title: FoxO1-AMPK-ULK1 Regulates Ethanol-Induced Autophagy in Muscle by Enhanced ATG14 Association with the BECN1-PIK3C3 Complex.
abstract: BACKGROUND
  Excessive alcohol (EtOH) consumption causes an imbalance in protein metabolism. EtOH impairs protein synthesis in C2C12 myoblasts via a FoxO1-AMPK-TSC2-mTORC1 pathway and also induces protein degradation. As the underlying regulatory signaling cascades for these processes are currently poorly defined, we tested the hypothesis that alcohol-induced autophagy is mediated via activation of the PIK3C3 complex that is regulated by FoxO1-AMPK.
  
  
  METHODS
  C2C12 myoblasts were incubated with EtOH for various periods of time, and autophagy pathway-related proteins were assessed by Western blotting and immunoprecipitation. Expression of targeted genes was suppressed using electroporation of specific siRNAs and chemical inhibitors.
  
  
  RESULTS
  Incubation of C2C12 myoblasts with 100 mM EtOH increased the autophagy markers LC3B-II and ATG7, whereas levels of SQSTM1/p62 decreased. The lysosomal inhibitor bafilomycin A1 caused a similar response, although there was no additive effect when combined with EtOH. EtOH altered ULK1 S555 and S757 phosphorylation in a time- and AMPK-dependent manner. The activation of AMPK and ULK1 was associated with increased BECN1 (S93, S14) and PIK3C3/VPS34 (S164) phosphorylation as well as increased total ATG14 and PIK3C3. These changes promoted formation of the ATG14-AMBRA1-BECN1-PIK3C3 proautophagy complex that is important in autophagosome formation. EtOH-induced changes were not associated with increased production of PtdIns3P, which may be due to enhanced PIK3C3 complex binding with 14-3-3θ. Reduction of AMPK using siRNA suppressed the stimulatory effect of EtOH on BECN1 S93, BECN1 S14, and PIK3C3 S164 phosphorylation in a time-dependent manner. Likewise, knockdown of AMPK or chemical inhibition of FoxO1 attenuated phosphorylation of ULK1 at both residues. Knockdown of ULK1 or BECN1 antagonized the effect of EtOH on LC3B-II, SQSTM1, and ATG7 protein expression.
  
  
  CONCLUSIONS
  EtOH-induced autophagy is mediated through changes in phosphorylation and interaction of various PIK3C3 complex components. This, in turn, is regulated either directly via FoxO1-AMPK or indirectly via the FoxO1-AMPK-ULK1 signaling cascade in a mTORC1-independent or mTORC1-dependent manner.
authors.affiliation: Department of Cellular and Molecular Physiology, Penn State College of Medicine, Hershey, Pennsylvania.
authors.forename: Ly Q
authors.initials: LQ
authors.lastname: Hong-Brown
authors.affiliation: Department of Cellular and Molecular Physiology, Penn State College of Medicine, Hershey, Pennsylvania.
authors.forename: C Randell
authors.initials: CR
authors.lastname: Brown
authors.affiliation: Department of Cellular and Molecular Physiology, Penn State College of Medicine, Hershey, Pennsylvania.
authors.forename: Maithili
authors.initials: M
authors.lastname: Navaratnarajah
authors.affiliation: Department of Cellular and Molecular Physiology, Penn State College of Medicine, Hershey, Pennsylvania.
authors.forename: Charles H
authors.identifier: http://orcid.org/0000-0002-3260-7363
authors.initials: CH
authors.lastname: Lang
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000502:physiology*; D000818:Animals; D001343:Autophagy / Q000187:drug effects / Q000502:physiology; D000071189:Autophagy-Related Protein-1 Homolog / Q000502:physiology*; D000071183:Autophagy-Related Proteins / Q000378:metabolism*; D000071186:Beclin-1 / Q000378:metabolism*; D002460:Cell Line; D058546:Class III Phosphatidylinositol 3-Kinases; D000431:Ethanol / Q000633:toxicity*; D000071161:Forkhead Box Protein O1 / Q000502:physiology*; D051379:Mice; D032446:Myoblasts / Q000187:drug effects / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D033921:Vesicular Transport Proteins / Q000378:metabolism*
chemical_list: C561107:Atg14 protein, mouse; D000071183:Autophagy-Related Proteins; D000071186:Beclin-1; C115010:Becn1 protein, mouse; D000071161:Forkhead Box Protein O1; C467200:Foxo1 protein, mouse; D033921:Vesicular Transport Proteins; D000431:Ethanol; D019869:Phosphatidylinositol 3-Kinases; D058546:Class III Phosphatidylinositol 3-Kinases; C000611846:PIK3C3 protein, mouse; D000071189:Autophagy-Related Protein-1 Homolog; C112444:Ulk1 protein, mouse; D055372:AMP-Activated Protein Kinases
keywords: Alcohol; Autophagy; Class III Phosphatidylinositol 3-Kinase; FoxO1-AMPK; Skeletal Muscle