FoxO1‐AMPK‐ULK1 Regulates Ethanol‐Induced Autophagy in Muscle by Enhanced ATG14 Association with the BECN1‐PIK3C3 Complex

Background Excessive alcohol (Et OH ) consumption causes an imbalance in protein metabolism. Et OH impairs protein synthesis in C2C12 myoblasts via a FoxO1‐ AMPK ‐ TSC 2‐ mTORC 1 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 PIK 3C3 complex that is regulated by FoxO1‐ AMPK . Methods C2C12 myoblasts were incubated with Et OH 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 si RNA s and chemical inhibitors. Results Incubation of C2C12 myoblasts with 100 mM Et OH increased the autophagy markers LC 3B‐ II and ATG 7, whereas levels of SQSTM 1/p62 decreased. The lysosomal inhibitor bafilomycin A1 caused a similar response, although there was no additive effect when combined with Et OH . Et OH altered ULK 1 S555 and S757 phosphorylation in a time‐ and AMPK ‐dependent manner. The activation of AMPK and ULK 1 was associated with increased BECN 1 (S93, S14) and PIK 3C3/ VPS 34 (S164) phosphorylation as well as increased total ATG 14 and PIK 3C3. These changes promoted formation of the ATG 14‐ AMBRA 1‐ BECN 1‐ PIK 3C3 proautophagy complex that is important in autophagosome formation. Et OH ‐induced changes were not associated with increased production of PtdIns3P, which may be due to enhanced PIK 3C3 complex binding with 14‐3‐3θ. Reduction of AMPK using si RNA suppressed the stimulatory effect of Et OH on BECN 1 S93, BECN 1 S14, and PIK 3C3 S164 phosphorylation in a time‐dependent manner. Likewise, knockdown of AMPK or chemical inhibition of FoxO1 attenuated phosphorylation of ULK 1 at both residues. Knockdown of ULK 1 or BECN 1 antagonized the effect of Et OH on LC 3B‐ II , SQSTM 1, and ATG 7 protein expression. Conclusions Et OH ‐induced autophagy is mediated through changes in phosphorylation and interaction of various PIK 3C3 complex components. This, in turn, is regulated either directly via FoxO1‐ AMPK or indirectly via the FoxO1‐ AMPK ‐ ULK 1 signaling cascade in a mTORC 1‐independent or mTORC 1‐dependent manner.