**4. AGE receptors**

Accumulation of AGEs *in vivo* causes a decrease in physiological function, leading to the onset and progression of various diseases. Recent studies revealed the existence of receptors involved in degrading and removing AGEs accumulated by glycation of such proteins and the intracellular signal transduction system via receptors [46]. AGEs are categorised into two groups based on their receptors; the first group includes the receptors AGE-R1, AGE-R3, scavenger receptor class A (SR-A) I, SR-AII, scavenger receptor-BI (SR-BI), cluster of differentiation 36 (CD36), FEEL1, FEEL2, and ezrin/radixin/moesin (ERM), which exert scavenger functions to removes AGE, and the second group includes RAGE, which is related to the enhancement of inflammation and oxidative stress (**Figure 4**).

AGE-R1 and AGE-R2 were identified as oligosaccharyltransferase-48 (OST-48) and 80-kDa protein kinase C (PKC) substrate (80 K-H), respectively, in rat livers [47]. Subsequently, AGE-R3 was identified as a protein that binds to AGE-1 and AGE-2 [48] to form a complex. AGE-R1 is also known as OST-48, belongs to the single transmembrane lectin family, and has a molecular weight of 48 kDa. AGE-R1 is expressed in endothelial cells, mesangial cells, macrophages, and mononuclear cells and functions by removing AGEs via endocytosis. AGE-R1, which enhances AGE removal, may also be a distinct receptor, as it suppresses AGE-mediated mesangial cell inflammatory injury by protecting against injury to the kidneys and other tissues due to diabetes [49]. Recent studies reported that AGE-R1 may be involved in lifespan extension [50, 51]. AGE-R2, also known as 80 K-H, is a tyrosine phosphorylated protein with a molecular weight of 80 kDa that was initially identified as a substrate for PKC and is expressed in the cytoplasm [47]. AGE-R2 is expressed

#### **Figure 4.**

*The receptors for AGEs. A schematic diagram of AGE receptors is shown [46]. The receptor of AGEs (RAGE) includes full-length RAGE (F-RAGE), N-terminally truncated RAGE (N-RAGE), and soluble RAGE (sRAGE), which are cleaved from the cell surface membrane by matrix metalloproteinases. The AGE receptor (AGE-R complex) contains AGE-R1 (OST-48), AGE-R2 (80K-H), and AGE-R3 (Galectin-3). Scavenger receptor class A (SR-A), cluster of differentiation 36 (CD36), fasciclin EGF-like, laminin-type EGF-like, and link domain-containing scavenger receptor 1 and its homolog 2 (FEEL1 and − 2) are indicated as scavenger receptors.*

#### *Advanced Glycation End Products and Oxidative Stress in a Hyperglycaemic Environment DOI: http://dx.doi.org/10.5772/intechopen.97234*

in mononuclear cells and in the kidneys, vascular endothelium, brain, and nerves. Importantly, AGE-R2 is involved in activating intracellular signals via receptors, such as fibroblast growth factor receptor [52, 53]. AGE-R3, also called galectin-3, is a receptor that belongs to the lectin family and has a molecular weight of 32 kDa [48]. AGE-R3 binds directly to AGEs via the carbohydrate recognition domain in cells and is expressed in macrophages, eosinophils, and mast cells as well as in the nerves and kidneys. AGE-R3 has been reported to suppress adhesion between cells and the matrix laminin [54], activate mast cells [55], and degrade AGEs via endocytosis [48]. In addition, when diabetes develops in AGE-R3-knockout mice, the expression of macrophage scavenger receptor A and AGE-R1, which is involved in degrading AGEs, is decreased, and the expression of AGE receptors related to cell damage, such as RAGE and AGE-R2, is increased [56]. Because the expression of AGE-R3 is enhanced in ageing and diabetes, this receptor may have protective effects against ageing [57].

SR-A has been identified as a macrophage scavenger receptor [58, 59] and has a wide range of functions, such as removal of acetylated or oxidised low-density lipoprotein (LDL), removal of apoptotic cells, biological defence from bacteria, and cell adhesion [60]. SR-A is highly expressed in peritoneal macrophages derived from humans and from diabetic mice after culture in high-glucose medium [61]. Furthermore, SR-A promotes macrophage infiltration and foaming by incorporating AGEs into cells from the cell surface of macrophages [62, 63]. SR-BI is expressed in macrophages and in the liver adrenal glands and ovaries, functioning to promote the uptake of the cholesterol ester of high-density lipoprotein (HDL) and subsequent return of HDL to the liver [64, 65]. CD36, also known as scavenger receptor-BII, is a highly expressed receptor for single-stranded glycoprotein of 88 kDa in macrophages, vascular endothelial cells, and adipocytes [66]. CD36 binds to fatty acids, collagen, and oxidised LDL and is responsible for the uptake of oxidised LDL into macrophages and transport of fatty acids to adipocytes. Because CD36 is involved in removing AGEs, this protein may play protective roles in atherosclerotic diseases [67, 68]. The fasciclin, EFG-like, laminin-type EGF-like, and link domaincontaining scavenger receptor-1 (FEEL-1) is expressed in the liver, vascular endothelial cells, and monocyte lineage cells, whereas FEEL-2 (a homologue of FEEL-1) is expressed in the spleen and lymph nodes. Despite the different tissue specificity, FEEL-1 and -2 are believed to be involved in the degradation of AGEs [69]. Megalin was identified as a 600-kDa glycoprotein (gp330) antigen expressed in glomerular epithelial cells (podocytes) of Heymann nephritis, a rat model of membranous nephropathy [70]. In recent studies, megalin was shown to bind to AGEs; AGEs that have passed through glomeruli are trapped and taken up by lysosomes to be decomposed [71]. AGEs bind to the N-terminus of the ERM protein family, which is a linker protein that crosslinks actin filaments and cell membrane proteins [72]. AGEs have been shown to promote angiogenesis through the hyperpermeability of human umbilical vein endothelial cells by inducing the phosphorylation of moesin via the RhoA/ROCK pathway [73].

RAGE is a single-pass 45-kDa transmembrane protein belonging to the immunoglobulin superfamily and was first isolated and identified from bovine lungs as a cell surface receptor that binds to AGEs [74]. RAGE is expressed in monocytes, macrophages, nerves, renal tubule cells, and mesangial cells [75]. In addition to AGEs, RAGE also binds to amyloid β protein, S100/calgranulins, and high-mobility group box 1 as ligands and is involved in the enhancement of inflammation and oxidative stress [76, 77]. RAGE is composed of a total of five domains: the extracellular domain of one V domain and two C domains, transmembrane domain, and intracellular domain [78]. When AGEs bind to this full-length RAGE, NADPH oxidase is activated, and the production of intracellular reactive oxygen species

#### **Figure 5.**

*AGE/RAGE signalling. NADPH oxidase is activated by the binding of AGE to RAGE, and intracellular ROS levels are elevated. Intracellular ROS activates the IκB kinase (IKK) complex and inhibitor of NF-κB (IκB), stimulating the translocation of the NF-κB subunits p65 and p50 and activating transcription. In addition, activation of PKCβ stimulates transcription via activator protein-1 (AP1) in the nucleus by phosphorylation of c-Jun N-terminal kinase (JNK). Enhancement of these inflammatory signals releases inflammatory cytokines, such as TNFα and IL-6, as well as VEGF, which is involved in angiogenesis, and B-cell lymphoma 2 (Bcl-2) and Bcl-2 associated X protein (Bax), which are involved in apoptosis. TNFα, an inflammatory cytokine, is released extracellularly and binds to the TNFα receptor, and activation of TGFβ activated kinase (TAK) reactivates JNK.*

(ROS) is promoted [79, 80]. ROS upregulate various inflammatory cytokines, growth factors, and adhesion molecules by activating nuclear factor-kappa B (NF-κB) signalling. In addition, c-Jun N-terminal kinase (JNK), a major subfamily of ROS-activated mitogen-activated protein kinase pathways, has been shown to cause cell apoptosis and dysfunction (**Figure 5**) [81]. In addition to full-length RAGE on the cell surface, RAGE can be expressed as two splice variants, i.e., the intracellular domain-deficient type (C-terminally truncated RAGE) and extracellular V domain-deficient type (N-terminally truncated RAGE) [82]. Of these, the intracellular domain-deficient RAGE is called soluble RAGE (sRAGE). sRAGE can further be divided into endogenous secretory RAGE (esRAGE) and soluble RAGE, which are cleaved by proteases such as matrix metalloproteinases [83]. sRAGE has a binding site for AGEs and is thought to function as a decoy receptor that captures extracellular AGEs and inhibits binding to RAGE on the cell surface, thereby blocking intracellular signals [84]. Blood esRAGE levels are significantly lower in patients with type 2 diabetes than in patients without diabetes, suggesting that this target is involved in the development of type 2 diabetes [85]. Moreover, blood esRAGE levels in patients with type 2 diabetes are inversely correlated with the severity of carotid atherosclerosis and coronary artery disease as complications [86, 87].

## **5. AGEs and oxidative stress**

Intracellular signal transduction of AGEs via RAGE increases intracellular ROS. ROS are oxygen-containing molecular derivatives that are in a more activated state than triplet oxygen, which is a ground-state oxygen molecule necessary for

#### *Advanced Glycation End Products and Oxidative Stress in a Hyperglycaemic Environment DOI: http://dx.doi.org/10.5772/intechopen.97234*

normal biological activities and is highly reactive, resulting in oxidative damage to various biological components. The main active oxygen species are singlet oxygen, superoxide, hydrogen peroxide, and hydroxyl radicals [88]; these molecules react with biopolymers, such as DNA, lipids, proteins, and enzymes, resulting in lipid peroxidation, DNA mutations, protein denaturation, and enzyme inactivation. Many amino acids are carbonylated and modified by ROS for detection of protein carbonylation using mass spectrometers [89]. Moreover, carbonylation of this protein is caused by addition reaction of aldehydes because of the peroxidation reaction of lipids and saccharification reaction of proteins described above [90, 91]. Highly reactive α-dicarbonyl compounds, such as 3-deoxyglucosone (3-DG), glyceraldehyde, and methylglyoxal, are produced from the Amadori compound generated by saccharification [91]. These AGEs then recombine with RAGE, creating a vicious cycle in which more ROS are generated. Such ROS are considered to have negative effects because overproduction of ROS is closely associated with ageing due to oxidative stress, cancer, and the development of lifestyle-related diseases [91]. However, ROS (e.g., superoxide and hydrogen peroxide) produced by white blood cells play important roles in biological defence and immune function [92]. ROS are also used in a wide range of tissues and cells as bioactive substances for intracellular signal transduction, fertilisation, cell differentiation, and apoptosis [93].

Because glucose is metabolised to obtain energy, the carboxyl group of glucose reacts with the amino group of the protein during the metabolic process to form AGEs in the body nonenzymatically via the Amadori compound. With ageing, these AGEs accumulate in various organs in the body, resulting in oxidative stress, ROS generation, and progression of organ stress. Thus, ageing is related to oxidative stress induced by AGEs. Additionally, AGEs-ised HbA1c levels in the blood have been used as an index for controlling blood glucose levels in clinical practice for patients with diabetes. Kusunoki et al. showed that fasting serum 3-DG levels in patients with diabetes were significantly higher than those in controls. Additionally, serum 3-DG levels tended to be higher in patients with diabetes showing low nerve conduction velocity [94]. In patients with diabetes, AGEs generated from excess glucose circulate throughout the body via the blood and increase oxidative stress in various organs. Therefore, in the hyperglycaemic environment associated with diabetes, oxidative stress due to excess glucose is thought to be significantly involved in the development of diabetic complications.
