AGE/RAGE as a Mediator of Insulin Resistance or Metabolic Syndrome: Another Aspect of Metabolic Memory?

Large randomized studies in diabetes have established that early intensive glycemic control reduces the risk of diabetic microvascular complications, with less impact on macrovascular complications 1, 2. In type 2 diabetic patients, further intensive therapy to target normal glycated hemoglobin levels also failed to reduce mortality and major cardiovascular events 3, 4, while it may be rather harmful 5. However, follow-up data of these trials reveal a longterm influence of early metabolic control on longer cardiovascular outcomes, even though the influence on glycemic control has been immediately disappeared after the trials 6, 7. This phenomenon has recently been defined as "metabolic memory". In at-risk patients with type 2 diabetes, intensive intervention with multiple drug combinations and behavior modification had similar sustained beneficial effects with respect to vascular complications and on rates of death from any cause and from cardiovascular causes 8. Similarly in patients with end-stage renal disease (ESRD), intensive interventions to the general risk factors, such as high LDL-cholesterol or C-reactive protein, have not been successful in improving their cardiovascular outcomes 9, 10, suggesting that the beneficial effect of risk reduction may be overwhelmed by accumulated “metabolic memory” by long-term exposure to oxidative stress during the progression of renal failure. Potential mechanisms for propagating this "memory" are the non-enzymatic glycation of cellular and tissue proteins which are conceptualized as advanced glycation end-products (AGEs), the generation of which has been implicated to be deeply associated with increased oxidative stress as well as hyperglycemia. AGEs, with their receptor (receptor for AGEs, RAGE), potentially mediate molecular and cellular pathway leading to metabolic memory. Moreover, interaction of the RAGE with AGEs leads to crucial biomedical pathway generating intracellular oxidative stress and inflammatory mediators, which could result in further amplification of the pathway involved in AGE generation. By utilizing genetically engineered mouse models, emerging evidence suggests that AGE/RAGE axis is also found to be profoundly associated with non-diabetic, non-uremic pathophysiological conditions including 1) atherogenesis, 2) angiogenic response, 3) vascular injury, and 4) inflammatory response (see review in 11), many of which are now implicated in metabolic syndrome. Numerous truncated forms of RAGE have also been described, and the


Introduction
Large randomized studies in diabetes have established that early intensive glycemic control reduces the risk of diabetic microvascular complications, with less impact on macrovascular complications 1,2 .In type 2 diabetic patients, further intensive therapy to target normal glycated hemoglobin levels also failed to reduce mortality and major cardiovascular events 3, 4 , while it may be rather harmful 5 .However, follow-up data of these trials reveal a longterm influence of early metabolic control on longer cardiovascular outcomes, even though the influence on glycemic control has been immediately disappeared after the trials 6,7 .This phenomenon has recently been defined as "metabolic memory".In at-risk patients with type 2 diabetes, intensive intervention with multiple drug combinations and behavior modification had similar sustained beneficial effects with respect to vascular complications and on rates of death from any cause and from cardiovascular causes 8 .Similarly in patients with end-stage renal disease (ESRD), intensive interventions to the general risk factors, such as high LDL-cholesterol or C-reactive protein, have not been successful in improving their cardiovascular outcomes 9,10 , suggesting that the beneficial effect of risk reduction may be overwhelmed by accumulated "metabolic memory" by long-term exposure to oxidative stress during the progression of renal failure.Potential mechanisms for propagating this "memory" are the non-enzymatic glycation of cellular and tissue proteins which are conceptualized as advanced glycation end-products (AGEs), the generation of which has been implicated to be deeply associated with increased oxidative stress as well as hyperglycemia.AGEs, with their receptor (receptor for AGEs, RAGE), potentially mediate molecular and cellular pathway leading to metabolic memory.Moreover, interaction of the RAGE with AGEs leads to crucial biomedical pathway generating intracellular oxidative stress and inflammatory mediators, which could result in further amplification of the pathway involved in AGE generation.By utilizing genetically engineered mouse models, emerging evidence suggests that AGE/RAGE axis is also found to be profoundly associated with non-diabetic, non-uremic pathophysiological conditions including 1) atherogenesis, 2) angiogenic response, 3) vascular injury, and 4) inflammatory response (see review in 11 ), many of which are now implicated in metabolic syndrome.Numerous truncated forms of RAGE have also been described, and the

AGEs and insulin resistance in vitro
Several evidences also suggest that AGEs affect the function of insulin-target cells in vitro.AGEs interact with CD36 in mouse 3T3 and human subcutaneous adipocytes, which is associated with down-regulation of leptin expression in adipocyte through reactive oxygen species (ROS) system 53 .Miele et al showed in L6 skeletal muscle cells that AGEs affect glucose metabolism by impairing insulin-induced insulin receptor substrate (IRS) signaling through protein kinase Cα-mediated mechanism 37 .The same research group also showed in the muscle cells that methylglyoxal, an essential source of intracellular AGEs, hampers a key insulin signaling molecule 54 .Recent observations by Unoki et al also showed that AGEs impair insulin signaling in adipocytes by increasing generation of intracellular ROS 55 .Thus, AGEs may not only induce the debilitating complications of diabetes, but may also contribute to the impairment of insulin signaling in insulin-target tissues which could be involved in pathophysiology of insulin resistance, metabolic syndrome and diabetes.

Endogenous RAGE ligands, insulin resistance and metabolic syndrome
RAGE also interacts with other endogenous non-glycated peptide ligands including S100/calgranulin 56 , amphoterin (also termed as high mobility group box 1 protein, HMGB1) 57,58 , amyloid fibrills 59 , transthyretin 60 , and a leukocyte integrin, Mac-1 61 , many of which are important inflammatory regulators.Some of these inflammatory ligands for RAGE may be involved in pathogenesis of obesity and metabolic syndrome.Early studies show expression of S100B protein in pre-and mature-adipocyte and is induced during adipogenesis 62,63 .Physiological S100B levels appear to closely reflect adipose tissue mass or insulin resistance in humans [64][65][66] .HMGB1 is also found to be expressed in human adipose tissue with the expression level associated with the fat mass and obesity-associated gene 67 .Moreover, growing evidences suggest that infiltration of inflammatory cells, including macrophages, play fundamental roles in adiposity and metabolic syndrome [68][69][70] .MAC-1, an integrin expressed in macrophage, can act as a RAGE ligand 61 , and may be involved in adipogenesis through interaction with RAGE.

RAGE and its potential link with insulin resistance and metabolic syndrome 4.1 Structure and function of RAGE
RAGE is a multiligand cell-surface protein that was isolated from bovine lung in 1992 by the group of Schmidt and Stern 71,72 .RAGE belongs to the immunoglobulin superfamily of cell surface molecules and has an extracellular region containing one "V"-type immunoglobulin domain and two "C"-type immunoglobulin domains 71,72 (Figure 1).The extracellular portion of the receptor is followed by a hydrophobic trans-membrane-spanning and then by a highly charged, short cytoplasmic domain which is essential for intracellular RAGE signaling.RAGE is initially identified as a receptor for CML-modified proteins 73 , a major AGE in vivo 74 .Three-dimensional structure of the recombinant AGE-binding domain by using multidimensional heteronuclear NMR spectroscopy revealed that the domain assumes a structure similar to those of other immunoglobulin V-type domains 75,76 .Three distinct surfaces of the V domain were identified to mediate AGE-V domain interactions 75 .The site-directed mutagenesis studies identified the basic amino acids which play a key role in the AGE binding activities 76 .As mentioned in the previous sentence, RAGE also interacts with other endogenous non-glycated peptide ligands, many of which are important inflammatory regulators.The common characteristics of these ligands are the presence of multiple β-sheets 61,77,78 .RAGE is thought to interact with these ligands through their shared three-dimensional structure.Fig. 1.Numerous truncated forms of RAGE.There are three major spliced variants of RAGE: full length, N-terminally truncated, and C-terminally truncated.The C-terminally truncated form of RAGE is secreted from the cell and is named endogenously secreted RAGE (esRAGE).esRAGE has a V-domain, which is essential for binding with ligands, and is capable of competing with RAGE signaling as a decoy receptor.There are other forms of soluble RAGE (sRAGE) that are cleaved from cell-surface RAGE by matrix metalloproteinases.The ELISA assay for sRAGE measures all soluble forms including esRAGE in human plasma, while the ELISA for esRAGE measures only esRAGE, using polyclonal antibody raised against the unique C-terminus of the esRAGE sequence.

Inflammatory signaling mediated by RAGE
Ligand engagement of RAGE leads to prolonged inflammation, resulting in a RAGEdependent expression of proinflammatory mediators such as monocyte chemoattractant protein-1 (MCP-1) and vascular cell adhesion molecule-1 (VCAM-1) 79,80 .RAGE-mediated proinflammatory signals could potentially converge with insulin signaling system (Figure 2).The engagement of RAGE has been reported to induce activation of the transcription factor nuclear factor-κB (NF-κB).Recent reports by Harja et al demonstrate that RAGE mediates upregulation of VCAM-1 in response to S100b and oxLDL and JNK MAP kinase underlies the RAGE ligand-stimulated molecular events 81 .It is not known at present whether this is also the case in classical insulin target cells.JNK activity is strikingly increased in critical metabolic sites (eg.adipose and liver tissues) 82 , and is shown to be crucial in IRS-1 phosphorylation and consequently insulin resistance 82,83 .Moreover, the main pathological consequence of RAGE ligation is the induction of intracellular reactive oxygen species (ROS) via NAD(P)H oxidases and other identified mechanisms such as mitochondrial electron transport chain 84 , which consequently results in oxidative stress in the cells 85 .Oxidative stress is emerging as a feature of obesity and an important factor in the development of insulin resistance 86,87 .Both the NF-kB and JNK pathways can be activated www.intechopen.comBiomedical Science, Engineering and Technology 96 under the conditions of oxidative stress, and this may be important for the ability of ROS to mediate insulin resistance.RAGE has a short cytosolic portion that contains 43 amino acids 72 .So far, adaptors and/or scaffold proteins that interact with the cytosolic tail of RAGE has barely been identified.The RAGE mutant lacking the 43-residue C-terminal tail fails to activate NF-κB, and expression of the mutant receptor results in a dominant negative effect against RAGE-mediated production of proinflammatory cytokines from macrophages 56,57 .Fig. 2. RAGE and insulin signaling.RAGE is known to activate JNK pathway, which could phosphorylate serine-residue of insulin receptor substrate (IRS) and inhibit its activity.RAGE mediated generation of reactive oxygen spices (ROS) may alternatively influence insulin signaing.

A. RAGE, adiposity and atherosclerosis in mouse model
Recent reports suggest that RAGE could be involved in progression of obesity.Recent study in humans shows RAGE mRNA expression in subcutaneous adipose tissues 88 .Although this study does not delineate which cells in adipose tissue express RAGE, our current animal study shows RAGE expression in adipocyte as wells as endothelial cells in adipose tissues 16 .We have shown by using apo E/RAGE double knockout mice that progression of atherosclerosis is closely associated with RAGE-regulated adiposity in non-diabetic conditions 16 .As shown in F i gur e 3 , apoE -/-RAGE -/-mice fed either with standard or atherogenic diet exhibited significantly decreased atherosclerotic plaque area in aorta as compared with apoE -/-RAGE +/+ mice.Importantly, apoE -/-RAGE -/-mice also exhibited significantly less body weight, epididymal fat weight and epididymal adipocyte size than apoE -/-RAGE +/+ mice at 20-weeks of age (Figure 4).Decreased body weight, epididymal fat weight, and adipocyte size are associated with higher plasma adiponectin levels and decreased atherosclerosis progression.RAGE is involved in adiposity even in apo E +/+ genetic background.At 20-weeks of age, epididymal adipocyte size of RAGE -/-mice was significantly smaller than that of RAGE +/+ mice (data not shown).Fig. 3. RAGE deficiency suppresses atherosclerotic progression in apoE deficient mice.Representative aortas from apoE -/-RAGE +/+ and apoE -/-RAGE -/-mice (20-weeks old) fed with atherogenic diet were shown in left panel.Right panel summarizes the quantitative analyses.Plaque area was represented as percentages of the total plaque area.Columns represent mean ± standard deviation.Black columns represent apoE -/-RAGE +/+ mice, and grey columns, apoE -/-RAGE -/-mice.P values were analyzed by Student's unpaired t-test.Reproduced from ref 16 .

B. Roles of inflammatory cells?
RAGE is also known to play fundamental role in functions of inflammatory cells 61,89,90 , raising an intriguing possibility that RAGE's function on adiposity may be mediated through its function in inflammatory cells infiltrated in adipose tissues.In our study in apoE -/-genetic background fed with atherogenic diet, numbers of Mac-3-positive inflammatory cells infiltrated in the epididymal adipose tissues of RAGE +/+ apoE -/-mice and RAGE -/-apoE -/-did not show significant differences, and crown-like structure were barely detected in epididymal adipose tissue in both groups even at 20-week of age.In standard diet-fed mice, even though the adiposity was significantly different between RAGE +/+ apoE - /-and RAGE -/-apoE -/-mice, crown-like structure were not detected in epididymal adipose tissues in both groups even at 20-week of age.Further in apoE +/+ genetic background at 10 week of age when significantly different pattern of gene expression was observed between WT and RAGE -/-mice, no marked differences in expressions of macrophage markers were observed as analyzed by gene microarray.At that age, macrophage infiltration in adipose tissues is also reported to be scant 91 .Thus, it appears infeasible to RAGE acting primarily at inflammatory cells at least in early phase of adiposity, while RAGE expressed in endothelial cells or adipocyte might play fundamental roles.

C. RAGE-regulated genes in adipose tissue: gene chip analysis
To explore potential mechanisms underlying RAGE-regulation of adiposity, mRNA expression profile in epididymal adipose tissue was compared between RAGE +/+ and RAGE -/-mice using Affymetrix GeneChip Mouse Genome 430 2.0.We isolated total RNA from epididymal adipose tissue at 10-weeks of age, at which phenotypic change in adipocyte size was not observed.Using 3 µg of total RNA, 59.8% and 61.4% of 45,037 genes w e r e r e v e a l e d t o b e p r e s e n t i n R A G E +/+ and RAGE -/-adipose tissue, respectively.Comparison analysis of the genes (RAGE +/+ adipose tissue as base line) revealed that 10.3% of the total genes were decreased, while 11.7% increased in RAGE -/-adipose tissue.As compared with RAGE +/+ adipose tissue, 623 genes were downregulated to less than a half, and 2,470 genes upregulated more than 2 fold in RAGE -/-adipose tissue.

D. RAGE-regulated genes in adipose tissue: ontology analysis
To mine specific group of genes involved in adiposity regulated by RAGE, gene ontology analyses were performed.Downregulated genes in RAGE -/-adipose tissue were significantly accumulated in the ontology terms of metabolic process including acetyl-CoA biosynthetic process, neutral lipid biosynthetic process, pyruvate metabolic process, gluconeogenesis, glycogen biosynthetic process, and NADPH regeneration.Interestingly, genes involved in fat cell differentiation were also identified to be accumulated as down-regulated in RAGE -/- adipose tissue.Ontology terms of glucose transport and neutral amino acid transport were also significantly extracted as downregulated in RAGE -/-adipose tissue.Insulin receptor signaling pathway was a highly significant ontology term downregulated in RAGE -/-adipose tissue.On the contrary, many of the genes upregulated in RAGE -/-adipose tissue were accumulated in ontology terms including cell adhesion, endocytosis, T cell activation, prostaglandin biosynthesis, protein binding, protein folding, processing and glycoprotein biosynthetic process, many of which are known be associated with cellular mechanisms for inflammation and defensive process.Nitrogen compound metabolic process, including amino acid metabolic process, was also identified to be a significant ontology term upregulated in RAGE -/-.Interestingly, upregulated genes in RAGE -/-tissue were also significantly accumulated in ontology term for cell redox homeostasis process.

E. RAGE-regulated genes in adipose tissue: pathway analysis
To further identify potential pathways involved in RAGE-regulation of adiposity, KEGG pathway analyses were performed (Table 1).In accordance with the ontology analyses, insulin signaling pathway, pyruvate metabolism, fatty acid biosynthesis and gluconeogenesis were identified to be downregulated pathways in RAGE -/-adipose tissue.PPAR signaling and adipocytokine signaling were also identified to be downregulated in RAGE -/-adipose tissue.Similar to gene ontology analyses, inflammatory pathways including cell adhesion molecules and leukocyte transendothelial migration were the significant pathways upregulated in RAGE -/-mice.Pathways including amino acid metabolic pathways, nitrogen metabolism, glycan biosynthesis, structure and degradation were the pathways significantly upregulated in RAGE -/-adipose tissues.

RAGE, endothelial dysfunction and insulin resistance
Impaired insulin action, when assessed by fasting serum insulin levels or the homeostasis model assessment of insulin resistance (HOMA-IR) 92 , is associated with atherosclerosis and an increased risk of myocardial infarction.Insulin resistance is associated with endothelial dysfunction 93 and may serve as a link between insulin resistance and atherosclerosis.Recent findings by Harja et al highlighted the involvement of RAGE in endothelial dysfunction 81 .Endothelium-dependent vasorelaxation was tested in isolated mouse aortic rings from apoE - /-and apoE -/-RAGE -/-mice, and relaxation response to acetylcholine was significantly improved in the RAGE deficient mouse.Similarly, impaired endothelial function in diabetic obese mice was also shown to be mediated by AGEs/RAGE system, since blockade of AGE-RAGE interaction by soluble RAGE significantly improved endothelial function 94 .Recent clinical observations by Linden et al 44 also implies AGEs/RAGE system is involved in impaired endothelial function in patients with chronic kidney diseases.Thus, not only by the interaction at the cellular signaling level, but RAGE appears to impair endothelial function and potentially blood flow in insulin target tissues, leading to insulin resistance in vivo.

C-terminally truncated form of RAGE (soluble RAGE, sRAGE) as potential biomarkers for cardiovascular diseases, metabolic syndrome and insulin resistance 5.1 Truncated form of RAGE
Numerous truncated forms of RAGE have recently been described 12,[95][96][97][98] (Figure 1).Two major spliced variants of RAGE mRNA, N-terminal and C-terminal truncated forms, have been most extensively characterized 12 .The N-truncated isoform of RAGE mRNA codes for a 303-amino-acid protein lacking the N-terminal signal sequence and the first V-like extracellular domain.The N-truncated form is incapable of binding with AGEs, since the V-domain is critical for binding of the ligand 71 .The N-truncated form of RAGE appears to be expressed on the cell surface similar to the full-length RAGE, although its biological roles remain to be elucidated 99 .It has been suggested that this form of RAGE could be involved in angiogenic regulation in a fashion independent of the classical RAGE signaling pathway 99 .

Endogenous secretory RAGE (esRAGE)
The C-terminal truncated form of RAGE lacks the exon 10 sequences encoding the transmembrane and intracytoplasmic domains 12 .This spliced variant mRNA of RAGE 103 encodes a protein consisting of 347 amino acids with a 22-amino-acid signal sequence, and is released from cells.This C-truncated form is now known to be present in human circulation and is named endogenous secretory RAGE (esRAGE) 12 .Regulation of alternative splicing of the RAGE is recently shown to be regulated through G-rich cis-elements and heterogenous nuclear ribonucleoprotein H 100 .esRAGE was found to be capable of neutralizing the effects of AGEs on endothelial cells in culture 12 .Adenoviral overexpression of esRAGE in vivo in mice reverses diabetic impairment of vascular dysfunction 101 .Thus, the decoy function of esRAGE may exhibit a feedback mechanism by which esRAGE prevents the activation of RAGE signaling.

Soluble RAGE generated by shedding
It has also been suggested that some sRAGE isoforms that could act as decoy receptors may be cleaved proteolytically from the native RAGE expressed on the cell surface 102 , suggesting heterogeneity of the origin and nature of sRAGE.This proteolytic generation of sRAGE was initially described as occurring in mice 103 .Recent studies suggest that ADAM10 and MMP9 to be involved in RAGE shedding 13,14 .ADAM is known as a shedase to shed several inflammatory receptors and can be involved in regulation of RAGE/sRAGE balance.A RAGE gene polymorphism is shown to be strongly associated with higher sRAGE levels, although the mechanism by which the polymorphism alters the sRAGE levels remains to be elucidated 104 .Thus, the molecular heterogeneity of the diverse types of sRAGE in human plasma could exert significant protective effects against RAGE-mediated toxicity.However, the endogenous action of sRAGE may not be confined to a decoy function against RAGE-signaling.In HMGB1-induced arthritis model, for example, sRAGE is found to interact with Mac-1, and act as an important proinflammatory and chemotactic molecule 105 .Further analyses are warranted to understand more about the endogenous activity of sRAGE.

A. Circulating sRAGE and cardiovascular diseases
Since sRAGE and esRAGE may be involved in feedback regulation of the toxic effects of RAGE-mediated signaling, recent clinical studies have focused on the potential significance of circulating sRAGE and esRAGE in a variety of pathophysiological conditions, including atherosclerotic disorders, diabetes, hypertension, Alzheimer's dieases and chronic kidney diseases (Table 2).First, Falcone et al 106

B. Circulating esRAGE and cardiovascular diseases
Following development of an ELISA system to specifically measure human esRAGE 108 , we measured plasma esRAGE level and cross-sectionally examined its association with atherosclerosis in 203 type 2 diabetic and 134 non-diabetic age-and gender-matched subjects 15 .esRAGE levels were inversely correlated with carotid and femoral atherosclerosis, as measured as intimal-medial thickness (IMT) by arterial ultrasound.
Stepwise regression analyses revealed that plasma esRAGE was the third strongest and an independent factor associated with carotid IMT, following age and systolic blood pressure 15 .Importantly however, when non-diabetic and diabetic groups were separately 105 analyzed, inverse correlation between plasma esRAGE level and IMT was significant in non-diabetic population only, suggesting a potential significance of esRAGE in nondiabetic condition.No association of plasma esRAGE with IMT in diabetes was also reported in other study with 110 Caucasian type 2 diabetic subjects 109 .Another Japanese research group found an inverse correlation between plasma esRAGE and carotid atherosclerosis in type 1 110 and type 2 diabetic subjects 111 .Recently, the same research group also longitudinally examined the predictive significance of plasma esRAGE and sRAGE on progression of carotid atherosclerosis, and found that low circulating esRAGE level as well as sRAGE level was an independent risk factor for the progression of carotid IMT in type 1 diabetic subjects 112 .In Chinese type 2 diabetic patients, plasma esRAGE is recently shown to be decreased in angiographically-proved patients with coronary artery disease than those without it 113 .

C. Low circulating sRAGE as a predictor of cardiovascular diseases
We also reported an observational cohort study in patients with end-stage renal disease (ESRD) and longitudinally evaluated the effect of plasma esRAGE on cardiovascular mortality 114 .The cohort in that study included 206 ESRD subjects, who had been treated by regular hemodialysis for more than 3 months.Even though the plasma esRAGE levels at baseline were higher in ESRD subjects than in those without kidney disease, the subjects in the lowest tertile of plasma esRAGE levels exhibited significantly higher cardiovascular mortality, but not non-cardiovascular mortality.Importantly, even in the subpopulation of non-diabetic subjects alone, low circulating esRAGE level was a predictor of cardiovascular mortality, independent of the other classical risk factors.Thus, low circulating esRAGE or sRAGE level is a potential predictor for atherosclerosis and cardiovascular diseases even in non-diabetic population.

D. Circulating sRAGE, esRAGE and metabolic syndrome
Several components of metabolic syndrome have been shown to be associated with altered plasma sRAGE or esRAGE levels.We first reported that plasma esRAGE levels are already decreased in patients with impaired glucose tolerance as compared with those with normal glucose tolerance (Figure 6A).Moreover, patients with metabolic syndrome showed significantly lower plasma esRAGE than those without it (Figure 6A).Plasma esRAGE levels are inversely correlated with many of the components of metabolic syndrome including body mass index (Figure 6B), blood pressures, fasting plasma glucose, serum triglyceride, and lower HDL-cholesterol levels 15 .The majorities of these correlations remained significant even when the non-diabetic or type 2 diabetic subpopulation was extracted for analyses.An inverse correaltion between esRAGE (or sRAGE) and body mass index was also found for control subjects 115 , those with type 1 diabetes 116 , and those with ESRD 114.Patients with hypertension have been found to have lower plasma sRAGE or esRAGE levels 15,117 .Importantly, our findings also showed that plasma esRAGE was also inversely associated with insulin resistance index, HOMA (Figure 6B), suggesting esRAGE and sRAGE as potential biomarkers for metabolic syndrome and insulin resistance, which could be associated with altered cardiovascular outcomes.Both sRAGE and esRAGE are found to be decreased in patients with liver steatosis 118,119 , which is know to be deeply associated with visceral fat accumulation and insulin resistance.

E. Circulating sRAGE and esRAGE in diabetes
The findings regarding plasma levels of the soluble form of RAGE in diabetes are quite confusing.We and other groups have found that plasma esRAGE level is significantly lower in type 1 and type 2 diabetic patients than in non-diabetic controls 15,110 .Plasma sRAGE levels have also been shown to be decreased in diabetic subjects 120,121 , although conflicting findings have also been reported for type 1 122 and type 2 diabetes 123,124 .We examined plasma sRAGE levels by different ELISA system using esRAGE as a standard protein and different sets of antibodies against whole RAGE molecule 125 .In our hand, type 2 diabetic subjects without overt nephropathy (0.60 ± 0.28 ng/ml) exhibited significantly (p<0.001,Student's t-test) lower plasma sRAGE level than non-diabetic controls (0.77 ± 0.34 ng/ml) 11 .Of note, when diabetic subjects alone were extracted for analyses, a direct association was not observed between plasma soluble RAGE (both sRAGE and esRAGE) levels and the status of glycemic control (i.e.glycated hemoglobin A1c) 15,109,116,120,126 .Thus, these complex findings in diabetic subjects suggest that levels of plasma soluble forms of RAGE are not determined simply by status of glycemic control, and that even plasma esRAGE and sRAGE levels may be under the control of distinct mechanisms.Recent study suggests that sRAGE levels may be significantly influenced by ethnicity 127 , which may partially explain controversial findings.

F. Circulating sRAGE and esRAGE in CKD
Another important component that can affect plasma sRAGE is the presence of chronic kidney disease.It has been shown that, in peripheral monocytes from subjects with varying severities of CKD, RAGE expression is closely associated with worsening of CKD and is strongly correlated with plasma levels of pentosidine, a marker for AGEs 128 .Circulating sRAGE levels have been shown to be increased in patients with decreased renal function, particularly those with ESRD 109,123,129 .Our observations revealed that plasma esRAGE levels in type 2 diabetic subjects without CKD are lower than non-diabetic controls, which is gradually elevated in accordance with progression of CKD 11 .Plasma sRAGE levels in diabetic subjects without CKD also exhibited significantly lower than those of non-diabetic controls 11 .Thus, plasma sRAGE and esRAGE are markedly affected by the presence of CKD, which might make the interpretation of the role of soluble RAGE quite complicated 130 .It remains to be determined whether the increase in plasma esRAGE in CKD is caused by decreased renal function alone or whether esRAGE levels are upregulated to protect against toxic effects of the RAGE ligands.Successful kidney transplantation resulted in significant decrease in plasma sRAGE 131 , implying that the kidneys play a role in sRAGE removal.

RAGE and Soluble RAGE as a therapeutic target against metabolic syndrome, insulin resistance and cardiovascular disease?
6.1 Soluble RAGE as a therapeutic tool in animal disease models Potential usefulness of soluble RAGE for prevention and treatment of inflammatory diseases has been demonstrated in many animal models.Blockade of RAGE by administration of genetically engineered sRAGE successfully prevented the development of micro- 132,133 and macrovascular complications in diabetes [134][135][136] .We have also shown that adenoviral overexpression of esRAGE successfully restored the impaired angiogenic response in diabetic mice 101 .Sakaguchi et al found that administration of sRAGE markedly suppressed neointimal formation following arterial injury in non-diabetic mice 137 .Soluble RAGE has also been shown to effectively prevent the development of diabetes 138 , protect against tumor growth and metastasis 58 , improve the outcome of colitis 56 , restore impaired wound healing 139 , and suppress Alzheimer disease-like conditions 140 .These effects of soluble RAGE in animal models could be explained by its decoy function, inhibiting RAGE interaction with its proinflammatory ligands, which might be applicable to human diseases as well.Since our findings strongly suggest the role of RAGE in adiposity, metabolic syndrome and atheroslcerosis 16 , RAGE/soluble RAGE axis could also be a potential therapeutic target against these pathophysiological conditions.

Potential regulatory mechanisms of circulating soluble RAGE
So far, limited findings are available regarding the mechanisms of regulation of circulating esRAGE or sRAGE in humans.A tissue microarray technique using a wide variety of adult normal human preparations obtained from surgical and autopsy specimens revealed that esRAGE was widely distributed in tissues, including vascular endothelium, monocyte/macrophage, pneumocytes, and several endocrine organs 141 .However, it is unclear at present from which organ or tissue plasma sRAGE or esRAGE originate.Circulating AGEs may be involved in regulation of the secretion or production of soluble RAGE, since AGEs are known to upregulate RAGE expression in vitro 142 .esRAGE could be simultaneously upregulated by AGEs and act as a negative feedback loop to compensate for the damaging effects of AGEs.We and others have found positive correlations between plasma sRAGE or esRAGE and AGEs 11,[114][115][116]123 . Signiicant positive correlation between plasma esRAGE and pentosidine was observed both in hemodialysis and non-hemodialysis subjects 11 .However, plasma CML did not significantly correlated with plasma esRAGE both in hemodialysis and non-hemodialysis subjects.AGEs-mediated regulation of soluble RAGE is also supported by the findings that the suppression of sRAGE expression in diabetic rat kidney is reversed by blockade of AGEs accumulation with alagebrium 143 .
Other inflammatory mediators, such as S100, tumor necrosis factor α, and C-reactive protein, could also be potential candidates for regulation of the plasma level of soluble RAGE in humans 120,142,144 .Moreover, Geroldi et al 145 showed that high serum sRAGE is associated with extreme longevity, suggesting that understanding the intrinsic regulation of RAGE and soluble RAGE is important for longevity/anti-aging strategies.Without doubt, further understanding of the regulation of soluble RAGE will be most helpful in delineating potential targets for therapeutic application of soluble RAGE.

A. Angiotensin-converting enzyme inhibitor
It would be essential to determine whether currently available pharmacological agents can regulate plasma sRAGE or esRAGE.Potential agents that may affect circulating soluble RAGE include the angiotensin-converting enzyme (ACE) inhibitor 146 , thiazolidinediones (TZD) 147 and statins [148][149][150] , which are known to modulate the AGEs-RAGE system in culture.Forbes et al 146 showed that inhibition of angiotensin-converting enzyme (ACE) in rats increased renal expression of sRAGE, and that this was associated with decreases in expression of renal full-length RAGE protein.They also showed that plasma sRAGE levels were significantly increased by inhibition of ACE in both diabetic rats and in human subjects with type 1 diabetes.Thus, one attractive scenario is that the protective effect of ACE inhibition against progression of renal dysfunction is mediated through regulation of RAGE versus soluble RAGE production.

B. Statin
Tam et al recently reported changes in serum levels of sRAGE and esRAGE in archived serum samples from a previous randomized double-blind placebo-controlled clinical trial that explored the cardiovascular effects of atorvastatin in hypercholesterolemic Chinese type 2 diabetic patients, and found that atorvastatin can increase circulating esRAGE levels 150 .

C. Thiazolidinedione
For thiazolidinedione, a randomised, open-label, parallel group study was performed with 64 participants randomised to receive add-on therapy with either rosiglitazone or sulfonylurea to examine the effect on plasma soluble RAGE 151 .At 6 months, both rosiglitazone and sulfonylurea resulted in a significant reduction in HbA1c, fasting glucose and AGE.However, significant increases in total sRAGE and esRAGE were only seen in the rosiglitazone group.In a recent study in type 2 diabetes mellitus patients, pioglitazone, but not rosiglitazone, significantly raised sRAGE levels 152 , suggesting that all thiazolidinedione may not act similarly.Nevertheless, thiazolidinedione could be one promising candidate which increase circulating levels of esRAGE and sRAGE, and RAGE/soluble RAGE regulation may be involved in thiazolidinedione-mediated improvement of insulin resistance.Finally, we have started the randomized clinical trial comparing the effect of pioglitazone with glimepiride on plasma sRAGE and esRAGE, expression of RAGE on peripheral mononuclear cells, and RAGE shedase gene expression in type 2 diabetic patients (UMIN000002055).This study will be of particular importance to understand the regulatory mechanisms of sRAGE and esRAGE in clinical setting.

Summary
The findings discussed here implicated pivotal role of RAGE system in initiation and progression of metabolic syndrome, insulin resistance and atherosclerosis.Provided that continuous RAGE activation represents the concept of "metabolic memory", metabolic syndrome might be conceptualized as memorized long-term subtle inflammation and oxidative stress using RAGE as an inflammatory scaffold.In this system, endogenous inflammatory RAGE ligands may be profoundly involved (Figure 7).Further, sRAGE or esRAGE could serve as a biomarker as well as a therapeutic target for these disease conditions.Obviously there are many missing parts to be veiled to further understand the role of RAGE/soluble RAGE axis in metabolic syndrome and insulin resistance.However, we believe our findings and this concept would open up a new research field which could further precede our understanding of the RAGE biology.Fig. 7. Metabolic syndrome may be an aspect of "metabolic memory" conceptualized as prolonged RAGE activation through subclinical information.

Fig. 4 .
Fig. 4. RAGE deficiency is associated with decreased body weight, epididymal fat weight and adipocyte size in apolipoprotein E (apoE)-deficient genetic background.(A) Comparisons of body weight between apoE -/-RAGE +/+ and apoE -/-RAGE -/-mice fed with standard or atherogenic diet.(B) Comparisons of epididymal fat weight between apoE -/- RAGE +/+ and apoE -/-RAGE -/-mice fed with standard or atherogenic diet.(C) Comparisons of adipocyte size in epididymal adipose tissues.Columns represent mean ± standard deviation.P values were analyzed by Student's t-test.Modified from ref 16 .

Fig. 6 .
Fig. 6.Plasma esRAGE levels are decreased in glucose intolerance, metabolic syndrome, obesity and insulin resistance (A) Left panel demonstrates the levels of plasma esRAGE in subjects with normal glucose tolerance (NGT) (n=118), impaired glucose tolerance (IGT) (n=16), and type 2 diabetes (DM) (n=203).Right panel compares the plasma esRAGE levels in subjects with (n-53) or without (n=282) metabolic syndrome (Met) as characterized by modified NCEP criteria.* p<0.05,ANOVA with multiple comparison (Scheffe's type).(B) Plasma esRAGE levels were inversely associated with body mass index or HOMA insulin resistance index.Logarithm-transformed HOMA index was used for the analyses because of the skewed distribution.Modified from ref 15 .

Table 1 .
Pathway analyses of the genes differentially expressed in WT vs. RAGE-/epididymal adipose tissue.

Table 1 .
Pathway analyses of the genes differentially expressed in WT vs. RAGE-/epididymal adipose tissue (continuation).
107orted that total sRAGE levels are significantly lower in patients with angiographically proven coronary artery disease (CAD) than in age-matched healthy controls.The association between circulating sRAGE and angiographic observations was shown to be dose-dependent, with individuals in the lowest quartile of sRAGE exhibiting the highest risk for CAD.Importantly, this cohort consisted of a non-diabetic population, suggesting that the potential significance of sRAGE is not confined to diabetes.Falcone et al also showed that the association between sRAGE and the risk of CAD was independent of other classical risk factors.Their findings are reproduced later by several research groups in larger numbers of subjects, and are also extended to other atherosclerotic diseases, such as carotid atherosclerosis, cerebral ischemia, and aortic valve stenosis (Table2).Patients with Alzheimer disease have also lower levels of sRAGE in plasma than patients with vascular dementia and controls, suggesting a role for the RAGE axis in this clinical entity as well107.

Table 2 .
Levels of circulating soluble RAGE in cardiovascular and metabolic diseases.