Succinate promotes skeletal muscle protein synthesis via Erk1/2 signaling pathway

Abstract

It is well known that endurance training is effective to attenuate skeletal muscle atrophy. Succinate is a typical TCA metabolite, of which exercise could dramatically increase the content. The present study aimed to investigate the effect of succinate on protein synthesis in skeletal muscle, and try to delineate the underlying mechanism. The in vitro study revealed that succinate dose‑dependently increased protein synthesis in C2C12 myotube along with the enhancement of phosphorylation levels of AKT Serine/Threonine Kinase 1(Akt), mammalian target of rapamycin, S6, eukaryotic translation initiation factor 4E, 4E binding protein 1 and forkhead box O (FoxO) 3a. Furthermore, it was demonstrated that 20 mM succinate markedly increased [Ca2+]i. Then, the phospho‑extracellular regulated kinase (Erk), ‑Akt level and the crosstalk between Erk and Akt were elevated in response to succinate. Notably, the Erk antagonist (U0126) or mTOR inhibitor (rapamycin) abolished the effect of succinate on protein synthesis. The in vivo study verified that succinate dose‑dependently increased the protein synthesis, in addition to phosphorylation levels of Erk, Akt and FoxO3a in gastrocnemius muscle. In summary, these findings demonstrated that succinate promoted skeletal muscle protein deposition via Erk/Akt signaling pathway.

Figure 1.

Succinate dose-dependently promoted protein synthesis in C2C12 myotubes. C2C12 cells were cultured in a differentiation medium for 6 days. Then C2C12 myotubes were treated with different concentrations of succinate (0, 0.05, 0.5, 1, 2 and 5 mM) for 48 h. (A) Total protein levels. (B) Puromycin incorporation detected by western blotting. (C) The statistical analyses result of the western blotting of the Puromycin. (D) The morphology of C2C12 myotubes. (E) The statistical analyses result of the C2C12 myotubes diameter. Data are presented as mean ± SEM. ^a-cSignificant differences between groups (P<0.05). β-actin served as a loading control.

Figure 2.

Akt/mTOR/FoxO cascade was involved in succinate-induced protein deposition in C2C12 myotubes. C2C12 cells were cultured for 6 days in a differentiation medium. C2C12 myotubes were then exposed to succinate (0.5 and 2 mM) for 48 h. (A) Western blot analysis of p-Akt, Akt, p-mTOR, mTOR, p-FoxO3a, FoxO3a, p-S6, S6, p-4EBP1, 4EBP1, p-eIF4e and eIF4e. (B-G) The statistical analyses results of the western blotting of the phosphorylation level of Akt, mTOR, FoxO3a, S6, 4E-BP1 and eIF4e. (H) The total protein level after C2C12 cells were co-treated with succinate and rapamycin. ^a,bSignificant differences between groups (P<0.05). *P<0.05 compared with the control. β-actin was used as a loading control.

Figure 3.

Succinate induced protein synthesis in C2C12 myotubes was mediated by Erk signaling pathway. C2C12 cells were cultured in a differentiation medium for 6 days. (A) [Ca²⁺]i in C2C12 cells in response to succinate (20 mM). (B-D) C2C12 myotubes were exposed to succinate (0.5 and 2 mM) for 48 h. Western blot analysis of p-Akt and p-Erk. (E) The binding of Erk with Akt was tested by co-Immunoprecipitation. (F and G) Erk inhibitor U0126 (10 µM) was co-treated with succinate (2 mM) for 48 h. Puromycin levels were detected. Data are presented as mean ± SEM. ^a,bSignificant differences between groups (P<0.05). *P<0.05 compared with the control or succinate group. β-actin was used as a loading control.

Figure 4.

Succinate increased protein synthesis in the skeletal muscle of C57 BL mice. Thirty 8-week-old male mice were randomly divided into three groups (n=10). Different concentrations of succinate (0, 150 and 300 mg/kg) were injected in intraperitoneal for 3 h. (A-D) Expression of p-Erk, p-Akt and p-FoxO3a were detected by western blotting. (E) Expression of p-S6 in the gastrocnemius of mice by immunoflurescence. (F and G) Puromycin incorporation levels in gastrocnemius detected by western blotting. (H) The mechanism picture that succinate promotes the protein synthesis of skeletal muscle myotubes. Data are presented as mean ± SEM. ^a,bSignificant differences between groups (P<0.05). β-actin was used as a loading control.