Ex vivo detection of histone H1 modified with advanced glycation end products

A number of oxidative stress agents cause DNA and protein damage, which may compromise genomic integrity. Whereas oxidant-induced DNA damage has been extensively studied, much less is known concerning the occurrence and fate of nuclear protein damage, particularly of proteins involved in the regulation and maintenance of chromatin structure. Protein damage may be caused by the formation of reactive carbonyl species such as glyoxal, which forms after lipid peroxide degradation. It may also result from degradation of early protein glycation adducts and from methylglyoxal, formed in the process of glycolytic intermediate degradation. Major adducts indicative of protein damage include the advanced glycation end product (AGE) carboxymethyllysine (CML) and argpyrimidine protein adducts. Thus, the formation of CML and argpyrimidine protein adducts represents potential biomarkers for nuclear protein damage deriving from a variety of sources. The purpose of this study was to identify and quantify AGE adducts formed in vivo in a nuclear protein, specifically histone H1, using CML and argpyrimidine as biomarkers. Histone H1 was isolated from calf thymus collected immediately after slaughter under conditions designed to minimize AGE formation before isolation. Using antibodies directed against oxidative protein adducts, we identified CML, argpyrimidine, and protein crosslinks present in the freshly isolated histone H1. Detailed mass spectroscopy analysis of histone H1 revealed the presence of two specific lysine residues modified by CML adducts. Our results strongly suggest that glycation of important nuclear protein targets such as histone H1 occurs in vivo and that these oxidative changes may alter chromatin structure, ultimately contributing to chronic changes associated with aging and diseases such as diabetes.     

Increased glyoxalase I levels inhibit accumulation of oxidative stress and an advanced glycation end product in mouse mesangial cells cultured in high glucose

Chronic high glucose levels lead to the formation of advanced glycation end-products (AGEs) as well as AGE precursors, such as methylglyoxal (MG) and glyoxal, via non-enzymatic glycation reactions in patients with diabetic mellitus. Glyoxalase 1 (GLO-1) detoxifies reactive dicarbonyls that form AGEs. To investigate the interaction between AGEs and GLO-1 in mesangial cells (MCs) under diabetic conditions, AGE levels and markers of oxidative stress were measured in GLO-1-overexpressing MCs (GLO-1-MCs) cultured in high glucose. Furthermore, we also examined levels of high glucose-induced apoptosis in GLO-1-MCs. In glomerular MCs, high glucose levels increased the formation of both MG and argpyrimidine (an MG-derived adduct) as well as GLO-1 expression. GLO-1-MCs had lower intracellular levels of MG accumulation, 8-hydroxy-deoxyguanosine (an oxidative DNA damage marker), 4-hydroxyl-2-nonenal (a lipid peroxidation product), and nitrosylated protein (a marker of oxidative-nitrosative stress) compared to control cells. Expression of mitochondrial oxidative phosphorylation complexes I, II, and III was also decreased in GLO-1-MCs. Furthermore, fewer GLO-1-MCs showed evidence of apoptosis as determined by terminal deoxynucleotidyl transferase-mediated dUTP nick labeling assay, and activation of both poly (ADP-ribose) polymerase 1 cleavage and caspase-3 was lower in GLO-1-MCs than in control cells cultured in high glucose. These results suggest that GLO-1 plays a role in high glucose-mediated signaling by reducing MG accumulation and oxidative stress in diabetes mellitus.     

Inhibition of advanced glycation end product formation on collagen by rutin and its metabolites

Several lines of evidence suggest that rutin, flavonoid in fruits and vegetables, or one of its metabolites may effectively modulate advanced glycation end product (AGE) formation. Following ingestion, rutin forms metabolites that include 3,4-dihydroxyphenylacetic acid (3,4-DHPAA), 3,4-dihydroxytoluene (3,4-DHT), m-hydroxyphenylacetic acid (m-HPAA), 3-methoxy-4-hydroxyphenylacetic acid (homovanillic acid, HVA) and 3,5,7,3',5'-pentahydroxyflavonol (quercetin). We studied the effects of rutin and its metabolites on the formation of AGE biomarkers such as pentosidine, collagen-linked fluorescence, N(epsilon)-carboxymethyllysine (CML) adducts, glucose autoxidation and collagen glycation, using an in vitro model where collagen I was incubated with glucose. Rutin metabolites containing vicinyl dihydroxyl groups, i.e., 3,4-DHT, 3,4-DHPAA and quercetin, inhibited the formation of pentosidine and fluorescent adducts, glucose autoxidation and glycation of collagen I in a dose-dependent manner, whereas non-vicinyl dihydroxyl group-containing metabolites, i.e., HVA and m-HPAA, were much less effective. All five metabolites of rutin effectively inhibited CML formation. In contrast, during the initial stages of glycation and fluorescent AGE product accumulation, only vicinyl hydroxyl group-containing rutin metabolites were effective. These studies demonstrate that rutin and circulating metabolites of rutin can inhibit early glycation product formation, including both fluorescent and nonfluorescent AGEs induced by glucose glycation of collagen I in vitro. These effects likely contribute to the beneficial health effects associated with rutin consumption.     

Protein glycation in Saccharomyces cerevisiae. Argpyrimidine formation and methylglyoxal catabolism

Methylglyoxal is the most important intracellular glycation agent, formed nonenzymatically from triose phosphates during glycolysis in eukaryotic cells. Methylglyoxal-derived advanced glycation end-products are involved in neurodegenerative disorders (Alzheimer's, Parkinson's and familial amyloidotic polyneurophathy) and in the clinical complications of diabetes. Research models for investigating protein glycation and its relationship to methylglyoxal metabolism are required to understand this process, its implications in cell biochemistry and their role in human diseases. We investigated methylglyoxal metabolism and protein glycation in Saccharomyces cerevisiae. Using a specific antibody against argpyrimidine, a marker of protein glycation by methylglyoxal, we found that yeast cells growing on d-glucose (100 mM) present several glycated proteins at the stationary phase of growth. Intracellular methylglyoxal concentration, determined by a specific HPLC based assay, is directly related to argpyrimidine formation. Moreover, exposing nongrowing yeast cells to a higher d-glucose concentration (250 mM) increases methylglyoxal formation rate and argpyrimidine modified proteins appear within 1 h. A kinetic model of methylglyoxal metabolism in yeast, comprising its nonenzymatic formation and enzymatic catabolism by the glutathione dependent glyoxalase pathway and aldose reductase, was used to probe the role of each system parameter on methylglyoxal steady-state concentration. Sensitivity analysis of methylglyoxal metabolism and studies with gene deletion mutant yeast strains showed that the glyoxalase pathway and aldose reductase are equally important for preventing protein glycation in Saccharomyces cerevisiae.     

The combined effect of acetylation and glycation on the chaperone and anti-apoptotic functions of human α-crystallin

N^ε-acetylation occurs on select lysine residues in α-crystallin of the human lens and alters its chaperone function. In this study, we investigated the effect of N^ε-acetylation on advanced glycation end product (AGE) formation and consequences of the combined N^ε-acetylation and AGE formation on the function of α-crystallin. Immunoprecipitation experiments revealed that N^ε-acetylation of lysine residues and AGE formation co-occurs in both αA- and αB-crystallin of the human lens. Prior acetylation of αA- and αB-crystallin with acetic anhydride (Ac₂O) before glycation with methylglyoxal (MGO) resulted in significant inhibition of the synthesis of two AGEs, hydroimidazolone (HI) and argpyrimidine. Similarly, synthesis of ascorbate-derived AGEs, pentosidine and N^ε-carboxymethyl lysine (CML), was inhibited in both proteins by prior acetylation. In all cases, inhibition of AGE synthesis was positively related to the degree of acetylation. While prior acetylation further increased the chaperone activity of MGO-glycated αA-crystallin, it inhibited the loss of chaperone activity by ascorbate-glycation in both proteins. BioPORTER-mediated transfer of αA- and αB-crystallin into CHO cells resulted in significant protection against hyperthermia-induced apoptosis. This effect was enhanced in acetylated and MGO-modified αA- and αB-crystallin. Caspase-3 activity was reduced in α-crystallin transferred cells. Glycation of acetylated proteins with either MGO or ascorbate produced no significant change in the anti-apoptotic function. Collectively, these data demonstrate that lysine acetylation and AGE formation can occur concurrently in α-crystallin of human lens, and that lysine acetylation improves anti-apoptotic function of α-crystallin and prevents ascorbate-mediated loss of chaperone function.     

Antiglycation effect of gliclazide on in vitro AGE formation from glucose and methylglyoxal

Gliclazide, a sulfonylurea widely used for treatment of diabetes mellitus, is known to scavenge reactive oxygen species. To clarify whether its antioxidative ability interferes with the glycation processes, we incubated bovine serum albumin (BSA) with 1 M glucose or 1 mM methylglyoxal, in the presence or absence of gliclazide, and observed the formation of advanced glycation end products (AGEs). AGE production was assessed by AGE-specific fluorescence, an enzyme-linked immunosorbent assay (ELISA), and Western blotting. The fluorescence at excitation/emission wavelengths of 320/383 nm and 335/385 nm was definitely increased by incubating BSA with 1 M glucose or 1 mM methylglyoxal, and 1 mM gliclazide significantly blunted the fluorescent augmentation, in both wavelengths, in a dose-dependent fashion. Gliclazide almost equaled to aminoguanidine, a putative antiglycation agent, in the inhibitory effect on the glucose-induced fluorescence, while the methylglyoxal-derived fluorescent formation was less suppressed by gliclazide than by aminoguanidine. The AGE concentrations determined by ELISA showed similar results. Incubation of BSA with 1 M glucose or 1 mM methylglyoxal yielded an apparent increase in carboxymethyllysine or argpyrimidine. Both AGEs were significantly lowered by 1 mM gliclazide and a reduction of glucose-derived carboxymethyllysine was comparable to that caused by aminoguanidine. The results of Western blotting supported the findings in ELISA. To our knowledge, the present study provides the first evidence of the antiglycation effect of gliclazide on in vitro AGE formation from glucose and methylglyoxal.     

DNA damage by carbonyl stress in human skin cells

Reactive carbonyl species (RCS) are potent mediators of cellular carbonyl stress originating from endogenous chemical processes such as lipid peroxidation and glycation. Skin deterioration as observed in photoaging and diabetes has been linked to accumulative protein damage from glycation, but the effects of carbonyl stress on skin cell genomic integrity are ill defined. In this study, the genotoxic effects of acute carbonyl stress on HaCaT keratinocytes and CF3 fibroblasts were assessed. Administration of the alpha-dicarbonyl compounds glyoxal and methylglyoxal as physiologically relevant RCS inhibited skin cell proliferation, led to intra-cellular protein glycation as evidenced by the accumulation of N(epsilon)-(carboxymethyl)-L-lysine (CML) in histones, and caused extensive DNA strand cleavage as assessed by the comet assay. These effects were prevented by treatment with the carbonyl scavenger D-penicillamine. Both glyoxal and methylglyoxal damaged DNA in intact cells. Glyoxal caused DNA strand breaks while methylglyoxal produced extensive DNA-protein cross-linking as evidenced by pronounced nuclear condensation and total suppression of comet formation. Glycation by glyoxal and methylglyoxal resulted in histone cross-linking in vitro and induced oxygen-dependent cleavage of plasmid DNA, which was partly suppressed by the hydroxyl scavenger mannitol. We suggest that a chemical mechanism of cellular DNA damage by carbonyl stress occurs in which histone glycoxidation is followed by reactive oxygen induced DNA stand breaks. The genotoxic potential of RCS in cultured skin cells and its suppression by a carbonyl scavenger as described in this study have implications for skin damage and carcinogenesis and its prevention by agents selective for carbonyl stress.     

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.     

Nuclear proteasome activation and degradation of carboxymethylated histones in human keratinocytes following glyoxal treatment

Nuclear DNA damage has been studied in detail, but much less is known concerning the occurrence and fate of nuclear protein damage. Glycoxidation, protein damage that results from a combination of protein glycation and oxidation, leads to the formation of protein-advanced glycation end products (AGE) of which N(epsilon)-carboxymethyllysine (CML) is a major AGE. We have used glyoxal, a product of environmental exposures that readily leads to the formation of CML, to study nuclear protein glycoxidation in HaCaT human keratinocytes. Glyoxal treatment that did not affect cell viability but inhibited cell proliferation in a dose-dependent manner that led to accumulation of CML-modified histones. Modified histones were slowly degraded but persisted for more than 3 days following treatment. Preincubation of cells with a proteasome inhibitor following glyoxal treatment led to an increase in CML-modified histones. While glyoxal treatment resulted in a slight decrease in total cellular proteasome activity, a dose dependent increase of up to 4-fold in nuclear proteasome activity was observed. The increase in nuclear proteasome activity was due to both increased nuclear proteasome protein content and increased activity, neither of which were affected by cyclohexamide. The increase also was unaffected by inhibitors of poly(ADP-ribose) polymerases, which have been previously implicated in nuclear proteasome activation by oxidizing agents. Accumulation of CML-modified histones over time may lead to epigenetic changes that contribute to various pathologies including aging and cancer, and upregulation of nuclear proteasome activity under conditions of glyoxidative stress may function to limit such damage.     

Heat-shock protein 27 is a major methylglyoxal-modified protein in endothelial cells

In endothelial cells cultured under high glucose conditions, methylglyoxal is the major intracellular precursor in the formation of advanced glycation endproducts. We found that endothelial cells incubated with 30 mM d-glucose produced approximately 2-fold higher levels of methylglyoxal but not 3-deoxyglucosone and glyoxal, as compared to 5 mM d-glucose. Under hyperglycaemic conditions, the methylglyoxal-arginine adduct argpyrimidine as detected with a specific antibody, but not N(e)-(carboxymethyl)lysine and N(e)-(carboxyethyl)lysine, was significantly elevated. The glyoxylase I inhibitor HCCG and the PPARgamma ligand troglitazone also increased argpyrimidine levels. Increased levels of argpyrimidine by glucose, HCCG and troglitazone are accompanied by a decrease in proliferation of endothelial cells. A 27 kDa protein was detected as a major argpyrimidine-modified protein. With in-gel digestion and mass spectrometric analysis, we identified this major protein as heat-shock protein 27 (Hsp27). This argpyrimidine modification of Hsp27 may contribute to changes in endothelial cell function associated to diabetes.     

Semicarbazide-sensitive amine oxidase in aortic smooth muscle cells mediates synthesis of a methylglyoxal-AGE: implications for vascular complications in diabetes

Semicarbazide-sensitive amine oxidase (SSAO) catalyzes formation of methylglyoxal (MG) from aminoacetone; MG then reacts with proteins to form advanced glycation end products or AGEs. Because of its potential to generate MG, SSAO may contribute to AGE-associated vascular complications of aging and diabetes. We developed a method to measure SSAO activity in bovine aortic smooth muscle cells (BASMC) based on the oxidation of 2',7'-dichlorofluorescin by hydrogen peroxide and horseradish peroxidase. The SSAO activity was completely inhibited by 10 mM semicarbazide. Argpyrimidine is a readily detectable fluorescent product of the reaction between MG and arginine. Cell lysates incubated with aminoacetone formed argpyrimidine in a reaction that was inhibited by 20 mM semicarbazide. Immunostaining of tissue sections showed that aminoacetone-treated rats (normal as well as diabetic) formed more argpyrimidine in aortic smooth muscle than untreated controls. We believe that SSAO can enhance AGE synthesis in the macrovasculature of diabetic individuals by production of MG.     

Inhibition of protein glycation by skins and seeds of the muscadine grape

The formation of advanced glycation end products (AGEs) leading to protein glycation and cross-linking is associated with the pathogenesis of diabetic complications. The inhibition of protein glycation by phenolic and flavonoid antioxidants demonstrates that the process is mediated, in part, by oxidative processes. In this study, the effects of seed and skin extracts of the muscadine grape on AGEs formation were examined. Seeds and skins were extracted (10% w/v) with 50% ethanol and incubated at 37 degrees C with a solution containing 250 mM fructose and 10 mg/ml albumin. After 72 h, fluorescence was measured at the wavelength pair of 370 and 440 nm as an index of the formation of AGEs. Both seed and skin extracts were found to be efficacious inhibitors of AGE formation. A 1:300 dilution of the seed extract decreased fluorescence by approximately 65%, whereas muscadine grape skin extract produced a 40% lowering. This difference correlates with the greater antioxidant activity found in muscadine seeds in comparison to skins, however, on a mass basis, the inhibitory activities of the seeds and skins were found to be nearly equivalent. Gallic acid, catechin and epicatechin, the three major polyphenols in the seeds, all significantly decreased the AGE product related fluorescence at a concentration of 50 microM. Neither muscadine seed extract nor skin extract inhibited the methylglyoxal-mediated glycation of albumin. These results suggest that consumption of the muscadine grape may have some benefit in altering the progression of diabetic complications.     

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.     

Resistance of L132 lung cell clusters to glyoxal-induced apoptosis

Glyoxal is a highly reactive glycating agent involved in the formation of advanced glycation end products (AGEs) and known to induce apoptosis. AGE-mediated apoptosis may be an important mechanism of alveolar epithelial remodelling in pulmonary fibrosis. In this study, we investigated the cytotoxic effect of glyoxal on the fetal human epithelial lung cell line L132 under serum-free conditions. This type of culture, which forces the cells to grow as spheroids, also excludes effects of preformed AGEs by the reaction of glyoxal with fetal calf serum proteins. Our results showed that in cells treated with 200 microM glyoxal, the intercellular contacts in spheroids were disrupted, i.e. cells became totally dissociated. Immunocytochemical analysis revealed a dose-dependent accumulation of the AGE product epsilonN-(carboxymethyl)lysine (CML) in cells detached from cell clusters. The loss of cell attachment was associated with decreased expression of beta1-integrins and CD44 as revealed by laser scanning cytometry (LSC). Increasing concentrations of glyoxal induced an increase in the number of apoptotic cells which were identified by the immunoreactivity for active caspase-3. Remaining cell clusters showed resistance to both CML formation and apoptosis. The present findings demonstrate that cells treated with glyoxal undergo possibly anoikis, a specific mode of apoptosis caused by loss of cell adhesion.     

Proteomic analysis of the site specificity of glycation and carboxymethylation of ribonuclease

Proteomic analysis using electrospray liquid chromatography-mass spectrometry (ESI-LC-MS) has been used to compare the sites of glycation (Amadori adduct formation) and carboxymethylation of RNase and to assess the role of the Amadori adduct in the formation of the advanced glycation end-product (AGE), N(epsilon)-(carboxymethyl)lysine (CML). RNase (13.7 mg/mL, 1 mM) was incubated with glucose (0.4 M) at 37 degrees C for 14 days in phosphate buffer (0.2 M, pH 7.4) under air. On the basis of ESI-LC-MS of tryptic peptides, the major sites of glycation of RNase were, in order, K41, K7, K1, and K37. Three of these, in order, K41, K7, and K37 were also the major sites of CML formation. In other experiments, RNase was incubated under anaerobic conditions (1 mM DTPA, N2 purged) to form Amadori-modified protein, which was then incubated under aerobic conditions to allow AGE formation. Again, the major sites of glycation were, in order, K41, K7, K1, and K37 and the major sites of carboxymethylation were K41, K7, and K37. RNase was also incubated with 1-5 mM glyoxal, substantially more than is formed by autoxidation of glucose under experimental conditions, but there was only trace modification of lysine residues, primarily at K41. We conclude the following: (1) that the primary route to formation of CML is by autoxidation of Amadori adducts on protein, rather than by glyoxal generated on autoxidation of glucose; and (2) that carboxymethylation, like glycation, is a site-specific modification of protein affected by neighboring amino acids and bound ligands, such as phosphate or phosphorylated compounds. Even when the overall extent of protein modification is low, localization of a high proportion of the modifications at a few reactive sites might have important implications for understanding losses in protein functionality in aging and diabetes and also for the design of AGE inhibitors.     

Advanced glycation end product recognition by the receptor for AGEs

Nonenzymatic protein glycation results in the formation of advanced glycation end products (AGEs) that are implicated in the pathology of diabetes, chronic inflammation, Alzheimer's disease, and cancer. AGEs mediate their effects primarily through a receptor-dependent pathway in which AGEs bind to a specific cell surface associated receptor, the Receptor for AGEs (RAGE). N(?)-carboxy-methyl-lysine (CML) and N(?)-carboxy-ethyl-lysine (CEL), constitute two of the major AGE structures found in tissue and blood plasma, and are physiological ligands of RAGE. The solution structure of a CEL-containing peptide-RAGE V domain complex reveals that the carboxyethyl moiety fits inside a positively charged cavity of the V domain. Peptide backbone atoms make specific contacts with the V domain. The geometry of the bound CEL peptide is compatible with many CML (CEL)-modified sites found in plasma proteins. The structure explains how such patterned ligands as CML (CEL)-proteins bind to RAGE and contribute to RAGE signaling.     

Activation of NADPH oxidase by AGE links oxidant stress to altered gene expression via RAGE

Engagement of the receptor for advanced glycation end products (RAGE) by products of nonenzymatic glycation/oxidation triggers the generation of reactive oxygen species (ROS), thereby altering gene expression. Because dissection of the precise events by which ROS are generated via RAGE is relevant to the pathogenesis of complications in AGE-related disorders, such as diabetes and renal failure, we tested the hypothesis that activation of NADPH oxidase contributed, at least in part, to enhancing oxidant stress via RAGE. Here we show that incubation of human endothelial cells with AGEs on the surface of diabetic red blood cells, or specific AGEs, (carboxymethyl)lysine (CML)-modified adducts, prompted intracellular generation of hydrogen peroxide, cell surface expression of vascular cell adhesion molecule-1, and generation of tissue factor in a manner suppressed by treatment with diphenyliodonium, but not by inhibitors of nitric oxide. Consistent with an important role for NADPH oxidase, although macrophages derived from wild-type mice expressed enhanced levels of tissue factor upon stimulation with AGE, macrophages derived from mice deficient in a central subunit of NADPH oxidase, gp91phox, failed to display enhanced tissue factor in the presence of AGE. These findings underscore a central role of NADPH oxidase in AGE-RAGE-mediated generation of ROS and provide a mechanism for altered gene expression in AGE-related disorders.     

Immunological detection of a novel advanced glycation end-product.

BACKGROUND: The advanced stage of 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. Recently, it has been proposed that the intermediates contributing to AGE formation include dicarbonyl intermediates such as glyoxal, methylglyoxal, and 3-deoxyglucosone (3-DG). In the present study, we developed a novel, non-carboxymethyllysine (CML) anti-AGE antibody that recognizes serum proteins and peptides modified by 3-DG in vivo. MATERIALS AND METHODS: AGE-modified serum albumins were prepared by incubation of rabbit serum albumin with 3-DG or D-glucose. After immunization of rabbits, anti-AGE antisera were subjected to affinity chromatography on a Sepharose 4B column coupled with CML-BSA, or AGE-BSA created by incubation with 3-DG (AGE-6) or D-glucose (AGE-1). The AGE-Ab-6 and AGE-Ab-1 thus obtained was used to investigate AGEs in serum from diabetic patients on hemodialysis. RESULTS: Characterization of the novel AGE-Ab-6 obtained by immunoaffinity chromatography was performed with a competitive ELISA and immunoblot analysis. This antibody specifically cross-reacted with proteins modified by 3-DG. AGE-6 was detected in diabetic serum as three peaks with apparent molecular weights of 200, 1.15, and 0.85 kD, while AGE-1 was detected as four peaks with apparent molecular weights of 200, 65, 1.15, and 0.85 kD. CONCLUSION: This study provides new data on the pathways of AGE formation from 3-DG and methods for the immunochemical detection of AGEs. We also provide immunochemical evidence for the existence of six distinct AGEs in vivo among the AGE-modified proteins and peptides in the serum of diabetic patients on hemodialysis.     

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.     

Advanced glycation endproducts: from precursors to RAGE: round and round we go

The formation of advanced glycation endproducts (AGEs) occurs in diverse settings such as diabetes, aging, renal failure, inflammation and hypoxia. The chief cellular receptor for AGEs, RAGE, transduces the effects of AGEs via signal transduction, at least in part via processes requiring the RAGE cytoplasmic domain binding partner, diaphanous-1 or mDia1. Data suggest that RAGE perpetuates the inflammatory signals initiated by AGEs via multiple mechanisms. AGE–RAGE interaction stimulates generation of reactive oxygen species and inflammation—mechanisms which enhance AGE formation. Further, recent data in type 1 diabetic kidney reveal that deletion of RAGE prevents methylglyoxal accumulation, at least in part via RAGE-dependent regulation of glyoxalase-1, a major enzyme involved in methylglyoxal detoxification. Taken together, these considerations place RAGE in the center of biochemical and molecular stresses that characterize the complications of diabetes and chronic disease. Stopping RAGE-dependent signaling may hold the key to interrupting cycles of cellular perturbation and tissue damage in these disorders.