FIG 2.

Hyperammonemia impairs skeletal muscle ribosomal biogenesis. (A) Relative fold changes in expression of ribosomal RPL5, RPL23, and RPL32 mRNAs, comparing myotubes treated with 10 mM AmAc for 6 or 24 h with untreated (UnT) controls, measured by RT-quantitative PCR (qRT-PCR). (B) Relative fold changes in expression of ribosomal RPS9, RPS19, and RPS26 mRNAs, comparing myotubes treated with 10 mM AmAc for 6 or 24 h with untreated controls, measured by qRT-PCR. (C to E) Representative immunoblotting and densitometry of expression of ribosomal proteins RPL5, RPL23, and RPL32 from untreated C2C12 myotubes or those treated with 10 mM AmAc for 24 h (C); from the gastrocnemius muscle of portocaval anastomosis (PCA) and sham-operated, pair-fed control (Sham) rats (n = 5 each) (D); and from the skeletal muscle of patients with cirrhosis (CIR) and controls (CTL) (n = 5 each) (E). (F) Representative immunoblotting and densitometry of ribosomal biogenesis markers RPL5, RPL23, and RPL32 in untreated HEK cells and those treated with 10 mM AmAc for 6 or 24 h. (G) Representative immunoblotting and densitometry of expression of ribosomal proteins RPL5, -23, and -32 show that with 24 h of treatment with the translational inhibitor cycloheximide (CHX), there is reduced expression of ribosomal proteins, which is blocked by proteasome inhibition. However, the expression of a housekeeping gene, β-actin, did not change in response to these interventions, and the expression of a regulatable, nonhousekeeping gene (P70S6K) did not change, except with cycloheximide treatment, which resulted in lower expression levels than in untreated cells. All cellular experiments were performed in at least 3 biological replicates, and all data were normalized to β-actin and are expressed as means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (versus untreated cells or controls for all panels).