| Biochem J. 2003 March 15; 370(Pt 3): 935–943. doi: 10.1042/BJ20021614. | PMCID: PMC1223222 |
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. Stefania Filosa, Annalisa Fico, Francesca Paglialunga, Marco Balestrieri, Almudena Crooke, Pasquale Verde, Paolo Abrescia, José M Bautista, and Giuseppe Martini IGB Adriano Buzzati Traverso CNR, Via G. Marconi 12, 80125 Napoli, Italy. filosa@iigb.na.cnr.it 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. Full Text The Full Text of this article is available as a PDF (260K). These references are in PubMed. This may not be the complete list of references from this article. - Hwang, C; Sinskey, AJ; Lodish, HF. Oxidized redox state of glutathione in the endoplasmic reticulum. Science. 1992 Sep 11;257(5076):1496–1502. [PubMed]
- Mareni, C; Gaetani, GF. NADP+ and NADPH in glucose-6-phosphate dehydrogenase-deficient erythrocytes under oxidative stimulation. Biochim Biophys Acta. 1976 Jun 8;430(3):395–398. [PubMed]
- Pandolfi, PP; Sonati, F; Rivi, R; Mason, P; Grosveld, F; Luzzatto, L. Targeted disruption of the housekeeping gene encoding glucose 6-phosphate dehydrogenase (G6PD): G6PD is dispensable for pentose synthesis but essential for defense against oxidative stress. EMBO J. 1995 Nov 1;14(21):5209–5215. [PubMed]
- Longo, Letizia; Vanegas, Olga Camacho; Patel, Meghavi; Rosti, Vittorio; Li, Haiqing; Waka, John; Merghoub, Taha; Pandolfi, Pier Paolo; Notaro, Rosario; Manova, Katia; Luzzatto, Lucio. Maternally transmitted severe glucose 6-phosphate dehydrogenase deficiency is an embryonic lethal. EMBO J. 2002 Aug 15;21(16):4229–4239. [PubMed]
- Zollo, M; D'Urso, M; Schlessinger, D; Chen, EY. Sequence of mouse glucose-6-phosphate dehydrogenase cDNA. DNA Seq. 1993;3(5):319–322. [PubMed]
- Chomczynski, P; Sacchi, N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. [PubMed]
- Gómez-Gallego, F; Garrido-Pertierra, A; Mason, PJ; Bautista, JM. Unproductive folding of the human G6PD-deficient variant A-. FASEB J. 1996 Jan;10(1):153–158. [PubMed]
- Smith, NC; Dunnett, M; Mills, PC. Simultaneous quantitation of oxidised and reduced glutathione in equine biological fluids by reversed-phase high-performance liquid chromatography using electrochemical detection. J Chromatogr B Biomed Appl. 1995 Nov 3;673(1):35–41. [PubMed]
- Shi, ZZ; Osei-Frimpong, J; Kala, G; Kala, SV; Barrios, RJ; Habib, GM; Lukin, DJ; Danney, CM; Matzuk, MM; Lieberman, MW. Glutathione synthesis is essential for mouse development but not for cell growth in culture. Proc Natl Acad Sci U S A. 2000 May 9;97(10):5101–5106. [PubMed]
- Reitzer, LJ; Wice, BM; Kennell, D. Evidence that glutamine, not sugar, is the major energy source for cultured HeLa cells. J Biol Chem. 1979 Apr 25;254(8):2669–2676. [PubMed]
- Delicado, E; Torres, M; Miras-Portugal, MT. Effects of insulin on glucose transporters and metabolic patterns in Harding-Passey melanoma cells. Cancer Res. 1986 Aug;46(8):3762–3767. [PubMed]
- Ohno, S; Payne, HW; Morrison, M; Beutler, E. Hexose-6-phosphate dehydrogenase found in human liver. Science. 1966 Aug 26;153(739):1015–1016. [PubMed]
- Mason, PJ; Stevens, D; Diez, A; Knight, SW; Scopes, DA; Vulliamy, TJ. Human hexose-6-phosphate dehydrogenase (glucose 1-dehydrogenase) encoded at 1p36: coding sequence and expression. Blood Cells Mol Dis. 1999 Feb;25(1):30–37. [PubMed]
- Martini, G; Ursini, MV. A new lease of life for an old enzyme. Bioessays. 1996 Aug;18(8):631–637. [PubMed]
- Naylor, CE; Rowland, P; Basak, AK; Gover, S; Mason, PJ; Bautista, JM; Vulliamy, TJ; Luzzatto, L; Adams, MJ. Glucose 6-phosphate dehydrogenase mutations causing enzyme deficiency in a model of the tertiary structure of the human enzyme. Blood. 1996 Apr 1;87(7):2974–2982. [PubMed]
- Au, SW; Gover, S; Lam, VM; Adams, MJ. Human glucose-6-phosphate dehydrogenase: the crystal structure reveals a structural NADP(+) molecule and provides insights into enzyme deficiency. Structure. 2000 Mar 15;8(3):293–303. [PubMed]
- Cohen, P; Rosemeyer, MA. Human glucose-6-phosphate dehydrogenase: purification of the erythrocyte enzyme and the influence of ions on its activity. Eur J Biochem. 1969 Mar;8(1):1–7. [PubMed]
- Babalola, AO; Beetlestone, JG; Luzzatto, L. Genetic variants of human erythrocyte glucose-6-phosphate dehydrogenase. Kinetic and thermodynamic parameters of variants A, B, and A- in relation to quaternary structure. J Biol Chem. 1976 May 25;251(10):2993–3002. [PubMed]
- Hendriksen, PJ; Hoogerbrugge, JW; Baarends, WM; de Boer, P; Vreeburg, JT; Vos, EA; van der Lende, T; Grootegoed, JA. Testis-specific expression of a functional retroposon encoding glucose-6-phosphate dehydrogenase in the mouse. Genomics. 1997 May 1;41(3):350–359. [PubMed]
- Lee, CY; Chasalow, F; Lee, SM; Lewis, S; Johnson, FM. A null mutation of cytoplasmic malic enzyme in mice. Mol Cell Biochem. 1980 May 7;30(3):143–149. [PubMed]
- Kosower, NS; Kosower, EM. Diamide: an oxidant probe for thiols. Methods Enzymol. 1995;251:123–133. [PubMed]
- D'Urso, M; Mareni, C; Toniolo, D; Piscopo, M; Schlessinger, D; Luzzatto, L. Regulation of glucose 6-phosphate dehydrogenase expression in CHO-human fibroblast somatic cell hybrids. Somatic Cell Genet. 1983 Jul;9(4):429–443. [PubMed]
- Warren, ST; Zhang, F; Licameli, GR; Peters, JF. The fragile X site in somatic cell hybrids: an approach for molecular cloning of fragile sites. Science. 1987 Jul 24;237(4813):420–423. [PubMed]
- Sato, N; Iwata, S; Nakamura, K; Hori, T; Mori, K; Yodoi, J. Thiol-mediated redox regulation of apoptosis. Possible roles of cellular thiols other than glutathione in T cell apoptosis. J Immunol. 1995 Apr 1;154(7):3194–3203. [PubMed]
- Kletzien, RF; Harris, PK; Foellmi, LA. Glucose-6-phosphate dehydrogenase: a "housekeeping" enzyme subject to tissue-specific regulation by hormones, nutrients, and oxidant stress. FASEB J. 1994 Feb;8(2):174–181. [PubMed]
- Préville, X; Salvemini, F; Giraud, S; Chaufour, S; Paul, C; Stepien, G; Ursini, MV; Arrigo, AP. Mammalian small stress proteins protect against oxidative stress through their ability to increase glucose-6-phosphate dehydrogenase activity and by maintaining optimal cellular detoxifying machinery. Exp Cell Res. 1999 Feb 25;247(1):61–78. [PubMed]
- Ursini, MV; Parrella, A; Rosa, G; Salzano, S; Martini, G. Enhanced expression of glucose-6-phosphate dehydrogenase in human cells sustaining oxidative stress. Biochem J. 1997 May 1;323 (:801–806. [PubMed]
- Fratelli, Maddalena; Demol, Hans; Puype, Magda; Casagrande, Simona; Eberini, Ivano; Salmona, Mario; Bonetto, Valentina; Mengozzi, Manuela; Duffieux, Francis; Miclet, Emeric; Bachi, Angela; Vandekerckhove, Joel; Gianazza, Elisabetta; Ghezzi, Pietro. Identification by redox proteomics of glutathionylated proteins in oxidatively stressed human T lymphocytes. Proc Natl Acad Sci U S A. 2002 Mar 19;99(6):3505–3510. [PubMed]
- Cancedda, R; Ogunmola, G; Luzzatto, L. Genetic variants of human erythrocyte glucose-6-phosphate dehydrogenase. Discrete conformational states stabilized by NADP + and NADPH. Eur J Biochem. 1973 Apr 2;34(1):199–204. [PubMed]
- Scopes, DA; Bautista, JM; Naylor, CE; Adams, MJ; Mason, PJ. Amino acid substitutions at the dimer interface of human glucose-6-phosphate dehydrogenase that increase thermostability and reduce the stabilising effect of NADP. Eur J Biochem. 1998 Jan 15;251(1-2):382–388. [PubMed]
- Hirono, A; Kuhl, W; Gelbart, T; Forman, L; Fairbanks, VF; Beutler, E. Identification of the binding domain for NADP+ of human glucose-6-phosphate dehydrogenase by sequence analysis of mutants. Proc Natl Acad Sci U S A. 1989 Dec;86(24):10015–10017. [PubMed]
- Casagrande, Simona; Bonetto, Valentina; Fratelli, Maddalena; Gianazza, Elisabetta; Eberini, Ivano; Massignan, Tania; Salmona, Mario; Chang, Geng; Holmgren, Arne; Ghezzi, Pietro. Glutathionylation of human thioredoxin: a possible crosstalk between the glutathione and thioredoxin systems. Proc Natl Acad Sci U S A. 2002 Jul 23;99(15):9745–9749. [PubMed]
- Ravichandran, V; Seres, T; Moriguchi, T; Thomas, JA; Johnston, RB., Jr S-thiolation of glyceraldehyde-3-phosphate dehydrogenase induced by the phagocytosis-associated respiratory burst in blood monocytes. J Biol Chem. 1994 Oct 7;269(40):25010–25015. [PubMed]
- Moley, KH; Mueckler, MM. Glucose transport and apoptosis. Apoptosis. 2000 Apr;5(2):99–105. [PubMed]
- Vander Heiden, MG; Plas, DR; Rathmell, JC; Fox, CJ; Harris, MH; Thompson, CB. Growth factors can influence cell growth and survival through effects on glucose metabolism. Mol Cell Biol. 2001 Sep;21(17):5899–5912. [PubMed]
- Gottlob, K; Majewski, N; Kennedy, S; Kandel, E; Robey, RB; Hay, N. Inhibition of early apoptotic events by Akt/PKB is dependent on the first committed step of glycolysis and mitochondrial hexokinase. Genes Dev. 2001 Jun 1;15(11):1406–1418. [PubMed]
- Pastorino, John G; Shulga, Nataly; Hoek, Jan B. Mitochondrial binding of hexokinase II inhibits Bax-induced cytochrome c release and apoptosis. J Biol Chem. 2002 Mar 1;277(9):7610–7618. [PubMed]
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