Alpha-ketoglutarate promotes skeletal muscle hypertrophy and protein synthesis through Akt/mTOR signaling pathways

Abstract

Skeletal muscle weight loss is accompanied by small fiber size and low protein content. Alpha-ketoglutarate (AKG) participates in protein and nitrogen metabolism. The effect of AKG on skeletal muscle hypertrophy has not yet been tested, and its underlying mechanism is yet to be determined. In this study, we demonstrated that AKG (2%) increased the gastrocnemius muscle weight and fiber diameter in mice. Our in vitro study also confirmed that AKG dose increased protein synthesis in C2C12 myotubes, which could be effectively blocked by the antagonists of Akt and mTOR. The effects of AKG on skeletal muscle protein synthesis were independent of glutamate, its metabolite. We tested the expression of GPR91 and GPR99. The result demonstrated that C2C12 cells expressed GPR91, which could be upregulated by AKG. GPR91 knockdown abolished the effect of AKG on protein synthesis but failed to inhibit protein degradation. These findings demonstrated that AKG promoted skeletal muscle hypertrophy via Akt/mTOR signaling pathway. In addition, GPR91 might be partially attributed to AKG-induced skeletal muscle protein synthesis.

Figure 1. Effects of α-ketoglutarate on skeletal muscle weight and fiber size in C57 BL mice.

Thirty 5-week-old mice were randomly divided into three groups (n = 10). Different concentrations of AKG (0%, 1% and 2%) were supplemented via drinking water for 9 weeks. (a) Serum AKG level examined by AKG assay kit. (b) Mice daily body weight gain. (c) Liver index, abdominal fat index, and gastrocnemius index. (d) Representative images of gastrocnemius muscle stained by H&E and gastrocnemius muscle fiber area distribution. The place of arrows direction represented muscle fiber. (e) Relative mRNA expression detected by qPCR. (f) Phosphorylation levels of Akt mTOR, S6, 4E-BP1, FoxO1, and the expression of MAFbx by Western blot. (g) The phospho-S6 protein expression examined by IHC in the gastrocnemius of mice. The place of arrows direction represented the expression of phospho-S6. Data are presented as mean ± SEM. Different superscripts “a”/“b”/“c” represent significant differences between groups (P < 0.05), and *means P < 0.05 compared with the control. β-actin served as a housekeeping gene.

Figure 2. AKG increased protein synthesis in C2C12 myotubes.

C2C12 cells were cultured for 6 d in a differentiation medium. C2C12 myotubes were then exposed to different concentrations of AKG (0, 0.5, and 2 mM) for 48 h. (a) Total protein levels. (b) Puromycin levels detected in C2C12 myotubes by Western blot. (c) The phosphorylation levels of mTOR, S6, 4E-BP1, eIF4E, eIF2a, and MyHC by Western blot. (d) IHC analysis for MHCII and phospho-S6 in C2C12 myotubes. Data are presented as mean±S.E.M. Different superscripts “a”/“b”/“c” represent significant differences between groups (P < 0.05), and * means P < 0.05 compared with the control. β-actin served as a housekeeping gene.

Figure 3. Glutamate failed to promote protein synthesis in C2C12 myotubes.

C2C12 cells were cultured for 6 d in a differentiation medium. C2C12 myotubes were then exposed to glutamate (2 mM) and AKG (2 mM) for 48 h. (a) Total protein levels. (b) The phosphorylation level of mTOR, S6, 4E-BP1, eIF2a, and MyHC. (c) The statistical analyses results of the Western blot of the phosphorylation level of mTOR, S6, 4E-BP1, eIF2a, and MyHC for (b). Data are presented as mean ± SEM. *means P < 0.05 compared with the control. β-actin served as a housekeeping gene control.

Figure 4. mTOR signaling pathway mediated AKG-induced protein synthesis in C2C12 myotubes.

(a) C2C12 myotubes were treated with 2 mM AKG for 1, 2, and 4 h. The expression levels of phosphorylation of mTOR, S6K, S6, 4E-BP1, eIF2a, and eIF4E were detected by Western blot. (b) C2C12 cells were cultured for 6 d in a differentiation medium. mTOR inhibitor rapamycin (1 μM) was used alone or co-treated with AKG (2 mM) for 48 h. Total protein levels. (c) The expression of puromycin determined by Western blot after C2C12 cells were co-treated with AKG and rapamycin. (d) The expression levels of phosphorylation of mTOR, S6, and 4E-BP1 detected by Western blot after C2C12 cells were co-treated with AKG and rapamycin. Data are presented as mean ± SEM. *means P < 0.05 compared with the control. β-actin served as a housekeeping gene control.

Figure 5. Akt was required to enable AKG to activate mTOR signaling pathway and promote protein synthesis in C2C12 myotubes.

(a) C2C12 myotubes were treated with AKG (2 mM) for 1, 2, and 4 h. The expression levels of MuRF1 and MAFbx and the phosphorylation of Akt FoxO1 and FoxO3a were then detected by Western blot. (b) C2C12 cells were cultured for 6 d in a differentiation medium. Akt inhibitor LY294002 (5 μM) was used alone or co-treated with AKG (2 mM) for 48 h. Total protein levels were detected. (c) The phosphorylation levels of Akt and FoxO1 by Western blot in C2C12 myotubes co-treated with AKG and LY294002. (d) The phosphorylation levels of mTOR, S6 and 4E-BP1 by Western blot in C2C12 myotubes co-treated with AKG and LY294002. Data are presented as mean ± SEM. *means P < 0.05 compared with the control. β-actin served as a housekeeping gene control.

Figure 6. Role of GPR91 in AKG-induced protein synthesis.

C2C12 cells were transfected with vector or siGPR91. C2C12 cells were cultured for 6 d in a differentiation medium and then treated with AKG (2 mM) for 48 h. (a) The expression of GPR91 and GPR99 in C2C12 cells. (b) AKG up-regulated the mRNA expression of GPR91. (c) Total protein levels in C2C12 myotubes. (d) The phosphorylation levels of mTOR, S6, and 4E-BP1 measured by Western blot. (e) The mRNA expression of MuRF1 and MAFbx by qPCR. Data are presented as mean ± SEM. Different superscripts “a”/“b” represent significant differences between groups (P < 0.05), and *means P < 0.05 compared with the control. β-actin served as a housekeeping gene control. n.d. not detectable.

Figure 7. AKG regulated the protein synthesis of the skeletal muscle of mice.

AKG (0.6 g/kg) and puromycin were co-injected for 3 h. Protein turnover associate protein expression was detected by Western blot. (a) The expression of puramycin was analyzed by Western blot. (b) The expression of MyHC and the phosphorylation levels of mTOR, S6, 4E-BP1, eIF4E, eIF2a in the gastrocnemius of mice. (c) The expression of Akt, MAFbx, and MuRF1. (d) The mRNA expression levels of protein turnover related genes were measured by qPCR. (e) The phosphorylation levels of Akt and mTOR 1 h after AKG injection detected by Western blot. Data are presented as mean ± SEM. *means P < 0.05 compared with the control. β-actin served as a housekeeping gene control.