非酶糖基化作用与衰老的关系

非酶糖基化(nonenzymatic glycation．NEG)，是以还原糖和生物大分子如蛋白质、脂质、核酸为核心反应物，不需要酶催化的一类复杂反应，最终生成不可逆的晚期糖基化终产物(advanced glycation end products，AGEs)。非酶糖基化反应又称为Maillard反应。近年来的研究发现，非酶糖基化作用及其终产物的广泛生理     

非酶糖基化致衰老的实验研究

目的为非酶糖基化（ＮＥＧ）致衰老理论提供实验依据。方法利用Ｄ半乳糖致小鼠衰老模型,通过血清中高级糖基化反应终产物（ＡＧＥ）含量测定、自主运动试验、避暗试验、脾淋巴细胞增殖及白细胞介素２（ＩＬ２）诱生实验、超氧化物歧化酶（ＳＯＤ）活力测定等检测ＮＥＧ反应致衰老效应。结果正常衰老小鼠及Ｄ半乳糖、Ｌ葡萄糖处理的青年小鼠血清中ＡＧＥ含量较高,分别为（６２１±１８４）、（５８７±１７０）及（５７５±１３６）Ｕ／ｍｌ,青年对照组为（３０３±１２８）Ｕ／ｍｌ。Ｄ半乳糖、ＡＧＥ、Ｌ葡萄糖都能引起类似衰老的效应。结论Ｄ半乳糖的拟衰老效应是因其在体内引发了ＮＥＧ反应及ＡＧＥ诱发了大量的自由基放大ＮＥＧ的效应而引起的,ＮＥＧ反应是生物体衰老的一个重要原因。     

Advanced nonenzymatic tissue glycosylation: cell-mediated interactions implicated in the complications associated with diabetes and aging

Tissue and cell surface proteins modified nonenzymatically by glucose are shown to be highly active in protein cross-linking and have been implicated in tissue damage. The production of such protein-glucose interactions called advanced glycosylation endproducts (AGE) are recently shown to be processed by macrophages through a recently characterized high-affinity receptor. Coupling of AGE proteins to their AGE receptor results in TNF and IL-1 synthesis and secretion. This suggests that AGE may act as a signal for growth-promoting factor secretion in a coordinated replacement process during tissue remodeling. A disturbance of this balance may lead to pathologic proliferative response such as in the vasculopathy of diabetes and aging. Since peritoneal surface proteins can be modified by AGE after exposure to high-glucose, a similar pathogenetic process may be involved in the peritoneal fibrosis associated with chronic peritoneal dialysis.     

Enhanced nonenzymatic glycation of eye lens proteins in experimental diabetes mellitus: an approach for the study of protein alterations as mediators of normal aging phenomena

The levels of advanced nonenzymatic glycation endproducts (ACE) were investigated by spectrofluorimetry in eye lens proteins obtained from rats with experimental diabetes of 3 and 6 months duration and from normal age-matched control rats. Diabetic animals showed higher AGE levels at both times studied. However the older control animals showed protein ACE levels comparable to those of the experimental 3 months diabetic group. These data suggest that a pathological phenomenon such as enhanced nonenzymatic glycation, associated to diabetic hyperglycemia, can be considered as a process leading to an accelerated aging of proteins. Thus experimental diabetes mellitus may be used as a model to investigate physiological protein senescence.     

Age-related acceleration of glycation of tissue proteins in rats

The Maillard reaction on proteins is a nonenzymatic glycosylation that is now termed glycation and produces a ketoamine linkage on the protein. The extent of this glycation was estimated by determining furosine (epsilon-N-(2-furoylmethyl)-L-lysine), which is derived from glycated lysine residues. Glycation of rat sciatic nerve and aorta increased with advancing age for up to 50 weeks and the high degree of glycation was maintained thereafter. The level of hemoglobin did not change significantly after 14 weeks. These results suggest that tissue glycation may increase with aging and may be involved in the mechanism of aging.     

Protein glycation: its role in the changes induced by diabetes in the properties of the serum insulin-like growth factor-I binding proteins.

The purpose of this work was to study the effect of diabetes on 125I-labelled insulin-like growth factor (IGF) binding to specific serum binding proteins (IGFBPs) and the possible role of protein glycation in such an effect. Accordingly, ligand blotting and fructosamine assays were performed in serum samples from diabetic and non-diabetic eSS rats as well as in samples of normal rat serum previously incubated with different concentrations of glucose. IGFBPs with molecular weights of 24, 30 and 40 kDa were identified in samples from diabetic and non-diabetic rats. 125I-Labelled IGF-I binding to each of these fractions increased significantly in the serum of diabetic rats. IGF-I binding to IGFBP-40 increased significantly as a function of the degree of glycation of serum proteins. Conversely, the increased binding of IGFBP-24 and IGFBP-30 was related only to the glucose concentration attained at 120 min during the oral glucose tolerance test. Glycation of proteins of normal serum and the binding of labelled IGF-I increased as a function of glucose concentration in the incubation media. In these in-vitro glycated normal sera, only the binding to IGFBP-40 increased significantly; this increase was closely related to the amount of protein glycation. No clear and reproducible changes occurred with the binding of 125I-labelled IGF-I to IGFBP-24 and IGFBP-30 fractions. These results confirm the increase in the binding capacity of IGFBPs reported in diabetic animals. They also show that the increase in IGF-I binding to each IGFBP fraction is regulated by a different mechanism; whereas protein glycation induces changes in IGFBP-40, this mechanism does not affect the binding properties of the other two IGFBPs. The increased binding of IGFBP might affect the availability of free IGF-I, and the consequent alterations in IGF-I-dependent metabolic processes could explain the role of this growth factor in the pathogenesis of chronic complications of diabetes.     

d-Lysine reduces the non-enzymatic glycation of proteins in experimental diabetes mellitus in rats

Summary d-Lysine, the non-physiological isomer of l-lysine, can competitively reduce protein non-enzymatic glycation in vitro. To study the effect of d-lysine in vivo, 6–8-week old Sprague-Dawley rats with streptozotocin-induced diabetes mellitus were treated from diagnosis for 45 days with two daily subcutaneous injections of d-lysine (0.5 g·ml^–1·day^–1). Another group of diabetic rats was only injected with equal volumes of physiological saline (0.9% NaCl). Glycated haemoglobin was measured by ion exchange chromatography, and glycated serum and lens proteins by boronate affinity gel chromatography. Serum and urinary creatinine concentrations were evaluated by the alkaline-picrate reaction. Urinary lysine concentrations at mid- and end-study were evaluated by cation exchange chromatography. Blood glucose concentrations, serum creatinine levels and creatinine clearances, measured at the end of the study, were similar in both diabetic groups (> 22.0 mmol/l, 106 mol/l and 0.02 ml/s, respectively). Urinary lysine concentration in d-lysine-treated diabetic animals was more than 50-fold higher than in placebo-treated diabetic rats. In d-lysine-treated vs placebo-treated diabetic animals, a statistically significant reduction was found in the levels of glycated haemoglobin (stable HbA₁; mean ± SD=3.00±0.74% vs 4.02±0.46%, p<0.05; labile HbA₁=3.92±0.89% vs 5.84±0.61%, p<0.005), glycated serum proteins (1.40±0.47% vs 2.52±1.15%, p<0.05) and glycated lens proteins (4.90±0.96% vs 5.98±0.65 %,p<0.05). Thus, d-lysine (i) is not nephrotoxic and (ii) causes a significant reduction of the early glycation products at the protein level. Therefore, the d-amino acid could be useful in attempting to control damaging phenomena associated with or due to an enhanced protein non-enzymatic glycation.     

Effect of a low protein diet on the relationship of nonenzymatic glycation to altered renal structure and function in diabetes mellitus

Renal functional parameters including creatinine clearance, urinary albumin excretion, basement membrane thickening, and levels of nonenzymatic glycation of glomerular basement membrane were studied in streptozotocin-induced diabetic rats and age-matched controls subjected to low protein diet. In addition, these parameters were also assessed in diabetic and streptozotocin injected nondiabetic animals fed a 24% protein diet, which served as “positive controls.” While diabetic animals from both diet groups had similar elevated glycated hemoglobin levels and increased levels of nonenzymatic glycation of glomerular basement membrane, these were significantly elevated as compared to insulin treated diabetic (euglycemic), age-matched controls on an 8% protein diet, and streptozotocin injected nondiabetic animals from both diet groups. However, urinary albumin excretion and creatinine clearance levels were significantly elevated only in the 24% protein diet fed diabetics over values seen in the various groups of animals on 8% and controls on 24% protein diet. In contrast, there were no statistical differences among diabetic, euglycemic and control (8% and 24% protein) animals with respect to creatinine clearance, urinary albumin excretion, and glomerular basement membrane thickness. Taken together these data cast some doubt on the role of nonenzymatic glycation in the development of diabetic nephropathy. Moreover, hyperglycemia per se causes a compensatory increase in kidney size irrespective of protein intake; a low protein diet, however, inhibits the hyperfiltration commonly seen in early diabetic nephropathy. The authors, thus, hypothesize that a low protein diet, by preventing compensatory increase in blood flow to surviving nephrons, in some fashion protects these functional units from subsequent damage and possibly delays the onset of renal failure.     

槲皮素对糖尿病大鼠主动脉胶原非酶糖化的抑制作用

观察了槲皮素（Qu）对不同病程糖尿病（DM）大鼠主动脉胶原非酶糖化的抑制作用，并与氨基胍（AG）作对比。结果：Qu（100mg·kg-1／d）灌胃治疗9周，能明显抑制主动脉胶原及胶原交联的AGEs含量，与等剂量AG作用相近，但起效要晚。Qu有一定的降压作用，但对血糖及血清果糖胺无影响。提示Qu能通过抑制胶原非酶糖化来防治DM血管病变。     

A study of the age-related acceleration of glycation of tissue proteins in rats

The relationship between age-related glycation and a fluorescent product attributable to the advanced Maillard reaction was investigated in the aortas of rats between 4 and 120 weeks of age. The early-stage product of the Maillard reaction was measured using furosine (epsilon-N-(2-furoylmethyl)-L-lysine) as a marker. The advanced product was measured by fluorescence high-performance liquid chromatography. The level of furosine in the aorta increased with aging to reach a maximum value in rats of 50 to 70 weeks of age, and decreased in rats from 90 to 120 weeks of age. By contrast, the level of the putative advanced product reached a maximum in 90-week-old rats and remained at the maximum level in 120-week-old rats. The level of glycated hemoglobin showed no significant change in rats of more than 14 weeks of age. The advanced Maillard products may play an important role in the pathophysiology of aging arteries.     

Clinical review: The role of advanced glycation end products in progression and complications of diabetes

CONTEXT: Diabetic complications appear to be multifactorial in origin, but in particular, the biochemical process of advanced glycation, which is accelerated in diabetes as a result of chronic hyperglycemia and increased oxidative stress, has been postulated to play a central role in these disorders. Advanced glycation involves the generation of a heterogenous group of chemical moieties known as advanced glycated end products (AGEs), this reaction occurring as a result of a nonenzymatic reaction with glucose interacting with proteins, lipids, and nucleic acids, and involves key intermediates such as methylglyoxal. EVIDENCE SYNTHESIS: In this review we report on how these AGEs may exert deleterious effects in diabetes, as well as address current strategies to interrupt the formation or action of AGEs. First, AGEs act directly to induce cross-linking of long-lived proteins such as collagen to promote vascular stiffness, and, thus, alter vascular structure and function. Second, AGEs can interact with certain receptors, such as the receptor for AGE, to induce intracellular signaling that leads to enhanced oxidative stress and elaboration of key proinflammatory and prosclerotic cytokines. Over the last decade, a large number of preclinical studies have been performed, targeting the formation and degradation of AGEs, as well as the interaction of these AGEs with receptors such as the receptor for AGE. CONCLUSION: It is hoped that over the next few years, some of these promising therapies will be fully evaluated in the clinical context with the ultimate aim to reduce the major economical and medical burden of diabetes, its vascular complications.     

Cross-linking in collagen by nonenzymatic glycation increases the matrix stiffness in rabbit achilles tendon

Nonenzymatic glycation of connective tissue matrix proteins is a major contributor to the pathology of diabetes and aging. Previously the author and colleagues have shown that nonenzymatic glycation significantly enhances the matrix stability in the Achilles tendon (Reddy et al., 2002, Arch. Biochem. Biophys., 399, 174-180). The present study was designed to gain further insight into glycation-induced collagen cross-linking and its relationship to matrix stiffness in the rabbit Achilles tendon. The glycation process was initiated by incubating the Achilles tendons (n = 6) in phosphate-buffered saline containing ribose, whereas control tendons (n = 6) were incubated in phosphate-buffered saline without ribose. Eight weeks following glycation, the biomechanical attributes as well as the degree of collagen cross-linking were determined to examine the potential associations between matrix stiffness and molecular properties of collagen. Compared to nonglycated tendons, the glycated tendons showed increased maximum load, stress, strain, Young's modulus of elasticity, and toughness indicating that glycation increases the matrix stiffness in the tendons. Glycation of tendons resulted in a considerable decrease in soluble collagen content and a significant increase in insoluble collagen and pentosidine. Analysis of potential associations between the matrix stiffness and degree of collagen cross-linking showed that both insoluble collagen and pentosidine exhibited a significant positive correlation with the maximum load, stress, and strain, Young's modulus of elasticity, and toughness (r values ranging from.61 to.94) in the Achilles tendons. However, the soluble collagen content present in neutral salt buffer, acetate buffer, and acetate buffer containing pepsin showed an inverse relation with the various biomechanical attributes tested (r values ranging from.22 to.84) in the Achilles tendons. The results of the study demonstrate that glycation-induced collagen cross-linking is directly associated with the increased matrix stiffness and other mechanical attributes of the tendon.     

Aging: role and control of glycation

PURPOSE: Advanced glycation end-products (AGEs) accumulate in aging tissues and organs during rheumatoid arthritis and Alzheimer disease. These aging toxins are especially involved in cell alteration during diabetes mellitus (glycotoxin) and renal failure (uremic toxin). AGEs participate to the endothelial dysfunction leading to diabetic macro but also micro-angiopathy. AGEs binding to cell receptors are critical steps in the deleterious consequences of AGE excess. AGE-receptor activation altered cell and organ functions by a pro-inflammatory, pro-coagulant and pro-fibrosis factors cell response. CURRENT KNOWLEDGE AND KEY POINTS: Non-enzymatic glycation and glycoxidation with glucose auto-oxidation represent the two main pathways resulting in AGE formation. No exclusive AGE classification is actually available. Pathophysiological mechanisms are described to explain AGE toxicity. AGEs bind to cell receptors inducing deleterious consequences such as endothelial dysfunction after endothelial RAGE activation. AGEs can also have deleterious effects through glycated protein accumulation or in situ protein glycation. FUTURE PROSPECTS AND PROJECTS: Many in vitro or animal studies demonstrated that AGE deleterious effects can be prevented by glycation inhibitors, AGE cross-link breakers or AGE-RAGE interaction inhibition. New molecules are actually studied as new strategy to prevent or treat the deleterious effects of these aging toxins.     

阿魏酸钠对糖尿病大鼠主动脉胶原非酶糖基化的影响

将链脲佐菌素诱发的糖尿病大鼠，以阿魏酸钠处理8周，可使糖尿病大鼠主动脉的胶原和胶原中糖基化终末产物的含量降低，其机制可能与阿魏酸钠保护抗氧化酶有关。     

Effects of nonenzymatic glycation and fatty acids on functional properties of human albumin

Human serum albumin nonenzymatically condenses with glucose to form stable Amadori adducts that are increased with the hyperglycemia of diabetes. The present study evaluated the influence of fatty acids, which are major endogenous ligands, on albumin glycation and of glycation on albumin conformation and exogenous ligand binding. Physiologic concentrations of palmitate, oleate, and linoleate reduced the ability of albumin to form glucose adducts, whereas glycation decreased intrinsic fluorescence, lowered the affinity for dansylsarcosine, and diminished the fatty acid–induced increase in limiting fluorescence of protein-bound warfarin that was observed with nonglycated albumin. The findings indicate that fatty acids impede the ability of albumin to undergo Amadori glucose modification and induce conformational changes affecting exogenous ligand binding, and that nonenzymatic glycation of albumin induces alterations in structural and functional properties that may have import in lipid transport and atherogenesis.     

Sodium nitrite de-stiffening of large elastic arteries with aging: role of normalization of advanced glycation end-products

We tested the hypothesis that sodium nitrite treatment reverses large elastic artery stiffening in old mice via reductions in collagen I, increases in elastin and/or decreases in advanced glycation end products (AGEs) mediated by reduced oxidative stress. Aortic pulse wave velocity (aPWV), a measure of large elastic artery stiffness, was greater in old (26-28months) compared with young (4-6months) control animals (520±9 vs. 405±6cm/s, p<0.05), and this was reversed by 3weeks of sodium nitrite treatment (50mg/L) (435±17cm/s). Age-related increases (p<0.05) in aortic superoxide production were associated with greater total and adventitial nitrotyrosine staining, all of which were reversed by nitrite treatment. Total and adventitial transforming growth factor β and collagen I were increased, and total and medial elastin were reduced with aging (p<0.05), but were unaffected by sodium nitrite. Aorta from old mice had increased total, adventitial and medial AGEs (p<0.05 vs. young), which were normalized by sodium nitrite treatment. In aortic segments from young mice in vitro, pyrogallol (10μM), a superoxide generator, induced an "aging-like" increase in AGEs, and direct treatment with AGEs induced vascular stiffening; these effects were prevented by incubation with sodium nitrite. De-stiffening of aged large elastic arteries by short-term sodium nitrite therapy is mediated in part by normalization of AGEs secondary to amelioration of oxidative stress.     

自然衰老与半乳糖衰老模型大鼠心肌与非酶糖基化相关性研究

目的以非酶糖基化和自由基学说探讨心肌老化的发生机制。方法用D-半乳糖制作老化模型与自然衰老组及成年对照组SD大鼠相比较,观察电镜下心肌超微结构的改变及心肌内AGEs含量,抗氧化指标的变化,及各组线粒体DNA(mtDNA)的缺失率。结果2组衰老组糖基化终末期产物(AGEs)、mtDNA缺失率及丙二醛(MDA)均较成年对照组显著增高,过氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-PX)在2组衰老模型较成年对照组显著降低,自然老化和半乳糖老化病理都表现为肌细胞肿大,脂褐素沉积,线粒体和肌质网等膜结构明显损伤。结论心肌的老化与非酶糖基化(NEG)相关,而其诱导自由基的损伤,导致心肌细胞的凋亡。     

Degradation products of proteins damaged by glycation, oxidation and nitration in clinical type 1 diabetes

Aims/hypothesis Hyperglycaemia in diabetes is associated with increased glycation, oxidative stress and nitrosative stress. Proteins modified consequently contain glycation, oxidation and nitration adduct residues, and undergo cellular proteolysis with release of corresponding free adducts. These free adducts leak into blood plasma for eventual renal excretion. The aim of this study was to perform a comprehensive quantitative analysis of protein glycation, oxidation and nitration adduct residues in plasma protein and haemoglobin as well as of free adducts in plasma and urine to quantify increased protein damage and flux of proteolytic degradation products in diabetes.Methods Type 1 diabetic patients (n=21) and normal healthy control subjects (n=12) were studied. Venous blood samples, with heparin anticoagulant, and 24-h urine samples were taken. Samples were analysed for protein glycation, oxidation and nitration adducts by a quantitative comprehensive screening method using liquid chromatography with triple quadrupole mass spectrometric detection.Results In type 1 diabetic patients, the concentrations of protein glycation, oxidation and nitration adduct residues increased up to three-fold in plasma protein and up to one-fold in haemoglobin, except for decreases in pentosidine and 3-nitrotyrosine residues in haemoglobin when compared with normal control subjects. In contrast, the concentrations of protein glycation and oxidation free adducts increased up to ten-fold in blood plasma, and urinary excretion increased up to 15-fold in diabetic patients.Conclusions/interpretation We conclude that there are profound increases in proteolytic products of glycated and oxidised proteins in diabetic patients, concurrent with much lower increases in protein glycation and oxidation adduct residues.     

The role of diabetes and aging in the determinism of hypertension and the related cerebrovascular complications

Epidemiological studies carried out on a large sample (3191 elderly and 640 centenarians) with identical criteria and applying the actual diagnostic standards, have evidenced a high, statistically significant prevalence of diabetes mellitus type 2 (DMT2) (18.84%) in the elderly, as compared to the centenarians (7.50%). This aspect is correlated with the major frequency of maturity onset diabetes in elderly (MODE), compared to the centenarians, correlated also to the mortality of diabetes mellitus (DM) of long duration. The DMT2 and the aging interact in the determinism of vascular alterations, i.e., of the hypertension, and related cardio-cerebrovascular complications. The most frequently occurring hypertension in both the elderly and centenarians was always the systolic-isolated one. The prevalence of hypertension and acute myocardial infarction (AMI) was statistically significantly higher in diabetics, compared to the normoglycemic patients, in both the elderly and the centenarians. In addition, in a group of 914 elderly patients, being diabetics or normoglycemic at the start of the studies, but having neither AMI nor stroke at the baseline studies, after 5 years, these complications were more prevalent, significantly in statistical terms, in the diabetic subjects, compared to the normoglycemic ones. The increase of life-span causes an increase of the age when the aging phenomena appear, resulting in that the equal-age elderly people today are of better clinical conditions, compared to the previous periods. The increased life span with a consequent progressive aging of the population causes a worse general clinical state of the elderly population, characterized by polypathologies, frailty, and appearance of cognitive deficits or incapabilities for performing manual or instrumental activities.