Fig. 2.

Hyperammonemia induces autophagy in murine myotubes. Differentiated C₂C₁₂ murine myotubes incubated in ammonium acetate (Am.Ac) for different time points to measure autophagy response. A: cell viability showed >94% viable C₂C₁₂ murine myotubes exposed to Am.Ac. B: proteasome 20S activity assay in C₂C₁₂ murine myotubes showed no significant effect of hyperammonemia. C: representative immunoblots of p62 degradation, beclin-1 overexpression, and LC3 lipidation in response to 10 mM Am.Ac over time. D: densitometry for the blots normalized to β-actin is shown. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with controls. All experiments were done in triplicate. E: representative confocal images of immunofluorescence of green fluorescent protein (GFP)-LC3 stably transfected C₂C₁₂ murine myotubes treated with Am.Ac and controls. F: quantification of GFP-LC3⁺ cells with >5 puncta (means ± SE) showed significant increase at 4 h and beyond. *P < 0.05 and **P < 0.01 compared with controls. G: representative confocal images of time course of immunofluorescence of GFP-mCherry-LC3 tandem reporter transfected C₂C₁₂ murine myotubes treated with Am.Ac. Initial green fluorescence transforms to yellow (red + green) with activation of autophagy, and the quenching of the green fluorescence in the lysosomes results in red puncta demonstrating the autophagolyosome formation with subsequent degradation of the vesicular contents. All experiments were done in triplicate. H: quantification of yellow and red punctae in GFP-mCherry-LC3 transfected cells treated with Am.Ac. *P < 0.05 and **P < 0.01 compared with controls. I: representative electron microscopy images (×15,000 and ×40,000 magnification) of C₂C₁₂ murine myotubes treated with Am.Ac alone, chloroquine (CQ) alone, and Am.Ac and CQ for 6 h and controls. Arrows show the double-membrane-enclosed autophagosomes. J: autophagosome area as a proportion of the total cytoplasmic area was significantly higher in cells treated with Am.Ac compared with controls. Increased autophagic flux was demonstrated using CQ with Am.Ac. *P < 0.05 compared with controls; ***P < 0.01 compared with controls and P < 0.05 compared to CQ alone and Am.Ac alone. All studies were done in triplicate. MuRF1, muscle RING finger 1. K: representative immunoblots for LC3 lipidation in protein extracts from murine myotubes treated with Am.Ac alone, CQ alone, and Am.Ac and chloroquine. L: densitometry of the immunoblots showed increased autophagic flux with hyperammonemia. AA, autophagic area.