PPARγ Protein Research Articles

PPARγ regulates lipid metabolism and viability of sheep trophoblast cells.

Peroxisome proliferator-activated receptor γ (PPARγ) is highly expressed in trophoblast tissues in pregnancy during which the protein participates in diverse events, including embryo implantation and placental formation. However, little is known about the role of PPARγ in embryonic development. This study investigated the function of PPARγ in sheep trophoblast cells. The coding sequence of sheep PPARγ encoded 475 amino acids and included one synonymou mutation compared with the sheep reference sequence for PPARγ. The PPARγ protein was localized in the nucleus and cytoplasm of sheep trophoblasts. The relative expression of PPARγ was elevated in cells treated with rosiglitazone and reduced following administration of GW9662. Activation of PPARγ promoted cell proliferation and mobility, but inhibited apoptosis. In addition, stimulation of PPARγ promoted the expression of lipid metabolism-related genes FABP4 and PLIN2. The expression of prostaglandin metabolism-related genes PLA2G4A, PTGS2 and PTGES also was upregulated significantly in trophoblast cells when PPARγ was activated. In contrast, activation of PPARγ did not impact expression of the prostaglandin-related genes PGFS and SLCO2A1. At the same time, activation of PPARγ activity increased the ratio of PGE2 to PGF2α. Furthermore, fluorescence labelling showed that the numbers of cell lipid droplets increased after stimulation of PPARγ activity, but decreased when PPARγ was inhibited. In conclusion, PPARγ is critical for the regulation of lipid metabolism and prostaglandin synthesis and secretion in sheep trophoblast cells and also has a potent effect on cell proliferation and viability.

Serelaxin inhibits LPS-induced inflammatory response in cardiac fibroblasts by activating PPAR-γ and suppressing NF-κB signaling pathway.

Serelaxin has an inhibitory effect on fibrosis. However, whether the anti-fibrotic effects of serelaxin are achieved by inhibiting the inflammatory response has not been clarified. This study aimed to investigate the role of serelaxin in lipopolysaccharide (LPS)-induced inflammation in cardiac fibroblasts (CFs) and elucidate the underlying mechanisms.CFs were isolated from adult rat hearts. The effect of serelaxin on the inhibition of the inflammatory response after LPS induction was examined. Cell viability was measured by MMT assay. Cell proliferation was determined using the cell-counting kit-8. The levels of inflammatory cytokines IL-1β, IL-6, TNF-α, and IL-10 were measured using an enzyme-linked immunosorbent assay (ELISA). The mRNA levels of α-SMA, collagen I/III, MMP-2, MMP-9, IL-1β, IL-6,TNF-α, IL-10, IκBα, p-IκBα, p65 subunit of nuclear factor-kappa B (NF-κB) and peroxisome proliferator-activated receptor-γ (PPAR-γ) were assessed by real-time quantitative PCR (RT-qPCR). The protein levels of α-SMA, collagen I/III, MMP-2, MMP-9, IκBα, p-IκBα, p65, p-p65 and PPAR-γ were examined by western blotting.Serelaxin inhibited LPS-induced IL-1β, IL-6, TNF-α, α-SMA, and collagen I/III, and elevated the expression of IL-10, MMP-2 and MMP-9. Moreover, LPS-induced activation of NF-κB pathway was suppressed by serelaxin treatment. Further studies showed that serelaxin did not significantly increase the expression of PPAR-γ mRNA and protein, but activated PPAR-γ activity, and the PPAR-γ inhibitor GW9662 reversed the inhibitory effect of serelaxin on IL-1β, IL-6, and TNF-α production. These results suggest that serelaxin alleviates cardiac fibrosis by stimulating PPAR-γ via a ligand-independent mechanism which subsequently abolish the expression of NF-κB signalling pathway.

Naringin may promote functional recovery following spinal cord injury by modulating microglial polarization through the PPAR-γ/NF-κB signaling pathway

ObjectiveThe flavonoid Naringin (Nar) has been extensively investigated and found to have multiple pharmacological properties, including neuroprotection. Although recent reports have shown that Nar can effectively treat spinal cord injury (SCI), its potential mechanism remains unknown. This study aimed to investigate the effects of Nar on motor recovery and inflammatory responses after SCI and to elucidate its mechanism. MethodsSCI rat models were established using Allen's weight-drop method. The rats were intragastrically given Nar (40 mg/kg) for 21 d, and their motor function before surgery and on the 1st, 3rd, 7th, 14th, 21st days after surgery was assessed by the Basso-Beattie-Bresnahan (BBB) scale and examined by the grid walking test (GWT). The enzyme linked immunosorbent assay (ELISA) was used to detect the interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and monocyte chemoattractant protein (MCP)-1 levels in rat spinal cord tissues, and quantitative reverse transcription polymerase chain reaction (qRT-PCR) to measure the mRNA expression levels of microglial activation markers CD68 and ionized calcium binding adaptor molecule 1 (Iba-1), M1 markers inducible nitric oxide synthase (iNOS) and IL-6, and M2 markers CD206 and Arginase 1 (Arg1). The expression levels of peroxisome proliferator-activated receptor gamma/nuclear factor kappa B (PPAR-γ/NF-κB) pathway-related proteins in rat spinal cord tissues were determined using western blotting. ResultsNar significantly increased the BBB score and decreased the mean error rate of GWT in SCI rats. Additionally, Nar effectively inhibited microglial activation and expression of M1 markers in spinal cord tissues. It also elevated M2 polarization-related gene expression and significantly lowered the levels of inflammatory factors. Further investigation showed that Nar enhanced the expression of PPAR-γ protein and inhibited NF-κB pathway activity. ConclusionNar promotes functional recovery by regulating microglial polarization and inhibiting the inflammatory response in SCI, and its mechanism may be related to the PPAR-γ/NF-κB signaling pathway activity.

Hepatoprotective and anti-hyperglycemic effects of ferulic acid in arsenic-exposed mice.

Arsenic is a toxic metalloid that increases the risk of hepatotoxicity and hyperglycemia. The objective of the present study was to assess the effect of ferulic acid (FA) in mitigating glucose intolerance and hepatotoxicity caused by sodium arsenite (SA). A total of six groups including control, FA 100mg/kg, SA 10mg/kg, and groups that received different doses of FA (10, 30, and 100mg/kg), respectively just before SA (10mg/kg) for 28 days were examined. Fasting blood sugar (FBS) and glucose tolerance tests were conducted on the 29th day. On day 30, mice were sacrificed and blood and tissues (liver and pancreas) were collected for further investigations. FA reduced FBS and improved glucose intolerance. Liver function and histopathological studies confirmed that FA preserved the structure of the liver in groups received SA. Furthermore, FA increased antioxidant defense and decreased lipid peroxidation and tumor necrosis factor-alpha level in SA-treated mice. FA, at the doses of 30 and 100mg/kg, prevented the decrease in the expression of PPAR-γ and GLUT2 proteins in the liver of mice exposed to SA. In conclusion, FA prevented SA-induced glucose intolerance and hepatotoxicity by reducing oxidative stress, inflammation, and hepatic overexpression of PPAR-γ and GLUT2 proteins.

Abdominal Massage Ameliorates Inguinal Fat Accumulation via Augmentation of PPARγ Signaling in High-Fat Diet-Induced Obese Mice.

With the increase in prevalence and decrease in age of the obese population, safer weight loss methods have attracted growing attention. While abdominal massage (AM) has been clinically proven for weight loss, the mechanism thereof has yet to be elucidated. We aimed to investigate the effect of AM on abdominal fat in obese mice fed a high-fat diet and explore the possible mechanisms involved. Male C57BL/6J mice were fed a high-fat diet for 16 weeks and then treated with AM for 5 weeks; mice fed a standard diet were used as normal controls. Blood and adipose tissue, including inguinal white adipose tissue (WAT) and epididymal WAT, were collected from the mice after the intervention. We explored the mechanism of weight reduction through inguinal WAT transcriptome sequencing, quantitative real-time polymerase chain reaction (PCR) validation, and Western blot. The results revealed that AM decreased fat mass, weight, glucose, and serum lipid levels. Meanwhile, AM enhanced the expression of the peroxisome proliferator-activated receptor gamma (PPARγ) and other downstream genes (Fabp4, Acox3, Pck1, and Aqp7) in inguinal WAT. In addition, AM increased the expression of PPARγ protein. AM may promote fatty acid oxidation, lipid metabolism, and glucose homeostasis by activating the PPARγ signaling pathway in inguinal WAT, thereby exhibiting therapeutic efficacy against obesity, even in the presence of a persistent high-fat diet.

Effect of Tongdu Tiaoshen electroacupuncture pretreatment on PPARγ-mediated pyroptosis of cerebral cortex in rats with cerebral ischemia reperfusion injury

To observe the effect of Tongdu Tiaoshen (promoting the circulation of the governor vessel and regulating the spirit) electroacupuncture (EA) pretreatment on pyroptosis mediated by peroxisome proliferators-activated receptor γ (PPARγ) of the cerebral cortex in rats with cerebral ischemia reperfusion injury (CIRI) and explore the potential mechanism of EA for the prevention and treatment of CIRI. A total of 110 clean-grade male SD rats were randomly divided into a sham-operation group, a model group, an EA group, an EA + inhibitor group and an agonist group, 22 rats in each group. In the EA group, before modeling, EA was applied to "Baihui" (GV 20), "Fengfu" (GV 16) and "Dazhui" (GV 14), with disperse-dense wave, 2 Hz/5 Hz in frequency, 1 to 2 mA in intensity, lasting 20 min; once a day, consecutively for 7 days. On the base of the intervention as the EA group, on the day 7, the intraperitoneal injection with the PPARγ inhibitor, GW9662 (10 mg/kg) was delivered in the EA + inhibitor group. In the agonist group, on the day 7, the PPARγ agonist, pioglitazone hydrochloride (10 mg/kg) was injected intraperitoneally. At the end of intervention, except the sham-operation group, the modified thread embolization method was adopted to establish the right CIRI model in the rats of the other groups. Using the score of the modified neurological severity score (mNSS), the neurological defect condition of rats was evaluated. TTC staining was adopted to detect the relative cerebral infarction volume of rat, TUNEL staining was used to detect apoptosis of cerebral cortical nerve cells and the transmission electron microscope was used to observe pyroptosis of cerebral cortical neural cells. The positive expression of PPARγ and nucleotide-binding to oligomerization domain-like receptor protein 3 (NLRP3) in the cerebral cortex was detected with the immunofluorescence staining. The protein expression of PPARγ, NLRP3, cysteinyl aspartate specific protease-1 (caspase-1), gasdermin D (GSDMD) and GSDMD-N terminal (GSDMD-N) in the cerebral cortex was detected with Western blot. Using the quantitative real-time fluorescence-PCR, the mRNA expression of PPARγ, NLRP3, caspase-1 and GSDMD of the cerebral cortex was detected. The contents of interleukin (IL)-1β and IL-18 in the cerebral cortex of rats were determined by ELISA. Compared with the sham-operation group, the mNSS, the relative cerebral infarction volume and the TUNEL positive cells rate were increased (P<0.01), pyroptosis was severe, the protein and mRNA expression levels of PPARγ, NLRP3, caspase-1 and GSDMD were elevated (P<0.01); and the protein expression of GSDMD-N and contents of IL-1β and IL-18 were increased (P<0.01) in the model group. When compared with the model group, the mNSS, the relative cerebral infarction volume and the TUNEL positive cells rate were decreased (P<0.01), pyroptosis was alleviated, the protein and mRNA expression levels of PPARγ were increased (P<0.01), the protein and mRNA expression levels of NLRP3, caspase-1 and GSDMD were decreased (P<0.01), the protein expression of GSDMD-N was reduced (P<0.01); and the contents of IL-1β and IL-18 were lower (P<0.01) in the EA group and the agonist group; while, in the EA + inhibitor group, the protein expression of PPARγ was increased (P<0.01), the protein and mRNA expression levels of NLRP3 and GSDMD were decreased (P<0.01, P<0.05), the mRNA expression of caspase-1 was reduced (P<0.01); and the contents of IL-1β and IL-18 were lower (P<0.01). When compared with the EA + inhibitor group, the mNSS, the relative cerebral infarction volume and the TUNEL positive cells rate were decreased (P<0.05, P<0.01), pyroptosis was alleviated, the protein and mRNA expression levels of PPARγ were increased (P<0.01), the protein and mRNA expression levels of NLRP3, caspase-1 and GSDMD were decreased (P<0.01), the protein expression of GSDMD-N was reduced (P<0.01); and the contents of IL-1β and IL-18 were declined (P<0.01) in the EA group. Compared with the agonist group, in the EA group, the relative cerebral infarction volume and the TUNEL positive cells rate were increased (P<0.05, P<0.01), the mRNA expression of PPARγ was decreased (P<0.01) and the protein expression of GSDMD-N was elevated (P<0.05); and the contents of IL-1β and IL-18 were higher (P<0.01). Tongdu Tiaoshen EA pretreatment can attenuate the neurological impairment in the rats with CIRI, and the underlying mechanism is related to the up-regulation of PPARγ inducing the inhibition of NLRP3 in the cerebral cortex of rats so that pyroptosis is affected.

Thermoneutrality or standard temperature: is there an ideal housing temperature to study the antisteatotic effects of green tea in obese mice?

Metabolic-associated fatty liver disease (MAFLD) is a condition characterized by excessive accumulation of triglycerides in hepatocytes, currently considered the number one cause of chronic liver disease. MAFLD is strongly associated with obesity, type 2 diabetes, hyperlipidaemia, and hypertension. Emphasis has been placed on the use of green tea (GT), produced from the Camellia sinensis plant, rich in antioxidants as polyphenols and catechins, on obesity and MAFLD treatment/prevention. Studies carried out in rodent models housed at a standard temperature (ST, 22°C) are being questioned as ST is a determining factor on generating changes in the physiology of immune response, and energy metabolism. On the other hand, it seems that thermoneutrality (TN, 28°C) represents a closer parallel to human physiology. In this perspective, we investigated the effects of GT (500 mg/kg of body weight, over 12 weeks, 5 days/week) by comparing mice housed at ST or TN in a model of MAFLD of diet-induced obese males C57Bl/6 mice. We show that the liver phenotype at TN exhibits a more severe MAFLD while GT ameliorates this condition. In parallel, GT restores the expression of genes involved in the lipogenic pathway, regardless of temperature, with slight modifications in lipolysis/fatty acid oxidation. We observed an increase promoted by GT in PPARα and PPARγ proteins independently of housing temperature and a dual pattern of bile acid synthesis. Thus, animals' conditioning temperature is a key factor that can interfere in the results involving obesity and MAFLD, although GT has beneficial effects against MAFLD independently of the housing temperature of mice.

Involvement of the peroxisome proliferator-activated receptor gamma (Pparγ) and matrix metalloproteinases-2 and -9 (Mmp-2 and -9) in the mechanism of action of di(2-ethylhexyl)phthalate (DEHP) in cultured mouse brain astrocytes and neurons

Di(2-ethylhexyl)phthalate (DEHP) is one of the most widely used phthalates in industry. It has been shown that, after entering the body, DEHP has the ability to cross the blood-placenta and blood-brain barriers. One of the proposed mechanisms of action of DEHP is the activation of peroxisome proliferator-activated receptors (PPARs). Many different functions of PPARγ in cells have been demonstrated, one of which is the modulation of the activation of matrix metalloproteinases (MMPs). The aim of this study was to investigate the role of Pparγ, Mmp-2, and Mmp-9 in the mechanism of action of DEHP. The experiments were performed on in vitro primary murine neurons and astrocytes. The results showed that DEHP has a pro-apototic effect on neurons, causing an increase in caspase-3 activity and in the number of apoptotic bodies. However, in astrocytes, the increase in caspase-3 activity was not related to the apoptosis process, as no increase in the formation of apoptotic bodies was observed. Moreover, DEHP increased the proliferation of astrocytes, which was confirmed by the increase in the amount and expression of the Ki-67 protein. In astrocytes, DEHP decreased the expression of the Pparγ and Mmp-9 proteins but increased the expression of the Mmp-2 protein. In DEHP neurons, it increased the expression of the Pparγ protein but decreased the expression of the Mmp-2 and Mmp-9 proteins.

The effect of aerobic training and hydroalcoholic extract of dill on skeletal muscle inflammation in a high-fat diet-induced obesity rat model

Abstract Aerobic training (AT) and herbal medicine interventions are effective in reducing chronic inflammation and achieving weight loss. The present study aimed to investigate the effect of 10 weeks of AT and hydroalcoholic extract of dill (HED) on tumour necrosis factor-alpha (TNF-α), interleukin-4 (IL-4), and peroxisome proliferator-activated receptor gamma (PPARγ) concentrations in gastrocnemius muscle and Lee index of obese male rats. Thirty-two obese male Wistar rats were randomly divided into AT (70-80% of maximal oxygen uptake, 5 times/week), HED (300 mg/kg body weight, 3 times/week), AT+HED, and obese control (Ctr), and eight non-obese rats were included in the non-obese control group (NCtr). Lee index was measured before and after 10 weeks of interventions. Also, rats were sacrificed 48 hs after the last intervention session and gastrocnemius muscle was removed to measure protein concentrations of TNF-α, IL-4, and PPARγ. When compared with the pre-intervention period, the Lee index decreased in the AT and AT+HED groups and increased in the NCtr group at the post-intervention period. Also, there was no significant difference between non-obese and obese rats in the concentrations of TNF-α and IL-4, but the AT+HED group showed a higher concentration of TNF-α than the AT group. Furthermore, the expression of PPARγ protein in the HED group was higher than in the Ctr and AT groups. Although we observed the beneficial effects of AT and its combination with HED in reducing the Lee index, we found that the administration of HED compared with AT can provide better protection against some metabolic complications in the skeletal muscles of obese rats caused by a high-fat diet. Lastly, it seems that the AT combined with HED interferes with possible anti-inflammatory effects of AT or HED intervention alone.

A combined network pharmacology and molecular biology approach to investigate the potential mechanisms of G-M6 on ovarian cancer

Ginsenoside 3β,12β,21α,22β-Hydroxy-24-norolean-12-ene (G-M6), a phase I metabolite of anti-tumor medication 20(R)-25-methoxyl-dammarane-3β,12β,20-triol (AD-1), beats the parent drug in anti-ovarian cancer efficacy. The mechanism of action for ovarian cancer, however, is uncertain. Using network pharmacology, human ovarian cancer cells and nude mouse ovarian cancer xenotransplantation model, the anti-ovarian cancer mechanism of G-M6 was preliminarily explored in this study. The PPAR signal pathway is the key signal pathway of the G-M6 anti-ovarian cancer mechanism, according to data mining and network analysis. Docking tests demonstrated that the bioactive chemical G-M6 was capable of forming a stable bond with the PPARγ target protein capsule. Using human ovarian cancer cells and xenograft model of ovarian cancer to evaluate the anticancer activity of G-M6. The IC50 value of G-M6 was 5.83±0.36, lower than AD-1 and Gemcitabine. The tumor weight of the RSG 80 mg/kg group (C), G-M6 80 mg/kg group (I), and RSG 80 mg/kg + G-M6 80 mg/kg group (J) after the intervention was as follows: C < I < J. The tumor inhibition rates of groups C, I, and J were 28.6%, 88.7%, and 92.6%, respectively. When RSG and G-M6 are combined to treat ovarian cancer, q = 1.00 is calculated according to King's formula, which indicates that RSG and G-M6 have additive effects. Its molecular mechanism may involve the up-regulation of PPARγ and Bcl-2 protein expressions, and the down-regulation of Bax, Cytochrome C (Cyt. C), Caspase-3, and Caspase-9 protein expressions. These findings serve as a reference for further research into the processes behind ginsenoside G-M6′s ovarian cancer therapy.

Effects of scutellarin on acute myocardial ischemia/reperfusion injury in isolated rat heart

Objectives This study aimed to evaluate the cardioprotective effects and mechanism of scutellarin (Scu) on heart ischaemia/reperfusion (I/R) injury by using an isolated Langendorff rat heart model. Methods Myocardial function was evaluated via measurements of cardiac haemodynamic parameters. Myocardial infarct size was macroscopically estimated by using TTC staining. The release of creatine kinase (CK) and lactate dehydrogenase (LDH) was used to evaluate cardiac injury. GSH/GSSG, SOD, and MDA were determined as being indicators of oxidative stress. CRP, IL-6, and TNF-α were also analysed to assess inflammation in the heart tissues. Cardiomyocyte apoptosis was quantified by using a TUNEL kit. PPARγ, Nrf2, and NF-κB were assayed via Western blotting. Results The results indicated that I/R induced significant cardiac dysfunction, myocardial infarct, and apoptosis, as well as decreasing the GSH/GSSG ratio, SOD activity, Nrf2 protein expression, and PPARγ protein expression, but increased levels of TNF-α, MDA, CRP, IL-6, and NF-κB. Conclusion These data suggest that Scu attenuates cardiac I/R.

Whether Carotenoids and Quercetin Affect the Proliferation andExpression of PPARγ Protein in Leukemia K562 Cells

Carotenoids are some of the most abundant pigments in nature, ranging from yellow to red. Carotenoids can divide intotwo categories: carotenoids composed of hydrocarbon elements and lutein-containing oxygen elements. They are lipidsoluble pigments, and some contain carbonyl or enol groups, which are water-soluble. Carotenoids absorb visible lightfrom 400 nm to 600 nm, while special ones such as octa-hydro lycopene and hexahydrolycopene only absorb ultravioletlight. Quercetin is a flavonoid compound widely found in fruits and vegetables. Its chemical structure phenolic hydroxylgroup is the basis of antioxidant action, which can be used for hydrogen to quench oxygen free radicals. It is oxidizedto form a highly stable catechol structure. It has a variety of biological activities and pharmacological effects, such asexpanding coronary blood vessels and inhibiting tumor cell proliferation. This paper shows that the K562 cell growthrate in carotenoids and quercetin treatment groups was slower than the control group.

Molecular docking analysis of PPARγ antagonists for obesity associated diabetes management.

Obesity is a major metabolic disorder in developed countries, with an increasing number of people affected globally. PPARγ is primarily expressed in adipose tissue with a lesser extent in other tissues. PPARγ is an important mediator in several metabolic processes such as insulin sensitivity and adipogenesis. Because of its critical role in these processes, PPARγ is regarded as a critical target for therapeutic intervention in obesity treatment. A library of 2,320 bioactive compounds was screened insilico to identify compounds that strongly interact with the PPARγ protein. The compounds Z1982689600, Z2235802137, Z2235801970, and Z2037275165, demonstrated notable binding affinity values towards the PPARγ protein with values of -12.1, -11.7, -11.4, and -11.4 kcal/mol, respectively, which were higher than the binding affinity value observed for the control compound (-10.5 kcal/mol). These compounds bind tightly to PPARγ and have several amino acid residue interactions in common with the control compound. In addition, these compounds meet the ADMET criteria. These compounds could aid in the development of PPARγ antagonists for the management of obesity associated diabetes. However, additional research is needed to optimize their efficacy in wet laboratory conditions.

Andrographolide exerts a neuroprotective effect by regulating the LRP1-mediated PPARγ/NF-κB pathway

Low-density lipoprotein receptor-associated protein 1 (LRP1) is widely expressed in neurons, microglia and astrocytes. Studies have revealed that the suppression of LRP1 expression in the brain significantly exacerbates Alzheimer's disease (AD)-related neuropathology. Andrographolide (Andro) has been demonstrated to possess neuroprotective properties, although its underlying mechanisms remain largely unknown. This study aims to investigate whether Andro can inhibit neuroinflammation in AD by modulating the LRP1-mediated PPARγ/NF-κB pathway. In Aβ-induced BV-2 cells, Andro was found to increase cell viability and enhance the expression of LRP1, while decreasing the expression of p-NF-κB (p65) and NF-κB(p65), as well as IL-1β, IL-6 and TNF-α levels. In addition, when Aβ was cotreatment with Andro to BV2 cells with either LRP1 or PPARγ knockdown, increased mRNA and protein expression of p-NF-κB(p65) and NF-κB(p65), NF-κB DNA binding activity as well as IL-1β, IL-6 and TNF-α levels were observed. These findings suggested that Andro could attenuate Aβ induced cytotoxicity by reducing neuroinflammation which may be partly attributed to its effects on this LRP1 mediated PPARγ/NF-κB pathway.

Prostaglandin D2 receptor expression in the lung and the effect of PPAR-γ, DP1, and DP2 receptor agonist treatment during respiratory viral infection

Abstract Prostaglandin D2 (PGD2) may play an anti-inflammatory role in the lung during respiratory viral infection, but little is known about the expression of its receptors (PPAR-γ, DP1, and DP2) in the lung or its mechanism of action. Adult C57BL/6 mice were infected with Sendai virus (500 pfu /g body weight), a natural mouse pathogen that infects airway epithelial cells. Immunostaining of uninfected lungs showed PPAR-γ protein in epithelial cells of larger airways, but not in the alveolar epithelial cells. After viral infection, PPAR-γ protein was no longer detectable in the lungs. Immunofluorescence showed DP1 present in parenchymal cells as well as in some CCSP+ airway epithelial cells, but levels did not appear to change during infection. DP2 expression increased after viral infection and was predominantly in CCSP+ epithelial cells 24 hours post-infection. DP2 also co-localized with MHC Class II+ cells in the parenchyma on days 3 and 7 post-infection. Viral infection of primary tracheal epithelial cells showed PPAR-γ mRNA expression was increased 48 and 72 hours post-infection. There was no change in DP1 or DP2 expression in vitro. To test the effect of each receptor in vivo, mice were infected and treated with PPAR-γ, DP1, and DP2 agonists, or vehicle every 12 hours. Mice treated with the PPAR-γ agonist were protected from body weight loss and had reduced IFN-γ expression on day 7, suggesting that PGD2 may be acting via PPAR-γ to reduce inflammation in the lung. Supported by grants from Sigma Xi Grant in Aid of Research and Saint Louis University College of Arts and Sciences Brennan Award.

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.

Breast cancer cell-derived exosome-delivered microRNA-155 targets UBQLN1 in adipocytes and facilitates cancer cachexia-related fat loss.

Cachexia occurrence and development are associated with loss of white adipose tissues, which may be involved with cancer-derived exosomes. This study attempted to characterize the functional mechanisms of bc cell-derived exosomes-loaded microRNA (miR)-155 in cancer cachexia-related fat loss. Exosomes were incubated with preadipocytes and cellular lipid droplet accumulation was observed using oil red O staining. Western blots evaluated the cellular levels of lipogenesis marker peroxisome proliferator activated receptor gamma (PPARγ) and adiponectin, C1Q and collagen domain containing (AdipoQ). Differentiated adipocytes were incubated with exosomes, and phosphate hormone sensitive lipase (P-HSL), adipose triglyceride lipase (ATGL), and glycerol were detected in adipocytes, in addition to uncoupling protein 1 (UCP1) and leptin levels. A mouse model of cancer cachexia was established where cancer exosomes were injected intravenously. The changes in body weight and tumor-free body weights were recorded and serum glycerol levels and lipid accumulation in adipose tissues were determined. Also, the relationship between miR-155 and UBQLN1 was predicted and verified. bc exosome treatment reduced PPARγ and AdipoQ protein levels, promoted the levels of P-HSL and ATGL proteins, facilitated glycerol release, increased UCP1 expression, and lowered leptin expression in adipocytes. Exosomal miR-155 inhibited lipogenesis in preadipocytes and boosted the browning of white adipose tissues. miR-155 downregulation alleviated cancer exosome-induced browning of white adipose tissues and fat loss. Mechanistically, miR-155 targeted UBQLN1, and UBQLN1 upregulation reversed the impacts of cancer exosomes. miR-155 loaded by bc cell-derived exosomes significantly affects white adipose browning and inhibition of cancer-derived exosomes.

Lipidomics of the erythrocyte membrane and network pharmacology to explore the mechanism of mangiferin from Anemarrhenae rhizoma in treating type 2 diabetes mellitus rats

Mangiferin, a natural C-glucoside xanthone, is one of the major bioactive ingredients derived from the dry rhizome of Anemarrhenae rhizome, which has been reported to exhibit various pharmacological effects, including anti-oxidant, anti-inflammatory, anti-fatty liver, anti-metabolic syndrome, and anti-diabetic. However, the precise molecular mechanisms underlying its impact on phospholipid metabolism in the erythrocyte membrane of type 2 diabetes mellitus (T2DM) remain unclear. The present research aimed to evaluate the effects of mangiferin on glucose and lipid metabolism in T2DM model rats and discuss the relationship between lipid metabolites and potential targets involved in the hypoglycemic effects by integrating lipidomics and network pharmacology method. After 8 consecutive weeks of treatment with mangiferin, the T2DM model rats exhibited significant improvements in several biochemical indices and cytokines, including fasting blood glucose (FBG) levels after 12 h of fasting, fasting insulin level (FINS), total cholesterol (T-CHO), triacylglycerols (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), homeostasis model assessment of insulin resistance (HMOA-IR), TNF-α and IL-6. A total of 22 differential lipid metabolites were selected from erythrocyte membrane phospholipids, which were closely associated with the processes of T2DM. These metabolites mainly belonged to glycerophospholipid metabolism and sphingolipid metabolism. Based on network pharmacology analysis, 22 genes were recognized as the potential targets of mangiferin against diabetes. Moreover, molecular docking analysis revealed that the targets of TNF, CASP3, PTGS2, MMP9, RELA, PLA2G2A, PPARA, and NOS3 could be involved in the modulation of inflammatory signaling pathways and arachidonic acid (AA) metabolism to improve IR and hyperglycemia. The combination of immunohistochemical staining and PCR showed that mangiferin could treat T2DM by regulating the expression of PPARγ protein and NF-κB mRNA expression to impact glycerophospholipids (GPs) and AA metabolism. The present study showed that mangiferin might alleviate IR and hyperglycemia of T2DM model rats via multiple targets and multiple pathways to adjust their phospholipid metabolism, which may be the underlying mechanism for mangiferin in the treatment of T2DM.

Metabolic regulation of the circadian clock in classically and alternatively activated macrophages.

Macrophages exhibit a range of functional pro- and anti-inflammatory states that induce changes in their cellular metabolism. We aimed to elucidate whether these changes affect the molecular properties of their circadian clock focusing on their anti-inflammatory phenotype. Primary cell cultures of bone marrow-derived macrophages (BMDMs; nonpolarized M0 BMDM) from PER2::LUC (fusion protein of PERIOD2 and LUCIFERASE) mice were polarized into the M1 (proinflammatory) or M2 (anti-inflammatory) phenotype, and PER2-driven bioluminescence was recorded in real-time at the cell-population and single-cell levels. Viability, clock gene expression profiles, polarization plasticity and peroxisome proliferator-activated receptor γ (PPARγ) protein levels were analyzed. The effects of pharmacological activation/inhibition of PPARγ (rosiglitazone/GW9662) and inhibition of fatty acid oxidation (FAO) by etomoxir in M2 BMDM cell cultures were examined. The parameters of PER2-driven bioluminescence rhythms differed between M0, M1 and M2 BMDM cultures at cell-population and single-cell levels. Compared with M0, polarization to M2 did not change the period but increased amplitude, mean bioluminescence level and rhythm persistence. Polarization to M1 shortened the period but had no effect on the amplitude of the rhythm. The same period changes were observed after a bidirectional switch between M1- and M2-polarized states in the same culture. Both PPARγ activation/inhibition and FAO inhibition modulated the clock in M2 BMDMs, suggesting metabolic regulation of the M2 clock. Our results indicate that bidirectional changes in the properties of BMDM circadian clocks in response to their actual polarization are mediated via changes in their metabolic state. They provide new information on the interrelationship between the BMDM polarization, their circadian clock and cellular metabolism.

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.