title: Availability of the key metabolic substrates dictates the respiratory response of cancer cells to the mitochondrial uncoupling.
abstract: Active glycolysis and glutaminolysis provide bioenergetic stability of cancer cells in physiological conditions. Under hypoxia, metabolic and mitochondrial disorders, or pharmacological treatment, a deficit of key metabolic substrates may become life-threatening to cancer cells. We analysed the effects of mitochondrial uncoupling by FCCP on the respiration of cells fed by different combinations of Glc, Gal, Gln and Pyr. In cancer PC12 and HCT116 cells, a large increase in O2 consumption rate (OCR) upon uncoupling was only seen when Gln was combined with either Glc or Pyr. Inhibition of glutaminolysis with BPTES abolished this effect. Despite the key role of Gln, addition of FCCP inhibited respiration and induced apoptosis in cells supplied with Gln alone or Gal/Gln. For all substrate combinations, amplitude of respiratory responses to FCCP did not correlate with Akt, Erk and AMPK phosphorylation, cellular ATP, and resting OCR, mitochondrial Ca(2+) or membrane potential. However, we propose that proton motive force could modulate respiratory response to FCCP by regulating mitochondrial transport of Gln and Pyr, which decreases upon mitochondrial depolarisation. As a result, an increase in respiration upon uncoupling is abolished in cells, deprived of Gln or Pyr (Glc). Unlike PC12 or HCT116 cells, mouse embryonic fibroblasts were capable of generating pronounced response to FCCP when deprived of Gln, thus exhibiting lower dependence on glutaminolysis. Overall, the differential regulation of the respiratory response to FCCP by metabolic environment suggests that mitochondrial uncoupling has a potential for substrate-specific inhibition of cell function, and can be explored for selective cancer treatment.
authors.affiliation: Biochemistry Department, University College Cork, Cavanagh Pharmacy Building, College Road, Cork, Ireland. Electronic address: a.zhdanov@ucc.ie.
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Zhdanov
authors.forename: Alicia H C
authors.initials: AH
authors.lastname: Waters
authors.forename: Anna V
authors.initials: AV
authors.lastname: Golubeva
authors.forename: Ruslan I
authors.initials: RI
authors.lastname: Dmitriev
authors.forename: Dmitri B
authors.initials: DB
authors.lastname: Papkovsky
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000235:genetics; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000737:chemistry / Q000378:metabolism*; D019069:Cell Respiration / Q000502:physiology; D004734:Energy Metabolism; D005690:Galactose / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism; D006019:Glycolysis / Q000235:genetics; D045325:HCT116 Cells; D006801:Humans; D051379:Mice; D008928:Mitochondria / Q000378:metabolism*; D009369:Neoplasms / Q000378:metabolism* / Q000473:pathology; D010085:Oxidative Phosphorylation; D010101:Oxygen Consumption / Q000502:physiology*; D016716:PC12 Cells; D019289:Pyruvic Acid / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity
chemical_list: D005973:Glutamine; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D019289:Pyruvic Acid; D005947:Glucose; D005690:Galactose
keywords: AMP-activated protein kinase; AMPK; Akt; BPTES; Cancer cell; D-galactose; D-glucose; DMEM; DMSO; Dulbecco's Modified Eagle's medium; ECA; ETC; Erk; FBS; FCCP; GLS1; GLUT; GSH; Gal; Glc; Gln; Glu; Glutaminolysis; Glycolysis; HS; L-glutamine; MEFs; Metabolic substrate; Mitochondrial respiration; NGF; OCR; OxPhos; PMF; PMPI; Pi; Pyr; ROS; RPMI; Roswell Park Memorial Institute; TMRM; Uncoupling; WM; bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide; carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; dimethyl sulphoxide; electron transport chain; extracellular acidification; fetal bovine serum; glucose transporter; glutamate; glutathione; horse serum; iO(2); inorganic phosphate; intracellular oxygen; kidney-type glutaminase; mitochondrial membrane potential; mitochondrial proton gradient; mitogen-activated protein kinase (MAPK); mouse embryonic fibroblasts; nerve growth factor; oxidative phosphorylation; oxygen consumption rate; plasma membrane potential; plasma membrane potential indicator; protein kinase B (PKB); proton motive force; pyruvate; reactive oxygen species; tetramethyl rhodamine methyl ester; working media; ΔpH; ΔΨm; ΔΨp; α-KG; α-ketoglutarate