Mass spectrometric study on the protein chemical modification of uremic patients in advanced Maillard reaction.

The Maillard reaction, initiated by the nonenzymatic reaction of reducing sugar with protein, is proposed to play a significant role in protein aging and the complications of aging and diabetes. In this study, we detected and quantified some advanced glycation endproducts (AGEs) in human serum proteins of control and uremic patients by a highly selective and specific assay, electrospray ionization liquid chromatography-mass spectrometry-mass spectrometry (ESI-LC-MS-MS). From our results, levels of each AGEs in serum of uremic patients were significantly elevated, compared to age-matched controls. These results provide the evidence for increased modifications of proteins by Maillard reaction in uremia.     

糖基化终产物(AGEs)的病理意义及其抑制性药物开发原理的研究进展

糖基化终产物(AGEs)是体内蛋白质与还原糖在无酶条件下发生反应后的产物。AGEs与糖尿病及其并发症、血管疾病、衰老以及肾脏疾病等密切相关。本文从AGEs的产生过程、生理作用的分子机制、病理意义以及采用药物干预糖基化终产物的形成和生物学效应等方面进行了综述。     

Immunofluorescence detection of advanced glycation end products (AGEs) in cookies and its correlation with acrylamide content and antioxidant activity

Food processing induces protein modifications by Maillard reactions. This generates advanced glycation end products (AGEs) that are known to affect human health. Therefore, it is of interest to monitor AGEs in food products. Currently Maillard products are detected by measuring fluorescence. However, several AGEs are non-fluorescent, while non-AGE components can exhibit autofluorescence. Therefore, specific AGE immunodetection was investigated. Immunofluorescence of AGEs as well as autofluorescence were determined in cookie extracts. Autofluorescence increases with baking time and sugar level, where AGE immunofluorescence increases with baking time until 20 minutes. Replacing sucrose by fructose confirmed the higher reactivity of fructose in AGE formation. The pattern of autofluorescence correlates well with the acrylamide and antioxidant activity. However, the immunodection of AGEs did not show such a correlation. At higher baking times the autofluorescence probably results from the generation of non-proteineious compounds. The immunofluorescence reduction likely results from the transient character of AGE epitopes.     

The Maillard reaction in the human body. The main discoveries and factors that affect glycation

Ever since the discovery of the Maillard reaction in 1912 and the discovery of the interaction between advanced glycation end-products and cellular receptors, impressive progress has been made in the knowledge of nonenzymatic browning of proteins in vivo. This reaction which leads to the accumulation of random damage in extracellular proteins is known to have deleterious effects on biological function, and is associated with aging and complication in chronic diseases. Despite a controlled membrane permeability and a protective regulation of the cells, intracellular proteins are also altered by the Maillard reaction. Two main factors, protein turnover and the concentration of carbonyls, are involved in the rate of formation of the Maillard products. This paper reviews the key milestones of the discovery of the Maillard reaction in vivo, better known as glycation, and the factors which are likely to affect it.     

3-Deoxyglucosone: metabolism, analysis, biological activity, and clinical implication.

3-Deoxyglucosone (3-DG) is synthesized via the Maillard reaction and the polyol pathway, and is detoxified to 3-deoxyfructose and 2-keto-3-deoxygluconic acid. 3-DG rapidly reacts with protein amino groups to form advanced glycation end products (AGEs) such as imidazolone, pyrraline, N'-(carboxymethyl)lysine and pentosidine, among which imidazolone is the AGE most specific for 3-DG. As demonstrated by using gas chromatography-mass spectrometry or high-performance liquid chromatography, plasma 3-DG levels are markedly increased in diabetes and uremia. Although the plasma 3-DG levels had been controversial, it was clearly demonstrated that its plasma level depends on the deproteinization method by which either free or total 3-DG, presumably bound to proteins, is measured. In diabetes, hyperglycemia enhances the synthesis of 3-DG via the Maillard reaction and the polyol pathway, and thereby leads to its high plasma and erythrocyte levels. In uremia, however, the decreased catabolism of 3-DG, which may be due to the loss of 3-DG reductase activity in the end-stage kidneys, may lead to high plasma 3-DG level. The elevated 3-DG levels in plasma and erythrocytes may promote the formation of AGEs such as imidazolone, as demonstrated by immunohistochemistry and immunochemistry using an anti-imidazolone antibody. Although AGE-modified proteins prepared in vitro exhibit a variety of biological activities, known AGE structures have not yet been demonstrated to show any biological activities. Because 3-DG is potent in the formation of AGEs and has some biological activities, such as cellular toxicity, it may be more important in the development of diabetic and uremic complications than the known AGE structures. By demonstrating that treatment with an aldose reductase inhibitor reduces the erythrocyte levels of 3-DG and AGEs, such as imidazolone, light is shed on the mystery of how aldose reductase inhibitors may prove beneficial in diabetic complications. These evidences suggest that 3-DG plays a principal role in the development of diabetic and uremic complications.     

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.     

Potential therapeutic implication of nifedipine, a dihydropyridine-based calcium antagonist, in advanced glycation end product (AGE)-related disorders

A non-enzymatic reaction between ketones or aldehydes and the amino groups of proteins contributes to the aging of proteins and to pathological complications of diabetes. Under hyperglycemic conditions in diabetes, this process begins with the conversion of reversible Schiff base adducts, and then to more stable, covalently-bound Amadori rearrangement products. Over a course of days to weeks, these early glycation products undergo further reactions and rearrangements to become irreversible crossed-linked, fluorescent protein derivatives termed advanced glycation end products (AGEs). Recent understanding of this process has confirmed that AGE-their receptor (RAGE) interaction-elicited oxidative stress generation was implicated in the pathogenesis of diabetic vascular complication, melanoma growth, expansion and metastasis. We have recently found that nifedipine, one of the most widely used dihydropyridine-based calcium antagonists (DHPs) for treatments of patients with angina pectoris and hypertension, inhibited RAGE overexpression in AGE-exposed endothelial cells by suppressing reactive oxygen species generation. Since RAGE is a signal-transducing receptor for AGEs and subsequently evokes inflammatory responses in various types of cells, thus eliciting angiogenesis and thrombogenesis, we hypothesize here that blockade of RAGE expression by nifedipine may have therapeutic potentials in treatment of patients with various AGE-related disorders. In this paper, we would like to propose the possible ways of testing our hypothesis. Does nifedipine treatment reduce the development and progression of diabetic vascular complications? If the answer is yes, is this beneficial effect of nifedipine superior than that of other DHPs with equihypotensive properties? Does nifedipine treatment decrease the incidence of melanoma and/or prolong the survival of patients with this devastating disorder? These prospective studies will provide further valuable information whether blockade by nifedipine of the AGE-RAGE signaling could be clinically relevant.     

Oxidative stress and protein glycation in diabetes mellitus

Metabolic alterations of diabetes mellitus are not only informative biological signs, but also factors of degenerative complications. Thus, hyperglycaemia increases non enzymatic glycation, characterized by the binding of simple oses (glucose) or by-products to amino groups of proteins. This reaction leads to the formation of complex compounds, advanced glycation end products (AGEs), which alter structure and functions of proteins. Glycation and oxidative stress are closely linked, and both phenomena are referred to as "glycoxidation". All steps of glycoxidation generate oxygen free radical production, some of them being common with lipidic peroxidation pathways. Besides, glycated proteins activate membrane receptors such as RAGE through AGEs, and induce an intracellular oxidative stress and a pro-inflammatory status. So, glycated proteins may modulate functions of cells involved in oxidative metabolism and induce inappropriate responses. Finally, some oxidative products (reactive aldehydes such as methylglyoxal) or lipid peroxidation products (malondialdehyde) may bind to proteins and amplify glycoxidation-generated lesions. The knowledge of glycoxidation mechanisms may lead to new therapeutic approaches.     

How can thermal processing modify the antigenicity of proteins?

This paper is a brief review of thermally induced covalent modifications to proteins in foods, focussing mainly on the advanced glycation end-products (AGE) of the Maillard reaction. Most foods are subjected to thermal processing, either in the home or during their production/manufacture. Thermal processing provides many beneficial effects, but also brings about major changes in allergenicity. Far from being a general way to decrease allergenic risk, thermal processing is as likely to increase allergenicity as to reduce it, through the introduction of neoantigens. These changes are highly complex and not easily predictable, but there are a number of major chemical pathways that lead to distinct patterns of modification. Perhaps the most important of these is through the reaction of protein amino groups with sugars, leading to an impressive cocktail of AGE-modified protein derivatives. These are antigenic and many of the important neoantigens found in cooked or stored foods are probably such Maillard reaction products. A deeper understanding of thermally induced chemical changes is essential for more advanced risk assessments, more effective QC protocols, production of more relevant diagnostic allergen extracts and the development of novel protein engineering and therapeutic approaches to minimise allergenic risk.     

Reaction of glycation and human skin: The effects on the skin and its components,reconstructed skin as a model

Skin is affected by the aging process and numerous modifications are observed. In human, with time the skin becomes drier, thinner, spots appear, elasticity decreases and stiffening increases, together with the appearance of wrinkles. These observations result from the overlapping of an intrinsic chronological aging (individual, genetic) and of an extrinsic aging (dependent on external factors like UV, pollution and lifestyle). One of the causes of aging is the appearance of the Advanced Glycosylation End Products (AGEs) during life. The glycation reaction results from a non-enzymatic reaction between a sugar and a free amine group of Lys, Arg amino acids in proteins. This reaction does not occur only in the skin, indeed, AGEs are also found in the kidney, lens, vessels, etc. These products are also responsible, because of their localization, of some pathologies related to diabetes. AGEs provoke biological modifications implying an activation of molecules synthesis (extracellular matrix, cytokines) and enzyme activation of matrix degradation (metalloproteinases). The UV effect on AGEs (like pentosidine) generates reactive oxygen species (ROS) in the extracellular matrix which could lead to additional deleterious effects. Molecules are described in the literature as inhibitors to this irreversible reaction i.e. aminoguanidine. To understand the consequences of the glycation in the skin, a system of reconstructed skin was developed with a collagen modified by glycation for the dermal component. In this system we observed that dermis and epidermis are both modified due to glycation (macromolecules synthesis, cytokines, metalloproteinases) and it is possible to test inhibitors of this reaction. In conclusion, in skin, glycation is involved in a very complex aging process and simultaneously affect, directly and indirectly, certain cells, their synthesis and the organization of the matrix.     

Crosslinking of α-synuclein by advanced glycation endproducts — an early pathophysiological step in Lewy body formation?

An excess of reactive carbonyl compounds (carbonyl stress) and their reaction products, advanced glycation endproducts (AGEs), are thought to play a decisive role in the pathogenesis of neurodegenerative disorders and Parkinson's disease (PD) in particular. Accumulation of AGEs in various intracellular pathological hallmarks of PD, such as Lewy bodies, densely crosslinked intracellular protein deposits formed from neurofilament components and α-synuclein, have already been described in patients in advanced stages of the disease. There is, however, no indication of the involvement of AGE-induced crosslinking of α-synuclein in very early stages of the disease. In this study, we observed that AGEs and α-synuclein are similarly distributed in very early Lewy bodies in the human brain in cases with incidental Lewy body disease. These cases might be viewed as pre-Parkinson patients, i.e. patients who came for autopsy before the possible development of clinical signs of PD. AGEs are both markers of transition metal induced oxidative stress as well as, inducers of protein crosslinking and free radical formation by chemical and cellular processes. Thus, it is likely that AGE promoted formation of Lewy bodies reflects very early causative changes rather than late epiphenomenons of PD.     

The sour side of neurodegenerative disorders: the effects of protein glycation

Neurodegenerative diseases are associated with the misfolding and deposition of specific proteins, either intra‐ or extracellularly in the nervous system. Although familial mutations play an important role in protein misfolding and aggregation, the majority of cases of neurodegenerative diseases are sporadic, suggesting that other factors must contribute to the onset and progression of these disorders. Post‐translational modifications are known to influence protein structure and function. Some of these modifications might affect proteins in detrimental ways and lead to their misfolding and accumulation. Reducing sugars play important roles in modifying proteins, forming advanced glycation end‐products (AGEs) in a non‐enzymatic process named glycation. Several proteins linked to neurodegenerative diseases, such as amyloid β, tau, prions and transthyretin, were found to be glycated in patients, and this is thought to be associated with increased protein stability through the formation of crosslinks that stabilize protein aggregates. Moreover, glycation may be responsible, via the receptor for AGE (RAGE), for an increase in oxidative stress and inflammation through the formation of reactive oxygen species and the induction of NF‐κB. Therefore, it is essential to unravel the molecular mechanisms underlying protein glycation to understand their role in neurodegeneration. Here, we reviewed the role of protein glycation in the major neurodegenerative disorders and highlight the potential value of protein glycation as a biomarker or target for therapeutic intervention. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Advanced glycation end‐products induce skeletal muscle atrophy and dysfunction in diabetic mice via a RAGE‐mediated,AMPK‐down‐regulated,Akt pathway

Diabetic myopathy, a less studied complication of diabetes, exhibits the clinical observations characterized by a less muscle mass, muscle weakness and a reduced physical functional capacity. Accumulation of advanced glycation end‐products (AGEs), known to play a role in diabetic complications, has been identified in ageing human skeletal muscles. However, the role of AGEs in diabetic myopathy remains unclear. Here, we investigated the effects of AGEs on myogenic differentiation and muscle atrophy in vivo and in vitro. We also evaluated the therapeutic potential of alagebrium chloride (Ala‐Cl), an inhibitor of AGEs. Muscle fibre atrophy and immunoreactivity for AGEs, Atrogin‐1 (a muscle atrophy marker) and phosphorylated AMP‐activated protein kinase (AMPK) expressions were markedly increased in human skeletal muscles from patients with diabetes as compared with control subjects. Moreover, in diabetic mice we found increased blood AGEs, less muscle mass, lower muscular endurance, atrophic muscle size and poor regenerative capacity, and increased levels of muscle AGE and receptor for AGE (RAGE), Atrogin‐1 and phosphorylated AMPK, which could be significantly ameliorated by Ala‐Cl. Furthermore, in vitro, AGEs (in a dose‐dependent manner) reduced myotube diameters (myotube atrophy) and induced Atrogin‐1 protein expression in myotubes differentiated from both mouse myoblasts and primary human skeletal muscle‐derived progenitor cells. AGEs exerted a negative regulation of myogenesis of mouse and human myoblasts. Ala‐Cl significantly inhibited the effects of AGEs on myotube atrophy and myogenesis. We further demonstrated that AGEs induced muscle atrophy/myogenesis impairment via a RAGE‐mediated AMPK‐down‐regulation of the Akt signalling pathway. Our findings support that AGEs play an important role in diabetic myopathy, and that an inhibitor of AGEs may offer a therapeutic strategy for managing the dysfunction of muscle due to diabetes or ageing. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Immunochemical crossreactivity of antibodies specific for "advanced glycation endproducts" with "advanced lipoxidation endproducts"

Antibodies against advanced glycation endproducts (AGEs) are used for their immunohistological localization in tissues, for example in Alzheimer's disease (AD) or diabetes. Many monoclonal and polyclonal antibodies have been used, and their specificity is unknown in most cases. Increased radical production, leading to the formation of lipid-derived reactive carbonyl species, such as malondialdehyde (MDA), acrolein, and glyoxal, is a characteristic aspect of age-related diseases like Alzheimer's disease or diabetic polyneuropathy. These reactive carbonyl species are able to modify proteins, resulting in AGE related structures, termed "advanced lipoxidation products" (ALEs). In this study, the monoclonal carboxymethyllysine-specific antibody 4G9 and the polyclonal AGE-antibody K2189 were tested for their immunoreactivity towards these carbonyl-derived protein modifications. To investigate which carbonyl-modified amino acid side chains are specifically recognized by these antibodies, peptide membranes were incubated with glyoxal, MDA and acrolein. As model proteins, microtubuli associated protein tau (MAP-tau), beta-amyloid, human serum albumin and chicken egg albumin were incubated likewise. It was found that both antibodies detected reaction products of these carbonyl compounds on lysine- and arginine residues and for the protein modification, it was found that some epitopes might not be detected. In conclusion, AGE-antibodies might not only detect sugar-derived AGEs but also structures derived from lipid peroxidation products (serving as markers of oxidative stress).     

Immunohistochemical distribution of advanced glycation end products (AGEs) in human osteoarthritic cartilage

Advanced glycation end products (AGEs) may be associated with osteoarthritis (OA), because the accumulation of AGEs in articular cartilage are among the most striking age-related changes. AGEs modify the tissue protein structure and function and stimulate the cellular responses mediated by a specific receptor for AGEs (RAGE). This study investigated the localization of AGEs in degenerated cartilage using newly identified epitope-specific antibodies to determine the linkage between the distribution of AGEs and the development and progression of OA. Osteochondral specimens of the tibial plateau from OA patients were immunostained by specific antibodies against N^?-(carboxymethyl)lysine (CML), N^?-(carboxyethyl)lysine (CEL), pentosidine, GA-pyridine, and RAGE. The immunohistochemical distribution of these epitopes was evaluated during cartilage degeneration. The immunoreactivity (IR) of AGEs and RAGE was stronger in cells rather than in the extracellular matrix. Higher IR of cellular CML and CEL was observed in both mild and severe OA cartilage in comparison to macroscopically intact cartilage. There was a strong association between GA-pyridine and RAGE in the pattern of increasing IR with the OA grade. These IR patterns of AGEs varying with cartilage degeneration indicate that AGE modified proteins are associated with cartilage degeneration. The coincidental up-regulation of GA-pyridine and RAGE suggests that GA-pyridine is the most significant AGE for cartilage degeneration via the RAGE pathway.     

Preferential recognition of epitopes on AGE–IgG by the autoantibodies in rheumatoid arthritis patients

Incubation of proteins with glucose lead to their non-enzymatic glycation ultimately resulting in the formation of advanced glycation end products (AGEs) in vivo. AGEs alter unique three dimensional structures of various plasma proteins such as IgG. The role of oxidative stress in the pathogenesis of rheumatoid arthritis (RA), a chronic inflammatory autoimmune disease, is well established. In view of this, commercially available human IgG was glycated in vitro with physiological concentration of glucose (5 mM) and the possible involvement of glycated IgG (AGE–IgG) in RA was evaluated. The RA patients were divided into two groups on the basis of disease onset with respect to age: group I (early onset: 20–32 years) and group II (late onset: 36–54 years). AGE–IgG and oxidative stress levels were detected in RA patients and normal healthy individuals by nitroblue tetrazolium (NBT) assay and carbonyl content estimation respectively. Binding characteristics and specificity of RA antibodies were analyzed by enzyme-linked immunosorbent assay (ELISA). We observed preferential binding of RA antibodies to AGE–IgG in comparison to native IgG. Band shift assay further substantiated the enhanced recognition of AGE–IgG by RA antibodies. The results suggest that glycation of IgG results in the generation of neo-epitopes, making it a potential immunogen. Our findings project AGE–IgG as one of the factors for induction of circulating RA autoantibodies.     

Stimulation of glucose utilization and inhibition of protein glycation and AGE products by taurine

AIM: Pathological effects of the process of non-enzymatic glycation of proteins are reflected in chronic complications of diabetes mellitus. We investigated the antiglycating effect of taurine in high fructose fed rats in vivo and the inhibiting potency of taurine in the process of in vitro glycation. Additionally, we investigated whether taurine enhances glucose utilization in the rat diaphragm. METHODS: Rats fed a high fructose diet (60% total calories) were provided 2% taurine solution for 30 days. The effects of taurine on plasma glucose, fructosamine, protein glycation and glycosylated haemoglobin in high fructose rats were determined. For in vitro glycation a mixture of 25 mm glucose and 25 mm fructose was used as glycating agent, bovine serum albumin as the model protein and taurine as the inhibitor. Incubations were carried out in a constant temperature bath at 37 degrees C for 3-30 days. Amadori products and advanced glycation end products (AGEs) formed were measured. In vitro utilization of glucose was carried out in the rat diaphragm in the presence and absence of insulin in which taurine was used as an additive. RESULTS: The contents of glucose, glycated protein, glycosylated haemoglobin and fructosamine were significantly lowered by taurine treatment to high fructose rats. Taurine prevented in vitro glycation and the accumulation of AGEs. Furthermore, taurine enhanced glucose utilization in the rat diaphragm. This effect was additive to that of insulin and did not interfere with the action of insulin. CONCLUSIONS: These results underline the potential use of taurine as a therapeutic supplement for the prevention of diabetic pathology.     

Crosslinking of alpha-synuclein by advanced glycation endproducts--an early pathophysiological step in Lewy body formation?

An excess of reactive carbonyl compounds (carbonyl stress) and their reaction products, advanced glycation endproducts (AGEs), are thought to play a decisive role in the pathogenesis of neurodegenerative disorders and Parkinson's disease (PD) in particular. Accumulation of AGEs in various intracellular pathological hallmarks of PD, such as Lewy bodies, densely crosslinked intracellular protein deposits formed from neurofilament components and alpha-synuclein, have already been described in patients in advanced stages of the disease. There is, however, no indication of the involvement of AGE-induced crosslinking of alpha-synuclein in very early stages of the disease. In this study, we observed that AGEs and alpha-synuclein are similarly distributed in very early Lewy bodies in the human brain in cases with incidental Lewy body disease. These cases might be viewed as pre-Parkinson patients, i.e. patients who came for autopsy before the possible development of clinical signs of PD. AGEs are both markers of transition metal induced oxidative stress as well as, inducers of protein crosslinking and free radical formation by chemical and cellular processes. Thus, it is likely that AGE promoted formation of Lewy bodies reflects very early causative changes rather than late epiphenomenons of PD.     

Advanced glycation endproducts regulate smooth muscle cells calcification in cultured HSMCs

Objective: To investigate the mechanism of Advanced glycation end products (AGEs) promoting the calcification of smooth muscle cells. Methods: The successfully cultured smooth muscle cells were divided into three groups: normal culture group (group A), calcified culture group (group B), calcification + AGEs group (group C); the concentration of intracellular calcium ion was detected in each group; the promotion of AGEs on the calcification of HSMCs was confirmed by VON KOSSA staining; and the expressions of β-catenin, RAGE, β-catenin, OPG and E-cadherin protein were detected by immunofluorescence and western blot. Results: The morphology of the cells in each group showed that the amount of calcified plaques in calcification + AGES group were significantly higher than the calcification group. VON KOSSA staining showed that with increasing concentrations of AGE-BSA, the amount of its calcification gradually increased. Calcium concentration in Calcification + 20 mg/L AGEs group was significantly higher, followed by 40 mg/L AGEs group. The expression of β-catenin increased with the increasing concentrations of AGEs. Conclusion: AGEs can promote the calcification of human femoral artery smooth muscle cells, with a concentration gradient effect. With increasing concentrations of AGEs, the expression of RAGE increased, indicating that AGEs-induced HSMCs proliferation was correlated with RAGE expression.     

Possible link of food-derived advanced glycation end products (AGEs) to the development of diabetes

China Da Qing diabetes prevention study has recently shown that group-based lifestyle interventions over six years can prevent or delay the development of diabetes in patients with impaired glucose tolerance (IGT) for up to 14 years after the active intervention. These findings suggest the sustained beneficial effects of lifestyle interventions to prevent diabetes in at-risk patients for diabetes. Therefore, the clinical study suggests that so-called 'metabolic memory' is involved in the development of diabetes. Potential mechanisms for propagating this 'metabolic memory' are the non-enzymatic glycation of proteins. The formation and accumulation of advanced glycation end products (AGEs) have been known to progress at an accelerated rate under diabetes, and there is accumulating evidence that AGEs play a role in the development of diabetes by inducing islet beta cell damage and/or insulin resistance. Further, there are several animal studies to suggest that dietary AGEs are involved in insulin resistance, visceral obesity and the development of diabetes. These findings led us to speculate that the long-term beneficial influence of early lifestyle interventions on the development of diabetes could be ascribed, at least in part, to their inhibitory effects on AGEs. That is, intake of food-derived AGEs may be suppressed in the lifestyle intervention group, which could reduce the risk for the development of diabetes in high-risk patients with IGT. Therefore, it is an interesting issue to clarify whether food-derived AGEs are actually restricted during the active intervention period and if circulating or tissue AGE levels at the closure of the China Da Qing diabetes prevention study could predict the risk for the development of diabetes 14 years after the trial. This additional, clinical investigation may provide us more information about whether restriction of food-derived AGEs is beneficial for the prevention of the development of diabetes in high-risk patients and may be a novel therapeutic target to prevent diabetes and its related complications.