC) by Müller cell inflammatory products. To test this, we used the PPARß/δ inhibitor, GSK0660, in primary human Müller cells (HMC), human retinal microvascular endothelial cells (HRMEC) and mouse retina. We found that palmitic acid (PA) activation of PPARß/δ in HMC leads to the production of pro-angiogenic and/or inflammatory cytokines that may constitute DR-relevant upstream paracrine inflammatory signals to EC and other retinal cells. Downstream, EC transduce these signals and increase their synthesis and release of chemokines such as CCL8 and CXCL10 that regulate leukostasis and other cellular events related to vascular inflammation in DR. Our results indicate that PPARß/δ inhibition mitigates these upstream (MC) as well as downstream (EC) inflammatory signaling events elicited by metabolic stimuli and inflammatory cytokines. Therefore, our data suggest that PPARß/δ inhibition is a potential therapeutic strategy against early DR pathology.

The Peroxisome Proliferator-Activated Receptor (PPAR)-γ Antagonist 2-Chloro-5-Nitro-N-Phenylbenzamide (GW9662) Triggers Perilipin 2 Expression via PPARδ and Induces Lipogenesis and Triglyceride Accumulation in Human THP-1 Macrophages.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor family, playing pivotal roles in regulating glucose and lipid metabolism as well as inflammation. While characterizing potential PPARγ ligand activity of natural compounds in macrophages, we investigated their influence on the expression of adipophilin [perilipin 2 (PLIN2)], a well-known PPARγ target. To confirm that a compound regulates PLIN2 expression via PPARγ, we performed experiments using the widely used PPARγ antagonist 2-chloro-5-nitro-N-phenylbenzamide (GW9662). Surprisingly, instead of blocking upregulation of PLIN2 expression in THP-1 macrophages, expression was concentration-dependently induced by GW9662 at concentrations and under conditions commonly used. We found that this unexpected upregulation occurs in many human and murine macrophage cell models and also primary cells. Profiling expression of PPAR target genes showed upregulation of several genes involved in lipid uptake, transport, and storage as well as fatty acid synthesis by GW9662. In line with this and with upregulation of PLIN2 protein, GW9662 elevated lipogenesis and increased triglyceride levels. Finally, we identified PPARδ as a mediator of the substantial unexpected effects of GW9662. Our findings show that: 1) the PPARγ antagonist GW9662 unexpectedly activates PPARδ-mediated signaling in macrophages, 2) GW9662 significantly affects lipid metabolism in macrophages, 3) careful validation of experimental conditions and results is required for experiments involving GW9662, and 4) published studies in a context comparable to this work may have reported erroneous results if PPARγ independence was demonstrated using GW9662 only. In light of our findings, certain existing studies might require reinterpretation regarding the role of PPARγ SIGNIFICANCE STATEMENT: Peroxisome proliferator-activated receptors (PPARs) are targets for the treatment of various diseases, as they are key regulators of inflammation as well as lipid and glucose metabolism. Hence, reliable tools to characterize the molecular effects of PPARs are indispensable. We describe profound and unexpected off-target effects of the PPARγ antagonist 2-chloro-5-nitro-N-phenylbenzamide (GW9662) involving PPARδ and in turn affecting macrophage lipid metabolism. Our results question certain existing studies using GW9662 and make better experimental design of future studies necessary.

PPAR δ inhibition protects against palmitic acid-LPS induced lipidosis and injury in cultured hepatocyte L02 cell.

Background: Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, and its pathogenesis and mechanism are intricate. In the present study, we aimed to evaluate the role of PPAR δ in LPS associated NAFLD and to investigate the signal transduction pathways underlying PPAR δ treatment in vitro. Material and Methods: L02 cells were exposed to palmitic acid (PA) and/or LPS in the absence or presence of PPAR δ inhibition and/or activation. Results: LPS treatment markedly increased lipid deposition, FFA contents, IL-6 and TNF-α levels, and cell apoptosis in PA treatment (NAFLD model). PPAR δ inhibition protects L02 cells against LPS-induced lipidosis and injury. Conversely, the result of PPAR δ activation showed the reverse trend. LPS+PA treatment group significantly decreases the relative expression level of IRS-1, PI3K, AKT, phosphorylation of AKT, TLR-4, MyD88, phosphorylation of IKKα, NF-κB, Bcl-2 and increases the relative expression level of Bax, cleaved caspase 3 and cleaved caspase 8, compared with the cells treated with NAFLD model. PPAR δ inhibition upregulated the related proteins' expression level in insulin resistance and inflammation pathway and downregulated apoptotic relevant proteins. Instead, PPAR δ agonist showed the reverse trend. Conclusion: Our data show that PPAR δ inhibition reduces steatosis, inflammation and apoptosis in LPS-related NAFLD damage, in vitro. PPAR δ may be a potential therapeutic implication for NAFLD.

Phytohormone abscisic acid ameliorates cognitive impairments in streptozotocin-induced rat model of Alzheimer's disease through PPARß/δ and PKA signaling.

Aim: Alzheimer's disease (AD) is characterized by oxidative stress, neuroinflammation and progressive cognitive decline. Abscisic acid (ABA) is produced in a variety of mammalian tissues, including brain. It has anti-inflammatory and antioxidant effects and elicits a positive effect on spatial learning and memory performance. Here, the possible protective effect of ABA was evaluated in streptozotocin (STZ)-induced AD rat model which were injected intracerebroventriculary (i.c.v.) with STZ (3 mg/kg). Material and Methods: The STZ-treated animals received ABA (10 µg/rat, i.c.v.), ABA plus PPARß/δ receptor antagonist (GSK0660, 80 nM/rat) or ABA plus selective inhibitor of PKA (KT5720, 0.5 µg/rat) for 14 d. Learning and memory were determined using Morris water maze (MWM) and passive avoidance (PA) tests. Results: The data showed that STZ produced a significant learning and memory deficit in both MWM and PA tests. ABA significantly prevented the learning and memory impairment in STZ-treated rats. However, ABA effects were blocked by GSK0660 and KT5720. Conclusion: The data indicated that ABA attenuates STZ-induced learning and memory impairment and PPAR-ß/δ receptors and PKA signaling are involved, at least in part, in the ABA mechanism.

PPARδ agonist prevents endothelial dysfunction via induction of dihydrofolate reductase gene and activation of tetrahydrobiopterin salvage pathway.

BACKGROUND AND PURPOSE: Impaired endothelium-dependent relaxation (EDR) is a hallmark of endothelial dysfunction. A deficiency of tetrahydrobiopterin (BH4 ) causes endothelial NOS to produce ROS rather than NO. PPARδ is an emerging target for pharmacological intervention of endothelial dysfunction. Thus, the present study examined the role of PPARδ in the regulation of dihydrofolate reductase (DHFR), a key enzyme in the BH4 salvage pathway. EXPERIMENTAL APPROACH: Gene expression was measured by using qRT-PCR and western blotting. Biopterins and ROS were determined by using HPLC. NO was measured with fluorescent dye and electron paramagnetic resonance spectroscopy. Vasorelaxation was measured by Multi Myograph System. KEY RESULTS: The PPARδ agonist GW501516 increased DHFR and BH4 levels in endothelial cells (ECs). The effect was blocked by PPARδ antagonist GSK0660. Chromatin immunoprecipitation identified PPAR-responsive elements within the 5'-flanking region of the human DHFR gene. The promoter activity was examined with luciferase assays using deletion reporters. Importantly, DHFR expression was suppressed by palmitic acid (PA, a saturated fatty acid) but increased by docosahexaenoic acid (DHA, a polyunsaturated fatty acid). GSK0660 prevented DHA-induced increased DHFR expression. Conversely, the suppressive effect of PA was mitigated by GW501516. In mouse aortae, GW501516 ameliorated the PA-impaired EDR. However, this vasoprotective effect was attenuated by DHFR siRNA or methotrexate. In EC-specific Ppard knockout mice, GW501516 failed to improve vasorelaxation. CONCLUSION AND IMPLICATIONS: PPARδ prevented endothelial dysfunction by increasing DHFR and activating the BH4 salvage pathway. These results provide a novel mechanism for the protective roles of PPARδ against vascular diseases.

Discovery of potent and selective PPARα/δ dual antagonists and initial biological studies.

We previously published on the design and synthesis of novel, potent and selective PPARα antagonists suitable for either i.p. or oral in vivo administration for the potential treatment of cancer. Described herein is SAR for a subsequent program, where we set out to identify selective and potent PPARα/δ dual antagonist molecules. Emerging literature indicates that both PPARα and PPARδ antagonism may be helpful in curbing the proliferation of certain types of cancer. This dual antagonism could also be used to study PPARs in other settings. After testing for selective and dual potency, off-target counter screening, metabolic stability, oral bioavailability and associated toxicity, compound 11, the first reported PPARα/δ dual antagonist was chosen for more advanced preclinical evaluation.

Role of PPAR-ß/δ/miR-17/TXNIP pathway in neuronal apoptosis after neonatal hypoxic-ischemic injury in rats.

Activation of peroxisome proliferator-activated receptor beta/delta (PPAR-ß/δ), a nuclear receptor acting as a transcription factor, was shown to be protective in various models of neurological diseases. However, there is no information about the role of PPAR-ß/δ as well as its molecular mechanisms in neonatal hypoxia-ischemia (HI). In the present study, we hypothesized that PPAR-ß/δ agonist GW0742 can activate miR-17-5p, consequently inhibiting TXNIP and ASK1/p38 pathway leading to attenuation of apoptosis. Ten-day-old rat pups were subjected to right common carotid artery ligation followed by 2.5 h hypoxia. GW0742 was administered intranasally 1 and 24 h post HI. PPAR-ß/δ receptor antagonist GSK3787 was administered intranasally 1 h before and 24 h after HI, antimir-17-5p and TXNIP CRISPR activation plasmid were administered intracerebroventricularly 24 and 48 h before HI, respectively. Brain infarct area measurement, neurological function tests, western blot, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), Fluoro-Jade C and immunofluorescence staining were conducted. GW0742 reduced brain infarct area, brain atrophy, apoptosis, and improved neurological function at 72 h and 4 weeks post HI. Furthermore, GW0742 treatment increased PPAR-ß/δ nuclear expression and miR-17-5p level and reduced TXNIP in ipsilateral hemisphere after HI, resulting in inhibition of ASK1/p38 pathway and attenuation of apoptosis. Inhibition of PPAR-ß/δ receptor and miR-17-5p and activation of TXNIP reversed the protective effects. For the first time, we provide evidence that intranasal administration of PPAR-ß/δ agonist GW0742 attenuated neuronal apoptosis at least in part via PPAR-ß/δ/miR-17/TXNIP pathway. GW0742 could represent a therapeutic target for treatment of neonatal hypoxic ischemic encephalopathy (HIE).

Peroxisome proliferator-activated receptor ß/δ does not regulate glucose uptake and lactose synthesis in bovine mammary epithelial cells cultivated in vitro.

The hypothesis of the study was that inhibition of PPARß/δ increases glucose uptake and lactose synthesis in bovine mammary epithelial cells by reducing the expression of the glucose transporter mRNA destabiliser calreticulin. Three experiments were conducted to test the hypothesis using immortalised bovine mammary alveolar (MACT) and primary bovine mammary (PBMC) cells. In Experiment 1, the most effective dose to inhibit PPARß/δ activity among two synthetic antagonists (GSK-3787 and PT-s58) was assessed using a gene reporter assay. In Experiment 2, the effect on glucose uptake and lactose synthesis was evaluated by measuring glucose and lactose in the media and expression of related key genes upon modulation of PPARß/δ using GSK-3787, the synthetic PPARß/δ agonist GW-501516, or a combination of the two in cells cultivated in plastic. In Experiment 3, the same treatments were applied to cells cultivated in Matrigel and glucose and lactose in media were measured. In Experiment 1 it was determined that a significant inhibition of PPARß/δ in the presence or absence of fetal bovine serum was achieved with ≥ 1000 nm GSK-3787 but no significant inhibition was observed with PT-s58. In Experiment 2, inhibition of PPARß/δ had no effect on glucose uptake and lactose synthesis but they were both increased by GW-501516 in PBMC. The mRNA abundance of PPARß/δ target gene pyruvate dehydrogenase kinase 4 was increased but transcription of calreticulin was decreased (only in MACT cells) by GW-501516. Treatment with GSK-3787 did not affect the transcription of measured genes. No effects on glucose uptake or lactose synthesis were detected by modulation of PPARß/δ activity on cells cultivated in Matrigel. The above data do not provide support for the original hypothesis and suggest that PPARß/δ does not play a major role in glucose uptake and lactose synthesis in bovine mammary epithelial cells.

Phytohormone abscisic acid elicits antinociceptive effects in rats through the activation of opioid and peroxisome proliferator-activated receptors ß/δ.

The phytohormone abscisic acid exists in animal tissues particularly in the brain. However, its neurophysiological effects have not yet been fully clarified. This study was designed to evaluate the possible antinociceptive effects of abscisic acid on animal models of pain and determine its possible signaling mechanism. Tail-flick, hot-plate and formalin tests were used to assess the nociceptive threshold. All experiments were carried out on male Wistar rats. To determine the role of Peroxisome proliferator-activated receptor ß/δ (PPARß/δ) and opioid receptors on the induction of abscisic acid antinociception, specific antagonists were injected 15 min before abscisic acid. The data showed that abscisic acid (5, 10 and 15 µg/rat, i.c.v.) significantly decreased pain responses in formalin test. In addition, it could also produce dose-dependent antinociceptive effect in tail-flick and hot-plate tests. Administration of PPARß/δ antagonist (GSK0660, 80 nM, i.c.v.) significantly attenuated the antinociceptive effect of abscisic acid in all tests. The antinociceptive effects of abscisic acid were completely inhibited by naloxone (6 µg, i.c.v.) during the time course of tail-flick and hot-plate tests. The results indicated that the central injection of abscisic acid has potent pain-relieving property which is mediated partly via the PPAR ß/δ and opioid signaling.