Induction of mycoplasmal pneumonia in experimentally infected pigs by means of different inoculation routes.

The purpose of this study was to assess the effect of three different inoculation routes into mycoplasmal pneumonia (MP) in pigs challenged with Mycoplasma hyopneumoniae (M. hyopneumoniae). Thirty six-week-old M. hyopneumoniae seronegative piglets were randomly assigned to four groups: three challenged groups with experimentally inoculated pigs by either the endotracheal (ET; n = 8), intranasal (IN; n = 8) or aerosol (AE; n = 8) routes and one uninfected group (Control; n = 6). Blood samples were collected 1 day before challenge and at necropsy, 28 days post-inoculation (dpi), to assess seroconversion. Laryngeal swabs were collected at -1, 7, 14, 21 and 28 dpi in order to evaluate colonization. At necropsy, lung lesions were scored and lung tissue was collected for histopathological studies and M. hyopneumoniae DNA detection. Broncho-alveolar lavage fluid (BALF) was also obtained to detect M. hyopneumoniae DNA, specific IgA antibodies and cytokines. MP was observed in all inoculated groups, but the ET group displayed a significantly higher number of animals affected by MP as well as a higher mean lung lesion score. These results were paralleled with an earlier seroconversion and upper respiratory tract colonization of M. hyopneumoniae. Additionally, in the ET group, higher levels of pro-inflammatory cytokines and specific IgA antibodies in BALF were found. Under the conditions of the present study, MP was reproduced by the three evaluated inoculation routes. Obtained results suggest that the ET route is the most effective in order to induce MP in pigs experimentally challenged with M. hyopneumoniae.

PPARß/δ activation blocks lipid-induced inflammatory pathways in mouse heart and human cardiac cells.

Owing to its high fat content, the classical Western diet has a range of adverse effects on the heart, including enhanced inflammation, hypertrophy, and contractile dysfunction. Proinflammatory factors secreted by cardiac cells, which are under the transcriptional control of nuclear factor-κB (NF-κB), may contribute to heart failure and dilated cardiomyopathy. The underlying mechanisms are complex, since they are linked to systemic metabolic abnormalities and changes in cardiomyocyte phenotype. Peroxisome proliferator-activated receptors (PPARs) are transcription factors that regulate metabolism and are capable of limiting myocardial inflammation and hypertrophy via inhibition of NF-κB. Since PPARß/δ is the most prevalent PPAR isoform in the heart, we analyzed the effects of the PPARß/δ agonist GW501516 on inflammatory parameters. A high-fat diet induced the expression of tumor necrosis factor-α, monocyte chemoattractant protein-1, and interleukin-6, and enhanced the activity of NF-κB in the heart of mice. GW501516 abrogated this enhanced proinflammatory profile. Similar results were obtained when human cardiac AC16 cells exposed to palmitate were coincubated with GW501516. PPARß/δ activation by GW501516 enhanced the physical interaction between PPARß/δ and p65, which suggests that this mechanism may also interfere NF-κB transactivation capacity in the heart. GW501516-induced PPARß/δ activation can attenuate the inflammatory response induced in human cardiac AC16 cells exposed to the saturated fatty acid palmitate and in mice fed a high-fat diet. This is relevant, especially taking into account that PPARß/δ has been postulated as a potential target in the treatment of obesity and the insulin resistance state.

Atorvastatin prevents carbohydrate response element binding protein activation in the fructose-fed rat by activating protein kinase A.

UNLABELLED: High fructose intake contributes to the overall epidemic of obesity and metabolic disease. Here we examined whether atorvastatin treatment blocks the activation of the carbohydrate response element binding protein (ChREBP) in the fructose-fed rat. Fructose feeding increased blood pressure (21%, P < 0.05), plasma free fatty acids (59%, P < 0.01), and plasma triglyceride levels (129%, P < 0.001) compared with control rats fed standard chow. These increases were prevented by atorvastatin. Rats fed the fructose-rich diet showed enhanced hepatic messenger RNA (mRNA) levels of glycerol-3-phosphate acyltransferase (Gpat1) (1.45-fold induction, P < 0.05), which is the rate-limiting enzyme for the synthesis of triglycerides, and liver triglyceride content (2.35-fold induction, P < 0.001). Drug treatment inhibited the induction of Gpat1 and increased the expression of liver-type carnitine palmitoyltransferase 1 (L-Cpt-1) (128%, P < 0.01). These observations indicate that atorvastatin diverts fatty acids from triglyceride synthesis to fatty acid oxidation, which is consistent with the reduction in liver triglyceride levels (28%, P < 0.01) observed after atorvastatin treatment. The expression of Gpat1 is regulated by ChREBP and sterol regulatory element binding protein-1c (SREBP-1c). Atorvastatin treatment prevented fructose-induced ChREBP translocation and the increase in ChREBP DNA-binding activity while reducing SREBP-1c DNA-binding activity. Statin treatment increased phospho-protein kinase A (PKA), which promotes nuclear exclusion of ChREBP and reduces its DNA-binding activity. Human HepG2 cells exposed to fructose showed enhanced ChREBP DNA-binding activity, which was not observed in the presence of atorvastatin. Furthermore, atorvastatin treatment increased the CPT-I mRNA levels in these cells. Interestingly, both effects of this drug were abolished in the presence of the PKA inhibitor H89. CONCLUSION: These findings indicate that atorvastatin inhibits fructose-induced ChREBP activity and increases CPT-I expression by activating PKA.

Atorvastatin inhibits GSK-3beta phosphorylation by cardiac hypertrophic stimuli.

In this study we examined the effect of the statin atorvastatin on the Akt/GSK-3beta pathway. Our findings indicate that atorvastatin treatment for 15 days inhibited pressure overload-induced cardiac hypertrophy and prevented nuclear translocation of GATA4 and c-Jun and AP-1 DNA-binding activity. In addition, atorvastatin treatment prevented the increase in the phosphorylation of Akt and GSK-3beta caused by cardiac hypertrophy, and this effect correlated with an increase in protein levels of phosphatase and tensin homolog on chromosome 10 (PTEN), which negatively regulates the phosphoinositide-3 kinase/Akt pathway. To test whether the inhibitory effect of atorvastatin on Akt and GSK-3beta phosphorylation was direct we performed in vitro studies using embryonic rat heart-derived H9c2 cells, human AC16 cardiomyoblasts and neonatal rat cardiomyocytes. Preincubation of cells with atorvastatin prevented Akt/GSK-3beta phosphorylation by different hypertrophic stimuli without affecting PTEN protein levels. However, atorvastatin prevented endogenous reactive oxygen species (ROS) generation and PTEN oxidation, a process that correlates with its inactivation, suggesting that atorvastatin prevents ROS-induced PTEN inactivation in acute treatments. These findings point to a new potential anti-hypertrophic effect of statins, which can prevent activation of the Akt/GSK-3beta hypertrophic pathway by modulating PTEN activation by different mechanisms in chronic and acute treatments.

Initial vaccination and revaccination with Type I PRRS 94881 MLV reduces viral load and infection with porcine reproductive and respiratory syndrome virus.

BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) causes respiratory distress in pigs, reproductive failure in breeding-age gilts and sows, and can have devastating economic consequences in domestic herds. Several PRRS vaccines are available commercially. This study compared the effectiveness of single-vaccination and revaccination schedules using the PRRS 94881 Type I modified live virus (MLV) vaccine ReproCyc® PRRS EU with no vaccination (challenge control) in protecting against a PRRS virus (PRRSV) challenge in non-pregnant gilts. RESULTS: Data were available from 48 gilts across three groups: a challenge control group (n = 16), which received no vaccination; a revaccination group (n = 16), which received ReproCyc® PRRS EU on Days 0 and 56; and a single vaccination group (n = 16), which received ReproCyc® PRRS EU on Day 56. All gilts were PRRSV RNA-negative (based on reverse transcription and quantitative polymerase chain reaction [RT-qPCR]) and PRRSV seronegative (based on enzyme-linked immunosorbent assay [ELISA]) at Day 0. All gilts were challenged with PRRSV strain 190136 on Day 91.Viral RNA loads in both vaccination groups were significantly reduced compared with the challenge control group on Days 98 (P < 0.0001) and 101 (P < 0.0001), indicating that vaccinated gilts were better able to respond to challenge than unvaccinated gilts. At all timepoints following challenge, mean viral RNA load and the percentage of PRRSV RNA-positive gilts were numerically higher in the single-vaccination group than in the revaccination group; these differences were statistically significant on Day 101 (P = 0.0434). Furthermore, viremia levels after challenge were significantly lower in the revaccination group than in the single-vaccination group based on median area under the curve (AUC) values for viral RNA load from Day 91 to Day 112, suggesting that revaccinated gilts had better protection from viral infection than gilts who received a single vaccination. Protection from viremia did not correlate with the proportion of seropositive gilts on Day 91. In the single-vaccination group, 94% of pigs were seropositive on Day 91 compared with 56% in the revaccination group. Vaccination was well tolerated and no safety concerns were identified. CONCLUSIONS: Both single-vaccination and revaccination with ReproCyc® PRRS EU were effective in reducing PRRSV viremia post-challenge. These findings have important implications for herd management as both the single-vaccination and revaccination schedules protect against PRRSV challenge, with revaccination appearing to provide better protection from viremia than single vaccination.

Palmitate-mediated downregulation of peroxisome proliferator-activated receptor-gamma coactivator 1alpha in skeletal muscle cells involves MEK1/2 and nuclear factor-kappaB activation.

The mechanisms by which elevated levels of free fatty acids cause insulin resistance are not well understood. Previous studies have reported that insulin-resistant states are characterized by a reduction in the expression of peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1, a transcriptional activator that promotes oxidative capacity in skeletal muscle cells. However, little is known about the factors responsible for reduced PGC-1 expression. The expression of PGC-1 mRNA levels was assessed in C2C12 skeletal muscle cells exposed to palmitate either in the presence or in the absence of several inhibitors to study the biochemical pathways involved. We report that exposure of C2C12 skeletal muscle cells to 0.75 mmol/l palmitate, but not oleate, reduced PGC-1alpha mRNA levels (66%; P < 0.001), whereas PGC-1beta expression was not affected. Palmitate led to mitogen-activated protein kinase (MAPK)-extracellular signal-related kinase (ERK) 1/2 (MEK1/2) activation. In addition, pharmacological inhibition of this pathway by coincubation of the palmitate-exposed cells with the MEK1/2 inhibitors PD98059 and U0126 prevented the downregulation of PGC-1alpha. Furthermore, nuclear factor-kappaB (NF-kappaB) activation was also involved in palmitate-mediated PGC-1alpha downregulation, since the NF-kappaB inhibitor parthenolide prevented a decrease in PGC-1alpha expression. These findings indicate that palmitate reduces PGC-1alpha expression in skeletal muscle cells through a mechanism involving MAPK-ERK and NF-kappaB activation.

Peroxisome proliferator-activated receptor alpha down-regulation is associated with enhanced ceramide levels in age-associated cardiac hypertrophy.

We used an experimental murine model of accelerated aging, the senescence-accelerated mouse (SAM), to examine the effect of age-associated cardiac hypertrophy on peroxisome proliferator-activated receptor alpha (PPARalpha) expression and activity in the heart. Senescence-accelerated prone mice (SAM-P8) showed cardiac hypertrophy compared with senescence-accelerated resistant mice (SAM-R1). Furthermore, a decrease in PPARalpha messenger RNA (mRNA; 28% reduction, p<.001) and protein (47%, p<.05) levels and in PPAR DNA-binding activity was observed in SAM-P8 hearts. Increased protein-protein interaction between PPARalpha and the p65 subunit of nuclear factor-kappaB (NF-kappaB) was found, suggesting that this mechanism may prevent PPARalpha from binding to its response elements. The mRNA levels of PPARalpha target genes involved in fatty acid use were strongly suppressed in SAM-P8, which was consistent with the accumulation of ceramide in SAM-P8 hearts (2.5-fold induction, p<.05). These findings suggest that NF-kappaB activation in SAM-P8 heart prevents PPARalpha from binding to its response elements leading to changes in gene expression that may lead to ceramide accumulation in the aged heart.

Potential use of local and systemic humoral immune response parameters to forecast Mycoplasma hyopneumoniae associated lung lesions.

Immunopathological events are key for the development of enzootic pneumonia (EP), which is macroscopically observed as cranioventral pulmonary consolidation (CVPC). This study aimed to investigate the putative association between the humoral immune response against Mycoplasma hyopneumoniae (M. hyopneumoniae) and prevalence and extension of CVPC in 1) experimentally infected pigs, 2) slaughtered pigs and 3) sequentially necropsied pigs in a longitudinal study. CVPC was scored by means of the European Pharmacopoeia recommended methodology. Specific IgG, IgG1 and IgG2 antibodies were assessed in serum. In addition, mucosal IgG and IgA antibodies were analyzed in broncho-alveolar lavage fluid (BALF) from experimentally challenged pigs. The systemic humoral immune response in experimentally infected pigs was delayed in onset whereas humoral respiratory mucosal immune response appeared more rapidly but declined earlier. Although low, BALF IgG antibodies showed the highest correlation with CVPC scores (r = 0.49, p<0.05). In slaughter-aged pigs, both percentage of lungs with CVPC and mean lung lesion score were significantly higher in M. hyopneumoniae seropositive farms compared to the seronegative ones (p<0.001). Similarly, seropositive sequentially necropsied pigs showed more severe CVPC than seronegative ones. Overall, mean serological values might help to forecast prevalence and severity of EP-like lung lesions using a population based approach. Remarkably, the specific systemic humoral immune response was found to be predominated by the IgG2 subclass, suggesting a dominant Th1-mediated immune response to M. hyopneumoniae.

PGC-1beta down-regulation is associated with reduced ERRalpha activity and MCAD expression in skeletal muscle of senescence-accelerated mice.

Mitochondrial dysfunction is involved in the development of aging. Here, we examined the effect of aging on the skeletal muscle expression of two isoforms of the transcriptional peroxisome proliferator-activated receptor gamma (PPARgamma) coactivator-1 (PGC-1) in an experimental murine model of accelerated aging, the senescence-accelerated mouse (SAM). The senescence-accelerated prone mice (SAM-P8) showed no changes in PGC-1alpha, but a decrease in PGC-1beta expression (52% reduction, p <.001) was observed compared to the senescence-accelerated resistant mice (SAM-R1). In agreement with the proposed role of PGC-1beta as an estrogen-related receptor (ERR) protein ligand, the expression of the ERRalpha target gene medium-chain acyl-coenzyme A dehydrogenase was strongly suppressed (85%, p <.001) in SAM-P8. The decrease in the expression of medium-chain acyl-coenzyme A dehydrogenase was consistent with the reduction in ERRalpha DNA-binding activity of SAM-P8. These findings indicate that the age-mediated decrease in PGC-1beta expression in SAM-P8 skeletal muscle affects the expression of genes involved in mitochondrial fatty acid oxidation.

Safety and early onset of immunity with a novel European porcine reproductive and respiratory syndrome virus vaccine in young piglets.

Porcine reproductive and respiratory syndrome virus (PRRSV) can be difficult to manage in commercial settings. A novel type I PRRSV vaccinal strain (94881) was evaluated for safety and efficacy/onset of immunity (OOI) in piglets. In 2 experiments, groups of piglets were vaccinated intramuscularly (IM) at approximately 14 d of age with a maximum-range commercial dose, an overdose, or a placebo in experiment 1 and either a minimum-range commercial dose or a placebo in experiment 2. The piglets in experiment 1 were evaluated for local and systemic reactions from days -2 through 14 after vaccination. The piglets in experiment 2 were challenged with a virulent heterologous type I PRRSV isolate 14 d after vaccination and observed once daily for general health from days -1 through 12 after vaccination and once daily for clinical signs associated with challenge from days 13 through 24 after vaccination. The average daily weight gain (ADWG) and the results of serologic and viremia testing were evaluated in experiments 1 and 2. Lung lesion scores and results of testing for PRRSV in lung tissue were evaluated in experiment 2. In experiment 1 the vaccine was shown to be safe, as there were no relevant differences between the vaccinated piglets and the piglets given a placebo. In experiment 2 the vaccine's efficacy, with an OOI of 14 d after vaccination, was established, as the vaccinated and challenged piglets exhibited significantly lower lung lesion scores, viremia, viral load in lung tissue, and total clinical sign scores, along with a significantly greater ADWG, compared with the placebo-vaccinated and challenged piglets.

La gestion du virus du syndrome reproducteur et respiratoire porcin (VSRRP) peut être difficile dans un environnement de production commerciale. Une nouvelle souche vaccinale du VSRRP de type 1 (94881) a été évaluée d'un point de vue sécurité et efficacité/début de l'immunité (DDI) chez des porcelets. Dans deux expériences, des groupes de porcelets ont été vaccinés par voie intramusculaire (IM) à l'âge d'environ 14 j avec une dose commerciale maximale, une surdose, ou un placebo dans l'expérience 1 et une dose commerciale minimale ou un placebo dans l'expérience 2. Les porcelets dans l'expérience 1 furent évalués pour des réactions locale et systémique à compter du jour −2 jusqu'au jour 14 post-vaccination. Les porcelets dans l'expérience 2 furent soumis à une infection défi avec un isolat virulent hétérologue de VSRRP de type 1 14 j après la vaccination et observés une fois par jour pour leur état de santé général du jour −1 jusqu'au jour 12 après la vaccination et une fois par jour pour des signes cliniques associés avec l'infection du jour 13 au jour 24 après l'infection. Le gain moyen quotidien (GMQ) et les résultats des analyses sérologiques et de virémie ont été évalués dans les expériences 1 et 2. Les pointages de lésions pulmonaires et les résultats de détection du VSRRP dans le tissu pulmonaire ont été évalués dans l'expérience 2. Dans l'expérience 1, le vaccin s'est montré sécuritaire étant donné qu'il n'y avait aucune différence significative entre les porcelets vaccinés et les porcelets ayant reçu un placebo. Dans l'expérience 2, l'efficacité du vaccin, avec un DDI de 14 j après la vaccination, a été établie, étant donné que les porcelets vaccinés, et soumis à une infection défi avaient des valeurs significativement moins élevées de pointage de lésions pulmonaires, de virémie, de charge virale dans le tissu pulmonaire, et des pointages de signes cliniques totaux, avec un GMQ significativement plus élevé, comparativement au porcs vaccinés avec un placebo et soumis à une infection défi.(Traduit par Docteur Serge Messier).

Safety and efficacy of a novel European vaccine for porcine reproductive and respiratory virus in bred gilts.

Porcine reproductive and respiratory syndrome virus (PRRSV) can be devastating to commercial breeding operations. The objective of this study was to evaluate a novel European PRRSV vaccinal strain for safety and efficacy in bred gilts. In 2 experiments, 110 gilts were vaccinated intramuscularly and the vaccine was evaluated for safety and efficacy. Gilts in Experiment 1 were evaluated for local and systemic reactions and gilts in both experiments were observed for clinical signs of disease through farrow. In both experiments, piglet clinical observations, piglet average daily weight gain (ADWG), gilt serology [determined by enzyme-linked immunosorbent assay (ELISA)], gilt and piglet viremia [determined by quantitative real-time polymerase chain reaction (qPCR)], as well as piglet lung lesion scores and PRRS virus in lung tissue (qPCR) were determined. The vaccine was shown to be safe as there were no significant differences among groups in either experiment. Efficacy was established in Experiment 2 as both vaccinated groups were associated with desirable significant differences in percentage of gilts with abnormal clinical findings; gilt viral load post-challenge [day 125, day of farrowing (DOF), and DOF + 13]; percentages of alive, healthy live, weak live, and mummified piglets per litter at farrowing and weaning; percentage of piglets per gilt that were positive for viremia; percentage of piglets per gilt with clinical disease; and piglet viral load on DOF. It was concluded that a vaccine formulated from the PRRSV modified live virus (MLV) strain 94881 is a safe and effective method of protection against the detrimental effects of virulent PRRSV infection in breeding female pigs.

Le virus du syndrome reproducteur et respiratoire porcin (VSRRP) peut être dévastateur pour les opérations de reproduction commerciales. L'objectif de la présente étude était d'évaluer l'innocuité et l'efficacité d'une nouvelle souche vaccinale européenne du VSRRP chez des cochettes saillies. Lors de deux expériences, 110 cochettes ont été vaccinées par voie intramusculaire afin d'évaluer l'innocuité et l'efficacité du vaccin. Les cochettes de l'Expérience 1 ont été évaluées pour la présence de réactions locales et systémiques et les cochettes dans les deux expériences ont été observées pour vérifier la présence de signes cliniques de maladie jusqu'au moment de la mise-bas. Lors des deux expériences on nota les observations cliniques des porcelets, le gain de poids quotidien moyen des porcelets (GMQ), les titres sérologiques des truies [déterminés par épreuve immuno-enzymatique (ELISA)], la virémie chez les cochettes et les porcelets [déterminées par réaction d'amplification en chaine par la polymérase quantitative (qACP)], ainsi que par les pointages de lésions pulmonaires des porcelets et la quantité de virus SRRP dans le tissu pulmonaire (qACP). Le vaccin s'est avéré sécuritaire et il n'y avait pas de différence significative entre les groupes dans les deux expériences. L'efficacité a été établie lors de l'Expérience 2 alors que les deux groupes vaccinés ont été associés à des différences significatives souhaitées dans le pourcentage de cochettes avec des trouvailles cliniques anormales; dans la charge virale post-challenge par cochette [jour 125, jour de la mise-bas (JMB), et JMB + 13]; pourcentages de porcelets vivants, vivants en santé, vivants faibles, et momifiés par portée au moment de la mise-bas et au sevrage; pourcentages de porcelets par truie qui étaient positifs pour une virémie; pourcentage de porcelets par cochette avec une maladie clinique; et charge virale des porcelets au JMB. Il a été conclu qu'un vaccin formulé à partir de la souche 94881 du VSRRP vivant modifié est une méthode sécuritaire et efficace de protection contre les effets néfastes d'une infection par le VSRRP chez des porcs femelles de reproduction.(Traduit par Docteur Serge Messier).

Cardiovascular disease predicts diabetic peripheral polyneuropathy in subjects with type 2 diabetes: A 10-year prospective study.

BACKGROUND: The relationship between cardiovascular disease and diabetic peripheral neuropathy is mainly sustained by data retrieved from cross-sectional studies focused on cardiovascular risk factors. We aimed to assess the presence of cardiovascular disease as a risk factor for developing diabetic peripheral neuropathy in a type 2 diabetes mellitus population. METHOD: A 10-year prospective, primary care, multicentre study in a randomly selected cohort. Cardiovascular disease presence included stroke, coronary artery disease and/or peripheral ischaemia. Diabetic peripheral neuropathy diagnosis was based on clinical neurological examination as well as the neuropathy symptoms score and nerve conduction studies. RESULTS: Three hundred and ten (N=310) patients were initially recruited. Two-hundred and sixty seven (N=267) patients were included in the study. Diabetic peripheral neuropathy cumulative incidence was 18.3% (95% confidence intervals 14.1-23.4; N=49). Diabetic peripheral neuropathy development was significantly more frequent in participants presenting with cardiovascular disease at baseline (P=0.01). In the final logistic regression analysis, the presence of cardiovascular disease remained associated with an increased risk for diabetic peripheral neuropathy (odds ratio 2.32, 95% confidence intervals 1.03-5.22) in addition to diabetes duration and low density lipoprotein-cholesterol levels. CONCLUSIONS: In our series, type 2 diabetes mellitus patients with cardiovascular disease at baseline present with an increased risk of developing diabetic peripheral neuropathy at 10 years of follow-up. Our results suggest that measures aimed at the prevention, control and treatment of cardiovascular disease can also help prevent diabetic peripheral neuropathy development.

Cyclooxygenase 2 inhibition exacerbates palmitate-induced inflammation and insulin resistance in skeletal muscle cells.

Palmitate-induced inflammation is involved in the development of insulin resistance in skeletal muscle cells. Here we evaluated the effect of the saturated fatty acid palmitate and the monounsaturated fatty acid oleate on Toll-like receptors (TLR)-2 and -4 and cyclooxygenase 2 (COX-2) expression and examined whether the inhibition of this enzyme modulates fatty acid-induced inflammation. Skeletal muscle cells exposed to palmitate showed enhanced TLR-2 and COX-2 mRNA levels, whereas oleate did not modify their expression. Palmitate-induced expression of these genes was dependent on nuclear factor (NF)-kappaB activation, because expression was reduced in the presence of the NF-kappaB inhibitor parthenolide. Coincubation of palmitate-exposed cells with oleate also prevented the increase in the expression of TLR-2 and COX-2, through a mechanism that may involve activation of peroxisome proliferator-activated receptor-alpha (PPAR alpha) by this monounsaturated fatty acid. COX-2 inhibition by NS-398 enhanced IL-6 and TNF-alpha expression and IL-6 protein secretion induced by palmitate. NF-kappaB binding activity and TNF-alpha mRNA levels were enhanced in palmitate-exposed cells in the absence or in the presence of NS-398, whereas coincubation of palmitate-exposed cells with NS-398 and prostaglandin E(2) (PGE(2)) prevented these changes. In contrast, 12-lypoxygenase and cytochrome P450 hydroxylase pathways were not involved in these changes. Similarly, COX-2 inhibition impaired insulin-stimulated Akt phosphorylation and 2-deoxy-D-[(14)C]glucose uptake in palmitate-exposed skeletal muscle cells, and this effect was abolished in the presence of PGE(2). These findings indicate that COX-2 activity, through the production of PGE(2), attenuates the fatty acid-induced inflammatory process and insulin resistance.

Activation of peroxisome proliferator-activated receptor-{delta} by GW501516 prevents fatty acid-induced nuclear factor-{kappa}B activation and insulin resistance in skeletal muscle cells.

Elevated plasma free fatty acids cause insulin resistance in skeletal muscle through the activation of a chronic inflammatory process. This process involves nuclear factor (NF)-kappaB activation as a result of diacylglycerol (DAG) accumulation and subsequent protein kinase Ctheta (PKCtheta) phosphorylation. At present, it is unknown whether peroxisome proliferator-activated receptor-delta (PPARdelta) activation prevents fatty acid-induced inflammation and insulin resistance in skeletal muscle cells. In C2C12 skeletal muscle cells, the PPARdelta agonist GW501516 prevented phosphorylation of insulin receptor substrate-1 at Ser(307) and the inhibition of insulin-stimulated Akt phosphorylation caused by exposure to the saturated fatty acid palmitate. This latter effect was reversed by the PPARdelta antagonist GSK0660. Treatment with the PPARdelta agonist enhanced the expression of two well known PPARdelta target genes involved in fatty acid oxidation, carnitine palmitoyltransferase-1 and pyruvate dehydrogenase kinase 4 and increased the phosphorylation of AMP-activated protein kinase, preventing the reduction in fatty acid oxidation caused by palmitate exposure. In agreement with these changes, GW501516 treatment reversed the increase in DAG and PKCtheta activation caused by palmitate. These effects were abolished in the presence of the carnitine palmitoyltransferase-1 inhibitor etomoxir, thereby indicating that increased fatty acid oxidation was involved in the changes observed. Consistent with these findings, PPARdelta activation by GW501516 blocked palmitate-induced NF-kappaB DNA-binding activity. Likewise, drug treatment inhibited the increase in IL-6 expression caused by palmitate in C2C12 and human skeletal muscle cells as well as the protein secretion of this cytokine. These findings indicate that PPARdelta attenuates fatty acid-induced NF-kappaB activation and the subsequent development of insulin resistance in skeletal muscle cells by reducing DAG accumulation. Our results point to PPARdelta activation as a pharmacological target to prevent insulin resistance.

The Role of Peroxisome Proliferator-Activated Receptor beta/delta on the Inflammatory Basis of Metabolic Disease.

The pathophysiology underlying several metabolic diseases, such as obesity, type 2 diabetes mellitus, and atherosclerosis, involves a state of chronic low-level inflammation. Evidence is now emerging that the nuclear receptor Peroxisome Proliferator-Activated Receptor (PPAR)beta/delta ameliorates these pathologies partly through its anti-inflammatory effects. PPARbeta/delta activation prevents the production of inflammatory cytokines by adipocytes, and it is involved in the acquisition of the anti-inflammatory phenotype of macrophages infiltrated in adipose tissue. Furthermore, PPARbeta/delta ligands prevent fatty acid-induced inflammation in skeletal muscle cells, avoid the development of cardiac hypertrophy, and suppress macrophage-derived inflammation in atherosclerosis. These data are promising and suggest that PPARbeta/delta ligands may become a therapeutic option for preventing the inflammatory basis of metabolic diseases.

The p65 subunit of NF-kappaB binds to PGC-1alpha, linking inflammation and metabolic disturbances in cardiac cells.

AIMS: Nuclear factor-kappaB (NF-kappaB) is a transcription factor induced by a wide range of stimuli, including hyperglycaemia and pro-inflammatory cytokines. It is associated with cardiac hypertrophy and heart failure. It was previously reported that the NF-kappaB-mediated inhibition of proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) might explain the shift in glucose metabolism during cardiac pathological processes induced by pro-inflammatory stimuli, although the specific mechanisms remain to be elucidated. We addressed the specific mechanisms by which exposure to tumour necrosis factor-alpha (TNF-alpha) results in PGC-1alpha down-regulation in cardiac cells and, as a consequence, in the metabolic dysregulation that underlies heart dysfunction and failure. METHODS AND RESULTS: By using coimmunoprecipitation studies, we report for the first time that the p65 subunit of NF-kappaB is constitutively bound to PGC-1alpha in human cardiac cells and also in mouse heart, and that NF-kappaB activation by TNF-alpha exposure increases this binding. Overexpression and gene silencing analyses demonstrated that the main factor limiting the degree of this association is p65, because only the modulation of this protein modified the physical interaction. Our data show that the increased physical interaction between p65 and PGC-1alpha after NF-kappaB activation is responsible for the reduction in PGC-1alpha expression and subsequent dysregulation of glucose oxidation. CONCLUSION: On the basis of these data, we propose that p65 directly represses PGC-1alpha activity in cardiac cells, thereby leading to a reduction in pyruvate dehydrogenase kinase 4 (PDK4) expression and the subsequent increase in glucose oxidation observed during the proinflammatory state.

Peroxisome proliferator-activated receptor (PPAR) beta/delta: a new potential therapeutic target for the treatment of metabolic syndrome.

Metabolic syndrome is defined as the clustering of multiple metabolic abnormalities, including abdominal obesity, dyslipidemia (high serum triglycerides and low serum HDL-cholesterol levels), glucose intolerance and hypertension. The pathophysiology underlying metabolic syndrome involves a complex interaction of crucial factors, but two of these, insulin resistance and obesity (especially visceral obesity), play a major role. The nuclear receptors Peroxisome Proliferator-Activated Receptors (PPAR)alpha and PPARgamma are therapeutic targets for hypertriglyceridemia and insulin resistance, respectively. Evidence is now emerging that the PPARbeta/delta; isotype is a potential pharmacological target for the treatment of disorders associated with metabolic syndrome. PPARbeta/delta; activation increases lipid catabolism in skeletal muscle, heart and adipose tissue and improves the serum lipid profile and insulin sensitivity in several animal models. In addition, PPARbeta/delta; ligands prevent weight gain and suppress macrophage-derived inflammation. These data are promising and indicate that PPARbeta/delta; ligands may become a therapeutic option for the treatment of metabolic syndrome. However, clinical trials in humans assessing the efficacy and safety of these drugs should confirm these promising perspectives in the treatment of the metabolic syndrome.

TNF-alpha reduces PGC-1alpha expression through NF-kappaB and p38 MAPK leading to increased glucose oxidation in a human cardiac cell model.

AIMS: Inflammatory responses in the heart that are driven by sustained increases in cytokines have been associated with several pathological processes, including cardiac hypertrophy and heart failure. Emerging data suggest a link between cardiomyopathy and myocardial metabolism dysregulation. To further elucidate the relationship between a pro-inflammatory profile and cardiac metabolism dysregulation, a human cell line of cardiac origin, AC16, was treated with tumour necrosis factor-alpha (TNF-alpha). METHODS AND RESULTS: Exposure of AC16 cells to TNF-alpha inhibited the expression of peroxisome proliferator-activated receptor coactivator 1alpha (PGC-1alpha), an upstream regulator of lipid and glucose oxidative metabolism. Studies performed with cardiac-specific transgenic mice (Mus musculus) overexpressing TNF-alpha, which have been well characterized as a model of cytokine-induced cardiomyopathy, also displayed reduced PGC-1alpha expression in the heart compared with that of control mice. The mechanism by which TNF-alpha reduced PGC-1alpha expression in vitro appeared to be largely mediated via both p38 mitogen-activated protein kinase and nuclear factor-kappaB pathways. PGC-1alpha downregulation resulted in an increase in glucose oxidation rate, which involved a reduction in pyruvate dehydrogenase kinase 4 expression and depended on the DNA-binding activity of both peroxisome proliferator-activated receptor beta/delta and estrogen-related receptor alpha transcription factors. CONCLUSION: These results point to PGC-1alpha downregulation as a potential contributor to cardiac dysfunction and heart failure in metabolic disorders with an inflammatory background.

Oleate reverses palmitate-induced insulin resistance and inflammation in skeletal muscle cells.

Here we report that in skeletal muscle cells the contribution to insulin resistance and inflammation of two common dietary long-chain fatty acids depends on the channeling of these lipids to distinct cellular metabolic fates. Exposure of cells to the saturated fatty acid palmitate led to enhanced diacylglycerol levels and the consequent activation of the protein kinase C/nuclear factor kappaB pathway, finally resulting in enhanced interleukin 6 secretion and down-regulation of the expression of genes involved in the control of the oxidative capacity of skeletal muscle (peroxisome proliferator-activated receptor (PPAR)gamma-coactivator 1alpha) and triglyceride synthesis (acyl-coenzyme A: diacylglycerol acyltransferase 2). In contrast, exposure to the monounsaturated fatty acid oleate did not lead to these changes. Interestingly, co-incubation of cells with palmitate and oleate reversed both inflammation and impairment of insulin signaling by channeling palmitate into triglycerides and by up-regulating the expression of genes involved in mitochondrial beta-oxidation, thus reducing its incorporation into diacylglycerol. Our findings support a model of cellular lipid metabolism in which oleate protects against palmitate-induced inflammation and insulin resistance in skeletal muscle cells by promoting triglyceride accumulation and mitochondrial beta-oxidation through PPARalpha- and protein kinase A-dependent mechanisms.

Activation of peroxisome proliferator-activated receptor beta/delta inhibits lipopolysaccharide-induced cytokine production in adipocytes by lowering nuclear factor-kappaB activity via extracellular signal-related kinase 1/2.

OBJECTIVE: Chronic activation of the nuclear factor-kappaB (NF-kappaB) in white adipose tissue leads to increased production of pro-inflammatory cytokines, which are involved in the development of insulin resistance. It is presently unknown whether peroxisome proliferator-activated receptor (PPAR) beta/delta activation prevents inflammation in adipocytes. RESEARCH DESIGN AND METHODS AND RESULTS: First, we examined whether the PPARbeta/delta agonist GW501516 prevents lipopolysaccharide (LPS)-induced cytokine production in differentiated 3T3-L1 adipocytes. Treatment with GW501516 blocked LPS-induced IL-6 expression and secretion by adipocytes and the subsequent activation of the signal transducer and activator of transcription 3 (STAT3)-Suppressor of cytokine signaling 3 (SOCS3) pathway. This effect was associated with the capacity of GW501516 to impede LPS-induced NF-kappaB activation. Second, in in vivo studies, white adipose tissue from Zucker diabetic fatty (ZDF) rats, compared with that of lean rats, showed reduced PPARbeta/delta expression and PPAR DNA-binding activity, which was accompanied by enhanced IL-6 expression and NF-kappaB DNA-binding activity. Furthermore, IL-6 expression and NF-kappaB DNA-binding activity was higher in white adipose tissue from PPARbeta/delta-null mice than in wild-type mice. Because mitogen-activated protein kinase-extracellular signal-related kinase (ERK)1/2 (MEK1/2) is involved in LPS-induced NF-kappaB activation in adipocytes, we explored whether PPARbeta/delta prevented NF-kappaB activation by inhibiting this pathway. Interestingly, GW501516 prevented ERK1/2 phosphorylation by LPS. Furthermore, white adipose tissue from animal showing constitutively increased NF-kappaB activity, such as ZDF rats and PPARbeta/delta-null mice, also showed enhanced phospho-ERK1/2 levels. CONCLUSIONS: These findings indicate that activation of PPARbeta/delta inhibits enhanced cytokine production in adipocytes by preventing NF-kappaB activation via ERK1/2, an effect that may help prevent insulin resistance.