Introduction
Skeletal muscle mass is regulated by the balance between protein synthesis and degradation. Leucine, an essential amino acid, is known to activate mechanistic/mammalian target of complex1 (mTORC1) activity and enhance muscle anabolic response (Atherton et al. 2010). Recently, lower serum level of vitamin B12 (VB12) (<350pg/ml) has been reported to be associated with the incidence of sarcopenia (Choi et al. 2023). One of the factors contributing to sarcopenia is believed to be blunted skeletal muscle anabolic response to protein and amino acids intake (Cuthbertson et al. 2005, Aragon et al. 2023). However, effect of VB12 on leucine-induced anabolic signaling has not been investigated. Therefore, this study aimed to investigate the effect of VB12 on leucine-induced responses on anabolic signaling pathways.
Methods
Mouse C2C12 cells were seeded at 0.5 × 105 cells/ml in 12-well plates and cultured in DMEM containing 2% horse serum for 6 days to form myotubes. After differentiation, myotubes were cultured in serum-free medium for 1 hour, followed by incubation in amino acids and serum-free medium for 1 hour. Cells were then cultured in (1) Control (Con), (2) Leucine 1.5mM (Leu), (3) Leucine 1.5mM + VB12 10μM (Leu + Low-VB12), (4) Leucine1.5mM + VB12 100μM (Leu + High-VB12) for 30 minutes and collected (n=4/group). Western blotting was performed to evaluate the expression levels of proteins involved in muscle protein synthesis. For statistical analysis, SPSS Statistics ver.28 was used. One-way ANOVA was performed, and multiple comparisons were conducted by Tukey only when significant differences were found. Significance level was set at p < 0.05.
Results
There was a significant difference in phosphorylation / total-p70S6K expression, a downstream factor of mTORC1, between groups (p < 0.001). Multiple comparisons showed that Leu + Low-VB12 group showed significantly lower phosphorylation / total-p70S6K expression than Leu group (Leu + Low-VB12: 1.02 ± 0.08 vs Leu: 1.46 ± 0.10, p = 0.016) and there is no significant difference between Leu + Low-VB12 group and Con group (Leu + Low-VB12: 1.02 ± 0.08 vs Con: 1.00 ± 0.06, p = 0.998). On the other hand, Leu + High-VB12 group showed significantly higher phosphorylation / total-p70S6K expression compared to Con and Leu group (Leu + High-VB12: 2.02 ± 0.10 vsCon: 1.00 ± 0.06 / Leu: 1.46 ± 0.10, p < 0.001, p = 0.003, respectively). Moreover, there was a significant difference in t-4E-BP1 γ form ratio between groups (p = 0.002). Multiple comparisons showed that Leu and Leu + High-VB12 group showed significantly higher t-4E-BP1 γ form ratio than Con group (Leu: 1.20 ± 0.02 / Leu + High-VB12 1.18 ± 0.03 vs Con: 1.00 ± 0.02, p = 0.002, p = 0.006, respectively).
Conclusion
These results suggest that low-level VB12 blunt anabolic signaling but high-level VB12 enhanced leucine-induced anabolic signaling in C2C12 cells.