GENERAL GUIDELINES Oxidative Stress and. Diabetes

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1 GENERAL GUIDELINES Oxidative Stress and THE ROLE OF OXIDATIVE STRESS IN HUMAN DISEASES Diabetes Definition and Description of Diabetes Mellitus 2 Diagnostic Criteria 3 The Role of Oxidative Stress 4 Glucose Autooxidation 5 Advanced Glycosylation End Products (AGEs) 5 The Polyol Pathway 5 Lipid Peroxidation and Antioxidant Patterns 6 Nitrosative Stress 6 Complications of diabetes and their relation to oxidative stress 7 Cardiovascular disease 7 Nephropathy 7 Neuropathy 8 Retinopathy 8 Antioxidant Therapeutic Approach 9 The information presented on the following pages is intended to be general and is not intended as a basis for diagnosis or treatment. This is definitely not intended to be a substitute for careful medical evaluation. Callegari company specifically disclaim any liability arising directly or indirectly from information contained on these pages. Version 1.0 September 2006

2 Definition and Description of Diabetes Mellitus Diabetes mellitus is a serious and growing health problem. A recent study by the World Health Organization (WHO) estimated that the worldwide prevalence of diabetes in 2002 was 170 million, with the number predicted to grow to 366 million by Diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both. It is a chronic condition, and the main risks to health are its characteristic long-term complications. These include damage, dysfunction, and failure of various organs, especially cardiovascular disease (doubled risk), chronic renal failure (it is the main cause for dialysis in developed world adults), retinal damage which can lead to blindness and is the most significant cause of adult blindness in the non-elderly in the developed world, nerve damage, erectile dysfunction (impotence), to gangrene with risk of amputation of toes, feet, and even legs. Patients with diabetes have an increased incidence of atherosclerotic cardiovascular, peripheral vascular, and cerebrovascular disease. Hypertension, abnormalities of lipoprotein metabolism, and periodontal disease are often found in people with diabetes. The emotional and social impact of diabetes and the demands of therapy may cause significant psychosocial dysfunction in patients and their families. Several pathogenic processes are involved in the development of diabetes. These range from autoimmune destruction of the β-cells of the pancreas with consequent insulin deficiency to abnormalities that result in resistance to insulin action. The basis of the abnormalities in carbohydrate, fat, and protein metabolism in diabetes is deficient action of insulin on target tissues. Deficient insulin action results from inadequate insulin secretion and/or diminished tissue responses to insulin at one or more points in the complex pathways of hormone action. Impairment of insulin secretion and defects in insulin action frequently coexist in the same patient, and it is often unclear which abnormality, if either alone, is the primary cause of the hyperglycemia. Glycation of tissue proteins and other macromolecules and excess production of polyol compounds from glucose are among the mechanisms thought to produce tissue damage from chronic hyperglycemia 2. Symptoms of marked hyperglycemia include polyuria, polydipsia, weight loss, sometimes with polyphagia, and blurred vision. Impairment of growth and susceptibility to certain infections may also accompany chronic hyperglycemia. These symptoms may develop quite fast in type 1, particularly in children (weeks or months), but may be subtle or completely absent - as well as developing much more slowly - in type 2. Acute, life-threatening consequences of diabetes are also ketoacidosis, an extreme state of dysregulation characterized by the smell of acetone on the patient's breath, or the nonketotic hyperosmolar syndrome, which is more common in type 2 diabetes, and is mainly the result of dehydration due to the polyuria. WHO recognizes three main forms of diabetes: type 1, type 2 and gestational diabetes (or type 3, occurring during pregnancy), which can be considered patterns of pancreatic failure 3. Type 1 is due to 1 Diabetes Care 27: , Diabetes Care 26(1): S5-S20,

3 autoimmune destruction of the pancreatic β-cells deriving an absolute deficiency of insulin production, while type 2 (the much more prevalent) and gestational diabetes are due to a combination of insulin resistance by tissues and an inadequate compensatory insulin secretory response. In the latter category, a degree of hyperglycemia sufficient to cause pathologic and functional changes in various target tissues, but without clinical symptoms, may be present for a long period of time before diabetes is detected. During this asymptomatic period, it is possible to demonstrate an abnormality in carbohydrate metabolism by measurement of plasma glucose in the fasting state or after a challenge with an oral glucose load. The WHO classification highlighted that the diabetes mellitus was an etiologically and clinically heterogeneous group of disorders that share hyperglycemia in common. Diagnostic criteria Diabetes constitutes a heavy burden for the patient and the society. It is associated with many complications and increased mortality. These further increase with smoking, obesity, hypertension, increased cholesterol and triglycerides, proteinuria, and poor glucose control. Early diagnosis, good glucose monitoring and control are crucial to reduce the burden of diabetes. Diabetes mellitus is diagnosed by demonstrating any one of the following 4 : fasting plasma glucose level at or above 126 mg/dl or 7.0 mmol/l plasma glucose at or above 200 mg/dl or 11.1 mmol/l two hours after a 75 g oral glucose load in a glucose tolerance test random plasma glucose at or above 200 mg/dl or 11.1 mmol/l. A positive result should be confirmed by any of the above-listed methods on a different day, unless there is no doubt as to the presence of significantly-elevated glucose levels. Most physicians prefer measuring a fasting glucose level because of the ease of measurement and time commitment of formal glucose tolerance testing, which can take two hours to complete. By definition, two fasting glucose measurements above 126 mg/dl or 7.0 mmol/l is considered diagnostic for diabetes mellitus. Also for monitoring of diabetes treatment blood glucose levels is a fundamental tool. Glucose levels, however fluctuates considerably depending on food intake and other factors, being lowest in the morning before breakfast and then increases more or less after meals, depending both on the diet and the individual glucose regulation with higher levels and greater variations diabetes. The fraction of hemoglobin that is bound to glucose - HbA1c - now serves as a powerful tool for assessing long-term glycemic control. HbA1c also correlates well with the risk of development of complications related to diabetes, which is a very important aspect. HbA1c is a useful tool also for the diagnosis and evaluation of all forms of diabetes mellitus and patients at risk of developing diabetes, making it possible to discover diabetes earlier. 3 World Health Organization: Diabetes mellitus: Report of a WHO Study Group. Geneva, WHO, 1985 (tech. Rep. Ser., no. 727). 4 World Health Organization: Definition, Diagnosis and Classification of Diabetes mellitus. Report of a WHO Consultation, 1999 (WHO/NCD/NCS/99.2). 3

4 Thus, early detection, and consequently early treatment, might well reduce the burden of type 2 diabetes and its complications. However, to increase the cost-effectiveness of testing undiagnosed, otherwise healthy individuals, testing should be considered in high-risk populations. Testing for diabetes 5 should be considered in individuals at age 45 years and above, particularly in those with a BMI 25 6 ; if normal, it should be repeated at 3-year intervals. Testing should be considered at a younger age or be carried out more frequently in individuals who are overweight and have additional risk factors: have a first-degree relative with diabetes are habitually physically inactive are members of a high-risk ethnic population (e.g., African-American, Hispanic American, Native American, Asian American, Pacific Islander) are hypertensive ( 140/90) have an HDL cholesterol level 35 mg/dl (0.90 mmol/l) and/or a triglyceride level 250 mg/dl (2.82 mmol/l) on previous testing, had IGT, Impaired Glucose Tolerance, or IFG, Impaired Fasting Glucose have a history of vascular disease. The Role of Oxidative Stress There is considerable evidence that oxidative stress (OS) is of importance in the etiology of diabetes. Hyperglycemia and/or insulin deficiency may precipitate an almost unique metabolic insult, being capable of simultaneously stimulating the production of free radical, while down-regulating the host s natural antioxidant defence systems. Due to these events, the balance normally present in cells is disturbed. This leads to a condition of oxidative stress 7 and oxidative damage of cell fundamental components such as proteins, lipids, and nucleic acids. Several studies demonstrate that patients with diabetes not only have increased levels of circulating markers of free radical-induced damage 8, but also have reduced antioxidant defences 9, suggesting a disturbed capacity of scavenging harmful free radicals. Hyperglycemia can induce oxidative stress via several mechanisms. These include glucose autoxidation, the formation of advanced glycation endproducts (AGE), and activation of the polyol pathway. Other circulating factors that are elevated in diabetics, such as free fatty acids and leptin, also contribute to increased reactive oxygen species (ROS) generation. The vascular complications of diabetes are conventionally divided into macrovascular (cardiovascular) and microvascular (such as retinopathy, neuropathy, and nephropathy) categories. 5 Diabetes Care 26(1): S5-S20, May be not correct for all ethnic groups. 7 Can J Appl Physiol 30(2): , 2005; J Intern Med 251: 69-76, Diabetes Care 19: , 1996; Diabetes 48: 1-9, Free Rad Biol Med 34: , 2003; J Diabetes Complications 17: 7 10, 2003; Clin Chim Acta 321: 89 96, 2002; Curr Opin Clin Nutr Metab Care 5: , 2002; Circulation 103: 1618, 2001; Eur J Clin Invest 27: , 1997; Diabetes 46: ,

5 Evidence of increased oxidative 10 and nitrosative 11 stress and impaired antioxidant defence enzymes 12 has been also reported in complications-prone tissues of diabetes. Glucose Autooxidation The increased metabolism of glucose due to intracellular hyperglycemia leads to the overproduction of nicotinamide adenine dinucleotide (NADH) and flavin adenosine dinucleotide (FADH), which are used by the electron transport chain to generate adenosine triphosphate 13. When NADH is in excess, an increase in the mitochondrial proton gradient is produced and electrons are transferred to oxygen, - producing superoxide radicals (O 2 ) 14. Advanced Glycosylation End Products (AGEs) The formation of AGEs begins with nonenzymatic covalent bonding of ketone or aldehyde groups of reducing sugars to the free amino groups of proteins and other molecules. A series of rearrangements and reactions occurs to irreversibly produce AGEs. They are proposed to contribute to damage by modifying the extracellular matrix and circulating lipoproteins, as well as binding to and activating the putative receptor for AGE (RAGE), which is present on many vascular cells 15. It is through their receptor-mediated effects that AGE have been shown to induce ROS production 16. Stimulation of the RAGE causes the production of ROS, perhaps via an NAD(P)H oxidase 17, and subsequent activation of redox-sensitive transcription factors and expression of inflammatory mediators 18. In addition, AGEs are resistant to enzymatic degradation and therefore very stable, thus their accumulation continues throughout aging. AGE accumulation causes arterial stiffening in the vessel wall, glomerulosclerosis in the kidney, and vascular hyperpermeability in the retina 19. The Polyol Pathway Two enzymes of the polyol pathway (chemical compounds containing multiple hydroxyl groups such as sorbitol, maltitol and other sugar alcohols) contribute to ROS generation. The first, aldose reductase, uses NADPH for the reduction of glucose to sorbitol. Under normal conditions, sorbitol production by aldose reductase is a minor reaction. However, under conditions of hyperglycemia, up to 30 35% of glucose is metabolized by this pathway. When this occurs, the availability of NADPH is reduced, which 10 Diabetologia 35: , Toxicol Lett : , Diabetes 49: , Curr Opin Clin Nutr Metab Care 5: , Nature 404: , 2000; Arterioscler Thromb Vasc Biol 24: , Circulation 108: , 2003; Cardiovasc Res 63: , J Biol Chem 269: , Am J Physiol Endocrinol Metab 280: E685 E694, J Clin Invest 96: , 1995; Atherosclerosis 157: , Curr Med Chem 13(15): ,

6 in turn reduces glutathione regeneration and NOS synthase activity, thus producing oxidative stress 20. The second enzyme, sorbitol dehydrogenase, oxidizes sorbitol to fructose with concomitant NADH production. Increased NADH may be used by NAD(P)H oxidases to produce superoxide 21. Lipid Peroxidation and Antioxidant Patterns Because free or nonesterified fatty acids are generally elevated in diabetic patients 22, an enhanced lipid peroxidation has been associated to hyperglycemia 23. The hypothesis that lipo-peroxidative processes occur in diabetes has recently been tested in several studies, mostly using indirect lipid peroxidation index such as susceptibility of LDL to in vitro oxidation 24 and the more conventional malondialdehyde (MDA) 25. Markers of lipid peroxidation, such as conjugated dienes (CDs) and lipid hydroperoxides (ROOHs), were significantly augmented 26, even in the absence of complications. A defective total antioxidant defence in subjects with type 1 diabetes mellitus has been reported in several studies 9,27. The total antioxidant capacity in plasma of type 1 diabetics was shown to be 16% lower than that of normal subjects 28.The apparent abnormality of chain-breaking antioxidant defence in diabetic subjects not only indicates a defect state of protection against oxidation but may also reflect a state of increased consumption of antioxidants owing to acute radical scavenging. Another possible explanation is glucose induced elevated fractional excretion of uric acid leading to hypouricemia 29. Decreased in the activity of enzymes that dispose of free radicals has been reported 30. The most common antioxidant deficiencies reported in diabetes are lower levels of ascorbate, glutathione and superoxide dismutase. Lower concentrations of reduced glutathione have been documented in diabetic neutrophils and monocytes 31. In addition, levels of some prooxidants such as ferritin and homocysteine are elevated in diabetes. Nitrosative Stress Hyperglicemia favours oxidative reactions especially in the microenvironment of the artery wall 32. Even increased superoxide generation is a key event in activating the other pathways involved in the pathogenesis of diabetic complications; it represents only a first step in the production of the endothelial 20 Curr Opin Clin Nutr Metab Care 5: , J Exp Biol 203(Pt. 10): , Diabetologia 45: , Circulation 99: , Diabetes 43: , 1994; Atherosclerosis 137(suppl.): 61-64, 1998; Atherosclerosis 136: , 1998; Diabetes Care 22: , J Intern Med 251: 69-76, Diabetes 46: , 1997; Diabetes Care 25(2): , Eur J Clin Invest 27: , 1997; Diabetes 46: , 1997; Diabetes 43: , 1994; Atherosclerosis 129: 89-96, J Intern Med 251: 69 76, Nephron 60: , IUBMB Life 49: 303 7, Free Radic Res 36(12): , J Mol Med 79: , 2001; J Clin Invest 112: ,

7 dysfunction in diabetes. Nitric oxide (NO) is a free radical and a key biological messenger, playing a role in a variety of biological process, including blood vessel relaxation. The superoxide radical may quench NO, thereby reducing its efficacy as vasodilator 33, and evidence suggests that during hyperglycemia, reduced NO availability exists 34. Superoxide overproduction, when accompanied by increased NO generation, favours the formation of the strong oxidant peroxynitrite (ONOO - ), a potent oxidant that lives for a long time. The peroxynitrite action is cytotoxic because it inhibits mitochondrial electron transport, oxidizes sulfhydryl groups in protein, initiates lipid peroxidation without requirement of transition metals, and nitrates amino acids such as tyrosine, which in turn affect many signal transduction pathways. Nitrosative stress trigger also DNA single-strand breakage, which is an obligatory stimulus for the activation of the nuclear enzyme poly-(adp-ribose) polymerase (PARP) 35 involved in a number of cellular processes including mainly DNA repair and programmed cell death. PARP activation in turn slows the rate of glycolysis, electron transport, and ATP formation, and also inhibits GAPDH 36, thereby impairing critical energy-producing pathways leading to the development of endothelial dysfunction 37 and chronic diabetic complications 38. Complications of Diabetes and Their Relation to Oxidative Stress Cardiovascular disease One of the major complications of diabetes is cardiovascular disease. The incidence of cardiovascular disease in people with diabetes mellitus is three to four times of that in non-diabetic individuals. Furthermore, established risk factors such as dyslipidemia, hypertension, and smoking cannot explain this increased prevalence of macrovascular disease in diabetes. Thus, the diabetic state itself is an independent risk factor for premature atherosclerosis. One of the potential mechanisms that mediates premature atherosclerosis in diabetes is oxidative stress. Oxidative stress plays a crucial role in atherogenesis and cause to oxidation of low density lipoprotein. Several lines of evidence support a proatherogenic role for oxidized LDL (Ox-LDL) and its in vivo existence. Ox-LDL is not recognized by the LDL receptor but by the scavenger receptor pathway on macrophages, which results in unregulated cholesterol accumulation, leading to foam cell formation 39. Nephropathy Diabetic nephropathy is one of the important microvascular complications of diabetes mellitus. It occurs in about one third of patients with insulin-dependent diabetes 40 and is the single largest cause of 33 Med Sci Monit 8: RA1 RA4, Am J Physiol 271: C1424, 1996; Circulation 95: , Nat Med 7: , 2001; J Mol Med 79: , Neurochem Int 30: , Circulation 106: , Antiox Redox Signal 7: , Free Radic Res 36(12): , IUBMB Life 49: 303 7,

8 end-stage renal disease requiring chronic dialysis or transplantation. The pathophysiology of diabetic nephropathy is not well defined. Recent studies have indicated that ROS play a key intermediate role in the development of diabetic nephropathy. High glucose directly increases hydrogen peroxide production by mesangial cells and lipid peroxidation of glomerular mesangial cells. Hyperglycemiainduced secondary mediators activation such as protein kinase C (PKC), mitogen-activated protein (MAP) kinases and cytokine production is also responsible for oxidative stress-induced renal injury in the diabetic condition 41. Hemodialysis (HD) is a life-saving procedure required in patients with renal insufficiency. However, contact of blood with a dialyser surface can produce inflammatory reactions and oxidative stress 42. In fact HD patients often have elevated levels of oxidation products and other markers of oxidative stress, such as LDL modification. Oxidation of LDL can take place in blood circulated through HD filters and this modification can be partially inhibited by specific antioxidants during HD. Neuropathy Another complication of diabetes is neuropathy. Hyperglycemia plays a critical role in the development and progression of diabetic neuropathy. One of the mechanisms by which hyperglycemia causes neural degeneration is via the increased oxidative stress that accompanies diabetes. Metabolic and oxidative insults often cause rapid changes in glial cells 43. Retinopathy Retinopathy is another complication of diabetes. In the pathogenesis of diabetic retinopathy, pericytes and endothelial cells are lost selectively before other histopathology is detectable. How these capillary cells die is unclear, but apoptosis is considered as one of the possible mechanisms in their death. Retinal capillary cell death precedes the development of other lesions characteristic of retinopathy in diabetes, and the frequency of early death of retinal capillaries can predict the development of histological lesions of retinopathy. A redox-sensitive nuclear transcriptional factor, NF-κB, is an important regulator of antioxidant enzymes. Activation of NF-κB is considered a key-signaling pathway by which high glucose induces apoptosis in endothelial cells. Activated NF-κB binds to nuclear DNA and modulates the expression of several genes, and this amplification cascade in turn, results in increased free radical production eventually leading to the cell death 44. Diabetic cataract is a major complication of diabetes mellitus, and is primarily caused by polyol accumulation and glycation within lens fibers and the epithelium. The polyol pathway may be related to hyperglycemia-induced oxidative stress, and there may be a metabolic connection between the polyol 41 Pharmacology 72: 42 50, Nephrol Dial Transplant 6: 24-30, 1991; Kidney Int 45: , 1994; Clin Nephrol 47: 37-46, 1997; J Immunol 161: , 1998; Nephrol Dial Transplant 18: , Free Radical Bio Med 35(7): , Free Radic Res 37: ,

9 pathway and oxidative stress 45. Oxidative stress can contribute to diabetic cataract formation, and diabetic human lenses have been found to be more susceptible to protein oxidation. Antioxidant Therapeutic Approach As previously stated, convincing evidence is now available about the role of oxidative stress in the development of diabetic complications. This raises the concept that an antioxidant therapy may be of great interest in these patients. It has recently been suggested that diabetic subjects with complications may have a defective cellular antioxidant response against the oxidative stress generated by hyperglycemia, which can predispose the patient to organ damage 46. According to the evidence previously discussed, it is suggested that interrupting the overproduction of superoxide by the mitochondrial electron-transport chain would normalize the pathways involved in the development of diabetic complications. It might, however, be difficult to accomplish this using conventional antioxidants, because these scavenge reactive oxygen species in a stechiometric manner. The use of traditional antioxidants such as vitamin E or C in large clinical trials has failed to demonstrate any beneficial effect on cardiovascular disease or all-cause mortality, even when only diabetic patients were analyzed 47. Furthermore, a recent meta-analysis of clinical trials studying vitamin E therapy suggests that the use of high-dose vitamin E (greater than 400 IU/day) may actually increase mortality 48. Many explanations for these findings have been put forward. These trials did not select patients on the basis of measurable oxidative stress biomarkers. It is conceivable that the possible beneficial effect of antioxidant therapy was diluted by subjects who had no or very low levels of oxidant stress and who may not have shown a benefit 49. Also, the use of antioxidant vitamins may have been initiated too late or for too short a time in the course of atherogenesis to demonstrate an effect. Finally, there is evidence that both vitamins C and E not only are ineffective at lowering biological markers of oxidative stress, but also can act as prooxidants 50. These findings have prompted a search for new antioxidants which may be able to impact diabetes complications. New low molecular mass compounds that act as SOD or catalase mimetics have the theoretical advantage of scavenging reactive oxygen species continuously by acting as catalysts with efficiencies approaching those of native enzymes 51. Another interesting compound is L-propionyl-carnitine. This substance has been shown to act as an intracellular superoxide scavenger, improving mitochondrial function and reducing DNA damage J Ethnopharmacol 3;101(1-3):49-54, Diabetes 49: , Lancet 360:23 33, 2002; Diabetes Care 25: , Ann Intern Med 142: 37 46, Heart 90: , 2004; Circulation 108: ; 2003; Circulation 106: e195; 2002 [Author reply p. e195]. 50 Circulation 94: 19 25, 1996; J Clin Invest 104: , 1999; Biochem. J. 363(Pt. 3): , 2002; Med Hypotheses 62: , Pharmacol Reviews 53: , Arch Biochem Biophys 288: , 1991; Metabolism 44: , 1995; J Mol Cell Cardiol 2: , 1996; Cell Biol Toxicol 16: , 2000; Diabetes Res Clin Pract 53: 17 24,

10 These properties have been shown to have beneficial effects on diabetic heart function, peripheral nerve function, and vascular blood flow in experimental diabetes. Recently, another substance has received much attention: lipoic acid (LA) 53. Its reduced form is dihydrolipoic acid (DHLA). The mechanisms of action of LA/DHLA redox couple are scavenging of free radicals, metal ion chelation and antioxidant recycling. It may have a unique self-regenerating capacity as a mitochondrial antioxidant, and the possibility that it restores endothelial dysfunction in both animal models of diabetes and in diabetic patients has been reported 54. In this regard, LA has been approved in Germany to manage diabetes-associated polyneuropathies 55. Other promising tools are LY (Ruboxistaurin), PJ34, and FP15, which block the protein C kinase (PKC)-β isoform, poly(adp-ribose) polymerase, and peroxynitrite, respectively. Not surprisingly, they have been shown to ameliorate the endothelial dysfunction induced by hyperglycemia 56. In particular, LY has been demonstrated to reduce oxidative stress generation in the retina 57. PJ34 is not an antioxidant, but a PARP inhibitor. Because mitochondria represent a principal source of reactive oxidants in endothelial cells placed in high glucose 58, may be that PARP suppresses mitochondrial oxidant generation 59. FP15 is a potent peroxynitrite decomposition catalyst 60 and then it is able to reduce the toxicity of peroxynitrite radicals. Another novel and promising antioxidant is AGI-1067 (the monosuccinic acid ester of probucol) which has shown promising results and is currently undergoing phase III trials 61. Therefore, a causal antioxidant therapy may include SOD and catalase mimetics, L-propionylcarnitine, lipoic acid, protein kinase C and poly(adp-ribose) polymerase inhibitors, and peroxynitrite catalysts. This combination would aim to block the noxious cascade activated by hyperglycemia through the overproduction of superoxide and NO. New insights into the mechanisms leading to the generation of oxidative stress in diabetes are now available. And these findings have led to the discovery and to the evaluation of new antioxidant molecules, such as those above described. While waiting for these new and specific compounds, it is reasonable to suggest that already-available substances, such as thiazolinediones, statins, ACE inhibitors, and ATI blockers, should also be used because they are effective causal antioxidants. Thiazolinediones significantly reduce peroxynitrite generation 62. Important trials have shown that these compounds are particularly efficacious in preventing diabetic complications such as retinopathy 63, nephropathy 64, and cardiovascular disease Nutrition 17: , 2001; Curr Med Chem 11: , Diabetes 50: , 2001; Free Radic Biol Med 31: 53 61, Exp Clin Endocrinol Diabetes 107(7): , Circ Res 89: , 2001; Diabetes 51: , 2002; Circ Res 90: , 2002; Mol Med 8: , Acta Diabetol 38: , Nature 414: , J Immunol 161: , Acta Diabetol 38: , Curr Opin Lipidol 14: , 2003; Am. J. Cardiol. 91: 41A 49A, 2003; Circulation 107: , Mol Cell Biol 20: , Lancet 351: 28 31,

11 Also ACE (Angiotensin Converting Enzyme) inhibitors and ATI (Angiotensin I) blockers, drugs used essentially in hypertension, have been demonstrated to act as causal antioxidants 66, and this property may account for their beneficial effect on diabetic complications. Interestingly, ACE were shown to prevent the onset of type 2 diabetes in the Heart Outcomes Prevention Evaluation Study 67. Recently, there has been a considerable interest in finding natural antioxidants from plant materials to replace synthetic ones. Data from both scientific reports and laboratory studies show that plants contain a large variety of substances that possess antioxidant activity 68. Phytochemicals with antioxidant effects include some cinnamic acids, coumarins, diterpenes, flavonoids, lignans, monoterpenes, phenylpropanoids, tannins and triterpenes 69. Injury of plant cells, as well as mammalian cells, is associated with the activation of lipoxygenases, which catalyse the formation of hydroperoxides of polyunsaturated fatty acids; hydroperoxide radicals may react with fatty acids to produce dioxoenes, which are regarded as plant defence compounds. The occurrence of oxidative mechanisms in plants may explain why an abundance of antioxidant compounds have been identified in plant tissue. Therefore it seems that plants particularly those with high levels and strong antioxidant compounds have an important role in improvement of disorders involving oxidative stress such as diabetes mellitus. There are many investigations which have studied the effects of these plants and their antioxidant ingredients on diabetes and its complications and achieved good results 70. Another strategy for diabetes therapy uses pharmacological agents that block AGE RAGE interactions or interrupt the formation of AGE themselves. It can possible to distinguish between drugs specifically developed as AGE inhibitors or AGE breakers such as pyridoxamine (phase II trials are ongoing 71 ; RAGE and receptor signalling blockers; and other therapeutic compounds which were found subsequently to possess also AGE inhibitor activity, including dietary antioxidants 72. Other approaches to prevent AGE-mediated damage include: trapping of circulating AGEs before their binding to AGE receptors, inhibition of AGE interaction with its receptor and inhibition of signal transduction mediated by AGE receptor activation 73. Possible tools for this strategy based on the inhibition of AGE RAGE interaction may be realized by anti-rage antibodies and soluble RAGE. Other strategies such as inhibition of the hexosamine pathway, vitamin therapy to reduce oxidation and AGE accumulation, reduction of the ROS, or blocking the actions of growth factors or intracellular messengers of cell differentiation are also currently under research. 64 Lancet 355: , 2000; N Engl J Med 345: , 2001; Lancet 359: , N Engl J Med 345: , 2001; Lancet 359: , Circulation 104: , 2001; Circulation 105: , 2002; IUBMB Life 49: 303 7, N Engl J Med 342(3):145-53, Food Chem 92:491 7, Vet Clin North Am Small Anim Pract 34(1): , Ann Nutr Metab 48:343 7, 2004; Biofactor 21:273 5, 2004; Pharmacol Res 51:117 23, 2005; Biomed & Pharmacother 59: , Ann N Y Acad Sci 1043: , Pathologie Biologie in press, Diabetes 53: 16672, 2004; Ann N Y Acad Sci 1043: , 2005; J Endocrinol 188: ,