ABSTRACTDiabetes is a multifactorial disease associated with serious comorbidities. This condition has been related to oxidative stress and, as a consequence, to overproduction of reactive oxygen species (ROS), which are known to be produced by different sources during diabetes. Excessive production of ROS can be harmful, making antioxidant defenses of vital importance. Dietary antioxidants, such as vitamin E or vitamin C, have been used to modulate the oxidative stress created in diabetes, producing contradictory results in clinical trials, perhaps as a consequence of the targets selected and/or the design of the studies in question. This chapter considers the process of diabetes with respect to oxidative stress and reviews the different antioxidants strategies employed to treat the disease.
Our writers will create one from scratch for
Key Words: Diabetes, mitochondria, oxidative stress, reactive oxygen species.
In physiological conditions there is a homeostatic control that ensures a balance between the generation of reactive oxygen species (ROS) and their scavenging by endogenous antioxidants. Oxidative stress is produced when there is excessive production of ROS, and has been implicated in many of the diseases that affect humans. These ROS include, among others, superoxide (O2.-), hydroxyl radicals (. HO), hydrogen peroxide (H2O2) and peroxynitrite (ONOO-). Oxidative stress is also associated with endothelial and mitochondrial dysfunction, both of which are related to risk factors of cardiometablic diseases, including diabetes. The mitochondrion is the principal source of ROS, though they are produced in other locations. In addition, there are various types of antioxidants that exert beneficial effects on the mitochondrial electron transport chain. The damaging effects of ROS target carbohydrates, proteins, lipids, DNA and enzymes, leading to cellular dysfunction. Therefore, mitochondria are extremely susceptible to oxidative damage and can accumulate oxidative damage more frequently than the rest of the cell. 1Mitochondrial impairment and damage may be involved in a high number of cellular pathologies. In this sense, several studies have demonstrated that the deleterious effects of ROS are counteracted by antioxidants such as -tocopherol, ascorbic acid, N-acetylcysteine and ubiquinol, all of which are capable of reducing mitochondrial oxidative damage. 2 However, given that these substances do not accumulate in the mitochondria, their effectiveness may be limited. 3
OXIDATIVE STRESS: ROS AND REACTIVE NITROGEN SPECIES (RNS)
The deleterious effect of ROS-mediated damage is evident in different disorders. 4 Oxidative stress can occur when there is an imbalance between ROS production and antioxidant defences, 4 and is associated with risk factors for multiple conditions, including diabetes, atherosclerosis, hypercholesterolaemia and cancer. 4The levels of ROS depend on the pathophysiological and physiological state of the organism; under physiological conditions, there is a homeostatic balance between the production of ROS and their elimination by antioxidants. 5 ROS are also secondary messengers generated in response to different forms of stress, and very slight changes in their intracellular levels can mediate cell communication and activate signal transduction pathways. ROS and reactive nitrogen species (RNS) have a seriously damaging property; both exert an effect on the mitochondrial lipid cardiolipin (CL) by which mitochondrial cytochrome c is released, leading to activation of the intrinsic death pathway.
ROS can be generated by biochemical reactions in the organisms. One of the main sources of mitochondrial ROS is the leakage of electrons from the mitochondrial electron transport chain, as a result of which O2 is the first ROS to be released. 6 O2 can be converted to H2O2 by the enzymes superoxide dismutase (SOD) and -ketoglutarate dehydrogenase, and pyruvate dehydrogenase complex generates both O2 and H2O2 via the oxidation pathway. 7 Although not itself a free radical, H2O2 is a very important biological marker of oxidative stress due to its ability to cross cellular membranes and even can act as an intracellular messenger that activates redox pathways. Excess H2O2 is converted to H2O through a harmless action exerted by catalase (CAT), GSH peroxidase (GPx) and other enzymes. OH is synthesized through the reaction of O2 with H2O2 in the presence of metal ions and is more reactive than O2 , which makes it highly deleterious for cellular membranes and molecules and an inducer of oxidative stress. Iron-catalysed OH generation requires Fe in its reduced, ferrous form (Fe2+), whereas the majority of Fe present in the organism is that of the oxidized form (Fe3+). O2 can also reduce Fe3+ to Fe2+, thereby promoting the production of OH. In physiological conditions, the majority of ROS are released from the electron transport chain and from the membrane NADPH oxidase in phagocytic cells (macrophages and neutrophils) during the inflammatory response. In diabetes, ROS are generated by several potential sources, including endothelial cells, muscle cells, nitric oxide synthase, release of iron and copper ions, metalloproteins, vascular damage caused by ischemia reperfusion, the mitochondrial respiratory electron transport chain, xanthine oxidase (XO) activation, the respiratory burst associated with immune cell activation, and arachidonic acid metabolism. NAD(P)H oxidase, which catalyzes the production of O2 by one-electron reduction of O2 using NADPH or NADH as the electron donor, is present mainly in leukocytes, particularly in phagocytes but also in non-phagocytes (epithelial cells, fibroblasts, chondrocytes and mesangial cells) and in microglial, endothelial and vascular smooth muscle cells. 8 This enzyme plays an important role in the development of diabetes due to the damage it causes to the endothelium by generating hypertension and vascular dysfunction. Importantly, as oxidants produced by immune cells, ROS and RNS have a dual function. On the one hand, they function as potent antimicrobicidal molecules by killing pathogens, while on the other they can act as signalling molecules that modulate different physiological signalling pathways in leukocytes. In the latter role, ROS and RNS are modulators of key enzymes, including proteins, lipid kinases and phosphatases, transporters, membrane receptors, ion channels and transcription factors including NF-B and HIF. In addition, they regulate proinflammatory cytokines and chemokines, thus controlling the inflammatory response, during which ROS and, in turn, RNS modulate the different functions of leukocytes (adhesion, migration and phagocytosis) and secretion, gene expression, autophagy and apoptosis. Under pathological circumstances, excess production of ROS can affect vicinal cells such as those found in the endothelium or epithelium, thereby contributing to inflammatory tissue injury and damage. 9 In this context, the classic behaviour of these cells when faced with oxidative stress implies alterations in different immune functions, such as an increase in adherence, ROS production and phagocytosis, and a decrease in chemotaxis. 2
Currently, research regarding NO and the oxides of nitrogen is of considerable biomedical interest due to the pathophysiological implications of these molecules. NO, nitrous oxide (N2O) and nitrogen dioxide (NO2) can be homeostatic or delererious. NO2 is an environmental pollutant produced in vivo in response to reactions against NO and is implicated in lipid peroxidation, cellular membrane damage and apoptotic processes. NO reacts slowly with the majority of molecules in the human body (non-radicals), but as a free radical reacts quickly with other molecules, including ROS such as O2, transition metals and amino acid radicals. The reaction between NO and O2 produces peroxynitrite (ONOO-). 10 ONOO- is implicated in a high number of human diseases; in fact, diminished availability of NO and increased ROS formation may constitute key events in the development of cardiovascular diseases (CVD) including atherosclerosis. iNOS expression is triggered as a consequence of the activation of macrophages, monocytes and endothelial cells and induces the transformation of L-arginine into NO, which can combine with O2 to form ONOO-. NO stimulates ROS – for example, H2O2 and O2 production by the mitochondria11 – possibly by inhibiting cytochrome c oxidase (COX), which promotes the leakage of electrons from the respiratory chain, and through irreversible inhibition of mitochondrial complex I. H2O2, in turn, is involved in the upregulation of iNOS expression via nuclear transcription factors such as NF-B activation. In tissue damage, inflammatory reactions play a key role that is mediated by adhesion and migration of leukocytes through the vessel, generation of RNS and ROS, and the production of several proinflammatory cytokines and chemokines by monocytes/macrophages. In addition, the local generation of RNS can contribute to tissue damage.
DIABETES, OXIDATIVE STRESS AND ANTIOXIDANTS
Diabetes is one of the leading health problems worldwide. In addition, it is associated with different comorbidities that affect the life expectancy and quality of life of patients. Nowdays, 270 million people around the world live with diabetes, and this figure will rise to 400 million over the coming years. The link between diabetes and cancer has been studied in great depth, and the majority of the evidence obtained suggests that diabetes enhances the risk of developing different types of cancer, which obviously magnifies the clinical implications of the former disease. However, there are contradictory results that deserve careful reinterpretation, as diabetes is a complex disease in which multiple metabolic pathways and nuclear transcription factors are involved. Taking this into account, the different types of diabetes that exist produce an array of metabolic and hormonal abnormalities that can affect patients in different manners. As a consequence, it is necessary to study diabetic patients as a homogeneous cohort. In addition, there are numerous parameters which may have a bearing on our understanding of the relationship between cancer and diabetes, including metabolic controls, population, obesity, statistical methods, diet and sex. Nevertheless, it is well demonstrated that oxidative stress is linked to diabetes and that the latter is characterised by mitochondrial impairment, 4 and both conditions are known to be related with cancer.
Diabetes, inflammation and oxidative stress
Diabetes is associated with oxidative stress and inflammation, which are related to a high production of adhesion molecules and proinflammatory cytokines, including IL-6 and TNF. Diabetes is, therefore, a chronic pro-inflammatory state that gradually reduces intracellular antioxidant stores, leaving cells vulnerable to damage. In fact, ROS at high concentrations can damage cell DNA by direct oxidation or by interfering with cell repair mechanisms. 12 ROS can also impair lipids and proteins, releasing molecules that alter cellular homeostasis and promoting the accumulation of key mutations, which eventually contributes to cancer. 13 Mitochondrial activity is crucial for DNA repair, as the impairment of mitochondria not only results in a low, insufficient energy supply, but also increases ROS production. 14 In addition, there are factors which correlate with insulin resistance, such as the proinflammatory cytokine TNF released by adipose tissue. 15 TNF can trigger the progression and development of different tumours16 by activating transcription factors such as nuclear factor-kappa B (NF-B), which is involved in the pro-tumoral effects of cytokines. In conclusion, diabetes promotes the biological aging processes that can induce cancerogenesis through mechanisms specific to both diabetes and other chronic degenerative diseases.
ROS PRODUCTION AND DIABETIC COMPLICATIONS
There is a wealth of evidence to suggest that oxidative stress plays a key role in the development and progression of diabetic complications such as nephropathy. The use of different agents to modulate ROS generation can reduce the cellular uptake of glucose and subsequently delay the feeding of glucose metabolites during oxidative phosphorylation. Nevertheless, oxidative phosphorylation and mitochondrial function are considered to be key targets in the modulation of said diabetic complications, among which nephropathy is included. High levels of glucose can induce mitochondrial ROS, which leads to activation of different biochemical pathways, hexosamine, increased flux through the polyol, enhanced AGE formation and activation of protein kinase C isoforms. 17 However, there are other important pathways, including NADPH oxidase and the uncoupling of eNOS, which require further investigation in order to determine their relative importance in progressive diabetic complications such as renal disease.
ANTIOXIDANTS AND DIABETES
Organisms have numerous antioxidant systems designed to ameliorate the deleterious effects of ROS production and oxidative stress. Superoxide dismutase (SOD) is one of the most important of these antioxidant enzymes. SOD has three isoenzymes – CuZnSOD (SOD1), MnSOD (SOD2) and extracellular (SOD3) – whose main function is the detoxification of O2 to H2O2 and water. Catalase and glutathione peroxidase (GPx) are other antioxidant enzymes that can catalyze the conversion of H2O2 to water. In addition, there are numerous antioxidants found in cells which play a homeostatic role, such as vitamins and glutathione. However, studies have shown that these antioxidants have no real beneficial effects in the treatment of diabetic complications. In one study, it was demonstrated that overexpression of CuZnSOD countered organ impairment in models of type 2 diabetic nephropathy. 18 In another study, MnTBAP (MnSOD mimetic) was shown to be effective in preventing ROS-induced injury in vitro, 17 while the in vivo use of such drugs has produced controversial data19 that do not confirm any substantial beneficial effects. It should be mentioned that some polymorphisms of the MnSOD gene are related to the development of diabetic nephropathy. 20 Interestingly, in an animal model of GPx-1–deficient mice, diabetic nephropathy and microvascular disease were not found to be more pronounced. 21 Overexpression of other antioxidant enzymes, such as catalase, in several models of type 2 diabetic nephropathy seems to be beneficial, 22 suggesting the importance of erradicating high levels of ROS. In human studies, however, no relationship has been detected between the incidence of nephropathy in diabetes and catalase gene polymorphisms. 23
As mentioned previously, the excessive energy intake owing to saturated fatty acids and high glycemic index foods is an important source of oxidative stress. In order to counteract this oxidative stress, there are several antioxidants (carotenoids, ascorbic acid, tocopherols and flavonoids) which can protect against oxidative damage and its complications, including diabetes and insulin resistance. Different in vitro and animal studies have shown that dietary antioxidants have beneficial effects on glucose metabolism, and can help to prevent diabetes. Polyphenols are one of the most studied of these antioxidants and have shown interesting results. For example, quercetin, one of the most consumed flavonoids in the human diet, has demonstrated beneficial effects on glucose metabolism by attenuating TNFmediated inflammation and insulin resistance in primary human adipocytes. 24 Cyanidin-3-O-β-glucoside has been shown to have insulin-like activities by activating PPAR in human adipocytes. 25 Other flavonoids, such as hesperetin and naringenin, inhibit TNF-stimulated free fatty acid secretion in cultured mouse adipocytes, 26 as well as increasing glucose uptake by AMPK in cultured skeletal muscle cells, 27 and enhancing insulin sensitivity by increasing tyrosine phosphorylation in fructose-fed rats. In one study, the flavonoid luteolin was shown to increase insulin sensitivity by activating PPAR transcriptional activity in 3T3-L1 adipocytes. 28 Caffeic acid phenethyl ester stimulates glucose uptake into cultured skeletal muscle cells through the AMPK pathway 29 and glucose uptake into insulin-resistant mouse hepatocytes, as does cinnamic acid. 30 Resveratrol can improve mitochondrial activity and energy balance, protecting mice against diet-induced obesity and insulin resistance. Therefore, resveratrol may have an important role to play in the prevention of metabolic diseases and diabetes. In addition to its antioxidant capacity, glucose metabolism exerts its actions through other molecules, such as sirtuin 1. 31There is considerable information available about the beneficial effects of dietary antioxidants in cells and animal models, but few intervention studies have been carried out to directly assess the effects of antioxidants on diabetes and glucose metabolism in humans. α-lipoic acid and vitamin E and C supplementation, alone or in association with other antioxidants, have been shown to have positive effects in some studies, 32 though others have failed to detect any benefits. Other research has demonstrated that a high dose of trans-resveratrol has favourable effects on glucose homeostasis in obese subjects by improving their HOMA index, thus exerting a positive effect on insulin sensitivity. Epidemiologic studies have reported that diets rich in antioxidants such as α-tocopherol, 33 vitamin C, 34 vitamin E 35 or β-carotene 33 are beneficial in that they prevent diabetes and improve glucose metabolism. Two meta-analyses have evaluated the association between the intake of fruit, vegetables and antioxidants and the risk of diabetes. The major finding of one was that consumption of antioxidants but not that of fruits and vegetables produced a 13% decrease in the risk of diabetes, which was attributed mainly to vitamin E. 36 In other, the intake of green leafy vegetables was associated with a 14% decrease in the risk of developing diabetes. 37
Antioxidants and mitochondria
Glucose is employed by the mitochondrial transport chain via oxidative phosphorylation. During this process, it is converted into various substrates (e. g. pyruvate) and NADH and FADH2 are released into the mitochondria via different transport systems. Hyperglycemia and the increase in the NADH/NAD ratio associated with diabetes lead to deleterious and serious complications. In addition, NADH is one of the main electron donors to the mitochondrial electron transport chain 38 and favours the presence of oxidative stress. For these reasons, it is of great importance to reduce hyperglycemia during diabetic complications 39 by decreasing the availability of the substrate fuel consumed by mitochondria. In fact, mitochondria consume other substrates such as free fatty acids (FFAs) as fuel, and their oxidation in the tricarboxylic acid cycle generates FADH2 and NADH. Therefore, the presence of high levels of FFAs mimick the effects of hyperglycemia on mitochondrial impairment. Oxidative stress and ROS production are recognized as key mediators of the development of diabetic complications, 17 and mitochondria are their main source. Therefore, therapies which use molecules to ameliorate mitochondrial ROS can be of benefit in the management of diabetic comorbidities. During oxidative phosphorylation, electrons from different substrates are transferred to O2 via the electron transport chain. Protons are then pumped across the mitochondrial membrane and the resulting voltage gradient generates ATP. Complex I and III of the electron transport chain are the two main sites of electron leakage. In diabetes, the excessive production of ROS as a consequence of high levels of glucose is believed to play an important role in mitochondrial membrane potential. 17 However, the majority of the studies exploring this aspect have been carried out in tissues, so in vivo investigation is now necessary. In this context, it has been hypothetized that mitochondrial dysfunction and damage of the mitochondrial respiratory chain can contribute to many pathologies; indeed, patients with deleterious genetic mutations that reduce the activity of different mitochondrial complexes display high levels of mitochondrial ROS. 40 Recently, diabetic patients have been reported to develop mitochondrial complex I dysfunction, which is followed by an increase in ROS production and decrease in membrane potential and antioxidant levels. 41 In relation with this idea, an impairment of mitochondrial complex I 42 and subsequent rise in oxidative stress have also been described in pathologies characterised by insulin resistance, such as polycystic ovary syndrome. In a recent study, mitochondrial function has been shown to be impaired and leukocyte-endothelium interactions to be more frequent among diabetic patients. These characteristics were evident in the lower membrane potential, mitochondrial O2 consumption, GSH/GSSG ratio and polymorphonuclear cell rolling velocity, and in the higher mitochondrial ROS production and rolling flux, leukocyte adhesion and vascular cell adhesion molecule-1 (VCAM-1) and E-selectin molecules observed in these subjects. 43 In addition, these alterations correlated with the presence of silent myocardial ischemia (SMI). The authors concluded that mitochondrial dysfunction, oxidative stress and endothelium-inducing leukocyte-endothelium interactions are key features of type 2 diabetes and correlate with SMI. Other evidence is available of the role of mitochondrial oxidative stress as one of the main factors in the development of diabetic complications such as Friedreich’s ataxia, a genetic disorder caused by mutations implicated in the down-regulation of mitochondrial complex I and a highly mitochondrial ROS generation. 44 In one study, mitochondria were shown to play a key role in diabetic nephropathy, as around 45% of children with mitochondrial dysfunction had renal diseases. 45 In addition, renal disease is the primary pathology in patients with oxidative phosphorylation defects. 46 These results highlight mitochondrial impairment as a priority for research into future therapies. In this sense, mitochondria-targeted antioxidants are a potential tool for the treatment of diabetes. As mentioned previously, mitochondrial ROS production can lead to a wide range of deleterious reactions that can damage several structures or molecules, including proteins, lipids and nucleic acids. Apart from being a source of ROS, mitochondria possess multiple enzymes that are susceptible to damage by ROS, which results in alterations of the membrane potential, impairment of cellular calcium storage and a diminution of ATP production (all related to reticulum stress, autophagy and the development of apoptosis or necrosis). Idebenone is a mitochondrial antioxidant that is highly available inside the organs. This kind of antioxidant is efficient and safe for protecting mitochondrial function from oxidative damage in humans with Friedreich’s ataxia. 47 Idebenone has been shown to reduce cardiomyopathy in these subjects, unlike dietary antioxidants such as vitamin E and alpha-tocopherol. 47MitoQ is a mitochondria-targeted antioxidant that is selectively uptaken into mitochondria due to the covalent attachment to the lipophilic triphenylphosphonium cation. This antioxidant can accumulate in mitochondria 1000-fold. 48 Although the efficacy of these mitochondria-targeted antioxidants in the treatment of insulin resistance and diabetes is yet to be determined, their targeted specificity for mitochondria makes them potential therapeutic agents of cardiometabolic diseases and diabetes. In this sense, it has been reported that MitoQ administered orally over a 12-week period improves tubular and glomerular function in Ins2(+/)⁻(AkitaJ) mice (an animal model of diabetes). In the study in question, MitoQ did not have a notable effect on plasma creatinine levels, but reduced urinary albumin levels to those of non-diabetic controls. Furthermore, glomerular damage and interstitial fibrosis were significantly reduced in treated animals. The nuclear accumulation of β-catenin and the transcription factor phospho-Smad2/3 observed in Ins2(+/)⁻(AkitaJ) mice was prevented by treatment with MitoQ. These results support the hypothesis that mitochondrially-targeted treatments are beneficial in the treatment of diabetic nephropathy. 49ConclusionsAlthough experimental data have been obtained in different cellular and animal models regarding the role of oxidative stress in insulin resistance and diabetes and the positive effects of dietary antioxidants, research carried in humans until now are contradictory. The poor outcome of trials with antioxidants is comprehensible if we bear in mind the magnitude and variety of oxidative events caused by ROS rather than focusing on classic antioxidants that are effective only against oxidative reactions. In this context, it is of vital importance to obtain future experimental evidence regarding the protection that scavengers of ROS offer against disease. There are several possible reasons for the discrepancies found among the data reported in the literature. First, the antioxidant actions of dietary antioxidants may be modified by environmental conditions such as metal ions and pH, and by their concentration and location. Indeed, they can even become pro-oxidant at high doses. Second, ROS can act as secondary messengers and are necessary for the transduction of insulin signals, which means that their excessive neutralization can be harmful. Third, the efficiency of dietary supplements and natural products is not restricted to their antioxidative capacity, which can be highly variable depending on the model studied. Fourth, given that the mitochondria are the main source of ROS and are key to the redox balance of the cell, mitochondria-targeted antioxidants are fundamental tools with which to control oxidative stress. We trust that work over the coming years will indicate in which organs these compounds are effective, whether they alleviate disease-related mitochondrial oxidative damage, and to what extent their use can positively affect the outcome of treatments.
We thank B Normanly for his editorial assistanceThis study was financed by grants PI10/1195, PI12/1984, CIBERehd CB06/04/0071, PROMETEO 2010/060, ACOMP/2012/042 and ACOMP/2012/045. V. M. V. is the recipient of a contract from the Ministry of Health of the Valencian Regional Government and Carlos III Health Institute (CES10/030).