Molecular and Cellular Biochemistry

, Volume 391, Issue 1–2, pp 37–46 | Cite as

Lysine suppresses protein degradation through autophagic–lysosomal system in C2C12 myotubes

  • Tomonori Sato
  • Yoshiaki Ito
  • Taku Nedachi
  • Takashi Nagasawa
Article

Abstract

Muscle mass is determined between protein synthesis and protein degradation. Reduction of muscle mass leads to bedridden condition and attenuation of resistance to diseases. Moreover, bedridden condition leads to additional muscle loss due to disuse muscle atrophy. In our previous study (Sato et al. 2013), we showed that administered lysine (Lys), one of essential amino acid, suppressed protein degradation in skeletal muscle. In this study, we investigated that the mechanism of the suppressive effects of Lys on skeletal muscle proteolysis in C2C12 cell line. C2C12 myotubes were incubated in the serum-free medium containing 10 mM Lys or 20 mM Lys, and myofibrillar protein degradation was determined by the rates of 3-methylhistidine (MeHis) release from the cells. The mammalian target of rapamycin (mTOR) activity from the phosphorylation levels of p70-ribosormal protein S6 kinase 1 and eIF4E-binding protein 1 and the autophagic–lysosomal system activity from the ratio of LC3-II/I in C2C12 myotubes stimulated by 10 mM Lys for 0–3 h were measured. The rates of MeHis release were markedly reduced by addition of Lys. The autophagic–lysosomal system activity was inhibited upon 30 min of Lys supplementation. The activity of mTOR was significantly increased upon 30 min of Lys supplementation. The suppressive effect of Lys on the proteolysis by the autophagic–lysosomal system was maintained partially when mTOR activity was inhibited by 100 nM rapamycin, suggesting that some regulator other than mTOR signaling, for example, Akt, might also suppress the autophagic–lysosomal system. From these results, we suggested that Lys suppressed the activity of the autophagic–lysosomal system in part through activation of mTOR and reduced myofibrillar protein degradation in C2C12 myotubes.

Keywords

Lysine Protein degradation Autophagy mTOR C2C12 myotubes 

References

  1. 1.
    Koopman R, van Loon LJ (2009) Aging, exercise, and muscle protein metabolism. J Appl Physiol 106:2040–2048CrossRefPubMedGoogle Scholar
  2. 2.
    Vary TC, Lynch CJ (2004) Biochemical approaches for nutritional support of skeletal muscle protein metabolism during sepsis. Nutr Res Rev 17:77–88CrossRefPubMedGoogle Scholar
  3. 3.
    Tisdale MJ (2009) Mechanisms of cancer cachexia. Physiol Rev 89:381–410CrossRefPubMedGoogle Scholar
  4. 4.
    Wolfe RR (2006) The underappreciated role of muscle in health and disease. Am J Clin Nutr 84:475–482PubMedGoogle Scholar
  5. 5.
    Nagasawa T, Yoshizawa F, Nishizawa N (1996) Plasma N-methylhistidine concentration is a sensitive index of myofibrillar protein degradation during starvation in rats. Biosci Biotechnol Biochem 60:501–502CrossRefPubMedGoogle Scholar
  6. 6.
    Nagasawa T, Kido T, Yoshizawa F, Ito Y, Nishizawa N (2002) Rapid suppression of protein degradation in skeletal muscle after oral feeding of leucine in rats. J Nutr Biochem 13:121–127CrossRefPubMedGoogle Scholar
  7. 7.
    Atherton PJ, Smith K (2012) Muscle protein synthesis in response to nutrition and exercise. J Physiol 590:1049–1057PubMedCentralCrossRefPubMedGoogle Scholar
  8. 8.
    Sugden PH, Fuller SJ (1991) Regulation of protein turnover in skeletal and cardiac muscle. Biochem J 273:21–37PubMedCentralCrossRefPubMedGoogle Scholar
  9. 9.
    Wolfe RR (2002) Regulation of muscle protein by amino acids. J Nutr 132:3219S–3224SPubMedGoogle Scholar
  10. 10.
    Paddon-Jones D, Sheffield-Moore M, Zhang XJ, Volpi E, Wolf SE, Aarsland A, Ferrando AA, Wolfe RR (2003) Amino acid ingestion improves muscle protein synthesis in the young and elderly. Am J Physiol Endocrinol Metab 286:E321–E328CrossRefPubMedGoogle Scholar
  11. 11.
    Kimball SR, Shantz LM, Horetsky RL, Jefferson LS (1999) Leucine regulates translation of specific mRNAs in L6 myoblasts though mTOR-mediated changes in availability of eIF4E and phosphorylation of ribosomal protein S6. J Biol Chem 274:11647–11652CrossRefPubMedGoogle Scholar
  12. 12.
    Lang SM, Kazi AA, Hong-Brown L, Lang CH (2012) Delayed recovery of skeletal muscle mass following hindlimb immobilization in mTOR heterozygous mice. PLoS ONE 7:e38910PubMedCentralCrossRefPubMedGoogle Scholar
  13. 13.
    Sugawara T, Ito Y, Nishizawa N, Nagasawa T (2007) Supplementation with dietary leucine to a protein-deficient diet suppresses myofibrillar protein degradation in rats. J Nutr Sci Vitaminol 53:552–555CrossRefPubMedGoogle Scholar
  14. 14.
    Sugawara T, Ito Y, Nishizawa N, Nagasawa T (2009) Regulation of muscle protein degradation, not synthesis, by dietary leucine in rats fed a protein-deficient diet. Amino Acids 37:609–616CrossRefPubMedGoogle Scholar
  15. 15.
    Lomonosova YN, Kalamkarov GR, Bugrova AE, Shevchenko TF, Kartashkina NL, Lysenko EA, Shvets VI, Nemirovskaya TL (2011) Protective effect of l-arginine administration on proteins of unloaded m.soleus. Biochemistry 76:701–712Google Scholar
  16. 16.
    Tesseraud S, Metayer-Coustard S, Boussaid S, Crochet S, Audouin E, Derouet M, Seiliez I (2007) Insulin and amino acid availability regulate atrogin-1 in avian QT6 cells. Biochem Biophys Res Commun 336:181–186CrossRefGoogle Scholar
  17. 17.
    Wu GY, Thompson JR (1990) The effect of glutamine on protein turnover in chick skeletal muscle in vitro. Biochem J 265:593–598PubMedCentralCrossRefPubMedGoogle Scholar
  18. 18.
    Bonetto A, Penna F, Minero VG, Reffo P, Costamagna D, Bonelli G, Baccino FM, Costelli P (2011) Glutamine prevents myostatin hyperexpression and protein hypercatabolism induced in C2C12 myotubes by tumor necrosis factor-α. Amino Acids 40:585–594CrossRefPubMedGoogle Scholar
  19. 19.
    Nagao K, Bannai M, Seki S, Kawai N, Mori M, Takahashi M (2010) Voluntary wheel running is beneficial to the amino acid profile of lysine-deficient rats. Am J Physiol Endocrinol Metab 298:E1170–E1178CrossRefPubMedGoogle Scholar
  20. 20.
    Ishida A, Kyoya T, Nakashima K, Katsumata M (2011) Muscle protein metabolism during compensatory growth with changing dietary lysine levels from deficient to sufficient in growing rats. J Nutr Sci Vitaminol 57:401–408CrossRefPubMedGoogle Scholar
  21. 21.
    Sato T, Ito Y, Nagasawa T (2013) Regulation of skeletal muscle protein degradation and synthesis by oral administration of lysine in rats. J Nutr Sci Vitaminol 59:412–419CrossRefPubMedGoogle Scholar
  22. 22.
    Young VR, Munro HN (1978) N-methylhistidine (3-methylhistidine) and muscle protein turnover: an overview. Fed Proc 37:2291–3000PubMedGoogle Scholar
  23. 23.
    Cowan JL, Morley SJ (2004) The proteasome inhibitor, MG132, promotes the reprogramming of translation in C2C12 myoblasts and facilitates the association of hsp25 with the eIF4F complex. Eur J Biochem 271:3596–3611CrossRefPubMedGoogle Scholar
  24. 24.
    Karim MR, Kanazawa T, Daigaku Y, Fujimura S, Miotto G, Kadowaki M (2007) Cytosolic LC3 ratio as a sensitive index of macroautophagy in isolated rat hepatocytes and H4-II-E cells. Autophagy 3:553–560CrossRefPubMedGoogle Scholar
  25. 25.
    Kang R, Zeh HJ, Lotze MT, Tang D (2011) The Beclin 1 network regulates autophagy and apoptosis. Cell Death Differ 18:571–580PubMedCentralCrossRefPubMedGoogle Scholar
  26. 26.
    Cao Y, Klionsky DJ (2007) Physiological functions of Atg6/Beclin 1: a unique autophagy-related protein. Cell Res 17:839–849CrossRefPubMedGoogle Scholar
  27. 27.
    McClung JM, Judge AR, Powers SK, Yan Z (2009) p38 MAPK links oxidative stress to autophagy-related gene expression in cachectic muscle wasting. Am J Physiol Cell Physiol 298:C542–C549PubMedCentralCrossRefPubMedGoogle Scholar
  28. 28.
    Yoshizawa F (2004) Regulation of protein synthesis by branched-chain amino acids in vivo. Biochem Biophys Res Commun 313:417–422CrossRefPubMedGoogle Scholar
  29. 29.
    Li F, Yin Y, Tan B, Kong X, Wu G (2011) Leucine nutrition in animals and humans: mTOR signaling and beyond. Amino Acids 41:1185–1193CrossRefPubMedGoogle Scholar
  30. 30.
    Nakashima K, Masaki S, Yamazaki M, Abe H (2004) Cysteine suppresses oxidative stress-induced myofibrillar proteolysis in chick myotubes. Biosci Biotechnol Biochem 68:2326–2331CrossRefPubMedGoogle Scholar
  31. 31.
    Sugawara T, Ito Y, Nishizawa N, Suzuki H, Kobayashi H, Nagasawa T (2009) Measurement of the rate of myofibrillar protein degradation using the alteriovenous difference in plasma 3-methylhistidine concentration of rats. J Nutr Sci Vitaminol 55:381–384CrossRefPubMedGoogle Scholar
  32. 32.
    Sandri M (2010) Autophagy in skeletal muscle. FEBS Lett 584:1411–1416CrossRefPubMedGoogle Scholar
  33. 33.
    Murton AJ, Constntin D, Greenhalf PL (2008) The involvement of the ubiquitin proteasome system in human skeletal muscle remodeling and atrophy. Biochim Biophys Acta 1782:730–743CrossRefPubMedGoogle Scholar
  34. 34.
    Foletta VC, White LJ, Larsen AE, Leger B, Russell AP (2011) The role and regulation of MAFbx/atrogin-1 and MuRF1 in skeletal muscle atrophy. Pflugers Arch 461:325–335CrossRefPubMedGoogle Scholar
  35. 35.
    Mordier S, Deval C, Bechet D, Tassa A, Ferrara M (2000) Leucine limitation induce autophagy and activation of lysosome-dependent proteolysis in C2C12 myotubes through a mammalian target of rapamycin-independent signaling pathway. J Biol Chem 275:29900–29906CrossRefPubMedGoogle Scholar
  36. 36.
    Jung CH, Ro SH, Cao J, Otto NM, Kim DH (2010) mTOR regulation of autophagy. FEBS Lett 584:1287–1295PubMedCentralCrossRefPubMedGoogle Scholar
  37. 37.
    Alessi DR, Kozlowski MT, Weng QP, Morrice N, Avruch J (1997) 3-Phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates and activates the p70 S6 kinase in vivo and in vitro. Curr Biol 8:69–81CrossRefGoogle Scholar
  38. 38.
    Pullen N, Dennis PB, Andjelkovic M, Dufner A, Kozma SC, Hemmings BA, Thomas G (1998) Phosphorylation and activation of p70s6k by PDK1. Science 279:707–710CrossRefPubMedGoogle Scholar
  39. 39.
    Tato I, Bartrons R, Ventura F, Rosa JL (2013) Amino acids activate mammalian target of rapamycin complex 2 (mTORC2) via PI3K/Akt signaling. J Biol Chem 25(286):6128–6142Google Scholar
  40. 40.
    Kanazawa T, Taneike I, Akashi R, Yoshizawa F, Furuya N, Fjujimura S, Kadowaki M (2004) Amino acids and insulin control autophagic proteolysis through different signaling pathway in relation to mTOR in isolated rat hepatocytes. J Biol Chem 279:8452–8459CrossRefPubMedGoogle Scholar
  41. 41.
    Mammucari C, Schiaffino S, Sandri M (2008) Downstream of Akt: FoxO3 and mTOR in the regulation of autophagy in skeletal muscle. Autophagy 4:524–526CrossRefPubMedGoogle Scholar
  42. 42.
    Haegens A, Schols AM, van Essen AL, van Loon LJ, Langen RC (2012) Leucine induces myofibrillar protein accretion in cultured skeletal muscle through mTOR dependent and -independent control of myosin heavy chain mRNA levels. Mol Nutr Food Res 56:741–752CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Tomonori Sato
    • 1
  • Yoshiaki Ito
    • 1
  • Taku Nedachi
    • 2
  • Takashi Nagasawa
    • 1
  1. 1.Department of Biological Chemistry and Food Science, Graduate School of AgricultureIwate UniversityMoriokaJapan
  2. 2.Department of Applied Biology, Faculty of Life SciencesToyo UniversityOura-gunJapan

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