Leucine attenuates muscle atrophy and autophagosome formation by activating PI3K/AKT/mTOR signaling pathway in rotator cuff tears

  • Rongzong Zheng
  • Shuming HuangEmail author
  • Junkun Zhu
  • Wei Lin
  • Huan Xu
  • Xiang Zheng
Regular Article


Rotator cuff tears (RCTs), the most common tendon injury, are always accompanied by muscle atrophy, which is characterized by excessive protein degradation. Autophagy–lysosome systems are the crucial proteolytic pathways and are activated in atrophying muscle. Thus, inhibition of the autophagy–lysosome pathway might be an alternative way to minimize skeletal muscle atrophy. In this present study, combined with a tendon transection-induced rat model of massive rotator cuff tears, HE staining and transmission electron microscopy methods, we found leucine supplementation effectively prevented muscle atrophy, muscle injury and autophagosome formation. Utilizing immunoblotting, we discovered that leucine supplementation is able to inhibit the rise in autophagy-related protein expression (including LC3, Atrogin-1, MuRF1, Bnip3 and FoxO3) driven by tendon transection. The PI3K/AKT/mTOR pathway that was essential in autophagosome formation and autophagy was blocked in atrophying muscle and reactivated in the presence of leucine. Once administrated with LY294002 (PI3K inhibitor) and Rapamycin (mTOR inhibitor), leucine mediated by the anti-atrophic effects was nearly blunted. These results suggest that leucine potentially attenuates tendon transection-induced muscle atrophy through autophagy inhibition via activating the PI3K/AKT/mTOR pathway.


Rotator cuff tears Skeletal muscle atrophy Leucine PI3K/AKT/mTOR pathway Autophagosome 


Author contributions

Dr. Rongzong Zheng (MD), the director of orthopedics, sports medicine and joint surgery sub-specialty conceived,designed and launched this research.

Dr. Shuming Huang (MD) performed the experiments and analyzed data, taking the responsibility of the chief instructor of orthopedics trauma sub-specialty, and shares the privilege of co-first authors and corresponding author.

Dr. Junkun Zhu (MD), the director of orthopedics rehabilitation sub-specialty, submitted the proposal and composed the manuscript and shares the privilege of co-first authors.

Wei Lin, Huan Xu and Xiang Zheng participated and collected the experiment data as well as completing statistics analysis.


This study is supported by Zhejiang Provincial Natural Science Foundation (grant number: Y16H060008, PR China) and Medical and Health Research Program of Zhejiang Province (grant number: 2012KYB249, 2017KY725, PR China).

Compliance with ethical standards

All animal care procedures were approved by laboratory animals of the Fifth Affiliated Hospital of Wenzhou Medical College in this study.


The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


  1. Anthony JC, Anthony TG, Kimball SR, Vary TC, Jefferson LS (2000) Orally administered leucine stimulates protein synthesis in skeletal muscle of postabsorptive rats in association with increased eIF4F formation. J Nutr 130:139–145CrossRefGoogle Scholar
  2. Anthony JC, Anthony TG, Layman DK (1999) Leucine supplementation enhances skeletal muscle recovery in rats following exercise. J Nutr 129:1102–1106CrossRefGoogle Scholar
  3. Bacurau AV, Jannig PR, de Moraes WM, Cunha TF, Medeiros A, Barberi L, Coelho MA, Bacurau RF, Ugrinowitsch C, Musaro A, Brum PC (2016) Akt/mTOR pathway contributes to skeletal muscle anti-atrophic effect of aerobic exercise training in heart failure mice. Int J Cardiol 214:137–147CrossRefGoogle Scholar
  4. Baptista IL, Leal ML, Artioli GG, Aoki MS, Fiamoncini J, Turri AO, Curi R, Miyabara EH, Moriscot AS (2010) Leucine attenuates skeletal muscle wasting via inhibition of ubiquitin ligases. Muscle Nerve 41:800–808CrossRefGoogle Scholar
  5. Baptista IL, Silva WJ, Artioli GG, Guilherme JP, Leal ML, Aoki MS, Miyabara EH, Moriscot AS (2013) Leucine and HMB differentially modulate proteasome system in skeletal muscle under different sarcopenic conditions. PLoS One 8:e76752CrossRefGoogle Scholar
  6. Baptista IL, Silvestre JG, Silva WJ, Labeit S, Moriscot AS (2017) FoxO3a suppression and VPS34 activity are essential to anti-atrophic effects of leucine in skeletal muscle. Cell Tissue Res 369:381–394CrossRefGoogle Scholar
  7. Bedi A, Dines J, Warren RF, Dines DM (2010) Massive tears of the rotator cuff. J Bone Joint Surg Am 92:1894–1908CrossRefGoogle Scholar
  8. Benson RT, McDonnell SM, Knowles HJ, Rees JL, Carr AJ, Hulley PA (2010) Tendinopathy and tears of the rotator cuff are associated with hypoxia and apoptosis. J Bone Joint Surg Br 92:448–453CrossRefGoogle Scholar
  9. Bolster DR, Vary TC, Kimball SR, Jefferson LS (2004) Leucine regulates translation initiation in rat skeletal muscle via enhanced eIF4G phosphorylation. J Nutr 134:1704–1710CrossRefGoogle Scholar
  10. Cofield RH, Parvizi J, Hoffmeyer PJ, Lanzer WL, Ilstrup DM, Rowland CM (2001) Surgical repair of chronic rotator cuff tears. A prospective long-term study. J Bone Joint Surg Am 83-A:71–77CrossRefGoogle Scholar
  11. Cruz B, Gomes-Marcondes MC (2014) Leucine-rich diet supplementation modulates foetal muscle protein metabolism impaired by Walker-256 tumour. Reprod Biol Endocrinol 12:2CrossRefGoogle Scholar
  12. Drummond MJ, Dreyer HC, Fry CS, Glynn EL, Rasmussen BB (2009) Nutritional and contractile regulation of human skeletal muscle protein synthesis and mTORC1 signaling. J Appl Physiol (1985) 106:1374–1384CrossRefGoogle Scholar
  13. Fuchs B, Weishaupt D, Zanetti M, Hodler J, Gerber C (1999) Fatty degeneration of the muscles of the rotator cuff: assessment by computed tomography versus magnetic resonance imaging. J Shoulder Elb Surg 8:599–605CrossRefGoogle Scholar
  14. Furuno K, Goodman MN, Goldberg AL (1990) Role of different proteolytic systems in the degradation of muscle proteins during denervation atrophy. J Biol Chem 265:8550–8557Google Scholar
  15. Galatz LM, Ball CM, Teefey SA, Middleton WD, Yamaguchi K (2004) The outcome and repair integrity of completely arthroscopically repaired large and massive rotator cuff tears. J Bone Joint Surg Am 86-A:219–224CrossRefGoogle Scholar
  16. Gerber C, Schneeberger AG, Hoppeler H, Meyer DC (2007) Correlation of atrophy and fatty infiltration on strength and integrity of rotator cuff repairs: a study in thirteen patients. J Shoulder Elb Surg 16:691–696CrossRefGoogle Scholar
  17. Gladstone JN, Bishop JY, Lo IK, Flatow EL (2007) Fatty infiltration and atrophy of the rotator cuff do not improve after rotator cuff repair and correlate with poor functional outcome. Am J Sports Med 35:719–728CrossRefGoogle Scholar
  18. Gomes MD, Lecker SH, Jagoe RT, Navon A, Goldberg AL (2001) Atrogin-1, a muscle-specific F-box protein highly expressed during muscle atrophy. Proc Natl Acad Sci U S A 98:14440–14445CrossRefGoogle Scholar
  19. Goutallier D, Postel JM, Gleyze P, Leguilloux P, Van Driessche S (2003) Influence of cuff muscle fatty degeneration on anatomic and functional outcomes after simple suture of full-thickness tears. J Shoulder Elb Surg 12:550–554CrossRefGoogle Scholar
  20. Hornberger TA, Sukhija KB, Chien S (2006) Regulation of mTOR by mechanically induced signaling events in skeletal muscle. Cell Cycle 5:1391–1396CrossRefGoogle Scholar
  21. Kim HM, Galatz LM, Lim C, Havlioglu N, Thomopoulos S (2012) The effect of tear size and nerve injury on rotator cuff muscle fatty degeneration in a rodent animal model. J Shoulder Elb Surg 21:847–858CrossRefGoogle Scholar
  22. Kim RJ, Hah YS, Sung CM, Kang JR, Park HB (2014) Do antioxidants inhibit oxidative-stress-induced autophagy of tenofibroblasts? J Orthop Res 32:937–943CrossRefGoogle Scholar
  23. Koopman R, Verdijk L, Manders RJ, Gijsen AP, Gorselink M, Pijpers E, Wagenmakers AJ, van Loon LJ (2006) Co-ingestion of protein and leucine stimulates muscle protein synthesis rates to the same extent in young and elderly lean men. Am J Clin Nutr 84:623–632CrossRefGoogle Scholar
  24. Liu X, Joshi SK, Samagh SP, Dang YX, Laron D, Lovett DH, Bodine SC, Kim HT, Feeley BT (2012) Evaluation of Akt/mTOR activity in muscle atrophy after rotator cuff tears in a rat model. J Orthop Res 30:1440–1446CrossRefGoogle Scholar
  25. Liu X, Manzano G, Kim HT, Feeley BT (2011) A rat model of massive rotator cuff tears. J Orthop Res 29:588–595CrossRefGoogle Scholar
  26. Maher A, Leigh W, Brick M, Young S, Caughey M (2017) Causes of pain and loss of function in rotator cuff disease: analysis of 1383 cases. ANZ J Surg 87:488–492CrossRefGoogle Scholar
  27. Mammucari C, Milan G, Romanello V, Masiero E, Rudolf R, Del Piccolo P, Burden SJ, Di Lisi R, Sandri C, Zhao J, Goldberg AL, Schiaffino S, Sandri M (2007) FoxO3 controls autophagy in skeletal muscle in vivo. Cell Metab 6:458–471CrossRefGoogle Scholar
  28. Mammucari C, Schiaffino S, Sandri M (2008) Downstream of Akt: FoxO3 and mTOR in the regulation of autophagy in skeletal muscle. Autophagy 4:524–526CrossRefGoogle Scholar
  29. Mizushima N, Yamamoto A, Matsui M, Yoshimori T, Ohsumi Y (2004) In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker. Mol Biol Cell 15:1101–1111CrossRefGoogle Scholar
  30. Morag Y, Jacobson JA, Miller B, De Maeseneer M, Girish G, Jamadar D (2006) MR imaging of rotator cuff injury: what the clinician needs to know. Radiographics 26:1045–1065CrossRefGoogle Scholar
  31. Pereira MG, Baptista IL, Carlassara EO, Moriscot AS, Aoki MS, Miyabara EH (2014) Leucine supplementation improves skeletal muscle regeneration after cryolesion in rats. PLoS One 9:e85283CrossRefGoogle Scholar
  32. Pereira MG, Silva MT, da Cunha FM, Moriscot AS, Aoki MS, Miyabara EH (2015) Leucine supplementation improves regeneration of skeletal muscles from old rats. Exp Gerontol 72:269–277CrossRefGoogle Scholar
  33. Reed SA, Sandesara PB, Senf SM, Judge AR (2012) Inhibition of FoxO transcriptional activity prevents muscle fiber atrophy during cachexia and induces hypertrophy. FASEB J 26:987–1000CrossRefGoogle Scholar
  34. Rom O, Reznick AZ (2016) The role of E3 ubiquitin-ligases MuRF-1 and MAFbx in loss of skeletal muscle mass. Free Radic Biol Med 98:218–230CrossRefGoogle Scholar
  35. Sandri M (2010) Autophagy in skeletal muscle. FEBS Lett 584:1411–1416CrossRefGoogle Scholar
  36. Schoenfeld BJ (2012) Does exercise-induced muscle damage play a role in skeletal muscle hypertrophy? J Strength Cond Res 26:1441–1453CrossRefGoogle Scholar
  37. Senf SM, Sandesara PB, Reed SA, Judge AR (2011) p300 Acetyltransferase activity differentially regulates the localization and activity of the FOXO homologues in skeletal muscle. Am J Physiol Cell Physiol 300:C1490–C1501CrossRefGoogle Scholar
  38. Sevivas N, Teixeira FG, Portugal R, Araújo L, Carriço LF, Ferreira N, Vieira da Silva M, Espregueira-Mendes J, Anjo S, Manadas B, Sousa N, Salgado AJ (2016) Mesenchymal stem cell secretome: a potential tool for the prevention of muscle degenerative changes associated with chronic rotator cuff tears. Am J Sports Med 45:179–188Google Scholar
  39. Sevivas N, Ferreira N, Andrade R, Moreira P, Portugal R, Alves D, Vieira da Silva M, Sousa N, Salgado AJ, Espregueira-Mendes J (2017) Reverse shoulder arthroplasty for irreparable massive rotator cuff tears: a systematic review with meta-analysis and meta-regression. J Shoulder Elb Surg 26:e265–e277CrossRefGoogle Scholar
  40. Shen PH, Lien SB, Shen HC, Lee CH, Wu SS, Lin LC (2008) Long-term functional outcomes after repair of rotator cuff tears correlated with atrophy of the supraspinatus muscles on magnetic resonance images. J Shoulder Elb Surg 17:1S–7SCrossRefGoogle Scholar
  41. Tracy K, Macleod KF (2007) Regulation of mitochondrial integrity, autophagy and cell survival by BNIP3. Autophagy 3:616–619CrossRefGoogle Scholar
  42. Viana LR, Gomes-Marcondes MC (2013) Leucine-rich diet improves the serum amino acid profile and body composition of fetuses from tumor-bearing pregnant mice. Biol Reprod 88:121CrossRefGoogle Scholar
  43. Williams GR Jr, Rockwood CA Jr, Bigliani LU, Iannotti JP, Stanwood W (2004) Rotator cuff tears: why do we repair them? J Bone Joint Surg Am 86-A:2764–2776CrossRefGoogle Scholar
  44. Wu M, Falasca M, Blough ER (2011) Akt/protein kinase B in skeletal muscle physiology and pathology. J Cell Physiol 226:29–36CrossRefGoogle Scholar
  45. Zhao J, Brault JJ, Schild A, Cao P, Sandri M, Schiaffino S, Lecker SH, Goldberg AL (2007) FoxO3 coordinately activates protein degradation by the autophagic/lysosomal and proteasomal pathways in atrophying muscle cells. Cell Metab 6:472–483CrossRefGoogle Scholar
  46. Zhao J, Brault JJ, Schild A, Goldberg AL (2008) Coordinate activation of autophagy and the proteasome pathway by FoxO transcription factor. Autophagy 4:378–380CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Rongzong Zheng
    • 1
  • Shuming Huang
    • 1
    Email author
  • Junkun Zhu
    • 2
  • Wei Lin
    • 1
  • Huan Xu
    • 1
  • Xiang Zheng
    • 1
  1. 1.Department of Orthopaedic SurgeryZhejiang University Lishui Hospital, The Fifth Affiliated Hospital of Wenzhou Medical University, Lishui Central HospitalLishuiPeople’s Republic of China
  2. 2.Department of Orthopaedic RehabilitationZhejiang University Lishui Hospital, The Fifth Affiliated Hospital of Wenzhou Medical University, Lishui Central HospitalLishuiPeople’s Republic of China

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