**4. Pathways of free radical generation in diabetes mellitus and its associated complications**

In diabetes, ROS is thought to be generated through increased polyol pathway [82], increased formation of advanced-glycation end products (AGEs) [83] and protein kinase C (PKC) activation [84].

#### **4.1. Aldose reductase pathway and ROS generation**

Aldose reductase is the rate limiting enzyme of the polyol pathway. The nicotinamide ade‐ nine dinucleotide phosphate (NAD(P)H)-requiring aldose reductase, catalyses the reduction of glucose to sorbitol followed by the oxidation of sorbitol to fructose by NAD+ dependent sorbitol dehydrogenase. At normal blood glucose concentration (5.5 mM), aldose reductase catalyzed reaction represents less than 3% of total glucose utilization [85]. However, hypergly‐ cemia results in saturation of hexokinase and more than 30% of glucose is directed into the polyol pathway [86]. In a diabetic state, polyol pathway increases in tissues that do not require insulin for cellular glucose uptake, such as retina, kidney, peripheral nerves and blood vessels [87].

The overall reaction of the polyol pathway leads to a shortage of intracellular NAD(P)H and a surplus of NADH, i.e, a reductive imbalance. Increased NADH generation during conversion of sorbitol to fructose provides substrate for NADH oxidase to generate ROS [88]. NADH serves as a source of electrons in complex 1 of the electron transport chain resulting in increased mitochondrial generation of superoxide radical. In diabetic cells, oxidative phosphorylation in mitochondria is enhanced due to increase flux of electron donors into the electron transport chain. This drives the inner mitochondrial membrane potential upward causing blockage of electron transfer inside complex III [89]. Electrons back up to coenzyme Q results and electrons are transferred one at a time to molecular oxygen, generating superoxide. DNA damage by superoxide and peroxynitrite results in the activation of poly (ADP-ribose) polymerase (PARP), a DNA repair enzyme. PARP reduces the activity of glyceraldehyde-3- phosphate dehydrogenase (GAPDH) (an enzyme of the glycolytic pathway which catalyses the conver‐ sion of glyceraldehydes -3 phosphate to 1, 3 biphosphoglycerate) by ADP- ribosylation [90, 91]. A consequence of GAPDH inhibition by PARP is an increase in triose phosphate pool, upstream of GAPDH and increase flux of intermediates into the damaging pathways of diabetic complications.

group of molecules formed by non-enzymatic reactions of reducing sugars, ascorbate and other carbohydrates with amino acids, lipids and nucleic acids [98, 99]. Glycation end product's adducts such as pyraline, pentosidine and N- Carboxy- methyl lysine (CML) are found to be

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Once formed, AGEs can cause tissue damage by two main pathways which are: (1) formation of cross links that alter protein structure and function and, (2) interaction of AGE with AGEcell surface receptors on the surfaces of various cells such as endothelial cells, macrophages, neurons, and smooth-muscle cells resulting in activation of cell signaling and gene expression that induces oxidative stress and inflammation [98, 99; 102-105]. Oxidative stress can accelerate AGE formation while AGE formation can also amplify the production of more ROS resulting

AGE's mediate some of their effect via interaction with some receptors that have been shown to bind to these chemical moieties. Among these receptors, Receptor for Advanced Glycation End products (RAGE) is the most extensively studied [106]. Evidence from numerous studies suggest that AGE's are involved in a vicious cycle of inflammation, generation of ROS and increased production of AGE's. Ligand RAGE interaction results in activation of pathways such as p21ras, erk1/2 (p44/p42), MAP kinases, p38 and SAPK/JNK MAP kinases [107-109]. A consequence of the activation of these pathways is the nuclear translocation of transcription factor, Nuclear Factor Kappa B (NF-КB). Translocation of NF-КB to the nucleus increases the transcription of a number of proteins such as, vascular endothelial growth factor (VEGF), monocyte chemoattractant protein-1 (MCP-1), vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1) and pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6, 1L-18 and tumour necrosis factor (TNF)-α which are centrally involved in the endothelial recruitment of neutrophil and subsequent development or

The gene regions of NF-КB are located at the promoter region of RAGE. Moreover, binding of NF-КB to the promoter region of RAGE results in up-regulation of RAGE itsel. Interaction of AGE with RAGE generates more oxidative stress and this further potentiates the formation of AGE's [109, 113]. Generation of ROS by ligand stimulated RAGE activation is mediated at least in part via activation of NADPH oxidase [114]. Other mechanisms by which AGE's may be linked to increased generation of ROS is by reducing the activities of enzymatic antioxidant such as SOD and CAT, lowering of glutathione stores, and activation of PKC [107, 115, 116].

Increased renal AGE in diabetic animals and patients have been linked to structural abnor‐ mality observed in diabetic nephropathy such as mesangial expansion, glomerular basement membrane thickening and tubulointerstitial fibrosis [117]. Advanced Glycation End Product's level is increased with decreased renal function in type 1 diabetic patients [118]. Evidence from clinical studies indicates a correlation between progression of diabetic retinopathy and the level of AGE in serum and retinal blood vessels of diabetic patients [100, 119]. In diabetes, increased AGE's are observed within retinal capillary cells and causes pericyte loss in diabetic retinopathy [120]. AGE's induce toxic effects on retinal pericytes by causing oxidative stress

elevated in diabetic tissues [100 - 102].

in a vicious cycle of AGE formation and oxidative stress.

progression of atherosclerotic plaque [109-112].

and subsequent apoptosis [121].

The polyol pathway also results in reduction in the bioavailability of NAD(P)H. The reduced bioavailability of NAD(P)H negatively affects the antioxidant defence system by depleting glutathione (GSH) a very important antioxidant. This is because the activity of GSH reductase, an antioxidant enzyme that generates GSH from its oxidized form (GSSH) depends on NAD(P)H. Depletion of NAD(P)H also decreases the synthesis of nitric oxide (NO), a vaculo‐ protective agent. NAD(P)H serves as a cofactor for nitric oxide synthase (NOS) which synthesizes NO from L-arginine. If endothelial nitric oxide synthase (eNOS) lack its substrate, L-arginine or one of its co-factor, it may produce superoxide radical (. O2 - ) instead of NO and this is referred to as ''uncoupled state of nitric oxide''[92]. Nitric oxide performs several physiological roles such as inhibition of platelet activation, vascular relaxation [93] and acts as an anti-inflammatory agent by reducing platelet aggregation and adhesion [94]. These properties inhibit atherogenesis and protect the blood vessel. Reduced bioavailability of NO level will therefore increase inflammation, enhance thrombosis and disrupt the integrity of endothelial cells. Reduction in NO has been documented in diabetes subjects with nephrop‐ athy [95]. Superoxide anion directly quenches NO by forming highly reactive peroxynitrite (ONOO- ) which initiates lipid peroxidation, oxidizes sulfhydryl group in protein and nitrates amino acids such as tyrosine, thereby affecting many signal transduction pathways. The polyol pathway serves as a main source of ROS generation in the retina [96]. In addition, sorbitol accumulation has been implicated in osmotic swelling of the eye lens and cataractogenesis [97].

#### **4.2. Advanced glycation end product (AGEs) formation and ROS generation in diabetic complications**

Glucose can react spontaneously with free amino groups of protein to form Schiff bases. These Schiff bases through complex reactions such as amadori rearrangement, dehydration and condensation forms cross-linked heterogeneous fluorescent derivatives called advanced glycation end products (AGEs). Advanced glycation end products constitute a heterogeneous group of molecules formed by non-enzymatic reactions of reducing sugars, ascorbate and other carbohydrates with amino acids, lipids and nucleic acids [98, 99]. Glycation end product's adducts such as pyraline, pentosidine and N- Carboxy- methyl lysine (CML) are found to be elevated in diabetic tissues [100 - 102].

The overall reaction of the polyol pathway leads to a shortage of intracellular NAD(P)H and a surplus of NADH, i.e, a reductive imbalance. Increased NADH generation during conversion of sorbitol to fructose provides substrate for NADH oxidase to generate ROS [88]. NADH serves as a source of electrons in complex 1 of the electron transport chain resulting in increased mitochondrial generation of superoxide radical. In diabetic cells, oxidative phosphorylation in mitochondria is enhanced due to increase flux of electron donors into the electron transport chain. This drives the inner mitochondrial membrane potential upward causing blockage of electron transfer inside complex III [89]. Electrons back up to coenzyme Q results and electrons are transferred one at a time to molecular oxygen, generating superoxide. DNA damage by superoxide and peroxynitrite results in the activation of poly (ADP-ribose) polymerase (PARP), a DNA repair enzyme. PARP reduces the activity of glyceraldehyde-3- phosphate dehydrogenase (GAPDH) (an enzyme of the glycolytic pathway which catalyses the conver‐ sion of glyceraldehydes -3 phosphate to 1, 3 biphosphoglycerate) by ADP- ribosylation [90, 91]. A consequence of GAPDH inhibition by PARP is an increase in triose phosphate pool, upstream of GAPDH and increase flux of intermediates into the damaging pathways of

The polyol pathway also results in reduction in the bioavailability of NAD(P)H. The reduced bioavailability of NAD(P)H negatively affects the antioxidant defence system by depleting glutathione (GSH) a very important antioxidant. This is because the activity of GSH reductase, an antioxidant enzyme that generates GSH from its oxidized form (GSSH) depends on NAD(P)H. Depletion of NAD(P)H also decreases the synthesis of nitric oxide (NO), a vaculo‐ protective agent. NAD(P)H serves as a cofactor for nitric oxide synthase (NOS) which synthesizes NO from L-arginine. If endothelial nitric oxide synthase (eNOS) lack its substrate,

this is referred to as ''uncoupled state of nitric oxide''[92]. Nitric oxide performs several physiological roles such as inhibition of platelet activation, vascular relaxation [93] and acts as an anti-inflammatory agent by reducing platelet aggregation and adhesion [94]. These properties inhibit atherogenesis and protect the blood vessel. Reduced bioavailability of NO level will therefore increase inflammation, enhance thrombosis and disrupt the integrity of endothelial cells. Reduction in NO has been documented in diabetes subjects with nephrop‐ athy [95]. Superoxide anion directly quenches NO by forming highly reactive peroxynitrite

) which initiates lipid peroxidation, oxidizes sulfhydryl group in protein and nitrates

amino acids such as tyrosine, thereby affecting many signal transduction pathways. The polyol pathway serves as a main source of ROS generation in the retina [96]. In addition, sorbitol accumulation has been implicated in osmotic swelling of the eye lens and cataractogenesis [97].

**4.2. Advanced glycation end product (AGEs) formation and ROS generation in diabetic**

Glucose can react spontaneously with free amino groups of protein to form Schiff bases. These Schiff bases through complex reactions such as amadori rearrangement, dehydration and condensation forms cross-linked heterogeneous fluorescent derivatives called advanced glycation end products (AGEs). Advanced glycation end products constitute a heterogeneous

O2 -

) instead of NO and

L-arginine or one of its co-factor, it may produce superoxide radical (.

diabetic complications.

30 Antioxidant-Antidiabetic Agents and Human Health

(ONOO-

**complications**

Once formed, AGEs can cause tissue damage by two main pathways which are: (1) formation of cross links that alter protein structure and function and, (2) interaction of AGE with AGEcell surface receptors on the surfaces of various cells such as endothelial cells, macrophages, neurons, and smooth-muscle cells resulting in activation of cell signaling and gene expression that induces oxidative stress and inflammation [98, 99; 102-105]. Oxidative stress can accelerate AGE formation while AGE formation can also amplify the production of more ROS resulting in a vicious cycle of AGE formation and oxidative stress.

AGE's mediate some of their effect via interaction with some receptors that have been shown to bind to these chemical moieties. Among these receptors, Receptor for Advanced Glycation End products (RAGE) is the most extensively studied [106]. Evidence from numerous studies suggest that AGE's are involved in a vicious cycle of inflammation, generation of ROS and increased production of AGE's. Ligand RAGE interaction results in activation of pathways such as p21ras, erk1/2 (p44/p42), MAP kinases, p38 and SAPK/JNK MAP kinases [107-109]. A consequence of the activation of these pathways is the nuclear translocation of transcription factor, Nuclear Factor Kappa B (NF-КB). Translocation of NF-КB to the nucleus increases the transcription of a number of proteins such as, vascular endothelial growth factor (VEGF), monocyte chemoattractant protein-1 (MCP-1), vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1) and pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6, 1L-18 and tumour necrosis factor (TNF)-α which are centrally involved in the endothelial recruitment of neutrophil and subsequent development or progression of atherosclerotic plaque [109-112].

The gene regions of NF-КB are located at the promoter region of RAGE. Moreover, binding of NF-КB to the promoter region of RAGE results in up-regulation of RAGE itsel. Interaction of AGE with RAGE generates more oxidative stress and this further potentiates the formation of AGE's [109, 113]. Generation of ROS by ligand stimulated RAGE activation is mediated at least in part via activation of NADPH oxidase [114]. Other mechanisms by which AGE's may be linked to increased generation of ROS is by reducing the activities of enzymatic antioxidant such as SOD and CAT, lowering of glutathione stores, and activation of PKC [107, 115, 116].

Increased renal AGE in diabetic animals and patients have been linked to structural abnor‐ mality observed in diabetic nephropathy such as mesangial expansion, glomerular basement membrane thickening and tubulointerstitial fibrosis [117]. Advanced Glycation End Product's level is increased with decreased renal function in type 1 diabetic patients [118]. Evidence from clinical studies indicates a correlation between progression of diabetic retinopathy and the level of AGE in serum and retinal blood vessels of diabetic patients [100, 119]. In diabetes, increased AGE's are observed within retinal capillary cells and causes pericyte loss in diabetic retinopathy [120]. AGE's induce toxic effects on retinal pericytes by causing oxidative stress and subsequent apoptosis [121].

High levels of serum AGE's have been documented in patients with type 2 diabetes mellitus and coronary heart disease [122]. Glycation increases susceptibility of low density lipoprotein (LDL) to oxidative modification which is considered a critical step in its atherogenicity [123]. Glycation end products can also enhance atherosclerosis by trapping LDL in the subendothe‐ lium and decrease the recognition of AGE-modified LDL by LDL receptor [124]. Modification of LDL and its increased localization in vessels increases foam cell production and accelerates atherosclerosis development [125]. Oxidative stress induces AGE's formation on collagen leading to cross-linking which is considered to play a role in diabetic cardiomyopathy [126]. The intermolecular collagen cross-linking caused by AGE increases vascular stiffness and interferes with arterial blood flow [127, 128] and this partly explains the diastolic dysfunction and systolic hypertension seen in diabetic subjects.

**5. Antioxidant as therapeutic agents in the management of diabetes**

Despite efforts to control blood glucose, tissue and organ damage are cumulative over many years in most diabetic patients. Varying degrees of hyperglycemia are virtually unavoidable in subjects with diabetes mellitus and glycemic memory has been used to describe the development of diabetes-related complications in diabetic patients even after normoglycemia has been restored and initial glycemic environment is remembered in the target organs [105,147]. It is noteworthy that ROS has been implicated as a major cause of the metabolic memory after glucose normalization due to the chains of reactions leading to cell damage and loss of cellular function. Due to the implication of hyperglycemia-induced oxidative stress in diabetes, these patients should in theory benefit from antioxidant supplementation. The beneficial effect of antioxidants has been reported in animal models of diabetes and in diabetic

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Vitamin E is a fat-soluble vitamin. It has been shown that plasma α-tocopherol concentrations are lower in diabetics compared to controls [58] and appear to be even lower in diabetics with complications such as microangiopathy than in diabetics without complications [81]. Admin‐ istration of Vitamin E has proven to be beneficial in preventing cellular damage by inhibition of lipid peroxidation, protein oxidation, protein glycations and platelet aggregation [149-151] Vitamin E supplementation for two weeks (600 mg/day) lowered urinary F2-isoprostanes (a marker of lipid oxidation) in type 2 diabetics [152]. It was shown in a study that a decrease in plasma F2-isoprostanes was seen in type 2 diabetic patients after six weeks supplementation

Oxidative stress in the kidney of diabetics is usually associated with tissue damage that interferes with proper organ function, causing an increase in urinary protein excretion and blood urea nitrogen (BUN) [154]. Vitamin E supplementation (1000 IU/kg diet) to diabetic rats for 4 weeks significantly reduced urinary protein excretion and BUN suggesting a beneficial effect on kidney function [154]. Inhibitory effect of Vitamin E on glycation of hemoglobin in type I and type 2 diabetic rats has been documented [151, 155]. The ability of vitamin E to inhibit AGE's might be due to its antioxidant effect on the autoxidative pathways of AGE formation [156]. Vitamin E administration has also reduced oxidation of low density lipopro‐

Numerous studies have shown that vitamin E normalized parameters of oxidative stress and inhibited vascular abnormalities caused by hyperglycemia-induced production of DAG and PKC activation in the retina, glomerulus and macrophages [158-160]. Supplementation with vitamin E reduced basement membrane thickening in diabetic rat retina and reduced vascular endothelial growth factor (VEGF) and aldose reductase activity, the abnormalities associated with diabetic retinopathy [161]. Dietary supplementation of vitamin E (2000 IU/kg) to diabetic

**mellitus**

patients [50, 148]

**6. Vitamins**

with Vitamin E [153].

tein (LDL) and development of atherosclerosis [157].

### **4.3. Protein kinase C (PKC) activation and ROS generation in diabetic complications**

PKC activation is related to vasoconstriction, proliferation and overgrowth of smooth muscle cells as well as accelerated synthesis of extracellular matrix proteins, and thus plays significant roles in the onset and progression of vascular cell dysfunction in diabetes mellitus [129-131]. Two major pathways have been implicated in the activation of PKC in hyperglycemia. Persistent and excessive activation of several PKC isoforms result primarily from enhanced *de novo* synthesis of diacylglycerol (DAG) from glucose via increase in triose phosphate availability [90, 105, 132, 133]. There is also evidence that the interaction between AGE's and their cell-surface receptors can result in enhanced activity of PKC isoforms [134, 135].

PKC likely regulates diabetic complications on multiple levels such as activation of eNOS, NAD(P)H oxidase, phospholipase A2 (PLA2), endothelin-1 (ET-1), Vascular endothelial growth factor (VEGF), Transforming growth factor-β (TGF-β), and by activating NF-KB. Diacylgly‐ cerol activated PKC alters the gene expression of key proteins leading to decrease blood flow, capillary occlusion, inflammation, free radicals generation and damage to cellular macromo‐ lecule [130-132, 136, 137].

High glucose levels can stimulate ROS production via a PKC-dependent activation of NAD(P)H oxidase in cultured aortic endothelial cells, smooth muscle cells, and renal mesan‐ gial cells [84]. Nicotinamide adenine dinucleotide phosphate oxidase, which is primarily found in phagocytic cells, is the main source of ROS in non-phagocytic cells such as mesangial cells, endothelial cells [138], fibroblasts [139], podocytes [140] and smooth muscle cells [141]. The expression of NAD(P)H oxidase components is up-regulated in vascular tissues from animal models of diabetes and in patients with diabetes and coronary artery disease [142-144]. Experimental evidence indicates that NAD(P)H oxidase-dependent production of ROS may cause DNA damage in diabetic renal tissues leading to the development of nephropathy [145]. Increased activity of the NAD(P)H oxidase has also been reported in the retina of diabetic rats suggesting its involvement in the development of diabetic retinopathy [146].
