In Vivo Effects of AICAR and Prodrug 39 (P39) on Anabolic Pathways Following Refeeding in Skeletal Muscle

Abstract

Exercise training is known to elicit many beneficial adaptations. However, the mechanisms by which this happens are not entirely understood. During muscle contraction, increased intracellular AMP levels mediate many exercise‐induced adaptations by activating AMP‐activated protein kinase (AMPK), among other targets. AMP mimicry may, therefore, be an attractive pharmacological approach to stimulate some of exercise’s benefits. The prodrug AICAR is a well‐established AMPK activator when phosphorylated in the cell to form ZMP, an AMP mimetic. However, it has limited pharmacological potential due to poor pharmacokinetics. Prodrug 39 (P39) uses an alternative approach to deliver ZMP to the cell. The purpose of this study was to compare AMPK and P39 effects on blood glucose levels, AMPK activation, and anabolic signaling in the fasted and refed states. C57BL/6 mice were subjected to a 12 hour fast, followed by 1 hour of refeeding. Following the 12‐hour fast, blood glucose was measured, and mice were injected with saline, AICAR or P39 (400 mg/kg BW). Mice were immediately transferred to cages with food, except for a fasted saline group. After 1 hour of refeeding, food was removed from cages. Blood glucose was measured 25 minutes later, and mice were sacrificed. Gastrocnemius‐plantaris‐soleus complex (GPS) muscles were quick‐frozen for protein analysis. Fasting blood glucose levels were 80 ± 4 mg/dL prior to refeeding and were elevated to 144 ± 8 mg/dL (p<0.0001) after food reintroduction. Both AICAR and P39 caused a significant reduction in blood glucose compared to refed controls (p<0.0001, 54 ± 9, and 56 ± 10 mg/dL, respectively). In GPS muscle, AMPK phosphorylation status was unaffected by any treatment. However, ACC phosphorylation, a downstream target of AMPK, was ~3‐fold higher in AICAR‐treated animals compared to refed controls (p<0.01), with no significant effect of P39. Refeeding induced an increase in anabolic signaling, as indicated by an increase in S6 phosphorylation (~4‐fold, p<0.01) and a trend for increased eIF4E‐binding protein (4E‐BP1) phosphorylation (p = 0.1). This increase was entirely prevented in AICAR and P39‐treated animals. Overall, these results suggest that AICAR and P39 are similarly effective in lowering blood glucose and dampening anabolic signaling following refeeding. However, AICAR was more effective in activating AMPK signaling in skeletal muscle at this dose.