PPARγ Antagonist Research Articles

DHA- and EPA-Enriched Phosphatidylcholine Suppress Human Lung Carcinoma 95D Cells Metastasis via Activating the Peroxisome Proliferator-Activated Receptor γ.

The antineoplastic effects of docosahexaenoic acid-containing phosphatidylcholine (DHA-PC) and eicosapentaenoic acid-containing phosphatidylcholine (EPA-PC) were explored, and their underlying mechanisms in the human lung carcinoma 95D cells (95D cells) were investigated. After treatment of 95D cells with DHA-PC or EPA-PC, cell biological behaviors such as growth, adhesion, migration, and invasion were studied. Immunofluorescence and western blotting were carried out to assess underlying molecular mechanisms. Results showed that 95D cells proliferation and adherence in the DHA-PC or EPA-PC group were drastically inhibited than the control group. DHA-PC and EPA-PC suppressed the migration and invasion of 95D cells by disrupting intracellular F-actin, which drives cell movement. The protein expression of PPARγ was induced versus the control group. Furthermore, critical factors related to invasion, including matrix metallopeptidase 9 (MMP9), heparanase (Hpa), and vascular endothelial growth factor (VEGF), were drastically downregulated through the PPARγ/NF-κB signaling pathway. C-X-C chemokine receptor type 4 (CXCR4) and cofilin were significantly suppressed via DHA-PC and EPA-PC through the PPARγ/phosphatase and tensin homolog (PTEN)/serine-threonine protein kinase (AKT) signaling pathway. DHA-PC and EPA-PC reversed the PPARγ antagonist GW9662-induced reduction of 95D cells in migration and invasion capacity, suggesting that PPARγ was directly involved in the anti-metastasis efficacy of DHA-PC and EPA-PC. In conclusion, DHA-PC and EPA-PC have great potential for cancer therapy, and the antineoplastic effects involve the activation of PPARγ. EPA-PC showed more pronounced antineoplastic effects than DHA-PC, possibly due to the more robust activation of PPARγ by EPA-PC.

Increasing brain glucose uptake by Gypenoside LXXV ameliorates cognitive deficits in a mouse model of diabetic Alzheimer's disease.

We have previously reported that Gypenoside LXXV (GP-75), a novel natural PPARγ agonist isolated from Gynostemma pentaphyllum, ameliorated cognitive deficits in db/db mice. In this study, we further investigated the beneficial effects on cognitive impairment in APP/PS1 mice and a mouse model of diabetic AD (APP/PS1xdb/db mice). Interestingly, intragastric administration of GP-75 (40 mg/kg/day) for 3months significantly attenuated cognitive deficits in APP/PS1 and APP/PS1xdb/db mice. GP-75 treatment markedly reduced the levels of glucose, HbA1c and insulin in serum and improved glucose tolerance and insulin sensitivity in APP/PS1xdb/db mice. Notably, GP-75 treatment decreased the β-amyloid (Aβ) burden, as measured by 11 C-PIB PET imaging. Importantly, GP-75 treatment increased brain glucose uptake as measured by 18 F-FDG PET imaging. Moreover, GP-75 treatment upregulated PPARγ and increased phosphorylation of Akt (Ser473) and GLUT4 expression levels but decreased phosphorylation of IRS-1 (Ser616) in the hippocampi of both APP/PS1 and APP/PS1xdb/db mice. Furthermore, GP-75-induced increases in GLUT4 membrane translocation in primary hippocampal neurons from APP/PS1xdb/db mice was abolished by cotreatment with the selective PPARγ antagonist GW9662 or the PI3K inhibitor LY294002. In summary, GP-75 ameliorated cognitive deficits in APP/PS1 and APP/PS1xdb/db mice by enhancing glucose uptake via activation of the PPARγ/Akt/GLUT4 signaling pathways.

Molecular Modeling of Allosteric Site of Isoform-Specific Inhibition of the Peroxisome Proliferator-Activated Receptor PPARγ.

The peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor and controls a number of gene expressions. The ligand binding domain (LBD) of PPARγ is large and involves two binding sites: orthosteric and allosteric binding sites. Increased evidence has shown that PPARγ is an oncogene and thus the PPARγ antagonists have potential as anticancer agents. In this paper, we use Glide Dock approach to determine which binding site, orthosteric or allosteric, would be a preferred pocket for PPARγ antagonist binding, though antidiabetic drugs such as thiazolidinediones (TZDs) bind to the orthosteric site. The Glide Dock results show that the binding of PPARγ antagonists at the allosteric site yielded results that were much closer to the experimental data than at the orthosteric site. The PPARγ antagonists seem to selectively bind to residues Lys265, Ser342 and Arg288 at the allosteric binding site, whereas PPARγ agonists would selectively bind to residues Leu228, Phe363, and His449, though Phe282 and Lys367 may also play a role for agonist binding at the orthosteric binding pocket. This finding will provide new perspectives in the design and optimization of selective and potent PPARγ antagonists or agonists.

The involvement of peroxisome proliferator-activated receptor gamma (PPARγ) in anti-inflammatory activity of N-stearoylethanolamine

BackgroundN-stearoylethanolamine (NSE) is a bioactive lipid amine with a wide range of biological activities. Anti-inflammatory properties of NSE were previously confirmed on multiple animal models. However, the molecular mechanisms of anti-inflammatory action of NSE remain unclear. In the current study, we examined the involvement of nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) in the NF-kB –dependent pathway of anti-inflammatory action of NSE using different methodological approaches. MethodsMolecular modeling calculated the possibility of NSE binding PPAR. Ex vivo experiment, using selective agonist of PPARα/γ - LY-171883 and antagonist of PPARγ - GW9662, examined the role of PPARα/γ in the NSE’s effect on nuclear NF-kB translocation in LPS-activated rat peritoneal macrophages. Finally, the NSE’s action on mRNA level of PPARγ-dependent genes was studied in the liver of insulin-resistant rats. ResultsThe results of molecular docking showed that NSE could bind to PPARγ and compete for the binding site with antagonist GW9662 and agonist LY-171883. These data was supported by in vitro study where pre-treatment with NSE prevented further LPS-induced NF-kB translocation into the nuclei of rat peritoneal macrophages. NSE treatment before GW9662 and LPS addition normalized the level of NF-kB translocation and IL-1β content. This finding confirmed a competitive binding of NSE with GW9662 for the ligand-binding domain of PPARγ. Additional in vivo study showed that NSE administration changed the mRNA expression of several PPARγ target genes, including SLC27A1 encoding fatty acid transport protein-1 and IL1RN - interleukin-1 receptor antagonist in insulin resistant rats. ConclusionNSE suppressed nuclear translocation of NF-κB in LPS-stimulated peritoneal macrophages via PPARγ and changed hepatic mRNA expression of PPARγ target genes (SLC27A1, IL1RN) in insulin resistant rats.

Protective Effects of the Chalcone-Based Derivative AN07 on Inflammation-Associated Myotube Atrophy Induced by Lipopolysaccharide.

Inflammation is a major cause of skeletal muscle atrophy in various diseases. 2-Hydroxy-4'-methoxychalcone (AN07) is a chalcone-based peroxisome-proliferator-activated receptor gamma (PPARγ) agonist with various effects, such as antiatherosclerosis, anti-inflammation, antioxidative stress, and neuroprotection. In this study, we examined the effects of AN07 on protein homeostasis pathway and mitochondrial function in inflammation-associated myotube atrophy induced by lipopolysaccharides (LPS). We found that AN07 significantly attenuated NF-κB activation, inflammatory factors (TNF-α, IL-1β, COX-2, and PGE2), Nox4 expression, and reactive oxygen species levels in LPS-treated C2C12 myotubes. Moreover, AN07 increased SOD2 expression and improved mitochondrial function, including mitochondrial membrane potential and mitochondrial oxygen consumption rate. We also demonstrated that AN07 attenuated LPS-induced reduction of myotube diameter, MyHC expression, and IGF-1/IGF-1R/p-Akt-mediated protein synthesis signaling. Additionally, AN07 downregulated LPS-induced autophagy-lysosomal protein degradation molecules (LC3-II/LC3-I and degraded p62) and ubiquitin-proteasome protein degradation molecules (n-FoxO1a/MuRF1/atrogin-1). However, the regulatory effects of AN07 on protein synthesis and degradation signaling were inhibited by the IGF-1R inhibitor AG1024 and the PI3K inhibitor wortmannin. In addition, the PPARγ antagonist GW9662 attenuated the effects of AN07 against LPS-induced inflammation, oxidation, and protein catabolism. In conclusion, our findings suggest that AN07 possesses protective effects on inflammation-induced myotube atrophy and mitochondrial dysfunction.

Chronic cAMP activation induces adipocyte browning through discordant biphasic remodeling of transcriptome and chromatin accessibility

ObjectiveAdipose tissue thermogenesis has been suggested as a new therapeutic target to promote energy metabolism for obesity and metabolic disease. Cold-inducible thermogenic adipocytes, called beige adipocytes, have attracted significant attention for their potent anti-obesity activity in adult humans. In this study, we identified the mechanisms underlying beige adipocyte recruitment, so-called adipocyte browning, by different stimuli. MethodsWe generated a new adipocyte cell line with enhanced browning potentials and determined its transcriptomic and epigenomic responses following cAMP (forskolin, FSK) versus PPARγ activation (rosiglitazone). We performed time-course RNA-seq and compared the treatments and in vivo adipocyte browning. We also developed an improved protocol for Assay for Transposase Accessible Chromatin-sequencing (ATAC-seq) and defined changes in chromatin accessibility in a time course. The RNA-seq and ATAC-seq data were integrated to determine the kinetics of their coordinated regulation and to identify a transcription factor that drives these processes. We conducted functional studies using pharmacological and genetic approaches with specific inhibitors and shRNA-mediated knockdown, respectively. ResultsFSK, not rosiglitazone, resulted in a biphasic transcriptomic response, resembling the kinetics of in vivo cold-induced browning. FSK promoted tissue remodeling first and subsequently shifted energy metabolism, concluding with a transcriptomic profile similar to that induced by rosiglitazone. The thermogenic effects of FSK were abolished by PPARγ antagonists, indicating PPARγ as a converging point. ATAC-seq uncovered that FSK leads to a significant chromatin remodeling that precedes or persists beyond transcriptomic changes, whereas rosiglitazone induces minimal changes. Motif analysis identified nuclear factor, interleukin 3 regulated (NFIL3) as a transcriptional regulator connecting the biphasic response of FSK-induced browning, as indicated by disrupted thermogenesis with NFIL3 knockdown. ConclusionsOur findings elucidated unique dynamics of the transcriptomic and epigenomic remodeling in adipocyte browning, providing new mechanistic insights into adipose thermogenesis and molecular targets for obesity treatment.

Pharmacological perturbation of peroxisome-proliferator-activated receptor gamma alters motility and mitochondrial function of bovine sperm.

Sperm transit through the female reproductive relies upon maintenance of sperm motility. Peroxisome-proliferator-activated receptor gamma (PPARγ) is a ligand-activated nuclear transcription factor with roles in glucose metabolism and reproductive processes including placental function. PPARγ roles in the mammalian postejaculatory sperm function are incompletely defined. Determine expression, localization, and functions of PPARγ in postejaculatory bovine sperm. Frozen-thawed bovine sperm from three to four different bulls were pooled and subjected to immunofluorescence and western blot for detection and localization of PPARγ. Functions in sperm energetics were explored through the addition of pharmacological inhibition (GW; GW9662) and activation (Ros; Rosiglitazone) in the culture medium at 0 and 24h under non-capacitating conditions. Samples were analyzed for sperm kinematics (CASA) and mitochondrial membrane potential (MMP; JC-1 fluorophore). PPARγ was detected in bovine sperm and co-localized to the acrosome with re-localization to the equatorial region in acrosome-compromised sperm. The addition of Ros 50µM for 24 h maintained superior total and progressive motility of sperm compared to vehicle control (VC-DMSO 0.01%). The PPARγ antagonist GW 1µM was detrimental to both total and progressive motility. A challenge experiment (Ros + GW) partially rescued total and progressive motility phenotypes observed with GW incubation. GW-treated samples had a lower number of sperm with high MMP at 24 h compared to Ros or VC. The negative GW MMP phenotype was reversed with the addition of Ros + GW. Likewise, GW-treated samples had more sperm with low MMP compared to VC and Ros, and this phenotype was partially restored with Ros + GW. PPARγ is expressed in post-ejaculatory bovine sperm with regulatory roles in sperm motility and MMP. These findings implicate PPARγ as a novel regulator of postejaculatory mammalian sperm energetics through non-canonical signaling mechanisms.

Cannabidiol inhibits microglia activation and mitigates neuronal damage induced by kainate in an in-vitro seizure model

BackgroundEpilepsy is one of the most common brain disorder and, despite the possible use of several therapeutic options, many patients continue to have seizures for their entire lifespan and they need new therapeutic approaches. In the last years the interest on the non-psychoactive compounds present in Cannabis sativa has massively increased, and cannabidiol (CBD) has been shown to be effective in the treatment of different types of neurological disorders and neurodegenerative diseases such as epilepsy, ischemia, multiple sclerosis and Alzheimer's Disease. MethodsWe investigated the effects of the selected cannabinoids, Δ9-tetrahydrocannabinol (THC), CBD and cannabigerol (CBG) in rat organotypic hippocampal slices exposed to kainate, an in vitro seizure model. Cell death in the cornu Ammonis 3 (CA3) hippocampal subregion was quantified by propidium iodide fluorescence. Morphological analysis and tissue organization were examined by immunohistochemistry and confocal microscopy and microglia activation and polarization was evaluated using flow cytometry and morphology analysis. ResultsWhen present in the incubation medium, cannabidiol reduced dose-dependent CA3 injury induced by kainate. Conversely, incubation with THC exacerbated hippocampal damage. The neuroprotective effects of cannabidiol were blocked by TRPV1, TRPV2, 5-HT1A, and PPARγ antagonists. Confocal microscopy confirmed that CBD but not THC had a significant protective effect against neuronal damage and tissue disorganization caused by kainate. Cannabidiol incubation significantly block the microglia activation from the M0 to M1 phenotype observed in the kainate in-vitro seizure model, pushing toward a transition from M0 to M2. ConclusionsOur results suggest that CBD mitigated neuronal damage induced by kainate and blocked the transition from the M0 to the M1 phenotype.

Increasing brain glucose metabolism by ligustrazine piperazine ameliorates cognitive deficits through PPARγ-dependent enhancement of mitophagy in APP/PS1 mice

PPARγ agonists have been proven to be neuroprotective in vitro and in vivo models of Alzheimer’s disease (AD). In the present study, we identified ligustrazine piperazine derivative (LPD) as a novel PPARγ agonist, which was detected by a dual-luciferase reporter assay system. LPD treatment dose-dependently reduced Aβ40 and Aβ42 levels in PC12 cells stably transfected with APP695swe and PSEN1dE9. Intragastric administration of LPD for 3 months dose-dependently reversed cognitive deficits in APP/PS1 mice. LPD treatment substantially decreased hippocampal Aβ plaques in APP/PS1 mice and decreased the levels of Aβ40 and Aβ42 in vivo and in vitro. Moreover, LPD treatment induced mitophagy in vivo and in vitro and increased brain 18F-FDG uptake in APP/PS1 mice. LPD treatment significantly increased OCR, ATP production, maximal respiration, spare respiratory capacity, and basal respiration in APP/PS1 cells. Mechanistically, LPD treatment upregulated PPARγ, PINK1, and the phosphorylation of Parkin (Ser65) and increased the LC3-II/LC3-I ratio but decreased SQSTM1/p62 in vivo and in vitro. Importantly, all these protective effects mediated by LPD were abolished by cotreatment with the selective PPARγ antagonist GW9662. In summary, LPD could increase brain glucose metabolism and ameliorate cognitive deficits through PPARγ-dependent enhancement of mitophagy in APP/PS1 mice.

Cisplatin-induced changes in calcitonin gene-related peptide or TNF-α release in rat dorsal root ganglia in vitro model of neurotoxicity are not reverted by rosiglitazone

IntroductionChemotherapy-induced peripheral neuropathy (CIPN) is a common side effect of anticancer drugs which affect the peripheral nervous system, as occurs with cisplatin treatment. Nowadays, one strategy in development to prevent, minimize and/or revert CIPN is neuroprotection. Therefore, we have evaluated the signaling pathways involved in CIPN and the effect of rosiglitazone, a peroxisome proliferator-activated receptor γ (PPAR-γ) agonist. MethodsDorsal Root Ganglia (DRG) were harvested from Wistar rats (Rattus norvegicus), the cells were dissociated, plated, and maintained with nerve growth factor for 9 days. On day 8, the cells were treated with cisplatin, rosiglitazone and/or T0070907 (PPAR-γ antagonist) for 24 h. The cell viability was measured by trypan blue exclusion method, the mRNA was quantified by real-time RT-PCRq and the release of TNF-α and calcitonin gene-related peptide (CGRP) was evaluated by ELISA. ResultsCisplatin, rosiglitazone or T0070907 treatments did not decreased the cell viability on the primary DRG cultures cells. Cisplatin treatment induced a decrease of PPAR-γ and -β/δ mRNA, while the co-treatment with rosiglitazone inhibited this cisplatin-induced effect. Moreover, T0070907 did not change the observed results, indicating that the rosiglitazone’s effect could be due to mechanisms beyond PPAR-γ activation. Also, the rosiglitazone effect is not exclusively to DRG cells since there was an increase of PPAR-γ mRNA expression in 3T3-L1 cells. Furthermore, rosiglitazone did not modulate the cisplatin decrease neuronal function of DRG cells (TNF-α and CGRP release). ConclusionCisplatin decreased the gene expression of PPAR-γ and -β/δ, while the rosiglitazone treatment inhibited these effects via PPAR-γ independent pathway. Rosiglitazone did not show improvement in modulation of TNF-α or CGRP release impaired by cisplatin.

PPARγ and NOTCH Regulate Regional Identity in the Murine Cardiac Outflow Tract.

The arterial pole of the heart is a hotspot for life-threatening forms of congenital heart defects (CHDs). Development of this cardiac region occurs by addition of Second Heart Field (SHF) progenitor cells to the embryonic outflow tract (OFT) and subsequently the base of the ascending aorta and pulmonary trunk. Understanding the cellular and genetic mechanisms driving arterial pole morphogenesis is essential to provide further insights into the cause of CHDs. A synergistic combination of bioinformatic analysis and mouse genetics as well as embryo and explant culture experiments were used to dissect the cross-regulatory transcriptional circuitry operating in future subaortic and subpulmonary OFT myocardium. Here, we show that the lipid sensor PPARγ (peroxisome proliferator-activated receptor gamma) is expressed in future subpulmonary myocardium in the inferior wall of the OFT and that PPARγ signaling-related genes display regionalized OFT expression regulated by the transcription factor TBX1 (T-box transcription factor 1). Modulating PPARγ activity in ex vivo cultured embryos treated with a PPARγ agonist or antagonist or deleting Pparγ in cardiac progenitor cells using Mesp1-Cre reveals that Pparγ is required for addition of future subpulmonary myocardium and normal arterial pole development. Additionally, the non-canonical DLK1 (delta-like noncanonical Notch ligand 1)/NOTCH (Notch receptor 1)/HES1 (Hes family bHLH transcription factor 1) pathway negatively regulates Pparγ in future subaortic myocardium in the superior OFT wall. Together these results identify Pparγ as a regulator of regional transcriptional identity in the developing heart, providing new insights into gene interactions involved in congenital heart defects.

Bergenin ameliorates cognitive deficits and neuropathological alterations in sodium azide-induced experimental dementia.

Background: Bergenin, 4-O-methyl gallic acid glucoside, is a bioactive compound found in the cortex of Mallotus japonicus (L.f.) Müll.Arg. along with many other natural resources including that from Bergenia species. The present study delineates the neuroprotective potential of bergenin through the modulation of PPAR-γ receptors. Method: Dementia was induced in the Wistar rats by intraperitoneal (i.p.) administration of sodium azide (12.5 mg/kg for the first 5 days followed by 10 mg/kg for the next 9 days). The rats were then exposed to the Morris water maze test to assess the effect on cognitive abilities followed by a series of biochemical and histopathological evaluations. Results: Sodium azide-treated rats exhibited a severe deterioration of memory as suggested by poor performance in the spatial learning task in addition to the enhancement of brain acetylcholinesterase potential, oxidative stress, inflammation, and amyloid-β (Aβ) accumulation. Administration of bergenin to sodium azide-treated rats significantly recovered cognition and related biochemical variations. Further, co-administration of Bisphenol A diglycidyl ether (BADGE), a PPAR-γ antagonist with bergenin challenged its neuroprotective effects. Conclusions: The findings of our study exhibit that the cognitive restoration potential of bergenin may be attributed to its modulatory effects against cholinesterase, oxidative stress, and inflammatory markers, as well as its neuroprotective actions, thus aligning it as a possible therapy for Alzheimer’s disease-related dementia. The study also fortifies the significance of PPAR-γ receptors in dementia.

Biochemical and structural basis for the pharmacological inhibition of nuclear hormone receptor PPARγ by inverse agonists

Recent studies have reported that the peroxisome proliferator-activated receptor gamma (PPARγ) pathway is activated in approximately 40% of patients with muscle-invasive bladder cancer. This led us to investigate pharmacological repression of PPARγ as a possible intervention strategy. Here, we characterize PPARγ antagonists and inverse agonists and find that the former behave as silent ligands, whereas inverse agonists (T0070907 and SR10221) repress downstream PPARγ target genes leading to growth inhibition in bladder cancer cell lines. To understand the mechanism, we determined the ternary crystal structure of PPARγ bound to T0070907 and the corepressor (co-R) peptide NCOR1. The structure shows that the AF-2 helix 12 (H12) rearranges to bind inside the ligand-binding domain, where it forms stabilizing interactions with the compound. This dramatic movement in H12 unveils a large interface for co-R binding. In contrast, the crystal structure of PPARγ bound to a SR10221 analog shows more subtle structural differences, where the compound binds and pushes H12 away from the ligand-binding domain to allow co-R binding. Interestingly, we found that both classes of compound promote recruitment of co-R proteins in biochemical assays but with distinct conformational changes in H12. We validate our structural models using both site-directed mutagenesis and chemical probes. Our findings offer new mechanistic insights into pharmacological modulation of PPARγ signaling.

Long-term IgE immunological tolerance to peanut allergens: An alternative to Noon’s daily desensitization paradigm

Herein, we show that profound afferent long-term peanut-allergen-specific IgE immunological tolerance for 3 to 9 months induced sustained unresponsiveness (SU) in naïve or peanut-sensitized rodents after peanut allergen immunization. Rodents were vaccinated sublingually with a peanut allergen extract or recombinant peanut allergen in chenodeoxycholate (CDCA), a fanesoid X receptor (FXR, NR1H4) agonist that downregulates SREBP-1c (sterol regulatory element binding protein-1c) and upregulates SHP in bone marrow-derived tolerogenic dendritic cells (DCs). Approximately 90 ∼ 95 % of the total circulating PE-potentiated IgE and Ara h1, Ara h 2, and Ara h 6 peanut allergen-specific IgE responses were suppressed by recombinant peanut allergen-conjugated solid magnetic beads (sensitivity of 0.2 IU/ml). In contrast, peanut allergen-specific IgG production was not affected. Similarly, oleoylethanolamine (OEA), a peroxisome proliferator-activator receptor alpha (PPARα) agonist, and GW9662, a PPARγ antagonist, induced long-term peanut-specific IgE tolerance when administered via the sublingual, oral or i.p. route. Prophylactic Ara h2 DNA immunization with caNRF2 and IL-35 coexpression induced Ara h2 IgE tolerance. In summary, peanut allergen vaccination with select natural molecular ligands of nuclear receptors induced long-term peanut allergen-specific IgE tolerance via the afferent limb, which indicates that vaccination is an immune tolerance-promoting strategy that is effective at the DC level and that differs from Noon’s daily desensitization program, which is effective at the mast cell level.

Rational design of stapled helical peptides as antidiabetic PPARγ antagonists to target coactivator site by decreasing unfavorable entropy penalty instead of increasing favorable enthalpy contribution.

Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated transcription factor belonging to the nuclear hormone receptor and has been exploited as a well-established druggable target for the treatment of diabetes mellitus (DM). Traditionally, small-molecule compounds have been developed to attack at the ligand site and Ser273 phosphorylation site of PPARγ. In this study, we derived helical peptide segments from the LXXLL motif region of coactivator proteins as antidiabetic PPARγ antagonists, which were expected to competitively disrupt the native interaction between PPARγ and its cognate coactivators by rebinding at PPARγ coactivator site. Structural analysis, dynamics simulation and energetics dissection revealed that these peptides cannot be well folded into active helical structure when splitting from the protein context of their parent coactivators and exhibit a large flexibility and intrinsic disorder in the free state, which would, therefore, incur a considerable entropy penalty upon rebinding to PPARγ. Hydrocarbon stapling strategy was employed to constrain these free coactivator peptides into ordered helical conformation, thus largely minimizing unfavorable entropy penalty but having only a moderate effect on favorable enthalpy contribution. The computational findings were further substantiated by fluorescence-based assays; the binding affinity of three potent SRC1, NCoA6 and p300 coactivator peptides to PPARγ was observed to be improved by 7.2-fold, 4.2-fold and 5.7-fold upon the stapling, which were also measured to have an efficient competitive potency with their unstapled counterparts for PPARγ coactivator site, with CC50 = 0.096, 0.12 and 0.18μM, respectively.

Influenza virus causes lung immunopathology through down-regulating PPARγ activity in macrophages.

Fatal influenza (flu) virus infection often activates excessive inflammatory signals, leading to multi-organ failure and death, also referred to as cytokine storm. PPARγ (Peroxisome proliferator-activated receptor gamma) agonists are well-known candidates for cytokine storm modulation. The present study identified that influenza infection reduced PPARγ expression and decreased PPARγ transcription activity in human alveolar macrophages (AMs) from different donors. Treatment with PPARγ agonist Troglitazone ameliorated virus-induced proinflammatory cytokine secretion but did not interfere with the IFN-induced antiviral pathway in human AMs. In contrast, PPARγ antagonist and knockdown of PPARγ in human AMs further enhanced virus-stimulated proinflammatory response. In a mouse model of influenza infection, flu virus dose-dependently reduced PPARγ transcriptional activity and decreased expression of PPARγ. Moreover, PPARγ agonist troglitazone significantly reduced high doses of influenza infection-induced lung pathology. In addition, flu infection reduced PPARγ expression in all mouse macrophages, including AMs, interstitial macrophages, and bone-marrow-derived macrophages but not in alveolar epithelial cells. Our results indicate that the influenza virus specifically targets the PPARγ pathway in macrophages to cause acute injury to the lung.

Angiotensin II receptor type 1 blocker candesartan improves morphine tolerance by reducing morphine‑induced inflammatory response and cellular activation of BV2 cells via the PPARγ/AMPK signaling pathway.

Morphine is the most common drug of choice in clinical pain management; however, morphine tolerance presents a significant clinical challenge. The pathogenesis of morphine tolerance is known to be closely associated with angiotensin II receptor type 1 (AT1R) in microglia. As an AT1R antagonist, candesartan may serve an important role in regulating morphine tolerance. Therefore, the present study aimed to investigate the role of candesartan in morphine tolerance, and to explore the underlying mechanism. To meet this aim, BV2 microglial cells were treated with morphine or candesartan alone, or as a combination, and the expression levels of AT1R in BV2 cells were detected by reverse transcription-quantitative PCR (RT-qPCR) and western blotting. The levels of the inflammatory cytokines tumor necrosis factor-α, interleukin (IL)-1β and IL-6 were subsequently detected by ELISA and western blotting. In addition, immunofluorescence analysis, western blotting and RT-qPCR were used to detect the expression levels of the BV2 cell activation marker, ionized calcium-binding adaptor molecule 1 (IBA-1). Western blotting was also used to detect the expression levels of peroxisome proliferator-activated receptor-γ/AMP-activated protein kinase (PPARγ/AMPK) signaling pathway-associated proteins. Finally, the cells were treated with the PPARγ antagonist GW9662 and the AMPK inhibitor compound C to further explore the mechanism underlying the effects of candesartan on improving morphine tolerance. The expression levels of AT1R were revealed to be significantly increased following morphine induction; however, candesartan treatment inhibited the expression levels of AT1R, the levels of inflammatory cytokines and the protein expression levels of IBA-1 in morphine-induced BV2 cells in a dose-dependent manner. These processes may be associated with activation of the PPARγ/AMPK signaling pathway. Taken together, the present study revealed that treatment with candesartan reduced morphine-induced inflammatory response and cellular activation of BV2 cells via PPARγ/AMPK signaling.

Intermittent fasting attenuates lipopolysaccharide-induced acute lung injury in mice by modulating macrophage polarization

Acute lung injury (ALI) is a spectrum of acute and life-threatening pulmonary inflammatory conditions. Treatment of ALI remains a clinical challenge. Recently, intermittent fasting (IF) has been shown to improve health and alleviate many diseases. In this study, we tested whether IF attenuated ALI and investigated the mechanism underlying this process. In vivo, the effects of IF on ALI were evaluated in a lipopolysaccharide (LPS)-induced murine ALI model. We found that two times of 24-h fasting in a week before ALI efficiently ameliorated LPS-induced lung injury in mice, characterized by alleviated lung lesions, wet-to-dry weight ratio, myeloperoxidase activity, malondialdehyde content, and lower levels of tumor necrosis factor-α, interleukin-6, and interleukin-1β. In vitro, functional assays were conducted to assess IF on the inflammatory response and macrophage polarization of bone marrow-derived macrophages (BMDMs) treated with LPS or IL-4. And PPARγ antagonist GW9662 and AMPK siRNA were used to test the role of PPARγ and AMPK in the IF-mediated improvement of ALI. The results showed that IF (serum deprivation) suppressed macrophage M1 activation and promoted M2 activation in LPS-treated BMDMs. While, IF also augmented macrophage M2 polarization in IL-4-treated BMDMs. Further mechanistic studies showed that the promotive effect of IF on M2 polarization was related to the activation of the PPARγ and AMPK pathways. In conclusion, this study suggests that IF enhances M2 polarization by activating the AMPK and PPARγ pathways, thus facilitating anti-inflammatory response and ameliorating ALI.

Gegen Qinlian decoction ameliorates murine colitis by inhibiting the expansion of Enterobacteriaceae through activating PPAR-γ signaling

Ulcerative colitis (UC) is a chronic and relapsing inflammatory disease of the intestine. Dysbiosis, especially the expansion of facultative anaerobic Enterobacteriaceae, maybe the main pathogenesis of UC. Gegen Qinlian decoction (GD), a traditional Chinese medicinal formula chronicled in the Shang Han Lun, is commonly used to treat UC and has shown an excellent effect on inducing disease remission. However, the role of GD in regulating gut microbiota has not been fully clarified. Herein, we investigated the potential effect of GD on inhibiting the expansion of Enterobacteriaceae and further explored the potential mechanism of this action. Our study demonstrated that GD remarkably reduced body weight loss of colitis mice, shortening of colon length, and inflammation of the colon. Peroxisome proliferator-activated receptor-γ (PPAR-γ) signaling was inactivated in colitis colon tissue, and the abundance of Escherichia coli (E. coli, family of Enterobacteriaceae) in colonic contents and the concentration of lipopolysaccharide (LPS) in colonic tissue were significantly upregulated after DSS-treatment. Notably, GD administration can result in the activation of PPAR-γ and inactivation of iNOS, which lead to the reduction of nitrate, the inhibition of E. coli, and less production of LPS. Combined GD with PPAR-γ antagonist, the effect of GD on the treatment of UC was weakened, and effectless in inhibiting the expansion of Enterobacteriaceae. Therefore, GD ameliorates UC by preventing a dysbiotic expansion of potentially pathogenic E. coli by reducing nitrate levels in the lumen through activating PPAR-γ signaling.

Lutein inhibits IL‑6 expression by inducing PPAR‑γ activation and SOCS3 expression in cerulein‑stimulated pancreatic acinar cells.

Acute pancreatitis is a severe inflammatory disease of the pancreas. In experimental acute pancreatitis, cerulein induces the expression of interleukin-6 (IL-6) by activating Janus kinase (JAK) 2/signal transducer and activator of transcription (STAT) 3 in pancreatic acinar cells. Ligands of peroxisome proliferator activated receptor-γ (PPAR-γ) and suppressor of cytokine signaling (SOCS) 3 inhibit IL-6 expression by suppressing JAK2/STAT3 in cerulein-stimulated pancreatic acinar AR42J cells. Lutein, an oxygenated carotenoid, upregulates and activates PPAR-γ to regulate inflammation in a renal injury model. The present study aimed to determine whether lutein activated PPAR-γ and induced SOCS3 expression in unstimulated AR42J cells, and whether lutein inhibited activation of JAK2/STAT3 and IL-6 expression via activation of PPAR-γ and SOCS3 expression in cerulein-stimulated AR42J cells. The anti-inflammatory mechanism of lutein was determined using reverse transcription-quantitative PCR, western blot analysis and enzyme-linked immunosorbent assay in AR42J cells stimulated with or without cerulein. In another experiment, cells were treated with lutein and the PPAR-γ antagonist GW9662 or the PPAR-γ agonist troglitazone prior to cerulein stimulation to determine the involvement of PPAR-γ activation. The results indicated that lutein increased PPAR-γ and SOCS3 levels in unstimulated cells. Cerulein increased phospho-specific forms of JAK2 and STAT3, and mRNA and protein expression of IL-6, but decreased SOCS3 levels in AR42J cells. Cerulein-induced alterations were suppressed by lutein or troglitazone. GW9662 alleviated the inhibitory effect of lutein on JAK2/STAT3 activation and IL-6 expression in cerulein-stimulated cells. In conclusion, lutein inhibited the activation of JAK2/STAT3 and reduced IL-6 levels via PPAR-γ-mediated SOCS3 expression in pancreatic acinar cells stimulated with cerulein.