Author’s Accepted Manuscript AMPK signaling in skeletal muscle during exercise: role of reactive oxygen and nitrogen species David Morales-Alamo, Jose A L Calbet www.elsevier.com

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S0891-5849(16)00013-7 http://dx.doi.org/10.1016/j.freeradbiomed.2016.01.012 FRB12723

To appear in: Free Radical Biology and Medicine Received date: 22 November 2015 Revised date: 15 January 2016 Accepted date: 18 January 2016 Cite this article as: David Morales-Alamo and Jose A L Calbet, AMPK signaling in skeletal muscle during exercise: role of reactive oxygen and nitrogen species, Free Radical Biology and Medicine, http://dx.doi.org/10.1016/j.freeradbiomed.2016.01.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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AMPK signaling in skeletal muscle during exercise: role of reactive oxygen and nitrogen species

David Morales-Alamo1,2* & Jose A L Calbet1,2

1

Department of Physical Education, University of Las Palmas de Gran Canaria, Campus Universitario de Tafira s/n,

Las Palmas de Gran Canaria, Canary Island, Spain.

2

Research Institute of Biomedical and Health Sciences (IUIBS), University of Las Palmas de Gran Canaria, Campus

Universitario de Tafira s/n, Las Palmas de Gran Canaria, Canary Island, Spain.

Running title: Exercise-release RONS and regulate AMPK activation

*Correspondence to: , David Morales-Alamo, Departamento de Educación Física, Campus Universitario de Tafira, , 35017 Las Palmas de Gran Canaria, Canary Island, Spain., Tel: 0034 928 458 896, Fax: 0034 928 458 867, email: [email protected]

Abstract Reactive oxygen and nitrogen species (RONS) are generated during exercise depending on intensity, duration and training status. A greater amount of RONS is released during repeated high-intensity sprint exercise and when the exercise is performed in hypoxia. By activating adenosine monophosphate-activated kinase (AMPK), RONS play a critical role in the regulation of muscle metabolism but also in the adaptive responses to exercise training. RONS may activate AMPK by direct an indirect mechanisms. Directly, RONS may activate or deactivate AMPK by modifying RONS-sensitive residues of the AMPK-α subunit. Indirectly, RONS may

2 activate AMPK by reducing mitochondrial ATP synthesis, leading to an increased AMP:ATP ratio and subsequent Thr172-AMPK phosphorylation by the two main AMPK kinases: LKB1 and CaMKKβ. In presence of RONS the rate of Thr172-AMPK dephosphorylation is reduced. RONS may activate LKB1 through Sestrin2 and SIRT1 (NAD+/NADH.H+-dependent deacetylase). RONS may also activate CaMKKβ by direct modification of RONS sensitive motifs and, indirectly, by activating the ryanodine receptor (Ryr) to release Ca2+. Both too high (hypoxia) and too low (ingestion of antioxidants) RONS levels may lead to Ser485-AMPKα1/ Ser491-AMPKα2 phosphorylation causing inhibition of Thr172-AMPKα phosphorylation. Exercise training increases muscle antioxidant capacity. When the same high-intensity training is applied to arm and leg muscles, arm muscles show signs of increased oxidative stress and reduced mitochondrial biogenesis, which may be explained by differences in RONS-sensing mechanisms and basal antioxidant capacities between arm and leg muscles. Efficient adaptation to exercise training requires optimal exposure to pulses of RONS. Inappropriate training stimulus may lead to excessive RONS formation, oxidative inactivation of AMPK and reduced adaptation or even maladaptation. Theoretically, exercise programs should be designed taking into account the intrinsic properties of different skeletal muscle, the specific RONS induction and the subsequent signalling responses.

Contents 1. Introduction 2. AMPK activation/inhibition mechanisms 2.1. Canonical AMPK activation 2.2. Non-Canonical AMPK activation 2.2.1. Calcium/Calmodulin-dependent kinase kinase β (CaMKKβ)

3 2.2.2. Calcium/Calmodulin-dependent kinase II (CaMKII) 2.2.3. Sirtuin 1 (SIRT1) 2.2.4. Transforming growth factor-β-activated kinase 1 (TAK-1) 2.2.5. v-ATPase (vacuolar H+-ATPase) 2.3. Activation of AMPK by RONS 2.4. Inhibition of AMPK by serine phosphorylation of the α subunits 3. Endurance exercise and AMPK activation 4. High-intensity exercise and AMPK activation 4.1. Regional differences in the muscular adaptations to sprint training are mediated by RONS-sensitive mechanisms 5. Future studies Acknowledgements References

1. Introduction Adenosine monophosphate-activated kinase (AMPK) is considered a metabolic master switch, which turns off several anabolic processes at the same time that turns on catabolic processes (Winder & Hardie, 1999). AMPK is usually activated, in response to hypoxia, exhausting exercise, and caloric restriction (Mantovani & Roy, 2011; Speakman & Mitchell, 2011). This is accompanied by activation of several metabolic pathways in an attempt to acutely increase the energy level of the cell (Merrill et al., 1997). Concomitantly, additional signalling pathways are activated to elicit chronic adaptations, which produce a switch to a more oxidative phenotype (Winder et al., 2000) ultimately increasing the chances of survival (Carling & Viollet, 2015).

4 AMPK has become a therapeutic target since AMPK activation promotes Glut4 translocation and mitochondrial biogenesis in skeletal muscle (Richter & Ruderman, 2009). However, the regulation of AMPK activation is modulated depending on the tissue and the microenvironment (Mantovani & Roy, 2011). Just in skeletal muscle, the response to one bout of high-intensity exercise may elicit phosphorylation in 1004 phosphosites on 562 proteins, several of which are related with AMPK but with a previous unknown implication on exercise signalling (Hoffman et al., 2015).

2. AMPK activation/inhibition mechanisms 2.1. Canonical AMPK activation AMPK is an enzyme with a heterotrimeric structure composed by one catalytic subunit (α) and two regulatory (β and γ). The γ subunit has a cystathionine β-synthase (CBS) motif capable of binding adenosyl nucleotides (ATP, ADP and AMP) (Cheung et al., 2000). The binding of AMP to AMPK causes an increase of AMPK activity by three mechanisms: 1) direct allosteric activation; 2) facilitation of phosphorylation of the 172 threonine residue; and 3) inhibition of Thr172 dephosphorylation (Hardie & Ashford, 2014). While AMP activates, ATP inhibits AMPK activation such that only an increase of AMP:ATP ratio produces AMPK activation. When AMP:ATP ratio increases, AMP binds to γ subunit an increase AMPK activity (allosteric activation) even in the absence of LKB1 (Gowans et al., 2013). In addition, binding of AMP to γ subunit induces a conformational change exposing the 172 threonine residue of the AMPK α subunit (Hardie et al., 2000). This allows the phosphorylation of 172 threonine residue by a heterotrimeric complex containing the tumour suppressor kinase LKB1, the main AMPK upstream kinase (Hawley et al., 2003), which increases further AMPK activity. Although LKB1

5 appears to be constitutively active (Sakamoto et al., 2004), LKB1 activity is also regulated by phosphorylation, deacetylation and compartmentalization (Baas et al., 2003; Song et al., 2008). The

subunit of AMPK has three specific binding sites for AMP, ADP or ATP (Hardie &

Ashford, 2014). Binding of AMP to site 1 elicits conformational changes in AMPK, which facilitate Thr172 phosphorylation by LKB1. When AMP or ADP binds to the site 3 of the γ 172

-AMPKα phosphorylation from the action of phosphatases 2A and 2C,

which dephosphorylate and deactivate AMPK (Suter et al., 2006; Powers & Jackson, 2008; Carling & Viollet, 2015). Wright et al. (2009) have shown that tyrosine and Ser/Thr phosphatases may be inhibited by exposure to RONS in skeletal muscle. RONS play a more important role in the regulation of the non-canonical AMPK activity (Carling & Viollet, 2015). As illustrated in Figure 1, LKB1 compartmentalization is regulated by STe20 Related ADaptor (STRAD). STRAD binds to and phosphorylates LKB1 facilitating the migration from the nucleus to the cytoplasm (Baas et al., 2003) necessary to produce an increase on LKB1 activity (Boudeau et al., 2003) since Ser431-LKB1 (orthologous to Ser428 in humans) is distal to the kinase domain (Fogarty & Hardie, 2009). The -terminal TrpGlu(Alessi et al., 2006) (Milburn et al., 2004)

-

where AMPK can be phosphorylated. Sestrin2 has been reported to increase directly AMPK activity (Budanov & Karin, 2008) (Fig. 1). Sestrins 1 to 3 constitute a family of proteins that are induced in mammalian cells in response to environmental stressors (Rhee & Bae, 2015). Sestrin2 expression is upregulated by

6 human myotubes exposed to H2O2 (Nascimento et al., 2013) and accumulates in cardiomyocytes during ischemia (Morrison et al., 2015). Sestrin2 stabilizes the LKB1/AMPK complex, which is involved in AMPK activation in response to ischemia (Morrison et al., 2015). Sestrin2 expression is modulated by p53 (Budanov & Karin, 2008) and upregulates NRf2 expression (Rhee & Bae, 2015). Nrf2 induces the expression of antioxidant enzymes (Tebay et al., 2015).

2.2. Non-Canonical AMPK activation 2.2.1. Calcium/Calmodulin-dependent kinase kinase β (CaMKKβ) CaMKKβ had been accepted, until recently, as the only proved AMPK upstream kinase regulated by Ca2+ release (Tokumitsu et al., 2001; Hawley et al., 2005). During muscle contraction, when sarcoplasmic Ca2+ levels raise, CaMKKβ activity is increased via the Ca2+/calmodulin complex (Tokumitsu et al., 2001) (Fig. 1). In cell-free experiments, AMPK activity can be increased by just adding CaMKKα/β (Hawley et al., 1995).

2.2.2. Calcium/Calmodulin-dependent kinase II (CaMKII) It has been suggested that calcium/calmodulin-dependent protein kinase II (CaMKII) could also act indirectly as an upstream activator of AMPK (Egan et al., 2010; Morales-Alamo et al., 2013). CaMKII has been reported to increase the autophagy marker microtubule-associated protein 1 light chain 3b-II protein expression (LC3b-II) in response to caffeine, in an AMPK-dependent manner (Mathew et al., 2014). Free radicals may activate CaMKII through modification of the Met281/282 pair within the regulatory domain, blocking reassociation with the catalytic domain

7 and preserving kinase activity via a similar but parallel mechanism to Thr286 autophosphorylation, a known mechanism of CaMKII activation (Erickson et al., 2008) (Fig. 1). Nevertheless, Thr286-CaMKII phosphorylation depends mainly on Ca2+ release (Hudmon & Schulman, 2002), which is regulated by conformational changes of the ryanodine receptor (Ryr) of the sarcoplasmic reticulum (MacIntosh et al., 2012) (Fig. 1). Ca2+ permeability of the Ryr depends on the superoxide production by the NADPH oxidase (Jackson et al., 2007). NADPH oxidase is likely the main source of ROS during the first 10-15 min of exercise (Jackson, 2015). Sprint exercise is a potent generator of free radicals in human skeletal muscle (Cuevas et al., 2005; Morales-Alamo & Calbet, 2014), particularly in untrained humans (Place et al., 2015). During sprint exercise free radicals, including superoxide, are also released by the mitochondria, causing Ryr fragmentation via calpain activation (Place et al., 2015). Ryr fragmentation causes increased SR Ca2+ leak and depressed force production due to impaired SR Ca2+ release upon stimulation (Place et al., 2015). The same type of exercise (30 s all-out sprint, called Wingate test) is a powerful stimulus for Thr172-AMPKα phosphorylation, and free radicals appear to play a critical role in this response (Morales-Alamo et al., 2013). As depicted in Figure 1, administration of antioxidants blunts the Thr172-AMPKα phosphorylation and Thr286CaMKII phosphorylation in response to the sprint exercise (Morales-Alamo et al., 2013).

2.2.3. Sirtuin 1 (SIRT1) SIRT1 is an NAD-dependent deacetylase that acts as a redox sensor (Hou et al., 2008). SIRT1 plays an important role in mitochondrial biogenesis, fatty acid oxidation and glucose homeostasis (Gerhart-Hines et al., 2007). SIRT1 activity is stimulated by the increase of the NAD+/NADH.H+ ratio, (Pillai et al., 2008) (Fig. 1). SIRT1 deacetylases LKB1, increasing its activity

8 (Hou et al., 2008). In cell cultures, exposure to H2O2 elicits C-Jun NH2-terminal kinase (JNK) phosphorylation, which phosphorylates SIRT1 (Nasrin et al., 2009), committing phosphorylated SIRT1 to proteasome degradation (Gao et al., 2011). It has been shown that sprint exercise in severe acute hypoxia elicits greater oxidative stress than the same exercise performed in normoxia (Morales-Alamo et al., 2012). Compared to normoxia, sprint exercise in severe hypoxia requires about 50% greater ATP production via the glycolysis (Morales-Alamo et al., 2012) and strong activation of the anaerobic metabolism has been associated with oxidative damage (Morales-Alamo & Calbet, 2014; Place et al., 2015). SIRT1 total protein amount and NAD+/NADH.H+ ratios are more reduced after sprint exercise in severe acute hypoxia than when sprint is performed in normoxia (Morales-Alamo et al., 2012).

2.2.4. Transforming growth factor-β-activated kinase 1 (TAK-1) Transforming growth factor-β-activated kinase 1 (TAK-1) is a kinase activated by Thr184-TAK-1 phosphorylation (Sakurai et al., 2000). TAK-1 has been shown to phosphorylate AMPK at Thr172 residues in vitro (Momcilovic et al., 2006). TAK-1 can act like an AMPK upstream kinase in cardiac myocytes (Xie et al., 2006). RONS released by reoxygenation produces an increase of JNK activity (Dougherty et al., 2004). JNK activation by free radicals is PKCα and TAK1 dependent (Frazier et al., 2007). In turn, JNK can regulate SIRT1/LKB1/AMPK pathway (Gao et al., 2011; Huang et al., 2014) (Fig. 1).

2.2.5. v-ATPase (vacuolar H+-ATPase) Another mechanism that may elicit AMPK activation during exercise involves v-ATPase (vacuolar H+-ATPase) (Carling & Viollet, 2015). v-ATPase-Ragulator is a late

9 endosomal/lysosomal protein complex. Nutrients abundance and growth factors phosphorylate TSC1-TSC2-TBC1D7 complex, a heterotrimeric GTPase-activating protein (GAP) for the small GTPase RHEB1. The phosphorylated GAP complex dissociates from RHEB1, rendering it GTPbound. The GTP-bound RHEB1 is located on the late endosome/lysosome surface where it binds to and activates mTORC1 (Roux et al., 2004; Ma et al., 2005; Dibble & Manning, 2013; Menon et al., 2014). Amino acids activate mTORC1 via v-ATPase-Ragulator-RAG complex, which mediates mTORC1 translocation to endosome surface (Kim et al., 2008; Sancak et al., 2008; Sancak et al., 2010) where it may be activated by RHEB1. Opposite to AMPK, activated mTORC1 shifts the metabolic program of the cell from catabolic to anabolic (Zhang et al., 2014). Glucose deprivation or AMP increase the binding between AXIN and LKB1, which begins the AXIN/LKB1AMPK complex formation and elicits Thr172-AMPKα phosphorylation, in a v-ATPase-Ragulator complex dependent manner (Zhang et al., 2013; Zhang et al., 2014). v-ATPase is a proton (H+) pump regulating pH, which is involved in cations uptake (Ca2+, Na+) (Dietz et al., 2001). For this reason, v-ATPase has been reported as a potent pH sensor (Recchi & Chavrier, 2006). Thr410PKCζ has been suggested to increase Ser428-LKB1 phosphorylation and its activity as well (Song et al., 2008). Moderate levels of RONS have been shown to activate PKC (Gopalakrishna & Anderson, 1989) and PKC enhances v-ATPase activity (Nordstrom et al., 1994). Although the regulation of AXIN/LKB1-AMPK via v-ATPasa-Ragulator has not been reported in response to RONS or acidosis, this mechanism could be a key redox-sensitive point between both pathways (mTOR and AMPK).

10 2.3. Activation of AMPK by RONS The addition of H2O2 or NO to cell cultures elicits AMPK activation (Zmijewski et al., 2010; Cardaci et al., 2012). RONS may activate AMPK by reducing mitochondrial ATP synthesis, leading to an increase of the AMP:ATP ratio and subsequent Thr172-AMPK phosphorylation by LKB1 and CaMKKβ (Choi et al., 2001; Irrcher et al., 2009; Auciello et al., 2014). Once Thr172AMPK phosphorylation has occurred, the rate of Thr172 dephosphorylation is markedly reduced by H2O2, through mechanisms that may involve AMP/ADP binding to the γ subunit or reduced activity of phosphatases (Auciello et al., 2014). Recent studies have shown that RONS may induce direct activating (Zmijewski et al., 2010) or deactivating (Shao et al., 2014) effects by modifying RONS-sensitive residues of the AMPK-α subunit. For example, exposure of recombinant AMPKαβγ complex or HEK 293 cells (HEK 293 cells do not express LKB1) to H2O2 resulted in oxidative modification of AMPK, including S-glutathionylation of the AMPKα and AMPKβ subunits associated with increased AMPK activity (Zmijewski et al., 2010). More specifically, Zmijewski et al. (2010) showed that oxidation (S-glutathionylation) of cysteine residues 299 and 304 is necessary for the enhancing effect of H2O2 on AMPK activity. In contrast, oxidation of Cys130/Cys174 in the AMPKα subunit causes aggregation of AMPK through intermolecular disulphide bonds formation, which prevents Thr172-AMPKα phosphorylation by upstream kinases in cardiomyocytes (Shao et al., 2014). Thus, RONS-elicited modifications of the AMPKα subunit may exert activating/deactivating effects on AMPK activity depending on the cell type. However, the magnitude of AMPK activation by direct oxidative modification of the AMPKα subunit is small compared to that mediated by the RONS-induced increase of AMP:ATP ratio (Auciello et al., 2014). Shao et al. (2014) have proposed that AMPK has three different statuses: oxidized (inactive), reduced (activatable), and reduced and phosphorylated (active). The relative proportions of each form are determined by the intracellular energy

11 status, ROS level, and Thioredoxin1 (Trx1), an important reducing enzyme that cleaves disulfides in proteins (Shao et al., 2014). It remains unknown whether oxidative modifications of the AMPKα subunit play a role in the regulation of AMPK during exercise and recovery.

2.4. Inhibition of AMPK by serine phosphorylation of the α subunits Phosphorylation of the 485 and 491 serine residues of the α1 and α2 subunits reduces Thr172AMPK phosphorylation (Dagon et al., 2012; Hawley et al., 2014). The Ser485 (or Ser487 in humans) lies in a region of the AMPK α1 subunit termed ST loop due to its high content of serine/threonine residues (Hardie & Ashford, 2014). In the α2 subunit, Ser491 plays a role similar to Ser485 in the α1. Insulin produces an inhibitory effect on AMPK activity via Akt (Kovacic et al., 2003). This Akt inhibitory effect on AMPK seems to be produced in a Ser 485-AMPKα1 phosphorylation-dependent manner (Horman et al., 2006; Hawley et al., 2014). In addition to Akt, protein kinase A (PKA) (cAMP-dependent protein kinase) can also elicit α1 Ser485 phosphorylation (Hurley et al., 2006). Recent studies have shown that glycogen synthase kinase 3 (GSK3) (Suzuki et al., 2013) can phosphorylate other sites (Thr481 and Ser477) of the ST loop to promote Thr172 dephosphorylation (Suzuki et al., 2013). Increased phosphorylation of the ST loop promotes interaction of the ST loop with the kinase domain, physically hindering the access of LKB1 and CaMKKβ to the Thr172, impeding phosphorylation of Thr172 (Hardie & Ashford, 2014; Hawley et al., 2014). Ser491 AMPKα2 phosphorylation reduces Thr172 AMPK phosphorylation in the hypothalamus (Quaresma et al., 2016) and likely in skeletal muscle (Guerra et al., 2010). In humans, ingestion of 75 g of glucose 1 hour before sprint exercise inhibits the expected Thr172-AMPKα phosphorylation response via Ser485-AMPKα1/ Ser491AMPKα2 phosphorylation (Guerra et al., 2010). In this experiment, there was a correlation

12 (r=0.84 P< 0.05) between Ser485-AMPKα1/ Ser491-AMPKα2 and Ser473-Akt phosphorylations (Guerra et al., 2010). Sprint exercise (30 s Wingate test) in severe acute hypoxia (FIO2: 0.105) increased Ser485-AMPKα1/ Ser491-AMPKα phosphorylation immediately at the end of the sprint (Morales-Alamo et al., 2012) (Fig. 2). The increase in Ser485-AMPKα1/ Ser491-AMPKα2 phosphorylation coincided with a blunted Thr172-AMPKα phosphorylation (Morales-Alamo et al., 2012). Recent experiments have shown that only Ser485-AMPKα1 phosphorylation, but not Ser491-AMPKα2 seems to be regulated via Akt (Hawley et al., 2014). Moreover, cell cultures experiments have shown that Ser491 may occur simultaneously with Thr172 phosphorylation in response to mitochondrial ATP synthesis inhibition with berberine, suggesting that Ser491 is caused by autophosphorylation (Hawley et al., 2014). This finding does not rule out a potential inhibitory effect of Ser491 α2 phosphorylation on Thr172 phosphorylation, as observed in the hypothalamus (Quaresma et al., 2016). In agreement, it has been reported that Ser491-AMPKα2 phosphorylation increases if the 30s-Wingate test is performed after antioxidant ingestion (Morales-Alamo et al., 2013), blunting the expected Thr172-AMPKα phosphorylation, despite no apparent role by Akt in this case (Morales-Alamo et al., 2013) (Fig. 2). Both too high (hypoxia) and too low (ingestion of antioxidants) RONS levels may lead to Ser485-AMPKα1/ Ser491-AMPKα2 phosphorylation and blunted on Thr172-AMPKα phosphorylation (Morales-Alamo et al., 2012; Morales-Alamo et al., 2013) (Fig 2).

3. Endurance exercise and AMPK activation AMPK activation in response to exercise was firstly observed after endurance exercise, and there is some evidence suggesting that RONS play a role in this process (Gomez-Cabrera et al., 2008; Irrcher et al., 2009). Exercise duration, intensity and training status are important

13 determinants of the AMPK response to endurance exercise (Coffey et al., 2006; Benziane et al., 2008). A common feature of exercises causing AMPK activation is the extension of exercise bout until exhaustion. For example, AMPK activation has been reported following one-hour at 75% VO2max until exhaustion (Wojtaszewski et al., 2000). Likewise, Thr172-AMPKα2 phosphorylation was increased when exercise at 45% VO2max was prolonged until exhaustion (about 3.5 hours) (Wojtaszewski et al., 2002). Thr172-AMPKα2 phosphorylation did not increase in response to cycling exercise when the absolute exercise intensity was low (111 W, 51% VO2peak) and the exercise was not performed until exhaustion, compared to an exercise bout of the same duration (30 min) performed at 171 W (73% of VO2peak) (Wadley et al., 2006). Interestingly, when the 111 W bout was performed in acute hypoxia (FIO2 = 11.5%) at a relative intensity of 72% of the VO2peak in hypoxia, there was a significant increase in Thr172-AMPKα phosphorylation, which was of lower magnitude than during the exercise in normoxia at the same relative intensity (Wadley et al., 2006). This response was associated with a lower increase of creatine, free ADP, and free AMP, concomitant with decreased phosphocreatine from rest during the exercise at the same relative intensity in hypoxia than normoxia. Thus, it seems that for endurance exercise to elicit AMPK activation a certain level of metabolite accumulation (AMP) must be reached, regardless of the exercise intensity, duration or level of oxygenation. The increase in AMP and ADP is more likely if the exercise is performed until exhaustion, regardless of the inspiratory oxygen fraction (Kjaer et al., 1999; Morales-Alamo et al., 2015). It is important to underline that RONS production is increased when the exercise is performed until exhaustion (Sastre et al., 1992; Vina et al., 2000). Moreover, ultra-endurance exercise increases mitochondrial RONS production in vitro (Sahlin et al., 2010). However, this effect is attenuated in well-trained athletes (Farney et al., 2012). If RONS play a critical role in

14 endurance-induced AMPK activation, administration of antioxidants or reduction of RONS production (or increased quenching) by adaptation to endurance training (Farney et al., 2012) should result in lowered AMPK activation, PGC1α expression and mitochondrial biogenesis (Richter & Ruderman, 2009). In rodents, endurance training with antioxidant supplementation (vitamin C) blunted the increase in mRNA coding PGC1α, nuclear respiratory factor 1, and mitochondrial transcription factor A, while citrate synthase protein expression was reduced, (Gomez-Cabrera et al., 2008). Similarly, Vitamin E and α-lipoic acid supplementation suppress skeletal muscle mitochondrial biogenesis in sedentary rats and blunts the expected increase after endurance training (Strobel et al., 2011). This has been confirmed in mice injected with Nacetyl-l-cysteine or placebo during treadmill training (Sun et al., 2015). The effect of and Nacetyl-l-cysteine was so intense that even endurance training could not impede the reduction of cytochrome c oxidase (COX) induced by N-acetyl-l-cysteine administration (Sun et al., 2015). In contrast, some rodent studies have also reported a lack of negative influence of vitamin C and E administration on the increase of muscle antioxidant enzymes (SOD 1 and 2) (Higashida et al., 2011). Likewise, in humans, 1000 mg of vitamin C and 235 mg of vitamin E or a placebo daily for 11 weeks, had no negative influence on the endurance-elicited improvement of VO2max, while COXIV and PGC1α only increased with training in the placebo group (Paulsen et al., 2014). Also in humans, antioxidants blunt the increase of PPARδ and PGC1α two AMPK downstream targets (Narkar et al., 2008), in response to 4 weeks of endurance training (Ristow et al., 2009). In the same experiment, insulin sensitivity enhancement was prevented by antioxidant supplementation (Ristow et al., 2009). Just ten sessions of endurance training reduced the AMPKα phosphorylation response observed after a 60 min bicycling at 70% of VO2peak (Benziane et al., 2008), with pre and posttraining exercise bouts performed at the same absolute intensity (164 W) (Benziane et al.,

15 2008). McConell et al. (2005) examined the effect of short-term exercise training on skeletal AMPK signalling and muscle metabolism during prolonged exercise in humans (120 min at 66% of VO2peak). The AMP:ATP ratio was increased 7-fold after 30 min of exercise and 22-fold at the end of the 120 min before training. After training the increase in the AMP:ATP was significantly smaller (4-fold at 30 min and 9-fold at 120 min). Skeletal muscle AMPK α1 and α2 activity increased progressively during exercise before but not after training. McConell et al. (2005) findings indicate that endurance training reduces AMPK activation in response to prolonged exercise, when pre and pot-training exercises are performed at the same absolute intensity, due to the lower increase of the AMP:ATP ratio during prolonged exercise after training. The reduction in AMPK activity in response to prolonged exercise observed after training cannot be explained by changes in the relative exercise load or glycogen levels (McConell et al., 2005). A reduced adrenergic response to prolonged exercise after training and/or reduced free radical release or increased quenching due to up-regulation of the antioxidant systems in muscles could explain the reduced activation of AMPK after training. In contrast, Clark et al. (2004) reported similar AMPK α1 and α2 activity response before and after high-intensity interval training, in well-trained athletes (VO2max of 65 ml.kg-1.min-1), in response to an exercise bout consisting of 20 min at 65% VO2peak followed by 8x5 min work bouts at 85%, with 60s recovery between bout at 100 W. Differences between species, muscles studied, training models, training status, type of antioxidants, dosage, pharmacodynamics and intracellular compartmentalization may explain differences between studies (Casuso et al., 2014; Leonardo-Mendonca et al., 2014; MoralesAlamo & Calbet, 2014; Paulsen et al., 2014). Given the protective influence that antioxidants may have under certain circumstances for specific populations (Braakhuis et al., 2014; Sanchis-

16 Gomar et al., 2015), more research is needed to establish when the administration of antioxidants should be advised in the context of exercise training and competition.

4. High-intensity exercise and AMPK activation Humans can sustain metabolic rates above 250% of VO2max during 30 s when developing the maximal power output possible since the start to the end of the exercise, a type of exercise called all-out. This type of exercise recruits all available energy pathways, i.e., ATP and phosphocreatine (phosphagens), glycogenolysis, glycolysis and oxidative phosphorylation (Medbo & Tabata, 1989; Calbet et al., 1997). During all-out exercise on the cycle ergometer, humans can reach ~80% of the VO2max in just 25s (Calbet et al., 2003; Calbet et al., 2015). If the all-out exercise last more than 60 s, 50% or more of the ATP needed is resynthesized by the oxidative phosphorylation (Medbo & Tabata, 1989). If several bouts of high-intensity exercise are repeated with short recovery periods in between, the aerobic contribution to the total energy expenditure in each bout increases (Dorado et al., 2004). All-out exercise elicits a fast increase of the AMP:ATP ratio and reduction of NAD+/NADH.H+ leading to activation of AMPK, which reaches maximal levels of phosphorylation during the first 30 min of the recovery (Chen et al., 2000; Cuevas et al., 2005; Guerra et al., 2010; Morales-Alamo et al., 2012). Both the aerobic and anaerobic energy pathways are important sources of RONS (Powers & Jackson, 2008; Morales-Alamo & Calbet, 2014). During a single sprint exercise muscle RONS are thought to be mainly produced by NADPH oxidase from T-tubules (Jackson, 2015), and from glycolysis via H+ accumulation, which accelerate the rate of dismutation of O2- to H2O2 and, in presence of Fe2+, generate OH· (for review see, (Morales-Alamo & Calbet, 2014)). However, recent research has shown that high

17 intensity exercise also causes a marked increase of RONS production by the mitochondria, particularly superoxide (Place et al., 2015). We have investigated the role played by RONS on the skeletal muscle signalling response to a single 30 s sprint by comparing the AMPK regulatory signalling responses after the ingestion of either placebo or an antioxidant cocktail containing α-lipoic acid, vitamin C and vitamin E, ingested 120 and 90 minutes before exercise. Performance and muscle metabolism were not significantly modified by the intake of antioxidants. In both conditions, the NAD+/NADH.H+ ratio was similarly reduced (84%), and the AMP/ATP ratio similarly increased (21 fold) immediately after the sprints. The antioxidants prevented Thr172-AMPKα phosphorylation by two potential mechanisms. First, they blunted Thr286-CaMKII phosphorylation, which may act as an indirect upstream kinase for AMPK in skeletal muscle (Raney & Turcotte, 2008; Asrih et al., 2013; Mathew et al., 2014). Secondly, antioxidants increased Ser485- AMPKα1/Ser491-AMPKα2 phosphorylation immediately after the sprint (Morales-Alamo et al., 2013). The mechanism by which antioxidants produce an increase on Ser485-AMPKα1/ Ser491-AMPKα2 phosphorylation has not been fully elucidated. In mast cells, high-affinity IgE receptor o Fc epsilon Ri (FcεRI) activation, via the Lyn-Syk-Akt cascade, can rapidly inhibit AMPK activation through increased Ser485-AMPKα1/ Ser491-AMPKα2 phosphorylation counteracting the LKB1/AMPK axis through Fyn activation (Lin et al., 2015). Fyn is a member of the Src kinase family, is a constitutively expressed, membrane-localized, nonreceptor, tyrosine kinase. Fyn phosphorylates tyrosine residues (Tyr261 and Tyr365) in LKB1 in the nucleus, which prevents LKB1 cytoplasmic translocation (Yamada et al., 2010), impeding AMPK activation. Oxidative injury caused by arachidonic acid and iron enhances Fyn activation by autophosphorylation at the Tyr420 (Koo et al., 2012) and this could explain why high levels of oxidative stress may result in reduced/blunted AMPKα phosphorylation during sprint exercise

18 in severe hypoxia (Morales-Alamo et al., 2012). Fyn overexpression blunts AMPK activation and results in reduced capacity to up-regulate the antioxidant system in response to oxidative stress (Kaspar & Jaiswal, 2011; Koo et al., 2012). It remains unknown whether Fyn is activated by sprint exercise, and how antioxidants may affect the interaction between Fyn and LKB1/AMPK axis. As mentioned previously, RONS may activate CaMKII through modification of the Met281/282 pair within the regulatory domain, blocking reassociation with the catalytic domain and preserving kinase activity via a similar but parallel mechanism to Thr286 autophosphorylation (Erickson et al., 2008). Free radicals may also modulate CaMKII activation via its effects on Ca2+ release (Hudmon & Schulman, 2002). Since Ca2+ release through Ryr is directly dependent to the O2- production of the NADPH oxidase (Jackson et al., 2007), antioxidants ingestion before sprint exercise may reduce sarcoplasmic Ca2+ release via inhibition of Ryr (MacIntosh et al., 2012). Consequently, quenching of superoxide by the antioxidants ingested before exercise could decrease Ca2+ transients eliciting lower stimulus for Ca2+-calmodulin-mediated Thr286-CaMKII phosphorylation. Despite that antioxidant ingestion before sprint exercise has a potential lowering effect on Ca2+ release this does not appear to have a negative influence on sprint performance. This may be due to the proximity of the triad to myofibers, which could prioritize Ca2+ delivery to muscle contractile structure; and also to the fact that Ca2+ affinity in muscle fibers is enhanced after high-intensity exercise (Gejl et al., 2015). Interestingly, if a similar power output can be generated with lower Ca2+ transients, lower energy would have to be expended to reestablished resting Ca2+ sarcoplasmic concentrations by the sarcoplasmic reticulum Ca2+-pumps, resulting in greater contraction efficiency. It is unknown, however, if antioxidants intake before sprint exercise increases the contraction efficiency during the sprint.

19 Sprint exercise in severe acute hypoxia (equivalent to 5300 m above sea level) elicits higher glycolytic rate, greater reductions of the NAD+/NADH.H+ ratio, lower muscle pH and increased protein carbonylation in skeletal muscle and plasma than a similar sprint in normoxia (Morales-Alamo et al., 2012). This suggests increased free radicals production in hypoxia or reduced quenching capacity, causing oxidative stress (Morales-Alamo et al., 2012). Despite this increased oxidative stress, Thr172-AMPKα phosphorylation was completely blunted after sprint exercise in hypoxia (Morales-Alamo et al., 2012). Since the free AMP/ATP ratio, was increased to the same extent after the sprint in hypoxia and normoxia, other mechanisms must intervene to explain how Thr172-AMPKα phosphorylation after sprint exercise in severe hypoxia is abrogated. One possibility is the observed reduction of SIRT1 protein after sprint exercise in hypoxia (Morales-Alamo et al., 2012). SIRT1 is an NAD-dependent deacetylase that activates (deacetylates) LKB1 (Hou et al., 2008). Both the reduction in SIRT1 protein and the lower NAD+/NADH.H+ after the sprint in hypoxia may result in lower SIRT1 activity (Gao et al., 2011). A lower SIRT1 activity could reduce activation of LKB1 blunting the expected Thr172-AMPKα phosphorylation. The AMPK inhibitory Ser485- AMPKα1/Ser491-AMPKα2 phosphorylation was also increased immediately after the sprints performed in severe hypoxia (Morales-Alamo et al., 2012), which could explain the observed abrogation of Thr172-AMPKα phosphorylation after the sprint exercise in severe hypoxia. In agreement, incubation of rat muscle with an excess of glucose or leucine elicits increased AMPKα phosphorylation at Ser 485/491 combined with a decrease of SIRT1 total protein an increase protein phosphatase 2A (PP2A) activity (Coughlan et al., 2015). PP2A dephosphorylates AMPKα at Thr172 (Wu et al., 2007). Another mechanism that could explain reduced Thr172-AMPKα phosphorylation by excessive RONS generation during sprint exercise in severe hypoxia is inactivation of AMPK by oxidative modification of AMPKα

20 subunits, which prevents Thr172-AMPKα phosphorylation by upstream kinases, as shown in cardiomyocytes (Shao et al., 2014) (Fig. 1). These experiments indicate that RONS play a critical role in the sprint exercise-induced Thr172 AMPKα and Thr286-CaMKII phosphorylation. Both kinases intervene in the regulation of mitochondrial biogenesis, fat oxidation, glucose uptake in skeletal muscle, and muscle fiber type phenotype (Guadalupe-Grau et al., 2015). Consequently, the ingestion of antioxidants immediately before exercise may attenuate or abrogate some of the expected adaptations to sprint exercise training (Malm et al., 1997).

4.1. Regional differences in the muscular adaptations to sprint training are mediated by RONS-sensitive mechanisms Even a single 30 s sprint bout is sufficient to elicit two hours later increased levels of PGC1α mRNA (Guerra et al., 2011). A greater response is expected with repeated sprints (Gibala et al., 2009). Most previous studies in humans have used biopsies from the muscle vastus lateralis for the analysis of the signalling responses to sprint-exercise. Larsen et al. have recently examined the response to the same sprint-training program applied to the arm (triceps brachii) and leg (vastus lateralis) muscles, with an intra-subject experimental design (Larsen et al., 2016). The m. vastus lateralis withstands greater loading during daily activities and as a consequence it displays a more aerobic profile as reflected by the greater percentage of type 1 fibers, mitochondrial density, and capillarisation than the triceps brachii (Ara et al., 2011; Gnaiger et al., 2015; Holmberg, 2015). Sprint-training is known to elicit oxidative stress in untrained subjects (Place et al., 2015). Excessive RONS production during sprint training inactivates aconitase (an enzyme of the tricarboxylic acid cycle, TCA) by 55-72%, resulting in inhibition of

21 mitochondrial respiration by 50-65%. Aconitase has an active [Fe4S4]2+ cluster that is rapidly inactivated into [Fe3S4]+ through oxidation by superoxide, peroxynitrite, and H2O2, constituting a redox-sensitive regulation of the rate-limiting enzyme in the TCA cycle. The location of a redox-sensitive enzyme in the TCA cycle implies that aconitase can act as a rheostat that modulates mitochondrial metabolism by its interaction with RONS (Armstrong et al., 2004). Aconitase inactivation was greater in the triceps than in the vastus lateralis. Compared to pretraining, mitochondrial ROS production was diminished after training in mitochondria isolated from the triceps whereas it was unchanged in the vastus lateralis mitochondria (Larsen et al., 2016). There was a trend toward more carbonylated proteins in the triceps but not in the vastus lateralis after training vs. before. In agreement with a greater exposure to oxidative stress in triceps than vastus lateralis despite the same training program, the increase of catalase protein levels (6-fold) occurred only in the triceps. Inactivation of aconitase caused accumulation of its substrate citrate in vastus lateralis (but not in triceps). Although citrate is known to inhibit phosphofructokinase (PFK) activity, sprint performance was increased after training, likely because the inhibitory effect of citrate on PFK is small at the concentration range observed in vivo, both at rest and during exercise (Peters & Spriet, 1995). Larsen et al. have also demonstrated that citrate protects mitochondrial proteins from oxidative damage (Larsen et al., 2016). Excessive inhibition of aconitase in the triceps, not only inhibited mitochondrial respiration but also blunted the increase in citrate and mitochondrial density and induced a robust parallel activation of the endogenous antioxidant catalase that restored redox homeostasis. This study provides a good example on how the same training program applied to different muscles of the same person results in different adaptations. This may explain why basal levels of Thr172-AMPKα are different between arm and leg muscles of the same person (Ponce-Gonzalez et al., 2016). It remains to be determined if the levels of RONS produced by

22 the triceps brachii during sprint exercise training reduces the expected mitochondrial biogenic response due to blunted AMPK-mediated signalling, by a mechanism similar to that observed in severe acute hypoxia. Larsen et al. (2016) and Morales-Alamo et al. (2012) experiments support the concept that optimal adaptation to sprint training requires repeated optimal exposure to pulses of RONS (Fig. 2). Inappropriate training may lead to excessive RONS and reduced adaptation or even maladaptation. Theoretically, exercise programs should be designed taking into account the intrinsic properties of different skeletal muscle, the metabolic state, the environmental conditions and the specific RONS-induced signalling responses.

5. Future studies Experimental evidence indicates that RONS play a critical role in AMPK activation and in maintaining AMPK active. However excessive RONS production may end in oxidative stress and AMPK inactivation. Since AMPK activation is needed for the adaptive response to exercise training, future research should identify how a proper level of RONS-mediated signalling can be achieved. Little is known about the temporal dynamics of the AMPK response to exercise or for how long should AMPK activity be increased for optimal adaptation to training. Likewise, it remains to be determined what the appropriate level is and how often RONS pulses should be generated for optimal adaptation to exercise. Several sources of RONS have been identified during exercise, but little is unknown about the role played by each specific source of RONS in AMPK activation and training adaptations. Recent experiments indicate that excessive RONS production during training may result in maladaptation. It remains unknown if excessive RONSmediated maladaptation could be prevented with antioxidants.

23 Acknowledgements This study was supported by a grant from the Ministerio de Economía y Competitividad (PI14/01509).

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Figure legends Figure 1. The main sources of reactive oxygen and nitrogen species (RONS) during muscle contraction are mainly NADPH oxidase (NOX) and Mitochondria. Optimal levels of RONS elicit AMPK activation by Thr172-AMPKα phosphorylation. Acute effects of RONS: JNK phosphorylates Thr184-Tak1, an upstream AMPK kinase. Ca2+ release through ryanodine receptors (Ryr) increases Thr286-CaMKII phosphorylation and CaMKKβ total protein amount. Moderate levels of RONS lead to PKC activation, which phosphorylates LKB1 at Ser431 activating LKB1. LKB1 activates AMPK by Thr172 phosphorylation. AMPK increases the protein expression of SIRT1. SIRT1 deacetylates and activates LKB1. Sestrin2 stabilizes the LKB1-AMPK complex and promotes Thr172-AMPKα phosphorylation. Excessive levels of RONS cause JNK phosphorylation (activation), which phosphorylates SIRT1 at Ser47 committing SIRT1 to proteasome degradation. A lower amount of SIRT1 reduces LKB1 deacetylation and hence, Thr172-AMPKα phosphorylation. High RONS levels increase Fyn autophosphorylation at Tyr420, which leads to AMPKα1/2 phosphorylation at Ser485/491 inhibiting AMPKα phosphorylation at Thr172. Tyr420-Fyn phosphorylates tyrosine residues (Tyr261 and Tyr365) in LKB1 in the nucleus preventing LKB1 cytoplasmic translocation, which is necessary for AMPK activation. AMP increases the binding between AXIN and LKB1, resulting in Thr172-AMPKα phosphorylation. Exogenous antioxidants

35 decrease RONS levels blunting Thr286-CaMKII phosphorylation and increase the inhibitory Ser485-AMPKα1/Ser491-AMPKα2 phosphorylation. Moderate RONS levels inhibit mitochondrial respiration causing an increase of the AMP:ATP ratio and Thr172-AMPKα phosphorylation. Moderate RONS may also activate directly AMPK. Excessive RONS may cause oxidative inactivation of AMPK. Chronic effects of RONS: repeated exposure to RONS pulses by endurance or high-intensity exercise (HIT) enhances the endogenous antioxidant defence mechanisms and nitric oxide synthase (NOS) levels. In the trained state, the endogenous antioxidant system blunts the RONS-mediated signalling resulting in lower or absent Thr172AMPKα phosphorylation in response to exercise, unless unaccustomed exercise bouts are performed. Dashed arrows indicate unknown mechanism. Figure 2. Optimal redox balance is required for Thr172-AMPKα phosphorylation in response to sprint exercise. The expected Thr172-AMPKα phosphorylation in response to high-intensity exercise (HIT) is blunted by changes in redox balance. Left: The ingestion of antioxidants before sprint exercise abrogates the expected Thr172-AMPKα phosphorylation by decreasing RONSmediated activation (autophosphorylation) of CaMKII, leading to reduced Thr 172-AMPKα phosphorylation by an unknown mechanism (dashed arrow). The reduction of ROS-mediated signalling is accompanied by increased Ser485-AMPKα1/Ser491-AMPKα2 phosphorylation. Right: In severe acute hypoxia (FIO2:0.105), excessive RONS production results in blunted AMPK phosphorylation by at least three potential mechanisms. Firstly, the increase in RONS is accompanied by a greater reduction of the NAD+/NADH.H+. Secondly, the total amount of SIRT1 protein is reduced. Thirdly, the sprint in hypoxia elicits an increase in Thr308-Akt phosphorylation, which increases Ser485-AMPKα1/Ser491-AMPKα2 blunting Thr172-AMPKα phosphorylation. Excessive RONS may cause maladaptation as observed during sprint training

36 with the arms, leading to increased catalase expression. In the middle: Normal Thr172-AMPKα phosphorylation in response to one bout of sprint exercise (30s Wingate test) in normoxia.

Highlights · Reactive oxygen and nitrogen species (RONS) are generated during exercise depending on intensity, duration and training status. · Efficient adaptation to exercise training requires optimal exposure to pulses of RONS. · RONS may activate or deactivate AMPK by modifying RONS-sensitive residues of the AMPKα subunit and by reducing mitochondrial ATP synthesis, leading to an increased AMP:ATP ratio and Thr172-AMPK phosphorylation by LKB1 and CaMKKβ. · Both too high (hypoxia) and too low (ingestion of antioxidants) RONS levels may lead to Ser485-AMPKα1/ Ser491-AMPKα2 phosphorylation causing inhibition of Thr172-AMPKα phosphorylation.