PPARalpha Activation Research Articles

Peroxisome proliferator-activated receptor and farnesoid X receptor ligands differentially regulate sebaceous differentiation in human sebaceous gland organ cultures in vitro

Nuclear hormone receptors are important in the regulation of epidermal differentiation and have been implicated in lipid metabolism. In particular, there is evidence suggesting that the activation of peroxisome proliferator-activated receptors (PPARs) is an important factor in the regulation of sebocyte lipogenesis. To determine the role of PPARs, farnesoid X receptor (FXR) and other orphan nuclear hormone receptors in sebaceous gland function in vitro by investigating the biochemical effects of appropriate ligands, and by establishing the RNA and protein expression patterns of a number of nuclear receptors in sebaceous glands ex vivo. Human chest sebaceous glands were maintained in vitro as freshly isolated and as 7-day cultured whole organs. We then studied the effects of appropriate ligands on the glandular rates of lipogenesis and DNA synthesis, as well as determining the mRNA (reverse transcription-polymerase chain reaction) and protein expression patterns (immunohistochemistry/immunoblotting) of the nuclear hormone receptors of interest. PPAR ligands, but not FXR ligands, inhibited sebaceous lipogenesis, in particular the PPARalpha ligands LY 171883 and WY 14643, and the PPARgamma ligands BRL 49653 and 15-deoxy-Delta-12,14-prostaglandin J(2). We detected RNA expression of PPARalpha, PPARbeta, PPARgamma, retinoid X receptor alpha, liver X receptor alpha (LXRalpha) and pregnane X receptor but not FXR in freshly isolated and 7-day maintained sebaceous glands. PPARalpha, PPARbeta, PPARgamma and LXRalpha protein were detected in nuclear extracts of sebaceous glands. We conclude that activation of nuclear hormone receptors, in particular activation of PPARalpha and PPARgamma, can regulate lipogenesis in human sebaceous glands. As suppression of sebum secretion is associated with reduced acne activity, the nuclear hormone receptors involved may open new avenues in the development of novel acne treatments.

PPAR alpha and gamma activators differently affect the inflammatory response by human vascular endothelium. Possible anti-angiogenic effects of rosiglitazone

PPAR alpha and gamma activators differently affect the inflammatory response by human vascular endothelium. Possible anti-angiogenic effects of rosiglitazone

Peroxisome proliferator-activated receptors and cardiovascular remodeling.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that heterodimerize with the retinoid X receptor and then modulate the function of many target genes. Three PPARs are known: alpha, beta/delta, and gamma. The better known are PPAR-alpha and PPAR-gamma, which may be activated by different synthetic agonists, although the endogenous ligands are unknown. PPAR-alpha is involved in fatty acid oxidation and expressed in the liver, kidney, and skeletal muscle, whereas PPAR-gamma is involved in fat cell differentiation, lipid storage, and insulin sensitivity. However, both have been shown to be present in variable amounts in cardiovascular tissues, including endothelium, smooth muscle cells, macrophages, and the heart. The activators of PPAR-alpha (fibrates) and PPAR-gamma (thiazolidinediones or glitazones) antagonized the actions of angiotensin II in vivo and in vitro and exerted cardiovascular antioxidant and anti-inflammatory effects. PPAR activators lowered blood pressure, induced favorable effects on the heart, and corrected vascular structure and endothelial dysfunction in several rodent models of hypertension. Activators of PPARs may become therapeutic agents useful in the prevention of cardiovascular disease beyond their effects on carbohydrate and lipid metabolism. Some side effects, such as weight gain, as well as documented aggravation of advanced heart failure through fluid retention by glitazones, may, however, limit their therapeutic application in prevention of cardiovascular disease.

Peroxisome Proliferator-Activated Receptor (PPAR)- : A Pharmacological Target with a Promising Future

Peroxisome proliferator-activated receptor (PPAR)-alpha is a ligand-activated transcriptional factor that belongs to the family of nuclear receptors. PPAR-alpha regulates the expression of genes involved in fatty acid beta-oxidation and is a major regulator of energy homeostasis. Fibrates are PPAR-alpha agonists and have been used to treat dyslipidemia for several decades because of their triglyceride (TG) lowering and high-density lipoprotein cholesterol (HDL-C) elevating effects. More recent research has demonstrated anti-inflammatory and anti-thrombotic actions of PPAR-alpha agonists in the vessel wall as well. Thus, PPAR-alpha agonists decrease the progression of atherosclerosis by modulating metabolic risk factors and by their anti-inflammatory actions on the level of the vascular wall. This is confirmed by several clinical studies, in which fibrates have shown to reduce atherosclerotic plaque formation and the event rate of coronary heart disease (CHD), especially in patients suffering from metabolic syndrome (MS). MS is characterized by a group of metabolic risk factors that include obesity, raised blood pressure, dyslipidemia, insulin resistance or glucose intolerance, and a prothrombotic state, and its incidence in the Western world is rising to epidemic proportions. This review paper will focus on the functions of PPAR-alpha in fatty acid beta-oxidation, lipid metabolism, and vascular inflammation. Furthermore, PPAR-alpha genetics, the clinical use of PPAR-alpha activators and their future perspective will be discussed.

Isohumulones, Bitter Acids Derived from Hops, Activate Both Peroxisome Proliferator-activated Receptor α and γ and Reduce Insulin Resistance

The peroxisome proliferator-activated receptors (PPARs) are dietary lipid sensors that regulate fatty acid and carbohydrate metabolism. The hypolipidemic effects of fibrate drugs and the therapeutic benefits of the thiazolidinedione drugs are due to their activation of PPARalpha and -gamma, respectively. In this study, isohumulones, the bitter compounds derived from hops that are present in beer, were found to activate PPARalpha and -gamma in transient co-transfection studies. Among the three major isohumulone homologs, isohumulone and isocohumulone were found to activate PPARalpha and -gamma. Diabetic KK-Ay mice that were treated with isohumulones (isohumulone and isocohumulone) showed reduced plasma glucose, triglyceride, and free fatty acid levels (65.3, 62.6, and 73.1%, respectively, for isohumulone); similar reductions were found following treatment with the thiazolidinedione drug, pioglitazone. Isohumulone treatment did not result in significant body weight gain, although pioglitazone treatment did increase body weight (10.6% increase versus control group). C57BL/6N mice fed a high fat diet that were treated with isohumulones showed improved glucose tolerance and reduced insulin resistance. Furthermore, these animals showed increased liver fatty acid oxidation and a decrease in size and an increase in apoptosis of their hypertrophic adipocytes. A double-blind, placebo-controlled pilot study for studying the effect of isohumulones on diabetes suggested that isohumulones significantly decreased blood glucose and hemoglobin A1c levels after 8 weeks (by 10.1 and 6.4%, respectively, versus week 0). These results suggest that isohumulones can improve insulin sensitivity in high fat diet-fed mice with insulin resistance and in patients with type 2 diabetes.

Oleoylethanolamide Stimulates Lipolysis by Activating the Nuclear Receptor Peroxisome Proliferator-activated Receptor α (PPAR-α)

Amides of fatty acids with ethanolamine (FAE) are biologically active lipids that participate in a variety of biological functions, including the regulation of feeding. The polyunsaturated FAE anandamide (arachidonoylethanolamide) increases food intake by activating G protein-coupled cannabinoid receptors. On the other hand, the monounsaturated FAE oleoylethanolamide (OEA) reduces feeding and body weight gain by activating the nuclear receptor PPAR-alpha (peroxisome proliferator-activated receptor alpha). In the present report, we examined whether OEA can also influence energy utilization. OEA (1-20 microm) stimulated glycerol and fatty acid release from freshly dissociated rat adipocytes in a concentration-dependent and structurally selective manner. Under the same conditions, OEA had no effect on glucose uptake or oxidation. OEA enhanced fatty acid oxidation in skeletal muscle strips, dissociated hepatocytes, and primary cardiomyocyte cultures. Administration of OEA in vivo (5 mg kg(-1), intraperitoneally) produced lipolysis in both rats and wild-type mice, but not in mice in which PPAR-alpha had been deleted by homologous recombination (PPAR-alpha(-/-)). Likewise, OEA was unable to enhance lipolysis in adipocytes or stimulate fatty acid oxidation in skeletal muscle strips isolated from PPAR-alpha mice. The synthetic PPAR-alpha agonist Wy-14643 produced similar effects, which also were dependent on the presence of PPAR-alpha. Subchronic treatment with OEA reduced body weight gain and triacylglycerol content in liver and adipose tissue of diet-induced obese rats and wild-type mice, but not in obese PPAR-alpha(-/-) mice. The results suggest that OEA stimulates fat utilization through activation of PPAR-alpha and that this effect may contribute to its anti-obesity actions.

Peroxisome Proliferator Activated Receptors α and γ Require Zinc for Their Anti-inflammatory Properties in Porcine Vascular Endothelial Cells

Zinc is an essential structural component of various proteins and is crucial for the integrity of the vascular endothelium. The present study focused on the effect of zinc deficiency on the anti-inflammatory properties of peroxisome proliferator activated receptor (PPAR) alpha and gamma agonists. Porcine pulmonary-arterial endothelial cells were deprived from zinc by chelator N,N,N',N'-tetrakis (2-pyridylmethyl)ethylene diamine. Cells were exposed to TNF-alpha for 2 h following pretreament with the PPARalpha agonists fenofibrate or ciprofibrate or the PPARgamma agonists thiazolidinedione or troglitazone. The inflammatory response was tested by measuring nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) binding activities as well as by measuring mRNA expression levels of inflammatory genes, such as vascular cell adhesion molecule-1 (VCAM-1) and IL-6. All PPAR agonists tested lost their potency to downregulate the TNF-alpha-induced inflammatory response in zinc-deficient cells. However, if zinc was added back, all PPAR agonists significantly downregulated the TNF-alpha-mediated induction of inflammatory transcription factors NF-kappaB and AP-1 and significantly reduced the expression of their target genes, VCAM-1 and IL-6. We therefore hypothesize that zinc is required for the PPARalpha and -gamma DNA binding activity. Indeed, zinc deficiency significantly reduced the agonist-induced binding activity of PPARalpha and -gamma to the PPAR response element. Our data demonstrate the importance of zinc in PPAR signaling and the requirement of zinc for the anti-inflammatory properties of PPARalpha and -gamma agonists.

?-Lipoic acid-based PPAR? agonists for treating inflammatory skin diseases

Novel thiazolidinedione derivatives of the potent antioxidant, alpha-lipoic (thioctic, 1,2-dithiolane) acid, were prepared. The prototype N-(2-[4-[2,4-dioxo(1,3-thiazolidin-5-yl)methyl]phenoxy]ethyl)-5-(1,2-dithiolan-3-yl)- N-methylpentanamide (designated BP-1003), and dithioester derivatives thereof were shown to be potent activators of peroxisome proliferator-activated receptor gamma (PPARgamma) (EC(50) range 15-101 nM) and modest activators of PPARalpha (EC(50) 5 microM). Both the relatively hydrophobic dithiolane prototype, BP-1003, and its water-soluble dithioglycinate derivative, BP-1017, were shown to inhibit the proliferation of human keratinocytes and suppress the production of interleukin-2 by human peripheral lymphocytes to a greater extent than the antidiabetic thiazolidinedione, rosiglitazone. Both oral and topical administration of BP-1017 showed significant antiinflammatory effects in the oxazolone-sensitized mouse model of allergic contact dermatitis (ACD). These findings suggest that water-soluble lipoic acid-based thiazolidinediones may be efficacious as oral and topical agents for treating inflammatory skin conditions such as contact dermatitis, atopic dermatitis, and psoriasis.

Differential regulation of cytosolic and peroxisomal bile acid amidation by PPARα activation favors the formation of unconjugated bile acids

In human liver, unconjugated bile acids can be formed by the action of bile acid-CoA thioesterases (BACTEs), whereas bile acid conjugation with taurine or glycine (amidation) is catalyzed by bile acid-CoA:amino acid N-acyltransferases (BACATs). Both pathways exist in peroxisomes and cytosol. Bile acid amidation facilitates biliary excretion, whereas the accumulation of unconjugated bile acids may become hepatotoxic. We hypothesized that the formation of unconjugated and conjugated bile acids from their common substrate bile acid-CoA thioesters by BACTE and BACAT is regulated via the peroxisome proliferator-activated receptor alpha (PPARalpha). Livers from wild-type and PPARalpha-null mice either untreated or treated with the PPARalpha activator WY-14,643 were analyzed for BACTE and BACAT expression. The total liver capacity of taurochenodeoxycholate and taurocholate formation was decreased in WY-14,643-treated wild-type mice by 60% and 40%, respectively, but not in PPARalpha-null mice. Suppression of the peroxisomal BACAT activity was responsible for the decrease in liver capacity, whereas cytosolic BACAT activity was essentially unchanged by the treatment. In both cytosol and peroxisomes, the BACTE activities and protein levels were upregulated 5- to 10-fold by the treatment. These effects caused by WY-14,643 treatment were abolished in PPARalpha-null mice. The results from this study suggest that an increased formation of unconjugated bile acids occurs during PPARalpha activation.

The protein kinase C signaling pathway regulates a molecular switch between transactivation and transrepression activity of the peroxisome proliferator-activated receptor alpha.

Peroxisome proliferator-activated receptor (PPAR) alpha is a nuclear receptor implicated in several physiological processes such as lipid and lipoprotein metabolism, glucose homeostasis, and the inflammatory response. PPARalpha is activated by natural fatty acids and synthetic compounds like fibrates. PPARalpha activity has been shown to be modulated by its phosphorylation status. PPARalpha is phosphorylated by kinases such as the MAPKs and cAMP-activated protein kinase A. In this report, we show that protein kinase C (PKC) inhibition impairs ligand-activated PPARalpha transcriptional activity. Furthermore, PKC inhibition decreases PPARalpha ligand-induction of its target genes including PPARalpha itself and carnitine palmitoyltransferase I. By contrast, PKC inhibition enhances PPARalpha transrepression properties as demonstrated using the fibrinogen-beta gene as model system. Finally, PKC inhibition decreases PPARalpha phosphorylation activity of hepatocyte cell extracts. In addition, PPARalpha purified protein is phosphorylated in vitro by recombinant PKCalpha and betaII. The replacement of serines 179 and 230 by alanine residues reduces the phosphorylation of the PPARalpha protein. The PPARalpha S179A-S230A protein displays an impaired ligand-induced transactivation activity and an enhanced trans-repression activity. Altogether, our data indicate that the PKC signaling pathway acts as a molecular switch dissociating the transactivation and transrepression functions of PPARalpha, which involved phosphorylation of serines 179 and 230.

The balance between concurrent activation of ERs and PPARs determines daidzein-induced osteogenesis and adipogenesis.

The soy phytoestrogen daidzein has biphasic dose responses, but the underlying mechanisms are not yet clear. Transcriptional and biochemical data show that PPARs, in addition to ERs, are molecular targets of daidzein, which divergently regulates osteogenesis and adipogenesis. Dose responses are the result of a balance among PPARs and between ERs and PPARs. Soy phytoestrogens have been used for the purposes of treatment and prevention of osteoporosis. Biphasic dose responses of daidzein, one of the main soy phytoestrogens, have long been recognized, but the underlying molecular mechanisms of action are not yet clear. Mouse bone marrow cells and mouse osteoprogenitor KS483 cells that concurrently differentiate into osteoblasts and adipocytes were cultured. Biochemical measurement of alkaline phosphatase (ALP) activity, RT-PCR, and gene reporter assays were used in this study. Daidzein, one of the major soy phytoestrogens, had biphasic effects on osteogenesis and adipogenesis. Daidzein stimulated osteogenesis (ALP activity and nodule formation) and decreased adipogenesis (the number of adipocytes) at concentrations below 20 microM, whereas it inhibited osteogenesis and stimulated adipogenesis at concentrations higher than 30 microM. When estrogen receptors (ERs) were blocked by ICI182,780, daidzein-induced effects were not biphasic. A decrease in osteogenesis and an increase in adipogenesis were observed at the concentrations higher than 20 and 10 microM, respectively. In addition to ERs, daidzein transactivated not only peroxisome proliferator-activate receptor gamma (PPARgamma), but also PPARalpha and PPARdelta at micromolar concentrations. Activation of PPARalpha had no direct effects on osteogenesis and adipogenesis. In contrast, activation of PPARdelta stimulated osteogenesis but had no effects on adipogenesis, whereas PPARgamma inhibited osteogenesis and stimulated adipogenesis. Transfection experiments show that an activation of PPARalpha or PPARgamma by daidzein downregulated its estrogenic transcriptional activity, whereas activation of PPARdelta upregulated its estrogenic transcriptional activity. Activation of ERalpha or ERbeta by daidzein downregulated PPARgamma transcriptional activity but had no influence on PPARalpha or PPARdelta transcriptional activity. Daidzein at micromolar concentrations concurrently activates different amounts of ERs and PPARs, and the balance of the divergent actions of ERs and PPARs determines daidzein-induced osteogenesis and adipogenesis.

Myocardial fibrosis and diastolic dysfunction in deoxycorticosterone acetate-salt hypertensive rats is ameliorated by the peroxisome proliferator-activated receptor-alpha activator fenofibrate, partly by suppressing inflammatory responses associated with the nuclear factor-kappa-b pathway

ObjectivesWe sought to clarify that a peroxisome proliferator-activated receptor-alpha (PPAR-alpha) activator inhibits myocardial fibrosis and its resultant diastolic dysfunction in hypertensive heart disease, as well as to investigate whether inflammatory mediators through the nuclear factor (NF)-kappa-B pathway are involved in the effects. BackgroundPatients with hypertensive heart disease often have diastolic heart failure without systolic dysfunction. Meanwhile, it has been well established in atherosclerosis that PPAR-alpha activation negatively regulates early inflammation. In hypertensive hearts, however, it is still unclear whether PPAR-alpha activation inhibits inflammation and fibrosis. MethodsTwenty-one rats were randomly separated into the following three groups: deoxycorticosterone acetate (DOCA)-salt hypertensive rats treated with a PPAR-alpha activator, fenofibrate (80 mg/kg/day for 5 weeks); DOCA-salt rats treated with vehicle only; and uni-nephrectomized rats as normotensive controls. ResultsFenofibrate significantly inhibited the elevation of left ventricular end-diastolic pressure and the reduction of the magnitude of the negative maximum rate of left ventricular pressure rise and decline, corrected by left ventricular pressure (−dP/dtmax/P), which are indicators of diastolic dysfunction. Next, fenofibrate prevented myocardial fibrosis and reduced the hydroxyproline content and procollagen I and III messenger ribonucleic acid expression. Finally, inflammatory gene expression associated with NF-kappa-B (interleukin-6, cyclooxygenase-2, vascular cell adhesion molecule-1, and monocyte chemoattractant protein-1), which is upregulated in DOCA-salt rats, was significantly suppressed by fenofibrate. Activation of NF-kappa-B and expression of I-kappa-B-alpha in DOCA-salt rats were normalized by fenofibrate. ConclusionsA PPAR-alpha activator reduced myocardial fibrosis and prevented the development of diastolic dysfunction in DOCA-salt rats. The effects of a PPAR-alpha activator may be mediated partly by prevention of inflammatory mediators through the NF-kappa-B pathway. These results suggest that treatment with PPAR-alpha activators will improve diastolic dysfunction in hypertensive heart disease.

Global Suppression of IL-6-induced Acute Phase Response Gene Expression after Chronic in Vivo Treatment with the Peroxisome Proliferator-activated Receptor-α Activator Fenofibrate

The peroxisome proliferator-activated receptor alpha (PPARalpha), which is highly expressed in liver, plays key roles in lipid metabolism and inflammation. Interleukin-6 (IL-6) is the principal inducer of acute phase response (APR) gene expression. In the present study, we demonstrate that chronic treatment with the PPARalpha agonist fenofibrate fully prevents the IL-6-induced APR gene expression in wild-type but not in PPARalpha-deficient mice. PPARalpha prevents the IL-6-induced expression of the positive APR genes fibrinogen-alpha, -beta, -gamma, haptoglobulin, and serum amyloid A and the IL-6-induced suppression of the negative APR gene, major urinary protein. Furthermore, the effect of PPARalpha on the APR gene expression does not simply consist in a delayed systemic response to IL-6 but occurs directly at the transcriptional level. This global suppression of acute phase gene transcription may be explained by two PPARalpha-dependent in vivo effects: 1) PPARalpha activation results in the down-regulation of the IL-6 receptor components gp80 and gp130 in the liver, thereby reducing the phosphorylation and activation of the downstream transcription factors STAT3 and c-Jun that transduce the IL-6 signal; and 2) PPARalpha reduces the basal expression of the transcription factors CCAAT enhancer-binding protein-alpha, -beta, -delta, which are responsible for immediate and maintained transcription of APR genes. A similar global effect of fenofibrate on acute phase protein expression is observed in hyperlipidemic patients chronically treated with fenofibrate, which displayed decreased plasma concentrations of the positive APR proteins fibrinogen, C-reactive protein, serum amyloid A, plasminogen, and alpha2-macroglobulin and increased plasma concentrations of the negative APR albumin, underlining the clinical significance of our findings.

Alcoholic liver injury in the rat is associated with reduced expression of peroxisome proliferator-alpha (PPARalpha)-regulated genes and is ameliorated by PPARalpha activation.

Alcoholic liver disease is associated with a state of hepatic fatty acid overload. We examined the effect of ethanol and different types of dietary fat on the expression of mRNA for liver fatty acid binding protein (L-FABP), peroxisome proliferator-activated receptor-alpha (PPARalpha), and peroxisomal fatty acyl CoA oxidase (FACO). Four groups of rats (n = 5) were fed intragastrically, a liquid diet with or without ethanol (10-16 g/kg/day) for 4 weeks. Pair-fed controls received isocaloric amounts of dextrose. The source of fat was either corn oil or fish oil. Ethanolfed rats developed fatty liver, necrosis, and inflammation; the changes were more severe in the fish oil-ethanol (FE) rats. PPARalpha mRNA levels were not different between groups, although there was a trend toward increased levels in ethanol-fed rats. We calculated L-FABP/PPARalpha and FACO/PPARalpha ratios as a measure of FACO and L-FABP up-regulation relative to PPARalpha expression. Both FACO/PPARalpha and L-FABP/PPARalpha ratios were significantly decreased in FE rats. However, only L-FABP/PPARalpha was decreased in corn oil plus ethanol rats. Also, the level of L-FABP/mRNA correlated inversely with the degree of fatty liver in ethanol-fed rats. Since expression of PPARalpha response genes was impaired in ethanol-fed rats, we determined whether activation of PPARalpha would normalize the PPARalpha response and prevent the pathological changes in ethanol-fed rats. Treatment with clofibrate, a PPARalpha-activating ligand, led to a marked decrease in fatty liver and complete abrogation of necroinflammatory changes in FE rats. Also, nuclear factor kappaB activation and up-regulation of tumor necrosis factor-alpha and cyclooxygenase-2 was also abolished in clofibrate-treated rats. We conclude that adaptive gene regulation of FACO and L-FABP by PPARalpha is impaired in ethanol-fed rats and that treatment with clofibrate, a PPARalpha ligand, prevents alcohol-induced pathological liver injury, possibly by reversing the above changes.

Transcriptional cofactors exhibit differential preference toward peroxisome proliferator-activated receptors alpha and delta in uterine cells.

We previously showed that peroxisome proliferator-activated receptor delta (PPARdelta) is crucial for embryo implantation as a receptor for cyclooxygenase-2-derived prostacyclin in mice. PPARs belong to the nuclear receptor superfamily. They form heterodimer with a retinoid X receptor, recruit transcriptional cofactors, and bind to a specific recognition element for regulation of target genes. Although cofactors are generally shared by various nuclear receptors, some are involved in cell-specific events. The objective of this investigation was to examine interactions of transcriptional cofactors with PPARdelta in uterine cells for its effectiveness in regulating gene expression. We chose two uterine cellular systems: periimplantation mouse uterus and AN(3)CA human uterine cell line. As examined by in situ hybridization, steroid receptor coactivator (SRC)-2, SRC-3, PPAR-interacting protein, receptor-interacting protein 140 (RIP140), nuclear receptor corepressor (N-CoR), and silencing mediator for retinoid and thyroid hormone receptor (SMRT) exhibit overlapping expression with that of PPARdelta in the periimplantation mouse uterus. Glutathione-S-transferase (GST) pull-down assays show that PPARdelta physically interacts with SRC 1-3, RIP140, PPAR-binding protein, N-CoR, and SMRT in the absence of ligands, suggesting their potent interactions with PPARdelta. Transient transfection assays in AN(3)CA cells show that among members of the SRC family, only SRC-2 serves as a true coactivator for PPARdelta, whereas all SRC members could enhance PPARalpha-induced transcriptional activation. Interestingly, N-CoR and SMRT potently repress PPARdelta-induced transcriptional activation but fail to repress PPARalpha activity. RIP140 is effective in repressing basal and PPAR-induced transcriptional activation. Collectively, the results suggest that gene regulation by PPARdelta in the uterine cells uniquely responds to SRC-2, N-CoR, SMRT, or RIP140, and these interactions may be operative during implantation when these cofactors are abundantly expressed.

Coadministration of a liver X receptor agonist and a peroxisome proliferator activator receptor-alpha agonist in Mice: effects of nuclear receptor interplay on high-density lipoprotein and triglyceride metabolism in vivo.

Liver X receptors (LXRs) are master transcription factors regulating cholesterol and fatty acid metabolism. Treatment of C57B6 mice with a specific synthetic LXR agonist, N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-trifluoro-1-hydroxy-1(trifluoromethyl)-ethyl]phenyl]-benzenesulfonamide (T0901317), resulted in elevated high-density lipoprotein (HDL) cholesterol as well as plasma and liver triglycerides. Peroxisome proliferator-activated receptor-alpha (PPARalpha) agonists are known to induce peroxisomal fatty acid beta-oxidation and also mediate HDL cholesterol metabolism. We have explored the hypothesis that simultaneous activation of PPARalpha and LXR may lead to additive effects on HDL cholesterol elevation as well as attenuation of triglyceride accumulation. Coadministration of T0901317 and the specific PPARalpha agonist [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid (Wy14643)] in mice led to synergistic elevation of HDL cholesterol that was primarily associated with enlarged HDL particles enriched with apoE and apoAI. Liver phospholipid transfer protein (PLTP) mRNA and plasma PLTP activity were additively elevated, suggesting a role of PLTP in the observed HDL cholesterol elevation. Moderate increases in plasma triglyceride levels induced by LXR activation was reduced, whereas the accumulation of triglyceride in the liver was not altered upon coadministration of the PPARalpha agonist. Peroxisomal fatty acid beta-oxidation in the liver was dramatically elevated upon PPARalpha activation as expected. Interestingly, activation of LXRs via T0901317 also led to a significant increase in peroxisomal fatty acid beta-oxidation. Sterol regulatory element binding protein 1c expression was dramatically up-regulated by the LXR agonist but was not changed with PPARalpha agonist treatment. Liver lipoprotein lipase expression was additively increased upon LXR agonist and PPARalpha agonist coadministration. Our studies mark the first exploration of nuclear receptor interplay on lipid homeostasis in vivo.

Endothelin-1-induced cardiac hypertrophy is inhibited by activation of peroxisome proliferator-activated receptor-alpha partly via blockade of c-Jun NH2-terminal kinase pathway.

Peroxisome proliferator-activated receptor-alpha (PPAR-alpha) is a lipid-activated nuclear receptor that negatively regulates the vascular inflammatory gene response by interacting with transcription factors, nuclear factor-kappaB, and AP-1. However, the roles of PPAR-alpha activators in endothelin (ET)-1-induced cardiac hypertrophy are not yet known. First, in cultured neonatal rat cardiomyocytes, a PPAR-alpha activator, fenofibrate (10 micromol/L), and PPAR-alpha overexpression markedly inhibited the ET-1-induced increase in protein synthesis. Second, fenofibrate markedly inhibited ET-1-induced increase in c-Jun gene expression and phosphorylation of c-Jun and JNK. These results suggest that this PPAR-alpha activator interferes with the formation and activation of AP-1 protein induced by ET-1 in cardiomyocytes. Third, fenofibrate significantly inhibited the increase of ET-1 mRNA level by ET-1, which was also confirmed by luciferase assay. Electrophoretic mobility shift assay revealed that fenofibrate significantly decreased the ET-1-stimulated or phorbol 12-myristate 13-acetate-stimulated AP-1 DNA binding activity, and the nuclear extract probe complex was supershifted by anti-c-Jun antibody. Fourth, 24 hours after aortic banding (AB) operation, fenofibrate treatment significantly inhibited left ventricular hypertrophy and hypertrophy-related gene expression pattern (ET-1, brain natriuretic peptide, and beta-myosin heavy chain mRNA) in AB rats. These results suggest that PPAR-alpha activation interferes with the signaling pathway of ET-1-induced cardiac hypertrophy through negative regulation of AP-1 binding activity, partly via inhibition of the JNK pathway in cultured cardiomyocytes. We also revealed that fenofibrate treatment inhibited left ventricle hypertrophy and phenotypic changes in cardiac gene expression in AB rats in vivo.

Stimulation of cardiac cardiolipin biosynthesis by PPARα activation

The role of peroxisome proliferator-activated receptor alpha (PPARalpha)-stimulated phospholipase A2 (PLA2) in cardiac mitochondrial cardiolipin (CL) biosynthesis was examined in both in vivo and in vitro models. Treatment of rat heart H9c2 cells with clofibrate increased the expression and activity of 14 kDa PLA2 but did not affect the pool size of CL. Clofibrate treatment stimulated de novo CL biosynthesis via an increase in phosphatidylglycerolphosphate (PGP) synthase activity, accounting for the unaltered CL content. Cardiac PLA2, PGP synthase, and CDP-1,2-diacyl-sn-glycerol synthase (CDS-2) activities and CDS-2 mRNA levels were elevated in mice fed clofibrate for 14 days compared with controls. In PPARalpha-null mice, clofibrate feeding did not alter cardiac PLA2, PGP synthase activities, or CDS-2 activity and mRNA level, confirming that these enzymes are regulated by PPARalpha activation. In contrast to mouse heart, clofibrate treatment did not affect the activity or mRNA levels of CDS-2 in H9c2 cells, indicating that CDS-2 is regulated differently in rat heart H9c2 cells in vitro and in mouse heart in vivo. These results clearly indicate that cardiac CL de novo biosynthesis is stimulated by PPARalpha activation in responsive rodent models and that CDS-2 is an example of an enzyme that exhibits alternative regulation in vivo and in cultured cell lines. This study is the first to demonstrate that CL de novo biosynthesis is regulated by PPARalpha activation.

Relationship between PPARalpha activation and NO on proximal tubular Na+ transport in the rat.

BackgroundNitric oxide (NO) regulates renal proximal tubular (PT) Na+ handling through modulation of Na+-K+ ATPase. Peroxisome Proliferator Activated Receptorα (PPARα), a nuclear transcription factor, is expressed in PTs and has been reported to influence NO generation/activity in renal tissues. This study tested the hypothesis that PPARα interacts with NO and thereby affects renal tubular Na+ transport. Urinary excretion of nitrite (UNOXV) and Na+ (UNaV) and PT Na+ transport (Na+-K+ ATPase activity) were determined in rats treated with clofibrate (250 mg/kg i.p) or WY14643 (45 mg/kg; i.p.), a PPARα ligand, 2% NaCl (orally), clofibrate/NaCl, L-NAME, an inhibitor of NO production (100 mg/kg; orally), L-NAME/Clofibrate.ResultsClofibrate or WY14643 increased PPARα expression by 106 ± 7% (p < 0.05) and 113 ± 8% (p < 0.05), respectively. Similarly, clofibrate and WY14643 increased expression of MCAD, a downstream target protein of PPARα by 123 ± 8% (p < 0.05) and 143 ± 8% (p < 0.05), respectively. L-NAME attenuated clofibrate-induced increase in PPARα expression by 27 ± 2% (p < 0.05) but did not affect MCAD expression. UNOXV excretion increased 3–4 fold in rats treated with clofibrate, WY14643 or NaCl from 44 ± 7 to 170 ± 15, 144 ± 18 or 132 ± 11 nmol/24 hr, respectively (p < 0.05). Similarly, clofibrate, WY14643 or NaCl elicited a 2–5 fold increase in UNaV. L-NAME significantly reduced basal UNOXV and UNaV and abolished the clofibrate-induced increase. Clofibrate, WY14643, NaCl or clofibrate + NaCl treatment reduced Na+-K+-ATPase activity in the PT by 89 ± 23, 62 ± 10, 43 ± 9 and 82 ± 15% (p < 0.05), respectively. On the contrary, L-NAME or ODQ, inhibitor of sGC, abolished the inhibition of Na+-K+-ATPase activity by clofibrate (p < 0.05). Clofibrate either alone or with NaCl elicited ~2-fold increase in the expression of the α1 subunit of Na+-K+ ATPase in the PT while L-NAME abolished clofibrate-induced increase in Na+-K+ ATPase expression.ConclusionThese data suggest that PPARα activation, through increased NO generation promotes renal excretion of Na+ through reduced Na+-K+ ATPase activity in the PT probably via post translational modification of Na+-K+-ATPase.

Correlation between thyroid hormone status and hepatic hyperplasia and hypertrophy caused by the peroxisome proliferator-activated receptor alpha agonist Wy-14,643

BackgroundThe metabolic inhibitor rotenone inhibits hepatocellular proliferation and the incidence of liver cancer resulting from exposure to the PPARα agonist Wy-14,643, via unknown mechanisms. Since the absence of thyroid hormones diminishes hepatomegaly, an early biomarker for the hepatocarcinogenicity induced by PPARα agonists, this study was undertaken to investigate whether rotenone might interference with the ability of Wy-14,643 to alter the animal thyroid status.MethodsMale B6C3F1 mice were given Wy-14,643 (100 ppm), rotenone (600 ppm) or a mixture of both, in the feed for 7 days. Bromodeoxyuridine (BrDU), marker of cell replication, was delivered through subcutaneously implanted osmotic mini-pumps. At the end of the experiment, sera were collected and corticosterone and thyroid hormone levels were measured by solid-phase radioimmunoassay kits. In addition, liver tissue samples were stained immunohistochemically for BrDU to determine percentages of labeled cells. Further, cell surface area was determined from images generated by a Zeiss Axioplan microscope equipped with a plan Neofluar ×40 0.75 na objective. Tracings of individual hepatocyte perimeters were then analyzed and cell-surface areas were calculated using MicroMeasure FL-4000.ResultsWy-14,643 caused a significant increase in liver weights, hepatocyte BrDU labeling index (LI), and hepatocyte surface area. In animals which received both Wy-14,643 and rotenone simultaneously, all of these effects were significantly less pronounced compared with mice that received Wy-14,643 alone. Rotenone alone decreased liver weights, LI and surface area. The Free Thyroid Index (FTI), which provides an accurate reflection of the animal's thyroid status, was 5.0 ± 0.3 in control mice. In animals exposed to rotenone, these values decreased to 2.0 ± 0.9, but in animals which received Wy-14,643, levels increased significantly to 7.7 ± 0.9. FTI values decreased to 3.4 ± 0.8 in mice receiving both rotenone and Wy-14,643.ConclusionA strong correlation was observed between the animal thyroid status and both, hepatocyte proliferation (r2 = 0.62), and hepatocyte surface area (r2 = 0.83). These results support the hypothesis that the thyroid status of the animal plays a role in PPARα-induced hepatocellular proliferation and liver cell enlargement. Both these events are known to contribute to the expression of liver cancer in response to the activation of PPARα.