Phosphodiesterase Type 5 Inhibitors, Sport and Doping

Phosphodiesterase type 5 inhibitors (PDE5i) (e.g., sildenafil, tadalafil, vardenafil, and avanafil) are drugs commonly used to treat erectile dysfunction, pulmonary arterial hypertension, and benign prostatic hyperplasia. PDE5i are not prohibited by the World Anti-Doping Agency (WADA) but are alleged to be frequently misused by healthy athletes to improve sporting performance. In vitro and in vivo studies have reported various effects of PDE5i on cardiovascular, muscular, metabolic, and neuroendocrine systems and the potential, therefore, to enhance performance of healthy athletes during training and competition. This suggests well-controlled research studies to examine the ergogenic effects of PDE5i on performance during activities that simulate real sporting situations are warranted to determine if PDE5i should be included on the prohibited WADA list. In the meantime, there is concern that some otherwise healthy athletes will continue to misuse PDE5i to gain an unfair competitive advantage over their competitors

Characterization of Keratinocyte Differentiation Induced by Ascorbic Acid: Protein Kinase C Involvement and Vitamin C Homeostasis11The authors declared not to have a conflict of interest.

Epidermal keratinocytes undergo differentiation in response to several stimuli to form the cornified envelope, a structure that contributes to the barrier function of skin. Although differentiation has been extensively analyzed, the precise role of vitamin C during this process is still not defined. Ascorbic acid, besides acting as a radical scavenger, has been shown to promote mesenchymal differentiation. In this study, we found that keratinocytes grown in ascorbate-supplemented medium developed a differentiated phenotype, as demonstrated by enhanced expression of marker genes and increase in cornified envelope content. The pro-differentiating effects of ascorbate were mediated by the protein-kinase-C-dependent induction of activating protein 1 DNA binding activity; indeed, down-modulation of protein kinase C activity abolished differentiation triggered by ascorbic acid. Although vitamin C appeared to regulate the same signaling pathway modulated by calcium, a classical in vitro inducer of epidermal differentiation, nonetheless terminally differentiated keratinocytes exhibited different ascorbate homeostasis and cellular antioxidant status. Indeed, we found that, unlike calcium, differentiation promoted by ascorbate was accompanied by (i) an enhanced ascorbate transport, due to overexpression of specific transporters, (ii) a great efficiency of dehydroascorbate uptake, and (iii) an increase in glutathione content with respect to proliferating cells. Ascorbic acid may be useful to promote epidermal differentiation, avoiding depletion of hydrophilic antioxidant stores

Moringa oleifera leaf extract influences oxidative metabolism in C2C12 myotubes through SIRT1-PPARα pathway

Abstract Background Moringa oleifera is an important traditional multipurpose plant, due to the presence of many bioactive compounds. Moringa oleifera leaf extracts (MOLE) have been shown to have many beneficial properties in pathological conditions including diabete. However, the lack of information about its exact molecular mechanism of action might hinder other potential use in different areas such as skeletal muscle physiology. Hypothesis/purpose Skeletal muscle represents about 40-50% of the total mass of a lean individual and is an insulin-sensitive tissue with wide variations in energy requirements. We aimed to test the effects of MOLE on oxidative metabolism and the molecular mechanism involved on myotubes by using C2C12 cell line, a well known model for in vitro skeletal muscle studies. Study design C2C12 myotubes were treated with MOLE at different working solutions for 24 and 48 hours and then culture media and cellular extracts were collected. MOLE was screened for phytochemicals determination. Methods Glucose and free fatty acids consumption along with lactate release were assessed in the culture media. Citrate sinthase, 3-hydroxy acylCoA dehydrogenase, alanine transglutaminase and creatine kinase enzyme activities, as well as the metabolic regulatory SIRT1 and PPARα protein levels were evaluated in cellular extracts. Results MOLE administration induced a dose and time dependent increase in substrates consumption accompanied by an increase in intracellular oxidative metabolism enzymatic activity levels. The extracts were also able to modulate positively the protein expression of SIRT1 and PPARα. Conclusion Altogether, these data indicate that MOLE could represent a valid nutritional support for improving skeletal muscle metabolism: in fact MOLE treatment increased oxidative energy metabolism and possibly favours mitochondrial biogenesis through SIRT1/PPARα pathway. future studies will clarify wether Moringa oleifera leaf extracts consumption may be useful to improve physical performance and metabolic-related skeletal muscle diseases

Explosive type of moderate-resistance training induces functional, cardiovascular, and molecular adaptations in the elderly

Current recommendations aimed at reducing neuromuscular and functional loss in aged muscle have identified muscle power as a key target for intervention trials, although little is known about the biological and cardiovascular systemic response in the elderly. This study investigated the effects of 12 weeks of low-frequency, moderate-intensity, explosive-type resistance training (EMRT) on muscle strength and powerin oldcommunity-dwellingpeople(70–75years), monitoring functional performance linked to daily liv- ing activities (ADL) and cardiovascular response, as well as biomarkers of muscle damage, cardiovascular risk, and cellular stress response. The present study provides the first evidence that EMRT was highly effective in achieving a significant enhancement in muscular strength and power as well as in functional performance without causing any detrimental modification in cardiovascular, inflammatory, and damage parameters. Moreover, trained elderly subjects showed an adaptive response at both systemic and cellular levels by modulation of antioxidant and stress-induced markers such as myeloperoxidase (MPO), heat shock protein 70 (Hsp70) and 27 (Hsp27), and thioredoxin reductase 1 (TrxR1)

Oxidative Stress and Skeletal Muscle Function

Skeletal muscle is continuously exposed during its activities to mechanical/oxidative damage [...

Muscles Journal: The New Home of Muscle Followers

In recent years, the number of scientific papers focused on the study of muscle and its physiopathology has grown significantly [...

Moringa oleifera Leaf Extract Upregulates Nrf2/HO-1 Expression and Ameliorates Redox Status in C2C12 Skeletal Muscle Cells

Moringa oleifera is a multi-purpose herbal plant with numerous health benefits. In skeletal muscle cells, Moringa oleifera leaf extract (MOLE) acts by increasing the oxidative metabolism through the SIRT1-PPARα pathway. SIRT1, besides being a critical energy sensor, is involved in the activation related to redox homeostasis of transcription factors such as the nuclear factor erythroid 2-related factor (Nrf2). The aim of the present study was to evaluate in vitro the capacity of MOLE to influence the redox status in C2C12 myotubes through the modulation of the total antioxidant capacity (TAC), glutathione levels, Nrf2 and its target gene heme oxygenase-1 (HO-1) expression, as well as enzyme activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and transferase (GST). Moreover, the impact of MOLE supplementation on lipid peroxidation and oxidative damage (i.e., TBARS and protein carbonyls) was evaluated. Our results highlight for the first time that MOLE increased not only Nrf2 and HO-1 protein levels in a dose-dependent manner, but also improved glutathione redox homeostasis and the enzyme activities of CAT, SOD, GPx and GST. Therefore, it is intriguing to speculate that MOLE supplementation could represent a valuable nutrition for the health of skeletal muscles

Oxidative stress resistance in skeletal muscle cells: role of vitamin C and redox sensitive transcription factors NF-KB and AP-1

Durante l’esercizio fisico le cellule muscolari sono continuamente esposte allo stress ossidativo e necessitano quindi di efficaci sistemi di difesa antiossidante. Tramite l’attivazione di fattori trascrizionali, quali AP-1 e NF-kB, le cellule muscolari possono compensare le variazioni ambientali incrementando le risposte adattative (antiossidanti endogeni, enzimatici e non). Alle difese cellulari contribuiscono anche gli antiossidanti esogeni, quali la vitamina C. Il contenuto di vitamina C nelle cellule muscolari è determinato sia dall’efficienza dei sistemi di trasporto sia dal mantenimento della vitamina nella sua forma ridotta. Lo scopo della tesi è stato quello di studiare in cellule muscolari scheletriche: i) specifici sistemi antiossidanti, quali il glutatione, la tioredossina reduttasi ed enzimi antiossidanti; ii) il ruolo di fattori di trascrizione sensibili allo stato redox quali AP-1 e NF-kB; iii) i meccanismi di trasporto sia per la forma ridotta (acido ascorbico, AA) che per la forma ossidata (acido deidroascorbico, DHA) della vitamina C; iiii) i sistemi enzimatici (NADH-, NADPH-, GSH-, acido lipoico-dipendenti) coinvolti nella rigenerazione della vitamina C. A tal fine sono state utilizzate cellule muscolari scheletriche di topo (C2C12) e di ratto (L6C5), sia in fase proliferativa che dopo differenziamento. I risultati ottenuti dimostrano che il differenziamento è correlato all'aumento della capacità antiossidante, e tale fenomeno è maggiormente evidente nelle cellule L6C5. Le cellule C2C12 (mioblasti e miotubi), possedendo elevati livelli di NF-kB, di complessi AP-1 di tipo inibitorio e di tioredossina reduttasi, mostrano una maggiore espressione di enzimi antiossidanti; perciò, tali cellule sono maggiormente resistenti allo stress ossidativo. Al contrario, i mioblasti L6C5 mostrano un fenotipo sensibile, correlato a bassi livelli di tioredossina reduttasi, catalasi ed attività di NF-kB, ed alti livelli di glutatione ossidato e di complessi AP-1 attivi. Tuttavia, questa linea cellulare, se indotta a differenziare, acquista un fenotipo resistente all’apoptosi, accompagnato da drastici cambiamenti nel bilancio fra specie ossidanti ed antiossidanti. I miotubi L6C5, infatti, aumentano le attività di catalasi e NF-kB, e cambiano i complessi AP-1 da attivanti ad inibitori. Gli esperimenti condotti per caratterizzare il metabolismo della vitamina C hanno evidenziato che il trasporto dell'AA è mediato dall'SVCT2 (trasportatore con elevata affinità), mentre il trasporto per il DHA è mediato dai trasportatori del glucosio GLUT1 e GLUT3. I mioblasti L6C5 sono più efficienti nel trasporto dell'acido ascorbico, mentre le cellule C2C12 sono più efficienti nel trasporto dell'acido deidroascorbico e mostrano maggiori attività DHA e AFR (radicale ascorbile) reduttasiche. Lo stress ossidativo, indotto dalla deplezione di glutatione, induce un aumento dell'espressione di SVCT2 e dell'attività DHA reduttasica tioredossina-dipendente, specialmente nelle cellule differenziate. Da questi dati appare evidente che il trasportatore SVCT2 e l'attività DHA reduttasica NADPH-tioredossina-dipendente appartengono ad un sistema inducibile attivato in risposta allo stress ossidativo.During physical exercise skeletal muscle cells are continuosly exposed to oxidative stress. Thus, they compensate environmental challenges by increasing adaptive responses, characterized by AP-1- and NF-kB-mediated transcriptional up-regulation of endogenous enzymatic and non-enzymatic antioxidants. Also exogenous antioxidants, such as vitamin C contribute to cellular defences. The skeletal muscle cells content of vitamin C is determined both by the efficiency of transport systems and by the ability to maintain the vitamin in its reduced form. Aim of this thesis was to study, in skeletal muscle cells: i) specific antioxidants systems, including glutathione (GSH), thioredoxin reductase (TxR) and antioxidants enzymes; ii) the role of redox sensitive transcription factors, such as AP-1 and NF-kB; iii) the transport of vitamin C, both in the reduced (ascorbic acid, AA) and oxidized form (dehydroascorbic acid, DHA); iiii) the enzymatic systems (NADH-, NADPH-, GSH-, lipoic acid-dependent) involved in vitamin C recycling. To this end, two skeletal muscle cell lines were used (mouse C2C12 and rat L6C5 cells), in both proliferating and differentiated conditions. We found that muscle cell differentiation was associated with increase in antioxidant ability, and this phenomenon was more evident in the L6C5 cell line. C2C12 myoblasts and myotubes show high levels of NF-kB and thioredoxin reductase, together with AP-1 inhibitory complexes. Furthermore, C2C12 cells have antioxidant enzymes highly active thus allowing survival after exposure to oxidative insults. On the contrary, L6C5 myoblasts show a sensitive phenotype, correlated to low levels of thioredoxin reductase, catalase and NF-kB activity, together with high levels of oxidized glutathione (GSSG) and activating AP-1 complexes. Interestingly, this cell line acquires an apoptosis-resistant phenotype, accompanied by drastic changes in the oxidant/antioxidant balance, when induced to differentiate. Indeed, L6C5 myotubes enhance catalase and NF-kB activities and shift AP-1 complexes from an activating to an inhibitory behaviour. Concerning the experiments on vitamin C metabolism, our data show that both cell lines import AA by the SVCT2 transporter, while DHA transport is mediated by glucose carriers GLUT1 and GLUT3. L6C5 myoblasts are more efficient in ascorbic acid transport, while C2C12 cells are more efficient in dehydroascorbic acid transport and ascorbyl free radical/dehydroascorbic acid reduction. Oxidative stress, induced by glutathione depletion, increased SVCT2 expression and thioredoxin reductase-mediated dehydroascorbic acid reduction, especially in differentiated cells. From these data, SVCT2 and NADPH-thioredoxin dependent DHA reduction appear to belong to an inducible system activated in response to oxidative injury

Oxidative stress resistance in skeletal muscle cells: role of vitamin C and redox sensitive transcription factors NF-KB and AP-1

Durante l’esercizio fisico le cellule muscolari sono continuamente esposte allo stress ossidativo e necessitano quindi di efficaci sistemi di difesa antiossidante. Tramite l’attivazione di fattori trascrizionali, quali AP-1 e NF-kB, le cellule muscolari possono compensare le variazioni ambientali incrementando le risposte adattative (antiossidanti endogeni, enzimatici e non). Alle difese cellulari contribuiscono anche gli antiossidanti esogeni, quali la vitamina C. Il contenuto di vitamina C nelle cellule muscolari è determinato sia dall’efficienza dei sistemi di trasporto sia dal mantenimento della vitamina nella sua forma ridotta. Lo scopo della tesi è stato quello di studiare in cellule muscolari scheletriche: i) specifici sistemi antiossidanti, quali il glutatione, la tioredossina reduttasi ed enzimi antiossidanti; ii) il ruolo di fattori di trascrizione sensibili allo stato redox quali AP-1 e NF-kB; iii) i meccanismi di trasporto sia per la forma ridotta (acido ascorbico, AA) che per la forma ossidata (acido deidroascorbico, DHA) della vitamina C; iiii) i sistemi enzimatici (NADH-, NADPH-, GSH-, acido lipoico-dipendenti) coinvolti nella rigenerazione della vitamina C. A tal fine sono state utilizzate cellule muscolari scheletriche di topo (C2C12) e di ratto (L6C5), sia in fase proliferativa che dopo differenziamento. I risultati ottenuti dimostrano che il differenziamento è correlato all'aumento della capacità antiossidante, e tale fenomeno è maggiormente evidente nelle cellule L6C5. Le cellule C2C12 (mioblasti e miotubi), possedendo elevati livelli di NF-kB, di complessi AP-1 di tipo inibitorio e di tioredossina reduttasi, mostrano una maggiore espressione di enzimi antiossidanti; perciò, tali cellule sono maggiormente resistenti allo stress ossidativo. Al contrario, i mioblasti L6C5 mostrano un fenotipo sensibile, correlato a bassi livelli di tioredossina reduttasi, catalasi ed attività di NF-kB, ed alti livelli di glutatione ossidato e di complessi AP-1 attivi. Tuttavia, questa linea cellulare, se indotta a differenziare, acquista un fenotipo resistente all’apoptosi, accompagnato da drastici cambiamenti nel bilancio fra specie ossidanti ed antiossidanti. I miotubi L6C5, infatti, aumentano le attività di catalasi e NF-kB, e cambiano i complessi AP-1 da attivanti ad inibitori. Gli esperimenti condotti per caratterizzare il metabolismo della vitamina C hanno evidenziato che il trasporto dell'AA è mediato dall'SVCT2 (trasportatore con elevata affinità), mentre il trasporto per il DHA è mediato dai trasportatori del glucosio GLUT1 e GLUT3. I mioblasti L6C5 sono più efficienti nel trasporto dell'acido ascorbico, mentre le cellule C2C12 sono più efficienti nel trasporto dell'acido deidroascorbico e mostrano maggiori attività DHA e AFR (radicale ascorbile) reduttasiche. Lo stress ossidativo, indotto dalla deplezione di glutatione, induce un aumento dell'espressione di SVCT2 e dell'attività DHA reduttasica tioredossina-dipendente, specialmente nelle cellule differenziate. Da questi dati appare evidente che il trasportatore SVCT2 e l'attività DHA reduttasica NADPH-tioredossina-dipendente appartengono ad un sistema inducibile attivato in risposta allo stress ossidativo.During physical exercise skeletal muscle cells are continuosly exposed to oxidative stress. Thus, they compensate environmental challenges by increasing adaptive responses, characterized by AP-1- and NF-kB-mediated transcriptional up-regulation of endogenous enzymatic and non-enzymatic antioxidants. Also exogenous antioxidants, such as vitamin C contribute to cellular defences. The skeletal muscle cells content of vitamin C is determined both by the efficiency of transport systems and by the ability to maintain the vitamin in its reduced form. Aim of this thesis was to study, in skeletal muscle cells: i) specific antioxidants systems, including glutathione (GSH), thioredoxin reductase (TxR) and antioxidants enzymes; ii) the role of redox sensitive transcription factors, such as AP-1 and NF-kB; iii) the transport of vitamin C, both in the reduced (ascorbic acid, AA) and oxidized form (dehydroascorbic acid, DHA); iiii) the enzymatic systems (NADH-, NADPH-, GSH-, lipoic acid-dependent) involved in vitamin C recycling. To this end, two skeletal muscle cell lines were used (mouse C2C12 and rat L6C5 cells), in both proliferating and differentiated conditions. We found that muscle cell differentiation was associated with increase in antioxidant ability, and this phenomenon was more evident in the L6C5 cell line. C2C12 myoblasts and myotubes show high levels of NF-kB and thioredoxin reductase, together with AP-1 inhibitory complexes. Furthermore, C2C12 cells have antioxidant enzymes highly active thus allowing survival after exposure to oxidative insults. On the contrary, L6C5 myoblasts show a sensitive phenotype, correlated to low levels of thioredoxin reductase, catalase and NF-kB activity, together with high levels of oxidized glutathione (GSSG) and activating AP-1 complexes. Interestingly, this cell line acquires an apoptosis-resistant phenotype, accompanied by drastic changes in the oxidant/antioxidant balance, when induced to differentiate. Indeed, L6C5 myotubes enhance catalase and NF-kB activities and shift AP-1 complexes from an activating to an inhibitory behaviour. Concerning the experiments on vitamin C metabolism, our data show that both cell lines import AA by the SVCT2 transporter, while DHA transport is mediated by glucose carriers GLUT1 and GLUT3. L6C5 myoblasts are more efficient in ascorbic acid transport, while C2C12 cells are more efficient in dehydroascorbic acid transport and ascorbyl free radical/dehydroascorbic acid reduction. Oxidative stress, induced by glutathione depletion, increased SVCT2 expression and thioredoxin reductase-mediated dehydroascorbic acid reduction, especially in differentiated cells. From these data, SVCT2 and NADPH-thioredoxin dependent DHA reduction appear to belong to an inducible system activated in response to oxidative injury

Tadalafil alters energy metabolism in C2C12 skeletal muscle cells

Phosphodiesterases (PDEs) are a family of enzymes that hydrolyze cyclic nucleotides, thereby modulating cell functions. Three highly selective PDE5 inhibitors (PDE5i), sildenafil, vardenafil and tadalafil, have been developed for treatment of erectile dysfunction (ED). Experimental evidence showed that chronic treatment with sildenafil PDE5i in a mouse model of diet-induced obesity and insulin resistance improved insulin action and decreased circulating fatty acid levels. It has recently been shown that healthy athletes use PDE5i as performance enhancers, hence in the present study we investigated whether the long-lasting PDE5i tadalafil influences energy metabolism in C2C12 skeletal muscle cells by evaluating lactate production, glucose consumption, and citrate synthase and 3-OH acyl CoA dehydrogenase activities. Our data demonstrate that tadalafil is able to modulate energy homeostasis in mouse skeletal muscle cells, depending on the treatment length and dose