Pathophysiology of type 2 diabetes complications

C H A P T E R

4 Pathophysiology of type 2 diabetes complications O U T L I N E 4.1 Acute emergencies

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4.3 Macrovascular diseases

4.2 Chronic complications of T2D and pathological hallmarks 70 4.2.1 Microvascular or microangiopathic damage of the capillaries 70 4.2.2 Diabetes retinopathy 73 4.2.3 Diabetic neuropathy 73 4.2.4 Diabetic nephropathy 74 4.2.5 The diabetic foot ulcer and amputation 75

4.4 Molecular mechanisms of diabetes complications 4.4.1 T2D and advanced glycated end products (AGEs) 4.4.2 Oxidative stress in T2D pathology 4.4.3 Low-grade inflammation as a link between obesity and T2D References

76 76 76 79 84 87

Before we describe the chronic pathological changes associated with type 2 diabetes (T2D), it is worth noting and revising the common symptoms that are outlined in Chapter 1. Diabetes complications could be both acute and chronic in nature. The acute diabetes complications are by and large related to the diabetes medical emergencies especially ketoacidosis and hypoglycaemia. The formation of ketone bodies and their role as energy source during diabetes are outlined in Chapter 3.

4.1 Acute emergencies The lack of insulin or its inability to induce its normal function means that peripheral cells cannot take up glucose from the blood. A state of starvation is thus set which intern initiates

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the mobilization of stored fats, carbohydrates and proteins to be used as a source of energy by converting them into glucose and ketone bodies. The dysregulation of glucose metabolism due to lack of insulin or its lack of action also means that ketone bodies level in the blood and their utilization as a main source of energy become significant, although the normal functioning of the body cannot be maintained in the long-term just by ketone bodies. This suggests that peripheral tissues are under starvation state despite persistent hyperglycaemia in T2D. The sorry state of the body with ketone bodies which are acidic in nature leading to acute ketoacidosis is one clinical feature of T2D though it is even more prevalent in type 1 diabetes (T1D). The physiological response to cellular starvation of glucose is to produce more glucose from the various sources (see Chapter 2) which further aggravate the persistent hyperglycaemia state of T2D. The excess glucose in the blood also means it cannot be fully reabsorbed by the kidney and hence the appearance of glucose in the urine—sweet urine. Water follows solutes and the excretion of more sugar by the kidney is coupled with excess water loss or urine and hence polyuria. As the physiological response to water loss is excessive thirst, polydipsia manifests as the initial symptoms of diabetes. In terms of medical emergencies, the most common case for T2D is perhaps the state of starvation or hypoglycaemia that could account to the clammy, pale and confused state of patients and could lead to full unconsciousness. The first manifestation of hypoglycaemia is the lack of glucose in the brain which could explain the confusion, unconsciousness and sometimes aggressive behavioural manifestation of the T2D patients.

4.2 Chronic complications of T2D and pathological hallmarks It is common knowledge that T2D is linked to a range of disease conditions such as cardiovascular problems and leads to blindness, kidney failure and limb amputation, etc. In this section we are looking at details of gross macro- and micro-changes that are common at biochemical, cellular, tissues and organ and system levels. A number of biochemical mechanisms including oxidative stress and inflammation play major role in the pathogenesis of macroand microvascular complications by hyperglycaemia associated with T2D. The pathogenic processes of the micro- and macrovascular changes are herein outlined first.

4.2.1 Microvascular or microangiopathic damage of the capillaries The microcirculatory system comprising of the arterioles, capillaries, and venules are the smallest functional unit of the circulatory system (Fig. 4.1). In this network of small blood vessels, often referred as the microvascular bed, oxygen and micronutrient coming from the arteriole side are delivered to tissues, while metabolic wastes are collected by the venule to be transported back to the heart. The ultimate exchange of nutrients/metabolites between tissues and the blood takes place in the capillaries and hence their anatomy and pathophysiological changes both in normal and diabetic states are given a lot more focus herein. Besides trafficking nutrients, gas and metabolites between tissues and the blood, the microcirculation also regulate blood flow to tissues/organs through myogenic responses that can dictate the volume of blood passing through the organ/tissue according to the local need. C. Pathophysiology of type 2 diabetes and therapeutic options

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FIG. 4.1 The microcirculatory system.

The permeability of the microvessels in the trafficking process is regulated by structural features of the endothelial cells (e.g. gap dimensions) and their immediate surroundings such the basement membrane which can considerably vary depending on organ or the microvascular beds. For example, the vascular bed in the kidney (glomeruli), the retina, the myocardium, the skin and the muscle have somehow specialist function requiring some physical or anatomical adaptation specific to their function. The biochemical changes that leads to microvascular damage will be discussed in the later sections and readers should know at this stage that hyperglycemia/diabetes induces not only cellular damage (e.g. endothelial cells) in the microvessels but also increase the development of diabetic microangiopathy. The cellular loss will affect a lot of haemostatic balance maintained by endothelial cells as well as the normal functioning of the vascular bed in nutrient/metabolite exchange between tissues and the blood. Such pathological changes ultimately attributes to hypoxia, wound healing delays, retinopathy and the various clinical features of T2D. The physiological response to hypoxia and diminished nutrient/metabolites exchange in the microvascular bed is neovascularization (e.g. diabetes retinopathy) while the platelet and vascular response to cellular damage could also lead to atherosclerosis. The cellular components of the capillaries are shown in Fig. 4.2 and constitute the endothelial cells monolayer that form the true cellular barrier between the blood and tissues. Endothelial cells are supported by a layer of protective matrix proteins forming the basement membrane. The specific function of the microvascular bed in the organ is governed by the nature of arrangement of endothelial cells in making the cellular barrier or gap junction between cells and the thickness of the basement membrane; both of which regulate materials to be exchanged between the blood and tissues. Small materials are exchanged by passing through the gap between endothelial cells which is very tight in C. Pathophysiology of type 2 diabetes and therapeutic options

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FIG. 4.2 The pericytes and other cellular components of the microvascular system.

the brain (blood–brain-barrier) and large in the kidneys (glomerular capillaries). Right outside the basement membranes are other cellular components of microvessels called the pericytes or mesangial cells forming the rather non-continuous cells which are now proven to play vital role in maintaining the normal function of the system. The key cellular alterations in diabetes are as follow: • The pericytes are group of cells closely associated with microvessels that regulate the physical maintenance of the vasculature. Through paracrine control, they are specifically involved in reparative process such as angiogenesis following endothelial damage. The increased apoptosis and degeneration of the pericytes have been shown to be associated in the common diabetic microangiopathy and/or microvascular damage in the retina, the kidney and the heart. Hence, diabetes hyperglycaemia initiate cellular damage including the pericytes; • Endothelial cells apoptosis is a common feature of diabetes in vital organs such as the eye. The first visible effect of such damage is the leakage of blood components to tissues. Moreover, diabetes is associated with impaired circulating endothelial progenitor cells and enhancing their function (by increasing their number or activity) has been suggested as one area of therapeutic options; • The basement membrane increase in thickness under diabetes condition. Increased production of extracellular matrix proteins, collagen and fibronectin, and of related enzymes (i.e. matrix metalloproteinases (MMPs)); • The above two (cellular loss and basement membrane thickness) restricts exchange of materials between the blood and tissues. Before the basement membrane gets too thickened, however, the barrier changes allow the leakage of content of capillaries into the tissues leading oedema that could alter the normal functioning of the organ. Good examples are the diabetes-associated retinal and macular oedema leading to visual problem and leakage of proteins from glomerular capillaries in the kidney; • Endothelial cell damage removes the paracrine and autocrine haemostasis control leading to cellular adhesion and platelet aggregation; • Microvessels eventually clogged up and blood circulation in the affected region ceased up—‘capillary drop out’;

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• Loss of capillaries leads to tissue ischaemia that promotes angiogenesis as commonly observed in diabetes retinopathy, maculopathy, and neuropathy. The common pathological feature of the above microcirculation abnormalities in diabetes results in the development of complications outlined in the following sections.

4.2.2 Diabetes retinopathy Diabetes retinopathy has been recognized as a major cause of blindness in adults in the developed world. After about 20 years life with diabetes, about 80% of T2D and nearly all T1D patients are expected to have developed some kind of retinopathy. Retinopathy in diabetes is primarily a widespread microvascular disease which includes vitreous haemorrhage, tractional retinal detachment, and endovascular glaucoma. The resulting hyperglycaemia in diabetes in known to be the cause for the micro-angiopathy and associated vascular leakage in the eye that results in diabetic macular oedema and capillary occlusion. The biochemical alterations that emanate from ischaemia and the release of vascular mediators such as the vascular endothelial growth factor (VEGF) triggers cellular proliferation and/or neovascularization. The development of these new blood vessels or neoangiogenesis has a tendency to bleed and vitreous haemorrhage is often accompanied by proliferation of other cell types such as fibroblasts, development of fibrotic membranes, retinal traction leading to retinal detachment. Hence, diabetic maculopathy and retinal ischaemia are two major factors that attribute to vision loss and/or disturbance in diabetes. With respect to the role of angiogenesis and the inflammation processes in diabetes retinopathy, several key cytokines have been proposed as therapeutic targets. Of these, the central role of VEGF has been established and anti-VEGF drugs including ranibizumab have already been approved (Boyer et al., 2009; Arevalo et al., 2010). The interleukin-1β (IL-1β), tumour necrosis factor-α (TNF-α), and IL-6, are key proinflammatory cytokines that are significantly upregulated in diabetes and also linked to insulin resistance. Inevitably, the adhesion molecules such as intercellular cell adhesion molecule-1 are expressed following proinflammatory cytokines release and are upregulated in diabetic retinopathy (Demircan et al., 2006). As the levels of cytokines such as VEGF and IL-1β increases in diabetic retinas, inflammatory cascades and cellular apoptosis increases as evidenced from experiments that showed intravitreal injection of cytokines such as IL-1β into normal rats could increase retinal capillary cell apoptosis (Vincent and Mohr, 2007).

4.2.3 Diabetic neuropathy As explained in the previous sections, microvessels or capillaries dysfunction leading to tissue ischaemia could lead to acute or chronic peripheral nerve pathology collectively called diabetes neuropathy. A number of clinical symptoms of either focal or diffuse neuropathicorigin, or diabetic-induced demyelination leading to classical symptoms of hyperesthesia and allodynia, and burning, stabbing, shooting pains may be encountered. Other symptoms including numbness which could also attributes to the loss of sensation and eventually foot ulceration are other complications of diabetes. The common symptoms encountered in diabetic peripheral neuropathy are as follow:

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• Pain—Damage to small nerve fibers causes pain. For some, even wearing socks and shoes or the touch of sheets and blankets on their feet at night is excruciating; • Burning, stabbing or electric-shock sensations; • Numbness (loss of feeling)—Damage to large fibers; • Tingling—Damage to large fibers; • Muscle weakness—Damage to large fibers; • Poor coordination; • Muscle cramping and/or twitching; • Insensitivity to pain and/or temperature; • Extreme sensitivity to even the lightest touch; • Symptoms get worse at night.

4.2.4 Diabetic nephropathy Diabetic nephropathy is a classic example of microvascular complications and constitute as the main cause of the end-stage renal disease that govern the morbidity and mortality of T2D. Some estimate suggests that 30–47% end-stage renal diseases trace their cause as diabetes nephropathy. Some 25–40% diabetics are generally expected to develop diabetic nephropathy at some stage (Giullian et al., 2008). The disease is characterized by a relatively higher level of albumen excretion often called proteinuria or microalbuminuria that in a severe case will lead to proteinuria of over 500 mg excretion in 24 h. The clinical stages of diabetic nephropathy include normoalbuminuria, microalbuminuria, overt proteinuria, and finally end-stage renal disease. If untreated, patients with persistent microalbuminuria of 30–300 mg/day could go to what is regarded as an overt nephropathy of over 300 mg/day with a risk of cardiovascular disease development. As the disease progresses, glomerular filtration rate (GFR) declines. Guidelines on the disease classification and assessment are provided by the various government and relevant associations. The American Diabetes association (ADA, 2014), for example advocate screening result of estimated GFR (eGFR) <60 mL/min/1.73 m2, and kidney damage, usually by estimation of albuminuria >30 mg/g creatinine as indicators of diabetes kidney disease. Caution for the assessment methods are also given and even the eGFR has its limitations (ADA, 2014). Blood tests for blood urea nitrogen (BUN) and creatinine levels are routinely measured to determine kidney function. The BUN level of 7–20 mg/dL and creatinine of 0.8–1.4 mg/dL are considered normal; with females being considered to have a slightly lower creatinine level (0.6–1.2 mg/dL). Considering 100–150 mL/min GFR as a normal range, eGFR is also routinely employed as a method of assessment for kidney function. Creatinine clearance with 24 h urine collection in conjunction with blood sample analysis may also be used although the reliability of this methodology particularly at the advanced stage of the disease is often in question, i.e. creatinine clearance may not be exactly the same as GFR for various reasons. At the molecular level, diabetic nephropathy also involves cellular death or apoptosis and inflammation that are described in Section 4.4. These include cascades of events following hyperglycaemia such as activation of protein kinase C (PKC), increased reactive oxygen species (ROS) and advanced glycation end-products (AGEs) generation, increased expression of proinflammatory cytokines and transforming growth factor β (TGFβ), enhanced flux into the

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polyol and hexosamine pathways and dysregulation of the mitogen-activated protein kinase (MAPK) signalling pathway, etc. All these mechanisms are associated with microvascular complications including podocyte loss, thickening of the glomerular basement membrane, endothelial cell death, tubular atrophy, dropout, etc.

4.2.5 The diabetic foot ulcer and amputation Around 25% of people with diabetes will develop a diabetic foot ulcer (DFU) during their lifetime and the aetiology of the condition seems to be closely related to the neuropathic, ischaemic and/or neuroischaemic condition that are already described in the previous sections. Hence, diabetic foot ulcer is the combined effect of micro- and macrovascular dysfunction leading to impaired perfusion and ischaemia developed in the limbs. The relationship between peripheral neuropathy and DFU development could be explained as follow: • Loss of sensation means being unaware and/or unresponsive to some physical, chemical or thermal stimuli/injury; • Motor neuropathy like those of hammer toes and claw foot can cause foot deformities; • Autonomic neuropathy could lead to dry skin that is prone to fissures, cracking and callus. The type and prevalence of DFU are further summarized in Table 4.1. Many reports (e.g. NICE, 2016) indicate that more than 80% of limb amputations in diabetic patients is preceded by DFU. About 70% of diabetic people die within 5 years after having amputation, while the figure for those dying within 5 years of developing a DFU is about 50%. The loss of capillaries in the vascular bed of the skin makes it difficult for wound to heal quickly. Wound infection is TABLE 4.1

Typical features of DFUs according to aetiology.

Feature

Neuropathic

Ischaemic

Neuroischaemic

Sensation

Sensory loss

Painful

Degree of sensory

Callus/ necrosis

Callus present and often thick

Necrosis common

Minimal callus prone to necrosis

Wound bed

Pink and granulating, surrounded by callus

Pale and sloughy with poor granulation

Poor granulation

Foot temperature and pulses

Warm with bounding pulses

Cool with absent pulses

Cool with absent pulses

Other

Dry skin and fissuring

Delayed healing

High risk of infection

Typical location

Weight-bearing areas of the foot, such as metatarsal heads, the heel and over the dorsum of clawed toes

Tips of toes, nail edges and between the toes and lateral borders of the foot

Margins of the foot and toes

Prevalence

35%

15%

50%

Source: International Best Practice Guidelines (2013).

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also the major factor contributing to limb amputation in diabetes patients and diabetic foot infection in general is considered to take up to one-quarter of all diabetic hospital admissions in both Europe and the United States (Mendes and Neves, 2012).

4.3 Macrovascular diseases Considered as the primary cause of death in diabetes, the chance of macrovascular damages increased in diabetes by two- to fourfold. The most common macrovascular disorders in diabetes are atherosclerosis and arteriosclerosis. In the case of atherosclerosis, plaques comprising of lipids and fibrovascular tissue (atheroma) are developing in the vessel wall of large arteries. As the plaque increases in size, the lumen of the arteries narrows making perfusion to tissues reduced, and hence leading to ischaemia development. The classical example is the coronary artery disease or angina pectoris where myocardial ischaemia resulted from reduced blood supply to the heart. In the severe case where complete blockade like that by platelet aggregation occur, myocardial infarction or cell death (or heart attack) is the ultimate consequence. In the second example of macrovascular damage, arteriosclerosis, rigidity of the arteries wall or sclerosis means that the blood vessel loses its elasticity leading to an increased blood pressure. Diabetes is also associated with lipid dysregulation and often characterized by hyperlipidaemia and/or hypercholesterolaemia that aggravates the macrovascular dysfunction in diabetes.

4.4 Molecular mechanisms of diabetes complications 4.4.1 T2D and advanced glycated end products (AGEs) One of the best characterized mechanism of diabetic complication is through the nonenzymatic post-translational modification of proteins called glycation reactions that yield products collectively called AGEs. Structurally, the AGEs represent a heterogeneous group of modified biological molecules formed through glycation reaction followed by oxidation, dehydration and/or carbonylation processes. The best characterized route of AGE formation is the so called Maillard reaction whereby the carbonyl moiety of reducing sugars reacts with amino groups of proteins to form a Schiff base. The reaction is non enzymatic, slow and occur in few hours but as the Schiff base product is unstable, it undergoes intramolecular rearrangement to form an Amadori product (Fig. 4.3). Glycated haemoglobin (haemoglobin A1c (HbA1c)) is an example of an Amadori product (from glucose reaction with the N-terminal amino residue of valine on the haemoglobin β chain) that is widely used in clinical practice for diagnosis and regulation of diabetes. The Amadori products themselves undergo a slow oxidation reaction to yield reactive dicarbonyl compounds such as glyoxal, methylglyoxal (MG), 3-deoxyglucosone (3-DG), and deoxyglucosones in a period of a week to months (see Fig. 4.4). The final stage of the glycation reaction involves further oxidation, dehydration, and cyclization steps resulting

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FIG. 4.3 Reaction of glucose to form Amdori product and AGEs.

O

O H

H O

Glyoxal

O CH 3

H O

Methylglyoxal (MG)

OH OH

H O

OH

3-Deoxyglucosone (3-DG)

FIG. 4.4 Common reactive dicarbonyl compounds.

in the formation of the irreversible products that we call AGEs which are yellow-brown in colour, often fluorescent and insoluble adducts. The accumulation of AGEs over a long period leads to a variety of physiological and pathological changes as evidenced in T2D. The AGEs are divided into three main groups: • Fluorescent cross-linked AGEs such as crossline and pentosidine; • Non-fluorescent cross-linked AGEs including the imidazolium dilysine, alkyl formyl glycosyl pyrrole (AFGP) and arginine-lysine imidazole (ALI) cross-links; • Non-cross-linked AGEs such as pyrraline and N-carboxymethyllysine (Nε-(carboxymethyl)-lysine CML). The three best characterized AGEs derived from this glycoxidation process are the pentosidine, Nε-carboxymethyl-lysine (CML) and glucosepane (Fig. 4.5). In addition to the Maillard reaction pathway of the AGEs formation, lipid peroxidation and the glycolysis pathways are also known to be the route of formation of AGEs. Peroxidation of lipids by ROS could lead to the formation of reactive carbonyl compounds that ultimately form AGEs or advanced lipid end products. In the latter case, the marker of lipid peroxidation product, malondialdehyde (MDA), is a classic example. Similarly, the glycolysis pathway involves the oxidation of glucose to yield reactive carbonyl compounds such as methylglyoxal that undergoes a serious of reactions with proteins to yield the AGEs such as hydroimidazolone (MG-H1). The structures of the common AGEs are shown in Fig. 4.5. In summary, AGEs are formed endogenously through three distinct reactions pathways (Fig. 4.6). They can also be introduced into the body by a variety of ways including cigarette smoking which contain reactive glycation products and intake of high-AGE food

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FIG. 4.5 Structures of some common AGEs.

products including those acquired by food processing steps (oven frying or high temperature could induce AGE formation). Hence, significant amount of the AGEs (
10%) are introduced via the food and drinks. The AGEs are metabolized by the liver and excreted by the kidney. Numerous experimental and clinical evidences suggest that the levels of protein bound AGEs and AGE-free adducts are not only elevated in diabetes but also contribute to the micro- and macrovascular damages associated with the disease. The intracellular accumulation of AGEs is mediated via their specific cellular receptors (RAGEs). A number of ligands and receptors of AGEs have also been characterized in various cell types (e.g. smooth muscle cells, macrophages, endothelial cells and astrocytes) and among them are the lactoferrin, scavenger receptors types I and II, oligosaccharyl transferase-48 (OST-48), 80K-H phosphoprotein, galectin-3, and CD36. Moreover, interaction of RAGEs with a number of proteins including amyloid-β peptide (Aβ), β-sheet fibrils, S100/calgranulins, amphoterin and Mac-1

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FIG. 4.6 Summary of AGEs formation inside and outside the cell through the three common routes.

integrins have been observed. Hence, RAGEs are involved in diverse cellular functions via discrete signal transduction pathways. With respect to stimulation of RAGEs by AGEs, nuclear factor-κB (NF-κB) activation and the MAPK signalling pathway have been shown to be involved. This leads to gene transcription for cytokines and other signalling molecules including the endothelin-1, tissue factor and thrombomodulin and IL-1α, IL-6 and TNF-α. Undoubtedly, the expression of proinflammatory cytokines (TNF, IL-1, and IL-6) which are also known to be associated with insulin resistance leads to the expression of adhesion molecules including vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). Hence, high levels of AGEs induce inflammation via activation of proinflammatory cytokines expression and adhesion molecules expression. The other common pathway linking diabetes and AGEs is through the generation of ROS that is implicated in diabetes pathology (see below). Moreover, the stable protein cross-linked products with extracellular matrix (ECM) proteins such as collagen alters the physical and functional characteristics of the proteins leading to the diabetes alterations in the basement membrane and/ or alterations of microvascular system that are discussed in the previous sections, i.e. excessive formation of AGEs can lead to a thickening of the microvessel, hypertension, endothelial dysfunction, loss of pericytes, decreased platelet survival and increased platelet aggregation. Such abnormalities undoubtedly promote procoagulation, tissue ischaemia and induction of growth factors (e.g. VEGF) with angiogenesis and neovascularization outcome. As a summary, Fig. 4.7 depicts the link between AGEs and diabetes.

4.4.2 Oxidative stress in T2D pathology Reactive oxygen species (ROS) is a terminology used in biology, chemistry and medicine to collectively describe oxygen derived radicals such as superoxide anion (O• 2 ), and hydroxyl radical (HO•) and other reactive non-radical species including hydrogen peroxide (H2O2) and

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FIG. 4.7 The link between diabetes and AGEs via oxidative stress and inflammation.

hypochlorous acid (HOCl). In a similar way, we also have reactive nitrogenous species (RNS) to describe the nitric oxide synthase (NOS) product, nitric oxide (NO), that possess multiple biological effects in the body; as well as other related compounds including peroxynitrite (OONO). Under normal physiological conditions, ROS and RNS are continuously produced to induce a variety of cellular functions mediated through a variety of direct effect on biological molecules or through signalling via activation/inhibition of enzymes and genes. In mammalian cell system, the best source of ROS is the cellular respiratory system of the mitochondrial electron transport system where leaked electrons may directly and prematurely reduce molecular oxygen to generate ROS via O• 2 . A number of enzymatic reactions such as the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, xanthine and cytochrome p450 systems, the arachidonic acid inflammatory pathway and many other biological processes do also generate ROS. A number of cytokines and death signalling molecules including apoptosis inducers (e.g. TNF-α) are also known to induce the generation of ROS. Both ROS and RNS are also generated through external factors such as drugs, toxins and other stimuli. Exposure to UV irradiation, for example, cleaves H2O2 to generate HO•. As a defence mechanism against microorganisms, white blood cells are employing ROS as a weapon and in this direction the enzyme myloperoxidase convert H2O2 to HOCl. Two of the most physiologically relevant ROS generating systems are further outlined below: 4.4.2.1 The NAD(P)H oxidase system in T2D The NAD(P)H oxidase is an efficient means of ROS generation in white blood cells but also found in vascular cells including podocytes and smooth muscle cells as well as in the kidneys. It sole purpose is to generate O• 2 and cytokines and AGEs which are implicated in diabetes are are known activate NAD(P)H oxidase (NOX) leading to ROS generation. Considering the primary function of this enzyme in neutrophils and macrophages is the ROS-mediated killing

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of microorganisms and therby serving as an inflammatory mediator, decades of research on this enzyme was related to oxidative burst phagocytosis. Several varities/isoforms of this enzyme has been discovered in recent years, however, with extensive variations in tissues distribution under normal and pathological conditions. Of the several isoforms known to date (NOX1, -2, -3, -4, and -5 and dual oxidase 1 and -2), Nox4 is predominantly found in the kidney where it mediate the generation of ROS under basal and pathological condition, particularly the diabetic nephropathy. Even though NOX2 (primarily of the leucocytes phagosomes) is also known to occur in the kidney, upregulation of expression of NOX4 appear to be the main renal oxidative stress and kidney injury that could be targeted by potential modulators. The overall reaction mechanism mediated by NOX is as follow: NADðPÞH + 2O2 $ NADP + + 2 O2  + H + Even though ROS genrated through the NAD(P)H system and others have profound effect in the body, the role played in the kidney with respect to diabetes is worth further scrutny. The kindeny is the second most important organ (next to the liver) for gluconeogenesis, an effect which is inhibited by insulin. In the proximal tubular cells, the regulation of glucose is also critical by reabsorbing filtered glucose into the blood. This glucose metabolism now understood to be regulated by NAD(P)H oxidase system and is dysregulated under T2D. Hence, considerable attention is now given to the modulation of the faulty glucose transport system in diabetic kidney tubules including the sodium/glucose cotransporters (SGLTs, see Chapter 2) across the brush border of the proximal tubules via NADPH oxidase. For extended review of the NAD(P)H oxidase role in kidneys under diabetic conditions, readers may refer review articles in the field (Rhee, 2016; Sedeek et al., 2013). The diverse role of ROS as cell signalling molecules to regulate numerous cellular functions such as cell growth, differentiation, survival mechanisms are also now extended to the NAD(P)H oxidase system. The role of NAD(P)H oxidase in cardiovascular system is also worth mentioning particularly angiotensin II being a known activator of the enzyme. Hence, it is relevant to hypertension and other common cardiovascular complications of T2D: further substantiating the role of ROS in diabetes pathology. 4.4.2.2 The mitochondrial respiratory system Mitochondrial respiratory chain employing complex chains I–III are the major sites of O• 2 generation (Fig. 4.8). The efficiency of mitochondria diminishes with age and pathological conditions such as diabetes suggesting the production of more ROS under disease state and/or old age. In the mitochondrial inner membrane where oxidative phosphorylation takes place, a gradient of proton (between the intermembrane space and inner mitochondrial region) is created as electrons are passed between careers (Fig. 4.8). While the electron being carried is eventually used to reduce molecular oxygen to form water at Complex IV, the proton gradient is used to couple the phosphorylation process to produce ATP. There is however a normal leakage of electron at complex I and III that directly form O• 2 from molecular oxygen. This makes the mitochondria as the major source of ROS that is even highly exaggerated under pathological conditions. The effects of various potential modulators of this ROS formation as antioxidants and/or antidiabetic mechanisms are discussed in the various chapters of this book.

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FIG. 4.8 The mitochondrial oxidative phosphorylation system and ROS generation. The mitochondrial electron transport system undertakes a sequence of oxidation-reduction reactions in four complexes (I-IV). Electrons are transferred from NAD(P)H through the system while the (H+) electrochemical gradient was created by pumping (complex I, II, and IV) it to the intermembrane space. While this electron transport is coupled with phosphorylation via the ATP synthase through the established (H+) gradient, O2 serve as a final electron acceptor at complex IV. On the other hand, a premature reduction of O2 at complexes I and III could lead to O• 2 formation.

4.4.2.3 Antioxidant defences Owing to the diverse pharmacology and toxicological effect of higher level of ROS, their production and elimination must be tightly regulated. As explained throughout this book, higher level of ROS are implicated in diabetes as well as a number of other pathologies. The regulation of ROS include a tight control of transition metals that promote ROS generation as a vast array of diseases including neurodegenerative disorders trace their origin thorough oxidative damage mediated by dysregulation of copper, iron and related transition metals. The best means of ROS regulation however remains through a range of antioxidant defenses as summarized below: Superoxide dismutase (SOD) enzymes primarily the CuZn and Mn isoforms acting on superoxide anion: SOD

2O2  + 2H + ƒ! H2 O2 + O2 Catalase (CAT) act on hydrogen peroxide to convert it to water CAT

2H2 O2 ƒ! 2H2 O + O2 The tripeptide glutathione (GSH) that removes ROS while itself oxidized to form GSSG (see Fig. 4.9). The two key oxidation-reduction enzymes are glutathione peroxidase (GPx) and reductase (GR). A number of dietary antioxidants and other nutritional factors that are discussed in this book also add to the antioxidant defense of the body. Hence, oxidative stress is a state when there is an imbalance between the formation of prooxidants (ROS/RNS) and antioxidant

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FIG. 4.9 Structures of reduced (GSH) and oxidized (GSSG) forms of glutathione.

defenses is in favour of the former, i.e. either the body antioxidant defense is weaker or exceptionally higher level of ROS/RNS are produced under pathological conditions. In T2D, generation as with other hyperglycaemia is directly associated with high level of O• 2 ROS. Hence, the above-mentioned diabetic nephropathy, microvascular injury and cellular death could be a direct result of oxidative stress. Throughout this book, emphasis is given to the role of natural products and other potential antidiabetic agents that modulate pharmacological effects via antioxidant mechanisms. This includes modulating the intricate balance of ROS and RNS generation and elimination as well as their diverse function in the body (Fig. 4.10). The various cellular and subcellular mechanisms including the role of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) are extensively discussed. Related to antioxidant defenses, transcriptional regulations of the cellular redox balance is critically important. As a valid therapeutic target for natural product, the transcription factor, erythroid 2-related factor 2 (Nrf2), is extensively discussed in the various chapters of this

FIG. 4.10 Summary of ROS/RNS generation and their inactivation.

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book. By binding to the antioxidant response element (ARE) of target genes, the Nrf2, regulates the expression of phase II detoxifying enzymes and antioxidants. These enzymes are collectively regarded as cytoprotective proteins and include the following: • Glutamate-cysteine ligase—critically involved in the synthesis of the antioxidant GSH; • GST enzymes at various cellular sites (mitochondrial and microsomal); • Multidrug resistance-associated proteins—membrane transporters for efflux and excretion of drugs and other compounds; • NAD(P)H quinone oxidoreductase 1 (Nqo1)—involved in the reduction and detoxification of highly reactive quinones. It ameliorate oxidative stress; • The UDP-glucuronosyltransferase (UGT)—enzyme for conjugation of drugs with glucuronic acid; • Heme oxygenase-1 (HO-1)—key enzyme in the production of the antioxidant and antiinflammatory biliverdin by breakdown haeme. The Nrf2/HO-1 axis as a regulator of both oxidative stress and inflammation will be highlighted in this book as it is a valid therapeutic target for diabetes and associated diseases. While breaking down haeme to release iron and carbon monoxide (CO), along with biliverdin, HO-1 boosts anti-inflammatory defenses by increasing the expression levels of IL-10 and IL-1 receptor antagonist. Extensive review articles describing the role of Nrf2/HO-1 in the various human pathologies have been published (e.g. Chen-Roetling and Regan, 2017; Furfaro et al., 2016; Landis et al., 2018; Loboda et al., 2016; Ndisang, 2017; Satoh et al., 2013; Wardyn et al., 2015).

4.4.3 Low-grade inflammation as a link between obesity and T2D Perhaps the best link between obesity and T2D is the common low grade but chronic inflammation associated with obesity that leads to insulin resistance. In a significant proportion of T2D sufferers, the level of inflammatory cytokines such as IL-6, and TNF-α as well as inflammation markers such as C-reactive protein (CRP) or high-sensitivity CRP (hs-CRP) and plasminogen activator inhibitor I are raised. The TNF-α gene expression is upregulated in adipose tissues during obesity, linking pro-inflammatory substances released from adipose tissues to insulin resistance in T2D. The role of proinflammatory cytokines in insulin resistance and T2D have been extensively studied and can be summarized as follow: • They inhibit insulin receptor signalling; • Promote hepatic fatty acid syntheses and induce the liver to produce more acute-phase proteins; • Recruit more inflammatory cells to adipose tissues; • Increase β-cell apoptosis and β-cell death; • Via the inflammatory mechanisms such as atherosclerosis, promote cardiovascular complications associated with diabetes; • Transcriptional activation such as NF-κB and activator protein-1 (AP-1) that are linked to diverse physiological and pathological conditions are activated by inflammatory mediators.

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In addition to the direct association between higher level of TNFα in obese subjects and under insulin resistance, the latter has been shown to be ameliorated by anti-TNF-α therapeutic approaches. IL-6 is the other best characterized cytokine that has been shown as a link between obesity and T2D. In recent years, the other potent proinflammatory cytokine, IL-1, is also emerging to play a role in insulin resistance. Virtually, all organs responding to insulin appear to be affected by the inflammatory mechanisms of diabetes which is outlined in the various sections of this book. The oxidative damage and inflammatory mechanisms should also be seen as a cooperative cascades of events that occur together in diabetes pathology. Pancreatic β-cell killing, for example, is associated with their extreme sensitivity to ROS (Wang and Wang, 2017) which cannot be seen separate from the inflammatory-mediated cell damage. The vascular injury in diabetes through oxidative stress, inflammation, and alteration of the haemodynamic balance (pro-coagulant cascades) have been shown to attribute to the micro- and macrovascular complications. The activation of NF-κB, for example, leads to the expression a range of adhesion molecules such as VCAM-1, ICAM-1 and E-selectin that mediate leucocyte infiltration and inflammation. The upregulation of expression of growth factors, such as VEGF, insulin-like growth factor-1 (IGF-1) and platelet-derived growth factor (PDGF) along with more inflammatory cytokines, such as IL-1 and TNF-α, and chemokines, such as monocyte chemoattractant protein-1 (MCP-1) further aggravate tissue injury in diabetes. The role of low density lipoprotein (LDL) in atherosclerosis and vascular injury is also known. For example, oxidized LDL is actively taken up by macrophage leading to their transformation into the foam cells in atherosclerosis development. The CRP concentration is also raised not only during inflammation but also under diabetes and cardiovascular damages. It is produced in the liver by stimulation of proinflammatory cytokines. Interestingly, adipocytes could also be stimulated by cytokines to release the CRP. The CRP intern stimulates various cells including endothelial cells to express adhesion molecules (e.g. ICAM-1) and hence facilitate leucocyte infiltration/inflammation. Hence, in the various antidiabetic assessments discussed in this book, the effect of potential modulators on CRP is discussed along with other obesity and cardiovascular disease markers such as low-density lipoprotein cholesterol (LDL-C) or high-density lipoprotein (HDL)-C. This kind of vascular injury through the combined action of oxidative stress and inflammation in diabetes has been reviewed (Domingueti et al., 2016). Overall, inflammation is now well accepted as a common link between obesity and insulin resistance and also attributes to the tissue damage that is observed under T2D pathology. Understanding the role of inflammation on T2D via the obesity pathway also requires the basic understanding of the adipose tissues cell population. Adipose tissues are largely composed of mature adipocytes, preadipocytes, fibroblasts, endothelial cells, histiocytes and macrophages. Preadipocytes can be induced to differentiate into macrophages-like cells but the adipocytes macrophage population are originated from monocytes of the blood and/or recruitment of blood monocytes bone-marrow progenitor cells of the haemopoisis process. As adipogenesis increases, the level of macrophage population and activation increases along with upregulated secretion of proinflammatory cytokines such as TNF-α, IL-1, and IL-6. This event accounts to the so called low grade inflammation associated with obesity that leads to increased insulin resistance. Furthermore, the production of adiponectin is suppressed as adipogenesis increased and low level of this hormone is known to be observed in obesity, T2D and during cardiovascular complications. This is associated with

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the known anti-inflammatory activity of adiponectin partly mediated by suppressing the levels of TNF-α and IL-6 output from macrophages and/or adipose tissues. On the other hand, leptin release from mature adipocytes increase with obesity and it is a proinflammatory mediator that promotes the release of TNF-α by macrophages. Readers should bear in mind that there are several other adipokines (e.g. omentin, visfatin, and vaspin) which are implicated in inflammation associated with obesity/T2D. The overall link between inflammation and obesity is depicted in Fig. 4.11. The increased levels of ROS, AGEs and angiotensin II are known to activate PKC that mediate the expression of VEGF to promote albuminurea and upregulation of TGF-β with implication of renal hypertrophy and sclerosis. This effect of TGF-β in diabetic nephropathy is also linked to increased collagen IV and fibronectin formation leading to enlargement of the basement membrane. PKC is also known to activate the NADPH oxidase thereby increasing ROS generation. These effects of PKC are also associated with other diabetes complications including diabetes retinopathy which is characterized by endothelial dysfunctions and capillary leakage. Various isoforms of PKC such as the α, β, and δ variants have also been characterized in recent years with distinct function in various tissues. The other pathway of diabetes pathology often cited in the literature is the polyol pathway (Fig. 4.12). The function of aldose dehydrogenase in removing aldehydes generated through the various mechanism of oxidative damage is also extended in the reduction of glucose to sorbitol. As sorbitol is oxidized by the enzymatic action of sorbitol dehydrogenase (SDH), the resulting fructose buildup initiates metabolic pathways leading to more AGEs and ROS generation. Furthermore, depletion of the antioxidant GSH and inactivation of glyceraldehyde-3-phosphate dehydrogenase (G3PD) leads to dysregulation of the pyruvate or glycolysis pathway (Fig. 4.12). Sorbitol is also linked to cellular damage through various

FIG. 4.11

The link between inflammation and T2D.

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References

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FIG. 4.12

The polyol and aldose reductase pathway in diabetes pathology. Increased level of glucose in diabetes leads to the generation of toxic levels of aldehydes that initiate the activity of aldose reductase. The enzyme also convert glucose to sorbitol which is further acted by sorbitol dehydrogenase (SDH) to produce fructose. The NADP+ generated through the process is removed by the GSH and hence over activity leads to the depletion of the antioxidant store (GSH) while the increasing NADH/NAD+ balance that inhibits the activity of glyceraldehyde-3-phosphate dehydrogenase (G3PD) which is driven by NAD+.

means including osmotic changes. Excessive level of glucose has also been implicated to the formation of glucosamine that leads to glycosylation of proteins as observed in diabetes complications.

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Chen-Roetling, J., Regan, R.F., 2017. Targeting the Nrf2-heme oxygenase-1 axis after intracerebral hemorrhage. Curr. Pharm. Des. 23 (15), 2226–2237. Demircan, N., Safran, B.G., Soylu, M., Ozcan, A.A., Sizmaz, S., 2006. Determination of vitreous interleukin-1 (IL-1) and tumour necrosis factor (TNF) levels in proliferative diabetic retinopathy. Eye 20, 1366–1369. Domingueti, C.P., Dusse, L.M., Carvalho, M.D., de Sousa, L.P., Gomes, K.B., Fernandes, A.P., 2016. Diabetes mellitus: the linkage between oxidative stress, inflammation, hypercoagulability and vascular complications. J. Diabetes Complications 30 (4), 738–745. Furfaro, A.L., Traverso, N., Domenicotti, C., Piras, S., Moretta, L., Marinari, U.M., Pronzato, M.A., Nitti, M., 2016. The Nrf2/HO-1 axis in cancer cell growth and chemoresistance. Oxid. Med. Cell. Longev. 20161958174. Giullian, J.A., Chuang, P., Lewis, J.B.L., 2008. Diabetic nephropathy. In: Brietzke, S.A. (Ed.), Endocrinology Board Review Manual, Endocrinology Vol. 7, Part 1, pp. 1–12. Available at: http://seminmedpract.com/pdf/brm_ Endo_V7P1.pdf. International Best Practice Guidelines, 2013. Wound Management in Diabetic Foot Ulcers. Wounds International, http://www.woundsinternational.com/media/best-practices/_/673/files/dfubestpracticeforweb.pdf. Landis, R.C., Quimby, K.R., Greenidge, A.R., 2018. M1/M2 Macrophages in Diabetic Nephropathy: Nrf2/HO-1 as Therapeutic Targets. Curr. Pharm. Des. 24 (20), 2241–2249. Loboda, A., Damulewicz, M., Pyza, E., Jozkowicz, A., Dulak, J., 2016. Role of Nrf2/HO-1 system in development, oxidative stress response and diseases: an evolutionarily conserved mechanism. Cell. Mol. Life Sci. 73 (17), 3221–3247. Mendes, J.J., Neves, J., 2012. Diabetic foot infections: current diagnosis and treatment. J. Diabetic Foot Complications 4 (2), 26–45. Ndisang, J.F., 2017. Synergistic interaction between heme oxygenase (HO) and nuclear-factor E2-related factor-2 (Nrf2) against oxidative stress in cardiovascular related diseases. Curr. Pharm. Des. 23 (10), 1465–1470. NICE (National Institute for health and Care Excellence), 2016. Diabetic foot problems: prevention and management. https://www.nice.org.uk/guidance/ng19/resources/diabetic-foot-problems-prevention-and-management1837279828933. (Accessed 8 January 2019). Rhee, E.P., 2016. NADPH oxidase 4 at the nexus of diabetes, reactive oxygen species, and renal metabolism. J. Am. Soc. Nephrol. 27 (2), 337–339. Satoh, T., McKercher, S.R., Lipton, S.A., 2013. Nrf2/ARE-mediated antioxidant actions of pro-electrophilic drugs. Free. Radic. Biol. Med. Dec. 65, 645–657. Sedeek, M., Nasrallah, R., Touyz, R.M., Hebert, R.L., 2013. NADPH oxidases, reactive oxygen species, and the kidney: friend and foe. JASN 24 (10), 1512–1518. Vincent, J.A., Mohr, S., 2007. Inhibition of caspase-1/interleukin-1beta signaling prevents degeneration of retinal capillaries in diabetes and galactosemia. Diabetes 56, 224–230. Wang, J., Wang, H., 2017. Oxidative stress in pancreatic beta cell regeneration. Oxid. Med. Cell. Longev.. 20171930261. Wardyn, J.D., Ponsford, A.H., Sanderson, C.M., 2015. Dissecting molecular cross-talk between Nrf2 and NF-κB response pathways. Biochem. Soc. Trans. 43 (4), 621–626.

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