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Les récepteurs activables par les proliférateurs de peroxysomes appartiennent à la superfamille des récepteurs nucléaires aux hormones. Trois isotypes de PPAR ont été identifiés¡: PPAR, PPAR et PPAR. Les PPAR sont impliqués dans la régulation du métabolisme lipidique, dans l homéostasie du glucose, la prolifération cellulaire et dans la réponse inflammatoire. Ils interviennent également dans la carcinogenèse colique et/ou la progression tumorale. Nous avons étudié l effet de l activation de PPAR et par les thiazolidinediones et les fibrates, respectivement, sur l adhérence et la migration de la lignée HT29 dérivant d adénocarcinome colique humain. Nos résultats montrent que les thiazolidinediones et le fénofibrate modifient l expression de gènes impliqués dans l adhérence et la migration des cellules HT29, en particulier lorsqu elles sont exposées de façon chronique. Un traitement long, quel que soit l agoniste, induit l expression de l intégrine 5. La modification de l expression des molécules d adhérence par les thiazolidinediones est, au moins en partie, due à un mécanisme PPAR -dépendant, et l ensemble des effets observés diffèrent selon le temps de traitement, la dose et la nature du ligand. In vivo, les thiazolidinediones inhibent la formation de métastases à distance et diminuent le volume tumoral. Administrée en prévention, la pioglitazone abolit la formation des tumeurs et métastases. Avec la même approche expérimentale, des résultats comparables sont obtenus en utilisant le fénofibrate, ligand de PPAR . En conclusion, un traitement par les agonistes de PPARg et pourrait être intéressant pour l amélioration des traitements actuels du cancer du colon. / Peroxisome proliferator-activated receptors (PPAR) belong to the nuclear hormone receptor family. Three isotypes, encoded by separate genes, have been identified: PPAR, PPAR and PPAR. They are involved in lipid metabolism, glucose homeostasis, cell proliferation and differentiation, and inflammatory response. They have also been implicated in colon carcinogenesis and/or tumour progression. We studied the effect of PPARg and activation by thiazolidinediones and fibrates, respectively, on adhesion and migration of colon adenocarcinoma HT29 cell line. Exposure to thiazolidinedione modifies expression of several genes involved in HT29 cell adhesion and migration, especially when cells are chronically treated with each drug. Of interest, long cell treatment either with pioglitazone, rosiglitazone or fenofibrate induced expression of integrin 5-chain. Our results suggest that the modulation of adhesion molecule expression by thiazolidinediones is partly through PPARg-dependent activation and that effects are different according to the dose and nature of ligand. In vivo, thiazolidinediones especially inhibit distant metastasis formation and diminish tumoral growth. In prevention, pioglitazone abolish tumoral and metastasis development. Using the same experimental approach, we demonstrated that fenofibrate as a ligand of PPAR raised similar results. Collectively, we conclude that treatment with PPARg or agonists may be interesting in the improvement of colon cancer treatment.

Our previous study identified one candidate transcript, chicken prostaglandin D2 synthase (cPGDS), significantly higher expressed in hens with high-egg production across several chicken strains including the Single-Comb White Leghorn layers. To further elucidate its underlying molecular mechanisms, the downstream effects of cPGDS in primary culture of chicken anterior pituitary cells were investigated. We successfully generated a highly specific rabbit anti-cPGDS polyclonal antibody. Of 13 examined tissues/cells, quantitative reverse transcription polymerase chain reaction and Western blotting analysis demonstrated that cPGDS mRNA and protein were highly expressed in intestine, kidney, liver and pituitary gland. In addition, significantly higher cPGDS mRNA and protein expression levels of the pituitary gland and ovary in hens than of the pituitary gland and testis in roosters with the same age were found. Transfection of pEGFP-cPGDS plasmid into primary culture of pituitary anterior cells in medium that contained 1 mM arachidonic acids induced luteinizing hormone beta subunit (LHB) transcription and subsequent translation. Treatments of two metabolites in the cyclooxygenase pathway, PGD2 and PGJ2 in the primary culture of chicken anterior pituitary cells induced LHB transcription and translation in both dose- and time-dependent manners. Treatment of peroxisome proliferator activated receptors (PPARs) agonist increased LHB transcription and translation in chicken anterior pituitary cells. On the other hand, specific inhibitions of PPARA and PPARG using antagonists dramatically suppressed PGJ2-induced LHB transcription level. Taken together; these data suggested PGDS upregulated LHB transcription in primary culture of chicken anterior pituitary cells via the PPAR signaling pathway.

The present study cloned full-length genes of peroxisome proliferators activated receptors (PPARs) of the cobia Rachycentron canadum and investigated their expressions in association with cobias¡¦ body adiposity and lipid-metabolism related physiological parameters. In addition to gene cloning, several studies evaluating the roles of PPARs were carried out, including: a time-series study on cage-farmed cobias from week 5 to week 52 post-hatching, a study comparing fish groups with contrasting growth performance and a study elucidating the effects of dietary fatty acids. Three isotypes, PPAR £\, PPAR£] and PPAR£^, that were cloned from cobia¡¦s cDNA contained 2046 bp, 2702 bp and 1943 bp, respectively. Their open reading frames encode 476, 510 and 531 amino acids, respectively. The identity in amino acid sequences between the PPARs are 52% (between PPAR£\ and PPAR£]), 52% (between PPAR£\ and PPAR£^), and 44% (between PPAR£] and PPAR£^), respectively. RT-PCR and real-time PCR (qPCR) analyses showed that expression of PPAR£\ mRNA predominated in red muscle, heart and liver, and at a lower level in the head kidney and dorsal muscle. PPAR£] transcripts were particularly abundant in the heart, liver, brain, and pyloric caeca. In contrast, PPAR£^ mRNA was detected primarily in the adipose tissues, liver, and pyloric caeca. In the time-series study, the PPARs expression was related to the body adiposity and lipid-metabolism related physiological parameters of the cobias that were raised for one year to approximately 4.5 Kg in a commercial cage-culture farm. Ten samplings were conducted on weeks 5, 7, 9, 14, 18, 23, 29, 34, 41, and 52 post-hatching. The cobias were raised in an outdoor nursery to 88 g before being transferred to an offshore cage on week 9. The adipocytes in the liver and ventral muscle showed a hypertrophic (increase in cell size) increase towards the end of the nursery phase. Their cell size decreased significantly after the cage transfer and was maintained afterwards a size spectrum dominated by small cells until week 34. The cobias grew rapidly after the offshore transfer and reached 330 g on week 14. They showed a concurrent increase in fat deposition in the liver and ventral muscle and a concurrent hyperplasia increase in density of adipocytes in the ventral and dorsal muscle. Adipocyte hypertrophy was obvious on week 41 and regressed afterwards. As the fish grew, serum phospholipids concentration increased significantly from approximately 380 to 750 mg/dL. Time-series pattern for the specific activity of two NADPH-generating enzymes, malic enzyme and glucose-6- phosphate dehydrogenase, were reciprocal and compensatory. The expression of liver PPAR£\ mRNA was negatively correlated to fat deposition and adiposity. There was a significant increase in body lipid deposition and hepatic PPAR£^ expression as the fish grew. Hepatic PPAR£^ expression could be a sufficient parameter describing its expression in whole body. These results showed that PPAR£^ and PPAR£\ played a pivotal role in the control of lipid metabolic and storage functions in the liver, muscle and visceral fat depot of the cobia. In the study comparing differential fish growth, two groups of cobias were selected based on their growth performance from a same batch of fish raised in a nursery. The large-size group that was regarded as superior grower was 54.1 cm in total length and 1,287 g in weight; while the small-size fish (inferior grower) was 36.8 cm and 386 g. Compared to large cobias, small cobias showed a similar hepatosomatic index and viscerasomatic index, but a significantly (p ¡Õ 0.05) smaller mesenteric fat index (MFI).The levels of crude lipid in the liver (35% vs. 26%) and the proportions of neutral lipids in lipid were higher in large cobias than in small cobias. Concentrations of serum phospholipids, free fatty acids and total protein of large cobias were significantly higher than those of small cobias. Adipocyte density of liver and ventral muscle was increased with increasing fish size. The PPAR£\ mRNA expression in the liver of small cobias was significantly higher (p ¡Õ 0.05) than large cobias, ascribing to possible stress effect from their inferior growth. The growth superiority obviously affected PPAR£\ mRNA expression and fat deposition in the liver. In general, the expression of liver PPAR£\ mRNA was negatively correlated to body weight, body length, MFI, and serum NEFA, as well as lipid concentration, adiposity (adipocyte density and adipocyte size), G6PDH enzyme activity in the liver. The PPAR£^ mRNA expression in the liver was positively correlated to size of the adipocytes size. The effects of dietary fatty acids on PPARs expression were evaluated in a 10-week growth trial, in which cobias with an initial weight of 80 g were fed diets containing 15% lipid. Among the lipids, 6% was fish oil and the remaining 9% were fish oil (rich in EPA and DHA), perilla oil (C18:3n-3), safflower oil (C18:2n-6), olive oil (C18:1n-9) or palm oil (C16:0). Significant difference was detected in PPARs mRNA expression among dietary treatments and among tissues. In the liver, among the dietary treatments, significantly higher expression levels of PPAR£\ mRNA were detected in perilla oil and olive oil group, PPAR£] mRNA in palm oil group and PPAR£^ mRNA in fish oil group. Linear regression analysis showed that liver PPAR£\ mRNA expression was positively (p ¡Õ 0.05) correlated with dietary C18:3n-3 levels and negatively with dietary C18:0 levels. Liver PPAR£] mRNA expression was positively correlated to C16:0 or C18:0 levels in diets. The PPAR£^ expression was positively (p ¡Õ 0.001) correlated to dietary levels of C20:1n-9, C20:5n-3 and C22:6n-3. In summary, the mRNA expression pattern of PPARs was tissue or organ-specific with the expression of PPAR£\ occurred predominantly in the liver and PPAR£^ in the adipose tissues. The expressions of PPARs in the liver were more related to their physiological roles than in other tissues or organs studied in the present study. The expression of PPAR£\ in the liver was shown correlated negatively to body fat deposition; and reciprocally, expression of PPAR£^ was positively correlated to fat deposition. PPARs mRNA expression was also associated with major dietary fatty acids. Increased dietary C18:0 levels down-regulated PPAR£\ and up-regulated PPAR£]. Up-regulation of PPAR£^ was significantly related to increased levels of highly (C>20) unsaturated fatty acid in diets. Dietary C16¡VC18 fatty acids on the other hand were more related to expressions of PPAR£\ and PPAR£]. These results suggest that fish oil could be partially replaced by plant oils as the lipid source in the diet of the cobia. In addition to highly unsaturated fatty acids, reduction in dietary C18:3n-3 and increase in C18:0 lead to increased fat deposition, implicating a possible strategy to modulate body lipid contents of the cobia through dietary manipulation.

PPAR (peroxisome proliferator activated receptors) is a transcription factor that regulates activity and transcription of enzymes of lipid metabolism and thought PPAR-PPRE (peroxisome proliferator activated receptors ¡V peroxisome prolierator response element) interaction. Cell toxicity was also tested by (3-(4,5-dimethylthiaz ol-2-yl)-2,5-diphenyltetrazolium bromide (MTT assay)). The effects of ethyl acetate (EA) extracts of algae and Echinodermata sp. on PPRE-luciferase activity in Huh-7 cell and cell cytotoxicity were conducted. EA extracts of Gracilaria coronopifolia can induce PPRE-luciferase activity without cytotoxity. EA extracts of Pterocladiella capillacea, Gelidium japonicum, and Euchema sp. showed PPRE-luciferase activity and cytotoxicity. Gelidium amansii and Porphyra sp. did not show PPRE-Luciferase activity but had cyotoxicity. EA extracts of Spirulina sp. had PPRE-Luciferase activity but no cytotoxicity, while that of Chlorella sorokiniana inhibited PPRE-Luciferase activity but did not show cytotoxicity. EA extracts of Echinodermata sp. can kill larva and repellent of mosquito, but had no PPRE-Luciferase activity and cytotoxicity. These results demonstrated that the effects of EA extracts on PPRE-luciferase activity are different between algae. EA extract of Gracilaria and Spirulina showed PPRE regulation activity and Echinodermata sp. EA extract have the potential to become green chemical.

Insulin resistacne and hyperinsulinemia are important risk factors for development of type 2 diabetes mellitus and hypertension. Recently, accumulating evidence has shown that endothelial dysfunction, increases in peripheral vessel resistnce and overactivation of the sympathetic neruvous system contribute to the development of insulin resistance-associated hypertension. The signigicance of cardiovascular regulatory center in the brain stem in pathophysiology of the insulin resistance-induced hypertension, however, has not been explored. Previously studies have proved that increases in superoxide anion (O2£»−) production in peripheral tissue and suppression of nitric oxide (NO) expression in the endothial cell are involved in insulin resistance and hypertension. The nucleus tractus solitarius (NTS) and rostral ventrolateral medulla (RVLM) are involved in neural regulation of blood pressure by serving respectively as the primary baroreceptor afferent terminal sites and the location of sympathetic premotor neurons for cardiovascular regulation in the brain stem. Clinically, the peroxisome proliferator-activated receptor (PPAR) agonist is commonly prescribed for the treatment of type 2 diabetes mellitus by activate PPAR£^ to enhance peripheral tissue insulin sensitizing ability, to maintain blood glucose homeostasis. Intriguingly, both animal and human studies revealed that PPAR£^ agonist also possesses blood pressure lowering effect, although the underlying mechanism is not clear. We therefore investigated in the present study the role of NO and O2£»− in the NTS and RVLM in the pathophysiology of the high fructose diet-induced insulin resistacne and hypertension, and to evaluate the potential central antihypertensive effect of PPAR£^ agonist in rats subjected to high fructose diet. The normotensive male Wistar Kyoto rats (WKY) were divided into 4 groups, including 3 experimental group that received 60% high fructose diet for 8 weeks and one control group that received regular chow diet for the same period of time. Within the 3 experimental groups, two of them received oral administration of rosiglitazone or pioglitazone (10 mg/kg/day) at the last two weeks (from week 6 to week 8) and the third group received saline ingestion. Systemic blood pressure was measured by tail vein sphygmomanometer very week and venous blood was drawn every other week to measure blood sugar and insulin level. At the end of the experiment, oral glucose tolerance test (OGTT) was tested and O2£»− and NO production in the NTS and RVLM were quantified. In adult male WKY rats I found that high fructose diet induced insulin resistance, hypertriglycemia and hypertension. Oral administration of rosiglitazone or pioglitazone significantly blunted the hypertension, hypertriglyceridemia, and ameliorated insulin resistance induced by high fructose diet. The high fructose diet also increased tissue level of O2£»− in the NTS and RVLM. PPAR£^ agonist treatment for two weeks did not affect the induced oxidative stress in these two nuclei. NO production was also increased in the NTS and RVLM after high fructose diet for 6 weeks. Oral treatment of rosiglitazone or pioglitazone significantly attenuated NO production after high fructose diet. At the molecular level, protein expressions of the NADPH oxdase subunits (p40phox, p47phox and gp91phox) and superoxide dismutase (cupper/zinc SOD, mitochondrial SOD, extracellular SOD) were not altered in the NTS or RVLM after high fructose diet alone or in addition with rosiglitazone or pioglitazone treatment. In the RVLM, there was a significant increase in neuronal NO synthase (nNOS) expression with concomitant decrease in inducible NOS (iNOS) expression. Oral treatment of PPAR£^ agonist for two weeks significantly suppressed the induced nNOS upregulation and attenuated the induced downregulation of iNOS expression in the RVLM. Together these results suggest that overproduction of O2£»− and NO in the NTS and RVLM may related to the development of insulin resistance-associated hypertension. Oral treatment of PPAR£^ agonist, including rosiglitazone and pioglitazone, may provide antihypertensive protection by superssing the induced-nNOS expression and increasing the induced-iNOS expression in the RVLM.

Pseudomonas aeruginosa is a significant pathogen of immunocompromised individuals, and the main mechanism by which it mediates virulence is through the coordination of gene expression by an intricate quorum sensing system. One of the signalling molecules of this system, N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL) has been shown to have immunomodulatory capabilities, discrete to its quorum sensing role. While the general focus of research in this area is on the physiological outcomes of this interaction on cell function, there is currently little concrete evidence identifying the receptor(s) for OdDHL in mammalian cells, and hence the biochemical mechanism behind the immunomodulation caused by OdDHL remains largely unknown. This study identifies peroxisome proliferator-activated receptor γ (PPARγ) as a mammalian target for OdDHL. PPARγ is a mammalian transcription factor involved in fatty acid metabolism and is heavily involved in the inflammatory response, being a negative regulator of inflammation. It is shown here that OdDHL is able to instigate signalling through PPARγ by activation of the ligand binding domain (LBD), suggesting that OdDHL may act as a PPARγ agonist. OdDHL is able to compete with the PPARγ agonist, rosiglitazone, causing a relative antagonism of PPARγ activity when given in tandem with the agonist. The bacterial signalling molecule is unable to displace the irreversible PPARγ antagonist GW9662. This effect on PPARγ is specific to OdDHL, as the smaller P. aeruginosa signalling molecule, N-butyryl-L-homoserine lactone had no significant effect on PPARγ activation. In order to confirm PPARγ as a putative receptor for OdDHL in mammalian cells, initial experiments were undertaken to optimise conditions to produce PPARγ LBD protein for binding interaction studies. The fidelity of the protein sequence was established and expression of the protein in an appropriate vector was confirmed. The protein produced was insoluble and hence not functional for binding studies, suggesting that additional optimisation of expression conditions, or manipulation and refolding of the protein is necessary before further experimentation can take place. The identification of PPARγ as a receptor for OdDHL in mammalian cells is an important step in understanding the nature and scope of the interaction between OdDHL and host cell physiology, especially the significance of this interaction during P. aeruginosa infection. Continuation of this research, in particular completion of protein-ligand binding studies between OdDHL and PPARγ has the potential to clarify the significance of the immunomodulation caused by OdDHL, while providing us with a platform from which we may exploit it.

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Exploration of peroxisome proliferator-activated receptor-gamma (PPAR") as a drug target holds applications for treating a wide variety of chronic inflammation-related diseases. Type 2 diabetes (T2D), which is a metabolic disease influenced by chronic inflammation, is quickly reaching epidemic proportions. Although some treatments are available to control T2D, more efficacious compounds with fewer side effects are in great demand. Drugs targeting PPAR" typically are compounds that function as agonists toward this receptor, which means they bind to and activate the protein. Identifying compounds that bind to PPAR" (i.e. binders) using computational docking methods has proven difficult given the large binding cavity of the protein, which yields a large target area and variations in ligand positions within the binding site. We applied a combined computational and experimental concept for characterizing PPAR" and identifying binders. The goal was to establish a time- and cost-effective way to screen a large, diverse compound database potentially containing natural and synthetic compounds for PPAR" agonists that are more efficacious and safer than currently available T2D treatments. The computational molecular modeling methods used include molecular docking, molecular dynamics, steered molecular dynamics, and structure- and ligand-based pharmacophore modeling. Potential binders identified in the computational component funnel into wet-lab experiments to confirm binding, assess activation, and test preclinical efficacy in a mouse model for T2D and other chronic inflammation diseases. The initial process used provided "-eleostearic acid as a compound that ameliorates inflammatory bowel disease in a pre-clinical trial. Incorporating pharmacophore analyses and binding interaction information improved the method for use with a diverse ligand database of thousands of compounds. The adjusted methods showed enrichment for full agonist binder identification. Identifying lead compounds using our method would be an efficient means of addressing the need for alternative T2D treatments. / Ph. D.

Background: Current targeted therapies in pancreatic cancer have been ineffective. The tumor stroma, including intra- and peri-tumoral inflammation and fibrosis, is increasingly implicated in pancreatic cancer. Pancreatic cancer is characterized by a highly fibrotic tumor environment resulting in stromal resistance to chemotherapy. Peroxisome proliferator-activated receptor-alpha (PPARα), a ligand-activated nuclear receptor/transcription factor, is a negative regulator of inflammation. In PPARα deficient mice, stromal processes inhibit tumor growth, resulting in dormant tumors. The presence of PPARα in the tumor cells as well as in the host is necessary for unabated tumor growth. Objective: We hypothesized that blocking the PPARα pathway with a small molecule PPARα antagonist (NXT) may prevent pancreatic cancer progression by targeting tumor cells as well as non-neoplastic cells in the tumor microenvironment. Methods: Growth inhibitory activity of the PPARα antagonist was assessed in murine as well as human pancreatic tumor cell lines (Panc0H7 and BxPC3) and in a murine macrophage cell line (RAW 264.7). Cell viability was determined by trypan blue exclusion assay. AKT, P-AKT, PCNA, BAX, and p27 levels were analyzed by western blot analysis. Cell cycle changes were detected by flow cytometry. Cellular senescence was determined by senescence-associated β-gal (SA-β-gal) staining. Results: The PPARα antagonist inhibited cell growth in macrophages and in pancreatic tumor cells as confirmed by reduced protein level expression of PCNA and activated AKT. Treatment of the PPARα antagonist was non-cytotoxic to tumor cells. Inhibition of PPARα induced cell cycle arrest at G0/G1 in tumor cells and macrophages. The induction of cellular senescence was observed in pancreatic cancer cells. Interestingly, we observed a reduction in protein level expression of BAX, a marker for apoptosis, and p27, an inhibitor of the cell cycle. Conclusion: We now demonstrate that a PPARα antagonist exerts its anti-growth activity by inducing G0/G1 cell cycle arrest, thereby inducing cellular senescence without cell death. These findings provide a mechanism for the anti-tumorigenic activity of PPARα inhibition, and the rationale to use PPARα antagonists as a novel therapeutic approach to pancreatic cancer. / 2016-11-03T00:00:00Z