Ginsenoside Rg1 prevents starvation-induced muscle protein degradation via regulation of AKT/mTOR/FoxO signaling in C2C12 myotubes

Abstract

Skeletal muscle atrophy is often caused by catabolic conditions including fasting, disuse, aging and chronic diseases, such as chronic obstructive pulmonary disease. Atrophy occurs when the protein degradation rate exceeds the rate of protein synthesis. Therefore, maintaining a balance between the synthesis and degradation of protein in muscle cells is a major way to prevent skeletal muscle atrophy. Ginsenoside Rg1 (Rg1) is a primary active ingredient in Panax ginseng, which is considered to be one of the most valuable herbs in traditional Chinese medicine. In the current study, Rg1 was observed to inhibit the expression of MuRF-1 and atrogin-1 in C2C12 muscle cells in a starvation model. Rg1 also activated the phosphorylation of mammalian target of rapamycin (mTOR), protein kinase B (AKT), and forkhead transcription factor O, subtypes 1 and 3a. This phosphorylation was inhibited by LY294002, a phosphatidylinositol 3-kinase inhibitor. These data suggest that Rg1 may participate in the regulation of the balance between protein synthesis and degradation, and that the function of Rg1 is associated with the AKT/mTOR/FoxO signaling pathway.

Keywords: C2C12; ginsenoside Rg1; proteolysis; ubiquitin.

Figure 1.

Effect of Rg1 on cell viability. On day 4, differentiated C2C12 myotubes were incubated for 48 h in serum-free Dulbecco's modified Eagle medium. Differences in their viability, following a MTT assay were measured vs. an untreated control. Treatment with 10⁻⁴, 10⁻³, 10⁻² and 10⁻¹ mM Rg1 for 48 h resulted in a dose-dependent enhancement of cell viability. Results are presented as mean ± standard deviation (n=5). ^#P<0.05 vs. the Con group; *P<0.05 vs. the SF group. Rg1, ginsenoside Rg1; OD, optical density; Con, control; SF, serum free.

Figure 2.

Effect of an increasing dose of Rg1 on the protein expression level of (A) MuRF1 and (B) atrogin-1 in serum-starved C2C12 cells. A significant dose-dependent suppressive effect on the expression of atrogin-1 and MuRF1 was observed following treatment with 10⁻⁴, 10⁻³ and 10⁻² mM Rg1. When 10⁻¹ mM Rg1 was used, no significant effect on the expression of atrogin-1 observed. Results are presented as means ± standard deviation, (n=6 for each group). ^#P<0.05 vs. the Con group; *P<0.05 vs. the SF group by analysis of variance and Tukey's post hoc tests. Rg1, ginsenoside Rg1; MuRF1, muscle ring finger protein; atrogin-1, muscle atrophy F-box; Con, control; SF, serum free; Actin, β-actin.

Figure 2.

Effect of an increasing dose of Rg1 on the protein expression level of (A) MuRF1 and (B) atrogin-1 in serum-starved C2C12 cells. A significant dose-dependent suppressive effect on the expression of atrogin-1 and MuRF1 was observed following treatment with 10⁻⁴, 10⁻³ and 10⁻² mM Rg1. When 10⁻¹ mM Rg1 was used, no significant effect on the expression of atrogin-1 observed. Results are presented as means ± standard deviation, (n=6 for each group). ^#P<0.05 vs. the Con group; *P<0.05 vs. the SF group by analysis of variance and Tukey's post hoc tests. Rg1, ginsenoside Rg1; MuRF1, muscle ring finger protein; atrogin-1, muscle atrophy F-box; Con, control; SF, serum free; Actin, β-actin.

Figure 3.

Rg1-induced phosphorylation of AKT in C2C12 cells over 24 h. Differentiated C2C12 cells were cultured with serum-free basal medium for 12 h prior to treatment with 10⁻⁴ mM Rg1 in the presence or absence of LY294002, a PI3K inhibitor. Cells were collected 5, 10, 30 and 60 min and 24 h later for western blot analysis of AKT phosphorylation at Ser473. The western blot analysis results are presented using representative images and the ratio of p-AKT to t-AKT levels at different time points is indicated using combined quantitative data. Results are presented as means ± standard deviation, (n=4 for each group). *P<0.01 vs. 0 min by analysis of variance and Tukey post hoc tests. AKT, protein kinase B; p-, phosphorylated; t-, total; Rg1, ginsenoside Rg1; PI3K, phosphatidylinositol 3-kinase; Ser, Serine; Actin, β-actin.

Figure 4.

Phosphorylation of FoxO1 and FoxO3a in C2C12 cells following Rg1 treatment. Levels of (A) FoxO1 and (B) FoxO3a phosphorylation in cultured C2C12 cells, incubated in the presence of 10⁻² mM Rg1 and/or the PI3K inhibitor LY294002. LY294002 was added 30 min prior to the addition of 10⁻² mM Rg1. Western blot analysis results are presented using representative images and the p-FoxO1:t-FoxO1 and p-FoxO3a:t-FoxO3a ratios are demonstrated using combined quantitative data. Results are presented as the mean ± standard deviation of four experiments. *P<0.05 vs. Rg1 group by analysis of variance and Tukey's post hoc tests. FoxO1, forkhead transcription factor O, subtype 1; FoxO3a, forkhead transcription factor O, subtype 3a; Rg1, ginsenoside Rg1; PI3K, phosphatidylinositol 3-kinase; LY294002, inhibitor of PI3K; p-, phosphorylated; t-, total; Actin, β-actin.

Figure 5.

Effect of Rg1 on the phosphorylation of AKT and mTOR in C2C12 cells. Phosphorylation of (A) AKT and (B) mTOR following 12 h culture in serum-free basal medium. Cells were incubated in the presence of 10⁻² mM Rg1 and/or the PI3K inhibitor LY294002. LY294002 was added 30 min prior to treatment with Rg1. The lysates were collected 60 min later for western blot analysis. Western blot analysis results are presented using representative images and the p-Akt:t-Akt and the p-mTOR:t-mTOR ratios are demonstrated using combined quantitative data. Results are presented as mean ± standard deviation (n=4 for each group). *P<0.05 vs. Rg1 group by analysis of variance and Tukey's post hoc tests. Rg1, ginsenoside Rg1; AKT, protein kinase B; mTOR, mammalian target of rapamycin; PI3K, phosphatidylinositol 3-kinase; LY294002, inhibitor of PI3K; p-, phosphorylated; t-, total; Actin, β-actin.