Immunological evidence that non-carboxymethyllysine advanced glycation end-products are produced from short chain sugars and dicarbonyl compounds in vivo

BACKGROUND: The Maillard reaction that leads to the formation of advanced glycation end-products (AGE) plays an important role in the pathogenesis of angiopathy in diabetic patients and in the aging process. Recently, it was proposed that AGE were not only created by glucose, but also by dicarbonyl compounds derived from the Maillard reaction, autoxidation of sugars and other metabolic pathways of glucose. In this study, we developed four types of non-carboxymethyllysine (CML) anti-AGE antibodies that recognized proteins modified by incubation with short chain sugars and dicarbonyl compounds. MATERIALS AND METHODS: AGE-modified serum albumins were prepared by incubation of rabbit serum albumin with glyceraldehyde, glycolaldehyde, methylglyoxal or glyoxal. After immunization of rabbits, four types of AGE-specific antisera were obtained that were specific for the AGE modification. To separate non-CML AGE antibodies (Ab) (non-CML AGE-Ab-2, -3, -4, and -5), these anti-AGE antisera were subjected to affinity chromatography on a matrix coupled with four kinds of AGE bovine serum albumin (BSA) or CML-BSA. These non-CML AGE antibodies were used to investigate the AGE content of serum obtained from diabetic patients on hemodialysis. RESULTS: Characterization of the four types of non-CML AGE antibodies obtained by immunoaffinity chromatography was performed by competitive ELISA and immunoblot analysis. Non-CML AGE-Ab-2 crossreacted with the protein modified by glyceraldehyde or glycolaldehyde. Non-CML AGE-Ab-3 and -Ab-4 specifically cross-reacted with protein modified by glycolaldehyde and methylglyoxal, respectively. NonCML AGE-Ab-5 cross-reacted with protein modified with glyoxal as well as methylglyoxal and glycolaldehyde. Three kinds of non-CML AGE (AGE-2, -4, and -5) were detected in diabetic serum as three peaks with apparent molecular weights of 200, 1.15, and 0.85 kD; whereas, AGE-3 was detected as two peaks with apparent molecular weights of 200 and 0.85 kD. CONCLUSION: We propose that various types of non-CML AGE are formed by the Maillard reaction, sugar autoxidation and sugar metabolism. These antibodies enable us to identify such compounds created by the Maillard reaction in vivo.     

Alternative routes for the formation of immunochemically distinct advanced glycation end-products in vivo

The advanced stage of the glycation process (also called the "Maillard reaction") that leads to the formation of advanced glycation end-products (AGEs) plays an important role in the pathogenesis of angiopathy in diabetic patients and in the aging process. AGEs elicit a wide range of cell-mediated responses that might contribute to diabetic complications, vascular disease, renal disease, and Alzheimer's disease. Recently, it has been proposed that AGE are not only created from glucose per se, but also from dicarbonyl compounds derived from glycation, sugar autoxidation, and sugar metabolism. However, this advanced stage of glycation is still only partially characterized and the structures of the different AGEs that are generated in vivo have not been completely determined. Because of their heterogeneity and the complexity of the chemical reactions involved, only some AGEs have been characterized in vivo, including N-carboxymethyllysine (CML), pentosidine, pyrraline, and crosslines. In this article, we provide a brief overview of the pathways of AGE formation and of the immunochemical methods for detection of AGEs, and we also provide direct immunological evidence for the existence of five distinct AGE classes (designated as AGE-1 to -5) within the AGE-modified proteins and peptides in the serum of diabetic patients on hemodialysis. We also propose pathways for the in vivo formation of various AGEs by glycation, sugar autoxidation, and sugar metabolism.     

Detection of noncarboxymethyllysine and carboxymethyllysine advanced glycation end products (AGE) in serum of diabetic patients.

BACKGROUND: The advanced stage of the Maillard reaction, which leads to the formation of advanced glycation end products (AGE), plays an important role in the pathogenesis of angiopathy in diabetic patients and in the aging process. N(epsilon)-(carboxymethyl)lysine (CML) is thought to be an important epitope for many of currently available AGE antibodies. However, recent findings have indicated that a major source of CML may be by pathways other than glycation. A distinction between CML and non-CML AGE may increase our understanding of AGE formation in vivo. In the present study, we prepared antibodies directed against CML and non-CML AGE. MATERIALS AND METHODS: AGE-rabbit serum albumin prepared by 4, 8, and 12 weeks of incubation with glucose was used to immunize rabbits, and a high-titer AGE-specific antiserum was obtained without affinity for the carrier protein. To separate CML and non-CML AGE antibodies, the anti-AGE antiserum was subjected to affinity chromatography on a column coupled with AGE-BSA and CML-BSA. Two different antibodies were obtained, one reacting specifically with CML and the other reacting with non-CML AGE. Circulating levels of CML and non-CML AGE were measured in 66 type 2 diabetic patients without uremia by means of the competitive ELISA. Size distribution and clearance by hemodialysis detected by non-CML AGE and CML were assessed in serum from diabetic patients on hemodialysis. RESULTS: The serum non-CML AGE level in type 2 diabetic patients was significantly correlated with the mean fasting blood glucose level over the previous 2 months (r = 0.498, p < 0.0001) or the previous 1 month (r = 0.446, p = 0. 0002) and with HbA(1c) (r = 0.375, p = 0.0019), but the CML AGE level was not correlated with these clinical parameters. The CML and non-CML AGE were detected as four peaks with apparent molecular weights of 200, 65, 1.15, and 0.85 kD. The hemodialysis treatment did not affect the high-molecular-weight protein fractions. Although the low-molecular-weight peptide fractions (absorbance at 280 nm and fluorescence) were decreased by hemodialysis, there was no difference before and after dialysis in the non-CML AGE- and CML-peptide fractions (1.15 and 0.85 kD fractions). CONCLUSIONS: We propose that both CML and non-CML AGE are present in the blood and that non-CML AGE rather than CML AGE should be more closely evaluated when investigating the pathophysiology of AGE-related diseases.     

3-Deoxyglucosone: metabolism,analysis, biological activity,and clinical implication

3-Deoxyglucosone (3-DG) is synthesized via the Maillard reaction and the polyol pathway, and is detoxified to 3-deoxyfructose and 2-keto-3-deoxygluconic acid. 3-DG rapidly reacts with protein amino groups to form advanced glycation end products (AGEs) such as imidazolone, pyrraline, N^ε-(carboxymethyl)1ysine and pentosidine, among which imidazolone is the AGE most specific for 3-DG. As demonstrated by using gas chromatography–mass spectrometry or high-performance liquid chromatography, plasma 3-DG levels are markedly increased in diabetes and uremia. Although the plasma 3-DG levels had been controversial, it was clearly demonstrated that its plasma level depends on the deproteinization method by which either free or total 3-DG, presumably bound to proteins, is measured. In diabetes, hyperglycemia enhances the synthesis of 3-DG via the Maillard reaction and the polyol pathway, and thereby leads to its high plasma and erythrocyte levels. In uremia, however, the decreased catabolism of 3-DG, which may be due to the loss of 3-DG reductase activity in the end-stage kidneys, may lead to high plasma 3-DG level. The elevated 3-DG levels in plasma and erythrocytes may promote the formation of AGEs such as imidazolone, as demonstrated by immunohistochemistry and immunochemistry using an anti-imidazolone antibody. Although AGE-modified proteins prepared in vitro exhibit a variety of biological activities, known AGE structures have not yet been demonstrated to show any biological activities. Because 3-DG is potent in the formation of AGEs and has some biological activities, such as cellular toxicity, it may be more important in the development of diabetic and uremic complications than the known AGE structures. By demonstrating that treatment with an aldose reductase inhibitor reduces the erythrocyte levels of 3-DG and AGEs, such as imidazolone, light is shed on the mystery of how aldose reductase inhibitors may prove beneficial in diabetic complications. These evidences suggest that 3-DG plays a principal role in the development of diabetic and uremic complications.     

Antibody-based detection of advanced glycation end-products: promises <Emphasis Type="Italic">vs</Emphasis>. limitations

Advanced glycation end-products (AGEs) of the Maillard reaction were originally measured according to their fluorescent and browning properties. A subsequent study with instrumental analyses such as high-performance liquid chromatography and gas chromatography mass spectrometry more clearly demonstrated the involvement of each AGE structure in pathological conditions. Furthermore, immunochemical methods have also been developed to clarify the localization of AGEs in tissues and measurement of AGEs in multiple clinical samples. Although the involvement of AGEs in age-related diseases has progressed due to immunochemical techniques, the relationship between AGE structure and diseases has not been clear because little was known about the epitope structure of each anti-AGE antibody. However, the development of epitope-identified antibodies against AGEs has made it possible to clarify AGE structures involved in diseases. This review discusses not only the usability of anti-AGE antibodies to evaluate AGEs and disease pathology and screen AGE inhibitors, but also describes their usage.     

Advanced glycation end-products and the progress of diabetic vascular complications

Epidemiological studies have confirmed that hyperglycemia is the most important factor in the onset and progress of vascular complications, both in Type 1 and 2 diabetes mellitus. The formation of advanced glycation end-products (AGEs) correlates with glycemic control. The AGE hypothesis proposes that accelerated chemical modification of proteins by glucose during hyperglycemia contributes to the pathogenesis of diabetic complications including nephropathy, retinopathy, neuropathy and atherosclerosis. Recent studies have shown that increased formation of serum AGEs exists in diabetic children and adolescents with or without vascular complications. Furthermore, the presence of diabetic complications in children correlates with elevated serum AGEs. The level of serum AGEs could be considered as a marker of later developments of vascular complications in children with Type 1 and 2 diabetes mellitus. The careful metabolic monitoring of young diabetics together with monitoring of serum AGEs can provide useful information about impending AGE-related diabetic complications. It is becoming clear that anti-AGE strategies may play an important role in the treatment of young and older diabetic patients. Several potential drug candidates such as AGE inhibitors have been reported recently.     

Structure of a synthetic glucose derived advanced glycation end product that is immunologically cross-reactive with its naturally occurring counterparts

Glucose reacts nonenzymatically with the amino groups of proteins to form stable, cross-linking adducts called advanced glycation end products or AGEs. While several lines of evidence have established that AGEs accumulate in tissues and contribute to the pathological sequelae of diabetes and aging, the identity of the major cross-link(s) that forms in vivo has remained enigmatic. This has been considered to be due to the labile nature and to the low fluorescence properties of this cross-link, despite the fact that fluorescence has been generally associated with AGE formation in vivo. Accordingly, the few AGE adducts that have been isolated thus far from proteins in vivo or from model systems in vitro have been found to account for only a fraction of the glucose-derived cross-links that occur in tissues. This situation has been further underscored by the development of a well-characterized class of antibodies that recognize in vivo AGEs but which fail to react with the structurally defined AGEs that have been identified to date. This particular class of anti-AGE antibodies has proven valuable in the quantification of AGE-modified forms of hemoglobin, low-density lipoprotein (LDL), and beta-amyloid peptide, and can provide prognostic information on the course of certain diabetic complications. To obtain insight into the structure of this immunoreactive, AGE adduct, we used an anti-AGE antibody (RU) as a probe to isolate novel AGE(s) that formed within a reaction mixture of glucose and the model glycation substrate, N(alpha)-CBZ-Arg-Lys. HPLC purification of an immunoreactive fraction that accumulated in this preparation showed the presence of a compound that was determined by (1)H NMR and electrospray ionization mass spectrometry (ESI-MS) to be a stable, imidazole-based cross-link (termed arginine-lysine imidazole or ALI). The properties of ALI, immunoreactivity, acid-lability, nonfluorescence, and inhibition of formation by aminoguanidine, suggest that ALI is likely to typify an important class of the AGE cross-links that form in vivo.     

Glycation of H1 Histone by 3-Deoxyglucosone: Effects on Protein Structure and Generation of Different Advanced Glycation End Products

Advanced glycation end products (AGEs) culminate from the non-enzymatic reaction between a free carbonyl group of a reducing sugar and free amino group of proteins. 3-deoxyglucosone (3-DG) is one of the dicarbonyl species that rapidly forms several protein-AGE complexes that are believed to be involved in the pathogenesis of several diseases, particularly diabetic complications. In this study, the generation of AGEs (N^ε-carboxymethyl lysine and pentosidine) by 3-DG in H1 histone protein was characterized by evaluating extent of side chain modification (lysine and arginine) and formation of Amadori products as well as carbonyl contents using several physicochemical techniques. Results strongly suggested that 3-DG is a potent glycating agent that forms various intermediates and AGEs during glycation reactions and affects the secondary structure of the H1 protein. Structural changes and AGE formation may influence the function of H1 histone and compromise chromatin structures in cases of secondary diabetic complications.     

Immunochemical detection of advanced glycosylation end products in vivo.

Reducing sugars react with protein amino groups to form a diverse group of protein-bound moieties with fluorescent and cross-linking properties. These compounds, called advanced glycosylation end products (AGEs), have been implicated in the structural and functional alterations of proteins that occur during aging and long-term diabetes. Although several AGEs have been identified on the basis of de novo synthesis and tissue isolation procedures, the measurement of AGE compounds in vivo has remained difficult. As an approach to the study of AGE formation in vivo, we prepared polyclonal antiserum to an AGE epitope(s) which forms in vitro after incubation of glucose with ribonuclease (RNase). This antiserum proved suitable for the detection of AGEs which form in vivo. Both diabetic tissue and serum known to contain elevated levels of AGEs readily competed for antibody binding. Cross-reactivity studies revealed the presence of a common AGE epitope(s) which forms after the incubation of diverse proteins with glucose. Cross-reactive epitopes also formed with glucose 6-phosphate or fructose. These data suggest that tissue AGEs which form in vivo appear to contain a common immunological epitope which cross-reacts with AGEs prepared in vitro, supporting the concept that immunologically similar AGE structures form from the incubation of sugars with different proteins (Horiuchi, S., Araki, N., and Morino, Y. (1991) J. Biol. Chem. 266, 7329-7332). None of the known AGEs, such as 4-furanyl-2-furoyl-1H-imidazole, 1-alkyl-2-formyl-3,4-diglycosylpyrrole, pyrraline, carboxymethyllysine, or pentosidine, were found to compete for binding to anti-AGE antibody. These data further suggest that the dominant AGE epitope which forms from the reaction of glucose with proteins under native conditions is immunologically distinct from the structurally defined AGEs described to date.     

Immunochemical approach to characterize advanced glycation end products of the Maillard reaction. Evidence for the presence of a common structure

Reaction of protein amino groups with glucose (the Maillard reaction) leads from early stage products such as Schiff base and Amadori products to advanced glycation end products (AGE), structures implicated in diabetic complications and the aging process. We have prepared the polyclonal anti-AGE antibody and the monoclonal anti-AGE antibody against AGE-bovine serum albumin and made an immunochemical approach to characterize AGE structures. Both polyclonal and monoclonal antibodies reacted with AGE-proteins such as AGE-bovine serum albumin, AGE-human serum albumin, and AGE-hemoglobin but not with unmodified counterparts. Treatments of these AGE-proteins with borohydride had no effect on the immunoreactivity. Moreover, fructosyl-epsilon-caproic acid, a synthetic Amadori compound, did not serve as an antigen, indicating that these antibodies were specific for AGE products but not for early stage products of the Maillard reaction. In addition, these antibodies were also able to recognize AGE products prepared either from alpha-tosyl-1-lysine, alpha-tosyl-1-lysine methyl ester, monoaminocarboxylic acid such as epsilon-aminocaproic acid, gamma-amino-n-butyric acid, and beta-alanine. Thus, these results strongly suggest the presence of a common structure in AGE preparations, regardless of whether AGE products are generated from proteins, amino acids, or monoaminocarboxylic acids.     

Advanced glycation end products and diabetic retinopathy

Studies have established hyperglycemia as the most important factor in the progress of vascular complications. Formation of advanced glycation end products (AGEs) correlates with glycemic control. The AGE hypothesis proposes that hyperglycemia contributes to the pathogenesis of diabetic complications including retinopathy. However, their role in diabetic retinopathy remains largely unknown. This review discusses the chemistry of AGEs formation and their patho-biochemistry particularly in relation to diabetic retinopathy. AGEs exert deleterious effects by acting directly to induce cross-linking of long-lived proteins to promote vascular stiffness, altering vascular structure and function and interacting with receptor for AGE, to induce intracellular signaling leading to enhanced oxidative stress and elaboration of key proinflammatory and prosclerotic cytokines. Novel anti-AGE strategies are being developed hoping that in next few years, some of these promising therapies will be successfully evaluated in clinical context aiming to reduce the major economical and medical burden caused by diabetic retinopathy.     

The pathogenic role of Maillard reaction in the aging eye

The proteins of the human eye are highly susceptible to the formation of advanced glycation end products (AGEs) from the reaction of sugars and carbonyl compounds. AGEs progressively accumulate in the aging lens and retina and accumulate at a higher rate in diseases that adversely affect vision such as, cataract, diabetic retinopathy and age-related macular degeneration. In the lens AGEs induce irreversible changes in structural proteins, which lead to lens protein aggregation and formation of high-molecular-weight aggregates that scatter light and impede vision. In the retina AGEs modify intra- and extracellular proteins that lead to an increase in oxidative stress and formation of pro-inflammatory cytokines, which promote vascular dysfunction. This review outlines recent advances in AGE research focusing on the mechanisms of their formation and their role in cataract and pathologies of the retina. The therapeutic action and pharmacological strategies of anti-AGE agents that can inhibit or prevent AGE formation in the eye are also discussed.     

Advanced glycation endproducts promote adhesion molecule (VCAM-1, ICAM-1) expression and atheroma formation in normal rabbits.

BACKGROUND: Reactive glucose-protein intermediates and advanced glycation endproducts (AGEs) are shown to colocalize with atheromatous lesions and to trigger complex chemical and biological responses through interaction with vessel wall elements. In diabetes and renal insufficiency, atherosclerosis is common, as are elevated levels of serum and vascular tissue AGEs. In the present study, AGEs supplied exogenously to normal animals elicited vascular and renal pathology. MATERIALS AND METHODS: Nondiabetic rabbits were injected intravenously with low doses of AGE-modified rabbit serum albumin (AGE-RSA, 16 mg/kg/day) for 4 months alone, or combined with a brief terminal period (2 weeks) of a cholesterol-rich diet (CRD) (2% cholesterol, 10% corn oil). AGE-RSA associated expression of vascular cell adhesion molecules and the development of atheromatous changes within the aorta were determined by immunohistology. RESULTS: The AGE content of aortic tissue increased by 2.2-fold in AGE-treated and by 3.2-fold in AGE + CRD-treated rabbits compared with normal saline-treated control rabbits (p < 0.025 and 0.001, respectively). Serum AGE levels in AGE groups rose up to 3-fold above the controls (p < 0.025 and p < 0.01). Ascending aortic sections from AGE-treated rabbits showed significant focal intimal proliferation, enhanced endothelial cell adhesion with infrequent intimal macrophages. oil-red-O staining lipid deposits and positive focal expression of vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1), a pattern not observed in controls. These AGE-induced changes were markedly enhanced in animals cotreated with AGEs and a brief period of CRD. Lesions consisted of multifocal atheromas, containing foam cells, massive lipid droplets, and strong endothelial expression of VCAM-1 and ICAM-1 restricted to the affected areas. CONCLUSIONS: This study provides in vivo evidence for a causal relationship between chronic AGE accumulation and atherosclerosis independent of diabetic hyperglycemia, and suggests the utility of this animal model for the study of diabetic vascular disease in relation to glycation.     

Increase in three alpha,beta-dicarbonyl compound levels in human uremic plasma: specific in vivo determination of intermediates in advanced Maillard reaction

Methylglyoxal (MGO), glypxal (GO) and 3-deoxyglucosone (3-DG) are reactive alpha,beta-dicarbonyl intermediates in advanced Maillard reaction, which form advanced glycation and oxidation end products (AGEs) by reaction with both lysine and arginine residues in protein. We measured these three dicarbonyl compound levels in human plasma to estimate the relationship between accumulation of alpha, beta-dicarbonyl compounds and AGE formation reactions in uremia and diabetes in human plasma by a highly selective and specific assay, electrospray ionization liquid chromatography mass spectrometry (ESI/LC/MS). We show that 3-DG and MGO levels are significantly higher in uremia and diabetes compared with age-matched healthy controls. Only the GO level in uremic plasma is significantly higher compared to diabetes and healthy controls. In both diabetic and uremic patients, these dicarbonyl compounds promote AGE accumulation in vivo, and especially in uremic patients, increased accumulation of GO could result from accelerating oxidative stress.     

GRP78 protects CHO cells from ribosylation

d-Ribose (Rib), a reactive glycation compound that exists in organisms, abnormally increases in the urine of diabetic patients and can yield large amounts of advanced glycation end products (AGEs), leading to cell dysfunction. However, whether cellular proteins are sensitive to this type of glycation is unknown. In this study, we found that cellular AGEs accumulate in Chinese hamster ovary (CHO) cells with increased Rib concentration and administration time. Mass spectrum analysis of isolated AGE-modified proteins from cell lysates showed that glucose-regulated protein 78?kD (GRP78) is one of the main ribosylated proteins. Co-immunoprecipitation assays further confirmed the interaction between AGEs and GRP78. Compared with d-glucose (Glc), Rib produced much more AGEs in cells. In kinetic studies, the first order rate constant of LDH released from CHO cells incubated with Rib was nearly 8-fold higher than that of Glc, suggesting that Rib is highly cytotoxic. Immunofluorescent co-localization analysis manifested partial superimposition of AGEs and GRP78, which were distributed throughout the endoplasmic reticulum. Western blotting showed that the expression of GRP78 is up-regulated and then down-regulated in CHO cells during Rib treatment. In the presence of Rib, the suppression of GRP78 expression either with transfected siRNA or with the inhibitor (-)-epigallocatechin gallate (EGCG) dramatically increased AGE levels and decreased cell viability compared with these parameters in the control groups. GRP78 overexpression decreased AGE levels and rescued the cells from Rib-induced cytotoxicity. These data indicate that GRP78 plays a role in preventing Rib-induced CHO cell cytotoxicity.     

Nepsilon-(Carboxymethyl)lysine and 3-DG-imidazolone are major AGE structures in protein modification by 3-deoxyglucosone

The levels of plasma 3-deoxyglucosone (3-DG) increase under hyperglycemic conditions and are associated with the pathogenesis of diabetic complications because of the high reactivity of 3-DG with proteins to form advanced glycation end products (AGE). To investigate potential markers for 3-DG-mediated protein modification in vitro and in vivo, we compared the yield of several 3-DG-derived AGE structures by immunochemical analysis and HPLC and measured their localization in human atherosclerotic lesions. When BSA was incubated with 3-DG at 37 degrees C for up to 4 wk, the amounts of N(epsilon)-(carboxymethyl)lysine (CML) and 3-DG-imidazolone steeply increased with incubation time, whereas the levels of pyrraline and pentosidine increased slightly by day 28. In contrast, significant amounts of pyrraline and pentosidine were also observed when BSA was incubated with 3-DG at 60 degrees C to enhance AGE-formation. In atherosclerotic lesions, CML and 3-DG-imidazolone were found intracellularly in the cytoplasm of most foam cells and extracellularly in the atheromatous core. A weak-positive immunoreaction with pyrraline was found in the extracellular matrix and a few foam cells in aortic intima with atherosclerotic lesions. Our results provide the first evidence that CML and 3-DG-imidazolone are major AGE structures in 3-DG-modified proteins, and that 3-DG-imidazolone provides a better marker for protein modification by 3-DG than pyrraline.     

D-ribose induces cellular protein glycation and impairs mouse spatial cognition

Background

D-Ribose, an important reducing monosaccharide, is highly active in the glycation of proteins, and results in the rapid production of advanced glycation end products (AGEs) in vitro. However, whether D-ribose participates in glycation and leads to production of AGEs in vivo still requires investigation.

Methodology/Principal Findings

Here we treated cultured cells and mice with D-ribose and D-glucose to compare ribosylation and glucosylation for production of AGEs. Treatment with D-ribose decreased cell viability and induced more AGE accumulation in cells. C57BL/6J mice intraperitoneally injected with D-ribose for 30 days showed high blood levels of glycated proteins and AGEs. Administration of high doses D-ribose also accelerated AGE formation in the mouse brain and induced impairment of spatial learning and memory ability according to the performance in Morris water maze test.

Conclusions/Significance

These data demonstrate that D-ribose but not D-glucose reacts rapidly with proteins and produces significant amounts of AGEs in both cultured cells and the mouse brain, leading to accumulation of AGEs which may impair mouse spatial cognition.     

Mechanistic targeting of advanced glycation end-products in age-related diseases

Glycative stress, caused by the accumulation of cytotoxic and irreversibly-formed sugar-derived advanced glycation end-products (AGEs), contributes to morbidity associated with aging, age-related diseases, and metabolic diseases. In this review, we summarize pathways leading to formation of AGEs, largely from sugars and glycolytic intermediates, and discuss detoxification of AGE precursors, including the glyoxalase system and DJ-1/Park7 deglycase. Disease pathogenesis downstream of AGE accumulation can be cell autonomous due to aggregation of glycated proteins and impaired protein function, which occurs in ocular cataracts. Extracellular AGEs also activate RAGE signaling, leading to oxidative stress, inflammation, and leukostasis in diabetic complications such as diabetic retinopathy. Pharmaceutical agents have been tested in animal models and clinically to diminish glycative burden. We summarize existing strategies and point out several new directions to diminish glycative stress including: plant-derived polyphenols as AGE inhibitors and glyoxalase inducers; improved dietary patterns, particularly Mediterranean and low glycemic diets; and enhancing proteolytic capacities of the ubiquitin-proteasome and autophagy pathways that are involved in cellular clearing of AGEs.     

CD36-mediated endocytic uptake of advanced glycation end products (AGE) in mouse 3T3-L1 and human subcutaneous adipocytes

Interaction of advanced glycation end products (AGE) with AGE receptors induces several cellular phenomena potentially relating to diabetic complications. We here show that AGE-modified bovine serum albumin (BSA) is endocytosed by adipocytes via CD36. Upon differentiation, 3T3-L1 and human subcutaneous adipose cells showed marked increases in endocytic uptake and subsequent degradation of [(125)I]AGE-BSA, which were inhibited effectively by the anti-CD36 antibody. Ligand specificity of CD36 for modified BSAs was compared with that of LOX-1 and scavenger receptor class A. Effect of fucoidan on [(125)I]AGE-BSA binding showed a sharp contrast to that on [(125)I]-oxidized low density lipoprotein. These results implicate that CD36-mediated interaction of AGE-modified proteins with adipocytes might play a pathological role in obesity or insulin-resistance.