PPARγ Activation Research Articles

Alpha-asaronol promoted oligodendrocyte precursor cell differentiation and improved myelination as an activator PPARγ

Preterm white matter injury (PWMI), characterized by oligodendrocyte precursor cell (OPC) differentiation disorder and dysmyelination, is a prevalent demyelinating disease of the central nervous system in premature infants, necessitating the development of mitigating strategies. Convincing evidence suggests that peroxisome proliferator-activated receptor γ (PPARγ) activation is a stimulative factor against the hindered process of oligodendrocyte (OL) differentiation. However, much remains unknown about its promotive mechanism. Our previous study indicated that alpha-asaronol (α-asaronol) could alleviate myelination disorder in a neonatal PWMI rat model, but the mechanism remained unclear. In this study, we demonstrated that α-asaronol attenuated cognitive deficits, repaired myelin damage, and stimulated OL differentiation in the corpus callosum of PWMI rats. Co-immunoprecipitation analysis confirmed that α-asaronol induced the binding of PPARγ with its coactivator peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), which in turn activated oligodendroglial PPARγ. This activation subsequently upregulated the expression of phosphatase and tensin homolog (PTEN) and pro-differentiation-associated genes of Cnp1 and Klk6 and downregulated the expression of Clk1. However, the benefits of α-asaronol were blocked by GW9662, an antagonist of PPARγ. Moreover, α-asaronol also promoted OPC differentiation under oxygen-glucose deprivation conditions. In conclusion, α-asaronol can promote OL differentiation and myelination and alleviate cognitive deficits in neonatal PWMI rats by activating PPARγ and modulating OL differentiation-associated gene expression. This study suggests that α-asaronol may be a potential therapeutic drug for myelination failure in PWMI.

Rosiglitazone-induced PPARγ activation promotes intramuscular adipocyte adipogenesis of pig

Intramuscular fat (IMF) positively influences various aspects of meat quality, while the subcutaneous fat (SF) has negative effect on carcass characteristics and fattening efficiency. Peroxisome proliferator-activated receptor gamma (PPARγ) is a key regulator of adipocyte differentiation, herein, through bioinformatic screen for the potential regulators of adipogenesis from two independent microarray datasets, we identified that PPARγ is a potentially regulator between porcine IMF and SF adipogenesis. Then we treated subcutaneous preadipocytes (SA) and intramuscular preadipocytes (IMA) of pig with RSG (1 µmol/L), and we found that RSG treatment promoted the differentiation of IMA via differentially activating PPARγ transcriptional activity. Besides, RSG treatment promoted apoptosis and lipolysis of SA. Meanwhile, by the treatment of conditioned medium, we excluded the possibility of indirect regulation of RSG from myocyte to adipocyte and proposed that AMPK may mediate the RSG-induced differential activation of PPARγ. Collectively, the RSG treatment promotes IMA adipogenesis, and advances SA lipolysis, this effect may be associated with AMPK-mediated PPARγ differential activation. Our data indicates that targeting PPARγ might be an effective strategy to promote intramuscular fat deposition while reduce subcutaneous fat mass of pig.

PO-02-246 PPARΓ ACTIVATION MEDIATES CARDIAC PATHOLOGIES IN TWO MOUSE MODELS OF ARRHYTHMOGENIC CARDIOMYOPATHY (ACM) AND ITS THERAPEUTIC IMPLICATIONS

Using ACM patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), we showed that abnormal peroxisome proliferator-activated receptor-gamma (PPARγ) activation on normal adult PPARα-mediated metabolic backgrounds in CMs resulted in pathological hallmarks of ACM. PPARγ inhibitors rescue all ACM pathologies effects (Nature 2013) but may carry systemic side effects. Here, we study the pathogenic role of PPARγ activation in 2 ACM mouse models and test therapeutic potentials of 2 angiotensin II-receptor blockers (ARBs) commonly used to treat cardiomyopathies: Valsartan and Losartan. Valsartan has minimal activity yet Losartan has strong activity for PPARγ gene activation, respectively. We used 2 established ACM mouse models in this study: 1) mice with tamoxifen (TAM)-inducible and CM-specific (by αγMHC-Cre) double knockout (DKO) of plakoglobin (JUP) and βcatenin (MCB 2012) that developed ACM pathologies at 2-3 months only after TAM injection; and 2) mice with CM-specific and homozygous knockout of JUP (JUP KO, Hum Mol Genet 2011) that develop ACM pathologies rapidly in 1-2 months after birth. Feeding DKO mice with 0.1 mg/ml rosiglitazone (Rosi, a PPARγ activator) for 2 weeks (wks) after TAM injection accelerated the decrease in left ventricular ejection fraction (LV EF) (from 67.4% to 27.1%) within 2-3 weeks after feeding Rosi. For JUP KO mice at 8-9 wks after birth, the pathological hearts showed hyperactivated PPARγ (at both mRNA and protein levels) and elevated apoptotic gene expressions (3-fold increases in caspase 3 and BAX/BCL2 ratios). Feeding both ACM mouse models with 0.2 mg/ml Valsartan for 6 wks reduced PPARγ proteins and apoptotic genes (BAX/BCL2 ratios) by 2-3 folds. In JUP KO mice, Valsartan prevented LV EF deterioration yet losartan at equivalent doses led to early death and reduced LV EF. Our study results support strongly that abnormal PPARγ activation in ACM CMs plays a key pathogenic role in both human iPSC-CM-based and 2 mouse models of ACM. An ARB with PPARγ agnostic activities can worsen ACM pathologies and only ARB with minimal PPARγ agonistic activity can prevent ACM disease progression. These results have significant implications in avoiding using any drug with PPARγ agnostic activities to treat human ACM.

Irisin drives macrophage anti-inflammatory differentiation via JAK2-STAT6-dependent activation of PPARγ and Nrf2 signaling.

Irisin is an exercise-induced myokine that alleviates inflammation and obesity. The induction of anti-inflammatory (M2) macrophage is facilitated for treatment of sepsis and associated lung damage. However, whether irisin drives macrophage M2 polarization remains unclear. Here, we found that irisin induced-macrophage anti-inflammatory differentiation in vivo using an LPS-induced septic mice model and in vitro using RAW64.7cells and bone marrow-derived macrophages (BMDMs). Irisin also promoted the expression, phosphorylation, and nuclear translocation of peroxisome proliferator-activated receptor gamma (PPAR-γ) and nuclear factor-erythroid 2-related factor 2 (Nrf2). Inhibition or knockdown of PPAR-γ and Nrf2 abolished irisin-induced accumulation of M2 macrophage markers, such as interleukin (IL)-10 and Arginase 1. Furthermore, dual-luciferase reporter and chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) assays confirmed that STAT6 boosts PPAR-γ and Nrf2 transcription by binding to their DNA promoters in irisin-stimulated macrophages. In contrast, STAT6 shRNA blocked the irisin-induced activation of Pparγ, Nrf2, and related downstream genes. Moreover, the interaction of irisin with its ligand integrin αVβ5 remarkably promoted Janus kinase 2 (JAK2) phosphorylation, while inhibition or knockdown of integrin αVβ5 and JAK2 attenuated the activation of STAT6, PPAR-γ, and Nrf2 signaling. Interestingly, co-immunoprecipitation (Co-IP) assay also revealed that the binding between JAK2 and integrin αVβ5 is critical for irisin-induced macrophage anti-inflammatory differentiation by enhancing the activation of the JAK2-STAT6 pathway. In conclusion, irisin boosted M2 macrophage differentiation by inducing JAK2-STAT6-dependent transcriptional activation of the PPAR-γ-related anti-inflammatory system and Nrf2-related antioxidant genes. The findings of this study suggest that the administration of irisin is a novel and promising therapeutic strategy for infectious and inflammatory diseases.

The nuclear receptor Nurr1 regulates the expression and activity of PPARγ in human pro-inflammatory macrophages

Abstract Nurr1 is a member of the orphan nuclear receptor family NR4A that modulates inflammation in several cell lineages through inhibition of the NF-κB signaling pathway. Nurr1 was found to be able to interact with the peroxisome proliferator-activated receptor (PPAR)γ, as well as to be recruited to the PPARG promoter in LPS-activated microglial cells; however, the functional relationship between Nurr1 and PPARγ is not clearly established. Here, we aimed to investigate the role of Nurr1 on PPARγ activity in human macrophages. Blood monocytes were cultured with GM-CSF or M-CSF to generate pro- (GM-MDMs) and anti-inflammatory (M-MDMs) macrophages, respectively. GM-MDMs showed enhanced expression of both Nurr1 and PPARγ compared to M-MDMs, and they were preferentially located at the cytoplasmic (Nurr1) and nuclear (PPARγ) compartments. PPARγ activation in GM-MDMs with the agonist rosiglitazone did not modify Nurr1 expression. Conversely, Nurr1 activation with the agonist C-DIM12 increased PPARγ expression. This was concomitant with enhanced expression of two PPARγ target genes, CD36 and PLIN2, while silencing Nurr1 expression led to a decrease in the amounts of CD36 transcripts. Co-treatment of GM-MDMs with C-DIM12 and rosiglitazone synergistically enhanced the transcriptional activity of PPARγ, whereas C-DIM12 alone had no effect. PPARγ and Nurr1 agonists alone exerted anti-inflammatory effects on GM-MDMs, and also showed synergistic activity in combined form. In sum, our data suggests that Nurr1 positively regulates PPARγ activity at least partially through induction of PPARγ expression, which may represent a novel mechanism of Nurr1 targeting metabolic pathways and inflammation. Supported by grants from the Secretaría de Educación Pública-Consejo Nacional de Ciencia y Tecnología (SEP-CONACYT, #A1-S-9430)

Juniper Berries Regulate Diabetes and Obesity Markers Through Modulating PPARα, PPARγ, and LXR: In Vitro and In Vivo Effects.

The berries of Juniperus communis have been traditionally used for therapeutic purposes. They have been reported to possess various pharmacological effects such as anti-inflammatory, hypoglycemic and hypolipidemic activities. In this study, a methanolic extract of J. communis berries (JB) was evaluated for its effects on peroxisome proliferator-activated receptors alpha and gamma (PPARα and PPARγ), liver X receptor (LXR), glucose uptake and lipid accumulation using various cellular systems. At a concentration of 25 μg/mL, JB caused 3.77-fold activation of PPARα, 10.90-fold activation of PPARγ, and 4.43-fold activation of LXR in hepatic cells. JB inhibited (11%) the adipogenic effect induced by rosiglitazone in adipocytes and increased glucose uptake (90%) in muscle cells. In high-fat diet (HFD) fed mice, JB at a dose of 25 mg/kg body weight exhibited a 21% decrease in body weight. Fasting glucose levels in mice treated with 12.5 mg/kg of JB were significantly decreased (39%) indicating its efficacy in regulating hyperglycemia and obesity induced by HFD thus ameliorating the symptoms of type 2 diabetes. A series of energy metabolic genes, including Sirt1 (2.00-fold) and RAF1 (2.04-fold), were upregulated by JB, while rosiglitazone regulated the hepatic PPARγ only. Phytochemical analysis of JB indicated presence of a number of flavonoids and biflavonoids which seem to be responsible for the observed activity. It was concluded that JB acted as a multiple agonist of PPARα, PPARγ and LXR without the undesired effect of adipogenesis and exhibited the property of enhancing glucose uptake. The regulation of PPARα, PPARγ and LXR seems to be through Sirt1 and RAF1. In vivo results confirmed the antidiabetic and antiobesity potential of JB and indicated its utility in metabolic disorder and type 2 diabetes.

Bifidobacterium bifidum prevents the IL-1B induced increase in intestinal permeability by a novel mechanism: TLR-2 dependent activation of PPAR-gamma and inhibition of NF-kB signaling pathway

Background: Bifidobacterium bifidum (BB) is one of the most common probiotic bacteria strains that have been shown to have protective effects against intestinal inflammation, primarily by enhancing the intestinal epithelial tight junction (TJ) barrier function. IL-1β contributes to the development of intestinal inflammation in inflammatory bowel disease (IBD) in part by causing an increase in intestinal permeability. The IL-1β induced increase in intestinal epithelial TJ permeability is mediated by an NF-κB-dependent activation of myosin light chain kinase (MLCK) gene and disruption of the intestinal TJ barrier. The peroxisome proliferator-activated receptor gamma (PPAR-γ) is an important nuclear receptor in enterocytes which is known to have anti-inflammatory activity by interfering with NF-κB activation. However, the role of BB/PPAR-γ (and the possible mechanism involved) in protecting against IL-1β increase in intestinal permeability remain unclear. Aims: The major purpose of this study was to delineate the protective effect of BB against the IL-1β induced increase in intestinal TJ permeability and the intracellular mechanism involved. Methods: Filter-grown Caco-2 monolayers ( in vitro) and recycling intestinal perfusion of live mice ( in vivo) were used to assess intestinal permeability by using a paracellular marker (dextran-10kDa). Results: IL-1β caused a rapid activation of NF-κB and NF-κB and MLCK-dependent increase in intestinal epithelial TJ permeability in vitro and in vivo. A specific strain of BB (1x108 CFU/ml) referred to as BB1 strain inhibited the IL-1β increase in intestinal TJ permeability while strain BB4 had no effect. Other BB strains examined (5 total strains including BB4) did not inhibit the IL-1β increase in intestinal TJ permeability, suggesting that the BB enhancement of Caco-2 TJ barrier function was strain-specific. BB1, not BB4 enhancement of the TJ barrier was associated with an increase in TLR-2 expression, and PPAR-γ activation; BB1 also inhibited NF-kB activation and MLCK activity in Caco-2 monolayers and in mouse enterocyte. The inhibitory effect of BB on 1L-1β induced increase intestinal permeability, NF-κB activation, MLCK expression and activity was blocked by siRNA-induced knockdown of TLR-2 or PPAR-γ in Caco-2 monolayers. We also generated Villin-cre intestinal epithelial specific PPAR-γ knockout mice to study the involvement of enterocytes TLR-2/PPAR-γ on BB1 effect. BB1 did not inhibit the IL-1β-induced activation of NF-κB, increase in MLCK expression and increase in mouse intestinal permeability in TLR-2 or PPAR-γ intestinal deficient mice. Conclusion: These studies provide a novel insight into the BB the protective mechanism against the IL-1β-induced increase in intestinal TJ permeability in -vitro and in-vivo. Our data show that BB protects against the IL-1β induced increase in intestinal TJ permeability by a novel mechanism involving TLR-2/PPAR-g mediated inhibition of NF-kB and MLCK gene activatiation. NIH funding This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract B031: Inhibition of mutant RAS via myotubularin related protein 7-mimicking peptide

Abstract The coiled coil domain of Myotubularin Related Protein 7 (MTMR7) and a peptide of the same sequence (M7CC) have been shown to have an anti-cancer effect via direct interaction with PPARγ (Weidner et al. 2020). The same studies also found an inhibition of ERK-mediated inactivation of PPARγ by the same peptide. In this work we have followed up on our previous findings and show a direct effect on mutant RAS activity, mediated by MTMR7 and M7CC. In the presence of MTMR7 or M7CC, a drop of active RAS-levels along with a delocalization from the plasma membrane can be observed. This goes along with a significant drop in the levels of phosphorylated ERK and the expression of RAS-controlled genes. This led to a decrease of cell proliferation in cell culture in different cancer cell lines and of tumor volume in mouse models. The effect of MTMR7 or M7CC on RAS levels was shown to be mediated by a direct interaction with RAS. Using biophysical studies including solution-state NMR spectroscopy, the interaction was in vitro shown to happen with the globular domain of K-Ras4B. Our results support a model for MTMR7 as part of a novel RAS-regulation machinery, and highlights MTMR7 as promising scaffold to be exploited for peptide-based cancer treatment.

Telmisartan Is a Promising Agent for Managing Neuropathic Pain and Delaying Opioid Analgesic Tolerance in Rats.

Despite the large arsenal of analgesic medications, neuropathic pain (NP) management is not solved yet. Angiotensin II receptor type 1 (AT1) has been identified as a potential target in NP therapy. Here, we investigate the antiallodynic effect of AT1 blockers telmisartan and losartan, and particularly their combination with morphine on rat mononeuropathic pain following acute or chronic oral administration. The impact of telmisartan on morphine analgesic tolerance was also assessed using the rat tail-flick assay. Morphine potency and efficacy in spinal cord samples of treated neuropathic animals were assessed by [35S]GTPγS-binding assay. Finally, the glutamate content of the cerebrospinal fluid (CSF) was measured by capillary electrophoresis. Oral telmisartan or losartan in higher doses showed an acute antiallodynic effect. In the chronic treatment study, the combination of subanalgesic doses of telmisartan and morphine ameliorated allodynia and resulted in a leftward shift in the dose-response curve of morphine in the [35S]GTPγS binding assay and increased CSF glutamate content. Telmisartan delayed morphine analgesic-tolerance development. Our study has identified a promising combination therapy composed of telmisartan and morphine for NP and opioid tolerance. Since telmisartan is an inhibitor of AT1 and activator of PPAR-γ, future studies are needed to analyze the effect of each component.

PSTPIP2 alleviates obesity associated adipose tissue inflammation and insulin resistance in diabetes mice through promoting M2 macrophage polarization via activation of PPARγ

BackgroundProline-serine-threonine phosphatase-interacting protein 2 (PSTPIP2) plays a role in inflammatory disease. In diabetes, very little is known about PSTPIP2 until now. Hence, this study aimed to determine PSTPIP2 functional role in diabetes. MethodsDiabetes mouse model was constructed by feeding high fat diet (HFD). Intraperitoneal glucose tolerance test and intraperitoneal insulin tolerance test were examined the glucose and insulin tolerance. The expression of genes and proteins was detected by quantitative real time PCR, immunohistochemistry and western blotting. The pathological changes of epididymal adipose tissues were examined by hematoxylin-eosin staining. RAW264.7 macrophages were treated with GW9662 (PPARγ antagonist). Flow cytometry examined the proportion of M1/M2 macrophages. ResultsHFD enhanced the body weight, glucose and insulin tolerance, and inhibited PSTPIP2 expression in mice. PSTPIP2 overexpression alleviated glucose and insulin tolerance, reduced inflammation and macrophage accumulation in the epididymal adipose tissues of diabetic mice. The expression of iNOS and TNF-α was increased, the expression of IL-10 and Arg-1 was decreased in diabetic mice, which was abrogated by PSTPIP2 overexpression. In vitro, PSTPIP2 overexpression reduced the proportions of iNOS-positive cells and enhanced the proportions of CD206-positive cells in RAW264.7 cells. PPARγ and p-STAT6 were up-regulated, STAT6 was down-regulated in RAW264.7 cells. GW9662 impaired PSTPIP2 overexpression-mediated up-regulation of Arg-1, YM-1 and FIZZ1 in RAW264.7 cells. ConclusionPSTPIP2 alleviates obesity associated adipose tissue inflammation and insulin resistance in diabetic mice through promoting M2 macrophage polarization via activation of PPARγ, suggesting that PSTPIP2 is a prospective target for diabetes treatment.

Docosahexaenoic acid protects against lipopolysaccharide-induced fetal growth restriction via inducing the ubiquitination and degradation of NF-κB p65 in placental trophoblasts

Lipopolysaccharide (LPS) could induce adverse birth outcomes by evoking inflammation. We investigated the effect and mechanism of docosahexaenoic acid (DHA) on LPS-induced placental inflammation and fetal growth restriction (FGR). In vivo, pregnant CD-1 mice were divided into four groups: Ctrl, DHA, LPS and DHA+LPS group. We found that DHA pretreatment reduced the incidence of FGR induced by LPS and activated the expression of peroxisome proliferators-activated receptor gamma (PPARγ) in placental tissue. Moreover, the LPS-induced increase of mRNA levels of Tnf-α, Il-6, Il-1β, Mip-2 and Kc in placental tissue was significantly attenuated by DHA pretreatment. A similar effect of DHA was observed in serum of pregnant mice and amniotic fluid. In contrast, the levels of the IL-10 were significantly increased after DHA pretreatment. In vitro, we clarified that DHA antagonized the activation of the NF-κB signaling pathway induced by LPS, which was dependent on PPARγ. Subsequently, CHX (translation inhibitor) was used to indicated that PPARγ significantly increased the degradation rate of p65, an effect that was inhibited by MG132 (proteasome inhibitor) treatment. Finally, it was confirmed that the activation of PPARγ could significantly promote the ubiquitination and degradation of p65. Our results suggested that DHA alleviated LPS-induced inflammatory responses and FGR by activating PPARγ expression, leading to p65 ubiquitination and degradation.

Anti-Diabetic Therapy and Heart Failure: Recent Advances in Clinical Evidence and Molecular Mechanism.

Diabetic patients have a two- to four-fold increase in the risk of heart failure (HF), and the co-existence of diabetes and HF is associated with poor prognosis. In randomized clinical trials (RCTs), compelling evidence has demonstrated the beneficial effects of sodium-glucose co-transporter-2 inhibitors on HF. The mechanism includes increased glucosuria, restored tubular glomerular feedback with attenuated renin-angiotensin II-aldosterone activation, improved energy utilization, decreased sympathetic tone, improved mitochondria calcium homeostasis, enhanced autophagy, and reduced cardiac inflammation, oxidative stress, and fibrosis. The RCTs demonstrated a neutral effect of the glucagon-like peptide receptor agonist on HF despite its weight-reducing effect, probably due to it possibly increasing the heart rate via increasing cyclic adenosine monophosphate (cAMP). Observational studies supported the markedly beneficial effects of bariatric and metabolic surgery on HF despite no current supporting evidence from RCTs. Bromocriptine can be used to treat peripartum cardiomyopathy by reducing the harmful cleaved prolactin fragments during late pregnancy. Preclinical studies suggest the possible beneficial effect of imeglimin on HF through improving mitochondrial function, but further clinical evidence is needed. Although abundant preclinical and observational studies support the beneficial effects of metformin on HF, there is limited evidence from RCTs. Thiazolidinediones increase the risk of hospitalized HF through increasing renal tubular sodium reabsorption mediated via both the genomic and non-genomic action of PPARγ. RCTs suggest that dipeptidyl peptidase-4 inhibitors, including saxagliptin and possibly alogliptin, may increase the risk of hospitalized HF, probably owing to increased circulating vasoactive peptides, which impair endothelial function, activate sympathetic tones, and cause cardiac remodeling. Observational studies and RCTs have demonstrated the neutral effects of insulin, sulfonylureas, an alpha-glucosidase inhibitor, and lifestyle interventions on HF in diabetic patients.

Identification of molecular initiating events (MIE) using chemical database analysis and nuclear receptor activity assays for screening potential inhalation toxicants

An adverse outcome pathway (AOP) framework can facilitate the use of alternative assays in chemical regulations by providing scientific evidence. Previously, an AOP, peroxisome proliferative-activating receptor gamma (PPARγ) antagonism that leads to pulmonary fibrosis, was developed. Based on a literature search, PPARγ inactivation has been proposed as a molecular initiating event (MIE). In addition, a list of candidate chemicals that could be used in the experimental validation was proposed using toxicity database and deep learning models. In this study, the screening of environmental chemicals for MIE was conducted using in silico and in vitro tests to maximize the applicability of this AOP for screening inhalation toxicants. Initially, potential inhalation exposure chemicals that are active in three or more key events were selected, and in silico molecular docking was performed. Among the chemicals with low binding energy to PPARγ, nine chemicals were selected for validation of the AOP using in vitro PPARγ activity assay. As a result, rotenone, triorthocresyl phosphate, and castor oil were proposed as PPARγ antagonists and stressor chemicals of the AOP. Overall, the proposed tiered approach of the database-in silico-in vitro can help identify the regulatory applicability and assist in the development and experimental validation of AOP.

Partial Activation of PPAR-γ by Synthesized Quercetin Derivatives Modulates TGF-β1-Induced EMT in Lung Cancer Cells.

Non-small cell lung cancer (NSCLC) has a very low survival rate due to poor response to chemotherapy and late detection. Epithelial to mesenchymal transition (EMT) is regarded as a major contributor to drive metastasis during NSCLC progression. Towards this, transforming growth factor-beta 1 (TGF-β1) is the key driver that endows cancer cells with increased aggressiveness. Recently, this group synthesized a series of Schiff base quercetin derivatives (QDs) and ascertained their effectiveness on EMT markers of A549 cell line. This study evidenced that the EMT process is counteracted via the partial activation of a nuclear hormone receptor, Peroxisome proliferator-activated receptor (PPAR)-γ through QDs. Here, that work is extended to investigate the interplay between PPAR-γ partial activation and TGF-β1-induced EMT in human lung cancer A549 cells. The results reveal that TGF-β1 plays a critical role in suppressing PPAR-γ, which is markedly reversed and increased by partial agonists: QUE2FH and QUESH at both protein and transcriptional levels. The partial agonists not only stimulate PPAR-γ in a balanced manner but also prevent the loss of E-cadherin and acquisition of TGF-β1-induced mesenchymal markers (Snail, Slug, Vimentin, and Zeb-1). Subsequently, the effects are accompanied by attenuation of TGF-β1-induced migratory ability of A549 cells.

Shikonin Binds and Represses PPARγ Activity by Releasing Coactivators and Modulating Histone Methylation Codes

Shikonin, a natural ingredient produced by Lithospermum erythrorhizon, has anti-inflammatory, anti-cancer, and anti-obesity effects. It also inhibits adipocyte differentiation; however, the underlying molecular and epigenetic mechanisms remain unclear. We performed RNA-sequencing of shikonin-treated 3T3-L1 cells. Gene ontology and gene set enrichment analysis showed that shikonin is significantly associated with genes related to adipogenesis, histone modification, and PPARγ. Shikonin treatment downregulated the mRNA expression of PPARγ-responsive genes and rosiglitazone-induced transcriptional activity of PPARγ. Microscale thermophoresis assays showed a KD value 1.4 ± 0.13 μM for binding between shikonin and PPARγ. Glutathione S-transferase pull-down assays exhibited that shikonin blocked the rosiglitazone-dependent association of PPARγ with its coactivator CBP. In addition, shikonin decreased the enrichment of the active histone code H3K4me3 and increased the repressive code H3K27me3 of PPARγ target promoters. Shikonin is a PPARγ antagonist that suppresses adipogenesis by regulating the enrichment of histone codes during adipogenesis. Therefore, it may be used to treat obesity-related disorders via epigenetic changes.

Design, Synthesis, and Biological Evaluation of Novel Quercetin Derivatives as PPAR-γ Partial Agonists by Modulating Epithelial-Mesenchymal Transition in Lung Cancer Metastasis.

Epithelial-to-mesenchymal transition (EMT) is responsible for driving metastasis of multiple cancer types including lung cancer. Peroxisome proliferator-activated receptor (PPAR)-γ, a ligand-activated transcription factor, controls expression of variety of genes involved in EMT. Although several synthetic compounds act as potent full agonists for PPAR-γ, their long term application is restricted due to serious adverse effects.Therefore, partial agonists involving reduced and balanced PPAR-γ activity are more effective and valued. A previous study discerned the efficacy of quercetin and its derivatives to attain favorable stabilization with PPAR-γ. Here this work is extended by synthesizing five novel quercetin derivatives (QDs) namely thiosemicarbazone (QUETSC)) and hydrazones (quercetin isonicotinic acid hydrazone (QUEINH), quercetin nicotinic acid hydrazone (QUENH), quercetin 2-furoic hydrazone (QUE2FH), and quercetin salicyl hydrazone (QUESH)) and their effects are analyzed in modulating EMT in lung cancer cell lines via PPAR-γ partial activation. QDs-treated A549 cells diminish cell proliferation strongly at nanomolar concentration compared to NCI-H460 cells. Of the five screened derivatives, QUETSC, QUE2FH, and QUESH exhibit the property of partial activation as compared to the overexpressive level of rosiglitazone. Consistently, these QDs also suppress EMT process by markedly downregulating the levels of mesenchymal markers (Snail, Slug, and zinc finger E-box binding homeobox 1) and concomitant upregulation of epithelial marker (E-cadherin).

Abstract 4849: PPARγ and FoxO1 expression and activity in aqueous cinnamon extract treated cells

Abstract Peroxisome-proliferator activated receptor (PPARγ) has been proven to have inhibitory effects on tumor angiogenesis. Research has demonstrated that natural PPARγ ligands are anti-tumorigenic due to growth inhibition and anti-proliferative effects. Cinnamon is one potential PPARγ ligand tested to explore its effects on PPARγ and FoxO1 expression and activity. Literature also suggests that breast cancer proliferation is increased with obesity and co-cultures of adipocytes with MCF-7 cells have suggested an increase in lipase activity. We therefore sought to explore the effects of cinnamon on these transcription factors and lipase activity in 3T3-L1 cells to determine if cinnamon is a plausible co-therapeutic for breast cancer patients. 3T3-L1 cells were cinnamon treated for 48-96 hours post differentiation. Nuclear protein was extracted for ELISA quantification of PPARγ and FoxO1 binding potential. Cytosolic and nuclear protein extracts were analyzed via Western blot of FoxO1 and PPARγ. Copy DNA was produced from the total RNA and expression levels of PPARγ and FoxO1 were examined by TaqMan real-time PCR assays. A lipase activity assay was performed on freshly extracted protein from cultured cells. Western blot data demonstrates an increase in FoxO1 protein with cinnamon treatment at 96 hours post differentiation. PPARγ data shows no significant changes in protein quantity, however ELISA data suggests increases in binding potential PPARγ as well as FoxO1 transcription factors with cinnamon treatment. Preliminary expression analysis suggests changes in both PPARγ and FoxO1. Lipase activity data suggests a significant increase in cinnamon treated adipocytes. Data from the study suggests that cinnamon activates PPARγ and that it may play a role in decreasing tumorigenic activity. However, with particular concern in breast cancer cells, the activation of PPARγ in adipocytes appears to increase the lipase activity, thus producing more free fatty acids and potentially enhancing energy dependent proliferation. Citation Format: Sean Mild, Cole Swartz, Amy Aulthouse, Amy Stockert. PPARγ and FoxO1 expression and activity in aqueous cinnamon extract treated cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4849.

Computational studies, synthesis, in-vitro binding and transcription analysis of novel imidazolidine-2,4‑dione and 2-thioxo thiazolidine-4-one based glitazones for central PPAR-γ agonism

Herein, we describe the structure-based designing and synthesis of novel glitazones with imidazolidine 2, 4‑dione and 2-thioxo thiazolidine-4-one ring structures as the fundamental scaffold for central PPAR-γ binding and activation. The central activation of PPAR-γ/coactivator PGC-1α induces a beneficial effect in neurodegenerative disorders. The compounds were rationally designed with particular emphasis on toxicity parameters and subjected to computational screening. Totally, four compounds were selected from an in-house library of designed glitazones based on imidazolidine 2, 4‑dione and 2-thioxo thiazolidine-4-one ring structures for further investigation for PPAR-γ activation. Molecular docking of designed glitazones with PPAR-γ proteins revealed more binding affinity with hydrogen bond interaction of protein residue at Ser289, His323, Tyr327, and Tyr473 compared with standard Pioglitazone. The structural stability of protein-ligand complexes was confirmed by molecular dynamic (MD) simulation (100 ns), and results indicate significant interactions with PPAR-γ active sites. Additionally, the relationship between geometry and electronic properties of chemical compounds were investigated using HOMO-LUMO, MEP, and NBO analysis. The compounds were synthesized by established synthetic schemes and structurally analyzed using IR, 1H NMR, 13C NMR and mass spectral analysis. The binding affinity of the compounds with the target protein (PPAR-γ) was further confirmed by an in vitro competitive binding assay with values ranging from 0.951μM- 4.768 μM. TR-FRET binding assay also concluded the ability of the novel compounds G5 and G7 to bind specifically at the LBD of PPAR-γ. Among the developed glitazones, the compound G7 activates PPAR-γ in a dose-dependent manner in transcriptional assay. Our results also indicated that compounds G5 and G7 exhibit nontoxicity (IC50>120 μM) against the SH-SY5Y cell lines and both could be a most bioactive lead candidates for further investigations.

Ursolic acid promotes microglial polarization toward the M2 phenotype via PPARγ regulation of MMP2 transcription

Microglia, which are the primary inflammatory cells of the brain, can undergo phenotypic switching between M1 and M2 polarization, which have opposing effects on inflammation. Peroxisome proliferator-activated receptor gamma (PPARγ) is a member of the nuclear receptor family of ligand-inducible transcription factors, and PPARγ is known to regulate M2 macrophage polarization. Previous studies have shown that the natural pentacyclic triterpenoid ursolic acid (3β-hydroxy-urs-12-en-28-oic acid; UA) influences microglial activation. Additionally, UA increases tissue inhibitor matrix metalloproteinase 1 (TIMP1), while greatly reducing the release of matrix metalloproteinase 2 (MMP2) and MMP9 in a PPARγ-dependent manner. Here, we examined the anti-inflammatory properties of UA by observing how well it promotes the phenotypic transition of lipopolysaccharide (LPS) and interferon gamma (IFNγ)-activated BV2 microglia from M1 to M2 polarization. To determine if PPARγ is involved in the underlying molecular pathway, we treated rats with UA and the PPARγ inhibitor BADGE. We also investigated the mechanisms by which PPARγ controls transcription from the MMP2 promoter. The in-vitro experiments showed that UA shifted LPS/IFNγ-activated BV2 microglia from the M1 to the M2 phenotype, which was associated with a reduction in the neurotoxic factors MMP2 and MMP9, and an increase in the anti-inflammatory factor TIMP1. Co-treatment with increased MMP2 and MMP9 synthesis while decreasing TIMP1 release, indicating that UA has anti-inflammatory effects on LPS/IFNγ-activated BV2 cells via activation of PPARγ. Next, we found that PPARγ directly influences MMP2 transcriptional activity by identifying the crucial peroxisome proliferator response element (PPRE) among five potential PPREs in the MMP2 promoter. These results suggest that UA has a protective anti-inflammatory effect against neuroinflammatory toxicity, which is exerted by direct activation of PPARγ and selectively modulates microglial polarization and suppresses MMP2 formation.

Inhibition of neuronal peroxisome proliferator-activated receptor-γ attenuates motor function improvement after spinal cord injury in rats.

Traumatic spinal cord injury (SCI) causes secondary damage in injured and adjacent regions due to temporal deprivation of oxygen and energy supply. Peroxisome proliferator-activated receptor γ (PPARγ) is known to regulate cell survival mechanisms such as hypoxia, oxidative stress, inflammation and energy homeostasis in various tissues. Thus, PPARγ has the potential to show neuroprotective properties. However, the role of endogenous spinal PPARγ in SCI is not well established. In this study, under isoflurane inhalation, a 10-g rod was freely dropped onto the exposed spinal cord after T10 laminectomy using a New York University impactor in male Sprague-Dawley rats. Cellular localization of spinal PPARγ, locomotor function and mRNA levels of various genes including NFκB-targeted pro-inflammatory mediators after intrathecal administration of PPARγ antagonists, agonists or vehicles in SCI rats were then analysed. In both sham and SCI rats, spinal PPARγ was presented in neurons but not in microglia or astrocytes. Inhibition of PPARγ induced IκB activation and increased mRNA levels of pro-inflammatory mediators. It also suppressed recovery of locomotor function with myelin-related gene expression in SCI rats. However, a PPARγ agonist showed no beneficial effects on the locomotor performances of SCI rats, although it further increased the protein expression of PPARγ. In conclusion, endogenous PPARγ has a role in anti-inflammation after SCI. Inhibition of PPARγ might have a negative influence on motor function recovery through accelerated neuroinflammation. Nonetheless, exogenous PPARγ activation does not appear to effectively help with functional improvement after SCI.