RIVIEW OF LITERATURE

Prevalence of Diabetes
The rapidly increasing prevalence of diabetes mellitus worldwide is one of the most serious and challenging health problems in the 21st century. Over the past 30 yr, the status of diabetes has changed from being considered as a mild disorder of the elderly to one of the major causes of morbidity and mortality affecting the youth and middle aged people. According to recent estimates, approximately 285 million people worldwide (6.6%) in the 2079 year age group will have diabetes in 2010 and by 2030, 438 million people (7.8%) of the adult population, is expected to have diabetes [International Diabetes Federation, 2009].The largest increases will take place in the regions dominated by developing economies. The global increase in the prevalence of diabetes is due to population growth, aging, urbanisation and an increase of obesity and physical inactivity. The primary determinants of the epidemic are the rapid epidemiological transition associated with changes in dietary patterns and decreased physical activity. Unlike in the West, where older populations are most affected, the burden of diabetes in Asian countries is disproportionately high in young to middle-aged adults [Chan et al., 2009; Ramachandran et al., 2010]. Roughly 80% of people with diabetes are in developing countries, of which India and China share the larger contribution [Ramachandran et al., 2010]. It is estimated that the total number of people with diabetes in 2010 to be around 50.8 million in India, rising to 87.0 million by 2030 [International Diabetes Foundation, 2009].

Diabetes mellitus and its types:

Diabetes mellitus (DM) is a metabolic disorder that either arrives during the early years of growth (Juvenile diabetes) or later in life or is called as maturity onset diabetes. It is observed as the bodys inability to effectively regulate the sugar balance which leads to severe complications such as hyperglycaemia, obesity, neuropathy, nephropathy, retinopathy, cardiopathy, osteoporosis and coma leading to death. Pancreatic damage resulting in the dysfunction of and cells causes disordered glucose homeostasis. In diabetic individuals the regulation of glucose levels by insulin is defective either due to defective insulin production which is called as Insulin Dependent Diabetes Mellitus (IDDM) or due to insulin resistance that is termed as Non Insulin Dependent Diabetes Mellitus (NIDDM). IDDM or Type 1 Diabetes mellitus (T1DM) occurs when the insulin-producing -cells in the pancreas are destroyed, typically by an autoimmune, T cell-mediated mechanism, resulting in the production of insufficient amounts of insulin [Rossini et al., 1985]. NIDDM or Type 2 Diabetes mellitus (T2DM) is caused by a resistance to insulin combined with a failure to produce sufficient insulin. T2DM is commonly linked to obesity, which can cause insulin resistance [Kahn, 2001; Kahn & Flier, 2000]. Despite the different pathogenic mechanisms of T1DM and T2DM, they share common symptoms including glucose intolerance, hyperglycaemia, hyperlipidaemia and similar complications. T1DM is an autoimmune disease characterized by a local inflammatory reaction in and around islets that is followed selective destruction insulin-secreting cells [Foulis, 1987; Gepts et al., 1985]. T1DM is characterized by islet inflammation (insulitis), which is the result of infiltration of pancreatic islets with macrophage, dendritic cells, B cells, CD4 + and CD8+ T cells. Prolonged insulitis leads to the preferential amplification of autoreactive CD8 + T cells with high affinity T cell receptors (TCR) [Trudeau et al., 2003]. These high affinity pre-cytotoxic T lymphocytes (CTLs) become differentiated into CTLs via TCR recognition of target peptide-MHC I complexes on local antigen presenting cells (APC). Co-stimulatory pathways involving CD28-B7 are essential in this process [Lenschow et al., 1996]. CD8+ CTLs initiate the immune response via the production of a key component perforin. Non obese diabetic mice lacking the perforin gene develop insulitis but significantly delayed and reduced diabetes, which supports a role of perforin [Kagi et al., 1997]. CD4+ T cells also play important role via Fas/FasL interaction and production of the inflammatory cytokines IFN- and TNF- [Eizirik et al., 2001]. Binding of TNF- and IFN- to their cognate receptors on -cells induces apoptosis through caspase cleavage or NO

production via regulation of inducible NO synthase (iNOS) [Stephens et al., 1999; Eizirik et al., 1996]. IFN- also enhances IL-1 production by APC which is also toxic to -cells [Papaccio et al., 2005]. Proinflammatory cytokines, TNF-, IFN-, and IL-1, can also upregulate Fas expression on -cells, facilitating cell recognition, and also stimulate Nitric oxide and reactive oxygen species (ROS) production, exacerbating cell death [McKenzie et al., 2006; Wachlin et al.,2003; Kwon et al., 1999; Kim et al., 2005]. Similar to T1DM, T2DM is also characterised by progressive loss of -cells through multiple pathways. These mechanisms include increased circulating cell nutrients [Maedler et al.,2001; Roehrich et al., 2003], endoplasmic reticulum (ER) stress [Harding & Ron, 2002], signalling factors from adipocytes [Zhao et al., 2006] and more recently, infiltration of immune cells. Plasma free fatty acid (FFA) levels are permanently increased in obesity, which causes insulin resistance and diminishes glucose uptake, resulting in transient postprandial hyperglycemic excursions. Elevated glucose causes oxidative stress due to increased production of mitochondrial ROS [Brownlee, 2001], nonenzymatic glycation of proteins [Brownlee & Cerami, 1981; Brownlee, 2000], and glucose autoxidation [Wolff & Dean, 1987; Wolff et al., 1991].The toxic effect of FFA is manifested as oxidative stress via formation of ceramide, increased nitric oxide production, mitochondrial uncoupling [Wojtczak & Schonfeld, 1993; Carlsson et al., 1999] and -oxidation [Yamagishi et al., 2001; Rao & Reddy, 2001], leading to activation of the apoptotic mitochondrial pathway [Maedler et al., 2003; Shimabukuro et al., 1998].This mild hyperglycemia could act on the -cells even before diabetes manifests itself or at the very early stages of the disease. Therefore both nutrients, FFAs and glucose, may interfere with -cell turnover and function influencing the onset and course of diabetes. ER is stressed by disturbances in cellular redox regulation, glucose deprivation, high fat diet and protein misfolding [Yoshida, 2007; Kim et al., 2008] that cause apoptosis of the -cells. Adipocyte secreted factors are leptin, TNF, IL-6 and IL-1 receptor antagonist (IL-1 Ra) [Fried et al., 1998; Hotamisligil et al., 1993; Meier et al., 2002; Zhang et al., 1994], whose expression are upregulated in obesity and have been causally linked to insulin resistance. Increase serum levels of IL-1, IL-6, and TNF- is also contributed by macrophages and endothelium in Type 2 diabetics patients [Pickup & Crook, 1998]. These cytokines may act on the pancreatic islets and impair -cell secretory function.

Insulin and Insulin like growth factor:

The effect of insulin receptor signaling on the -cell has been assessed in both in vivo and in vitro models. Insulin infusion in mice induces -cell proliferation and increases -cell mass and this effect is augmented by concomitant glucose infusion [Paris et al., 2003]. Mice deficient in the insulin receptor in -cells exhibit hyperglycemia and reduced -cell mass with age [Kulkarni et al., 1999]. MIN6 cells with 80% knockdown of the insulin receptor exhibit decreased proliferation, suggesting that insulin is a growth factor at least for this insulinoma cell line [Ohsugi et al., 2005]. Insulin and Insulin like growth factor I (IGF-I) bind to the insulin and IGF-I receptor respectively, but can each also cross-react with the other receptor. Glucose enhances IGF-I mediated proliferation of insulinoma cells in culture and this process is phosphoinositide 3-kinase (PI3K) dependent [Hugl et al., 1998]. Interestingly, overexpression of IGF-I in -cells is associated with increased -cell proliferation, but not mass [George et al., 2002]. In contrast, transgenic mice overexpressing Insulin like growth factor II (IGF-II) exhibit an increase in -cell mass due to augmented proliferation [Petrik et al., 1999]. IRS2 signaling leads to stimulation of the PI3 kinase/Akt and ERK1/2 pathways [Kulkarni, 2005]. Akt, also known as protein kinase B (PKB), has been proposed to be one of the critical mediators of many IRS2 signals in -cells. Mice deficient in AKT2 develop insulin resistance and diabetes due to impaired adaptation of -cells, providing evidence for the importance of Akt signaling in cells [Garofalo et al., 2003]. Moreover, overexpression of Akt in -cells induces -cell mass by augmented proliferation, cell size and resistance to apoptosis [Tuttle et al., 2001; Bernal-Mizrachi et al., 2001].Taken together; there is clear evidence that the IGF molecules are important factors in -cell proliferation and mass.

Insulin regulates gene expression through MAP Kinase pathway. Insulin initiates a signal that travels a branched pathway from the plasma membrane receptor to insulin-sensitive enzymes in the cytosol and to the nucleus, where it stimulates the transcription of specific gene. The Insulin receptor INS-R is a receptor enzyme with tyrosine kinase activity. Insulin signalling pathway begins when binding of insulin to INS-R activates its Tyrosine kinase activity and each subunit phosphorylates three critical tyrosine residues near the carboxyl terminus of the other subunit in the () 2 dimer. The kinase catalyzes the phosphorylation of tyrosine residue on other protein such as IRS-1. Phosphotyrosine residues of IRS-1 binds to the SH2 (Src homology-2) domain of the protein Grb2, which act as an adaptor protein to bring together IRS-1 and Sos protein. Sos bound to Grb2 catalyzes GDP-GTP exchange on Ras (a small G protein), which in turn activates a MAPK cascade that ends with the phosphorylation of target proteins in the cytosol and nucleus. The result is specific metabolic changes and altered gene expression. The enzyme PI-3K (Phosphoinositide 3 kinase), activated by interaction with IRS-1, converts the membrane lipid PIP2 (Phosphatidylinositide 4,5-bisphosphate) to PIP3 (Phosphatidylinositide 3,4,5triphosphate), which becomes the point of nucleation for proteins in a second and third branch of insulin signalling [Lehninger 5th Edition, 2008 A].

Diabetes (Type 1 & 2) and Oxidative Stress

The human body is exposed to free radicals from outside the body (exogenous) and inside the body (endogenous). Some of the factors that lead to free radicals are smog, cigarette smoke, radiation, consumption of excessive amounts of alcohol, and even sunlight. Yet, some factors that led to free radicals come from within the body. The cells necessitate oxygen to produce the energy they need to work properly. In the process known as mitochondrial respiration, the cells take in oxygen, burn it, and release energy. During the process, free radicals are produced. Oxidative stress occurs when free radical production exceeds the bodys ability to neutralize them. This imbalance happens for one of two reasons: a) when the antioxidant production is decreased, or, b) when the free radicals are produced in excess. For instance diabetes, or the aging process itself, can direct to increased speed of the production of these endogenous free radicals and reduced antioxidant resistance. The cause of oxidative stress in diabetes has been, and continues to be, the object of considerable clinical investigation. Oxidative stress has the ability to lower the insulin sensitivity and injure the insulin producing cells within the pancreas. Adipose tissue and muscle are the most significant tissues participating in the development of insulin resistance. Oxidative stress modifies the signaling pathway within a cell installing insulin resistance [Evans et al., 2003]. In both type 1 and type 2 diabetes, diabetic complications in target organs arise from chronic elevations of glucose. The pathogenic effect of high glucose, possibly in concert with fatty acids, is mediated to a significant extent via increased production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) and subsequent oxidative stress. ROS and RNS directly oxidize and damage DNA, proteins, and lipids. In addition to their ability to directly inflict damage on macromolecules, ROS and RNS indirectly induce damage to tissues by activating a number of cellular stress sensitive pathways. These pathways include nuclear factor-kappaB, p38 mitogen-activated protein kinase, NH (2)-terminal Jun kinases/stress-activated protein kinases, hexosamines, and others. In addition, there is evidence that in type 2 diabetes, the activation of these same pathways by elevations in glucose and free fatty acid (FFA) levels leads to both insulin resistance and impaired insulin secretion. Therefore, it is proposed that the hyperglycemia-induced, and possibly FFA-induced, activation of stress pathways plays a key role in the development of not only the late complications in type 1 and type 2 diabetes, but also the insulin resistance and impaired insulin secretion seen in type 2 diabetes [Evans et al., 2003]. Oxidative stress functions on both sides, meaning that it help the progression and the development of diabetes and its complications [Ha and Lee, 2000]. Oxidative stress is one of the important mediators of vascular complications in diabetes including nephropathy [Ha and Lee, 2000]. High glucose produces reactive oxygen species as a result of glucose auto-oxidation, metabolism, and the development of advanced glycosylation end products. The concept of reactive oxygen species-induced tissue injury has currently been modified with the appreciation of new roles for reactive oxygen species in signaling pathways and gene expression.

Mechanisms of Hyperglycemia-Induced Damage:

The hyperglycemia causes damage of the vascular tissues of diabetic patients through five major mechanisms. All these five mechanisms are activated by a single upstream event: mitochondrial overproduction of the ROS. These mechanisms are briefly discussed below:

Increased Polyol Pathway Flux

The polyol pathway is based on a family of aldo-keto reductase enzymes such as aldose reductase that use a wide variety of carbonyl compounds as substrates and reduce these by NADPH to their respective sugar alcohols (polyols). In tissues such as nerve, retina, lens, glomerulus, and vascular cells, aldose reductase convert glucose to sorbitol, which then oxidized to fructose by the enzyme sorbitol dehydrogenase (SDH), with NAD+ as a cofactor [Ramasamy et al., 2010]. Redox stress is increased in polyol pathway because of NADPH consumption, which is required to regenerate reduced glutathione (GSH), and GSH is an important scavenger of ROS, this could induce or exacerbate intracellular oxidative stress.

Increased Intracellular Advanced Glycation End (AGE) Product

AGEs are formed by the nonenzymatic reaction of glucose and other glycating compounds derived both from glucose and from increased fatty acid oxidation in arterial endothelial cells and most likely heart (eg, dicarbonyls such as 3deoxyglucosone, methylglyoxal, and glyoxal) with proteins [Wautier & Schmidt, 2004; Candido et al., 2003]. Plasma proteins modified by AGE precursors bind to AGE receptors on cells such as macrophages, vascular endothelial cells, and vascular smooth muscle cells. Receptor for AGE (RAGE) binding induces the production of ROS, which in turn activates the pleiotropic transcription factor nuclear factor (NF)-B, causing multiple pathological changes in gene expression [Goldin et al., 2006].

Increased Protein kinase C (PKC) Activation

PKC family proteins are widely distributed in mammalian tissues and phosphorylate various target proteins. The activity of PKC is dependent on both Ca2+ ions and phosphatidyl serine and is greatly enhanced by diacylglycerol (DAG) [Geraldes et al., 2010]. Persistent and excessive activation of several PKC isoforms operates as a third common pathway mediating tissue injury induced by diabetes-induced ROS. This results primarily from enhanced de novo synthesis of DAG from glucose via triose phosphate, whose availability is increased because increased ROS inhibit activity of the glycolytic enzyme GAPDH, raising intracellular levels of the DAG precursor triose phosphate [Geraldes & King, 2010; Inoguchi et al., 1992; Craven et al., 1990; Shiba et al., 1993; Scivittaro et al., 2000]. The interaction between AGEs and their cell-surface receptors also enhance the activity of PKC [Derubertis & Craven, 1994]. In the diabetic retina, hyperglycemia persistently activates protein kinase C and p38 mitogen-activated protein kinase (MAPK) to increase the expression of a target of PKC signaling, SHP-1 (Src homology-2 domaincontaining phosphatase-1), a protein tyrosine phosphatase. This signaling cascade leads to platelet-derived growth factor (PDGF) receptor- dephosphorylation and a reduction in downstream signaling from this receptor, resulting in pericyte apoptosis [Geraldes et al., 2009]. The same pathway, activated by increased fatty acid oxidation in insulin-resistant arterial endothelial cells and heart, may play an equally important role in diabetic atherosclerosis and cardiomyopathy.

Increased Hexosamine Pathway Flux

Hyperglycemia and insulin resistanceinduced excess fatty acid oxidation contribute to the pathogenesis of diabetic complications by increasing the flux of fructose 6-phosphate into the hexosamine pathway [Sayeski & Kudlow, 1996; Kolm-Litty et al., 1998; Chen et al., 1998; Du et al., 2000]. In hexosamine pathway, glutamine:fructose 6-phosphate amidotransferase (GFAT) converts fructose 6-phosphate to glucosamine 6-phosphate, which is then converted to UDP-

N-acetylglucosamine. Specific O-linked N-acetylglucosamine (O-GlcNAc) transferases use this for posttranslational modification of specific serine and threonine residues on cytoplasmic and nuclear proteins by O-GlcNAc.

Role of Mitochondria on Oxidative Stress

Specific inhibitors of aldose reductase activity, AGE formation, RAGE ligand binding, PKC activation, and hexosamine pathway flux each ameliorate various diabetes-induced abnormalities in cell culture or animal models, but it has not been clear whether these processes are interconnected or might have a common cause [Ganz & Seftel, 2000; Engerman et al., 1994; Sima et al., 1990; Lee et al., 1995; Browlee, 1995] It has now been established that all of the different pathogenic mechanisms described above stem from a single hyperglycemia-induced process, namely overproduction of superoxide by the mitochondrial electron-transport chain [Du et al., 2000; Nishikawa et al., 2000] . Superoxide is the initial oxygen free radical formed by the mitochondria, which is then converted to other more reactive species that can damage cells in numerous ways [Wallace, 1992]. Normally, electron transfer through complexes I, III, and IV extrudes protons outward into the intermembrane space, generating a proton gradient that drives ATP synthase (complex V) as protons pass back through the inner membrane into the matrix. In contrast, in diabetic cells with high intracellular glucose concentration, there is more glucose-derived pyruvate being oxidized in the TCA cycle, increasing the flux of electron donors (NADH and FADH 2) into the electron transport chain. As a result, the voltage gradient across the mitochondrial membrane increases until a critical threshold is reached. At this point, electron transfer inside complex III is blocked [Trumpower, 1990], causing the electrons to back up to coenzyme Q, which donates the electrons one at a time to molecular oxygen, superoxide thereby (Figure generating 1). The

mitochondrial isoform of the enzyme SOD degrades this oxygen free radical to hydrogen peroxide, Figure 1: Production of ROS by the mitochondrial electron transport chain. which is then converted to H2O and O2 by other enzymes. In primary arterial endothelial cells in culture, intracellular hyperglycemia increases the voltage across the mitochondrial membrane above the critical threshold necessary to increase superoxide formation [Korshunov, 1997] and, subsequently, increases production of ROS. It has been also demonstrated that dynamic changes in mitochondrial morphology are associated with high glucose-induced overproduction of ROS. Inhibition of mitochondrial fission prevented periodic fluctuation of ROS production during high glucose exposure [Yu et al., 2006]. Neither hyperglycemia nor increased fatty acid oxidation in vascular endothelium increases ROS nor activates any of the pathways when either the voltage gradient across the mitochondrial membrane is collapsed by uncoupling protein 1 (UCP-1) or when the superoxide produced is degraded by manganese super oxide dismutase (MnSOD) [Du et al., 2001]. Although it thus appears that the mitochondria are required for the initiation of hyperglycemia-induced superoxide production, much evidence indicates that this, in turn, can activate a number of other superoxide production pathways that may amplify the original damaging effect of hyperglycemia. These include redox changes, NADPH oxidases, and uncoupled endothelial nitric oxide synthase (eNOS). Diabetes in animals and patients, and hyperglycemia in cells, all decrease the activity of the key glycolytic enzyme Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in cell types that develop intracellular hyperglycemia. Hyperglycemia-induced superoxide inhibits GAPDH activity in vivo by modifying the enzyme with polymers of ADP-

ribose [Du et al., 2003]. Inhibition of GAPDH activity by hyperglycemia does not occur when mitochondrial overproduction of superoxide is prevented by either UCP-1 or MnSOD [Nascimento et al., 2006]. When GAPDH activity is inhibited, the levels of all the glycolytic intermediates those are upstream of GAPDH increase. This then increases the flux into the 5 pathways described earlier. Along with UCP-1 or MnSOD, both modification of GAPDH by poly (ADP-ribose) and reduction of its activity by hyperglycemia were prevented by a specific inhibitor of poly (ADP-ribose) polymerase (PARP), the enzyme that makes these polymers of ADP-ribose. Normally, PARP resides in the nucleus in an inactive form, waiting for DNA damage to activate it. When increased intracellular glucose generates increased ROS in the mitochondria, free radicals induce DNA strand breaks, thereby activating PARP. Once activated, PARP splits the NAD molecule into its 2 component parts: nicotinic acid and ADP-ribose. PARP then proceeds to make polymers of ADP-ribose, which accumulate on GAPDH and other nuclear proteins.

Oxidative damage to DNA, Lipids and Proteins

At high concentrations, ROS can be important mediators of damage to cell structures, nucleic acids, lipids and proteins [Valko et al., 2006]. The hydroxyl radical is known to react with all components of the DNA molecule, damaging both the purine and pyrimidine bases and also the deoxyribose backbone [Halliwell & Gutteridge, 1999]. The most extensively studied DNA lesion is the formation of 8-OH-G. Permanent modication of genetic material resulting from these oxidative damage incidents represents the rst step involved in mutagenesis, carcinogenesis, and ageing. It is known that metal-induced generation of ROS results in an attack not only on DNA, but also on other cellular components involving polyunsaturated fatty acid residues of phospholipids, which are extremely sensitive to oxidation [Siems, et al., 1995]. Once formed, peroxyl radicals (ROO) can be rearranged via a cyclisation reaction to endoperoxides (precursors of malondialdehyde) with the nal product of the peroxidation process being malondialdehyde (MDA) [Fedtke et al., 1990; Fink et al., 1997; Marnett, 1999; Wang et al., 1996].The major aldehyde product of lipid peroxidation other than malondialdehyde is 4-hydroxy- 2-nonenal (HNE). Mechanisms involved in the oxidation of proteins by ROS were elucidated by studies in which amino acids, simple peptides and proteins were exposed to ionising radiations under conditions where hydroxyl radicals or a mixture of hydroxyl/superoxide radicals are formed [Stadtman, 2004]. The side chains of all amino acid residues of proteins, in particular cysteine and methionine residues of proteins are susceptible to oxidation by the action of ROS/RNS [Stadtman, 2004]. Oxidation of cysteine residues may lead to the reversible formation of mixed disulphides between protein thiol groups (SH) and low molecular weight thiols, in particular GSH (S-glutathiolation). The concentration of carbonyl groups, generated by many different mechanisms is a good measure of ROS-mediated protein oxidation.

Biomarkers of Oxidative Stress

Lipid Peroxidation
Hydroperoxides have toxic effects on cells both directly and through degradation to highly toxic hydroxyl radicals. They may also react with transition metals like iron or copper to form stable aldehydes such as malondialdehydes (MDA) that will damage cell membranes. Peroxyl radicals can remove hydrogen from lipids, producing hydroperoxides that further propagate the free-radical pathway . Induction of diabetes in rats with streptozotocin (STZ) or alloxan uniformly results in an increase in thiobarbituric acid reactive substances (TBARS), an indirect evidence of intensied free-radical production. Preventing the formation of hydroxyl radicals would be an efcient means to reduce hydroxyl induced damage, and several compounds have been tested as antioxidants in diabetic animals with varying success.

Glutathione Peroxidase and Glutathione Reductase

Glutathione peroxidase and reductase are two enzymes that are found in the cytoplasm, mitochondria, and nucleus. Glutathione peroxidase metabolizes hydrogen peroxide to water by using reduced glutathione as a hydrogen donor.

Glutathione disulde is recycled back to glutathione by glutathione reductase, using the cofactor NADPH generated by glucose 6-phosphate dehydrogenase. There is not total agreement about the effects of diabetes on the activities of these enzymes. However, glutathione peroxidase activity is seen to be elevated in liver [Rauscher et al., 2001; Rauscher et al., 2001; Rauscher et al., 2000; Rauscher et al., 2000; Aragno et al., 1999; Sanders et al., 2001], kidney [Rauscher et al., 2001; Rauscher et al., 2001; Rauscher et al., 2000; Aragno et al., 1999; Kedziora-Kornatowska et al., 2000], aorta [Kocak et al., 2000], pancreas [Jang et al., 2000], blood [Mohan & Das, 1998; El-Khatib et al., 2001; Kedziora-Kornatowska iet al., 1998], and red blood cells [Sailaja et al., 2000], whereas decreased activity was seen in heart [Kaul et al., 1995; Kaul et al., 1996] and retina [Obrosova et al., 2000].

Super Oxide Dismutase (SOD)

Isoforms of SOD are variously located within the cell. CuZn-SOD is found in both the cytoplasm and the nucleus. Mn-SOD is conned to the mitochondria, but can be released into extracellular space [Reiter et al., 2000]. SOD converts superoxide anion radicals produced in the body to hydrogen peroxide, thereby reducing the likelihood of superoxide anion interacting with nitric oxide to form reactive peroxynitrite. Catalase Catalase, located in peroxisomes, decomposes hydrogen peroxide to water and oxygen [88]. Catalase activity is consistently found to be elevated in heart [Kaul et al., 1995; Kaul et al., 1996; Rauscher et al., 2001; Rauscher et al., 2001; Rauscher et al., 2000; Sanders et al., 2001; Stefek et al., 2000] and aorta [Ozansoy et al., 2001; Kocak et al., 2000], as well as brain [Aragno et al., 1999] of diabetic rats. In contrast to decreased renal [Maritim et al., 1999; Kedziora-Kornatowska et al., 2000; Mekinova et al., 1995], hepatic [Rauscher et al., 2001; Maritim et al., 1999; Sanders et al., 200] and red blood cell [Kedziora-Kornatowska et al., 1998] catalase activity, catalase activity in liver [Aragno et al., 1999; Caballero et al., 2000] and kidney [Aragno et al., 1999] of diabetic animals is increased.

Glutathione level
Reduced glutathione is a major intracellular redox buffer that may approach concentrations up to 10 mM. Glutathione functions as a direct free-radical scavenger, as a co-substrate for glutathione peroxidase activity, and as a cofactor for many enzymes, and forms conjugate in endo- and xenobiotic reactions. Glutathione concentration is found to be decreased in the liver [Ewis & Abdel-Rahman, 1995], kidney [Ha & Lee, 2000], pancreas, plasma, red blood cells, nerve, and pre-cataractous lens of chemically induced diabetic animals. However, there is also some contradictory evidence of increased glutathione concentration in diabetic rat kidney and lens.

Vitamins
Vitamins A, C, and E are diet-derived and detoxify free radicals directly. They also interact in recycling processes to generate reduced forms of the vitamins. -Tocopherol is reconstituted when ascorbic acid recycles the tocopherol radical; dihydroascorbic acid, which is generated, is recycled by glutathione. These vitamins also foster toxicity by producing prooxidants under some conditions. Vitamin E, a component of the total peroxyl radical-trapping antioxidant system, reacts directly with peroxyl and superoxide radicals and singlet oxygen and protects membranes from lipid per-oxidation. The deciency of vitamin E is concurrent with increased peroxides and aldehydes in many tissues. There have been conicting reports about vitamin E levels in diabetic animals and human subjects. Plasma and /or tissue levels of vitamin E are reported to be unaltered, increased, or decreased by diabetes.

Diabetes, Oxidative Stress and Apoptosis

Hyperglycemia has been linked to microvascular disease in patients with diabetes [The Diabetes Control and Complications Trial 1993; U.K. Perspective Diabetes Study 1998]. Although less clear-cut, a relationship between hyperglycemia and macrovascular disease [Laakso, 1999] has also been reported. Hyperglycemia increases apoptosis in cultured endothelium was first demonstrated by Barmgartner-Parzer [Barmgartner-Parzer et al., 1995] in human umbilical vein endothelial cells (HUVECs) and since then by many others [Du et al., 1998; Ho et al., 2000; Wu et al., 1999]. It has been reported that hyperglycemia directly induces apoptosis of endothelial cells [Barmgartner-Parzer et al., 1995] neural cells [Barber et al., 1998]. In diabetic mouse retina, neurons ganglion cell layer die, and this death occurs thrugh an apoptotic pathway [Pamela et al., 2004]. The retinal endothelial cells incorporate glucose via glucose transporter-1 [Boado & Pardridge, 1990], leading to intracellular accumulation of glucose which leads to the activation of various pathways, that mirror other microvascular complications. This endothelial cell dysfunction arises as a result of impaired NO bioavailability, oxidative stress, increased polyol pathway activation and AGE formation, which in combination lead to activation of intracellular signalling pathways involving Protein kinase C (PKC), Protein kinase B (PKB), and Mitogen activated protein kinase (MAPK), finally culminating in induction of the pro-inflammatory transcription factors such as NFB. NF-B is a widely expressed inducible transcription factor that is an important regulator of many genes involved in mammalian inflammatory and immune responses, proliferation and apoptosis. Chronic hyperglycemia induced oxidative stress has been cited as another critical mediator of cell death, and may either trigger or modulate apoptosis of pancreatic -cell. Transient increases in glucose levels within physiological range induce insulin secretion and potentially beneficial signals. In contrast, glucotoxicity induced by prolonged hyperglycemia causes -cell dysfunction and altered -cell mass. In animal models and humans, chronic hyperglycemia is associated with alterations in -cell mass and function. The -cell has an incredible ability to adapt and compensate for chronic hyperglycemia, as seen in the Zucker diabetic fatty (ZDF) rat, but ultimately, obesity, chronic hyperglycemia, and worsening insulin resistance lead to increased -cell apoptosis. Proposed mechanisms by which glucotoxicity act include mitochondrial dysfunction with production of reactive oxygen species (ROS), endoplasmic reticulum (ER) stress and increased levels of intracellular calcium. Chronic hyperglycemia leads to long-term increases in cytosolic Ca2+ that may be proapoptotic and induce -cell dysfunction. Incresed Apoptosis has been observed in the hearts of Streptozotocin-induced diabetic rats, which was prevented by angiotensin II blockade in vivo [Fiordaliso et al., 2000]. Hyperglycemia directly induces apoptotic cell death in the mice myocardium in vivo [Lu cai et al., 2002]. Enhanced apoptosis has also been demonstrated in adult rat ventricular myocyters (AVRM) cultured cells induced by hyperglycemia in a PKC- dependent manner. Reactive oxygen species (ROS) production regulated by PKC- is in part responsible for the induction of apoptosis by hyperglycemia [Yukitaka et al., 2002]

The cell death machine: Apoptosis

The word Apoptosis comes from the ancient Greek word , meaning the falling off of petals from a flower or of leaves from a tree in autumn. The name was first introduced by John Kerr [Kerr et al., 1972] in 1972 and refers to the morphological feature of formation of apoptotic bodies from a cell. Apoptosis is associated with a distinct set of biochemical and physical changes involving the cytoplasm, nucleus and plasma membrane. Early in apoptosis, the cells round up, losing contact with their neighbours, and shrink. In the cytoplasm, the endoplasmic reticulum dilates and the cisternae swell to form vesicles and vacuoles. In the nucleus, chromatin condenses and aggregates into dense compact masses, and fragmented internucleosomally by endonucleases, which can be analyzed by the typical DNA ladder formation in apoptosis, for which DNA (either total or cytosolic) is extracted from the cells and separated in an agarose gel [Johnson et al., 1996]. The nucleus becomes convoluted and buds off

into several fragments, which are encapsulated within the forming apoptotic bodies. In the plasma membrane, cell junctions are disintegrated, whereby the plasma membrane becomes active and convoluted, eventually blebbing. The cells Breaks up in a florid manner leading to the falling away of several membrane spheres containing the packaged cellular contents identified as apoptotic bodies of various size [Kerr et al., 1994]. Since the apoptotic bodies are surrounded by an intact plasma membrane, apoptosis is usually occurs without leakage of cell content and usually without inflammation. This form of physiological cell death is morphologically quite different from oncosis, in which the cell swell and disintegrates in an unordered manner, eventually leading to the destruction of cellular organelles and finally rupture of the plasma membrane and leakage of the cell content, which signs necrosis. Apoptosis process requires two very important proteins to occur, without which it is impossible. They are Bcl-2 family protein and Caspase. The Bcl-2 proteins are again subdivided into anti-apoptotic members, Bcl-2 and Bcl-XL, and pro-apoptotic members, Bax, Bak, Bad, Bim, Bid, and Bik. Bcl-2 is the first discovered proto-oncogene and its oncogene characteristics are due to its ability to prevent apoptosis (rather than stimulate proliferation). It was reported to act in an antioxidant manner. The anti-apoptotic protein, Bcl-2 and Bcl-XL contains a C-terminal membrane anchor and the four Bcl-2 homology (BH1-BH2) domains. The pro-apoptotic members, Bax and Bak lack some of the four BH domains and Bad, Bik, Bid, and Bim contains only the BH3 domain. The relative levels of pro- and anti-apoptotic proteins determine a cells susceptibility to apoptosis [Korsmeyer, 1995]. There are fourteen mammalian caspases identified to date [Strasser et al., 2000]. Caspase is so named because it contain a key cysteine residue in the catalytic site and selectively cleave proteins at sites just C-terminal to aspartate residues. They are synthesized as pro-enzymes, which usually undergo proteolysis and activation by other caspases in a cascade [Earnshaw et al., 1999]. Caspases can be grouped into subclasses in various ways. Functionally, we can distinguish three classes of caspases; (i) the initiator caspases that are characterized by long prodomains (> 90 amino acids) containing either death effector domain (DED) domains (caspase-8 and caspase-10) or a caspase recruitment domain (CARD) (caspase2 and caspase-9); (ii) the executioner or effector caspases containing short prodomains (caspase-3, caspase-6 and caspase-7) and (iii) the remaining caspases which main role lies in cytokine maturation rather than apoptosis [Grutter, 2000]. Apoptosis can be triggered by a wide variety of cellular stresses, including DNA damage, radiation, ionizing radiation, heat shock and oxidative stress, as well as by extracellular stimuli acting through cell-surface receptors. Nevertheless, most cells undergoing apoptosis exhibit a similar series of changes, suggesting that these signals converge to engage a common pathway leading to cell death. The regulatory mechanisms that trigger apoptosis involve some of the same proteins that regulate the cell cycle. The signal for suicide often comes from outside, through a surface receptor. Tumor necrosis factor (TNF), produced by cells of the immune system, interacts with cells through specific TNF receptors. These receptors have TNFbinding sites on the outer face of the plasma membrane and a "death domain" (-80 amino acid residues) that carries the self-destruct signal through the membrane to cytosolic proteins such as TRADD (TNF receptor-associated death domain). Another receptor, Fas, has a similar death domain that allows it to interact with the cytosolic protein FADD (flas-associated death domain), which activates the cytosolic protease caspase 8. When caspase 8, an "initiator" caspase, is Figure 2: Extrinsic pathway of apoptosis mediated

activated by an apoptotic signal carried through FADD, it further self-activates by cleaving its own proenzyrne form. Mitochondria are one target of active caspase 8. The protease causes the release of certain proteins contained between the inner and outer mitochondrial membranes:cytochrome c and several effector caspases. Cytochrome c binds to the proenzyrne form of the effector enzyme caspase 9 and stimulates its proteolytic activation. The activated caspase 9 in turn catalyzes wholesale destruction of cellular proteins-a major cause of apoptotic cell death. This pathway of apoptosis is known as extrinsic or receptor mediated pathway (Figure 2) [Lehninger 5th Edition 2008, B].

Another pathway (also called intrinsic or Bcl-2 regulated) involved in the activation of apoptosis is carried out through mitochondria under oxidative stress and treatment with cytotoxic drugs. Mitochondria play a critical role in triggering

apoptosis. When a stressor gives the signal for cell death, one early consequence is an increase in the permeability of the outer mitochondrial membrane, allowing cytochrome c to escape from the intermembrane space into the cytosol (Figure 3)

The increased permeability is due to the opening of the permeability transition pore complex (PTPC), a multisubunit protein in the outer membrane; its opening and closing are affected by several proteins that stimulate or suppress apoptosis. When released into the cytosol,

cytochrome c interacts with monomers of the protein Apaf-l (apoptosis protease activating factor-l), causing the formation of an apoptosome composed of seven Apaf-l and seven cytochrome c molecules.The

apoptosome provides the platform on which the protease procaspase-9 is activated to caspase-9, and activated caspase-9 initiates a cascade of proteolytic activations, with one caspase activating a second, and it in turn activating a third, and so forth [Lehninger 5th Edition 2008, C] Figure 3: Intrinsic pathway of apoptosis Endoplasmic reticulum (ER), a highly dynamic organelle with a central role in lipid and protein biosynthesis, also plays important role in apoptosis. The translation of proteins is performed by ribosomes on the cytosolic surface of the ER [Adesnik et al., 1976], and the unfolded polypeptide chains are translocated into the ER lumen, where they are often Nglycosylated and folded into secondary and tertiary structures that are stabilized by disulfide bonds [Kornfeld & Kornfeld, 1985]. The unique oxidizing environment of the ER and the numerous protein chaperones present in the organelle are crucial for the proper folding of proteins and protein complexes [Gething & Sambrook, 1992] The ER is exquisitely sensitive to alterations in homeostasis, and proteins formed in the ER may fail to attain correct conformation due to: 1) lack of chaperones or cellular energy to promote chaperone-protein interactions; 2) Ca2 depletion; 3) disruption of redox state; 4) protein mutations that hamper adequate folding; and 5) reduction of disulfide bonds. Accumulation of misfolded proteins that aggregate in the ER lumen causes ER stress and activation of a signal response termed the unfolded protein response (UPR) [Patil & Walter, 2001; Marciniak & Ron, 2006; Zhang & Kaufman, 2006; Ron & Walter, 2007]. The aim of the UPR is to alleviate ER stress, restore ER homeostasis, and prevent cell death. To achieve these goals, the UPR induces several coordinated responses, including: 1) a decrease in the arrival of new proteins into the ER, thus preventing additional protein misfolding and overloading of the organelle; 2) an increase in the amount of ER chaperones, thus augmenting the folding capacity of the ER to deal with misfolded proteins; 3) an increase in the extrusion of irreversibly misfolded proteins from the ER and subsequently degradation of these proteins in the proteasome; and 4) in case the steps described above fail, apoptosis is triggered. In case the UPR fails to solve ER stress, the apoptosis pathway will be activated.
+

Antioxidant treatment and Diabetes

Given the involvement of oxidative stress in diabetes complications, supplementation with antioxidants could be of interest, by allowing a delay in the appearance or in the development of vascular complications[Hayoz et al., 1998 ]. Some information is available on the effects of treatments with classical antioxidants such as vitamin E, vitamin C or lipoic acid. Treatment of streptozotocin-induced diabetes pregnant rats with vitamin E resulted in a decrease of plasma lipid peroxidation in comparison with untreated animals, associated with higher cell glutathione content and SOD activity, thus suggesting that vitamin E supplementation could in part reduce the imbalance between oxidants and antioxidants [Kinalski et al., 1999 ]. It is noteworthy that vitamin E supplementation in streptozotocin-induceddiabetic rats protects against lipid peroxidation and contributes to avoid elevation of plasma glucose levels, whereas vitamin E deciency leads to increased hepatic TBARS levels in these diabetic rats [Kinalski et al., 1999]. A vitamin E supplementation in diabetic patients results in an improvement of the insulin effect and a better glycemic control [Sharma et al., 2000; Jain et al., 1996; Paolisso et al., 1993], by reducing glucose, hemoglobin A1c and fructosamin values, by decreasing plasma lipid peroxidation and LDL oxidizability [Anderson et al., 1999; Jain et al., 1996]. In addition, an antioxidant treatment using vitamin E, vitamin C and N-acetylcysteine in diabetic mice can preserve beta-cell function [Kaneto et al., 1999]. Thus, this antioxidant treatment preserves the amounts of insulin content and insulin mRNA, and expression of a -cell-specic transcription factor is more clearly visible in the nuclei of islets cells after this treatment. Since the advanced protein glycation and oxidative stress could result in a lot of metabolic disorders in diabetes [Kennedy & Lyons, 1997; Brownlee, 1992], the design of molecules which can inhibit AGE formation and scavenge free radicals could be of great interest. Aminoguanidine (also called pimagedine) is a nucleophilic hydrazine which reacts with early protein glycation products, such as Amadori products. It inhibits the formation of AGEs such as pentosidine [Nilsson, 1999 ] which lead to intra- and inter-molecular bindings in proteins with long half-life such as collagen [Brownlee et al.,1986 ]. Aminoguanidine and betaresorcylene aminoguanidine reduce the formation of AGEs, inhibit malondialdehyde formation in erythrocytes incubated with hydrogen peroxide, and thus may have therapeutic potential [Jakus et al., 1999]. In vivo, a treatment with aminoguanidine in animals inhibits the development and the progression of the main diabetes complications such as retinopathy, nephropathy and neuropathy [Guillausseau, 1994]. Finally, oral antidiabetic agents themselves can have a potential antioxidant activity. Thus, in high fructose-fed rats (a diet that leads to insulin resistance), metformin is able to improve erythrocyte antioxidant activities (Cu, Zn-SOD, GSHPx) [Faure et al ., 1999]. It also increases blood glutathione level that is classically low in diabetic animals [Faure et al., 1999; Ewis & Abdel-Rahman, 1995; Ewis & Abdel-Rahman, 1995], independently of its effect on insulin activity. In addition, metformin is able to prevent collagen glycation in a canine diabetic model [Jyothirmayi et al., 1998]. Due to the fact that guanidino compounds can block dicarbonyl groups [Ruggiero-Lopez et al., 1999], the effects of the diamino biguanide metformin have been investigated on the methylglyoxal formation in type 2 diabetes. Indeed, metformin reduces the levels of methylglyoxal (above mentioned as a reactive alpha-dicarbonyl thought to contribute to diabetic complications as a precursor of AGEs) [Beisswenger et al., 1999]. Thus, metformin treatment can prevent diabetic complications not only by lowering plasma glucose, but also by inhibiting AGE formation [Tanaka et al., 1999]. Thiazolidinediones are other antidiabetic drugs which are able to possess antioxidant properties. As an example, troglitazone has a combined chemical structure of thiazolidinedione and -tocopherol-like structure (chroman ring). It has been shown to inhibit galactose-induced cataractogenesis in rat lens culture and lipid peroxide formation associated with this process [Yokoyama et al., 1999 ]. In a similar way, troglitazone is able to inhibit Cu2+-induced oxidation of LDLs and the subsequent uptake and degradation of these LDLs by macrophages, by acting as an aqueous phase antioxidant in addition to its effect on glucose homeostasis [Crawford et al., 1999 ]. Similarly, sulfonylureas can exhibit antioxidant activity. Thus, gliclazide decreases LDL oxidation and monocyte adhesion to endothelial cells, suggesting a benecial effect of this drug in the prevention of atherosclerosis associated with type 2 diabetes [Renier et al., 2000 ]. Treatment with gliclazide also induces a decrease in plasma lipid

peroxides and an enhancement of erythrocyte Cu, Zn-SOD activity, this effect resulting from a free radical scavenging activity independent of glycemic control [Jennings & Belch, 2000]. In recent years there has been an increased interest in areas related to newer developments in the prevention of disease especially those involving natural compounds with antioxidant activities. These antioxidants neutralize free radicals or their activities. Such compounds, especially derived from natural sources and capable of minimizing oxidative damage in situ, have potential uses to control human diseases. These compounds, capable of interacting with DNA, cellular membranes and other biomolecules have potential protective properties against deleterious free radicals [Sies, 1996; Packer & Ong, 1998; Surh, 2003]. Chlorophyllin (CHL) is a water-soluble analogue of chlorophyll, the ubiquitous photosynthetic green pigment present in food materials of plant origin as well as in nutritional supplements such as extracts from Spirulina and Chlorella vulgaris. Chlorophyll has been credited with several beneficial properties. Chlorophyllin, has proved to be better than the parent compound as evident from studies employing model systems [Negishi, 1997; Dashwood et al, 1998]. It possesses highly potent antioxidant activity [Sato et al, 1984; Sato et al, 1985; Kamat et al, 2000; Kumar et al, 2001]. It has been used, without apparent toxic side effects, to treat a number of human ailments. Chlorophyllin is a potent antioxidant against oxidative stress induced by radiation, photosensitization and peroxynitrite as measured by inhibition of lipid peroxidation, protein oxidation, DNA damage and restoration of endogenous antioxidants [Kamat et al, 2000; Kumar et al, 2001]. It is therefore considered apropriate to investigate protective effects of CHL agaist oxidative damage under different forms of oxidative stress such as hyperglycemia induced oxidative stress. To summarize, Free radicals have been implicated in the etiology of large number of major diseases. They can adversely alter many crucial biological molecules leading to loss of form and function. Such undesirable changes in the body can lead to diseased conditions. Antioxidants can protect against the damage induced by free radicals acting at various levels. Dietary and other components of plants form major sources of antioxidants. The relation between free radicals, antioxidants and functioning of various organs and organ systems is highly complex and the discovery of redox signaling is a milestone in this crucial relationship. Recent research centers around various strategies to protect crucial tissues and organs against oxidative damage induced by free radicals. Many novel approaches are made and significant findings have come to light in the last few years. Coordinated research involving biomedical scientists, nutritionists and physicians can make significant difference to human health in the coming decades. Research on free radicals and antioxidants involving these is one such effort in the right direction.

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Role of Chlorophyllin on Oxidative Stress and Apoptosis of diabetic mice

A literature review submitted to the Department of Biochemistry for the partial fulfilment of the pre Ph.D course work (2012)

Department of Biochemistry North-Eastern Hill University Shillong 793 022

Submitted by Abani Kumar Patar Roll no. BC-01