PPARδ Expression Research Articles

Increase of Peroxisome Proliferator-activated Receptor δ (PPARδ) by Digoxin to Improve Lipid Metabolism in the Heart of Diabetic Rats

Increase of peroxisome proliferator-activated receptor δ (PPARδ) expression by digoxin in the heart of diabetic rats has been documented. The present study investigated the mediation of PPARδ in lipid metabolism improved by digoxin in the heart of diabetic rats and in the hyperglycemia-treated cardiomyocytes using the primary cultured cardiomyocytes from neonatal rat. The lipid deposition within the heart section was assessed in diabetic rats by oil red O staining. The fatty acid oxidation genes in cardiomyocytes were also examined. Inhibitor of calcium ions and siRNA-PPARδ were employed to investigate the potential mechanisms. After a 20-day digoxin treatment, the PPARδ expression was elevated in hearts of diabetic rats while the cardiac lipid deposition was reduced. In neonatal cardiomyocytes, digoxin also caused an increase in expressions of PPARδ and fatty acid oxidation genes. But both actions of digoxin were blocked by BAPTA-AM to chelate calcium ions and by siRNA-PPARδ in cardiomyocytes. The obtained results show that increase of PPARδ by digoxin is related to regulation of fatty acid oxidation genes in cardiac cells mediated by calcium-triggered signals.

IL-15 Overexpression Promotes Endurance, Oxidative Energy Metabolism, and Muscle PPARδ, SIRT1, PGC-1α, and PGC-1β Expression in Male Mice

Endurance exercise initiates a pattern of gene expression that promotes fat oxidation, which in turn improves endurance, body composition, and insulin sensitivity. The signals from exercise that initiate these pathways have not been completely characterized. IL-15 is a cytokine that is up-regulated in skeletal muscle after exercise and correlates with leanness and insulin sensitivity. To determine whether IL-15 can induce any of the metabolic adaptations associated with exercise, substrate metabolism, endurance, and molecular expression patterns were examined in male transgenic mice with constitutively elevated muscle and circulating IL-15 levels. IL-15 transgenic mice ran twice as long as littermate control mice in a run-to-exhaustion trial and preferentially used fat for energy metabolism. Fast muscles in IL-15 transgenic mice exhibited high expression of intracellular mediators of oxidative metabolism that are induced by exercise, including sirtuin 1, peroxisome proliferator-activated receptor (PPAR)-δ, PPAR-γ coactivator-1α, and PPAR-γ coactivator-1β. Muscle tissue in IL-15 transgenic mice exhibited myosin heavy chain and troponin I mRNA isoform expression patterns indicative of a more oxidative phenotype than controls. These findings support a role for IL-15 in induction of exercise endurance, oxidative metabolism, and skeletal muscle molecular adaptations induced by physical training.

Lipoic acid prevents liver metabolic changes induced by administration of a fructose-rich diet

BackgroundTo evaluate whether co-administration of R/S-α-lipoic acid can prevent the development of oxidative stress and metabolic changes induced by a fructose-rich diet (F). MethodsWe assessed glycemia in the fasting state and during an oral glucose tolerance test, triglyceridemia and insulinemia in rats fed with standard diet (control) and fructose without or with R/S-α-lipoic acid. Insulin resistance and hepatic insulin sensitivity were also calculated. In liver, we measured reduced glutathione, protein carbonyl groups, antioxidant capacity by ABTS assay, antioxidant enzymes (catalase and superoxide dismutase 1 and 2), uncoupling protein 2, PPARδ and PPARγ protein expressions, SREBP-1c, fatty acid synthase and glycerol-3-phosphate acyltransferase-1 gene expression, and glucokinase activity. ResultsR/S-α-lipoic acid co-administration to F-fed rats a) prevented hyperinsulinemia, hypertriglyceridemia and insulin resistance, b) improved hepatic insulin sensitivity and glucose tolerance, c) decreased liver oxidative stress and increased antioxidant capacity and antioxidant enzymes expression, d) decreased uncoupling protein 2 and PPARδ protein expression and increased PPARγ levels, e) restored the basal gene expression of PPARδ, SREBP-1c and the lipogenic genes fatty acid synthase and glycerol-3-phosphate acyltransferase, and f) decreased the fructose-mediated enhancement of glucokinase activity. ConclusionsOur results suggest that fructose-induced oxidative stress is an early phenomenon associated with compensatory hepatic metabolic mechanisms, and that treatment with an antioxidant prevented the development of such changes. General significanceThis knowledge would help to better understand the mechanisms involved in liver adaptation to fructose-induced oxidative stress and to develop effective strategies to prevent and treat, at early stages, obesity and type 2 diabetes mellitus.

Inhibition of Doxorubicin-Induced Senescence by PPARδ Activation Agonists in Cardiac Muscle Cells: Cooperation between PPARδ and Bcl6

Senescence and apoptosis are two distinct cellular programs that are activated in response to a variety of stresses. Low or high doses of the same stressor, i.e., the anticancer drug doxorubicin, may either induce apoptosis or senescence, respectively, in cardiac muscle cells. We have demonstrated that PPARδ, a ligand-activated transcriptional factor that controls lipid metabolism, insulin sensitivity and inflammation, is also involved in the doxorubicin-induced senescence program. This occurs through its interference with the transcriptional repressor protein B cell lymphoma-6 (Bcl6). Low doses of doxorubicin increase the expression of PPARδ that sequesters Bcl6, thus preventing it from exerting its anti-senescent effects. We also found that L-165041, a specific PPARδ activator, is highly effective in protecting cardiomyocytes from doxorubicin-induced senescence through a Bcl6 related mechanism. In fact, L-165041 increases Bcl6 expression via p38, JNK and Akt activation, and at the same time it induces the release of Bcl6 from PPARδ, thereby enabling Bcl6 to bind to its target genes. L-165041 also prevented apoptosis induced by higher doses of doxorubicin. However, while experiments performed with siRNA analysis techniques very clearly showed the weight of Bcl6 in the cellular senescence program, no role was found for Bcl6 in the anti-apoptotic effects of L-165041, thus confirming that senescence and apoptosis are two very distinct stress response cellular programs. This study increases our understanding of the molecular mechanism of anthracycline cardiotoxicity and suggests a potential role for PPARδ agonists as cardioprotective agents.

Role of TNF-α in Renal Damage in Mice Showing Hepatic Steatosis Induced by High Fat Diet

The present study was designed to investigate the role of TNF-α in renal damage observed in mice with hepatic steatosis. We induced hepatic steatosis in mice using high fat diet and treated mice with ectanercept at the dose sufficient to block TNF-α receptors or vehicle for 1 month. Plasma TNF-α, total cholesterol (TC), triglyceride (TG), LDL-cholesterol (LDL-C), and HDL-cholesterol (HDL-C) were determined at the end of this treatment. Renal damage was identified by histologic observation and the higher of serum blood urea nitrogen (BUN) and creatinine. Also, changes of PPAR-δ in kidney and renal mesangial cell (RMC) were analyzed using Western blot. Plasma TNF-α was markedly raised in mice showing hepatic steatosis. However, the levels of blood lipids (TC, TG, HDL-C, and LDL-C) and TNF-α were not modified by the treatment of etanercept although the hepatic steatosis has been improved. Etanercept shows renal protection from histological identification and recovery of serum BUN and creatinine levels. Moreover, restoration of PPAR-δ expression by etanercept was observed in mice kidney. Direct effect of TNF-α on PPAR-δ expression was also characterized in RMC cell. We suggest that renal damage in mice with hepatic steatosis is mainly induced by increase of TNF-α through the decrease of renal PPAR-δ. Etanercept could block TNF-α receptors to restore PPAR-δ and improve renal function in mice with hepatic steatosis.

Abstract 73: Selective Inhibition of COX-1 & 2 Results in Reprogramming of Adipocyte Gene Expression and Adipogenesis in Mesenchymal Stem Cells: Role of 20-HETE-derived 20-OH-PGE2

Eicosanoids are increasingly recognized to play a central role in modulating the process of adipogenesis in human bone marrow derived mesenchymal stem cells (MSCs) where prostaglandin E2 (PGE2) has been shown to negatively regulate adipogenesis. 20-Hydroxyeicosatetraenoic acid (20-HETE), a product of the cytochrome P450 (CYP)-catalyzed ω-hydroxylation of arachidonic acid, induces oxidative stress, and in clinical studies, is associated with increased BMI and metabolic syndrome. In this study we sought to characterize the effects of exogenous 20-HETE on MSC-derived adipogenesis. 20-HETE dose-dependently (0.01-1 μM) increased (p<0.05) adipogenesis (0.11±0.001, 0.12±0.007, 0.14±0.015 and 0.16±0.013) in MSCs, an effect not attenuated by concurrent administration of a putative 20-HETE antagonist (DKB-H1-40-32). However, a selective cyclooxygenase-2 (COX-2) inhibitor prevented (0.071±0.004 vs 0.16±0.013) this pro-adipogenic effect of 20-HETE thus raising the possibility that a metabolic product of 20-HETE is mediating its effects on MSCs. We next exposed these MSCs to exogenous 20-OH-PGE2, an endogenous COX-2 derived product of 20-HETE. Treatment with 20-OH-PGE2 also dose-dependently enhanced (0.08±0.02, 0.14±0.01, 0.21±0.01 and 0.4±0.02) adipogenesis and lipid accumulation in MSC-derived adipocytes. In addition, in cells undergoing COX-1 &2 blockade, treatment of MSCs with PGE2 shifted the dose response curve of 20-OH-PGE2 to the right, indicative of a competitive antagonistic interactions of COX-1 derived PGE2 and COX-2-derived 20-OH-PGE2 on the process of adipogenesis. Both 20-HETE and 20-OH-PGE2-induced adipocyte hypertrophy was accompanied with increased expression of adipogenic regulators, PPARδ, C/EBPα and MEST1. Taken together we show for the first time that 20-HETE-derived COX-2-dependent 20-OH-PGE2 could enhance lipid accumulation and induces adipocyte hypertrophy in MSC undergoing adipogenic differentiation. We also propose that effects of COX-1 derived PGE2, a known inhibitor of adipocyte differentiation, are opposed by endogenous 20-OH-PGE2 production which appears to predominantly COX-2 dependent.

T.P.37 Changes in muscle expression of PGC-1α and related signalling molecules in the mdx mouse: Possible impact on adaptive fiber phenotype transition and implication for drug studies

<h2>Abstract</h2> In Duchenne muscular dystrophy (DMD), the absence of dystrophin causes a complex pathogenetic cascade also involving sarcolemmal fragility and metabolic distress. Increasing evidences in mdx mouse model outline the potential interest of therapeutic strategies to reinforce pathways leading to activation of peroxisome proliferator-activated receptor (PPAR) γ coactivator 1α (PGC-1α), a key modulator of oxidative metabolism and mitochondrial biogenesis. Accordingly, we recently found beneficial effects of resveratrol, a PGC-1α activator via sirtuin 1 (Sirt1), in exercised mdx mice. To better understand the basal regulation of this mechano-sensitive metabolic pathway in dystrophic skeletal muscle we performed real-time PCR experiments in gastrocnemius (GC) muscles of mdx mice at different ages. We found slightly higher levels of PGC-1α mRNA in mdx mice of 8weeks of age vs. age-matched C57BL/10 (wt) mice. The increase in PGC-1α became highly significant at 16weeks of age and was paralleled by a 40% rise in Sirt1 mRNA. Focusing on 16weeks of age, we then assessed the gene expression of PPARs since PGC-1α cooperates with members of this receptor family to regulate expression of mitochondrial and β-oxidation enzymes. Interestingly, the expression of PPAR-δ was markedly higher in mdx GC muscle vs. wt one, while PPAR-γ had a comparable expression in the two genotypes. Afterwards the impact of the above changes on phenotype and glycolytic/oxidative metabolism of dystrophic muscle has been evaluated on genes expression of myosin heavy chain (MHC) isoforms. A significant increase of slow-oxidative type I MHC and a decrease of fast-glycolytic IIb MHC were found in mdx GC muscle vs. wt ones. Then a modification of PGC-1α pathway, which likely triggers a protective slow-oxidative myofiber program, naturally occurs in the mdx mouse muscle and may modulate the outcome of physiological stimuli and of pharmacological strategies (Supported by Duchenne Parent Project NL).

Increase of myocardial performance by Rhodiola–ethanol extract in diabetic rats

Ethno pharmacological relevanceRhodiola rosea (also known as golden root or roseroot) is a perennial plant of the Crassulaceae family that grows in the Arctic and in the mountainous regions of Europe, Asia, and North America. The rhizome and roots of this plant have been long used as traditional medicine in Eastern Europe and Asia for enhancing physical and mental performance. Aim of the studyThe present study is designed to investigate the cardiac action of Rhodiola–ethanol extract in streptozotocin-induced diabetic rats (STZ-diabetic rats) with heart failure. Materials and methodsDiabetes was induced in Wistar rats by injection of streptozotocin. We measured the changes of body weight, water intake, and food intake in three groups of age-matched rats; the normal control received vehicle, STZ-diabetic rat received Rhodiola–ethanol extract or vehicle. Cardiac output, heart rate, blood pressure, and hemodynamic dP/dt in addition to plasma insulin and glucose level were also determined. The mRNA and protein levels of PPARδ were measured using real-time PCR and Western blotting, respectively. ResultsFood intake, water intake and blood glucose were raised in STZ-diabetic rats showing lower body weight and plasma insulin, as compared with the control. Also, cardiac output, heart rate, blood pressure and hemodynamic dP/dt were markedly reduced in STZ-diabetic rats indicating the heart failure physiologically. After a 21-day treatment with Rhodiola–ethanol extract, cardiac output was raised in STZ-rats while the diabetic parameters were not modified. The PPARδ expression of both mRNA and protein was markedly elevated in the heart of STZ-rats receiving treatment with Rhodiola–ethanol extract. Also, the increased phosphorylation level of cardiac troponin-I was restored by this treatment with Rhodiola–ethanol extract. Otherwise, increase of cardiac output by Rhodiola–ethanol extract was blocked by antagonist of PPARδ in STZ-diabetic rats. ConclusionsOur results suggest that ethanol extract of Rhodiola has an ability to increase the cardiac output in STZ-diabetic rats showing heart failure. Also, increase of PPAR-δ is responsible for this action of Rodiola–ethanol extract.

Neuron-Specific Deletion of Peroxisome Proliferator-Activated Receptor Delta (PPARδ) in Mice Leads to Increased Susceptibility to Diet-Induced Obesity

Central nervous system (CNS) lipid accumulation, inflammation and resistance to adipo-regulatory hormones, such as insulin and leptin, are implicated in the pathogenesis of diet-induced obesity (DIO). Peroxisome proliferator-activated receptors (PPAR α, δ, γ) are nuclear transcription factors that act as environmental fatty acid sensors and regulate genes involved in lipid metabolism and inflammation in response to dietary and endogenous fatty acid ligands. All three PPAR isoforms are expressed in the CNS at different levels. Recent evidence suggests that activation of CNS PPARα and/or PPARγ may contribute to weight gain and obesity. PPARδ is the most abundant isoform in the CNS and is enriched in the hypothalamus, a region of the brain involved in energy homeostasis regulation. Because in peripheral tissues, expression of PPARδ increases lipid oxidative genes and opposes inflammation, we hypothesized that CNS PPARδ protects against the development of DIO. Indeed, genetic neuronal deletion using Nes-Cre loxP technology led to elevated fat mass and decreased lean mass on low-fat diet (LFD), accompanied by leptin resistance and hypothalamic inflammation. Impaired regulation of neuropeptide expression, as well as uncoupling protein 2, and abnormal responses to a metabolic challenge, such as fasting, also occur in the absence of neuronal PPARδ. Consistent with our hypothesis, KO mice gain significantly more fat mass on a high-fat diet (HFD), yet are surprisingly resistant to diet-induced elevations in CNS inflammation and lipid accumulation. We detected evidence of upregulation of PPARγ and target genes of both PPARα and PPARγ, as well as genes of fatty acid oxidation. Thus, our data reveal a previously underappreciated role for neuronal PPARδ in the regulation of body composition, feeding responses, and in the regulation of hypothalamic gene expression.

Peroxisome proliferator-activated receptor- activation ameliorates albuminuria by preventing nephrin loss and restoring podocyte integrity in Type 2 diabetes

Peroxisome proliferator-activated receptor (PPAR)-δ is a ligand-activated transcription factor in regulating gene expression and is believed to play an important role in various kidney diseases including diabetic nephropathy. This study investigated the efficacy of GW610742, a highly specific agonist for PPAR-δ, for the treatment of diabetic nephropathy. Type 2 diabetic Otsuka Long-Evans Tokushima Fatty rats were randomized into an untreated diabetic group (n = 9) and a GW610742-treated diabetic group (n= 9). The GW610742 was administered (10 mg/kg/day) orally for 11 weeks. Long-Evans Tokushima Otsuka rats (n = 9) were used as a non-diabetic control. Albuminuria was markedly increased and renal PPAR-δ expression was decreased in diabetes. Diabetic albuminuria and renal injury markers, such as glomerular basement membrane thickening, decreased number of slit pores between podocyte foot processes, decreased nephrin expression, increased desmin expression and increased CCL2 expression, were significantly reversed through the treatment with GW610742. PPAR-δ agonist GW610742 markedly increased nephrin expression in cultured podocytes. Nephrin mRNA expression was markedly decreased in response to high glucose in cultured podocytes and effectively prevented by GW610742. PPAR-δ activation by GW610742 ameliorates albuminuria by preventing diabetes-induced nephrin loss and restoring podocyte integrity, implying that GW610742 may be a potential therapeutic agent for diabetic nephropathy.

Abstract 3028: Peroxisome proliferator-activated receptor ≤ (PPARδ) reveals a critical role in promoting the TGFβ-induced EMT and metastasis of non-small cell lung cancer cell (H460)

Abstract Although transforming growth factor ≤ (TGFβ) is anti-cancer identified, it was also known to promote tumor malignancy by enhancing the growth, epithelial to mesenchymal transition (EMT), tissue invasion and metastasis in a variety of cancer cells, including non-small cell lung cancers (NSCLCs). The aim of this study was to know the contribution of peroxisome proliferator-activated receptor ≤ (PPARδ) to TGFβ-promoted EMT and metastasis in NSCLCs. The degree of EMT, cell scattering, migration and invasion, and protein expression of PPARδ were first analyzed and compared between H460 and CH27 cells with or without TGFβ treatment (both are NSCLC cell lines). Chemical or shRNA blocking assay was also utilized to further confirm the importance of PPARδ in promoting TGFβ-induced tumor metastasis. Finally, signaling pathway(s) responsible for TGFβ/PPARδ-mediated metastasis was evaluated. The results demonstrated that TGFβ can effectively triggered the EMT, cell scattering, migration and invasion in H460 cell, but only a slight EMT was seen in TGFβ-treated CH27 cell. Meanwhile, PPARδ expression was induced by TGFβ in H460 cells but remained unchanged in CH27 cell. This indicated that PPARδ may involve in TGFβ-mediated metastasis. Co-treatment of TGFβ with GW9662 (a specific PPARδ inhibitor) significantly blocked TGFβ-promoted metastasis of H460 cell indicating the presence of PPARδ was indeed critical for TGFβ-attributed tumor metastasis. TGFβ appeared to act through P38 signaling pathway to trigger the expression of PPARδ and the subsequent activation of epidermal growth factor receptor (EGFR)/c-mesenchymal-epithelial transition factor (c-MET) signaling pathway and the reduction of E-cadherin that eventually led to the EMT and metastasis of H460 cell. Additionally, TGFβ also act through P38 to trigger the expression of β-catenin which could bind to and up-regulate the expression of PPARδ. Blocking of β-catenin activity, however, did not interfere with the metastasis indicating β-catenin may not play a direct role in promoting the metastasis of H460 cell. Nevertheless, we can not preclude the possibility that β-catenin may still act through PPARδ to regulate the metastasis of H460 cell, indirectly. In summary, TGFβ-induced EMT and metastasis in H460 cell was PPARδ-dependent and GW9662 revealed a therapeutical value in the control of NSCLCs (H460). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3028. doi:1538-7445.AM2012-3028

Abstract 4537: Tumoral expression on peroxisome proliferator-activated receptor ≤ is an independent prognostic marker in intestinal type of gastric cancer

Abstract Introduction: Peroxisome proliferator-activated receptor ≤ (PPARδ) is a member of the nuclear hormone receptor family that regulates the expression of mediators of the inflammatory response. Metaplastic change secondary to chronic inflammation of gastric mucosa is regarded as a premalignant condition of intestinal-type gastric cancer (IGC). We aimed to evaluate the prognostic significance of PPARδ in IGC patients. Methods: The clinical significance of PPARδ was explored using tissue microarray methods and immunohistochemical staining of specimens from 306 IGC patients. The Kaplan-Meier method was used to calculate survival curves, and differences between the survival curves were assessed using the log-rank test. Cox proportional hazards regression was used to assess the effect of PPARδ expression on survival while controlling for other confounding covariates. Results: Among 306 patients with IGC (stage I-IV), PPARδ expression was detected by immunohistochemistry in the tumors of 209 (68.3%). The patients with PPARδ-positive tumors had significantly lower overall and gastric cancer-specific mortalities (P = 0.0010 and 0.0016, respectively, by the log-rank test) than did those with PPARδ-negative tumors. A positive PPARδ expression was a significant independent prognostic factor for gastric cancer-specific mortality by the Cox regression analysis in patients with IGC (adjusted hazard ratio, 0.42; 95% confidence interval, 0.22-0.81). The relationship between PPARδ and lower mortality was not modified by other clinical variables including tumor stage (all Pinteraction &amp;gt; 0.05). Conclusions: tumor expression of PPARδ was independently associated with lower mortality of patients with IGC. Keywords: Gastric cancer, Intestinal-type gastric cancer, Peroxisome proliferator-activated receptor ≤ (PPARδ), Prognostic marker, Survival Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4537. doi:1538-7445.AM2012-4537

Abstract 2580: Induction of estrogen receptor alpha by GW9662 sensitizes mammary glands to tamoxifen treatment in organ culture

Abstract Peroxisome proliferator-activated receptor gamma (PPARδ) are ligand activated nuclear receptor and binds to natural and synthetic ligands. Earlier we had reported that PPARδ agonist troglitazone inhibited the development of precancerous lesions induced by DMBA in mouse mammary gland organ culture (MMOC). In the present study we evaluated the effects of PPARδ agonist GW7845 and antagonist GW9662 on the development of 7,12 dimethylbenz(a)anthracene (DMBA)-induced mammary alveolar lesions (MAL) in ER negative mammary glands in MMOC. The results showed that both GW7845 and GW9662 inhibited development of ER- MAL in MMOC. Since PPARδ and ER cross talk has been reported in the literature, we determined the effects of GW9662 on the induction of ER and PR in this model. Real-time PCR studies and immunohistochemical analyses showed that GW9662 induced ERα in the absence of estradiol, whereas GW7845 did not induce ERα under these conditions. Both GW7845 and GW9662 failed to suppress mRNA expression of PPARδ in the mammary glands that are otherwise ER negative. However GW9662 suppressed the expression of PPARδ response genes adipsin and aP2. Conversely, GW7845 an agonist of PPARδ enhanced the expression of aP2 and adipsin. These results suggested that the effects of these agents are mediated by PPARδ in MMOC. Finally, Tamoxifen does not inhibit MAL in MMOC, however sequential treatments with GW9662 followed by Tamoxifen suppressed development of DMBA-induced MAL in these glands. Since the ER negative breast cancer patients are generally not treated with SERM, inducing ERα in the mammary cells by GW9662 possibly could make them responsive to Tamoxifen or other SERM. [This work was supported by N01 CN433303] Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2580. doi:1538-7445.AM2012-2580

Acute Exercise Remodels Promoter Methylation in Human Skeletal Muscle

DNA methylation is a covalent biochemical modification controlling chromatin structure and gene expression. Exercise elicits gene expression changes that trigger structural and metabolic adaptations in skeletal muscle. We determined whether DNA methylation plays a role in exercise-induced gene expression. Whole genome methylation was decreased inskeletal muscle biopsies obtained from healthy sedentary men and women after acute exercise. Exercise induced a dose-dependent expression of PGC-1α, PDK4, and PPAR-δ, together with a marked hypomethylation on each respective promoter. Similarly, promoter methylation of PGC-1α, PDK4, and PPAR-δ was markedly decreased in mouse soleus muscles 45min after exvivo contraction. In L6 myotubes, caffeine exposure induced gene hypomethylation in parallel with an increase in the respective mRNA content. Collectively, our results provide evidence that acute gene activation is associated with a dynamic change in DNA methylation in skeletal muscle and suggest that DNA hypomethylation is an early event in contraction-induced gene activation.

PARVALBUMIN AFFECTS FISH ATHLETICISM

How quickly you escape a predator or whether you catch the next meal is one of the key factors that decides whether you contribute to the greatest experiment on earth: evolution. Athletic performance varies enormously from individual to individual, and muscle performance is probably the main determinant of whether you’re a Usain Boult or one of the stragglers. Frank Seebacher and Isabel Walter from the University of Sydney, Australia, reasoned that the biochemical machinery that regulates how hard and fast muscle contracts determines whether zebrafish are elite athletes or laggards. The duo decided to investigate the muscle biochemistry of zebrafish to identify key components that determine a fish’s athleticism (p. 663).Setting the fish a swimming challenge, the duo compared the animals’ endurance and sprint performance, expecting to find that the best sprinters had the poorest endurance. However, they found no correlation, so the fish had not traded off one performance against another.Next, they looked to see if increasing the fish’s levels of a calcium handling protein – parvalbumin, which is involved in the regulation of muscular contraction – affected the fish’s endurance and sprint performance; and it did, improving their performance measurably. ‘This is the first demonstration that parvalbumin plays a role in determining whole-animal performance’, say Seebacher and Walter.Finally, the duo investigated the fish’s metabolic machinery and found that the high performance fish – the fast sprinters and the fish with the greatest endurance – had higher expression of the muscle biochemistry regulators PPARδ and PGC-1α. They also found that the fastest sprinters were able to release energy faster because they had higher levels of the ATP-release enzyme creatine kinase, as well as having higher expression levels of dihydropyridine receptor and sarcoplasmic reticulum calcium ATPase (SERCA) 2, both of which regulate the release of calcium. And when they looked at the muscles of the high endurance swimmers, they found that those fish had higher levels of ryanodine receptor and SERCA 1, which are also involved in calcium regulation in the muscle.Seebacher and Walter say, ‘We have identified a number of molecular traits that can explain differences in sprint and sustained locomotor performance in individuals’. They add, ‘Understanding how these traits function and what regulates their expression will provide new insights into locomotion, its evolution and into associated diseases’.

Oleic acid activates peroxisome proliferator-activated receptor δ to compensate insulin resistance in steatotic cells

Nonalcoholic fatty liver disease is frequently associated with type 2 diabetes; however, this idea is challenged by recent studies because hepatic steatosis is not always associated with insulin resistance (IR). Oleic acid (OA) is known to induce hepatic steatosis with normal insulin sensitivity; however, the mechanism is still unknown. Previous studies depict that activation of peroxisome proliferator-activated receptor δ (PPARδ) improves hepatic steatosis and IR, whereas the role of PPARδ in the improvement of insulin sensitivity by OA is unknown. Here we induced steatosis in HepG2 cells by incubation with OA and OA significantly increased the expression of PPARδ through a calcium-dependent pathway. OA also induced the expression of G protein-coupled receptor 40 (GPR40), and deletion of GPR40 by small interfering ribonucleic acid transfection partially reversed the effect of OA on PPARδ. Inhibition of phospholipase C (PLC) by U73122 also reversed OA-induced PPARδ expression. Otherwise, deletion of PPARδ augmented the OA-induced steatosis in HepG2 cells. Furthermore, IR was developed in OA-treated HepG2 cells with PPARδ deletion, while insulin-related signals and insulin-stimulated glycogen synthesis were reduced through increase of phosphatase and tensin homolog (PTEN) expression. In conclusion, OA activates GPR40-PLC-calcium pathway to increase the expression of PPARδ and PPARδ further decreased the expression of PTEN to regulate insulin sensitivity in hepatic steatosis.

Functional Role of PPARδ in Corneal Epithelial Wound Healing

The peroxisome proliferator-activated receptor (PPAR) δ is involved in tissue repair. In this study, we investigated the functional role of PPARδ in corneal epithelial wound healing. In an in vivo corneal wound-healing model, the changes of PPARδ expression in corneal epithelia were examined by immunofluorescence microscopy, and the effect of topical administrations of a PPARδ agonist on corneal wound healing was also evaluated. The inhibitory effect of a PPARδ agonist on the cytokine-induced death of human corneal epithelial cells was evaluated using a DNA fragmentation assay kit. The changes of PPARδ expression and epithelial cell death were also investigated using human corneoscleral tissues ex vivo. Our findings showed that PPARδ expression was temporally up-regulated in corneal epithelial cells during experimental wound healing and that topical administration of a PPARδ agonist significantly promoted the healing of experimental corneal epithelial wounds. In human corneal epithelial cells, up-regulation of PPARδ and DNA fragmentation was demonstrated by stimulation with cytokines, and the DNA fragmentation was significantly inhibited by pretreatment with a PPARδ agonist. By using human corneoscleral tissues ex vivo, PPARδ was up-regulated in both healthy corneal epithelia (during re-epithelialization) and diseased corneal epithelia. Inflammatory stimulation-induced corneal epithelial cell death was inhibited by pretreatment with a PPARδ agonist. These results strongly suggest that PPARδ is involved in the corneal epithelial wound healing.

Role of Lipid Peroxidation and PPAR-δ in Amplifying Glucose-Stimulated Insulin Secretion

OBJECTIVEPrevious studies show that polyunsaturated fatty acids (PUFAs) increase the insulin secretory capacity of pancreatic β-cells. We aimed at identifying PUFA-derived mediators and their cellular targets that are involved in the amplification of insulin release from β-cells preexposed to high glucose levels.RESEARCH DESIGN AND METHODSThe content of fatty acids in phospholipids of INS-1E β-cells was determined by lipidomics analysis. High-performance liquid chromatography was used to identify peroxidation products in β-cell cultures. Static and dynamic glucose-stimulated insulin secretion (GSIS) assays were performed on isolated rat islets and/or INS-1E cells. The function of peroxisome proliferator–activated receptor-δ (PPAR-δ) in regulating insulin secretion was investigated using pharmacological agents and gene expression manipulations.RESULTSHigh glucose activated cPLA2 and, subsequently, the hydrolysis of arachidonic and linoleic acid (AA and LA, respectively) from phospholipids in INS-1E cells. Glucose also increased the level of reactive oxygen species, which promoted the peroxidation of these PUFAs to generate 4-hydroxy-2E-nonenal (4-HNE). The latter mimicked the GSIS-amplifying effect of high glucose preexposure and of the PPAR-δ agonist GW501516 in INS-1E cells and isolated rat islets. These effects were blocked with GSK0660, a selective PPAR-δ antagonist, and the antioxidant N-acetylcysteine or by silencing PPAR-δ expression. High glucose, 4-HNE, and GW501516 also induced luciferase expression in a PPAR-δ–mediated transactivation assay. Cytotoxic effects of 4-HNE were observed only above the physiologically effective concentration range.CONCLUSIONSElevated glucose levels augment the release of AA and LA from phospholipids and their peroxidation to 4-HNE in β-cells. This molecule is an endogenous ligand for PPAR-δ, which amplifies insulin secretion in β-cells.

Inhibition of adipogenic differentiation by myostatin is alleviated by arginine supplementation in porcine-muscle-derived mesenchymal stem cells

Porcine mesenchymal stem cells in postnatal muscle have been demonstrated to differentiate into adipocytes. This increases adipocyte number and lipid accumulation, and is thought to be the origin of intramuscular fat. In this study, the effects of myostatin and arginine on adipogenic differentiation in mesenchymal stem cells derived from porcine muscle (pMDSCs) were investigated in vitro. Intracellular triglyceride levels were reduced by exogenous myostatin and increased by arginine supplementation or myostatin antibody (P<0.01). The inhibition of lipid accumulation by myostatin in pMDSCs was alleviated by arginine supplementation (P<0.01). Expression patterns of adipogenic transcription factors showed that exogenous myostatin suppressed PPARγ2 and aP2 expression (P<0.01), while supplemental arginine or myostatin antibody promoted ADD1 expression (P<0.01). Furthermore, compared with the addition of either myostatin protein or antibody alone, ADD1 and PPARδ expression were promoted by the combination of arginine and myostatin (P<0.01), and arginine combined with myostatin antibody promoted the expression of ADD1, PPARδ, C/EBPα, PPARγ2 and LPL in pMDSCs (P<0.05). These results suggest that myostatin inhibits adipogenesis in pMDSCs, and that this can be alleviated by arginine supplementation, at least in part, through promoting ADD1 and PPARδ expression.

Immunomodulatory action of dietary fish oil and targeted deletion of intestinal epithelial cell PPARδ in inflammation-induced colon carcinogenesis

The ligand-activated transcription factor peroxisome proliferator-activated receptor (PPAR)-δ is highly expressed in colonic epithelial cells; however, the role of PPARδ ligands, such as fatty acids, in mucosal inflammation and malignant transformation has not been clarified. Recent evidence suggests that the anti-inflammatory/chemoprotective properties of fish oil (FO)-derived n-3 polyunsaturated fatty acids (PUFAs) may be partly mediated by PPARδ. Therefore, we assessed the role of PPARδ in modulating the effects of dietary n-3 PUFAs by targeted deletion of intestinal epithelial cell PPARδ (PPARδ(ΔIEpC)). Subsequently, we documented changes in colon tumorigenesis and the inflammatory microenvironment, i.e., local [mesenteric lymph node (MLN)] and systemic (spleen) T cell activation. Animals were fed chemopromotive [corn oil (CO)] or chemoprotective (FO) diets during the induction of chronic inflammation/carcinogenesis. Tumor incidence was similar in control and PPARδ(ΔIEpC) mice. FO reduced mucosal injury, tumor incidence, colonic STAT3 activation, and inflammatory cytokine gene expression, independent of PPARδ genotype. CD8(+) T cell recruitment into MLNs was suppressed in PPARδ(ΔIEpC) mice. Similarly, FO reduced CD8(+) T cell numbers in the MLN. Dietary FO independently modulated MLN CD4(+) T cell activation status by decreasing CD44 expression. CD11a expression by MLN CD4(+) T cells was downregulated in PPARδ(ΔIEpC) mice. Lastly, splenic CD62L expression was downregulated in PPARδ(ΔIEpC) CD4(+) and CD8(+) T cells. These data demonstrate that expression of intestinal epithelial cell PPARδ does not influence azoxymethane/dextran sodium sulfate-induced colon tumor incidence. Moreover, we provide new evidence that dietary n-3 PUFAs attenuate intestinal inflammation in an intestinal epithelial cell PPARδ-independent manner.