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Biochem J. Jun 1, 2002; 364(Pt 2): 349–359.
PMCID: PMC1222579

Metabolic control of resistance of human epithelial cells to H2O2 and NO stresses.

Abstract

The carbon flux through the oxidative branch of the pentose phosphate pathway (PPP) can be viewed as an integrator of the antioxidant mechanisms via the generation of NADPH. It could therefore be used as a control point of the cellular response to an oxidative stress. Replacement of glucose by galactose sensitized the human epithelial cell line HGT-1 to H2O2 stress. Here we demonstrate that, due to the restricted galactose flux into the PPP, the H2O2 stress led to early cellular blebbing followed by cell necrosis, these changes being associated with a fall in the NADPH/NADP+ ratio and GSH depletion. H2O2 cytotoxicity was prevented by adding 2-deoxyglucose (2dGlc). This protection was associated with an increased flow of 2-deoxyglucose 6-phosphate into the oxidative branch of the PPP together with the prevention of the NADPH/NADP+ fall and the maintenance of intracellular GSH redox homoeostasis. Inhibitors of enzyme pathways connecting the PPP to GSH recycling abolished the 2dGlc protection. In carbohydrate-free culture conditions, 2dGlc dose-dependent protective effect was paralleled by a dose-dependent influx of 2dGlc into the PPP leading to the maintenance of the intracellular redox status. By contrast, in Glc-fed cells, the PPP was not a control point of the cellular resistance to H2O2 stress as they maintained a high NADPH/NADP+ ratio. Both 2dGlc and Glc inhibited, through the maintenance of GSH redox status, NO cytotoxicity on galactose-containing Dulbecco's modified Eagle's medium (Gal-DMEM)-fed cells. 2dGlc did not prevent the fall of ATP content in NO-treated Gal-DMEM-fed cells, indicating that NO cytotoxicity was essentially due to the disruption of GSH redox homoeostasis and not to the alteration of ATP production by the mitochondrial respiratory chain. The maintenance of ATP content in NO-treated glucose-fed cells was due to their ability to derive their energy from anaerobic glycolysis. In conclusion, Gal-DMEM and 2dGlc-supplemented Gal-DMEM provide a useful system to decipher and organize into a hierarchy the targets of several stresses at the level of intact barrier epithelial cells.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Parks DA. Oxygen radicals: mediators of gastrointestinal pathophysiology. Gut. 1989 Mar;30(3):293–298. [PMC free article] [PubMed]
  • Aw TY, Rhoads CA. Glucose regulation of hydroperoxide metabolism in rat intestinal cells. Stimulation of reduced nicotinamide adenine dinucleotide phosphate supply. J Clin Invest. 1994 Dec;94(6):2426–2434. [PMC free article] [PubMed]
  • Aw TY. Determinants of intestinal detoxication of lipid hydroperoxides. Free Radic Res. 1998 Jun;28(6):637–646. [PubMed]
  • Nguyen TD, Canada AT. Modulation of human colonic T84 cell secretion by hydrogen peroxide. Biochem Pharmacol. 1994 Jan 20;47(2):403–410. [PubMed]
  • Rao RK, Baker RD, Baker SS, Gupta A, Holycross M. Oxidant-induced disruption of intestinal epithelial barrier function: role of protein tyrosine phosphorylation. Am J Physiol. 1997 Oct;273(4 Pt 1):G812–G823. [PubMed]
  • DuVall MD, Guo Y, Matalon S. Hydrogen peroxide inhibits cAMP-induced Cl- secretion across colonic epithelial cells. Am J Physiol. 1998 Nov;275(5 Pt 1):C1313–C1322. [PubMed]
  • van Gorp RM, Broers JL, Reutelingsperger CP, Bronnenberg NM, Hornstra G, van Dam-Mieras MC, Heemskerk JW. Peroxide-induced membrane blebbing in endothelial cells associated with glutathione oxidation but not apoptosis. Am J Physiol. 1999 Jul;277(1 Pt 1):C20–C28. [PubMed]
  • Orrenius S. Oxidative stress studied in intact mammalian cells. Philos Trans R Soc Lond B Biol Sci. 1985 Dec 17;311(1152):673–677. [PubMed]
  • Spragg RG, Schraufstatter IU, Hyslop PA, Hinshaw DB, Cochrane CG. Oxidant injury of cultured cells: biochemical consequences. Prog Clin Biol Res. 1987;236A:253–258. [PubMed]
  • Luperchio S, Tamir S, Tannenbaum SR. NO-induced oxidative stress and glutathione metabolism in rodent and human cells. Free Radic Biol Med. 1996;21(4):513–519. [PubMed]
  • Le Goffe C, Vallette G, Jarry A, Bou-Hanna C, Laboisse CL. The in vitro manipulation of carbohydrate metabolism: a new strategy for deciphering the cellular defence mechanisms against nitric oxide attack. Biochem J. 1999 Dec 15;344(Pt 3):643–648. [PMC free article] [PubMed]
  • Laboisse CL, Augeron C, Couturier-Turpin MH, Gespach C, Cheret AM, Potet F. Characterization of a newly established human gastric cancer cell line HGT-1 bearing histamine H2-receptors. Cancer Res. 1982 Apr;42(4):1541–1548. [PubMed]
  • Krippeit-Drews P, Kramer C, Welker S, Lang F, Ammon HP, Drews G. Interference of H2O2 with stimulus-secretion coupling in mouse pancreatic beta-cells. J Physiol. 1999 Jan 15;514(Pt 2):471–481. [PMC free article] [PubMed]
  • Tietze F. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal Biochem. 1969 Mar;27(3):502–522. [PubMed]
  • Baker MA, Cerniglia GJ, Zaman A. Microtiter plate assay for the measurement of glutathione and glutathione disulfide in large numbers of biological samples. Anal Biochem. 1990 Nov 1;190(2):360–365. [PubMed]
  • Bartos D, Vlessis AA, Muller P, Mela-Riker L, Trunkey DD. Microassay of decarboxylation reactions in cultured cells. Anal Biochem. 1993 Sep;213(2):241–244. [PubMed]
  • Zerez CR, Lee SJ, Tanaka KR. Spectrophotometric determination of oxidized and reduced pyridine nucleotides in erythrocytes using a single extraction procedure. Anal Biochem. 1987 Aug 1;164(2):367–373. [PubMed]
  • Lemaire G, Alvarez-Pachon FJ, Beuneu C, Lepoivre M, Petit JF. Differential cytostatic effects of NO donors and NO producing cells. Free Radic Biol Med. 1999 May;26(9-10):1274–1283. [PubMed]
  • Molina y Vedia L, McDonald B, Reep B, Brüne B, Di Silvio M, Billiar TR, Lapetina EG. Nitric oxide-induced S-nitrosylation of glyceraldehyde-3-phosphate dehydrogenase inhibits enzymatic activity and increases endogenous ADP-ribosylation. J Biol Chem. 1992 Dec 15;267(35):24929–24932. [PubMed]
  • Arnelle DR, Stamler JS. NO+, NO, and NO- donation by S-nitrosothiols: implications for regulation of physiological functions by S-nitrosylation and acceleration of disulfide formation. Arch Biochem Biophys. 1995 Apr 20;318(2):279–285. [PubMed]
  • Padgett CM, Whorton AR. S-nitrosoglutathione reversibly inhibits GAPDH by S-nitrosylation. Am J Physiol. 1995 Sep;269(3 Pt 1):C739–C749. [PubMed]
  • Padgett CM, Whorton AR. Glutathione redox cycle regulates nitric oxide-mediated glyceraldehyde-3-phosphate dehydrogenase inhibition. Am J Physiol. 1997 Jan;272(1 Pt 1):C99–108. [PubMed]
  • Mohr S, Hallak H, de Boitte A, Lapetina EG, Brüne B. Nitric oxide-induced S-glutathionylation and inactivation of glyceraldehyde-3-phosphate dehydrogenase. J Biol Chem. 1999 Apr 2;274(14):9427–9430. [PubMed]
  • Wink DA, Grisham MB, Mitchell JB, Ford PC. Direct and indirect effects of nitric oxide in chemical reactions relevant to biology. Methods Enzymol. 1996;268:12–31. [PubMed]
  • WICK AN, DRURY DR, NAKADA HI, WOLFE JB. Localization of the primary metabolic block produced by 2-deoxyglucose. J Biol Chem. 1957 Feb;224(2):963–969. [PubMed]
  • Suzuki M, O'Dea JD, Suzuki T, Agar NS. 2-Deoxyglucose as a substrate for glutathione regeneration in human and ruminant red blood cells. Comp Biochem Physiol B. 1983;75(2):195–197. [PubMed]
  • Woost PG, Griffin CC. Transport and metabolism of 2-deoxy-D-glucose by Rhodotorula glutinis. Biochim Biophys Acta. 1984 Apr 16;803(4):284–289. [PubMed]
  • Eggleston LV, Krebs HA. Regulation of the pentose phosphate cycle. Biochem J. 1974 Mar;138(3):425–435. [PMC free article] [PubMed]
  • Kehrer JP, Lund LG. Cellular reducing equivalents and oxidative stress. Free Radic Biol Med. 1994 Jul;17(1):65–75. [PubMed]
  • Sener A, Blachier F, Malaisse WJ. Crabtree effect in tumoral pancreatic islet cells. J Biol Chem. 1988 Feb 5;263(4):1904–1909. [PubMed]
  • Greiner EF, Guppy M, Brand K. Glucose is essential for proliferation and the glycolytic enzyme induction that provokes a transition to glycolytic energy production. J Biol Chem. 1994 Dec 16;269(50):31484–31490. [PubMed]
  • Brun T, Roche E, Kim KH, Prentki M. Glucose regulates acetyl-CoA carboxylase gene expression in a pancreatic beta-cell line (INS-1). J Biol Chem. 1993 Sep 5;268(25):18905–18911. [PubMed]
  • Roche E, Assimacopoulos-Jeannet F, Witters LA, Perruchoud B, Yaney G, Corkey B, Asfari M, Prentki M. Induction by glucose of genes coding for glycolytic enzymes in a pancreatic beta-cell line (INS-1). J Biol Chem. 1997 Jan 31;272(5):3091–3098. [PubMed]
  • Susini S, Roche E, Prentki M, Schlegel W. Glucose and glucoincretin peptides synergize to induce c-fos, c-jun, junB, zif-268, and nur-77 gene expression in pancreatic beta(INS-1) cells. FASEB J. 1998 Sep;12(12):1173–1182. [PubMed]
  • Hillgartner FB, Charron T. Glucose stimulates transcription of fatty acid synthase and malic enzyme in avian hepatocytes. Am J Physiol. 1998 Mar;274(3 Pt 1):E493–E501. [PubMed]
  • Robinson BH. Use of fibroblast and lymphoblast cultures for detection of respiratory chain defects. Methods Enzymol. 1996;264:454–464. [PubMed]
  • McKay ND, Robinson B, Brodie R, Rooke-Allen N. Glucose transport and metabolism in cultured human skin fibroblasts. Biochim Biophys Acta. 1983 Apr 5;762(2):198–204. [PubMed]
  • Clementi E, Brown GC, Feelisch M, Moncada S. Persistent inhibition of cell respiration by nitric oxide: crucial role of S-nitrosylation of mitochondrial complex I and protective action of glutathione. Proc Natl Acad Sci U S A. 1998 Jun 23;95(13):7631–7636. [PMC free article] [PubMed]
  • Clementi E, Brown GC, Foxwell N, Moncada S. On the mechanism by which vascular endothelial cells regulate their oxygen consumption. Proc Natl Acad Sci U S A. 1999 Feb 16;96(4):1559–1562. [PMC free article] [PubMed]
  • Brown GC. Nitric oxide and mitochondrial respiration. Biochim Biophys Acta. 1999 May 5;1411(2-3):351–369. [PubMed]
  • Brookes PS, Bolaños JP, Heales SJ. The assumption that nitric oxide inhibits mitochondrial ATP synthesis is correct. FEBS Lett. 1999 Mar 12;446(2-3):261–263. [PubMed]
  • Beltrán B, Orsi A, Clementi E, Moncada S. Oxidative stress and S-nitrosylation of proteins in cells. Br J Pharmacol. 2000 Mar;129(5):953–960. [PMC free article] [PubMed]

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