Glucose-derived Advanced Glycation Endproducts Research Articles

Shikimic acid from Artemisia absinthium inhibits protein glycation in diabetic rats

This study investigated the impact of Shikimic Acid (SA) obtained from leaves of Artemisia absinthium on protein glycation in the retina of diabetic rats. The GC/MS analysis of A. absinthium showed that the most abundant bioactive compound was SA (C7H10O5) with a measured retention Index (RI) of 1960 compared to that of the reference sample (1712). Male albino rats were divided into two main groups, Group I (control) and Group II (diabetic); Group II was further divided into four subgroups: Group IIa (diabetic control), Group IIb (diabetic rats were given SA orally [50 mg/kg, body weight (bw)/day], Group IIc diabetic rats were given SA orally [100 mg/kg, bw/day], and Group IId (diabetic rats were given metformin orally [100 mg/kg, bw/day] as positive control). The data obtained suggested that SA reduced glucose and glycated hemoglobin levels. In addition, SA also decreased the formation of glucose-derived advanced glycation end products. Interestingly, SA showed interference with the release of inflammatory mediators in retina and possess antioxidant potential. In conclusion, SA protected the tissues from detrimental effects of hyperglycemia and enhanced antioxidant activity. SA could be a potential lead in the process of drug development in the future to prevent retinopathy in diabetic subjects.

M-Coumaric acid attenuates non-catalytic protein glycosylation in the retinas of diabetic rats.

In this study, we investigated the inhibitory effects of m-coumaric acid on the glycosylation of proteins in the retinas of diabetic rats. Male rats were divided into two main groups, Group I (normal control) and Group II (diabetic); Group II was further divided into four subgroups: Group IIa (diabetic control), Group IIb (diabetic rats were given m-coumaric acid orally [150 mg/kg, body weight (bw)/day]), Group IIc (diabetic rats were given HCA m-coumaric acid orally [300 mg/kg bw/day]), and Group IId (diabetic rats were given insulin [10 units/kg bw/day]) as a positive control). The treatment lasted for six weeks, and the data obtained suggested that m-coumaric acid reduced glucose and glycated hemoglobin levels, which further decreased the formation of glucose-derived advanced glycation end products. Hence, it protected the tissues from the detrimental effects of hyperglycemia and enhanced antioxidant activity. In conclusion, m-coumaric acid could be a potential candidate to prevent the onset and progression of retinopathy in diabetic patients.

Serum Levels of Toxic AGEs (TAGE) May Be a Promising Novel Biomarker for the Onset/Progression of Lifestyle-Related Diseases.

Advanced glycation end-products (AGEs) generated with aging or in the presence of diabetes mellitus, particularly AGEs derived from the glucose/fructose metabolism intermediate glyceraldehyde (Glycer-AGEs; termed toxic AGEs (TAGE)), were recently shown to be closely involved in the onset/progression of diabetic vascular complications via the receptor for AGEs (RAGE). TAGE also contribute to various diseases, such as cardiovascular disease; nonalcoholic steatohepatitis; cancer; Alzheimer’s disease, and; infertility. This suggests the necessity of minimizing the influence of the TAGE-RAGE axis in order to prevent the onset/progression of lifestyle-related diseases (LSRD) and establish therapeutic strategies. Changes in serum TAGE levels are closely associated with LSRD related to overeating, a lack of exercise, or excessive ingestion of sugars/dietary AGEs. We also showed that serum TAGE levels, but not those of hemoglobin A1c, glucose-derived AGEs, or Nε-(carboxymethyl)lysine, have potential as a biomarker for predicting the progression of atherosclerosis and future cardiovascular events. We herein introduce the usefulness of serum TAGE levels as a biomarker for the prevention/early diagnosis of LSRD and the evaluation of the efficacy of treatments; we discuss whether dietary AGE/sugar intake restrictions reduce the generation/accumulation of TAGE, thereby preventing the onset/progression of LSRD.

Assessment of the concentrations of various advanced glycation end-products in beverages and foods that are commonly consumed in Japan.

Dietary consumption has recently been identified as a major environmental source of pro-inflammatory advanced glycation end-products (AGEs) in humans. It is disputed whether dietary AGEs represent a risk to human health. Nε-(carboxymethyl)lysine (CML), a representative AGE compound found in food, has been suggested to make a significant contribution to circulating CML levels. However, recent studies have found that the dietary intake of AGEs is not associated with plasma CML concentrations. We have shown that the serum levels of glyceraldehyde-derived AGEs (Glycer-AGEs), but not hemoglobin A1c, glucose-derived AGEs (Glu-AGEs), or CML, could be used as biomarkers for predicting the progression of atherosclerosis and future cardiovascular events. We also detected the production/accumulation of Glycer-AGEs in normal rats administered Glu-AGE-rich beverages. Therefore, we assessed the concentrations of various AGEs in a total of 1,650 beverages and foods that are commonly consumed in Japan. The concentrations of four kinds of AGEs (Glu-AGEs, fructose-derived AGEs (Fru-AGEs), CML, and Glycer-AGEs) were measured with competitive enzyme-linked immunosorbent assays involving immunoaffinity-purified specific antibodies. The results of the latter assays indicated that Glu-AGEs and Fru-AGEs (especially Glu-AGEs), but not CML or Glycer-AGEs, are present at appreciable levels in beverages and foods that are commonly consumed by Japanese. Glu-AGEs, Fru-AGEs, CML, and Glycer-AGEs exhibited concentrations of ≥85%, 2–12%, <3%, and trace amounts in the examined beverages and ≥82%, 5–15%, <3%, and trace amounts in the tested foods, respectively. The results of the present study indicate that some lactic acid bacteria beverages, carbonated drinks, sugar-sweetened fruit drinks, sports drinks, mixed fruit juices, confectionery (snacks), dried fruits, cakes, cereals, and prepared foods contain markedly higher Glu-AGE levels than other classes of beverages and foods. We provide useful data on the concentrations of various AGEs, especially Glu-AGEs, in commonly consumed beverages and foods.

MK615 decreases RAGE expression and inhibits TAGE-induced proliferation in hepatocellular carcinoma cells

To investigate the proliferative effect of advanced glycation end-products (AGEs) and the role of their cellular receptor (RAGE) on hepatocellular carcinoma (HCC) cells, and the inhibitory effects of MK615, an extract from Japanese apricot, against AGEs were also evaluated. Two HCC cell lines, HuH7 and HepG2, were used. Expression of RAGE was investigated by polymerase chain reaction, Western blotting, and flow cytometry (FACS). The effect of MK615 on RAGE expression was also evaluated by FACS. The proliferative effects of a control (unglycated bovine serum albumin), glucose-derived AGEs (Glc-AGE), and glyceraldehyde-derived AGEs (Glycer-AGE), and the anti-proliferative effect of MK615 against AGEs, were evaluated using MTT assays. Expression of RAGE was confirmed at both the mRNA and protein levels in both HuH7 and HepG2. FACS revealed that the level of RAGE expression was higher in HuH7 than in HepG2. Treatment with 0.1 μg/mL MK615 decreased the expression level of RAGE from 24.3% to 3.7% in HuH7 and from 6.2% to 4.8% in HepG2. The growth indices for the control, Glc-AGE, and Glycer-AGE were 1.06 ± 0.08, 0.99 ± 0.04, and 1.38 ± 0.05, respectively, in HuH7 (P = 0.037), and were 1.03 ± 0.04, 1.04 ± 0.03, and 1.07 ± 0.05, respectively, in HepG2 (P > 0.05). When the cells were cultured simultaneously with Glycer-AGE and MK615, MK615 abrogated the proliferative effect of Glycer-AGE in HuH7. Only Glycer-AGE has a proliferative effect on HuH7, which expresses a higher level of RAGE. MK615 suppresses the proliferative effect of Glycer-AGE on HuH7 by decreasing the expression of RAGE.

The Role of the Amadori Product in the Complications of Diabetes

Strong evidence has emerged in recent years in support of an association between advanced glycation and the complications of diabetes, whereby both glycoxidation products and oxoaldehydes have been implicated. In contrast, except for the fact that skin collagen-linked fructosamine (Amadori product) is a strong predictor of the risk of progression of microvascular disease in humans, Amadori products have not been associated with complications in most animal experiments. Below we develop the hypothesis that glucose-derived advanced glycation end products (AGEs), such as glucosepane, may inflict sustained damage to the extracellular matrix in diabetes and contribute to tissue stiffening and accelerated sclerosis in arteries, kidneys, and other organs as supported by immunochemical studies using a glucosepane antibody. We also hypothesize that many more structures derived from Amadori products with nucleophiles, such as primary amines and thiols, are expected. The selective prevention of Amadori-derived AGEs using deglycating enzymes would be desirable. However, x-ray diffraction studies of Amadoriase I crystals show that the active site of the enzyme is deeply embedded, explaining why this approach is unlikely to succeed in vivo. Preliminary experiments with nucleophiles show that aminoguanidine and other compounds block glucosepane in vitro.

Photosensitizing Activity of Advanced Glycation Endproducts on Tryptophan, Glucose 6-phosphate Dehydrogenase, Human Serum Albumin and Ascorbic Acid Evaluated at Low Oxygen Pressure†

A comparative study of the photosensitizing activity of advanced glycation endproducts (AGEs) prepared by incubating glucose (Glc), threose (Threo) and ascorbate (AH-) in the presence of lysine (Lys) was performed. Photochemical activity was evaluated under low oxygen pressure with the aim to simulate the conditions of the eye lens. AGE-sensitized tryptophan and AH- photodecomposition and glucose 6-phosphate dehydrogenase inactivation were studied. In all systems, glucose-derived AGEs showed the highest photosensitizing efficiency, followed by ascorbate and threose. The presence of different sensitizers in glycation products mixtures was investigated. For this purpose, Trp decomposition quantum yields were determined at 344 and 367 nm. The values obtained at 344 nm are between three and six times higher than those observed at 367 nm, confirming the presence of at least two compounds with different photosensitizing activities in the mixtures. The chemiluminescence associated with the AGE-mediated oxidation of free Trp and Trp residues in human serum albumin was also studied, and a good correlation between the emission of light and the extent of Trp decomposition was found. In conclusion, it is demonstrated that glucose derived AGEs, which can be formed in vivo in the eye lens of diabetic patients and are accumulated in elderly lenses, have a higher photosensitizing efficiency, at low oxygen pressure, than those arising from ascorbate and threose. This high efficiency is especially significant when proteins are employed as photochemical targets, indicating that protein-sensitizer interaction and the local environment around the sensitizers play an important role.

Impact of mitochondrial ROS production on diabetic vascular complications

Vascular complications are the leading cause of morbidity and mortality in patients with diabetes. Four main molecular mechanisms have been implicated in glucose-mediated vascular disease. There are: glucose-induced activation of protein kinase C (PKC) isoforms; increased formation of glucose-derived advanced glycation end-products (AGE); increased glucose flux through the aldose reductase pathway; and increased production of reactive oxygen species (ROS). Here we demonstrate that hyperglycemia-induced production of ROS is abrogated by inhibitors of mitochondrial metabolism, or by overexpression of uncoupling protein-1 or manganese superoxide dismutase. Normalization of mitochondrial ROS production by each of these agents prevents glucose-induced activation of PKC, formation of AGE, and accumulation of sorbitol in bovine vascular endothelial cells. We also claim that 8-hydroxydeoxyguanosine, which represents mitochondrial oxidative damage was elevated in patients with either retinopathy, albuminuria or increased intima-media thickness of carotid arteries. These results suggest that hyperglycemia induces mitochondrial ROS production, and which can associate to the pathogenesis of diabetic vascular complications.

Human serum albumin minimally modified by methylglyoxal binds to human mononuclear leukocytes via the RAGE receptor and is displaced by Nε-carboxymethyl-lysine and hydroimidazolone AGE epitopes

Proteins modified by advanced glycation endproducts (AGEs) may activate functional responses in cells by binding to one or more cell surface AGE receptors. We investigated the binding of human serum albumin (HSA) minimally modified by methylglyoxal (MGmin–HSA) to peripheral human mononuclear leukocytes (HMLs) in vitro. MGmin–HSA bound to HMLs specifically with a dissociation constant KD of 265±4 nM and a mean receptor number of 6.5±0.9×105 receptors per cell. Antibodies to the receptor for advanced glycation endproducts (RAGE) inhibited the specific binding of MGmin–HSA to HMLs but antibodies to 80K-H, galectin-3 and control antibodies did not. This suggests that MGmin–HSA binds to HMLs via the RAGE receptor. Human serum albumin minimally modified by glucose-derived AGEs and Nε-carboxymethyl-lysine, and a synthetic hydroimidazolone, 2-ethylamino-5-hydro-5-methylimidazolon-4-one (EtMG-H1), displaced MGmin–HSA from specific binding sites. This suggests that both hydroimidazolone- and CML-like AGE epitopes influenced the binding of AGE-modified proteins to the RAGE receptor.

Oxidative Stress-Inducing Carbonyl Compounds From Common Foods: Novel Mediators of Cellular Dysfunction

The general increase in reactive oxygen species generated from glucose-derived advanced glycation endproducts (AGEs) is among the key mechanisms implicated in tissue injury due to diabetes. AGE-rich foods could exacerbate diabetic injury, at least by raising the endogenous AGE. Herein, we tested whether, prior to ingestion, diet-derived AGEs contain species with cell activating (TNFalpha), chemical (cross-linking) or cell oxidative properties, similar to native AGEs. Glutathione (GSH) and GSH peroxidase (GPx) were assessed after exposure of human umbilical vein endothelial cell (HUVECs) to affinity-purified food-AGE extracts, each exposed to 250 degrees C, for 10 min, along with synthetic AGEs. Animal product-derived AGE, like synthetic methylglyoxal-bovine serum albumin (MG-BSA), AGE-BSA, and AGE-low density lipoprotein (AGE-LDL), induced a dose- and time-dependent depletion of GSH (()60-75%, p, 0.01) and an increase in GPx activity (()500-600%, p < 0.01), consistent with marked TNFalpha and cross-link formation (p < 0.05); this contrasted with the low bioreactivity of starch/vegetable AGE-extracts, which was similar to that of control BSA and CML- BSA and BSA (p:NS). Anti-AGE-R1,2,3 and -RAGE IgG each inhibited cell-associated (125) I-dAGE by approximately 30-55%; GSH/GPx were effectively blocked by N-acetyl-cysteine (NAC, 800 uM, p < 0.01) and aminoguanidine-HCl (AG, 100 uM, p < 0.01). Thus, food-derived AGE, prior to absorption, contain potent carbonyl species, that can induce oxidative stress and promote inflammatory signals.

The missing link: A single unifying mechanism for diabetic complications

A causal relationship between chronic hyperglycemia and diabetic microvascular disease, long inferred from various animal and clinical studies, has now been definitely established by data from the Diabetes Control and Complications Trial (DCCT), a multicenter, randomized, prospective, controlled clinical study. A relationship between chronic hyperglycemia and diabetic macrovascular disease in patients with non-insulin-dependent diabetes mellitus (NIDDM) is also supported by the Kumamoto study. How does hyperglycemia induce the functional and morphologic changes that define diabetic complications? Vascular endothelial cells are a major target of hyperglycemic damage, but the mechanisms underlying this damage remain incompletely understood. Three seemingly independent biochemical pathways are involved in the pathogenesis: glucose-induced activation of protein kinase C (PKC) isoforms: increased formation of glucose-derived advanced glycation end products; and increased glucose flux through the aldose reductase pathway. The relevance of each of these three pathways is supported by animal studies in which pathway-specific inhibitors prevent various hyperglycemia-induced abnormalities. Hyperglycemia increases reactive oxygen species (ROS) production inside cultured bovine aortic endothelial cells. In this paper, we show that ROS may activate aldose reductase, induce diacylglycerol, activate PKC, induce advanced glycation end product formation, and activate the pleiotropic transcription factor nuclear factor-kappa B (NF-kappaB). These data demonstrate that a single unifying mechanism of induction, increased production of ROS, serves as a causal link between elevated glucose and each of the three major pathways responsible for diabetic damage.

Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage.

Diabetic hyperglycaemia causes a variety of pathological changes in small vessels, arteries and peripheral nerves. Vascular endothelial cells are an important target of hyperglycaemic damage, but the mechanisms underlying this damage are not fully understood. Three seemingly independent biochemical pathways are involved in the pathogenesis: glucose-induced activation of protein kinase C isoforms; increased formation of glucose-derived advanced glycation end-products; and increased glucose flux through the aldose reductase pathway. The relevance of each of these pathways is supported by animal studies in which pathway-specific inhibitors prevent various hyperglycaemia-induced abnormalities. Hyperglycaemia increases the production of reactive oxygen species inside cultured bovine aortic endothelial cells. Here we show that this increase in reactive oxygen species is prevented by an inhibitor of electron transport chain complex II, by an uncoupler of oxidative phosphorylation, by uncoupling protein-1 and by manganese superoxide dismutase. Normalizing levels of mitochondrial reactive oxygen species with each of these agents prevents glucose-induced activation of protein kinase C, formation of advanced glycation end-products, sorbitol accumulation and NFkappaB activation.

Synthesis and secretion of tumour necrosis factor- α by human monocytic THP-1 cells and chemotaxis induced by human serum albumin derivatives modified with methylglyoxal and glucose-derived advanced glycation endproducts

Human serum albumin minimally-modified by methylglyoxal (MG min-HSA) stimulated the synthesis and secretion of tumour necrosis factor- α (TNF- α) from human monocytic THP-1 cells in vitro. Human serum albumin minimally-modified by glucose-derived advanced glycation endproducts (AGE min-HSA) and human serum albumin highly-modified by glucose-derived advanced glycation endproducts (AGE-HSA) stimulated markedly lower synthesis and secretion of TNF- α from THP-1 cells than did MG min-HSA. The median effective concentration EC 50 value of MG min-HSA for the secretion of TNF- α was 5.8±0.3 μM and the maximal secretion was 0.28±0.01 ng TNF- α/ml ( n=12) for incubations containing 5×10 5 cells/ml. MG min-HSA (0.2–2.0 μM) also stimulated chemotaxis of THP-1 cells in vitro but AGE-HSA did not in this concentration range. The EC 50 value of MG min-HSA for the chemotactic response was 0.44±0.07 μM ( n=15). Similar induction of the synthesis and secretion of TNF- α and chemotaxis by monocytes in response to MG min-HSA in vivo may contribute to atherosclerosis in macro- and micro-angiopathy, particularly in the development of chronic clinical complications of diabetes mellitus.

Methylglyoxal-modified arginine residues — a signal for receptor-mediated endocytosis and degradation of proteins by monocytic THP-1 cells

Non-enzymatic glycosylation or glycation of proteins to form advanced glycation endproducts (AGE) has been proposed as a process which provides a signal for the degradation of proteins. Despite this, the AGE which act a recognition factor for receptor-mediated endocytosis and degradation of glycated proteins by monocytes and macrophages has not been identified. Methylglyoxal, a reactive α-oxoaldehyde and physiological metabolite, reacted irreversibly with arginine residues in proteins to form N δ -(5-hydro-5-methyl-4-imidazolon-2-yl)ornithine and N δ -(5-methyl-4-imidazolon-2-yl)ornithine residues. Human serum albumin minimally-modified with methylglyoxal (MG min-HSA) was bound by cell surface receptors of human monocytic THP-1 cells in vitro at 4°C: the binding constant K d value was 377±35 nM and the number of receptors per cell was 5.9±0.2×10 5 ( n=12). N α -Acetyl- N δ -(5-hydro-5-methyl-4-imidazolon-2-yl)ornithine displaced MG min-HSA from THP-1 cells, suggesting that the N δ -(5-hydro-5-methyl-4-imidazolon-2-yl)ornithine residue was the receptor recognition factor. At 37°C, MG min-HSA was internalised by THP-1 cells and degraded. Similar binding and degradation of human serum albumin modified by glucose-derived AGE was found but only when highly modified. MG min-HSA, therefore, is the first example of a protein minimally-modified by AGE-like compounds that binds specifically to monocyte receptors. The irreversible modification of proteins by methylglyoxal is a potent signal for the degradation of proteins by monocytic cells in which the arginine derivative, N δ -(5-hydro-5-methyl-4-imidazolon-2-yl)ornithine, is the receptor recognition factor. This factor is not present in glucose-modified proteins.

Synthesis and secretion of macrophage colony stimulating factor by mature human monocytes and human monocytic THP-1 cells induced by human serum albumin derivatives modified with methylglyoxal and glucose-derived advanced glycation endproducts

Human serum albumin minimally-modified by methylglyoxal (MG min-HSA) stimulated the synthesis and secretion of macrophage-colony stimulating factor (M-CSF) by mature human monocytes in vitro. Human serum albumin minimally-modified by glucose-derived advanced glycation endproducts (AGE min-HSA) and human serum albumin highly-modified by glycose-derived advanced glycation endproducts (AGE-HSA) stimulated much lower secretion of M-CSF from human monocytes than did MG min-HSA. MG min-HSA and AGE-HSA but not AGE min-HSA also stimulated the growth of human monocytic THP-1 cells in vitro which was inhibited by polyclonal antibodies to human M-CSF. For MG min-HSA, the median growth stimulatory concentration EC 50 value was 0.24 ± 0.07 μM and the maximal increase in cell growth was 36% of control cell growth ( n = 24). Similar induction of secretion of M-CSF from monocytes in vivo may contribute to atherosclerosis in macro- and micro-angiopathy, particularly in the development of diabetic complications.

First Evidence for Accumulation of Protein-Bound and Protein-Free Pyrraline in Human Uremic Plasma by Mass Spectrometry

Glucose-derived advanced glycation end products (AGEs) cross-link proteins and cause various biological tissue damage. One of them, pyrraline [ϵ-2-(formyl-5-hydroxymethyl-pyrrol-1-yl)-L-norleucine], has been demonstrated by utilizing antibody to accumulate in plasma and sclerosed matrix of diabetic individuals, suggesting responsibility for diabetic complications. To elucidate the involvement of pyrraline in uremia, we examined the pyrraline levels in patients with chronic renal failure by a mass spectrometric approach. Here we show that protein-free pyrraline as well as pyrraline with binding protein are significantly increased in non-diabetic uremic plasma compared to healthy subjects. Our results suggest that circulating pyrraline could be a substance contributing to complications in uremia.

Induction of synthesis and secretion of interleukin 1β in the human monocytic THP-1 cells by human serum albumins modified with methylglyoxal and advanced glycation endproducts

Human serum albumin modified with 1–2 methylglyoxal residues per molecule of protein (MG min-HSA) stimulated the synthesis and secretion of interleukin 1β (IL-1β) from human monocytic THP-1 cells in vitro. It was a more potent inducer of IL-1β synthesis than human serum albumin highly-modified with glucose-derived advanced glycation endproducts (AGE-HSA). With 20 μM ligand, IL-1β synthesis was (pg/10 6 cells): MG min-HSA 484.5 ± 50.3; AGE-HSA 30.6 ± 2.0 ( n = 3). IL-1β synthesis increased markedly with MG min-HSA concentrations > 5 μM. IL-1β synthesis and secretion from monocytes in response to methylglyoxal-modifed proteins in vivo may contribute to the development of macro- and micro-angiopathy, particularly in diabetes mellitus.

Advanced glycation end-products and atherosclerosis.

The late rearrangements of the covalent nonenzymatic modification of proteins by glucose, called advanced glycation end-products (AGEs), have been shown to accumulate in diabetic and ageing tissues. AGEs elicit a wide range of cell-mediated responses leading to vascular dysfunction, matrix expansion and athero- and glomerulosclerosis. Cellular responses are thought to be largely induced through an AGE-specific cell-surface receptor complex (AGEr). Interaction of AGE-modified proteins with these cells may serve diverse purposes, including disposal of senescent AGE-modified molecules and initiation of tissue repair and protein turnover. In humans, the normal renal clearance rate for the AGE-degradation products found in serum, AGE peptides (AGEp), correlates inversely with renal creatinine clearance rate. Of note, circulating AGEp include reactive intermediates which readily attach covalently to either insoluble matrix collagen or serum proteins, e.g. low-density lipoproteins (LDL), to form AGEp collagen and AGEp-LDL. Consistent with this, diabetic and nondiabetic patients with renal failure (a group highly susceptible to accelerated atherosclerosis) exhibit markedly elevated AGE-modified serum LDL. In summary, in addition to glucose-derived AGEs, the endogenously produced degradation products, AGE peptides, can amplify tissue damage and thus account as distinct toxins. The effects may particularly accelerate glucose toxicity in certain individuals that are genetically susceptible to diabetic renal and extrarenal disease.