Effect of the new thiazolidinedione-pioglitazone on the development of oxidative stress in liver and kidney of diabetic rabbits

Impaired homeostasis under diabetic conditions is connected with the increased production of free radicals and deficiency of antioxidative systems. The aim of this study was to analyze the effect of new oral antidiabetic drug-pioglitazone on activity of antioxidant factors and lipid peroxidation in vivo. The liver and kidney of alloxan-induced diabetic rabbits were examined after 4 and 8 weeks of treatment. After 4 weeks of diabetes the superoxide dismutase (Cu,Zn-SOD) activity in the liver was diminished while the catalase (CAT) activity and the level of ascorbic acid (AA) were elevated in comparison with the control group. Pioglitazone treatment during 4 weeks decreased the catalase activity in relation to the control diabetic animals. After 8 weeks of diabetes the CAT activity in the liver was elevated in comparison with the control group. Pioglitazone treatment during 8 weeks decreased the CAT activity and the level of lipid peroxidation products (LPO), and increased the Cu,Zn-SOD activity in relation to control diabetic animals. After 4 weeks of diabetes in the kidney the Cu,Zn-SOD activity and the level of ascorbic acid (AA) were diminished while the CAT activity and the LPO level were elevated in comparison with the control group. Pioglitazone treatment during 4 weeks increased the AA and decreased the LPO levels in relation to non-treated diabetic animals. After 8 weeks of disease the Cu,Zn-SOD activity in the kidney was diminished in comparison with the control group. Pioglitazone during 8 weeks decreased the LPO level in relation to non-treated diabetic animals. This study shows that diabetic animals undergo an important oxidative stress, which is partially corrected by pioglitazone treatment.     

Modification and inactivation of human Cu,Zn-superoxide dismutase by methylglyoxal

Methylglyoxal (MG) has been identified as an intermediate in non-enzymatic glycation, and increased levels have been reported in patients with diabetes. In this study, the effect of MG on the structure and function of human Cu,Zn-superoxide dismutase (SOD) was investigated. MG modifies Cu,Zn-SOD, as indicated by the formation of fluorescent products. When Cu, Zn-SOD was incubated with MG, covalent crosslinking of the protein increased progressively. MG-mediated modification of Cu,Zn-SOD led to loss of enzymatic activity and release of copper ions from the protein. Radical scavengers inhibited the crosslinking of Cu,Zn-SOD. When Cu,Zn-SOD that had been exposed to MG was analyzed, glycine, histidine, lysine, and valine residues were found to be particularly sensitive. It is suggested that oxidative damage to Cu,Zn-SOD by MG may perturb cellular antioxidant defense systems and damage cells. This effect may account, in part, for organ deterioration in diabetes.     

Delta aminolevulinate dehydratase (ALA-D) activity in human and experimental diabetes mellitus

The haem pathway is impaired in porphyrias and a frequent coexistence of diabetes mellitus and porphyria disease has been reported. We have therefore decided to investigate delta-aminolevulinate dehydratase, one of the more sensitive enzymes in the haem pathway, in both human diabetic patients and diabetic rats. We have studied 131 diabetes mellitus patients, 32 insulin dependent and 99 non-insulin dependent. The latter group was further subdivided according to treatment: diet alone (n = 24), diet plus oral hypoglycemic agents (n = 28) and diet plus insulin (n = 47). We have also performed similar studies in the rat model of diabetes mellitus, induced in 11 Wistar rats by streptozotocin. Control groups of both humans and animals were used. Erythrocytic aminolevulinate dehydratase activity was reduced in both insulin dependent and non-insulin dependent diabetic patients as compared to their controls (p < 0.001). This activity was only partially restored by addition of zinc and thiols to the incubation media. In insulin-dependent diabetes mellitus, reduction of enzyme activity was related to the glycosilated hemoglobin concentration (p < 0.05) and in non-insulin dependent diabetes mellitus to the glycemia (p < 0.01). In the diabetic rat, aminolevulinate dehydratase activity was diminished on both erythrocytes (p < 0.01) and hepatic tissue (p < 0.01) when compared to the control group. The decrease in activity of erythrocyte aminolevulinate dehydratase observed in diabetic patients, may represent an additional and useful parameter for the assessment of the severity of carbohydrate metabolism impairment.     

Copper and zinc concentrations and the activities of ceruloplasmin and superoxide dismutase in atherosclerosis obliterans

The relationships among concentrations of copper and zinc, the oxidase activity of ceruloplasmin (Cp) in serum, and Cu,Zn-SOD (superoxide dismutase) activity in erythrocytes were investigated in men with atherosclerosis obliterans (AO) and a control group. The oxidase activity of Cp was measured with o-dianisidine dihydrochloride as a substrate, and Cu,Zn-SOD activity in erythrocytes by using the RANSOD kit. The lipid profile and uric acid concentration were determined in AO and control groups. The results showed higher copper and zinc concentrations in serum in the AO group (20.0±3.5 and 18.0±3.2 μmol/L, respectively) in comparison with the control group (15.6±2.3 and 14.7±1.9 μmol/L). The Cp activity in serum was higher in the AO group (174.2±61.8 U/L) than in the control group (93.7±33.9 U/L), and a significant difference was found in the activity of Cu,Zn-SOD in erythrocytes (2389±1396 and 1245±365 U/g Hb, respectively) between both groups. The activity of Cu,Zn-SOD was positively correlated with copper in the control group (r=0.73), but not in AO, and negatively with uric acid concentration (r=−0.63) in the AO group. The oxidase activity of Cp was correlated with copper, but not zinc, in AO and control groups (r≥0.65). Negative correlation coefficients were calculated for uric acid and copper and zinc concentrations in the AO group (−r≥0.61). Increased copper concentrations and oxidase activity of Cp in serum in AO and the activity of Cu,Zn-SOD in erythrocytes could result from atherosclerotic disease, accompanied by chronic ischemia of a lower limb. These results suggest also that relationship between copper concentration and Cu,Zn-SOD activity in erythrocytes found in the serum of healthy subjects may be disturbed in pathologic conditions.     

L-Cysteine influx in type 2 diabetic erythrocytes

Erythrocyte oxidative stress has been implicated in the pathogenesis of diabetes mellitus, and the deficiency of antioxidant defense by the glutathione (GSH) pathway is thought to be one of the factors responsible for development of complications in diabetes. Erythrocytes require L-cysteine for the synthesis of GSH and the rate of synthesis is determined only by L-cysteine availability. In the present study we have found that the L-cysteine influx in erythrocytes from type 2 diabetic patients was significantly lower compared to age-matched controls. The decreased influx may be one of the factors leading to low GSH concentration observed in type 2 diabetes. Since L-cysteine is the limiting amino acid in GSH synthesis, any strategy aimed to increase L-cysteine influx in erythrocytes may be beneficial for type 2 diabetic patients.     

Eryptosis and oxidative damage in type 2 diabetic mellitus patients with chronic kidney disease

It has been suggested that oxidative stress may participate in the progression of diabetes and its complications. Long-term complications of type 2 diabetes mellitus (T2DM) include retinopathy, atherosclerosis, shortened life span of erythrocytes, nephropathy, and chronic kidney disease (CKD). Oxidative damage has been associated with erythrocyte apoptosis induction in other pathological conditions. Our aim was to study the presence of eryptosis and its possible relationship with oxidative damage in patients with T2DM without CKD (T2DM/CKD(-)) and in patients with T2DM and CKD (T2DM/CKD(+)).Oxidative damage of lipids erythrocytes were increased in diabetic patients. The highest lipoperoxidation was found in T2DM/CKD(+). Likewise, the lower plasma total antioxidant capacity, GSH/GSSG ratio, and GSH in erythrocytes were found in T2DM/CKD(+) patients. A negative correlation was found between plasma total antioxidant capacity and oxidative damage. Phosphatidylserine (PS) externalization was measured in erythrocytes to evaluate eryptosis. Annexin binding in erythrocytes of T2DM/CKD(+) patients was higher than in healthy subjects and T2DM/CKD(-) patients. A positive correlation between lipoperoxidation and PS externalization in erythrocytes was found. This work showed that the erythrocytes of diabetic patients have increased oxidative damage, a reduction of antioxidant systems and more erythrocyte PS externalization. The duration of diabetes and the presence of CKD increase both oxidative damage and eryptosis. It is possible that a longer time of evolution induces an increase in erythrocyte oxidative damage and the consumption of blood antioxidant systems, adding to the osmotic stress in CKD and so contributes to an increase in PS externalization in diabetic patients.     

Association between dietary fat and antioxidant status of Tunisian type 2 diabetic patients

BACKGROUND: The antioxidant enzymes: superoxide dismutase (Cu/Zn SOD) and glutathione peroxidase (GSH-Px) provide a defense against the damage of cells by reactive oxygen species, which increased in diabetic state. It was demonstrated that dietary treatment could improve the antioxidant status in patients with type 2 diabetes mellitus. This study was undertaken to determine if erythrocyte Cu/Zn SOD and GSH-Px activities correlate with dietary nutrients in 35 selected type 2 diabetic patients (21 women and 14 men) without diabetic complications. RESULTS: We found that erythrocyte Cu/Zn SOD was diminished in patients with poor controlled diabetes and GSH-Px activity was significantly decreased in obese compared with non-obese type 2 diabetic patients (1.07+/-0.87 and 2.36+/-1.99 U/ml, respectively; P=0.024). Both erythrocyte Cu/Zn SOD and GSH-Px activities were positively correlated to erythrocyte omega3-polyunsaturated fatty acids (PUFA). In non-obese diabetic patients, only GSH-Px activity was correlated negatively to the fraction of linoleic acid (18:2omega6) and arachidonic acid (20:4omega6) in erythrocytes phospholipids. CONCLUSIONS: The data of this study reveal that activities of erythrocyte antioxidant enzymes were altered in type 2 diabetic patients. Further studies are needed to determine if diet supplemented with omega3-PUFA is required to improve antioxidant defense system in diabetic state.     

Insulin binding to circulating erythrocytes in nonobese diabetic mice

Insulin binding to circulating erythrocytes was studied in nonobese diabetic (NOD) mice which develop insulinopenic diabetes mellitus spontaneously. NOD mice with a short duration of diabetes mellitus and mild insulinopenia did not show any change in insulin binding, while those with a long duration of diabetes mellitus and severe insulinopenia showed an increase in insulin binding compared with nondiabetic NOD mice (6.85 +/- 0.38% bound vs. 4.19 +/- 0.24% bound, p less than 0.01). This increase in insulin binding was due to an increase in the number of receptors. Insulin treatment of diabetic NOD mice significantly reduced the insulin binding by 64%, which resulted from a decrease in the number of the receptors. These results indicate that insulin binding to erythrocytes in NOD mice is controlled mostly by up-and-down regulation.     

The Evaluation of the Oxidative Stress Parameters in Nondiabetic and Diabetic Senile Cataract Patients

The aim of the present study was to evaluate the copper (Cu), zinc (Zn), malondialdehyde (MDA), glutathione (GSH), and advanced oxidation protein products (AOPP) levels and superoxide dismutase (SOD) activities in diabetic senile cataract. Ten patients with diabetic senile cataract and ten patients with nondiabetic senile cataract (control group) were included in this study. AOPP, MDA, and GSH levels and SOD activity were measured by a spectrophotometric method. Serum, lens Cu, and Zn levels were measured by an atomic absorption spectrophotometric method. Both the lens and serum Zn and Cu levels between the two groups were not significantly different (p > 0.05). GSH, AOPP, and MDA levels and the SOD activities in the diabetic senile cataract group were significantly increased as compared to the control group (p < 0.05). Oxidative stress is one of the major factors which may lead to the early cataract formation. Oxidative events are of great importance in diabetic complications and, particularly in the lens, may have a role in the pathogenesis of cataract associated with diabetes mellitus as exhibited in this study.     

Increased Superoxide Dismutase Activity Improves Survival of Cultured Postnatal Midbrain Neurons

Abstract: Copper/zinc superoxide dismutase (Cu/Zn-SOD) is a major free radical scavenging enzyme. Increased Cu/Zn-SOD activity protects cells against oxidative stress mediated by different mechanisms. However, there is also in vitro and in vivo evidence that, in the absence of abnormal oxidative stress, chronic increased Cu/Zn-SOD activity is detrimental to living cells. To address this issue, we examined the fate of mature midbrain neurons from transgenic mice expressing human Cu/Zn-SOD and from their nontransgenic littermates. Midbrain from transgenic pups had about threefold higher Cu/Zn-SOD activity than that from nontransgenic pups. Virtually all transgenic neurons were strongly immunoreactive for human Cu/Zn-SOD protein in their cell bodies and processes. The number of midbrain neurons decreased over time in both transgenic and nontransgenic cultures, but to a significantly smaller extent in the transgenic cultures. Postnatal midbrain neurons died by either necrosis or apoptosis, and increased Cu/Zn-SOD activity attenuated both forms of cell death. Furthermore, increased Cu/Zn-SOD activity better prevented the loss of dopaminergic neurons than GABAergic neurons. We also found that neuronal processes were dramatically denser in transgenic cultures than in nontransgenic cultures. These results indicate that chronic increased Cu/Zn-SOD activity does not appear to be detrimental, but rather promotes cell survival and neuronal process development in postnatal midbrain neurons, probably by providing more efficient detoxification of free radicals. They also show that increased Cu/Zn-SOD activity does not seem to play a critical role in determining the mode of cell death in this culture system.     

Selected metals status in patients with noninsulin-dependent diabetes mellitus

In order to investigate the relationships between metals zinc [Zn], copper [Cu], magnesium [Mg], or Calcium [Ca] and noninsulin-dependent diabetes mellitus, 65 patients of newly diagnosed noninsulin-dependent diabetes mellitus and 54 nondiabetic healthy controls were studied. The concentrations of selected metals in fasting blood samples and 24-h urine collections were determined. Hyperzincuria and hypermagnesuria were detected in diabetic patients (p<0.01). The diabetics also had lower Zn and Mg, and higher Cu, and Ca levels in their plasma than those of the controls, but the statistical differences in Ca and Mg were not significant.Significantly lower Zn and higher Ca levels in erythrocytes were found in diabetic patients (p<0.01). There is evidence of a significant difference in metals status between diabetic patients with or without the specific complications. This study further indicates that patients with NIDDM on Taiwan also have distinct changes in their metals status, and these perturbations are associated with some diabetic complications.     

DNA oxidation and superoxide dismutase in the kidney of diabetic animals: effects of pioglitazone and repaglinide

In the present study, DNA oxidative damage was elevated and superoxide dismutase (Cu,Zn-SOD) metabolism was disturbed in the kidney of alloxan-induced diabetic animals. The effects of pioglitazone and repaglinide, new oral antidiabetics, on 8-hydroxy-2′-deoxyguanosine (8-OHdG) and Cu,Zn-SOD were studied. Diabetic versus control levels (mean ± SE) of 8-OHdG were 24.9 ± 0.2 vs. 21.8 ± 0.1 and 21.5 ± 0.2 vs 20.1 ± 0.2 pmol/μg DNA after 4 and 8 weeks, respectively. At p<0.05, pioglitazone diminished this parameter in diabetic animals (22.0 ± 0.2 and 20.1 ± 0.3 pmol/μg DNA). The level was not affected in diabetic groups receiving repaglinide (24.9 ± 0.2 and 21.5 ± 0.3 pmol/μg DNA). In diabetic kidney, Cu,Zn-SOD mRNA was diminished relative to control animals and was modulated by pioglitazone and repaglinide treatments. Simultaneously, Cu,Zn-SOD activity was also diminished (1.5 ± 0.2 vs. 2.8 ± 0.3 and 1.8 ± 0.1 vs 2.9 ± 0.3 U/mg protein after 4 and 8 weeks, respectively) and partly changed after pioglitazone (2.1 ± 0.4 and 2.3 ± 0.3 U/mg protein) and repaglinide (2.0 ± 0.1 and 2.4 ± 0.2 U/mg protein). These results suggest that a reduction in oxidative stress in diabetic kidney can be achieved with the administration of pioglitazone and to some extent using repaglinide treatment.     

Modification of oxidative stress by pioglitazone in the heart of alloxan-induced diabetic rabbits

Summary The study was undertaken to analyze the effect of pioglitazone on superoxide dismutase (Cu, Zn-SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GSSG-R), glutathione (GSH), ascorbic acid (AA), lipid peroxidation products (LPO) and protein carbonyl groups (PCG) in the heart of alloxan-induced diabetic rabbits after 4 and 8 weeks of pioglitazone treatment. In diabetic animals, Cu, Zn-SOD and CAT were elevated by 60 and 55%, and 90 and 77% as compared to controls at 4 and 8 weeks, respectively. GSH-Px, GSSG-R and GSH were diminished by 11, 14 and 33% as compared to controls at 4 or 8 weeks. AA was diminished by 52 and 41%. At P <0.05, pioglitazone normalized the activities of Cu, Zn-SOD, GSH-Px and GSSG-R. The activity of CAT was modified as compared to diabetic non-treated rabbits. After pioglitazone treatment, GSH and AA were increased as compared to diabetic non-treated animals. In diabetic rabbits, LPO was elevated by 52 and 111% and normalized by pioglitazone treatment. PCG was elevated by 72 and 133% and diminished as compared to diabetic non-treated animals at 8 weeks. The study shows that pioglitazone reduces oxidative stress in the heart of diabetic rabbits. In therapy, similar action can improve the cardiovascular system of diabetic patients.     

Flow cytometric analysis of glyoxalase-1 expression in human leukocytes

Altered glyoxalase‐1 (GLO‐1) activity and expression is associated with the development of late diabetic complications, malignancy and oxidative stress‐ and aging‐related diseases. In the present study, we developed a flow cytometry method for GLO‐1 detection in human leukocytes isolated from peripheral blood samples to investigate GLO‐1 expression in leukocyte subsets from type 1 and 2 diabetes mellitus patients (n = 11) and healthy subjects (n = 8). The flow cytometry analysis of GLO‐1 in leukocytes showed that expression index of GLO‐1‐positive cells was slightly increased in mononuclear leukocytes from diabetic patients. This result correlated with the increase in GLO‐1 activity in the whole blood samples of type 2 diabetes patients. In conclusion, the present study demonstrates that flow cytometry is suitable for the detection of the GLO‐1 enzyme in human leukocytes and that this method could be used to investigate the fast adaptation of the glyoxalase system related to the pathogenesis of late complications of diabetes mellitus and other glycation stress‐related disorders. Copyright © 2011 John Wiley & Sons, Ltd.     

The antioxidant status during maturation of reticulocytes to erythrocytes in type 2 diabetics

Free radical-induced lipid peroxidation has been associated with numerous disease processes including diabetes mellitus. The extent of lipid peroxidation (LPO) and antioxidant defense system [i.e., levels of glutathione (GSH), glucose-6-phosphate dehydrogenase (G6PDH), glutathione reductase (GR), glutathione peroxidase (GPx), glutathione-S-transferase (GST), and catalase (CAT)] were evaluated in reticulocytes and erythrocytes of type 2 diabetic males and age-matched controls. Type 2 diabetics have shown increased lipid peroxidation and decreased levels of GSH, GR, GPx, G6PDH, and GST both in reticulocytes and erythrocytes compared to controls, indicating the presence of oxidative stress and defective antioxidant systems in these patients. CAT activity is found to be enhanced in both the reticulocytes and erythrocytes of diabetics, with a greater percentage enhancement in reticulocytes. The extent of increase in lipid peroxidation is greater in erythrocytes compared to reticulocytes in these patients. Furthermore, the maturation of reticulocytes to erythrocytes resulted in decreased GSH and decreased activities of all antioxidant enzymes (except CAT) both in normals and type 2 diabetes individuals, indicating decreased scavenging capacity as reticulocytes mature to erythrocytes. These maturational alterations are further intensified in type 2 diabetics. The present study reveals that the alterations in lipid peroxidation and antioxidant system lean toward early senescence of erythrocytes in type 2 diabetic patients.     

The effect of agmatine on L-arginine metabolism in erythrocytes under streptozotocin-induced diabetes in rats

The effects of agmatine on oxidative and nonoxidative metabolic pathways of L-arginine were investigated both in plasma and erythrocytes under experimental diabetes mellitus. It was indicated, that agmatine prevents the development of oxidative-nitrosative stress in diabetic rats. After treatment of animals by agmatine NO-synthase methabolic pathway of L-arginine is depressed whereas arginase one increases in erythrocytes of rats with experimental diabetes mellitus.     

Selenium and glycogen levels in diabetic patients

Selenium in serum and selenium and glycogen in erythrocytes were determined in diabetic patients divided into noninsulin-dependent (n=50) and insulin-dependent (n=31) groups according to the etiopathogenesis of their diabetes. Selenium was determined by the method of atomic absorption spectrometry. Serum level of selenium was statistically significantly different in patients with either noninsulin-dependent (59.23±12.2 μg/L) or insulin-dependent (58.23±16.7 μg/L) diabetes mellitus as compared with the control group of 62 subjects (64.2±11.5 μg/L; p<0.05). There was no statistically significant difference in the serum levels of selenium between the groups of patients with noninsulin-dependent and insulin-dependent diabetes mellitus. The levels of erythrocyte glycogen were 2.0580±1.326, 2.0380±1.735, and 2.0036±1.3537 μg/g Hb in the control group, noninsulin-dependent group, and insulin-dependent group, respectively, with no statistically significant between-group difference. The decreased levels of selenium in serum and erythrocytes of diabetic patients suggest the possible role of glutathione peroxidase activity.     

Effect of overexpression of human Cu/Zn-SOD on activation-induced lymphocyte proliferation and apoptosis

The process of lymphocyte proliferation and apoptosis is known to be linked to oxidative stress. In the present study, we have used a new transgenic mouse model to investigate the effect of human Cu/Zn superoxide dismutase (Cu/Zn-SOD) overexpression on activation-induced lymphocytes proliferation and apoptosis. Cu/Zn-SOD activity was 3.5-fold higher in the spleen of the transgenic mice overexpressing Cu/Zn-SOD (Tg-Cu/Zn-SOD) compared to the wild-type littermates. Proliferative response of lymphocytes to lipopolysaccharide (LPS), Concanavalin A (Con A), and anti-CD3 was measured by [3H]-thymidine incorporation. Activation-induced apoptosis was determined by incubating the T cells with anti-CD3 (primary stimulus) for 72 h, followed by restimulation with Con A (secondary stimulus) for various times. Apoptosis was assessed by measuring DNA fragmentation using a spectrofluorimetric assay and monitoring the expression of the specific apoptotic markers (Fas/CD95 receptor and Fas/CD95 ligand (Fas-L) using flow cytometry. There was no significant difference in proliferative response of lymphocytes to LPS, Con A, or anti-CD3 in transgenic mice overexpressing human Cu/Zn superoxide dismutase (Tg-Cu/Zn-SOD) compared to wild-type littermates. In addition, no significant difference was observed in lymphocyte populations and subsets between Tg-Cu/Zn-SOD mice and wild-type littermates. However, splenic T cells from Tg-Cu/Zn-SOD mice exhibited a significantly (p <.05) higher level of activation-induced DNA fragmentation than T cells from wild-type littermates. The increase in DNA fragmentation was paralleled with an increase in the proportion of T cells expressing Fas and Fas-L molecules. The possible consequences of Cu/Zn-SOD overproduction on activation-induced apoptosis are discussed.     

In vitro upregulation of erythrocytes glucose uptake by Rhaphnus sativa extract in diabetic patients

In diabetes, both the increase in the oxidative stress and the decrease in the antioxidant defense may elevate the susceptibility of diabetic patients to many pathological complications. So, the aim of the present study was to investigate the effect of superoxide dismutase (SOD) like activity protein, partially purified from radish (Rhaphnus sativa) on uptake of glucose in vitro by erythrocytes of diabetic patients. In hyperglycemic patients, erythrocytes malondialdehyde level was highly significantly increased (P < 0.0001) than that of the control. However, the erythrocytes glutathione content and glutathione reductase activity, were both highly significantly decreased (P < 0.0001) compared to that corresponding control values. The glucose uptake by erythrocytes of diabetic patients was highly significantly decreased (P < 0.0001) with increasing hyperglycemia, while it was highly significantly elevated (p < 0.0001) after addition of the partially purified SOD like activity protein. On the other hand, the malondialdehyde concentration was highly significantly reduced (p < 0.001) on adding the partially purified protein. It thus can be concluded that, an appropriate support for enhancing antioxidant supply, such as SOD like activity protein from natural sources, may help control blood glucose level and may prevent clinical complications of diabetes.