AMPK in skeletal muscle function and metabolism

The FASEB JournalVolume 32, Issue 4 p. 1741-1777 ReviewOpen Access AMPK in skeletal muscle function and metabolism Rasmus Kjøbsted, Corresponding Author Rasmus Kjøbsted rasmus.kjobsted@nexs.ku.dk Section of Molecular Physiology, Department of Nutrition, Exercise, and Sports, Faculty of Science, University of Copenhagen, Copenhagen, Denmark Correspondance: Department of Nutrition, Exercise and Sports, Section of Molecular Physiology, Faculty of Science, University of Copenhagen, Universitetsparken 13, DK-2100 Copenhagen, Denmark. E-mail: rasmus.kjobsted@nexs.ku.dk Correspondance: Department of Molecular Physiology and Biophysics, Vanderbilt University, 823 Light Hall, 2215 Garland Ave., Nashville, TN 37232, USA. E-mail: louise.lantier@vanderbilt.eduSearch for more papers by this authorJanne R. Hingst, Janne R. Hingst Section of Molecular Physiology, Department of Nutrition, Exercise, and Sports, Faculty of Science, University of Copenhagen, Copenhagen, DenmarkSearch for more papers by this authorJoachim Fentz, Joachim Fentz Section of Molecular Physiology, Department of Nutrition, Exercise, and Sports, Faculty of Science, University of Copenhagen, Copenhagen, DenmarkSearch for more papers by this authorMarc Foretz, Marc Foretz INSERM, Unité 1016, Institut Cochin, Paris, France Centre National de la Recherche Scientifique (CNRS), Unité Mixte de Recherche (UMR) 8104, Paris, France Université Paris Descartes, Sorbonne Paris Cité, Paris, FranceSearch for more papers by this authorMaria-Nieves Sanz, Maria-Nieves Sanz Department of Cardiovascular Surgery, Inselspital, Bern University Hospital, Bern, Switzerland Department of Biomedical Research, University of Bern, Bern, SwitzerlandSearch for more papers by this authorChristian Pehmøller, Christian Pehmøller Internal Medicine Research Unit, Pfizer Global Research and Development, Cambridge, Massachusetts, USASearch for more papers by this authorMichael Shum, Michael Shum Axe Cardiologie, Quebec Heart and Lung Research Institute, Québec, Canada Institute for Nutrition and Functional Foods, Laval University, Québec, CanadaSearch for more papers by this authorAndré Marette, André Marette Axe Cardiologie, Quebec Heart and Lung Research Institute, Québec, Canada Institute for Nutrition and Functional Foods, Laval University, Québec, CanadaSearch for more papers by this authorRemi Mounier, Remi Mounier Institute NeuroMyoGène, Université Claude Bernard Lyon 1, INSERM Unite 1217, CNRS UMR, Villeurbanne, FranceSearch for more papers by this authorJonas T. Treebak, Jonas T. Treebak Section of Integrative Physiology, Novo Nordisk Foundation Center for Basic Metabolic Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, DenmarkSearch for more papers by this authorJørgen F. P. Wojtaszewski, Jørgen F. P. Wojtaszewski Section of Molecular Physiology, Department of Nutrition, Exercise, and Sports, Faculty of Science, University of Copenhagen, Copenhagen, DenmarkSearch for more papers by this authorBenoit Viollet, Benoit Viollet INSERM, Unité 1016, Institut Cochin, Paris, France Centre National de la Recherche Scientifique (CNRS), Unité Mixte de Recherche (UMR) 8104, Paris, France Université Paris Descartes, Sorbonne Paris Cité, Paris, FranceSearch for more papers by this authorLouise Lantier, Corresponding Author Louise Lantier louise.lantier@vanderbilt.edu Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee, USA Mouse Metabolic Phenotyping Center, Vanderbilt University, Nashville, Tennessee, USA Correspondance: Department of Nutrition, Exercise and Sports, Section of Molecular Physiology, Faculty of Science, University of Copenhagen, Universitetsparken 13, DK-2100 Copenhagen, Denmark. E-mail: rasmus.kjobsted@nexs.ku.dk Correspondance: Department of Molecular Physiology and Biophysics, Vanderbilt University, 823 Light Hall, 2215 Garland Ave., Nashville, TN 37232, USA. E-mail: louise.lantier@vanderbilt.eduSearch for more papers by this author Rasmus Kjøbsted, Corresponding Author Rasmus Kjøbsted rasmus.kjobsted@nexs.ku.dk Section of Molecular Physiology, Department of Nutrition, Exercise, and Sports, Faculty of Science, University of Copenhagen, Copenhagen, Denmark Correspondance: Department of Nutrition, Exercise and Sports, Section of Molecular Physiology, Faculty of Science, University of Copenhagen, Universitetsparken 13, DK-2100 Copenhagen, Denmark. E-mail: rasmus.kjobsted@nexs.ku.dk Correspondance: Department of Molecular Physiology and Biophysics, Vanderbilt University, 823 Light Hall, 2215 Garland Ave., Nashville, TN 37232, USA. E-mail: louise.lantier@vanderbilt.eduSearch for more papers by this authorJanne R. Hingst, Janne R. Hingst Section of Molecular Physiology, Department of Nutrition, Exercise, and Sports, Faculty of Science, University of Copenhagen, Copenhagen, DenmarkSearch for more papers by this authorJoachim Fentz, Joachim Fentz Section of Molecular Physiology, Department of Nutrition, Exercise, and Sports, Faculty of Science, University of Copenhagen, Copenhagen, DenmarkSearch for more papers by this authorMarc Foretz, Marc Foretz INSERM, Unité 1016, Institut Cochin, Paris, France Centre National de la Recherche Scientifique (CNRS), Unité Mixte de Recherche (UMR) 8104, Paris, France Université Paris Descartes, Sorbonne Paris Cité, Paris, FranceSearch for more papers by this authorMaria-Nieves Sanz, Maria-Nieves Sanz Department of Cardiovascular Surgery, Inselspital, Bern University Hospital, Bern, Switzerland Department of Biomedical Research, University of Bern, Bern, SwitzerlandSearch for more papers by this authorChristian Pehmøller, Christian Pehmøller Internal Medicine Research Unit, Pfizer Global Research and Development, Cambridge, Massachusetts, USASearch for more papers by this authorMichael Shum, Michael Shum Axe Cardiologie, Quebec Heart and Lung Research Institute, Québec, Canada Institute for Nutrition and Functional Foods, Laval University, Québec, CanadaSearch for more papers by this authorAndré Marette, André Marette Axe Cardiologie, Quebec Heart and Lung Research Institute, Québec, Canada Institute for Nutrition and Functional Foods, Laval University, Québec, CanadaSearch for more papers by this authorRemi Mounier, Remi Mounier Institute NeuroMyoGène, Université Claude Bernard Lyon 1, INSERM Unite 1217, CNRS UMR, Villeurbanne, FranceSearch for more papers by this authorJonas T. Treebak, Jonas T. Treebak Section of Integrative Physiology, Novo Nordisk Foundation Center for Basic Metabolic Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, DenmarkSearch for more papers by this authorJørgen F. P. Wojtaszewski, Jørgen F. P. Wojtaszewski Section of Molecular Physiology, Department of Nutrition, Exercise, and Sports, Faculty of Science, University of Copenhagen, Copenhagen, DenmarkSearch for more papers by this authorBenoit Viollet, Benoit Viollet INSERM, Unité 1016, Institut Cochin, Paris, France Centre National de la Recherche Scientifique (CNRS), Unité Mixte de Recherche (UMR) 8104, Paris, France Université Paris Descartes, Sorbonne Paris Cité, Paris, FranceSearch for more papers by this authorLouise Lantier, Corresponding Author Louise Lantier louise.lantier@vanderbilt.edu Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee, USA Mouse Metabolic Phenotyping Center, Vanderbilt University, Nashville, Tennessee, USA Correspondance: Department of Nutrition, Exercise and Sports, Section of Molecular Physiology, Faculty of Science, University of Copenhagen, Universitetsparken 13, DK-2100 Copenhagen, Denmark. E-mail: rasmus.kjobsted@nexs.ku.dk Correspondance: Department of Molecular Physiology and Biophysics, Vanderbilt University, 823 Light Hall, 2215 Garland Ave., Nashville, TN 37232, USA. E-mail: louise.lantier@vanderbilt.eduSearch for more papers by this author First published: 05 January 2018 https://doi.org/10.1096/fj.201700442RCitations: 28AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract Skeletal muscle possesses a remarkable ability to adapt to various physiologic conditions. AMPK is a sensor of intracellular energy status that maintains energy stores by fine-tuning anabolic and catabolic pathways. AMPK's role as an energy sensor is particularly critical in tissues displaying highly changeable energy turnover. Due to the drastic changes in energy demand that occur between the resting and exercising state, skeletal muscle is one such tissue. Here, we review the complex regulation of AMPK in skeletal muscle and its consequences on metabolism (e.g., substrate uptake, oxidation, and storage as well as mitochondrial function of skeletal muscle fibers). We focus on the role of AMPK in skeletal muscle during exercise and in exercise recovery. We also address adaptations to exercise training, including skeletal muscle plasticity, highlighting novel concepts and future perspectives that need to be investigated. Furthermore, we discuss the possible role of AMPK as a therapeutic target as well as different AMPK activators and their potential for future drug development.— Kjøbsted, R., Hingst, J. R., Fentz, J., Foretz, M., Sanz, M.-N., Pehmøller, C., Shum, M., Marette, A., Mounier, R., Treebak, J. T., Wojtaszewski, J. F. P., Viollet, B., Lantier, L. AMPK in skeletal muscle function and metabolism. FASEB J. 32, 1741–1777 (2018). www.fasebj.org ABBREVIATIONS 4E-BP1 4E binding protein 1 ACC2 acetyl-CoA carboxylase 2 AICAR 5-amino-1-β-d-ribofuranosyl-imidazole-4-carboxamide BDNF brain-derived neurotrophic factor C2 5-(5-hydroxyl-isoxazol-3-yl)-furan-2-phosphonic acid CaMK Ca2+/calmodulin-dependent protein kinase CaMKKβ Ca2+/calmodulin-dependent protein kinase kinase β CD36 cluster of differentiation 36 DMD Duchenne muscular dystrophy ERR estrogen-related receptor FOXO3a Forkhead box protein O3a GBD glycogen-binding domain β-GPA β-guanidinopropionic acid GS glycogen synthase HDAC5 class II histone deacetylase 5 HFD high-fat diet KD kinase domain KO knockout LIF leukemia inhibitory factor LKB1 liver kinase B1 MAFbx muscle atrophy F-box mdKO muscle-specific double knockout MEF2 myocyte enhancer factor 2 mTORC1 mammalian target of rapamycin complex 1 MuRF1 muscle RING finger 1 MuSC muscle stem cell NAM nicotinamide NAMPT nicotinamide phosphoribosyltransferase NR4A nuclear hormone receptor 4A PDH pyruvate dehydrogenase PGC-1α peroxisome proliferator-activated receptor γ coactivator 1α PKD protein kinase D PLIN2 perilipin 2 ROS reactive oxygen species SIRT NAD-dependent sirtuin SNARK sucrose nonfermenting AMPK-related kinase TSC tuberous sclerosis complex ULK1 uncoordinated 51-like kinase 1 WADA World Anti-Doping Agency WT wild type ZMP 5-aminoimidazole-4-carboxamide ribonucleotide One fundamental function of skeletal muscle is to generate mechanical force to support body posture and to facilitate a wide variety of movements. Besides this role in body motility, skeletal muscle has been shown to be important for regulating whole-body metabolism. Skeletal muscle demonstrates high malleability and can adapt its contractile composition and metabolic properties in response to a number of physiologic conditions, including exercise. Such adaptations are reflected by changes in muscle size, fiber type distribution, contractile velocity, force production, and endurance capacity, being the result of the functional demands of the contractile activity (1, 2). This plasticity may involve short- and long-term mechanisms, leading to changes in protein abundance and activity (1–3). These changes are mediated by activation and repression of specific intracellular signaling events that govern effectors involved in metabolic pathways and transcription/translation processes of exercise-responsive genes (4). The intracellular signaling mechanisms that modify skeletal muscle function in response to exercise are regulated by perturbations in muscle cell homeostasis, including alterations in tissue perfusion, oxygen tension, redox state, calcium (Ca2+) dynamics, and ATP turnover (5). Evidence suggests that ATP turnover in skeletal muscle may increase by >100-fold in response to exercise (6). Keeping cellular ATP concentrations fairly constant during such conditions represents a major challenge to the cell and highlights the vast dynamics of muscle energy metabolism. Because skeletal muscle ATP consumption increases during exercise, intracellular AMP concentrations may accumulate as a result of the adenylate kinase reaction. This increases cellular AMP/ATP and ADP/ATP ratios, leading to activation of AMPK (7). This kinase is considered a central sensor of intracellular energy status and maintains energy stores by regulating anabolic and catabolic pathways, thereby ensuring a balance between energy supply and demand (8). In skeletal muscle, acute pharmacological activation of AMPK has been shown to promote glucose transport and fatty acid oxidation (9) while suppressing glycogen synthase activity and protein synthesis (10, 11). In addition, chronic activation of AMPK reduces markers of skeletal muscle fragility (12) and enhances muscle fiber oxidative capacity by stimulating mitochondrial biogenesis (13–15). These events are initiated by AMPK downstream phosphorylation of key metabolic enzymes as well as transcription factors that modulate cellular metabolism in order to handle both current and future metabolic challenges. Several excellent reviews have examined the role of AMPK in regulating skeletal muscle function and metabolism (16–49). Therefore, this review addresses novel concepts and future perspectives of AMPK in skeletal muscle that need to be experimentally validated and tested. AMPK STRUCTURE AND EXPRESSION AMPK is a heterotrimeric protein complex that consists of a catalytic subunit (α) and 2 regulatory subunits (β and γ), of which several isoforms have been found (α1, α2, β1, β2, γ1, γ2, and γ3) (50) (Fig. 1). The a subunit contains the kinase domain, activity of which is highly dependent on thereversiblephosphorylationofa-Thr172 (51–53). The β subunit acts as a scaffold for binding the α and γ subunits (54) and contains a glycogen-binding domain (GBD) that likely targets the heterotrimeric complex to glycogen particles (55, 56). The γ subunit functions as a sensor of intracellular energy status through its direct binding of adenosine nucleotides (57). Besides these well-established functions, all 3 subunits contain different domains or posttranslational modifications that may locate AMPK to distinct subcellular compartments. In the a subunit, a nuclear export sequence has been found (58), and myristoylation of the β subunit has been suggested to facilitate AMPK translocation to perinuclear speckles and mitochondrial membranes (59–61). The γ subunit isoforms differ in their N-terminal extensions, which also appear to determine AMPK localization (62–64). Thus, in skeletal muscle fibers, the γ1 isoform is localized to the z disk, whereas the g3 isoform is found in the nucleus and along the z disk and I-band in a pattern that closely resembles the T-tubule/sarcoplasmic reticulum structures (64). The functional role of AMPK in different subcellular locations has not received much attention, but recent findings indicate that it may represent another way of regulating AMPK activity. For example, the unspecific AMPK activator PT-1 appears to only activate γ1 complexes in mouse skeletal muscle, whereas it displays no isoform selectivity in HEK293 cells stably expressing each of the 3 γ isoforms (65). Because PT-1 activates AMPK by inhibiting the mitochondrial respiratory chain (65), this may signify an important role of the γ1-associated complexes in monitoring ATP synthesis, and the highly contraction-responsive γ3-containing complex may serve as the major sensor of ATP consumption in skeletal muscle. On the other hand, the PT-1 concentration previously used to stimulate isolated skeletal muscle (65) may have been insufficient to activate γ3-containing complexes. Recent evidence from cell-based studies also indicates that AMPK subunit composition influences sensitivity to AMP, which likely contributes to the specialized functions of AMPK heterotrimeric subtypes (57, 66-68). The 7 different AMPK subunit isoforms give rise to 12 heterotrimeric combinations that seem to be expressed in a tissue-specific manner. Thus, in skeletal muscle preparations from human and mouse, all subunit isoforms have been detected, but only a subset of possible heterotrimeric complexes seems to exist (69, 70). In human skeletal muscle (vastus lateralis), 3 different complexes have been described (α2β2γ1, α2β2γ3, and α1β2γ1) (69), whereas 5 complexes have been identified in mouse skeletal muscle (α2β2γ1, α2β2γ3, α2β1γ1, α1β2γ1, and α1β1γ1) (70) (Table 1). In addition, some AMPK subunits are expressed in a fiber type-dependent manner (71), which may explain the relative distribution of complexes between different muscles (70). Currently, it is not known why mouse skeletal muscle appears to express two additional complexes (β1-associated) compared with human skeletal muscle. Based on findings in muscle-specific AMPKα and AMPKβ double-knockout (KO) mice (AMPKα mdKO and AMPK β1β2M-KO mice, respectively) (72–74), the majority of β1-associated complexes detected in a crude sample of skeletal muscle seem to derive from nonmuscle tissue (e.g., connective tissue, neuronal cells, adipocytes, endothelial cells, etc.). This is in line with the notion that the α1β1γ1 complex is the most ubiquitously expressed complex of AMPK (68, 75). Interestingly, the β1 subunit is also detected in sample preparations of human skeletal muscle, but, in light of findings from coimmunoprecipitations of AMPKα2, -α1, -γ1, and -γ3, it does not seem to engage in stable complex formation or contribute to any measurable AMPK activity (69, 76). Although it is generally thought that all 3 AMPK subunits must be present to form a stable complex, it has been demonstrated in cell models that stable β1γ1 complexes can form in the absence of catalytic subunits (77). Whether formation of βγ heterodimer complexes also occurs in mature skeletal muscle may be derived from observations in two muscle-specific AMPK double-KO mouse models. Thus, in the AMPKβ1β2M-KO model it seems evident that expression of β2 protein is restricted to the myocytes (74). Interestingly, significant amounts of β2 protein have been detected in a skeletal muscle sample preparation from the AMPKα mdKO mouse model (73), which may suggest the formation of stable βγ complexes in mature skeletal muscle, assuming that single unbound subunits of AMPK are targeted for degradation. This may also be inferred from the AMPKβ1β2M-KO mouse model, which does not express α2 protein in skeletal muscle (74), indicating that the β subunit is vital for maintaining AMPKα muscle protein expression. Collectively, these observations may suggest that the AMPK heterodimer (βγ) exists in skeletal muscle tissue and raises the possibility of a regulatory mechanism facilitating the association of catalytic subunits with regulatory complexes. Alternatively, and somewhat speculatively, other proteins may bind to the regulatory heterodimer complex to regulate their activity or cellular localization. In this context, 12 protein kinases related to AMPKα1andAMPKα2 have been detected in the human kinome (78). These are known as AMPK-related kinases, and, with a single exception, these are activated by upstream kinase liver kinase B1 (LKB1) (79). Although green fluorescent protein-transporter associated with antigen processing-tagged versions of these kinases do not appear to bind AMPK β and γ subunits (80), the sucrose nonfermenting AMPK-related kinase (SNARK/NUAK2) is activated in skeletal muscle by 5-amino-1-β-d-ribofuranosyl-imidazole-4-carboxamide (AICAR), contraction, and exercise (81, 82), indicating that SNARK activity is regulated similarly to AMPK. Is it possible that AMPK βγ subunits form heterotrimeric complexes with SNARK, facilitating its regulation by adenine nucleotides? If so, it could be anticipated that AMPKβ-deficient skeletal muscle exhibits a phenotype different from that of AMPKα-deficient skeletal muscle. Indeed, in skeletal muscle several phenotypic differences have been observed between AMPKα mdKO and AMPKβ1β2M-KO mice, including muscle mass, ATP levels, mitochondrial DNA and structure, citrate synthase activity, and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) mRNA levels (72, 74). In light of these observations, it has recently been reported that SNARK may be involved in the maintenance of muscle mass with age (83). Assuming that the potential binding of SNARK to AMPKβγ subunits induces an increase in SNARK activity and/or protects SNARK from degradation and that the association between SNARK and AMPKβγ is enhanced in AMPKα-deprived muscle, this could explain the increase in muscle mass observed in skeletal muscle deprived of AMPKα subunits (72). Table 1. Relative distribution and basal activity of AMPK heterotrimeric complexes detected in human and mouse skeletal muscle Mus musculus Trimer complex Homo sapiens vastus lateralis Extensor digitorum longus Soleus Relative expression Relative basal activity Relative expression Relative basal activity Relative expression Relative basal activity α2β2γ1 ~65% ~30% ~70% ~50% ~60% ~35% α2β2γ3 ~20% <5% ~20% ~20% <2% <2% α1β2γg1 ~15% ~65% <5% ~25% ~20% ~50% α2β1γ1 N.D. N.D. <3% <5% ~10% <15% α1β1γ1 N.D. N.D. <2% <8% The composition of AMPK heterotrimeric complexes was estimated from immunoprecipitation experiments in extensor digitorum longus and soleus from C57BL/6 mice as well as human male vastus lateralis. Values adapted from references 70, 76, and 487. N.D., nondetectable. Figure 1Open in figure viewer Structure of mammalian AMPK subunits. AMPK is a heterotrimeric protein consisting of 1 catalytic subunit (α subunit) and 2 regulatory subunits (β and γ subunit). The a subunit contains a kinase domain (KD), the activity of which relies on the phosphorylation of Thr172 by upstream AMPK kinases. The KD is followed by an autoinhibitory domain (AID) and an α-hook (αH), which seem to be important for AMP-regulated catalytic activity. At the α-C terminus, a β-interacting domain (β-ID) has been detected that binds to the C-terminal domain of the β subunit. Phosphorylation of Ser485/491 in the β-ID during insulin stimulation has been suggested to regulate kinase activity. The β subunit is subjected to myristoylation at the N terminus, which enhances phosphorylation of α-Thr172 by AMP/ADP and facilitates AMPK translocation to specific intracellular compartments. At the center, the β subunit contains a GBD that causes AMPK to bind to glycogen particles. Within the GBD, 2 phosphorylation sites have been found (Ser108 and Thr148) that seem to regulate binding capacity to glycogen particles as well as kinase activity. An α- and γ-interaction domain (α-ID, γ-ID) is located at the β-C terminus that acts as a scaffold keeping the heterotrimeric complex together. The γ-subunit contains 4 cystathionine-β-synthase (CBS) domains. These occur in tandem pairs, also known as Bateman domains, and are involved in adenosine nucleotide binding. A β-ID is located close to the γ-N terminus. An asterisk denotes that the 3 γ isoforms contain different N-terminal extensions. REGULATION AND ACTIVATION OF AMPK IN SKELETAL MUSCLE During skeletal muscle contraction, the adenylate energy charge in muscle is decreased depending on the duration and intensity of exercise (84, 85). As a result, the intracellular AMP/ATP and ADP/ATP ratios increase, which leads to activation of AMPK (7). AMPK activation occurs in two steps: stimulatory allosteric binding of AMP within the γ subunit and covalent activation through reversible phosphorylation on Thr172 in the catalytic a subunit (Fig. 2). AMPK activity is stimulated by AMP and ADP and inhibited by ATP binding to the two regulatory Bateman domains of the γ subunit. This competitive binding means that increases in cellular AMP/ATP and ADP/ATP ratios stimulate AMPK allosterically (86–90). The allosteric stimulation has a moderate effect on AMPK activity (<10-fold) (91). More importantly, binding of AMP and/or ADP to the γ subunit induces conformational changes that promote phosphorylation of α-Thr172 (67, 90) and permit protection against dephosphorylation by protein phosphatases PP1, PP2A, and PP2C (41, 92-95). The combined effect of allosteric activation and phosphorylation on α-Thr172 induces a >1000-fold increase in AMPK activity (91). In skeletal muscle, LKB1 is the primary upstream kinase responsible for the phosphorylation of α2-containing AMPK complexes in response to contraction and pharmacological AMPK activators (96–99). To a lesser extent, Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) likely phosphorylates and activates AMPKα1 complexes during long-term low-intensity exercise/contraction (100, 101) (Fig. 3). LKB1 appears constitutively active (102, 103), whereas CaMKKβ activates AMPK upon an increase in intracellular Ca2+ concentrations, even in the absence of adenine nucleotide content imbalance (104, 105). In addition, glycogen has been shown to influence AMPK activity through its interaction with the β subunit. The β subunit contains a GBD that causes AMPK complexes to associate with glycogen particles in cell-free systems and cultured cells, and this association inhibits AMPK activity (56, 106). This inhibition by glycogen seems to affect mainly α2-containing AMPK complexes (107). Furthermore, in vivo studies report an inverse relationship between muscle glycogen content and AMPK activation in rodents (108, 109) and humans (107), although this inhibition by glycogen in vivo is not consistently found (110). Interestingly, the β subunit is autophosphorylated at Ser108 and Thr148, which seems to regulate AMPK activity and its binding capacity to glycogen, respectively (59, 111). In addition, findings suggest that insulin reduces AMPK activity in rat skeletal muscle likely through Aktmediated phosphorylation of Ser485/491 on the α1/α2 subunit (112). Figure 2Open in figure viewer Regulation of AMPK in skeletal muscle during contractile activity. Exercise induces an energy imbalance in muscle, which leads to a rise in intracellular AMP and ADP concentrations. Binding of ADP and AMP at the Bateman domains of the γ subunit causes a conformational change that activates AMPK by up to 10-fold via an allosteric mechanism. This conformational change also triggers Thr172 phosphorylation of the a catalytic subunit by the upstream LKB1 and protects against dephosphorylation by protein phosphatases, increasing the activity 100-fold. Together the allosteric effect and α-Thr172 phosphorylation lead to a >1000-fold activation. AMPK is also activated by a rise in the intracellular Ca2+ concentration through α-Thr172 phosphorylation catalyzed by CaMKKβ. After exercise and energy repletion, AMPK is converted back to an inactive form by dephosphorylation catalyzed by protein phosphatases (PP1A, PP2A, and PP2C) and undergoes inhibition by glycogen via binding to the GBD of the β subunit. The activation of AMPK in rodent skeletal muscle during exercise was initially described by Winder and Hardie (113), and the first reports of AMPK activation during exercise in human skeletal muscle were published some years later (114–116). Several studies have been carried out to determine the activation profile of the different AMPK complexes during exercise bouts depending on intensity and duration. Typically, AMPK activation is observed only at exercise intensities of a minimum of 60% Vo2peak (107, 116-119). However, low-intensity exercise at 30-40% of Vo2peak but performed until exhaustion also activates AMPK in skeletal muscle (119). Moreover, AMPK activation seems dependent on exercise duration (115, 118, 119), although not all studies report this (120). During exercise at high intensity and moderate duration, α2-containing AMPK complexes are predominantly activated (115–118), whereas activity of AMPKα1 complexes have been found to increase, stay unchanged, or decrease in an intensity- and duration-dependent manner (76, 101, 110, 117). Specifically, in human vastus lateralis muscle during a short (up to 20 min) and intense exercise bout, AMPK activation is restricted to γ3-containing complexes (α2β2γ3), whereas activity of the other complexes (α2β2γ1 and α1 β2γ1) appears unchanged or even decreased (76). When exercise is prolonged, α2β2γ1 heterotrimers are activated (101). During exercise at lower intensities and of longer duration, a moderate increase in α2β2γ1 activity and a weak increase in α1β2γ1 activity have been observed (101). Thus, in skeletal muscle the different AMPK heterotrimer complexes are regulated in a distinct manner during contraction depending on exercise intensity and duration, which may cause different functional responses. How this differential activation of the specific AMPK complexes is accomplished during exercise needs further investigation. One hypothesis could be that it may in part reflect differences