NMR-Based Metabolomic Analysis for the Effects of α-Ketoglutarate Supplementation on C2C12 Myoblasts in Different Energy States

Abstract

α-Ketoglutarate (AKG) is attracting much attention from researchers owing to its beneficial effects on anti-aging and cancer suppression, and, more recently, in nutritional supplements. Given that glucose is the main source of energy to maintain normal physiological functions of skeletal muscle, the effects of AKG supplementation for improving muscle performance are closely related to the glucose level in skeletal muscle. The differences of AKG-induced effects in skeletal muscle between two states of normal energy and energy deficiency are unclear. Furthermore, AKG-induced metabolic changes in skeletal muscles in different energy states also remain elusive. Here, we assessed the effects of AKG supplementation on mouse C2C12 myoblast cells cultured both in normal medium (Nor cells) and in low-glucose medium (Low cells), which were used to mimic two states of normal energy and energy deficiency, respectively. We further performed NMR-based metabolomic analysis to address AKG-induced metabolic changes in Nor and Low cells. AKG supplementation significantly promoted the proliferation and differentiation of cells in the two energy states through glutamine metabolism, oxidative stress, and energy metabolism. Under normal culture conditions, AKG up-regulated the intracellular glutamine level, changed the cellular energy status, and maintained the antioxidant capacity of cells. Under low-glucose culture condition, AKG served as a metabolic substrate to reduce the glutamine-dependence of cells, remarkably enhanced the antioxidant capacity of cells and significantly elevated the intracellular ATP level, thereby ensuring the normal growth and metabolism of cells in the state of energy deficiency. Our results provide a mechanistic understanding of the effects of AKG supplements on myoblasts in both normal energy and energy deficiency states. This work may be beneficial to the exploitation of AKG applications in clinical treatments and nutritional supplementations.

Keywords: AKG supplementation; biomolecular NMR; metabolic profile; metabolomics; myoblasts.

Figure 1

Proliferation and differentiation abilities of C2C12 myoblasts under the conditions of normal culture and low-glucose culture. (A) Myoblasts morphologies. (B) Cell numbers corresponding to panel A (n = 4). (C) Cell viabilities relative to Nor cells analyzed by MTS cell proliferation assay (n = 5). (D) MyoD1 expressions in myoblasts analyzed by western blot. The anti-GAPDH antibody was used to standardize the amount of protein in each lane. (E) Statistical analyses corresponding to the panels (D) (n = 4). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 2

Average 850 MHz ¹H nuclear magnetic resonance (NMR) spectra recorded on aqueous extracts derived from the Nor, Nor-A, Low, and Low-A groups of C2C12 myoblasts. (A) Comparison of the average NMR spectra of the four groups. The vertical scales were kept constant in all the ¹H NMR spectra. The water region (4.7–5.2 ppm) was removed. (B) Local amplified regions of α-Ketoglutarate (AKG) peaks. Blue/green/yellow/red line: spectral regions from the Nor/Nor-A/Low/Low-A groups. AKG, α-ketoglutarate; PC, O-phosphocholine; GPC, sn-glycero-3-phosphocholine; UDP-glucose, Uridine diphosphate glucose; GTP, sn-glycero-3-phosphocholine; NAD+, nicotinamide adenine dinucleotide; AXP, adenine mono/di/tri phosphate.

Figure 3

Multivariate analyses for ¹H NMR spectra recorded on aqueous extracts derived from C2C12 myoblasts of the Nor, Nor-A, Low, and Low-A groups. (A–C) PCA scores plots of the Low and Nor groups, the Low-A and Low groups, the Nor-A and Nor groups; (D–F) OPLS-DA scores plots of the Low and Nor groups (R2: 0.999; Q2: 0.996), the Low-A and Low groups (R2: 0.918; Q2: 0.761), the Nor-A and Nor groups (R2: 0.927; Q2: 0.838). The ellipses indicate the 95% confidence limits.

Figure 4

Relative intensities of differential metabolites identified from pairwise comparisons between the four groups of C2C12 myoblasts. (A) Low vs. Nor; (B) Low-A vs. Low; (C) Nor-A vs. Nor. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. n = 9 for each group.

Figure 5

VIP scores of important metabolites identified from pairwise comparisons between the four groups of C2C12 myoblasts. (A) Low vs. Nor.; (B) Low-A vs. Low; (C) Nor-A vs. Nor. Red/Blue font denotes increased/decreased level of the metabolite.

Figure 6

Schematic representation of significantly altered metabolic pathways identified from pairwise comparisons of Nor-A vs. Nor, Low vs. Nor, Low-A vs. Low. The up/down arrow highlights metabolites with significantly increased/decreased levels compared with the control group; dotted arrow indicates multiple biochemical reactions; solid arrow denotes a single biochemical reaction. The significantly altered metabolic pathways were identified based on the KEGG database using the MetaboAnalyst webserver.

Figure 7

Antioxidant capacities and energy states of the four groups of C2C12 myoblasts. (A) Western blot analyses of antioxidant-related proteins in myoblasts. The anti-GAPDH antibody was used to standardize the amount of protein in each lane. (B) Expressions of the catalase (CAT) protein; (C) Expressions of the superoxide dismutase (SOD) protein; (D) Total antioxidant capacities; (E) Ratios of p-AMPK/AMPK; (F) ATP content. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. n = 4 for each group.