Tomato paste fraction inhibiting the formation of advanced glycation end-products

A water-soluble and low-molecular-weight fraction (SB) was obtained from tomato paste. The effects of SB on the formation of advanced glycation end-products (AGE) in protein glycation were studied by the methods of specific fluorescence, ELISA and a Western blot analysis, using the anti-AGE antibody after incubating protein with sugar. The results suggest that SB had strong inhibitory activity, in comparison with aminoguanidine as a positive control, and that the inhibitory mechanism of SB differed from that of aminoguanidine to involve trapping of reactive dicarbonyl intermediates in the early stage of glycation. SB contained an antioxidant, rutin, which showed potent inhibitory activity. The results also suggest that rutin chiefly contributed to inhibiting the formation of AGE, and that other compounds in SB may also have been related to the activity.     

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.     

Comparison of distinct protein isoforms of the receptor for advanced glycation end-products expressed in murine tissues and cell lines

The receptor for advanced glycation end-products (RAGE) is thought to be expressed ubiquitously as various protein isoforms. Our objective was to use Northern blotting, immunoblotting, and sensitivity to N-glycanase digestion to survey RAGE isoforms expressed in cell lines and mouse tissues in order to obtain a more comprehensive view of the RAGE expressome. Pulmonary RAGE mRNA (1.4 kb) was smaller than cell-line and tissue RAGE mRNA (6 kb-10 kb). Three anti-RAGE antibodies that recognized three distinct RAGE epitopes were used for protein studies (N-16, H-300, and αES). Lung expressed three predominant protein isoforms with apparent molecular masses of 45.1, 52.6, and 57.4 kDa (N-16/H-300) and four isoforms at 25.0, 46.9, 52.5, and 54.2 kDa (αES). These isoforms were expressed exclusively in lung. Heart, ileum, and kidney expressed a 44.0-kDa isoform (N-16), whereas aorta and pancreas expressed a 53.3-kDa isoform (αES). Each of these isoforms were absent in tissue extracts prepared from RAGE^−/− mice. Cell lines expressed a 70.0-kDa isoform, and a subset expressed a 30.0-kDa isoform (αES). Lung RAGE appeared to contain two N-linked glycans. Tissue and cell-line RAGE isoforms were completely insensitive to PNGase F digestion. Thus, numerous RAGE protein isoforms are detectable in tissues and cell lines. Canonical transmembrane and soluble RAGE appear to be expressed solely in lung (N-16/H-300). Non-pulmonary tissues and cell lines, regardless of the source tissue, both express distinct RAGE protein isoforms containing the N-terminal N-16 epitope or the αES RAGE epitope encoded by alternate exon 9, but lacking the H-300 epitope. This work was supported by NIH grants R01 GM37631 and GM68481.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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)     

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.