Effect of aldose reductase inhibition on glutathione redox status in erythrocytes of diabetic patients

doi:10.1016/0026-0495(94)90175-9

Metabolism

Volume 43, Issue 8, July 1994, Pages 965-968

https://doi.org/10.1016/0026-0495(94)90175-9 Get rights and content

Abstract

Diabetic patients undergo a chronic oxidative stress. This phenomenon is demonstrated by low levels of reduced glutathione (GSH) levels. The NADPH used by glutathione reductase for the reduction of oxidized glutathione (GSSG) to GSH is also used by aldose reductase for the reduction of glucose to sorbitol through the polyol pathway. The competition for NADPH could be responsible for the decreased glutathione levels found in non-insulin-dependent diabetic patients. For this purpose, we investigated the effect of polyol pathway inhibition on the glutathione redox status in these patients. We measured GSH and GSSG levels in erythrocytes of non-insulin-dependent diabetic patients (n = 15) before and after 1 week of treatment with placebo, followed by 1 week of treatment with an aldose reductase inhibitor (tolrestat 200 mg/d). We found lower GSH levels (7.7 ± 1.4 μmol/g hemoglobin [Hb]), higher GSSG levels (0.35 ± 0.09 μmol/g Hb), and lower GSH/GSSG ratios (23.9 ± 7.7) in diabetics compared with controls (n = 15; 9.8 ± 0.8 μmol/g Hb, P < .001; 0.17 ± 0.02, P < .001; and 58.3 ± 9.1, P < .001, respectively). We did not demonstrate any statistical difference after 1 week of treatment with placebo. In contrast, the treatment with tolrestat induced a significant increase in GSH (8.9 ± 0.7 μmol/g Hb, P < .01), a decrease in GSSG (0.25 ± 0.06 μmol/g Hb, P < .02), and an increase in the GSH/GSSG ratio (37.3 ± 8.4, P < .01). These data strongly support the hypothesis that the polyol pathway plays an important role in the impairment of the glutathione redox status in diabetic patients.

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Several mechanisms have been proposed for the heart dysfunction during hyperglycemia. The aim of the present in vitro study is to elucidate the role of alterations in redox homeostasis in the induction of apoptosis during hyperglycemia in H9c2 cells via dysfunction in mitochondria and polyol pathway and evaluation of the beneficial effect of cinnamic acid against the same. The H9c2 cells were incubated with 33 mM glucose for 48 h to simulate the diabetic condition. Cell injury was confirmed with a significant increase of atrial natriuretic peptide and lactate dehydrogenase release. Alterations in the innate antioxidant system, polyol pathway, mitochondrial integrity, dynamics and apoptosis were investigated. Hyperglycemic insult has significantly affected redox homeostasis via depletion of superoxide dismutase, glutathione and enhanced reactive oxygen species generation. It also caused dysregulation in mitochondrial dynamics (fusion, fission proteins), dissipation of mitochondrial transmembrane potential and increased sorbitol accumulation. Finally, apoptosis was observed with upregulation of Bax, activation of caspase-3 and downregulation of Bcl-2. Cinnamic acid cotreatment increased the innate antioxidant status, improved mitochondrial function and prevented apoptosis in H9c2 cardiomyoblasts. Moreover, this in vitro model is found to be ideal for the elucidation of mechanisms at the cellular and molecular level of any physiological, pharmacological and toxicological incidents in H9c2 cells.
Effect of oral N-acetyl cysteine supplementation in type 2 diabetic patients on intracellular glutathione content and innate immune responses to Burkholderia pseudomallei
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Diabetics have been reported to have depleted GSH levels, as well as a lower GSH ratio [14–18]. One major reason for this is an increased glucose flux though the polyol pathway due to hyperglycemia where excess cellular glucose is reduced to sorbitol by aldose reductase [19,20]. This depletes the intracellular NADPH pool, which is required to regenerate free GSH from oxidized GSH.
Type 2 diabetic patients have increased susceptibility to melioidosis, an infectious disease caused by Burkholderia pseudomallei. We had previously shown that peripheral blood mononuclear cells (PBMCs) from diabetic patients with poor glycemic control had a defective IL-12 and IFNγ response to B. pseudomallei infection, resulting in poor intracellular bacterial control. The impaired IL-12 response was due to glutathione (GSH) deficiency characterized by a low reduced to oxidized glutathione ratio (GSH ratio) and could be restored by the addition of reduced GSH to the infected cells. Our goal is to determine whether N-acetyl cysteine (NAC, a GSH pro-drug) supplementation in diabetic patients could improve their immune control of B. pseudomallei. Type 2 diabetic patients with poor glycemic control were given oral supplementation of NAC for six weeks at 1200 mg daily. Their PBMCs and subsets of immune cells showed a significant increase in free GSH concentration. However, the GSH ratio, IL-12 and IFNγ production, and intracellular bacterial killing upon ex-vivo infection did not improve. Thus, oral NAC supplementation in diabetic patients is sufficient to increase intracellular GSH content in blood cells. However, modulating the free GSH content is not sufficient to improve infection outcome as it is the GSH ratio that regulates the IL-12 response in monocytes.
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In contrast, under conditions of chronic hyperglycemia, activity of the polyol pathway increases dramatically and uses large amounts of NADPH, thus competing with the pentose pathway for scarce NADPH stores [24]. Accordingly, inhibition of aldose reductase was found to enhance GSH concentration in erythrocytes from patients with T2D [25]. The main finding of the current study was the inability of an acute improvement in blood glucose control to restore GSH.
Depletion of blood glutathione (GSH), a key antioxidant, is associated with type 1 diabetes mellitus (T1D) and contributes to the pathophysiology of diabetes complications. The aim of the current study was to determine whether acute normalization of blood glucose would restore GSH kinetics in adolescents with poorly controlled T1D. Ten 16.9 ± 1.5-year-old (SE) adolescents who had had T1D for 8.5 ± 1.9 years and were free of complications but were in poor control (hemoglobin A_1c, 9.2% ± 0.5%) received two 5-hour intravenous infusions of L-[3,3-²H₂]cysteine in the postabsorptive state on 2 separate days after blood glucose had been maintained overnight at 246 ± 24 mg/dL (hyperglycemia) or 118 ± 23 mg/dL (euglycemia) using intravenous insulin infusion. Blood GSH fractional synthesis rates were determined by mass spectrometry from ²H₂-cysteine incorporation into GSH. Neither blood GSH (551 ± 169 vs 541 ± 232 μmol/L, P = .629) nor GSH fractional synthesis rate (84% ± 30% vs 82% ± 33% d⁻¹, P = .965) was altered by the short-term change in glycemic control. This finding suggests that, in adolescents with poorly controlled T1D, either (a) blood glucose per se does not regulate GSH metabolism or (b) GSH may only respond to sustained, more chronic changes in blood glucose level.
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Hyperglycemia is known to promote free radicals production and impairment of the antioxidant systems, such as glutathione reduced form (GSH) and vitamin C [1–6].
To evaluate the relationship between oxidative stress and endothelial dysfunction (ED) in diabetic patients without clinical macrovascular complications.
In 27 type 1, 56 type 2 diabetic patients and 35 healthy controls the redox state (GSH, GSSG; enzymatic method), endothelin-1 (ET-1; ELISA) and von Willebrand factor (vWF; ELISA) plasma levels, urinary vascular endothelial growth factor (VEGF; ELISA) were measured.
Decreased GSH levels (p < 0.05, type 1 and type 2), GSH/GSSG ratio (p < 0.05 type 1, p < 0.001 type 2) and elevated vWF levels (p < 0.001, type 1 and type 2) were observed in diabetic patients in comparison with controls. A negative correlation between GSH and vWF (p < 0.02 and p < 0.001, in type 1 and type 2, respectively) and GSH and BMI (p < 0.02 in type 1 and type 2) was observed. ET-1 was positively correlated to age (p < 0.05) and diabetes duration (p < 0.03) in type 1, while vWF was correlated to systolic blood pressure (p < 0.05) in type 2 diabetic patients. Urinary VEGF was higher in type 2 (p < 0.05) in comparison with type 1 diabetic patients and was correlated to glycemia (p < 0.05) and systolic blood pressure (p < 0.05).
These data might indicate that markers of oxidative stress and ED are altered in diabetic patients without clinical macrovascular complications.
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The glutathione redox cycle plays a major role in scavenging hydrogen peroxide (H₂O₂) under physiological conditions. Recently, we demonstrated that a high glucose concentration in the culture medium reduced the level of H₂O₂ scavenging activity of human vascular smooth muscle cells (hVSMCs). We also showed that a high glucose concentration reduced the intracellular glutathione (GSH) content and the rate of uptake of cystine, which itself is a rate-limiting factor that maintains the GSH level (FEBS Lett.421: 19–22,1998). In the present study, we investigated whether the hyperglycemic condition in diabetic rats impairs the glutathione content in the aortic tissue in vivo. Wistar rats were divided into the following three groups: streptozotocin-induced diabetic rats (STZ-D, n=7), insulin-treated STZ-D rats (I-STZ-D, n=8), and non-diabetic controls (C, n=7). Fourteen days after streptozotocin injection, the aortic tissue was extracted and the GSH content in the aortic tissue was measured. Furthermore, the relationship between the GSH content in the aortic tissue and blood glucose level in Otsuka Long-Evans Tokushima Fatty (OLETF) rats aged 30 weeks, which developed diabetes spontaneously, was investigated. The GSH content in the aortic tissue of the STZ-D group (0.99±0.14 nmol/mg protein) was significantly lower than that of the control group (1.68±0.15 nmol/mg protein). Insulin treatment to the diabetic rats restored the GSH content in the aortic tissue (I-STZ-D group; 1.45±0.11 nmol/mg protein). Among the 22 Wistar rats, the GSH content in the aortic tissue was negatively correlated with the blood glucose level (r = − 0.69, p<0.01, n=22). Among the OLETF rats, a similar negative correlation between the GSH content in the aortic tissue and blood glucose level was seen (r = − 0.64, p<0.05, n=10). We demonstrated in vivo that the hyperglycemic condition in STZ-induced diabetic Wistar rats and OLETF rats reduced the GSH content in aortic tissue. This suggested reduced glutathione redox cycle function of aorta.
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Effect of aldose reductase inhibition on glutathione redox status in erythrocytes of diabetic patients

Abstract

Free Radical Biol Med

Blood

Metabolism

Biochem Biophys Res Commun

J Biol Chem

Biochim Biophys Acta

Anal Biochem

Metabolism

Increased diene conjugates in diabetic subjects with microangiopathy

Diabetic Med

Lipid peroxide level in plasma of diabetic patients

Biochem Med

Free radical activity in type 2 diabetes

Diabetic Med

Role of glutathione in protecting endothelial cells against hydrogen peroxide oxidant injury

J Lab Clin Med

The metabolic behavior of glutathione in human and avian erythrocytes

J Biol Chem

Vitamin E and oxidative stress

Free Radical Biol Med