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.     

Glyceraldehyde-derived advanced glycation end-products having pyrrolopyridinium-based crosslinks

Reducing sugars and reactive aldehydes, such as glyceraldehyde, non-enzymatically react with amino or guanidino groups of proteins to form advanced glycation end-products (AGEs) by the Maillard reaction that involves Schiff base formation followed by Amadori rearrangement. AGEs are found relatively in abundance in the human eye and to accumulate at a higher rate in diseases that impair vision such as cataract, diabetic retinopathy or age-related macular degeneration. We identified two novel AGEs of pyrrolopyridinium lysine dimer derived from glyceraldehyde, PPG1 and PPG2, in the Maillard reaction of N^α-acetyl-l-lysine with glyceraldehyde under physiological conditions. Having fluorophores similar to that of vesperlysine A, which was isolated from the human lens, PPGs were found to act as photosensitizers producing singlet oxygen in response to blue light irradiation. Moreover, PPG2 interacts with receptor for AGE (RAGE) in vitro with a higher binding affinity than GLAP, a well-known ligand of the receptor. We also proposed a pathway to form PPGs and discussed how they would be formed in vitro. As glyceraldehyde-derived AGEs have been studied extensively in connection with various hyperglycemia-related diseases, further studies will be required to find PPGs in vivo such as in the lens or other tissues.     

Glycosphingolipid changes induced by advanced glycation end-products

The effects of advanced glycation end-products (AGEs) on retinal microvascular cell glycosphingolipids were investigated as a potential pathogenic mechanism of diabetic retinopathy. The results obtained showed that, in microvascular retinal endothelial cells and pericytes, AGEs increased the amount of all glycosphingolipids studied (from 25 to 115% depending on the glycosphingolipid species), except for a specific ganglioside, GD3, which decreased by 35% only in pericytes. Glycosphingolipid profiles and GM3 fatty acid analysis did not show any qualitative differences after incubation with AGEs, suggesting that AGEs only induced quantitative changes in cell glycosphingolipids. These results show a new metabolic effect of AGEs, which could be involved in the microvascular alterations observed in diabetic retinopathy.     

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.     

The role of the receptor for advanced glycation end-products in lung fibrosis

The pathogenesis of pulmonary fibrosis remains unclear. The receptor for advanced glycation end-products (RAGE) is a multi-ligand receptor known to be involved in the process of fibrotic change in several organs, such as peritoneal fibrosis and kidney fibrosis. The aim of this study was to examine the contribution of RAGE during the acute inflammation and chronic fibrotic phases of lung injury induced by intratracheal instillation of bleomycin in mice. Bleomycin-induced lung fibrosis was evaluated in wild-type and RAGE-deficient (RAGE-/-) mice. Bleomycin administration to wild-type mice caused an initial pneumonitis that evolved into fibrosis. While RAGE-/- mice developed a similar early inflammatory response, the mice were largely protected from the late fibrotic effects of bleomycin. The protection afforded by RAGE deficiency was accompanied by reduced pulmonary levels of the potent RAGE-inducible profibrotic cytokines transforming growth factor (TGF)-beta and PDGF. In addition, bleomycin administration induced high mobility group box 1 (HMGB-1) production, one of the ligands of RAGE, from inflammatory cells that accumulated within the air space. Coculture with HMGB-1 induced epithelial-mesenchymal transition (EMT) in alveolar type II epithelial cells from wild-type mice. However, alveolar type II epithelial cells derived from RAGE-/- mice did not respond to HMGB-1 treatment, such that the RAGE/HMGB-1 axis may play an important role in EMT. Also, bleomycin administration induced profibrotic cytokines TGF-beta and PDGF only in wild-type mouse lungs. Our results suggested that RAGE contributes to bleomycin-induced lung fibrosis through EMT and profibrotic cytokine production. Thus, RAGE may be a new therapeutic target for pulmonary fibrosis.     

Analysis of DNA-bound advanced glycation end-products by LC and mass spectrometry

Sugars and sugar degradation products readily react in vitro with guanine derivatives, resulting in the formation of DNA-bound advanced glycation end-products (DNA-AGEs). The two diastereomers of N(2)-(1-carboxyethyl)-2'-deoxyguanosine (CEdG(A,B)) and the cyclic adduct of methylglyoxal and 2'-deoxyguanosine (mdG) (N(2)-7-bis(1-hydroxy-2-oxopropyl)-2'-deoxyguanosine have also been detected in cultured cells and/or in vivo. LC-MS/MS methods have been developed to analyze sensitively DNA adducts in vitro and in vivo. In this paper, the chemical structures of possible DNA-AGEs and the application of LC-MS/MS to measure DNA-AGEs are reviewed.     

N-Terminal pyrazinones: a new class of peptide-bound advanced glycation end-products

Summary. The reaction of peptide Gly-Ala-Phe with the -dicarbonyl compounds glyoxal and methylglyoxal was studied under physiological conditions (pH=7.4, 37°C). Using HPLC with UV and fluorescence detection, a rapid derivatization of the peptide and the concomitant formation of well-defined products were observed. The products, which showed characteristic UV absorbance (_max=320 to 340nm) and fluorescence (_ex=330 to 340nm, _em=395 to 405nm), were identified by ESI-MS and NMR spectroscopic analysis as the N-terminally pyrazinone-modified peptides I (N-[2-(2-oxo-2H-pyrazin-1-yl)-propyl]-phenylalanine) and II (N-[2-(5-methyl-2-oxo-2H-pyrazin-1-yl)-propionyl]-phenylalanine). Model experiments revealed that the reactivity of the N-termini of peptides towards a derivatization by glyoxal is in the same order of magnitude as that of arginine, which generally is attributed as main target for -dicarbonyl compounds in proteins. Incubation of insulin with glyoxal proved the protein-bound formation of pyrazinones, with the N-terminus of the B-chain as the main target. According to these results, we conclude that N-terminal pyrazinones represent a new type of advanced glycation end-products (AGEs) with significance for biological systems and foods.     

Effects of advanced glycation end-products on the proliferation and differentiation of osteoblast-like cells

Dextran M20 was added to isolated rat liver mitochondria to mimic cytosolic macromolecules. Under these conditions, the morphological changes in the mitochondrial periphery that occur upon isolation of the organelle are restored, i.e. the volume of the intermembrane space decreases and the contact site frequency increases. The ADP routing from mitochondrial kinases at various locations was investigated by using the activities of oxidative phosphorylation and externally added pyruvate kinase as sensors for ADP transport into the matrix and extramitochondrial compartment, respectively. The studies reveal that a significant fraction of the ADP generated by either adenylate kinase in the intermembrane space or by outer membrane bound hexokinase isozyme I, is not accessible to extramitochondrial pyruvate kinase. Quantitative information on the ADP compartmentation in rat liver mitochondria was obtained by comparing the ADP supply from mitochondrial kinases to oxidative phosphorylation with that of non-bound, extramitochondrially located kinases. This approach allowed us to estimate the ADP diffusion gradients which were present across the outer membrane and between the compartment formed by bound hexokinase and the extramitochondrial compartment. In the presence of 10% dextran M20 these ADP gradients amounted to approximately 12 µM. The possible role of mitochondrial kinases in ADP transport into mitochondria in vivo is discussed. (Mol Cell Biochem 174: 43–51, 1997)     

Early and advanced glycation end-products are increased in dietary copper deficiency

The hypothesis that nonenzymatic glycosylation of proteins (glycation) contributes to damage associated with dietary copper deficiency has depended largely on indirect evidence. Thus far, the observation of an elevated percentage of glycated hemoglobin in copper-deficient rats has provided the only direct evidence of an increase in glycation. We sought further direct evidence of increased glycation in copper deficiency. Male weanling rats were fed a copper-adequate (CuA, 6.4 mg Cu/kg diet) or copper-deficient diet (CuD, 0.4 mg Cu/kg diet) for 5 weeks. Rats fed the CuD diet were copper deficient as judged by depressed organ copper concentrations and a variety of indirect indices. Measurements of hemoglobin A(1) and serum fructosamine (both early glycation end-products) as well as serum pentosidine (an advanced glycation end-product) indicated that all three compounds were elevated in CuD rats relative to CuA rats. This finding further supports the view that glycation is enhanced and thus may contribute to defects associated with dietary copper deficiency.     

Advanced glycation end-products and advanced oxidation protein products in patients with diabetes mellitus

Accelerated glycoxidation takes part in the development of diabetic complications. We determined advanced glycation end-products (AGEs) and advanced oxidation protein products (AOPP) in the sera of 52 patients with diabetes mellitus (DM) - 18 with DM Type 1 and 34 with DM Type 2 and examined their relationship to the compensation of the disease. AGEs were estimated spectrofluorimetrically (350 nm/440 nm) whereas AOPP were determined spectro-photometrically (340 nm). AGEs were elevated only in DM Type 2 (DM2 5.11+/-1.15 x 10(3) AU/g vs controls 4.08+/-0.71 x 10(3) AU/g, p<0.001, vs DM1 4.14+/-0.86 x 10(3) AU/g, p<0.005, DM1 vs controls were not significant). AOPP were elevated significantly in both types of DM with higher levels in DM Type 2 (DM2 157.50+/-75.15 micromol/l vs healthy subjects 79.80+/-23.72 micromol/l, p<0.001, vs DM1 97.50+/-30.91 micromol/l, p<0.005, DM1 vs controls p<0.05). There was a tight correlation between AGEs and AOPP in both types of DM (DM1 r=0.75, DM2 r=0.47 (p<0.05)) and both AGEs and AOPP correlated with triglycerides. In DM Type 1 only, AGEs correlated with HbA1c r=0.47 (p<0.05) and with blood glucose. Slight but not significant differences in AGEs and AOPP levels were observed in patients with or without diabetic complications. Oxidative stress is increased in both types of DM, more in Type 2 where it contributes to the formation of glycoxidation products.     

Lipocalin-2 elicited by advanced glycation end-products promotes the migration of vascular smooth muscle cells

Advanced glycation end-products (AGEs) play key roles in the development of diabetic vascular complications by activating the proliferation and migration of vascular smooth muscle cells. Here, we identified an increase of the migratory properties of human aortic smooth muscle cells (HASMC) through AGE-induced expression of lipocalin-2 (LCN2). Because the AGE-elicited expression of LCN2 was diminished by an antibody against the AGE receptor (RAGE), diphenylene iodonium (DPI), N-acetyl cysteine, LY294002, and SP600125, we suggest that AGEs enhance the expression of LCN2 via a RAGE-NADPH oxidase-reactive oxygen species pathway, leading to the phosphorylation of PI3K-Akt and JNK in HASMCs. In addition, a chromatin immunoprecipitation assay and promoter assay revealed that CCAAT/enhancer binding protein β is crucial for AGE-induced expression of LCN2. However, any other AGE-related signaling pathway, including ERK1/2, p38, NF-κB, and AP-1, did not affect the AGE- induced expression of LCN2. Knockdown of LCN2 expression by shRNA showed that AGE-elicited LCN2 expression enhanced the invasive and migratory properties of HASMCs, but showed no effect on cell proliferation. Considering the importance of HASMC migration in the development of atherosclerosis, our study provides a novel insight into diabetic vascular complications.     

Lack of the receptor for advanced glycation end-products attenuates E. coli pneumonia in mice

Background

The receptor for advanced glycation end-products (RAGE) has been suggested to modulate lung injury in models of acute pulmonary inflammation. To study this further, model systems utilizing wild type and RAGE knockout (KO) mice were used to determine the role of RAGE signaling in lipopolysaccharide (LPS) and E. coli induced acute pulmonary inflammation. The effect of intraperitoneal (i.p.) and intratracheal (i.t.) administration of mouse soluble RAGE on E. coli injury was also investigated.

Methodology/Principal Findings

C57BL/6 wild type and RAGE KO mice received an i.t. instillation of LPS, E. coli, or vehicle control. Some groups also received i.p. or i.t. administration of mouse soluble RAGE. After 24 hours, the role of RAGE expression on inflammation was assessed by comparing responses in wild type and RAGE KO. RAGE protein levels decreased in wild type lung homogenates after treatment with either LPS or bacteria. In addition, soluble RAGE and HMGB1 increased in the BALF after E. coli instillation. RAGE KO mice challenged with LPS had the same degree of inflammation as wild type mice. However, when challenged with E. coli, RAGE KO mice had significantly less inflammation when compared to wild type mice. Most cytokine levels were lower in the BALF of RAGE KO mice compared to wild type mice after E. coli injury, while only monocyte chemotactic protein-1, MCP-1, was lower after LPS challenge. Neither i.p. nor i.t. administration of mouse soluble RAGE attenuated the severity of E. coli injury in wild type mice.

Conclusions/Significance

Lack of RAGE in the lung does not protect against LPS induced acute pulmonary inflammation, but attenuates injury following live E. coli challenge. These findings suggest that RAGE mediates responses to E. coli-associated pathogen-associated molecular pattern molecules other than LPS or other bacterial specific signaling responses. Soluble RAGE treatment had no effect on inflammation.     

Chemoselective synthesis of peptides containing major advanced glycation end-products of lysine and arginine.

Useful methodologies have been developed, enabling the straightforward synthesis of peptides containing N(epsilon)-(carboxymethyl)-L-lysine (CML) and N(epsilon)-(carboxyethyl)-L-lysine (CEL), the major glycation end-products of lysine. These lysine derivatives were successfully incorporated into growing peptide chains via standard Fmoc/Ot-Bu peptide synthesis procedures. For the synthesis of peptides containing major glycation end-products of arginine, synthetic routes have been developed enabling the transformation of ornithine residues in peptides into the well-known arginine-derived advanced glycation end-products (AGEs) Glarg, carboxymethyl-L-arginine (CMA), MG-H1, MG-H2, MG-H3, and carboxyethyl-L-arginine (CEA), respectively, by means of special modifying agents. Furthermore, it was shown that Glarg-containing peptides become quantitatively hydrolyzed into CMA-peptides under physiologic conditions. A similar reaction was observed in case of a MG-H3-peptide, which turned into a CEA-peptide under these conditions.     

Inhibitors of aldose reductase and advanced glycation end-products formation from the leaves of Stelechocarpus cauliflorus R.E. Fr.

Two dihydroflavonol glycosides, engeletin and astilbin, were isolated from an EtOAc extract of the leaves of Stelechocarpus cauliflorus R.E. Fr. (Annonaceae). The inhibitory activity of engeletin against a recombinant human aldose reductase (IC₅₀ value=1.16 μM) was twice that of quercetin as a positive control (2.48 μM), and 23 times greater than that of astilbin (26.7 μM). Engeletin inhibited the enzyme uncompetitively. Astilbin was about as potent as the positive control, quercetin, in its inhibition of advanced glycation end-products formation. These flavonoids displayed therapeutic potential in the prevention and treatment of diabetic complications.     

S100 to receptor for advanced glycation end-products binding assay: Looking for inhibitors

Secreted by tumor and stromal cells, S100 proteins exert their biological functions via the interaction with surface receptors. The most described receptor is the receptor for advanced glycation end-products (RAGE), thereby participating in the S100-dependent cell migration, invasion, tumor growth, angiogenesis and metastasis. Several approaches have been described for determining this interaction. Here we describe an easy, specific and highly reproducible ELISA-based method, by optimizing several parameters such as the binding and blocking buffer, interaction time and concentrations, directed to screen chemical and biological inhibitors of this interaction for S100A4, S100A7 and S100P proteins. The efficiency of the protocol was validated by using well described neutralizing agents of the RAGE receptor and of the S100A4 activity. The methodology described here will allow future works with other members of the S100 protein family and their receptors.     

Combined immunoelectron microscopic and computer-assisted image analyses to detect advanced glycation end-products in human myocardium

Advanced glycation end-products (AGEs) result from oxidation–reduction reactions that ensue when a sugar becomes adducted to a protein. AGEs cause various complications of diabetes mellitus (DM). Experimental and clinical evidence suggest that AGEs also contribute to the complications of hypertension (HTN). Little is known about the abundance and localization of AGEs in human myocardium. In a few light microscopic studies, the AGE carboxymethyl lysine (CML) has been immunolabeled and localized virtually exclusively to the walls of small arteries. To more precisely delineate the abundance and localization of CML, we developed an immunoelectron microscopic (IEM) detection method using anti-CML monoclonal antibody 6D12 in conjunction with computer-assisted image analysis. Antibody was pre-absorbed with purified AGE-bovine serum albumin to assure specificity. Antigen–antibody (ag–ab) complexes were individually identified with protein A-conjugated colloidal gold and counted with an automated system. We applied this method in 21 patients (pts) undergoing epicardial biopsy during coronary bypass grafting (CBG) [20 M, 1 F; mean age 65 ± 7.4 (± SEM) years]. Seven pts had neither DM nor HTN, seven had HTN, and seven had DM + HTN. In contrast to the prior light microscopic studies, we detected CML scattered throughout the cardiomyocyte in all pts, but in widely varying amounts. Ag–ab complexes were abundant in sections through myofilaments (mean count 23.6 ± 9.2 per μm², range 9.4–48) and even more so in mitochondria (mean count 34.4 ± 11.9 per μm², range 14.1–68.2, P < 0.001 vs. myofilaments). CML was also detected in vascular endothelial cells. There were no statistically significant differences based on presence or absence of HTN or DM. In conclusion, our IEM method is the first to provide detailed delineation of the localization and abundance of CML in myocardium. CML is very prevalent in CBG pts, suggesting that AGEs could play a role in abnormal cardiomyocyte function, including altered energy metabolism.