Nonenzymatic glycation of DNA nucleosides with reducing sugars

Reducing sugars can react with the free amino groups of proteins to form a heterogeneous group of compounds known as advanced glycation endproducts (AGEs) or Maillard reaction products. The objective of this investigation was to monitor the nonenzymatic glycation of DNA nucleosides and to characterize the formation of nucleoside AGEs using capillary electrophoresis (CE), high-performance liquid chromatography (HPLC), UV fluorescence spectroscopy, and mass spectrometry. Deoxyguanosine, deoxyadenosine, deoxythymidine, and deoxycytidine were used as the model nucleosides and were incubated over time with glucose, galactose, or glyceraldehyde. Under increasing concentrations and time, deoxyguanosine exhibited the highest rate of glycation with glyceraldehyde. Deoxyadenosine and deoxycytidine exhibited comparable reactivity with glyceraldehyde and no appreciable reactivity with galactose or glucose. No reactivity was observed between deoxythymidine and the sugars. A combination of CE, HPLC, UV fluorescence spectroscopy, and mass spectrometry provided a convenient method for characterizing nucleoside AGEs and for monitoring the physical factors that influence the formation of sugar adducts of DNA nucleosides.     

Advanced glycation and endothelial functions: a link towards vascular complications in diabetes

The formation of advanced glycation end-products (AGEs), also called the Maillard reaction, occurs ubiquitously and irreversibly in patients with diabetes mellitus, and its consequences are especially relevant to vascular dysfunctions. The interaction of AGEs with their receptors (RAGE) has been implicated in the development of vascular complications. This interaction elicits remarkable vascular cell changes analogous to those observed in diabetes mellitus, including angiogenic and thrombogenic responses of endothelial cells, increased oxidative stress, and functional alterations in vascular tone control. This review focuses on AGEs formation, the interaction with their specific receptors and how the triggered intracellular events determine functional alterations of vascular endothelium. Finally, some potential pharmacological approaches undertaken to circumvent the deleterious effects of AGEs are also discussed.     

Extract of Cassiae semen attenuates diabetic nephropathy via inhibition of advanced glycation end products accumulation in streptozotocin-induced diabetic rats

Chronic hyperglycemia leads to the formation of advanced glycation end products (AGEs), which accelerates the development of diabetic complications. Previous studies have shown that extract of Cassiae semen (CS), the seed of Cassia tora, has inhibitory activity on AGEs formation in vitro and reduces transforming growth factor-beta1 (TGF-β1) and extracellular matrix protein expression via inhibition of AGEs-mediated signaling in glomerular mesangial cells. In this study, to examine the preventive effects of CS extract on the development of diabetic nephropathy in vivo, streptozotocin (STZ)-injected diabetic rats were orally administered CS extract (200 mg/kg body weight/day) for 12 weeks. Serum glucose, triglycerides, and total cholesterol in diabetic rats were significantly higher compared to control rats. CS or aminoguanidine (AG) treatment significantly reduced these factors. Proteinuria and creatinine clearance were also significantly decreased in the CS-treated group compared with the untreated diabetic group. The CS-treated group had significantly inhibited COX-2 mRNA and protein, which mediates the symptoms of inflammation in the renal cortex of diabetic rats. Furthermore, histopathological studies of kidney tissue showed that in diabetic rats, AGEs, the receptor for AGEs, TGF-β1, and collagen IV were suppressed by CS treatment. Our data suggest that oral treatment of CS can inhibit the development of diabetic nephropathy via inhibition of AGEs accumulation in STZ-induced diabetic rats.     

Nonenzymatic glycation of guanosine 5'-triphosphate by glyceraldehyde: an in vitro study of AGE formation

Guanosine 5'-triphosphate (GTP) plays a significant role in the bioenergetics, metabolism, and signaling of cells; consequently, any modifications to the structure of the molecule can have profound effects on a cell's survival and function. Previous studies in our laboratory demonstrated that like proteins, purines, and pyrimidines can nonenzymatically react with sugars to generate advanced glycation endproducts (AGEs) and that these AGEs can form in vitro under physiological conditions. The objective of this investigation was twofold. First, it was to evaluate the susceptibility of ATP, GTP, CTP, and TTP to nonenzymatic modification by D-glucose and DL-glyceraldehyde, and second to assess the effect of various factors such as temperature, pH and incubation time, and sugar concentration on the rate and extent of nucleotide triphosphate AGE formation. Of the four nucleotide triphosphates that were studied, only GTP was significantly reactive forming a heterogeneous group of compounds with DL-glyceraldehyde. D-Glucose exhibited no significant reactivity with any of the nucleotide triphosphates, a finding that was supported by UV and fluorescence spectroscopy. Capillary electrophoresis, high-performance liquid chromatography and mass spectrometry allowed for a thorough analysis of the glycated GTP products and demonstrated that the modification of GTP by dl-glyceraldehyde occurred via the classical Amadori pathway.     

Pyridoxamine, an inhibitor of advanced glycation and lipoxidation reactions: a novel therapy for treatment of diabetic complications

Pyridoxamine (PM), originally described as a post-Amadori inhibitor of formation of advanced glycation end-products (AGEs), also inhibits the formation of advanced lipoxidation end-products (ALEs) on protein during lipid peroxidation reactions. In addition to inhibition of AGE/ALE formation, PM has a strong lipid-lowering effect in streptozotocin (STZ)-induced diabetic and Zucker obese rats, and protects against the development of nephropathy in both animal models. PM also inhibits the development of retinopathy and neuropathy in the STZ-diabetic rat. Several products of reaction of PM with intermediates in lipid autoxidation have been identified in model reactions in vitro and in the urine of diabetic and obese rats, confirming the action of PM as an AGE/ALE inhibitor. PM appears to act by a mechanism analogous to that of AGE-breakers, by reaction with dicarbonyl intermediates in AGE/ALE formation. This review summarizes current knowledge on the mechanism of formation of AGE/ALEs, proposes a mechanism of action of PM, and summarizes the results of animal model studies on the use of PM for inhibiting AGE/ALE formation and development of complications of diabetes and hyperlipidemia.     

Reduced formation of advanced glycation endproducts via interactions between glutathione peroxidase 3 and dihydroxyacetone kinase 1

Dihydroxyacetone (DHA) induces the formation of advanced glycation endproducts (AGEs), which are involved in several diseases. Earlier, we identified dihydroxyacetone kinase 1 (Dak1) as a candidate glutathione peroxidase 3 (Gpx3)-interacting protein in Saccharomyces cerevisiae. This finding is noteworthy, as no clear evidence on the involvement of oxidative stress systems in DHA-induced AGE formation has been found to date. Here, we demonstrate that Gpx3 interacts with Dak1, alleviates DHA-mediated stress by upregulating Dak activity, and consequently suppresses AGE formation. Based on these results, we propose that defense systems against oxidative stress and DHA-induced AGE formation are related via interactions between Gpx3 and Dak1.     

Aminosalicylic acid reduces the antiproliferative effect of hyperglycaemia,advanced glycation endproducts and glycated basic fibroblast growth factor in cultured bovine aortic endothelial cells: Comparison with aminoguanidine

Hyperglycaemia reduces proliferation of bovine aortic endothelial cells in vitro. A similar effect in vivo may contribute to long-term complications of diabetes such as impaired wound-healing and retinopathy.We report the effect of increased glucose concentrations, glycated basic fibroblast growth factor (FGF-2) and bovine serum albumin-derived advanced glycation endproducts (BSA-AGE) on the proliferation of bovine aortic endothelial cells.Glucose (30 and 50 mmol/l) had an antiproliferative effect on endothelial cells. This effect may be mediated through reduced mitogenic activity of FGF-2. The glycation of FGF-2 with 250 mmol/l glucose-6-phosphate led to reduced mitogenic activity compared to native FGF-2. BSA-AGE at concentrations of 10, 50 and 250 g/ml had an antiproliferative effect on cultured endothelial cells.Aminosalicylic acid at a concentration of 200 mol/l proved to be more effective than equimolar concentrations of aminoguanidine in protecting endothelial cells against the antiproliferative effects of both high (30 mmol/l) glucose and 50 g/ml BSA-AGE. FGF-2 glycated in the presence of 4 mmol/l aminosalicylic acid or aminoguanidine retained mitogenic activity compared to that glycated in their absence.Compounds like aminoguanidine and, in particular, aminosalicylic acid protect endothelial cells against glucose-mediated toxicity and may therefore have therapeutic potential.     

Reduced cell replication and induction of apoptosis by advanced glycation end products in rat Schwann cells

We investigated the effects of advanced glycation end products (AGEs) derived from glucose, glyceraldehyde, and glycolaldehyde (designated as AGE-1, -2, and -3, respectively) on the viability, replication rate, and cytokine production of cultured Schwann cells. AGE-2 and -3, but not AGE-1, induced apoptosis, and significantly decreased the viability measured by MTT assay. The decrease was prevented completely by antioxidant alpha-lipoic acid and was prevented partially by p38 mitogen-activated protein kinase inhibitor SB202190. The decrease in mitochondrial membrane potential by AGE-2 and -3 was also observed. In addition, AGE-2 and -3 significantly suppressed the replication rate as shown by reduced bromodeoxyuridine uptake, whereas they enhanced the release of TNF-alpha and IL-1beta into the medium and activated nuclear factor-kappaB. The effects of AGE-1 on these measures were equivocal. The series of events elicited by AGE-2 and -3 may be responsible for some of the aspects of pathogenetic mechanisms in patients with diabetic neuropathy.     

Molecular characteristics of methylglyoxal-modified bovine and human serum albumins. Comparison with glucose-derived advanced glycation endproduct-modified serum albumins

The amino acid modification, gel filtration chromatographic, and electrophoretic characteristics of bovine and human serum albumins irreversibly modified by methylglyoxal (MG-SA) and by glucose-derived advanced glycation endproducts (AGE-SA) were investigated. Methylglyoxal selectively modified arginine residues at low concentration (1 mM); at high methylglyoxal concentration (100 mM), the extent of arginine modification increased and lysine residues were also modified. Both arginine and lysine residues were modified in AGE-SA. Analytical gel filtration HPLC of serum albumin derivatives suggested that the proportion of dimers and oligomers increased with modification in both low and highly modified MG-SA and AGE-SA derivatives relative to unmodified serum albumins. In SDS-PAGE analysis, dimers and oligomers of low-modified MG-SA were dissociated into monomers, but not in highly modified MG-SA. MG-SA had increased anodic electrophoretic mobility under nondenaturing conditions atpH 8.6, indicating an increased net negative charge, which increased with extent of modification; highly modified MG-SA and AGE-SA had similar high electrophoretic mobilities. MG-SA derivatives were fluorescent: the fluorescence was characteristic of the arginine-derived imidazoloneN -(5-methyl-4-imidazolon-2-yl)ornithine, but other fluorophores were also present. AGE-SA had similar fluorescence, attributed, in part, to glucose-derived imidazolones. AGE formed from glucose-modified proteins and AGE-like compounds formed from methylglyoxal-modified proteins may both be signals for recognition and degradation of senescent macromolecules.Abbreviations AGE advanced glycation endproduct - BSA bovine serum albumin - HSA human serum albumin - MG-SA methylglyoxal-modified serum albumin - MG-BSA methylglyoxal-modified bovine serum albumin - MG-HSA methylglyoxal-modified human serum albumin - AGE-SA AGE-modified serum albumin - AGE-BSA AGE-modified bovine serum albumin - AGE-HSA AGE-modified human serum albumin - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - HPLC high-performance liquid chromatography - FFI 2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole     

Expression and purification of the soluble isoform of human receptor for advanced glycation end products (sRAGE) from Pichia pastoris

RAGE is a multi-ligand receptor involved in various human diseases including diabetes, cancer or Alzheimer's disease. Engagement of RAGE by its ligands triggers activation of key cellular signalling pathways such as the MAP kinase and NF-kappaB pathways. Whereas the main isoform of RAGE is a transmembrane receptor with both extra- and intracellular domains, a secreted soluble isoform (sRAGE), corresponding to the extracellular part only, has the ability to block RAGE signalling and suppress cellular activation. Administration of sRAGE to animal models of cancer or multiple sclerosis blocked successfully tumour growth and the course of the autoimmune disease. These findings demonstrate that sRAGE may have a potential as therapeutic. We present here a fast and simple purification protocol of sRAGE from the yeast Pichia pastoris. The identity of the protein was confirmed by mass spectrometry and Western blot. The protein was N-glycosylated and 95-98% pure as judged by SDS-PAGE.     

Minimum stable structure of the receptor for advanced glycation end product possesses multi ligand binding ability

The receptor for advanced glycation end products (RAGE) is a multi-ligand receptor involved in the development of diabetic complications. Although the soluble form of the extracellular domain maintains the ability to bind multi-ligands, it is unstable and degrades into several peptide species during storage. Proteolysis with thrombin or factor Xa revealed several protease sensitive sites. Most sensitive site is located between Arg228 and Val229, and peptide bond next to Arg216, Arg116, Arg114 and Trp271 are also cleaved. Seven truncated extracellular domains of RAGE were engineered in order to obtain a stable soluble fragment. RAGE 143 (Ala23-Thr143) is not only protease resistant but also shows the same ligand-binding ability as that of the full-length extracellular domain. The resultant minimum RAGE 143 works as a stable recognition devise to detect advanced glycation end products (AGEs).     

Studies on advanced glycation end products by recent mass spectrometric techniques

Summary The results obtained by different mass spectrometric approaches in the field of advanced glycation of proteins are reported and discussed in detail in comparison with those obtained by other analytical methodologies (fluorescence and absorbance spectroscopies, radioimmunoassay, enzyme-linked immunosorbent assay). They have been subdivided in three main groups: analysis on degraded glycated proteins, direct analysis of glycated proteins and studies on the reaction between protected lysine and glucose. The general overview so achieved indicate mass spectrometry as a particularly valid analytical method in this field of research.     

Cellular oxidant stress and advanced glycation endproducts of albumin: caveats of the dichlorofluorescein assay

In order to understand the mechanism by which advanced glycation endproducts (AGEs) elicit oxidative stress, macrophage-like RAW264.7 cells were exposed to various AGE-albumins, and oxidant stress was estimated from the fluorescence of oxidized dichlorofluorescein using the microtiter plate assay. Strongest fluorescence was observed with methylglyoxal modified albumin (MGO-BSA) compared with native albumin. Similar effects that were prevented by arginine coincubation were seen with phenylglyoxal-BSA. MGO-BSA had increased affinity for Cu(2+) and Ca(2+), but was conformationally similar to native albumin. Surprisingly, the mere addition of unmodified albumin to cells suppressed the fluorescence of oxidized DCF. While, several site-directed mutants of human serum albumin (HSA), including C34S and recombinant domains II and III retained fluorescence suppressing activity, proteolytic digests, recombinant domain I, and several nonalbumin proteins failed to suppress. Kinetic and ANS binding studies suggested albumin quenches DCF fluorescence by binding to hydrophobic pockets in domains II and III and that MGO-BSA is less hydrophobic than BSA. Finally, BSA also prevented H(2)O(2) catalyzed DCF fluorescence more potently than MGO-BSA. These studies reveal important caveats of the widely used dichlorofluorescein assay and suggest methods other than the microtiter plate assay are needed to accurately assess cellular oxidant stress in presence of native or modified albumin.     

Advanced glycation endproducts and pro-inflammatory cytokines in transgenic Tg2576 mice with amyloid plaque pathology

Increased expression and altered processing of the amyloid precursor protein (APP) and generation of beta-amyloid peptides is important in the pathogenesis of amyloid plaques in Alzheimer's disease (AD). Transgenic Tg2576 mice overexpressing the Swedish mutation of human APP exhibit beta-amyloid deposition in the neocortex and limbic areas, accompanied by gliosis and dystrophic neurites. However, murine plaques appear to be less cross-linked and the mice show a lower degree of inflammation and neurodegeneration than AD patients. 'Advanced glycation endproducts (AGEs)', formed by reaction of proteins with reactive sugars or dicarbonyl compounds, are able to cross-link proteins and to activate glial cells, and are thus contributing to plaque stability and plaque-induced inflammation in AD. In this study, we analyze the tissue distribution of AGEs and the pro-inflammatory cytokines IL-1beta and TNF-alpha in 24-month-old Tg2576 mice, and compare the AGE distribution in these mice with a younger age group (13 months old) and a typical Alzheimer's disease patient. Around 70% of the amyloid plaque cores in the 24-month-old mice are devoid of AGEs, which might explain their solubility in physiological buffers. Plaque associated glia, which express IL-1beta and TNF-alpha, contain a significant amount of AGEs, suggesting that plaques, i.e. Abeta as its major component, can induce intracellular AGE formation and the expression of the cytokines on its own. In the 13-month-old transgenic mice, AGEs staining can neither be detected in plaques nor in glial cells. In contrast, AGEs are present in high amounts in both plaques and glia in the human AD patient. The data obtained in this show interesting differences between the transgenic mouse model and AD patients, which should be considered using the transgenic approach to test therapeutical strategies to eliminate plaques or to attenuate the inflammatory response in AD.     

The receptor for advanced glycation end products promotes bacterial growth at distant body sites in Staphylococcus aureus skin infection

The receptor for advanced glycation endproducts (RAGE) has been implicated in the regulation of skin inflammation. We here sought to study the role of RAGE in host defense during skin infection caused by Staphylococcus (S.) aureus, the most common pathogen in this condition. Wild-type (Wt) and RAGE deficient (rage^−/−) mice were infected subcutaneously with S. aureus and bacterial loads and local inflammation were quantified at regular intervals up to 8 days after infection. While bacterial burdens were similar in both mouse strains at the primary site of infection, rage^−/− mice had lower bacterial counts in lungs and liver. Skin cytokine and chemokine levels did not differ between groups. In accordance with the skin model, direct intravenous infection with S. aureus was associated with lower bacterial loads in lungs and liver of rage^−/− mice. Together these data suggest that RAGE does not impact local host defense during S. aureus skin infection, but facilitates bacterial growth at distant body sites.     

Plasma protein advanced glycation end products, carboxymethyl cysteine, and carboxyethyl cysteine, are elevated and related to nephropathy in patients with diabetes

In Diabetes Mellitus (DM), glucose and the aldehydes glyoxal and methylglyoxal modify free amino groups of lysine and arginine of proteins forming advanced glycation end products (AGEs). Elevated levels of these AGEs are implicated in diabetic complications including nephropathy. Our objective was to measure carboxymethyl cysteine (CMC) and carboxyethyl cysteine (CEC), AGEs formed by modification of free cysteine sulfhydryl groups of proteins by these aldehydes, in plasma proteins of patients with diabetes, and investigate their association with the albumin creatinine ratio (ACR, urine albumin (mg)/creatinine (mmol)), an indicator of nephropathy. Blood was collected from forty-two patients with type 1 and 2 diabetes (18–36 years) and eighteen individuals without diabetes (17–35 years). A liquid chromatography-mass spectrophotometric method was developed to measure plasma protein CMC and CEC levels. Values for ACR and hemoglobin A1C (HbA1C) were obtained. Mean plasma CMC (μg/l) and CEC (μg/l) were significantly higher in DM (55.73 ± 29.43, 521.47 ± 239.13, respectively) compared to controls (24.25 ± 10.26, 262.85 ± 132.02, respectively). In patients with diabetes CMC and CEC were positively correlated with ACR, as was HbA1C. Further, CMC or CEC in combination with HbA1C were better predictors of nephropathy than any one of these variables alone. These results suggest that glucose, glyoxal, and methylglyoxal may all be involved in the etiology of diabetic nephropathy.     

Novel inhibitors of advanced glycation endproducts

A number of natural or synthetic compounds as AGE inhibitors have been proposed, discovered or currently being advanced by others and us. We have identified two new classes of aromatic compounds; aryl- (and heterocyclic) ureido and aryl (and heterocyclic) carboxamido phenoxyisobutyric acids, and benzoic acid derivatives and related compounds, as potential inhibitors of glycation and AGE formation. Some of these novel compounds also showed "AGE-breaking" activities in vitro. Current evidence is that chelation of transition metals and/or trapping or indirect inhibition of formation of reactive carbonyl compounds are involved in the mechanisms of action of these novel AGE inhibitors and breakers. Here, we review the inhibitors of glycation and AGE-breakers published to date and present the results of our in vitro and in vivo investigations on a number of these novel AGE inhibitors. These AGE-inhibitors and AGE-breakers may find therapeutic use in the treatment of diseases that AGE formation and accumulation may be responsible for their pathogenesis such as diabetes, Alzheimer's, rheumatoid arthritis, and atherosclerosis.     

Advanced glycation end product ligands for the receptor for advanced glycation end products: biochemical characterization and formation kinetics

Advanced glycation end products (AGEs) accumulate with age and at an accelerated rate in diabetes. AGEs bind cell-surface receptors including the receptor for advanced glycation end products (RAGE). The dependence of RAGE binding on specific biochemical characteristics of AGEs is currently unknown. Using standardized procedures and a variety of AGE measures, the present study aimed to characterize the AGEs that bind to RAGE and their formation kinetics in vitro. To produce AGEs with varying RAGE binding affinity, bovine serum albumin (BSA) AGEs were prepared with 0.5M glucose, fructose, or ribose at times of incubation from 0 to 12 weeks or for up to 3 days with glycolaldehyde or glyoxylic acid. The AGE-BSAs were characterized for RAGE binding affinity, fluorescence, absorbance, carbonyl content, reactive free amine content, molecular weight, pentosidine content, and N-epsilon-carboxymethyl lysine content. Ribose-AGEs bound RAGE with high affinity within 1 week of incubation in contrast to glucose- and fructose-AGE, which required 12 and 6 weeks, respectively, to generate equivalent RAGE ligands (IC50=0.66, 0.93, and 1.7 microM, respectively). Over time, all of the measured AGE characteristics increased. However, only free amine content robustly correlated with RAGE binding affinity. In addition, detailed protocols for the generation of AGEs that reproducibly bind RAGE with high affinity were developed, which will allow for further study of the RAGE-AGE interaction.