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

Abstract

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, gamma-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 gamma-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 gamma-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 mumol/l vs 840 +/- 29 mumol/l, p < 0.01). Expression of gamma-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.

Restoration of glutathione levels in vascular smooth muscle cells exposed to high glucose conditions

“…A decrease in ␥-GCS mRNA expression and increase in DNA damage accompany this reduction. The decrease observed is in agreement with published results; Yoshida et al [15] found GSH levels 77% of controls in erythrocytes from diabetic patients, Urata et al [14] reported GSH levels 59% of normal glucose after 7 d in 22 mM glucose, and Tachi et al [25] found a decrease of 20% in HVSMC exposed to 27.5 mM glucose for 10 d. The depletion of GSH may be brought about through a variety of mechanisms, including an increased breakdown of GSH by ␥-glutamyltranspeptidase, an increased export of GSSG from the cell, or a reduction in de novo synthesis through reduced activity of ␥-GCS or availability of substrate. In human vascular smooth muscle cells, there is no significant difference in the rate of GSH efflux between cells exposed to high glucose compared to those in normal glucose [25].…”

“…An aliquot (2 l) of the subsequent cDNA was added directly to the PCR mastermix containing: 100 pmoles of sense and antisense primers, 200 M dNTP, 1.5 U Taq DNA polymerase, 10ϫ PCR buffer (200 mM Tris-HCl (pH 8.4), 500 mM KCl, 2.5 mM MgCl 2 ), and 10 Ci 32 P-dCTP (NEN Life Sciences, Hounslow, UK, 370 MBq/ml). During PCR, aliquots were withdrawn at regular intervals (15,20,25,30,35,40, or 45 cycles) of amplification. An aliquot was electrophoresed in 1% agarose and radioactivity was determined on a Bio-Rad Molecular Imager system (Bio-Rad, Richmond, CA, USA).…”

“…Several studies have demonstrated the reductions in gene expression of the heavy subunit of ␥-GCS and activity of ␥-GCS in high-glucose conditions [13][14][15]. However, regulation of ␥-GCS activity at the mRNA level with respect to both the light and heavy subunits has not been investigated in high glucose conditions.…”

Increased glutathione conjugate transport: a possible compensatory protection mechanism against oxidative stress in obesity?

“…DNP-SG impairment in cells increases oxidative stress, and this is in agreement with the data reported previously. 33,43 However, the accumulation of MDA in cells in a timedependent manner was very similar in control and obese subjects, after exposure of DNP-SG-loaded erythrocytes to vanadate. Together, these findings suggest that the regulation at the glutathione S-conjugate efflux step in obesity may attenuate the susceptibility to oxidative stress of cells.…”

Effects of some analgesic anaesthetic drugs on human erythrocyte glutathione reductase: anin vitrostudy

“…[3]. Decreased glutathione levels have also been reported in several diseases, such as acquired immune deficiency syndrome [4], Parkinson's disease [5] and diabetes [6,7]. A major role of GSH in erythrocytes is prevention of hemoglobin denaturation, preserving the integrity of erythrocyte membrane sulphydryl groups and detoxification of the xenobiotics and reactive oxygen species in red blood cells [8].…”