NMR-Based Metabolic Profiling of the Effects of α-Ketoglutarate Supplementation on Energy-Deficient C2C12 Myotubes

Abstract

Skeletal muscle is closely linked to energy metabolism, but it is inevitably deprived of energy. Cellular differentiation is an essential and energy-demanding process in skeletal muscle development. Much attention has been paid to identifying beneficial factors that promote skeletal muscle satellite cell differentiation and further understanding the underlying regulatory mechanisms. As a critical metabolic substrate or regulator, α-ketoglutarate (AKG) has been recognized as a potential nutritional supplement or therapeutic target for skeletal muscle. We have previously found beneficial effects of AKG supplementation on the proliferation of C2C12 myoblasts cultured under both normal and energy-deficient conditions and have further elucidated the underlying metabolic mechanisms. However, it remains unclear what role AKG plays in myotube formation in different energy states. In the present study, we investigated the effects of AKG supplementation on the differentiation of C2C12 myoblasts cultured in normal medium (Nor myotubes) and low glucose medium (Low myotubes) and performed NMR-based metabonomic profiling to address AKG-induced metabolic changes in both Nor and Low myotubes. Significantly, AKG supplementation promoted myotube formation and induced metabolic remodeling in myotubes under normal medium and low glucose medium, including improved energy metabolism and enhanced antioxidant capacity. Specifically, AKG mainly altered amino acid metabolism and antioxidant metabolism and upregulated glycine levels and antioxidase expression. Our results are typical for the mechanistic understanding of the effects of AKG supplementation on myotube formation in the two energy states. This study may be beneficial for further exploring the applications of AKG supplementation in sports, exercise, and therapy.

Keywords: AKG supplementation; biomolecular NMR; energy deficiency; metabolomics; myotubes.

Figure 1

Differentiation abilities of the Nor, Nor-A, Low, and Low-A groups of C2C12 myotubes. (A) Morphology of the four groups of myotubes on Days 2, 4, and 6. (B) Western blot analysis of MYH expressions in the four groups of myotubes. The anti-GAPDH antibody was used to standardize the amount of the measured protein in each lane. (C) Statistical analysis of the protein expressions shown in panel B. Statistical significance: ns, p > 0.05; *, p < 0.05; **, p < 0.01 (n = 3).

Figure 2

Average 600 MHz 1D ¹H-NMR spectra recorded on aqueous extracts from the Nor, Nor-A, Low, and Low-A groups of myotubes. The vertical scales were kept constant in all the NMR spectra. The water region (4.7–4.9 ppm) was removed. Green/blue/red/orange lines: the spectral region from the Nor/Nor-A/Low/Low-A groups. Abbreviations: AKG, α-ketoglutarate; GPC, sn-glycerol-3-phosphocholine; UDP-glucose, uridine diphosphate glucose; GTP, guanosine triphosphate; NAD+, nicotinamide adenine dinucleotide; AXP, adenine mono/di/tri phosphate.

Figure 3

Multivariate statistical analysis for 1D ¹H-NMR spectra recorded on aqueous extracts from the Nor, Nor-A, Low, and Low-A groups of myotubes. (A–C) PCA scores plots of Low and Nor, Low-A and Low, and Nor-A and Nor; (D–F) OPLS-DA scores plots; and (G–I) OPLS-DA cross-validation plots of Low vs. Nor, Low-A vs. Low, and Nor-A vs. Nor. The ellipses indicate the 95% confidence limits. The cross-validation plots were generated from permutation tests (n = 200).

Figure 4

Relative levels of differential metabolites identified from pairwise comparisons between the four groups of myotubes. (A) Low vs. Nor; (B) Low-A vs. Low; and (C) Nor-A vs. Nor. Differential metabolites were identified from Student’s t-test analysis with a criterion of p < 0.05. Statistical significances: *, p < 0.05, **, p < 0.01, ***, p < 0.001, ****, p < 0.0001.

Figure 5

VIP scores of significant metabolites identified from pairwise comparisons between the four groups of myotubes. (A) Low vs. Nor; (B) Low-A vs. Low; and (C) Nor-A vs. Nor. Significant metabolites were identified from the OPLS-DA models with a criterion of VIP > 1. Red/blue font indicates an increased/decreased level of the metabolite.

Figure 6

Expressions of proteins corrected with energy metabolism and antioxidant capacities of the four groups of myotubes. (A) Western blot analysis of energy metabolism-corrected and antioxidant-corrected proteins. The anti-β-actin antibody and anti-GAPDH proteins were used to standardize amounts of the SOD and CAT proteins, respectively. (B) ratio of p-AMPK (T172)/AMPK; (C) expressions of the catalase (CAT) protein; (D) expressions of the superoxide dismutase (SOD) protein; (E) levels of reactive oxygen species (ROS); (F) total antioxidant capacities; and (G) ATP contents. Statistical significances: ns, p > 0.05; *, p < 0.05, **, p < 0.01, ***, p < 0.001, **** p < 0.0001. n = 4 for each group.

Figure 7

Schematic representation of significantly altered metabolic pathways identified from pairwise comparisons between the four groups of myotubes. The up/down arrow highlights the increased/decreased metabolite; the dotted arrow indicates multiple biochemical reactions; and the solid arrow denotes a single biochemical reaction. Significantly altered metabolic pathways were identified using the KEGG database and the MetaboAnalyst webserver.