Time course changes in signaling pathways and protein synthesis in C ₂C₁₂ myotubes following AMPK activation by AICAR

The role of the AMP-activated kinase (AMPK) as a metabolic sensor in skeletal muscle has been far better characterized for glucose and fat metabolism than for protein metabolism. Therefore, the studies presented here were designed to examine the effects of 5-aminoimidazole-4-carboxamide-1-β-D- ribonucleoside (AICAR)-induced AMPK signaling on effector mechanisms of mRNA translation and protein synthesis in cultures of C₂C₁₂ myotubes. The findings show that, following AICAR (2 mM) treatment, AMPK phosphorylation was increased within 15 min and remained elevated throughout a 60-min time course. In association with the increase in AMPK phosphorylation, global rates of protein synthesis declined to 90, 70, and 63% of the control values at the 15-, 30-, and 60-min time points, respectively. By 60 min, polysomes disaggregated into free ribosomal subunits, suggesting an inhibition of initiation of mRNA translation. However, phosphorylation of eukaryotic elongation factor 2 was increased at 15 and 30 min but then declined to control values by 60 min, suggesting a transient inhibition of translation elongation. The decline in protein synthesis and changes in mRNA translation were associated with a repression of the mammalian target of rapamycin (mTOR) signaling pathway, as indicated by increased association of Hamartin with Tuberin, increased association of regulatory associated protein of mTOR with mTOR, and dephosphorylation of the downstream targets ribosomal protein S6 kinase-1 and eukaryotic initiation factor 4E-binding protein-1. They were also associated with activation of the MAPK signaling pathway, as indicated by increased phosphorylation of MEK1/2 and ERK1/2 and the downstream target eIF4E. Overall, the data support the conclusion that AICAR-induced AMPK activation suppresses protein synthesis through concurrent repression of mTOR signaling and activation of MAPK signaling, the combination of which modulates transient changes in the initiation and elongation phases of mRNA translation.