Selective PPARγ Antagonist GW9662 Research Articles

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.

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.

Andrographis paniculata and Its Main Bioactive Ingredient Andrographolide Decrease Alcohol Drinking and Seeking in Rats Through Activation of Nuclear PPARγ Pathway.

Andrographis paniculata is an annual herbaceous plant which belongs to the Acanthaceae family. Extracts from this plant have shown hepatoprotective, anti-inflammatory and antidiabetic properties, at least in part, through activation of the nuclear receptor Peroxisome Proliferator-Activated Receptor-gamma (PPAR γ). Recent evidence has demonstrated that activation of PPARγ reduces alcohol drinking and seeking in Marchigian Sardinian (msP) alcohol-preferring rats. The present study evaluated whether A. paniculata reduces alcohol drinking and relapse in msP rats by activating PPARγ. Oral administration of an A. paniculata dried extract (0, 15, 150mg/kg) lowered voluntary alcohol consumption in a dose-dependent manner and achieved ~65% reduction at the dose of 450mg/kg. Water and food consumption were not affected by the treatment. Administration of Andrographolide (5 and 10mg/kg), the main active component of A. paniculata, also reduced alcohol drinking. This effect was suppressed by the selective PPARγ antagonist GW9662. Subsequently, we showed that oral administration of A. paniculata (0, 150, 450mg/kg) prevented yohimbine- but not cues-induced reinstatement of alcohol seeking. Results point to A. paniculata-mediated PPARγactivation as a possible therapeutic strategy to treat alcohol use disorder.

Further evidence for the involvement of the PPARγ system on alcohol intake and sensitivity in rodents.

Peroxisome Proliferator Activator receptors (PPARs) are intracellular receptors that function as transcription factors, which regulate specific metabolic and inflammatory processes. PPARs are broadly distributed in the body and are also expressed in the central nervous system, especially in areas involved in addiction-related behavioral responses. Recent studies support a role of PPARs in alcoholism and pioglitazone: a PPARγ agonist used for treatment of type 2 diabetes showed efficacy in reducing alcohol drinking, stress-induced relapse, and alcohol withdrawal syndrome in rats. In the current work, we tested the pharmacological effects of pioglitazone on binge-like alcohol consumption using an intermittent two-bottle choice paradigm in Wistar rats and on the "drinking in the dark" (DID) model in mice with selective deletion of PPARγ in neurons. Our data show that repeated administration of pioglitazone (10, 30mg/kg) reduces high voluntary alcohol consumption in Wistar rats. Pre-treatment with the selective PPARγ antagonist GW9662 (5mg/kg) completely prevented the effect of pioglitazone, demonstrating that its action is specifically mediated by activation of PPARγ. In line with this result, repeated administration of pioglitazone (30mg/kg) attenuated binge alcohol consumption in PPARγ(+/+) mice. Whereas in PPARγ(-/-) mice, which exhibit reduced alcohol consumption, pioglitazone had no effect. Of note, PPARγ(-/-) mice exhibited lower patterns of alcohol drinking without showing difference in sucrose (control) intake. Interestingly, PPARγ(-/-) mice displayed a higher sensitivity to the sedative and ataxic effect of alcohol compared with their wild-type counterpart. Collectively, these data suggest that PPARγ agonists, and specifically pioglitazone, could be potential therapeutics for the treatment of binge alcohol drinking.

Promoting differentiation and lipid metabolism are the primary effects for DINP exposure on 3T3-L1 preadipocytes

Diisononyl phthalate (DINP) is a high-molecular-weight phthalate, and has been recently introduced as di-(2-ethyl hexyl) phthalate (DEHP) substitute and commonly used in a large variety of plastic items. The fat tissue is an important target for DINP exposure, however, very little is understood about its toxicity and mechanism(s) in adipocyte cells. Therefore, the present work aimed to investigate the role of DINP in adipogenesis using 3T3-L1 preadipocytes. DINP exposure for 10 days extensively induced adipogenesis in 3T3-L1 preadipocytes to adipocytes as assessed by lipid accumulation and gene expression of adipogenic markers. The RT-qPCR results showed that DINP could upregulate the expression of peroxisome proliferator-activated receptor-gamma (PPARγ), CCAAT/enhancer-binding protein alpha (C/EBPα) and C/EBPβ, while the expression of sterol regulatory element binding transcription factor 1 (SREBF1) and C/EBPδ was not affected. The DINP-induced adipogenesis could be inhibited by using the selective PPARγ antagonist GW9662. The RNA-seq analysis was used to study the systemic toxicities of DINP on preadipocytes. A total of 1181 differently expressed genes (DEGs) (640 genes were up-regulated, 541 genes were down-regulated) were detected in 3T3-L1 preadipocytes under 50 μM DINP. The GO enrichment showed the GO term of “fat cell differentiation” was the most significantly affected metabolic functions, and the KEGG pathway enrichment showed the PPAR pathway was the top affected pathway. The interactive pathway (iPath) analysis showed that the changed metabolic pathways were focus on the lipid metabolism.

Pseudolaric acid B attenuates atherosclerosis progression and inflammation by suppressing PPARγ-mediated NF-κB activation

Aims/objectiveAtherosclerosis is a progressive disease of large arteries characterized with chronic inflammation and aberrant immune response. Pseudolaric acid B (PB) has been found to exert multiple effects by inhibiting inflammatory response. However, there is no comprehensive assessment of the effects of PB on atherosclerosis using relevant in vivo and in vitro models. Material and methodsMale ApoE−/− mice were treated with PB orally with a high fat diet (HFD) to clarify its anti-atherosclerotic activities. RAW264.7 macrophage line, a well-accepted cell model of atherosclerosis, was used to investigate anti-inflammatory effects and molecular mechanisms of PB. ResultsPB significantly attenuated atherosclerotic lesions by modulating plasma lipid profiles as well as inhibiting inflammatory responses in macrophages of atherosclerotic mice. Meanwhile, PB markedly suppressed the expression of pro-inflammatory cytokines, and regulated cholesterol efflux related genes in oxidative low density lipoprotein (ox-LDL)-loaded macrophages. The cellular uptake of Dil-labeled ox-LDL was significantly inhibited by PB either. Moreover, the ability of PB to suppress nuclear factor kappa B (NF-κB) and activate peroxisome proliferator-activated receptor gamma (PPARγ) was confirmed using luciferase reporter assays. Conversely, the selective PPARγ antagonist GW9662 reversed the influence of PB in macrophages. ConclusionTogether, these findings indicate that PB exerts its protective effects on atherosclerosis by inhibiting macrophage-mediated inflammatory response and cellular ox-LDL uptake, and promoting cholesterol efflux by suppressing NF-κB activation PPARγ-dependently. Therefore, PB may be a promising agent for inflammatory and atherosclerotic diseases.

Troglitazone Inhibits Matrix Metalloproteinase-9 Expression and Invasion of Breast Cancer Cell through a Peroxisome Proliferator-Activated Receptor γ-Dependent Mechanism.

PurposePeroxisome proliferator-activated receptor γ (PPARγ) is involved in the pathology of numerous diseases including atherosclerosis, diabetes, obesity, and cancer. Matrix metalloproteinases (MMPs) play a significant role in tissue remodeling related to various processes such as morphogenesis, angiogenesis, tissue repair, invasion, and metastasis. We investigated the effects of PPARγ on MMP expression and invasion in breast cancer cells.MethodsMCF-7 cells were cultured and then cell viability was monitored in an MTT assay. Western blotting, gelatin zymography, real-time polymerase chain reaction, and luciferase assays were performed to investigate the effect of the synthetic PPARγ ligand troglitazone on MMP expression. Transcription factor DNA binding was analyzed by electrophoretic mobility shift assay. A Matrigel invasion assay was used to assess the effects of troglitazone on MCF-7 cells.ResultsTroglitazone did not affect MCF-7 cell viability. 12-O-tetradecanoylphorbol-13-acetate (TPA) induced MMP-9 expression and invasion in MCF-7 cell. However, these effects were decreased by troglitazone. TPA increased nuclear factor κB and activator protein-1 DNA binding, while troglitazone inhibited these effects. The selective PPARγ antagonist GW9662 reversed MMP-9 inhibition by troglitazone in TPA-treated MCF-7 cells.ConclusionTroglitazone inhibited nuclear factor κB and activator protein-1-mediated MMP-9 expression and invasion of MCF-7 cells through a PPARγ-dependent mechanism.

Firemaster® 550 and its components isopropylated triphenyl phosphate and triphenyl phosphate enhance adipogenesis and transcriptional activity of peroxisome proliferator activated receptor (Pparγ) on the adipocyte protein 2 (aP2) promoter.

Firemaster® 550 (FM550) is a chemical mixture currently used as an additive flame retardant in commercial products, and is comprised of 2-ethylhexyl-2,3,4,5-tertrabromobenzoate (TBB), bis(2-ethylhexyl) tetrabromophthalate (TBPH), triphenyl phosphate (TPP), and isopropylated triphenyl phosphate (IPTP). Animal and in vitro studies suggest that FM550, TPP and IPTP may have adipogenic effects and may exert these effects through PPARγ activation. Using murine 3T3-L1 preadipocytes, we investigated the detailed expression of transcription factors and adipogenic markers in response to FM550 and its components. Further we investigated the mechanism of action of the peroxisome proliferator-activated receptor gamma (PPARγ) on downstream targets of the receptor by focussing on the mature adipocyte marker, adipocyte protein 2 (aP2). In addition, we set to elucidate the components responsible for the adipogenic effects seen in the FM550 mixture. We show that FM550 and its components TPP, IPTP, and TBPH, but not TBB induced lipid accumulation in a dose-dependent manner. Interestingly, despite displaying enhanced lipid accumulation, TBPH did not alter the mRNA or protein expression of terminal differentiation markers. In contrast, FM550, TPP, and IPTP treatment enhanced lipid accumulation, and mRNA and protein expression of terminal differentiation markers. To further delineate the mechanisms of action of FM550 and its components we focussed on aP2 promoter activity. For this purpose we used the enhancer region of the mouse aP2 promoter using a 584-bp reporter construct containing an active PPRE located 5.4 kb away from the transcription start site of aP2. Exposure to FM550, IPTP, and TPP significantly increased PPARγ mediated aP2 enhancer activity. Furthermore, we show that TPP- and IPTP-dependent upregulation of aP2 was significantly inhibited by the selective PPARγ antagonist GW9662. In addition, chromatin immunoprecipitation experiments showed that IPTP and TPP treatment led to the recruitment of PPARγ to the regulatory region of aP2.

Bisphenol S- and bisphenol A-induced adipogenesis of murine preadipocytes occurs through direct peroxisome proliferator-activated receptor gamma activation.

The use of bisphenol A (BPA) in consumer products and food packaging has been associated under certain conditions with a risk of negative health outcomes. This prompted its removal from many products and replacement with structural analogs. Bisphenol S (BPS) is one such analog, but its metabolic effects have not been fully characterized. The objective of our study was to determine whether BPS functions similarly to BPA at inducing adipogenesis. Murine 3T3-L1 preadipocytes were used to evaluate and compare the adipogenic potential of BPS to BPA. Cells were treated with 0.01-50 μM BPS or 0.01-50 μM BPA and adipogenic effects were measured. Further, their ability to activate peroxisome proliferator-activated receptor gamma (PPARγ), an adipogenic transcription factor, was also determined. Our results indicate that treatment of 3T3-L1 cells with BPS induced lipid accumulation and increased mRNA and protein expression of key adipogenic markers (1-50 μM; P<0.05). BPS treatment resulted in a higher expression of adipogenic markers as well as greater lipid accumulation when compared with BPA treatment. We showed that BPS can upregulate lipoprotein lipase, adipocyte protein 2, PPARγ, perilipin, adipsin and CCAAT/enhancer-binding protein alpha mRNA expression levels. Furthermore, using transcriptional assays, we showed that BPS and BPA can modestly activate PPARγ using a PPRE (PPARγ response element)-dependent luciferase construct by 1.5-fold (P<0.05). However, BPS but not BPA was able to competitively inhibit rosiglitazone (ROSI)-activated PPARγ, suggesting that BPS interacts with PPARγ distinctly from BPA. Co-treatment of cells with the selective PPARγ antagonist GW9662 inhibits BPS-, BPA-, ROSI- but not dexamethasone-dependent adipogenic differentiation. Both BPA and BPS can enhance 3T3-L1 adipocyte differentiation in a dose-dependent manner and require PPARγ to induce adipogenesis. Through direct comparison, we show that BPS is a more potent adipogen than BPA.

Telmisartan attenuated LPS-induced neuroinflammation in human IMR-32 neuronal cell line via SARM in AT1R independent mechanism.

The aim of this study was to find the protective role of Telmisartan (TS) in LPS intoxicated neuronal cells and elucidate the possible neuroprotective mechanism of action. TLR4 and AT1R specific primers were designed and used in rtPCR to confirm the receptor expression in IMR-32 and Neuro2A cell lines. The protective effect of TS was assayed by MTT assay. The mechanism of action of TS was elucidated by assessing the expression and activation of TLR4 specific adaptor proteins SARM and MyD88, phosphorylated NFκB, PPARγ, MAPK p38, c-JNK, ERK by Western blotting. Selective PPARγ antagonist GW9662 was used to confirm the link between PPARγ activation and TLR4 mediated NFκB inflammatory mechanisms. The pro-inflammatory cytokines TNFα, IL1β, and IL-6 and anti-inflammatory cytokine IL10 release were measured by ELISA. IMR-32 cells expressed TLR4 receptor and Neuro2A cells expressed both AT1R and TLR4 receptors. TS significantly protected both the cell lines from LPS intoxication. TS significantly suppressed the TLR4 mediated inflammatory response by PPARγ and SARM protein activation and the effect was reversed significantly by selective PPARγ antagonist GW9662, confirming the existence of link between PPARγ activation and TLR4 mediated inflammation via SARM. LPS intoxicated human neuronal IMR-32 cells can be a good in vitro model to study inflammatory mediated neurodegeneration due to TLR4 receptor expression. Our study strongly recommends that the PPARγ activating pleiotropic effect of TS is responsible for the protective effect in LPS induced TLR4 mediated inflammation via SARM adaptor protein in the IMR-32 cell line.

Impact of peroxisome proliferator-activated receptor γ on angiotensin II type 1 receptor-mediated insulin sensitivity, vascular inflammation and atherogenesis in hypercholesterolemic mice.

IntroductionThe angiotensin II type 1 receptor (AT1R) and the peroxisome proliferator-activated receptor γ (PPARγ) have been implicated in the pathogenesis of atherosclerosis. A number of studies have reported that AT1R inhibition or genetic AT1R disruption and PPARγ activation inhibit vascular inflammation and improve glucose and lipid metabolism, underscoring a molecular interaction of AT1R and PPARγ. We here analyzed the hypothesis that vasculoprotective anti-inflammatory and metabolic effects of AT1R inhibition are mediated by PPARγ.Material and methodsFemale ApoE–/–/AT1R–/– mice were fedwith a high-fat and cholesterol-rich diet and received continuous treatment with the selective PPARγ antagonist GW9662 or vehicle at a rate of 700 ng/kg/min for 4 weeks using subcutaneously implanted osmotic mini-pumps. Additionally, one group of female ApoE–/– mice served as a control group. After treatment for 4 weeks mice were sacrificed and read-outs (plaque development, vascular inflammation and insulinsensitivity) were performed.ResultsUsing AT1R deficient ApoE–/– mice (ApoE–/–/AT1R–/– mice) we found decreased cholesterol-induced endothelial dysfunction and atherogenesis compared to ApoE–/– mice. Inhibition of PPARγ by application of the specific PPARγ antagonist GW9662 significantly abolished the anti-atherogenic effects of AT1R deficiency in ApoE–/–/AT1R–/– mice (plaque area as % of control: ApoE–/–: 39 ±5%; ApoE–/–/AT1R–/–: 17 ±7%, p = 0.044 vs. ApoE–/–; ApoE–/–/AT1R–/– + GW9662: 31 ±8%, p = 0.047 vs. ApoE–/–/AT1R–/–). Focusing on IL6 as a pro-inflammatory humoral marker we detected significantly increased IL-6 levels in GW9662-treated animals (IL-6 in pg/ml: ApoE–/–: 230 ±16; ApoE–/–/AT1R–/–: 117 ±20, p = 0.01 vs. ApoE–/–; ApoE–/–/AT1R–/– + GW9662: 199 ±20, p = 0.01 vs. ApoE–/–/AT1R–/–), while the anti-inflammatory marker IL-10 was significantly reduced after PPARγ inhibition in GW9662 animals (IL-10 in pg/ml: ApoE–/–: 18 ±4; ApoE–/–/AT1R–/–: 55 ±12, p = 0.03 vs. ApoE–/–; ApoE–/–/AT1R–/– + GW9662: 19 ±4, p = 0.03 vs. ApoE–/–/AT1R–/–). Metabolic parameters of glucose homeostasis (glucose and insulin tolerance test) were significantly deteriorated in ApoE–/–/AT1R–/– mice treated with GW9662 as compared to vehicle-treated ApoE–/–/AT1R–/– mice. Systolic blood pressure and plasma cholesterol levels were similar in all groups.ConclusionsGenetic disruption of the AT1R attenuates atherosclerosis and improves endothelial function in an ApoE–/– mouse model of hypercholesterolemia-induced atherosclerosis via PPARγ, indicating a significant role of PPARγ in reduced vascular inflammation, improvement of insulin sensitivity and atheroprotection of AT1R deficiency.

Role of macrophage PPARγ in experimental hypertension

Targeted disruption of the Alox15 gene makes mice resistant to angiotensin II-, DOCA/salt-, and N(ω)-nitro-L-arginine methyl ester (L-NAME)-induced experimental hypertension. Macrophages, a primary source of Alox15, are facilitating this resistance, but the underlying mechanism is not known. Because Alox15 metabolites are peroxisome proliferator-activated receptor (PPAR)γ agonists, we hypothesized that activation of macrophage PPARγ is the key step in Alox15 mediation of hypertension. Thioglycollate, used for macrophage elicitation, selectively upregulated PPARγ and its target gene CD36 in peritoneal macrophages of both wild-type (WT) and Alox15(-/-) mice. Moreover, thioglycollate-injected Alox15(-/-) mice became hypertensive upon L-NAME treatment. A similar hypertensive effect was observed with adoptive transfer of thioglycollate-elicited Alox15(-/-) macrophages into Alox15(-/-) recipient mice. The role of PPARγ was further specified by using the selective PPARγ antagonist GW9662. WT mice treated with 50 μg/kg daily dose of GW9662 for 12 days became resistant to L-NAME-induced hypertension. The PPARγ antagonist treatment also prevented L-NAME-induced hypertension in thioglycollate-injected Alox15(-/-) mice, indicating a PPARγ-mediated effect in macrophage elicitation and the resultant hypertension. These results indicate a regulatory role for macrophage-localized PPARγ in L-NAME-induced experimental hypertension.

AT1-receptor-deficiency induced atheroprotection in diabetic mice is partially mediated via PPARγ

ObjectivePeroxisome-proliferator–activated-receptor-γ (PPARγ) acts as a transcriptional regulator of multiple genes involved in glucose and lipid metabolism. In vitro studies showed that activated PPARγ suppresses AT1R-gene expression and vice versa. However, it has not yet been determined in vivo, whether AT1R-PPARγ-interactions play a relevant role in the pathogenesis of diabetic complications and specifically in accelerated atherosclerosis.Methods and resultsApoE−/− and ApoE−/−/AT1R−/−-mice were rendered diabetic by intraperitoneal injections of streptozotocin. Diabetic and non-diabetic ApoE−/−-mice were further randomized to receive the AT1R antagonist telmisartan, the selective PPARγ antagonist GW9662, telmisartan and GW9662 or vehicle for 18 weeks. Diabetic and non-diabetic ApoE−/−/AT1R−/−-mice were randomized to receive either GW9662 or vehicle. GW9662 treatment in diabetic ApoE−/− and diabetic ApoE−/−/AT1−/−-mice resulted in the highest elevation of fasting blood glucose levels, whereas telmisartan treatment and AT1 deficiency in ApoE−/−-mice showed the lowest fasting blood glucose levels. Diabetic ApoE−/−-mice displayed severe impairment of endothelial function, enhanced oxidative stress and increased atherosclerotic lesion formation. ApoE−/−/AT1R−/− and telmisartan-treated ApoE−/−-mice showed a significantly better endothelial function, decreased oxidative stress and reduced atherosclerotic lesion formation. Treatment of diabetic ApoE−/− and ApoE−/−/AT1R−/−-mice with the selective PPARγ antagonist GW9662 omitted the atheroprotective effects of AT1R deficiency or AT1 antagonism.ConclusionGenetic disruption or pharmacological inhibition of the AT1R attenuates atherosclerosis and improves endothelial function in diabetic ApoE−/−-mice via the PPARγ pathway.

Lycium barbarum (Goji Berry) extracts and its taurine component inhibit PPAR-γ-dependent gene transcription in human retinal pigment epithelial cells: Possible implications for diabetic retinopathy treatment

The peroxisome proliferator activated receptor-γ (PPAR-γ) is involved in the pathogenesis of diabetic retinopathy. Diabetic retinopathy is a preventable microvascular diabetic complication that damages human retinal pigment epithelial cells. Taurine is abundant in the fruit of Lycium barbarum (Goji Berry), and is reportedly beneficial for diabetic retinopathy. However, the mechanism of its action is unknown. Hence, we have investigated the mechanism of action of an extract from L. barbarum on a model of diabetic retinopathy, the retinal ARPE-19 cell line, and identified the receptor function of taurine, an active component of L. barbarum (Goji Berry) extract, which is potentially responsible for the protective effect on diabetic retinopathy. We demonstrate for the first time that L. barbarum extract and its taurine component dose-dependently enhance PPAR-γ luciferase activity in HEK293 cell line transfected with PPAR-γ reporter gene. This activity was significantly decreased by a selective PPAR-γ antagonist GW9662. Moreover, L. barbarum extract and taurine dose-dependently enhanced the expression of PPAR-γ mRNA and protein. In an inflammation model where ARPE-19 cells were exposed to high glucose L. barbarum extract and taurine down-regulated the mRNA of pro-inflammatory mediators encoding MMP-9, fibronectin and the protein expression of COX-2 and iNOS proteins. The predicted binding mode of taurine in the PPAR-γ ligand binding site mimics key electrostatic interactions seen with known PPAR-γ agonists. We conclude that PPAR-γ activation by L. barbarum extract is associated with its taurine content and may explain at least in part its use in diabetic retinopathy progression.

Activation of Nuclear PPARγ Receptors by the Antidiabetic Agent Pioglitazone Suppresses Alcohol Drinking and Relapse to Alcohol Seeking

Pioglitazone and rosiglitazone belong to the class of thiazolidinediones (TZDs). They were first developed as antioxidants and then approved for the clinical treatment of insulin resistance and Type 2 diabetes. TZDs bind with high affinity and activate peroxisome proliferator-activated receptor-gamma (PPARγ) receptors, which in the brain are expressed both in neurons and in glia. We evaluated the effect of PPARγ activation by TZDs on alcohol drinking, relapse-like behavior, and withdrawal in the rat. We also tested the effect of TZDs on alcohol and saccharin self-administration. We showed that activation of PPARγ receptors by pioglitazone (0, 10, and 30 mg/kg) and rosiglitazone (0, 10 and 30 mg/kg) given orally selectively reduced alcohol drinking. The effect was blocked by pretreatment with the selective PPARγ antagonist GW9662 (5 μg/rat) given into the lateral cerebroventricle, suggesting that this TZD's effect is mediated by PPARγ receptors in the central nervous system. Pioglitazone abolished reinstatement of alcohol seeking, a relapse-like behavior, induced by yohimbine, a pharmacologic stressor, but did not affect cue-induced relapse. In the self-administration experiments, pioglitazone reduced lever pressing for alcohol but not for saccharin. Finally, pioglitazone prevented the expression of somatic signs of alcohol withdrawal. These findings provide new information about the role of brain PPARγ receptors and identify pioglitazone as candidate treatments for alcoholism and possibly other addictions.

Rosiglitazone Abrogates Bleomycin-Induced Scleroderma and Blocks Profibrotic Responses Through Peroxisome Proliferator-Activated Receptor-γ

The nuclear hormone receptor, peroxisome proliferator-activated receptor (PPAR)-gamma, originally identified as a key mediator of adipogenesis, is expressed widely and implicated in diverse biological responses. Both natural and synthetic agonists of PPAR-gamma abrogated the stimulation of collagen synthesis and myofibroblast differentiation induced by transforming growth factor (TGF)-beta in vitro. To characterize the role of PPAR-gamma in the fibrotic process in vivo, the synthetic agonist rosiglitazone was used in a mouse model of scleroderma. Rosiglitazone attenuated bleomycin-induced skin inflammation and dermal fibrosis as well as subcutaneous lipoatrophy and counteracted the up-regulation of collagen gene expression and myofibroblast accumulation in the lesioned skin. Rosiglitazone treatment reduced the induction of the early-immediate transcription factor Egr-1 in situ without also blocking the activation of Smad2/3. In both explanted fibroblasts and skin organ cultures, rosiglitazone prevented the stimulation of collagen gene transcription and cell migration elicited by TGF-beta. Rosiglitazone-driven adipogenic differentiation of both fibroblasts and preadipocytes was abrogated in the presence of TGF-beta; this effect was accompanied by the concomitant down-regulation of cellular PPAR-gamma mRNA expression. Collectively, these results indicate that rosiglitazone treatment attenuates inflammation, dermal fibrosis, and subcutaneous lipoatrophy via PPAR-gamma in a mouse model of scleroderma and suggest that pharmacological PPAR-gamma ligands, widely used as insulin sensitizers in the treatment of type-2 diabetes mellitus, may be potential therapies for scleroderma.

Does the inhibition of c-myc expression mediate the anti-tumor activity of PPAR’s ligands in prostate cancer cell lines?

Peroxisome proliferator-activated receptor gamma (PPARγ) ligands seem to induce anticancer effects on prostate cancer cells, but the mechanism is not clear. The effect of PPARγ ligands ω-6 fatty acids and ciglitazone (2–15 μM)—on proliferation, and apoptosis of LNCaP, PC-3, DU145, CA-K and BPH-K cells was studied. PPARγ ligands led to: (1) reduction of proliferation (20–50%) of all the studied cell lines, (2) stimulation of differentiation of prostate cancer cells through an increased expression (1.5–3-fold: LNCaP, DU145, BPH-K) or reexpression (PC-3, CA-K) of E-cadherin with parallel inhibition of N-cadherin expression (PC-3, CA-K) and (3) down-regulation (1–2-fold) of β-catenin and c-myc expression. The selective PPARγ antagonist GW9662 abolished the effect of those ligands on prostate cancer cells. These results suggest that inhibition of β-catenin and in effect c-myc expression through activation of PPARγ may help prostate cancer cells to restore several characteristics of normal prostate cells phenotype.

PPARγ-independent induction of growth arrest and apoptosis in prostate and bladder carcinoma

BackgroundAlthough PPARγ antagonists have shown considerable pre-clinical efficacy, recent studies suggest PPARγ ligands induce PPARγ-independent effects. There is a need to better define such effects to permit rational utilization of these agents.MethodsWe have studied the effects of a range of endogenous and synthetic PPARγ ligands on proliferation, growth arrest (FACS analysis) and apoptosis (caspase-3/7 activation and DNA fragmentation) in multiple prostate carcinoma cell lines (DU145, PC-3 and LNCaP) and in a series of cell lines modelling metastatic transitional cell carcinoma of the bladder (TSU-Pr1, TSU-Pr1-B1 and TSU-Pr1-B2).Results15-deoxy-prostaglandin J2 (15dPGJ2), troglitazone (TGZ) and to a lesser extent ciglitazone exhibited inhibitory effects on cell number; the selective PPARγ antagonist GW9662 did not reverse these effects. Rosiglitazone and pioglitazone had no effect on proliferation. In addition, TGZ induced G0/G1 growth arrest whilst 15dPGJ2 induced apoptosis.ConclusionTroglitazone and 15dPGJ2 inhibit growth of prostate and bladder carcinoma cell lines through different mechanisms and the effects of both agents are PPARγ-independent.

The selective PPARγ antagonist GW9662 reverses the protection of LPS in a model of renal ischemia-reperfusion

We have recently reported that pretreatment of rats with endotoxin (lipopolysaccharide, LPS) and selective agonists of the nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARgamma) protect the kidney against ischemia/reperfusion (I/R) injury. Here we investigate the hypothesis that the renoprotective effects of LPS may be due to an enhanced formation of endogenous ligands of PPARgamma, rather than an up-regulation of PPARgamma expression. Rats were pretreated with LPS (1 mg/kg, IP, 24 hours prior to ischemia) in the absence (control) or presence of the selective PPARgamma antagonist GW9662 (1 mg/kg, IP, 24 and 12 hours prior to ischemia). Twenty-four hours after injection of LPS, rats were subjected to 60 minutes of bilateral renal ischemia, followed by 6 hours of reperfusion. Serum and urinary indicators of renal injury and dysfunction were measured, specifically serum creatinine, aspartate aminotransferase, and gamma-glutamyl-transferase, creatinine clearance, urine flow, and fractional excretion of sodium. Kidney PPARgamma1 mRNA levels were determined by reverse transcriptase-polymerase chain reaction. Pretreatment with LPS significantly attenuated all markers of renal injury and dysfunction caused by I/R. Most notably, GW9662 abolished the protective effects of LPS. Additionally, I/R caused an up-regulation of kidney PPARgamma1 mRNA levels compared to sham animals, which were unchanged in rats pretreated with LPS. We document here for the first time that endogenous ligands of PPARgamma may contribute to the protection against renal I/R injury afforded by LPS pretreatment in the rat.

THE PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR-γ LIGAND 15-DEOXYD12,14 PROSTAGLANDIN J2 REDUCES THE ORGAN INJURY IN HEMORRHAGIC SHOCK

The cyclopentenone prostaglandin 15-deoxyDelta12,14PGJ2 (15d-PGJ2) exerts potent anti-inflammatory effects in vivo, which are in part caused by the activation of peroxisome proliferator-activated receptor-gamma (PPAR-gamma). Here we investigate the effects of 15d-PGJ2 on the multiple organ injury/dysfunction associated with severe hemorrhage and resuscitation. Male Wistar rats were subjected to hemorrhage (to lower mean arterial blood pressure to 45 mmHg) for 90 min and subsequently resuscitated with shed blood for 4 h. Rats were treated with either 15d-PGJ2 (0.3 mg/kg i.v.) or its vehicle (10% dimethyl sulfoxide) at 30 min before the hemorrhage. In some experiments, the selective PPAR-gamma antagonist GW9662 (1 mg/kg i.v.) or its vehicle (10% dimethyl sulfoxide) was given 45 min before the hemorrhage. Hemorrhage and resuscitation resulted in an increase in serum levels of (a) urea and creatinine and, hence renal dysfunction; alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and, hence, hepatic injury. The potent PPAR-gamma agonist 15d-PGJ2 abolished the renal dysfunction and largely reduced the liver injury caused by hemorrhagic shock. In addition, 15d-PGJ2 also attenuated the lung and intestinal injury (determined by histology) caused by hemorrhage and resuscitation. The specific PPAR-gamma antagonist GW9662 reduced the protective effects afforded by 15d-PGJ2. 15d-PGJ2 did not affect the delayed fall in blood pressure caused by hemorrhage and resuscitation. The mechanisms of the protective effect of this cyclopentenone prostaglandin are, at least in part, PPAR-gamma dependent, as the protection afforded by 15d-PGJ2 was reduced by the PPAR-gamma antagonist GW9662. We propose that 15d-PGJ2 or other ligands for PPAR-gamma may be useful in the therapy of the organ injury associated with hemorrhagic shock.