Sports Med DOI 10.1007/s40279-015-0366-z

REVIEW ARTICLE

Activation of AMPK and its Impact on Exercise Capacity Ellen Niederberger1 • Tanya S. King1 • Otto Quintus Russe1 • Gerd Geisslinger1

Ó Springer International Publishing Switzerland 2015

Abstract Activation of the adenosine monophosphate (AMP)-activated kinase (AMPK) contributes to beneficial effects such as improvement of the hyperglycemic state in diabetes as well as reduction of obesity and inflammatory processes. Furthermore, stimulation of AMPK activity has been associated with increased exercise capacity. A study published in 2008, directly before the Olympic Games in Beijing, showed that the AMPK activator AICAR (5-amino1-b-D-ribofuranosyl-imidazole-4-carboxamide) increased the running capacity of mice without any training and thus, prompted the World Anti-Doping Agency (WADA) to include certain AMPK activators in the list of forbidden drugs. This raises the question as to whether all AMPK activators should be considered for registration or whether the increase in exercise performance is only associated with specific AMPK-activating substances. In this review, we intend to shed light on currently published AMPK-activating drugs, their working mechanisms, and their impact on body fitness. Key Points Adenosine monophosphate (AMP) activation is associated with beneficial effects such as lowering of blood glucose. Some, but not all, AMP kinase activators lead to improved exercise capacity.

& Ellen Niederberger [email protected] 1

Pharmazentrum Frankfurt/ZAFES, Institut fu¨r Klinische Pharmakologie, Klinikum der Goethe-Universita¨t Frankfurt, Theodor Stern Kai 7, 60590 Frankfurt am Main, Germany

1 Introduction Exercise training, particularly endurance exercise, has been frequently associated with beneficial health effects. As endurance exercise improves metabolic as well as cardiovascular disturbances, patient-adapted endurance exercise is often recommended as a basic therapeutic action for these diseases. Physiological effects of endurance training comprise remodeling of muscles, which are composed of a number of different fibers, including red oxidative slowtwitch (type I), white oxidative/glycolytic fast-twitch (type IIa), oxidative fast-twitch (type IIx), and glycolytic fasttwitch (type IIb) myofibers. The slow-twitch fibers express high levels of fat-oxidizing enzymes, mitochondria, and slow contractile proteins. Fast-twitch myofibers are fastresistance or fast-fatigue fibers that express the fast contractile proteins but contain only a few mitochondria and metabolize glucose anaerobically. Endurance exercise leads to an increase in slow-twitch contractile proteins, mitochondrial biogenesis, and fatty acid oxidation, which change the skeletal muscles towards an oxidative slowtwitch phenotype with higher aerobic capacity [1, 2]. These changes are associated with increased insulin sensitivity and an overall improvement in glucose homeostasis and therefore, positively influence diabetes and obesity [3, 4]. However, quite a large number of people and/or patients with these diseases are unable or unwilling to perform even a minimum of physical activity to support their therapy, thus, indicating that drugs mimicking endurance exercise are highly desirable. One of the proteins activated in skeletal muscles during exercise training is the adenosine monophosphate (AMP)activated kinase (AMPK), a heterotrimeric protein consisting of two regulatory (b and c) and one catalytic (a) subunit, which is ubiquitously expressed (e.g. in liver,

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heart, skeletal muscle, brain). It acts as an ‘energy sensor’ and constitutes an important regulator of cellular metabolism. Its activation occurs in states of adenosine triphosphate (ATP) deficit, as after heat stress, excessive training, hypoxia/ischemia, and starvation [5] and has been associated with a number of beneficial effects in metabolic diseases. Activation of AMPK leads to processes that inhibit ATP consumption, such as gluconeogenesis in the liver and fatty acid release in liver and adipose cells. On the other hand, AMPK activation supports ATP production by increasing glucose uptake in the muscles and fatty acid oxidation in liver and muscle cells. Thus, AMPK activation might provide a promising tool for the treatment of obesity and diabetes [5–8]. Accordingly, several pharmacological AMPK activators have been reported to increase glucose uptake [6, 9–13]. Interestingly, directly before the Olympic Games in Beijing 2008, AMPK activation was reported to contribute to a drastic increase in exercise capacity in mice [8]. This report fueled the debate on the usage of AMPK activators for doping and prompted the World Anti-Doping Agency (WADA) to include AICAR, an AMPK activator, in the prohibited list of substances in the class ‘Hormone and metabolic modulators’. Obviously, it is now of interest whether other AMPK activators might also be able to enhance exercise performance. This review, based mainly on a literature search in PubMed (http://www.ncbi.nlm.nih. gov/pubmed), aims to summarize studies describing effects on endurance capacity after AMPK activation by naturally occurring substances, small molecules, or pharmaceutical agents (Table 1).

2 Structure and Regulation of Adenosine Monophosphate (AMP)-Activated Kinase (AMPK) AMPK is a heterotrimeric enzyme that consists of three subunits, a, b, and c. Two isoforms are known for the a and b subunits (a1, a2, b1, b2) while the c-subunit exists in three isoforms (c1, c2, c3) [14–16]. So far, the function of the different isoforms, which can form several different complexes, is not clear, but the specificity of isoforms for the subcellular localization of the complexes is under discussion. The catalytical a-subunit contains the serinethreonine kinase domain at the N-terminus and a regulatory domain at the C-terminus. The kinase domain carries the Thr172 residue in the activation loop, which is phosphorylated upon activation of the kinase [17]. While the a1 isoform is ubiquitously expressed, a2 is specifically localized in skeletal and cardiac muscles and the nervous system [14]. The C-terminus of the regulatory b-subunit builds a scaffold for the heterotrimeric complex through binding domains that interact with the a and the c subunits

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[18]. The c subunit carries two pairs of Bateman domains and the AMP and ATP binding sites. It is suggested that conditions with high ATP levels support binding of ATP to the enzyme, which then remains inactive [19]. In low energy states, AMP binding to the Bateman domains stimulates AMPK activity by three different mechanisms: (1) allosterical activation of the kinase, which, however, causes only slight activation; (2) stimulation of upstream kinases leading to increased phosphorylation of Thr172 of the a subunit; (3) inhibition of dephosphorylation by phosphatases, indicating that AMP binding to the c subunit induces maximal AMPK activation by increasing the extent of phosphorylation of the a subunit by inhibition of dephosphorylation (reviewed in Carling et al. [20]). This complex activation system results in an extremely sensitive regulation of AMPK in response to small changes in the AMP/ATP ratio. The two major AMPK kinases in mammalian cells are liver kinase B1 (LKB1) and calciumcalmodulin-dependent protein kinase kinase (CamKK) a and b. LKB1 is associated, in a heterotrimeric complex, with the two proteins, Ste20-related adaptor protein (STRADa/b) and mouse protein 25 (MO25a/b) and is then constitutively active in most cells [21, 22]. In the skeletal muscle, LKB1 has been suggested to be the most important AMPK kinase during high cellular energy stress, since LKB1 deficiency in the muscle results in reduced basal AMPK activity and a lack of AMPK activation by drugs or muscle contraction [23]. CamKK exists in the two isoforms, a and b, which share 70 % sequence homology [24]. CamKK is activated by increasing cellular concentrations of calcium and calmodulin and is able to phosphorylate AMPK in the brain, endothelium, lymphocytes, and in skeletal muscle during contraction [25]. Transforming growth factor b-activated kinase 1 (TAK1) has been described recently as a third AMPK kinase [26]; however, the complete mechanism and its in vivo role are not yet clear.

3 AMPK and Skeletal Muscle Endurance as well as strength exercise training is associated with several changes in gene expression and oxidative metabolism in skeletal muscles. Exercise and skeletal muscle contraction induce modulation of the cellular energy status, which varies depending on the intensity and duration of exercise. In particular, concentrations of creatine phosphate and ATP decrease while those of creatine and AMP increase when exercise is performed acutely at high intensity or for a prolonged period at lower intensity [27]. The increase in the AMP/ATP ratio may then activate AMPK, which has been demonstrated in several animal and human studies [28–31]. a1b2c1, a2b2c1, and a2b2c3

AMPK and Exercise Table 1 Adenosine monophosphate-activated kinase activators and their impact on exercise capacity Drug class/drug

AMPK activation mechanism

Effect on exercise capacity

Literature

AICAR (5-amino-1-b-Dribofuranosyl-imidazole4-carboxamide)

Binding of ZMP (5-amino-1-b-D-ribofuranosylimidazole-4-carboxamide monophosphate) to the AMP-binding site in the c subunit

Enhances endurance capacity

[8, 12, 55]

Metformin/phenformin

Inhibition of mitochondrial respiratory chain; increase of intracellular AMP levels

Metformin: no effect; enhanced exercise duration

[62–64]

Pioglitazone/rosiglitazone

Inhibition of mitochondrial respiratory chain; Increase of intracellular AMP levels

Pioglitazone: no effect

[75, 76]

AMP mimics

Rosiglitazone: increased maximal exercise capacity

Salicylate/aspirin

Allosteric activation

No effect

Phenobarbital

Inhibition of mitochondrial respiratory chain; Increase of intracellular AMP levels

Not investigated

[66]

Telmisartan

PPARd-dependent

Increased running endurance

[79]

Resveratrol

Inhibition of mitochondrial respiratory chain; Increase of intracellular AMP levels

Enhanced running performance in high-capacity runner rats

[88–94]

Reversal of positive training effects in low-capacity runner rats Contradictory results in clinical studies EGCG

Inhibition of mitochondrial respiratory chain; Increase of intracellular AMP levels

Capsaicin

Not clarified

Increase in exercise performance in mice

[100–102]

No effect in human athletes Enhanced exercise capacity (AMPKdependent?)

[106]

MAFs

Only possible in combination with exercise

Improved running time and distance

[107]

Chitooligosaccharide

Not clarified

Increased running capacity

[110]

Arctigenin

LKB1/CamKK dependent

Nootkatone

Increased endurance capacity

[108, 109]

Increased swimming endurance

[111]

A769662

Allosteric activation

Not investigated

R118

Disruption of mitochondrial respiratory chain

Enhanced endurance capacity and running speed

[114]

AMPK adenosine monophosphate-activated kinase, CamKK calcium-calmodulin-dependent kinase kinase, EGCG epigallocatechingallate, LKB1 liver kinase B1, MAFs mitochondria activating factors, PPAR peroxisome proliferator-activated receptor

heterotrimers are expressed in human skeletal muscle. The a2b2c3 complex is predominantly activated during shortterm intense exercise [32], while long-term moderate exercise increases the activity of the a2b2c1 complex [33]. AMPK activation by exercise has been associated with increased muscle glucose uptake, an increase in fatty acid oxidation and decreased protein synthesis [34]. Mice with a kinase-dead form of AMPKa2 in skeletal and cardiac muscles displayed an impaired capacity for voluntary running compared with wild-type mice. Since hexose uptake in skeletal muscle was only partially diminished, it was concluded that the lack of AMPKa2 in cardiac muscles might contribute to the decreased physical activity [35]. AMPKa1a2 double knock-out mice showed a reduced mitochondrial oxidative capacity, associated with reduced expression of mitochondrial genes such as peroxisome proliferator-activated receptor gamma (PPARc), coactivator 1 alpha (PGC-1a), cytochrome C, or citrate synthase. These effects are suggested to contribute to

decreased exercise tolerance. However, contraction-stimulated glucose uptake remained unchanged in the extensor digitorum longus muscle of male and female mice while glucose uptake was impaired in the soleus muscle of male but not female mice [36]. This observation corresponds to the smaller exercise-induced AMPK activation in women than in men and hints to sex differences in muscle cells during exercise [37]. In an exercise capacity test using progressive treadmill running, mice with skeletal musclespecific AMPKb1 knock-out did not differ from wild-type littermates, while b2-/--mice showed a *25 % reduced running capacity. In contrast, mice with a double knock-out of both b1 and b2 subunits were extremely inactive and revealed strong reductions in their maximal running speed (*57 %) and distance covered (*94 %) compared with wild-type control animals. This effect was associated with a reduction of the mitochondrial content and a loss of contraction-induced glucose uptake [38]. Decreased voluntary running behavior could also be shown in mice with

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a skeletal-muscle-specific deficiency of LKB1, the major AMPK kinase in skeletal muscles [39]. In these mice, AMPK activation in response to electrical stimulation was completely diminished and contraction-induced glucose uptake, as well as substrate phosphorylation, was strongly inhibited. The effects were associated with a reduction of muscle mitochondrial oxidative enzymes, such as citrate synthase and cytochrome C, indicating reduced muscle oxidative capacity. Moreover, blood glucose availability was decreased, which might contribute to the reduction of endurance capacity [39]. However, a further study showed that prolonged exercise training led to a strong increase in running capacity in the knock-out mice, indicating that the LKB1 deficiency does not impair the improvement in exercise capacity [40]. On the other hand, gain of function mutants of the regulatory c subunit (AMPKc1R70Q, c3R225Q, c3R200Q) showed an increased mitochondrial and exercise capacity [41–43]. In summary, these data support the hypothesis that a loss of several components of the AMPK activation cascade is associated with poor exercise performance, indicating that AMPK in the skeletal muscle is required for metabolic adaptation in response to exercise [44]. This is in good accordance with the fact that several AMPK subunits, as well as AMPK activity, are increased in the muscle after endurance training. The enhanced level of AMPK might, thus, additionally contribute to an increase in the insulin sensitivity of trained individuals [45–47].

4 Molecular Mechanisms of AMPK-Mediated Increase in Exercise Capacity

PPARd signal transduction in the skeletal muscle. In this pathway, most of the regulated genes are involved in oxidative metabolism [8]. On the other hand, it should be noted that, in PGC-1a knock-out mice, mitochondrial content, isolated muscle contraction capacity, fiber-type composition, in-cage ambulation, and voluntary running capacity are all preserved [51], indicating that other factors must participate in AMPK-mediated exercise effects. This assumption is supported by further studies that show that AMPK activation by AICAR upregulates GLUT4 transporters, mitochondrial proteins, and cellular glycogen content [52, 53], all of which have already been associated with increased endurance capacity.

5 AMPK Activators and their Effect on Exercise Capacity A number of various AMPK activators with differing mechanisms have been described in the literature. These activators comprise AMP and the closely related substance, AICAR, well-known approved drugs such as metformin or aspirin, natural compounds such as resveratrol and epigallocatechingallate, as well as novel, small-molecular synthetic drugs such as A769662. The mechanism of AMPK activation by most of these drugs has been extensively studied using cells with mutations/deletions of specific AMPK subunits. In particular, mutation of the gamma subunit, which leads to insensitivity of the complex to AMP and adenosine diphosphate (ADP), provides important hints concerning the influence of drugs on cellular AMP and ADP levels [54]. 5.1 AMP Mimics

In 2008, Narkar et al. [8] found that activation of AMPK by AICAR increased the running capacity of mice without any exercise training compared with untreated mice. The animals were able to run for a 22 % longer period of time and 45 % further than control mice. This effect was associated with an increased oxygen consumption and a decrease in body fat [8]. Some of these effects might be related to direct phosphorylation and activation of AMPK target genes, such as the transcription factors and coactivators p53, transducer of regulated cAMP response elementbinding protein (CREB) protein 2 (TORC2) or PGC-1a [48–50] indicating that long-term changes are probably mediated by changes in gene transcription. PGC-1a appears to be particularly important in the regulation of AMPK signaling in the skeletal muscle, since gene expression of glucose transporter 4, mitochondrial genes, and PGC-1a itself are dependent on cellular PGC-1a expression [50]. Further experiments with PPARd knockout mice revealed that the AMPK-induced effects on exercise capacity are mediated by PGC-1a-dependent

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5.1.1 AICAR To date, AICAR is the most popular AMPK activator and has been used in the majority of scientific reports to stimulate AMPK activity in cells and animals. AICAR is rapidly processed into ZMP (5-amino-1-b-D-ribofuranosylimidazole-4-carboxamide monophosphate) by adenosine kinase activity. ZMP is an AMP analogue and is able to bind to the AMP-binding site in the c subunit of AMPK, which leads to subsequent AMPK activation. In isolated skeletal muscle preparations, AICAR enhances AMPK activation [55], which was confirmed in muscle tissue of AICAR-treated mice. As indicated above, treatment with AICAR increased endurance capacity of untrained mice to about 45 % [8]. In a clinical study which, however, did not investigate AICAR effects on endurance capacity, it was shown that AICAR, similar to exercise training, increases the muscular uptake of 2-deoxyglucose [12], indicating that AICAR might also stimulate exercise effects in humans.

AMPK and Exercise

Nevertheless, AICAR, besides activating AMPK, might interfere with other signal transduction pathways through the interaction of ZMP with other AMP-regulated enzymes, such as fructose-1,6-bisphosphatase (FBPase) [56], glycogen phosphorylase [57], or adenosine receptors [58]. Therefore, it cannot be ruled out that the observed AICAR-induced effects are mediated not only through AMPK but also via one or more of these targets. 5.2 Pharmacological Drugs 5.2.1 Metformin/Phenformin The blood glucose-lowering biguanides, metformin and phenformin, are well known AMPK activators; however, their distinct mechanisms of AMPK activation are not completely understood. Metformin is thought to activate AMPK in an indirect manner [59], possibly via inhibition of the complex I of the mitochondrial respiratory chain [60, 61] and modification of the intracellular AMP:ATP ratio rather than direct phosphorylation or dephosphorylation of the catalytical subunit. So far, it has not been clarified whether metformin or phenformin are able to increase body fitness and endurance capacity. In rats, metformin treatment had no effect on twitch characteristics or tetanic contractions of extensor digitorum longus and soleus muscles [62]. Clinical studies have been carried out on the effect of metformin on exercise capacity in healthy individuals. One study, focusing on peak aerobic performance, showed that metformin slightly but significantly decreased volume oxygen (VO2) peak, peak heart rate, peak ventilation, peak respiratory exchange ratio, and exercise duration [63]. In contrast, another report showed that a single metformin dose did not acutely influence maximal oxygen consumption or ventilatory threshold in healthy active males [64]. Thus, it is not possible at present to draw a clear conclusion on biguanide effects on exercise capacity. 5.2.2 Salicylic Acid (Salicylate) Salicylate is the active metabolite of the non-steroidal antiinflammatory drug acetylsalicylic acid (aspirin), which is a very popular drug for the treatment of inflammation, fever, and pain and, in low doses, as an antiplatelet agent. In addition to its well known mechanism of cyclooxygenase inhibition, recent studies have shown that it is also able to activate AMPK. Similarly to the small-molecule AMPK activator, A769662 (see below), it induces allosteric activation independently of AMP and inhibits dephosphorylation of the a-subunit. Salicylate does not require the c subunit but fails to activate AMPK in mice with a deletion of the b1-subunit, indicating that this subunit is obligatory to the activation mechanism [65]. The effects of salicylate

on exercise performance have not been intensively studied. A relatively old clinical study indicated that 1 g of aspirin prior to a progressive maximal exercise test on a cycle ergometer has no effect on the physical performance during exercise [66]. Nevertheless, in rat skeletal muscles, treatment with 5 mM salicylate led to salicylate concentrations of 3.1 ± 0.2 mM in the epitrochlearis and 1.8 ± 0.1 mM in the soleus muscle and contributed to glucose uptake. For comparison, plasma concentrations of salicylate are in the range of 1 mM after uptake of 1 g in humans [67]. Since ATP levels, and creatinine and glycogen contents simultaneously decreased, it has been suggested that salicylate stimulates AMPK and glucose transport via energy deprivation [68]. 5.2.3 Thiazolidinediones Rosiglitazone and pioglitazone are ‘insulin sensitizers’ with a long tradition as antidiabetic drugs. Their main mechanism of action is considered to be activation of the transcription factor, PPARc, thus inducing the expression of several genes that regulate glucose and fatty acid metabolism, thereby facilitating insulin effects, such as lowering blood glucose and increasing free fatty acid absorption. Furthermore, these drugs are able to activate AMPK in isolated skeletal muscles and in a mouse model of type 2 diabetes (ob/ob mice). Since activation starts rapidly after drug treatment, it is not very likely that PPARc is involved in this mechanism [69]. It has been suggested that thiazolidinedione effects are mediated by adiponectin-dependent and -independent mechanisms, which include an increase in the AMP/ATP ratio by inhibition of the mitochondrial respiratory chain [10, 70, 71]. Adiponectin is an adipokine that inhibits glucose production in the liver [72] and stimulates an increase in cellular AMP [73]. Adiponectin treatment of mice induced AMPK activation in skeletal muscles and the liver [74]. Pioglitazone, in contrast to training alone, did not enhance maximal aerobic velocity, endurance capacity, and grip strength. Furthermore, expression of mitochondrial biogenesis markers, such as PGC-1a and citrate synthase activity in the soleus muscle, were not changed after pioglitazone treatment of mice [75]. In contrast, a clinical study showed that rosiglitazone (4 mg daily for a period of 4 months) increased maximal exercise capacity in individuals with type 2 diabetes. Since no mechanistic data were provided in that study, the impact of AMPK activation is not clear [76]. 5.2.4 Phenobarbital Phenobarbital is a barbiturate that has sedative properties and is administered for the treatment of seizure. It is known

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to be an inducer of cytochrome P450 (CYP) enzymes, catalyzing oxidation of hydrophobic drugs and thereby improving their water solubility. Several studies suggest that the induction of CYP, which has also been observed with other AMPK activators, is mediated via activation of AMPK [77, 78]. The mechanism of phenobarbital-induced AMPK activation relies, as for metformin and thiazolidinediones, on inhibition of the respiratory chain [54]. Given the sedative effects of phenobarbital, it is not very likely that the drug will enhance exercise performance. In accordance with this, no study has been published so far that has investigated endurance effects after phenobarbital uptake. 5.2.5 Telmisartan The angiotensin II receptor type 1 blocker, telmisartan, activates AMPK in a PPARd-dependent manner. AMPK activation was associated with an increase in running endurance, post-exercise oxygen consumption, and slowtwitch muscle fibers. Furthermore, telmisartan treatment prevented weight gain [79]. AMPK activation has also been reported after administration of other angiotensin receptor blockers (‘sartans’) such as losartan, olmesartan, or candesartan [80–82]. Some of these drugs were also associated with improved exercise capacity [83–85], but this effect was not directly linked to AMPK activation. 5.3 Natural Products Several phytochemicals have been described as AMPK activators. Most of these plant products have been associated with beneficial health effects or are already used in traditional Chinese medicine (TCM). In particular, resveratrol, an ingredient of grapes and red wine (*1.2–7.2 mg/l depending on wine type [86]), or epigallocatechin-3-gallate (EGCG) in green tea are popular examples of natural compounds. Both impair the respiratory chain by inhibition of the F1 ATPase [87]. Since they failed to activate AMPK in cells with a mutated c subunit, their mechanism of action relies on an increase in the AMP/ATP ratio [54]. 5.3.1 Resveratrol A study in low-capacity runner (LCR) rats focused on effects of training and administration of resveratrol (100 mg/kg body weight) in these animals. Unexpectedly, resveratrol in combination with exercise training reversed the positive gain in endurance and mitochondrial biogenesis of training alone. Interestingly, exercise training in these animals led to reduced AMPK activation, which was also the case after resveratrol treatment [88]. A clinical study in aged men showed that resveratrol (250 mg daily

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for 8 weeks) did not enhance, but rather attenuated, the positive effects of endurance exercise on inflammatory and oxidative stress markers. From these results, it was concluded that resveratrol does not act as an exercise mimetic [89]. Further clinical studies supported these findings with the observation that exercise training induces improvement of cardiovascular health parameters and strong angiogenic effects, which were impaired in the resveratrol-treated group [90, 91]. In contrast, high-capacity runner (HCR) rats showed enhanced exercise performance and strength of upper limbs after treatment with resveratrol. These effects were associated with activation of AMPK and the histone deacetylase SIRT1 [92]. A further study showed that resveratrol (400 mg/kg body weight)-induced SIRT1 activation in mice led to a decrease in the acetylation of PGC1a and thereby to an increase in its activation state. This effect was associated with protection against diet-induced obesity and insulin resistance as well as with an increase in aerobic capacity [93]. In another study in rats, resveratrol (146 mg/ kg body weight) enhanced training-induced fatty acid oxidation and improved exercise performance by 21 %, which was associated with increased twitch and tetanic forces generated during isometric contraction. These effects were associated with increased resting of the left ventricular ejection fraction and decreased left ventricular wall stress [94]. In view of these contradictory results, it is difficult to judge whether resveratrol might have beneficial effects on endurance capacity. Naturally, it must be taken into account that the experiments were performed in different species and with different treatment strategies (treatment period, dose, exercise type), which could account for some of the differences. In addition, resveratrol effects can be highly selective and might vary depending on the metabolic status of the treated subjects [95]. Thus, further studies will be necessary to clearly evaluate the role of resveratrol in exercise performance. 5.3.2 Epigallocatechin Gallate EGCG is the pharmacologically most active component of green tea and is probably responsible for many health benefits after frequent consumption of green tea, including weight loss [96], improved blood glucose and lipid regulation [96–98], and prevention of cancer and cardiovascular disturbances [99]. In addition, EGCG consumption has been associated with increased exercise capacity. Mice treated with a standard diet supplemented with green tea extract showed a 30 % longer treadmill-running time-tofatigue compared with control animals [100]. Similar effects could also be observed in a swimming test with mice [101]. A double-blind, placebo-controlled clinical

AMPK and Exercise

study with eight cyclists was performed to investigate the effect of EGCG on endurance capacity. EGCG was administered orally over a period of 7 days. At 1 h after the last administration, the athletes performed three cycling exercises. The results showed no significant effect of EGCG on exercise performance, indicating that EGCG does not provide benefit for human athletes [102].

5.3.6 Chito-Oligosaccharide Chito-oligosaccharide (COS), derived from chitin in the exoskeleton of Crustaceae, activates PGC-1a in an SIRT1and AMPK-dependent manner, thereby increasing mitochondrial biogenesis. These effects were associated with an increased running capacity in COS-treated compared with control mice [110].

5.3.3 Capsaicin Capsaicin, an agonist of the transient receptor potential vanilloid (TRPV) 1, has also been associated with activation of AMPK in cell culture [103–105]. Furthermore, it was found to increase exercise endurance, for instance, by regulation of PGC-1a [106], but a direct impact on AMPK activation remains to be demonstrated. However, since AMPK activation as well as PGC-1a have been repeatedly associated with enhanced exercise capacity, it might be assumed that AMPK is at least partially involved in capsaicin-induced improvement of performance. 5.3.4 Mitochondria-Activating Factors High molecular weight polyphenols from black tea, which are also referred to as mitochondria-activating factors (MAFS), have shown AMPK-activating properties when combined with exercise training, while MAF alone had no effect on AMPK activation. The increase in AMPK activation after MAF treatment of mice was associated with an improved running time and distance. It has been suggested that MAF effects are mediated by AMPK-dependent signaling pathways [107]. 5.3.5 Arctigenin Arctigenin, a lignin in certain Asteraceae, e.g. burdocks, used in TCM, promotes AMPK activation in an LKB1- and CamKK-dependent manner. In addition, it enhanced mitochondrial biogenesis as well as expression of genes responsible for fatty acid synthesis and oxidation, respectively, and thereby improved treadmill endurance capacity in mice. The authors suggested that this mechanism might provide more fatty acids in skeletal muscles, which can then be immediately oxidized to supply energy during exercise. Since AMPK activity increases fatty acid oxidation but reduces fatty acid synthesis, it must be assumed that the latter is mediated by other AMPK-independent mechanisms of arctigenin [108]. In rats, arctigenin has been shown to increase the transcription of antioxidantrelated genes, such as PGC-1a, PPARa, and uncoupling protein (UCP)-2 transcription in the gastrocnemius and quadriceps muscles via AMPK and strongly increased swimming endurance [109].

5.3.7 Nootkatone Nootkatone, a constituent of grapefruit, activates AMPK in cell culture as well as in liver and muscles of mice and increases whole body energy expenditure. In mice, symptoms of type 2 diabetes and obesity were reduced and the capacity for swimming endurance increased by 21 % compared with control mice [111]. 5.4 Small Molecule Activators 5.4.1 A769662 The small molecule A769662 was identified by in vitro analyses of chemical compounds that had been synthesized as potential new AMPK activators [112]. A769662 is a non-nucleoside thienopyridine that potently activates AMPK in a reversible manner (half maximal effective concentration [EC50] *116 nM). Its effects are mediated by a similar pathway to that induced by AMP: allosteric activation and inhibition of dephosphorylation of Thr172 in the a subunit. In contrast to the substances shown above, A769662 does not increase AMP levels, and therefore works in cells with a mutated AMPKc subunit [54]. A769662 exclusively activates AMPK complexes that contain the b1 subunit [113]. So far, no studies have been published that have investigated a relationship between AMPK activation by A769662 and effects on exercise endurance. 5.4.2 R118 R118, a novel heterocyclic small-molecule AMPK activator, modulates AMPK activity by disrupting the mitochondrial respiratory chain. In vivo, R118 increased AMPK phosphorylation in muscles of mice and improved exercise capacity in wheel-running and treadmill tests. A high dose of R118 enhanced the wheel-running counts twofold, decreased the frequency of resting by 50 %, and increased the average running speed [114]. R118 treatment was associated with increased glycolysis and lipolysis, mitochondrial biogenesis, and improved microvascular function in the skeletal muscle [114, 115].

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6 Discussion Activation of AMP-activated kinase is associated with regulation of glucose and lipid metabolism as well as redox signaling, leading to a number of positive effects such as decreased obesity and an improvement in glucose homeostasis in diabetes [116]. In addition, a number of recent reports hint towards a role of AMPK activators in the improvement of endurance exercise capacity. AMPK activators comprise well known approved drugs, many natural products, as well as small molecules that have been specifically synthesized as potential AMPK-activating drugs. As described above, findings on the exercise-improving capacities of these drugs are controversial, and a number of the AMPK activators described have not even been investigated in the context of endurance exercise. The anti-diabetic drugs, metformin/phenformin, as well as rosiglitazone/pioglitazone, resulted in increased exercise capacity in some studies and no effects in other studies. Similarly, no effects on body fitness have been reported for salicylate. Natural products have been described as AMPK activators and enhancers of aerobic capacity in most of the reports. However, a direct link between AMPK activation and improvement in exercise capacity is missing in most of the studies. Furthermore, it is clear that these natural products, which are mostly ingredients of food, are not specific for AMPK activation, but affect multiple signal transduction pathways. Thus, several of their targets might influence training success. AICAR and R118, two drugs that activate AMPK relatively specifically, have been reported to improve endurance capacity and might therefore appear suitable as a kind of AMPK-activating ‘exercise mimetic’. Basically, exercise mimetics would be expected to show the same effects as those gained with normal endurance exercise; namely, increased metabolism in the skeletal muscles as well as changes in their contractile properties. Since these adaptations, particularly in skeletal muscles, are very complex, many researchers deny the existence of ‘exercise pills’ that are able to exactly replicate exercise-induced physiological changes associated with gene regulation in cells, tissues, and organs in response to different types of exercise [117–119]. This attitude is supported by studies investigating AMPK and exercise effects. AMPK can be activated by both exercise and the potential ‘exercise mimetic’ AICAR, but subsequent signal transduction differs between the two activation pathways. While plasma fatty acid and glycerol strongly increase after exercise, AICAR induced a decrease of fatty acids and did not affect glycerol levels [120]. Furthermore, studies with AMPKa knock-out mice showed that a number of genes important for the improvement of

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oxygen utilization and oxidative capacity are not necessarily dependent on AMPK activation [121]. Thus, AMPK activation exemplifies the fact that drugs or genetic modifications that target only single molecules may not mimic exercise-induced physical modifications. Nevertheless, although it might not be possible to exactly mimic the exercise-induced physical adaptations, it has clearly been shown that AICAR can improve running capacity in mice without any exercise training, which prompted the WADA to add this compound and AMPK activators in general to the doping list of prohibited substances. However, several results in this review support the hypothesis that there is no need to ban all AMPK activators for athletes, since a number of AMPK-activating drugs do not influence endurance capacity, indicating that AMPKactivating properties are not mandatorily associated with exercise-mimicking properties. This might be due to the low specificity or low potency of the drugs; however, this remains to be clarified. Furthermore, it is to be expected that administration of these drugs will not have a drastic impact on sports performance in well-trained individuals. In the rodent studies, effects of AICAR were investigated in sedentary animals with low training-induced AMPK activity. Due to ongoing intense exercise training, AMPK is already highly activated in human athletes, thus making a further strong increase after uptake of AMPK activators unlikely [47]; therefore, the doping capacity might be low. On the other hand, one study showed that HCR rats, an experimental model of endurance sport, had improved running endurance after resveratrol treatment [92], which contradicts the assumption that no further enhancement of exercise effects is possible. However, resveratrol effects may not be mediated in an AMPK-specific manner. In clinical practice, mimicking of exercise by pharmacological AMPK activation, if indeed possible, might be particularly interesting and beneficial for individuals with diseases in which the therapy is supported by exercise training. A number of these patients are not able to perform exercise to a sufficient degree. These include obese patients, patients with diabetes-induced neuropathies, severe cardiovascular conditions, or musculoskeletal disorders. Supportive therapy with pharmacologically active drugs would be helpful for such patient groups. As an example, patients with Duchenne’s muscular dystrophy are thought to benefit from muscle remodeling towards a more slower oxidative fiber type that is resistant to dystrophic effects. Activators of AMPK, PPARc, and SIRT1 might contribute to this remodeling and are under consideration as potential therapy for these patients [122–124]. In any case, all clinical considerations should take into account that AMPK activation has various beneficial effects, but is not completely hazard free. In the heart, AMPK activation

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might occasionally result in ventricular pre-excitation and hypertrophic cardiomyopathy in patients with naturally occurring mutations in the c subunit of AMPK. In the nervous system, AMPK activation has been associated with Alzheimer‘s disease [125, 126] and amyotrophic lateral sclerosis [127]. AMPK activation in the hypothalamus might lead to increased consumption of food and thereby exacerbate type 2 diabetes and obesity. Isoform-specific and tissue-specific activators might circumvent such complications but are not available so far.

7 Conclusion Data summarized in this review show that activation of AMPK by specific drugs may support effects of exercise training in special cases. However, it is unlikely to completely replace individual body workouts, certainly not in well trained athletes with pre-activated AMPK. A number of further clinical studies will be necessary to clarify the impact of AMPK activation and its possible role as a ‘doping target’ in humans. Acknowledgments This work was supported by the ‘Landesoffensive zur Entwicklung wissenschaftlich-o¨konomischer Exzellenz (LOEWE), Zentrum fu¨r Translationale Medizin und Pharmakologie’ and a grant from the Deutsche Forschungsgemeinschaft (DFG) to Ellen Niederberger (NI/705/3-3). The authors would like to thank Professor Michael Parnham for carefully reading and correcting the manuscript. Gerd Geisslinger is an anti-doping expert of the Landessportbund Hessen, Frankfurt, Germany. Compliance with Ethical Standards Conflict of interest Ellen Niederberger, Tanya S. King, Otto Q. Russe, and Gerd Geisslinger declare that they have no conflicts of interest.

References 1. Holloszy JO, Coyle EF. Adaptations of skeletal muscle to endurance exercise and their metabolic consequences. J Appl Physiol Respir Environ Exerc Physiol. 1984;56(4):831–8. 2. Gundersen K. Excitation-transcription coupling in skeletal muscle: the molecular pathways of exercise. Biol Rev Camb Philos Soc. 2011;86(3):564–600. 3. Mikines KJ, Sonne B, Farrell PA, Tronier B, Galbo H. Effect of physical exercise on sensitivity and responsiveness to insulin in humans. Am J Physiol. 1988;254(3 Pt 1):E248–59. 4. Koval JA, Maezono K, Patti ME, Pendergrass M, DeFronzo RA, Mandarino LJ. Effects of exercise and insulin on insulin signaling proteins in human skeletal muscle. Med Sci Sports Exerc. 1999;31(7):998–1004. 5. Hardie DG. AMP-activated/SNF1 protein kinases: conserved guardians of cellular energy. Nat Rev Mol Cell Biol. 2007;8(10):774–85.

6. Merrill GF, Kurth EJ, Hardie DG, Winder WW. AICA riboside increases AMP-activated protein kinase, fatty acid oxidation, and glucose uptake in rat muscle. Am J Physiol. 1997;273(6 Pt 1):E1107–12. 7. Andersson U, Filipsson K, Abbott CR, Woods A, Smith K, Bloom SR, et al. AMP-activated protein kinase plays a role in the control of food intake. J Biol Chem. 2004;279(13):12005–8. 8. Narkar VA, Downes M, Yu RT, Embler E, Wang YX, Banayo E, et al. AMPK and PPARdelta agonists are exercise mimetics. Cell. 2008;134(3):405–15. 9. Zhou G, Myers R, Li Y, Chen Y, Shen X, Fenyk-Melody J, et al. Role of AMP-activated protein kinase in mechanism of metformin action. J Clin Invest. 2001;108(8):1167–74. 10. Fryer LG, Parbu-Patel A, Carling D. The Anti-diabetic drugs rosiglitazone and metformin stimulate AMP-activated protein kinase through distinct signaling pathways. J Biol Chem. 2002;277(28):25226–32. 11. Koistinen HA, Galuska D, Chibalin AV, Yang J, Zierath JR, Holman GD, et al. 5-amino-imidazole carboxamide riboside increases glucose transport and cell-surface GLUT4 content in skeletal muscle from subjects with type 2 diabetes. Diabetes. 2003;52(5):1066–72. 12. Cuthbertson DJ, Babraj JA, Mustard KJ, Towler MC, Green KA, Wackerhage H, et al. 5-aminoimidazole-4-carboxamide 1-betaD-ribofuranoside acutely stimulates skeletal muscle 2-deoxyglucose uptake in healthy men. Diabetes. 2007;56(8):2078–84. 13. Boon H, Bosselaar M, Praet SF, Blaak EE, Saris WH, Wagenmakers AJ, et al. Intravenous AICAR administration reduces hepatic glucose output and inhibits whole body lipolysis in type 2 diabetic patients. Diabetologia. 2008;51(10):1893–900. 14. Stapleton D, Mitchelhill KI, Gao G, Widmer J, Michell BJ, Teh T, et al. Mammalian AMP-activated protein kinase subfamily. J Biol Chem. 1996;271(2):611–4. 15. Thornton C, Snowden MA, Carling D. Identification of a novel AMP-activated protein kinase beta subunit isoform that is highly expressed in skeletal muscle. J Biol Chem. 1998;273(20):12443–50. 16. Cheung PC, Salt IP, Davies SP, Hardie DG, Carling D. Characterization of AMP-activated protein kinase gamma-subunit isoforms and their role in AMP binding. Biochem J. 2000;15(346 Pt 3):659–69. 17. Crute BE, Seefeld K, Gamble J, Kemp BE, Witters LA. Functional domains of the alpha1 catalytic subunit of the AMP-activated protein kinase. J Biol Chem. 1998;273(52):35347–54. 18. Iseli TJ, Walter M, van Denderen BJ, Katsis F, Witters LA, Kemp BE, et al. AMP-activated protein kinase beta subunit tethers alpha and gamma subunits via its C-terminal sequence (186-270). J Biol Chem. 2005;280(14):13395–400. 19. Xiao B, Heath R, Saiu P, Leiper FC, Leone P, Jing C, et al. Structural basis for AMP binding to mammalian AMP-activated protein kinase. Nature. 2007;449(7161):496–500. 20. Carling D, Thornton C, Woods A, Sanders MJ. AMP-activated protein kinase: new regulation, new roles? Biochem J. 2012;445(1):11–27. 21. Baas AF, Boudeau J, Sapkota GP, Smit L, Medema R, Morrice NA, et al. Activation of the tumour suppressor kinase LKB1 by the STE20-like pseudokinase STRAD. EMBO J. 2003;22(12):3062–72. 22. Boudeau J, Baas AF, Deak M, Morrice NA, Kieloch A, Schutkowski M, et al. MO25alpha/beta interact with STRADalpha/beta enhancing their ability to bind, activate and localize LKB1 in the cytoplasm. EMBO J. 2003;22(19):5102–14. 23. Sakamoto K, McCarthy A, Smith D, Green KA, Grahame Hardie D, Ashworth A, et al. Deficiency of LKB1 in skeletal

123

E. Niederberger et al.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

muscle prevents AMPK activation and glucose uptake during contraction. EMBO J. 2005;24(10):1810–20. Kitani T, Okuno S, Fujisawa H. Molecular cloning of Ca2?/calmodulin-dependent protein kinase kinase beta. J Biochem. 1997;122(1):243–50. Hawley SA, Selbert MA, Goldstein EG, Edelman AM, Carling D, Hardie DG. 5’-AMP activates the AMP-activated protein kinase cascade, and Ca2?/calmodulin activates the calmodulindependent protein kinase I cascade, via three independent mechanisms. J Biol Chem. 1995;270(45):27186–91. Momcilovic M, Hong SP, Carlson M. Mammalian TAK1 activates Snf1 protein kinase in yeast and phosphorylates AMPactivated protein kinase in vitro. J Biol Chem. 2006;281(35):25336–43. Broberg S, Sahlin K. Adenine nucleotide degradation in human skeletal muscle during prolonged exercise. J Appl Physiol. 1989;67(1):116–22. Winder WW, Hardie DG. Inactivation of acetyl-CoA carboxylase and activation of AMP-activated protein kinase in muscle during exercise. Am J Physiol. 1996;270(2 Pt 1):E299–304. Chen ZP, McConell GK, Michell BJ, Snow RJ, Canny BJ, Kemp BE. AMPK signaling in contracting human skeletal muscle: acetyl-CoA carboxylase and NO synthase phosphorylation. Am J Physiol Endocrinol Metab. 2000;279(5):E1202–6. Fujii N, Hayashi T, Hirshman MF, Smith JT, Habinowski SA, Kaijser L, et al. Exercise induces isoform-specific increase in 50 AMP-activated protein kinase activity in human skeletal muscle. Biochem Biophys Res Commun. 2000;273(3):1150–5. Wojtaszewski JF, Nielsen P, Hansen BF, Richter EA, Kiens B. Isoform-specific and exercise intensity-dependent activation of 50 -AMP-activated protein kinase in human skeletal muscle. J Physiol. 2000;1(528 Pt 1):221–6. Birk JB, Wojtaszewski JF. Predominant alpha2/beta2/gamma3 AMPK activation during exercise in human skeletal muscle. J Physiol. 2006;577(Pt 3):1021–32. Treebak JT, Birk JB, Rose AJ, Kiens B, Richter EA, Wojtaszewski JF. AS160 phosphorylation is associated with activation of alpha2beta2gamma1- but not alpha2beta2gamma3AMPK trimeric complex in skeletal muscle during exercise in humans. Am J Physiol Endocrinol Metab. 2007;292(3):E715–22. Richter EA, Ruderman NB. AMPK and the biochemistry of exercise: implications for human health and disease. Biochem J. 2009;418(2):261–75. Mu J, Brozinick JT Jr, Valladares O, Bucan M, Birnbaum MJ. A role for AMP-activated protein kinase in contraction- and hypoxia-regulated glucose transport in skeletal muscle. Mol Cell. 2001;7(5):1085–94. Lantier L, Fentz J, Mounier R, Leclerc J, Treebak JT, Pehmoller C, et al. AMPK controls exercise endurance, mitochondrial oxidative capacity, and skeletal muscle integrity. FASEB J. 2014;28(7):3211–24. Roepstorff C, Thiele M, Hillig T, Pilegaard H, Richter EA, Wojtaszewski JF, et al. Higher skeletal muscle alpha2AMPK activation and lower energy charge and fat oxidation in men than in women during submaximal exercise. J Physiol. 2006;574(Pt 1):125–38. O’Neill HM, Maarbjerg SJ, Crane JD, Jeppesen J, Jorgensen SB, Schertzer JD, et al. AMP-activated protein kinase (AMPK) beta1beta2 muscle null mice reveal an essential role for AMPK in maintaining mitochondrial content and glucose uptake during exercise. Proc Natl Acad Sci. 2011;108(38):16092–7. Thomson DM, Porter BB, Tall JH, Kim HJ, Barrow JR, Winder WW. Skeletal muscle and heart LKB1 deficiency causes decreased voluntary running and reduced muscle mitochondrial

123

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

marker enzyme expression in mice. Am J Physiol Endocrinol Metab. 2007;292(1):E196–202. Tanner CB, Madsen SR, Hallowell DM, Goring DM, Moore TM, Hardman SE, et al. Mitochondrial and performance adaptations to exercise training in mice lacking skeletal muscle LKB1. Am J Physiol Endocrinol Metab. 2013;305(8):E1018–29. Barre L, Richardson C, Hirshman MF, Brozinick J, Fiering S, Kemp BE, et al. Genetic model for the chronic activation of skeletal muscle AMP-activated protein kinase leads to glycogen accumulation. Am J Physiol Endocrinol Metab. 2007;292(3):E802–11. Rockl KS, Hirshman MF, Brandauer J, Fujii N, Witters LA, Goodyear LJ. Skeletal muscle adaptation to exercise training: AMP-activated protein kinase mediates muscle fiber type shift. Diabetes. 2007;56(8):2062–9. Scheffler TL, Scheffler JM, Park S, Kasten SC, Wu Y, McMillan RP, et al. Fiber hypertrophy and increased oxidative capacity can occur simultaneously in pig glycolytic skeletal muscle. Am J Physiol Cell Physiol. 2014;306(4):C354–63. Lee-Young RS, Griffee SR, Lynes SE, Bracy DP, Ayala JE, McGuinness OP, et al. Skeletal muscle AMP-activated protein kinase is essential for the metabolic response to exercise in vivo. J Biol Chem. 2009;284(36):23925–34. Frosig C, Jorgensen SB, Hardie DG, Richter EA, Wojtaszewski JF. 50 -AMP-activated protein kinase activity and protein expression are regulated by endurance training in human skeletal muscle. Am J Physiol Endocrinol Metab. 2004;286(3):E411–7. Iglesias MA, Ye JM, Frangioudakis G, Saha AK, Tomas E, Ruderman NB, et al. AICAR administration causes an apparent enhancement of muscle and liver insulin action in insulin-resistant high-fat-fed rats. Diabetes. 2002;51(10):2886–94. Nielsen JN, Mustard KJ, Graham DA, Yu H, MacDonald CS, Pilegaard H, et al. 50 -AMP-activated protein kinase activity and subunit expression in exercise-trained human skeletal muscle. J Appl Physiol. 2003;94(2):631–41. Imamura K, Ogura T, Kishimoto A, Kaminishi M, Esumi H. Cell cycle regulation via p53 phosphorylation by a 50 -AMP activated protein kinase activator, 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside, in a human hepatocellular carcinoma cell line. Biochem Biophys Res Commun. 2001;287(2):562–7. Shaw RJ, Lamia KA, Vasquez D, Koo SH, Bardeesy N, Depinho RA, et al. The kinase LKB1 mediates glucose homeostasis in liver and therapeutic effects of metformin. Science. 2005;310(5754):1642–6. Jager S, Handschin C, St-Pierre J, Spiegelman BM. AMP-activated protein kinase (AMPK) action in skeletal muscle via direct phosphorylation of PGC-1alpha. Proc Natl Acad Sci. 2007;104(29):12017–22. Rowe GC, Patten IS, Zsengeller ZK, El-Khoury R, Okutsu M, Bampoh S, et al. Disconnecting mitochondrial content from respiratory chain capacity in PGC-1-deficient skeletal muscle. Cell Rep. 2013;3(5):1449–56. Holmes BF, Kurth-Kraczek EJ, Winder WW. Chronic activation of 50 -AMP-activated protein kinase increases GLUT-4, hexokinase, and glycogen in muscle. J Appl Physiol. 1999;87(5):1990–5. Winder WW, Holmes BF, Rubink DS, Jensen EB, Chen M, Holloszy JO. Activation of AMP-activated protein kinase increases mitochondrial enzymes in skeletal muscle. J Appl Physiol. 2000;88(6):2219–26. Hawley SA, Ross FA, Chevtzoff C, Green KA, Evans A, Fogarty S, et al. Use of cells expressing gamma subunit variants to identify diverse mechanisms of AMPK activation. Cell Metab. 2010;11(6):554–65.

AMPK and Exercise 55. Hayashi T, Hirshman MF, Kurth EJ, Winder WW, Goodyear LJ. Evidence for 50 AMP-activated protein kinase mediation of the effect of muscle contraction on glucose transport. Diabetes. 1998;47(8):1369–73. 56. Vincent MF, Marangos PJ, Gruber HE, Van den Berghe G. Inhibition by AICA riboside of gluconeogenesis in isolated rat hepatocytes. Diabetes. 1991;40(10):1259–66. 57. Longnus SL, Wambolt RB, Parsons HL, Brownsey RW, Allard MF. 5-Aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside (AICAR) stimulates myocardial glycogenolysis by allosteric mechanisms. Am J Physiol Regul Integr Comp Physiol. 2003;284(4):R936–44. 58. Bullough DA, Zhang C, Montag A, Mullane KM, Young MA. Adenosine-mediated inhibition of platelet aggregation by acadesine. A novel antithrombotic mechanism in vitro and in vivo. J Clin Invest. 1994;94(4):1524–32. 59. Hawley SA, Gadalla AE, Olsen GS, Hardie DG. The antidiabetic drug metformin activates the AMP-activated protein kinase cascade via an adenine nucleotide-independent mechanism. Diabetes. 2002;51(8):2420–5. 60. Owen MR, Doran E, Halestrap AP. Evidence that metformin exerts its anti-diabetic effects through inhibition of complex 1 of the mitochondrial respiratory chain. Biochem J. 2000;15(348 Pt 3):607–14. 61. El-Mir MY, Nogueira V, Fontaine E, Averet N, Rigoulet M, Leverve X. Dimethylbiguanide inhibits cell respiration via an indirect effect targeted on the respiratory chain complex I. J Biol Chem. 2000;275(1):223–8. 62. McGuire M, MacDermott M. The influence of streptozotocin diabetes and metformin on erythrocyte volume and on the membrane potential and the contractile characteristics of the extensor digitorum longus and soleus muscles in rats. Exp Physiol. 1999;84(6):1051–8. 63. Braun B, Eze P, Stephens BR, Hagobian TA, Sharoff CG, Chipkin SR, et al. Impact of metformin on peak aerobic capacity. Appl Physiol Nutr Metab. 2008;33(1):61–7. 64. Johnson ST, Robert C, Bell GJ, Bell RC, Lewanczuk RZ, Boule NG. Acute effect of metformin on exercise capacity in active males. Diabetes Obes Metab. 2008;10(9):747–54. 65. Hawley SA, Fullerton MD, Ross FA, Schertzer JD, Chevtzoff C, Walker KJ, et al. The ancient drug salicylate directly activates AMP-activated protein kinase. Science. 2012;336(6083):918–22. 66. Roi GS, Garagiola U, Verza P, Spadari G, Radice D, Zecca L, et al. Aspirin does not affect exercise performance. Int J Sports Med. 1994;15(5):224–7. 67. de Gaetano G, Cerletti C, Dejana E, Latini R. Pharmacology of platelet inhibition in humans: implications of the salicylateaspirin interaction. Circulation. 1985;72(6):1185–93. 68. Serizawa Y, Oshima R, Yoshida M, Sakon I, Kitani K, Goto A, et al. Salicylate acutely stimulates 50 -AMP-activated protein kinase and insulin-independent glucose transport in rat skeletal muscles. Biochem Biophys Res Commun. 2014;453(1):81–5. 69. LeBrasseur NK, Kelly M, Tsao TS, Farmer SR, Saha AK, Ruderman NB, et al. Thiazolidinediones can rapidly activate AMP-activated protein kinase in mammalian tissues. Am J Physiol Endocrinol Metab. 2006;291(1):E175–81. 70. Brunmair B, Staniek K, Gras F, Scharf N, Althaym A, Clara R, et al. Thiazolidinediones, like metformin, inhibit respiratory complex I: a common mechanism contributing to their antidiabetic actions? Diabetes. 2004;53(4):1052–9. 71. Kubota N, Terauchi Y, Kubota T, Kumagai H, Itoh S, Satoh H, et al. Pioglitazone ameliorates insulin resistance and diabetes by both adiponectin-dependent and -independent pathways. J Biol Chem. 2006;281(13):8748–55.

72. Berg AH, Combs TP, Du X, Brownlee M, Scherer PE. The adipocyte-secreted protein Acrp30 enhances hepatic insulin action. Nat Med. 2001;7(8):947–53. 73. Yamauchi T, Kamon J, Minokoshi Y, Ito Y, Waki H, Uchida S, et al. Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase. Nat Med. 2002;8(11):1288–95. 74. Yamauchi T, Nio Y, Maki T, Kobayashi M, Takazawa T, Iwabu M, et al. Targeted disruption of AdipoR1 and AdipoR2 causes abrogation of adiponectin binding and metabolic actions. Nat Med. 2007;13(3):332–9. 75. Sanchis-Gomar F, Pareja-Galeano H, Martinez-Bello VE. PPARgamma agonist pioglitazone does not enhance performance in mice. Drug Test Anal. 2014;6(9):922–9. 76. Regensteiner JG, Bauer TA, Reusch JE. Rosiglitazone improves exercise capacity in individuals with type 2 diabetes. Diabetes Care. 2005;28(12):2877–83. 77. Rencurel F, Stenhouse A, Hawley SA, Friedberg T, Hardie DG, Sutherland C, et al. AMP-activated protein kinase mediates phenobarbital induction of CYP2B gene expression in hepatocytes and a newly derived human hepatoma cell line. J Biol Chem. 2005;280(6):4367–73. 78. Rencurel F, Foretz M, Kaufmann MR, Stroka D, Looser R, Leclerc I, et al. Stimulation of AMP-activated protein kinase is essential for the induction of drug metabolizing enzymes by phenobarbital in human and mouse liver. Mol Pharmacol. 2006;70(6):1925–34. 79. Feng X, Luo Z, Ma L, Ma S, Yang D, Zhao Z, et al. Angiotensin II receptor blocker telmisartan enhances running endurance of skeletal muscle through activation of the PPAR-delta/AMPK pathway. J Cell Mol Med. 2011;15(7):1572–81. 80. Hernandez JS, Barreto-Torres G, Kuznetsov AV, Khuchua Z, Javadov S. Crosstalk between AMPK activation and angiotensin II-induced hypertrophy in cardiomyocytes: the role of mitochondria. J Cell Mol Med. 2014;18(4):709–20. 81. Vazquez-Medina JP, Popovich I, Thorwald MA, Viscarra JA, Rodriguez R, Sonanez-Organis JG, et al. Angiotensin receptormediated oxidative stress is associated with impaired cardiac redox signaling and mitochondrial function in insulin-resistant rats. Am J Physiol Heart Circ Physiol. 2013;305(4):H599–607. 82. Ribeiro-Oliveira A, Jr, Marques MB, Vilas-Boas WW, Guimaraes J, Coimbra CC, Anjos AP, et al. The effects of chronic candesartan treatment on cardiac and hepatic adenosine monophosphate-activated protein kinase in rats submitted to surgical stress. JRAAS. 2013. doi:10.1177/1470320313499199 83. Dayi SU, Akbulut T, Akgoz H, Terzi S, Sayar N, Aydin A, et al. Long-term combined therapy with losartan and an angiotensinconverting enzyme inhibitor improves functional capacity in patients with left ventricular dysfunction. Acta Cardiol. 2005;60(4):373–7. 84. De Rosa ML, Chiariello M. Candesartan improves maximal exercise capacity in hypertensives: results of a randomized placebo-controlled crossover trial. J Clin Hypertens. 2009;11(4):192–200. 85. Takada S, Kinugawa S, Hirabayashi K, Suga T, Yokota T, Takahashi M, et al. Angiotensin II receptor blocker improves the lowered exercise capacity and impaired mitochondrial function of the skeletal muscle in type 2 diabetic mice. J Appl Physiol. 2013;114(7):844–57. 86. Mattivi F. Solid phase extraction of trans-resveratrol from wines for HPLC analysis. Zeitschrift fur Lebensmittel-Untersuchung und -Forschung. 1993;196(6):522–5. 87. Zheng J, Ramirez VD. Inhibition of mitochondrial proton F0F1ATPase/ATP synthase by polyphenolic phytochemicals. Br J Pharmacol. 2000;130(5):1115–23.

123

E. Niederberger et al. 88. Hart N, Sarga L, Csende Z, Koch LG, Britton SL, Davies KJ, et al. Resveratrol attenuates exercise-induced adaptive responses in rats selectively bred for low running performance. Dose Response. 2014;12(1):57–71. 89. Olesen J, Gliemann L, Bienso R, Schmidt J, Hellsten Y, Pilegaard H. Exercise training, but not resveratrol, improves metabolic and inflammatory status in skeletal muscle of aged men. J Physiol. 2014;592(Pt 8):1873–86. 90. Gliemann L, Olesen J, Bienso RS, Schmidt JF, Akerstrom T, Nyberg M, et al. Resveratrol modulates the angiogenic response to exercise training in skeletal muscles of aged men. Am J Physiol Heart Circ Physiol. 2014;307(8):H1111–9. 91. Gliemann L, Schmidt JF, Olesen J, Bienso RS, Peronard SL, Grandjean SU, et al. Resveratrol blunts the positive effects of exercise training on cardiovascular health in aged men. J Physiol. 2013;591(Pt 20):5047–59. 92. Hart N, Sarga L, Csende Z, Koltai E, Koch LG, Britton SL, et al. Resveratrol enhances exercise training responses in rats selectively bred for high running performance. Food Chem Toxicol. 2013;61:53–9. 93. Lagouge M, Argmann C, Gerhart-Hines Z, Meziane H, Lerin C, Daussin F, et al. Resveratrol improves mitochondrial function and protects against metabolic disease by activating SIRT1 and PGC-1alpha. Cell. 2006;127(6):1109–22. 94. Dolinsky VW, Jones KE, Sidhu RS, Haykowsky M, Czubryt MP, Gordon T, et al. Improvements in skeletal muscle strength and cardiac function induced by resveratrol during exercise training contribute to enhanced exercise performance in rats. J Physiol. 2012;590(Pt 11):2783–99. 95. Louis XL, Thandapilly SJ, MohanKumar SK, Yu L, Taylor CG, Zahradka P, et al. Treatment with low-dose resveratrol reverses cardiac impairment in obese prone but not in obese resistant rats. J Nutr Biochem. 2012;23(9):1163–9. 96. Nagao T, Hase T, Tokimitsu I. A green tea extract high in catechins reduces body fat and cardiovascular risks in humans. Obesity (Silver Spring). 2007;15(6):1473–83. 97. Tsuneki H, Ishizuka M, Terasawa M, Wu JB, Sasaoka T, Kimura I. Effect of green tea on blood glucose levels and serum proteomic patterns in diabetic (db/db) mice and on glucose metabolism in healthy humans. BMC Pharmacol. 2004;26(4):18. 98. Maron DJ, Lu GP, Cai NS, Wu ZG, Li YH, Chen H, et al. Cholesterol-lowering effect of a the aflavin-enriched green tea extract: a randomized controlled trial. Arch Intern Med. 2003;163(12):1448–53. 99. Nakachi K, Matsuyama S, Miyake S, Suganuma M, Imai K. Preventive effects of drinking green tea on cancer and cardiovascular disease: epidemiological evidence for multiple targeting prevention. BioFactors. 2000;13(1–4):49–54. 100. Murase T, Haramizu S, Shimotoyodome A, Tokimitsu I, Hase T. Green tea extract improves running endurance in mice by stimulating lipid utilization during exercise. Am J Physiol Regul Integr Comp Physiol. 2006;290(6):R1550–6. 101. Murase T, Haramizu S, Shimotoyodome A, Nagasawa A, Tokimitsu I. Green tea extract improves endurance capacity and increases muscle lipid oxidation in mice. Am J Physiol Regul Integr Comp Physiol. 2005;288(3):R708–15. 102. Dean S, Braakhuis A, Paton C. The effects of EGCG on fat oxidation and endurance performance in male cyclists. Int J Sport Nutr Exerc Metab. 2009;19(6):624–44. 103. Hwang JT, Park IJ, Shin JI, Lee YK, Lee SK, Baik HW, et al. Genistein, EGCG, and capsaicin inhibit adipocyte differentiation process via activating AMP-activated protein kinase. Biochem Biophys Res Commun. 2005;338(2):694–9. 104. Kim SH, Hwang JT, Park HS, Kwon DY, Kim MS. Capsaicin stimulates glucose uptake in C2C12 muscle cells via the reactive

123

105.

106.

107.

108.

109.

110.

111.

112.

113.

114.

115.

116. 117.

118.

119.

120.

oxygen species (ROS)/AMPK/p38 MAPK pathway. Biochem Biophys Res Commun. 2013;439(1):66–70. Lee GR, Jang SH, Kim CJ, Kim AR, Yoon DJ, Park NH, et al. Capsaicin suppresses the migration of cholangiocarcinoma cells by down-regulating matrix metalloproteinase-9 expression via the AMPK-NF-kappaB signaling pathway. Clin Exp Metastasis. 2014;31(8):897–907. Luo Z, Ma L, Zhao Z, He H, Yang D, Feng X, et al. TRPV1 activation improves exercise endurance and energy metabolism through PGC-1alpha upregulation in mice. Cell Res. 2012;22(3):551–64. Eguchi T, Kumagai C, Fujihara T, Takemasa T, Ozawa T, Numata O. Black tea high-molecular-weight polyphenol stimulates exercise training-induced improvement of endurance capacity in mouse via the link between AMPK and GLUT4. PLoS One. 2013;8(7):e69480. Tang X, Zhuang J, Chen J, Yu L, Hu L, Jiang H, et al. Arctigenin efficiently enhanced sedentary mice treadmill endurance. PLoS One. 2011;6(8):e24224. Wu RM, Sun YY, Zhou TT, Zhu ZY, Zhuang JJ, Tang X, et al. Arctigenin enhances swimming endurance of sedentary rats partially by regulation of antioxidant pathways. Acta Pharmacol Sin. 2014;35(10):1274–84. Jeong HW, Cho SY, Kim S, Shin ES, Kim JM, Song MJ, et al. Chitooligosaccharide induces mitochondrial biogenesis and increases exercise endurance through the activation of Sirt1 and AMPK in rats. PLoS One. 2012;7(7):e40073. Murase T, Misawa K, Haramizu S, Minegishi Y, Hase T. Nootkatone, a characteristic constituent of grapefruit, stimulates energy metabolism and prevents diet-induced obesity by activating AMPK. Am J Physiol Endocrinol Metab. 2010;299(2):E266–75. Cool B, Zinker B, Chiou W, Kifle L, Cao N, Perham M, et al. Identification and characterization of a small molecule AMPK activator that treats key components of type 2 diabetes and the metabolic syndrome. Cell Metab. 2006;3(6):403–16. Scott JW, van Denderen BJ, Jorgensen SB, Honeyman JE, Steinberg GR, Oakhill JS, et al. Thienopyridone drugs are selective activators of AMP-activated protein kinase beta1containing complexes. Chem Biol. 2008;15(11):1220–30. Baltgalvis KA, White K, Li W, Claypool MD, Lang W, Alcantara R, et al. Exercise performance and peripheral vascular insufficiency improve with AMPK activation in high-fat diet-fed mice. Am J Physiol Heart Circ Physiol. 2014;306(8):H1128–45. Jenkins Y, Sun TQ, Li Y, Markovtsov V, Uy G, Gross L, et al. Global metabolite profiling of mice with high-fat diet-induced obesity chronically treated with AMPK activators R118 or metformin reveals tissue-selective alterations in metabolic pathways. BMC Res Notes. 2014;7:674. Viollet B, Andreelli F. AMP-activated protein kinase and metabolic control. Handb Exp Pharmacol. 2011;203:303–30. Booth FW, Laye MJ. Lack of adequate appreciation of physical exercise’s complexities can pre-empt appropriate design and interpretation in scientific discovery. J Physiol. 2009;587(Pt 23):5527–39. Carey AL, Kingwell BA. Novel pharmacological approaches to combat obesity and insulin resistance: targeting skeletal muscle with ‘exercise mimetics’. Diabetologia. 2009;52(10):2015–26. Mercken EM, Carboneau BA, Krzysik-Walker SM, de Cabo R. Of mice and men: the benefits of caloric restriction, exercise, and mimetics. Ageing Res Rev. 2012;11(3):390–8. Rantzau C, Christopher M, Alford FP. Contrasting effects of exercise, AICAR, and increased fatty acid supply on in vivo and skeletal muscle glucose metabolism. J Appl Physiol. 2008;104(2):363–70.

AMPK and Exercise 121. Jorgensen SB, Wojtaszewski JF, Viollet B, Andreelli F, Birk JB, Hellsten Y, et al. Effects of alpha-AMPK knockout on exerciseinduced gene activation in mouse skeletal muscle. FASEB J. 2005;19(9):1146–8. 122. Ljubicic V, Burt M, Jasmin BJ. The therapeutic potential of skeletal muscle plasticity in Duchenne muscular dystrophy: phenotypic modifiers as pharmacologic targets. FASEB J. 2014;28(2):548–68. 123. Ljubicic V, Jasmin BJ. AMP-activated protein kinase at the nexus of therapeutic skeletal muscle plasticity in Duchenne muscular dystrophy. Trends Mol Med. 2013;19(10):614–24. 124. Jahnke VE, Van Der Meulen JH, Johnston HK, Ghimbovschi S, Partridge T, Hoffman EP, et al. Metabolic remodeling agents show beneficial effects in the dystrophin-deficient mdx mouse model. Skelet Muscle. 2012;2(1):16.

125. Vingtdeux V, Davies P, Dickson DW, Marambaud P. AMPK is abnormally activated in tangle- and pre-tangle-bearing neurons in Alzheimer’s disease and other tauopathies. Acta Neuropathol. 2011;121(3):337–49. 126. Vingtdeux V, Chandakkar P, Zhao H, d’Abramo C, Davies P, Marambaud P. Novel synthetic small-molecule activators of AMPK as enhancers of autophagy and amyloid-beta peptide degradation. FASEB J. 2011;25(1):219–31. 127. Lim MA, Selak MA, Xiang Z, Krainc D, Neve RL, Kraemer BC, et al. Reduced activity of AMP-activated protein kinase protects against genetic models of motor neuron disease. J Neurosci. 2012;32(3):1123–41.

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