Activated Protein Phosphatase 2A Disrupts Nutrient Sensing Balance Between Mechanistic Target of Rapamycin Complex 1 and Adenosine Monophosphate-Activated Protein Kinase, Causing Sarcopenia in Alcohol-Associated Liver Disease

Abstract

Background and aims: Despite the high clinical significance of sarcopenia in alcohol-associated cirrhosis, there are currently no effective therapies because the underlying mechanisms are poorly understood. We determined the mechanisms of ethanol-induced impaired phosphorylation of mechanistic target of rapamycin complex 1 (mTORC1) and adenosine monophosphate-activated protein kinase (AMPK) with consequent dysregulated skeletal muscle protein homeostasis (balance between protein synthesis and breakdown).

Approach and results: Differentiated murine myotubes, gastrocnemius muscle from mice with loss and gain of function of regulatory genes following ethanol treatment, and skeletal muscle from patients with alcohol-associated cirrhosis were used. Ethanol increases skeletal muscle autophagy by dephosphorylating mTORC1, circumventing the classical kinase regulation by protein kinase B (Akt). Concurrently and paradoxically, ethanol exposure results in dephosphorylation and inhibition of AMPK, an activator of autophagy and inhibitor of mTORC1 signaling. However, AMPK remains inactive with ethanol exposure despite lower cellular and tissue adenosine triphosphate, indicating a "pseudofed" state. We identified protein phosphatase (PP) 2A as a key mediator of ethanol-induced signaling and functional perturbations using loss and gain of function studies. Ethanol impairs binding of endogenous inhibitor of PP2A to PP2A, resulting in methylation and targeting of PP2A to cause dephosphorylation of mTORC1 and AMPK. Activity of phosphoinositide 3-kinase-γ (PI3Kγ), a negative regulator of PP2A, was decreased in response to ethanol. Ethanol-induced molecular and phenotypic perturbations in wild-type mice were observed in PI3Kγ^-/- mice even at baseline. Importantly, overexpressing kinase-active PI3Kγ but not the kinase-dead mutant reversed ethanol-induced molecular perturbations.

Conclusions: Our study describes the mechanistic underpinnings for ethanol-mediated dysregulation of protein homeostasis by PP2A that leads to sarcopenia with a potential for therapeutic approaches by targeting the PI3Kγ-PP2A axis.

Fig. 1.. Ethanol causes simultaneous reduction in protein synthesis and increased autophagy in myotubes and mouse muscle.

A. Representative immunoblots and densitometry (entire lane) for puromycin incorporation in C2C12 myotubes treated with 100mM ethanol for varying time points. B. Representative immunoblots and densitometry of LC3II lipidation, Beclin1 and p62 in myotubes treated with and without 100mM ethanol and/or chloroquine n=3 biological replicates). C. Representative immunoblots and densitometry of LC3 lipidation, Beclin1 and p62 in the gastrocnemius muscle of ethanol or pair-fed mice . Data: mean±SD. *p<0.05; **p<0.01; ***p<0.001 vs. untreated control myotubes (ANOVA) or pair-fed mice (unpaired Student’s ‘t’ test). E:ethanol-treated; EF:ethanol-fed mice (n=4); PF:pair-fed mice (n=6); UnT:untreated myotubes.

Fig. 2.. Ethanol impairs regulatory signaling in myotubes and skeletal muscle from mice and human subjects.

A,B. Representative immunoblots and densitometry of mTOR phosphorylation and mTORC1 signaling in ethanol-treated C2C12 myotubes and gastrocnemius muscle of ethanol or pair-fed mice. C-E. Representative immunoblots and densitometry of phosphorylated AMPK in ethanol-treated myotubes, muscle from ethanol or pair-fed mice and skeletal muscle from alcoholic cirrhosis and controls. F. ATP content in myotubes, mouse or human skeletal muscle. All data expressed as mean±SD from n=3 biological replicates of myotubes, n=4 in PF and n=6 EF mice; human cirrhotics and controls n=5 each. *p<0.05; **p<0.01; ***p<0.001 vs. untreated control myotubes (ANOVA) or pair-fed mice/control subjects (unpaired Student’s ‘t’ test). CIR:alcoholic cirrhotics; CTL:control subjects, E, EtOH:ethanol; EF:ethanol-fed mice; PF:pair-fed mice; UnT:untreated myotubes.

Fig. 3.. Ethanol-mediated increase in PP2A activity dephosphorylates specific signaling phosphoproteins.

A.PP2A activity-fold change in myotubes and mouse muscle. B.Representative immunoblots and densitometry of mTOR phosphorylation and mTORC1 signaling response to fostriecin in ethanol-treated myotubes. C.Representative immunoblots and densitometry of phosphorylated AMPK with and without fostriecin. D.Representative immunoblots and densitometry of mTORC1 activation and signaling in ethanol-treated myotubes transfected with shPP2A or scrambled construct. E.Representative immunoblots and densitometry of phosphorylation of AMPK in ethanol-treated myotubes transfected with shPP2A or scrambled construct. F.Protein synthesis in ethanol-treated myotubes transfected with shPP2A. G.Representative immunoblots densitometry of autophagy markers in myotubes transfected with shPP2A or scrambled construct. H.Representative photomicrographs of myotubes transfected with GFP-LC3 treated with ethanol and lysosomal inhibitor, chloroquine. Bars graphs show number of myotubes with ≥5 punctae. Data: mean±SD from n=3 biological replicates for myotubes; quantification of punctae from at least 100 myotubes from 5 independent slides. and n=4 PF and n=6 EF mice. *p<0.05; **p<0.01; ***p<0.001 vs. untreated control myotubes (ANOVA) or pair-fed mice (unpaired Student’s ‘t’ test). E:ethanol-treated myotubes; EF:ethanol-fed mice; PF:Pair-fed mice; Scr:scrambled construct; UnT:untreated myotubes.

Fig. 4.. Ethanol activates PP2A via inhibitory I2PP2A and targets specific signaling molecules via the B56 regulatory subunit in myotubes and muscle tissue from mice and human subjects.

A-C.Representative immunoblots and densitometry of methylated and phosphorylated PP2A in myotubes and skeletal muscle from mice and human subjects. D-F.Immunoprecipitate of AMPK and mTOR immunoblotted for PP2A regulatory subunit, B56 in myotubes and skeletal muscle from mice and human subjects. Data: mean±SD from n=3 biological replicates for myotubes; n=4 PF and n=6 for EF mice and n=5 each for human subjects. **p<0.01; ***p<0.001 vs. untreated control myotubes (ANOVA) or pair-fed mice/control subjects (unpaired Student’s ‘t’ test). CIR:cirrhosis patients; CTL:control subjects; E:ethanol-treated myotubes; EF:ethanol-fed mice; PF:pair-fed mice; UnT:untreated myotubes.

Fig. 5.. Ethanol increases PP2A by inhibition of the kinase domain of PI3KY in myotubes and skeletal muscle from mice and human subjects.

A-B.Immunoprecipitate of I2PP2A, from myotubes and gastrocnemius muscle from mice, probed for PP2A Catalytic subunit. C-D.Immunoblots of phosphorylated and total I2PP2A from skeletal muscle from mice and human subjects. E-F.Representative thin layer chromatograms and densitometry of phosphatidyl-inositol-3-phosphate as a measure of PI3KY activity in myotubes and gastrocnemius muscle from mice. Data: mean±SD from n=3 biological replicates for myotubes; n=4 PF and n=6 for EF mice and n=5 each for human subjects. *p<0.05; **p<0.01; ***p<0.001 vs. untreated control myotubes (ANOVA) or pair-fed mice/control subjects (unpaired Student’s ‘t’ test). CIR:cirrhosis patients; CTL:control subjects; E:ethanol-treated myotubes; EF:ethanol-fed mice; PF:pair-fed mice; PIP:phosphatidyl inositol phosphate; Ori:origin; UnT:untreated myotubes.

Fig. 6.. Ethanol-induced increase in PP2A activity and its consequences are mediated via PI3KY.

A.PP2A activity in PI3KY-overexpressing myotubes. B-F.Representative immunoblots and densitometry of mTOR phosphorylation and mTORC1 signaling, phosphorylated AMPK, protein synthesis, autophagy flux, and myotube size (representative photomicrographs and diameter) in PI3KY-overexpressing myotubes. G.Immunoprecipitate of I2PP2A immunoblotted for PP2A Catalytic subunit; immunoprecipitates of AMPK and mTOR immunoblotted for PP2A regulatory subunit, B56; immunoprecipitate of PP2A regulatory subunit, B56 immunoblotted for AMPK and mTOR. H,I. Representative immunoblots of mTORC1 signaling and autophagy flux in muscle from ethanol and pair-fed PI3KY^+/+ and PI3KY^−/− female mice. J. Fractional (FSR) and total (TSR) muscle protein synthesis rates in female ethanol and pair-fed PI3KY^+/+ and PI3KY^−/− female mice. Data: mean±SD from n=3 biological replicates for myotubes; n=4 PF and n=5 for EF mice (each with and without colchicine) and n=5 each for human subjects. *p<0.05; **p<0.01; ***p<0.001 vs. untreated control myotubes (ANOVA) or pair-fed mice (unpaired Student’s ‘t’ test). E:ethanol-treated myotubes; EF:ethanol-fed mice; PF:pair-fed mice; UnT:untreated myotubes. All immunoprecipitates from myotubes.

Fig. 7.

Schematic model of ethanol mediated PI3KY-PP2A axis with phosphatase dependent regulation of regulatory kinases.