Abstract Advanced Glycation End Products Research Articles

1302-P: Inhibitory Effect of Advanced Glycation End Products on Proinsulin Processing

Advanced glycation end-products (AGEs) form at an accelerated rate in diabetes and are associated with the development of diabetic complications. So far, the effect of AGEs on proteases was mostly investigated with substrates modified non-specifically with hexoses amongst others in combination with proteases of broad specificity. Here, we investigated the effect of AGEs on the proinsulin processing peptidases preprotein convertase 1 (PC1) and carboxypeptidase E (CPE) via two ways: - Measuring of activity towards peptide substrates. These contained chemically defined AGEs carboxymethyllysine (CML) or methylglyoxyl-hydroimidazolone-1 (MGH1) or unmodified amino acids. - Recombinant human proinsulin was incubated with methylglyoxal for 24 h and was processed by addition of PC1 and CPE in vitro. Formation of insulin was compared to unmodified proinsulin. In parallel formation of AGE-modified proinsulin was quantified. Artificial peptide substrates were synthesized by solid phase peptide synthesis and purified by HPLC. A protected building block was used for MGH1 incorporation while CML was introduced on the resin. Protease activity towards artificial substrates was measured by fluorescence assay or LC MS/MS. Proinsulin, insulin and AGE-modified proinsulin were quantified by LC MS. Strong activity of PC1 and CPE was seen towards arginine containing substrates. Activity was completely absent towards modified amino acids MGH1 and CML for both, PC1 and CPE. Incubation of proinsulin with MG resulted in MGH1 modified proinsulin. At low MG (µM) concentrations this was associated with a 25 % reduction of insulin formation while high MG concentrations (95 µM) led to a 75 % reduction in insulin formation. Here we demonstrate for the first time the effect of substrate modifications with AGEs on physiologically important proteases PC1 and CPE. The latter are not only involved in the processing of proinsulin but a range of endocrinologically and neurophysiologically important peptide hormones. Disclosure W. Mier: None. B. Beijer: None. J.M. Szendroedi: Consultant; Boehringer Ingelheim International GmbH. T.H. Fleming: None. Funding DFG (SFB 1118)

Inhibitory Effects of Saururus chinensis Extract on Receptor for Advanced Glycation End-Products-Dependent Inflammation and Diabetes-Induced Dysregulation of Vasodilation

Advanced glycation end-products (AGEs) and the receptor for AGEs (RAGE) are implicated in inflammatory reactions and vascular complications in diabetes. Signaling pathways downstream of RAGE are involved in NF-κB activation. In this study, we examined whether ethanol extracts of Saururus chinensis (Lour.) Baill. (SE) could affect RAGE signaling and vascular relaxation in streptozotocin (STZ)-induced diabetic rats. Treatment with SE inhibited AGEs-modified bovine serum albumin (AGEs-BSA)-elicited activation of NF-κB and could compete with AGEs-BSA binding to RAGE in a dose-dependent manner. Tumor necrosis factor-α (TNF-α) secretion induced by lipopolysaccharide (LPS)—a RAGE ligand—was also reduced by SE treatment in wild-type Ager+/+ mice as well as in cultured peritoneal macrophages from Ager+/+ mice but not in Ager−/− mice. SE administration significantly ameliorated diabetes-related dysregulation of acetylcholine-mediated vascular relaxation in STZ-induced diabetic rats. These results suggest that SE would inhibit RAGE signaling and would be useful for the improvement of vascular endothelial dysfunction in diabetes.

Deletion of RAGE fails to prevent hepatosteatosis in obese mice due to impairment of other AGEs receptors and detoxifying systems

Advanced glycation endproducts (AGEs) are involved in several diseases, including NAFLD and NASH. RAGE is the main receptor mediating the pro-inflammatory signalling induced by AGEs. Therefore, targeting of RAGE has been proposed for prevention of chronic inflammatory diseases. However, the role of RAGE in the development of NAFLD and NASH remains poorly understood. We thus aimed to analyse the effect of obesity on AGEs accumulation, AGE-receptors and AGE-detoxification, and whether the absence of RAGE might improve hepatosteatosis and inflammation, by comparing the liver of lean control, obese (LeptrDb−/−) and obese RAGE-deficient (RAGE−/− LeptrDb−/−) mice. Obesity induced AGEs accumulation and RAGE expression with hepatosteatosis and inflammation in LeptrDb−/−, compared to lean controls. Despite the genetic deletion of RAGE in the LeptrDb−/− mice, high levels of intrahepatic AGEs were maintained accompanied by decreased expression of the protective AGE-receptor-1, impaired AGE-detoxifying system glyoxalase-1, and increased expression of the alternative AGE-receptor galectin-3. We also found sustained hepatosteatosis and inflammation as determined by persistent activation of the lipogenic SREBP1c and proinflammatory NLRP3 signalling pathways. Thus, RAGE targeting is not effective in the prevention of NAFLD in conditions of obesity, likely due to the direct liver specific crosstalk of RAGE with other AGE-receptors and AGE-detoxifying systems.

Accumulation of Nε-(carboxyethyl) lysine in Caenorhabditis elegans is correlated with the formation of ketone body.

Advanced glycation end-products (AGEs) are formed when proteins react with carbonyl compounds, and they gradually accumulate with age. However, AGE accumulation with ageing is not fully understood because longevity studies in mammals are time-consuming. Therefore, we used Caenorhabditis elegans to evaluate the correlation between ageing and AGE accumulation. Age-synchronized C.elegans nematodes were cultured for 3 and 12 days. The levels of Nε-(carboxymethyl) lysine, Nω-(carboxymethyl) arginine, Nδ-(5-hydro-5-methyl-4-imidazolone-2-yl) ornithine and Nε-(carboxyethyl) lysine (CEL) were compared. Glucose, methylglyoxal and acetol were incubated with human serum albumin, and CEL formation was evaluated. The levels of methylglyoxal and ketone bodies in C.elegans were quantified. CEL accumulation increased significantly with culture duration. Methylglyoxal and ketone bodies-possible forerunners of AGE accumulation-were also quantified with respect to culture duration. The levels of ketone bodies increased significantly during culture, and correlated closely with CEL accumulation (R2 = 0.72, P = 0.0008), whereas the levels of methylglyoxal did not increase over time. CEL was formed in vitro in a time-dependent manner from methylglyoxal and acetol when incubated with human serum albumin (HSA) at the same temperature as C.elegans culture, suggesting that increased levels of CEL in C.elegans are attributable to ketone bodies.

Molecular recognition of a single-chain Fv antibody specific for GA-pyridine, an advanced glycation end-product (AGE), elucidated using biophysical techniques and synthetic antigen analogues.

Advanced glycation end-products (AGEs) are a heterogeneous group of compounds formed by non-enzymatic reaction between reducing-sugar and Arg/Lys in proteins and are involved in various diabetic complications. GA-pyridine is derived from glycolaldehyde and is one of the most cytotoxic AGEs. Here, we established a single-chain Fv (scFv) antibody against GA-pyridine, 73MuL9-scFv, and examined the details of its specificity and antigen recognition by using various techniques involving biophysics, chemical biology and structural biology. We also synthesized several compounds that differ slightly in regard to the position and number of GA-pyridine substituent groups, and revealed that GA-pyridine was specifically bound to 73MuL9-scFv. Thermodynamic analysis revealed that the association of GA-pyridine to 73MuL9-scFv was an exothermic and enthalpy driven reaction, and thus that the antigen recognition involved multiple specific interactions. Crystallographic analysis of the Fv fragment of 73MuL9-scFv revealed that several CH-π and hydrogen bond interactions took place between the Fv-fragment and GA-pyridine, which was consistent with the results of thermodynamic analysis. Further studies using 73MuL9-scFv as a tool to clarify the relevance of GA-pyridine to diabetic complications are warranted.

Plasma concentrations of advanced glycation end-products and colorectal cancer risk in the EPIC study.

Advanced glycation end-products (AGEs) are a heterogeneous group of compounds formed by the non-enzymatic reaction between amino acids and reducing sugars, or dicarbonyls as intermediate compounds. Experimental studies suggest that AGEs may promote colorectal cancer, but prospective epidemiologic studies are inconclusive. We conducted a case-control study nested within a large European cohort. Plasma concentrations of three protein-bound AGEs-Nε-(carboxy-methyl)lysine (CML), Nε-(carboxy-ethyl)lysine (CEL) and Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H1)-were measured by ultra-performance liquid chromatography-tandem mass spectrometry in baseline samples collected from 1378 incident primary colorectal cancer cases and 1378 matched controls. Multivariable-adjusted odds ratios (ORs) and 95% confidence intervals (CIs) were computed using conditional logistic regression for colorectal cancer risk associated with CML, CEL, MG-H1, total AGEs, and [CEL+MG-H1: CML] and [CEL:MG-H1] ratios. Inverse colorectal cancer risk associations were observed for CML (OR comparing highest to lowest quintile, ORQ5 versus Q1 = 0.40, 95% CI: 0.27-0.59), MG-H1 (ORQ5 versus Q1 = 0.73, 95% CI: 0.53-1.00) and total AGEs (OR Q5 versus Q1 = 0.52, 95% CI: 0.37-0.73), whereas no association was observed for CEL. A higher [CEL+MG-H1: CML] ratio was associated with colorectal cancer risk (ORQ5 versus Q1 = 1.91, 95% CI: 1.31-2.79). The associations observed did not differ by sex, or by tumour anatomical sub-site. Although individual AGEs concentrations appear to be inversely associated with colorectal cancer risk, a higher ratio of methylglyoxal-derived AGEs versus those derived from glyoxal (calculated by [CEL+MG-H1: CML] ratio) showed a strong positive risk association. Further insight on the metabolism of AGEs and their dicarbonyls precursors, and their roles in colorectal cancer development is needed.

In-Depth AGE and ALE Profiling of Human Albumin in Heart Failure: Ex Vivo Studies.

Advanced glycation end-products (AGEs) and advanced lipoxidation end-products (ALEs), particularly carboxymethyl-lysine (CML), have been largely proposed as factors involved in the establishment and progression of heart failure (HF). Despite this evidence, the current literature lacks the comprehensive identification and characterization of the plasma AGEs/ALEs involved in HF (untargeted approach). This work provides the first ex vivo high-resolution mass spectrometry (HR-MS) profiling of AGEs/ALEs occurring in human serum albumin (HSA), the most abundant protein in plasma, characterized by several nucleophilic sites and thus representing the main protein substrate for AGE/ALE formation. A set of AGE/ALE adducts in pooled HF-HSA samples was defined, and a semi-quantitative analysis was carried out in order to finally select those presenting in increased amounts in the HF samples with respect to the control condition. These adducts were statistically confirmed by monitoring their content in individual HF samples by applying a targeted approach. Selected AGEs/ALEs proved to be mostly CML derivatives on Lys residues (i.e., CML-Lys12, CML-Lys378, CML-Lys402), and one deoxy-fructosyl derivative on the Lys 389 (DFK-Lys 389). The nature of CML adducts was finally confirmed using immunological methods and in vitro production of such adducts further confirmed by mass spectrometry.

RasGRP2 inhibits glyceraldehyde-derived toxic advanced glycation end-products from inducing permeability in vascular endothelial cells

Advanced glycation end-products (AGEs) are formed by the non-enzymatic reaction of sugars and proteins. Among the AGEs, glyceraldehyde-derived toxic AGEs (TAGE) are associated with various diseases, including diabetic complications such as diabetic retinopathy (DR). The risk of developing DR is strongly associated with poor glycemic control, which causes AGE accumulation and increases AGE-induced vascular permeability. We previously reported that Ras guanyl nucleotide releasing protein 2 (RasGRP2), which activates small G proteins, may play an essential role in the cell response to toxicity when exposed to various factors. However, it is not known whether RasGRP2 prevents the adverse effects of TAGE in vascular endothelial cells. This study observed that TAGE enhanced vascular permeability by disrupting adherens junctions and tight junctions via complex signaling, such as ROS and non-ROS pathways. In particular, RasGRP2 protected adherens junction disruption, thereby suppressing vascular hyper-permeability. These results indicate that RasGRP2 is an essential protective factor of vascular permeability and may help develop novel therapeutic strategies for AGE-induced DR.

Аdvanced glycation end-products as novel biomarkers of eosinophil-derived lung inflammatory diseases (literature review)

Advanced glycation end-products (AGEs) are created during the process of glycation of cells from various tissues and fluids and are a heterogeneous group of molecules formed from the nonenzymatic reaction of reducing sugars with the amino group of proteins, lipids, and nucleic acid. In normal conditions, they play the immunoregulatory role. In pathologic conditions AGEs activate the receptors for advanced glycation end products (RAGE) and cause long-lasting inflammation. RAGE participates actively in various disorders such as rheumatoid arthritis, diabetes, etc. However, there is relatively small number of scientific studies on the possibility of using the role of AGE in the pathogenesis of allergic diseases. RAGE transcript and protein are expressed in the lung by pulmonary type I alveolar epithelial cells, suggesting that RAGE has an important role in lung pathophysiology. They repress some endogenous autoregulatory functions leading to many diseases, including allergy. Oxidative stress increases the inflammatory reaction in asthma and allergies. Long-lasting inflammation followed by free radicals production are important factors involved in allergic reactions, they negatively influence the incidence and prognosis of allergy. RAGEs are expressed on circulating immune cells, they activate NF kappaB and intracellular oxidative stress also increases the inflammatory reaction in asthma and allergies. The membrane RAGE (mRAGE) signaling is proinflammatory, whereas soluble RAGE (sRAGE), a secreted form of RAGE, is generally anti-inflammatory. The study of AGEs, soluble RAGE, ligands of RAGE HMGB1, and S100A8/A913 and IL-33 is useful in the context of their considering as biomarkers to the differentiation diagnostic between eosinophils-derived and neutrophil-derived asthma/AAD. The mean serum levels of RAGE may be the target of new therapeutic interventions.

Dietary Advanced Glycation End-products (AGE) and Risk of Breast Cancer in the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial (PLCO).

Advanced glycation end-products (AGEs) are implicated in the pathogenesis of several chronic diseases including cancer. AGEs are produced endogenously but can also be consumed from foods. AGE formation in food is accelerated during cooking at high temperatures. Certain high fat or highly processed foods have high AGE values. The objective of the study was to assign and quantify Nϵ-carboxymethyl-lysine (CML)-AGE content in food and investigate the association between dietary AGE intake and breast cancer risk in the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial. The study included women enrolled in the intervention arm who were cancer-free at baseline and completed a baseline questionnaire and food frequency questionnaire (DQX). CML-AGE values were assigned and quantified to foods in the DQX using a published AGE database. Cox proportional hazards models were used to estimate the hazard ratios (HR) and 95% confidence intervals (CI) of breast cancer among all women, and stratified by race/ethnicity, invasiveness of disease, and hormone receptor status. After a median 11.5 years of follow-up, 1,592 women were diagnosed with breast cancer. Higher CML-AGE intake was associated with increased risk of breast cancer among all women (HRQ5VSQ1, 1.30; 95% CI, 1.04-1.62; P trend = 0.04) and in non-Hispanic white women (HRT3VST1, 1.21; 95% CI, 1.02-1.44). Increased CML-AGE intake was associated with increased risk of in situ (HRT3VST1, 1.49; 95% CI, 1.11-2.01) and hormone receptor-positive (HRT3VST1, 1.24; 95% CI, 1.01-1.53) breast cancers. In conclusion, high intake of dietary AGE may contribute to increased breast cancer.

717-P: The Levels and Related Factors of Advanced Glycation End Products in Korean Patients with Type 2 Diabetes Mellitus

Advanced glycation end-products (AGEs) are known to be produced by the Maillard reaction, ingested through food. AGEs are actively generated and accumulated in the circulating blood and various tissues in diabetes mellitus patients by chronic hyperglycemia. AGEs play an important role in the development of diabetic vascular complications. The aim of this study was to assess levels and related factors of AGEs in Korean patients with type 2 diabetes mellitus. A total of 310 Korean men (n=177) and women (n=133) with T2DM subjects were participated. This study was measured and analyzed anthrometric data, body composition, and biochemical parameters. Tissue accumulated AGEs can be evaluated a non-invasive methodology by skin autofluorescence reader. The means age of total subjects was 58.9 ± 11.0 years, BMI was 25.8 ± 4.5 kg/m2. There is no difference between male and female patients in levels of AGEs, pulse wave velocity (PWV) and vein age. There is a significant correlation among AGEs, height, weight, body mass index, skeletal muscle mass, diastolic blood pressure and HDL cholesterol for male patients and among fasting blood sugar, glycated hemoglobin A1c, total cholesterol, LDL cholesterol, and systolic blood pressure for female patients. The correlation of AGEs for both male and female subjects is highly associated with age, duration of diabetes and PWV. This study shows possibility of using AGEs as an index for clinical diagnosis, complications prediction, and diabetes management in Korean patients with type 2 diabetes mellitus. However, it is considered that a change in AGEs may be influenced by gender, age, and duration of diabetes. Disclosure J. Yim: None. Y. Kim: None. J. Park: None.

Abstract B047: Advanced glycation end products promote prostate tumor growth and are a potential biologic consequence of lifestyle factors contributing to cancer disparity

Abstract Advanced glycation end-products (AGEs), are reactive metabolites produced endogenously as a consequence of glucose uptake during glycolysis. AGEs accumulate in tissues and organs as we grow older to promote multiple chronic disease phenotypes. AGE pathogenic effects are mediated through modification of protein function, genetic fidelity, stress responses and cellular signaling pathways. Critically, cancer disparity factors such as a sedentary lifestyle, obesity and an unhealthy diet are external influences that also contribute to the AGE accumulation pool in the body. This research group examined circulating and tumor AGE levels in clinical specimens of prostate cancer and identified a race specific, tumor-dependent pattern of accumulation. AGE levels were highest in aggressive tumors, especially those from men with African ancestry. Increased AGE levels correlated with an upregulation in the receptor for advanced glycation end products (RAGE) and activated NFkB. In a syngeneic subcutaneous prostate cancer mouse model, chronic consumption of AGE resulted in a 3-fold increase in tumor growth. Strikingly, dietary-AGE mediated increases in tumor growth were accompanied by a cytoplasmic accumulation of AR, elevation in MYC, RAGE, and AGE as well as increased cell proliferation. Cytoplasmic accumulation of AR is elevated in CRPC tissue and is an independent predictor of biochemical recurrence. Our data show that treatment of prostate cancer cells with AGEs induces a neuroendocrine differentiated phenotype by promoting a more mesenchymal morphology and increasing the expression of associated marker genes including ENO2, MYC and SYP and the prostate cancer stem cell marker CD44 as well as the downregulation of AR. The aberrant activation and recruitment of immune cells is a major pathogenic consequence of AGE accumulation, and a series of studies have highlighted the tumor-associated immune response as a critical pathway contributing to cancer disparity. Using patient-derived primary tumor cells, the investigators found that AGEs released into the extracellular matrix may recapitulate the tumor-associated immune response observed in ancestry-specific prostate tumor tissues. Further preliminary studies indicate that AGE treatment of prostate cancer cells can alter how cancer cells metabolize glucose to promote an aggressive phenotype. The investigators' studies support the concept that AGE metabolites represent a biologic consequence of the socioeconomic and environmental factors that promote cancer health disparity. As our understanding of tumor biology advances, it is becoming increasingly clear that these inter-related lifestyle factors have distinct molecular consequences on the biologic makeup of tumors, altering cell signaling events and gene expression profiles to contribute to cancer disparity outcomes such as its earlier development or its progression to more aggressive disease. Citation Format: Pamela Woods, Bradley A. Krisanits, Dion Foster, Lourdes M. Nogueira, Laura Spruill, Marvella E. Ford, Michael B. Lilly, Victoria J. Findlay, David P. Turner. Advanced glycation end products promote prostate tumor growth and are a potential biologic consequence of lifestyle factors contributing to cancer disparity [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr B047.

Abstract C031: Regular physical activity can prevent the oncogenic effects of lifestyle-associated advanced glycation end products

Abstract Advanced glycation end-products (AGEs), are reactive metabolites produced endogenously as a consequence of glucose metabolism. AGEs accumulate in tissues and organs as we grow older to promote multiple chronic disease phenotypes. AGE pathogenic effects are mediated through modification of protein function, genetic fidelity, stress responses and cellular signaling pathways. Critically, cancer disparity factors such as a sedentary lifestyle, obesity and an unhealthy diet are external influences that have been shown to contribute to the accumulation of AGEs. This research group examined circulating and tumor AGE levels in clinical specimens of prostate cancer and identified a race specific, tumor-dependent pattern of accumulation. AGE levels were highest in aggressive tumors, especially those from men with African ancestry. As our understanding of tumor biology advances, it is becoming increasingly clear that these lifestyle factors have distinct molecular consequences on the biologic make up of tumors, altering cell signaling events and gene expression profiles to contribute to cancer disparity outcomes such as earlier development or its progression to more aggressive disease. Increased AGE levels correlated with an up-regulation in the receptor for advanced glycation end products (RAGE) and activated NFkB. In a syngeneic sub-cutaneous prostate cancer mouse model, chronic consumption of AGE resulted in a 3-fold increase in tumor growth. Dietary-AGE mediated increases in tumor growth were accompanied by a cytoplasmic accumulation of AR, elevation in MYC, RAGE, and AGE as well as increased cell proliferation. Given the links between lifestyle and AGEs we examined the effects of regular physical activity on AGE induced tumor growth in our syngeneic sub-cutaneous dietary-AGE prostate cancer model. Mice exposed to physical activity for 1 hour, 5 days per week showed a significant decrease in AGE induced tumor growth. We also examined the effects of dietary-AGEs on tumor progression using the FVB-Tg(C3-1-TAg)cJeg/JegJ (C3-Tag) transgenic spontaneous prostate cancer mouse model. This model progresses to low grade prostate intraepithelial neoplasia (PIN) at 24 weeks. However, chronic consumption of AGE resulted in increased progression towards moderate to high grade PIN at this same time point. When regular physical activity was introduced, we observed delayed progression of PIN in both dietary groups, but most significantly in the high AGE fed mice. These studies support the concept that AGEs represent a biological consequence of the socioeconomic and environmental factors that promote cancer disparity, which may be at least in part reversed via physical activity. This may have the greatest impact for African American patients who tend to have poorer survival, and where a lack of physical activity, poor diet, and high obesity rates are more prevalent. Citation Format: Bradley A Krisanits, Pamela M Woods, Dion Foster, Lourdes M Nogueira, Laura Spruill, Marvella E Ford, Victoria J Findlay, David P Turner. Regular physical activity can prevent the oncogenic effects of lifestyle-associated advanced glycation end products [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr C031.

B procyanidins of Annona crassiflora fruit peel inhibited glycation, lipid peroxidation and protein-bound carbonyls, with protective effects on glycated catalase

Advanced glycation end-products (AGEs) have been reported as results of increased oxidative stress. Consequently, the search for new antioxidant and anti-glycating agents is under intense investigation. Plant-derived procyanidins have previously demonstrated anti-glycation properties. Thus, this study aimed to isolate procyanidins from Annona crassiflora fruit peel, a species from the Brazilian Savanna, and investigate their antioxidant and anti-glycation effects. Free radical scavenging and quenching properties, formation of reactive oxygen species (ROS), AGEs, protein carbonyl and thiol groups, lipid peroxidation, crosslinked AGEs, as well as glycated catalase activity, were analyzed. In addition, in silico assessment of absorption, distribution, metabolism, excretion and toxicity was carried out. The procyanidins-enriched fraction, named here as F7, showed high antioxidant and anti-glycation capacities, with inhibitory activities against lipid peroxidation, and AGEs and ROS formation. In addition, there were reductions in AGEs-induced crosslinks and protein carbonyls and protective effects against oxidation of thiol groups and glycated-catalase. ADMET predictions of F7 showed favorable absorption and distribution, with no hepatotoxicity or mutagenicity. Together, our results support the anti-glycation activities of the procyanidins-enriched fraction from A. crassiflora, and suggest that these effects are triggered, at least in part, by scavenging free radical and dicarbonyls intermediates.

Identification of a novel advanced glycation end product derived from lactaldehyde.

Advanced glycation end products (AGEs) are implicated in the development of diabetic complications via the receptor for AGEs (RAGE). We have reported that the 3-hydroxypyridinium (3HP)-containing AGEs derived from α-hydroxyaldehydes physically interact with RAGE and show cytotoxicity. Lactaldehyde (LA) is formed from a reaction between threonine and myeloperoxidase, but no LA-derived AGEs have been characterized. Here, we identify the structure and physiological effects of an AGE derived from LA. We isolated a novel 3HP derivative, 2-acetamido-6-(3-hydroxy-5-methyl-pyridin-1-ium-1-yl)hexanoate, named as N-acetyl-LAPL (lactaldehyde-derived pyridinium-type lysine adduct), from a mixture of LA with Nα-acetyl-L-lysine. LAPL was also detected in the LA-modified protein. LAPL elicited toxicity in PC12 neuronal cells, but the effect was suppressed by the soluble form of RAGE as a decoy receptor. Moreover, surface plasmon resonance-based analysis revealed that LAPL specifically binds to recombinant RAGE. These results indicate that LA generates an AGE containing the 3HP moiety and contributes to RAGE-dependent cytotoxicity. Abbreviations: AGEs: advanced glycation end products; RAGE: receptor for advanced glycation end products; 3HP: 3-hydroxypyridinium; LA: lactaldehyde; LAPL: lactaldehyde-derived pyridinium-type lysine adduct; BSA: bovine serum albumin; GLAP: glyceraldehyde-derived pyridinium; MPO: myeloperoxidase; HFBA: heptafluorobutyric acid; TFA: trifluoroacetic acid; HPLC: high performance liquid chromatography; LC-ESI-QTOF-MS: liquid chromatography-electrospray ionization-quadrupole time-of-flight-mass spectrometry; NMR: nuclear magnetic resonance; LA-BSA: lactaldehyde-modified bovine serum albumin; PBS: phosphate buffered saline, GST, glutathione S-transferase; SPR: surface plasmon resonance; OP-lysine: 2-ammonio-6-(3-oxidopyridinium-1-yl)hexanoate; GLO1: glyoxalase 1; MG, methylglyoxal.

2215-PUB: Serum Levels of the Advanced Glycation End Product (AGE) Pentosidine Do Not Correlate with the Extent of Coronary Artery Disease Assessed by SYNTAX Score

Advanced glycation end-products (AGEs) are endogenously produced or exogenously derived glycated proteins and lipoproteins. The accumulation of AGEs may have a variety of adverse effects since they cause oxidative stress and thus may act as contributing factors to the development of disease states such as, for example, complications of diabetes mellitus. The aim of this study was to investigate whether the levels of three AGEs (pentosidine, N(6)-carboxymethyllysine [CML], and methylglyoxal [MGO]) and the soluble form of the AGE receptor (sRAGE) may be used as markers for the presence and extent of coronary artery disease (CAD). We recruited 363 consecutive patients with suspected CAD who presented for cardiac catheterization at Lenox Hill Hospital in New York City (54% of patients were male, 48% of the patients had diabetes). At the time of this abstract submission 169 patients had circulating pentosidine levels measured (74 of these patients had diabetes). We examined relations between pentosidine levels, HbA1c, LDL, and the extend of CAD (measured by SYNTAX score). Serum pentosidine levels did not significantly correlate with any of other parameters. There was, however, a positive correlation between HbA1c levels and the SYNTAX score (Spearman correlation coefficients of 0.26, ρ = 0.0001 for the entire group; 0.18, ρ = 0.01 for patients without diabetes; and 0.29, ρ= 0.0001 for patients with diabetes). In conclusion, our preliminary results demonstrate that in patients with suspected CAD undergoing cardiac catheterization, SYNTAX score does not correlate with pentosidine levels but correlates positively with HbA1c. The data on all three AGEs and sRAGE in the entire cohort of 363 patients will be presented at the meeting. Disclosure C. Basman: None. D. Avtanski: None. K. Ziskovich: None. R. Jonas: None. S. Fishman: None. H. Sonmez: None. U. Rashid: None. A. Kodra: None. G. Stoffels: None. M. Lesser: None. V. Singh: None. L. Poretsky: None. Funding The Gerald J. and Dorothy R. Friedman New York Foundation for Medical Research

English

Advanced glycation end-products (AGEs) have been shown to be implicated in many degenerative disorders as well as diabetic pathology. Of interest, several natural compounds including phenolic constituents are capable of inhibiting the formation of AGEs. Bio-guided fractionation of a methanol extract from Mangifera indica leaves led to the characterization of six antiglycative constituents including gallic acid (1), iriflophenone-3-C-β-D-glucoside (2), mangiferin (3), iriflophenone-3-C-(2'-O-galloyl)-β-D-glucoside (4), hyperoside (5) and isoquercitrin (6). With IC50 values lower than that of aminoguanidine, the six identified phenolic compounds can be regarded as remarkable inhibitors of advanced glycation end products formation and M. indica leaves should be considered as a potential nutraceutical resource to prevent carbonyl stress-related diseases. Key words: Mangifera indica, antiglycation, radical scavenging, phenolics, benzophenone.

Advanced glycation end-products disrupt human endothelial cells redox homeostasis: new insights into reactive oxygen species production

Advanced glycation end-products (AGEs) trigger multiple metabolic disorders in the vessel wall that may in turn lead to endothelial dysfunction. The molecular mechanisms by which AGEs generate these effects are not completely understood. Oxidative stress plays a key role in the development of deleterious effects that occur in endothelium during diabetes. Our main objectives were to further understand how AGEs contribute to reactive oxygen species (ROS) overproduction in endothelial cells and to evaluate the protective effect of an antioxidant plant extract. The human endothelial cell line EA.hy926 was treated with native or modified bovine serum albumin (respectively BSA and BSA-AGEs). To monitor free radicals formation, we used H2DCF-DA, dihydroethidium (DHE), DAF-FM-DA and MitoSOX Red dyes. To investigate potential sources of ROS, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and mitochondrial inhibitors were used. The regulation of different types of ROS by the polyphenol-rich extract from the medicinal plant Doratoxylon apetalum was also studied for a therapeutic perspective. BSA-AGEs exhibited not only less antioxidant properties than BSA, but also pro-oxidant effects. The degree of albumin glycoxidation directly influenced oxidative stress through a possible communication between NADPH oxidase and mitochondria. D. apetalum significantly decreased intracellular hydrogen peroxide and superoxide anions mainly detected by H2DCF-DA and DHE respectively. Our results suggest that BSA-AGEs promote a marked oxidative stress mediated at least by NADPH oxidase and mitochondria. D. apetalum plant extract appeared to be an effective antioxidant compound to protect endothelial cells.

Advanced glycation endproducts induce self- and cross-tolerance in monocytes.

Advanced glycation endproducts (AGEs) are well-known inflammatory mediators, which are recognized by immune cells through their corresponding receptor RAGE and have been shown to participate in the pathophysiology of a variety of acute as well as chronic inflammatory diseases. Nevertheless, no data are available on the aftermath of AGE recognition on immune cells. We used the monocytic cell line MonoMac6 as well as primary human monocytes for double stimulation experiments. We measured secreted as well as intracellular levels of TNF-α using ELISA and flow cytometry. In addition, gene expression of surface receptors (RAGE and TLR4) and TNF were measured by qPCR. Stimulation with AGE leads to a dose-dependent induction of self- and cross-tolerance in both primary monocytes as well as the MonoMac6 cell line. The AGE tolerance depended neither on a decreased expression of RAGE or TLR4, nor on a decrease of TNF-α expression. Nevertheless, intracellular TNF-α was decreased, hinting towards a posttranscriptional regulation. High levels of AGEs are capable to activate immune cellsat first, but induce a secondary state of hypo-responsiveness in these cells. Based on the origin of its causal agent, we propose this phenomenon to be "metabolic tolerance".

Rosella Flower Decreases the CML Serum and Liver Inflammation of Rats Given Baked-Food Diet

Advanced Glycation Endproducts (AGEs) or so-called glycotoxin can be triggered by heated food in which the Maillard reaction occurs. One type of glycotoxins is CML. Accumulated N-Carboxymethyl-Lysine (CML) can cause inflammation of organs, e.g. liver. Rosella flowers contain anthocyanin compound that has anti-glycation and antioxidant effects. This study aimed to determine the effect of ethanol extract of Rosella on CML serum level, IL-6 level, and NF-ƙB activation in the liver of mice fed with baked food. This study used post-test design using 25 Wistar rats aged 3-4 months old that were divided into 5 groups, namely negative control group, positive control group, treatment groups given Rosella extract dose of 200mg.kgBW -1 , 300mg.kgBW -1 , and 400mg.kgBW -1 . Baked feed was given for 12 weeks, and Rosella ethanol extract was administered in the 9 th to 12 th week. The examination on CML serum and IL-6 of the liver was using ELISA method. Immunofluorescent staining was used to determine NF-ƙB activation in the liver using a confocal microscope. CML serum is proven to increase significantly (p = 0.000). The effective dose of Rosella flower extract to prevent CML Serum increase is 200 mg.kgBW -1 , whereas a dose of 400 mg.kgBW -1 can decrease IL-6 level and NF-ƙB activation. Ethanol extract of Rosella flower decrease the levels of N-carboxymethyl-lysine serum, IL-6, and NF-ƙB activation in the liver of rats given baked-food diet. Keywords: Advanced Glycation Endproducts (AGEs), Interleukin-6, N-Carboxymethyl-lysine, Rosella.