Islets and their antioxidant defense

REVIEW

review

Islets 2:4, 225-235; July/August 2010; © 2010 Landes Bioscience

Islets and their antioxidant defense Jhankar D. Acharya and Saroj S. Ghaskadbi* Department of Zoology; University of Pune; Pune, India

Key words: islets, oxidative stress, reactive oxygen species, antioxidant defense

Pancreatic β-cells secrete insulin in response to changes in extracellular glucose concentration. Persistent hyperglycemia during diabetes exerts toxic effects on islets by creating redox imbalance arising from overproduction of reactive oxygen species (ROS). ROS accumulation disturbs the integrity and physiological function of cellular biomolecules impairing viability and functionality of cells. Susceptibility of an organ to oxidative stress (OS) is determined by its defense mechanism and ability to repair DNA damage caused by ROS. Weak defense status of islets along with its inefficiency to repair oxidative DNA damage as compared to other tissues renders it extraordinarily sensitive to OS. Realizing the vulnerability of islet cells to oxidative damage, several efforts to boost their defense mechanism in the form of oral administration of antioxidants and overexpression of genes responsible for antioxidant enzymes have proven successful. Recently accountability for this low antioxidant defense of islets has been given by correlating it with its metabolic evolution.

Pancreatic Islets Pancreatic islets discovered in 1869 by Paul Langerhans were named after him in 1894 by Laguesse as islets of Langerhans. They are scattered throughout the pancreas and comprise only 2–3% of the pancreatic mass. Islets contain four major types of cells: a, β, δ and PP cells, secreting glucagon, insulin, somatostatin and pancreatic polypeptide, respectively, in the ratio of 68:20:10:2% in adult islets.1 Pancreatic β-cells act as glucose sensor which interpret various signals in the form of nutrients, hormones and neurotransmitters and thereby influence the synthesis and secretion of insulin. Insulin, though the primary regulator of blood glucose level, is also essential for normal growth and storage of fuels in body tissues. The primary regulator for glucagon secretion is just the opposite. Hypoglycemia stimulates glucagon secretion whereas hyperglycemia inhibits it. Somatostatin secretion is stimulated by glucose and amino acids, whereas pancreatic polypeptide is stimulated by hypoglycemia as well as by another hormone, secretin. The interplay of regulators of hormone secretion from the pancreatic islets provides a means by which these hormones regulate overall body metabolism.2 *Correspondence to: Saroj S. Ghaskadbi; Email: [email protected] Submitted: 10/30/09; Revised: 04/26/10; Accepted: 04/28/10 Previously published online: www.landesbioscience.com/journals/islets/article/12219

Since their discovery, islets have been viewed as a possible in vitro system for studying syndromes that cannot be mimicked very effectively using cell lines. Islets can be cultured as miniature organ systems wherein they retain their architecture, differentiated state and ability of insulin secretion upon glucose stimulation, independent of nervous control. Ability to maintain islets in vitro has promoted several studies related to the pathophysiology of type I and II diabetes, islet transplantation, screening of hypoglycemic drugs, probing into causes of and mechanisms involved in diabetes and to device effective means of prevention.3 Oxidative Stress during Diabetes Diabetes mellitus which is a complex metabolic disorder resulting due to insulin insufficiency or insulin dysfunction is characterized by hyperglycemia. Two major types of Diabetes mellitus include Type I or Insulin-dependent diabetes mellitus (IDDM) accounting for 10–15% of individuals suffering from the disease and is characterized by an absolute insulin insufficiency due to destruction of pancreatic β-cells. Type II or non-insulin dependent diabetes mellitus (NIDDM), accounting for 90% of all cases of diabetes is characterized by insulin resistance and defective insulin secretion. Type II diabetes is the progressive failure of pancreatic β-cells to secrete sufficient insulin to compensate for insulin resistance leading to hyperglycemia, which in turn exerts deleterious effects on β-cells. Experimental evidences have demonstrated that reactive oxygen species (ROS) are involved in pathogenesis of diabetes and more importantly in the development of secondary complications of diabetes. Formation of ROS such as superoxide anion (•O2), hydrogen peroxide (H2O2), hydroxyl radicals (•OH), and the concomitant generation of nitric oxide (NO)4,5 have been implicated in β-cell death and dysfunction in both Type I6 and Type II7 diabetes. Under physiological conditions, glucose metabolism through tricarboxylic acid (TCA) cycle generates electron donors which transfer electrons across the four complexes (Complex I, II, III and IV) of electron transport chain (ETC) creating voltage gradient across mitochondrial membrane. The energy from this voltage gradient drives the synthesis of ATP by ATP synthase. However, in the diabetic cells with excess glucose inside the cells, more glucose is oxidized by TCA cycle which in effect pushes more electron donors (NADH and FADH2) into the ETC thereby overwhelming complex III of ETC where the transfer of electrons is blocked. Excess electrons, thus generated, backfire at coenzyme Q, which in turn donates the electrons one at a time to molecular oxygen generating superoxide. Although the mitochondrial isoform of SOD degrades superoxide to hydrogen peroxide which

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is then converted to water and molecular oxygen by catalase or glutathione peroxidase, excess superoxide which manages to escape this defense mechanism causes severe ­complications during diabetes. Several in vitro and in vivo studies have confirmed continuous generation of superoxide radicals due to persistent hyperglycemia during diabetes.8-11 Involvement of ROS in the complications of diabetes such as retinopathy, neuropathy and nephropathy has also been proven by showing that inhibition of ROS production by using inhibitors of electron transport chain complex II, or by upregulating expression of uncoupling protein-1 (UCP-1) and mitochondrial SOD (MnSOD), prevents the development of secondary complications in bovine aortic endothelial cells.12 Biochemical pathways activated as a consequence of excess superoxide generation during hyperglycemia include polyol pathway, hexosamine pathway, AGEs pathway and PKC pathway. These pathways are known to be responsible for generation of secondary complications during diabetes and additionally contribute to oxidative stress. Polyol pathway is activated when excess glucose is converted to sorbitol in the presence of aldose reductase (AR). In the process of reducing high intracellular glucose to sorbitol, AR consumes the cofactor nicotinamide adenine dinucleotide phosphate (NADPH), an essential cofactor for regenerating a critical intracellular pool of reduced glutathione, increasing the cytosolic NADH:NAD + ratio.13 Thus, by reducing the amount of reduced glutathione, the polyol pathway increases susceptibility to intracellular oxidative stress. Overexpression of AR gene in pancreatic β-cells induced apoptosis by creating a redox imbalance due to decreased NADH:NAD + ratio.14 In the hexosamine biosynthetic pathway (HBP), glutamine: fructose-6-phosphate amidotransferase (GFAT) converts fructose-6-phosphate to N-acetylglucosamine-6-phosphate which in turn is converted to N-acetylglucosamine-1,6-phosphate and to UDP (uridine diphosphate)-GlcNAc. UDP-GlcNAc serves as a substrate for UDP-N-acetylglucosaminyl transferase (OGT) which causes O-glycosylation (O-GlcNAcylation) of numerous cytosolic and nuclear proteins including transcription factors and proteins involved in signal transduction. Induction of O-glycosylation of signaling molecules due to Increased routing of glucose through this pathway results in impaired activation of Insulin receptor/insulin receptor substrates/PI3 Kinase survival pathway.15 Kaneto et al.16 have demonstrated that glucosamine increases hydrogen peroxide levels in isolated rat islets β-cells indicating that it is a toxic reactive species in β-cells and causes β-cell dysfunction during hyperglycemia by generating oxidative stress. This effect was seen to be reversed in the presence of antioxidant like N-acetyl-l-cysteine. Advanced glycosylation end-products (AGEs) are formed as a result of the non-enzymatic protein glycation under hyperglycemic condition. AGEs are initially formed through the process of nucleophilic addition reaction of glucose with proteins forming a Schiff base followed by the formation of an Amadori compound which undergoes further reactions, rearrangements, dehydrations and cleavage resulting in brown insoluble, cross-linked complexes called AGEs. H 2O2 liberated during this process17 also contributes to oxidative

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stress. Deleterious effects of AGEs by triggering oxidative modifications in pancreatic β-cell derived HIT and INS-1 cell lines have been demonstrated.18,19 Increased production of diacylglycerol (DAG) under hyperglycemic condition activates protein kinase C which subsequently stimulates reactive oxygen species (ROS) production through a PKC-dependent activation of NAD(P)H oxidase.20 Additionally under hyperglycemic condition glucose auto-oxidation generates free radicals contributing to oxidative stress. In its enediol form, glucose is oxidized in a transition-metal-dependent reaction to an enediol radical anion that is converted into reactive ketoaldehydes and to super oxide anion radicals. The superoxide anion radicals undergo dismutation to hydrogen peroxide, which further in the presence of transition metals, can lead to production of extremely reactive hydroxyl radicals generating oxidative stress.21,22 Another source of ROS especially in Type 1 diabetes is several inflammatory cytokines such as interleukin (IL)-1β, interferon (IFN)γ and Tumor necrosis factor (TNF)α which are produced by auto-aggressive T lymphocytes and are therefore responsible for inducing β-cell apoptosis. This cytokine induced β-cell dysfunction and apoptosis is achieved through concerted action of a series of intracellular signaling pathways, including the generation of ROS and c-Jun N-terminal kinase (JNK/SAPK) activation. These cytokines under in vitro conditions in islets generate free radicals that cause expression of inducible nitric oxide synthase (iNOS) leading to the generation of NO and translocation of nuclear factor-κB (NFκB).23 NFκB is a transcription factor that controls NADPH oxidase which in turn is a major source of superoxide generation thus generating oxidative stress. The cytokine IL-1β, alone or in combination with interferon (IFN)γ and TNFα induced the expression of iNOS in primary rodent β-cells and insulin-producing cell lines, and by blocking of both iNOS activity, or iNOS gene expression by creating gene knockout, deleterious effects of cytokines in β-cells can be prevented.24,25 Increased expression of Mn-SOD also prevented cytokine induced NFκB activation and iNOS gene expression in rat insulinoma cells.26 Several stress kinases activated by cytokines in turn activate the expression of various pro-inflammatory mediators such as TNFα, IL-6 and monocyte chemoattractant protein-1 (MCP-1) which further aggravate the production of ROS, thus potentiating a positive feedback loop. A great majority of type II diabetic patients are obese and often have elevated free fatty acids (FFAs). Several studies have indicated that chronic exposure of β-cells to elevated level of FFA is associated with the loss of GSIS and a decrease in total insulin content.27-30 Ex vivo transcriptional profiling of human islets after chronic exposure to FFA also led to an increase in ROS generation and therefore an increase in expression of antioxidant enzymes along with activation of genes associated with inflammatory and metabolic pathways. This demonstrated that cytokines are the mediators of FFA induced oxidative stress and GSIS in human islets.31 FFA is also known to cause oxidative stress by its adverse effects on mitochondrial uncoupling of oxidative phosphorylation and β-oxidation leading to increased production of ROS, including superoxide generation.8

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Prolonged exposure of FFAs to β-cell preparations caused decreased mitochondrial membrane potential and increased uncoupling proteins, leading to the opening of K+ sensitive ATP channels and selective impairment of glucose-stimulated, but not K+ stimulated insulin secretion.32,33 The combined action of increased glucose and FFA levels in turn leads to activation of stress sensitive signaling pathways mediated by NFκB, p38 MAPK, JNK and PKC leading to cellular dysfunction and damage. HIT-T15 β-cells or isolated islets when exposed to chronic elevated glucose and FFA levels exhibited a decrease in insulin mRNA and the activation of an insulin gene-reporter construct.34 Thus although several studies have linked glucose induced β-cell dysfunction to oxidative stress,10 involvement of ROS, such as H 2O2 in glucose stimulated insulin signaling (GSIS) cannot be overlooked. The production of superoxide in response to glucose have been studied by Bindokas et al.10 by quantitating the rate of mitochondrial superoxide production in islets isolated from Zucker Lean fatty (ZLF) and Zucker diabetic fatty (ZDF) rats. Stimulation with glucose led to a large increase in superoxide production in the islets of ZLF rats as compared to islets of ZDF rats, which exhibit an elevated basal rate of superoxide, and was also associated with perturbed mitochondrial morphology contributing to abnormal insulin signaling. These findings clearly suggested that development and progression of diabetes is associated with defective mechanism regulating ROS content within islet cells. On the other hand Pi et al. 2007,35 have demonstrated that isolated mouse islets and INS-1(832/13) cells, when stimulated with glucose, generate excess H2O2 which serves as a signaling molecule for GSIS. This was further confirmed by demonstrating that addition of exogenous H2O2, hypochlorous acid, 4-hydroxy2-nonenal and diethyl maleate, which increase intracellular H2O2, led to increased insulin secretion whereas H2O2 scavengers like Catalase, N-Acetyl-L-Cysteine and α-Lipoic acid led to decreased H2O2 production and impaired GSIS. Another important mechanism which has recently been linked with the generation of oxidative stress in pancreatic islets during hyperglycemia is Renin-Angiotensin System (RAS). Pancreatic islets express prorenin, renin, angiotensin II (Ang II), and the Ang II type 1 receptor (AT1R).36,37 AT1 receptor and angiotensinogen expression is markedly upregulated in islets or pancreas from animal models of TIIDM37,38 as well as in islets or pancreatic stellate cells exposed to hyperglycaemia.39,40 It has been observed that in response to high glucose concentration Ang II dose-dependently inhibits insulin release from isolated mouse islets.41 Ang II also upregulates [NAD(P)H] oxidase, which produces superoxide anion which on interaction with nitric oxide forms peroxynitrite (ONOO-) thereby causing oxidative stressinduced pancreatic β-cell apoptosis and fibrosis. Therefore, blockade of the RAS could contribute to the development of novel therapeutic strategies in the prevention and treatment of patients with diabetes. Inhibition of RAS using irbesartan, an angiotensin receptor (AT1R) antagonist has been shown to attenuate NADPH oxidase activity and reduce ROS and associated pancreatic fibrosis in the Zucker obese insulin-resistant diabetic rat37 and the skeletal muscle of transgenic Ren2 rats.42

Although implications of ROS in pathogenesis of diabetes have been studied extensively, the importance of ROS as second messenger in GSIS cannot be overlooked. Regulatory Role of ROS The rise in blood glucose induces an increase in β-cell glucose metabolism, resulting in increased production of ATP from several sources such as glycolysis, mitochondrial glucose oxidation, and active shuttling of reducing equivalents from the cytosol to the mitochondrial electron transport chain. The resultant increase in ATP/ADP ratio inhibits ATP-sensitive K+ (K ATP) channels, resulting in plasma membrane depolarization, activation of voltage-gated Ca 2+ channels, and influx of extracellular Ca 2+, which serves to activate insulin granule exocytosis thus stimulating insulin secretion by β-cells in response to glucose43,44 also referred to as GSIS. However, inhibition of ATP generation in mitochondria using agents such as rotenone, antimycin A, oligomycin led to decreased GSIS inspite of increased mitochondria derived ROS. This indicates that glucose induced rise in ROS alone is not enough for insulin release and elevation of glucose induced ATP is also essential.45,46 H2O2 produced during glucose metabolism is considered to be a second messenger in GSIS35,47-50 and several signal transduction molecules act as downstream targets of H2O2. Involvement of H2O2 in Ca 2+ influx has also been proven by demonstrating that H2O2 and alloxan treatment caused a rapid elevation in intracellular Ca 2+ and increased insulin release in rat islets at basal, non-stimulatory glucose concentration.51,52 H2O2 activates tyrosine phosphorylation cascade in cultured cells in a manner that mimics ligand-mediated signaling by PDGF and EGF and this has been proven by demonstrating that the distal signaling effect of PDGF and EGF is dependent upon phosphorylation by H2O2.53-55 Involvement of ROS in GSIS has been proven by Ebelt et al. 2000,56 by demonstrating that exposure to alloxan and xanthine oxidase/hypoxanthine (•O2 generating system) led to temporary increase in insulin in isolated rat islets. Increasing the production of ROS by attenuating the expression of glutamate-cysteine ligase (GCL) which reduces GSH synthesis raised intracellular ROS and enhanced insulin secretion in MIN6 cells.57 Thus, ROS generated from glucose metabolism seems to serve as a metabolic signal facilitating insulin secretion. The production of H2O2 is not only necessary in β-cells for insulin secretion but it also serves as a second messenger in insulin responsive cells for insulin mediated signal transduction. The generation of cellular ROS enhances insulin signal transduction by inhibiting cellular protein-tyrosine phosphatases (PTPases) that negatively regulate the insulin action pathway by catalyzing the tyrosine dephosphorylation of the insulin receptor (IR) and its substrate proteins. The intracellular single-domain enzyme protein tyrosine phosphatase 1B (PTP1B) is a physiological negative regulator of insulin action. Mice lacking PTP1B exhibit enhanced insulin sensitivity due to increased IR phosphorylation in liver and muscle.58,59 Additionally, antisense oligonucleotides suppressing PTP1B expression in mouse and rat animal models of insulin resistance enhanced insulin sensitivity and normalized blood glucose levels.60

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PTPases have a common redox-sensitive catalytic cysteine residue, which is susceptible to oxidative damage by hydrogen peroxide and other oxidants, producing sulfenic acid intermediates which can further react with thiols to form catalytically inactive PTP disulfides, thus enhancing insulin signaling cascade.61-63 Though H2O2 has been implicated to have role in GSIS in β-cells, continuous production of H2O2 due to persistent hyperglycemia results in chronic oxidative stress which impairs β-cell survival and function,64 referred to as glucose toxicity. Destructive Role of ROS ROS directly or indirectly disturbs the integrity and physiological function of cellular biomolecules like lipids, proteins and DNA leading to generation of chronic oxidative stress. Excessive ROS induces lipid peroxidation that results in the formation of conjugated dienes, lipid hydroperoxides and degradation products such as alkanes, aldehydes and isoprostanes. ROS degrades polyunsaturated fatty acids, incorporated in all biological membranes, to peroxyl radicals (ROO.) which is further converted to malondialdehyde (MDA) through a series of chain reactions.65-67 MDA is a major aldehyde product of lipid peroxidation other than 4-hydroxy2-nonenal (HNE) and is shown to be mutagenic in bacterial and mammalian cells and carcinogenic in rats.68 ROS damages proteins by attacking amino acids, particularly cysteine and methionine.69 Direct oxidation of lysine, arginine, proline and threonine residues yield carbonyl derivatives.70 Protein carbonyl derivatives are also formed when proteins react with aldehydes like HNE and MDA formed during lipid peroxidation.71 Damage to proteins is of particular importance in vivo as it affects the function of several receptors, enzymes, transport proteins and also generates antigens that can provoke immune response. The hydroxyl radical is known to react with all the components of DNA, damaging both purine and pyrimidine bases and also the deoxyribose backbone72 generating a wide range of oxidative DNA lesions. These mainly include C-8 hydroxylation of guanine to form 8-Oxo-7, 8-dehydro-2'-deoxyguanosine (8-OHdG), 2-OH-adenine, thymine glycol, 8-OH adenine and cytosine glycol etc.73 Persistent hyperglycemia also results in the glycosylation and therefore unfolding of several proteins, lipids and nucleic acids including b-cell derived amylin, also termed as islet amyloid polypeptide (IAPP). Amylin is a 37 amino acid polypeptide which parallels insulin synthesis, secretion and excretion. In presence of hyperglycemia due to excessive ROS generation glycosylation of IAPP causes formation of the secondary conformational antiparallel crossed β-pleated sheet structure called amylin derived islet amyloid (ADIA) which in turn undergoes excessive free radical polymerization due to the presence of ROS.74,75 Islet amyloid is the pathological hallmark of the pancreatic islet in type 2 diabetes. Several evidences indicate that IAPP mediated cytotoxicity in islet β-cells is responsible for the gradual loss of islet cells in Type II diabetes. The role of oxidative stress in the mechanism of IAPP mediated cytotoxicity has been shown by Konarkowska et al.76 in RINmF5 cells by challenging them with IAPP led to the intracellular accumulation of ROS and also an increase in

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cellular lipid peroxidation and increased activity of membrane NADPH oxidase.77 This cytotoxicity was prevented by treatment with several antioxidants like catalase, n-propyl gallate, NAC, GSH and dithiothrietol thus re-emphasisng the role of free radical production by IAPP. Extent of amyloid β-cell lesions has also been correlated with oxidative-stress related tissue ­damage in type II diabetic patients which is identified by an intensified expression of biomarkers of oxidative stress like 8OHdG and HNE and reduced expression of SOD.78,79 Deposition of amyloid led to generation of oxidative stress which is also evident from in vitro studies where islets from IAPP transgenic mice showed time-dependent increase in ROS levels and treatment with antioxidant like NAC reversed ROS associated β-cell apoptosis.80 Therefore, in addition to controlling hyperglycemia, it is necessary to minimize the oxidative stress to prevent further oxidative damage to cells. Defense Mechanism against Oxidative Stress In an attempt to neutralize ROS and thereby to cope up with the oxidative stress a cell is provided with intrinsic antioxidant defense mechanism in the form of various scavenging enzymes and antioxidant molecules. The primary defense includes free radical scavenging enzymes like catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and several disulfide reductases namely, thioredoxin (Trx) and glutaredoxin (Grx), Peroxiredoxins (Prxs), Glutamate-cysteine ligase (GCL), etc. SOD, the first line of defense against free radicals, catalyzes the dismutation of superoxide anion radical (O2•-) into hydrogen peroxide (H2O2) by reduction reaction. The oxidant formed (H2O2) is transformed into water and oxygen (O2) by CAT or GPx or Prxs. Peroxiredoxin family of thiol-peroxidases catalyzes the reduction of H2O2 and alkyl hydroperoxides to water and alcohol, respectively, with the use of reducing equivalents provided by thiol-containing proteins resulting in the generation of sulfenic acid. Hyperoxidation of peroxiredoxins occurs when H2O2 concentration exceeds the capacity of Prx regeneration by Trx rendering Prx inactive resulting in complete inactivation of its peroxidase activity.81,82 This inactivation of Prxs is reversed by sulfiredoxins (Srxs) which catalyze ATP-dependent reduction of the hyper-oxidized peroxiredoxin (PrxSO2) into sulfenic peroxiredoxin (PrxSOH). Thioredoxin is a ubiquitous disulfide reductase responsible for maintaining proteins in their reduced state, which is reduced by electrons from NADPH via thioredoxin reductase (TRX).83 TRX is induced under oxidative stress conditions and can protect proteins and DNA from oxidation by ROS by scavenging them. The Grx system consists of Grx enzymes, GSH, glutathione reductase and NADPH. The selenoprotein GPx enzyme and Glutaredoxin reductase 2 (Grx2) reduces H2O2 and lipid peroxides to water and lipid alcohols by utilizing reduced glutathione (GSH) which is converted into oxidized glutathione (GSSG).72 Glutathione reductase, a flavoprotein enzyme, though not directly involved in scavenging free radicals, is essential for regenerating GSH from GSSG at the expense of NADPH. GSH is considered to be the major thiol disulfide redox buffer of the cell and measurement of

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Figure 1. Oxidative stress induced β-cell dysfunction. ROS (e.g., H2O2) generated during glucose metabolism is essential for GSIS. Antioxidant defense upregulated in response to excessive ROS not only scavenges free radicals but also dampens the ROS signaling essential for insulin secretion. Excess ROS which manage to escape the defense mechanism diminish β-cell functionality by damaging proteins, lipids and DNA out of which only DNA can be repaired and lipids and proteins are turned over.

GSH/GSSG ratio is used to estimate the redox environment of the cell.84 Glutathione peroxidase/glutathione systems are very crucial during oxidative stress. Secondary defense is in the form of small non-enzymatic antioxidant molecules like glutathione, uric acid, ascorbate, α-tocopherol, etc. GSH is the principle non-protein thiol and has antioxidant properties since the thiol group in its cysteine moiety is a reducing agent and can be reversibly oxidized and reduced. GSH also participates in the detoxification of xenobiotics as a substrate for the enzyme glutathione-S-transferase.85 Glutathione, maintained in the reduced state by glutathione reductase, transfers its reducing equivalents to several metabolites and enzymes such as ascorbate in the glutathione-ascorbate cycle, GPx, glutathione reductases and glutaredoxins.86 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 peroxidation.9 Uric acid is a well-known antioxidant that scavenges peroxides, hydroxyl radicals and hypochlorous acid87,88 and can also react chemically with singlet oxygen, superoxide and hydroxyl radicals and therefore function directly as a free radical scavenger. Induction of antioxidant enzymes like CAT, SOD, g-glutamylcysteine synthetase (GCS) is regulated at the transcriptional level by a transcription factor NF-E2-related factor 2 (Nrf2) 89,90

which is mediated by a specific enhancer, the antioxidant response element (ARE), found in the promoter of the enzymes that help control the cellular redox status and defend the cell against oxidative damage.91,92 Nrf2 is a central regulator in both constitutive and inducible antioxidant response elements (ARE)-related gene expression93 and activates gene transcription constitutively or in response to an oxidative stress signal by interacting with ARE. Nrf2 knockout mice exhibit a higher susceptibility to oxidative damage.94 Although induction of endogenous antioxidants is necessary to scavenge excess ROS, persistent induction of these antioxidant defenses could inhibit metabolic function of ROS such as the one in GSIS. Thus endogenous antioxidant defense no doubt protects cells from the oxidative damage but continuous presence of elevated antioxidants may also blunt glucose triggered ROS signaling. Therefore, a delicate balance has to be maintained between ROS production and induction of antioxidant defense of cells in order to maintain cellular homeostasis (Fig. 1). Defense Status of Islets Under Oxidative Stress The susceptibility of an organ against damage due to oxidative stress is determined by the antioxidant defense of the cell. Pancreatic β-cells express glucose transporter GLUT2 abundantly

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Table 1. Comparison of gene expression profile of antioxidant enzymes in several tissues from albino mouse in comparison with liver Tissues

CuZn SOD

MnSOD

Catalase

GPx

(% of liver)

(% of liver)

(% of liver)

(% of liver)

Liver

100 ± 7

100 ± 17

100 ± 10

100 ± 5

Kidney

99 ± 7

125 ± 19

78 ± 8

91 ± 9

Brain

77 ± 8

67 ± 16

36 ± 10

39 ± 8

Lung

80 ± 12

66 ± 17

50 ± 10

58 ± 9

Skeletal muscle

59 ± 7

95 ± 14

41 ± 12

40 ± 10

Heart muscle

70 ± 10

142 ± 9

72 ± 11

39 ± 7

Pituitary gland

79 ± 19

47 ± 11

23 ± 2

66 ± 11

Adrenal Gland

175 ± 16

239 ± 25

45 ± 7

77 ± 12

Pancreatic islets

38 ± 9

30 ± 5

Not detectable

15 ± 6

Adapted from Lenzen et al., 1996.

and thereby display highly efficient glucose uptake when exposed to high glucose concentration. Persistent exposure to high levels of glucose during diabetes can therefore have more serious implications in islets. Pancreatic islets have been shown to express low activity of free radical detoxifying enzymes and redox-regulating enzymes such as catalase, SOD and glutathione peroxidase as compared to other tissues.95,96 This has been additionally confirmed by Lenzen et al.97 by studying the gene expression of the antioxidant enzymes in various mouse tissues like liver, brain, kidney, lung, skeletal muscle, heart muscle, adrenal gland and pituitary gland. Gene expression profiling revealed that pancreatic islets express substantially low amounts of SOD, CAT and GPx in comparison with other tissues (Table 1). The levels of Cu/Zn SOD and MnSOD gene expression were in the range of 30–40% of those in the liver whereas glutathione peroxidase gene expression was 15% and catalase gene expression was not detectable in pancreatic islets.98 An increase in the expression of oxidative stress markers like, 8-hydroxy-2'-deoxyguanosine (8-OHdG) and 4-hydroxy-2,3-nonenal (4-HNE) in islets under diabetic conditions also suggests the vulnerability of islets to oxidative stress.99-102 Thus, excessive ROS assault in the face of low antioxidant defense status of islets leads to oxidative stress during hyperglycemia and β-cell dysfunction. ROS under normal levels are neutralized by intrinsic antioxidant defense and those which escape this damage biomolecules. Though the damage inflicted to proteins and lipids is irreversible, the damage caused to DNA can be repaired by DNA repair mechanism. Nearly all oxidatively damaged modified bases are repaired by the base excision repair mechanism (BER). Islets are also very poor in rectifying the oxidative damage to DNA compared to liver cells. Cell free extracts from islets and liver cells when examined for their ability to remove 8-OHdG from oxidatively damaged plasmid pBR322 in vitro revealed that liver cells were much more efficient as compared to islet cells. Liver cells removed 89% of the DNA damage within two hours as compared to only 23% by islets.102 Thus, both low intrinsic antioxidant defense system, and very poor DNA repair capacity render islets extraordinarily sensitive to damage caused by oxidative stress. This clearly suggests that oxidative stress plays a pivotal role in islet deterioration

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in terms of its viability and functionality during persistent hyperglycemia. Response of Islets Under Oxidative Stress Chronic exposure of the β-cells to supraphysiologic concentrations of glucose inducing oxidative stress causes defective insulin gene expression accompanied by marked decrease in insulin ­content and abnormal insulin secretion. Excessive ROS, in addition to oxidizing proteins, lipids and DNA also activate various stress-sensitive signal transduction pathways like c-Jun N-terminal kinase (JNK), p38 Mitogen activated protein kinase (p38MAPK)103 and protein kinase C.104 The JNK (also referred to as SAPK) and p38MAPK are members of the complex super-family of MAP serine/threonine protein kinases. It has been shown that the activation of JNK pathway during oxidative stress results in decreased insulin gene expression by affecting the DNA binding activity of PDX-1. Adenoviral overexpression of dominant-negative c-Jun NH2-terminal kinase (DN-JNK) protected both PDX-1 binding to DNA and insulin gene mRNA from hydrogen peroxide-induced oxidative stress in β-cells as well as from the adverse effects of hyperglycemia.105 PDX-1 is a member of homeodomain-containing transcription factor family and plays an important role in pancreatic development,106,107 β-cell differentiation108-112 and in the maintenance of normal β-cell function by transactivating insulin gene and genes involved in glucose sensing and metabolism such as GLUT2, glucokinase and islet amyloid polypeptide.113-117 Oxidative stress induces nucleo-cytoplasmic translocation of PDX-1 through activation of JNK pathway. Since PDX-1 gene is transactivated by PDX-1 itself, it has been proposed that the decrease of PDX-1 expression in nuclei results in further decrease of PDX-1 expressed in the cells.118 Thus, the oxidative stress-induced nucleo-cytoplasmic translocation of PDX-1 plays a crucial role in suppression of insulin gene expression and its biosynthesis under diabetic conditions. Strategies against Oxidative Stress Though cells are usually well endowed with an intrinsic antioxidant defense mechanism, administration of several antioxidant supplements can help strengthen the defense status of the cell.

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This is of more relevance to islets cells as they are intrinsically not well equipped in terms of antioxidant defense mechanism. Several studies have reported that oral administration of antioxidants increases the islets capacity to cope up with oxidative stress. Antioxidants like vitamin C, vitamin E along with N-acetyl-Lcysteine have been used for reducing the oxidative damage caused to islet cells of C57BL/KsJ-db/db mice.119 Other antioxidants like Curcumin,120 Quercetin121 and Probucol100 have also been proven to be efficient in rescuing islet cells from damage caused by free radicals. Resveratrol (3,4',5-trihydroxy stilbene), a phytoalexin, augments cellular antioxidant defense capacity through induction of HO-1 via Nrf2-ARE signaling, thereby protecting PC12 cells from oxidative stress.122 This has been further confirmed in our laboratory by checking the ability of Syndrex, a formulated anti-diabetic drug with strong antioxidant capacity to improve the functionality and viability of islet cells in vitro by conferring them better protection against oxidative damage.123 In addition to oral administration of antioxidants, overexpression of genes responsible for antioxidant enzymes in islets also exhibit better protection against damage caused by free radicals and this strategy could possibly serve as a valuable pharmacological approach towards treating diabetes. Adenoviral based overexpression of glutathione peroxidase in human islets increased GPx activity and protected islets against ribose induced oxidative stress124,125 and also enhanced the resistance of rat β-cell line to both ROS and RNS cytotoxicity.126 Overexpression of cellular enzymes like Cu/Zn SOD and Mn-SOD is also shown to have protective effect on islets against oxidative damage in RINm5F cells and INS-1 insulin secreting cells.127-129 Adenoviral vector mediated overexpression of glutamyl cysteine ligase catalytic subunit, a primary regulator of de novo synthesis of glutathione (GSH) in mammalian cells protected pancreatic islets against oxidative stress by increasing GSH levels in islets.130 Overexpression of catalase, however, could allow only partial reduction of islet susceptibility to oxidative stress and a preservation of insulin secretory capacity in human and rat pancreatic islets.131 Overexpressing the mitochondrial catalase gene expression conferred better protection against oxidative injury as compared to the cytoplasmic catalase in insulin producing RINm5F cells.132 Co-expression of several antioxidant enzymes like glutathione peroxidase along with two isoforms of superoxide dismutase133 and catalase along with glutathione peroxidase or catalase with SOD133-135 provided better protection to islets from oxidative injury when compared with overexpression of individual antioxidant enzyme. Non-obese diabetic (NOD) transgenic mice that overexpress TRX in their pancreatic β-cells also attenuate induction of diabetes on treatment with STZ, a ROS-generating system.136,137 Thus, it seems that by enhancing the antioxidant defense status of islets, they exhibit better ability to cope up with oxidative stress. Oxidative Stress during Islet Transplantation Although insulin is used as a standard therapy to control blood glucose levels in patients with type I diabetes, an alternative therapy for restoration of endogenous insulin production is

transplantation of islets which is preferred over multiple, daily insulin injections. One of the most important areas of research in islet transplantation is the isolation procedure, which remains a major concern in islet transplant studies. A significant number of transplanted islets are lost because of apoptosis and necrosis,138 mediated by oxidative stress generated at the time of transplantation. Hypoxia/ reoxygenation which occurs during the islet transplantation procedure initiates a cascade of biochemical reactions which results in the production of ROS including superoxide radicals (•O2), hydrogen peroxide (H2O2), and hydroxyl radical (•HO) causing necrosis and apoptosis via intracellular pathways.139 ROS generated as a result of hypoxia/reoxygenation damages enzymes, lipids and DNA within the cell thus leading to cell death and therefore functional islet graft failure.140 Low levels of antioxidants in isolated islets,141 as described earlier, worsen the situation more by weakly defending the cells against ROS assault. Thus, protection of transplanted islets against damage by ROS is essential. The relevance of oxidative stress during transplantation is also demonstrated by Mysore et al.133 by showing that transgenic mouse islets co-expressing glutathione peroxidase along with superoxide dismutase when transplanted into streptozotocin induced diabetic recipients in a syngeneic marginal islet mass model, improved the functionality of islet grafts. Overexpression of TRX in islets using a lentiviral vector before transplantation prolonged islet graft survival when transplanted in diabetic NOD mice.142 These studies have demonstrated the importance of oxidative stress in islet transplantation and have encouraged the usage of antioxidants to maintain islet functionality and viability and also to optimize islet isolation and transplantation. Several antioxidants like Curcumin,143 (-)-epigallocatechin-3-gallate,144 catalytic antioxidant probes AEOL10150 manganese [III] 5, 10, 15, 20-tetrakis [1,3,-diethyl-2imidazoyl] manganese-porphyrin pentachloride [TDE-2, 5-IP]145 and metallothionein146 have been proven to be useful in protecting islets from hypoxia and improve islet graft survival. All the available data demonstrate that treatment with antioxidants protects islets from damage induced by ROS produced during the early phase of islet transplantation and improves their survival and functionality. Poor Defense Mechanism of Islets: Boon or Bane? Although the inefficiency of islet cells to cope up against oxidative stress owing to the poor intrinsic antioxidant defense has been investigated extensively, the reasons for this are not known till date. If islet cells are to play an important role in maintaining glucose levels by secreting insulin then why is it that islets are not as well equipped with strong defense mechanism as other tissues to cope up under stressful conditions? Does this poor defense status of islets have any other role to play? In an attempt to answer these questions recently, Rashidi et al.147 have hypothesized that ROS in β-cells, by their negative effect on insulin secretion, plays a significant fitness enhancing role for the whole organism. During early stages of evolution i.e., around 500 million years ago, β-cells first evolved as scattered insulin-producing cells in the intestinal tissue of primitive proto-chordates to sense gut

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glucose, and later evolved into endocrine pancreas of vertebrates to sense blood glucose. This evolution of pancreas from scattered insulin producing cells to present day endocrine pancreas was also concurrent with the evolution of complex brain and nervous system where β-cells “provided an extra degree of freedom for the brain to evolve without having to think of getting energy supply.”148 In order to nourish insulin-independent brain and also maintain its functionality, a high glucose concentration is needed under several stressful conditions like predation, competition, infection etc which were prevalent at the time of evolution. This led to increased energy demands of brain to maintain its functionality, and the development of pancreas alone was not sufficient to fulfill this additional energy demand. Interestingly at around the same time, coinciding with the evolution of brain and endocrine pancreas, cortisol and corticosteroid receptors also evolved. Cortisol, a stress hormone released at the time of stress reduces the responsiveness of insulin-dependent tissues to insulin,149 a condition described as insulin resistance. The ensuing hyperglycemia that develops during stress is partially corrected by β-cells, owing to the regulatory role of ROS, which prevent β-cells from secreting insulin to the extent required to maintain homeostasis. This allows reallocation of additional amount of glucose to insulin-independent tissues like brain and fetus but this was achieved at the cost of β-cells losing some part of their anti-oxidant defense. This evolutionary trade-off between brain and β-cells allowed for the optimization of anti-oxidant defense and also facilitated the metabolic demands during stress. However, instead of making such frequent adjustments during stressful conditions and its normalization afterwards, it would have been more efficient and cost-effective to tune-down antioxidant defense system of β-cells for the entire life-span of an individual. It has been hypothesized by Rashidi et al.147 that this well-tuned co-evolution of brain, corticosteroid response and β-cells in the endocrine pancreas has resulted in the present day compromised antioxidant defense status of islets. A further reduction in the antioxidant defense of β-cells occurred around 100 million years ago when placental mammals evolved. Since pregnancy is also described as a normal period of physiological stress, therefore maternal insulin resistance that develops during pregnancy, by increasing the cortisol concentration, helps divert more glucose to the increasing demands of fetus. ROS plays an important role in causing maternal insulin resistance and the low levels of antioxidants in islets thereby allow ROS to perform its regulatory role. This explanation corroborates with the observation that females have lower antioxidant levels as compared to males in mice150 and also in humans.141 References 1.

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Rahier J. The Diabetic pancreas: a pathologist’s view. In: Lefebvre PJ, Pipeleers DG, eds. The pathology of endocrine pancreas in diabetes. Berlin: Springer 1988; 17-40. Robertson RP, Harmon JS. Pancreatic islet beta-cell and oxidative stress: the importance of glutathione peroxidase. FEBS Lett 2007; 581:3743-8. Bhonde R, Shukla RC, Kanitkar M, Shukla R, Banerjee M, Datar S. Isolated islets in diabetes research. Indian J Med Res 2007; 125:425-40.

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Since natural selection favors the strategy to maximize fitness of the whole organism and offspring production, β-cells till date have retained their weak defense mechanism. This hypothesis has been proven by demonstrating that birds that have higher reproductive success (by increasing their brood size) also have lower levels of antioxidant enzyme expression and that senescence acceleration by increased reproductive effort is at least in part mediated by oxidative stress.151 Thus, by tuning down its antioxidant defense system, β-cells had to pay a heavy price in exchange of an increase in efficiency of brain as corticosteroid response and β-cells in the pancreas co-evolved. Therefore, although increasing the defense mechanism by oral administration of antioxidants and overexpression of antioxidant levels in islets seems to be a better pharmacological strategy to alleviate oxidative stress, the benefits from increased resistance to ROS-induced damage, derived from such an attempt would mean an interference with the physiological ROS signaling. Therefore while using antioxidant therapy emphasis should be given on protecting islets from ROS induced oxidative damage without causing an interference with basal ROS levels required for insulin secretion. Conclusion Oxidative stress is known to play a major role in the pathogenesis of both type I and type II diabetes mellitus which is evident by several in vitro and in vivo studies. On the contrary, several emerging evidences also suggest the importance of ROS, such as H2O2, as metabolic signaling molecule for GSIS in pancreatic β-cells. Owing to poor antioxidant defense and inefficiency to repair oxidatively damaged DNA β-cells are predisposed to ROS assault during hyperglycemia and cause β-cell dysfunction. Several strategies to boost the antioxidant defense of islets by implementing antioxidant therapy and overexpressing antioxidant enzymes have been proven beneficial in alleviating oxidative stress. However, continuous use of antioxidant therapy may also interfere with ROS signaling required for GSIS. The present day compromised antioxidant defense status of islets seems to have retained during evolution as it offers advantage to insulinindependent tissues during stressful conditions. Acknowledgements

The authors acknowledge financial support from DAE, DBT, DST-FIST and UGC-CAS of Department of Zoology. Jhankar Acharya is a recipient of JRF under UGC-CAS program of Department of Zoology, University of Pune.

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127. Lortz S, Tiedge M, Nachtwey T, Karlsen AE, Nerup J, et al. Protection of insulin-producing RINm5F cells against cytokine-mediated toxicity through overexpression of antioxidant enzymes. Diabetes 2000; 49:1123-30. 128. Hohmeier HE, Thigpen A, Tran VV, Davis R, Newgard CB. Stable expression of manganese superoxide dismutase (MnSOD) in insulinoma cells prevents IL-1beta-induced cytotoxicity and reduces nitric oxide production. J Clin Invest 1998; 101:1811-20. 129. Moriscot C, Pattou F, Kerr-Conte J, Richard MJ, Lemarchand P, et al. Contribution of adenoviral-mediated superoxide dismutase gene transfer to the reduction in nitric oxide-induced Cytotoxicity on human islets and INS-1 insulin-secreting cells. Diabetologia 2000; 43:625-31. 130. Tran PO, Parker SM, LeRoy E, Franklin CC, Kavanagh TJ, et al. Adenoviral overexpression of the glutamylcysteine ligase catalytic subunit protects pancreatic islets against oxidative stress. J Biol Chem 2004; 279:53988-93. 131. Benhamou PY, Moriscot C, Richard MJ, Beatrix O, Badet L, et al. Adenovirus-mediated catalase gene transfer reduces oxidant stress in human, porcine and rat pancreatic islets. Diabetologia 1998; 41:1093-100. 132. Gurgul E, Lortz S, Tiedge M, Jorns A, Lenzen S. Mitochondrial catalase overexpression protects insulinproducing cells against toxicity of reactive oxygen species and proinflammatory cytokines. Diabetes 2004; 53:2271-80. 133. Mysore TB, Shinkel TA, Collins J, Salvaris EJ, Fisicaro N, et al. Overexpression of glutathione peroxidase with two isoforms of superoxide dismutase protects mouse islets from oxidative injury and improves islet graft function. Diabetes 2005; 54:2109-16. 134. Tiedge M, Lortz S, Munday R, Lenzen S. Complementary action of antioxidant enzymes in the protection of bioengineered insulin-producing RINm5F cells against the toxicity of reactive oxygen species. Diabetes 1998; 47:1578-85.

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