Protein-bound Advanced Glycation Endproducts Research Articles

Protein-bound AGEs derived from methylglyoxal induce pro-inflammatory response and barrier integrity damage in epithelial cells by disrupting the retinol metabolism.

Advanced glycation end-products (AGEs) are complex and heterogeneous compounds widely present in processed foods. Previous studies evidenced the adverse effects of AGEs on gut homeostasis, but the precise pathological mechanisms and molecular pathways responsible for the disruption of intestinal barrier integrity by AGEs remain incompletely elucidated. In this study, protein-bound AGEs (BSA-MGO), the most common type of dietary AGE, were prepared by methylglyoxal-mediated glycation, and an in vitro human epithelial colorectal adenocarcinoma (Caco-2) cell model was employed to evaluate the impact of protein-bound AGEs on gut epithelial function. Results showed that exposure to BSA-MGO significantly increased the permeability of Caco-2 cell monolayers as evidenced by the decreased transepithelial electrical resistance value, increased paracellular transport of FITC-dextran, and down-regulated tight-junction proteins. In parallel, BSA-MGO induced pro-inflammatory responses and oxidative stress in the monolayers. Transcriptomic profiling further revealed that BSA-MGO disrupted the retinol metabolism, thereby contributing to the barrier integrity damage in epithelial cells. Overall, these results provide valuable insights into the disrupting effects of dietary AGEs on intestinal barrier function, and the perturbed pathways present potential targets for further exploration of the molecular mechanisms underlying the detrimental effect of processed foods on gut health.

Investigation of Advanced Glycation End-Products, α-Dicarbonyl Compounds, and Their Correlations with Chemical Composition and Salt Levels in Commercial Fish Products.

The contents of free and protein-bound advanced glycation end-products (AGEs) including Nε-carboxymethyllysine (CML) and Nε-carboxyethyllysine (CEL), along with glyoxal (GO), methylglyoxal (MGO), chemical components, and salt in commercially prepared and prefabricated fish products were analyzed. Snack food classified as commercially prepared products exhibited higher levels of GO (25.00 ± 3.34-137.12 ± 25.87 mg/kg of dry matter) and MGO (11.47 ± 1.39-43.23 ± 7.91 mg/kg of dry matter). Variations in the contents of free CML and CEL increased 29.9- and 73.0-fold, respectively. Protein-bound CML and CEL in commercially prepared samples were higher than those in raw prefabricated ones due to the impact of heat treatment. Levels of GO and MGO demonstrated negative correlations with fat (R = -0.720 and -0.751, p < 0.05) in commercially prepared samples, whereas positive correlations were observed (R = 0.526 and 0.521, p < 0.05) in raw prefabricated ones. The heat-induced formation of protein-bound CML and CEL showed a negative correlation with the variations of GO and MGO but was positively related to protein levels in prefabricated products, suggesting that GO and MGO may interact with proteins to generate AGEs during heating. The influence of NaCl on the formation of GO and MGO exhibited variations across different fish products, necessitating further investigation.

Isotope dilution-HPLC-MS/MS to investigate the production patterns and possible pathways of free and protein-bound AGEs and 4-MI in cookies

A qualitative and quantitative method for detecting free and protein-bound advanced glycation end products (AGEs) and 4-methylimidazole (4-MI) was established using isotope dilution-HPLC-MS/MS, and successfully applied in cookies and model systems. The effects of different temperatures (160–220 °C) on the formation of free and protein-bound AGEs and 4-MI in cookies were discussed, and the possible model systems (Maillard reaction pathway 1 using wheat gluten protein + glucose + sucrose; direct addition pathway 1 using wheat gluten protein + CML/CEL/4-MI) of protein-bound AGEs and 4-MI were verified. The results showed that the contents of protein-bound CML, CEL, and 4-MI were higher than free content with a tendency of increasing first and subsequently decreasing with temperature, reaching a maximum at 200 °C in cookies. In the model systems, the levels of protein-bound CML, CEL, and 4-MI are higher than those of free CML, CEL, and 4-MI. The protein-bound CML, CEL, and 4-MI accounted for 90.73, 87.64, and 97.56% of the total amount in the model system 1, while accounting for 68.19, 59.00, and 50.96% in the model system 2, respectively. In comparison, protein-bound CML, CEL, and 4-MI could be easily generated directly by Maillard reaction.

Plasma advanced glycation end products and the subsequent risk of microvascular complications in type 1 diabetes in the DCCT/EDIC

IntroductionTo assess impact of glycemic control on plasma protein-bound advanced glycation end products (pAGEs) and their association with subsequent microvascular disease.Research design and methodsEleven pAGEs were measured by liquid chromatography-mass...

Advanced glycation endproducts and dicarbonyls in end-stage renal disease: associations with uraemia and courses following renal replacement therapy.

BackgroundEnd-stage renal disease (ESRD) is strongly associated with cardiovascular disease (CVD) risk. Advanced glycation endproducts (AGEs) and dicarbonyls, major precursors of AGEs, may contribute to the pathophysiology of CVD in ESRD. However, detailed data on the courses of AGEs and dicarbonyls during the transition of ESRD patients to renal replacement therapy are lacking.MethodsWe quantified an extensive panel of free and protein-bound serum AGEs [N∈-(carboxymethyl)lysine (CML), N∈-(carboxyethyl)lysine (CEL), Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)ornithine (MG-H1)], serum dicarbonyls [glyoxal (GO), methylglyoxal (MGO), 3-deoxyglucosone (3-DG)] and tissue AGE accumulation [estimated by skin autofluorescence (SAF)] in a combined cross-sectional and longitudinal observational study of patients with ESRD transitioning to dialysis or kidney transplantation (KTx), prevalent dialysis patients and healthy controls. Cross-sectional comparisons were performed with linear regression analyses, and courses following renal replacement therapy were analysed with linear mixed models.ResultsFree and protein-bound AGEs, dicarbonyls and SAF were higher in chronic kidney disease (CKD) Stage 5 non-dialysis (CKD 5-ND; n = 52) and CKD Stage 5 dialysis (CKD 5-D; n = 35) than in controls (n = 42). In addition, free AGEs, protein-bound CML, GO and SAF were even higher in CKD 5-D than in CKD5-ND. Similarly, following dialysis initiation (n = 43) free and protein-bound AGEs, and GO increased, whereas SAF remained similar. In contrast, following KTx (n = 21), free and protein-bound AGEs and dicarbonyls, but not SAF, markedly declined.ConclusionsAGEs and dicarbonyls accumulate in uraemia, which is even exaggerated by dialysis initiation. In contrast, KTx markedly reduces AGEs and dicarbonyls. Given their associations with CVD risk in high-risk populations, lowering AGE and dicarbonyl levels may be valuable.

Hepatic Fat Content and Liver Enzymes Are Associated with Circulating Free and Protein-Bound Advanced Glycation End Products, Which Are Associated with Low-Grade Inflammation: The CODAM Study.

Advanced glycation end products (AGEs) accumulate in fatty livers and may contribute to low-grade inflammation (LGI), potentially via their receptor, RAGE. It is unknown if the AGE accumulation in fatty livers results in elevated circulating AGEs. In a cohort study, we investigated the association of liver fat and hepatocellular damage with circulating AGEs and soluble RAGE (sRAGE) and subsequently the association of circulating AGEs and sRAGE with LGI. Cross-sectional associations of liver fat percentage (eLF%; ln-transformed) and liver enzymes (LE score; standardized) with circulating AGEs (free CML, CEL, and MG-H1 in nM and protein-bound CML, CEL, and pentosidine in nmol/mmol lysine; ln-transformed) and sRAGE (pg/ml, ln-transformed) and additionally of AGEs and sRAGE with LGI (standardized) were determined by multiple linear regression. eLF% was positively associated with circulating free CEL (β = 0.090; 95% CI 0.041; 0.139) but inversely with protein-bound CML (β = −0.071; 95% CI -0.108; -0.034). Similarly, the LE score was positively associated with free CML (β = 0.044; 95% CI 0.012; 0.076) and CEL (β = 0.040; 95% CI 0.009; 0.072) but inversely with protein-bound CML (β = −0.037; 95% CI -0.060; -0.013). Free CML (β = 0.297; 95% CI 0.049; 0.545) was positively associated with LGI, while protein-bound CML (β = −0.547; 95% CI -0.888; -0.207) was inversely associated, although this association was absent after adjustment for BMI. eLF% and LE score were not associated with sRAGE and sRAGE not with LGI after adjustment for BMI. Liver fat and enzymes were positively associated with circulating free AGEs, which were associated with LGI. In contrast, inverse relations were observed of liver fat and enzymes with circulating protein-bound AGEs and of protein-bound AGEs with LGI. These data suggest that hepatic steatosis and inflammation affect the formation and degradation of hepatic protein-bound AGEs resulting in elevated circulating free AGE levels. These alterations in AGE levels might influence LGI, but this is likely independent of RAGE.

Nutritional supplements modulate fluorescent protein-bound advanced glycation endproducts and digestive enzymes related to type 2 diabetes mellitus.

BackgroundChronic hyperglycemia enhances the formation of advanced glycation endproducts (AGEs) and reactive oxygen species (ROS), contributing to diabetic complications. Thus, controlling blood glucose levels, inhibiting the formation of AGEs and reducing ROS are key therapeutic targets in early stage type 2 diabetes.MethodsThe inhibitory effects of seven commercial liquid nutritional supplements against carbohydrate hydrolysing enzymes, α-amylase and α-glucosidase, was determined by dinitrosalicylic (DNS) reagent and p-nitrophenyl-α-D-glucopyranoside solution, respectively. Antiglycation activity was determined using the formation of fluorescent protein-bound AGEs. Total phenolic and flavonoid content and antioxidant properties (1,1-diphenyl-2-picrylhydrazyl antioxidant activity (DPPH) and ferric reducing antioxidant power (FRAP)) were determined for correlation among these components and inhibitory activities.ResultsSamoan noni juice showed the greatest inhibitory effects against α-amylase, whereas chlorophyll extracts showed the greatest inhibitory effect against α-glucosidase. Inhibition of α-glucosidase correlated with TFC (r2 = 0.766; p < 0.01) and FRAP (r2 = 0.750; p < 0.01) whereas no correlation was observed for α-amylase inhibition. All supplements inhibited fluorescent protein-bound AGEs, with the greatest effect exerted by Olive Leaf Extract, Blood Sugar Support (IC50 = 0.5 mg/ml). The IC50 values negatively correlated with TPC (r2 = −0.707; p < 0.001) and DPPH scavenging activities (r2 = 0.515; p < 0.05).ConclusionThe findings of this study highlight the potential of liquid nutritional supplements in managing and treating type 2 diabetes mellitus.

Serum advanced glycation endproducts are associated with left ventricular dysfunction in normal glucose metabolism but not in type 2 diabetes: The Hoorn Study.

To investigate whether serum advanced glycation endproducts are associated with left ventricular systolic and diastolic function in participants with normal glucose metabolism, impaired glucose metabolism and type 2 diabetes mellitus. Participants from a cross-sectional, population-based study (n = 280 with normal glucose metabolism, n = 171 with impaired glucose metabolism, n = 242 with type 2 diabetes mellitus) underwent echocardiography. Serum protein-bound advanced glycation endproducts [i.e. Nε-(carboxymethyl)lysine, pentosidine and Nε-(carboxyethyl)lysine] were measured. Linear regression analyses were used and stratified according to glucose metabolism status. In normal glucose metabolism, higher Nε-(carboxymethyl)lysine and pentosidine levels were associated with worse diastolic function (left atrial volume index and left atrial volume × left ventricular mass index product term) and higher Nε-(carboxymethyl)lysine and Nε-(carboxyethyl)lysine levels with worse systolic function (ejection fraction). In impaired glucose metabolism, a similar pattern emerged, though less consistent. In type 2 diabetes mellitus, these associations were non-existent for diastolic function or even reversed for systolic function. This suggests that serum advanced glycation endproducts are associated with impaired left ventricular function in normal glucose metabolism, but that with deteriorating glucose metabolism status, serum advanced glycation endproducts may not mirror heart failure risk.

Markers of and Risk Factors for the Development and Progression of Diabetic Kidney Disease

Diabetic kidney disease (DKD) occurs in 25%-40% of patients with diabetes. Given the dual problems of a significant risk of progression from DKD to end-stage renal disease (ESRD) and increased cardiovascular morbidity and mortality, it is important to identify patients at risk of DKD and ESRD and initiate protective renal and cardiovascular therapies. The importance of preventive therapy is emphasized further by worldwide increases in the incidence of diabetes. This review summarizes the evidence regarding the prognostic value and benefits of targeting established and novel risk markers for DKD development and progression. Family history of DKD, smoking history, and glycemic, blood pressure, and plasma lipid level control are established factors for identifying people at greatest risk of DKD development and progression. Absolute albumin excretion rate (AER) and glomerular filtration rate (GFR) measurements also are important, although AER categorization generally lacks the necessary specificity and sensitivity, and estimates of declining GFR are compromised by methodological limitations for GFRs in the normal-to-high range. Emerging risk markers for progressive loss of kidney function include markers of oxidation and inflammation, profibrotic cytokines, uric acid, advanced glycation end products, functional and structural markers of vascular dysfunction, kidney structural changes, and tubular biomarkers. Among these, the most promising are serum uric acid and soluble tumor necrosis factor receptor (type 1 and type 2) levels, especially in relation to GFR changes. At present, these can only be considered as risk markers because they only identify an individual at increased risk of progressive DKD and not necessarily related to the causal pathway promoting kidney damage. Further work is needed to establish whether modulating these factors improves the prognosis in DKD. Although change in urinary peptidome levels also is a promising marker, there currently is neither a clinical assay nor adequate studies defining its prognostic value. Until these or other novel markers become available for clinical use, predictive accuracy often may be increased with greater attention to established markers.

Protein-bound advanced glycation endproducts (AGEs) as bioactive amino acid derivatives in foods

The Maillard reaction or nonenzymatic browning is of outstanding importance for the formation of flavour and colour of heated foods. Corresponding reactions, also referred to as "glycation", are known from biological systems, where the formation of advanced glycation endproducts (AGEs) shall play an important pathophysiological role in diabetes and uremia. In this review, pathways leading to the formation of individual protein-bound lysine and arginine derivatives in foods are described and nutritional consequences resulting from this posttranslational modifications of food proteins are discussed.

Inhibitors of advanced glycation end product-associated protein cross-linking

The reaction of lens proteins with sugars over time results in the formation of protein-bound advanced glycation end products (AGEs). The most damaging element of AGE formation may be the synthesis of protein-protein cross-links in long-lived proteins, such as collagen or lens crystallins. A quantitative cross-linking assay, involving the sugar-dependent incorporation of [U- 14C]lysine into protein, was employed to determine the efficacy of a variety of potential cross-linking inhibitors. Reaction mixtures contained 5.0 mM l-threose, 2.5 μCi [ 14C]lysine (1.0 mCi/mmole), 5.0 mg/ml bovine lens proteins, 0–10 mM inhibitor and 1.0 mM DTPA in 100 mM phosphate buffer, pH 7.0. Of 17 potential inhibitors tested, 11 showed 50% inhibition or less at 10 mM. The dicarbonyl-reactive compounds 2-aminoguanidine, semicarbazide and o-phenylenediamine inhibited 50% at 2.0 mM, whereas 10 mM dimethylguanidine had no effect. Several amino acids failed to compete effectively with [ 14C]lysine in the cross-linking assay; however, cysteine inhibited 50% at 1.0 mM. This was likely due to the sulfhydryl group of cysteine, because 3-mercaptopropionic acid and reduced glutathione exhibited similar activity. Sodium metabisulfite had the highest activity, inhibiting 50% at only 0.1–0.2 mM. Protein dimer formation, as determined by SDS-PAGE, was inhibited in a quantitatively similar manner. The dicarbonyl-reactive inhibitors and the sulfur-containing compounds produced similar inhibition curves for [ 14C]lysine incorporation over a 3 week assay with 250 mM glucose. A much lesser effect was observed on either the incorporation of [ 14C]glucose, or on fluorophore formation (360/420 nm), suggesting that non-cross-link fluorophores were also formed. The inhibitor data were consistent with cross-linking by a dicarbonyl intermediate. This was supported by the fact that the inhibitors were uniformly less effective when the 5.0 mM threose was replaced by either 3.0 mM 3-deoxythreosone or 3.0 mM threosone.

Non-enzymatic glycosylation of a type I collagen matrix: effects on osteoblastic development and oxidative stress

BackgroundThe tissue accumulation of protein-bound advanced glycation endproducts (AGE) may be involved in the etiology of diabetic chronic complications, including osteopenia. The aim of this study was to investigate the effect of an AGE-modified type I collagen substratum on the adhesion, spreading, proliferation and differentiation of rat osteosarcoma UMR106 and mouse non-transformed MC3T3E1 osteoblastic cells. We also studied the role of reactive oxygen species (ROS) and nitric oxide synthase (NOS) expression on these AGE-collagen mediated effects.ResultsAGE-collagen decreased the adhesion of UMR106 cells, but had no effect on the attachment of MC3T3E1 cells. In the UMR106 cell line, AGE-collagen also inhibited cellular proliferation, spreading and alkaline phosphatase (ALP) activity. In preosteoblastic MC3T3E1 cells (24-hour culture), proliferation and spreading were significantly increased by AGE-collagen. After one week of culture (differentiated MC3T3E1 osteoblasts) AGE-collagen inhibited ALP activity, but had no effect on cell number. In mineralizing MC3T3E1 cells (3-week culture) AGE-collagen induced a decrease in the number of surviving cells and of extracellular nodules of mineralization, without modifying their ALP activity. Intracellular ROS production, measured after a 48-hour culture, was decreased by AGE-collagen in MC3T3E1 cells, but was increased by AGE-collagen in UMR106 cells. After a 24-hour culture, AGE-collagen increased the expression of endothelial and inducible NOS, in both osteoblastic cell lines.ConclusionsThese results suggest that the accumulation of AGE on bone extracellular matrix could regulate the proliferation and differentiation of osteoblastic cells. These effects appear to depend on the stage of osteoblastic development, and possibly involve the modulation of NOS expression and intracellular ROS pathways.