NEWS & VIEWS M E TA B O L I S M
One step forward for exercise John A. Hawley and Anna Krook Exercise training has positive effects on disease risk and health outcomes through mechanisms that have not been fully characterized. Newly published data reveal that a single bout of exercise regulates the phosphoproteome via activation of a global network of kinases and 5ʹ-AMP-activated protein kinase substrates — targets with therapeutic potential for chronic metabolic diseases. Refers to Hoffman, N. J. et al. Global phosphoproteomic analysis of human skeletal muscle reveals a network of exercise-regulated kinases and AMPK substrates. Cell Metab. 22, 1–14 (2015)
Regular physical activity (exercise training) confers numerous, wide-ranging health bene fits and can prevent or delay the progression of chronic metabolic states including insulin resistance, obesity, type 2 diabetes mellitus and cardiovascular disease1. In both asymptomatic individuals and those with cardiovascular disease, exercise capacity and/or physical activity status is a stronger predictor of allcause mortality than established risk factors such as hypertension, smoking and diabetes2. However, although physical activity reduces disease risk and improves health outcomes, the precise molecular and cellular mechanisms underpinning such benefits are not completely understood. In a 2015 commentary3 on the health benefits of exercise, the authors stated that deciphering the mechanisms that underlie the responses to physical activity holds enormous discovery potential for human health, and that a major step toward this goal will be identification of the molecules that are altered in key organs and tissues in response to this activity. Newly published results4 now provide the first clues to discovering some of the ‘molecular transducers’ that link contractile events in skeletal muscle during exercise to whole-body health. With paired skeletal muscle biopsy samples obtained from four healthy men before and after a single bout of intense cycling, Hoffman and colleagues4 used phospho proteomics, and biochemical and bioinformatic approaches to identify the exercise-activated phosphoproteome. More
than 8,500 unique phosphorylation sites were analysed, and exercise induced changes in ~1,000 of them (FIG. 1). The nature of the phosphopeptides that were identified enabled prediction of the kinases most likely to have been activated in response to the exercise. Reassuringly, the exercise-mediated ‘phosphopeptide signature’ identified by Hoffman et al.4 corroborated previous data implicating
several major pathways in the coordination of skeletal muscle adaptations, including the 5ʹ-AMP-activated protein kinase (AMPK), calcium/calmodulin-dependent kinases, calcineurin, mitogen-activated protein kinase and mammalian target of rapamycin5. However, phosphopeptides that could be coupled to these kinases represented only 10% of the total changes in phosphorylation status, meaning that many unknown exercise-induced signalling pathways remain to be identified. The role of AMPK, an energy-sensitive protein kinase implicated in exercise-trainingmediated adaptations and metabolic regulation, was further investigated with a second phosphopeptide analysis of rat muscle cells exposed to a chemical activator of AMPK (AICAR) in culture. The phosphopeptides identified in this screen were interrogated using a novel approach based on machine learning, to identify kinase–substrate relationships. Integration of the exercise-mediated and AICARmediated proteomes enabled prediction of a number of novel AMPK targets, including A‑kinase anchor protein 1, mitochondrial (AKAP1), a scaffold protein that — when phosphorylated — increases mitochondrial respiration. AKAP1 provides a mechanistic link
Scan
Mitochondrial Transcription/ respiration translation
Improved health
Histone modification
Acetylation/ methylation
AKAP1
AMPK
P P P P P
?
?
P P P P P
?
?
P P P P P
Figure 1 | Deciphering the dance of a thousand phosphates. Phosphoproteomic analysis reveals Nature Reviews | Endocrinology that a single bout of intense aerobic exercise activates >1,000 unique phosphosites on 562 proteins in human skeletal muscle, including substrates of 5ʹ-AMP-activated protein kinase (AMPK). Although new data demonstrate that AMPK regulates mitochondrial respiration via the novel AMPK substrate A‑kinase anchor protein 1, mitochondrial (AKAP1), numerous additional pathways await further validation, to enable understanding of the contribution of exercise to reduction of disease risk and improvement of whole-body health.
NATURE REVIEWS | ENDOCRINOLOGY
ADVANCE ONLINE PUBLICATION | 1 © 2015 Macmillan Publishers Limited. All rights reserved
NEWS & VIEWS between skeletal muscle contraction, AMPK activation and metabolism. These novel results4 demonstrate the complexity and magnitude of an array of networks induced by an acute bout of strenuous aerobic exercise. The multi-tiered approach enabled a more dynamic and functionally based snapshot of exercise-induced events in skeletal muscle than had previously been possible with array-based analysis, and highlights the need to develop even-more-sophisticated bioinformatic tools to probe and integrate large and complex data sets. Many phosphory lation events are transient, and a comprehensive understanding of cellular regulation will require time-course studies involving a range of exercise intensities and modalities. Exercise–nutrient interactions are another fertile area for research, because nutrient availability is a potent signal that can modulate the acute cellular responses to a single bout of exercise6. Detailed study of other biochemical modifications (such as acetylation and methy lation) should also be undertaken, as these events are likely to have roles in the mediation of intracellular signalling.
…many unknown exerciseinduced signalling pathways remain to be identified Although AMPK is clearly an important signalling mediator, the health benefits of even low-intensity exercise (such as walking), which does not markedly challenge skeletal muscle energetics, are increasingly being appreciated. In this regard, identification of several signalling pathways not previously linked to exercise or muscle contraction provides novel entry points for future analysis. In addition, these results4 give new insight into mitochondrial biochemistry, increasing the number of pathways known to be in the ‘exercise kinome network’. The response to intense short-term exercise in human
skeletal muscle is clearly not governed by a single kinase, but instead involves the interplay and synchronous activation of multiple kinases. Unravelling the hierarchical organization to reveal the kinases that confer the greatest health benefits of exercise, so that targeted ‘molecular cocktails’ (so‑called ‘exercise mimetics’) can be developed, will be a major challenge. Contracting skeletal muscle releases ‘myokines’ — cytokines and other peptides that exert autocrine, paracrine or endocrine effects in ‘crosstalk’ with other organs (adipose tissue, bone, liver, pancreas and the brain)7. Because many of the beneficial ‘whole-body’ effects of exercise are likely to be mediated through such crosstalk (provoking widespread perturbations in numerous cells, tissues and organs), identifying a single pharmacological agent that mimics the wide-ranging effects of exercise is somewhat unlikely5. Future investigations should incorporate other ‘omics’ approaches8 to identify whether the adaptive responses to exercise in people with metabolic diseases (such as type 2 dia betes mellitus) differ from those of the healthy male individuals selected for this study 4. The networks induced by resistance-based exercise urgently need to be determined, as sarcopenia (the age-related decline in skeletal muscle quality and quantity) is commonly exacerbated in individuals with overweight or obesity, and has a growing effect on healthylife expectancy and health-care costs in developed nations. Individual differences in responses to standardized exercise training programmes — at both the whole-body and skeletal muscle levels — are often large, and it would be worthwhile determining whether targeted phosphoproteomic analyses can aid in the identification of individuals likely to be poor responders to fitness programmes, so that training can be personalized to confer maximum health benefits. Finally, the effects of chronic training protocols (lasting several months or years) in both healthy and diseased populations should be assessed to determine whether exercise-induced signalling networks
2 | ADVANCE ONLINE PUBLICATION
are transiently upregulated (to preserve cellular homeostasis) or represent stable changes in skeletal muscle biochemistry and physio logical function. Answering these questions could keep molecular and cellular biologists with interests in exercise science busy for many years to come. In the meantime, it seems prudent to continue to endorse public-health policies recommending that individuals participate in regular physical activity, to exploit the numerous physiological and psychological benefits of exercise training, and to improve health outcomes at the population level. John A. Hawley is at the Centre for Exercise & Nutrition, Mary MacKillop Institute for Health Research, Australian Catholic University, 8–18 Brunswick St, Fitzroy, Melbourne, Victoria 3065, Australia. Anna Krook is at the Department of Physiology & Pharmacology, Von Eulers Väg 4a, Karolinska Instituet, 171 77 Stockholm, Sweden. Correspondence to J.A.H. ([email protected]) doi:10.1038/nrendo.2015.201 Published online 27 November 2015 1. Booth, F. W., Gordon, S. E., Carlson, C. J. & Hamilton, M. T. Waging war on modern chronic diseases: primary prevention through exercise biology. J. Appl. Physiol. 88, 774–787 (2000). 2. Myers, J. et al. Exercise capacity and mortality among men referred for exercise testing. N. Engl. J. Med. 346, 793–801 (2002). 3. Neufer, P. D. et al. Understanding the cellular and molecular mechanisms of physical activity-induced health benefits. Cell Metab. 22, 4–11 (2015). 4. Hoffman, N. J. et al. Global phosphoproteomic analysis of human skeletal muscle reveals a network of exercise-regulated kinases and AMPK substrates. Cell Metab. 22, 1–14 (2015). 5. Hawley, J. A., Hargreaves, M., Joyner, M. J. & Zierath, J. R. Integrative biology of exercise. Cell 159, 738–749 (2014). 6. Hawley, J. A. & Morton, J. P. Ramping up the signal: promoting endurance training adaptation in skeletal muscle by nutritional manipulation. Clin. Exp. Pharmacol. Physiol. 41, 608–613 (2014). 7. Pedersen, B. K. & Febbraio, M. A. Muscles, exercise and obesity: skeletal muscle as a secretory organ. Nat. Rev. Endocrinol. 8, 457–465 (2012). 8. Zierath, J. R. & Wallberg-Henriksson, H. Looking ahead perspective: where will the future of exercise biology take us? Cell Metab. 22, 25–30 (2015).
Competing interests statement
The authors declare no competing interests.
www.nature.com/nrendo © 2015 Macmillan Publishers Limited. All rights reserved
One step forward for exercise John A. Hawley and Anna Krook Exercise training has positive effects on disease risk and health outcomes through mechanisms that have not been fully characterized. Newly published data reveal that a single bout of exercise regulates the phosphoproteome via activation of a global network of kinases and 5ʹ-AMP-activated protein kinase substrates — targets with therapeutic potential for chronic metabolic diseases. Refers to Hoffman, N. J. et al. Global phosphoproteomic analysis of human skeletal muscle reveals a network of exercise-regulated kinases and AMPK substrates. Cell Metab. 22, 1–14 (2015)
Regular physical activity (exercise training) confers numerous, wide-ranging health bene fits and can prevent or delay the progression of chronic metabolic states including insulin resistance, obesity, type 2 diabetes mellitus and cardiovascular disease1. In both asymptomatic individuals and those with cardiovascular disease, exercise capacity and/or physical activity status is a stronger predictor of allcause mortality than established risk factors such as hypertension, smoking and diabetes2. However, although physical activity reduces disease risk and improves health outcomes, the precise molecular and cellular mechanisms underpinning such benefits are not completely understood. In a 2015 commentary3 on the health benefits of exercise, the authors stated that deciphering the mechanisms that underlie the responses to physical activity holds enormous discovery potential for human health, and that a major step toward this goal will be identification of the molecules that are altered in key organs and tissues in response to this activity. Newly published results4 now provide the first clues to discovering some of the ‘molecular transducers’ that link contractile events in skeletal muscle during exercise to whole-body health. With paired skeletal muscle biopsy samples obtained from four healthy men before and after a single bout of intense cycling, Hoffman and colleagues4 used phospho proteomics, and biochemical and bioinformatic approaches to identify the exercise-activated phosphoproteome. More
than 8,500 unique phosphorylation sites were analysed, and exercise induced changes in ~1,000 of them (FIG. 1). The nature of the phosphopeptides that were identified enabled prediction of the kinases most likely to have been activated in response to the exercise. Reassuringly, the exercise-mediated ‘phosphopeptide signature’ identified by Hoffman et al.4 corroborated previous data implicating
several major pathways in the coordination of skeletal muscle adaptations, including the 5ʹ-AMP-activated protein kinase (AMPK), calcium/calmodulin-dependent kinases, calcineurin, mitogen-activated protein kinase and mammalian target of rapamycin5. However, phosphopeptides that could be coupled to these kinases represented only 10% of the total changes in phosphorylation status, meaning that many unknown exercise-induced signalling pathways remain to be identified. The role of AMPK, an energy-sensitive protein kinase implicated in exercise-trainingmediated adaptations and metabolic regulation, was further investigated with a second phosphopeptide analysis of rat muscle cells exposed to a chemical activator of AMPK (AICAR) in culture. The phosphopeptides identified in this screen were interrogated using a novel approach based on machine learning, to identify kinase–substrate relationships. Integration of the exercise-mediated and AICARmediated proteomes enabled prediction of a number of novel AMPK targets, including A‑kinase anchor protein 1, mitochondrial (AKAP1), a scaffold protein that — when phosphorylated — increases mitochondrial respiration. AKAP1 provides a mechanistic link
Scan
Mitochondrial Transcription/ respiration translation
Improved health
Histone modification
Acetylation/ methylation
AKAP1
AMPK
P P P P P
?
?
P P P P P
?
?
P P P P P
Figure 1 | Deciphering the dance of a thousand phosphates. Phosphoproteomic analysis reveals Nature Reviews | Endocrinology that a single bout of intense aerobic exercise activates >1,000 unique phosphosites on 562 proteins in human skeletal muscle, including substrates of 5ʹ-AMP-activated protein kinase (AMPK). Although new data demonstrate that AMPK regulates mitochondrial respiration via the novel AMPK substrate A‑kinase anchor protein 1, mitochondrial (AKAP1), numerous additional pathways await further validation, to enable understanding of the contribution of exercise to reduction of disease risk and improvement of whole-body health.
NATURE REVIEWS | ENDOCRINOLOGY
ADVANCE ONLINE PUBLICATION | 1 © 2015 Macmillan Publishers Limited. All rights reserved
NEWS & VIEWS between skeletal muscle contraction, AMPK activation and metabolism. These novel results4 demonstrate the complexity and magnitude of an array of networks induced by an acute bout of strenuous aerobic exercise. The multi-tiered approach enabled a more dynamic and functionally based snapshot of exercise-induced events in skeletal muscle than had previously been possible with array-based analysis, and highlights the need to develop even-more-sophisticated bioinformatic tools to probe and integrate large and complex data sets. Many phosphory lation events are transient, and a comprehensive understanding of cellular regulation will require time-course studies involving a range of exercise intensities and modalities. Exercise–nutrient interactions are another fertile area for research, because nutrient availability is a potent signal that can modulate the acute cellular responses to a single bout of exercise6. Detailed study of other biochemical modifications (such as acetylation and methy lation) should also be undertaken, as these events are likely to have roles in the mediation of intracellular signalling.
…many unknown exerciseinduced signalling pathways remain to be identified Although AMPK is clearly an important signalling mediator, the health benefits of even low-intensity exercise (such as walking), which does not markedly challenge skeletal muscle energetics, are increasingly being appreciated. In this regard, identification of several signalling pathways not previously linked to exercise or muscle contraction provides novel entry points for future analysis. In addition, these results4 give new insight into mitochondrial biochemistry, increasing the number of pathways known to be in the ‘exercise kinome network’. The response to intense short-term exercise in human
skeletal muscle is clearly not governed by a single kinase, but instead involves the interplay and synchronous activation of multiple kinases. Unravelling the hierarchical organization to reveal the kinases that confer the greatest health benefits of exercise, so that targeted ‘molecular cocktails’ (so‑called ‘exercise mimetics’) can be developed, will be a major challenge. Contracting skeletal muscle releases ‘myokines’ — cytokines and other peptides that exert autocrine, paracrine or endocrine effects in ‘crosstalk’ with other organs (adipose tissue, bone, liver, pancreas and the brain)7. Because many of the beneficial ‘whole-body’ effects of exercise are likely to be mediated through such crosstalk (provoking widespread perturbations in numerous cells, tissues and organs), identifying a single pharmacological agent that mimics the wide-ranging effects of exercise is somewhat unlikely5. Future investigations should incorporate other ‘omics’ approaches8 to identify whether the adaptive responses to exercise in people with metabolic diseases (such as type 2 dia betes mellitus) differ from those of the healthy male individuals selected for this study 4. The networks induced by resistance-based exercise urgently need to be determined, as sarcopenia (the age-related decline in skeletal muscle quality and quantity) is commonly exacerbated in individuals with overweight or obesity, and has a growing effect on healthylife expectancy and health-care costs in developed nations. Individual differences in responses to standardized exercise training programmes — at both the whole-body and skeletal muscle levels — are often large, and it would be worthwhile determining whether targeted phosphoproteomic analyses can aid in the identification of individuals likely to be poor responders to fitness programmes, so that training can be personalized to confer maximum health benefits. Finally, the effects of chronic training protocols (lasting several months or years) in both healthy and diseased populations should be assessed to determine whether exercise-induced signalling networks
2 | ADVANCE ONLINE PUBLICATION
are transiently upregulated (to preserve cellular homeostasis) or represent stable changes in skeletal muscle biochemistry and physio logical function. Answering these questions could keep molecular and cellular biologists with interests in exercise science busy for many years to come. In the meantime, it seems prudent to continue to endorse public-health policies recommending that individuals participate in regular physical activity, to exploit the numerous physiological and psychological benefits of exercise training, and to improve health outcomes at the population level. John A. Hawley is at the Centre for Exercise & Nutrition, Mary MacKillop Institute for Health Research, Australian Catholic University, 8–18 Brunswick St, Fitzroy, Melbourne, Victoria 3065, Australia. Anna Krook is at the Department of Physiology & Pharmacology, Von Eulers Väg 4a, Karolinska Instituet, 171 77 Stockholm, Sweden. Correspondence to J.A.H. ([email protected]) doi:10.1038/nrendo.2015.201 Published online 27 November 2015 1. Booth, F. W., Gordon, S. E., Carlson, C. J. & Hamilton, M. T. Waging war on modern chronic diseases: primary prevention through exercise biology. J. Appl. Physiol. 88, 774–787 (2000). 2. Myers, J. et al. Exercise capacity and mortality among men referred for exercise testing. N. Engl. J. Med. 346, 793–801 (2002). 3. Neufer, P. D. et al. Understanding the cellular and molecular mechanisms of physical activity-induced health benefits. Cell Metab. 22, 4–11 (2015). 4. Hoffman, N. J. et al. Global phosphoproteomic analysis of human skeletal muscle reveals a network of exercise-regulated kinases and AMPK substrates. Cell Metab. 22, 1–14 (2015). 5. Hawley, J. A., Hargreaves, M., Joyner, M. J. & Zierath, J. R. Integrative biology of exercise. Cell 159, 738–749 (2014). 6. Hawley, J. A. & Morton, J. P. Ramping up the signal: promoting endurance training adaptation in skeletal muscle by nutritional manipulation. Clin. Exp. Pharmacol. Physiol. 41, 608–613 (2014). 7. Pedersen, B. K. & Febbraio, M. A. Muscles, exercise and obesity: skeletal muscle as a secretory organ. Nat. Rev. Endocrinol. 8, 457–465 (2012). 8. Zierath, J. R. & Wallberg-Henriksson, H. Looking ahead perspective: where will the future of exercise biology take us? Cell Metab. 22, 25–30 (2015).
Competing interests statement
The authors declare no competing interests.
www.nature.com/nrendo © 2015 Macmillan Publishers Limited. All rights reserved
