, Volume 38, Issue 2, pp 201–210 | Cite as

Weakened cellular scavenging activity against oxidative stress in diabetes mellitus: regulation of glutathione synthesis and efflux

  • K. Yoshida
  • J. Hirokawa
  • S. Tagami
  • Y. Kawakami
  • Y. Urata
  • T. Kondo


Glutathione functions to scavenge oxidants or xenobiotics by covalently binding them and transporting the resulting metabolites through an adenosine 5′-triphosphate-dependent transport system. It has been reported that the intracellular concentration of glutathione decreases in diabetes mellitus. In order to elucidate the physiological significance and the regulation of anti-oxidants in diabetic patients, changes in the activity of the glutathione-synthesizing enzyme, γ-glutamylcysteine synthetase, and transport of thiol [S-(2,4-dinitrophenyl)glutathione] were studied in erythrocytes from patients with non-insulin-dependent diabetes and K562 cells cultured with 27 mmol/l glucose for 7 days. The activity of γ-glutamylcysteine synthetase, the concentration of glutathione, and the thiol transport were 77%, 77% and 69%, respectively in erythrocytes from diabetic patients compared to normal control subjects. Treatment of patients with an antidiabetic agent for 6 months resulted in the restoration of γ-glutamylcysteine synthetase activity, the concentration of glutathione, and the thiol transport. A similar impairment of glutathione metabolism was observed in K562 cells with high glucose levels. The cytotoxicity by a xenobiotic (1-chloro-2,4-dinitrobenzene) was higher in K562 cells with high glucose than in control subjects (50% of inhibitory concentration. 300±24 Μmol/l vs 840±29 Μmol/l, p<0.01). Expression of γ-glutamylcysteine synthetase protein was augmented in K562 cells with high glucose, while enzymatic activity and expression of mRNA were lower than those in the control subjects. These results suggest that inactivation of glutathione synthesis and thiol transport in diabetic patients increases the sensitivity of the cells to oxidative stresses, and these changes may lead to the development of some complications in diabetes mellitus.

Key words

Glutathione γ-glutamylcysteine synthetase thiol transport erythrocytes cytotoxicity non-insulin-dependent diabetes mellitus K562 cells 



Adenosine 5′-triphosphate


non-insulin-dependent diabetes mellitus


γ-glutamylcysteinyl glycine


glutathione disulphide


γ-glutamylcysteine synthetase


messenger ribonucleic acid


deoxyribonucleic acid


50% inhibitory concentration






photostimulated luminescence


  1. 1.
    Wolf SP (1987) The potential role of oxidative stress in the diabetic complications: novel implications for theory and therapy. In: Crabbe MJC (ed) Diabetic complications, scientific and clinical aspects. Churchill Livingstone, Edinburgh, pp 167–220Google Scholar
  2. 2.
    Lyons TJ, Silvestri G, Dunn JA, Dyer DG, Baynes JW (1991) Role of glycation in modification of lens crystallins in diabetic and nondiabetic senile cataracts. Diabetes 40: 1010–1015Google Scholar
  3. 3.
    Meister A (1985) Methods for the selective modification of glutathione metabolism and study of glutathione transport. Methods Enzymol 113: 571–585Google Scholar
  4. 4.
    Richman P, Meister A (1975) Regulation of γ-glutamylcysteine synthetase by nonallosteric feedback inhibition by glutathione. J Biol Chem 250: 1422–1426Google Scholar
  5. 5.
    Yan N, Meister A (1990) Amino acid sequence of rat kidney γ-glutamylcysteine synthetase. J Biol Chem 265: 1588–1593Google Scholar
  6. 6.
    Goldwin AK, Meister A, O'Dwyer PJ, Haung CS, Hamilton TC, Anderson ME (1992) High resistance to cisplatin in human ovarial cancer cell lines is associated with marked increase of glutathione synthesis. Proc Natl Acad Sci USA 89: 1070–1074Google Scholar
  7. 7.
    Fujii S, Dale GL, Beutler E (1984) Glutathione-dependent protection against oxidative damage of the human red cell membrane. Blood 63: 1094–1101Google Scholar
  8. 8.
    Murakami K, Kondo T, Ohtsuka Y, Fujiwara Y, Shimada M, Kawakami Y (1989) Impairment of glutathione metabolism in erythrocytes from patients with diabetes mellitus. Metabolism 38: 753–758Google Scholar
  9. 9.
    Tagami S, Kondo T, Yoshida K, Hirokawa J, Ohtsuka Y, Kawakami Y (1992) Effect of insulin on impaired antioxidants in aortic endothelial cells from diabetes mellitus. Metabolism 41: 1053–1058Google Scholar
  10. 10.
    Pickett CB, Lu AYH (1989) Glutathione S-transferases: Gene structure, regulation, and biological function. Ann Rev Biochem 58: 743–764Google Scholar
  11. 11.
    Board PG (1981) Transport of glutathione S-conjugate from human erythrocytes. FEBS Lett 124: 163–165Google Scholar
  12. 12.
    Kondo T, Murao M, Taniguchi N (1982) Glutathione S-conjugate transport using inside-out vesicles from human erythrocytes. Eur J Biochem 125: 551–554Google Scholar
  13. 13.
    Awasthi YC, Misra G, Rassin DK, Srivastava SK (1983) Detoxification of xenobiotics by glutathione S-transferase in erythrocytes: the transport conjugate of glutathione and 1-chloro-2, 4-dinitrobenzene. Br J Haematol 55: 419–425Google Scholar
  14. 14.
    Ishikawa T, Sies H (1984) Cardiac transport of glutathione disulfide and S-conjugate. J Biol Chem 259: 3838–3843Google Scholar
  15. 15.
    Kobayashi K, Sogame Y, Hara H, Hayashi K (1990) Mechanism of glutathione S-conjugate transport in canalicular and basolateral rat liver plasma membrane. J Biol Chem 265: 7737–7741Google Scholar
  16. 16.
    Kondo T, Dale GL, Beutler E (1980) Glutathione transport by inside-out vesicles from human erythrocytes. Proc Natl Acad Sci USA 77: 6359–6362Google Scholar
  17. 17.
    LaBelle EF, Singh SV, Ahmad H, Wronski L, Srivastava SK, Awasthi YC (1988) A novel dinitrophenyl glutathione stimulated ATPase is present in human erythrocyte membranes. FEBS Lett 228: 53–56Google Scholar
  18. 18.
    Kondo T, Yoshida K, Urata Y, Goto S, Gasa S, Taniguchi N (1993) γ-Glutamylcysteine synthetase and active transport of glutathione S- conjugate transport are responsive to heat shock in K562 erythroid cells. J Biol Chem 268: 20366–20372Google Scholar
  19. 19.
    Kondo T, Sakai M, Isobe H et al (1991) Induction of carbonic anhydrase I isozyme precedes the globin synthesis during erythropoiesis in K562 cells. Am J Hematol 38: 201–206Google Scholar
  20. 20.
    Beutler E, West C, Blume RV (1976) The removal of leukocytes and most platelets from whole blood. J Lab Clin Med 88: 328–333Google Scholar
  21. 21.
    Kondo T, Ohtsuka Y, Shimada M et al. (1987) Erythrocyte-oxidized glutathione transport in pyrimidine 5′-nucleotidase deficiency. Am J Hematol 26:37–45Google Scholar
  22. 22.
    Lunn G, Dale GL, Beutler E (1979) Transport accounts for glutathione turnover in human erythrocytes. Blood 47: 645–650Google Scholar
  23. 23.
    Vettore L, DeMatteis MC, Zampini P (1980) A new density gradient system for the separation of human red blood cells. Am J Hematol 8: 291–296Google Scholar
  24. 24.
    Kondo T (1989) Preparation of microcapsule from human erythrocytes: use in transport experiments of glutathione and its S-conjugate. Methods Enzymol 171: 217–225Google Scholar
  25. 25.
    Wahllander A, Sies H (1979) Glutathione S-conjugate formation from 1-chloro-2, dinitrobenzene and biliary S-conjugate excretion in the perfused rat liver. Eur J Biochem 98: 441–446Google Scholar
  26. 26.
    Beutler E, Gelbart T (1986) Improved assay of the enzymes of glutathione synthesis: γ-glutamylcysteine synthetase and glutathione synthetase. Clin Chim Acta 158: 115–123Google Scholar
  27. 27.
    Beutler E (1984) A manual of biochemical methods. In: Beutler E (ed) Red cell metabolism, 3rd edn. Grune and Stratton, Orlando, pp 77–136Google Scholar
  28. 28.
    Kaplow LS (1979) Leukocyte peroxidase and nonspecific erastase. In: Melamed MR, Mullaney PF, Mendelsohn ML (eds) Flow cytometry and sorting. John Willey & Sons, New York, pp 531–545Google Scholar
  29. 29.
    Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. In: Sambrook J, Fritsch EF, Maniatis T (eds) A Laboratory manual, 2nd. Cold Spring Harbor Laboratory Press, New York, pp 7–39Google Scholar
  30. 30.
    Amemiya Y, Miyahara J (1988) Imaging plate illuminates many fields. Nature 336: 89–90Google Scholar
  31. 31.
    Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65: 55–63Google Scholar
  32. 32.
    Meister A (1983) Selective modification of glutathione metabolism. Science 220: 472–477Google Scholar
  33. 33.
    Martensson J, Steinherz R, Jain A, Meister A (1989) Glutathione ester prevents buthionine sulfoximine-induced cataracts and lens epithelial cell damage. Proc Natl Acad Sci USA 86: 8727–8731Google Scholar
  34. 34.
    Oogawara T, Kawamura N, Kitagawa Y, Taniguchi N (1992) Site-specific and random augmentation of Cu, Zn-superoxide dismutase by glycation reaction: implication of reaction oxygen species. J Biol Chem 267: 18505–18510Google Scholar
  35. 35.
    Kondo T, Murakami K, Ohtsuka Y et al. (1987) Estimation and characterization of glycosylated carbonic anhydrase I in erythrocytes from patients with diabetes mellitus. Clin Chim Acta 166: 227–236Google Scholar
  36. 36.
    Mazzanti L, Faloia E, Rabin RA (1992) Diabetes mellitus induces red blood cell plasma membrane alterations possibly affecting the aging process. Clin Med 25: 41–46Google Scholar
  37. 37.
    Schwartz RS, Madsen JW, Rybicki RC, Negel RL (1991) Oxidation of spectrin and deformability defects in diabetic erythrocytes. Diabetes 40: 701–708Google Scholar
  38. 38.
    Hasegawa H, Shigeta Y, Egawa K, Kobayashi M (1991) Impaired autophosphorylation of insulin receptors from abdominal skeletal muscles in nonobese subjects with NIDDM. Diabetes 40: 815–819Google Scholar
  39. 39.
    Brownlee M, Vlassara H, Kooney A et al. (1986) Aminoguanidine prevents diabetes-induced arterial wall protein cross-linking. Nature 232: 1629–1632Google Scholar
  40. 40.
    Oxlund H, Andreassen TT (1992) Aminoguanidine treatment reduces the increase in collagen stability of rats with experimental diabetes mellitus. Diabetologia 35: 19–25Google Scholar
  41. 41.
    Mazzanti L, Faloia E, Rabin RA (1992) Diabetes mellitus induces red blood cell plasma membrane alterations possibly affecting the aging process. Clin Med 25: 41–46Google Scholar
  42. 42.
    Schwartz RS, Madsen JW, Rybicki RC, Negel RL (1991) Oxidation of spectrin and deformability defects in diabetic erythrocytes. Diabetes 40: 701–708Google Scholar

Copyright information

© Springer-Verlag 1995

Authors and Affiliations

  • K. Yoshida
    • 1
  • J. Hirokawa
    • 1
  • S. Tagami
    • 1
  • Y. Kawakami
    • 1
  • Y. Urata
    • 2
  • T. Kondo
    • 2
  1. 1.First department of MedicineHokkaido University School of MedicineSapporoJapan
  2. 2.Department of Pathological Biochemistry, Atomic Disease InstituteNagasaki University School of MedicineNagasakiJapan

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