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Effects of contraction and insulin on protein synthesis, AMP-activated protein kinase and phosphorylation state of translation factors in rat skeletal muscle

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Abstract

In rat epitrochlearis skeletal muscle, contraction inhibited the basal and insulin-stimulated rates of protein synthesis by 75 and 70%, respectively, while increasing adenosine monophosphate-activated protein kinase (AMPK) activity. Insulin, on the other hand, stimulated protein synthesis (by 30%) and increased p70 ribosomal protein S6 kinase (p70S6K) Thr389, 40S ribosomal protein S6 (rpS6) Ser235/236, rpS6 Ser240/244 and eukaryotic initiation factor-4E-binding protein-1 (4E-BP1) Thr37/46 phosphorylation over basal values. Electrical stimulation had no effect on mammalian target of rapamycin complex 1 (mTORC1) signalling, as reflected by the lack of reduction in basal levels of p70S6K, rpS6 Ser235/236, rpS6 Ser240/244 and 4E-BP1 phosphorylation, but did antagonize mTORC1 signalling after stimulation of the pathway by insulin. Eukaryotic elongation factor-2 (eEF2) Thr56 phosphorylation increased rapidly on electrical stimulation reaching a maximum at 1 min, whereas AMPK Thr172 phosphorylation slowly increased to reach threefold after 30 min. Eukaryotic elongation factor-2 kinase (eEF2K) was not activated after 30 min of contraction when AMPK was activated. This could not be explained by the expression of a tissue-specific isoform of eEF2K in skeletal muscle lacking the Ser398 AMPK phosphorylation site. Therefore, in this skeletal muscle system, the contraction-induced inhibition of protein synthesis could not be attributed to a reduction in mTORC1 signalling but could be due to an increase in eEF2 phosphorylation independent of AMPK activation.

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Abbreviations

4E-BP1:

eukaryotic initiation factor-4E-binding protein-1

ACC:

acetyl-CoA carboxylase

AICAR:

5-aminoimidazole-4-carboxamide riboside

AMPK:

AMP-activated protein kinase

eEF2:

eukaryotic elongation factor-2

eEF2K:

eukaryotic elongation factor-2 kinase

mTOR:

mammalian target of rapamycin

mTORC1:

mammalian target of rapamycin complex 1

PRAS40:

proline-rich Akt/PKB substrate 40 kDa

p70S6K:

p70 ribosomal protein S6 kinase

rp S6:

40S ribosomal protein S6

References

  1. Aslesen R, Jensen J (1998) Effects of epinephrine on glucose metabolism in contracting rat skeletal muscle. Am J Physiol 275:E448–E456

    PubMed  CAS  Google Scholar 

  2. Atherton PJ, Babraj J, Smith K, Singh J, Rennie MJ, Wackerhage H (2005) Selective activation of AMPK-PGC-1alpha or PKB-TSC2-mTOR signaling can explain specific adaptive responses to endurance or resistance training-like electrical muscle stimulation. FASEB J 19:786–788

    PubMed  CAS  Google Scholar 

  3. Biolo G, Declan Fleming RY, Wolfe RR (1995) Physiologic hyperinsulinemia stimulates protein synthesis and enhances transport of selected amino acids in human skeletal muscle. J Clin Invest 95:811–819

    PubMed  CAS  Google Scholar 

  4. Bolster DR, Crozier SJ, Kimball SR, Jefferson LS (2002) AMP-activated protein kinase suppresses protein synthesis in rat skeletal muscle through downregulated mTOR signalling. J Biol Chem 277:23977–23980

    Article  PubMed  CAS  Google Scholar 

  5. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  PubMed  CAS  Google Scholar 

  6. Browne GJ, Finn SG, Proud CG (2004) Stimulation of the AMP-activated protein kinase leads to activation of eukaryotic elongation factor 2 kinase and to its phosphorylation at a novel site, serine 398. J Biol Chem 279:12220–12231

    Article  PubMed  CAS  Google Scholar 

  7. Buttgereit F, Brand MD (1995) A hierarchy of ATP-consuming processes in mammalian cells. Biochem J 312:163–167

    PubMed  CAS  Google Scholar 

  8. Bylund-Fellenius AC, Ojamaa KM, Flaim KE, Li JB, Wassner SJ, Jefferson LS (1984) Protein synthesis versus energy state in contracting muscles of perfused rat hindlimb. Am J Physiol 246:E297–E305

    PubMed  CAS  Google Scholar 

  9. Carraro F, Stuart CA, Hartl WH, Rosenblatt J, Wolfe RR (1990) Effect of exercise and recovery on muscle protein synthesis in human subjects. Am J Physiol 259:E470–E476

    PubMed  CAS  Google Scholar 

  10. Cheng SWY, Fryer LGD, Carling D, Shepherd PR (2004) T2446 is a novel mTOR phosphorylation site regulated by nutrient status. J Biol Chem 279:15719–15722

    Article  PubMed  CAS  Google Scholar 

  11. Davies SP, Carling D, Hardie DG (1989) Tissue distribution of the AMP-activated protein kinase, and lack of activation by cyclic-AMP-dependent protein kinase, studied a specific and sensitive peptide assay. Eur J Biochem 186:123–128

    Article  PubMed  CAS  Google Scholar 

  12. Dreyer HC, Fujita S, Cadenas JG, Chinkes DL, Volpi E, Rasmussen BB (2006) Resistance exercise increases AMPK activity and reduces 4E-BP1 phosphorylation and protein synthesis in human skeletal muscle. J. Physiol 576:613–624

    Article  PubMed  CAS  Google Scholar 

  13. Dubbelhuis PF, Meijer AJ (2002) Hepatic amino acid-dependent signaling is under control of AMP-dependent protein kinase. FEBS Lett 521:39–42

    Article  PubMed  CAS  Google Scholar 

  14. Gelfand RA, Barrett EJ (1987) Effect of physiologic hyperinsulinemia on skeletal muscle protein synthesis and breakdown in man. J Clin Invest 80:1–6

    PubMed  CAS  Google Scholar 

  15. Hait WN, Ward MD, Trakht IN, Ryazanov AG (1996) Elongation factor-2 kinase: immunological evidence for the existence of tissue-specific isoforms. FEBS Lett 397:55–60

    Article  PubMed  CAS  Google Scholar 

  16. Hardie DG, Carling D, Carlson M (1998) The AMP-activated/SNF1 protein kinase subfamily: metabolic sensors of the eukaryotic cell? Annu Rev Biochem 67:821–855

    Article  PubMed  CAS  Google Scholar 

  17. Hardie DG, Sakamoto K (2006) AMPK: a key sensor of fuel and energy status. Physiology 21:48–60

    Article  PubMed  CAS  Google Scholar 

  18. Horman S, Beauloye C, Vertommen D, Vanoverschelde J-L, Hue L, Rider MH (2003) Myocardial ischemia and increased heart work modulate the phosphorylation state of eukaryotic elongation factor-2. J Biol Chem 278:41970–41976

    Article  PubMed  CAS  Google Scholar 

  19. Horman S, Browne GJ, Krause U, Patel JV, Vertommen D, Bertrand L, Lavoine A, Hue L, Proud CG, Rider MH (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

    Article  PubMed  CAS  Google Scholar 

  20. Horman S, Hussain N, Dilworth SM, Storey KB, Rider MH (2005) Evaluation of the role of AMP-activated protein kinase and its downstream targets in mammalian hibernation. Comp Biochem Physiol B 142:374–382

    Article  PubMed  CAS  Google Scholar 

  21. Inoki K, Zhu T, Guan K-L (2003) TSC2 mediates cellular energy response to control cell growth and survival. Cell 115:577–590

    Article  PubMed  CAS  Google Scholar 

  22. Jakobsen SN, Hardie DG, Morrice N, Tornqvist HE (2001) 5″-AMP-activated protein kinase phosphorylates IRS-1 on Ser-789 in mouse C2C12 myotubes in response to 5-aminoimidazole-4-carboxamide riboside. J Biol Chem 276:46912–46916

    Article  PubMed  CAS  Google Scholar 

  23. Kahn BB, Alquier T, Carling D, Hardie DG (2005) AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism. Cell Metab 1:15–25

    Article  PubMed  CAS  Google Scholar 

  24. Kemp BE, Mitchelhill KI, Stapleton D, Michell BJ, Chen ZP, Witters LA (1999) Dealing with energy demand: the AMP-activated protein kinase. Trends Biochem Sci 24:22–25

    Article  PubMed  CAS  Google Scholar 

  25. Kimball SR, Farrell PA, Jefferson LS (2002) Role of insulin in translational control of protein synthesis in skeletal muscle by amino acids or exercise. J Appl Physiol 93:1168–1180

    PubMed  CAS  Google Scholar 

  26. Krause U, Bertrand L, Hue L (2002) Control of p70 ribosomal protein S6 kinase and acetyl-CoA carboxylase by AMP-activated protein kinase and protein phosphatases in isolated hepatocytes. Eur J Biochem 269:3751–3759

    Article  PubMed  CAS  Google Scholar 

  27. Long X, Lin Y, Ortiz-Vega S, Yonezawa K, Avruch J (2005) Rheb binds and regulates the mTOR kinase. Curr Biol 15:702–713

    Article  PubMed  CAS  Google Scholar 

  28. Marin P, Nastiuk KL, Daniel N, Girault J, Czernik AJ, Glowinski J, Nairn AC, Premont J (1997) Glutamate-dependent phosphorylation of elongation factor-2 and inhibition of protein synthesis in neurons. J Neurosci 17:3445–3454

    PubMed  CAS  Google Scholar 

  29. Plumb-Rudewiez N, Clotman F, Strick-Marchand H, Pierreux CE, Weiss MC, Rousseau GG, Lemaigre F (2004) Transcription factor HNF-6/OC-1 inhibits the stimulation of the HNF-3a/Foxa1 gene by TGF-b in mouse liver. Hepatology 40:1266–1274

    Article  PubMed  CAS  Google Scholar 

  30. Proud CG (2007) Signalling to translation: how signal transduction pathways control the protein synthesis machinery. Biochem J 403:217–234

    Article  PubMed  CAS  Google Scholar 

  31. Redpath NT, Price NT, Proud CG (1996) Cloning and expression of cDNA encoding protein synthesis elongation factor-2 kinase. J Biol Chem 271:17547–17554

    Article  PubMed  CAS  Google Scholar 

  32. Rennie MJ (1996) Influence of exercise on protein and amino acid metabolism. In: Rowell LB, Shepherd JT (eds) Handbook of physiology; exercise: regulation and integration of multiple systems. Oxford University Press, New York, pp. 995–1035

  33. Reynolds TH, Bodine SC, Lawrence JC (2002) Control of Ser2448 phosphorylation in the mammalian target of rapamycin by insulin and skeletal muscle load. J Biol Chem 277:17657–17662

    Article  PubMed  CAS  Google Scholar 

  34. Rider MH, Hussain N, Horman S, Dilworth SM, Storey KB (2006) Stress-induced activation of the AMP-activated protein kinase in the freeze-tolerant frog Rana sylvatica. Cryobiology 53:297–309

    Article  PubMed  CAS  Google Scholar 

  35. Rose AJ, Broholm C, Kiillerich K, Finn SG, Proud CG, Rider MH, Richter EA, Kiens B (2005) Exercise rapidly increases eukaryotic elongation factor 2 phosphorylation in skeletal muscle of men. J Physiol 569:223–228

    Article  PubMed  CAS  Google Scholar 

  36. Roskoski R Jr (1983) Assays of protein kinase. Methods Enzymol 99:3–6

    Article  PubMed  CAS  Google Scholar 

  37. Ruvinsky I, Meyuhas O (2006) Ribosomal protein S6 phosphorylation: from protein synthesis to cell size. Trends Biochem Sci 31:342–348

    Article  PubMed  CAS  Google Scholar 

  38. Ryder JW, Fahlman R, Wallberg-Henriksson H, Alessi DR, Krook A, Zierath JR (2000) Effect of contraction on mitogen-activated protein kinase signal transduction in skeletal muscle. Involvement of the mitogen- and stress-activated protein kinase 1. J Biol Chem 275:1457–1462

    Article  PubMed  CAS  Google Scholar 

  39. Sancak Y, Thoreen CC, Peterson TR, Lindquist RA, Kang SA, Spooner E, Carr SA, Sabatini DM (2007) PRAS40 is an insulin-regulated inhibitor of the mTORC1 protein kinase. Mol Cell 25:903–915

    Article  PubMed  CAS  Google Scholar 

  40. Sanders MJ, Grondin PO, Hegarty BD, Snowden MA, Carling D (2007) Investigating the mechanism for AMP activation of the AMP-activated protein kinase cascade. Biochem J 403:139–148

    Article  PubMed  CAS  Google Scholar 

  41. Smith K, Barua JM, Watt PW, Scrimgeour CM, Rennie MJ (1992) Flooding with L-[1–13C]leucine stimulates human muscle protein incorporation of continuously infused L-[1–13C]valine. Am J Physiol 262:E372–E376

    PubMed  CAS  Google Scholar 

  42. Stirewalt WS, Low RB (1983) Effects of insulin in vitro on protein turnover in rat epitrochlearis muscle. Biochem J 210:323–330

    PubMed  CAS  Google Scholar 

  43. Tzatsos A, Tsichlis PN (2007) Energy depletion inhibits phosphatidylinositol 3-kinase/akt signaling and induces apoptosis via amp-activated protein kinase-dependent phosphorylation of IRS-1 at Ser-794. J Biol Chem 282:18069–18082

    Article  PubMed  CAS  Google Scholar 

  44. Wagenmakers AJ (1998) Protein and amino acid metabolism in human muscle. Adv Exp Med Biol 441:307–319

    PubMed  CAS  Google Scholar 

  45. Whitehead JP, Soos MA, Aslesen R, O, Rahilly S, Jensen J (2000) Contraction inhibits insulin-stimulated insulin receptor substrate-1/2-associated phosphoinositide 3-kinase activity, but not PKB activation or glucose uptake in rat muscle. Biochem J 349:775–781

    PubMed  CAS  Google Scholar 

  46. Wieser G, Krumschnabel G (2001) Hierarchies of ATP-consuming processes: direct compared with indirect measurements, and comparative aspects. Biochem J 355:389–395

    Article  PubMed  CAS  Google Scholar 

  47. Williamson DL, Bolster DR, Kimball SR, Jefferson LS (2006) Time course changes in signaling pathways and protein synthesis in C2C12 myotubes following AMPK activation by AICAR. Am J Physiol Endocrinol Metab 291:80–89

    Article  CAS  Google Scholar 

  48. Witters LA, Kemp BE, Means AR (2006) Chutes and Ladders: the search for protein kinases that act on AMPK. Trends Biochem Sci 31:13–16

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

We thank Ada Ingvaldsen and Jorid Thrane Stuenæs for technical assistance. L.M. was supported by the Université catholique de Louvain. The work was supported by the Interuniversity Attraction Poles Program—Belgian Science Policy (P5/05 and P6/28), the Directorate General Higher Education and Scientific Research, French Community of Belgium, the Fund for Medical Scientific Research (Belgium), the EXGENESIS Integrated Project (LSHM-CT-2004-005272) from the European Commission, the Research Council of Norway and the Novo Nordisk Foundation.

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  1. Hormone and Metabolic Research Unit, de Duve Institute, Université catholique de Louvain, Avenue Hippocrate, 75, 1200, Brussels, Belgium

    Lisa Miranda, Sandrine Horman, Isabelle De Potter, Louis Hue & Mark H. Rider

  2. National Institute of Occupational Health, Pb 8149 Dep, Oslo, Norway

    Jørgen Jensen

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  1. Lisa Miranda
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Correspondence to Mark H. Rider.

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Miranda, L., Horman, S., De Potter, I. et al. Effects of contraction and insulin on protein synthesis, AMP-activated protein kinase and phosphorylation state of translation factors in rat skeletal muscle. Pflugers Arch - Eur J Physiol 455, 1129–1140 (2008). https://doi.org/10.1007/s00424-007-0368-2

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