TY - JOUR
T1 - Hyperammonemia-mediated autophagy in skeletal muscle contributes to sarcopenia of cirrhosis
AU - Qiu, Jia
AU - Tsien, Cynthia
AU - Thapalaya, Samjhana
AU - Narayanan, Arvind
AU - Weihl, Conrad Chris
AU - Ching, James K.
AU - Eghtesad, Bijan
AU - Singh, Kamini
AU - Fu, Xiaoming
AU - Dubyak, George
AU - Mcdonald, Christine
AU - Almasan, Alex
AU - Hazen, Stanley L.
AU - Naga Prasad, Sathyamangla V.
AU - Dasarathy, Srinivasan
PY - 2012/10/15
Y1 - 2012/10/15
N2 - Hy-perammonemia 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 C2C12 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. C2C12 cells stably expressing GFP-LC3 or GFP-mCherry-LC3 constructs showed increased formation of mature au-tophagosomes supported by electron microscopic studies. Hyperam-monemia 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 C2C12 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 C2C12 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 sar-copenia of cirrhosis.
AB - Hy-perammonemia 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 C2C12 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. C2C12 cells stably expressing GFP-LC3 or GFP-mCherry-LC3 constructs showed increased formation of mature au-tophagosomes supported by electron microscopic studies. Hyperam-monemia 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 C2C12 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 C2C12 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 sar-copenia of cirrhosis.
KW - Autophagometer
KW - Human
KW - Tyrosine nitration
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UR - http://www.scopus.com/inward/citedby.url?scp=84867593236&partnerID=8YFLogxK
U2 - 10.1152/ajpendo.00183.2012
DO - 10.1152/ajpendo.00183.2012
M3 - Article
C2 - 22895779
AN - SCOPUS:84867593236
SN - 0193-1849
VL - 303
SP - E983-E993
JO - American Journal of Physiology - Endocrinology and Metabolism
JF - American Journal of Physiology - Endocrinology and Metabolism
IS - 8
ER -