Glyoxylic Acid, an α-Keto Acid Metabolite Derived from Glycine, Promotes Myogenesis in C2C12 Cells

Abstract

α-Keto acids may help prevent malnutrition in patients with chronic kidney disease (CKD), who consume protein-restricted diets, because they serve as amino acid sources without producing nitrogenous waste compounds. However, the physiological roles of α-keto acids, especially those derived from non-essential amino acids, remain unclear. In this study, we examined the effect of glyoxylic acid (GA), an α-keto acid metabolite derived from glycine, on myogenesis in C2C12 cells. Differentiation and mitochondrial biogenesis were used as myogenesis indicators. Treatment with GA for 6 d resulted in an increase in the expression of differentiation markers (myosin heavy chain II and myogenic regulatory factors), mitochondrial biogenesis, and intracellular amounts of amino acids (glycine, serine, and alanine) and their metabolites (citric acid and succinic acid). In addition, GA treatment suppressed the 2.5-µM dexamethasone (Dex)-induced increase in mRNA levels of ubiquitin ligases (Trim63 and Fbxo32), muscle atrophy markers. These results indicate that GA promotes myogenesis, suppresses Dex-induced muscle atrophy, and is metabolized to amino acids in muscle cells. Although further in vivo experiments are needed, GA may be a beneficial nutrient for ameliorating the loss of muscle mass, strength, and function in patients with CKD on a strict dietary protein restriction.

Keywords: C2C12 cells; glyoxylic acid; muscle atrophy; myogenesis; α-keto acid.

Figure 1

Effect of GA on the viability and differentiation of C2C12 cells. C2C12 cells grown to subconfluence in a growth medium (without GA) were further cultured in a differentiation medium with or without GA for 6 d. (a) Cell viability was measured using the neutral red assay. (b) Western blot images of MyHC II, a biomarker protein for differentiation, and β-actin, an internal control. (c–e) mRNA levels of MyHCII encoding isoforms (Myh1, Myh2, and Myh4). (f) mRNA levels of Pax7, a satellite cell marker. (g,h) mRNA levels of myogenic regulatory factors (Myod1 and Myog). mRNA levels were measured using qPCR. Values are represented as mean ± SD of three independent experiments. Data were analyzed using a one-way analysis of variance followed by Dunnett’s post hoc comparison tests. The mean value was significantly different from that of the control: ** p < 0.01, * p < 0.05.

Figure 2

Effect of GA on mitochondrial biogenesis in C2C12 cells. C2C12 cells grown to subconfluence in a growth medium (without GA) were further cultured in differentiation medium with or without GA for 6 d. (a) Relative mtDNA copy number measured using qPCR. Results are shown as fold change values compared with the results of the control group. (b) MtMP measured via Rho123 uptake. Results are shown as fold change values compared with the results of the control group. (c) mRNA levels of Cs. The mRNA levels were measured using qPCR. (d) Western blot images of CS and β-actin, the internal control. Values are presented as mean ± SD of three independent experiments. Data were analyzed using a one-way ANOVA followed by Dunnett’s post hoc comparison tests. Mean value was significantly different from that of the control: ** p < 0.01, * p < 0.05.

Figure 3

Effect of GA on the levels of mRNAs involved in mitochondrial biogenesis in C2C12 cells. C2C12 cells grown to subconfluence in a growth medium (without GA) were further cultured in a differentiation medium with or without GA for 6 d. mRNA levels of (a) Sirt1, (b) Ppargc1a, (c) Nrf1, (d) Nfe2l2 (e) Tfam (f) Tfb1m were measured using qPCR. Values are presented as mean ± SD of three independent experiments. Data were analyzed using a one-way ANOVA followed by Dunnett’s post hoc comparison tests. Mean value was significantly different from that of the control: ** p < 0.01, * p < 0.05.

Figure 4

Changes in the intracellular content of several amino acids and their metabolites caused by GA. C2C12 cells grown to subconfluence in a growth medium (without GA) were further cultured in a differentiation medium with or without GA for 6 d. Relative metabolite changes shown in the graphs were obtained using GC-MS analysis. Values are presented as mean ± SD of three independent experiments. Statistical differences were determined using Student’s t-test. Differences with ** p < 0.01 was considered significant.

Figure 5

Effect of GA and Dex on the mRNA expression levels involved in muscle atrophy in C2C12 cells. (a,b) C2C12 cells grown to subconfluence in a growth medium (without GA) were further cultured in a differentiation medium without GA for 6 d. The cells were further cultured for 1 d in a differentiation medium with or without Dex (2.5 µM) and GA (0–0.8 mM). (c,d) C2C12 cells grown to subconfluence in a growth medium (without GA) were further cultured for the indicated days in a differentiation medium with or without Dex (2.5 µM) and GA (0.8 mM). (e,f) L6 cells grown to subconfluence in a growth medium (without GA) were further cultured in a differentiation medium without GA for 6 d. The cells were further cultured for 1 d in a differentiation medium with or without Dex (2.5 µM) and GA (0.8 mM). mRNA levels of ubiquitin ligases (Trim63 and Fbxo32), markers of muscle atrophy, were measured using qPCR. Values are presented as mean ± SD of three independent experiments. (a,b,e,f) Data were analyzed using a one-way ANOVA followed by Dunnett’s post hoc comparison test. Mean value was significantly different from that of Dex: (c,d) Data were analyzed using a one-way ANOVA followed by Tukey post hoc comparison test. ** p < 0.01, * p < 0.05.