Failure to increase glucose consumption through the pentose-phosphate pathway results in the death of glucose-6-phosphate dehydrogenase gene-deleted mouse embryonic stem cells subjected to oxidative stress.
Mouse embryonic stem (ES) glucose-6-phosphate (G6P) dehydrogenase-deleted cells ( G6pd delta), obtained by transient Cre recombinase expression in a G6pd -loxed cell line, are unable to produce G6P dehydrogenase (G6PD) protein (EC 1.1.1.42). These G6pd delta cells proliferate in vitro without special requirements but are extremely sensitive to oxidative stress. Under normal growth conditions, ES G6pd delta cells show a high ratio of NADPH to NADP(+) and a normal intracellular level of GSH. In the presence of the thiol scavenger oxidant, azodicarboxylic acid bis[dimethylamide], at concentrations lethal for G6pd delta but not for wild-type ES cells, NADPH and GSH in G6pd delta cells dramatically shift to their oxidized forms. In contrast, wild-type ES cells are able to increase rapidly and intensely the activity of the pentose-phosphate pathway in response to the oxidant. This process, mediated by the [NADPH]/[NADP(+)] ratio, does not occur in G6pd delta cells. G6PD has been generally considered essential for providing NADPH-reducing power. We now find that other reactions provide the cell with a large fraction of NADPH under non-stress conditions, whereas G6PD is the only NADPH-producing enzyme activated in response to oxidative stress, which can act as a guardian of the cell redox potential. Moreover, bacterial G6PD can substitute for the human enzyme, strongly suggesting that a relatively simple mechanism of enzyme kinetics underlies this phenomenon.
Filosa
Stefania
S
IGB Adriano Buzzati Traverso CNR, Via G. Marconi 12, 80125 Napoli, Italy. filosa@iigb.na.cnr.it
Fico
Annalisa
A
Paglialunga
Francesca
F
Balestrieri
Marco
M
Crooke
Almudena
A
Verde
Pasquale
P
Abrescia
Paolo
P
Bautista
José M
JM
Martini
Giuseppe
G
D000818:Animals; D002245:Carbon Dioxide / Q000378:metabolism; D016923:Cell Death; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D002952:Citric Acid Cycle / Q000502:physiology; D003958:Diamide / Q000494:pharmacology; D004622:Embryo, Mammalian / Q000166:cytology; D017353:Gene Deletion; D005947:Glucose / Q000378:metabolism*; D005954:Glucosephosphate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D019426:Integrases / Q000378:metabolism; D008297:Male; D051379:Mice; D009249:NADP / Q000378:metabolism; D016877:Oxidants / Q000378:metabolism; D018384:Oxidative Stress; D010427:Pentose Phosphate Pathway / Q000502:physiology*; D012313:RNA / Q000378:metabolism; D013234:Stem Cells / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014764:Viral Proteins / Q000378:metabolism
D016877:Oxidants; D013439:Sulfhydryl Reagents; D014764:Viral Proteins; D003958:Diamide; D002245:Carbon Dioxide; D009249:NADP; D012313:RNA; D005954:Glucosephosphate Dehydrogenase; C045073:Cre recombinase; D019426:Integrases; D005978:Glutathione; D005947:Glucose