Expression Of PGC-1 Research Articles

“Cold training” affects rat liver responses to continuous cold exposure

Continuous exposure of homeothermic animals to low environmental temperatures elicits physiological adaptations necessary for animal survival, which are associated to higher generation of pro-oxidants in thermogenic tissues. It is not known whether intermittent cold exposure (cold training) is able to affect tissue responses to continuous cold exposure. Therefore, we investigated whether rat liver responses to continuous cold exposure of 2 days are modified by cold training (1h daily for 5 days per week for 3 consecutive weeks). Continuous cold increased liver oxidative metabolism by increasing tissue content of mitochondrial proteins and mitochondrial aerobic capacity. Cold training did not affect such parameters, but attenuated or prevented the changes elicited by continuous cold exposure. Two-day cold exposure increased lipid hydroperoxide and protein-bound carbonyl levels in homogenates and mitochondria, whereas cold training decreased such effects although it decreased only homogenate protein damage in control rats. The activities of the antioxidant enzymes GPX and GR and H2O2 production were increased by continuous cold exposure. Despite the increase in GPX and GR activities, livers from cold-exposed rats showed increased susceptibility to in vitro oxidative challenge. Such cold effects were decreased by cold training, which in control rats reduced only H2O2 production and susceptibility to stress. The changes of PGC-1, NRF-1, and NRF-2 expression levels were consistent with those induced by cold exposure and cold training in mitochondrial protein content and antioxidant enzyme activities. However, the mechanisms by which cold training attenuates the effects of the continuous cold exposure remain to be elucidated.

PGC-1β regulates HER2-overexpressing breast cancer cells proliferation by metabolic and redox pathways.

Breast cancer is a prevalent neoplastic disease among women worldwide which treatments still present several side effects and resistance. Considering that cancer cells present derangements in their energetic homeostasis, and that peroxisome proliferator-activated receptor- gamma coactivator 1 (PGC-1) is crucial for cellular metabolism and redox signaling, the main objective of this study was to investigate whether there is a relationship between PGC-1 expression, the proliferation of breast cancer cells and the mechanisms involved. We initially assessed PGC-1β expression in complementary DNA (cDNA) from breast tumor of patients bearing luminal A, luminal B, and HER2-overexpressed and triple negative tumors. Our data showed that PGC-1β expression is increased in patients bearing HER2-overexpressing tumors as compared to others subtypes. Using quantitative PCR and immunoblotting, we showed that breast cancer cells with HER2-amplification (SKBR-3) have greater expression of PGC-1β as compared to a non-tumorous breast cell (MCF-10A) and higher proliferation rate. PGC-1β expression was knocked down with short interfering RNA in HER2-overexpressing cells, and cells decreased proliferation. In these PGC-1β-inhibited cells, we found increased citrate synthase activity and no marked changes in mitochondrial respiration. Glycolytic pathway was decreased, characterized by lower intracellular lactate levels. In addition, after PGC-1β knockdown, SKBR-3 cells showed increased reactive oxygen species production, no changes in antioxidant activity, and decreased expression of ERRα, a modulator of metabolism. In conclusion, we show an association of HER2-overexpression and PGC-1β. PGC-1β knockdown impairs HER2-overexpressing cells proliferation acting on ERRα signaling, metabolism, and redox balance.

Vitamin E-enriched diet reduces adaptive responses to training determining respiratory capacity and redox homeostasis in rat heart

ABSTRACTWe investigated whether reactive oxygen species (ROS) are involved in heart adaptive responses administering a vitamin E-enriched diet to trained rats. Using the homogenates and/or mitochondria from rat hearts we determined the aerobic capacity, tissue level of mitochondrial proteins, and expression of cytochrome c and factors (PGC-1, NRF-1, and NRF-2) involved in mitochondrial biogenesis. We also determined the oxidative damage, glutathione peroxidase (GPX) and reductase activities, glutathione content, mitochondrial ROS release rate, and susceptibility to in vitro oxidative challenge. Glutathione (GSH) content was not affected by both training and antioxidant supplementation. Conversely, antioxidant supplementation prevented metabolic adaptations to training, such as the increases in oxidative capacity, tissue content of mitochondrial proteins, and cytochrome c expression, attenuated some protective adaptations, such as the increase in antioxidant enzyme activities, and did not modify the decrease in ROS release by succinate supplemented mitochondria. Moreover, vitamin E prevented the training-linked increase in tissue capacity to oppose an oxidative attach. The antioxidant effects were associated with decreased levels of PGC-1, NRF-1, and NRF-2 expression. Our results support the idea that some heart adaptive responses to training depend on ROS produced during the exercise sessions and are mediated by the increase in PGC-1 expression which is involved in both the regulation of respiratory capacity and antioxidant protection. However, vitamin inability to prevent some adaptations suggests that other signaling pathways impinging on PGC-1 can modify the response to the antioxidant integration.

PGC-1 coactivators in β-cells regulate lipid metabolism and are essential for insulin secretion coupled to fatty acids.

ObjectivesPeroxisome proliferator-activated receptor γ coactivator 1 (PPARGCA1, PGC-1) transcriptional coactivators control gene programs important for nutrient metabolism. Islets of type 2 diabetic subjects have reduced PGC-1α expression and this is associated with decreased insulin secretion, yet little is known about why this occurs or what role it plays in the development of diabetes. Our goal was to delineate the role and importance of PGC-1 proteins to β-cell function and energy homeostasis.MethodsWe investigated how nutrient signals regulate coactivator expression in islets and the metabolic consequences of reduced PGC-1α and PGC-1β in primary and cultured β-cells. Mice with inducible β-cell specific double knockout of Pgc-1α/Pgc-1β (βPgc-1 KO) were created to determine the physiological impact of reduced Pgc1 expression on glucose homeostasis.ResultsPgc-1α and Pgc-1β expression was increased in primary mouse and human islets by acute glucose and palmitate exposure. Surprisingly, PGC-1 proteins were dispensable for the maintenance of mitochondrial mass, gene expression, and oxygen consumption in response to glucose in adult β-cells. However, islets and mice with an inducible, β-cell-specific PGC-1 knockout had decreased insulin secretion due in large part to loss of the potentiating effect of fatty acids. Consistent with an essential role for PGC-1 in lipid metabolism, β-cells with reduced PGC-1s accumulated acyl-glycerols and PGC-1s controlled expression of key enzymes in lipolysis and the glycerolipid/free fatty acid cycle.ConclusionsThese data highlight the importance of PGC-1s in coupling β-cell lipid metabolism to promote efficient insulin secretion.

Stress triggers mitochondrial biogenesis to preserve steroidogenesis in Leydig cells

Adaptability to stress is a fundamental prerequisite for survival. Mitochondria are a key component of the stress response in all cells. For steroid-hormones-producing cells, including also Leydig cells of testes, the mitochondria are a key control point for the steroid biosynthesis and regulation. However, the mitochondrial biogenesis in steroidogenic cells has never been explored. Here we show that increased mitochondrial biogenesis is the adaptive response of testosterone-producing Leydig cells from stressed rats. All markers of mitochondrial biogenesis together with transcription factors and related kinases are up-regulated in Leydig cells from rats exposed to repeated psychophysical stress. This is followed with increased mitochondrial mass. The expression of PGC1, master regulator of mitochondrial biogenesis and integrator of environmental signals, is stimulated by cAMP-PRKA, cGMP, and β-adrenergic receptors. Accordingly, stress-triggered mitochondrial biogenesis represents an adaptive mechanism and does not only correlate with but also is an essential for testosterone production, being both events depend on the same regulators. Here we propose that all events induced by acute stress, the most common stress in human society, provoke adaptive response of testosterone-producing Leydig cells and activate PGC1, a protein required to make new mitochondria but also protector against the oxidative damage. Given the importance of mitochondria for steroid hormones production and stress response, as well as the role of steroid hormones in stress response and metabolic syndrome, we anticipate our result to be a starting point for more investigations since stress is a constant factor in life and has become one of the most significant health problems in modern societies.

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Objectives There is a growing body of evidence indicating that mitochondrial dysfunction is a pathogenic mediator of oxidative stress in preeclampsia and can modulate the clinical characteristics of the syndrome. The health of mitochondria is in part mediated by their biogenesis. The inducible transcriptional co-activator peroxisome proliferator activated receptor γ co-activator 1- α (PGC-1 α ) is a well characterized pleiotropic orchestrator of mitochondrial biogenesis and homeostasis. Furthermore, PGC-1 α is a powerful regulator of ROS metabolism. Aim: To investigate the role of mitochondrial-specific ROS in coordinating vascular dysfunction in pre-eclampsia. Methods HUVEC were cultured for 24 hours with 3% pooled plasma from women with preeclampsia ( n = 6) and matched controls with uncomplicated pregnancies ( n = 6) from the SCOPE ( www.scopestudy.net ) biobank. Mitochondrial-specific superoxide was detected by fluorescent microscopy of MitoSOX probe upon its oxidation by mitochondrial O2−. The expression profile of PGC-1 α and its target genes SOD2, HO-1 and mitochondrial uncoupling protein UCP-1 were evaluated by real-time PCR. Markers of endothelial activation including endothelin-1 were measured by real-time PCR. Results Levels of plasma-induced mitochondrial superoxide production were increased in endothelial cells incubated with plasma from women with pre-eclampsia compared with matched controls (15.28 vs. 11.32 MFI, P ⩽ 0.01). The expression of PGC-1 α (1 vs. 1.5 ± 0.19 fold, P P P Conclusions Mitochondrial superoxide regulates plasma mediator-induced endothelial dysfunction in pre-eclampsia. The concurrent increase in PGC-1 α expression may co-ordinate a potential protective response to the elevation in mitochondrial ROS through the induction of uncoupling protein antioxidants. Disclosures C. McCarthy: None. L.C. Kenny: None.

Mechanistic study on the cardioprotective and chemosensitizing effects of a novel danshensu derivative

Doxorubicin (Dox) is an anthracycline antibiotic which is widely used in clinics as an anticancer agent. However, the use of Dox is limited by its cardiotoxicity. Our previous studies have shown that a Danshensu derivative ADTM displayed strong cardioprotective effects in an oxidative stress-induced cell injury model in H9c2 cardiomyocytes and an acute myocardial ischemia model in rat [1]. We are developing more potent cardioprotective danshensu derivative based on ADTM structure for management of the cardiotoxic side effect of Dox. In the present study, we investigated the protective and chemosensitizing effects of a novel Danshensu derivative D006 against cardiotoxicity induced by Dox and in increasing the antitumor activity of Dox respectively. Our results showed that D006 was approximately 30-fold more potent than ADTM in ameliorating Dox-induced cardiotoxicity in H9c2 cells. In the zebrafish model, D006 but not ADTM or Danshensu alone, significantly preserved stroke volume of heart function after Dox treatment. In addition, D006 enhanced Dox-induced apoptosis as well as ROS production in MCF-7 tumor cells. Mitochondrial biogenesis, a process involved in the formation of new mitochondrial, which depends on the expression of PGC-1. Recent studies showed that suppressed cardiac mitochondrial biogenesis and decreased mtDNA contents may play vital roles in the pathogenesis of Dox induced cardiomyopathy [2]. Further mechanistic studies verified that D006 increased the expression of PGC-1β in H9c2 cells. Moreover, D006 upregulated mitochondrial biogenesis and increased mtDNA copy number after Dox treatment in H9c2 cells. Our results revealed that a new danshensu derivative displayed promising cardioprotective effects against Dox induced cardiotoxicity both in vivo and in vitro, at least partially through activating mitochondrial biogenesis. In addition, the new danshensu derivative also potentiated the anticancer effects of Dox in breast tumor cells.

Vitamin E supplementation modifies adaptive responses to training in rat skeletal muscle

Aim of the present study was to test, by vitamin E treatment, the hypothesis that muscle adaptive responses to training are mediated by free radicals produced during the single exercise sessions. Therefore, we determined aerobic capacity of tissue homogenates and mitochondrial fractions, tissue content of mitochondrial proteins and expression of factors (PGC-1, NRF-1, and NRF-2) involved in mitochondrial biogenesis. Moreover, we determined the oxidative damage extent, antioxidant enzyme activities, and glutathione content in both tissue preparations, mitochondrial ROS production rate. Finally we tested mitochondrial ROS production rate and muscle susceptibility to oxidative stress. The metabolic adaptations to training, consisting in increased muscle oxidative capacity coupled with the proliferation of a mitochondrial population with decreased oxidative capacity, were generally prevented by antioxidant supplementation. Accordingly, the expression of the factors involved in mitochondrial biogenesis, which were increased by training, was restored to the control level by the antioxidant treatment. Even the training-induced increase in antioxidant enzyme activities, glutathione level and tissue capacity to oppose to an oxidative attach were prevented by vitamin E treatment. Our results support the idea that the stimulus for training-induced adaptive responses derives from the increased production, during the training sessions, of reactive oxygen species that stimulates the expression of PGC-1, which is involved in mitochondrial biogenesis and antioxidant enzymes expression. On the other hand, the observation that changes induced by training in some parameters are only attenuated by vitamin E treatment suggests that other signaling pathways, which are activated during exercise and impinge on PGC-1, can modify the response to the antioxidant integration.

The effect of exercise on epigenetic modifications of PGC1: The impact on type 2 diabetes

The worldwide prevalence of diabetes type 2 is increasing and intramuscular accumulation of fatty acid metabolites is gradually becoming recognized as core features of this condition as lipotoxicity induces insulin resistance. Emerging evidences suggest that defects in mitochondria, key organelle in lipid metabolism, play a central role on insulin resistance. Mitochondria homeostasis is tightly regulated by a nucleus-mitochondria signaling pathway and peroxisome proliferator-activated receptor γ coactivator-1α (PGC1) is the master regulator of important mitochondria process. PGC1 is down regulated in insulin resistant skeletal muscle and abnormal posttranslational modification at histone, epigenetic modifications, is an important factor. Studies have demonstrated the benefits of regular exercise on improving insulin sensitivity however the mechanism for this outcome is not entirely identified. Moreover evidences point out the increase in PGC1 expression induced by exercise as an important element for the improvement of insulin sensitivity in skeletal muscle via increase in mitochondria density and glucose transporter expression (GLUT4). Therefore, we here proposed that aerobic exercise attenuates epigenetic modifications at PGC1 induced by high-energy diets and reduced physical activity, and that leads to inhibition/delay of type 2 diabetic onset.

Evaluation of the Influence of thiosemicarbazone–triazole hybrids on genes implicated in lipid oxidation and accumulation as potential anti-obesity agents

A series of thiosemicarbazone–triazole hybrids 1a–h are efficiently synthesised and evaluated for their influence on the expression of genes, cpt-1, acc-1 and pgc-1, which are essential in lipid metabolism. The test results show that hybrids 1c and 1g exhibited relatively high influence on the expression of cpt-1 and pgc-1 and suppression of acc-1 as desired.

Peroxisome Proliferator-activated Receptor γ Coactivator 1 (PGC-1)- and Estrogen-related Receptor (ERR)-induced Regulator in Muscle 1 (PERM1) Is a Tissue-specific Regulator of Oxidative Capacity in Skeletal Muscle Cells

Mitochondrial oxidative metabolism and energy transduction pathways are critical for skeletal and cardiac muscle function. The expression of genes important for mitochondrial biogenesis and oxidative metabolism are under the control of members of the peroxisome proliferator-activated receptor γ coactivator 1 (PGC-1) family of transcriptional coactivators and the estrogen-related receptor (ERR) subfamily of nuclear receptors. Perturbations in PGC-1 and/or ERR activities have been associated with alterations in capacity for endurance exercise, rates of muscle atrophy, and cardiac function. The mechanism(s) by which PGC-1 and ERR proteins regulate muscle-specific transcriptional programs is not fully understood. We show here that PGC-1α and ERRs induce the expression of a so far uncharacterized muscle-specific protein, PGC-1- and ERR-induced regulator in muscle 1 (Perm1), which regulates the expression of selective PGC-1/ERR target genes. Perm1 is required for the basal as well as PGC-1α-enhanced expression of genes with roles in glucose and lipid metabolism, energy transfer, and contractile function. Silencing of Perm1 in cultured myotubes compromises respiratory capacity and diminishes PGC-1α-induced mitochondrial biogenesis. Our findings support a role for Perm1 acting downstream of PGC-1α and ERRs to regulate muscle-specific pathways important for energy metabolism and contractile function. Elucidating the function of Perm1 may enable novel approaches for the treatment of disorders with compromised skeletal muscle bioenergetics, such as mitochondrial myopathies and age-related/disease-associated muscle atrophies.

Metabolic Stress Modulates Alzheimer’s β-Secretase Gene Transcription via SIRT1-PPARγ-PGC-1 in Neurons

Classic cardio-metabolic risk factors such as hypertension, stroke, diabetes, and hypercholesterolemia all increase the risk of Alzheimer's disease. We found increased transcription of β-secretase/BACE1, the rate-limiting enzyme for Aβ generation, in eNOS-deficient mouse brains and after feeding mice a high-fat, high-cholesterol diet. Up- or downregulation of PGC-1α reciprocally regulated BACE1 invitro and invivo. Modest fasting in mice reduced BACE1 transcription in the brains, which was accompanied by elevated PGC-1 expression and activity. Moreover, the suppressive effect of PGC-1 was dependent on activated PPARγ, likely via SIRT1-mediated deacetylation in a ligand-independent manner. The BACE1 promoter contains multiple PPAR-RXR sites, and direct interactions among SIRT1-PPARγ-PGC-1 at these sites were enhanced with fasting. The interference on the BACE1 gene identified here represents a unique noncanonical mechanism of PPARγ-PGC-1 in transcriptional repression in neurons in response to metabolic signals that may involve recruitment of corepressor NCoR.

Sirtuin 1-mediated Effects of Exercise and Resveratrol on Mitochondrial Biogenesis

The purpose of this study was to evaluate the role of sirtuin 1 (SirT1) in exercise- and resveratrol (RSV)-induced skeletal muscle mitochondrial biogenesis. Using muscle-specific SirT1-deficient (KO) mice and a cell culture model of differentiated myotubes, we compared the treatment of resveratrol, an activator of SirT1, with that of exercise in inducing mitochondrial biogenesis. These experiments demonstrated that SirT1 plays a modest role in maintaining basal mitochondrial content and a larger role in preserving mitochondrial function. Furthermore, voluntary exercise and RSV treatment induced mitochondrial biogenesis in a SirT1-independent manner. However, when RSV and exercise were combined, a SirT1-dependent synergistic effect was evident, leading to enhanced translocation of PGC-1α and SirT1 to the nucleus and stimulation of mitochondrial biogenesis. Thus, the magnitude of the effect of RSV on muscle mitochondrial biogenesis is reliant on SirT1, as well as the cellular environment, such as that produced by repeated bouts of exercise.

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Introduction: Autophagy is a highly conserved process involved in lysosomal degradation of cellular contents and plays an important role in various pathophysiologic conditions. Formation of LC3B-II is a downstream product and a marker of autophagy. Tumor necrosis factor-α (TNFα) is a cytokine, which is overexpressed during the systemic inflammatory response of sepsis, trauma and ischemia and reperfusion injury and contributes to myocardial depression. AMP-activated protein kinase (AMPK) is an important homeostasis regulator, which activates autophagy and mitochondrial recovery. Hypothesis: TNFα induces autophagy through activation of AMPK in rat cardiac myocytes. Methods: H9C2 cardiac myocytes were stimulated with TNFα (100 U/ml) or medium alone for 24 hrs. Western blot analysis was used to evaluate expression of LC3B-II in the cytosol, expression of AMPK and its phosphorylated active form pAMPK in the cytosol, and PPAR gamma coactivator-1? (PGC-1?) in the nucleus. Data was analyzed by t-test. Results: Treatment with TNFα increased expression of LC3B-II in the cytosol (3.32 ± 0.15 fold increase) when compared with basal levels of cells treated with medium alone (p<0.05), thus suggesting formation of autophagosome. To determine the mode of regulation of autophagy, we evaluated the cytosol expression of the upstream kinase AMPK. TNFα also increased both total AMPK and active pAMPK (1.75 ± 0.19 and 2.61 ± 0.30 fold increase, respectively) when compared with basal levels (p<0.05). Since AMPK influences metabolic homeostasis also through regulation of the transcriptional coactivator PGC-1?, we determined the nuclear expression of PGC-1?. Treatment with TNFα increased PGC-1? expression (1.31 ± 0.18 fold increase) when compared with basal levels of cells treated with medium alone (p<0.05). Conclusions: These data indicate that TNFα induces autophagy in cardiac myocytes through activation of AMPK. This event might represent a metabolic adaptation since TNFα-induced autophagy is associated with increased expression of the energy regulator PGC-1?. Supported by NIH grants (R01 GM067202 and R01 AG027990).

Abstract 292: Isoform-Specific Expression of Estrogen-Related Receptors Regulates Cardiomyocyte Metabolic Adaptation to Oxidative Stress

In the ischemic heart, metabolic adaptation of cardiomyocytes exposed to prolonged hypoxia is critical to sustain myocardial energy production. Metabolic gene expression is regulated by Estrogen-Related Receptor (ERR) transcription factors and Peroxisome Proliferator-Activated Receptor γ Coactivator-1 (PGC-1) co-activators. ERRs do not require ligand to achieve an active conformation and regulate an array of target genes involved in myocardial energy metabolism. These studies aim to determine the regulatory mechanisms governing expression of ERR isoforms (ERRα and ERRβ) in cardiomyocytes undergoing oxidative stress. We hypothesize that metabolic energy production in myocardium initially adapts to hypoxia by inducing transient ERRβ expression, and maintains this compensatory expression through induction of sustained ERRα expression. Adult feline cardiomyocytes in primary culture were electrically stimulated to contract continuously at 1 Hz under normoxia (21% [O2]), hypoxia (0.5% [O2]), or hypoxia followed by reoxygenation. Expression of ERR, PGC-1 and target genes was measured by QRT-PCR (n≥5). QRT-PCR arrays were used to screen for prospective target genes. In response to 24 hours of hypoxia, mRNA levels increased for ERRα (3-fold by 24 hours), ERRβ (11-fold by 12 hours), PGC-1α (16-fold by 24 hours) and PGC-1β (8-fold by 24 hours). Subsequent reoxygenation for 24 hours decreased ERRβ, PGC-1α and PGC-1β mRNA levels to that of normoxia controls. However, reoxygenation did not markedly decrease (∼25%; 0.7 of 3.0-fold) ERRα mRNA levels. Cytochrome C Oxidase Subunit VIc (Cox6c) and Fatty Acid Binding Protein 3 (Fabp3) were identified as potential ERRα target genes in adult cardiomyocytes based on correlation of expression. Moreover, hypoxia-induced increases in Cox6c and Fabp3 mRNA levels were partially blocked by treatment with XCT-790, an inhibitor of ERRα activity. We conclude that hypoxia and subsequent reoxygenation elicit isoform-specific changes in ERR and PGC-1 mRNA expression in adult cardiomyocytes. This observed regulation of expression for ERR isoforms and their target genes may play a critical role in metabolic adaptation to oxidative stress.

PGC-1α determines light damage susceptibility of the murine retina.

The peroxisome proliferator-activated receptor γ coactivator 1 (PGC-1) proteins are key regulators of cellular bioenergetics and are accordingly expressed in tissues with a high energetic demand. For example, PGC-1α and PGC-1β control organ function of brown adipose tissue, heart, brain, liver and skeletal muscle. Surprisingly, despite their prominent role in the control of mitochondrial biogenesis and oxidative metabolism, expression and function of the PGC-1 coactivators in the retina, an organ with one of the highest energy demands per tissue weight, are completely unknown. Moreover, the molecular mechanisms that coordinate energy production with repair processes in the damaged retina remain enigmatic. In the present study, we thus investigated the expression and function of the PGC-1 coactivators in the healthy and the damaged retina. We show that PGC-1α and PGC-1β are found at high levels in different structures of the mouse retina, most prominently in the photoreceptors. Furthermore, PGC-1α knockout mice suffer from a striking deterioration in retinal morphology and function upon detrimental light exposure. Gene expression studies revealed dysregulation of all major pathways involved in retinal damage and apoptosis, repair and renewal in the PGC-1α knockouts. The light-induced increase in apoptosis in vivo in the absence of PGC-1α was substantiated in vitro, where overexpression of PGC-1α evoked strong anti-apoptotic effects. Finally, we found that retinal levels of PGC-1 expression are reduced in different mouse models for retinitis pigmentosa. We demonstrate that PGC-1α is a central coordinator of energy production and, importantly, all of the major processes involved in retinal damage and subsequent repair. Together with the observed dysregulation of PGC-1α and PGC-1β in retinitis pigmentosa mouse models, these findings thus imply that PGC-1α might be an attractive target for therapeutic approaches aimed at retinal degeneration diseases.

Sustained expression of PGC-1 in the rat nigrostriatal system selectively impairs dopaminergic function

Mitochondrial dysfunction and oxidative stress have been implicated in the etiology of Parkinson's disease. Therefore, pathways controlling mitochondrial activity rapidly emerge as potential therapeutic targets. Here, we explore the neuronal response to prolonged overexpression of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α), a transcriptional regulator of mitochondrial function, both in vitro and in vivo. In neuronal primary cultures from the ventral midbrain, PGC-1α induces mitochondrial biogenesis and increases basal respiration. Over time, we observe an increasing proportion of the oxygen consumed by neurons which are dedicated to adenosine triphosphate production. In parallel to enhanced oxidative phosphorylation, PGC-1α progressively leads to a decrease in mitochondrial polarization. In the adult rat nigrostriatal system, adeno-associated virus (AAV)-mediated overexpression of PGC-1α induces the selective loss of dopaminergic markers and increases dopamine (DA) catabolism, leading to a reduction in striatal DA content. In addition, PGC-1α prevents the labeling of nigral neurons following striatal injection of the fluorogold retrograde tracer. When PGC-1α is expressed at higher levels following intranigral AAV injection, it leads to overt degeneration of dopaminergic neurons. Finally, PGC-1α overexpression does not prevent nigrostriatal degeneration in pathologic conditions induced by α-synuclein overexpression. Overall, we find that lasting overexpression of PGC-1α leads to major alterations in the metabolic activity of neuronal cells which dramatically impair dopaminergic function in vivo. These results highlight the central role of PGC-1α in the function and survival of dopaminergic neurons and the critical need for maintaining physiological levels of PGC-1α activity.

P2-05-02: PGC1, Peroxisome Proliferator Activated Receptor-gamma (PPAR-gamma) Coactivator-1, Is Necessary in PPAR-gamma Modulated Angiogenesis.

Abstract Background. Peroxisome proliferator activated receptor- gamma (PPAR-gamma), a nuclear hormone receptor, has been shown to be an important regulator of gene expression. PPAR-gamma agonists have been shown to have anti-cancer properties by ways of inducing expression of tumour suppressor genes. It has been indicated that this action of PPAR-gamma is manifested by forming a complex with the PPAR-gamma coactivators (PGC1 &amp;2) and in association with other transcription factors such as SRC-1 and CREB binding proteins. Both PPAR-gamma and PGC-1 have been shown to be aberrantly expressed in human cancer including human breast cancer (1). PPAR-gamma also plays a role in regulating angiogenesis and that PPAR-gamma agonists have been shown to have anti-angiogenic functions. In the present study, we investigated the role of PGC-1 in PPAR-gamma mediated actions on vascular endothelial cells. Materials and methods. Mammalian expression systems for human full length PGC-1 ereonstructed. Anti-PGC-1 transgenes were constructed again using human mammalian expression vector in order to knock down PGC-1 transcript from the cells. Human vascular endothelial cell, HECV, was used in the study. Cell functions linked to angiogenesis including cell adhesion and cellular migration was evaluated using the electric cell impedance sensing method. PPAR-gamma agonists and antagonists were used in the cell models during the analyses. Results. HECV cells weakly expressed PGC-1 and PPAR-gamma. Using the transgenes created, sublines over-expressing PGC1 (HECVPGC1exp) or with PGC-1 expression knockdown (HECVPGC1KD) were established. Loss of PGC1 showed marked increase in both adhesion and cellular migration, opposite was true forHECVPGC1exp cells. PPAR-gamma agonist, pioglitizone and ciglitizone, reduced the adhesion of control cells. In contrast, PPAR-gamma antagonist SR202 exhibited a concentration dependent stimulation of both adhesion and migration of the control cells. Knocking down PGC1 in endothelial cells rendered cells lost response to SR202. Conclusions. PGC-1 is essential in during the modulation of endothelial cells by PPAR-gamma. Together with the aberrant expression of both PPAR-gamma and PGC-1 in breast cancer, it is concluded that PGC-1 is a central player in PPAR-gamma mediated action in both cancer cells and angiogenesis. 1. Jiang WG, Douglas-Jones A, Mansel RE. Expression of peroxisome-proliferator activated receptor-gamma (PPARgamma) and the PPARgamma co-activator, PGC-1, in human breast cancer correlates with clinical outcomes. Int J Cancer. 2003, 106(5):752–757. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-05-02.

P196 - Dynamic regulation of mitochondrial network and oxidative functions during 3T3-L1 fat cell differentiation

Introduction Mitochondria have been shown to be impaired in insulin resistance-related diseases, but have not been extensively studied during the first steps of adipose cell development. This study aimed to describe the sequence of changes of the mitochondrial network and function during the first days of adipogenesis. Materiels et methodes 3T3-L1 preadipocytes were differentiated into adipocytes. At days 0, 3, 6, 9 and 12, mitochondrial network imaging, mitochondrial oxygen consumption, membrane potential, and oxidative phosphorylation efficiency were assessed in permeabilized cells. Gene and protein expressions related to fatty acid metabolism and mitochondrial network were also determined. Resultats Compared to preadipocytes (day 0), new adipocytes (day 6 and 9) displayed profound changes of their mitochondrial network that underwent fragmentation and redistribution around lipid droplets. Drp1 and mitofusin 2 displayed a progressive increase in their gene expression and protein content during the first 9 days of differentiation. In parallel with the mitochondrial network redistribution, mitochondria switched to uncoupled respiration with a tendency towards decreased membrane potential, with no variation of mtTFA and NRF1 gene expression. The expression of PGC1 (and NRF2 genes and genes involved in lipid oxidation (UCP2, CD36, CPT1) was increased. ROS production displayed a nadir at day 6 with a concomitant increase in antioxidant enzyme gene expression. Conclusion This 3T3-L1 based in vitro model of adipogenesis showed that mitochondria adapted to the increased number of lipid droplets by network redistribution and uncoupling respiration. The timing and regulation of lipid oxidation-associated ROS production appeared to play an important role in these changes.

Morphofunctional and Biochemical Approaches for Studying Mitochondrial Changes during Myoblasts Differentiation

This study describes mitochondrial behaviour during the C2C12 myoblast differentiation program and proposes a proteomic approach to mitochondria integrated with classical morphofunctional and biochemical analyses. Mitochondrial ultrastructure variations were determined by transmission electron microscopy; mitochondrial mass and membrane potential were analysed by Mitotracker Green and JC-1 stains and by epifluorescence microscope. Expression of PGC1α, NRF1α, and Tfam genes controlling mitochondrial biogenesis was studied by real-time PCR. The mitochondrial functionality was tested by cytochrome c oxidase activity and COXII expression. Mitochondrial proteomic profile was also performed. These assays showed that mitochondrial biogenesis and activity significantly increase in differentiating myotubes. The proteomic profile identifies 32 differentially expressed proteins, mostly involved in oxidative metabolism, typical of myotubes formation. Other notable proteins, such as superoxide dismutase (MnSOD), a cell protection molecule, and voltage-dependent anion-selective channel protein (VDAC1) involved in the mitochondria-mediated apoptosis, were found to be regulated by the myogenic process. The integration of these approaches represents a helpful tool for studying mitochondrial dynamics, biogenesis, and functionality in comparative surveys on mitochondrial pathogenic or senescent satellite cells.