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Cell and Tissue Research

, Volume 369, Issue 2, pp 381–394 | Cite as

FoxO3a suppression and VPS34 activity are essential to anti-atrophic effects of leucine in skeletal muscle

  • Igor L. Baptista
  • João G. Silvestre
  • William J. Silva
  • Siegfried Labeit
  • Anselmo S. MoriscotEmail author
Regular Article

Abstract

Our aim is to gain insight into the mechanisms underlying the anti-atrophic effects of leucine, namely, the way that this amino acid can restrain the up-regulation of MuRF1 and Mafbx/Atrogin-1 in muscle atrophy. Male rats received dietary leucine supplementation for 1–3 days, during which time their hind limbs were immobilized. Our results showed that leucine inhibited Forkhead Box O3 (FoxO3a) translocation to cell nuclei. In addition, leucine was able to reverse the expected reduction of FoXO3a ubiquitination caused by immobilization. Unexpectedly, leucine promoted these effects independently of the Class I PI3K/Akt pathway. Vacuolar protein sorting 34 (VPS34; a Class III PI3K) was strongly localized in nuclei after immobilization and leucine supplementation was able to prevent this effect. In experiments on cultured primary myotubes, dexamethasone led to the localization of VPS34 in the nucleus. In addition, the pharmacological inhibition of VPS34 blocked VPS34 nuclear localization and impaired the protective effect of leucine upon myotube trophicity. Finally, the pharmacological inhibition of VPS34 in primary myotubes prevented the protective effects of leucine upon MuRF1 and Mafbx/Atrogin-1 gene expression. Autophagy-related target genes were not responsive to leucine. Thus, we demonstrate that the anti-atrophic effect of leucine is dependent upon FoxO3a suppression and VPS34 activity.

Keywords

Atrophy FoxO3a Class III PI3K Skeletal muscle Atrogene expression 

Abbreviations

3MA

3-Methylaldenine

BNIP3

BCL2/adenovirus E1B 19 kDa protein-interacting protein 3

FoxO3a

Forkhead BoxO3

VPS34

Vacuolar protein sorting 34

Im

Immobilization procedure

Leu

Leucine-supplemented animal

Dexa

Dexamethasone-treated myotubes

DAPI

4′,6-Diamidino-2-phenylindole

MuRF1

Muscle-specific ring finger protein 1

PI3K

Phosphatidylinositol-3-kinase

mTOR

Mammalian target of rapamycin

Poly-UB

Poly-ubiquitin

Notes

Acknowledgments

We thank Fundação de Amparo à Pesquisa do Estado de São Paulo (Research Grant no. 2015/04090-0), Conselho Nacional de Desenvolvimento Científico e Tecnológico (Fellowship/grant no. 306915/2014-6) the Research Executive Agency (FP7 network Sarcosi no. 291834) and the Leducq Fondation.

Supplementary material

441_2017_2614_MOESM1_ESM.doc (7 mb)
ESM 1 a–i Immunodetection of FoxO1 with identification of nuclei (DAPI, blue) in cross sections from immobilized soleus. The animals had one leg immobilized for 1 day (d–f) and were supplemented with leucine (g–i). Bar (in a) 100 μm. j Real-time polymerase chain reaction analysis of MuRF1 in soleus muscle of animals supplemented with leucine (Leu), hind limb immobilized (Im) for 3 days, and concomitantly immobilized and supplemented with leucine (Im+Leu) for 3 days. k Soleus mass of animals supplemented with leucine (Leu), hind limb immobilized (Imob) for 7 days, and concomitantly immobilized and supplemented with leucine (Im+Leu) for 7 days. Bars indicate mean plus standard deviation. Letters above indicate P < 0.05 vs Control (a), P < 0.05 vs Leu group (b), P < 0.05 vs Im or Imob group (c). n = 5. (DOC 89 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Igor L. Baptista
    • 1
  • João G. Silvestre
    • 1
  • William J. Silva
    • 1
  • Siegfried Labeit
    • 2
  • Anselmo S. Moriscot
    • 1
    Email author
  1. 1.Department of Anatomy, Institute of Biomedical SciencesUniversity of Sao PauloSão PauloBrazil
  2. 2.Department of Integrative PathophysiologyMedical Faculty MannheimMannheimGermany

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