WY-14,643 Research Articles

In vitro activation of peroxisome proliferator activated receptor α by some extracts from food materials

Peroxisome proliferator activated receptor α (PPARα) is a ligand-dependent transcription factor that regulates the expression of genes involved in lipid metabolism and transport. Activators of PPARα may be potential hypolipidemic agents, like the fibrate-type drugs. This study was therefore aimed at identifying potential hypolipidemic functional foods by screening activators of PPARα from food materials. CHO-K1 cells were stably transfected with vectors (pBKCMV) carrying cDNAs of a (UAS)4-tk-alkaline phosphatase reporter and a chimeric receptor Gal4-PPARα LBD successively. Clones of cells responsive to Wy-14643, a known PPARα activator were selected, characterized and used for testing the PPARα activating potency of fractions extracted from food materials. Food materials were extracted with ethyl acetate and the extract (EAE) was cold-saponified and tested for the PPARα activating ability using the selected stable transfected clone of cells. 10 µM Wy-14643 (WY) was used as the positive control. Among food materials tested, EAE of bitter gourd showed the highest activity that exceeded Wy-14643, although the activity significantly decreased after saponification. The non-saponifiable fraction of onion EAE had a high maximal fold of activation equivalent to Wy-14643, while that of peanut, lychee, ricinus seed and longan showed moderate activity. The saponifiable fraction of ricinus seed, sesame and peanut also showed moderate activity. Further investigation is needed to explore the health benefit of the PPARα activating food extracts found in this study.

Role of peroxisome proliferator-activated receptor-α on the synthesis of monounsaturated fatty acids in goat mammary epithelial cells.

A key member of the nuclear receptor superfamily is the peroxisome proliferator-activated receptor alpha (PPARA) isoform, which in nonruminants is closely associated with fatty acid oxidation. Whether PPARA plays a role in milk fatty acid synthesis in ruminants is unknown. The main objective of the present study was to use primary goat mammary epithelial cells (GMEC) to activate PPARA via the agonist WY-14643 (WY) or to silence it via transfection of small-interfering RNA (siRNA). Three copies of the peroxisome proliferator-activated receptor response element (PPRE) contained in a luciferase reporter vector were transfected into GMEC followed by incubation with WY at 0, 10, 20, 30, 50, or 100 µM. A dose of 50 µM WY was most effective at activating PPRE without influencing PPARA mRNA abundance. Transfecting siRNA targeting PPARA decreased its mRNA abundance to 20% and protein level to 50% of basal levels. Use of WY upregulated FASN, SCD1, ACSL1, DGAT1, FABP4, and CD36 (1.1-, 1.5-, 2-, 1.4-, 1.5-, and 5-fold, respectively), but downregulated DGAT2 and PGC1A (-20% and -40%, respectively) abundance. In contrast, triacylglycerol concentration decreased and the content and desaturation index of C16:1 and C18:1 increased. Thus, activation of PPARA via WY appeared to channel fatty acids away from esterification. Knockdown of PPARA via siRNA downregulated ACACA, SCD1, AGPAT6, CD36, HSL, and SREBF1 (-43%, -67%, -16%, -56%, -26%, and -29%, respectively), but upregulated ACSL1, DGAT2, FABP3, and PGC1A (2-, 1.4-, 1.3-, and 2.5-fold, respectively) mRNA abundance. A decrease in the content and desaturation index of C16:1 and C18:1 coupled with an increase in triacylglycerol content accompanied those effects at the mRNA level. Overall, data suggest that PPARA could promote the synthesis of MUFA in GMEC through its effects on mRNA abundance of genes related to fatty acid synthesis, oxidation, transport, and triacylglycerol synthesis.

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.

Perfluoroalkyl acids-induced liver steatosis: Effects on genes controlling lipid homeostasis.

Persistent presence of perfluoroalkyl acids (PFAAs) in the environment is due to their extensive use in industrial and consumer products, and their slow decay. Biochemical tests in rodent demonstrated that these chemicals are potent modifiers of lipid metabolism and cause hepatocellular steatosis. However, the molecular mechanism of PFAAs interference with lipid metabolism remains to be elucidated. Currently, two major hypotheses are that PFAAs interfere with mitochondrial beta-oxidation of fatty acids and/or they affect the transcriptional activity of peroxisome proliferator-activated receptor α (PPARα) in liver. To determine the ability of structurally-diverse PFAAs to cause steatosis, as well as to understand the underlying molecular mechanisms, wild-type (WT) and PPARα-null mice were treated with perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), or perfluorohexane sulfonate (PFHxS), by oral gavage for 7days, and their effects were compared to that of PPARα agonist WY-14643 (WY), which does not cause steatosis. Increases in liver weight and cell size, and decreases in DNA content per mg of liver, were observed for all compounds in WT mice, and were also seen in PPARα-null mice for PFOA, PFNA, and PFHxS, but not for WY. In Oil Red O stained sections, WT liver showed increased lipid accumulation in all treatment groups, whereas in PPARα-null livers, accumulation was observed after PFNA and PFHxS treatment, adding to the burden of steatosis observed in control (untreated) PPARα-null mice. Liver triglyceride (TG) levels were elevated in WT mice by all PFAAs and in PPARα-null mice only by PFNA. In vitro β-oxidation of palmitoyl carnitine by isolated rat liver mitochondria was not inhibited by any of the 7 PFAAs tested. Likewise, neither PFOA nor PFOS inhibited palmitate oxidation by HepG2/C3A human liver cell cultures. Microarray analysis of livers from PFAAs-treated mice indicated that the PFAAs induce the expression of the lipid catabolism genes, as well as those involved in fatty acid and triglyceride synthesis, in WT mice and, to a lesser extent, in PPARα-null mice. These results indicate that most of the PFAAs increase liver TG load and promote steatosis in mice We hypothesize that PFAAs increase steatosis because the balance of fatty acid accumulation/synthesis and oxidation is disrupted to favor accumulation.

Protective effect of WY14643 in hepatic ischemia/reperfusion injury via suppressing the activation of TLR4/NF-KB P65 signal pathway.

Background : Hepatic ischemia/reperfusion (I/R) injury is a disaster common critical event which frequently occurs in a variety of clinical scenarios. To investigate the protective effect of Wy14643 (WY) precondition against hepatic ischemia/ reperfusion (I/R) injury in rats and its potential mechanism. Methods : Thirty Sprague–Dawley male rats weighing 220-250 g were randomly divided into three groups (n=10) including the sham-operated +saline group (Sham), the ischemic-reperfusion+ vehicle (IRI), and the WY14643 preconditioning group (WY). The three groups were pretreated with saline, ethanol and WY, at 1 h before ischemia with a concentration of 10 mg/kg , respectively. Hepatic ischemia-reperfusion (I/R) was induced by clamping blood supply to the left lateral and median lobes of the liver for 90 min. Blood samples and liver tissues were obtained at the end of 4h reperfusion. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) were measured. The histological changes of hepatic tissues were examanied by HE staining. The expression of interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) were detected by ELISA and RT-PCR. Moreover, the expression of TLR-4 , IκΒ-α, Myd88, p65 and PPAR-α were detected by western blotting. Result : After 4 h reperfusion, compared with the IRI group, the liver dysfunction (ALT, AST and LDH), damage score, the expression of TNF-α , IL-6, and IL-1β, the protein expression level of TLR4, Myd88, the phosphorylation level of IκB-α and p65 in the WY group was significantly decreased (all P 0.05) . Conclusion : Wy14643 precondition could protect liver againest I/R injury and the protective effect is associated with suppressing inflammation by reducing the activation of TLR-4/NF-κB signaling. Key words : hepatic ischemia reperfusion injury; inflammation; Wy14643; TLR-4/NF-κB

PPAR-α activation reduced LPS-induced inflammation in alveolar epithelial cells

ABSTRACTPurpose of the study: Acute respiratory distress syndrome (ARDS) represents a major cause of mortality in intensive care patients. Activation of peroxisome proliferator-activated receptor-α (PPAR-α) by fibrates, such as WY-14643 (WY), has been described to beneficially influence inflammation and experimental lung injury. The impact of PPAR-α activation on alveolar epithelial cells (AEC) has not been studied yet. Materials and Methods: To investigate the effect of PPAR-α activator WY in wild-type (WT) and in PPAR-α knockout (PPAR-α−/−) animals, mice were treated in different regimes: mice received chow enriched with or without WY for 14 days prior AEC isolation (in-vivo treatment). Furthermore, isolated AEC from both groups were subsequently cultured with or without WY (in-vitro treatment). AEC were stimulated with lipopolysaccharide (LPS). Cell culture supernatant and cell lysate were used for analysis of pro-inflammatory mediators. Results: AEC challenged with LPS showed a significantly increased generation of pro-inflammatory mediators. After in-vivo WY-exposure, AEC displayed significantly reduced concentration of TNF-α, MIP-2, and TxB2 after LPS stimulation. This beneficial effect was abrogated in PPAR-α−/− animals. Interestingly, sole in-vitro application of WY-14643 failed to reduce levels of pro-inflammatory mediators whereas we found an additive effect of a combined in-vivo and in-vitro PPAR-α activation. PGE2 concentration remained high after LPS challenge and was unaffected by WY treatment. Conclusion: PPAR-α activation by in-vivo exposure to fibrates reduced the inflammatory response in isolated AEC. These findings may facilitate further studies investigating the translation of pharmacological PPAR-α activation into clinical therapy of ARDS.

PROTECTIVE EFFECT OF WY14643 IN HEPATIC ISCHEMIA/REPERFUSION INJURY VIA SUPPRESSING THE ACTIVATION OF TLR4/NF-ΚB P65 SIGNAL PATHWAY.

Background: Hepatic ischemia/reperfusion (I/R) injury is a disaster common critical event which frequently occurs in a variety of clinical
 scenarios. To investigate the protective effect of Wy14643 (WY) precondition against hepatic ischemia/ reperfusion (I/R) injury in rats and its
 potential mechanism.
 Methods: Thirty Sprague–Dawley male rats weighing 220-250 g were randomly divided into three groups (n=10) including the sham-operated
 +saline group (Sham), the ischemic-reperfusion+ vehicle (IRI), and the WY14643 preconditioning group (WY). The three groups were
 pretreated with saline, ethanol and WY, at 1 h before ischemia with a concentration of 10 mg/kg , respectively. Hepatic ischemia-reperfusion
 (I/R) was induced by clamping blood supply to the left lateral and median lobes of the liver for 90 min. Blood samples and liver tissues were
 obtained at the end of 4h reperfusion. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate
 dehydrogenase (LDH) were measured. The histological changes of hepatic tissues were examanied by HE staining. The expression of
 interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) were detected by ELISA and RT-PCR. Moreover, the
 expression of TLR-4 , I-, Myd88, p65 and PPAR- were detected by western blotting.
 Result: After 4 h reperfusion, compared with the IRI group, the liver dysfunction (ALT, AST and LDH), damage score, the expression
 of TNF-α , IL-6, and IL-1β, the protein expression level of TLR4, Myd88, the phosphorylation level of IκB- and p65 in the WY group was
 significantly decreased (all P

Abstract 642: Wy14643 and Fenofibrate Reduce Acetylcholine-induced Contractions in Spontaneously Hypertensive Rat Aortae

Introduction and hypothesis: Hypertension is associated with endothelial dysfunction, which is defined as an imbalanced release of endothelium-derived relaxing factors (EDRF) and endothelium-derived contracting factors (EDCF). Oxidative stress contributes to endothelial dysfunction by increasing the production of EDCF. Research studies have demonstrated that peroxisome proliferator-activated receptor alpha agonists, Wy14643 (WY) and fenofibrate (FF), improve endothelial function. Therefore, we assessed the hypothesis that WY and FF inhibited acetylcholine-induced endothelium-dependent contractions through reduction of oxidative stress in aortae of spontaneously hypertensive rats (SHR). Method: Thoracic aortic rings, with and without endothelium, were isolated from 40-44 weeks old male SHR and their normotensive counterparts, Wistar Kyoto rats (WKY). They were suspended in organ chambers for isometric tension recording. Responses to the endothelium-dependent vasoactive agent, acetylcholine (10 -8 - 10 -4 M), and to the oxidizing agent, hydrogen peroxide (10 -8 - 10 -4 M), were examined in the presence of N ω -nitro-L-arginine methyl ester (L-NAME, 3*10 -4 M; nitric oxide synthase inhibitor). Results: Acetylcholine caused significantly greater contractions in aortic rings, with endothelium, of SHR than in those of WKY. The contraction was significantly inhibited by the combination of Tiron (10 -3 M; intracellular superoxide anion scavenger) and DETCA (10 -4 M; superoxide dismutase inhibitor), suggesting that the reactive oxygen species, hydrogen peroxide, was involved in vascular dysfunction in hypertension. Both WY (10 -5 M) and FF (10 -5 M) significantly inhibited contractions to acetylcholine in SHR aorta, suggesting that they may reduce the amount of hydrogen peroxide or inhibit its downstream signaling. Moreover, they inhibited contractions induced by hydrogen peroxide in SHR aortic rings with, but not in those without, endothelium, indicating that their inhibitory effect was endothelium-dependent. Conclusion: The peroxisome proliferator-activated receptor alpha agonists act on the endothelium to reduce acetylcholine-induced contractions that are mediated by hydrogen peroxide in aortae of hypertensive rats.

PPARα-independent actions of omega-3 PUFAs contribute to their beneficial effects on adiposity and glucose homeostasis.

Excess dietary lipid generally leads to fat deposition and impaired glucose homeostasis, but consumption of fish oil (FO) alleviates many of these detrimental effects. The beneficial effects of FO are thought to be mediated largely via activation of the nuclear receptor peroxisomal-proliferator-activated receptor α (PPARα) by omega-3 polyunsaturated fatty acids and the resulting upregulation of lipid catabolism. However, pharmacological and genetic PPARα manipulations have yielded variable results. We have compared the metabolic effects of FO supplementation and the synthetic PPARα agonist Wy-14,643 (WY) in mice fed a lard-based high-fat diet. In contrast to FO, WY treatment resulted in little protection against diet-induced obesity and glucose intolerance, despite upregulating many lipid metabolic pathways. These differences were likely due to differential effects on hepatic lipid synthesis, with FO decreasing and WY amplifying hepatic lipid accumulation. Our results highlight that the beneficial metabolic effects of FO are likely mediated through multiple independent pathways.

A Toxicogenomic Approach for the Prediction of Murine Hepatocarcinogenesis Using Ensemble Feature Selection

The current strategy for identifying the carcinogenicity of drugs involves the 2-year bioassay in male and female rats and mice. As this assay is cost-intensive and time-consuming there is a high interest in developing approaches for the screening and prioritization of drug candidates in preclinical safety evaluations. Predictive models based on toxicogenomics investigations after short-term exposure have shown their potential for assessing the carcinogenic risk. In this study, we investigated a novel method for the evaluation of toxicogenomics data based on ensemble feature selection in conjunction with bootstrapping for the purpose to derive reproducible and characteristic multi-gene signatures. This method was evaluated on a microarray dataset containing global gene expression data from liver samples of both male and female mice. The dataset was generated by the IMI MARCAR consortium and included gene expression profiles of genotoxic and nongenotoxic hepatocarcinogens obtained after treatment of CD-1 mice for 3 or 14 days. We developed predictive models based on gene expression data of both sexes and the models were employed for predicting the carcinogenic class of diverse compounds. Comparing the predictivity of our multi-gene signatures against signatures from literature, we demonstrated that by incorporating our gene sets as features slightly higher accuracy is on average achieved by a representative set of state-of-the art supervised learning methods. The constructed models were also used for the classification of Cyproterone acetate (CPA), Wy-14643 (WY) and Thioacetamid (TAA), whose primary mechanism of carcinogenicity is controversially discussed. Based on the extracted mouse liver gene expression patterns, CPA would be predicted as a nongenotoxic compound. In contrast, both WY and TAA would be classified as genotoxic mouse hepatocarcinogens.

PPARα-mediated responses in human adult liver stem cells: In vivo/in vitro and cross-species comparisons

The peroxisome proliferator-activated receptor α (PPARα) is a ligand-activated transcription factor that regulates a variety of biological processes including lipid metabolism and energy homeostasis. Peroxisome proliferators (PPs) are carcinogens in rodents, while humans are resistant to peroxisome proliferation and carcinogenesis. In this study, we examined the differential gene expression elicited by clofibrate (CLO) and WY-14,643 (WY) in C57BL/6 mouse liver compared to responses in human HepG2 hepatoma and HL1-1 adult stem cells. Mice were gavaged with sesame oil, 300mg/kg CLO or WY for 2, 4, 8, 12, 18 or 24h, or daily for 4 or 14 days. Although no significant changes in body weight gain were observed, WY induced relative liver weight at 4 and 14 days. Genome-wide hepatic gene expression analysis identified 719 and 1443 differentially expressed unique genes elicited by CLO and WY, respectively (|fold change|>1.5, P1(t)>0.99). Functional analysis associated the gene expression changes with lipid metabolism, transport, cell cycle and immune response. Most differentially expressed genes were in common to both treatments and clustered together only at early time points (2–8h). Complementary QRT-PCR studies in human HL1-1 and HepG2 cells treated with 50μM WY or DMSO for 1, 2, 4, 8, 12, 24 or 48h identified a minimal number of conserved orthologous responses (e.g., Pdk4, Adfp and Angptl4) while some genes (i.e., Bmf, a tumor suppressor) exhibited induction in human cells but repression in mice. These data suggest that PPs elicit species-specific PPARα-mediated gene expression.

Delayed cardioprotective effects of WY-14643 are associated with inhibition of MMP-2 and modulation of Bcl-2 family proteins through PPAR-α activation in rat hearts subjected to global ischaemia–reperfusion

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors regulating cardiac lipid metabolism and energy homeostasis. Although the activation of PPARs has been implicated in cardioprotection, the molecular mechanisms are largely unexplored. In this study, we aimed to investigate the effect of the PPAR-α agonist WY-14643 (WY), mimicking a delayed effect of preconditioning in rat hearts exposed to acute ischaemia-reperfusion (I/R) 24 h later, and to define whether antioxidative and antiapoptotic mechanisms are involved. Treatment with WY markedly attenuated post-ischaemic contractile dysfunction (as evidenced by the reduced infarct size), the higher left ventricular developed pressure (LVDP) recovery, and the decreased occurrence of arrhythmias. These effects were abolished in the presence of the PPAR-α antagonist MK886. Heme oxygenase-1, a key antioxidative enzyme implicated in cytoprotection, was upregulated in response to WY at baseline, but was markedly reduced after I/R, indicating reduced oxidative stress. WY treatment was also associated with decreased mRNA levels and enzymatic activity of matrix metalloproteinase-2, and increased ratios of Bcl-2:Bax proteins. These results indicate that PPAR-α activation by its selective ligand WY may confer delayed preconditioning-like protection in rat hearts subjected to I/R by modulating oxidative stress, activation of matrix metalloproteinase-2, and expression of Bcl-2 and Bax.

Peroxisome Proliferator-Activated ReceptorαAgonists Differentially Regulate Inhibitor of DNA Binding Expression in Rodents and Human Cells

Inhibitor of DNA binding (Id2) is a helix-loop-helix (HLH) transcription factor that participates in cell differentiation and proliferation. Id2 has been linked to the development of cardiovascular diseases since thiazolidinediones, antidiabetic agents and peroxisome proliferator-activated receptor (PPAR) gamma agonists, have been reported to diminish Id2 expression in human cells. We hypothesized that PPARα activators may also alter Id2 expression. Fenofibrate diminished hepatic Id2 expression in both late pregnant and unmated rats. In 24 hour fasted rats, Id2 expression was decreased under conditions known to activate PPARα. In order to determine whether the fibrate effects were mediated by PPARα, wild-type mice and PPARα-null mice were treated with Wy-14,643 (WY). WY reduced Id2 expression in wild-type mice without an effect in PPARα-null mice. In contrast, fenofibrate induced Id2 expression after 24 hours of treatment in human hepatocarcinoma cells (HepG2). MK-886, a PPARα antagonist, did not block fenofibrate-induced activation of Id2 expression, suggesting a PPARα-independent effect was involved. These findings confirm that Id2 is a gene responsive to PPARα agonists. Like other genes (apolipoprotein A-I, apolipoprotein A-V), the opposite directional transcriptional effect in rodents and a human cell line further emphasizes that PPARα agonists have different effects in rodents and humans.

Expression and regulation of GPAT isoforms in cultured human keratinocytes and rodent epidermis

Phospholipids are required for epidermal lamellar body formation. Glycerol 3-phosphate acyltransferases (GPATs) catalyze the initial step in the biosynthesis of glycerolipids. Little is known about the expression and regulation of GPATs in epidermis/keratinocytes. Here, we demonstrate that GPAT 1, 3, and 4 are expressed in epidermis/keratinocytes, whereas GPAT2 is not detected. In mouse epidermis, GPAT 3 and 4 are mainly localized to the upper layers whereas GPAT1 is found in both the upper and lower layers. GPAT1 and 3 mRNA increase during fetal rat epidermal development. No change in GPAT expression was observed in adult mice following acute permeability barrier disruption. Calcium-induced human keratinocyte differentiation increased GPAT3 mRNA whereas both GPAT1 and 4 mRNA levels decreased. In parallel, total GPAT activity increased 2-fold in differentiated keratinocytes attributable to an increase in N-ethylmaleimide (NEM) sensitive GPAT activity localized to microsomes with little change in NEM resistant activity, consistent with an increase in GPAT3. Furthermore, PPARγ or PPARδ activators increased GPAT3 mRNA, microsomal GPAT activity, and glycerol lipid synthesis without affecting the expression of GPAT1 or 4. Finally, both PPARγ and PPARδ activators increased GPAT3 mRNA via increasing its transcription. Thus, multiple isoforms of GPAT are expressed and differentially regulated in epidermis/keratinocytes.

Evaluation of in vivo liver genotoxic potential of Wy-14,643 and piperonyl butoxide in rats subjected to two-week repeated oral administration

Wy-14,643 (WY), a peroxisome proliferator-activated receptor-alpha agonist, and piperonyl butoxide (PBO), a pesticide synergist, induce oxidative stress and promote hepatocarcinogenesis in the liver of rodents. These chemicals belong to a class of non-genotoxic carcinogens, but DNA damage secondary to the oxidative stress resulting from reactive oxygen species generation is suspected in rodents given these chemicals. To examine whether WY or PBO have DNA-damaging potential in livers of rats subjected to repeated oral administration for 14 days, the in vivo liver comet assay was performed in partially hepatectomized rats, and the expression of some DNA-repair genes was examined. Then, to examine whether they have genotoxic potential, the in vivo liver initiation assay was performed in rats. In the comet assay, positive results were obtained at 3 h after the last treatment of WY, and some DNA-repair genes such as Apex1, Mlh1, Xrcc5, and Gadd45 were up-regulated in the liver. In the liver initiation assay, negative results were obtained for both WY and PBO. The results of the present study suggest that WY, but not PBO, causes some DNA damage in livers of rats, but such DNA damage was repaired by the increased activity of some DNA repair genes and may not lead to a DNA mutation.

Analysis of the heat shock response in mouse liver reveals transcriptional dependence on the nuclear receptor peroxisome proliferator-activated receptor α (PPARα)

BackgroundThe nuclear receptor peroxisome proliferator-activated receptor alpha (PPARα) regulates responses to chemical or physical stress in part by altering expression of genes involved in proteome maintenance. Many of these genes are also transcriptionally regulated by heat shock (HS) through activation by HS factor-1 (HSF1). We hypothesized that there are interactions on a genetic level between PPARα and the HS response mediated by HSF1.ResultsWild-type and PPARα-null mice were exposed to HS, the PPARα agonist WY-14,643 (WY), or both; gene and protein expression was examined in the livers of the mice 4 or 24 hrs after HS. Gene expression profiling identified a number of Hsp family members that were altered similarly in both mouse strains. However, most of the targets of HS did not overlap between strains. A subset of genes was shown by microarray and RT-PCR to be regulated by HS in a PPARα-dependent manner. HS also down-regulated a large set of mitochondrial genes specifically in PPARα-null mice that are known targets of PPARγ co-activator-1 (PGC-1) family members. Pretreatment of PPARα-null mice with WY increased expression of PGC-1β and target genes and prevented the down-regulation of the mitochondrial genes by HS. A comparison of HS genes regulated in our dataset with those identified in wild-type and HSF1-null mouse embryonic fibroblasts indicated that although many HS genes are regulated independently of both PPARα and HSF1, a number require both factors for HS responsiveness.ConclusionsThese findings demonstrate that the PPARα genotype has a dramatic effect on the transcriptional targets of HS and support an expanded role for PPARα in the regulation of proteome maintenance genes after exposure to diverse forms of environmental stress including HS.

Peroxisome proliferator-activated receptorα (PPARα) agonists down-regulate α2-macroglobulin expression by a PPARα-dependent mechanism

Fibrates are peroxisome proliferator-activated receptor alpha (PPARα) ligands used to normalize lipid and glucose parameters and exert anti-inflammatory effects. The acute-phase response (APR) is an important inflammatory process. One of the most important acute-phase proteins in rats is α2-macroglobulin (A2Mg). Whereas normal adult rats present low serum levels, pregnant rats display high amounts. Therefore, we used pregnant rats to detect the effect of fenofibrate on hepatic A2Mg expression by RT-PCR and Northern blot. Virgin rats were used as controls. The expression of other APR genes, a known fibrate-responder gene, gamma-chain fibrinogen (γ-Fib), and one gene from the same family as A2Mg, complement component 3 (C3), were also measured in liver. In order to determine whether the fibrate-effects were mediated by PPARα, wild-type mice and PPARα-null mice were also used and treated with WY-14,643 (WY) or di-2-ethylhexyl phthalate (DEHP). Fenofibrate depressed A2Mg expression in virgin rats, but expression was decreased more sharply in pregnant rats. Expression of C3 and γ-Fib was diminished after treatment only in pregnant rats. On the other hand, WY, but not DEHP, reduced A2Mg and γ-Fib expression in the livers of wild-type mice, without any effect in PPARα-null mice. WY or DEHP did not affect C3 expression. Therefore, A2Mg expression is modified by PPARα agonists not only in pregnant rats under augmented APR protein synthesis, but also in virgin rats and mice under basal conditions. Interestingly, our results also identify A2Mg as a novel PPARα agonist-regulated gene.

Long-chain fatty acid effects on peroxisome proliferator-activated receptor-α-regulated genes in Madin-Darby bovine kidney cells: Optimization of culture conditions using palmitate

Studying long-chain fatty acid (LCFA) effects on gene network expression in bovine cells could provide useful information for future practical applications. An optimized in vitro system that does not require tissue collection or cell isolation could fill a niche in the study of PPARα activity in ruminants. Specific aims were to optimize culture conditions in Madin-Darby bovine kidney (MDBK) cells to achieve maximal mRNA expression of known peroxisome proliferator-activated receptor-α (PPARα) target genes using palmitate (16:0) as a representative LCFA. Variables included length of incubation time, use of albumin-bound (4:1 molar proportion) 16:0 (A16:0), or addition of insulin. A first time-course experiment tested culturing cells in Dulbecco's modified Eagle's medium with 150μM PPAR ligand Wy-14643 (WY) and A16:0. A second experiment tested the effects of albumin and insulin using 150μM of 16:0 without albumin or insulin (−Alb/−Ins), 16:0 without albumin plus 5 mg/L of bovine insulin (−Alb/+Ins), A16:0 without insulin (+Alb/−Ins), or a control. A third experiment was a preliminary metabolic characterization of cells and assessed intracellular lipid droplet formation after treatment with 150μM of 16:0 or an ethanol control. For all experiments, cells were harvested at 0, 6, 12, 18, and 24h posttreatment. In experiments 1 and 2, mRNA expression was assessed by quantitative PCR of selected PPARα target genes as well as PPARα coactivators (ACOX1, CPT1A, ACADVL, ACSL1, PPARA, PPARGC1A, LPIN1). In experiment 1, there was a linear increase in mRNA expression of CPT1A (∼500%) and ACSL1 (50 to 200%) by 6h of incubation with both WY and A16:0. The LPIN1 mRNA increased by >100% by 6h only with A16:0. Further, there was a linear increase in expression of PPARA (∼100%) with A16:0 through 24h of incubation. In experiment 2, insulin increased, and coupling LCFA with albumin tended to delay the response in expression of CPT1A and ACSL1 to 16:0. Data indicated a toxic effect of 150μM free 16:0 as assessed by cell counts after 12h of incubation. In experiment 3, MDBK cells appeared to use glucose and AA as energy sources and were able to secrete triglycerides. In addition, MDBK cells cultured with 150μM of 16:0 had a substantial uptake of LCFA and synthesized intracellular lipid droplets. Overall, results indicated that a 6-h incubation with free LCFA and addition of insulin was suitable to detect marked effects on mRNA expression of PPARα target genes in MDBK cells.

Regulation of Cpt1b Gene Transcription in liver and muscle cell lines

Regulation of mitochondrial fatty acid oxidation resides at the outer membrane enzyme carnitine palmitoyltransferase‐1 (Cpt1). The Cpt1b isoform is expressed in skeletal muscle cells while liver cells express Cpt1a. Fatty acids stimulate expression of Cpt1 genes through PPARa response elements (PPRE). We found a similar expression spectrum of Cpt1b‐luciferase activities in C2C12, L6 and H4IIE cell lines. WY‐14,643 (WY) stimulated expression in H4IIE cells, but not in C2C12 or L6 cells. Measurement of PPARa mRNA by real‐time PCR indicated the muscle cell lines had low levels of PPARa as well as PGC1a. After cotransfection of L6 cells with PPARa and PGC1a expression vectors, luciferase activity was increased by PPARa and additionally enhanced by PGC1a. WY further stimulated expression in cells with PPARa overexpression. Mutation of the PPRE in the ‐391/+80 Cpt1b construct eliminated WY stimulation of transcription, but co‐transfection with the PGC1a still increased luciferase activity in the mutant construct. Results show that activation through PPRE is highly dependent on PPARa binding, but PGC1a can stimulate through PPARa and independently of PPARa.

PPAR-α activation protects the type 2 diabetic myocardium against ischemia-reperfusion injury: involvement of the PI3-Kinase/Akt and NO pathway

Several clinical studies have shown the beneficial cardiovascular effects of fibrates in patients with diabetes and insulin resistance. The ligands of peroxisome proliferator-activated receptor-alpha (PPAR-alpha) reduce ischemia-reperfusion injury in nondiabetic animals. We hypothesized that the activation of PPAR-alpha would exert cardioprotection in type 2 diabetic Goto-Kakizaki (GK) rats, involving mechanisms related to nitric oxide (NO) production via the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. GK rats and age-matched Wistar rats (n >or= 7) were given either 1) the PPAR-alpha agonist WY-14643 (WY), 2) dimethyl sulfoxide (DMSO), 3) WY and the NO synthase inhibitor N(G)-nitro-l-arginine (l-NNA), 4) l-NNA, 5) WY and the PI3K inhibitor wortmannin, or 6) wortmannin alone intravenously before a 35-min period of coronary artery occlusion followed by 2 h of reperfusion. Infarct size (IS), expression of endothelial NO synthase (eNOS), inducible NO synthase, and Akt as well as nitrite/nitrate were determined. The IS was 75 +/- 3% and 72 +/- 4% of the area at risk in the Wistar and GK DMSO groups, respectively. WY reduced IS to 56 +/- 3% in Wistar (P < 0.05) and to 46 +/- 5% in GK rats (P < 0.001). The addition of either l-NNA or wortmannin reversed the cardioprotective effect of WY in both Wistar (IS, 70 +/- 5% and 65 +/- 5%, respectively) and GK (IS, 66 +/- 4% and 64 +/- 4%, P < 0.05, respectively) rats. The expression of eNOS and eNOS Ser1177 in the ischemic myocardium from both strains was increased after WY. The expression of Akt, Akt Ser473, and Akt Thr308 was also increased in the ischemic myocardium from GK rats following WY. Myocardial nitrite/nitrate levels were reduced in GK rats (P < 0.05). The results suggest that PPAR-alpha activation protects the type 2 diabetic rat myocardium against ischemia-reperfusion injury via the activation of the PI3K/Akt and NO pathway.