Effect of phytoecdysteroids on protein synthesis and Akt/mTOR signaling after downhill running in skeletal muscle of mice

Abstract

Phytoecdysteroids are natural plant steroids synthesized by a variety of hardy plants. Phytoecdysteroids, such as 20‐hydroxyecdysone (20E), possess anabolic properties and previous research indicates that 20E stimulates protein synthesis in cultured muscle cells and increases grip strength in young rats after 28 days of supplementation. The purpose of this study was to investigate the extent to which 20E stimulates protein synthesis and Akt/mTOR signaling in skeletal muscle after an acute bout of downhill running (DHR). Male C57BL6 mice (3–6 mo old) were assigned to four groups. Mice in the DHR groups performed an acute bout of DHR to exhaustion on a rodent treadmill at 17 m•min−1 and −20° decline. After completion of the DHR bout and recovery, mice received an oral gavage treatment of either 50 mg•kg−1 body mass (BM) of 20E (DHR + 20E; n=5) or vehicle (DHR + vehicle; n=6), and then treated daily for the next four consecutive days. Two groups that did not perform DHR, but were treated for five consecutive days with either 50 mg•kg−1 BM of 20E (No DHR + 20E; n=8) or vehicle (No DHR + vehicle; n=6), were used as controls. On the fifth day post‐DHR, mice were not treated with 20E or vehicle; however, an IP injection of puromycin (0.040 μmol•g−1 BM) was administered 30 min prior to sacrifice to assess protein synthesis. Skeletal muscles were harvested and activation of protein synthesis was assessed using the SUnSET method and Western blot and Akt/mTOR signaling activation was measured via Western blot. Mice in the DHR + vehicle group ran for 58.5 min, while mice in the DHR + 20E group ran for 57.0 min. Compared to No DHR + vehicle, DHR + vehicle increased skeletal muscle puromycin incorporation 14%, but DHR + 20E increased puromycin incorporation 32%, five days post‐DHR. Puromycin incorporation was not different between No DHR + vehicle and No DHR + 20E groups. DHR resulted in ~25% increase in phosphorylation of both Akt and p70S6K, compared to No DHR + vehicle; but there was no difference in Akt or p70S6K phosphorylation between DHR + 20E and DHR + vehicle groups, five days post‐DHR. Compared to No DHR + vehicle, DHR + vehicle increased 4EBP1 phosphorylation 35%, but DHR + 20E increased 4EBP1 phosphorylation 70%, and DHR + vehicle increased ribosomal protein S6 (rpS6) phosphorylation 40%, but DHR + 20E increased rpS6 phosphorylation 119%, five days post‐DHR. In conclusion, these findings suggest that DHR stimulates protein synthesis and activates Akt/mTOR signaling five days after DHR, but 20E treatment enhances the pattern of response to an acute bout of DHR in skeletal muscle of young mice.