Low Glucose but Not Galactose Enhances Oxidative Mitochondrial Metabolism in C2C12 Myoblasts and Myotubes

Abstract

BackgroundSubstituting galactose for glucose in cell culture media has been suggested to enhance mitochondrial metabolism in a variety of cell lines. We studied the effects of carbohydrate availability on growth, differentiation and metabolism of C2C12 myoblasts and myotubes.Methodology/Principal FindingsWe measured growth rates, ability to differentiate, citrate synthase and respiratory chain activities and several parameters of mitochondrial respiration in C2C12 cells grown in media with varying carbohydrate availability (5 g/l glucose, 1 g/l glucose, 1 g/l galactose, and no added carbohydrates). C2C12 myoblasts grow more slowly without glucose irrespective of the presence of galactose, which is not consumed by the cells, and they fail to differentiate without glucose in the medium. Cells grown in a no-glucose medium (with or without galactose) have lower maximal respiration and spare respiratory capacity than cells grown in the presence of glucose. However, increasing glucose concentration above physiological levels decreases the achievable maximal respiration. C2C12 myotubes differentiated at a high glucose concentration showed higher dependency on oxidative respiration under basal conditions but had lower maximal and spare respiratory capacity when compared to cells differentiated under low glucose condition. Citrate synthase activity or mitochondrial yield were not significantly affected by changes in the available substrate concentration but a trend towards a higher respiratory chain activity was observed at reduced glucose levels.Conclusions/SignificanceOur results show that using galactose to increase oxidative metabolism may not be applicable to every cell line, and the changes in mitochondrial respiratory parameters associated with treating cells with galactose are mainly due to glucose deprivation. Moderate concentrations of glucose (1 g/l) in a growth medium are optimal for mitochondrial respiration in C2C12 cell line while supraphysiological concentrations of glucose cause mitochondrial dysfunction in C2C12 myoblasts and myotubes.

Nanoformulated copper/zinc superoxide dismutase exerts differential effects on glucose vs lipid homeostasis depending on the diet composition possibly via altered AMPK signaling

“…We used both low and high glucose media to determine the effect of Nano on mitochondrial respiration. As previously reported [30], the presence of high glucose actually reduced mitochondrial respiration as evident from a reduction in basal respiration, maximal respiration, ATP production, and spare respiratory capacity (Fig. 9A).…”

Analysis of Mammalian Cell Proliferation and Macromolecule Synthesis Using Deuterated Water and Gas Chromatography-Mass Spectrometry

“…As seen in Figure 2A, 2 H enrichment in DNA-derived deoxyribose and protein-derived alanine occurred rapidly, and while the absolute amount of labelling differed between the two molecules, the rate of rise in enrichment, referred to as the rise to plateau, was remarkably similar and occurred within 72 h. Accordingly, using non-linear least squares fitting of plots, the calculated DNA ( k = 0.044 h −1 ) and protein (0.043 h −1 ) turnover constants were very similar. The t 1/2 for protein, which represents the time taken to achieve 50% turnover, was 16.16 h. In the case of DNA labelling, the t 1/2 , which represents the cell doubling time, was calculated to be 15.69 h, which is consistent with previously published C2C12 doubling times of 15.2–19.7 h [49], as assessed using manual cell counting. The maximum level of 2 H-labeling in deoxyribose was higher than in alanine (Figure 2A), because there are more opportunities for 2 H to be incorporated into deoxyribose (through the pentose phosphate pathway, hexose phosphate isomerization) [35], compared to alanine (transamination of pyruvate) [30,31,32].…”

Inhibition of ubiquitin‐specific protease 2 causes accumulation of reactive oxygen species, mitochondria dysfunction, and intracellular ATP decrement in C2C12 myoblasts

“…Thus, the production of ATP in C2C12 cells may be highly dependent on the presence of glucose. To support this, a previous study demonstrated that C2C12 cells exhibited lower maximal respiration and drastic inhibition of proliferation under glucose‐free conditions (Elkalaf et al, ). In any case, USP2 may contribute to maintaining the supply of ATP in C2C12 cells in the presence of glucose.…”

“…In the present study, we demonstrated that USP2 contributes to the generation of sufficient ATP by OXPHOS in myoblasts. On the other hand, Usp2 KO cells exhibited impairments in proliferation and differentiation, both of which require an adequate glucose‐derived energy supply by the mitochondria (Elkalaf et al, ; Riddle et al, ). Collectively, these results suggest that USP2 might control proliferation and differentiation in myoblasts through sustained activation of mitochondria.…”