Increased intracellular generation of reactive oxygen species in mononuclear leukocytes from patients with diabetes mellitus type 2

Summary:

Since increased cellular production of reactive oxygen species is a source of oxidative stress and thus may contribute to the development of diabetic complications, the baseline and stimulated concentrations of intracellular reactive oxygen species were measured in 16 patients with diabetes mellitus type 2 and 19 healthy control subjects. Reactive oxygen species and cytosolic calcium were monitored spectrophotometrically using dihydrorhodamine-123 and fura-2, respectively, in a suspension of mononuclear leukocytes. Measurements were made in the presence or absence of superoxide dismutase, sodium azide, genistein, or bisindolylmaleimide 1. Baseline reactive oxygen species concentrations were significantly higher in diabetic patients compared with control (p < 0.001). Activation of mononuclear leukocytes by formyl-Met-Leu-Phenylalanine and phytohemagglutinin significantly increased reactive oxygen species in diabetic patients compared with control (p < 0.05). The formyl-Met-Leu-Phenylalanine-induced increases were unchanged in the presence of superoxide dismutase, but dropped significantly in the presence of sodium azide by 80% and 73% in diabetic patients and control, respectively (each p < 0.01). The formyl-Met-Leu-Phenylalanine-induced responses were significantly inhibited by genistein and bisindolylmaleimide 1 (p < 0.01), suggesting the involvement of tyrosine kinase and protein kinase C. Resting calcium (p < 0.05) and stimulated calcium were significantly greater in diabetic patients than in control. The results show that patients with diabetes mellitus type 2 generate increased reactive oxygen species under stimulated conditions, suggesting increased risk for oxidative stress and associated complications.

Key words:

Reactive oxygen species - calcium - diabetes mellitus type 2 - mononuclear leukocytes

References

• 1 Alexiewicz J M, Kumar D, Smogorzewski M, Massry S G. Elevated cytosolic calcium and impaired proliferation of B lymphocytes in type II diabetes mellitus.  Am J Kidney Dis. 30 98-104 1997; 
• 2 Baynes J W. Role of oxidative stress in development of complications in diabetes.  Diabetes. 40 405-412 1991; 
• 3 Bokoch G M. Regulation of the human neutrophil NADPH oxidase by the Rac GTP-binding proteins.  Curr Opinion Cell Biol. 6 212-218 1994; 
• 4 Bueb J L, Gallois A, Schneider J C, Parini J P, Tschirhart E. A double-labelling fluorescent assay for concomitant measurements of [Ca²⁺]_i and O^2- production in human macrophages.  Biochim Biophys Acta. 1244 79-84 1994; 
• 5 Burdon R H. Superoxide and hydrogen peroxide in relation to mammalian cell proliferation.  Free Radical Biol Med. 4 775-794 1995; 
• 6 Chang F Y, Shaio M F. Respiratory burst activity of monocytes from patients with non-insulin-dependent diabetes mellitus.  Diabetes Res Clinl Prac. 29 121-127 1995; 
• 7 Dean R T, Fu S, Stocker R, Davies M J. Biochemistry and pathology of radical-mediated protein oxidation.  Biochem J. 324 1-18 1997; 
• 8 Fuller C J, Agil A, Lender D, Jialal I. Superoxide production and LDL oxidation by diabetic neutrophils.  J Diabet Complications. 10 206-210 1996; 
• 9 Giugliano D, Ceriello A, Paolisso G. Oxidative stress and diabetic vascular complications.  Diabet Care. 19 257-267 1996; 
• 10 Grynkiewicz G, Poenie M, Tsien R Y. A new generation of Ca²⁺ indicators with greatly improved fluorescence properties.  J Biol Chem. 360 3440-3450 1985; 
• 11 Hollan S. Free radicals in health and disease.  Haematologia. 26 177-189 1995; 
• 12 Moslen M T. Reactive oxygen species in normal physiology, cell injury and phagocytosis.  Adv Exp Med Biol. 366 17-27 1994; 
• 13 National Diabetes Data Group . Classification and diagnosis of diabetes mellitus and other categories of glucose intolerance.  Diabetes. 28 1039-1057 1979; 
• 14 Noritake M, Katsura Y, Shinomiya N, Kanatani M, Uwabe Y, Nagata N, Tsuru S. Intracellular hydrogen peroxide production by peripheral phagocytes from diabetic patients. Dissociation between polymorphonuclear leukocytes and monocytes.  Clin Exp Immunol. 88 269-274 1992; 
• 15 Oberley L W. Free radicals and diabetes.  Free Radical Biol Med. 5 113-124 1988; 
• 16 Schreck R, Rieber P, Bauerle P A. Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-kappa B transcription factor and HIV-1.  EMBO J. 10 2247-2258 1991; 
• 17 Sinclair A J, Lunec J, Girling A J, Barnett A H. Modulators of free radical activity in diabetes mellitus: role of ascorbic acid.  EXS. 62 342-352 1992; 
• 18 Stohls S J. The role of free radicals in toxicity and disease.  Journal of Basic Clin Physiol Pharmacol. 6 205-228 1995; 
• 19 Sundarean M, Yu Z X, Ferrans J, Sulciner D J, Gutkind J S, Irani K, Goldschmidt-Clermont P J, Finkel T. Regulation of reactive-oxygen species generation in fibroblasts by Rac1.  Biochem J. 318 379-382 1996; 
• 20 Tepel M, Kühnapfel S, Theilmeier G, Teupe C, Schlotmann R, Zidek W. Filling state of intracellular Ca²⁺ pools triggers trans plasma membrane Na⁺ and Ca²⁺ influx by a tyrosine kinase-dependent pathway.  J Biol Chem. 269 26239-26242 1994; 
• 21 Watanabe A, Tomino Y, Yokoyama K, Koide H. Production of hydrogen peroxide by neutrophilic polymorphonuclear leukocytes in patients with diabetic nephropathy.  J Clin Lab Anal. 7 209-213 1993; 
• 22 Wykretowicz A, Wierusz-Wysocka B, Wysocki J, Szczepanik A, Wysocki H. Impairment of the oxygen-dependent microbicidal mechanisms of polymorphonuclear neutrophils in patients with type 2 diabetes is not associated with increased susceptibility to infection.  Diabetes Res Clin Pract. 19 195-201 1993;