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

Abstract

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.

Changes in the glucose-6-phosphate dehydrogenase activity in granulosa cells during follicular atresia in ewes

“…The over-expression of G6PD protects against H 2 O 2 -induced cellular death through GSH production (Salvemini et al 1999) and, by contrast, the deficiency of G6PD increases susceptibility to oxidative stress and apoptosis (Pandolfil et al 1995, Filosa et al 2003, Jiang et al 2003. This indicates that cells with high G6PD activity respond efficiently to NADPH requirements for maintaining cellular redox states, antioxidant systems, and cell survival.…”

“…G6PD as a supplier of NADPH is indispensable for replenishing GSH in oxidative stress conditions of diverse cells (Leopold & Loscalzo 2000, Filosa et al 2003, Jiang et al 2003. The over-expression of G6PD protects against H 2 O 2 -induced cellular death through GSH production (Salvemini et al 1999) and, by contrast, the deficiency of G6PD increases susceptibility to oxidative stress and apoptosis (Pandolfil et al 1995, Filosa et al 2003, Jiang et al 2003.…”

“…G6PD activity increases in growing cells and prevents cell death during oxidative stress (Tian et al 1998(Tian et al , 1999; hence, its inhibition brings about a drop in NADPH levels, a decrease in cell growth (Endo et al 1993), and an exacerbation of damage caused by free radicals (Filosa et al 2003). Likewise, it has been demonstrated that an increase in its expression provides better protection against cell death caused by H 2 O 2 , through the increase in GSH (Salvemini et al 1999, Filosa et al 2003. The aforementioned information led us to suppose that a decrease in G6PD activity must be associated with the start and progression of apoptosis in follicular atresia.…”

“…In this context, glucose-6-phosphate dehydrogenase (G6PD) has been proposed as a cell enzyme aiding in survival against oxidative stress by generating NADPH required for GSH regeneration (Leopold & Loscalzo 2000, Filosa et al 2003, Jiang et al 2003, Díaz-Flores et al 2006. G6PD activity increases in growing cells and prevents cell death during oxidative stress (Tian et al 1998(Tian et al , 1999; hence, its inhibition brings about a drop in NADPH levels, a decrease in cell growth (Endo et al 1993), and an exacerbation of damage caused by free radicals (Filosa et al 2003). Likewise, it has been demonstrated that an increase in its expression provides better protection against cell death caused by H 2 O 2 , through the increase in GSH (Salvemini et al 1999, Filosa et al 2003.…”

Secrets of the lac Operon

“…Since NADPH is the only major source of reducing equivalents for antioxidant defense, this profile would be expected to enhance antioxidant defenses. For example, elevation of glucose-6-phosphate dehydrogenase, the rate-limiting step in the pentose pathway, produces dramatic resistance to oxidative damage without changing levels of catalase or SOD [91,92], whereas reduction of this enzyme greatly enhances cellular sensitivity to oxidative stress [93]. The pentose pathway absolutely requires carbons derived from glucose, so for this essential source of cytoplasmic NADPH to function in the presence of low glucose, alternative metabolic pathways for glucose carbons must be inhibited by low glucose, which they robustly are at several rate-limiting steps.…”