Effects of a novel AMP‐mimicking prodrug on AMPK and mTORC1 activity in C2C12 cells

The beneficial effects of exercise on skeletal muscle health and function are well‐established. The intracellular signaling that mediates exercise’s effects on muscle are undoubtedly complex and are not entirely understood. Nonetheless, increasing adenosine monophosphate (AMP) levels during muscle contraction signals a declining energy charge in the cell and this certainly drives many of the beneficial adaptations of exercise. AMP‐activated protein kinase (AMPK) is one of AMP’s best‐defined targets and has been described as the cell’s master energy regulator. Because it is activated by exercise, AMPK has gained recent attention as an attractive pharmacological target in harnessing at least some of the benefits of exercise for those who cannot exercise due to old age or other disorders. The prodrug AICAR effectively activates AMPK as it is taken up into the cell and phosphorylated to form ZMP, an AMP mimetic. However, AICAR’s therapeutic potential is severely limited by its poor pharmacokinetics. Here, we describe a novel approach to ZMP delivery in a novel prodrug (Prodrug‐39; P39). We found that similar to AICAR, P39 concentrations above ~400 μM effectively achieved significant AMPK activation and glycogen depletion. Surprisingly, given AMPK’s well‐documented inhibition of the mechanistic target of rapamycin (mTORC1), P39 was also effective at stimulating p70S6k and rpS6 phosphorylation concentrations as low as 10 uM and early in the timecourse after administration of P39 at higher concentrations. Given this evidence, we conclude that Prodrug‐39 is indeed an effective AMPK activator and may also have some utility in mTOR activation at lower concentrations.