Induction of GM-CSF Production of Macrophages by Advanced Glycation End Products of the Maillard Reaction

We previously reported that AGEs can induce macrophage growth. In this paper, we examined whether advanced glycation end products (AGE) of protein induced GM-CSF production of macrophages. AGE of bovine serum albumin markedly stimulated not only the expression of GM-CSF mRNA, but also GM-CSF secretion in macrophage supernatant. Thus GM-CSF is suggested to be an endogenous signal for macrophage growth induction by AGEs.     

Phosphatidylinositol 3'-kinase-dependent activation of renal mesangial cell Ki-Ras and ERK by advanced glycation end products

Advanced glycation end products (AGEs) are produced by the non-enzymatic glycation of proteins and lipids. AGE levels are pathologically elevated in a number of inflammatory diseases and in diabetes mellitus. There is evidence that AGEs, acting through the receptor for AGEs, contribute to diabetic complications. Nephropathy is a major complication of diabetes mellitus. However, the initiating molecular events that trigger diabetic renal disease are unknown. Renal mesangial cells produce excess extracellular matrix in response to treatment with transforming growth factor-beta, and excess mesangial cell matrix production, by impairing glomerular filtration, contributes to diabetic nephropathy. AGEs are known to trigger the autocrine production and release of transforming growth factor-beta. However, it is unclear how AGEs signal in mesangial cells. Here we show that treatment of mesangial cells with AGEs and with the receptor for AGEs agonist S100 triggers activation of the extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3'-kinase (PI3K) pathways. AGEs trigger the GTP loading of mesangial cell Ras, and AGE activation of ERK requires Ras. We observe that Ki-Ras, but not Ha-Ras, is the target of AGE action. Surprisingly, inhibition of PI3K blocks both ERK and Ki-Ras activation. We also observe that activation of ERK and the PI3K target kinase protein kinase-B is blocked with free radical scavengers, indicating a role for reactive oxygen species in AGE recruitment of PI3K. Thus, AGEs signal to Ki-Ras and ERK through reactive oxygen species-dependent activation of PI3K.     

Antibody-based detection of advanced glycation end-products: promises <Emphasis Type="Italic">vs</Emphasis>. limitations

Advanced glycation end-products (AGEs) of the Maillard reaction were originally measured according to their fluorescent and browning properties. A subsequent study with instrumental analyses such as high-performance liquid chromatography and gas chromatography mass spectrometry more clearly demonstrated the involvement of each AGE structure in pathological conditions. Furthermore, immunochemical methods have also been developed to clarify the localization of AGEs in tissues and measurement of AGEs in multiple clinical samples. Although the involvement of AGEs in age-related diseases has progressed due to immunochemical techniques, the relationship between AGE structure and diseases has not been clear because little was known about the epitope structure of each anti-AGE antibody. However, the development of epitope-identified antibodies against AGEs has made it possible to clarify AGE structures involved in diseases. This review discusses not only the usability of anti-AGE antibodies to evaluate AGEs and disease pathology and screen AGE inhibitors, but also describes their usage.     

Advanced glycation end products suppress osteoblastic differentiation of stromal cells by activating endoplasmic reticulum stress

Advanced glycation end products (AGEs) are involved in bone quality deterioration in diabetes mellitus. We previously showed that AGE2 or AGE3 inhibited osteoblastic differentiation and mineralization of mouse stromal ST2 cells, and also induced apoptosis and decreased cell growth. Although quality management for synthesized proteins in endoplasmic reticulum (ER) is crucial for the maturation of osteoblasts, the effects of AGEs on ER stress in osteoblast lineage are unknown. We thus examined roles of ER stress in AGE2- or AGE3-induced suppression of osteoblastogenesis of ST2 cells. An ER stress inducer, thapsigargin (TG), induced osteoblastic differentiation of ST2 cells by increasing the levels of Osterix, type 1 collagen (Col1), alkaline phosphatase (ALP) and osteocalcin (OCN) mRNA. AGE2 or AGE3 suppressed the levels of ER stress sensors such as IRE1α, ATF6 and OASIS, while they increased the levels of PERK and its downstream molecules, ATF4. A reduction in PERK level by siRNA did not affect the AGEs-induced suppression of the levels of Osterix, Col1 and OCN mRNA. In conclusion, AGEs inhibited the osteoblastic differentiation of stromal cells by suppressing ER stress sensors and accumulating abnormal proteins in the cells. This process might accelerate AGEs-induced suppression of bone formation found in diabetes mellitus.     

Immunochemical detection of advanced glycosylation end products in vivo.

Reducing sugars react with protein amino groups to form a diverse group of protein-bound moieties with fluorescent and cross-linking properties. These compounds, called advanced glycosylation end products (AGEs), have been implicated in the structural and functional alterations of proteins that occur during aging and long-term diabetes. Although several AGEs have been identified on the basis of de novo synthesis and tissue isolation procedures, the measurement of AGE compounds in vivo has remained difficult. As an approach to the study of AGE formation in vivo, we prepared polyclonal antiserum to an AGE epitope(s) which forms in vitro after incubation of glucose with ribonuclease (RNase). This antiserum proved suitable for the detection of AGEs which form in vivo. Both diabetic tissue and serum known to contain elevated levels of AGEs readily competed for antibody binding. Cross-reactivity studies revealed the presence of a common AGE epitope(s) which forms after the incubation of diverse proteins with glucose. Cross-reactive epitopes also formed with glucose 6-phosphate or fructose. These data suggest that tissue AGEs which form in vivo appear to contain a common immunological epitope which cross-reacts with AGEs prepared in vitro, supporting the concept that immunologically similar AGE structures form from the incubation of sugars with different proteins (Horiuchi, S., Araki, N., and Morino, Y. (1991) J. Biol. Chem. 266, 7329-7332). None of the known AGEs, such as 4-furanyl-2-furoyl-1H-imidazole, 1-alkyl-2-formyl-3,4-diglycosylpyrrole, pyrraline, carboxymethyllysine, or pentosidine, were found to compete for binding to anti-AGE antibody. These data further suggest that the dominant AGE epitope which forms from the reaction of glucose with proteins under native conditions is immunologically distinct from the structurally defined AGEs described to date.     

Identification of AGE-precursors and AGE formation in glycation-induced BSA peptides

The glycation of BSA leads to protein/peptide modifications that result in the formation of AGEs. AGEs react with the amino groups of N-terminal amino acid residues, particularly arginine and lysine residues. Enhanced AGE formation exists in the blood and tissues of diabetics, as well as in aging and other disorders. The Identification of AGEs is of great importance. Mass spectrometry has been applied to identify and structurally elucidate AGEs. Here, we report on the identification of AGE- peptides and AGE-precursors based on relative mass changes as a result of specific AGE formation. HPLC-ESIMS, ESI-MS/MS, and the Mascot database were used. The relative mass changes due to the specific type of AGE formation were added to the identified peptides followed by a manual search of the glycated samples, which resulted in the identification of seven peptides for the formation of five AGEs, namely CML, pyrraline, imidazolone A, imidazolone B, and AFGP. Four glycated peptides (FPK, ECCDKPLLEK, IETMR, and HLVDEPQNLIK) were identified in the formation of AGE-precursors.     

Effect of dietary advanced glycation end products on mouse liver

The exact pathophysiology of non-alcoholic steatohepatitis (NASH) is not known. Previous studies suggest that dietary advanced glycation end products (AGEs) can cause oxidative stress in liver. We aim to study the effects of dietary AGEs on liver health and their possible role in the pathogenesis of NASH. METHODS: Two groups of mice were fed the same diet except the AGE content varied. One group was fed a high AGE diet and the second group was fed a regular AGE diet. Liver histology, alanine aminotransferase, aspartate aminotransferase, fasting glucose, fasting insulin, insulin resistance and glucose tolerance were assessed. RESULTS: Histology revealed that neutrophil infiltration occurred in the livers of the high AGE group at week 26; steatosis did not accompany liver inflammation. At week 39 livers from both groups exhibited macro- or micro-steatosis, yet no inflammation was detected. Higher insulin levels were detected in the regular AGE group at week 26 (P = 0.034), compared to the high AGE group. At week 39, the regular AGE group showed higher levels of alanine aminotransferase (P<0.01) and aspartate aminotransferase (P = 0.02) than those of the high AGE group. CONCLUSIONS: We demonstrate that a high AGE diet can cause liver inflammation in the absence of steatosis. Our results show that dietary AGEs could play a role in initiating liver inflammation contributing to the disease progression of NASH. Our observation that the inflammation caused by high AGE alone did not persist suggests interesting future directions to investigate how AGEs contribute to pro-oxidative and anti-oxidative pathways in the liver.     

Prognostic potential and tumor growth-inhibiting effect of plasma advanced glycation end products in non-small cell lung carcinoma

The plasma fluorescence related to the standard fluorescence of advanced glycation end products (AGEs) is a simple measurable blood parameter for distinct diseases but its importance in human cancer, including non-small cell lung carcinoma (NSCLC), is unknown. Plasma samples of 70 NSCLC patients who underwent resection surgery of the tumor were analyzed for the distinct AGE-related fluorescence at 370 nm excitation/440 nm emission. In a retrospective study, we tested the prognostic relevance of this AGE-related plasma fluorescence. The effect of circulating AGEs on the NSCLC growth was studied experimentally in vitro and in vivo. NSCLC patients with high (> median) AGE-related plasma fluorescence were characterized by a later reoccurrence of the tumor after curative surgery and a higher survival rate compared with patients with low plasma fluorescence (25% versus 47% 5-y survival, P = 0.011). Treating NSCLC cell spheroids with patients' plasma showed an inverse correlation between the growth of spheroids in vitro and the individual AGE-related fluorescence of each plasma sample. To confirm the impact of circulating AGEs on the NSCLC progression, we studied the NSCLC growth in mice whose circulating AGE level was elevated by AGE-rich diet. In vivo tumorigenicity assays demonstrated that mice with higher levels of circulating AGEs developed smaller tumors than mice with normal AGE levels. The AGE-related plasma fluorescence has prognostic relevance for NSCLC patients in whom the tumor growth-inhibiting effect of circulating AGEs might play a critical role.     

晚期糖基化终产物对人脐静脉内皮细胞的肝细胞生长因子表达的影响

目的探讨晚期糖基化终产物(AGEs)对人脐静脉内皮细胞的肝细胞生长因子(HGF)mRNA及蛋白表达的影响。方法体外培养人脐静脉内皮细胞,予不同浓度(100mg/L、200mg/L、400mg/L)的AGEs刺激24h及400mg/LAGEs作用6h、12h、24h及48h,采用RT-PCR及免疫细胞化学法检测内皮细胞HGFmRNA及蛋白的表达水平。结果在一定范围内随着AGEs浓度增加,内皮细胞HGF表达逐渐增高;AGEs早期作用内皮细胞,促进HGFmR-NA及蛋白的表达,随着AGEs的持续作用,HGF表达减弱。结论随着AGEs作用时间的延长,HGF对受损内皮细胞的修复作用先增强后减弱。     

In vitro glycation of human serum albumin by dihydroxyacetone and dihydroxyacetone phosphate

Amino groups in proteins can non-enzymatically react with reducing sugars to generate a structurally diverse group of compounds referred to as advanced glycation end products (AGEs). The in vivo formation of AGEs contributes to some of the complications of diabetes including atherosclerosis, cataract formation, and renal failure. The formation of AGEs is dependent on both sugar and protein concentrations. Increases in temperature, pH, and exposure time of sugars to the proteins also play a significant role in the rate of AGE formation. This study focuses on the use of a combination of analytical techniques to study the in vitro AGE formation of HSA with dihydroxyacetone phosphate (DHAP), a ketose generated during glycolysis, and its dephosphorylated analog, dihydroxy acetone (DHA), commonly used as a browning reagent in skin tanning preparations. The extent of AGE formation was affected by DHAP and DHA concentrations and by the duration of HSA exposure to these glycating agents. Increases in temperature and pH sped the glycation process and enhanced the formation of the AGEs of HSA. MALDI-TOF mass spectroscopic data provided a reliable result to evaluate the extent of the AGE formation.     

Advanced glycation end products-induced apoptosis and overexpression of vascular endothelial growth factor in bovine retinal pericytes.

The influence of advanced glycation end products (AGEs) on apoptotic cell death and vascular endothelial growth factor (VEGF) gene expression in cultured bovine retinal pericytes was investigated. When pericytes were incubated with three immunochemically distinct AGEs, which were prepared in vitro by incubating bovine serum albumin with glucose, glyceraldehyde, or glycolaldehyde, apoptotic cell death and DNA ladder formation were significantly induced. The cytopathic effects of glyceraldehyde- or glycolaldehyde-derived AGEs were significantly enhanced in AGE receptor-transfected pericytes. Furthermore, all of these AGEs were found to upregulate the secretory forms of VEGF mRNA levels in retinal pericytes. These results suggest that AGEs disturbed retinal microvascular homeostasis by inducing pericyte apoptosis and VEGF overproduction and thus were involved in the pathogenesis of early phase diabetic retinopathy.     

Non-enzymatic glycoxidation linked with nutrition enhances the tumorigenic capacity of prostate cancer epithelia through AGE mediated activation of RAGE in cancer associated fibroblasts

The molecular implications of food consumption on cancer etiology are poorly defined. The rate of nutrition associated non-enzymatic glycoxidation, a reaction that occurs between reactive carbonyl groups on linear sugars and nucleophilic amino, lysyl and arginyl groups on fats and proteins, is rapidly increased by food cooking and manufacturing processes. In this study, we assign nutrition-associated glycoxidation with significant oncogenic potential, promoting prostate tumor growth, progression, and metastasis in vivo. Advanced glycation end products (AGEs) are the final irreversible product of non-enzymatic glycoxidation. Exogenous treatment of prostate tumor cells with a single AGE peptide replicated glycoxidation induced tumor growth in vivo. Mechanistically, receptor for AGE (RAGE) deficiency in the stroma inhibited AGE mediated tumor growth. Functionally, AGE treatment induced RAGE dimerization in activated fibroblasts which sustained and increased the migratory potential of tumor epithelial cells. These data identify a novel nutrition associated pathway that can promote a tissue microenvironment conducive for aggressive tumor growth. Targeted and/or interventional strategies aimed at reducing AGE bioavailability as a consequence of nutrition may be viewed as novel chemoprevention initiatives.     

Advanced glycation endproducts influence the mRNA expression of RAGE, RANKL and various osteoblastic genes in human osteoblasts

Glycation reactions resulting in the generation and accumulation of advanced glycation endproducts (AGEs) are potential mechanisms by which bone protein may be altered in vivo. AGEs accumulate in the bone increasingly with age come into close contact with osteoblasts or osteoclasts. The direct effect of AGEs on bone cells has not been thoroughly investigated. This study aimed to examine whether glycated bovine serum albumin (AGE - BSA) as an AGE modulate the mRNA expression of various genes in primary human osteoblast cultures. The following parameters were included: RAGE (receptor for AGEs), alkaline phosphatase, osteocalcin, osterix and RANKL (receptor activator of nuclear factor-kappaB ligand). Primary human osteoblast cultures were obtained from bone specimens of six patients with osteoarthrosis. Human osteoblasts were treated in AGE - BSA or control-BSA (non-glycated BSA) containing medium (5 mg/ml each) over a time course of seven days. After RT-PCR the mRNA expression was measured by real-time PCR. Related to control - BSA exposure, the mRNA expression of RAGE, RANKL and osterix increased during AGE - BSA treament. For alkaline phosphatase and osteocalcin a tendency of down-regulation was found. In summary, the study presents evidence that advanced glycation end products accumulated in bone alter osteoblasts by activation the AGE - RAGE pathway (RAGE mRNA up-regulation), inducing enhanced osteoclastogenesis (RANKL mRNA up-regulation) and impaired matrix mineralization (down-regulation of alkaline phosphatase and osteocalcin mRNA). Thus, AGEs may play a functional role in the development of bone diseases (e.g. osteoporosis).     

Mechanistic targeting of advanced glycation end-products in age-related diseases

Glycative stress, caused by the accumulation of cytotoxic and irreversibly-formed sugar-derived advanced glycation end-products (AGEs), contributes to morbidity associated with aging, age-related diseases, and metabolic diseases. In this review, we summarize pathways leading to formation of AGEs, largely from sugars and glycolytic intermediates, and discuss detoxification of AGE precursors, including the glyoxalase system and DJ-1/Park7 deglycase. Disease pathogenesis downstream of AGE accumulation can be cell autonomous due to aggregation of glycated proteins and impaired protein function, which occurs in ocular cataracts. Extracellular AGEs also activate RAGE signaling, leading to oxidative stress, inflammation, and leukostasis in diabetic complications such as diabetic retinopathy. Pharmaceutical agents have been tested in animal models and clinically to diminish glycative burden. We summarize existing strategies and point out several new directions to diminish glycative stress including: plant-derived polyphenols as AGE inhibitors and glyoxalase inducers; improved dietary patterns, particularly Mediterranean and low glycemic diets; and enhancing proteolytic capacities of the ubiquitin-proteasome and autophagy pathways that are involved in cellular clearing of AGEs.     

AGE/RAGE signalling regulation by miRNAs: Associations with diabetic complications and therapeutic potential

Excessive formation of advanced glycation end-products (AGEs) presents the most important mechanism of metabolic memory that underlies the pathophysiology of chronic diabetic complications. Independent of the level of hyperglycaemia, AGEs mediate intracellular glycation of the mitochondrial respiratory chain proteins leading to excessive production of reactive oxygen species (ROS) and amplification of their formation. Additionally, AGEs trigger intracellular damage via activation of the receptor for AGEs (RAGE) signalling axis that leads to elevation of cytosolic ROS, nuclear factor kappaB (NF-κB) activation, increased expression of adhesion molecules and cytokines, induction of oxidative and endoplasmic reticulum stress. Recent studies have identified novel microRNAs (miRNAs) involved in the regulation of AGE/RAGE signalling in the context of diabetic micro- and macrovascular complications. The aim of this review is to discuss the emerging role of miRNAs on AGE/RAGE pathway and the potential use of several miRNAs as novel therapeutic targets.     

The combination of high glucose and advanced glycation end-products (AGEs) inhibits the mineralization of osteoblastic MC3T3-E1 cells through glucose-induced increase in the receptor for AGEs.

Type 1 diabetes mellitus is known to be associated with reduced bone mass and increased bone fractures. This is thought to be due to a decrease in osteoblastic bone formation rather than an increase in osteoclastic bone resorption, but the precise mechanism is unknown. In this study, we examined whether or not high glucose or advanced glycation end-products (AGEs), which play key roles in the pathogenesis and complications of diabetes, affect the differentiation of osteoblastic MC3T3-E1 cells. First, MC3T3-E1 cells were incubated in media containing either 22 mM glucose, 22 mM mannitol, 300 microg/ml AGE2, or 300 microg/ml AGE3. Each of these agents alone did not affect the mineralization of the cells by von Kossa staining and Alizarin red staining. However, high glucose but not mannitol or AGEs markedly increased mRNA expression of AGE receptor (RAGE) by real-time PCR. Next, we examined the combined effects of high glucose and AGEs on the differentiation of MC3T3-E1 cells. The combination of 22 mM glucose and 300 microg/ml AGE2 significantly inhibited the mineralization of MC3T3-E1 cells, and 22 mM glucose in combination with either 300 microg/ml AGE2 or AGE3 apparently decreased osteocalcin mRNA expression. These results suggest that high glucose or AGEs alone might have no effect on osteoblastic differentiation, but their combination could additionally or synergistically inhibit osteoblastic mineralization through glucose-induced increase in RAGE expression.     

Effect of collagen turnover on the accumulation of advanced glycation end products

Collagen molecules in articular cartilage have an exceptionally long lifetime, which makes them susceptible to the accumulation of advanced glycation end products (AGEs). In fact, in comparison to other collagen-rich tissues, articular cartilage contains relatively high amounts of the AGE pentosidine. To test the hypothesis that this higher AGE accumulation is primarily the result of the slow turnover of cartilage collagen, AGE levels in cartilage and skin collagen were compared with the degree of racemization of aspartic acid (% d-Asp, a measure of the residence time of a protein). AGE (N(epsilon)-(carboxymethyl)lysine, N(epsilon)-(carboxyethyl)lysine, and pentosidine) and % d-Asp concentrations increased linearly with age in both cartilage and skin collagen (p < 0.0001). The rate of increase in AGEs was greater in cartilage collagen than in skin collagen (p < 0.0001). % d-Asp was also higher in cartilage collagen than in skin collagen (p < 0.0001), indicating that cartilage collagen has a longer residence time in the tissue, and thus a slower turnover, than skin collagen. In both types of collagen, AGE concentrations increased linearly with % d-Asp (p < 0.0005). Interestingly, the slopes of the curves of AGEs versus % d-Asp, i.e. the rates of accumulation of AGEs corrected for turnover, were identical for cartilage and skin collagen. The present study thus provides the first experimental evidence that protein turnover is a major determinant in AGE accumulation in different collagen types. From the age-related increases in % d-Asp the half-life of cartilage collagen was calculated to be 117 years and that of skin collagen 15 years, thereby providing the first reasonable estimates of the half-lives of these collagens.     

Alternative routes for the formation of immunochemically distinct advanced glycation end-products in vivo

The advanced stage of the glycation process (also called the "Maillard reaction") that leads to the formation of advanced glycation end-products (AGEs) plays an important role in the pathogenesis of angiopathy in diabetic patients and in the aging process. AGEs elicit a wide range of cell-mediated responses that might contribute to diabetic complications, vascular disease, renal disease, and Alzheimer's disease. Recently, it has been proposed that AGE are not only created from glucose per se, but also from dicarbonyl compounds derived from glycation, sugar autoxidation, and sugar metabolism. However, this advanced stage of glycation is still only partially characterized and the structures of the different AGEs that are generated in vivo have not been completely determined. Because of their heterogeneity and the complexity of the chemical reactions involved, only some AGEs have been characterized in vivo, including N-carboxymethyllysine (CML), pentosidine, pyrraline, and crosslines. In this article, we provide a brief overview of the pathways of AGE formation and of the immunochemical methods for detection of AGEs, and we also provide direct immunological evidence for the existence of five distinct AGE classes (designated as AGE-1 to -5) within the AGE-modified proteins and peptides in the serum of diabetic patients on hemodialysis. We also propose pathways for the in vivo formation of various AGEs by glycation, sugar autoxidation, and sugar metabolism.