PPARα Agonist WY-14643 Research Articles

Weighted gene co-expression network analysis identified GBP2 connected to PPARα activity and liver cancer

Liver cancer is the fourth leading cause of cancer-related deaths with a steadily increasing rate worldwide, as a well-known hallmark of liver cancer, metabolic alterations are related to liposomal changes, a common characteristic of primary liver cancers based on recent lipidomics studies. Peroxisome proliferator-activated receptor α (PPARα) is a ligand-activated transcription factor with important lipid homeostasis function, therefore we aimed to understand the molecular mechanisms and pathways that activate PPARα after using PPAR-α agonist WY-14643 and identify candidate biomarkers related to PPARα activity and evaluate their effects in liver cancer. The data from differently expressed genes (DEGs) between liver cancer tissue from obese subjects alone and liver tissue after treatment were evaluated by DESeq2 and module genes were analyzed using weighted gene co-expression network analysis (WGCNA). Final candidate genes were identified by intersecting genes among highly ranked DEGs and the brown module, which demonstrated a significant negative correlation with drug treatments. We conducted a protein–protein interaction network, and KEGG enrichment analysis, and core hub genes (CD40, CXCL9, CXCL10, TNFSF14, GBP2, GBP3, APOL3, CLDN1) were identified using the cyto-hubba plugin, among them we focused on GBP2 that plays key roles in oncogenesis and evaluate its expressional with clinical outcomes. In conclusion, the WGCNA-based co-expression network identified GBP2 as one of the hub genes with a negative relation with PPARα agonist treatments. higher expression of GBP2 was closely associated with HCC progression. Therefore, GBP2 might be a potential candidate for the study of PPARα activity in HCC.

Impaired autophagy contributes to the aggravated deterioration of osteoarthritis articular cartilage by peroxisome proliferator-activated receptor α deficiency, associated with decreased ERK and Akt activation

BackgroundAlthough the chondroprotection of peroxisome proliferator-activated receptor α (PPARα) activation against osteoarthritis (OA) has been revealed, the regulatory mechanism of PPARα deficiency to aggravate osteoarthritic cartilage deterioration remains unclear. Here, we aimed to investigate whether and how autophagy is involved in OA pathological progression.MethodsModel of experimental OA was established using destabilization of the medial meniscus in PPARα-KO 129S4/SvJae male mice, followed by histopathological detection of articular cartilage and immunohistochemistry detection of extracellular matrix (ECM) or autophagy-related signal molecules. Meanwhile, human OA chondrocytes obtained from total knee replacement surgery patients with OA were cultured with the pretreatment of IL-1β, followed with the treatment of PPARα agonist WY14643 and the detection of related signal molecules.ResultsPPARα deficiency aggravated cartilage damage with decreased LC3B level in combination with an increase in P62 level, accompanied with reduced p-Akt and p-ERK levels in PPARα-KO mouse model of experimental OA. On the contrary, PPARα activation by WY14643 promoted ECM synthesis in IL-1β-treated human OA chondrocytes, accompanied with increased LC3B-II/I ratio and Beclin 1 level and decreased P62 and Bcl2 levels. Meanwhile, it was observed that activated ERK and Akt by PPARα activation contributed to the enhancement of autophagy and ECM synthesis in human OA chondrocytes.ConclusionsImpaired autophagy contributed to the aggravated deterioration of osteoarthritis articular cartilage by PPARα deficiency associated with the suppression of ERK and Akt, with an implication that triggering PPARα activation ought to be a potential promising therapeutic target for OA therapy.

Cardiomyocyte peroxisome proliferator-activated receptor α prevents septic cardiomyopathy via improving mitochondrial function.

Clinically, cardiac dysfunction is a key component of sepsis-induced multi-organ failure. Mitochondria are essential for cardiomyocyte homeostasis, as disruption of mitochondrial dynamics enhances mitophagy and apoptosis. However, therapies targeted to improve mitochondrial function in septic patients have not been explored. Transcriptomic data analysis revealed that the peroxisome proliferator-activated receptor (PPAR) signaling pathway in the heart was the most significantly decreased inthe cecal ligation puncture-treated mouse heart model, and PPARα was the most notably decreased among the three PPAR family members. Male Pparafl/fl (wild-type), cardiomyocyte-specific Ppara-deficient (PparaΔCM), and myeloid-specific Ppara-deficient (PparaΔMac) mice were injected intraperitoneally with lipopolysaccharide (LPS) to induce endotoxic cardiac dysfunction. PPARα signaling was decreased in LPS-treated wild-type mouse hearts. To determine the cell type in which PPARα signaling was suppressed, the cell type-specific Ppara-null mice were examined. Cardiomyocyte- but not myeloid-specific Ppara deficiency resulted in exacerbated LPS-induced cardiac dysfunction. Ppara disruption in cardiomyocytes augmented mitochondrial dysfunction, as revealed by damaged mitochondria, lowered ATP contents, decreased mitochondrial complex activities, and increased DRP1/MFN1 protein levels. RNA sequencing results further showed that cardiomyocyte Ppara deficiency potentiated the impairment of fatty acid metabolism in LPS-treated heart tissue. Disruption of mitochondrial dynamics resulted in increased mitophagy and mitochondrial-dependent apoptosis in Ppara△CM mice. Moreover, mitochondrial dysfunction caused an increase of reactive oxygen species, leading to increased IL-6/STAT3/NF-κB signaling. 3-Methyladenine (3-MA, an autophagosome formation inhibitor) alleviated cardiomyocyte Ppara disruption-induced mitochondrial dysfunction and cardiomyopathy. Finally, pre-treatment with the PPARα agonist WY14643 lowered mitochondrial dysfunction-induced cardiomyopathy in hearts from LPS-treated mice. Thus, cardiomyocyte but not myeloid PPARα protects against septic cardiomyopathy by improving fatty acid metabolism and mitochondrial dysfunction, thus highlighting that cardiomyocyte PPARα may be a therapeutic target for the treatment of cardiac disease.

Peroxisome proliferator-activated receptor α agonist induces mouse hepatomegaly through the spatial hepatocyte enlargement and proliferation.

Peroxisome proliferator-activated receptor alpha (PPARα) activation-induced hepatomegaly is accompanied by hepatocyte hypertrophy around the central vein (CV) area and hepatocyte proliferation around the portal vein (PV) area. However, the molecular mechanisms underlying this spatial change of hepatocytes remains unclear. In this study, we examined the characteristics and possible reasons for the zonation distinction of hypertrophy and proliferation during PPARα activation-induced mouse liver enlargement. Mice were injected with corn oil or a typical mouse PPARα agonist WY-14643 (100 mg·kg-1·d-1, i.p.) for 1, 2, 3, 5 or 10 days. At each time point, the mice were sacrificed after the final dose, and liver tissues and serum were harvested for analysis. We showed that PPARα activation induced zonal changes in hepatocyte hypertrophy and proliferation in the mice. In order to determine the zonal expression of proteins related to hepatocyte hypertrophy and proliferation in PPARα-induced liver enlargement, we performed digitonin liver perfusion to separately destroy the hepatocytes around the CV or PV areas, and found that PPARα activation-induced increase magnitude of its downstream targets such as cytochrome P450 (CYP) 4 A and acyl-coenzyme A oxidase 1 (ACOX1) levels around the CV area were higher compared with those around the PV area. Upregulation of proliferation-related proteins such as cell nuclear antigen (PCNA) and cyclin A1 (CCNA1) after WY-14643-induced PPARα activation mainly occurred around the PV area. This study reveals that the zonal expression of PPARα targets and proliferation-related proteins is responsible for the spatial change of hepatocyte hypertrophy and proliferation after PPARα activation. These findings provide a new insight into the understanding of PPARα activation-induced liver enlargement and regeneration.

Effects of peroxisome proliferator activated receptor-α agonist on growth performance, blood profiles, gene expression related to liver fat metabolism in broilers fed diets containing corn naturally contaminated with mycotoxins.

This study was conducted to determine the subclinical symptom of broilers exposure to mycotxoins from corn naturally contaminated, and the preventive effect with peroxisome proliferator activated receptor-α (PPARα) agonist (Wy-14643) supplementation. A total of 360 one-day -old male Arbor Acres broilers were randomly distributed into 4 treatments with 9 replicates of 10 birds. Dietary treatments included: treatment 1, normal corn diets group, treatment 2, normal corn + Wy-14643 diets group, treatment 3, mycotoxin-contaminated corn diets group, treatment 4, mycotoxin-contaminated corn + Wy-14643 diets group. The supplementation of Wy-14643 was added at the expense of 1 and 2 mg/kg in starter and grower diets, respectively. Birds fed mycotoxin diets had lower (P < 0.05) final body weight (BW), Body weight gain (BWG), feed intake (FI), and had higher (P < 0.05) feed conversion ratio (FCR). Feeding mycotoxin diets reduced (P < 0.05) the levels of serum superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), catalase (CAT), total antioxidative capacity (T-AOC) and high-density lipoprotein cholesterol (HDL-C), but higher malondialdehyde (MDA), triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and fatty acid synthetase (FAS). The supplementation of Wy-14643 increased (P < 0.05) the level of serum T-AOC, but reduced (P < 0.05) TG and LDL-C. Interactive effect was not observed (P > 0.05) in growth performance and blood profiles. The relative expression of PPARα mRNA and 3-Hydroxy-3-MethylGlutaryl-CO enzyme A (HMGCoA) mRNA was higher (P < 0.05) in treatment 3 and treatment 4 than treatment 1 and treatment 2, and there was significant difference (P <0.05) between treatment 3 and treatment 4. There was significant difference (P < 0.05) between groups of the relative expression of recombinant carnitine palmitoyl transferase 1 (CPT1) mRNA. The relative expression of acyl CoA oxidase (ACO) mRNA was higher (P < 0.05) in treatment 1 and treatment 4 than treatment 2 and treatment 3, and there was significant difference (P < 0.05) between treatment 1 and treatment 4. The relative expression of apolipoprotein A (APO-A) mRNA was higher (P < 0.05) in treatment 1 and treatment 4 than treatment 2 and treatment 3. The relative expression of sterol regulatory element binding protein (SREBP) mRNA was lower (P < 0.05) in treatment 2, treatment 3 and treatment 4 than treatment 1, and there was significant difference (P < 0.05) between treatment 3 and treatment 4. Overall, feeding naturally contaminated mycotoxin diets caused negative effects on growth performance and blood profiles, while diet supplementation with Wy-14643 alleviate the detrimental effects on gene and expression related to liver fat metabolism in broilers.

The effect of PPAR isoform (de)activation on the lipid composition in full-thickness skin models.

Human skin equivalents (HSEs) are 3D-cultured human skin models that mimic many aspects of native human skin (NHS). Although HSEs resemble NHS very closely, the barrier located in the stratum corneum (SC) is impaired. This is caused by an altered lipid composition in the SC of HSEs compared with NHS. One of the most pronounced changes in this lipid composition is a high level of monounsaturation. One key enzyme in this change is stearoyl-CoA desaturase-1 (SCD1), which catalyses the monounsaturation of lipids. In order to normalize the lipid composition, we aimed to target a group of nuclear receptors that are important regulators in the lipid synthesis. This group of receptors are known as the peroxisome proliferating activating receptors (PPARs). By (de)activating each isoform (PPAR-α, PPAR-δ and PPAR-γ), the PPAR isoforms may have normalizing effects on the lipid composition. In addition, another PPAR-α agonist Wy14643 was included as this supplement demonstrated normalizing effects in the lipid composition in a more recent study. After PPAR (ant)agonists supplementation, the mRNA of downstream targets, lipid synthesis genes and lipid composition were investigated. The PPAR downstream targets were activated, indicating that the supplements reached the keratinocytes to trigger their effect. However, minimal impact was observed on the lipid composition after PPAR isoform (de) activation. Only the highest concentration Wy14643 resulted in strong, but negative effects on CER composition. Although the novel tested modifications did not result in an improvement, more insight is gained on the nuclear receptors PPARs and their effects on the lipid barrier in full-thickness skin models.

Chronic Treatment With WY-14643 Induces Tumorigenesis and Triglyceride Accumulation in Mouse Livers.

Peroxisome proliferator-activated receptor α (PPARα) is closely related to lipid metabolism and various liver diseases. Previous study has shown that chronic treatment with PPARα agonist WY-14643 can induce liver tumors in rodents, but the implications of this process on lipid metabolism in the liver remain unclear. Thus, this study aimed to explore the influences of chronic treatment with WY-14643 on the liver and hepatic lipid metabolism. Wild-type C57BL/6 mice were treated with WY-14643 (100 mg/kg/week, i.p.) or corn oil, and liver and serum samples were collected for testing after 42 weeks of WY-14643 treatment. The results showed that hepatomegaly, liver tumors with mild liver injury, and hepatocyte proliferation were induced in mice treated with WY-14643. The mRNA and protein expression levels of PPARα downstream targets acyl-CoA oxidase 1 and cytochrome P450 4A were significantly upregulated in the WY-14643-treated group. Lipidomic analysis revealed that chronic treatment with WY-14643 disturbed lipid homeostasis, especially triglycerides (TGs), which were significantly elevated after WY-14643 treatment. Moreover, TG homeostasis-related genes were significantly increased in the WY-14643-treated group. In conclusion, these findings demonstrated that hepatomegaly and liver tumors induced by chronic treatment with WY-14643 in mice are accompanied by hepatocyte proliferation and TG accumulation. SIGNIFICANCE STATEMENT: The present study clearly demonstrated that sustained peroxisome proliferator-activated receptor α (PPARα) activation by chronic treatment with WY-14643 induces hepatomegaly and liver tumors with triglyceride accumulation by regulating lipid homeostasis-related genes in mice. These findings may help to clarify the influences of sustained PPARα activation on liver lipid homeostasis and provide data for the clinically rational use of drugs that can activate PPARα.

Peroxisome Proliferator-Activated Receptor Activation in Precision-Cut Bovine Liver Slices Reveals Novel Putative PPAR Targets in Periparturient Dairy Cows.

Metabolic challenges experienced by dairy cows during the transition between pregnancy and lactation (also known as peripartum), are of considerable interest from a nutrigenomic perspective. The mobilization of large amounts of non-esterified fatty acids (NEFA) leads to an increase in NEFA uptake in the liver, the excess of which can cause hepatic accumulation of lipids and ultimately fatty liver. Interestingly, peripartum NEFA activate the Peroxisome Proliferator-activated Receptor (PPAR), a transcriptional regulator with known nutrigenomic properties. The study of PPAR activation in the liver of periparturient dairy cows is thus crucial; however, current in vitro models of the bovine liver are inadequate, and the isolation of primary hepatocytes is time consuming, resource intensive, and prone to errors, with the resulting cells losing characteristic phenotypical traits within hours. The objective of the current study was to evaluate the use of precision-cut liver slices (PCLS) from liver biopsies as a model for PPAR activation in periparturient dairy cows. Three primiparous Jersey cows were enrolled in the experiment, and PCLS from each were prepared prepartum (−8.0 ± 3.6 DIM) and postpartum (+7.7± 1.2 DIM) and treated independently with a variety of PPAR agonists and antagonists: the PPARα agonist WY-14643 and antagonist GW-6471; the PPARδ agonist GW-50156 and antagonist GSK-3787; and the PPARγ agonist rosiglitazone and antagonist GW-9662. Gene expression was assayed through RT-qPCR and RNAseq, and intracellular triacylglycerol (TAG) concentration was measured. PCLS obtained from postpartum cows and treated with a PPARγ agonist displayed upregulation of ACADVL and LIPC while those treated with PPARδ agonist had increased expression of LIPC, PPARD, and PDK4. In PCLS from prepartum cows, transcription of LIPC was increased by all PPAR agonists and NEFA. TAG concentration tended to be larger in tissue slices treated with PPARδ agonist compared to CTR. Use of PPAR isotype-specific antagonists in PCLS cultivated in autologous blood serum failed to decrease expression of PPAR targets, except for PDK4, which was confirmed to be a PPARδ target. Transcriptome sequencing revealed considerable differences in response to PPAR agonists at a false discovery rate-adjusted p-value of 0.2, with the most notable effects exerted by the PPARδ and PPARγ agonists. Differentially expressed genes were mainly related to pathways involved with lipid metabolism and the immune response. Among differentially expressed genes, a subset of 91 genes were identified as novel putative PPAR targets in the bovine liver, by cross-referencing our results with a publicly available dataset of predicted PPAR target genes, and supplementing our findings with prior literature. Our results provide important insights on the use of PCLS as a model for assaying PPAR activation in the periparturient dairy cow.

Adipose triglyceride lipase-mediated lipid catabolism is essential for bronchiolar regeneration.

The lung airways are constantly exposed to inhaled toxic substances, resulting in cellular damage that is repaired by local expansion of resident bronchiolar epithelial club cells. Disturbed bronchiolar epithelial damage repair lies at the core of many prevalent lung diseases, including chronic obstructive pulmonary disease, asthma, pulmonary fibrosis, and lung cancer. However, it is still not known how bronchiolar club cell energy metabolism contributes to this process. Here, we show that adipose triglyceride lipase (ATGL), the rate-limiting enzyme for intracellular lipolysis, is critical for normal club cell function in mice. Deletion of the gene encoding ATGL, Pnpla2 (also known as Atgl), induced substantial triglyceride accumulation, decreased mitochondrial numbers, and decreased mitochondrial respiration in club cells. This defect manifested as bronchiolar epithelial thickening and increased airway resistance under baseline conditions. After naphthalene‑induced epithelial denudation, a regenerative defect was apparent. Mechanistically, dysfunctional PPARα lipid-signaling underlies this phenotype because (a) ATGL was needed for PPARα lipid-signaling in regenerating bronchioles and (b) administration of the specific PPARα agonist WY14643 restored normal bronchiolar club cell ultrastructure and regenerative potential. Our data emphasize the importance of the cellular energy metabolism for lung epithelial regeneration and highlight the significance of ATGL-mediated lipid catabolism for lung health.

MTORC2 Facilitates Liver Regeneration Through Sphingolipid-Induced PPAR-α-Fatty Acid Oxidation.

During liver regeneration after partial hepatectomy, the function and metabolic pathways governing transient lipid droplet accumulation in hepatocytes remain obscure. Mammalian target of rapamycin 2 (mTORC2) facilitates de novo synthesis of hepatic lipids. Under normal conditions and in tumorigenesis, decreased levels of triglyceride (TG) and fatty acids (FAs) are observed in the mTORC2-deficient liver. However, during liver regeneration, their levels increase in the absence of mTORC2. Rictor liver-specific knockout and control mice underwent partial hepatectomy, followed by measurement of TG and FA contents during liver regeneration. FA metabolism was evaluated by analyzing the expression of FA metabolism-related genes and proteins. Intraperitoneal injection of the peroxisome proliferator-activated receptor α (PPAR-α) agonist, p53 inhibitor, and protein kinase B (AKT) activator was performed to verify the regulatory pathways involved. Lipid mass spectrometry was performed to identify the potential PPAR-α activators. The expression of FA metabolism-related genes and proteins suggested that FAs are mainly transported into hepatocytes during liver regeneration. The PPAR-α pathway is down-regulated significantly in the mTORC2-deficient liver, resulting in the accumulation of TGs. The PPAR-α agonist WY-14643 rescued deficient liver regeneration and survival in mTORC2-deficient mice. Furthermore, lipidomic analysis suggested that mTORC2 deficiency substantially reduced glucosylceramide (GluCer) content. GluCer activated PPAR-α. GluCer treatment in vivo restored the regenerative ability and survival rates in the mTORC2-deficient group. Our data suggest that FAs are mainly transported into hepatocytes during liver regeneration, and their metabolism is facilitated by mTORC2 through the GluCer-PPAR-α pathway, thereby establishing a novel role for mTORC2 in lipid metabolism.

PPARα Agonist WY-14643 Relieves Neuropathic Pain through SIRT1-Mediated Deacetylation of NF-κB.

Inflammation caused by neuropathy contributes to the development of neuropathic pain (NP), but the exact mechanism still needs to be understood. Peroxisome proliferator-activated receptor α (PPARα), an important inflammation regulator, might participate in the inflammation in NP. To explore the role of PPARα in NP, the effects of PPARα agonist WY-14643 on chronic constriction injury (CCI) rats were evaluated. The results showed that WY-14643 stimulation could decrease inflammation and relieve neuropathic pain, which was relative with the activation of PPARα. In addition, we also found that the SIRT1/NF-κB pathway was involved in the WY-14643-induced anti-inflammation in NP, and activation of PPARα increased SIRT1 expression, thus reducing the proinflammatory function of NF-κB. These data suggested that WY-14643 might serve as an inflammation mediator, which may be a potential therapy option for NP.

SIRT1 alleviates hepatic ischemia-reperfusion injury via the miR-182-mediated XBP1/NLRP3 pathway

The hepatoprotection of histone deacetylase sirtuin 1 (SIRT1) has been identified to attenuate ischemia-reperfusion (IR)-triggered inflammation and liver damage. This study was performed to characterize the function of SIRT1 in hepatic IR injury. In in vivo assays on liver-specific knockout mice of SIRT1, we first validated the effect of SIRT1 knockout on liver damage and XBP1/NLRP3 inflammasome activation. Next, we examined whether knockdown of XBP1/NLRP3 or miR-182 agomir could reverse the effect of SIRT1 knockout. In in vitro assays, NCTC1469 cells subjected to hypoxia/reoxygenation (H/R) were transduced with small interfering RNA (siRNA)/activator of SIRT1 or miR-182 agomir to confirm the effect of SIRT1 on NCTC1469 cell behaviors as well as the regulation of miR-182 and the XBP1/NLRP3 signaling pathway. Hepatic IR injury was appreciably aggravated in SIRT1 knockout mice, and SIRT1 knockdown abolished the inhibition of XBP1/NLRP3 inflammasome activation, which was reversed by NLRP3 knockdown, XBP1 knockdown, or miR-182 agomir. Mechanistically, miR-182 expression was positively regulated by SIRT1 in hepatic IR injury in mice, and miR-182 inhibited the expression of XBP1 by binding to the 3′ untranslated region (UTR) of XBP1. The histone deacetylase SIRT1 inhibits the downstream XBP1/NLRP3 inflammatory pathway by activating miR-182, thus alleviating hepatic IR injury in mice.

Peroxisome proliferator-activated receptor α (PPARα)-dependent regulation of fibroblast growth factor 23 (FGF23).

Bone cells secrete fibroblast growth factor 23 (FGF23), a hormone that inhibits the synthesis of active vitamin D (1,25(OH)2D3) and induces phosphate excretion in the kidney. In addition, it exerts paracrine effects on other cells including hepatocytes, cardiomyocytes, and immune cells. The production of FGF23 is controlled by different factors including parathyroid hormone, 1,25(OH)2D3, alimentary phosphate, insulin, inflammation, and AMP-dependent kinase (AMPK) regulation of store-operated Ca2+ entry (SOCE). Peroxisome proliferator-activated receptor α (PPARα) is a transcription factor with anti-inflammatory properties regulating lipid metabolism. Fibrates, PPARα agonists, are used in the treatment of dyslipidemia and activate AMPK. Here, we tested whether PPARα is a regulator of FGF23. Fgf23 gene expression was analyzed in UMR106 rat osteoblast-like cells by qRT-PCR, AMPK phosphorylation by Western blotting, and SOCE assessed by fluorescence optics. PPARα agonists fenofibrate and WY-14643 suppressed, whereas PPARα antagonist GW6471 and siRNA-mediated knockdown of PPARα induced Fgf23 gene expression. Fenofibrate induced AMPK activity in UMR106 cells and lowered SOCE. AMPK inhibitor compound C abrogated the PPARα effect on FGF23 in large part. Silencing of Orai-1 resulted in failure of PPARα to significantly influence Fgf23 expression. Taken together, PPARα is a potent regulator of FGF23. PPARα agonists down-regulate FGF23 formation at least in part through AMPK-mediated suppression of SOCE.

Peroxisome proliferator-activated receptor alpha regulates the expression of the immune response mediators in the porcine endometrium during the estrous cycle and early pregnancy.

Cytokines are immune response mediators that play an important role in the regulation of reproductive functions. An association between cytokines and peroxisome proliferator receptors (PPARs) has been reported in various tissues, including the endometrium. The present study aimed to evaluate the impact of PPARα ligands on the expression of nuclear factor kappa B (NF-κB) and cytokines (interleukin [IL]-1β, IL-4, IL-6, IL-8, IL-10, and LIF) in the porcine endometrium in different reproductive stages. Endometrial slices were collected from gilts on days 10-12 or 14-16 of the estrous cycle and pregnancy. Endometrial tissue explants were incubated in vitro in the presence or absence of PPARα agonist WY-14643 and antagonist MK886. Expression of mRNA and protein for NF-ĸB and selected cytokines was evaluated by real-time PCR and immunoblot. PPARα agonist WY-14643 decreased the mRNA expression of NF-κB in most of the analyzed stages (excluding days 10-12 of the estrous cycle), but increased the expression of NF-κB protein (excluding days 14-16 of pregnancy). The WY-14643 increased expression of IL-1β and IL-6 proteins, and the mRNA expression of IL-8 and LIF, decreased IL-4 expression, and did not affect the mRNA and protein expression of IL-10. The obtained results demonstrate that PPARα is involved in the regulation of NF-κB and cytokine expression in the porcine endometrium. PPARα ligands exert a varied influence on immune system components, which could be attributed to differences in the receptivity of porcine endometrial tissue during the reproductive cycle.

The role of Ap2a2 in PPARα-mediated regulation of lipolysis in adipose tissue.

Adipose tissue plays a major role in the regulation of systemic metabolic homeostasis, with the AP2 adaptor complex being important in clathrin-mediated endocytosis (CME) of various cell surface receptors, including glucose transporter 4, the insulin receptor, and β-adrenergic receptors (ARs). One of the AP2 subunits, adaptor-related protein complex 2, α2 subunit (Ap2a2), has recently been identified as a peroxisome proliferator-activated receptor (PPAR)α target gene. The effects of PPARα on the AP2 adaptor complex and CME are unknown. We generated adipocyte-specific Ap2a2 knockout mice and investigated their metabolism when fed a standard chow or high-fat diet, without and with supplementation with the PPARα-agonist WY-14643 (WY). Although Ap2a2 deletion had only minor effects on glycaemic control, it led to substantial impairment in β-adrenergic activation of lipolysis, as evidenced by a loss of cAMP response, PKA activation, and glycerol/fatty acid release. These differences were related to increased cell surface localization of the β2- and β3-ARs. Lipolytic defects were accompanied by impaired WY-mediated loss of fat mass and whole-body fat oxidation. This study demonstrates a novel role for PPARα in β-adrenergic regulation of adipose tissue lipolysis and for adipose tissue in supplying adequate substrate to other peripheral tissues to accommodate the increase in systemic fatty acid oxidation that occurs upon treatment with PPARα agonists.-Montgomery, M. K., Bayliss, J., Keenan, S., Rhost, S., Ting, S. B., Watt, M. J. The role of Ap2a2 in PPARα-mediated regulation of lipolysis in adipose tissue.

Hepatic peroxisome proliferator-activated receptor alpha mediates the major metabolic effects of Wy-14643.

Peroxisome proliferator-activated receptor alpha (PPARα) is a molecular target of various fibrate drugs clinically used to lower serum lipids. However, the tissue-specific functions of PPARα remain to be elucidated. This study aimed to explore the tissue-specific functions of PPARα in response to Wy-14643. A hepatocyte-specific Ppara knockout mouse line was used to explore the impact of hepatic PPARα activity on the systemic response to treatment with the potent PPARα agonist Wy-14643. Wy-14643 mainly activated hepatic PPARα and regulated the expression of PPARα target genes in liver. Hepatic Ppara disruption abolished the triglyceride lowering effects of Wy-14643, prevented agonist-induced hypophagia, and ablated PPARα target gene response in the liver. These findings indicate that Wy-14643 treatment mainly activates hepatic PPARα, and the hypolipidemic and hypophagic effects of Wy-14643 are dependent on PPARα activation within hepatocytes.

PPAR-α agonists acutely inhibit Ca2+-independent PLA2 to reduce H2O2-induced contractions in aortae of spontaneously hypertensive rats.

Hypertension is associated with endothelial dysfunction, which favors the release of endothelium-derived contracting factors, including vasoconstrictor prostanoids and reactive oxygen species. Peroxisome proliferator-activated receptor-α (PPAR-α) agonists, clinically used as lipid-lowering drugs, possess antioxidant properties and exert beneficial effects in the vascular system. The present study aimed to identify the mechanism(s) underlying the acute effects of the PPAR-α agonists Wy14643 and fenofibate on endothelium-dependent contractions, in particular those related to oxidative stress, in the aorta of the spontaneously hypertensive rat (SHR). Aortic rings with and without endothelium of male SHRs and normotensive Wistar-Kyoto rats were suspended in organ chambers for isometric tension measurements and homogenized for enzyme activity assays. Contractions to acetylcholine in quiescent SHR aortae with endothelium were reduced by tiron (superoxide anion scavenger), diethyldithiocarbamic acid (superoxide dismutase inhibitor), and acute treatment with either Wy14643 or fenofibrate. Similarly to contractions evoked by acetylcholine, H2O2-induced increases in tension in SHR aortae involved, in succession, phospholipase A2 (PLA2), cyclooxygenase, and thromboxane-prostanoid receptors. Wy14643 or fenofibrate, by decreasing the activity of endothelial Ca2+-independent PLA2, attenuated the contractions to H2O2. In conclusion, the increased oxidative stress in the SHR aorta (mainly increased production of H2O2 and its partially reduced product, hydroxyl radical) contributed to acetylcholine-induced, endothelium-dependent contractions; PPAR-α agonists likely inhibit the H2O2-mediated contractions by inhibiting endothelial Ca2+-independent PLA2. The present study highlights the prospective therapeutic effects of PPAR-α agonists in improving endothelial function in hypertension and other vascular implications due to oxidative stress. NEW & NOTEWORTHY Peroxisome proliferator-activated receptor-α agonists, which are used clinically as lipid-lowering drugs, acutely reduce H2O2-induced contractions in aortae of hypertensive rats by inhibiting the activity of endothelial Ca2+-independent phospholipase A2. These vascular effects of peroxisome proliferator-activated receptor-α agonists suggest that they may help to prevent vascular complications under pathological conditions associated with oxidative stress.

Alleviation of Toxicity Caused by Overactivation of Pparα through Pparα-Inducible miR-181a2.

Widely varied compounds, including certain plasticizers, hypolipidemic drugs (e.g., ciprofibrate, fenofibrate, WY-14643, and clofibrate), agrochemicals, and environmental pollutants, are peroxisome proliferators (PPs). Appropriate dose of PPs causes a moderate increase in the number and size of peroxisomes and the expression of genes encoding peroxisomal lipid-metabolizing enzymes. However, high-dose PPs cause varied harmful effects. Chronic administration of PPs to mice and rats results in hepatomegaly and ultimately carcinogenesis. Nuclear receptor protein peroxisome proliferator-activated receptor-α (Pparα) was shown to be required for this process. However, biological adaptations to minimize this risk are poorly understood. In this study, we found that miR-181a2 expression was induced by the Pparα agonist WY-14643. Moreover, exogenous expression of miR-181a-5p dramatically alleviated the cell toxicity caused by overactivation of Pparα. Further studies showed that miR-181a-5p directly targeted the Pparα 3′ untranslated region and depressed the Pparα protein level. This study identified a feedback loop between miR-181a-5p and Pparα, which allows biological systems to approach a balance when Pparα is overactivated.

Involvement of nutrients and nutritional mediators in mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase gene expression.

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase (HMGCS2) catalyses the first step of ketogenesis and is critical in various metabolic conditions. Several nutrient molecules were able to differentially modulate HMGCS2 expression levels. Docosahexaenoic acid (DHA, C22:6, n-3), eicosapentaenoic acid (EPA, C20:5, n-3), arachidonic acid (AA, C20:4, n-6), and glucose increased HMGCS2 mRNA and protein levels in HepG2 hepatoma cells, while fructose decreased them. The effect of n-6 AA resulted significantly higher than that of n-3 PUFA, but when combined all these molecules were far less efficient. Insulin reduced HMGCS2 mRNA and protein levels in HepG2 cells, even when treated with PUFA and monosaccharides. Several nuclear receptors and transcription factors are involved in HMGCS2 expression regulation. While peroxysome proliferator activated receptor α (PPAR-α) agonist WY14643 increased HMGCS2 expression, this treatment was unable to affect PUFA-mediated regulation of HMGCS2 expression. Forkhead box O1 (FoxO1) inhibitor AS1842856 reduced HMGCS2 expression and suppressed induction promoted by fatty acids. Cells treatment with liver X receptor alpha (LXRα) agonist T0901317 reduced HMGCS2 mRNA, indicating a role for this transcription factor as suppressor of HMGCS2 gene. Previous observations already indicated HMGCS2 expression as possible nutrition status reference: our results show that several nutrients as well as specific nutritional related hormonal conditions are able to affect significantly HMGCS2 gene expression, indicating a relevant role for PUFA, which are mostly derived from nutritional intake. These insights into mechanisms of its regulation, specifically through nutrients commonly associated with disease risk, indicate HMGCS2 expression as possible reference marker of metabolic and nutritional status.

Inhibition of JNK signalling mediates PPARα‐dependent protection against intrahepatic cholestasis by fenofibrate

Fenofibrate, a PPARα agonist, is the most widely prescribed drug for treating hyperlipidaemia. Although fibrate drugs are reported to be beneficial for cholestasis, their underlying mechanism has not been determined. Wild-type mice and Pparα-null mice were pretreated orally with fenofibrate for 3 days, following which α-naphthylisothiocyanate (ANIT) was administered to induce cholestasis. The PPARα agonist WY14643 and JNK inhibitor SP600125 were used to determine the role of PPARα and the JNK pathway, respectively, in cholestatic liver injury. The same fenofibrate regimen was applied to investigate its beneficial effects on sclerosing cholangitis in a DDC-induced cholestatic model. Fenofibrate, 25mg·kg-1 twice a day, totally attenuated ANIT-induced cholestasis and liver injury as indicated by biochemical and histological analyses. This protection occurred in wild-type, but not in Pparα-null, mice. Alterations in bile acid synthesis and transport were found to be an adaptive response rather than a direct effect of fenofibrate. WY14643 attenuated ANIT-induced cholestasis and liver injury coincident with inhibition of JNK signalling. Although SP600125 did not affect cholestasis, it inhibited liver injury in the ANIT model when the dose of fenofibrate used was ineffective. Fenofibrate was also revealed to have a beneficial effect in the sclerosing cholangitis model. These data suggest that the protective effects of fenofibrate against cholestasis-induced hepatic injury are dependent on PPARα and fenofibrate dose, and are mediated through inhibition of JNK signalling. This mechanism of fenofibrate protection against intrahepatic cholestasis may offer additional therapeutic opportunities for cholestatic liver diseases.