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Activation of AMPK/TSC2/PLD by alcohol regulates mTORC1 and mTORC2 assembly in C2C12 myocytes

Ly Q Hong-Brown1, C Randell Brown, Maithili Navaratnarajah

  • 1Department of Cellular and Molecular Physiology, Penn State College of Medicine, Hershey, Pennsylvania.

Alcoholism, Clinical and Experimental Research
|July 31, 2013
PubMed
Abstract

Insights

Ethanol (EtOH) increases phospholipase D (PLD) and phosphatidic acid (PA) in muscle cells, which may counteract EtOH’s negative effects. PA stimulates mTORC1 signaling and inhibits mTORC2, suggesting a feedback loop.

Area of Science:

  • Cellular signaling pathways
  • Muscle protein synthesis
  • Molecular biology

Background:

  • Ethanol (EtOH) exposure impairs muscle protein synthesis by altering mammalian target of rapamycin complex (mTORC) signaling.
  • Phospholipase D (PLD) and phosphatidic acid (PA) are known to positively regulate mTORC1, but their role in EtOH-induced signaling changes, particularly concerning mTORC2, is less understood.

Purpose of the Study:

  • To investigate the specific roles of PLD and PA in EtOH-mediated regulation of mTORC1 and mTORC2 signaling pathways in muscle cells.

Main Methods:

  • C2C12 myoblasts were treated with EtOH, and PLD activity and PA levels were measured.
  • The effects of PLD inhibition and PA addition on protein phosphorylation, including Akt and S6K1, were assessed via immunoblotting.
  • Protein-protein interactions within mTORC2 complexes were analyzed using immunoprecipitation.

Main Results:

  • EtOH exposure increased PLD activity and PA levels in C2C12 myocytes, an effect inhibited by AMPK and PI3K inhibitors.
  • PLD inhibition prevented EtOH-induced Akt phosphorylation and altered mTORC2 complex member interactions.
  • PA addition decreased Akt phosphorylation but increased S6K1 phosphorylation, indicating mTORC1 stimulation and mTORC2 suppression, potentially via a feedback mechanism.

Conclusions:

  • EtOH-induced increases in PLD and PA may partially mitigate EtOH's detrimental effects on muscle cells.
  • A signaling cascade involving PI3K, AMPK, TSC2, and PLD mediates EtOH and PA's regulation of mTORC1.
  • PA acts to stimulate mTORC1 and suppress mTORC2, establishing an mTORC1/2 feedback loop.
Keywords:
14-3-3 ProteinsEthanolPhosphatidic AcidProtein SynthesisRag GTPases

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