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COMMENTARY

The role of nNOS and PGC-1a in skeletal muscle cells

Sara Baldelli1, Daniele Lettieri Barbato2, Giuseppe Tatulli1, Katia Aquilano2,* and Maria Rosa Ciriolo3,*

ABSTRACT

Neuronal nitric oxide synthase (nNOS) and peroxisome proliferator

activated receptor c co-activator 1a (PGC-1a) are two fundamental

factors involved in the regulation of skeletal muscle cell metabolism.

nNOS exists as several alternatively spliced variants, each having

a specific pattern of subcellular localisation. Nitric oxide (NO)

functions as a second messenger in signal transduction pathways

that lead to the expression of metabolic genes involved in oxidative

metabolism, vasodilatation and skeletal muscle contraction. PGC-

1a is a transcriptional coactivator and represents a master regulator

of mitochondrial biogenesis by promoting the transcription of

mitochondrial genes. PGC-1a can be induced during physical

exercise, and it plays a key role in coordinating the oxidation of

intracellular fatty acids with mitochondrial remodelling. Several lines

of evidence demonstrate that NO could act as a key regulator of

PGC-1a expression; however, the link between nNOS and PGC-1a

in skeletal muscle remains only poorly understood. In this

Commentary, we review important metabolic pathways that

are governed by nNOS and PGC-1a, and aim to highlight how

they might intersect and cooperatively regulate skeletal muscle

mitochondrial and lipid energetic metabolism and contraction.

KEY WORDS: Nitric oxide, Lipid metabolism, Mitochondrial

biogenesis, Mitochondrial metabolism

IntroductionNitric oxide (NO) is a free radical molecule that isphysiologically synthesised in a tightly controlled manner by afamily of cytochrome-P450-like flavo-haem proteins, the NOsynthases (NOSs) (Forstermann and Sessa, 2012). In mammals,

three quite distinct NOS isoforms have been described. NeuronalNOS (nNOS or NOS1) was the first isoform to be reported; it ispredominantly found in neuronal tissue and is also highly

expressed in skeletal muscle. Inducible NOS (iNOS or NOS2)is expressed on demand in a wide range of cells (particularlymacrophages) and tissues, whereas endothelial NOS (eNOS or

NOS3) was initially described in vascular endothelial cells buthas subsequently been shown to also be expressed in other celltypes, including cardiomyocytes and adipocytes (Forstermann

and Sessa, 2012; Tanaka et al., 2003; Wei et al., 1996). NOSs arethe products of distinct genes and have different regulation,catalytic properties and inhibitor sensitivities. The three NOSisoforms function as homodimers and catalyse the two-step

oxidation of L-arginine to citrulline and NO. The NOS monomer

exhibits a bidomain structure; it has an N-terminal oxygenasedomain, which contains the binding sites for haem and L-

arginine, and a C-terminal reductase domain, which containsbinding sites for the essential cofactors flavin adeninedinucleotide (FAD), flavin mononucleotide (FMN) and

nicotinamide adenine dinucleotide phosphate (NADPH). nNOScontains an additional PDZ (postsynaptic density protein 95/discslarge/ZO-1 homology domain) domain located at the N-terminus,through which it can directly interact with the PDZ domains of

other proteins. These interactions determine the subcellulardistribution and activity of nNOS (Fig. 1) (Zhou and Zhu,2009; Aquilano et al., 2014).

The mechanism by which NO exerts its physiological action ispeculiar and considerably different from that of other second

messengers. Once produced, NO can exist as reduction andoxidation products (i.e. NO2, NO+) and, owing to its highdiffusibility and permeability, it does not need to be transported

through membrane carriers or pores. Furthermore, NO does notneed to interact with intracellular or extracellular receptors, as isthe case for other second messengers, in order to exert itsbiological function. NO has been implicated in several

processes, including fertilisation, embryogenesis, modulation ofneurotransmission, vasodilatation and inflammatory response(Alderton et al., 2001).

In the past two decades, NO has been receiving increasingattention. Besides canonical NO signalling, which consists of the

selective activation of soluble guanylate cyclase (sGC) andinhibition of cytochrome c oxidase through the interaction of NOwith haem, other signalling modes have been discovered,

including the inhibition of mitochondrial respiratory complexesand the Krebs cycle enzyme aconitase (Cooper, 1999; Valdez etal., 2000). Moreover, S-nitrosylation of certain reactive proteincysteines has been recognised as another fundamental aspect of

NO-mediated signalling (Stamler et al., 1992). This has opened anew area of research and has tremendously increased thespectrum of biological activities of NO and its targets. For

instance, NO has been implicated in mitochondrial biogenesis andlipid metabolism (Aquilano et al., 2014; Schild et al., 2006). Amaster regulator of both mitochondrial function and lipid

catabolism is peroxisome proliferator activated receptor ccoactivator 1a (PGC-1a) (Nisoli et al., 2004; Piantadosi andSuliman, 2012); it acts as a coactivator of a wide array of

transcription factors that are involved in the expression ofoxidative phosphorylation (OXPHOS) and mitochondrial fattyacid oxidation genes (Aquilano et al., 2013b; Lettieri Barbato etal., 2014). Particularly in skeletal muscle, PGC-1a plays a key

role in coordinating the oxidation of intracellular fatty acids withmitochondrial remodelling (Scarpulla et al., 2012) and, for thisreason, modulation of NO metabolism and/or targeting of PGC-

1a could be a promising strategy to combat several myopathiesthat are associated with altered mitochondrial metabolism.However, in our opinion, the intersection between nNOS- and

PGC-1a-governed pathways in skeletal muscle remains only

1Scientific Institute for Research, Hospitalization and Health Care, UniversitaTelematica San Raffaele Roma, Via di Val Cannuta, 00167, Rome, Italy.2Department of Biology, University of Rome ‘Tor Vergata’, Via della RicercaScientifica, 00133 Rome, Italy. 3IRCCS San Raffaele ‘La Pisana’, Via di ValCannuta, 00166, Rome, Italy.

*Authors for correspondence (katia.aquilano@uniroma2.it;ciriolo@bio.uniroma2.it)

� 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 4813–4820 doi:10.1242/jcs.154229

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poorly emphasised at present. Here, we review the mostimportant metabolic routes that are governed by nNOS and

PGC-1a, in order to highlight how they cooperatively modulateskeletal muscle contraction and oxidative metabolism.

Localisation and function of NOS in skeletal muscleIn skeletal muscle, nNOS and eNOS isoforms are expressedconstitutively, whereas iNOS is only expressed during

inflammatory responses, such as those occurring in individualswith type 2 diabetes (Torres et al., 2004). Signalling by the nNOSsplice variant nNOSm (Fig. 1) is essential for skeletal musclehealth and is commonly reduced in neuromuscular diseases,

including Duchennes’s dystrophy (Brenman et al., 1995). nNOSmis thought to be the predominant source of NO in skeletal muscle.However, it has been reported that the nNOSb variant, lacking

the PDZ domain, is also present and has a crucial role in cellsignalling (Percival et al., 2010). In skeletal muscle, the differentnNOS splicing variants have important roles in the regulation of

many muscle functions, including blood flow, contraction andmuscle metabolism (Suhr et al., 2013).

The physiological functions of NO have been initiallyexplained by it being a freely diffusible messenger that acts on

targets that are distant from its site of synthesis (Lancaster,1994). However, skeletal muscle displays high concentrations ofpotent NO scavengers, such as myoglobin or glutathione (GSH),

which could considerably limit diffusion-based NO signalling(Flogel et al., 2001). In addition, NO is extremely reactive with awide range of biomolecules; therefore, the specificity of NO

signalling requires that NOSs are located in the vicinity of NOeffector protein targets (Sullivan and Pollock, 2003; Zhou andZhu, 2009). For example, in myotubes, anchoring of nNOSm at

the plasma membrane through specific interaction with thescaffold protein a-syntrophin, which forms part of thedystrophin-associated glycoprotein complex (Fig. 2i), assuresthe coupling of NO production to Ca2+ influx, which is essential

for skeletal muscle contraction (Brenman et al., 1996; Thomaset al., 1998). The sarcolemmal nNOSm has a higher activity intype II (fast twitch) fibres than in type I (slow twitch) fibres.

Furthermore, by acting in synergy with eNOS that is expressed

in the blood vessels, sarcolemmal NOSm enhances bloodflow and oxygen delivery, thereby more efficiently matchingthe blood supply to the metabolic demands of active

muscle (Thomas et al., 2003). In particular, NO that is derivedfrom sarcolemmal nNOSm finely attenuates a-adrenergicvasoconstriction and increases blood supply in contracting

skeletal muscle (Thomas et al., 1998; Thomas et al., 2003).Indeed, mice with reduced sarcolemmal nNOSm show aconsiderable decrease in blood supply in electrically evoked

muscle contraction, with consequent functional ischemia andfatigue, as well as reduced exercise capacity (Thomas et al.,2003). nNOSm has also been shown to be present in amembrane-unbound form in gastrocnemius homogenates

(Thomas et al., 2003). However, in the absence ofsarcolemmal nNOSm, the soluble form is not able tocompensate for the loss of its sarcolemmal counterpart in

contractile performance (Thomas et al., 1998). However, theexact function of cytoplasmic nNOSm remains to be elucidatedfully.

COOHNH2

17 99 730 774 1434

FMN NADPHFAD

CaM

HaemPDZ

Oxygenasedomain

Reductasedomain

nNOSa (160 kDa)

BH4NOSIP1 205

163 245PIN

nNOSb (136 kDa)

nNOSg (125 kDa)

236 336

844

+34aanNOSm (165 kDa)

Fig. 1. Molecular structure of nNOS and its splice variants. Active NOSenzymes are homodimers that are formed of monomers containing a haem-oxygenase domain and a reductase domain (as shown). The reductase domaincomprises binding sites for NADPH, FAD and FMN. The binding oftetrahydrobiopterin (BH4) and Ca2+–calmodulin (CaM) to specific domains is alsoessential for the catalytic activity of nNOS. Different splice variants of nNOS exist,with different molecular masses, as illustrated in the figure. The skeletal musclevariant nNOSm contains an insertion of 34 amino acids (aa) with respect to nNOSa.nNOS can be inhibited by interaction with nitric oxide synthase-interacting protein(NOSIP) and protein inhibitor of neuronal nitric oxide synthase (PIN, also known asDYNLL1) through the nNOSN-terminal domain. The illustrated primary structure ofhuman nNOS is based on UniProt data (entry P29475).

Fig. 2. Schematic representation of the subcellular distribution andfunction of nNOS isoforms in skeletal muscle. (i) PDZ-containing nNOS(nNOSm) is located at the sarcolemma and is part of the dystrophin complexthrough its interaction with a-syntrophin (a-synt). NO that is produced herefacilitates vasodilatation, thus ensuring the correct delivery of oxidisablesubstrates for energy production. (ii) PDZ-lacking nNOS (nNOSb) retains fullNO synthesising activity; it localises at the Golgi and is involved in thecontractile activity of the muscle cell. (iii) The dystrophin complex alsolocalises to the inner membrane of the nuclear envelope (Gonzalez-Ramırezet al., 2008) and recruits nNOSm to the nucleus through the interaction of itsPDZ domain with a-syntrophin. The a-syntrophin-mediated nNOSmrecruitment and subsequent NO synthesis promotes S-nitrosylation ofnuclear proteins, including the transcription factor CREB . (iv) S-nitrosylatedCREB (CREB-SNO) induces the transcription of PGC-1a and of thedownstream mitochondrial oxidative phosphorylation genes, therebyultimately resulting in mitochondrial biogenesis. (v) The mitochondrialisoform of nNOS (mtNOS) locates inside mitochondria and modulates theactivity of the electron transport chain (ETC) owing to the reversibleinteraction of NO with haem and iron–sulphur groups and S-nitrosylation ofmitochondrial protein complexes. DGs, dystroglycans; OXPHOS, oxidativephosphorylation proteins; SGs, sarcoglycans.

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As mentioned above, skeletal muscle cells also express thePDZ-lacking nNOS splice variant nNOSb, which is mostly

soluble but has also been found to localise at the Golgi complexin mice (Percival et al., 2010) (Fig. 2ii). In contrast to musclesthat lack only nNOSm, muscles lacking both nNOSm and nNOSbshow severe myopathy and exhibit microstructural cellular

defects of the cytoskeleton, Golgi complex and mitochondria(Percival et al., 2010). Morphologically, skeletal muscles thatlack both nNOSm and nNOSb have a reduced mass, are

intrinsically weak and highly susceptible to fatigue, andmanifest marked post-exercise weakness (Percival et al., 2010),suggesting that nNOSb also modulates the ability of skeletal

muscle to maintain force production during and after exercise.It has been reported that nNOS also localises in the nucleus

(Rothe et al., 1999; Riefler and Firestein, 2001). Work from our

group has confirmed this by showing the presence of nNOS in thenucleus of skeletal muscle cells, where it is involved intranscriptional regulation by acting as an inhibitor of theconstitutive transcription factor Sp1 (Baldelli et al., 2008). The

nuclear expression of nNOS impairs the binding of Sp1 to thepromoter of the sod1 gene, which encodes copper, zincsuperoxide dismutase (SOD1). However, inhibition of SOD1

expression is not triggered by NO signalling, because theadministration of specific nNOS inhibitors did not restoreSOD1 expression (Baldelli et al., 2008). We have also

demonstrated that the formation of the nNOS–Sp1 complex atthe sod1 promoter is promoted by the PDZ domain of nNOS.Indeed, the expression of an nNOS mutant that lacks the PDZ

domain (DnNOS nNOSb) dramatically reduces the interaction ofnNOS with Sp1. Interestingly, we found that the loss of nNOS–Sp1 complex formation is due to the inability of DnNOS nNOSbto translocate to the nucleus (Baldelli et al., 2011), raising the

intriguing question of the importance of nNOS splicing inregulating its nuclear function. More recently, we havedemonstrated that nNOS recruitment to the nucleus through its

PDZ domain is generally applicable, as nNOS can be targeted tothe nucleus in neuroblastoma and HeLa cells and, moreimportantly, in myocytes (Aquilano et al., 2014).

The regulation of gene expression by NO is well established,and S-nitrosylation of transcription factors has been suggested tobe the main mechanism through which their activity is affected.S-nitrosylation of nuclear proteins has been predominantly

ascribed to the trans-S-nitrosylation activity of glyceraldehyde3-phosphate dehydrogenase (GAPDH) (Kornberg et al., 2010).However, the exact function of nuclear nNOS has remained

elusive even though compelling evidence has shown that many ofthe factors that are involved in its activity (e.g. sGC subunits,calmodulin and enzymes involved in tetrahydrobiopterin

synthesis) are present in the nucleus (Bachs et al., 1992;Elzaouk et al., 2004; Pifarre et al., 2009). Besides regulatingtranscription by interacting with transcription factors (i.e. by the

formation of nNOS–Sp1 complex), we have found that, duringmyocyte differentiation, nuclear nNOS-derived NO functions bydriving mitochondrial biogenesis (Aquilano et al., 2014). In fact,in differentiating myocytes, nNOS expression is induced, which

is accompanied by its redistribution to the nucleus and increasedS-nitrosylation of nuclear proteins, including cAMP responseelement-binding protein (CREB). S-nitrosylated CREB (CREB-

SNO) more efficiently engages with the promoter of the geneencoding PGC-1a, which contains a CREB consensus sequence.This results in a strong induction of mitochondrial biogenesis,

which is crucial for myocyte differentiation and function

(Fig. 2iii,iv). Although nNOSb (lacking the PDZ domain) fullyretains the ability to synthesise NO, this construct is unable to

induce mitochondrial biogenesis because it fails to be recruited tothe nucleus and is thus unable to favour CREB S-nitrosylationand subsequent PGC-1a-mediated mitochondrial biogenesis(Aquilano et al., 2014).

We have also shown that nuclear a-syntrophin is the genuinemediator of nuclear recruitment of nNOS (Aquilano et al., 2014)(Fig. 2). By knocking down a-syntrophin in differentiating

myocytes, we have shown that lack of nNOS targeting to thenucleus through a-syntrophin was the underlying cause ofimpaired mitochondrial biogenesis in these cells.

In conclusion, these findings suggest that an adequate level ofexpression of both nNOSm and nNOSb is important for theregulation of contractile activity and in controlling fatigue.

Furthermore, in addition to sarcolemmal anchoring, a-syntrophin-mediated nNOS anchoring to the nuclear envelope alsoparticipates in NO signalling that promotes mitochondrialbiogenesis and differentiation of skeletal muscle cell precursors.

Therefore, the control of nNOS splicing and its subcellularlocalisation in skeletal muscle could represent a novel therapeuticavenue to prevent or treat myopathies.

The role of nitric oxide in skeletal muscle cell metabolismNO influences myocyte metabolism by multiple means; it

promotes glucose uptake, while inhibiting mitochondrialrespiration, glycolysis and phosphocreatine breakdown. Each ofthe activities of NO is likely to involve a distinct molecular target

and has been shown to be reversible (Stamler and Meissner, 2001).

Regulation of mitochondrial electron transport chain complexesThe best-known effect of NO on skeletal muscle metabolism is its

capacity to inhibit mitochondrial respiration directly byinterfering with electron transport chain complexes in severalways; (1) NO inhibits cytochrome c oxidase activity by

competing with O2, (2) NO inhibits electron transfer betweencytochrome b and c (both belonging to Complex III) andincreases mitochondrial production of O2N2, and (3) NO inhibits

electron transfer and NADH-dehydrogenase function at the levelof Complex I (Grozdanovic, 2001; Sarti et al., 2012a).

The decrease in mitochondrial respiration has a crucial role inmany pathological states, including conditions that are linked to

skeletal muscle abnormalities. In fact, reduced mitochondrialenergy production and decreased energy expenditure contributelargely to metabolic dysfunctions that are typical of aging,

including insulin resistance and diabetes, which ultimately lead tolipid and glycogen accumulation, in turn further increasinginsulin resistance (Derave et al., 2000; Finocchietto et al., 2008).

At low concentrations of O2, NO binds to the haem ofcytochrome c oxidase, thus acting as a competitive inhibitor ofO2. By contrast, at high O2 concentrations, NO binds to oxidised

cytochrome c oxidase through the copper moiety of the binuclearcentre, instead of the iron moiety, thus producing nitrite (NO2

2)and consuming O2 (Brown, 1995; Sarti et al., 2012b). At the earlyphase of NO production, the persistent inhibition of cytochrome c

oxidase by NO can promote the release of small amounts ofhydrogen peroxide, which itself acts as a signalling molecule andinduces adaptive defensive responses. However, at later phases of

NO generation, hydrogen peroxide is produced at higherconcentrations, resulting in the formation of peroxynitrite thatinduces degeneration of skeletal muscle mass (Cleeter et al.,

1994).

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NO inhibits succinate-cytochrome c reductase and NADH-cytochrome c reductase in skeletal muscle (Cadenas, 2004;

Poderoso et al., 1996). NO is also able to reversibly reducecytochrome b, owing to its interaction with the iron–sulphurcentre (Boveris et al., 2000). Even though the mechanisms are notcompletely clear, several studies have shown that prolonged

exposure to NO can result in the inhibition of Complex I activityin skeletal muscle (Brown and Borutaite, 2004; Stamler andMeissner, 2001), which might be mediated by tyrosine nitration,

S-nitrosylation and damage to Fe–S centres that displaceselemental sulphur or cysteine residues (Brown and Borutaite,2004).

The importance of NO in regulating mitochondrial activity hasbeen emphasised by the discovery of the presence of amitochondrial NOS isoform (mtNOS) (Fig. 2v) (Elfering et al.,

2002). mtNOS localises to mitochondria of several tissues,including skeletal muscle, where it regulates oxygen consumption(Aguirre et al., 2012; Finocchietto et al., 2008). At least inskeletal muscle, mtNOS appears to be a mitochondrial form of

nNOS and can regulate mitochondrial respiration. In particular, ithas been demonstrated that, under certain circumstances (i.e.upon hyperinsulinemia), mtNOS can be activated in situ by Akt,

leading to decreased mitochondrial respiration in the muscle(Finocchietto et al., 2008). Hence, persistent mtNOS activationcould contribute to mitochondrial dysfunction upon insulin

resistance and, therefore, to the progression of the metabolicsyndromes (Finocchietto et al., 2008).

Regulation of carbohydrate metabolismAnother aspect of NO-mediated regulation of skeletal musclemetabolism is at the level of glucose uptake. NO stimulatesglucose transport by activating upstream signalling events that

result in increased amounts of the glucose transporter GLUT4 atthe cell surface (Etgen et al., 1997). GLUT4-mediated glucoseuptake is mainly achieved through the activation of cGMP- and

59-AMP-activated protein kinase (AMPK) (Lira et al., 2007). Inaddition, inhibition of NOSs limits the uptake of glucose inskeletal muscle, both under basal conditions and during physical

activity, thereby impairing anaerobic skeletal muscle ATPproduction (Balon and Nadler, 1997; Higaki et al., 2001; Rosset al., 2007). Moreover, under resting conditions, NO mightregulate carbohydrate metabolism by reversibly inhibiting the

glycolytic enzyme GAPDH by mediating its S-nitrosylation (Haraet al., 2005; Hara et al., 2006). Finally, NO inhibits thebreakdown of phosphocreatine, which is mediated by creatine

kinase, thereby decreasing ATP synthesis through this pathway(Gross et al., 1996; Wolosker et al., 1996).

The functional role of PGC-1a in skeletal muscle cellsPGC-1a is a transcriptional coactivator, encoded by thePPARGC1A gene, that was first identified as an interacting

partner of the peroxisome proliferator-activated receptor gamma(PPARc) in adipocytes of brown adipose tissue (Puigserver et al.,1998). Later, PGC-1a was found to be expressed in other tissuesthat are rich in mitochondria and have high energy demands, such

as cardiac and skeletal muscle, kidney, liver and brain (Austinand St-Pierre, 2012). In these tissues, PGC-1a controls theexpression of genes that are tailored to the immediate energy

demands of the organism and are involved in gluconeogenesis,lipogenesis and peroxisomal and mitochondrial fatty acidoxidation. Undoubtedly, PGC-1a plays a crucial role in

maintaining muscle metabolic function and controls numerous

genes that impact on muscle morphology and physiologicalfunction (Baar, 2004; Pilegaard et al., 2003).

PGC-1a represents the master regulator of mitochondrialbiogenesis, as it is an upstream inducer of mitochondrialmetabolism, acting to positively influence the activity of somehormone nuclear receptors (PPARs and ERRa) and nuclear

transcription factors [nuclear respiratory factor 1 and 2 (NRF-1and NRF-2)], which enhance the expression of OXPHOScomponents and mitochondrial transporters and transcription

factors [mitochondrial transcription factor A (TFAM), TFBM1and TFBM2]. For a long time, the coordination of the twogenomes (nuclear versus mitochondrial) was considered to be

exclusively achieved by the nucleus-encoded proteins TFAM,TFB1M and TFB2M; among these, TFAM was believed to beessential for transcription, replication and maintenance of

mitochondrial DNA (mtDNA) (Lu et al., 2013). Importantly,we have demonstrated that PGC-1a also resides in mitochondriaand, at the mitochondrial matrix, associates with TFAM and withmtDNA, thereby coactivating the transcription of mtDNA-

encoded genes (Pagliei et al., 2013).When expressed ectopically, PGC-1a induces mitochondrial

biogenesis and increases cellular respiration in cell cultures. For

instance, Wu and colleagues have shown that ectopic expressionof PGC-1a in the skeletal muscle cell lineage induces asignificant increase in mtDNA content and, in parallel, a rise in

mitochondrial density (Wu et al., 1999). Here, the PGC-1a-mediated mitochondrial biogenesis occurs in parallel with anincrease in the basal oxygen consumption and in the expression of

OXPHOS genes (i.e. nucleus-encoded cytochrome c oxidasesubunit IV and mitochondria-encoded cytochrome c oxidasesubunit II) (Wu et al., 1999).

PGC-1a is upregulated by muscle contraction and is involved

in most of the metabolic adaptations that occur in muscle (Genget al., 2010; Lira et al., 2010a; Lira et al., 2010b). A bout ofexercise increases the nuclear abundance of PGC-1a, which is

responsible for the activation of the entire programme ofmitochondrial and metabolic adaptations, including an enhancedcapacity for substrate transport and oxidation (Fig. 3) (Little

et al., 2011). Accordingly, overexpression of PGC-1a in skeletalmuscle recapitulates many aspects of endurance trainingadaptation (Koves et al., 2005). Moreover, an acute enduranceexercise in mice promotes the fast import of PGC-1a into

mitochondria and increases the expression of TFAM-inducedmitochondrial genes (Safdar et al., 2011). This mitochondrialevent has been found to be associated with an increased activity

of nuclear PGC-1a, thereby facilitating the concomitanttranscription of nucleus-encoded mitochondrial genes (Fig. 3).In skeletal muscle, mitochondria exist as two subcellular

populations (Smith et al., 2013) – subsarcolemmal andintermyofibrillar mitochondria – among which the former moreefficiently respond to exercise training. Recently, it has been

reported that, in humans, as well as in rats, acute exerciseincreases the levels of both PGC-1a and TFAM exclusively insubsarcolemmal mitochondria (Smith et al., 2013). One of themost important regulators of PGC-1a is AMPK, which serves as

an energy sensor under conditions of low energy charge (i.e.increased AMP:ATP ratio). Thus, conditions that causesignificant cellular energy stress, such as exercise, are able to

increase AMPK activity and promote mitochondrial biogenesisthrough AMPK-mediated PGC-1a phospho-activation (Reznickand Shulman, 2006; Steinberg and Kemp, 2009). The use of

AMPK agonists or AMPK inhibition in rodents has made it

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possible to identify the signalling mechanisms that regulate thetranslocation of PGC-1a to mitochondria during exercise

(Smith et al., 2013). In particular, the exposure of rats tothe AMPK agonist 5-aminoimidazole-4-carboxamide-1-beta-ribofuranoside recapitulates the effect of exercise in

increasing the levels of PGC-1a in only subsarcolemmalmitochondria, indicating that AMPK signalling is likely to beinvolved. In addition, the inactivation of AMPK prevents

exercise-induced PGC-1a translocation to subsarcolemmalmitochondria, further suggesting that AMPK has a pivotalrole in modulating PGC-1a translocation (Smith et al., 2013).Thus, taken together, these findings place PGC-1a at centre

stage of mitochondrial–nuclear communications that coordinatethe efficient expression of mitochondrial genes in order to meetthe higher metabolic needs of exercised skeletal muscle

(Fig. 3).The regulation of proteins involved in fat metabolism is

another process in which PGC-1a is involved in skeletal muscle.

In fact, once overexpressed, PGC-1a enhances the transcriptionof mRNA encoding enzymes involved in fat oxidation, includingcarnitine palmitoyltransferase and medium-chain acyl-coenzyme

A dehydrogenase (Calvo et al., 2008; Vega et al., 2000).In response to exercise and other metabolic stresses (e.g.

ischemia), PGC-1a controls an angiogenic pathway that is

aimed at delivering oxygen and substrates to skeletal musclefor maintaining adequate energy production upon increased

energetic needs (Arany et al., 2008). PGC-1a mediatesthe induction of vascular endothelial growth factor (VEGF), theprimary mediator of the angiogenic process. Remarkably, theinduction of VEGF by PGC-1a does not involve the canonical

hypoxia response pathway or hypoxia inducible factor (HIF).Instead, PGC-1a coactivates the orphan nuclear receptor ERRa,which binds to conserved binding sites located in the promoter of

the VEGF gene. Mice lacking PGC-1a show a dramatic energeticfailure upon ischemic insult, whereas PGC-1a-overexpressingmice more rapidly reconstitute blood flow in skeletal muscle

(Arany et al., 2008).

The intersection of PGC-1a and the NO pathway in skeletalmuscle metabolismPGC-1a and NO in the regulation of mitochondrial biogenesisIt is well established that NO donors and cGMP analoguesincrease mitochondrial biogenesis in muscle cells. Initially, NO

was implicated in this process based on work that found a role foreNOS-derived NO in the differentiation of brown adipose tissue(BAT) (Nisoli et al., 2003). Several groups have shown that

treatment of muscle cells with NO donors increasesmitochondrial markers, demonstrating that NO induces thesynthesis of new mitochondria that are able to generate ATP by

oxidative phosphorylation (Lira et al., 2010b; McConell et al.,2010). It has been demonstrated that, in cultured skeletal musclecells, NO donors increase mitochondrial biogenesis and function

(Nisoli et al., 2004; Tengan et al., 2007). These effects of NO onskeletal muscle mitochondrial biogenesis appear to be mainlymediated by AMPK and Ca2+ (Lira et al., 2010b). However, theexact molecular pathways involved in mitochondrial biogenesis

in response to NO production are not entirely understood, but inmany tissues and cells, including skeletal muscle cells, they havebeen ascribed to PGC-1a (Lira et al., 2010b). As discussed above,

stimulation of mitochondrial biogenesis by NO also requires theexpression of transcription factors, including CREB (Ventura-Clapier et al., 2008). Although the serine/threonine kinase Akt

and protein kinase A (PKA) are able to phosphorylate andactivate eNOS, thus leading to an increase in NO production,phosphorylation of CREB by PKA results in expression of itstarget gene PPARGC1A and thus in mitochondrial biogenesis.

Furthermore, we have shown that CREB binding to thePPARGC1A promoter is greatly increased when CREB is alsoS-nitrosylated (Aquilano et al., 2014). In addition, Akt might also

induce mitochondrial biogenesis through the phosphorylation ofNRF-1 and CREB, thus enabling their nuclear translocation andactivation of target genes such as TFAM (Piantadosi and

Suliman, 2012).AMPK is also involved in NO-dependent regulation of PGC-

1a and mitochondrial biogenesis. In particular, treatment of

myotubes with NO donors results in the inhibition ofmitochondrial ATP production and an increase in theAMP:ATP ratio (Lira et al., 2010b). This event leads toAMPK phospho-activation, which is responsible for PGC-1aphosphorylation and PGC-1a-mediated induction of theexpression of mitochondrial genes (Lira et al., 2010b) (Fig. 3).Moreover, Ca2+-mediated signalling pathways are also able to

induce mitochondrial biogenesis by activating NO synthesis orstimulating Ca2+-dependent transcription factors. In fact, exercisein humans induces phosphorylation of nNOSm by AMPK, leading

to nNOSm activation, increased NO production and mitochondrial

Fig. 3. Role of PGC-1a in coordinating the expression of mitochondrialgenes during physical exercise. During physical exercise, PGC-1ainteracts with the transcription factors NRF1 and NRF2 and induces theexpression of nucleus-encoded mitochondrial genes, including thoseencoding TFAM and proteins involved in oxidative phosphorylation(OXPHOS). In parallel, the increase in NO, due to nNOS and eNOSactivation, can limit mitochondrial respiration, increase the ratio betweenAMP and ATP and lead to AMPK phospho-activation. Activated AMPK isresponsible for the phosphorylation of PGC-1a, thus mediating itstranslocation to the subsarcolemmal mitochondrial matrix of skeletal muscle.Here, PGC-1a interacts with TFAM on mtDNA and coactivates thetranscription of mitochondria-encoded mitochondrial genes.

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biogenesis, and increased glucose uptake (Chen et al., 2003).AMPK is also able to phosphorylate and activate eNOS during

contraction of human skeletal muscle (Chen et al., 1999). Takentogether, these findings strongly indicate that phosphorylation ofboth nNOSm and eNOS by AMPK might be involved inmitochondrial biogenesis. Accordingly, treatment with a NO

donor could increase the activation of AMPK and mitochondrialbiogenesis, whereas the pharmacological inhibition of NOSattenuates these effects (McConell et al., 2010).

However, it is worth mentioning that upregulation of PGC-1aexpression does not always require NO signalling. For example,Wadley et al. (Wadley et al., 2007) have reported that normal

skeletal muscle of nNOS- and eNOS-knockout mice harboursbaseline levels of PGC-1a expression. Furthermore, normalupregulation of PGC-1a was elicited in these mice in response

to acute exercise, indicating that NOS-mediated NO signalling ispart of a redundant system of metabolic regulation in skeletalmuscle (Wadley et al., 2007).

PGC-1a and NO in the regulation of oxidative stressThe production of reactive oxygen species (ROS) and NO-derived reactive species (RNS) is an integral part of skeletal

muscle metabolism (Westerblad and Allen, 2011). At lowconcentrations, ROS and RNS might function as signallingmolecules; thus NO can act as an antioxidant by directly

scavenging more-damaging species, such as hydroxyl radicals(Aquilano et al., 2007a). Physiologically, the cell counteracts anexcess of ROS or RNS by enhancing its antioxidant defence

system, which includes superoxide dismutases and the enzymesinvolved in GSH metabolism (e.g. GSH peroxidase, c-glutamylcysteine ligase). An imbalance between ROS or RNS levels andantioxidant defence leads to oxidative stress and might result in

cell death (Aquilano et al., 2007b). However, a controlled mildflux of ROS or RNS is a central means of inducing redox-sensitive signalling pathways that act to fine-tune the metabolic

adaptive response by enhancing energy request during musclecontraction and regulating the cellular defence against thedamaging effects of oxidative stress. Indeed, upregulation of

endogenous antioxidant defence systems and complex regulationof repair systems such as those involving heat shock proteins(HSP70, HSP27, HO-1) are seen in response to training andexercise (Fehrenbach and Northoff, 2001). For this reason, it has

become evident that physical exercise can ameliorate not onlyskeletal muscle function but can also have beneficial effects onsystemic physiology, subsequently leading to improved health

and increased lifespan (Mercken et al., 2012).Nutrient starvation or caloric restriction is broadly applicable

and extends the life of most species through the retardation of

aging, thereby exerting beneficial effects on virtually all cells andtissues (Lettieri Barbato et al., 2012). It is well established thatcaloric restriction improves skeletal muscle function to levels that

are comparable to that of physical exercise, thus favouringmitochondrial biogenesis and oxidative metabolism andrestraining insulin resistance (Mercken et al., 2012). Thebeneficial effect of caloric restriction on lifespan has been

linked to the increase in NO production. In particular, caloricrestriction increases both eNOS and nNOS activity (Nisoli et al.,2005; Fusco et al., 2012). In our laboratory, we have shown that,

in skeletal muscle, caloric restriction also increases NObioavailability independently of NOS upregulation (Aquilanoet al., 2013a). In particular, caloric restriction reduces the level of

GSH, the main intracellular NO buffer (Aquilano et al., 2011a;

Aquilano et al., 2011b; Baldelli et al., 2008). The resultingincreased NO bioavailability is the genuine mediator of the

upregulation of PGC-1a, which, in turn, favours the expression ofantioxidant proteins SOD2 and c-GCS (also known as glutamatecysteine ligase) (Aquilano et al., 2013a).

ConclusionsIt has become increasingly clear that NO is a crucial player inskeletal muscle physiology, as it regulates a variety of highly

relevant pathways to maintain both skeletal muscle integrity andproper signalling mechanisms during adaptation to mechanicaland metabolic stimulation (i.e. exercise and caloric restriction).

The expression and localisation of a specific nNOS isoform isessential for modulating the activity of the electron transportchain, mitochondrial biogenesis, glucose uptake and utilisation,

and fatty acid oxidation. Together with these effects, and morestrictly related to energetic metabolism, nNOS in skeletal musclecan also regulate the delivery of local oxygen and nutrients byinducing vasodilatation as well as angiogenesis (Huber-Abel

et al., 2012). The metabolic regulator PGC-1a is at the crossroadsof nNOS-mediated signalling and associated adaptationprocesses, as NO directly induces PGC-1a expression,

thus augmenting mitochondrial biogenesis (Aquilano et al.,2014), while, at the same time, favouring VEGF-mediatedvascularisation (Arany et al., 2008; Rowe et al., 2011). With

the finding that exercise and caloric restriction exert particularlybeneficial outcomes in the activation of the nNOS enzyme andPGC-1a, it will be interesting in future studies to focus on the

underlying mechanisms that might determine the adaptiveresponse of skeletal muscle to physical exercise underpathological conditions.

AcknowledgementsM.R.C. and K.A. contributed equally to this work.

Competing interestsThe authors declare no competing interests.

FundingThis work was partially supported by Ministero dell’Istruzione, dell’Universita edella Ricerca (PRIN 2012) and Ministero della Salute [grant number GR-2008-1138121].

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