Hyperammonemia-mediated autophagy in skeletal muscle contributes to sarcopenia of cirrhosis.
Hyperammonemia and sarcopenia (loss of skeletal muscle) are consistent abnormalities in cirrhosis and portosystemic shunting. We have shown that muscle ubiquitin-proteasome components are not increased with hyperammonemia despite sarcopenia. This suggests that an alternative mechanism of proteolysis contributes to sarcopenia in cirrhosis. We hypothesized that autophagy could be this alternative pathway since we observed increases in classic autophagy markers, increased LC3 lipidation, beclin-1 expression, and p62 degradation in immunoblots of skeletal muscle protein in cirrhotic patients. We observed similar changes in these autophagy markers in the portacaval anastamosis (PCA) rat model. To determine the mechanistic relationship between hyperammonemia and autophagy, we exposed murine C(2)C(12) myotubes to ammonium acetate. Significant increases in LC3 lipidation, beclin-1 expression, and p62 degradation occurred by 1 h, whereas autophagy gene expression (LC3, Atg5, Atg7, beclin-1) increased at 24 h. C(2)C(12) cells stably expressing GFP-LC3 or GFP-mCherry-LC3 constructs showed increased formation of mature autophagosomes supported by electron microscopic studies. Hyperammonemia also increased autophagic flux in mice, as quantified by an in vivo autophagometer. Because hyperammonemia induces nitration of proteins in astrocytes, we quantified global muscle protein nitration in cirrhotic patients, in the PCA rat, and in C(2)C(12) cells treated with ammonium acetate. Increased protein nitration was observed in all of these systems. Furthermore, colocalization of nitrated proteins with GFP-LC3-positive puncta in hyperammonemic C(2)C(12) cells suggested that autophagy is involved in degradation of nitrated proteins. These observations show that increased skeletal muscle autophagy in cirrhosis is mediated by hyperammonemia and may contribute to sarcopenia of cirrhosis.
Departments of Pathobiology, Cleveland Clinic, Cleveland, Ohio 44195, USA.
Jia
J
Qiu
Cynthia
C
Tsien
Samjhana
S
Thapalaya
Arvind
A
Narayanan
Conrad Chris
CC
Weihl
James K
JK
Ching
Bijan
B
Eghtesad
Kamini
K
Singh
Xiaoming
X
Fu
George
G
Dubyak
Christine
C
McDonald
Alex
A
Almasan
Stanley L
SL
Hazen
Sathyamangla V
SV
Naga Prasad
Srinivasan
S
Dasarathy
D000818:Animals; D001343:Autophagy / Q000502:physiology*; D002460:Cell Line; D002478:Cells, Cultured; D005455:Fluorescent Antibody Technique; D006801:Humans; D006863:Hydrogen-Ion Concentration; D022124:Hyperammonemia / Q000473:pathology*; D008103:Liver Cirrhosis / Q000473:pathology*; D008297:Male; D051379:Mice; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D009124:Muscle Proteins / Q000378:metabolism; D018482:Muscle, Skeletal / Q000473:pathology*; D011167:Portacaval Shunt, Surgical; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D012313:RNA / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D060888:Real-Time Polymerase Chain Reaction; D055948:Sarcopenia / Q000473:pathology*; D014162:Transfection; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
C480860:Map1lc3b protein, mouse; D008869:Microtubule-Associated Proteins; D009124:Muscle Proteins; C002744:3-nitrotyrosine; D014443:Tyrosine; D012313:RNA; D046988:Proteasome Endopeptidase Complex