Diabetes: Oxidative Stress and Dietary Antioxidants

Spain

Preface

In the past few decades there have been major advances in our understanding of the etiology of disease and its causative mechanisms. Increasingly it is becoming evident that free radicals are contributory agents: either to initiate or propagate pathologies or to create an overall cellular and metabolic imbalance. Furthermore, a reduced intake of dietary antioxidants can also lead to an increased risk of specific diseases. On the other hand, there is abundant evidence that naturally occurring antioxidants can be used to prevent, ameliorate, or impede such disease risks. The science of oxidative stress and free radical biology is rapidly advancing and new approaches include examining the roles of genetics and molecular biology.

However, most textbooks on dietary antioxidants do not have material on the fundamental biology of free radicals, especially their molecular and cellular effects on pathology. They also fail to include material on the nutrients and foods that contain antioxidative activity. In contrast, most books on free radicals and disease have little or no text on the usage of natural antioxidants.

In the present volume Diabetes: Oxidative Stress and Dietary Antioxidants, Second Edition, holistic information is imparted within a structured format of three main sections.

Section I: Oxidative Stress and Diabetes

Section II: Antioxidants and Diabetes

Section III: Techniques and Resources

Section I: Oxidative Stress and Diabetes covers the basic biology of oxidative stress from molecular biology to physiological pathology. In Section II: Antioxidants and Diabetes we describe agents and their actions. The caveat of these chapters in Section II is that there needs to be further in-depth analysis of these components in terms of safety and efficacy as some material is exploratory or preclinical. A cautionary and critical approach is needed. Nevertheless, the material in Section II can provide the framework for further in-depth analysis or studies. This would be via well-designed clinical trials or via the analysis of pathways, mechanisms, and components in order to devise new therapeutic strategies. Section III: Techniques and Resources provides a practical source of information. Both preclinical and clinical studies are embraced using an evidence-based approach. However, the science of oxidative stress is not described in isolation but in concert with other processes such as apoptosis, cell signaling, and receptor-mediated responses. This approach recognizes that diseases are often multifactorial and oxidative stress is a single component of this.

Diabetes: Oxidative Stress and Dietary Antioxidants, Second Edition is designed for dietitians and nutritionists, food scientists, as well as healthcare workers and research scientists. In this book the target audience also includes diabetologists, biochemists and food scientists, clinicians, basic science researchers, medical students, healthcare industry workers, endocrinologists, family medicine physicians, diabetes nurse practitioners, and drug developers. Contributions are from leading national and international experts including those from world-renowned institutions.

Professor Victor R. Preedy,

King’s College London

Section I

Oxidative stress and diabetes

Outline

Chapter 1 Oxidative stress markers in diabetes

Chapter 2 Oxidative stress and diabetic neuropathy

Chapter 3 Diabetic enteric neuropathy: imbalance between oxidative and antioxidative mechanisms

Chapter 4 Hyperglycemia-induced oxidative stress in the development of diabetic foot ulcers

Chapter 5 Oxidative stress in diabetic retinopathy

Chapter 6 Cerebral ischemia in diabetics and oxidative stress

Chapter 7 Gingival wound healing in diabetes

Chapter 8 Oxidative stress in gestational diabetes mellitus

Chapter 9 Epigenetics, oxidative states and diabetes

Chapter 10 MicroRNAs linking oxidative stress and diabetes

Chapter 11 Polymorphism of MnSOD 47C/T antioxidant enzymes and type 1 diabetes

Chapter 12 Sodium-glucose cotransporter 2 inhibitors, diabetes, and oxidative stress

Chapter 13 NADPH oxidases, nuclear factor kappa B, NF-E2-related factor2, and oxidative stress in diabetes

Chapter 14 Antioxidant properties of drugs used in type 2 diabetes management

Chapter 1

Oxidative stress markers in diabetes

Eugene Butkowski,    School of Community Health, Charles Sturt University, Thurgoona, NSW, Australia

Abstract

Oxidative stress (OS) plays a significant role in the development and pathophysiology of type 2 diabetes mellitus (T2DM) and cardiovascular disease (CVD). Studies indicate the inference between OS and its impact on a variety of biochemical and physiological responses result in changes to normal metabolic activities, including alterations to enzyme systems and lipid peroxidation with inflammatory responses, just to name a few. These cascading changes are responsible for disease progression; however studies to date are still not entirely definitive. What is evident is a requirement that we continue toward obtaining a greater understanding of how an increase in free radicals/reactive oxidation species and the consequential OS imbalance, along with the hyperglycemic state, contributes to T2DM and CVD disease progression and associated comorbidities.

Keywords

Oxidative stress; inflammation; type 2 diabetes mellitus; cardiovascular disease; oxidative and inflammatory biomarkers

List of abbreviations

8-OhdG 8-hydroxy-2′-deoxyguanosine

CVD cardiovascular disease

GSH reduced glutathione

GSSG glutathione disulfide

IL interleukin

MDA malondialdehyde

OS oxidative stress

ROS reactive oxygen species

T2DM type 2 diabetes mellitus

WBV whole blood viscosity

Introduction

This chapter explores several known and emerging biomarkers that have vastly contributed to increasing our knowledge to the understanding of the pathophysiological changes and events occurring as a result of our response to hyperglycemia. Most importantly, there is the requirement to acknowledge the link that oxidative stress (OS) and inflammatory processes have with diabetes and its associated comorbidities. Studies in OS have reinforced its mechanistic relationship with hyperglycemia, the consequential development of type 2 diabetes mellitus (T2DM) and its associated emergence of cardiac disease, atherosclerosis, neuropathy, retinopathy, nephropathy, and other comorbidities. The rise of metabolic syndrome (MetS), a constellation of five risk factors, of which three are required for a definitive diagnosis (see Table 1.1), appears to have an inextricable link with its potential progression to T2DM and cardiovascular disease (CVD). This further corroborates the requirement and need to fully grasp the importance that OS and the consequential inflammatory process as a probable predisposition to disease progression.

Table 1.1

>3 risk factors are indicative of MetS.

HDLC, high density lipoprotein cholesterol.

While it is viewed that CVD complications of T2DM are a major worldwide health problem, knowledge of the progression of T2DM and degree of CVD risk associated with T2DM still requires further exploration. Profound and critical to global health is the impact of T2DM and CVD, including heart disease, vascular disease, and atherosclerosis, which it is purported to contribute to over one third of global morbidity.¹ Current estimates are that globally, there are 79.1 million deaths occurring annually as a result of CVD.² T2DM can therefore be considered a pandemic clinical disorder with no distinction between developing and developed countries, and continues to contribute to the burden of CVD.³ A major concern is that as many countries emerge from third-world conditions with an ensuing increase in a middle-class population, there will be a further exacerbation of this insidious disease state. The Daily Sun reported in 2006 that some 12 million Nigerians were now suffering from diabetes.⁴ This would indicate that we are not necessarily dealing only with a lifestyle epidemic, but other factors are also contributing. Cardiovascular risk assessment in prediabetes is important in further establishing any causal link to the recognized morbidity attributed to T2DM and CVD.⁵

The heterogeneous nature of T2DM contributes to the diverse views of how elevated levels of glucose contribute to the endothelial dysfunction evident in CVD associated with T2DM. There is strong epidemiology evidence showing that T2DM is a major risk factor for CVD, with several plausible mechanisms underlying their association and underpinning possible treatment regimen.⁶ While the diagnostic capability of elevated blood glucose is a predictor of Diabetes Mellitus (DM) - types 1 and 2 and to some extent insulin resistance, it is a relatively poor predictor of CVD. Cardiac autonomic dysfunction, assessed by heart rate variability (HRV), is a known risk factor for the development of CVD⁷ and is associated with hyperglycemia and a demonstrated decrease in the prevalence of HRV.⁸ There is, however, a significant relationship between blood glucose and CVD, but no reliable threshold level of glucose and identification with CVD.⁹ Some microvascular complications, in particular retinopathy, do evidence a clearer threshold with glycemia.¹⁰ The effects of hyperglycemia are multifactorial, as HRV is subject to autonomic control and structurally, the cardiovascular system is impacted by the OS and inflammatory response. All these effects, along with our incomplete understanding of how OS and inflammatory mechanisms participate in hyperglycaemia and CVD, provide further reinforcement in the need to investigate innovative markers to further our understanding of this insidious global dilemma.

The World Health Organization (WHO) has long recognized the link between obesity, CVD, and T2DM and has classified a constellation of factors to include at least three of the five characteristics (Table 1.1) to MetS.¹¹–¹³ The MetS risk for the development of CVD and T2DM is dramatically increased in people who meet the diagnostic criteria for MetS.¹⁴ Table 1.1 emphasizes the National Cholesterol Education Program (NCEP) Adult Treatment Panel III criteria.

Oxidative stress: an overview

OS may be described as a disturbance in the oxidant/antioxidant balance within the cell, where the oxidant prevails.¹⁵ It is a state in which a cell is experiencing alteration of cellular components, due to its being under high exposure to free radicals and ROS beyond its antioxidant capacity. Although the reaction of ROS is essential for cellular functions, such as the utilization of the chemical energy of nutrients for production of adenosine triphosphate (ATP), excess in OS. The generation of oxygen-free radicals is often formed through the stimuli of physical agents such as chemical, radiation, pollutants, and physiological processes which have the capacity to disrupt mitochondrial function.¹⁶ While the literature portrays ample information regarding the presence of OS in a variety of pathophysiological processes, the clear correlation between many disease states and OS is still lacking due to insufficiencies still present in our full understanding of contributing molecular mechanisms. Recognizing that persistent hyperglycaemia initiates OS by an increase in both intracellular and extracellular free radical levels,¹⁷ progressive biochemical reactions inclusive of procoagulation and inflammation contribute to the development of atherosclerosis. Thus, because of OS, a constellation of biochemical activity (see Fig. 1.1) is important when gauging our understanding of disease progression and observed relationships with T2DM, CVD, and comorbidities. The traditional and novel OS and inflammatory and procoagulant markers changes, and whole blood viscosity (WBV) requires discussion and mention as they are all inextricably related in the pathophysiological changes operating within, and because of, the hyperglycemic state. The drive of the inflammatory process due to the increased perturbations of the OS state at an anatomical level provides insight as to how diabetes contributes to CVD, reduced blood flow, and plaque formation. Plaque formation and potential rupture may occur as a result of increased oxidative and inflammatory pathological changes which are intimately associated with increased macrophage cellular oxidative activity.¹⁸ Although macrophages are diverse in functionality their immunological role in OS and atherosclerosis from formation through to rupture is indicative of their contributory role in all requisite stages of the inflammatory atherosclerotic process. Consumption of oxidized LDL (ox-LDL) results in lipid-laden macrophages deposited underneath the endothelium of arteries as part of the plaque formation, which then may become obstructive. Macrophage cellular oxidation, hydroperoxides, autophagy protein, and thiol OS also contribute to accelerated atherosclerotic plaque progression.

Figure 1.1 Processes associated with the development of atherosclerosis in type 2 diabetes mellitus (T2DM) and cardiovascular disease (CVD). The formation of the atherosclerotic lesion linking the biochemical and pathophysiological changes which occur due to the interactions of OS, inflammation, and coagulation/fibrinolysis. The pathology is linked directly to T2DM and CVD.

Oxidative stress in type 2 diabetes mellitus and cardiovascular disease

OS is likely implicated in diabetes and vascular disease states. The endothelium plays an integral role in many disease states by regulation of vascular tone, platelet activity, leukocyte adhesion, and thrombosis, and can therefore be intricately linked to the development of atherosclerosis. Increasing evidence is highly suggestive of OS contributing significantly to endothelial dysfunction.¹⁹ Increased production of oxygen-derived free radicals, such as the superoxide anion, has been linked to impaired endothelial vasomotor function in clinical and experimental models of atherosclerosis.²⁰

It is now widely accepted that OS contributes to and plays a central role in the pathophysiology of T2DM and associated vascular and neurological changes, possibly via mediating the diversion of glycolytic intermediates.²¹ Several oxidative markers discussed intra provide further insight into their contribution and how their measurement assists in providing evidenced-based information of their biochemical contribution to our current knowledge. It is important to recognize that the markers discussed are far from exhaustive, but as we gain more understanding, the number of biomarkers will no doubt increase.

Protein kinase C and reactive oxygen species

All forms of diabetes are characterized by hyperglycemia. As a consequence, the metabolic abnormalities result in endothelial superoxide production, which in turn produces pathological processes, including the activation of the protein kinase C (PKC) isoforms.²² The family groups of PKC which feature in many cellular signal transduction pathways, are enzymes involved in the control of protein functionality through the phosphorylation of the hydroxyl groups of the residue groups of threonine and serine. ROS may trigger various PKC isoforms (PKC-α, β1/2 and PKC-δ) implicated in CVD, through redox signaling, and has been associated with vascular alterations related to permeability, contractility, extracellular matrix synthesis, cell growth and apoptosis, angiogenesis, leukocyte adhesion, and cytokine activation and inhibition.²³

Reduced glutathione, glutathione disulfide, and glutathione/glutathione disulfide

Reduced glutathione (GSH), one of the major cellular antioxidants, is a linear tripeptide consisting of L-glutamine, L-cysteine, and glycine, possessing a sulfydryl (SH) group on the cysteinyl portion accounting for its strong proton-donating character. The synthesis of the tripeptide is catalyzed in two steps by L-glutamyl-L-cysteine: glycine ligase and glutathione synthetase.²⁴ The biological activity of GSH occurs in the proton-donor SH group of the cysteine moiety; the loss of the GSH electrons results in the oxidized form, consisting of a reversible-linked dimerized glutathione disulfide (GSSG). A tight homeostatic control exists, both intracellular and extracellular, with a dynamic balance maintained between GSH synthesis,²⁵ the recycled GSSG/oxidized GSH, and its utilization.²⁶

Despite the diverse nature of systemic issues with T2DM and the recognition of the multifactorial pathways involved in mediating tissue damage, a common feature is increased OS marked by demonstratively elevated levels of ROS. The generation of OS and increased ROS in hyperglycemia appears to be diminished by lowering the concentration of blood glucose. Notably, intracellular cysteine and glycine have also been demonstrated to be depleted with replenishment of the amino acids showing levels of GSH approaching normality after 14 days.²⁴ Furthermore, a proinflammatory response in adipose tissue induced by hyperglycemia has been shown to be reduced by the introduction of N-acetylcysteine (NAC).²⁷ Interestingly, glycine depletion, possibly due to glutathione consumption driven by OS, may deplete glycine, reflecting increased gluconeogenesis, or it could also be indicative of abundant, incompletely oxidized fuels that are excreted as urinary acylglycine conjugates.²⁸ The introduction of NAC would therefore provide a reversal role and possibly decrease gluconeogenesis.

A decrease in GSH with a concomitant increase in GSSG has been demonstrated in T2DM patients, resulting in a significant decrease in the GSH:GSSG ratio.²⁹ It has also been found that in patients with impaired fasting glucose (IFG), despite no significant changes in GSH and GSSG levels, the GSH:GSSG ratio is significantly decreased (P = .04) when compared to a normal control group, suggesting that an impaired redox balance can be evident even when minor increases in blood glucose levels are demonstrable.³⁰ Decreasing OS through glycemic control will therefore potentially be an important mechanism in diminishing the incidence of microvascular complications associated with T2DM.

Antioxidant defense is important in the removal of free radicals, providing the maximal protection of biological sites, such as thiol groups, which are part of active sites in some metabolizing enzymes. A good antioxidant should specifically quench free radicals, chelate redox metals, interact with other antioxidants within an antioxidant network, have a positive effect on gene expression, be readily absorbed, and have a concentration in tissues and biofluids at a physiologically relevant level. The antioxidant should be functional in both aqueous and/or membrane domains. The most efficient enzymatic antioxidants involve superoxide dismutase, catalase, and glutathione peroxidase. Nonenzymatic antioxidants involve thiol antioxidants (glutathione, thioredoxin, and lipoic acid), vitamin C, vitamin E, carotenoids, natural flavonoids, melatonin, and other compounds (selenium).³¹

8-Hydroxy-2′-deoxyguanosine

8-Hydroxy-2′-deoxyguanosine (8-OhdG) is a product of oxidatively damaged DNA formed by hydroxyl radical, singlet oxygen, and direct photodynamic action. 8-OhdG can be detected in tissue, serum, urine, and other biological material.³² Amongst the purine and pyridines, the guanine base appears to be the most susceptible to oxidation. It is widely recognized that the most important oxygen-free radical causing biomolecular damage is the hydroxyl radical. This radical can be produced by a variety of mechanisms, including the Fenton reaction of hydrogen peroxide and metals of other endogenous and exogenous ROS. The interaction of the hydroxyl radical with a nucleobase, such as guanine, results in the formation of C8-hydroxyguanine, or its nucleoside 8-OhdG.³² Upon the oxidation of in vivo guanine to the free radical, 8-OhdG is readily excreted into urine by glomerular filtration, thus serving as a useful indicator for the quantification of systemic OS. Markedly increased levels of urinary 8-OhdG have been shown in T2DM and are positively correlated with hemoglobin A1c (HbA1c)³³ and have been shown to be an early indicator for the development of microvascular and macrovascular complications in T2DM.³⁴ Increased levels of 8-OhdG levels in both prediabetes and diabetes have also revealed statistically significant results upon comparison with cholesterol, Malondialdehyde (MDA), and erythrocyte-reduced GSH levels, further indicating the utility of 8-OhdG as a potentially useful early marker of vascular damage as a consequence of OS.³⁵ This is further confirmed in recent studies conducted by the author,¹⁷ where it was demonstrated that a higher risk of CVD is presented with increased 8-OhdG. These findings do support previous studies that reflect the antioxidant function of GSH, where the erythrocyte GSH pool decreases at first with increased Blood glucose (BGL) and CVD risk, but increase due to de novo synthesis in response to continued elevated fasting BGL in the erythrocytes increase.³⁰,³⁶

F2-isoprostanes

The F2-isoprostanes are a group of prostaglandin-like compounds formed in vivo by the nonenzymatic free radical catalyzed peroxidation of nonesterified arachidonic acid. This group of isomers is formed independently from the action of cyclooxygenases (COX-1 and COX-2).³⁷ The autoxidation process lacks specificity and leads to the formation of many different structural and stereoisomers. Isoprostanes, while possessing relatively short half-lives, demonstrate potent biological activities, especially in the lungs and kidneys with possible normal physiological functions.³⁸ While isoprostanes may resemble normal prostanoids, the most abundant form is analogous to prostaglandin F2α, while analogs of prostaglandin D2 and prostaglandin E2 are also found, differing in many aspects of their stereochemistry. Among the sequelae of hyperglycemia, it is widely recognized that excessive OS is associated with increased vascular disease in DM.³⁹ Early experimental results with thiobarbituric acid-reactive substances and lipid hydroperoxides have established the link with atherosclerosis and lipid peroxidation, both within the vasculature and the plasma, providing the evidential link between hyperglycemia-induced OS and diabetic vascular disease.⁴⁰ More recent evaluations on the utility of isoprostanes have established their accuracy and sensitivity as a useful analytical tool for assessing OS in a variety of pathologies, with some isoprostanes exhibiting potent biological activity and therefore likely mediators in oxidant injury.⁴¹ A meta-analysis of elevated F2-isoprostanes with coronary artery disease, stroke, and peripheral artery disease has further consolidated the utility of this biomarker, albeit as a nonspecific indicator of CVD.⁴²,⁴³ Further detailed ongoing analysis of isoprostane levels may therefore provide useful information as a biomonitor when developing strategies for minimizing vascular complications.⁴⁴

Malondialdehyde

ROS degrade polyunsaturated lipids to form MDA. The aldehyde MDA, as a reactive electrophile species, has the capacity to cause toxic stress by producing lipid peroxidation end products, with the measurement of MDA formation considered to be one of the most frequently quantitative assessments.⁴⁵ As lipids are primary targets in the peroxidation, the formation of the peroxide radical is considered extremely harmful to the cell membrane. While the actual mechanism of MDA as an endothelial cytotoxic agent is poorly understood, it is still considered a useful primary biomarker in the assessment of free radical-mediated lipid damage and OS.⁴⁶ Diabetic studies utilizing MDA as a marker of OS have demonstrated increased levels.⁴⁷ A recent Veterans Affairs Diabetes Trial investigated the association with MDA and diabetes, demonstrating that circulating levels of the immune complex MDA-LDL may predict the occurrence of myocardial infarction and acute cardiovascular events in patients with T2DM.⁴⁸ Interestingly, ox-LDL has been shown to accumulate in atherosclerotic lesions with increasing evidence pointing to a pathogenic role in CAD, ACS, and vulnerable plaque. Measurement of MDA has therefore provided further insight into progressive atherosclerosis. Ox-LDL has been shown to have suppressive effects on genes regulating endothelial function, such as nitrous oxide synthase, and the downregulation of fibroblast growth factor 2 (FG2F). This downregulation is now a recognized form of endothelial dysfunction.⁴⁹ While we are considering OS, we cannot separate the strong emphasis on hyperglycemia and oxidative damage as underlying mechanisms in macrovascular pathogenicity and their link with the inflammatory response and altered coagulative response. The array of anthropometric, traditional, and emerging biomarkers (OS and inflammatory) may possibly reflect different biochemical processes, and not necessarily imply the same pathophysiological pathways.

Whole blood viscosity

Hemorheology, or blood rheology, is the study of the flow properties of blood and its plasma protein constituents. Normal tissue perfusion occurs within certain rheological limits with any alteration contributing significantly to a disease state.⁵⁰ While it is not within the scope of this chapter to provide a full discussion of WBV, a brief commentary is warranted due to WBV implications in metabolic disturbances as a result of T2DM or MetS.⁵¹ The measurement of WBV is assessed by viscometry, however, for routine use, it may be calculated via extrapolation using the hematocrit expressed as % and plasma protein level in g/L.⁵²

Any significant increase in WBV has the potential to impair the microvasculature, resulting in hyperviscosity syndrome.⁵³ Of interest and relevance is the recognition that WBV has an association with developing CVD. The hyperglycemic state may mediate its adverse effects through several metabolic pathways with resultant OS. The emergent erythrocyte oxidative stress (EOS) has the capacity to compromise the vasculature (micro and macro). Generated erythrocytic ROS may expose the red cell membrane, leading to decreased membrane fluidity and resultant increased aggregation and diminished blood flow.¹⁹ WBV is an established phenomenon of Virchow’s triad: vascular stasis, endothelial function, and atherothrombosis, which may ultimately lead to and/or result from CVD complications.⁵⁴ Assessment of WBV and its association with anticoagulant and/or antiplatelet therapy have a positive and negative correlation respectively.⁵⁵ In addition, an algorithmically derived WBV may be useful in monitoring patients on antiplatelet therapy (aspirin).⁵⁶ Treatment for CVD may incorporate one or both treatment regimens, therefore WBV may be useful, as much conjecture still exists on the use of aspirin treatment in T2DM.⁵⁷ As more studies are required on its efficacy, WBV and its association with OS, inflammatory response, hypercoagulation, hyperglycemia, T2DM, and CVD will possibly provide more definitive information.

Inflammatory biomarkers

We cannot escape a discussion of the inflammatory process due to its intrinsic link with the OS state in hyperglycemia. Table 1.2 lists a number of current and emerging biomarkers depicting their cell source, target, and action with their target and resultant action. The inflammatory process is a second-tier natural physiological reaction and defense mechanism occurring in response to any insult upon tissue or organs considered foreign to the organism. The response is as diverse as it is predictable. There is wide recognition and evidence that in T2DM and CVD, just as OS is implicated, so is the inflammatory process. Traditional and emerging biomarkers considered to be contributory to this process are mentioned. Additionally, as already mentioned when discussing MetS, the importance of the obese state as a contributor to the inflammatory process cannot be underestimated. Adipose (fat) tissue consists of adipocytes and other cell types embedded in loose connective tissue. Adipose tissue was originally thought of as an energy storage repository, however, it is now recognized that adipocytes release, amongst other things, a group of bioactive mediators. These substances are known to modulate hemostasis, BP, lipid and glucose metabolism, inflammation, and atherosclerosis.⁵⁸ With obesity, a dysregulated secretion of a number of proinflammatory cytokines, such as adiponectin and IL-10, and antiinflammatory cytokines including IL-1, IL-1β, IL-6, MCP-1, TNF-α, TGF-β, leptin, resistin, and angiotensin occurs.⁵⁹ Macrophages also provide a major contribution by way of their response to the inflammatory challenge.

Table 1.2

8-OHdG, 8 Hydroxy-deoxy-guanosine; GSH, glutathione; GSSG, glutathione disulfide; IGF-1, insulin-like growth factor-1; IL-10, interleukin 10; IL-1β, interleukin 1 beta; IL-6, interleukin 6; MCP-1, monocyte chemotactic protein 1.

The interleukins

The interleukins, a large group of cytokines, gained their nomenclature from their discovery of expression in white blood cells; however, the naming is somewhat of a relic as many cells are now known to produce these cytokines. In general, the term interleukin is now used to describe a group of cytokines which possess complex immunomodulatory functions inclusive of cell proliferation, maturation, migration, and adhesion Therefore, the functionality of the immune system is very dependent upon these mediators.⁶⁰ A list of relevant biomarkers is provided in Table 1.2.

Insulin-like growth factor-1

The insulin-like growth factor (IGF-1) may not be considered an inflammatory factor, as its role in cellular function is more posited in the development of cell growth, differentiation, and tissue repair along with potential mechanisms for atherogenic protection. IGF-1 has therefore attracted a great deal of interest due to its potential implications as a protectant through its interaction with OS, the proinflammatory response, and apoptosis.

Hyperglycemia and coagulability

In unraveling the pathophysiological processes, T2DM-increased incidences of atherothrombotic and venous thrombosis occur. OS and diminished levels of nitrous oxide (NO) result from the impairment of endothelial nitric oxide synthase; all of which contribute to the proatherogenic state.⁶¹ Hypercoagubility and hypofibrinolytic impairment are evident in T2DM.⁶² The underlying mechanisms for the increased likelihood of thrombotic events in T2DM are multifactorial with premature atherosclerotic lesions predisposing subjects to plaque rupture and thrombus (Fig. 1.1). Endothelial dysfunction and increased thrombotic episodes arise as a consequence of the imbalance between coagulation, fibrinolysis and platelet activation, along with architectural changes due to smooth muscle proliferation within the vasculature.⁴³

Conclusion

Recognition of the interaction between the OS state and inflammatory response, and the interaction with both the hypercoagulable and fibrinolytic process does invite the reader to consider the role that OS, along with the inflammatory process, contributes to the pathophysiology of T2DM and CVD and their comorbidities. Further exploration of the unique relationship between the increased OS state in hyperglycemia is required. Its ensuing interaction with a variety of physiological and biochemical cellular micro and macro responses, with the concomitant contribution to the inflammatory response, will contribute to our understanding of OS and its association with T2DM, CVD and comorbidities. Certainly, more research and identification of reliable biomarkers will continue to increase our understanding in the role OS has in disease and aid in predictive and preventative risk management in our quest to curtail T2DM and CVD progression.

Summary points

• This chapter focuses on OS and inflammatory biomarkers associated with T2DM and CVD.

• Data suggests that T2DM and CVD and associated comorbidities are increasing globally.

• OS has a central role in the pathophysiology of T2DM and alterations to physiological changes.

• Heterogeneous and the multifactorial nature of elevated glucose levels and its contribution, via OS and inflammation, is a major contributor to endothelial dysfunction.

• The measurement of specific OS and inflammatory markers can assist in monitoring disease progression.

• Recognition of the interaction between OS, inflammation, and the interaction with both the hypercoagulable and fibrinolytic state invites further discovery into their contribution to the pathophysiology of T2DM, CVD, and their comorbidities.

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Chapter 2

Oxidative stress and diabetic neuropathy

Hajung Chun¹ and Yongsoo Park¹, ²,    ¹Department of Radiation Oncology and Bioengineering, Hanyang University College of Medicine and Engineering, Seoul, South Korea,    ²Health Insurance Review and Assessment Service, Uijeongbu, South Korea

Abstract

Diabetic neuropathy (DN) is a heterogeneous diabetic complication of nerve dysfunction found in diabetic patients. Chronic hyperglycemia increases the incidence and severity of DN. Although the disease process of DN may affect different sets of nerve fibers to different degrees in different individuals, surrogate markers of polyneuropathy and/or detailed neurologic assessment, including quantitative sensory testing, may predict mortality in diabetic patients. Increased activation of the polyol and PKC pathway, AGE formation, nerve hypoxia/ischemia, and reduction of NGF support have been proposed to link chronic hyperglycemia to the development of DN. As an underlying mechanism, overproduction of ROS in mitochondria leads to oxidative stress (OS), which in turn leads to neuronal damage. Besides the mitochondria, the ER is another crucial place where OS is generated and the emerging role of miRNAs has become important in their involvement in OS. As OS is found in various tissues under diabetic conditions, and is involved in the development of DN, antioxidant therapy has long been explored for the treatment of DN. However, results are disappointing because it is difficult to maintain a consistent antioxidant level and adequate tissue distribution let alone insufficient suitable exogenous antioxidants. In contrast, antioxidant treatment delivered in a new format may improve neuronal repair in patients with DN. Moreover, exercise training coupled with dietary restriction is an effective nonpharmacological strategy to reduce DN development.

Keywords

Diabetic neuropathy; oxidative stress; chronic hyperglycemia; miRNA; antioxidant treatment

List of abbreviations

AGE advanced glycation end-products

AMPK 5′AMP-activated protein kinase

DRG dorsal root ganglion

DRP1 dynamin-related protein 1

DTR deep tendon reflux

ED endothelial dysfunction

ER endoplasmic reticulum

ERK extracellular signal-regulated kinase

GPx glutathione peroxidase

GSH glutathione

HO-1 heme oxygenase 1

IR insulin resistance

JNK c-Jun N-terminal protein kinase

Keap1 Kelch-like ECH-associated protein 1

Maf musculo-aponeurotic fibrosarcoma

MAPK mitogen-activated protein kinase

MD mitochondrial dysfunction

MiRNA microRNA

NADPH nicotinamide adenine dinucleotide phosphate

NCV nerve conduction velocity

NF-κB nuclear factor kappa B

NGF nerve growth factor

NQO-1 NADPH quinone dehydrogenase 1

Nrf2 NFE2-related factor 2

OS oxidative stress

PI3K phosphatidylinositol-4,5-bisphosphate-3-kinase

PKC protein kinase C

QST quantitative sensory testing

RNS reactive nitrogen species

ROS reactive oxygen species

SOD superoxide dismutase

TPT thermal perception threshold

VPT vibration perception threshold

Introduction

Diabetic neuropathy (DN) is a heterogeneous condition found in both type 1 and type 2 diabetic patients that encompasses a wide range of nerve dysfunction, and whose development might be attributable to diabetes mellitus, per se, or to factors associated with the disease.¹ Specifically, diabetic peripheral neuropathy (DPN) is a chronic microvascular complication of diabetes affecting both somatic and autonomic peripheral nerves.¹ It may be defined as the presence of symptoms (numbness, sensory loss, and stabbing or burning pain in the hands and feet) and/or signs of peripheral nerve dysfunction in people with diabetes, after the exclusion of other causes of neuropathy. The most common form of DPN is distal symmetric polyneuropathy (DSP), which can affect somatic sensory or motor nerves and the autonomic nervous system. Focal or multifocal forms are asymmetric and affect cranial, trunk, or limb innervations. Most often, DPN represents an insidious and progressive disorder which begins with a long asymptomatic stage. Ultimately, late stage DPN may lead to serious consequences, such as foot ulceration, gangrene, amputation, and neuropathic pain. The loss of sensation is what predisposes diabetic patients to ulceration, infection, and ultimately limb loss, accounting for high morbidity and mortality rates.

DPN is a complex disorder in which the disease process may affect different sets of nerve fibers to different degrees in different individuals.¹ The signs, symptoms, and neurological deficits vary depending on the classes of nerve fibers involved. Thus, one individual may have an abnormality of large-fiber sensory function, which could be detected by measuring the VPT, while another may have a predominantly small-fiber neuropathy that can only be detected by the TPT. To understand if a given subject is affected by DPN, several different tests should be performed, and DN should only be diagnosed when more than one is abnormal.¹,² Therefore multiple testing of different neurological functions is recommended as the gold standard.¹ Complex patterns of sensation in DSP with decreased VPT (hypoesthesia) and heat stimulus-induced hyperesthesia (low TPT thresholds) are known to be characteristics of mild DSP as they correlate with neuropathic symptoms and deficits, whereas panmodality hypoesthesia is typical of severe DSP.³

The major confirmed risk factors are poor glycemic control, diabetes duration and height with possible roles for hypertension, age, smoking, hypoinsulinemia, and dyslipidemia. Additional risk factors for DPN are represented by alcohol consumption or other drug abuse, and classic cardiovascular risk factors including obesity and albuminuria. There is a long way to go in finding a universal definition of neuropathy that can be widely used.⁴

Natural history

It is difficult to describe the natural history of DN since the signs, symptoms, and neurological deficits found in a diabetic patient may vary depending on the classes of nerve fibers impaired. Small nerve fiber damage usually precedes large nerve fiber damage and is manifested first in the lower limbs, with pain and hyperalgesia, followed by a loss of thermal sensitivity and reduced light touch and pinprick sensation (Fig. 2.1). When pain occurs, nerve conduction velocity is often normal or minimally reduced.⁵ However, the slowing of NCVs is one of the earliest neuropathic abnormalities in diabetes and is often present even at diagnosis,⁶ especially in patients with T2D. After diagnosis, the slowing of NCVs usually progresses at a steady rate by approximately 1 m/s/year, and the level of impairment is positively correlated with the duration of diabetes.⁷ Sensory fibers are usually affected first, followed by motor fibers, necessitating multiple measures of sensory function if early intervention is to be possible.⁸ Although slowing of NCV is common in diabetes and often occurs early in the course of the disease, there is considerable uncertainty as to the relevance of these abnormalities to the future development of either subclinical manifestation or clinically apparent DN,⁸,⁹ and NCV does not appear to correlate with the severity of symptoms.¹⁰ Progressive reduction of VPT and loss of DTR, characteristics of which implies large nerve fiber damage, have been observed in patients who at the same time reported an improvement in pain symptoms.

Figure 2.1 A simplified view of the peripheral nervous system according to the fiber diameter, degree of myelination, and function of each component.

Symmetrical neuropathies of the sensory and autonomic nervous systems affect an increasing proportion of diabetic patients as diseases progress.¹¹ There is accumulating evidence to suggest that not only surrogate markers of microangiopathy, such as albuminuria, but also those used for polyneuropathy, such as NCV and VPT, may predict mortality in diabetic patients,¹² but further studies are needed to assess the prognostic role of polyneuropathy in diabetes. In diabetic patients with ultimate clinical endpoints of neuropathy, such as foot ulcers, the risk of death was increased to 12 per 100 person-years of follow-up, compared to 5 per 100 person-years in those without foot ulcers.¹³

DN is considered a common complication affecting more than half of T2D patients in their lifetime.⁴ Neurological abnormalities affecting the distal lower extremities are known to appear in the early stages of T2D and even in patients with prediabetes and abnormal glucose tolerance. Although it is difficult to determine the prevalence of DN precisely, several large studies have examined the prevalence in hospital-based populations and found the prevalence of DSP in T2D at approximately 30%, among both European and African populations.⁴,⁵ DN is also one of the most frequently encountered complications in T1D, occurring in about 60% of diabetic patients over the lifetime.¹²

Hyperglycemia is a crucial cause of diabetic neuropathy

Chronic hyperglycemia is known to be the main contributor to diabetic tissue damage,¹⁴,¹⁵ and ultimately to DPN development. Incidence and severity of neuropathy are increased by poor control of glycemia,¹¹,¹² indicating that excess glucose may be the biochemical trigger in pathogenesis. Increasing evidence suggests that consequent OS and ED may be the key mediators of the deleterious effects of hyperglycemia. An important contribution can be attributed to genetic risk factors, some of which are common for all microvascular complications.⁴,¹⁶ Additional factors are represented by some accelerators such as hemodynamic (hypertension) and metabolic (insulin resistance (IR), dyslipidemia) components.

Current data suggest that DPN results from the damage of the vasa nervorum associated with the direct damage of neuronal components. Both are the result of the interaction between metabolic (chronic hyperglycemia, dyslipidemia, OS), hemodynamic (microangiopathy of the vasa nervorum) factors, and impairment of the nerve fiber repair mechanisms.¹⁶ The vascular and metabolic mechanisms act simultaneously and have an additive effect.

As the primary crucial factor, persistent hyperglycemia is the main contributor to DPN development.) by the mitochondrial electron transport chain (ETC) during the process of oxidative phosphorylation (OP).¹⁸,¹⁹ It was postulated that hyperglycemia induces increased mitochondrial production of ROS/RNS followed by nuclear DNA strand breaks that, in turn, activate the enzyme poly ADP-ribose polymerase and may associate with and lead to a cascade of processes that finally activate major pathways of diabetic complications (Fig. 2.2):

1. Increased aldose reductase activity and activation of the polyol pathway leading to increased sorbitol accumulation with osmotic effects, resulting in impaired nerve conduction and, finally, apoptosis.⁴

2. Activation of PKC with subsequent activation of NF-κB pathway.¹⁷,²⁰

3. Intracellular AGE generation,¹⁸ formed from the nonenzymatic reaction of reducing sugars with free amino groups of proteins, lipids, and nucleic acids.

4. Activation of the hexosamine pathway.

Figure 2.2 Major pathways of diabetic (microvascular) complication developments from increased glucose turnover by way of oxidative stress.

Oxidative stress is a key mediator of diabetic neuropathy

), hydroxyl radical (•OH) and hydrogen peroxide (H2O2)] are produced by the mitochondrial ETC, NADPH oxidase and xanthine reductase, while RNS [nitric oxide (NO) and peroxynitrite (ONOO−)] are produced by NO synthase. OS plays important roles for the determination of neuronal cell fate by eliciting a wide variety of cellular responses, such as proliferation, differentiation, and cell death depending on cell types, cellular contexts, and amounts and duration of ROS/RNS generation.²¹ Importantly, most ROS, as well as RNS, have been shown to be involved in the development of DPN. They can cause protein dysfunction and DNA damage, leading to gene mutations and neuronal cell death.²¹ In fact, they also have the beneficial functions in normal cellular signaling of proliferation, gene expression, adhesion, differentiation, senescence, apoptosis, and necrosis, which are mainly mediated by MAPKs.²² In addition, ROS can modulate various stress-induced signaling pathways of Wnt, NF-κB, PI3K, Sirt-1/AMPK and others.²³ Nonetheless, many studies have revealed that OS plays an important role in the pathogenesis of various diseases, including DN. MAPK signalings such as ERK1/2, JNK, and p38 MAP kinases are demonstrated to participate in DN pathogenesis.²³,²⁴

There are many hypotheses regarding the origins of OS in diabetes in addition to the inevitable OP, including free radical accumulation related to glycation of proteins,¹⁸ consumption of NADPH through the polyol pathway,²⁵ glucose autoxidation,²⁶ hyperglycemia-induced pseudohypoxia,²⁷ or activation of PKC.²⁰ There are several sources of ROS productions in cells such as high glucose, hypoxia, and AGEs. Most importantly, neuropathy is characterized by an activation of MAPK in different neuronal and Schwann cells induced by overproduction of ROS, which then leads to MD, neuronal damage, and finally, apoptosis of these cells.²⁴,²⁸ Under diabetic conditions, ER stress is also increased in various tissues, including neuronal cells.²⁹ Although several pathways contribute to the development of DN, including increased activation of polyol pathway, OS, AGE formation, nerve hypoxia/ischemia, PKC, and reduction of NGF support, OS is the main important underlying factor found in various tissues under diabetic conditions, and is involved in the development of