Modulation of mitochondrial bioenergetics in a skeletal muscle cell line model of mitochondrial toxicity.
Mitochondrial toxicity is increasingly being implicated as a contributing factor to many xenobiotic-induced organ toxicities, including skeletal muscle toxicity. This has necessitated the need for predictive in vitro models that are able to sensitively detect mitochondrial toxicity of chemical entities early in the research and development process. One such cell model involves substituting galactose for glucose in the culture media. Since cells cultured in galactose are unable to generate sufficient ATP from glycolysis they are forced to rely on mitochondrial oxidative phosphorylation for ATP generation and consequently are more sensitive to mitochondrial perturbation than cells grown in glucose. The aim of this study was to characterise cellular growth, bioenergetics and mitochondrial toxicity of the L6 rat skeletal muscle cell line cultured in either high glucose or galactose media. L6 myoblasts proliferated more slowly when cultured in galactose media, although they maintained similar levels of ATP. Galactose cultured L6 cells were significantly more sensitive to classical mitochondrial toxicants than glucose-cultured cells, confirming the cells had adapted to galactose media. Analysis of bioenergetic function with the XF Seahorse extracellular flux analyser demonstrated that oxygen consumption rate (OCR) was significantly increased whereas extracellular acidification rate (ECAR), a measure of glycolysis, was decreased in cells grown in galactose. Mitochondria operated closer to state 3 respiration and had a lower mitochondrial membrane potential and basal mitochondrial O2 (•-) level compared to cells in the glucose model. An antimycin A (AA) dose response revealed that there was no difference in the sensitivity of OCR to AA inhibition between glucose and galactose cells. Importantly, cells in glucose were able to up-regulate glycolysis, while galactose cells were not. These results confirm that L6 cells are able to adapt to growth in a galactose media model and are consequently more susceptible to mitochondrial toxicants.
Department of Cardiovascular Sciences, University of Leicester, Glenfield Hospital, Leicester LE3 9QP, UK.
William
W
Dott
Syngenta Ltd., Jealott's Hill, UK.
Pratibha
P
Mistry
Syngenta Ltd., Jealott's Hill, UK.
Jayne
J
Wright
MRC Toxicology Unit, University of Leicester, Leicester, UK.
Kelvin
K
Cain
Department of Cardiovascular Sciences, University of Leicester, Glenfield Hospital, Leicester LE3 9QP, UK.
Karl E
KE
Herbert
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000968:Antimycin A / Q000494:pharmacology; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D004734:Energy Metabolism; D005690:Galactose / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism*; D056945:Hep G2 Cells; D006801:Humans; D008931:Mitochondria, Muscle / Q000378:metabolism*; D008954:Models, Biological; D018482:Muscle, Skeletal / Q000378:metabolism*; D010101:Oxygen Consumption; D051381:Rats
D000968:Antimycin A; D000255:Adenosine Triphosphate; D005947:Glucose; D005690:Galactose
AA, antimycin A; ANT, adenine nucleotide translocase; CPD, cumulative population doublings; ECAR, extracellular acidification rate; ETC, electron transport chain; Extracellular flux analysis; FCCP, Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; FSC, forward scatter; Galactose; Mitochondria; O2•–, superoxide; OCR, oxygen consumption rate; OXPHOS, oxidative phosphorylation; Oligo, oligomycin; PD, population doublings; PPP, pentose phosphate pathway; RCR, respiratory control ratio; SSC, side scatter; Skeletal muscle toxicity; TCA, tricarboxylic acid cycle; UCPs, uncoupling proteins; XF, extracellular flux