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Drug Discovery Today Volume 00, Number 00 July 2011
AMP kinase in exercise adaptation of skeletal muscle Niels Jessen1,2, Elias I.O. Sundelin1 and Andreas Buch Møller1 1 2
Research Laboratory for Biochemical Pathology, Institute for Clinical Medicine, Aarhus University, Denmark Department of Molecular Medicine, Aarhus University Hospital, Denmark
Regular physical exercise has undisputed health benefits in the prevention and the treatment of many diseases. Understanding the mechanisms that regulate adaptations to exercise training therefore has obvious clinical perspectives. Several lines of evidence suggest that the AMP-activated protein kinase (AMPK) has a central role as a master metabolic regulator in skeletal muscle. Exercise is a potent activator of AMPK, and AMPK signaling can play a key part in regulating protein turnover during and after exercise training.
Introduction Physical activity has long been known to provide people with type 2 diabetes with remarkable health benefits, and moderate-intensity regular physical activity has been demonstrated to prevent or delay the onset of diabetes in high-risk subjects [1]. Skeletal muscle plays a key part in this beneficial adaptive response by changing its size as well as its substrate metabolism. These outcomes are brought about by changes in gene expression, biochemical and metabolic properties, and understanding the mechanisms that regulate these processes could open possibilities for future interventions that will prevent type 2 diabetes. In this short review, we will discuss new insights into the role of AMP-activated protein kinase (AMPK) in regulation of protein turnover in skeletal muscle in response to physical exercise.
AMPK expression and activation in skeletal muscle AMPK is an evolutionally conserved serine/threonine kinase that is ubiquitously expressed in human cells. The enzyme is a heterotrimer that consists of three subunits: a, b and g, each expressed in two or more isoforms [2]. The a subunit contains the typical serine/ threonine protein kinase domain, whereas the b and g subunits are regulatory subunits. The isoform-specific composition of the heterotrimer varies among species and tissue types, but expression of all three isoforms is required for full enzymatic activity. Two isoforms of the catalytic a subunit have been identified (a1 and a2), and they
Corresponding author:. Jessen, N. ([email protected])
have broad tissue distribution including in skeletal muscle. In fact, in comparison with all other tissues, the highest expression level of the a2 isoform is found in skeletal muscle, but both a subunits have been suggested to have a physiological role in response to exercise, reviewed in [2,3]. AMPK is activated by phosphorylation on Thr172 of the a1 or a2 subunit in response to various cellular energy stressors. When AMP:ATP and ADP:ATP ratios rise, AMP and ADP bind to the g subunit of AMPK and this causes a conformational change in the heterotrimer that makes the a subunit a substrate for upstream kinases. Several AMPK-related kinases have been identified, but liver kinase B1 (LKB1) (in a complex with the proteins Ste20related adaptor protein (STRAD) and Mouse protein-25 (MO25) and calmodulin-dependent protein kinase kinase (CaMKKb) appear to be the only important upstream kinases in mammalian cells [4–7]. Binding of AMP and ADP to the g subunit also leads to a decrease in accessibility of protein phosphatases to the a subunit of AMPK [8], and this could potentiate the activation of AMPK in response to energy deprivation. Activation of AMPK in skeletal muscle during exercise of different intensities and durations has been extensively investigated. Resistance exercise, characterized by short duration and high intensity, increases AMPK activity in human skeletal muscle [9,10]. Similarly, AMPK activation is consistently reported during endurance exercise when the exercise is performed at intensities over 60% VO2 peak. At lower intensities AMPK might not necessarily be activated; however, as little as 40% of VO2
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peak can activate AMPK if the exercise is continued until exhaustion [11]. The activation of AMPK by exercise is transient and usually returns to basal levels after resting a few minutes, but increased phosphorylation up to 60 min into the postexercise recovery period after endurance exercise has been reported [12]. An important question about the role of AMPK in adaptations to exercise training has been the degree to which AMPK is activated in trained individuals. In humans, examined before and after 10 days of endurance exercise training, a single bout of exercise was only able to increase AMPK activity in the untrained state [13]. However, in this study exercise in the trained state did raise AMP levels and was associated with increased phosphorylation of the AMPK target acetyl-CoA carboxylase (ACC) [13], and this raises the possibility that in the trained state AMPK activity was below the detection level of the activity assay. In agreement with this, we have found that endurance and resistance exercise can increase AMPK phosphorylation in trained subjects who have been accustomed to the specific form of exercise [9]. It is therefore probable that AMPK is involved in training adaptations in trained and untrained individuals but exercise-induced AMPK signaling is attenuated when subjects adapt to exercise training.
AMPK and protein synthesis It is widely accepted that adaptations to exercise training in muscle involves gradual alterations in protein content in skeletal muscle brought about partly by increased protein expression. The synthesis of specific proteins can be divided into several levels such as transcription of mRNA, content of ribosomes and translation of mRNA into peptides, and these processes are acutely regulated by the action of mammalian target of rapamycin (mTOR) complex 1 (mTORC1). mTORC1 is composed of the catalytic subunit mTOR in complex with the regulatory-associated protein of mTOR (Raptor) and mammalian lethal with SEC13 protein 8 (MLST8) [14]. mTORC1 modulates the rate of protein translation in part through phosphorylation of the 70 kDa ribosomal protein S6 kinase (P70S6K), eukaryotic initiation factors (eIF) and 4E binding protein (4E-BP1). Protein synthesis is an energy-consuming process and, in agreement with the role as an energy sensor, activation of AMPK can suppress protein synthesis by inhibiting mTOR activity on several levels: (i) AMPK can act directly on mTORC1 by phosphorylating mTOR on Thr2446, and thereby inhibit phosphorylation on the activating Ser2448 site [15]; (ii) AMPK can inhibit mTORC1 activity indirectly by phosphorylating and activating tuberin (TSC2), an inhibitor of mTOR activity [16]; (iii) AMPK can phosphorylate Raptor on Ser722 and Ser792 which causes dissociation of 14-3-3 and thereby further inhibition of mTORC1 activity [17]; (iv) AMPK can inhibit translation elongation through activation of eukaryotic translation elongation factor 2 kinase (eEF2K), an inhibitor of translation elongation factor eEF2 [18]. Complete inhibition of AMPK signaling can therefore be expected to have pronounced effects on protein synthesis, but inhibition of protein synthesis following muscle contractions is normal in transgenic mice expressing a dominant-negative kinase dead AMPK isoform [19]. It is important to notice that these transgenic mice do have residual AMPK activity, and this could be enough to transmit 2
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the inhibition of mTORC1 signaling. Alternatively, redundant signaling through AMPK-related kinases could compensate for the inhibition of AMPK activity in the transgenic mice. Support for this theory comes from muscle-specific LKB1 knockout mice. LKB1 is the upstream activator of 11 of the 12 AMPK-related kinases [20], and muscle-specific LKB1 knockout animals have increased mTORC1 signaling in cardiac muscle and develop cardiac hypertrophy [21,22]. In humans, a role of AMPK as an inhibitor of protein synthesis is supported by reports of decreased phosphorylation of 4E-BP1 at Thr37/46, a bona fide mTOR phosphorylation site, immediately after exercise [10,12]. However, not all human studies report reductions in TSC2/mTORC1 signaling immediately after exercise despite increased AMPK activity [9,23]. The mTORC1 complex integrates the input from many upstream pathways, including growth factors and nutrients, and these factors could modulate the AMPK effects on mTOR. In situations of severe energy deprivation AMPK can inhibit protein synthesis in human skeletal muscle but under low-to-moderate intensity exercise AMPK plays a minor part in regulating protein synthesis in humans. In contrast to the attenuation of protein synthesis during and immediately after exercise, the post-exercise recovery period is characterized by increased signaling through mTORC1 [9,12]. Trained muscle increases expression of mitochondrial oxidative enzymes, glucose transporter type 4 (GLUT4) and hexokinase, and these mechanisms have obvious implications for treatment and prevention of diabetes [24]. Chronic treatment with the pharmacological AMPK activators 5-aminoimidazole-4-carboxamide-b-Dribofuranoside (AICAR) and b-guanidinopropionic acid (b-GPA) in rodents mimics these adaptations to exercise [25–28], and these effects are ablated in transgenic mice expressing a dominantnegative form of AMPK or in AMPK a2 knockout animals [28– 30]. However, increases in protein expression after exercise training are normal in AMPK transgenic animal models with impaired AMPK signaling [29,30]. These observations suggest that AMPK is not necessary for these adaptations to exercise, but effects of residual AMPK activity in the transgenic animal models cannot be excluded. In the perspectives of disease prevention it might not be of importance if redundant pathways can compensate for the effects of AMPK in exercise adaptation. The effects of chronic pharmacological activators in animals clearly indicates that AMPK has potential as a mediator of at least some of the beneficial increases in enzyme expression induced by exercise training [31].
AMPK and protein degradation Physical exercise constitutes a major metabolic and mechanical stress that causes considerable damage to the involved muscles. Post-exercise tissue repair therefore involves several molecular mechanisms, including protein synthesis and protein degradation, that serve to remove and replace the damaged tissue. Autophagy is a catabolic process that enables removal of damaged organelles and proteins through the lysosomes. Growing evidence suggests adaptation to exercise training is reliant on normally regulated autophagy, and acute physical exercise has been identified as a potent inducer of autophagy in human and rodent skeletal muscle [32–35]. Recently, it was shown that normally regulated autophagy is required for beneficial metabolic effects of exercise
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training in a transgenic mouse model [32]. In these mice native B cell lymphoma 2 (BCL2) was replaced with an inactive mutant that was unable to initiate autophagy and, as a result, exercise was no longer able to induce autophagy [32]. To investigate the role of exercise-induced autophagy in response to exercise training, wildtype and BCL2-deficient mice were fed on a high-fat diet (HFD) for 4 weeks before they were exposed to a continuous HFD with or without combined treadmill training for 8 weeks. The results showed that exercise training in the BCL2-deficient mice failed to protect against HFD-induced glucose intolerance [32]. Another study in genetically haplodeficient mice for the autophagy-related gene 6 (Atg6/Beclin1), a class III phosphoinositide 3 kinase (PI3K) that is essential for autophagosome formation, has proven that adaptation in terms of physical performance is reliant on normally regulated autophagy [33]. In this study, autophagy-deficient mice failed to improve endurance capacity after 5 weeks endurance training, whereas the wild-type mice improved their performance significantly. Moreover, autophagy-deficient mice failed to increase skeletal muscle cytochrome c and cytochrome c oxidase 4 protein levels, suggesting that autophagy is also required for mitochondrial adaptation. Interestingly, these results were obtained without any observed differences in total training volume. Because exercise strongly increases AMPK activity, it is obvious to speculate whether AMPK might be involved in exercise-induced autophagy. In fact, strong evidence from studies conducted in cultured cells has revealed a major role of AMPK in autophagy initiation [36–38]. The underlying mechanism of AMPK-mediated autophagy is not completely understood, but accumulating evidence suggests Unc-51-like kinase 1 (ULK1) as a crucial direct intermediate in AMPK-mediated autophagy [36,38–41]. ULK1 Ser317, Ser555 and Ser777 have been identified as direct targets of AMPK, and the biological function of these phosphorylations in autophagosome formation has been demonstrated [36,39,40]. Several other AMPK sites exist at ULK1, including Ser467, Thr574,
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Ser637 and Thr659, and the biological roles of these have been attributed to important events in autophagy initiation, such as delivering membranes to the expanding phagophore [36,41]. Thus, AMPK appears to be involved as an important enzyme in the regulation of protein degradation through the lysosomes by its ability to regulate autophagy through ULK1. In addition to the direct phosphorylation of ULK1, AMPK could also stimulate autophagy by suppression of mTORC1 activity. Activated mTORC1 can phosphorylate ULK1 at Ser757 and this inhibits autophagosome formation [39]. These findings demonstrate several roles of AMPK in autophagy induction and, interestingly, a negative feedback mechanism involving ULK1mediated phosphorylation of AMPK has also been identified [42]. In vitro, ULK1 has been demonstrated to phosphorylate all three AMPK subunits at unknown sites, and this is associated with decreased phosphorylation at the activating Thr172 site on AMPK. The biological relevance of this feedback mechanism in autophagy induction has not yet been verified but, based on the major role of ULK1 in autophagy initiation, it is probable that AMPK is not only involved in the induction of autophagy but also in termination of signaling events that stimulate autophagy.
Concluding remarks The mechanisms that regulate the adaptations to the trained state have important perspectives for developing new drugs that can be effective in the treatment of lifestyle-related diseases such as type 2 diabetes. AMPK is involved in regulating these processes but, to appreciate the function of AMPK fully, it is crucial to understand protein turnover as a balance between protein synthesis and degradation. AMPK is involved in regulating both aspects of protein turnover, and care must be taken not to tip the balance in future treatments that target AMPK to achieve as many of the beneficial adaptations that are associated with exercise training.
References 1 Tuomilehto, J. et al. (2001) Prevention of type 2 diabetes mellitus by changes in lifestyle among subjects with impaired glucose tolerance. N. Engl. J. Med. 344, 1343–1350 2 Carling, D. et al. (2012) AMP-activated protein kinase: new regulation, new roles? Biochem. J. 445, 11–27 3 Fujii, N. et al. (2006) AMP-activated protein kinase and the regulation of glucose transport. Am. J. Physiol. Endocrinol. Metab. 291, E867–E877 4 Sakamoto, K. et al. (2005) Deficiency of LKB1 in skeletal muscle prevents AMPK activation and glucose uptake during contraction. EMBO J. 24, 1810–1820 5 Koh, H.J. et al. (2006) Skeletal muscle-selective knockout of LKB1 increases insulin sensitivity, improves glucose homeostasis, and decreases TRB3. Mol. Cell. Biol. 26, 8217–8227 6 Woods, A. et al. (2005) Ca2+/calmodulin-dependent protein kinase kinase-beta acts upstream of AMP-activated protein kinase in mammalian cells. Cell Metab. 2, 21–33 7 Hawley, S.A. et al. (2005) Calmodulin-dependent protein kinase kinase-beta is an alternative upstream kinase for AMP-activated protein kinase. Cell Metab. 2, 9–19 8 Xiao, B. et al. (2011) Structure of mammalian AMPK and its regulation by ADP. Nature 472, 230–233 9 Vissing, K. et al. (2013) Differentiated mTOR but not AMPK signaling after strength vs endurance exercise in training-accustomed individuals. Scand. J. Med. Sci. Sports 23, 355–366 10 Dreyer, H.C. et al. (2006) Resistance exercise increases AMPK activity and reduces 4E-BP1 phosphorylation and protein synthesis in human skeletal muscle. J. Physiol. 576, 613–624
11 Wojtaszewski, J.F. et al. (2002) Dissociation of AMPK activity and ACCbeta phosphorylation in human muscle during prolonged exercise. Biochem. Biophys. Res. Commun. 298, 309–316 12 Camera, D.M. et al. (2010) Early time course of Akt phosphorylation after endurance and resistance exercise. Med. Sci. Sports Exerc. 42, 1843–1852 13 McConell, G.K. et al. (2005) Short-term exercise training in humans reduces AMPK signalling during prolonged exercise independent of muscle glycogen. J. Physiol. 568, 665–676 14 Wullschleger, S. et al. (2006) TOR signaling in growth and metabolism. Cell 124, 471–484 15 Cheng, S.W. et al. (2004) Thr2446 is a novel mammalian target of rapamycin (mTOR) phosphorylation site regulated by nutrient status. J. Biol. Chem. 279, 15719–15722 16 Inoki, K. et al. (2003) TSC2 mediates cellular energy response to control cell growth and survival. Cell 115, 577–590 17 Gwinn, D.M. et al. (2008) AMPK phosphorylation of raptor mediates a metabolic checkpoint. Mol. Cell 30, 214–226 18 Horman, S. et al. (2002) Activation of AMP-activated protein kinase leads to the phosphorylation of elongation factor 2 and an inhibition of protein synthesis. Curr. Biol. 12, 1419–1423 19 Rose, A.J. et al. (2009) A Ca(2+)-calmodulin-eEF2K-eEF2 signalling cascade, but not AMPK, contributes to the suppression of skeletal muscle protein synthesis during contractions. J. Physiol. 587, 1547–1563
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20 Lizcano, J.M. et al. (2004) LKB1 is a master kinase that activates 13 kinases of the AMPK subfamily, including MARK/PAR-1. EMBO J. 23, 833–843 21 Jessen, N. et al. (2010) Ablation of LKB1 in the heart leads to energy deprivation and impaired cardiac function. Biochim. Biophys. Acta 1802, 593–600 22 Ikeda, Y. et al. (2009) Cardiac-specific deletion of LKB1 leads to hypertrophy and dysfunction. J. Biol. Chem. 284, 35839–35849 23 Coffey, V.G. et al. (2006) Early signaling responses to divergent exercise stimuli in skeletal muscle from well-trained humans. FASEB J. 20, 190–192 24 Holloszy, J.O. (2003) A forty-year memoir of research on the regulation of glucose transport into muscle. Am. J. Physiol. Endocrinol. Metab. 284, E453–E467 25 Winder, W.W. et al. (2000) Activation of AMP-activated protein kinase increases mitochondrial enzymes in skeletal muscle. J. Appl. Physiol. 88, 2219–2226 26 Buhl, E.S. et al. (2001) Chronic treatment with 5-aminoimidazole-4-carboxamide-1beta-D-ribofuranoside increases insulin-stimulated glucose uptake and GLUT4 translocation in rat skeletal muscles in a fiber type-specific manner. Diabetes 50, 12–17 27 Jessen, N. et al. (2003) Effects of AICAR and exercise on insulin-stimulated glucose uptake, signaling, and GLUT-4 content in rat muscles. J. Appl. Physiol. 94, 1373– 1379 28 Zong, H. et al. (2002) AMP kinase is required for mitochondrial biogenesis in skeletal muscle in response to chronic energy deprivation. Proc. Natl. Acad. Sci. U. S. A. 99, 15983–15987 29 Jorgensen, S.B. et al. (2005) Effects of alpha-AMPK knockout on exercise-induced gene activation in mouse skeletal muscle. FASEB J. 19, 1146–1148 30 Holmes, B.F. et al. (2004) AMP kinase is not required for the GLUT4 response to exercise and denervation in skeletal muscle. Am. J. Physiol. Endocrinol. Metab. 287, E739–E743
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31 Pold, R. et al. (2005) Long-term AICAR administration and exercise prevents diabetes in ZDF rats. Diabetes 54, 928–934 32 He, C. et al. (2012) Exercise-induced BCL2-regulated autophagy is required for muscle glucose homeostasis. Nature 481, 511–515 33 Lira, V.A. et al. (2013) Autophagy is required for exercise training-induced skeletal muscle adaptation and improvement of physical performance. FASEB J. 27, 4184– 4193 34 Grumati, P. et al. (2011) Physical exercise stimulates autophagy in normal skeletal muscles but is detrimental for collagen VI-deficient muscles. Autophagy 7, 1415–1423 35 Jamart, C. et al. (2012) Autophagy-related and autophagy-regulatory genes are induced in human muscle after ultraendurance exercise. Eur. J. Appl. Physiol. 112, 3173–3177 36 Egan, D.F. et al. (2011) Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy. Science 331, 456–461 37 Sanchez, A.M. et al. (2012) AMPK promotes skeletal muscle autophagy through activation of forkhead FoxO3a and interaction with Ulk1. J. Cell. Biochem. 113, 695– 710 38 Lee, J.W. et al. (2010) The association of AMPK with ULK1 regulates autophagy. PLoS ONE 5, e15394 39 Kim, J. et al. (2011) AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat. Cell Biol. 13, 132–141 40 Bach, M. et al. (2011) The serine/threonine kinase ULK1 is a target of multiple phosphorylation events. Biochem. J. 440, 283–291 41 Mack, H.I. et al. (2012) AMPK-dependent phosphorylation of ULK1 regulates ATG9 localization. Autophagy 8, 1197–1214 42 Loffler, A.S. et al. (2011) Ulk1-mediated phosphorylation of AMPK constitutes a negative regulatory feedback loop. Autophagy 7, 696–706
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Drug Discovery Today Volume 00, Number 00 July 2011
AMP kinase in exercise adaptation of skeletal muscle Niels Jessen1,2, Elias I.O. Sundelin1 and Andreas Buch Møller1 1 2
Research Laboratory for Biochemical Pathology, Institute for Clinical Medicine, Aarhus University, Denmark Department of Molecular Medicine, Aarhus University Hospital, Denmark
Regular physical exercise has undisputed health benefits in the prevention and the treatment of many diseases. Understanding the mechanisms that regulate adaptations to exercise training therefore has obvious clinical perspectives. Several lines of evidence suggest that the AMP-activated protein kinase (AMPK) has a central role as a master metabolic regulator in skeletal muscle. Exercise is a potent activator of AMPK, and AMPK signaling can play a key part in regulating protein turnover during and after exercise training.
Introduction Physical activity has long been known to provide people with type 2 diabetes with remarkable health benefits, and moderate-intensity regular physical activity has been demonstrated to prevent or delay the onset of diabetes in high-risk subjects [1]. Skeletal muscle plays a key part in this beneficial adaptive response by changing its size as well as its substrate metabolism. These outcomes are brought about by changes in gene expression, biochemical and metabolic properties, and understanding the mechanisms that regulate these processes could open possibilities for future interventions that will prevent type 2 diabetes. In this short review, we will discuss new insights into the role of AMP-activated protein kinase (AMPK) in regulation of protein turnover in skeletal muscle in response to physical exercise.
AMPK expression and activation in skeletal muscle AMPK is an evolutionally conserved serine/threonine kinase that is ubiquitously expressed in human cells. The enzyme is a heterotrimer that consists of three subunits: a, b and g, each expressed in two or more isoforms [2]. The a subunit contains the typical serine/ threonine protein kinase domain, whereas the b and g subunits are regulatory subunits. The isoform-specific composition of the heterotrimer varies among species and tissue types, but expression of all three isoforms is required for full enzymatic activity. Two isoforms of the catalytic a subunit have been identified (a1 and a2), and they
Corresponding author:. Jessen, N. ([email protected])
have broad tissue distribution including in skeletal muscle. In fact, in comparison with all other tissues, the highest expression level of the a2 isoform is found in skeletal muscle, but both a subunits have been suggested to have a physiological role in response to exercise, reviewed in [2,3]. AMPK is activated by phosphorylation on Thr172 of the a1 or a2 subunit in response to various cellular energy stressors. When AMP:ATP and ADP:ATP ratios rise, AMP and ADP bind to the g subunit of AMPK and this causes a conformational change in the heterotrimer that makes the a subunit a substrate for upstream kinases. Several AMPK-related kinases have been identified, but liver kinase B1 (LKB1) (in a complex with the proteins Ste20related adaptor protein (STRAD) and Mouse protein-25 (MO25) and calmodulin-dependent protein kinase kinase (CaMKKb) appear to be the only important upstream kinases in mammalian cells [4–7]. Binding of AMP and ADP to the g subunit also leads to a decrease in accessibility of protein phosphatases to the a subunit of AMPK [8], and this could potentiate the activation of AMPK in response to energy deprivation. Activation of AMPK in skeletal muscle during exercise of different intensities and durations has been extensively investigated. Resistance exercise, characterized by short duration and high intensity, increases AMPK activity in human skeletal muscle [9,10]. Similarly, AMPK activation is consistently reported during endurance exercise when the exercise is performed at intensities over 60% VO2 peak. At lower intensities AMPK might not necessarily be activated; however, as little as 40% of VO2
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peak can activate AMPK if the exercise is continued until exhaustion [11]. The activation of AMPK by exercise is transient and usually returns to basal levels after resting a few minutes, but increased phosphorylation up to 60 min into the postexercise recovery period after endurance exercise has been reported [12]. An important question about the role of AMPK in adaptations to exercise training has been the degree to which AMPK is activated in trained individuals. In humans, examined before and after 10 days of endurance exercise training, a single bout of exercise was only able to increase AMPK activity in the untrained state [13]. However, in this study exercise in the trained state did raise AMP levels and was associated with increased phosphorylation of the AMPK target acetyl-CoA carboxylase (ACC) [13], and this raises the possibility that in the trained state AMPK activity was below the detection level of the activity assay. In agreement with this, we have found that endurance and resistance exercise can increase AMPK phosphorylation in trained subjects who have been accustomed to the specific form of exercise [9]. It is therefore probable that AMPK is involved in training adaptations in trained and untrained individuals but exercise-induced AMPK signaling is attenuated when subjects adapt to exercise training.
AMPK and protein synthesis It is widely accepted that adaptations to exercise training in muscle involves gradual alterations in protein content in skeletal muscle brought about partly by increased protein expression. The synthesis of specific proteins can be divided into several levels such as transcription of mRNA, content of ribosomes and translation of mRNA into peptides, and these processes are acutely regulated by the action of mammalian target of rapamycin (mTOR) complex 1 (mTORC1). mTORC1 is composed of the catalytic subunit mTOR in complex with the regulatory-associated protein of mTOR (Raptor) and mammalian lethal with SEC13 protein 8 (MLST8) [14]. mTORC1 modulates the rate of protein translation in part through phosphorylation of the 70 kDa ribosomal protein S6 kinase (P70S6K), eukaryotic initiation factors (eIF) and 4E binding protein (4E-BP1). Protein synthesis is an energy-consuming process and, in agreement with the role as an energy sensor, activation of AMPK can suppress protein synthesis by inhibiting mTOR activity on several levels: (i) AMPK can act directly on mTORC1 by phosphorylating mTOR on Thr2446, and thereby inhibit phosphorylation on the activating Ser2448 site [15]; (ii) AMPK can inhibit mTORC1 activity indirectly by phosphorylating and activating tuberin (TSC2), an inhibitor of mTOR activity [16]; (iii) AMPK can phosphorylate Raptor on Ser722 and Ser792 which causes dissociation of 14-3-3 and thereby further inhibition of mTORC1 activity [17]; (iv) AMPK can inhibit translation elongation through activation of eukaryotic translation elongation factor 2 kinase (eEF2K), an inhibitor of translation elongation factor eEF2 [18]. Complete inhibition of AMPK signaling can therefore be expected to have pronounced effects on protein synthesis, but inhibition of protein synthesis following muscle contractions is normal in transgenic mice expressing a dominant-negative kinase dead AMPK isoform [19]. It is important to notice that these transgenic mice do have residual AMPK activity, and this could be enough to transmit 2
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the inhibition of mTORC1 signaling. Alternatively, redundant signaling through AMPK-related kinases could compensate for the inhibition of AMPK activity in the transgenic mice. Support for this theory comes from muscle-specific LKB1 knockout mice. LKB1 is the upstream activator of 11 of the 12 AMPK-related kinases [20], and muscle-specific LKB1 knockout animals have increased mTORC1 signaling in cardiac muscle and develop cardiac hypertrophy [21,22]. In humans, a role of AMPK as an inhibitor of protein synthesis is supported by reports of decreased phosphorylation of 4E-BP1 at Thr37/46, a bona fide mTOR phosphorylation site, immediately after exercise [10,12]. However, not all human studies report reductions in TSC2/mTORC1 signaling immediately after exercise despite increased AMPK activity [9,23]. The mTORC1 complex integrates the input from many upstream pathways, including growth factors and nutrients, and these factors could modulate the AMPK effects on mTOR. In situations of severe energy deprivation AMPK can inhibit protein synthesis in human skeletal muscle but under low-to-moderate intensity exercise AMPK plays a minor part in regulating protein synthesis in humans. In contrast to the attenuation of protein synthesis during and immediately after exercise, the post-exercise recovery period is characterized by increased signaling through mTORC1 [9,12]. Trained muscle increases expression of mitochondrial oxidative enzymes, glucose transporter type 4 (GLUT4) and hexokinase, and these mechanisms have obvious implications for treatment and prevention of diabetes [24]. Chronic treatment with the pharmacological AMPK activators 5-aminoimidazole-4-carboxamide-b-Dribofuranoside (AICAR) and b-guanidinopropionic acid (b-GPA) in rodents mimics these adaptations to exercise [25–28], and these effects are ablated in transgenic mice expressing a dominantnegative form of AMPK or in AMPK a2 knockout animals [28– 30]. However, increases in protein expression after exercise training are normal in AMPK transgenic animal models with impaired AMPK signaling [29,30]. These observations suggest that AMPK is not necessary for these adaptations to exercise, but effects of residual AMPK activity in the transgenic animal models cannot be excluded. In the perspectives of disease prevention it might not be of importance if redundant pathways can compensate for the effects of AMPK in exercise adaptation. The effects of chronic pharmacological activators in animals clearly indicates that AMPK has potential as a mediator of at least some of the beneficial increases in enzyme expression induced by exercise training [31].
AMPK and protein degradation Physical exercise constitutes a major metabolic and mechanical stress that causes considerable damage to the involved muscles. Post-exercise tissue repair therefore involves several molecular mechanisms, including protein synthesis and protein degradation, that serve to remove and replace the damaged tissue. Autophagy is a catabolic process that enables removal of damaged organelles and proteins through the lysosomes. Growing evidence suggests adaptation to exercise training is reliant on normally regulated autophagy, and acute physical exercise has been identified as a potent inducer of autophagy in human and rodent skeletal muscle [32–35]. Recently, it was shown that normally regulated autophagy is required for beneficial metabolic effects of exercise
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training in a transgenic mouse model [32]. In these mice native B cell lymphoma 2 (BCL2) was replaced with an inactive mutant that was unable to initiate autophagy and, as a result, exercise was no longer able to induce autophagy [32]. To investigate the role of exercise-induced autophagy in response to exercise training, wildtype and BCL2-deficient mice were fed on a high-fat diet (HFD) for 4 weeks before they were exposed to a continuous HFD with or without combined treadmill training for 8 weeks. The results showed that exercise training in the BCL2-deficient mice failed to protect against HFD-induced glucose intolerance [32]. Another study in genetically haplodeficient mice for the autophagy-related gene 6 (Atg6/Beclin1), a class III phosphoinositide 3 kinase (PI3K) that is essential for autophagosome formation, has proven that adaptation in terms of physical performance is reliant on normally regulated autophagy [33]. In this study, autophagy-deficient mice failed to improve endurance capacity after 5 weeks endurance training, whereas the wild-type mice improved their performance significantly. Moreover, autophagy-deficient mice failed to increase skeletal muscle cytochrome c and cytochrome c oxidase 4 protein levels, suggesting that autophagy is also required for mitochondrial adaptation. Interestingly, these results were obtained without any observed differences in total training volume. Because exercise strongly increases AMPK activity, it is obvious to speculate whether AMPK might be involved in exercise-induced autophagy. In fact, strong evidence from studies conducted in cultured cells has revealed a major role of AMPK in autophagy initiation [36–38]. The underlying mechanism of AMPK-mediated autophagy is not completely understood, but accumulating evidence suggests Unc-51-like kinase 1 (ULK1) as a crucial direct intermediate in AMPK-mediated autophagy [36,38–41]. ULK1 Ser317, Ser555 and Ser777 have been identified as direct targets of AMPK, and the biological function of these phosphorylations in autophagosome formation has been demonstrated [36,39,40]. Several other AMPK sites exist at ULK1, including Ser467, Thr574,
REVIEWS
Ser637 and Thr659, and the biological roles of these have been attributed to important events in autophagy initiation, such as delivering membranes to the expanding phagophore [36,41]. Thus, AMPK appears to be involved as an important enzyme in the regulation of protein degradation through the lysosomes by its ability to regulate autophagy through ULK1. In addition to the direct phosphorylation of ULK1, AMPK could also stimulate autophagy by suppression of mTORC1 activity. Activated mTORC1 can phosphorylate ULK1 at Ser757 and this inhibits autophagosome formation [39]. These findings demonstrate several roles of AMPK in autophagy induction and, interestingly, a negative feedback mechanism involving ULK1mediated phosphorylation of AMPK has also been identified [42]. In vitro, ULK1 has been demonstrated to phosphorylate all three AMPK subunits at unknown sites, and this is associated with decreased phosphorylation at the activating Thr172 site on AMPK. The biological relevance of this feedback mechanism in autophagy induction has not yet been verified but, based on the major role of ULK1 in autophagy initiation, it is probable that AMPK is not only involved in the induction of autophagy but also in termination of signaling events that stimulate autophagy.
Concluding remarks The mechanisms that regulate the adaptations to the trained state have important perspectives for developing new drugs that can be effective in the treatment of lifestyle-related diseases such as type 2 diabetes. AMPK is involved in regulating these processes but, to appreciate the function of AMPK fully, it is crucial to understand protein turnover as a balance between protein synthesis and degradation. AMPK is involved in regulating both aspects of protein turnover, and care must be taken not to tip the balance in future treatments that target AMPK to achieve as many of the beneficial adaptations that are associated with exercise training.
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