Age-related changes in the glycation of human aortic elastin

Non-enzymatic glycation of proteins is a consequence of hyperglycemia in diabetes and correlates with aging. The aim of the study was to investigate age-related changes in the glycation of human aortic elastin in healthy subjects by two approaches: (1) assessment by fluorescence method of formed in vivo advanced glycation end products (AGEs) of elastins, purified from human aortas, obtained from different age groups; (2) in vitro glycation of elastins from different age groups and investigation of their capacity to form early (by colorimetric nitroblue tetrazolium method) and AGEs (fluorescence method). Human insoluble elastins were prepared from macro- and microscopic unaltered regions of thoracic aortas, obtained from 68 accident victims, distributed in 15 age-groups, using the method of Starcher and Galione. Soluble alpha-elastins were obtained by the method of Partridge et al. The direct assessment of Maillard reaction related fluorescence in the age groups showed increase of the fluorescence with age. The 'young' elastin had the highest capacity to form both fructosamine and AGEs under glycation in vitro. The glycation of 'old' elastin did not increase markedly during the incubation. These results are consistent with the interpretation that because of its long biological half-life, elastin is susceptible to the slow process of glycation and the following modifications would contribute to the age-related changes of connective tissue.     

Advanced glycation end products and their receptors co-localise in rat organs susceptible to diabetic microvascular injury

Summary Advanced glycation end products (AGEs) are believed to play an important role in the development of diabetic complications. AGEs are increased in experimental diabetes and treatment with the inhibitor of advanced glycation end products, aminoguanidine, has been shown to attenuate the level of these products in tissues undergoing complications. Recently, an AGE-binding protein has been isolated from bovine lung endothelial cells and termed the receptor for advanced glycated end products (RAGE). The present study sought to determine the distribution of AGE and RAGE in tissues susceptible to the long-term complications of diabetes including the kidney, eye, nerve, arteries as well as in a tissue resistant to such complications, the lung. Using polyclonal antisera both AGE and RAGE were found to co-localize in the renal glomerulus. AGE staining was clearly increased with age and was further increased by diabetes. Aminoguanidine treatment reduced AGE accumulation in the kidney. Co-localisation of AGE and RAGE was demonstrated in the inner plexiform layer and the inner limiting membrane of the retina and in nerve bundles from mesenteric arteries. In the aorta, both AGE and RAGE were found in the intima, media and adventitia. Medial staining was increased in diabetes and was reduced by aminoguanidine treatment. A similar pattern was observed for RAGE in the aorta. In the lung, RAGE was found widely distributed throughout the lung whereas the distribution of AGE staining was more limited, primarily localising to macrophages. The co-localisation of AGEs and RAGE in sites of diabetic microvascular injury suggests that this ligand-receptor interaction may represent an important mechanism in the genesis of diabetic complications. [Diabetologia (1997) 40: 619–628] Received: 16 October 1996 and in final revised form: 17 February 1997     

A novel advanced glycation index and its association with diabetes and microangiopathy

Formation of advanced glycation end products (AGEs) is an important mechanism by which chronic exposure to high glucose levels leads to vascular complications. Measurement of AGEs is hence of great importance for clinicians and researchers concerned with the management and prevention of diabetic vascular disease. The aim of this study was to evaluate a simple methodology to detect AGEs in the serum and to correlate their levels with diabetes and microangiopathy, specifically retinopathy and nephropathy. We studied 157 subjects, which included nondiabetic control subjects (n = 38), type 2 diabetic patients without microangiopathy (n = 65), and type 2 diabetic subjects with retinopathy (n = 29) or both retinopathy and nephropathy (n = 25). All subjects were assessed for their glycemic and lipid status. Serum AGEs were monitored by recording the Maillard-specific fluorescence that resulted from sequential addition of serum into the buffer. The resultant linear regression was modeled to yield the slope values that were termed advanced glycation index (AGI) in arbitrary units. The serum levels of AGI (mean +/- SD) were higher in diabetic subjects without complications (6.0 +/- 1.6 units) compared with nondiabetic subjects (4.6 +/- 1.0 units), still higher among diabetic subjects with retinopathy (7.6 +/- 1.2 units) and highest in diabetic subjects with both retinopathy and nephropathy (8.3 +/- 2.0 units). Among diabetic subjects, AGI had a significant positive correlation with duration of diabetes (r = 0.25, P = .006), glycated hemoglobin (r = 0.27, P = .004), cholesterol (r = 0.24, P = .009), triglycerides (r = 0.23, P = .014), and serum creatinine (r = 0.30, P = .001), and a significant negative correlation with creatinine clearance (r = -0.27, P = .003). Logistic regression analysis using diabetic microangiopathy as the dependent variable showed an association with AGI even after including age, duration of diabetes, and glycated hemoglobin (P < .001) into the model. Advanced glycation index is a simple method to detect AGEs, and it correlates well with diabetes, particularly with microangiopathy.     

Glycation amplifies lipoprotein(a)-induced alterations in the generation of fibrinolytic regulators from human vascular endothelial cells

Increased lipoprotein(a) [Lp(a)] in plasma is an independent risk factor for premature cardiovascular diseases. The levels of glycated Lp(a) are elevated in diabetic patients. The present study demonstrated that glycation enhanced Lp(a)-induced production of plasminogen activator inhibitor-1 (PAI-1), and further decreased the generation of tissue-type plasminogen activator (t-PA) from human umbilical vein endothelial cells (HUVEC) and human coronary artery EC. The levels of PAI-1 mRNA and its antigen in the media of HUVEC were significantly increased following treatments with 5 μg/ml of glycated Lp(a) compared to equal amounts of native Lp(a). The secretion and de novo synthesis of t-PA, but not its mRNA level, in EC were reduced by glycated Lp(a) compared to native Lp(a). Treatment with aminoguanidine, an inhibitor for the formation of advanced glycation end products (AGEs), during glycation normalized the generation of PAI-1 and t-PA induced by glycated Lp(a). Butylated hydroxytoluene, a potent antioxidant, inhibited native and glycated Lp(a)-induced changes in PAI-1 and t-PA generation in EC. The results indicate that glycation amplifies Lp(a)-induced changes in the generation of PAI-1 and t-PA from venous and arterial EC. This may attenuate fibrinolytic activity in blood circulation and potentially contributes to the increased incidence of cardiovascular complications in diabetic patients with hyperlipoprotein(a). EC-mediated oxidative modification and the formation of AGEs may be implicated in glycated Lp(a)-induced alterations in the generation of fibrinolytic regulators from vascular EC.     

Non-enzymatic glycation of epidermal proteins of the stratum corneum in diabetic patients

A selected group of diabetic patients showed a statistically significant increase in levels of glycated proteins in the stratum corneum compared with a control group. The values of glycated proteins correlated with those of glycohaemoglobin (GHb), and in diabetic patients also with serum glucose concentrations. The values of glycated proteins (and GHb) exhibited a positive correlation with age both in a control group and in diabetic patients. The average values of glycated proteins (and GHb) were slightly higher in women than in men. Determination of glycated proteins levels of the stratum corneum can serve as a stable parameter for long-term monitoring of the course of non-enzymatic glycation in structural and connective tissues and thus also for the prognosis of the development of dermatological complications related to diabetes mellitus. In vitro incubation of stratum corneum proteins and keratin with glucose resulted in an increase of their glycation. The values of glycated proteins and glycated keratin increased proportionally to the glucose concentration and duration of incubation. Glucose binding to keratin and proteins of the insoluble stratum corneum fraction appeared to occur at practically the same rate, and it is a first-order reaction with regard to the glucose concentration. Water-soluble proteins of the stratum corneum undergo non-enzymatic glycation preferentially (on average 83.4% of the total amount of glycated proteins is present in the soluble fraction), regardless of the initial content of glycated proteins in the sample. The content of glycated soluble proteins of a higher molecular weight significantly increased after 4 weeks of incubation with glucose.     

Role of advanced glycation end products (AGEs) and receptor for AGEs (RAGE) in vascular damage in diabetes

A non-enzymatic reaction between ketones or aldehydes and the amino groups of proteins, lipids and nucleic acids contributes to the aging of macromolecules and to the development and progression of various age-related disorders such as vascular complications of diabetes, Alzheimer's disease, cancer growth and metastasis, insulin resistance and degenerative bone disease. Under hyperglycemic and/or oxidative stress conditions, 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 irreversibly crossed-linked, fluorescent protein derivatives termed advanced glycation end products (AGEs). There is a growing body of evidence that AGE and their receptor RAGE (receptor for AGEs) interaction elicits oxidative stress, inflammatory reactions and thrombosis, thereby being involved in vascular aging and damage. These observations suggest that the AGE-RAGE system is a novel therapeutic target for preventing diabetic vascular complications. In this paper, we review the pathophysiological role of the AGE-RAGE-oxidative stress system and its therapeutic intervention in vascular damage in diabetes. We also discuss here the potential utility of the restriction of food-derived AGEs in diabetic vascular complications.     

Synergistic action of advanced glycation end products and endogenous nitric oxide leads to neuronal apoptosis in vitro: A new insight into selective nitrergic neuropathy in diabetes

Aims/hypothesis We have previously shown that in diabetes nitrergic neurones innervating the urogenital and gastrointestinal organs undergo a selective degenerative process. This comprises an initial insulin-reversible decrease in neuronal nitric oxide synthase (nNOS) in the axons, followed by apoptosis of the nitrergic neurones, a process that is not reversible by insulin. Since apoptosis was independent of serum glucose concentrations, and advanced glycation endproducts (AGEs) have been implicated in the pathogenesis of diabetic complications, we have now measured AGEs in the serum and penis, pyloric sphincter and pelvic ganglia of diabetic animals at different times after streptozotocin treatment. Furthermore, we have studied their effect in vitro on human neuroblastoma (SH-SY5Y) cells in the presence or absence of nNOS expression.Methods Serum AGEs were measured using fluorometry and ELISA. Accumulation of AGEs in the tissues was evaluated with immunohistochemistry. The viability, apoptosis and oxidative stress in SH-SY5Y cells were measured upon exposure to AGEs or high concentrations of glucose.Results AGEs increased gradually in the serum and tissues of streptozotocin-induced diabetic rats; this process was not affected by delayed insulin treatment. In SH-SY5Y cells, AGEs, but not high glucose concentrations, increased the reactive oxygen species and caspase-3-dependent apoptosis in a synergistic fashion with endogenous nitric oxide (NO). Apoptosis was prevented by treatment with a NOS inhibitor, a pan-caspase inhibitor, a soluble receptor of AGEs or an anti-oxidant, but not an inhibitor of soluble guanylate cyclase.Conclusions/interpretation The synergistic actions of NO and AGEs account for the irreversible nitrergic degeneration in diabetes.Abbreviations AGEs advanced glycation endproducts - eNOS endothelial nitric oxide synthase - HSA-AGEs advanced glycated human serum albumin - iNOS inducible nitric oxide synthase - L-NAME N^G-nitro-L-arginine methyl ester (NOS inhibitor) - MPG major pelvic ganglion - NAC N-acetyl-L-cysteine - NO nitric oxide - NOS nitric oxide synthase - nNOS neuronal nitric oxide synthase - ODQ 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (sGC inhibitor) - RA retinoic acid - ROS reactive oxygen species - sRAGE soluble receptor of advanced glycation endproduct - STZ streptozotocin - Z-VAD-FMK Z-Val-Ala-Asp(OMe)-CH₂F (pan-caspase inhibitor)     

Glycated albumin modifies platelet adhesion and aggregation responses

A diabetic vasculature is detrimental to cardiovascular health through the actions of advanced glycation end products (AGEs) on endothelial cells and platelets. Platelets activated by AGEs agonize endothelial responses promoting cardiovascular disease (CVD) development. While it has been established that AGEs can alter platelet functions, little is known about the specific platelet pathways that AGEs modify. Therefore, we evaluated the effects of AGEs on specific salient platelet pathways related to CVDs and whether the effects that AGEs elicit are dependent on glycation extent. To accomplish our objective, platelets were incubated with reversibly or irreversibly glycated albumin. A time course for adhesion and aggregation agonist receptor expression was assessed. Optical platelet aggregometry was used to confirm the functional activity of platelets after AGE exposure. In general, platelets subjected to glycated albumin had a significantly enhanced adhesion and aggregation potential. Furthermore, we observed an enhancement in dense body secretion and intracellular calcium concentration. This was especially prevalent for platelets exposed to irreversibly glycated albumin. Additionally, functional aggregation correlated well with receptor expression, suggesting that AGE-induced altered receptor sensitivity translated to altered platelet functions. Our findings indicate that under diabetic vascular conditions platelets become more susceptible to activation and aggregation due to an overall enhanced receptor expression, which may act to promote CVD development.     

A quick,simple method for detecting circulating fluorescent advanced glycation end-products: Correlation with in vitro and in vivo non-enzymatic glycation

ObjectiveAdvanced glycation end-products (AGEs) constitute a highly heterogeneous family of compounds, relevant in the pathogenesis of diabetic complications, which could represent efficient biomarkers of disease progression and drug response. Unfortunately, due to their chemical heterogeneity, no method has been validated to faithfully monitor their levels in the course of the disease. In this study, we refine a procedure to quantitatively analyze fluorescent AGEs (fAGEs), a subset considered remarkably representative of the entire AGE family, and measure them in in vitro glycated BSA (gBSA) and in plasma and vitreous of diabetic rats, for testing its use to possibly quantify circulating AGEs in patients, as markers of metabolic control.MethodsfAGE levels were evaluated by spectrofluorimetric analysis in in vitro and in vivo experimental models. BSA was glycated in vitro with increasing D-glucose concentrations for a fixed time or with a fixed D-glucose concentration for increasing time. In in vivo experiments, streptozotocin-induced diabetic rats were studied at 1, 3, 6 and 12 weeks to analyze plasma and vitreous. To confirm the presence of AGEs in our models, non-diabetic rat retinal explants were exposed to high glucose (HG), to reproduce short-term effects, or in vitro gBSA, to reproduce long-term effects of elevated glucose concentrations. Rat retinal explants and diabetic retinal tissues were evaluated for the receptor for advanced glycation end-product (RAGE) by Western blot analysis.ResultsIn in vitro experiments, fluorescence emission showed glucose concentration- and time-dependent increase of fAGEs in gBSA (p ≤ 0.05). In streptozotocin-induced diabetic rats, fAGE in plasma and vitrei showed an increase at 6 (p ≤ 0.005) and 12 (p ≤ 0.05) weeks of diabetes, with respect to control. RAGE was time-dependently upregulated in retinas incubated with gBSA, but not with HG, and in diabetic retinal tissue, substantiating exposure to AGEs.ConclusionsApplying the proposed technique, we could show that fAGEs levels increase with glucose concentration and time of exposure in vitro. Furthermore, in diabetic rats, it showed that circulating fAGEs are similarly upregulated as those in vitreous, suggesting a correlation between circulating and tissue AGEs. These results support the use of this method as a simple and reliable test to measure circulating fAGEs and monitor diabetes progression.     

Pathophysiological role of Amadori-glycated proteins in diabetic microangiopathy

Early and advanced nonenzymatic glycation of proteins are increased in diabetes. Although Amadori-glycated proteins are the major glycated modifications, most studies so far have focused on the role of advanced glycation end-products (AGEs) in diabetes-related vascular complications. It was only recently that the role of Amadori-glycated proteins has come under consideration. Here we review data that point to an important role of Amadori-modified glycated serum proteins in diabetic microangiopathy. Amadori-glycated albumin induces the activation of glomerular mesangial and endothelial cells to a phenotype that may be linked to the pathogenesis of diabetic microangiopathy, that is, by the stimulation of protein kinase C, activation of transforming growth factor beta, and the expression of extracellular matrix proteins. In type 1 diabetic patients, levels of Amadori-glycated proteins are independently associated with nephropathy and retinopathy. Reduction of Amadori-glycated albumin levels in diabetic animal models ameliorates the progression of nephropathy and retinopathy, indicating a causal role of Amadori-glycated proteins in the pathogenesis of diabetic nephropathy and retinopathy. Based on these data, inhibition of Amadori-glycated albumin may be a target for reduction of diabetic vascular complications.     

Glycated hemoglobin and its spinoffs: Cardiovascular disease markers or risk factors?

Atherosclerosis is a major complication of diabetes, increasing the risk of cardiovascular related morbidities and mortalities. The hallmark of diabetes is hyperglycemia which duration is best predicted by elevated glycated haemoglobin A_1C (HbA_1C) levels. Diabetic complications are usually attributed to oxidative stress associated with glycation of major structural and functional proteins. This non-enzymatic glycation of long lived proteins such as collagen, albumin, fibrinogen, liver enzymes and globulins result in the formation of early and advanced glycation end products (AGEs) associated with the production of myriads of free radicles and oxidants that have detrimental effects leading to diabetic complications. AGEs have been extensively discussed in the literature as etiological factors in the advancement of atherogenic events. Mechanisms described include the effects of glycation on protein structure and function that lead to defective receptor binding, impairment of immune system and enzyme function and alteration of basement membrane structural integrity. Hemoglobin (Hb) is a major circulating protein susceptible to glycation. Glycated Hb, namely HbA_1C is used as a useful tool in the diagnosis of diabetes progression. Many studies have shown strong positive associations between elevated HbA_1C levels and existing cardiovascular disease and major risk factors. Also, several studies presented HbA_1C as an independent predictor of cardiovascular risk. In spite of extensive reports on positive associations, limited evidence is available considering the role of glycated Hb in the etiology of atherosclerosis. This editorial highlights potential mechanisms by which glycated hemoglobin may contribute, as a causative factor, to the progression of atherosclerosis in diabetics.     

Effect of glycated LDL on microvascular tone in mice: a comparative study with LDL modified in vitro or isolated from diabetic patients

Aims/hypothesis In vitro studies have suggested that glycation of LDL might be implicated in diabetic microangiopathy. We therefore investigated the in vivo effects of LDL glycated in vitro on the mouse skeletal muscle arteriolar tone. Since glycation naturally occurs during diabetes, we also tested the effects of LDL isolated from diabetic patients.Methods In anaesthetized mice, the spinotrapezius muscle microcirculation was observed, in situ, using the orthogonal polarization spectral imaging technology. The diameter of terminal (<20 µm) and small arterioles (20–40 µm) was measured before and after a bolus intravenous injection of glycated LDL followed by a continuous perfusion (115 µg/kg/min).Results A slight decrease of terminal and small arterioles diameter (<10%) was observed with native LDL and LDL isolated from healthy subjects. In contrast, mildly glycated LDL induced a clear vasoconstriction of arterioles (>15%), which was further increased when highly glycated LDL was perfused (>22%). LDL isolated from diabetic patients mimicked the vasoconstriction obtained with in vitro mildly glycated LDL, which underwent similar glycation as those isolated from diabetic patients.Conclusion/Interpretation Our results show in vivo that acute perfusion of both types of glycated LDL (artificially or naturally modified), cause major microvascular modification by enhancing arteriolar tone in skeletal muscle. These findings highlight a new role of glycated LDL at the level of microvessels. We suggest that glycation of LDL could contribute to the impaired vascular reactivity observed in diabetes.Abbreviations OPS orthogonal polarization spectral - ACEi angiotensin converting enzyme inhibitors - N-LDL native LDL - mG-LDL mildly glycated LDL - hG-LDL highly glycated LDL - H-LDL LDL isolated from healthy subjects - D1-LDL LDL isolated from Type 1 diabetic patients - D2-LDL LDL isolated from Type 2 diabetic patients - TBARS thiobarbituric acid-reactive substances - TNBS trinitrobenzenesulfonic acid - MDA malondialdehyde-(bis(dimethyl acetal))     

Age-related decrease in susceptibility of human articular cartilage to matrix metalloproteinase-mediated degradation: the role of advanced glycation end products.

OBJECTIVE: Progressive destruction of articular cartilage is a hallmark of osteoarthritis (OA) and rheumatoid arthritis (RA). Age-related changes in cartilage may influence tissue destruction and thus progression of the disease. Therefore, the effect of age-related accumulation of advanced glycation end products (AGEs) on cartilage susceptibility to proteolytic degradation by matrix metalloproteinases (MMPs) present in synovial fluid (SF) of OA and RA patients was studied. METHODS: Cartilage was incubated with APMA-activated SF obtained from OA or RA patients, and tissue degradation was assessed by colorimetric measurement of glycosaminoglycan (GAG) release. Cartilage degradation was related to the level of AGEs in cartilage from donors of different ages (33-83 years) and in cartilage with in vitro-enhanced AGE levels (by incubation with ribose). MMP activity in SF was measured using a fluorogenic substrate. AGE levels were assessed by high-performance liquid chromatography measurement of the glycation product pentosidine. RESULTS: In cartilage from donors ages 33-83 years, a strong correlation was found between the age-related increase in pentosidine and the decrease in MMP-mediated tissue degradation (r = -0.74, P < 0.0005). Multiple regression analysis showed pentosidine to be the strongest predictor of the decreased GAG release (P < 0.0005); age did not contribute (P > 0.8). In addition, decreased MMP-mediated GAG release was proportional to increased pentosidine levels after in vitro enhancement of glycation (r = -0.27, P < 0.01). This was demonstrated for both OA and RA SF (for control versus glycated, P < 0.002 for all SF samples tested). CONCLUSION: Increased cartilage AGEs resulted in decreased cartilage degradation by MMPs from SF, indicating that aged cartilage is less sensitive than young cartilage to MMP-mediated cartilage degradation, such as occurs in OA and RA. Therefore, the level of cartilage glycation may influence the progression of these diseases.     

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.     

In vitro Glycation of Human IgG and Its Effect on Interaction with Anti-IgG

Non-enzymatic glycosylation of proteins is one of the key mechanisms in the pathogenesis of diabetic complications. Glycation of IgG is of special interest due to its possible influence on the functionality of immunoglobulins and overall immuno-competence. The aim of this study was to clarify more details of in vitro glycation of IgG and to study the effect of this modification on its interation with anti-IgG. Purified human IgG was glycated in the presence of 50 and 100 mM glucose. Glycation was measured using spectrophotometric thiobarbituric acid method. To study the effect of glycation on interaction with anti IgG the Single Radial Immunodiffusion (SRID) was used and the diameters of precipitation rings of glycated IgG and non-glycated IgG were measured and compared. The results showed that IgG was glycated in presence of 50 and 100 mM glucose at 27 degrees and 37 degrees C and the extent of glycation was dependent on glucose concentration and time of incubation. In higher concentration of glucose and longer period of incubation glycation was higher at 27 degrees C (p<0.01). Similar results were obtained at 37 degrees C.The results of SRID indicated that glycated IgG showed reduced interaction with anti-IgG. The diameters of precipitated rings for glycated IgG were significantly lower than those of non-glycated IgG (p < 0.01). It can be concluded that modification that occurred in IgG structure due to glycation can be the reason of the reduction of its interaction with anti-IgG.     

Glycated fetal calf serum affects the viability of an insulin-secreting cell line in vitro

The purpose of the present study was to evaluate the direct effects of advanced glycation end products (AGEs) on beta-cells by their exposure to a glycated serum to estimate the cellular viability and the related insulin secretion. Glycation of fetal calf serum was obtained by incubation with 50 mol/L ribose at 37 degrees C for 7 days; at the end of this incubation period, the pentosidine content ranged between 15 and 16 x 10(5) pmol/L. HIT-T15 cells, a pancreatic islet cell line, were grown and cultured for 5 days in Roswell Park Memorial Institute (RPMI) medium containing either not glycated (NGS) or glycated (GS) fetal calf serum. Cellular oxidative stress (ie, thiobarbituric acid-reactive substances) was assessed by high-performance liquid chromatography. Cellular viability was evaluated by detection of proliferation, cell necrosis, and cell apoptosis rate. The insulin secretion and the related intracellular content were evaluated by enzyme-linked immunosorbent assay. The present study reported, after 5 days of exposure to the glycation environment, a moderately reduced cellular proliferation (-20.44% +/- 2.92%) with a corresponding increase of cell necrosis (+67.7% +/- 1.56%) and cell apoptosis (+39.83% +/- 2.92%) rate in comparison with the untreated cells. Oxidative intracellular stress was higher in GS conditions compared with the NGS ones (+293.3% +/- 87.53%). Insulin release from GS-treated HIT-T15 cells was lower than that of NGS-treated cells both when cells were stimulated with low glucose concentration (2.8 mmol/L, -30.3% +/- 4.91%) or when they were challenged with high glucose concentration (16.7 mmol/L, -29.2% +/- 5.82%). Incubation of HIT-T15 cells with glycated serum also caused a significant decrease of insulin intracellular content (-44.47% +/- 9.98%). Thus, AGEs were shown to exert toxic effects on insulin-secreting cells. Chronically high intracellular oxidative stress, due to accumulation of AGEs, affects the insulin secretion machinery. The present data suggest a pivotal role of the non-enzymatic glycation process in the onset and progression of diabetes during aging and a direct adverse effect of a glycated environment on the pancreatic islet cells.     

Prevention and repair of protein damage by the Maillard reaction in vivo

The aging human extracellular matrix (ECM) and tissues rich in long-lived proteins undergo extensive changes with age that include increased stiffening, loss of elasticity, insolubilization, and decreased proteolytic digestibility. Most if not all these changes can be duplicated by the Maillard reaction in vitro, that is, the incubation of the proteins with reducing sugars and oxoaldehydes. These carbonyls eventually form advanced glycation end products (AGEs) and crosslinks that impair proteolytic digestibility and alter protein conformation. To date, close to 20 AGEs have been found in the human skin, of which ornithine is the single major result of damage to arginine residues, and glucosepane the single major crosslink. Although redox active metals and oxoaldehydes appear to play an important role in protein damage in experimental diabetes, their role in diabetic humans is still poorly understood. Evidence for the existence of deglycating enzymes has been found in vertebrates, bacteria, and fungi. However, only the vertebrate enzymes can deglycate larger, intracellular proteins via an ATP-dependent mechanism. Protein engineering will thus be needed to adapt Amadoriase enzymes toward deglycation of ECM proteins for purpose of probing the role of advanced glycation in animal models of diabetes and age-related diseases. The blocking of the reactivity of the glucosepane precursor using potent nucleophiles may be useful in preventing age-related changes in ECM proteins. However, there currently is no evidence in support of the proposed ability of so-called "AGE breakers" to cleave existing crosslinks of the Maillard reaction in vivo, and other mechanisms of action should be sought for this class of compounds.     

Hepatocyte growth factor has a role in the amelioration of diabetic vascular complications via autophagic clearance of advanced glycation end products: Dispo85E, an HGF inducer, as a potential botanical drug

The aim of this study was to evaluate the effect and elucidate the potential mechanism of the extract of rhizomes from Dioscorea alata L. cv. Phyto, Dispo85E, on accelerating the elimination of advanced glycation end products (AGEs) in vitro and in vivo. Primary mouse nonparenchymal cells (NPCs) were used to evaluate the drug effect on AGEs clearance and autophagic-lysosomal activity. In an animal study, we used AGEs-induced diabetic mice to evaluate the drug effect on AGEs-induced vascular complications. Our results indicated that Dispo85E enhanced the endocytosis and degradation activity of AGEs in hepatic NPCs. Furthermore, the hepatocyte growth factor (HGF) expression level was positively correlated with the clearance capacity of the AGEs in NPCs after Dispo85E treatment. In addition, the effects of Dispo85E on the degradation and uptake capability of ¹⁴C-AGEs were abolished in the presence of an anti-HGF neutralizing antibody. We further demonstrated that recombinant mouse HGF could enhance the endocytosis and autophagic clearance of AGEs in NPCs. The in vivo data indicated that Dispo85E increased hepatic HGF messenger RNA expression levels and decreased serum AGEs level in diabetic mice. Moreover, the function of retina and kidneys was improved by Dispo85E treatment in AGEs-induced diabetic mice. These results suggest that HGF may have an important role in the elimination of AGEs. This study suggests that Dispo85E is a botanical drug with a novel mechanism that enhances the clearance of AGEs through HGF-induced autophagic-lysosomal pathway and is a candidate drug for the treatment of diabetic vascular complications.