Non-enzymatic Glycation Of Proteins Research Articles

Effects of Short-Term Streptozotocin-Induced Diabetes and Vitamin C on Platelet Non-Enzymatic Glycation

Diabetes mellitus is one of the most prevalent metabolic syndromes worldwide. Glycation, a chemical modification of proteins with reducing sugars, indicates a possible explanation for the association between hyperglycemia and the wide variety of tissue pathologies. Non-enzymatic glycation (NEG) of platelet proteins is one of the key mechanisms in the pathogenesis of diabetic complications and may be significant in diabetic atherothrombosis. The aim of this study was to investigate the effects of streptozotocin (STZ)-induced short-term experimental diabetes on the glycation of platelets and to find out if vitamin C affected this glycation. A total of 40 male Wistar albino rats, 200–250 g, were randomly divided into 4 groups (2 diabetic and 2 control groups). The diabetic groups were made diabetic by intraperitoneal injection of STZ (65 mg/kg, citrate buffer pH 4.5). By daily intraperitoneal injection, 80 mg/kg vitamin C (Roche, Turkey) was administered until the end of the experiment. Blood glucose levels of the diabetic groups were significantly higher than those at day 0 and also higher than those of the non-diabetic control groups. The changes in total protein, NEG and vitamin C levels were not statistically significant. Although the differences among the groups were not statistically significant, vitamin C administration increased NEG levels in the diabetic group. The results of this study demonstrate that 8 days of STZ-induced short-term diabetes did not cause a significant increase in NEG of platelets. However, the effect of vitamin C on platelet NEG needs to be further investigated.

The structural feature surrounding glycated lysine residues in human hemoglobin

Complications derived from diabetes mellitus are caused by nonenzymatic protein glycation at the specific sites. LC/MS/MS was performed for the identification of the tryptic peptides of glycated hemoglobins using glyceraldehyde. After the identification of the glycation or non-glycation site, computer analysis of the structure surrounding the sites was carried out using PDB data (1BZ0). Five glycated lysine residues (Lys-16(α), -56(α), -8(β), -82(β), and -144(β)) and four non-glycated lysine residues (Lys-7(α), -40(α), -99(α), and -132(β)) were identified. The non-glycated lysine residues, Lys-7(α), -40(α), and -132(β), are most likely to form electrostatic interactions with the β carboxyl group of Asp-74(α), C-terminal His-146(β), and Glu-7(β) by virtue of their proximity, which is 2.67-2.91 Å (N-O). Additionally, there are histidine residues within 4.55-7.38 Å (N-N) around eight sites except for Lys-7(α). We conclude that the following factors seem to be necessary for glycation of lysine residues: (i) the apparent absence of aspartate or glutamate residues to inhibit the glycation reaction by forming an electrostatic interaction, (ii) the presence of histidine residues for acid-base catalysis of the Amadori rearrangement, and (iii) the presence of an amino acid residue capable of stabilizing a phosphate during proton transfer.

Glycated lysine residues: a marker for non-enzymatic protein glycation in age-related diseases.

Nonenzymatic glycosylation or glycation of macromolecules, especially proteins leading to their oxidation, play an important role in diseases. Glycation of proteins primarily results in the formation of an early stage and stable Amadori-lysine product which undergo further irreversible chemical reactions to form advanced glycation endproducts (AGEs). This review focuses these products in lysine rich proteins such as collagen and human serum albumin for their role in aging and age-related diseases. Antigenic characteristics of glycated lysine residues in proteins together with the presence of serum autoantibodies to the glycated lysine products and lysine-rich proteins in diabetes and arthritis patients indicates that these modified lysine residues may be a novel biomarker for protein glycation in aging and age-related diseases.

Glycated Lysine Residues: A Marker for Non-Enzymatic Protein Glycation in Age-Related Diseases

Nonenzymatic glycosylation or glycation of macromolecules, especially proteins leading to their oxidation, play an important role in diseases. Glycation of proteins primarily results in the formation of an early stage and stable Amadori-lysine product which undergo further irreversible chemical reactions to form advanced glycation endproducts (AGEs). This review focuses these products in lysine rich proteins such as collagen and human serum albumin for their role in aging and age-related diseases. Antigenic characteristics of glycated lysine residues in proteins together with the presence of serum autoantibodies to the glycated lysine products and lysine-rich proteins in diabetes and arthritis patients indicates that these modified lysine residues may be a novel biomarker for protein glycation in aging and age-related diseases.

Effect of Micronutrients on Methylglyoxal-Mediated In Vitro Glycation of Albumin

Nonenzymatic glycation of long-lived proteins has been implicated in several complications related to age and diabetes. Dicarbonyl compounds such as methylglyoxal (MGO) have been identified as the predominant source for the formation of advanced glycation end-products (AGEs) in various tissues. We investigated the effect of 13 micronutrients on MGO-mediated in vitro glycation of bovine serum albumin (BSA), as formation of AGEs and protein carbonyls. BSA (10 mg/ml) was incubated at 37°C with 100 mM MGO for 24 hours, in presence of ascorbic acid, Trolox (water-soluble α-tocopherol analog), β-carotene, retinol, riboflavin, thiamin, folic acid, niacin, pyridoxine, zinc, iron, manganese, and selenium. Fluorescence was measured at the wavelength pair of 370 and 440 nm as an index of the formation of AGEs and spectra were recorded for promising interactions at λex=280 nm and λex=370 nm. Within four standard antiglycating agents, aminoguanidine showed highest inhibitory response for BSA glycation followed by quercetin, gallic acid, and tannic acid. Promising antiglycation potential was seen for Trolox, riboflavin, Zn, and Mn as evidenced by decrease in the formation of AGEs and protein carbonyls.

Impairment of human keratinocyte mobility and proliferation by advanced glycation end products-modified BSA

The migration and proliferation of keratinocytes is critical to wound re-epithelialization and defects in this function are associated with the clinical phenomenon of chronic non-healing wounds. Advanced glycation end products (AGEs) occur through non-enzymatic glycation of long-lived proteins in diabetes and play important roles in diabetic complications. However, specific roles for AGEs in keratinocyte migration and proliferation, and the underlying molecular mechanisms, have not been fully established. The aim of the current study was to elucidate the interaction between AGE-modified bovine serum albumin (AGE-BSA) and keratinocytes. As a result, we found that AGE-BSA had no effect on the viability of keratinocytes for up to 48h of incubation with 50μg/ml of AGE-BSA. AGE-BSA (but not non-glycated BSA) exerted a concentration-dependent suppression of keratinocyte migration at a range of concentrations. The expression of matrix metalloproteinase-9 (MMP-9) was significantly up-regulated in keratinocytes incubated with increasing AGE-BSA, but tissue inhibitor of metalloproteinases-1 (TIMP-1) expression was down-regulated. AGE-BSA also profoundly depressed phospho-focal adhesion kinase-Tyr397 (p-FAK) and α2β1 integrin expression, while total-FAK expression levels remained constant, in keratinocytes. The proliferative capacity of keratinocytes was diminished after 72h AGE-BSA incubation. Taken together, these findings suggested that in the presence of AGE-BSA, keratinocytes lose their migratory and proliferation abilities. These data also indicated that, in the context of the chronic hyperglycemia in diabetes, the effects of AGE-BSA on keratinocyte migration might be mediated through MMP-9/TIMP-1, p-FAK and α2β1 integrin.

S100 proteins in health and disease

One class of proteins that has been increasingly emerging as a potentially important group of both molecular key players and biomarkers in the etiology, progression, manifestation and therapy of various inflammatory, neurodegenerative, metabolic and neoplastic disorders is the S100 family. S100 proteins are small, acidic, calcium-binding proteins, characterized by the presence of two calcium-binding EF-hand motifs, and found exclusively in vertebrates. The first member was identified in the bovine nervous system by Moore (1965). The name S100 was derived from the observation of the protein fraction remaining soluble after precipitation with 100% saturated ammonium sulfate at neutral pH. Subsequent studies demonstrated that this fraction contained predominantly two proteins, S100A1 and S100B. Since the first isolation and characterization of these 2 S100 proteins, at least 23 additional proteins have been assigned as members of the S100 family in humans (Marenholz et al. 2004, 2006). Twenty-one of them (S100A1–S100A18, and the multidomain proteins trichohyalin, filaggrin and repetin) are coded by genes clustered at chromosome locus 1q21, known as the epidermal differentiation complex, while the other genes belonging to the subfamilies of S100B, S100P, S100Z and S100G are, respectively, located at chromosome loci 21q22, 4p16, 5q14 and Xp22 (Santamaria-Kisiel et al. 2006). S100 proteins form homodimer, heterodimer and even oligomeric molecular assemblies and are expressed in a tissueand cell-specific manner, suggesting that each S100 protein may perform different functions (Donato 1999; Fritz and Heizmann 2006). Functional complexity and diversification of S100 proteins have been further achieved by differences in localization, e.g., intracellularly in cytoplasm and/or nucleus, extracellularly in various body fluid compartments, by action as autocrine or paracrine effectors, and by exhibition of different affinities toward calcium ions resulting in various degrees of conformational change and modes of interaction with a whole host of specific target proteins (Donato 1999; Leclerc et al. 2009). Moreover, several S100 proteins are known to bind to other divalent metal ions, such as magnesium, zinc and the transition metal copper with high affinity, perhaps implicating them additionally in the homeostasis of toxic metals and related pathophysiological conditions (Moroz et al. 2009). All of these result in a tremendous spectrum of pleiotropic intraand extracellular functions. In this regard, through, e.g., inhibition of protein phosphorylation, regulation of transcriptional factors, modulation of enzyme activity and cytoskeletal dynamics, S100 proteins have been linked to vital cellular processes, including cell cycle regulation, cell growth and differentiation, transcription, cell motility and invasion, extracellular signal transduction and intercellular adhesion (Santamaria-Kisiel et al. 2006, Leclerc et al. 2009). Among natural targets of extracellular S100 proteins, the multiligand or pattern recognition receptor for advanced glycation end products (RAGE) has gained significant importance (Hofmann et al. 1999; Leclerc et al. 2009). RAGE is a signal transduction receptor of the immunoglobulin superfamily and was first described as a receptor for end products of non-enzymatic glycation and glycoxidation of proteins (Schmidt et al. 1992). Importantly, RAGE also transduces signals stimulated by non-glycated proteins that J. Pietzsch (&) Department of Radiopharmaceutical Biology, Institute of Radiopharmacy, Research Center Dresden-Rossendorf, POB 510119, 01314 Dresden, Germany e-mail: j.pietzsch@fzd.de

Advanced glycation end products and its receptor (RAGE) are increased in patients with COPD

Advanced Glycation End products (AGEs) are the products of nonenzymatic glycation and oxidation of proteins and lipids. Formation of AGEs is increased in response to hyperglycaemia, reactive oxygen species and ageing. AGEs are proinflammatory and can modify the extracellular matrix. RAGE (Receptor for Advanced Glycation End Products) mediates some of the effects of AGEs. Formalin-fixed lung tissue from patients who had lobectomy for bronchial carcinoma was used to investigate the presence of AGEs and RAGE. Subjects were divided into those with COPD and controls. Immunostaining for AGEs and RAGE was performed and the intensity of staining measured. Subjects with COPD and controls were similar in age and smoking history but FEV(1)% predicted was lower for COPD than controls. Intensity of staining for AGEs was greater in the airways (p = 0.025) and alveolar walls (p = 0.004) in COPD. Intensity of staining for RAGE was also significantly increased in alveolar walls (p = 0.03) but not the airways. FEV(1)% predicted was correlated with the intensity of staining for AGEs in the airways and alveoli. The increased staining for both AGEs and RAGE in COPD lung raises the possibility that the RAGE-AGEs interaction may have a role in the pathogenesis of COPD.

Study of Hemoglobin and Human Serum Albumin Glycation with Electrochemical Techniques

AbstractHigh concentration glucose in diabetes mellitus may stimulate nonenzymatic glycation of proteins. Hemoglobin (Hb) and human serum albumin (HSA) are among the most sensitive proteins for the modification by glucose. In this paper, we report our study of Hb and HSA modification by glucose using electrochemical methods. Compared with native Hb, it is found that highly glycated Hb presents lower electron transfer reactivity and electrocatalytic activity toward O2 and H2O2, and the glycation is glucose concentration and time dependent. Meanwhile, the changes of the electrochemical signal of heme after binding with HSA and glycated HSA have also suggested that proteins modified by high concentration glucose lasting for months and years in diabetes mellitus might be the reason for diabetes mellitus complication.

Comparison of modification sites formed on human serum albumin at various stages of glycation

Background Many of the complications encountered during diabetes can be linked to the non-enzymatic glycation of proteins, including human serum albumin (HSA). However, there is little information regarding how the glycation pattern of HSA changes as the total extent of glycation is varied. The goal of this study was to identify and conduct a semi-quantitative comparison of the glycation products on HSA that are produced in the presence of various levels of glycation. Methods Three glycated HSA samples were prepared in vitro by incubating physiological concentrations of HSA with 15 mmol/l glucose for 2 or 5 weeks, or with 30 mmol/l glucose for 4 weeks. These samples were then digested and examined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify the glycation products that were formed. Results It was found that the glycation pattern of HSA changed with its overall extent of total glycation. Many modifications including previously-reported primary glycation sites (e.g., K199, K281, and the N-terminus) were consistently found in the tested samples. Lysines 199 and 281, as well as arginine 428, contained the most consistently identified and abundant glycation products. Lysines 93, 276, 286, 414, 439, and 524/525, as well as the N-terminus and arginines 98, 197, and 521, were also found to be modified at various degrees of HSA glycation. Conclusions The glycation pattern of HSA was found to vary with different levels of total glycation and included modifications at the 2 major drug binding sites on this protein. This result suggests that different modified forms of HSA, both in terms of the total extent of glycation and glycation pattern, may be found at various stages of diabetes. The clinical implication of these results is that the binding of HSA to some drug may be altered at various stages of diabetes as the extent of glycation and types of modifications in this protein are varied.

Advanced glycation end‐products and the kidney

Advanced glycation end-products (AGEs) are increased in situations with hyperglycemia and oxidative stress such as diabetes mellitus. They are products of nonenzymatic glycation and oxidation of proteins and lipids. The kidney plays an important role in clearance and metabolism of AGEs. Medline and other relevant databases were searched. In addition, key review articles were scanned for relevant original publication. Finally, original data from our research group were also included. Kidney podocytes and endothelial cells express specific receptors for AGEs. Their activation leads to multiple pathophysiological effects including hypertrophy with cell cycle arrest and apoptosis, altered migration, and generation of proinflammatory cytokines. AGEs have been primarily implicated in the pathophysiology of diabetic nephropathy and diabetic microvascular complications. AGEs are also involved in other primary renal diseases as well as in the development and progression of atherosclerosis. However, serum or plasma concentrations of AGEs do not correlate well with cardiovascular events in patients with chronic kidney disease (CKD). This is likely due to the fact that serum concentrations failed to correlate with AGEs deposited in target tissues. Several inhibitors of the AGE-RAGE axis are currently tested for various indications. AGEs and their receptors are involved in the pathogenesis of vascular and kidney disease. The role of circulating AGEs as biomarkers for cardiovascular risk estimation is questionable. Whether putative inhibitors of AGEs will get the maturity for its therapeutic use in the future remains open.

010 Advanced glycation end products induce functional impairment in neonatal rat cardiomyocytes through enhanced production of reactive oxygen species

Advanced Glycation End products (AGEs) are formed through the non enzymatic glycation of proteins in conditions such as diabetes and may influence cellular function through actions on cell surface receptors, the most common of which (RAGE) belongs to the immunoglobulin superfamily of receptors. AGEs are implicated in the development of diabetic micro and macrovascular disease and are thought to be important in the development of heart failure in diabetic patients. For example, serum AGE levels have been correlated with a reduced survival and an increase in severity of heart failure. It is unclear is whether AGEs contribute to myocardial abnormalities observed in diabetes through direct myocardial actions. We have therefore investigated the effects of AGEs on cardiomyocytes and their potential mechanisms of action. Calcium (Ca2+) transient amplitude was measured using field stimulation and Indo-1 dye, western blotting used for protein quantification, NF-KB luciferase assay to detect NF-KB activity and DHCF (dihydrochlorofluorscene) dye for ROS (reactive oxygen species) measurement. Western blot analysis showed that RAGE receptor is expressed in neonatal rat cardiomyocytes (NRCM) at 45 KDa. Incubation of NRCM with AGEs for 24 h at different concentrations, showed a dose response reduction of Ca2+ transient amplitude with a maximum reduction (52±12%) at a concentration of 1 gm/l (p

Does Accumulation of Advanced Glycation End Products Contribute to the Aging Phenotype?

Aging is a complex multifactorial process characterized by accumulation of deleterious changes in cells and tissues, progressive deterioration of structural integrity and physiological function across multiple organ systems, and increased risk of death. We conducted a review of the scientific literature on the relationship of advanced glycation end products (AGEs) with aging. AGEs are a heterogeneous group of bioactive molecules that are formed by the nonenzymatic glycation of proteins, lipids, and nucleic acids. Humans are exposed to AGEs produced in the body, especially in individuals with abnormal glucose metabolism, and AGEs ingested in foods. AGEs cause widespread damage to tissues through upregulation of inflammation and cross-linking of collagen and other proteins. AGEs have been shown to adversely affect virtually all cells, tissues, and organ systems. Recent epidemiological studies demonstrate that elevated circulating AGEs are associated with increased risk of developing many chronic diseases that disproportionally affect older individuals. Based on these data, we propose that accumulation of AGEs accelerate the multisystem functional decline that occurs with aging, and therefore contribute to the aging phenotype. Exposure to AGEs can be reduced by restriction of dietary intake of AGEs and drug treatment with AGE inhibitors and AGE breakers. Modification of intake and circulating levels of AGEs may be a possible strategy to promote health in old age, especially because most Western foods are processed at high temperature and are rich in AGEs.

Advanced glycation end products inhibit Na + K + ATPase in proximal tubule epithelial cells: Role of cytosolic phospholipase A 2α and phosphatidylinositol 4-phosphate 5-kinase γ

Chronic hyperglycaemia during diabetes leads to non-enzymatic glycation of proteins to form advanced glycation end products (AGEs) that contribute to nephropathy. In diabetes, renal Na + K + ATPase (NKA) activity is downregulated and phosphoinositide metabolism is upregulated. We examined the effects of AGEs on NKA activity in porcine LLC-PK1 and human HK2 proximal tubule epithelial cells. AGE-BSA increased cellular phosphoinositol 4,5 bisphosphate (PIP2) production as determined by immunofluorescence microscopy and thin layer chromatography. AGE-BSA (40 μM) induced 3H-arachidonic acid release and reactive oxygen species (ROS) production via cytosolic phospholipase A 2 (cPLA 2) activation. Within minutes, AGE-BSA significantly inhibited NKA surface expression and activity in a dose- and time-dependent manner as determined by immunofluorescence staining and [ 86Rb +] uptake, respectively, suggesting AGEs inhibit NKA by stimulating its endocytosis. The AGE-BSA-induced decrease in cell surface NKA was reversed by a cPLA 2α inhibitor, neomycin, a PIP2 inhibitor, and PP2, a Src inhibitor. AGE-BSA increased binding of NKA to the α-adaptin but not β2- or μ2-adaptin subunits of the AP-2 clathrin pit adaptor complex. Transfection of HK2 cells with PIP5Kγ siRNA prevented AGE-BSA inhibition of NKA activity. AGEs may stimulate PIP5Kγ to increase PIP2 production, which may enhance AP-2 localisation to clathrin pits, increase clathrin pit formation, enhance NKA cargo recognition by AP-2 and/or stimulate cPLA 2α activity. These results suggest AGEs modulate arachidonic acid and phosphoinositide metabolism to inhibit NKA via clathrin-mediated endocytosis. Elucidation of new intracellular AGE signaling pathways may lead to improved therapies for diabetic nephropathy.

Abstract 521: Impaired migration of human fibrosarcoma cells HT1080 on glycated collagen type I

Abstract Although a strict link does exist between cancer and aging, elderly patients present less invasive tumors than their younger counterparts. During aging, the fibrillar collagen type I, which is an abundant component of the extracellular matrix, becomes a prominent target of post-translational modifications such as glycation. Our present study is mainly designed to investigate the effect of non-enzymatic glycation of this long-lived protein on the proliferation and migration of human fibrosarcoma cells HT1080. For this purpose, collagen type I extracted from rat tail tendons was used for in vitro glycoxydation with ribose. Tumor cell behaviour was then evaluated on 2-dimensional plastic surfaces coated with native or glycated collagen type I. Results showed that the proliferation rate of HT1080 cells on glycated collagen type I did not differ from that on native collagen I after 1, 2 and 3 days of culture. Furthermore, the migration ability of HT1080 cells was studied by quantifying cell speed and trajectories via time-lapse videomicroscopy. At day 1, glycated collagen type I distinctly slowed down the cell locomotion by decreasing the mean cell speed by 47% (20.2 μm/h for glycated versus 38.3 μm/h for its native form). Continuous single cell tracking also permitted the investigation of cell trajectories. Cells grown on glycated collagen exhibited shorter or circular trajectories around their starting point in accordance with the cell speed inhibition. Because actin and vinculin appeared as key proteins involved in cell locomotion, we next investigated the effect of glycated collagen on the organization of these proteins in individual cells in motion. Cells exhibited a dramatic disorganization of F-actin cytoskeleton with loss of stress fibers as compared to native collagen. Actin was principally localized at the rim of the cell. Disturbance of actin integrity was accompanied with a disorganization of vinculin that also localized at the cell periphery. The impact of glycated collagen on the non-receptor protein tyrosine kinase FAK, which plays a crucial role in focal adhesion formation and cell locomotion, was also evaluated. We found that at day 1, glycated collagen significantly inhibits FAK expression by 39% but did not affect their phosphorylation state. These data clearly suggest that age-related changes of extracellular matrix proteins may contribute to a less invasive behaviour of tumor cells. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 521.

Glycation Isotopic Labeling with 13C-Reducing Sugars for Quantitative Analysis of Glycated Proteins in Human Plasma

Non-enzymatic glycation of proteins is a post-translational modification produced by a reaction between reducing sugars and amino groups located in lysine and arginine residues or in the N-terminal position. This modification plays a relevant role in medicine and food industry. In the clinical field, this undesired role is directly linked to blood glucose concentration and therefore to pathological conditions derived from hyperglycemia (>11 mm glucose) such as diabetes mellitus or renal failure. An approach for qualitative and quantitative analysis of glycated proteins is here proposed to achieve the three information levels for their complete characterization. These are: 1) identification of glycated proteins, 2) elucidation of sugar attachment sites, and 3) quantitative analysis to compare glycemic states. Qualitative analysis was carried out by tandem mass spectrometry after endoproteinase Glu-C digestion and boronate affinity chromatography for isolation of glycated peptides. For this purpose, two MS operational modes were used: higher energy collisional dissociation-MS2 and CID-MS3 by neutral loss scan monitoring of two selective neutral losses (162.05 and 84.04 Da for the glucose cleavage and an intermediate rearrangement of the glucose moiety). On the other hand, quantitative analysis was based on labeling of proteins with [(13)C(6)]glucose incubation to evaluate the native glycated proteins labeled with [(12)C(6)]glucose. As glycation is chemoselective, it is exclusively occurring in potential targets for in vivo modifications. This approach, named glycation isotopic labeling, enabled differentiation of glycated peptides labeled with both isotopic forms resulting from enzymatic digestion by mass spectrometry (6-Da mass shift/glycation site). The strategy was then applied to a reference plasma sample, revealing the detection of 50 glycated proteins and 161 sugar attachment positions with identification of preferential glycation sites for each protein. A predictive approach was also tested to detect potential glycation sites under high glucose concentration.

Detection of glycation sites in proteins by high-resolution mass spectrometry combined with isotopic labeling

The products of nonenzymatic glycation of proteins are formed in a chemical reaction between reducing sugars and the free amino group located either at the N terminus of the polypeptide chain or in the lysine side chain. Glycated proteins and their fragments could be used as markers of the aging process as well as diabetes mellitus and Alzheimer’s disease, making them an object of interest in clinical chemistry. In this article, we propose a new method for the identification of peptide-derived Amadori products in the mixtures obtained by enzymatic hydrolysis of glycated proteins. Two proteins, ubiquitin and human serum albumin (HSA), were modified with an equimolar mixture of glucose and [ 13C 6]glucose and were subjected to enzymatic hydrolysis. The obtained enzymatic digests were analyzed by high-resolution mass spectrometry (HRMS), and the peptide-derived Amadori products were identified on the basis of specific isotopic patterns resulting from 13C substitution. The number of glycated peptides in the digest of HSA detected by our procedure was in agreement with the data recently reported in the literature.

Glycation of PDGF results in decreased biological activity

Advanced glycation end products (AGEs) are formed by the non-enzymatic glycation of proteins by reducing carbohydrates or alpha-oxo-aldehydes such as glyoxal and methylglyoxal and further rearrangements, eliminations and oxidations. AGE-modifications alter peptide structure, function and stability and accumulate under several pathophysiological conditions such as diabetes and are considered a biomarker of ageing. PDGF is a major regulator of wound healing, which is impaired in hyperglycaemia and ageing. We analyzed whether glycated PDGF has impaired activity in cell culture models and occurs in human subjects. PDGF was AGE-modified by the alpha-oxo-aldehydes glyoxal and methylglyoxal, which was shown by Western-blotting using alpha-carboxymethyllysine (CML) or alpha-arginine-pyrimidine (Arg-Pyr) antibodies. In mouse AKR-2B fibroblasts, this AGE-modified PDGF exhibited reduced signalling to AKT and ERK resulting in decreased cell proliferation. In the human osteosarcoma cell line 143B, PDGF signalling towards the AKT-kinase was decreased when using modified PDGF-AA, -AB, and -BB whereas the constitutive active ERK was not affected. Secreted proteins from collagen-activated platelets from diabetic subjects contained more CML-modified proteins compared to healthy controls. PDGF protein as a platelet protein coprecipitated in immunoprecipitation experiments with alpha-CML-antiserum. In summary, our data suggest that AGE-modification of PDGF contributes to reduced wound healing in diabetic patients.

Non-enzymatic glycation of proteins: from diabetes to cancer

Incubation of proteins with glucose leads to their non-enzymatic glycation and formation of Amadori products known as an early glycation product. Oxidative cleavage of Amadori products is considered as a major route to advanced glycation endproducts (AGEs) formation in vivo. Nonenzymatic glycation of proteins or Maillard reaction is increased in diabetes mellitus due to hyperglycemia and leads to several complications such as blindness, heart disease, nerve damage and kidney failure. Accumulation of the early and advanced glycation products in plasma and tissues of diabetic patients and causes production of autoantibodies against corresponding products. The advanced glycation products are also associated with other diseases like cancer. This review summarizes current knowledge of these stage specific glycated products as common and early diagnostic biomarkers for the associated diseases and the complications with the aim of a novel therapeutic target for the diseases.

Polyol pathway and RAGE: a central metabolic and signaling axis in diabetic complications

There are multiple metabolic and molecular consequences of hyperglycemia. This review will focus on the roles of the polyol pathway and the receptor for advanced glycation end products (RAGE) in the pathogenesis of diabetic complications. The lead enzyme of the polyol pathway, aldose reductase, transduces maladaptive effects of hyperglycemia by multiple mechanisms, at least in part via the generation of the products of nonenzymatic glycation of proteins, the advanced glycation end products (AGEs). Furthermore, seminal shifts in metabolic flux in the intracellular space stimulated by aldose reductase action activate signal transduction pathways, which alter gene expression and change cellular phenotype. Among the ligands of the multi-ligand receptor RAGE are the AGEs. AGE–RAGE stimulation mediates vascular and target cell dysfunction. The intersection and interdependence of the polyol pathway–RAGE connection suggest that targeting this axis may provide benefit in reducing the complications of diabetes.