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Targeting peroxisome proliferator-activated receptor-gamma (PPAR-[gamma]) is an approved strategy in facing insulin resistance (IR) for diabetes mellitus (DM) type 2. The PPAR-[gamma] modulators display improvements in the insulin-sensitizing and adverse effects of the traditional thiazolidinediones. Nitazoxanide (NTZ) is proposed as a PPAR-[gamma] receptor ligand with agonistic posttranscriptional effects. Currently, NTZ antidiabetic activities versus pioglitazone (PIO) in a high-fat diet/streptozotocin rat model of type 2 diabetes was explored. Diabetic adult male Wistar rats were treated orally with either PIO (2.7 mg x [kg.sup.- 1] x day-1) or NTZ (200 mg x [kg.sup.-1] x [day.sup.-1]) for 14, 21, and 28 days. Body masses, fasting blood glucose, IR, lipid profiles, and liver and kidney functions of rats were assayed. Hepatic glucose metabolism and PPAR-[gamma] protein expression levels as well as hepatic, pancreatic, muscular, and renal histopathology were evaluated. Significant time-dependent euglycemic and insulin-sensitizing effects with preservation of liver and kidney functions were offered by NTZ. Higher hepatic levels of glucose-6-phosphatase and glucose-6- phosphate dehydrogenase enzymes and PPAR-[gamma] protein expressions were acquired by NTZ and PIO, respectively. NTZ could be considered an oral therapeutic strategy for DM type 2. Further systematic NTZ/PPAR-[gamma] receptor subtype molecular activations are recommended. Simultaneous use of NTZ with other approved antidiabetics should be explored. Key words: high-fat diet/streptozotocin-induced diabetes, insulin resistance, nitazoxanide, peroxisome proliferator activated receptor-gamma, pioglitazone. Le recepteur gamma active par les proliferateurs de peroxysomes (PPAR-[gamma] pour peroxisome << proliferator- activated receptor-gamma >>) est une cible homologuee pour contrer la resistance a l'insuline (RI) dans le diabete de type 2 (DT2). Les modulateurs du PPAR-[gamma] permettent d'obtenir des ameliorations sur le plan des effets indesirables et de la sensibilisation a l'insuline lies aux thiazolidinediones classiques. On propose d'utiliser la nitazoxanide (NTZ), dotee d'une activite posttranscriptionnelle agoniste, comme ligand des PPAR-[gamma]. Dans les presents travaux, nous avons compare l'activite antidiabetique de la NTZ a celle de la pioglitazone (PIO) dans un modele de DT2 engendre par un regime alimentaire a teneur elevee en matieres grasses et la streptozotocine chez le rat. Chez des rats Wistar males adultes diabetiques, nous avons administre par voie orale de la PIO (2,7 mg x [kg.sup.-1] x [jour.sup.-1]) ou de la NTZ (200 mg-[kg.sup.-1]-jour-1) pendant 14, 21 et 28 jours. Nous avons mesure le poids corporel, la glycemie a jeun, la RI, le profil lipidique et les fonctions renale et hepatique des rats. Nous avons evalue le metabolisme hepatique du glucose, les taux d'expression proteique du PPAR-[gamma], ainsi que l'histopathologie du foie, du pancreas, des muscles et des reins. La NTZ permettait d'obtenir des effets marques et proportionnels a la duree d'euglycemie, de sensibilisation a l'insuline et de preservation des fonctions renale et hepatique. Avec la NTZ et la PIO, nous avons observe respectivement une augmentation des concentrations hepatiques des enzymes glucose-6-phosphatase et glucose-6-phosphate deshydrogenase, ainsi que de l'expression proteique du PPAR-[gamma]. On pourrait considerer la NTZ comme constituant une strategie therapeutique orale contre le DT2. Nous recommandons d'etudier plus avant l'activation moleculaire de sous-types du PPAR-[gamma] par la NTZ de maniere systematique. Il conviendrait aussi d'etudier l'utilisation simultanee de NTZ avec d'autres antidiabetiques homologues. [Traduit par la Redaction] Mots-cles : diabete engendre par un regime alimentaire a teneur elevee en matieres grasses et la streptozotocine, resistance a l'insuline, nitazoxanide, recepteur gamma active par les proliferateurs de peroxysomes, pioglitazone., Introduction Insulin resistance (IR) is a disease where cellular failure to insulin responses (Kubota et al. 2017; Sam et al. 2017) and obesity are the main contributors of diabetes mellitus [...]

This study was designed to evaluate the effect of beta-sitosterol (BS) on the peroxisome proliferator-activated receptor gamma (PPAR-[gamma]) gene expression role in the activity of paraoxonase (PON-1) enzyme in oxidative stress status of irradiated rats. Animals were exposed to whole body [gamma]-radiation single dose 6 Gy and received BS dose (40 mg x [(kg body mass).sup.-1] x [day.sup.-1], orally). In liver tissue, gene expression of PPAR-[gamma] ligand was determined. Oxidative stress marker (malondialdehyde, MDA) and antioxidant enzyme activities (superoxide dismutase (SOD), catalase (CAT), PON-1, and arylesterase (ARE)) were assayed in serum and liver tissue. Also, serum lipid profile (cholesterol, triglycerides (TG), low-density lipoprotein cholesterol (LDL-c), and high-density lipoprotein cholesterol (HDL-c)) was measured. In irradiated animals that received BS, expression of PPAR-[gamma] ligand increase significantly associated with increase in PON-1 and ARE enzyme activities. Also, the activities of SOD, CAT enzymes, and HDL-c levels display elevation. By contrast, significant decrease in MDA content, cholesterol, TG, and LDL-c levels were revealed after BS administration. Our findings in this study provide the evidence that BS has radio-protective effect via regulating the gene expression of PPAR-[gamma], causing an increase in PON-1 and ARE enzyme activities. This action of BS is due to its free radical scavenging properties, antioxidant effect, lowering of cholesterol, and PPAR-[gamma] agonist properties. Key words: [beta]-sitosterol, [gamma]-radiation, PPAR-[gamma], PON-1, MDA. Nous avons concu cette etude en vue d'evaluer les effets du beta-sitosterol (BS) sur le role de l'expression du gene PPAR-[gamma] (pour << peroxisome proliferator-activated receptor gamma >>) dans l'activite de l'enzyme PON-1 sur l'etat de stress oxydatif chez le rat irradie. Nous avons expose le corps entier des animaux a des rayons [gamma] a 6 Gy et administre du BS a 40 mg x [(kg de masse corporelle).sup.-1] x [jour.sup.-1] par voie orale. Nous avons etabli l'expression du gene du ligand du PPAR-[gamma] dans le foie. Nous avons dose un marqueur du stress oxydatif (malondialdehyde, MDA) et l'activite d'enzymes anti-oxydantes (superoxyde dismutase (SOD), catalase (CAT), PON-1 et arylesterase (ARE)) dans le serum et dans le tissu hepatique. De plus, nous avons evalue le profil lipidique dans le serum (cholesterol, triglycerides (TG), cholesterol associe a des lipoproteines de faible densite (LDL-c) et cholesterol associe a des lipoproteines de haute densite (HDL-c)). Chez les animaux irradies qui ont recu du BS, l'expression du ligand du PPAR-[gamma] augmentait nettement en association avec une augmentation de l'activite des enzymes PON-1 et ARE. De plus, l'activite des enzymes SOD et CAT, et les taux de HDL-c ont augmente. Par contre, nous avons observe une augmentation importante des taux de MDA, de cholesterol, de TG et de LDL-c apres l'administration de BS. Dans cette etude, nos resultats apportent des donnees probantes selon lesquelles le BS aun effet radio-protecteur par l'intermediaire de la regulation de l'expression genique du PPAR-[gamma], qui entraine une augmentation de l'activite des enzymes PON-1 et ARE. Le BS agit par ses proprietes de piegeur des radicaux libres, ses effets anti-oxydants, ainsi que ses proprietes d'abaissement du cholesterol et d'agoniste du PPAR-[gamma]. [Traduit par la Redaction] Mots-cles: [beta]-sitosterol, rayons [gamma], PPAR-[gamma], PON-1, MDA., Introduction The exposure of living organisms to ionizing radiation-induced damage has a lot in common with other metabolic stress and diseases. The underlying mechanism, in most cases, is oxidative stress [...]

Infarct-like lesion induced by isoprenaline is a well-known model to study myocardial infarction (MI). Vitamin D has been shown to have anti-inflammatory and antioxidant effects. Recent studies highlighted cross talk between vitamin D and per oxisome proliferator-activated receptor gamma (PPAR-[gamma]). The present study was designed to investigate the effect of pretreatment with vitamin D on the isoprenaline-induced infarct-like lesion in rats and the role of PPAR-[GAMMA] as a novel mechanism in vitamin-D-mediated cardio protective effect. Markers chosen to assess cardiac damage included serum level of creatine kinase (CK), lactate dehydrogenase (LDH), tumor necrosis factor-alpha (TNF-[alpha]), and interleukin-6 (IL-6). Cardiac contents of malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH) were also assessed. Furthermore, ECG monitoring and measurement of injury extension were carried out. Is oprena line increased the level of cardiac enzymes, as well as inflammatory and oxidative stress biomarkers. In addition, it produced ST-segment elevation. Pretreatment with vitamin D significantly improved previous parameters. The prior treatment with bisphenol A diglycidyl ether (BADGE), a PPAR-[GAMMA] antagonist, significantly attenuated the protective effect of vitamin D. In conclusion, vitamin D can be demonstrated as a promising cardio protective agent in MI and PPAR-[GAMMA] significantly contributes toward vitamin-D-mediated protection. Key words: vitamin D, isoprenaline, myocardial infarction, peroxisome proliferator activated receptor-[gamma], oxidative stress. Les lesions s' apparentant a l' in farctus qu'induit l'isoprenaline constituent un modele bien connu pour l'etude de l' infarctus du myocarde (IM). Par ailleurs, on a montre que la vitamine D a des effets anti-inflammatoires et anti-oxydants. De recentes etudes ont mis l'accent sur des interrelations (cross-talk ') entre la vitamine D et le recepteur gamma active par les proliferateurs de peroxysome (ou PPAR-[GAMMA] pour peroxisome proliferator-activated receptor gamma '). Nous avons concu la presente etude en vue d'examiner les effets de l'exposition prealable a la vitamine D sur les lesions s 'apparentant a l'infarctus qu'induit l'isoprenaline chez le rat et le role du PPAR-[GAMMA] dans un nouveau mode d 'action quant aux effets cardio-protecteurs dont la vitamine D assure la mediation. Les marqueurs que nous avons choisis pour evaluer les dommages cardiaques etaient les taux seriques de crea tine kinase (CK), de deshydrogenase lactique (LDH), du facteur de necrose tumorale alpha (TNF-[alpha]) et d'interleukine 6 (IL-6). Nous avons aussi evalue les taux de malondialdehyde (MDA), de superoxyde dismutase (SOD) et de glutathion peroxydase (GSH) dans le creur. En outre, nous avons effectue une surveillance de l'ECG ( monitoring ' card iaque) et mesure l 'etendue des lesions. L'isoprenaline entrainaitune augmentation des taux d'enzymes cardiaques, ainsi que de bio-marqueurs de l' inflammation et du stress oxydatif. De plus, elle entrainait une elevation du segment ST. L'exposition anterieure a la vitamin D permettait d'ameliorer nettement les resultats quant aux parametres precedents. Par contre, l'exposition anterieure a un ether de bisphenol A et de diglycidyle (BADGE), un antagoniste du PPAR-[GAMMA], entrainait une attenuation marquee des effets protecteurs de la vitamine D. En conclusion, on peut montrer que la vitamine D est un agent cardio protecteur prometteur dans l'IM et que le PPAR-[GAMMA] contribue de maniere appreciable a la protection dont la vitamine D assure la mediation. [Traduit par la Redaction] Mots-cles : vitamine D, isoprenaline, infarctus du myocarde, recepteur 7 active par les proliferateurs de peroxysome, stress oxydatif., Introduction Despite great efforts during the last decades, cardiovascular diseases remain the major cause of death worldwide, increasing their prevalence every year (Basak et al. 2015). Myocardial infarction (MI) is [...]

Blocking ERK/MAP kinases improves insulin sensitivity thorough a mechanism similar to the actions of the anti-diabetic thiazolidinediones drugs on PPAR[gamma]. Role of MEK/ERK pathway in glucose metabolism The nuclear receptor PPAR[gamma] is required for glucose metabolism and is a drug target in diabetes. Earlier work has suggested that PPAR[gamma] phosphorylation has an adverse effect on insulin sensitivity and that the kinase Cdk5 is the main kinase responsible for phosphorylation at this site. Now Bruce Spiegelman and colleagues report the paradoxical observation that insulin resistance is worsened in mice lacking Cdk5 in adipose tissue. The authors further show that Cdk5 suppresses the MEK/ERK signalling pathway that is central to the regulation of cell growth and proliferation, and that ERK directly phosphorylates PPAR[gamma]. MEK/ERK inhibitors can improve insulin resistance in obese wild-type and ob/ob mice. This work suggests that MEK/ERK inhibitors -- already approved for cancer treatment -- might also be effective against diabetes. Obesity-linked insulin resistance is a major precursor to the development of type 2 diabetes. Previous work has shown that phosphorylation of PPAR[gamma] (peroxisome proliferator-activated receptor [gamma]) at serine 273 by cyclin-dependent kinase 5 (Cdk5) stimulates diabetogenic gene expression in adipose tissues.sup.1. Inhibition of this modification is a key therapeutic mechanism for anti-diabetic drugs that bind PPAR[gamma], such as the thiazolidinediones and PPAR[gamma] partial agonists or non-agonists.sup.2. For a better understanding of the importance of this obesity-linked PPAR[gamma] phosphorylation, we created mice that ablated Cdk5 specifically in adipose tissues. These mice have both a paradoxical increase in PPAR[gamma] phosphorylation at serine 273 and worsened insulin resistance. Unbiased proteomic studies show that extracellular signal-regulated kinase (ERK) kinases are activated in these knockout animals. Here we show that ERK directly phosphorylates serine 273 of PPAR[gamma] in a robust manner and that Cdk5 suppresses ERKs through direct action on a novel site in MAP kinase/ERK kinase (MEK). Importantly, pharmacological inhibition of MEK and ERK markedly improves insulin resistance in both obese wild-type and ob/ob mice, and also completely reverses the deleterious effects of the Cdk5 ablation. These data show that an ERK/Cdk5 axis controls PPAR[gamma] function and suggest that MEK/ERK inhibitors may hold promise for the treatment of type 2 diabetes., Author(s): Alexander S. Banks [sup.1] , Fiona E. McAllister [sup.2] , João Paulo G. Camporez [sup.3] , Peter-James H. Zushin [sup.1] , Michael J. Jurczak [sup.3] , Dina Laznik-Bogoslavski [sup.4] [...]

In adipose tissue, muscle, liver and macrophages, signaling by the nuclear receptor peroxisome proliferator--activated receptor-γ (PPAR-γ) is a determinant of insulin sensitivity and this receptor mediates the insulin-sensitizing effects of thiazolidinediones (TZDs) (1-4). As PPAR-γ is also expressed in neurons (5), we generated mice with neuron-specific Pparg knockout (Pparg brain knockout (BKO)) to determine whether neuronal PPAR-γ signaling contributes to either weight gain or insulin sensitivity. During high-fat diet (HFD) feeding, food intake was reduced and energy expenditure increased in Pparg-BKO mice compared to [Pparg.sup.f/f] mice, resulting in reduced weight gain. Pparg-BKO mice also responded better to leptin administration than [Pparg.sup.f/f] mice. When treated with the TZD rosiglitazone, Pparg-BKO mice were resistant to rosiglitazone-induced hyperphagia and weight gain and, relative to rosiglitazone-treated [Pparg.sup.f/f] mice, experienced only a marginal improvement in glucose metabolism. Hyperinsulinemic euglycemic clamp studies showed that the increase in hepatic insulin sensitivity induced by rosiglitazone treatment during HFD feeding was completely abolished in Pparg-BKO mice, an effect associated with the failure of rosiglitazone to improve liver insulin receptor signal transduction. We conclude that excess weight gain induced by HFD feeding depends in part on the effect of neuronal PPAR-γ signaling to limit thermogenesis and increase food intake. Neuronal PPAR-γ signaling is also required for the hepatic insulin sensitizing effects of TZDs., TZDs are a class of drugs that activate PPAR-g and improve blood glucose control and systemic insulin sensitivity in patients with type 2 diabetes mellitus (T2DM) (6). In addition to [...]

Pex19p, a soluble cytoplasmic transport protein, is required for the traffic of the peroxisomal membrane proteins Pex3p and Pex15p from the endoplasmic reticulum (ER) to the peroxisome. We documented Pex15p traffic from the ER using a chimeric protein containing a C-terminal glycosylation acceptor peptide. Pex15Gp expressed in wild-type yeast cells is N-glycosylated and functions properly in the peroxisome. In contrast, pex19[DELTA]-mutant cells accumulate the glycoprotein Pex15Gp in the ER. We developed a cell-free preperoxisomal vesicle-budding reaction in which Pex15Gp and Pex3p are packaged into small vesicles in the presence of cytosol, Pex19p, and ATP. Secretory vesicle budding (COPll) detected by the packaging of a SNARE protein (soluble N-ethylmaleimide-sensitive attachment protein receptor) occurs in the same incubation but does not depend on Pex19p. Conversely a dominant GTPase mutant Sar1p which inhibits COPII has no effect on Pex3p packaging. Pex15Gp and Pex3p budded vesicles sediment as low-buoyant-density membranes on a Nycodenz gradient and copurify by affinity isolation using native but not Triton X-100--treated budded vesicles. ER--peroxisome transport vesicles appear to rely on a novel budding mechanism requiring Pex19p and additional unknown factors. doi/ 10.1073/pnas.1013397107

Retinoic acid (RA) was found to be a ligand for peroxisome proliferation-activated receptor [delta] (PPAR[delta]) as well as the classical RA receptor (RAR). Carrier proteins that move the RA from the cytosol into the nucleus are the fatty acid-binding protein 5 (FABP5), activating PPAR[delta], and the cellular retinoic acid-binding protein II (CRABPII), activating RAR. The ratio of FABPS/CRABPII concentrations determines which receptor is activated. By activating PPAR[delta], RA was found to induce expression of genes affecting lipid and glucose homeostasis, in particular, leading to expression of the insulin-signaling gene PDK1 and improvement of insulin action. Hence, RA stimulates lipolysis and reduces triglyceride content. In vivo, obesity has led to downregulation of adipose PPAR[delta] expression. RA implantation into obese mice has caused upregulation of levels of PPAR[delta] and consequent weight loss as well as increased expression of PPAR[delta] target genes, including the insulin-signaling gene PDK1. doi: 10.1111/j.1753-4887.2009.00261.x

Am J Physiol Enclocrinol Metab 298: E68-E79, 2010. First published October 27, 2009; doi: 10.1152/ajpendo.00367.2009.--Very-low-density lipoprotein receptor (VLDLR) is a member of the low-density receptor family, highly expressed in adipose tissue, heart, and skeletal muscle. It binds apolipoprotein E-triglyceride-rich lipoproteins and plays a significant role in triglyceride metabolism. PPAR[gamma] is a primary regulator of lipid metabolism in adipocytes and controls the expression of an array of genes involved in lipid trafficking in adipocytes. However, it is not known whether VLDLR is also under the control of PPAR[gamma]. In this study, we investigated the role of PPAR[gamma] in the regulation of VLDLR expression and function in vivo and in vitro. During the differentiation of 3T3-L1 preadipocytes, the levels of VLDLR protein and mRNA increased in parallel with the induction of PPAR[gamma] expression and reached maximum in mature adipocytes. Treatment of differentiated adipocytes with PPAR[gamma] agonist pioglitazone upregulated VLDLR expression in dose- and time-dependent manners. In contrast, specific inhibition of PPAR[gamma] significantly downregulated the protein level of VLDLR. Induction of VLDLR is also demonstrated in vivo in adipose tissue of wild-type (WT) mice treated with pioglitazone. In addition, pioglitazone increased plasma triglyceride-rich lipoprotein clearance and increased epididymal fat mass in WT mice but failed to induce similar effects in [vldlr.sup.-/-] mice. These results were further corroborated by the finding that pioglitazone treatment enhanced adipogenesis and lipid deposition in preadipocytes of WT mice, while its effect in VLDLR-null preadipocytes was significantly blunted. These findings provide direct evidence that VLDLR expression is regulated by PPAR[gamma] and contributes in lipid uptake and adipogenesis. white adipose tissue; triglyceride; triglyceride-rich lipoproteins; triglyceride clearance

Saccharomyces cerevisiae Pex3p has been shown to act at the ER during de novo peroxisome formation. However, its steady state is at the peroxisomal membrane, where its role is debated. Here we show that Pex3p has a dual function: one in peroxisome formation and one in peroxisome segregation. We show that the peroxisome retention factor Inp 1p interacts physically with Pex3p in vitro and in vivo, and split-GFP analysis shows that the site of interaction is the peroxisomal membrane. Furthermore, we have generated PEX3 alleles that support peroxisome formation but fail to support recruitment of Inp 1p to peroxisomes, and as a consequence are affected in peroxisome segregation. We conclude that Pex3p functions as an anchor for Inp1 p at the peroxisomal membrane, and that this function is independent of its role at the ER in peroxisome biogenesis. doi 10.1083/jcb.200906161

Vascular endothelial cells express the ligand-activated transcription factor, peroxisome proliferator-activated receptor-[gamma] (PPAR[gamma]), which participates in the regulation of metabolism, cell proliferation, and inflammation. PPAR[gamma] ligands attenuate, whereas the loss of function mutations in PPAR[gamma] stimulate, endothelial dysfunction, suggesting that PPAR[gamma] may regulate vascular endothelial nitric oxide production. To explore the role of endothelial PPAR[gamma] in the regulation of vascular nitric oxide production in vivo, mice expressing Cre recombinase driven by an endothelial-specific promoter were crossed with mice carrying a floxed PPAR[gamma] gene to produce endothelial PPAR[gamma] null mice ([ePPAR[gamma].sup.-/-]). When compared with littermate controls, [ePPAR[gamma].sup.-/-] animals were hypertensive at baseline and demonstrated comparable increases in systolic blood pressure in response to angiotensin II infusion. When compared with those of control animals, aortic ring relaxation responses to acetylcholine were impaired, whereas relaxation responses to sodium nitroprusside were unaffected in [ePPAR[gamma].sup.-/-] mice. Similarly, intact aortic segments from [ePPAR[gamma].sup.-/-] mice released less nitric oxide than those from controls, whereas endothelial nitric oxide synthase expression was similar in control and [ePPAR[gamma].sup.-/-] aortas. Reduced nitric oxide production in [ePPAR[gamma].sup.-/-] aortas was associated with an increase in the parameters of oxidative stress in the blood and the activation of nuclear factor-[kappa]B in aortic homogenates. These findings demonstrate that endothelial PPAR[gamma] regulates vascular nitric oxide production and that the disruption of endothelial PPAR[gamma] contributes to endothelial dysfunction in vivo. endothelial nitric oxide synthase doi: 10.1152/ajpheart.00148.2009.

In Saccharomyces cerevisiae, peroxisomal inheritance from mother cell to bud is conducted by the class V myosin motor, Myo2p. However, homologues of S. cerevisiae Myo2p peroxisomal receptor, Inp2p, are not readily identifiable outside the Saccharomycetaceae family. Here, we demonstrate an unexpected role for Pex3 proteins in peroxisome inheritance. Both Pex3p and Pex3Bp are peroxisomal integral membrane proteins that function as peroxisomal receptors for class V myosin through direct interaction with the myosin globular tail. In cells lacking Pex3Bp, peroxisomes are preferentially retained by the mother cell, whereas most peroxisomes gather and are transferred en masse to the bud in cells overexpressing Pex3Bp or Pex3p. Our results reveal an unprecedented role for members of the Pex3 protein family in peroxisome motilily and inheritance in addition to their well-established role in peroxisome biogenesis at the endoplasmic reticulum. Our results point to a temporal link between peroxisome formation and inheritance and delineate a general mechanism of peroxisome inheritance in eukaryotic cells. doi 10.1083/jcb.200902117

Restraint stress induces permeability changes in the small intestine, but little is known about the role of endogenous peroxisome proliferator-activated receptor-[alpha] (PPAR-[alpha]) ligand in the defects of the tight junction function. In the present study, we used PPAR-[alpha] knockout mice to understand the roles of endogenous PPAR-[alpha] on ileum altered permeability function in models of immobilization stress. The absence of a functional PPAR-[alpha] gene in PPAR-[alpha] knockout mice resulted in a significant augmentation of the degree of 1) TNF-[alpha] production in ileum tissues; 2) the alteration of zonula occludens-1, occludin, and [beta]-catenin (immunohistochemistry); and 3) apoptosis (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling staining, Bax, Bcl-2 expression). Taken together, our results demonstrate that endogenous PPAR-[alpha] ligands reduce the degree of tight junction permeability in the ileum tissues associated with immobilization stress, suggesting a possible role of endogenous PPAR-[alpha] ligands on ileum barrier dysfunction. apoptosis; peroxisome proliferator-activated receptor-[alpha]-deficient mice; zonula occludens-l; occludin; [beta]-catenin

The effect of early intervention with a peroxisome proliferator-activated receptor-[gamma] (PPAR[gamma]) agonist on skeletal muscle GLUT4 translocation and insulin signaling was examined in intrauterine (IUGR) and postnatal (PNGR) growth-restricted pregestational female rat offspring. Rosiglitazone [11 [micro]mol/ day provided from postnatal day (PN)21 to PN60] improved skeletal muscle insulin sensitivity and GLUT4 translocation in prenatal nutrient restriction [50% calories from embryonic day (e)11 to e21; IUGR] with (IUGR+PNGR) and without (IUGR) postnatal nutrient restriction (50% calories from PN1 to PN21; PNGR) similar to that of control (ad libitum feeds throughout; Con) (n = 6 each). This was accomplished by diminished basal and improved insulin-responsive GLUT4 association with the plasma membrane in IUGR, IUGR+PNGR, and PNGR mimicking that in Con (P < 0.005). While no change in p85-phosphatidylinositol 3-kinase (PI3-K) and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) was observed, a decrease in protein tyrosine phosphatase 1B (PTP1B; P < 0.0002) and SH2-containing protein tyrosine phosphatase 2 (SHP2; P < 0.05) contributing to the rosiglitazone-induced insulin sensitivity was seen only in IUGR+PNGR. In contrast, an increase in phosphorylated 5'-adenosine monophosphate kinase (pAMPK; P < 0.04) and insulin responsiveness of phosphorylated phosphoinositide-dependent protein kinase-1 (pPDK1; P < 0.05), pAkt (P < 0.01), and particularly pPKC[zeta] (P < 0.0001) and its corresponding enzyme activity (P < 0.005) were observed in all four experimental groups. We conclude that early introduction of PPAR[gamma] agonist improved skeletal muscle activation of AMPK and insulin signaling, resulting in insulin-independent AMPK and insulin-responsive GLUT4 association with plasma membranes in IUGR, IUGR+PNGR, and PNGR adult off-spring, similar to that of Con. These findings support a role for insulin sensitizers in preventing the subsequent development of gestational or type 2 diabetes mellitus in intrauterine and postnatal growth-restricted offspring. fetal origins of adult disease; glucose transporter; metabolic programming; protein kinase C[zeta]

Thiazolidinediones (TZDs) are synthetic peroxisome proliferator-activated receptor-[gamma] (PPAR[gamma]) ligands that are widely used in type II diabetes treatment. In addition to their ability to improve glucose homeostasis, TZDs possess antiinflammatory properties and inhibit growth of many cells, particularly cancerous airway epithelial cells. However, the functional effects of PPAR[gamma] ligands on nonmalignant human bronchial epithelial cells have never been investigated. In the present study, we questioned whether PPAR[gamma] ligands may regulate proliferation of human bronchial epithelial cells, and we studied their potential molecular mechanisms. We found that synthetic PPAR[gamma] agonists, rosiglitazone (RGZ) and troglitazone (TGZ), induced proliferation of human bronchial epithelial cells, whereas the endogenous PPAR[gamma], ligand, 15-deoxy-[[DELTA].sup.12,14]-prostaglandin [J.sub.2] (15d-[PGJ.sub.2]), inhibited cell growth. RGZ and TGZ (10 [micro]M) induced a rapid and transient intracellular [Ca.sup.2+] mobilization from thapsigargin-sensitive intracellular stores, whereas 15d-[PGJ.sub.2] (5 [micro]M) did not induce any [Ca.sup.2+] signal. The PPAR[gamma] antagonist GW-9662 did not inhibit any biological responses, but it reversed the effect of 15d-[PGJ.sub.2] on cell growth. Using RT-PCR, we detected mRNA expression of the GPR40 receptor, a G proteincoupled receptor recently identified as a receptor for free fatty acids and TZDs, in human bronchial epithelial cells. Downregulation of GPR40 by small-interfering RNA led to a significant inhibition of TZD-induced [Ca.sup.2+] mobilization and proliferation. This study provides evidence for the proliferative effect of anti-diabetic drug TZDs in nonmalignant human bronchial epithelial cells through GPR40 receptor activation, involving an intracellular [Ca.sup.2+] signaling pathway. airway epithelium; glitazone; calcium; peroxisome proliferator-activated receptor-[gamma]; G protein receptor 40

Peroxisomes are essential organelles exerting key functions in fatty acid metabolism such as the degradation of very long-chain fatty acids (VLCFAs). VLCFAs accumulate in X-adrenoleukodystrophy (X-ALD), a disease caused by deficiency of the Abcdl peroxisomal transporter. Its closest homologue, Abcd2, exhibits a high degree of functional redundancy on the catabolism of VLCFA, being able to prevent X-ALD-related neurodegeneration in the mouse. In the search for specific roles of Abcd2, we screened fatty acid profiles in organs and primary neurons of mutant knockout mice lacking Abcd2 in basal conditions and under dietary challenges. Our results indicate that ABCD2 plays a role in the degradation of long-chain saturated and [omega]9-monounsaturated fatty acids and in the synthesis of docosahexanoic acid (DHA). Also, we demonstrated a defective VLCFA [beta]-oxidation ex vivo in brain slices of Abcd1 and Abcd2 knockouts, using radiolabeled hexacosanoic acid and the precursor of DHA as substrates. As DHA levels are inversely correlated with the incidence of Alzheimer's and several degenerative conditions, we suggest that ABCD2 may act as modulator/modifier gene and therapeutic target in rare and common human disorders. adrenoleukodystrophy; adenosine 5'-triphosphate-binding cassette transporters; peroxisome; polyunsaturated fatty acid; docosahexanoic acid

The glyoxylate cycle, a metabolic pathway required for generating [C.sub.4] units from [C.sub.2] compounds, is an important factor in virulence, in both animal and plant pathogens. Here, we report the localization of the key enzymes of this cycle, isocitrate lyase (Icl1; EC 4.1.3.1) and malate synthase (Mls1; EC 2.3.3.9), in the human fungal pathogen Candida albicans. Immunocytochemistry in combination with subcetlular fractionation showed that both Icl1 and Mls1 are localized to peroxisomes, independent of the carbon source used. Although Icl1 and Mls1 lack a consensus type I peroxisomal targeting signal (PTS1), their import into peroxisomes was dependent on the PTS1 receptor Pex5p, suggesting the presence of non-canonical targeting signals in both proteins. Peroxisomal compartmentalization of the glyoxylate cycle is not essential for proper functioning of this metabolic pathway because a pex5[DELTA]/[DELTA] strain, in which Icl1 and Mls1 were localized to the cytosol, grew equally as well as the wild-type strain on acetate and ethanol. Previously, we reported that a fox2[DELTA]/[DELTA] strain that is completely deficient in fatty acid [beta]- oxidation, but has no peroxisomal protein import defect, displayed strongly reduced growth on non-fermentable carbon sources such as acetate and ethanol. Here, we show that growth of the fox2[DELTA]/[DELTA] strain on these carbon compounds can be restored when Icl1 and Mls1 are relocated to the cytosol by deleting the PEX5 gene. We hypothesize that the fox2[DELTA]/[DELTA] strain is disturbed in the transport of glyoxylate cycle products and/or acetyI-CoA across the peroxisomal membrane and discuss the possible relationship between such a transport defect and the presence of giant peroxisomes in the fox2[DELTA]/[DELTA] mutant.

The objectives of these experiments were to compare differentiation of bovine stromal-vascular (S-V) cells isolated from i.m. and s.c. adipose tissues in response to a glucocorticoid and a peroxisome proliferator-activated receptor [gamma] agonist. Stromal-vascular cells were isolated from i.m. and s.c. fat depots of 3 Angus steers and propagated in culture. Cells were exposed to differentiation media containing 0.25 [micro]M dexamethasone (DEX), a glucocorticoid analog, and 40 [micro]M troglitazone (TRO), a peroxisome proliferator-activated receptor [gamma] agonist, or both. Cells treated with DEX and TRO had greater (P < 0.02) glycerol-3-phosphate dehydrogenase activity than control cells. No interactions between DEX, TRO, and depot (P > 0.59) or depot differences (P = 0.41) in glycerol-3-phosphate dehydrogenase activity were found. Morphological assessment of adipogenic colonies showed that DEX induced a 1.8-fold increase in the percentage of adipogenic colonies (P = 0.03), whereas TRO increased the proportion of adipogenic colonies by 1.9-fold (P = 0.02) compared with those not treated with DEX or TRO, respectively. Depots had a similar percentage of adipogenic colonies (P = 0.18); however, the percentage of differentiated cells within adipogenic colonies was found to be 6.4-fold greater in s.c. isolates compared with i.m. (P < 0.001). Addition of TRO increased the proportion of differentiated cells within colonies by 10-fold compared with those of nontreated colonies (P < 0.001), whereas the percentage of differentiated cells within adipogenic colonies only tended to be increased by DEX (P = 0.10). These data indicate that bovine i.m. and s.c. S-V cells are capable of enhanced differentiation in response to DEX and TRO, and these effects were additive. Most importantly, inherent differences in the capacity to differentiate exist between adipogenic bovine i.m. and s.c. S-V cells. Key words: bovine, differentiation, glycerol-3-phosphate dehydrogenase, glucocorticoid, peroxisome proliferator-activated receptor gamma, stromal-vascular

Pharmacological administration 1 of the natural lipid amide, oleoylethanolamide (OEA), inhibits food intake in flee-feeding rodents by prolonging latency to feed and postmeal interval. This anorexic effect is mediated by activation of type-[alpha] peroxisome proliferator-activated receptors (PPAR-[alpha]. Food intake stimulates mucosal cells in duodenum and jejunum to generate OEA, suggesting that this lipid-derived messenger may act as a local to satiety hormone. As a test of this hypothesis, here, we examined whether targeted enhancement of OEA production in the small intestine affects feeding behavior in rats. We constructed an adenoviral vector (Ad-NPLD) that directs overexpression of the enzyme N-acylphosphatidylethanolamine (NAPE)-phospholipase D (PLD), which catalyzes the hydrolysis of NAPE to generate OEA. Intraduodenal injection of the Ad-NPLD vector resulted in a time-dependent increase in NAPE-PLD expression and OEA production, which was restricted to the proximal small intestine. No such effect was observed after administration of a control adenoviral vector. Enhanced OEA production in Ad-NPLD-injected animals was temporally associated with increased expression of two PPAR-[alpha] target genes (PPAR-[alpha] and CD36) and with decreased food intake. The hypophagic phenotype of Ad-NPLD-injected rats was attributable to increase feeding latency and postmeal interval, rather than decreased meal size. The results suggest that localized changes in OEA production in the small intestine, such as those produced by food intake, are sufficient to induce in rats a state of across-meal satiety similar to that elicited by systemic administration of exogenous OEA. N-acylphosphatidylethanolamine-phospholipase D; peroxisome pro-liferator-activated receptor-[alpha]; adenovirus

The pathogenic bacterium Pseudomonas aeruginosa utilizes the 3-oxododecanoyl homoserine lactone (3O[C.sub.12]-HSL) autoinducer as a signaling molecule to coordinate the expression of virulence genes through quorum sensing. 3O[C.sub.12]-HSL also affects responses in host cells, including the upregulation of genes encoding inflammatory cytokines. This proinflammatory response may exacerbate underlying disease during P. aeruginosa infections. The specific mechanism(s) through which 3O[C.sub.12]-HSL influences host responses is unclear, and no mammalian receptors for 3O[C.sub.12]-HSL have been identified to date. Here, we report that 3O[C.sub.12]-HSL increases mRNA levels for a common panel of proinflammatory genes in murine fibroblasts and human lung epithelial cells. To identify putative 3O[C.sub.12]-HSL receptors, we examined the expression patterns of a panel of nuclear hormone receptors in these two cell lines and determined that both peroxisome proliferator-activated receptor beta/delta (PPAR[beta]/[delta]) and PPAR[gamma] were expressed. 3O[C.sub.12]-HSL functioned as an agonist of PPAR[beta]/[delta] transcriptional activity and an antagonist of PPAR[gamma], transcriptional activity and inhibited the DNA binding ability of PPAR[gamma]. The proinflammatory effect of 3O[C.sub.12]-HSL in lung epithelial cells was blocked by the PPAR[gamma], agonist rosiglitazone, suggesting that 3O[C.sub.12]-HSL and rosiglitazone are mutually antagonistic negative and positive regulators of PPAR[gamma] activity, respectively. These data identify PPAR[beta]/[delta] and PPAR[gamma] as putative mammalian 3O[C.sub.12]-HSL receptors and suggest that PPAR[gamma] agonists may be employed as anti-inflammatory therapeutics for P. aeruginosa infections.

Author(s): Ayumu Sugiura [1]; Sevan Mattie [1]; Julien Prudent [1]; Heidi M. McBride (corresponding author) [1] Peroxisomes function together with mitochondria in a number of essential biochemical pathways, from bile [...]

Peroxisomal oxidation yields metabolites that are more efficiently utilized by mitochondria. This is of potential clinical importance because reduced fatty acid oxidation is suspected to promote excess lipid accumulation in obesity-associated insulin resistance. Our purpose was to assess peroxisomal contributions to mitochondrial oxidation in mixed gastrocnemius (MG), liver, and left ventricle (LV) homogenates from lean and fatty (fa/fa) Zucker rats. Results indicate that complete mitochondrial oxidation (C[O.sub.2] production) using various lipid substrates was increased approximately twofold in MG, unaltered in LV, and diminished ~50% in liver of fa/fa rats. In isolated mitochondria, malonyl-CoA inhibited C[O.sub.2] production from palmitate 78%, whereas adding isolated peroxisomes reduced inhibition to 21%. These data demonstrate that peroxisomal products may enter mitochondria independently of CPT I, thus providing a route to maintain lipid disposal under conditions where malonyl-CoA levels are elevated, such as in insulin-resistant tissues. Peroxisomal metabolism of lignoceric acid in fa/fa rats was elevated in both liver and MG (LV unaltered), but peroxisomal product distribution varied. A threefold elevation in incomplete oxidation was solely responsible for increased hepatic peroxisomal oxidation (C[O.sub.2] unaltered). Alternatively, only C[O.sub.2] was detected in MG, indicating that peroxisomal products were exclusively partitioned to mitochondria for complete lipid disposal. These data suggest tissue-specific destinations for peroxisome-derived products and emphasize a potential role for peroxisomes in skeletal muscle lipid metabolism in the obese, insulin-resistant state. fatty acid; lipid metabolism; liver; heart; skeletal muscle; Zucker rat

Achieving efficient in vivo delivery of siRNA to the appropriate target cell would be a major advance in the use of RNAi in gene function studies and as a therapeutic modality. Hepatocytes, the key parenchymal cells of the liver, are a particularly attractive target cell type for siRNA delivery given their central role in several infectious and metabolic disorders. We have developed a vehicle for the delivery of siRNA to hepatocytes both in vitro and in vivo, which we have named siRNA Dynamic PolyConjugates. Key features of the Dynamic PolyConjugate technology include a membrane-active polymer, the ability to reversibly mask the activity of this polymer until it reaches the acidic environment of endosomes, and the ability to target this modified polymer and its siRNA cargo specifically to hepatocytes in vivo after simple, low-pressure i.v. injection. Using this delivery technology, we demonstrate effective knockdown of two endogenous genes in mouse liver: apolipoprotein B (apoB) and peroxisome proliferator-activated receptor alpha (ppara). Knockdown of apoB resulted in clear phenotypic changes that included a significant reduction in serum cholesterol and increased fat accumulation in the liver, consistent with the known functions of apoB. Knockdown of ppara also resulted in a phenotype consistent with its known function, although with less penetrance than observed in apoB knockdown mice. Analyses of serum liver enzyme and cytokine levels in treated mice indicated that the siRNA Dynamic PolyConjugate was nontoxic and well tolerated. pH labile bonds | nonviral siRNA delivery | siRNA-polymer conjugates | endosomolytic polymers

Activation of AMP-activated kinase (AMPK) in skeletal muscle increases glucose uptake, fatty acid oxidation, and mitochondrial biogenesis by increasing gene expression in these pathways. However, the transcriptional components that are directly targeted by AMPK are still elusive. The peroxisome-proliferator-activated receptor [gamma] coactivator 1[alpha] (PGC-1[alpha]) has emerged as a master regulator of mitochondrial biogenesis; furthermore, it has been shown that PGC-1[alpha] gene expression is induced by exercise and by chemical activation of AMPK in skeletal muscle. Using primary muscle cells and mice deficient in PGC-1[alpha], we found that the effects of AMPK on gene expression of glucose transporter 4, mitochondrial genes, and PGC-1[alpha] itself are almost entirely dependent on the function of PGC-1[alpha] protein. Furthermore, AMPK phosphorylates PGC-1[alpha] directly both in vitro and in cells. These direct phosphorylations of the PGC-1[alpha] protein at threonine-177 and serine-538 are required for the PGC-1[alpha]-dependent induction of the PGC-1[alpha] promoter. These data indicate that AMPK phosphorylation of PGC-1[alpha] initiates many of the important gene regulatory functions of AMPK in skeletal muscle. mitochondria | respiration

Glyoxysomes are a subclass of peroxisomes involved in lipid mobilization. Two distinct peroxisomal targeting signals (PTSs), the C-terminal PTS1 and the N-terminal PTS2, are defined. Processing of the PTS2 on protein import is conserved in higher eukaryotes. The cleavage site typically contains a Cys at P1 or P2. We purified the glyoxysomal processing protease (GPP) from the fat-storing cotyledons of watermelon (Citrullus vulgaris) by column chromatography, preparative native isoelectric focusing, and 2D PAGE. The GPP appears in two forms, a 72-kDa monomer and a 144-kDa dimer, which are in equilibrium with one another. The equilibrium is shifted on [Ca.sup.2+] removal toward the monomer and on [Ca.sup.2+] addition toward the dimer. The monomer is a general degrading protease and is activated by denatured proteins. The dimer constitutes the processing protease because the substrate specificity proven for the monomer ([phi]-Arg/Lys [down arrow]) is different from the processing substrate specificity (Cys-Xxx [down arrow]/Xxx-Cys [down arrow]) found with the mixture of monomer and dimer. The Arabidopsis genome analysis disclosed three proteases predicted to be in peroxisomes, a Deg-protease, a pitrilysin-like metallopeptidase, and a Lonprotease. Specific antibodies against the peroxisomal Degprotease from Arabidopsis (Deg15) identify the watermelon GPP as a Deg15. A knockout mutation in the DEG15 gene of Arabidopsis (Atlg28320) prevents processing of the glyoxysomal malate dehydrogenase precursor to the mature form. Thus, the GPP/Deg15 belongs to a group of trypsin-like serine proteases with Escherichia coli DegP as a prototype. Nevertheless, the GPP/Deg15 possesses specific characteristics and is therefore a new subgroup within the Deg proteases. Arabidopsis thaliana / [Ca.sup.2+] signal / Citrullus vulgaris / monomer/dimer equilibrium

Epoxyeicosatrienoic acids (EETs), which function primarily as autocrine and paracrine mediators in the cardiovascular and renal systems, are synthesized from arachidonic acid by cytochrome P-450 epoxygenases. They activate smooth muscle large-conductance [Ca.sup.2+]-activated [K.sup.+] channels, producing hyperpolarization and vasorelaxation. EETs also have anti-inflammatory effects in the vasculature and kidney, stimulate angiogenesis, and have mitogenic effects in the kidney. Many of the functional effects of EETs occur through activation of signal transduction pathways and modulation of gene expression, events probably initiated by binding to a putative cell surface EET receptor. However, EETs are rapidly taken up by cells and are incorporated into and released from phospholipids, suggesting that some functional effects may occur through a direct interaction between the EET and an intracellular effector system. In this regard, EETs and several of their metabolites activate peroxisome proliferator-activated receptor [alpha] (PPAR [alpha]) and PPAR [gamma], suggesting that some functional effects may result from PPAR activation. EETs are metabolized primarily by conversion to dihydroxyeicosatrienoic acids (DHETs), a reaction catalyzed by soluble epoxide hydrolase (sEH). Many potentially beneficial actions of EETs are attenuated upon conversion to DHETs, which do not appear to be essential under routine conditions. Therefore, sEH is considered a potential therapeutic target for enhancing the beneficial functions of EETs. soluble epoxide hydrolase; eicosanoids; dihydroxyeicosatrienoic acids; cytochrome P-450; peroxisome proliferator-activated receptor

Plant peroxisomes perform multiple vital metabolic processes including lipid mobilization in oil-storing seeds, photorespiration, and hormone biosynthesis. Peroxisome biogenesis requires the function of peroxin (PEX) proteins, including PEX10, a [C.sub.3]H[C.sub.4] Zn RING finger peroxisomal membrane protein. Loss of function of PEX10 causes embryo lethality at the heart stage. We investigated the function of PEX10 with conditional sublethal mutants. Four T-DNA insertion lines expressing pex10 with a dysfunctional RING finger were created in an Arabidopsis WT background ([DELTA]Zn plants). They could be normalized by growth in an atmosphere of high C[O.sub.2] partial pressure, indicating a defect in photorespiration. [beta]-Oxidation in mutant glyoxysomes was not affected. However, an abnormal accumulation of the photorespiratory metabolite glyoxylate, a lowered content of carotenoids and chlorophyll a and b, and a decreased quantum yield of photosystem II were detected under normal atmosphere, suggesting impaired leaf peroxisomes. Light and transmission electron microscopy demonstrated leaf peroxisomes of the [DELTA]Zn plants to be more numerous, multilobed, clustered, and not appressed to the chloroplast envelope as in WT. We suggest that inactivation of the RiNG finger domain in PFX10 has eliminated protein interaction required for attachment of peroxisomes to chloroplasts and movement of metabolites between peroxisomes and chloroplasts. [beta]-oxidation | biogenesis | glyoxysome

Peroxisome proliferator-activated receptor-[gamma] (PPAR[gamma]) are ligand-activated transcription factors that regulate cell growth, inflammation, lipid metabolism, and insulin sensitivity. We recently demonstrated that PPAR[gamma] agonists limit high glucose-induced inflammation in a model of proximal tubular cells (PTC; Panchapakesan U, Pollock CA, and Chen XM. Am J Physiol Renal Physiol 287: F528-F534, 2004). However, the role of PPAR[gamma] in the excess extracellular matrix production is largely unknown. We evaluated the effect of 24- to 48-h 8 [micro]M L-805645 or 10 [micro]M pioglitazone on 25 mM o-glucose-induced markers of fibrosis in HK-2 cells. High D-glucose induced nuclear binding of activator protein-1 (AP-1) to 140.8 [+ or -] 10.9% (P < 0.05), which was attenuated with L-805645 and pioglitazone to 82.3 [+ or -] 14.4 (P < 0.01 vs. high o-glucose) and 99.3 [+ or -] 12.2% (P < 0.05 vs. high D-glucose), respectively. High D-glucose increased total production of transforming growth factor (TGF)-[beta]1 139.6 [+ or -] 6.5% (P < 0.05), which was reversed with L-805645 and pioglitazone to 68.73 [+ or -] 5.7 (P < 0.01 vs. high D-glucose) and 112 [+ or -] 13.6% (P < 0.05 vs. high D-glucose). L-805645 and pioglitazone reduced high D-glucose-induced fibronectin from 156.0 [+ or -] 24.9 (P < 0.05) to 81.9 [+ or -] 16.0 and 57.4 [+ or -] 12.7%, respectively (both P < 0.01 vs. high D-glucose). Collagen IV was not induced by high D-glucose. L-805645 and pioglitazone suppressed collagen IV to 68.0 [+ or -] 14.5 (P < 0.05) and 46.5 [+ or -] 11.6% (P < 0.01) vs. high D-glucose, respectively. High D-glucose increased the nuclear binding of NF-[kappa]B to 167 [+ or -] 22.4% (P < 0.05), which was not modifed with PPAR[gamma] agonists. In conclusion, PPAR[gamma] /agonists exert antifibrotic effects in human PTC in high glucose by attenuating the increase in AP-1, TGF-[[beta].sub.1], and the downstream production of the extracellular matrix protein fibronectin. thiazolidinediones; diabetic nephropathy; proximal tubular cells

Peroxisome proliferator-activated receptor [alpha] (PPAR[alpha]), a key regulator of fatty acid oxidation, is essential for adaptation to fasting in rats and mice. However, physiological functions of PPAR[alpha] in other species, including humans, are controversial. A group of PPAR[alpha] ligands called peroxisome proliferators (PPs) causes peroxisome proliferation and hepatocarcinogenesis only in rats and mice. To elucidate the role of PPAR[alpha] in adaptation to fasting in nonproliferating species, we compared gene expressions in pig liver from lasted and clofibric acid (a PP)-fed groups against a control diet-ted group. As in rats and mice, fasting induced genes involved with mitochondrial fatty acid oxidation and ketogenesis in pigs. Those genes were also induced by clofibric acid feeding, indicating that PPAR[alpha] mediates the induction of these genes. In contrast to rats and mice, little or no induction of genes for peroxisomal or microsomal fatty acid oxidation was observed in clofibric acid-fed pigs. Histology showed no significant hyperplasia or hepatomegaly in the clofibric acid-led pigs, whereas it showed a reduction of glycogen by clofibric acid, an effect of PPs also observed in rats. Copy number of PPAR[alpha] mRNA was higher in pigs than in mice and rats, suggesting that peroxisomal proliferation and hyperresponse of several genes to PPs seen only in rats and mice are unrelated to the abundance of PPAR[alpha]. In conclusion, PPAR[alpha] is likely to play a central role in adaptation to fasting in pig liver as in rats and mice. clofibric acid; fatty acid oxidation; rats; mice; microarray

Pulmonary fibrosis is a progressive life-threatening disease for which no effective therapy exists. Myofibroblasts are one of the key effector cells in pulmonary fibrosis and are the primary source of extracellular matrix production. Drugs that inhibit the differentiation of fibroblasts to myofibroblasts have potential as antifibrotic therapies. Peroxisome proliferator-activated receptor (PPAR)-[gamma] is a transcription factor that upon ligation with PPAR[gamma] agonists activates target genes containing PPAR response elements. PPAR[gamma] agonists have anti-inflammatory activities and may have potential as antifibrotic agents. In this study, we examined the abilities of PPAR[gamma] agonists to block two of the most important profibrotic activities of TGF[beta] on pulmonary fibroblasts: myofibroblast differentiation and production of excess collagen. Both natural (15d-PG[J.sub.2]) and synthetic (ciglitazone and rosiglitazone) PPAR[gamma] agonists inhibited TGF-[beta]-driven myofibroblast differentiation, as determined by [alpha]-smooth muscle actin-specific immunocyto-chemistry and Western blot analysis. PPAR[gamma] agonists also potently attenuated TGF[beta]-driven type I collagen protein production. A dominant-negative PPAR[gamma] partially reversed the inhibition of myofibroblast differentiation by 15d-PG[J.sub.2] and rosiglitazone, but the irreversible PPAR[gamma] antagonist GW-9662 did not, suggesting that the antifibrotic effects of the PPAR[gamma] agonists are mediated through both PPAR[gamma]-dependent and independent mechanisms. Thus PPAR[gamma] agonists have novel and potent antifibrotic effects in human lung fibroblasts and may have potential for therapy of fibrotic diseases in the lung and other tissues. peroxisome proliferator-activated receptor-[gamma]; myofibroblasts; 15d-PG[J.sub.2]; ciglitazone; rosiglitazone

Peroxisome proliferator-activated receptor-[gamma], (PPAR[gamma],) is considered to be one of the master regulators of adipocyte differentiation. PPAR[gamma]2 is abundantly expressed in mature adipocytes and is elevated in the livers of animals that develop fatty livers. The aim of this study was to determine the ability of PPAR[gamma]2 to induce lipid accumulation in hepatocytes and to delineate molecular mechanisms driving this process. The hepatic cell line AML-12 was used to generate a cell line stably expressing PPAR[gamma]2. Oil Red O staining revealed that PPAR[gamma]2 induces lipid accumulation in hepatocytes. This phenotype is accompanied by a selective upregulation of several adipogenic and lipogenic genes including adipose differentiation-related protein (ADRP), adipocyte fatty acid-binding protein 4, sterol regulatory element-binding protein-1 (SREBP-1), fatty acid synthase (FAS), and acetyl-CoA carboxylase, genes whose expression levels are known to increase in steatotic livers of ob/ob mice. Furthermore, the PPAR[gamma]2-regulated induction of both SREBP-1 and FAS parallels an increase in de novo triacylglycerol synthesis in hepatocytes. Triacylglycerol synthesis and lipid accumulation are further enhanced by culturing hepatocytes with troglitazone in the absence of exogenous lipids. These results correspond with an increase in the lipid droplet protein, ADRP, and the data demonstrate that ADRP functions to coat lipid droplets in hepatocytes as observed by confocal microscopy. Taken together, these observations propose a role for PPAR[gamma]2 as an inducer of steatosis in hepatocytes and suggest that this phenomenon occurs through an induction of pathways regulating de novo lipid synthesis. adipose differentiation-related protein; lipid droplets; nonalcoholic fatty liver disease; steatosis; triacylglycerol; peroxisome proliferator-activated receptor-[gamma]2

Recently, we demonstrated that the peroxisome proliferator-activated receptor-[gamma] (PPAR-[gamma]) ligands, either 15-deoxy-[[DELTA].sup.12,14]- prostaglandin [J.sub.2] (15d-[PGJ.sub.2]) or ciglitazone, increased endothelial nitric oxide (xNO) release without altering endothelial nitric oxide synthase (eNOS) expression (4). However, the precise molecular mechanisms of PPAR-[gamma]-stimulated endothelial xNO release remain to be defined. Superoxide anion radical ([O.sup.-.sub.2]x) combines with -NO to decrease x NO bioavailability. NADPH oxidase, which produces [O.sup.-.sub.2], and Cu/Zn-superoxide dismutase (Cu/Zn-SOD), which degrades [O.sup.-.sub.2], thereby contribute to regulation of endothelial cellxNO metabolism. Therefore, we examined the ability of PPAR-[gamma] ligands to modulate endothelial [O.sup.-.sub.2] x metabolism through alterations in the expression and activity of NADPH oxidase or Cu/Zn-SOD. Treatment with 10 [micro]M 15d-[PGJ.sub.2] or ciglitazone for 24 h decreased human umbilical vein endothelial cell (HUVEC) membrane NADPH-dependent [O.sup.-.sub.2] x production detected with electron spin resonance spectroscopy. Treatment with 15d-[PGJ.sub.2] or ciglitazone also reduced relative mRNA levels of the NADPH oxidase subunits, nox-1, [gp91.sup.phox] (nox-2), and nox-4, as measured using real-time PCR analysis. Concordantly, Western blot analysis demonstrated that 15d-[PGJ.sub.2] or ciglitazone decreased nox-2 and nox-4 protein expression. PPAR-[gamma] ligands also stimulated both activity and expression of Cu/Zn-SOD in HUVEC. These data suggest that in addition to any direct effects on endothelial x NO production, PPAR-[gamma] ligands enhance endothelial x NO bioavailability, in part by altering endothelial [O.sup.-.sub.2] x metabolism through suppression of NADPH oxidase and induction of Cu/Zn-SOD. These findings further elucidate the molecular mechanisms by which PPAR-[gamma] ligands directly alter vascular endothelial function. reduced nicotinamide adenine dinucleotide phosphate oxidase; copper/zinc superoxide dismutase; nitric oxide; endothelial cells

The peroxisome proliferator-activated receptor [gamma] (PPAR-[gamma]) has been the focus of intense research during the past decade because ligands for this receptor have emerged as potent insulin sensitizers used in the treatment of type 2 diabetes. Recent advances include the discovery of novel genes that are regulated by PPAR-[gamma], which helps explain how activation of this adipocytepredominant transcription factor regulates glucose and lipid homeostasis. Increased levels of circulating free fatty acids and lipid accumulation in non-adipose tissue have been implicated in the development of insulin resistance. This situation is improved by PPAR-[gamma] ligands, which promote fatty acid storage in fat depots and regulate the expression of adipocyte-secreted hormones that impact on glucose homeostasis. The net result of the pleiotropic effects of PPAR-[gamma] ligands is improvement of insulin sensitivity, although undesired side-effects limit the utility of this therapy. It might be possible to dissociate the anti-diabetic and adverse effects through selective modulation of PPAR-[gamma] activity.

Skeletal muscle adapts to chronic physical activity by inducing mitochondrial biogenesis and switching proportions of muscle fibers from type II to type I. Several major factors involved in this process have been identified, such as the calcium/calmodulin-dependent protein kinase IV (CaMKIV), calcineurin A (CNA), and the transcriptional component peroxisome proliferator-activated receptor [gamma] coactivator 1[alpha] (PGC-1[alpha]). Transgenic expression of PGC-1[alpha] recently has been shown to dramatically increase the content of type I muscle fibers in skeletal muscle, but the relationship between PGC-1[alpha] expression and the key components in calcium signaling is not clear. In this report, we show that the PGC-1[alpha] promoter is regulated by both CaMKIV and CnA activity. CaMKIV activates PGC-1[alpha] largely through the binding of cAMP response element-binding protein to the PGC-1[alpha] promoter. Moreover, we show that a positive feedback loop exists between PGC-1[alpha] and members of the myocyte enhancer factor 2 (MEF2) family of transcription factors. MEF2s bind to the PGC-1[alpha] promoter and activate it, predominantly when coactivated by PGC-1[alpha]. MEF2 activity is stimulated further by CnA signaling. These findings imply a unified pathway, integrating key regulators of calcium signaling with the transcriptional switch PGC-1[alpha]. Furthermore, these data suggest an autofeedback loop whereby the calcium-signaling pathway may result in a stable induction of PGC-1[alpha], contributing to the relatively stable nature of muscle fiber-type determination.

Natural and synthetic agonists of the peroxisome proliferator-activated receptor [gamma] (PPAR[gamma]) regulate adipocyte differentiation, glucose homeostasis, and inflammatory responses. Although effects on adipogenesis and glucose metabolism are genetically linked to PPAR[gamma], the PPAR[gamma] dependence of antiinflammatory responses of these substances is less clear. Here, we have used a combination of mRNA expression profiling and conditional disruption of the PPAR[gamma] gene in mice to characterize programs of transcriptional activation and repression by PPAR[gamma] agonists in elicited peritoneal macrophages. Natural and synthetic PPAR[gamma] agonists, including the thiazolidinedione rosiglitazone (Ro), modestly induced the expression of a surprisingly small number of genes, several of which were also induced by a specific PPAR[delta] agonist. The majority of these genes encode proteins involved in lipid homeostasis. In contrast, Ro inhibited induction of broad subsets of lipopolysaccharide and IFN-[gamma] target genes in a gene-specific and PPAR[gamma]-dependent manner. At high concentrations, Ro inhibited induction of lipopolysaccharide target genes in PPAR[gamma]-deficient macrophages, at least in part by activating PPAR[delta] These studies establish overlapping transactivation and transrepression functions of PPAR[gamma] and PPAR[delta] in macrophages and suggest that a major transcriptional role of PPAR[gamma] is negative regulation of specific subsets of genes that are activated by T helper 1 cytokines and pathogenic molecules that signal through pattern recognition receptors. These findings support a physiological role of PPAR[gamma] in regulating both native and acquired immune responses.

Peroxisome proliferator-activated receptor (PPAR)[alpha], and PPAR[gamma] agonists lower lipid accumulation in muscle and liver by different mechanisms. We investigated whether benefits could be achieved on insulin sensitivity and lipid metabolism by the dual PPAR[alpha]/[gamma] agonist ragaglitazar in high fat-fed rats. Ragaglitazar completely eliminated high-fat feeding-induced liver triglyceride accumulation and visceral adiposity, like the PPAR[alpha] agonist Wy-14643 but without causing hepatomegaly. In contrast, the PPAR[gamma] agonist rosiglitazone only slightly lessened liver triglyceride without affecting visceral adiposity. Compared with rosiglitazone or Wy-14643, ragaglitazar showed a much greater effect (79%, P < 0.05) to enhance insulin's suppression of hepatic glucose output. Whereas all three PPAR agonists lowered plasma triglyceride levels and lessened muscle long-chain acyl-CoAs, ragaglitazar and rosiglitazone had greater insulin-sensitizing action in muscle than Wy-14643, associated with a threefold increase in plasma adiponectin levels. There was a significant correlation of lipid content and insulin action in liver and particularly muscle with adiponectin levels (P < 0.01). We conclude that the PPAR[alpha]/[gamma] agonist ragaglitazar has a therapeutic potential for insulin-resistant states as a PPAR[gamma] ligand, with possible involvement of adiponectin. Additionally, it can counteract fatty liver, hepatic insulin resistance, and visceral adiposity generally associated with PPAR, activation, but without hepatomegaly. peroxisome proliferator-activated receptor subtypes; adipokines; tissue lipids; insulin resistance

The myocyte enhancer factor-2 (MEF2) transcription factor regulates muscle development and calcium-dependent gene expression. MEF2 activity is repressed by class II histone deacetylases (HDACs), which dissociate from MEF2 when phosphorylated on two serine residues in response to calcium signaling. To explore the potential importance of MEF2/HDAC interactions in the heart, we generated transgenic mice expressing a signal-resistant form of HDAC5 under cardiac-specific and doxycycline-inducible regulation. Transgene expression resulted in sudden death in male mice accompanied by loss and morphologic changes of cardiac mitochondria and down-regulation of mitochondrial enzymes. The transcriptional coactivator PGC-1[alpha], a master regulator of mitochondrial biogenesis and fatty acid oxidation, was also down-regulated in response to HDAC5 expression. Examination of the PGC-1[alpha] promoter revealed two MEF2-binding sites that mediate transcriptional activation by MEF2 and repression by HDAC5. These findings identify PGC-1[alpha] as a key target of the MEF2/HDAC regulatory pathway and demonstrate this pathway's importance in maintenance of cardiac mitochondrial function.

To explore the role of peroxisome proliferator-activated receptor [alpha] (PPAR[alpha])-mediated derangements in myocardial metabolism in the pathogenesis of diabetic cardiomyopathy, insulinopenic mice with PPAR[alpha] deficiency (PPAR[[alpha].sup.-/-]) or cardiac-restricted overexpression [myosin heavy chain (MHC)-PPAR] were characterized. Whereas PPAR[[alpha].sup.-/-] mice were protected from the development of diabetes-induced cardiac hypertrophy, the combination of diabetes was exacerbated in MHC-PPAR mice fed a diet enriched in triglyceride containing long-chain fatty acid, an effect that was reversed by discontinuing the high-fat diet and absent in mice given a medium-chain triglyceride-enriched diet. Reactive oxygen intermediates were identified as candidate mediators of cardiomyopathic effects in MHC-PPAR mice. These results link dysregulation of the PPAR[alpha] gene regulatory pathway to cardiac dysfunction in the diabetic and provide a rationale for serum lipid-lowering strategies in the treatment of diabetic cardiomyopathy.

To test the hypothesis that endothelial dysfunction in hyperhomocysteinemia was due to increased levels of nitrotyrosine and matrix metalloproteinase (MMP) activity in response to antagonism of peroxisome proliferator-activated receptor-[alpha] (PPAR-[alpha]), cystathionine [beta]-synthase (CBS) -/+ mice were bred, tail tissue was analyzed for genotype by PCR, and tail vein blood was analyzed for homocysteine (Hcy) by spectrofluorometry. To induce PPAR-[alpha], mice were administered 8 [micro]g/ml of ciprofibrate (CF) and grouped: 1) wild type (WT), 2) WT + CF, 3) CBS, 4) CBS + CF (n = 6 in each group). In these four groups of mice, plasma Hcy was 3.0 [+ or -] 0.2, 2.5 [+ or -] 1.2, 15.2 [+ or -] 2.6 (P < 0.05 compared with WT), 11.0 [+ or -] 2.9 [micro]mol/l. Mouse urinary protein was 110 [+ or -] 11, 86 [+ or -] 6, 179 [+ or -] 13, 127 [+ or -] 9 [micro]g*[day.sup.-1]*[kg.sup.-1] by Bio-Rad dye binding assay. Aortic nitrotyrosine was 0.099 [+ or -] 0.012, 0.024 [+ or -] 0.004, 0.132 [+ or -] 0.024 (P < 0.01 compared with WT), 0.05 [+ or -] 0.01 (scan unit) by Western analysis. MMP-2 activity was 0.053 [+ or -] 0.010, 0.024 [+ or -] 0.002, 0.039 [+ or -] 0.009, 0.017 [+ or -] 0.006 (scan unit) by zymography. MMP-9 was specifically induced in CBS -/+ mice and inhibited by CF treatment. Systolic blood pressure (SPB) was 90 [+ or -] 2, 88 [+ or -] 16, 104 [+ or -] 8 (P < 0.05 compared with WT), 96 [+ or -] 3 mmHg. Aortic wall stress [(SPB*[radius.sup.2]/wall thickness)/2(radius + wall thickness)] was 10.2 [+ or -] 1.9, 9.7 [+ or -] 0.2, 16.6 [+ or -] 0.8 (P < 0.05 compared with WT), 13.1 [+ or -] 2.1 dyn/[cm.sup.2]. The results suggest that Hcy increased aortic wall stress by increasing nitrotyrosine and MMP-9 activity. extracellular matrix; matrix metalloproteinase; tissue inhibitor of metalloproteinase; collagen; elastin; cystathionine [beta]-synthase; nitric oxide; arteriosclerosis; fibrate

Liver regeneration after partial hepatectomy (PH) involves several signaling mechanisms including activation of the small GTPases Ras and RhoA in response to mitogens leading to DNA synthesis and cell proliferation. Peroxisome proliferator-activated receptor-[alpha] (PPAR[alpha]) regulates the expression of several key enzymes in isoprenoid synthesis, which are key events for membrane association of Ras and RhoA. Thus the role of PPAR[alpha] in cell proliferation after PH was tested. After PH, an increase in PPAR[alpha] DNA binding was observed in wild-type mice, correlating with an increase in the PPAR[alpha]-regulated enzyme acyl-CoA oxidase. In addition, the PPAR[alpha]-regulated genes farnesyl pyrophosphate synthase and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase were significantly increased in wild-type mice. However, these increases were not observed in PPAR[alpha] knockout (PPAR[alpha] -/-) mice. The peak in DNA synthesis observed 42 h after PH was reduced by ~60% in PPAR[alpha] -/- mice, despite increases in TNF-[alpha] and IL-1. Also, under these conditions, membrane association of Ras was high in wild-type mice after PH but was impaired in PPAR[alpha] -/- mice. Accordingly, Ras was significantly elevated in the cytosol in PPAR[alpha] -/- mice. This observation correlated with lower levels of active GTP-bound Ras after PH in PPAR[alpha] -/- mice compared with wild-type mice. Similar observations were made for RhoA. Moreover, deletion of PPAR[alpha] blunted the activation of cyclindependent kinase (cdk)2/cyclin E and cdk4/cyclin D complexes. Collectively, these results support the hypothesis that PPAR[alpha] is necessary for cell cycle progression in regenerating mouse liver via mechanisms involving prenylation of small GTPases Ras and RhoA. hepatocyte proliferation; cell cycle regulation; RhoA; peroxisome proliferation-activated receptor-[alpha]

Lysophosphatidic acid (LPA) is a pluripotent lipid mediator acting through plasma membrane-associated [LPA.sub.x] receptors that transduce many, but not all, of its effects. We identify peroxisome proliferator-activated receptor [gamma] (PPAR[gamma]) as an intracellular receptor for LPA. The transcription factor PPAR[gamma], is activated by several lipid ligands, but agonists derived from physiologic signaling pathways are unknown. We show that LPA, but not its precursor phosphatidic acid, displaces the drug rosiglitazone from the ligand-binding pocket of PPAR[gamma]. LPA and novel LPA analogs we made stimulated expression of a PPAR-responsive element reporter and the endogenous PPAR[gamma]-controlled gene CD36, and induced monocyte lipid accumulation from oxidized low-density lipoprotein via the CD36 scavenger receptor. The synthetic LPA analogs were effective PPAR[gamma], agonists, but were poor ones for [LPA.sub.1], [LPA.sub.2], or [LPA.sub.3] receptor transfected cells. Transfection studies in yeast, which lack nuclear hormone and [LPA.sub.x] receptors, show that LPA directly activates PPAR[gamma]. A major growth factor of serum is LPA generated by thrombin-activated platelets, and media from activated platelets stimulated PPAR[gamma] function in transfected RAW264.7 macrophages. This function was suppressed by ectopic LPA-acyltransferase expression. LPA is a physiologic PPAR[gamma] ligand, placing PPAR[gamma] in a signaling pathway, and PPAR[gamma] is the first intracellular receptor identified for LPA. Moreover, LPA produced by stimulated plasma platelets activates PPAR[gamma], in nucleated cells.