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Selective activation of AMPK-PGC-1 or PKB-TSC2-mTOR signaling can explain specific adaptive responses to endurance or resistance training-like electrical muscle stimulation

P. J. Atherton^*,,, J. Babraj^*, K. Smith, J. Singh^,1, M. J. Rennie and H. Wackerhage^*,

^* School of Life Sciences, University of Dundee;
Department of Biological Sciences, University of Central Lancashire; and
Clinical Physiology Laboratory, University of Nottingham, UK

¹ Correspondence: Department of Biological Sciences, University of Central Lancashire, Preston PR1 2HE, UK. E-mail jsingh3{at}uclan.ac.uk

SPECIFIC AIMS

Endurance training induces a partial fast-to-slow muscle phenotype transformation and mitochondrial biogenesis, but it usually induces no growth. In contrast, resistance training stimulates muscle growth but has little effect on phenotype. We used 3 h of low-frequency stimulation (LFS) of isolated rat skeletal muscle to mimic endurance training and sixty 3 s bursts of high-frequency stimulation (HFS) to mimic resistance training. The specific aims were to identify signaling events that are activated by either LFS or HFS and that can explain the specific muscle adaptations to such stimulation patterns.

PRINCIPAL FINDINGS

LFS and HFS had specific effects on adaptation markers and on the activation of numerous signal transduction proteins.

1. LFS, but not HFS, increases AMPK Thr172 phosphorylation and induces PGC-1 and UCP-3
AMP kinase (AMPK) Thr172 phosphorylation and peroxisome proliferator-activated receptor gamma coactivator-1 (PGC-1) increased significantly to 2.02 ± 0.11 and 1.30 ± 0.04 of control directly after LFS, respectively (all signaling data relative to control; n=8; ANOVA, P<0.05; mean±SE). In contrast, AMPK Thr172 was 1.01 ± 0.03 and PGC-1 significantly decreased to 0.82 ± 0.03 directly after HFS, showing that only LFS can activate AMPK-PGC-1 signaling. We measured the expression of uncoupling protein-3 (UCP3) as a marker because has been reported to acutely increase after endurance training and to be AMPK dependent. In line with our hypothesis, UCP3 mRNA increased significantly 11.70 ± 0.96 times 3 h after LFS but it remained at 1.25 ± 0.12 of control 3 h after HFS, confirming that only LFS induced this marker. A study in which PGC-1 was overexpressed in mice suggests that the increase in PGC-1 can explain many of the specific adaptations to chronic LFS and endurance training.

2. HFS, but not LFS, activates PKB-TSC2-mTOR-dependent translation initiation, elongation and protein synthesis
A specific adaptation to resistance training is a prolonged increase in protein synthesis resulting in skeletal muscle hypertrophy. Myofibrillar protein synthesis increased significantly from a control value of 0.23 ± 0.01%·h^–1 by 5-fold to 1.24 ± 0.09%·h^–1 3 h after HFS and to 0.33 ± 0.02%·h^–1 3 h after LFS, which was not significant. The large increase in protein synthesis induced by HFS can be explained by a selective activation of the protein kinase B-tuberin mammalian target of rapamycin (PKB-TSC2-mTOR)-signaling cascade and of downstream regulators of translation initiation and elongation. PKB Ser473 phosphorylation significantly increased to 5.22 ± 1.38 directly after HFS but was unchanged after LFS. Phosphorylation of the PKB-sensitive TSC2 Thr1462 and mTOR Ser2448 sites significantly increased directly after HFS but not LFS and was not significantly changed 3 h later. Glycogen synthase kinase-3ß (GSK-3ß) Ser9 phosphorylation was significantly increased to 1.58 ± 0.05 directly after LFS and 3.08 ± 0.12 after HFS. Phosphorylation of translational regulators downstream of PKB, mTOR, and GSK-3ß suggested that they were all significantly activated directly after HFS and 3 h after HFS, with the exception of eIF4E binding protein 1 (4E-BP1). The prolonged activation of most translational regulators can explain that protein synthesis was increased 3 h after HFS. For example, p70 ribosomal S6 kinase (p70 S6k) Thr389 phosphorylation increased significantly to 6.85 ± 0.94 and 9.76 ± 0.60 of control directly and 3 h after HFS, respectively. The activation of the PKB-TSC2-mTOR-signaling cascade and the prolonged activation of multiple downstream regulators of translation initiation and elongation can explain the increase in protein synthesis that is a specific adaptation to HFS and resistance training.

3. LFS inhibits translation initiation and elongation
LFS reduced TSC2 Thr1462 phosphorylation significantly to 0.24 ± 0.02 directly and to 0.55 ± 0.06 3 h after. AMPK has recently been reported to directly phosphorylate TSC2 at the Thr1227 and Ser1345 sites, which are close to the PKB-phosphorylated Thr1462 site. Therefore, interference between the AMPK- and PKB-phosphorylated sites could potentially explain the decreased phosphorylation of Thr1462 after LFS. mTOR Ser2448 is directly phosphorylated by PKB but not TSC2, which can explain why this site is not affected when TSC2 Thr1462 is dephosphorylated after LFS. Downstream regulators of translation initiation and elongation were all significantly inhibited directly and 3 h after LFS. An example is p70 S6k Thr389 phosphorylation, which was 0.22 ± 0.03 directly and 0.17 ± 0.03 of control 3 h after LFS. However, protein synthesis was not significantly changed 3 h after LFS. This was likely due to the need of amino acid supplementation in order to be able to measure muscle protein synthesis compared with the "fasted" muscles used to measure the phosphorylation state of signaling analysis. Amino acids can activate mTOR via a phosphoinositide 3-kinase-phosphoinositide-dependent protein kinase-1 (PI3K-PDK1)-PKB-independent signaling pathway that can explain why protein synthesis was restored to control levels 3 h after LFS.

Finally, none of the mitogen-activated protein kinase pathway (ERK1/2, p38, or JNK) investigated was specifically activated by either LFS or HFS.

CONCLUSIONS AND SIGNIFCANCE

The major result of our study is that electrical muscle stimulation mimicking endurance or resistance training can switch signaling to either a catabolic AMPK-PGC-1 or anabolic PKB-TSC2-mTOR-dominated state. We term this behavior the AMPK-PKB switch (Fig. 1 ). We hypothesize that the selective activation of AMPK and increased expression of PGC-1 is a major response to the prolonged catabolism associated with LFS or endurance training. Previous results suggest that the increased expression of PGC-1 can at least partially explain increases in mitochondrial biogenesis and a progression toward a slower muscle phenotype. In a fasted muscle, LFS activation of AMPK suppresses TSC2-dependent, energy-consuming translation. This could possibly explain why endurance training does not usually stimulate muscle growth. We hypothesize that HFS and resistance training induce an anabolic signaling state without the need for systemic effectors or feeding. The observed effects of HFS on the PKB-TSC2-mTOR signaling cascade can potentially explain the observed increase in protein synthesis 3 h after HFS because fibers expressing a constitutively active PKB construct hypertrophy. The novelty of our study is the demonstration that LFS and HFS can selectively activate either AMPK-PGC-1 or PKB-TSC2-mTOR-signaling, respectively. We hypothesize that these two signaling states can at least partially explain specific adaptations to electrical muscle stimulation, endurance, and resistance training. This does not exclude that AMPK and PKB can both be activated by some forms of contractile activity such as combinations of resistance and endurance training or specific stimulation protocols. Future work needs to integrate myostatin-Smad2/3, calcineurin-NFAT-c1, and CamK IV signaling into a larger model because these pathways are all activated by contractile activity and regulate fiber phenotype, mitochondrial biogenesis, or muscle growth.

View larger version (41K): [in this window] [in a new window]   Figure 1. Schematic diagram summarizing the "AMPK-PKB switch" hypothesis. 1) LFS specifically increases AMP and will reduce glycogen causing AMPK activation and 2) an increased expression of PGC-1 that can partially explain a progression toward a slow phenotype and increased mitochondrial biogenesis. 3) In contrast, it is possible that the high tension generated as a result of HFS will induce IGF-1 and will, via a PI3K-PDK1,2-dependent mechanism (not measured), activate PKB. 4) PKB will then, directly or via TSC2, regulate activity of mTOR, which also depends 5) on nutrients via a PI3K-independent pathway. 6) Regulators of translation initiation and elongation will be activated by PKB and mTOR and will stimulate a prolonged increase in protein synthesis. 7) PKB increases protein synthesis by inhibiting GSK-3ß, which eliminates the inhibition of eIF2B, activating translation initiation further. 8) AMPK can directly activate TSC2, which then inhibits mTOR and downstream regulators of translation initiation and elongation.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2179fje;

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