**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

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.

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 64-66. 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 68-70. MAC-1, an integrin expressed in macrophage, can act as a RAGE ligand 61, and may be involved in

**3.4 Endogenous RAGE ligands, insulin resistance and metabolic syndrome** 

**4. RAGE and its potential link with insulin resistance and metabolic** 

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

**3.3 AGEs and insulin resistance in vitro** 

adipogenesis through interaction with RAGE.

**4.1 Structure and function of RAGE** 

**syndrome** 

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.
