The Corepressor NCoR1 Antagonizes PGC-1α and Estrogen-Related Receptor α in the Regulation of Skeletal Muscle Function and Oxidative Metabolism

Skeletal muscle exhibits a high plasticity and accordingly can quickly adapt to different physiological and pathological stimuli by changing its phenotype largely through diverse epigenetic mechanisms. The nuclear receptor corepressor 1 (NCoR1) has the ability to mediate gene repression; however, its role in regulating biological programs in skeletal muscle is still poorly understood. We therefore studied the mechanistic and functional aspects of NCoR1 function in this tissue. NCoR1 muscle-specific knockout mice exhibited a 7.2% higher peak oxygen consumption (VO(2peak)), a 11% reduction in maximal isometric force, and increased ex vivo fatigue resistance during maximal stimulation. Interestingly, global gene expression analysis revealed a high overlap between the effects of NCoR1 deletion and peroxisome proliferator-activated receptor gamma (PPARγ) coactivator 1α (PGC-1α) overexpression on oxidative metabolism in muscle. Importantly, PPARβ/δ and estrogen-related receptor α (ERRα) were identified as common targets of NCoR1 and PGC-1α with opposing effects on the transcriptional activity of these nuclear receptors. In fact, the repressive effect of NCoR1 on oxidative phosphorylation gene expression specifically antagonizes PGC-1α-mediated coactivation of ERRα. We therefore delineated the molecular mechanism by which a transcriptional network controlled by corepressor and coactivator proteins determines the metabolic properties of skeletal muscle, thus representing a potential therapeutic target for metabolic diseases

PGC-1β modulates catabolism and fiber atrophy in the fasting-response of specific skeletal muscle beds

Skeletal muscle is a pivotal organ for the coordination of systemic metabolism, constituting one of the largest storage site for glucose, lipids and amino acids. Tight temporal orchestration of protein breakdown in times of fasting has to be balanced with preservation of muscle mass and function. However, the molecular mechanisms that control the fasting response in muscle are poorly understood.; We now have identified a role for the peroxisome proliferator-activated receptor γ coactivator 1β (PGC-1β) in the regulation of catabolic pathways in this context in muscle-specific loss-of-function mouse models.; Muscle-specific knockouts for PGC-1β experience mitigated muscle atrophy in fasting, linked to reduced expression of myostatin, atrogenes, activation of AMP-dependent protein kinase (AMPK) and other energy deprivation signaling pathways. At least in part, the muscle fasting response is modulated by a negative effect of PGC-1β on the nuclear factor of activated T-cells 1 (NFATC1).; Collectively, these data highlight the complex regulation of muscle metabolism and reveal a new role for muscle PGC-1β in the control of proteostasis in fasting

The coactivator PGC-1α regulates skeletal muscle oxidative metabolism independently of the nuclear receptor PPARβ/δ in sedentary mice fed a regular chow diet

Aims/hypothesis: Physical activity improves oxidative capacity and exerts therapeutic beneficial effects, particularly in the context of metabolic diseases. The peroxisome proliferator-activated receptor (PPAR) γ coactivator-1α (PGC-1α) and the nuclear receptor PPARβ/δ have both been independently discovered to play a pivotal role in the regulation of oxidative metabolism in skeletal muscle, though their interdependence remains unclear. Hence, our aim was to determine the functional interaction between these two factors in mouse skeletal muscle in vivo. Methods: Adult male control mice, PGC-1α muscle-specific transgenic (mTg) mice, PPARβ/δ muscle-specific knockout (mKO) mice and the combination PPARβ/δ mKO + PGC-1α mTg mice were studied under basal conditions and following PPARβ/δ agonist administration and acute exercise. Whole-body metabolism was assessed by indirect calorimetry and blood analysis, while magnetic resonance was used to measure body composition. Quantitative PCR and western blot were used to determine gene expression and intracellular signalling. The proportion of oxidative muscle fibre was determined by NADH staining. Results: Agonist-induced PPARβ/δ activation was only disrupted by PPARβ/δ knockout. We also found that the disruption of the PGC-1α-PPARβ/δ axis did not affect whole-body metabolism under basal conditions. As expected, PGC-1α mTg mice exhibited higher exercise performance, peak oxygen consumption and lower blood lactate levels following exercise, though PPARβ/δ mKO + PGC-1α mTg mice showed a similar phenotype. Similarly, we found that PPARβ/δ was dispensable for PGC-1α-mediated enhancement of an oxidative phenotype in skeletal muscle. Conclusions/interpretation: Collectively, these results indicate that PPARβ/δ is not an essential partner of PGC-1α in the control of skeletal muscle energy metabolism

Regulation of skeletal muscle plasticity by the transcriptional coregulators PGC-1α and NCoR1

Skeletal muscle plasticity is regulated by a wide range of factors, among which environmental stimuli such as exercise play a central role. Importantly, changes in skeletal muscle phenotype exert a direct impact on health and risk to premature death. In fact, physical inactivity promotes the development of diseases like cancer and heart diseases. In contrast, exercise training has been shown to lower the risk of these pathologies, mainly by enhancing the metabolic fitness, mass and function of skeletal muscle. Skeletal muscle remodelling is regulated at the transcriptional level by the coordinated interplay between transcription factors and coregulators. The transcription factors estrogen-related receptor alpha (ERRalpha) and proliferator-activated receptor beta/delta (PPARbeta/delta) play a key regulatory function of skeletal muscle metabolism, while their coactivator PPARgamma coactivator 1alpha (PGC-1alpha) and corepressor nuclear receptor corepressor 1 (NCoR1) have emerged as potential modulators of skeletal muscle plasticity. However, the physiological role and the mechanisms by which PGC-1alpha and NCoR1 regulates skeletal muscle phenotype and function are not fully understood. To define the role of NCoR1 in skeletal muscle plasticity and to identify its potential interplay with PGC-1alpha, we initially characterized NCoR1 muscle-specific knockout (mKO) mice. We observed that the deletion of NCoR1 in skeletal muscle resulted in enhanced oxygen consumption (VO2) during exercise, lower maximal force and increased ex vivo fatigue resistance. Interestingly, microarray analysis of NCoR1 mKO and PGC-1alpha muscle-specific transgenic (mTg) mice skeletal muscle revealed an up-regulation of genes related to oxidative metabolism in both mouse models. Consistently, we found that PGC-1alpha knockdown in cultured myotubes inhibited the up-regulation of mitochondrial enzymes induced by NCoR1 knockdown. Moreover, ERRalpha and PPARbeta/delta were identified as direct targets of both NCoR1 and PGC-1alpha. However, only the inhibition of ERRalpha was able to block the effects of NCoR1 knockdown in myotubes. Next, during the second study, the interplay between PGC-1alpha and PPARbeta/delta was determined by using different genetic mouse models. Surprisingly, our data demonstrated that the PGC-1alpha-PPARbeta/delta axis does not control whole body metabolism under basal conditions. Actually, PPARbeta/delta was found to be dispensable for the positive effects of PGC-1alpha on whole body (e.g. VO2) and skeletal muscle oxidative metabolism. Altogether, these studies demonstrate that, under basal conditions, NCoR1 and PGC-1alpha modulate skeletal muscle oxidative metabolism specifically by controlling ERRalpha-mediated gene expression. Finally, skeletal muscle remodelling induced by chronic overload was studied by using the model of synergist ablation (SA). Interestingly, SA has been shown to induce skeletal muscle hypertrophy through the activation of the mammalian target of rapamycin complex 1 (mTORC1), while mTORC1 can enhance skeletal muscle oxidative metabolism by regulating the PGC-1alpha-Ying Yang 1 complex. Accordingly, in the last study of this thesis the potential function of the mTORC-1-PGC-1alpha axis in SA-induced skeletal muscle remodelling was defined by using PGC-1alpha mTg and mKO mice. As expected, SA strongly induced mTORC1 activation and skeletal muscle hypertrophy, though these effects were independent of PGC-1alpha. Moreover, SA down-regulated PGC-1alpha mRNA levels, consistent thus with the global repression of glycolytic and oxidative metabolism. Functional analyses further demonstrated that, SA promoted a switch toward a slow-contractile phenotype characterized by lower peak force and higher fatigue resistance, which was not altered in PGC-1alpha mTg mice. However, genetic ablation of PGC-1alpha preserved peak force after SA, an effect that seems to be related to the regulation of myosin heavy chain 2B, myosin regulatory light chain (MLC) and MLC kinase 2 by PGC-1alpha. Hence, we have found that PGC-1alpha is not involved in skeletal muscle hypertrophy and metabolic remodelling induced by SA, while this coactivator seem to be partially involved in the functional adaptations to SA. However, SA did not fully resemble the effects of resistance exercise in human skeletal muscle, thus the relevance of PGC-1alpha as a therapeutic target aiming at promoting skeletal muscle growth remains to be further explored under different conditions. Therefore, the studies performed during this thesis have revealed new molecular mechanisms by which coregulators mediate skeletal muscle plasticity, especially related with the control of oxidative metabolism. Considering the relevance of skeletal muscle metabolic fitness in the development and prevention of metabolic diseases, these data has direct biomedical relevance. However, the therapeutic potential of the mechanisms here described remain to be defined in future studies

The transcriptional coactivator PGC-1α is dispensable for chronic overload-induced skeletal muscle hypertrophy and metabolic remodeling

Skeletal muscle mass loss and dysfunction have been linked to many diseases. Conversely, resistance exercise, mainly by activating mammalian target of rapamycin complex 1 (mTORC1), promotes skeletal muscle hypertrophy and exerts several therapeutic effects. Moreover, mTORC1, along with peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), regulates skeletal muscle metabolism. However, it is unclear whether PGC-1α is required for skeletal muscle adaptations after overload. Here we show that although chronic overload of skeletal muscle via synergist ablation (SA) strongly induces hypertrophy and a switch toward a slow-contractile phenotype, these effects were independent of PGC-1α. In fact, SA down-regulated PGC-1α expression and led to a repression of energy metabolism. Interestingly, however, PGC-1α deletion preserved peak force after SA. Taken together, our data suggest that PGC-1α is not involved in skeletal muscle remodeling induced by SA

PDE2 activity differs in right and left rat ventricular myocardium and differentially regulates β2 adrenoceptor-mediated effects

The important regulator of cardiac function, cAMP, is hydrolyzed by different cyclic nucleotide phosphodiesterases (PDEs), whose expression and activity are not uniform throughout the heart. Of these enzymes, PDE2 shapes β1 adrenoceptor-dependent cardiac cAMP signaling, both in the right and left ventricular myocardium, but its role in regulating β2 adrenoceptor-mediated responses is less well known. Our aim was to investigate possible differences in PDE2 transcription and activity between right (RV) and left (LV) rat ventricular myocardium, as well as its role in regulating β2 adrenoceptor effects. The free walls of the RV and the LV were obtained from Sprague-Dawley rat hearts. Relative mRNA for PDE2 (quantified by qPCR) and PDE2 activity (evaluated by a colorimetric procedure and using the PDE2 inhibitor EHNA) were determined in RV and LV. Also, β2 adrenoceptor-mediated effects (β2-adrenoceptor agonist salbutamol + β1 adrenoceptor antagonist CGP-20712A) on contractility and cAMP concentrations, in the absence or presence of EHNA, were studied in the RV and LV. PDE2 transcript levels were less abundant in RV than in LV and the contribution of PDE2 to the total PDE activity was around 25% lower in the microsomal fraction of the RV compared with the LV. β2 adrenoceptor activation increased inotropy and cAMP levels in the LV when measured in the presence of EHNA, but no such effects were observed in the RV, either in the presence or absence of EHNA. These results indicate interventricular differences in PDE2 transcript and activity levels, which may distinctly regulate β2 adrenoceptor-mediated contractility and cAMP concentrations in the RV and in the LV of the rat heart

Characterization of regulatory transcriptional mechanisms in hepatocyte lipotoxicity

Non-alcoholic fatty liver disease is a continuum of disorders among which non-alcoholic steatohepatitis (NASH) is particularly associated with a negative prognosis. Hepatocyte lipotoxicity is one of the main pathogenic factors of liver fibrosis and NASH. However, the molecular mechanisms regulating this process are poorly understood. The main aim of this study was to dissect transcriptional mechanisms regulated by lipotoxicity in hepatocytes. We achieved this aim by combining transcriptomic, proteomic and chromatin accessibility analyses from human liver and mouse hepatocytes. This integrative approach revealed several transcription factor networks deregulated by NASH and lipotoxicity. To validate these predictions, genetic deletion of the transcription factors MAFK and TCF4 was performed, resulting in hepatocytes that were better protected against saturated fatty acid oversupply. MAFK- and TCF4-regulated gene expression profiles suggest a mitigating effect against cell stress, while promoting cell survival and growth. Moreover, in the context of lipotoxicity, some MAFK and TCF4 target genes were to the corresponding differentially regulated transcripts in human liver fibrosis. Collectively, our findings comprehensively profile the transcriptional response to lipotoxicity in hepatocytes, revealing new molecular insights and providing a valuable resource for future endeavours to tackle the molecular mechanisms of NASH

New insights in the regulation of skeletal muscle PGC-1α by exercise and metabolic diseases

Skeletal muscle energy metabolism is severely impaired in insulin resistant and type 2 diabetic patients. In particular, deregulated transcription of oxidative metabolism genes has been linked to the development of non-communicable metabolic diseases. The peroxisome proliferator-activated receptor γ (PPARγ) coactivator-1α (PGC-1α) is a key molecule in the regulation of oxidative metabolism in different tissues, including skeletal muscle. In this tissue, physical exercise is one of the most dominant physiological stimuli to induce PGC-1α. In addition, exercise training efficiently prevents the development of metabolic diseases. Hence, better knowledge about the regulation of PGC-1α by exercise would significantly help to design effective treatments for these diseases