However not all oxygen containing radicals have high oxidative potential .ROS are neutralized by a battery of AOS, which can be divided into mainly two categories: enzymes (ex: superoxide dismutase SOD, glutathione peroxidase GPX, and catalase) and non-enzymatic systems (ex: glutathione GSH, vitamins A, C and E.)Some are located in cell membranes, others in cytosol, and in blood plasma. Due to its particularly important since ever modest decreases in SOD are sufficient to provoke cell damage. Quantitatively, however, albumin and uric acid are the main AOS³.

Mechanism for increased oxidative stress in diabetes

Hyperglycemia: Hyperglycemia generates oxidative stress. It induces OS by various mechanisms. The steps and mechanism in hyperglycemia induced oxidative stress is explained in figure-1 and figure-2.

Advanced glycation products:

Advanced glycation or glycosylation end products (AGEs) are the products of glycation and oxidation (glycoxidation) .They are increased with age and at an accelerated rate in diabetes mellitus^{4, 5}. In vitro studies have shown that glycation results in the production of superoxide ^6,
7.Oxidation results in the generation of superoxide, hydrogen peroxide (H₂O₂) and through transition metal catalysis, hydroxyl radicals⁸. Catalase and other antioxidants decrease cross linking and AGE formation^{9, 10}.

Alterations in glutathione metabolism:

Tissue glutathione plays a central role in antioxidant defense^{11, 12}. Reduced glutathione detoxifies ROS such as hydrogen peroxide and lipid peroxides directly or in a GPX catalyzed mechanism. Glutathione also regenerates the major aqueous and lipid phase antioxidants, ascorbate and α-tocopherol. Glutathione reductase (GRD) catalyzes the nicotinamide adenine dinucleotide phosphate (NADPH) dependent reduction of oxidized glutathione, serving to maintain intracellular glutathione stores and a favorable redox status. Glutathione –S - transferase (GST) catalyzes the reaction between the thiol (–SH) group and potential alkylating agents, rendering them more water soluble and suitable for transport out of the cell¹³.

Glutathione homeostasis:

In type 2 diabetes there is decreased erythrocyte GSH and increased GSSG levels^{14, 15}. Blood GSH is significantly decreased in different phases of type 2 diabetes mellitus (DM) such as: glucose intolerance and early hyperglycemia ¹⁶, within two years of diagnosis and before development of complications ¹⁷, and in poor glycemic control ¹⁸.Red cells in type 2 diabetes mellitus (DM) showed decreased GSH levels, impaired gammaglutamyl transferase activity and impaired thiol transport ¹⁹. And there is an inverse correlation between erythrocyte GSH levels and the presence of DM complications in type 1 and 2 DM patients ²⁰.Decreased blood or red cell glutathione levels are found in type 2 DM.

Glutathione dependent enzymes:

There is no difference in whole blood GRD activity in type 1 and type 2 DM patients compared to non-diabetics²¹. The normal red cell GRD enzyme kinetics in type 1 DM patients was observed²². On the other hand, blood GRD activity was lower in children with type 1 DM compared to healthy children²³. Both in type 1 and type 2 diabetes mellitus the red blood cell, whole blood and leukocyte, glutathione peroxidase (GPX) activity was similar^24,25. On the other hand, erythrocyte GPX activity was also impaired in Asian diabetic patients²⁶. In type 1 DM plasma selenium levels is normal, but red cell selenium content and GPX activity were decreased²⁷. Normal red cell GST enzyme kinetics is found in type 1 DM patients. GST activity has been reported to be decreased in heart and liver²⁸. Changes in glutathione dependent enzymes in experimental diabetic models have been contradictory.

Impairment of superoxide dismutase and catalase activity:

The major antioxidants enzymes are SOD and catalase. SOD exists in three different isoforms. Cu, Zn-SOD and Mn-SOD.

Cu, Zn-SOD is mostly found in the cytosol and dismutates superoxide to hydrogen peroxide. Extracellular (EC) SOD is found in the plasma and extracellular space. Mn-SOD is located in mitochondria. Catalase is a hydrogen peroxide decomposing enzyme mainly localized to peroxisomes or microperoxisomes. Superoxide may react with other reactive oxygen species such as nitric oxide to form highly toxic species such as peroxynitrite, in addition to direct toxic effects²⁹. Peroxynitrite reacts with the tyrosine residues in proteins resulting with the nitrotyrosine production in plasma proteins, which is considered as an indirect evidence of peroxynitrite production and increased oxidative stress. Nitrotyrosine was found in the plasma of all type 2 diabetes. Depending on these, plasma nitrotyrosine values were correlated with plasma glucose concentrations³⁰.

Exposure of endothelial cells to high glucose leads to augmented production of superoxide anion, which may quench nitric oxide. Decreased nitric oxide levels result with impaired endothelial functions, vasodilation and delayed cell replication ³¹.Superoxide can be dismutated to much more reactive hydrogen peroxide, which through the Fenton reaction can then lead to highly toxic hydroxyl radical formation. Decreased activity of cytoplasmic Cu,Zn-SOD and especially mitochondrial (Mn-) SOD in diabetic neutrophils is seen.

As a result of decreased SOD activity the superoxide levels estimated indirectly by cytochrome reduction were elevated in neutrophils of diabetic patients³². Major reason for the decreased SOD activity is the glycosylation of Cu,Zn-SOD which has been shown to lead to enzyme inactivation both in vivo and in vitro ³³. Also Cu, Zn-SOD

cleavage and release of cupric ions (Cu++) in vitro resulted in transition metal catalyzed ROS formation ³⁴. Erythrocyte Cu, Zn-SOD activity correlated inversely with indices of glycemic control in DM patients²⁶. Red cell Cu, Zn/SOD activity has also been found to be decreased in DM^33,35. Glycation may decrease cell associated EC-SOD, which could predispose to oxidative damage. They had found decreased red cell Cu, Zn-SOD activity in type 1 DM patients with retinopathy compared to type1 DM patients without micro vascular complications and non-diabetic control subjects³⁶. Red cell Cu, Zn-SOD activity was similar in type 1 and 2 DM³⁷. Leukocyte SOD activity was similar between type 2 DM and healthy control subjects, despite increased lipid peroxidation and decreased ascorbate levels²⁵.Further more, increased red cell SOD activity and serum malonaldehyde (MDA) levels were reported in type 1 DM with normo- microalbuminuria and retinopathy compared to healthy individuals^{38, 39}. Red cell superoxide and catalase activities were decreased in individuals with impaired glucose tolerance (IGT) and early hyperglycemia and also in type 2 DM. EC-SOD activity was found to be similar in type 1 DM, despite somewhat higher plasma EC-SOD levels^{40, 41}.

The polyol pathway:

Hyperglycemia induces the polyol pathway, resulting in induction of aldose reductase and production of sorbitol. Importances of the polyol pathway vary among tissues. Induction of oxidative stress may occur through many different mechanisms, including depletion of NADPH and consequent disturbance of glutathione and nitric oxide metabolism. Mean red cell GSH and NADPH levels and NADPH/NADP+ and GSH/GSSG ratios are decreased in type 2 diabetes⁴². One week of treatment with the aldose reductase inhibitor Tolrestat improved the NADPH and GSH levels in diabetics whose NADPH levels were Thus in at least a subset of type 2 DM patients activation of the polyol pathway appears to deplete erythrocyte NADPH and GSH. Similarly in a recent study aldose reductase inhibitor sorbinil restored nerve concentrations of antioxidants reduced glutathione (GSH) and ascorbate, and normalized diabetes induced lipid peroxidation in streptozotocin-diabetic rats⁴³.

Hyperglycemia reduces antioxidant potential:

OS acts on signal transduction and it affects gene expression. There by the expression of antioxidant enzyme can be reduced. Moreover hyperglycemia can simply inactivate existing enzymes by glycating these proteins, glycation of SOD. For example, it also leads to DNA cleavage ⁴⁴. From this we come to know that OS develops insufficient AOS activity, even if ROS production is within a physiological range.

Factors generating oxidative stress:

Hormones:

Most type 2 diabetes patients are hyperinsulinemic for a long period. Insulin can stimulate OS by various mechanisms - the hormone induces production of H₂O₂ when activating its receptors and although hydrogen peroxide is not a strong oxidant itself, it can indirectly activate oxidative reactions. Insulin also stimulates the sympathetic nervous system, which leads to activation of neurotransmitters and their enzymatic systems, several of which induce OS. For example diabetic vessel walls contain high levels of NAD (P) H oxidase ⁴⁵.Leptin is another hormone reportedly stimulating OS ⁴⁶.

Lipids:

Increased fasting and postprandial plasma levels of triglycerides, free fatty acids and cholesterol are common in type 2 diabetes. They are known to generate ROS ⁴⁷. In the vessel wall, import of or local formation of oxLDL (oxidized low-density lipoprotein) is a cardinal mechanism involving OS in the atherosclerotic process.

Angiotensin II:

Angiotensin II generates OS in blood vessels by stimulating nicotinamide adenine dinucleotide (NADH) oxidase and is claimed to mediate the effect of hyperinsulinemia. A major source of OS in vascular pathophysiology is the alternance of ischemia/reperfusion, since the hypoxic period characterizing ischemia is followed by a brutal oxidative burst upon refilling of the vessels with blood during reactive hyperemia. Thus, diabetic patients suffering from complications such as arteritis or diabetic foot experience numerous daily repetitive episodes of ischemia/reperfusion⁴⁸. Nitric oxide (NO), is both a scavenger and a prooxidant when it is attacked by radical such as becoming transformed into peroxynitrite⁴⁹.It may represent an important contributor to OS because NO levels are frequently elevated in early stages of diabetes.

Diabetes: targets of oxidative stress:

As diabetes is characterized by defects in both metabolic and vascular domains, this disease represents a privileged situation for OS exerting harmful effects.

Effects of OS on diabetic metabolism:

The development from prediabetes to fasting hyperglycemia is now considered to be due mainly to the development of cell failure, a process being aggravated by the duration of the disease. An implication of OS has been first suggested when it was found that alloxan and STZ, used to induce diabetes in animals, destroyed pancreas by OS. In fact, OS induces β-cell death, this is favoured by an obvious low antioxidant potential of native β-cells⁵⁰. In vitro, OS decreases the insulin gene promoter activity in hamster islet β – cell line (HIT) cells⁵¹. It was recently found that addition of a SOD mimetic increased human islet survival⁵². OS may be the mediator whereby free fatty acids (FFA) induce β-cell apoptosis. Moreover amyloid deposition in the pancreas is linked with OS⁵³. OS can also impair the internalisation of insulin by endothelial, thus limiting hormone delivery to targets tissues and interfere with GLUT-4-mediated glucose transport⁵⁴.

Effects of OS in blood vessels:

As they are located at the interface between blood and tissue, vessels walls are particularly exposed to OS. Not only do they have constitutive ROS-generating enzymes (cyclooxygenase COX 1, lipoxygenases, NADH oxidase, cytochrome P450) but they also contain extravasated cells such as monocytes when atherosclerotic damage is present. When these cells are activated, NADH(P)H oxidase and myeloperoxidase are stimulated. Activated leucocytes/ monocytes as well as glycation of endothelial cells induce OS, which favours the expression of adhesion molecules and subsequent cell infiltration. In diabetic capillaries activated leucocytes stick to the endothelium, plug the vessel and stimulate permeability. Endothelium-produced NO can be transformed into the oxidant peroxynitrite but, although this substance can induce apoptosis, its relevance in vivo is controversial⁵⁵. There is also evidence that glucose can directly scavenge NO and, although there are data showing stimulation of NO formation by high glucose and several studies have shown that acute hyperglycemia reduces endothelial-dependent vasodilatation⁵⁶. Interestingly it has been found that tetrahydrobiopterin, an important cofactor of NO synthesis, is reduced in insulin resistant, fructose-fed rats, generating superoxide and reducing endothelial vasodilatation⁵⁷. Cyclic strain, exerting tension on vessel walls, generates OS in endothelium and secretes plasminogen activator inhibitor (PAI-1), an inhibitor of the fibrinolytic system largely involved in the metabolic syndrome and in diabetes⁵⁸. Such a mechanism may be an important contributor to the development of atherosclerotic lesions at arterial bifurcations. In organs like the heart, OS may lead to cardiomyocyte apoptosis. Finally, a provocative hypothesis has recently been proposed, implying the competitive inhibition by hyperglycemia of DHA (dehydroascorbate, the uncharged form of vitamin C) uptake at the level of the glucose transporters. By preventing entry of DHA and consequent reconversion into ascorbic acid, cells would lose their antioxidant potential. Since DHA uptake occurs in micro vessels, this defect might be the common denominator of the typical small vessel complications of diabetes⁵⁹. More extensive data can be found in several recent reviews⁶⁰. An important aspect must be evoked, because it could influence the outcome of AOS treatments: the basal antioxidant equipment can vary drastically among cell types. Thus the reaction to hyperglycemia – induced OS is different in cells from large (smooth muscle cells) and small vessels (pericytes). This difference can be observed even between cell types of the same vessel (endothelial cells vs pericytes)^61,
62. OS may also affect vessel integrity by disrupting intercellular junctions through a stimulation of matrix metalloproteinase, in particular matrix mettallopeptidase (MMP-9)⁶³.

Is OS harmful?

In view of the evidence for elevated OS in diabetes, this question might sound provocative. It is however a frequent trend in medicine to assimilate abnormal levels of a parameter with harmfulness. Although many studies show indeed an abnormal shift of the OS/AOS balance in favour of the former, there is still lacking proof that levels of OS observed in diabetic patients are harmful to tissues to an extent that its inhibition would save the organ structure or the biological function. Conceivably there could exist thresholds for OS harmfulness and this remains to be demonstrated. The fact that intensive OS can easily injure or kill cells in vitro must be considered with great caution because the culture conditions are frequently unphysiological in respect of oxygen environment or AOS levels in the medium. Thus the concept and the true role of OS are exaggerated ⁶⁴.

The choice of the antioxidant:

An antioxidant is a molecule capable of slowing or preventing the oxidation of other molecules. Oxidation reactions are crucial for life. Plants and animals maintain complex systems of multiple types of antioxidants, such as glutathione, vitamin C, and vitamin E as well as enzymes such as catalase, superoxide dismutase and various peroxides. Low levels of antioxidants or inhibition of the antioxidant enzymes, cause oxidative stress and may damage or kill cells. Thus the choice of selecting the antioxidants is more important .Most human trials were performed with vitamin E, which raises many questions as to this choice. Thus, there is no proof that the orally administered vitamin E reaches the adequate target cells in sufficient concentrations. Conversely there has been concern about the dosage (usually very high), because most AOS, including vitamin E can behave as prooxidants at higher dosage⁶⁵. Finally, it has been suggested that vitamin E, for example, has other biological properties possibly responsible for the observed positive effects in vitro ⁶⁶. However a recent report using vitamin C also failed to show any improvement in glycemia, blood pressure, markers of oxidative stress and endothelial function in type 2 patients⁶⁷. Thus, vitamins may have simply been the wrong choice! Alternatively pharmacological intervention with oxidant chain breakers may reveal insufficient and highlight the need for interfering directly with ROS production⁶⁸.

CONCLUSION:

Diabetes mellitus is associated with a markedly increased mortality from coronary heart disease, not explainable by traditional risk factors. Although data are not yet conclusive, oxidative stress has been increasingly implicated in the pathogenesis of diabetic micro- and macrovascular disease⁶⁹. If antioxidants can show a protective effect against stress in DM, this may have direct impact on the use of antioxidants as a safe therapeutic modality in diabetes⁷⁰.

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Received on 04.02.2010

Accepted on 24.03.2010

Research J. Pharmacology and Pharmacodynamics. 2(3): May-June 2010, 221-227