3179
J Physiol 594.12 (2016) pp 3179–3180
JOURNAL CLUB
Exercise and muscle protein synthesis: not all roads lead to mTORC1 S. Engelke, F. Koch and Q. Sciascia Leibniz Institute for Farm Animal Biology (FBN), Institute of Nutritional Physiology ‘Oskar Kellner’, Dummerstorf, Germany
The Journal of Physiology
Email: [email protected]
Exercise has been shown to positively impact an increasing number of diseases in humans, such as obesity, diabetes and cardiovascular disease, and offset sarcopenia-related decreases in quality of life. Whilst the effects are global and impact all organs in the body, the major positive impacts are thought to result directly from how skeletal muscle adapts to exercise. Adaptation occurs through rapid transcriptional and translational remodelling of skeletal muscle, leading to increased mitochondrial biogenesis and enhanced oxidative capacity. A pathway known to link cellular regulation of transcription, translation and mitochondrial biogenesis during skeletal muscle growth and exercise is the highly conserved mechanistic target of rapamycin complex 1 (mTORC1). High intensity aerobic exercise in human males (Di Donato et al. 2014) and high intensity interval training in female rats (Edgett et al. 2013) has been shown to increase mTORSer2448 phosphorylation (p-mTORSer2448 ), with corresponding effects on protein synthesis. Yet, a causal link between p-mTORSer2448 and protein synthesis in response to endurance exercise in skeletal muscle has not been elucidated. Rapamycin is a lipophilic macrolide isolated from a strain of Streptomyces hygroscopicus indigenous to Easter Island. Studies show that rapamycin binds the mTORC1 component FK506-binding protein (FKBP12), and that this rapamycin– FKBP12 complex binds to the mTOR protein and disrupts the activity of the mTORC1 by potentially disrupting the interaction between regulatory associated protein of mTOR (RAPTOR) and mTOR. Thus rapamycin has been utilized in a vast number of studies, in a wide range of organisms, from yeast to humans, to specifically disrupt and inhibit mTORC1 signalling (under some experimental
conditions and in some cell types rapamycin has been shown to disrupt mTORC2 formation), and enable investigation of the causal link between experimental conditions and mTORC1 signalling. In a recent publication in The Journal of Physiology, Philp et al. (2015) utilized rapamycin to investigate the causal link between myofibrillar (MyoPS) and mitochondrial (MitoPS) protein synthesis, mitochondrial biogenesis and mTORC1 activation in response to moderate endurance exercise. In a well-constructed experimental design, Philp et al. (2015) gave female mice, 1 h prior to exercise, an I.P. injection of either rapamycin/PBS or DMSO/PBS (vehicle control). All mice then performed 1 h mild jogging (18 m min−1 at 5 deg gradient), and gastrocnemius muscles were collected under anaesthesia at 0.5, 3 and 6 h post-exercise in the recovery period (n = 4 per time point) for Western blot analysis of mTORC1 signalling pathway proteins, S6K1 and AMPK activity assays and real time PCR of mitochondrial biogenesis marker transcripts. Labelled [ring-13 C6 ] phenylalanine was also used in a separate group of female mice to assess the gastrocnemius muscle protein fractional synthetic rate (FSR). As expected, MyoPS and MitoPS increased in gastrocnemius muscle following endurance exercise. However, contrary to the working hypothesis, ‘inhibition of mTORC1 with rapamycin did not blunt MyoPS and MitoPS following moderate endurance exercise during the 6 h post-exercise recovery period’, only a transient reduction in MyoPS (30 min post-exercise) was observed. In addition, p-mTORSer2448 abundance was unaffected in the vehicle or rapamycin groups, which Philp et al. (2015) conclude to mean that a p-mTORSer2448 -independent mechanism regulates MyoPS and MitoPS in the post-recovery period following mild endurance exercise. To support this conclusion Philp et al. (2015) show that the ratio of downstream mTORC1 signalling targets, p-S6KThr389 , p-S6Ser235/236 and Ser240/244 and p-4EBP1Thr37/46 , to their respective protein totals, are blunted in both the rapamycin and vehicle groups during the post-exercise recovery period. Interestingly, the activity of S6K1 in the vehicle group increases back to basal levels during the 6 h recovery period, but remains
C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society
blunted in the rapamycin group. The final step of S6K1 activation is p-S6K1Thr229 by phosphoinositide-dependent protein kinase 1 (PDK1). This cannot occur without prior p-S6K1Thr389 by mTORC1, which decreases during the recovery period in the vehicle group and is completely blunted in the rapamycin group. The disconnection between p-S6K1Thr389 and S6K1 activation present only in the vehicle groups suggests both groups may increase FSR through different mechanisms or independently of mTORC1. Philp et al. (2015) also hypothesized that activation of AMPK, ACC, ERK1/2 or eEF2, known repressors of mTORC1, may be responsible for the blunted p-mTORSer2448 response observed in both the vehicle and rapamycin groups. Contrary to their hypothesis, no changes in the ratios of p-AMPKThr172 , p-ACCSer79 , p-ERK1/2Thr202/Tyr204 or p-eEF2Thr56 to their respective protein totals in the rapamycin-treated group were observed (Philp et al. 2015). However, the abundance of regulated in development and DNA damage response 1 (REDD1) was increased (30 min to 3 h post-exercise; relative to GAPDH) in only the vehicle group. REDD1 suppresses mTORC1 activity by releasing tumor suppressor complex 2 (TSC2) from its association with inhibitory 14-3-3 proteins, and the result suggests that p-mTORSer2448 suppression occurs by two different mechanisms in the vehicle and rapamycin groups. Comparative studies investigating the effect of exercise on mTORC1 signalling show increased p-mTORSer2448 abundance post-exercise (Edgett et al. 2013; Di Donato et al. 2014), which is transient and uncoupled from p-S6K1Thr389 abundance. Philp et al. (2015) suggest the exercise intensity used in the study was not enough to elicit a change in p-mTORSer2448 abundance and thus the increased MyoPS and MitoPS are regulated independently of p-mTORSer2448 abundance. A rate-limiting step in the translation of mRNA into protein is initiation, where cytoplasmic eIF4E binds to eIF4G and recruits the multiprotein eIF4F complex to the 5 -m7G cap of the mRNA. The activity of eIF4E is regulated by phosphorylation at Ser209 and it has been reported that rapamycin treatment increases p-eIF4ESer209
DOI: 10.1113/JP272006
3180 abundance and the overexpression of eIF4E has been shown to elevate protein synthesis in mammalian cells undergoing oncogenic transformation. This model was proposed in a comparative exercise study conducted by Williamson et al. (2006) and occurs via mitogen-activated protein kinase (MAPK) MAPK-interacting serine/threonineprotein kinase 1/2 (MNK1/2) signalling that is independent of mTORC1. Additionally, nuclear eIF4E is responsible for selectively promoting translation of nucleus-encoded mitochondria-related mRNAs and a special class of 4E-SE-containing mRNAs involved in cellular growth and proliferation. Through these interactions nuclear eIF4E functions as a central node of an RNA regulon that coordinates the expression of genes directly under its translational control and may explain why the expression of PGC-1α, PDK4 and TFAM were increased in the rapamycin group. Thus, it would be interesting to determine if MAPK– MNK1/2–cytoplasmic eIF4E signalling is responsible for the increased MyoPS and MitoPS in both groups.
Journal Club In summary, moderate endurance exercise (1 h of treadmill running at 18 m min−1 , 5 deg gradient) increased skeletal muscle MyoPS and MitoPS in both vehicle- and rapamycin-treated groups, with no apparent change in p-mTORSer2448 , suggesting regulation independent of mTORC1. Studies suggest that mTORC1-independent signalling of protein synthesis may occur via MAPK–MNK1/2–eIF4E. The authors should be commended for conducting a well-designed and rigorous investigation to identify a causal link between mTORC1 and skeletal muscle protein synthesis in response to endurance exercise – it does appear that not all roads lead to mTORC1.
References
J Physiol 594.12
Edgett BA, Fortner ML, Bonen A & Gurd BJ (2013). Mammalian target of rapamycin pathway is up-regulated by both acute endurance exercise and chronic muscle contraction in rat skeletal muscle. Appl Physiol Nutr Metab 38, 862–869. Philp A, Schenk S, Perez-Schindler J, Hamilton DL, Breen L, Laverone E, Jeromson S, Phillips SM & Baar K (2015). Rapamycin does not prevent increases in myofibrillar or mitochondrial protein synthesis following endurance exercise. J Physiol 593, 4275–4284. Williamson DL, Kubica N, Kimball SR & Jefferson LS (2006). Exercise-induced alterations in extracellular signal-regulated kinase 1/2 and mammalian target of rapamycin (mTOR) signalling to regulatory mechanisms of mRNA translation in mouse muscle. J Physiol 573, 497–510. Additional information
Di Donato DM, West DWD, Churchward-Venne TA, Breen L, Baker SK & Phillips SM (2014). Influence of aerobic exercise intensity on myofibrillar and mitochondrial protein synthesis in young men during early and late postexercise recovery. Am J Physiol Endocrinol Metab 306, E1025–E1032.
Competing interests
None declared. Acknowledgements
We would like to acknowledge PD Dr Metges and PD Dr Kuhla for their comments and edits during the preparation of this manuscript.
C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society
J Physiol 594.12 (2016) pp 3179–3180
JOURNAL CLUB
Exercise and muscle protein synthesis: not all roads lead to mTORC1 S. Engelke, F. Koch and Q. Sciascia Leibniz Institute for Farm Animal Biology (FBN), Institute of Nutritional Physiology ‘Oskar Kellner’, Dummerstorf, Germany
The Journal of Physiology
Email: [email protected]
Exercise has been shown to positively impact an increasing number of diseases in humans, such as obesity, diabetes and cardiovascular disease, and offset sarcopenia-related decreases in quality of life. Whilst the effects are global and impact all organs in the body, the major positive impacts are thought to result directly from how skeletal muscle adapts to exercise. Adaptation occurs through rapid transcriptional and translational remodelling of skeletal muscle, leading to increased mitochondrial biogenesis and enhanced oxidative capacity. A pathway known to link cellular regulation of transcription, translation and mitochondrial biogenesis during skeletal muscle growth and exercise is the highly conserved mechanistic target of rapamycin complex 1 (mTORC1). High intensity aerobic exercise in human males (Di Donato et al. 2014) and high intensity interval training in female rats (Edgett et al. 2013) has been shown to increase mTORSer2448 phosphorylation (p-mTORSer2448 ), with corresponding effects on protein synthesis. Yet, a causal link between p-mTORSer2448 and protein synthesis in response to endurance exercise in skeletal muscle has not been elucidated. Rapamycin is a lipophilic macrolide isolated from a strain of Streptomyces hygroscopicus indigenous to Easter Island. Studies show that rapamycin binds the mTORC1 component FK506-binding protein (FKBP12), and that this rapamycin– FKBP12 complex binds to the mTOR protein and disrupts the activity of the mTORC1 by potentially disrupting the interaction between regulatory associated protein of mTOR (RAPTOR) and mTOR. Thus rapamycin has been utilized in a vast number of studies, in a wide range of organisms, from yeast to humans, to specifically disrupt and inhibit mTORC1 signalling (under some experimental
conditions and in some cell types rapamycin has been shown to disrupt mTORC2 formation), and enable investigation of the causal link between experimental conditions and mTORC1 signalling. In a recent publication in The Journal of Physiology, Philp et al. (2015) utilized rapamycin to investigate the causal link between myofibrillar (MyoPS) and mitochondrial (MitoPS) protein synthesis, mitochondrial biogenesis and mTORC1 activation in response to moderate endurance exercise. In a well-constructed experimental design, Philp et al. (2015) gave female mice, 1 h prior to exercise, an I.P. injection of either rapamycin/PBS or DMSO/PBS (vehicle control). All mice then performed 1 h mild jogging (18 m min−1 at 5 deg gradient), and gastrocnemius muscles were collected under anaesthesia at 0.5, 3 and 6 h post-exercise in the recovery period (n = 4 per time point) for Western blot analysis of mTORC1 signalling pathway proteins, S6K1 and AMPK activity assays and real time PCR of mitochondrial biogenesis marker transcripts. Labelled [ring-13 C6 ] phenylalanine was also used in a separate group of female mice to assess the gastrocnemius muscle protein fractional synthetic rate (FSR). As expected, MyoPS and MitoPS increased in gastrocnemius muscle following endurance exercise. However, contrary to the working hypothesis, ‘inhibition of mTORC1 with rapamycin did not blunt MyoPS and MitoPS following moderate endurance exercise during the 6 h post-exercise recovery period’, only a transient reduction in MyoPS (30 min post-exercise) was observed. In addition, p-mTORSer2448 abundance was unaffected in the vehicle or rapamycin groups, which Philp et al. (2015) conclude to mean that a p-mTORSer2448 -independent mechanism regulates MyoPS and MitoPS in the post-recovery period following mild endurance exercise. To support this conclusion Philp et al. (2015) show that the ratio of downstream mTORC1 signalling targets, p-S6KThr389 , p-S6Ser235/236 and Ser240/244 and p-4EBP1Thr37/46 , to their respective protein totals, are blunted in both the rapamycin and vehicle groups during the post-exercise recovery period. Interestingly, the activity of S6K1 in the vehicle group increases back to basal levels during the 6 h recovery period, but remains
C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society
blunted in the rapamycin group. The final step of S6K1 activation is p-S6K1Thr229 by phosphoinositide-dependent protein kinase 1 (PDK1). This cannot occur without prior p-S6K1Thr389 by mTORC1, which decreases during the recovery period in the vehicle group and is completely blunted in the rapamycin group. The disconnection between p-S6K1Thr389 and S6K1 activation present only in the vehicle groups suggests both groups may increase FSR through different mechanisms or independently of mTORC1. Philp et al. (2015) also hypothesized that activation of AMPK, ACC, ERK1/2 or eEF2, known repressors of mTORC1, may be responsible for the blunted p-mTORSer2448 response observed in both the vehicle and rapamycin groups. Contrary to their hypothesis, no changes in the ratios of p-AMPKThr172 , p-ACCSer79 , p-ERK1/2Thr202/Tyr204 or p-eEF2Thr56 to their respective protein totals in the rapamycin-treated group were observed (Philp et al. 2015). However, the abundance of regulated in development and DNA damage response 1 (REDD1) was increased (30 min to 3 h post-exercise; relative to GAPDH) in only the vehicle group. REDD1 suppresses mTORC1 activity by releasing tumor suppressor complex 2 (TSC2) from its association with inhibitory 14-3-3 proteins, and the result suggests that p-mTORSer2448 suppression occurs by two different mechanisms in the vehicle and rapamycin groups. Comparative studies investigating the effect of exercise on mTORC1 signalling show increased p-mTORSer2448 abundance post-exercise (Edgett et al. 2013; Di Donato et al. 2014), which is transient and uncoupled from p-S6K1Thr389 abundance. Philp et al. (2015) suggest the exercise intensity used in the study was not enough to elicit a change in p-mTORSer2448 abundance and thus the increased MyoPS and MitoPS are regulated independently of p-mTORSer2448 abundance. A rate-limiting step in the translation of mRNA into protein is initiation, where cytoplasmic eIF4E binds to eIF4G and recruits the multiprotein eIF4F complex to the 5 -m7G cap of the mRNA. The activity of eIF4E is regulated by phosphorylation at Ser209 and it has been reported that rapamycin treatment increases p-eIF4ESer209
DOI: 10.1113/JP272006
3180 abundance and the overexpression of eIF4E has been shown to elevate protein synthesis in mammalian cells undergoing oncogenic transformation. This model was proposed in a comparative exercise study conducted by Williamson et al. (2006) and occurs via mitogen-activated protein kinase (MAPK) MAPK-interacting serine/threonineprotein kinase 1/2 (MNK1/2) signalling that is independent of mTORC1. Additionally, nuclear eIF4E is responsible for selectively promoting translation of nucleus-encoded mitochondria-related mRNAs and a special class of 4E-SE-containing mRNAs involved in cellular growth and proliferation. Through these interactions nuclear eIF4E functions as a central node of an RNA regulon that coordinates the expression of genes directly under its translational control and may explain why the expression of PGC-1α, PDK4 and TFAM were increased in the rapamycin group. Thus, it would be interesting to determine if MAPK– MNK1/2–cytoplasmic eIF4E signalling is responsible for the increased MyoPS and MitoPS in both groups.
Journal Club In summary, moderate endurance exercise (1 h of treadmill running at 18 m min−1 , 5 deg gradient) increased skeletal muscle MyoPS and MitoPS in both vehicle- and rapamycin-treated groups, with no apparent change in p-mTORSer2448 , suggesting regulation independent of mTORC1. Studies suggest that mTORC1-independent signalling of protein synthesis may occur via MAPK–MNK1/2–eIF4E. The authors should be commended for conducting a well-designed and rigorous investigation to identify a causal link between mTORC1 and skeletal muscle protein synthesis in response to endurance exercise – it does appear that not all roads lead to mTORC1.
References
J Physiol 594.12
Edgett BA, Fortner ML, Bonen A & Gurd BJ (2013). Mammalian target of rapamycin pathway is up-regulated by both acute endurance exercise and chronic muscle contraction in rat skeletal muscle. Appl Physiol Nutr Metab 38, 862–869. Philp A, Schenk S, Perez-Schindler J, Hamilton DL, Breen L, Laverone E, Jeromson S, Phillips SM & Baar K (2015). Rapamycin does not prevent increases in myofibrillar or mitochondrial protein synthesis following endurance exercise. J Physiol 593, 4275–4284. Williamson DL, Kubica N, Kimball SR & Jefferson LS (2006). Exercise-induced alterations in extracellular signal-regulated kinase 1/2 and mammalian target of rapamycin (mTOR) signalling to regulatory mechanisms of mRNA translation in mouse muscle. J Physiol 573, 497–510. Additional information
Di Donato DM, West DWD, Churchward-Venne TA, Breen L, Baker SK & Phillips SM (2014). Influence of aerobic exercise intensity on myofibrillar and mitochondrial protein synthesis in young men during early and late postexercise recovery. Am J Physiol Endocrinol Metab 306, E1025–E1032.
Competing interests
None declared. Acknowledgements
We would like to acknowledge PD Dr Metges and PD Dr Kuhla for their comments and edits during the preparation of this manuscript.
C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society
