Non-enzymatic glycation of proteins: a cause for complications in diabetes

Diabetes mellitus is one of the most common non-communicable diseases, and is the fifth leading cause of death in most of the developed countries. It can affect nearly every organ and system in the body and may result in blindness, end stage renal disease, lower extremity amputation and increase risk of stroke, ischaemic heart diseases and peripheral vascular disease. Hyperglycemia in diabetes causes non-enzymatic glycation of free amino groups of proteins (of lysine residues) and leads to their structural and functional changes, resulting in complications of the diabetes. Glycation of proteins starts with formation of Shiff's base, followed by intermolecular rearrangement and conversion into Amadori products. When large amounts of Amadori products are formed, they undergo cross linkage to form a heterogeneous group of protein-bound moieties, termed as advanced glycated end products (AGEs). Rate of these reactions are quite slow and only proteins with large amounts of lysine residues undergo glycation with significant amounts of AGEs. The formation of AGEs is a irreversible process, causing structural and functional changes in protein leading to various complications in diabetes like nephropathy, retinopathy, neuropathy and angiopathy. The present review discusses about role of glycation in various complications of diabetes.     

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. Non-enzymatic 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. The early and advanced glycation products are accumulated in plasma and tissues of diabetic patients and cause 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.     

Binding of long-term glycated low density lipoprotein and AGE-albumin by peripheral monocytes and endothelial cells

Modification of low density lipoprotein (LDL) and plasma or tissue proteins by non-enzymatic glycation culminating in the formation of advanced glycation endproducts (AGEs) is one of the essential pathomechanisms leading to diabetes-associated long-term complications. We compared binding of glycated, glycoxidated and oxidated LDL by peripheral monocytes in activated and quiescent form. Interaction via specific receptors was different for glycated as compared to (glyc)oxidated LDL-modifications. In addition, binding of glycated LDL to quiescent and activated human umbilical vein endothelial cells was studied. In patients with insulin-dependent diabetes mellitus (IDDM), AGE-binding was significantly increased as compared to healthy individuals. Specific and non-specific monocyte binding mechanisms were detected, and both were significantly increased in IDDM patients. Specific and non-specific binding strategies possibly act in concert to eliminate circulating AGEs, which are instrumental in the development and progress of microangiopathic and macroangiopathic complications of diabetes mellitus.     

The assessment of protein glycation in human atherosclerotic plaques by affinity chromatography

SUMMARY

Human atherosclerotic plaques are characterized by a massive deposition of lipid within arterial walls. The lipids accumulated are partly oxidized, as assessed by gas chromatography of lipids and their oxidation products. Both advancing age and diabetes mellitus are associated with an increased prevalence and severity of atherosclerosis.

In diabetes mellitus the development of secondary complications appear to be increased by poor glucose control. Indeed, the post-translational modification of protein by non-enzymatic glycation may provide the link between abnormal glucose control and diabetic complications. For atherosclerosis however, the relationship between glucose control and disease is unclear, with evidence available to support and discount such a link. To study protein glycation in a condition associated with a significant level of lipid oxidation products poses several methodological problems, most of which are associated with interference by lipid-derived aldehydes. Many chemical assays of protein glycation monitor aldehydic products common to the chemistry of both protein glycation and lipid oxidation. Studies of protein glycation in human atheroma, obtained at necropsy, are presented which make use of a commercially available boronic acid affinity-based chromatographic assay of glycated protein. The commercially available affinity-based chromatographic assay of glycated protein appears to be free from such interference and may well prove useful in the study of other conditions in which the non-enzymatic glycation of protein is suspected.     

肌红蛋白的非酶糖化及其生物学效应

蛋白质的非酶糖化（non－enzymatic glycation）在糖尿病及其并发症的发生发展中起着重要作用。糖化血红蛋白（hemoglobin Alc,HbAlC）已作为糖尿病患者血糖控制的“金标准”。相对而言,同为血红素蛋白的肌红蛋白（myoglobin,Mb）,其非酶糖化的研究相对较少。研究发现：非酶糖化能改变Mb的空间构象,且对Mb血红素活性中心产生一定的影响,从而导致Mb各种生物学功能发生一定程度的改变。本文综述非酶糖化对Mb结构与功能的影响的研究现状,旨在阐明Mb非酶糖化后的结构．性质-功能之间的关系及其生物学意义,为糖尿病及其并发症的发病机制研究提供新的线索。     

Molecular susceptibility to glycation and its implication in diabetes mellitus and related diseases

The modification of free amino groups on proteins, lipids, and nucleic acids by non-enzymatic glycosylation produce a variety of complex structures named advanced glycation end products (AGEs). Glycation of these molecules participate in the development of diabetic complications and related diseases. Diabetes mellitus is characterized by short-term metabolic changes in lipid and protein metabolism, and long-term irreversible changes in vascular and connective tissue. AGEs are directly implicated in the development of chronic complications in diabetes such as nephropathy, rethinopathy, neuropathy, and other related diseases such as atherosclerosis, heart disease, stroke, and peripheral vascular disease. In this review, we aim to explain how glycation occurs in different molecules and what the pathological consequence of AGE formation in diabetes mellitus and other diseases are.     

Comprehensive identification of glycated peptides and their glycation motifs in plasma and erythrocytes of control and diabetic subjects

Nonenzymatic glycation of proteins sets the stage for formation of advanced glycation end-products and development of chronic complications of diabetes. In this report, we extended our previous methods on proteomics analysis of glycated proteins to comprehensively identify glycated proteins in control and diabetic human plasma and erythrocytes. Using immunodepletion, enrichment, and fractionation strategies, we identified 7749 unique glycated peptides, corresponding to 3742 unique glycated proteins. Semiquantitative comparisons showed that glycation levels of a number of proteins were significantly increased in diabetes and that erythrocyte proteins were more extensively glycated than plasma proteins. A glycation motif analysis revealed that some amino acids were favored more than others in the protein primary structures in the vicinity of the glycation sites in both sample types. The glycated peptides and corresponding proteins reported here provide a foundation for potential identification of novel markers for diabetes, hyperglycemia, and diabetic complications in future studies.     

NMR analysis of synthetic human serum albumin alpha-helix 28 identifies structural distortion upon amadori modification

The non-enzymatic reaction between reducing sugars and long-lived proteins in vivo results in the formation of glycation and advanced glycation end products, which alter the properties of proteins including charge, helicity, and their tendency to aggregate. Such protein modifications are linked with various pathologies associated with the general aging process such as Alzheimer disease and the long-term complications of diabetes. Although it has been suggested that glycation and advanced glycation end products altered protein structure and helicity, little structural data and information currently exist on whether or not glycation does indeed influence or change local protein secondary structure. We have addressed this problem using a model helical peptide system containing a di-lysine motif derived from human serum albumin. We have shown that, in the presence of 50 mm glucose and at 37 degrees C, one of the lysine residues in the di-lysine motif within this peptide is preferentially glycated. Using NMR analysis, we have confirmed that the synthetic peptide constituting this helix does indeed form a alpha-helix in solution in the presence of 30% trifluoroethanol. Glycation of the model peptide resulted in the distortion of the alpha-helix, forcing the region of the helix around the site of glycation to adopt a 3(10) helical structure. This is the first reported evidence that glycation can influence or change local protein secondary structure. The implications and biological significance of such structural changes on protein function are discussed.     

Glycated collagen alters endothelial cell actin alignment and nitric oxide release in response to fluid shear stress

People with diabetes suffer from early accelerated atherosclerosis, which contributes to morbidity and mortality from myocardial infarction, stroke, and peripheral vascular disease. Atherosclerosis is thought to initiate at sites of endothelial cell injury. Hyperglycemia, a hallmark of diabetes, leads to non-enzymatic glycosylation (or glycation) of extracellular matrix proteins. Glycated collagen alters endothelial cell function and could be an important factor in atherosclerotic plaque development. This study examined the effect of collagen glycation on endothelial cell response to fluid shear stress. Porcine aortic endothelial cells were grown on native or glycated collagen and exposed to shear stress using an in vitro parallel plate system. Cells on native collagen elongated and aligned in the flow direction after 24 h of 20 dynes/cm(2) shear stress, as indicated by a 13% decrease in actin fiber angle distribution standard deviation. However, cells on glycated collagen did not align. Shear stress-mediated nitric oxide release by cells on glycated collagen was half that of cells on native collagen, which correlated with decreased endothelial nitric oxide synthase (eNOS) phosphorylation. Glycated collagen likely inhibited cell shear stress response through altered cell-matrix interactions, since glycated collagen attenuated focal adhesion kinase activation with shear stress. When focal adhesion kinase was pharmacologically blocked in cells on native collagen, eNOS phosphorylation with flow was reduced in a manner similar to that of glycated collagen. These detrimental effects of glycated collagen on endothelial cell response to shear stress may be an important contributor to accelerated atherosclerosis in people with diabetes.     

Papaverine increases human serum albumin glycation

Glycation is a non-enzymatic reaction that is initiated by the primary addition of sugars to amino groups of proteins. In the early phase of glycation, the synthesis of intermediates leads to formation of Amadori compounds. In the last phase, advanced glycation end products (AGE) are irreversibly formed following a complex cascade of reactions. It has recently been shown that glycation also affects diabetes-related complications and Alzheimer’s disease. In this study, human serum albumin at a concentration of 10 mg/ml was incubated in PBS with 40 mM of glucose and in different concentrations of papaverine (25, 100, 250, 500 μM) for 42 days at 37 °C. HSA with no additives as well as with glucose 40 mM were incubated as a control and as a glycated sample, respectively. Following the incubation, the samples were prepared for circular dichroism, fluorescence and absorbance techniques. The results showed that in presence of papaverine and glucose, the glycation of HSA increased notably compared with the glycated sample. In conclusion, in this work, we showed that papaverine affects HSA and increases its glycation level.     

Effect of glycation on alpha-crystallin structure and chaperone-like function

The chaperone-like activity of alpha-crystallin is considered to play an important role in the maintenance of the transparency of the eye lens. However, in the case of aging and in diabetes, the chaperone function of alpha-crystallin is compromized, resulting in cataract formation. Several post-translational modifications, including non-enzymatic glycation, have been shown to affect the chaperone function of alpha-crystallin in aging and in diabetes. A variety of agents have been identified as the predominant sources for the formation of AGEs (advanced glycation end-products) in various tissues, including the lens. Nevertheless, glycation of alpha-crystallin with various sugars has resulted in divergent results. In the present in vitro study, we have investigated the effect of glucose, fructose, G6P (glucose 6-phosphate) and MGO (methylglyoxal), which represent the major classes of glycating agents, on the structure and chaperone function of alpha-crystallin. Modification of alpha-crystallin with all four agents resulted in the formation of glycated protein, increased AGE fluorescence, protein cross-linking and HMM (high-molecular-mass) aggregation. Interestingly, these glycation-related profiles were found to vary with different glycating agents. For instance, CML [N(epsilon)-(carboxymethyl)lysine] was the predominant AGE formed upon glycation of alpha-crystallin with these agents. Although fructose and MGO caused significant conformational changes, there were no significant structural perturbations with glucose and G6P. With the exception of MGO modification, glycation with other sugars resulted in decreased chaperone activity in aggregation assays. However, modification with all four sugars led to the loss of chaperone activity as assessed using an enzyme inactivation assay. Glycation-induced loss of alpha-crystallin chaperone activity was associated with decreased hydrophobicity. Furthermore, alpha-crystallin isolated from glycated TSP (total lens soluble protein) had also increased AGE fluorescence, CML formation and diminished chaperone activity. These results indicate the susceptibility of alpha-crystallin to non-enzymatic glycation by various sugars and their derivatives, whose levels are elevated in diabetes. We also describe the effects of glycation on the structure and chaperone-like activity of alpha-crystallin.     

Effects of oxidative modifications induced by the glycation of bovine serum albumin on its structure and on cultured adipose cells

Non-enzymatic glycosylation (glycation) and oxidative damages represent major research areas insofar as such modifications of proteins are frequently observed in numerous states of disease. Albumin undergoes structural and functional alterations, caused by increased glycosylation during non insulin-dependent diabetes mellitus, which is closely linked with the early occurrence of vascular complications. In this work, we first characterized structural modifications induced by the glycation of bovine serum albumin (BSA). A pathophysiological effect of glycated BSA was identified in primary cultures of human adipocytes as it induces an accumulation of oxidatively modified proteins in these cells. BSA was incubated in the presence or absence of physiological, pathological or supra-physiological concentrations of glucose at 37 degrees C for 7 weeks. Enhanced BSA glycation percentages were determined using boronate affinity columns. The occurrence of oxidative modifications was found to be enhanced in glycated BSA, after determination of the free thiol groups content, electrophoretic migration and infrared spectrometry spectra. An accumulation of carbonyl-modified proteins and an increased release of isoprostane were observed in cell media following the exposure of adipocytes to glycated albumin. These results provide a new possible mechanism for enhanced oxidative damages in diabetes.     

Localization of the ezrin binding epitope for advanced glycation endproducts

Glycated proteins/advanced glycation endproducts contribute to the development of diabetic complications but the precise pathway from glycated proteins to complications is still being delineated. The ezrin, radixin and moesin protein family is a new class of advanced glycation endproduct-binding protein and we hypothesize that advanced glycation endproducts mediate some of their detrimental effects leading to diabetic complications by inhibiting ezrin's actions. Our previous study revealed that glycated proteins bind to the N-terminal domain of ezrin (aa 1–324) and this study further defines the ezrin binding epitope. Binding of glycated albumin to recombinant N-ezrin deletion constructs (aa 1–280, 1–170 and 1–144) and glutathione-S-transferase-N-ezrin fusion proteins, (aa 200–324 and 270–324) was analysed using ligand and far Western blotting, and surface plasmon resonance. Glycated albumin binding was markedly reduced on removal of amino acids 280–324, while binding was preserved in the fusion proteins. A series of peptides based on residues 280–324 was synthesized and those containing residues 277–299 of ezrin bound maximally. Peptide binding to glycated albumin was glycation-specific. An ezrin peptide (aa 277–299) dose-dependently reversed the inhibitory effect of glycated albumin on ezrin (1–324) phosphorylation in vitro, suggesting that binding of advanced glycation endproducts to ezrin changes the conformation of the latter sufficiently to alter binding interactions distant from the advanced glycation endproduct-binding site. This may have consequences for subcellular ezrin localization and signalling pathways. Altogether, these studies provide important structural knowledge for developing peptide antagonists that may be therapeutically useful in preventing advanced glycation endproduct:ezrin interactions in diabetes.     

Clinical,pathophysiological and structure/function consequences of modification of albumin by Amadori-glucose adducts

Background

The nonenzymatic condensation of glucose with albumin results in the formation of albumin modified by Amadori glucose adducts, the principal form in which glycated albumin exists in vivo.

Scope of review

This review focuses on (a) the utility of measurement of Amadori-modified glycated albumin (AGA) as a biomarker in diabetes, where elevated levels attend the hyperglycemic state; (b) the role of AGA as a causal factor in the pathogenesis of complications of diabetes; (c) effects on transport properties; and (d) structural and functional consequences of the modification of albumin by Amadori glucose adducts. It does not discuss counterparts with respect to Advanced Glycation Endproducts (AGE), which may be found in other publications.

Major Conclusions

Nonenzymatic glycation of albumin, which is increased in diabetes, has clinical relevance and pathophysiologic importance, with ramifications for the management of this disease, the development of its complications, and the transport of endogenous and exogenous ligands.

General significance

Appreciation of the manifold consequences of AGA has afforded new avenues for assessing clinical management of diabetes, awareness of the impact of nonenzymatic glycation on albumin biology, insights into the pathogenesis of vascular complications of diabetes, and avenues of investigation of and intervention strategies for these complications. This article is part of a Special Issue on albumin. This article is part of a Special Issue entitled Serum Albumin.     

Fisetin lowers methylglyoxal dependent protein glycation and limits the complications of diabetes

The elevated glycation of macromolecules by the reactive dicarbonyl and α-oxoaldehyde methylglyoxal (MG) has been associated with diabetes and its complications. We have identified a rare flavone, fisetin, which increases the level and activity of glyoxalase 1, the enzyme required for the removal of MG, as well as the synthesis of its essential co-factor, glutathione. It is shown that fisetin reduces two major complications of diabetes in Akita mice, a model of type 1 diabetes. Although fisetin had no effect on the elevation of blood sugar, it reduced kidney hypertrophy and albuminuria and maintained normal levels of locomotion in the open field test. This correlated with a reduction in proteins glycated by MG in the blood, kidney and brain of fisetin-treated animals along with an increase in glyoxalase 1 enzyme activity and an elevation in the expression of the rate-limiting enzyme for the synthesis of glutathione, a co-factor for glyoxalase 1. The expression of the receptor for advanced glycation end products (RAGE), serum amyloid A and serum C-reactive protein, markers of protein oxidation, glycation and inflammation, were also increased in diabetic Akita mice and reduced by fisetin. It is concluded that fisetin lowers the elevation of MG-protein glycation that is associated with diabetes and ameliorates multiple complications of the disease. Therefore, fisetin or a synthetic derivative may have potential therapeutic use for the treatment of diabetic complications.     

Protein glycation in vivo: functional and structural effects on yeast enolase

Protein glycation is involved in structure and stability changes that impair protein functionality, which is associated with several human diseases, such as diabetes and amyloidotic neuropathies (Alzheimer's disease, Parkinson's disease and Andrade's syndrome). To understand the relationship of protein glycation with protein dysfunction, unfolding and beta-fibre formation, numerous studies have been carried out in vitro. All of these previous experiments were conducted in non-physiological or pseudo-physiological conditions that bear little to no resemblance to what may happen in a living cell. In vivo, glycation occurs in a crowded and organized environment, where proteins are exposed to a steady-state of glycation agents, namely methylglyoxal, whereas in vitro, a bolus of a suitable glycation agent is added to diluted protein samples. In the present study, yeast was shown to be an ideal model to investigate glycation in vivo since it shows different glycation phenotypes and presents specific protein glycation targets. A comparison between in vivo glycated enolase and purified enolase glycated in vitro revealed marked differences. All effects regarding structure and stability changes were enhanced when the protein was glycated in vitro. The same applies to enzyme activity loss, dimer dissociation and unfolding. However, the major difference lies in the nature and location of specific advanced glycation end-products. In vivo, glycation appears to be a specific process, where the same residues are consistently modified in the same way, whereas in vitro several residues are modified with different advanced glycation end-products.     

Thermal aggregation of glycated bovine serum albumin

Aggregation and glycation processes in proteins have a particular interest in medicine fields and in food technology. Serum albumins are model proteins which are able to self-assembly in aggregates and also sensitive to a non-enzymatic glycation in cases of diabetes. In this work, we firstly reported a study on the glycation and oxidation effects on the structure of bovine serum albumin (BSA). The experimental approach is based on the study of conformational changes of BSA at secondary and tertiary structures by FTIR absorption and fluorescence spectroscopy, respectively. Secondly, we analysed the thermal aggregation process on BSA glycated with different glucose concentrations. Additional information on the aggregation kinetics are obtained by light scattering measurements. The results show that glycation process affects the native structure of BSA. Then, the partial unfolding of the tertiary structure which accompanies the aggregation process is similar both in native and glycated BSA. In particular, the formation of aggregates is progressively inhibited with growing concentration of glucose incubated with BSA. These results bring new insights on how aggregation process is affected by modification of BSA induced by glycation.     

Intracellular protein glycation in cultured retinal capillary pericytes and endothelial cells exposed to high-glucose concentration.

There is now increasing evidence suggesting that non-enzymatic glycation (NEG) of proteins is involved in the pathogenesis of chronic diabetic complication. In this study we demonstrate that chronic exposure to high-glucose concentration leads to intracellular protein glycation in cultured bovine retinal capillary pericytes and endothelial cells. The level of intracellular protein glycation, as measured using a competitive enzyme-linked immunoabsorbant assay (ELISA), was found to increase in both pericytes and endothelial cells as function of time. As expected products of NEG were only detected when the Schiff base and the Amadori products were chemically reduced to glucitollysine by sodium borohydride. Despite the accumulation of early glycation products on cellular proteins there was no further rearrangement reaction into advanced glycation endproducts (AGEs), even after 12 days of incubation in high-glucose medium. Immunofluorescence microscopy demonstrated that the monoclonal antibody reacting with glucitollysine stains the cytoplasm of both pericytes and endothelial cells in a finely punctate pattern. Further studies using Western blot analysis suggested that a number of cellular proteins, including smooth muscle actin in pericytes, become rapidly glycated. The results from this in vitro study suggest that excessive accumulation of early products of non-enzymatic glycation in pericytes and endothelial cells may play an important role in the pathogenesis of diabetic retinopathy.     

The in vitro inhibition effect of 2 nm gold nanoparticles on non-enzymatic glycation of human serum albumin

The reaction of amino groups of protein and the carbonyl groups of reducing sugar molecules, non-enzymatically induce a series of chemical reactions that form a heterogeneous group of compounds known as advanced glycation end products (AGEs). The accumulation of AGEs is associated with various disease conditions that include complications in diabetes, Alzheimer's disease and aging. The current study monitored the extent of non-enzymatic glycation of human serum albumin (HSA) in order to estimate the formation of HSA related AGEs in the presence of 2 nm gold nanoparticles. The rate of glycation was evaluated using several analytical methods. Physiological concentrations of HSA and glyceraldehyde mixtures, incubated with various concentrations of negatively charged 2 nm gold nanoparticles, resulted in a lower reaction rate than mixtures without 2GNP. Moreover, increasing concentrations of gold nanoparticles exhibited a pronounced reduction in AGE formation. High performance liquid chromatography, UV-visible spectroscopy and circular dichroism analytical methods provide reliable techniques for evaluating AGE formation of HSA adducts.     

Glycated albumin modified by amadori adducts modulates aortic endothelial cell biology

Increased protein glycation has been mechanistically linked to accelerated vascular pathobiology in diabetes. To test the influence of protein modified by Amadori glucose adducts on vascular cell biology, we examined the effect of glycated albumin on replicative capacity and basement membrane collagen production by aortic endothelial cells in culture. Relative to carbohydrate-free albumin, which supported cell proliferation and Type IV collagen synthesis, glycated albumin significantly inhibited³H-thymidine incorporation and Type IV collagen production. The glycated albumin-induced effects were prevented by monoclonal antibodies (A717) that specifically react with Amadori-modified albumin, but not by IgG that was unreactive with glycated albumin. A717 had no effect on thymidine incorporation or collagen synthesis by cells cultured in the presence of nonglycated albumin. The findings indicate that the interaction of glycated albumin with endothelial cells, which have been shown to display dose-responsive, saturable receptors, limits cell replication and triggers maladaptive biosynthetic programs, which may contribute to degenerative macrovascular disease in diabetes.