Below the radar: advanced glycation end products that detour "around the side". Is HbA1c not an accurate enough predictor of long term progression and glycaemic control in diabetes?

Advanced glycation is the irreversible attachment of reducing sugars onto the free amino groups of proteins. Its physiological roles are thought to include the identification of senescent proteins and hence there is a time dependent accumulation of advanced glycation end products (AGEs). AGE labelled proteins are catabolised by cells into low molecular weight peptides and amino acids and excreted primarily via the kidneys. This process appears to be tightly controlled by AGE clearance receptor complexes containing AGE-R1, AGE-R2 and AGE-R3 and scavenger receptors such as CD36, SR-AII and SR-BI. Conditions such as diabetes, however, which have a metabolic overload of reducing sugars, rapidly accelerate AGE formation. In addition, advanced glycation is facilitated by oxidative stress and renal disease even in the absence of increases in reducing sugar concentrations. As part of our western diet, we also ingest AGEs of which approximately 50-80% are absorbed, catabolised and excreted over a period of two days. As AGE levels rise during diabetes, interruption of normal function occurs via three distinct mechanisms, namely AGE induced cross-linking of extracellular matrices, stiffening elastic fibres, disturbing cellular adhesion and preventing turnover. The second is by intracellular formation of AGEs, which causes generalised cellular dysfunction. The third is via the chronic activation of specific receptors such as RAGE, the receptor for advanced glycation end products, which produces excesses in inflammatory molecule production. Due to the range of dysfunction produced by the accumulation of AGEs in diabetes, there is a growing need for early recognition and intervention in this process.     

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.     

Inhibitory effect of some acetyl esters and acetamides on glycation of the histone H1

Non-enzymatic glycosylation (glycation) is a spontaneous set of reactions between reducing sugars and free amino groups in proteins or other biomolecules leading to the formation of fluorescent and coloured compounds known as advanced glycation end products (AGEs). AGEs cause structural changes of key proteins in humans, and therefore they are related with a number of physiological processes and diseases such as aging, atherosclerosis, cataract, arthritis, Alzheimer's disease. Two main strategies have been employed to prevent the formation of AGEs: a) low carbohydrate diet and b) pharmacological intervention. The latter includes treatment with reactive compounds which might be either sugar competitors (type A), carbonyl traps (type B) or free radical trapping antioxidants (type C). Acetylsalicylic acid (ASA, aspirin) is a good example of sugar competitor capable of inhibiting glycation by acetylating epsilon-amino groups of lysine residues in proteins. Taking into consideration the inhibiting effect of ASA on glycation we designed to study the antiglycation activity of other acetyl group-containing compounds (acetamides and acetyl esters) using the lysine-rich protein histone H1 as a model. The glycation of the histone H1 was carried out by either fructose or a complex mixture of glycating agents obtained from E. coli and monitored by fluorescent spectroscopy, SDS-PAGE and measurement of the content of reactive carbonyl groups in the target protein. Our results showed that the inhibitory effect of phenyl acetate, acetanilide, 4-acetamidophenylacetic acid and isopropenyl acetate was comparable to that of ASA. Based on the obtained results we conclude that these compounds act as free radical scavengers protecting proteins from the damaging effect of reactive oxygen species produced during the formation of AGEs.     

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.     

Molecular susceptibility to glycation and its implication in diabetes mellitus and related diseases

The modification of free amino groups on proteins, lipids, and nucleic acids by non-enzymatic glycosylation produce a variety of complex structures named advanced glycation end products (AGEs). Glycation of these molecules participate in the development of diabetic complications and related diseases. Diabetes mellitus is characterized by short-term metabolic changes in lipid and protein metabolism, and long-term irreversible changes in vascular and connective tissue. AGEs are directly implicated in the development of chronic complications in diabetes such as nephropathy, rethinopathy, neuropathy, and other related diseases such as atherosclerosis, heart disease, stroke, and peripheral vascular disease. In this review, we aim to explain how glycation occurs in different molecules and what the pathological consequence of AGE formation in diabetes mellitus and other diseases are.     

Ellagic acid, a new antiglycating agent: its inhibition of Nϵ-(carboxymethyl)lysine

Non-enzymatic glycation is a complex series of reactions between reducing sugars and amino groups of proteins. Accumulation of AGEs (advanced glycation end-products) due to non-enzymatic glycation has been related to several diseases associated with aging and diabetes. The formation of AGEs is accelerated in hyperglycaemic conditions, which alters the structure and function of long-lived proteins, thereby contributing to long-term diabetic complications. The present study describes AGE inhibition and the mechanism of action of a new antiglycating agent, EA (ellagic acid), a flavonoid present in many dietary sources. Inhibition of AGE formation by EA was demonstrated with different proteins, namely eye lens TSP (total soluble protein), Hb (haemoglobin), lysozyme and BSA, using different glycating agents such as fructose, ribose and methylglyoxal by a set of complementary methods. These results suggest that the antiglycating action of EA seems to involve, apart from inhibition of a few fluorescent AGEs, predominantly inhibition of CEL [N?-(carboxyethyl)lysine] through scavenging of the dicarbonyl compounds. Furthermore, MALDI-TOF-MS (matrix-assisted laser-desorption ionisation-time-of-flight MS) analysis confirms inhibition of the formation of CEL on lysozyme on in vitro glycation by EA. Prevention of glycation-mediated β-sheet formation in Hb and lysozyme by EA confirm its antiglycating ability. Inhibition of glycosylated Hb formation in human blood under ex vivo high-glucose conditions signifies the physiological antiglycating potential of EA. We have also determined the effectiveness of EA against loss of eye lens transparency through inhibition of AGEs in the lens organ culture system. These findings establish the antiglycating potential of EA and its in vivo utility in controlling AGE-mediated diabetic pathologies.     

Rutin metabolites: Novel inhibitors of nonoxidative advanced glycation end products

Glycation is a nonenzymatic condensation reaction between reducing sugars and amino groups of proteins that undergo rearrangements to stable ketoamines, leading to the formation of advanced glycation end products (AGEs) including fluorescent (argpyrimidine) and nonfluorescent (N^ε-carboxymethyllysine; CML) protein adducts and protein cross-links. AGEs are formed via protein glycation and correlate with processes resulting in aging and diabetes complications. Reactive carbonyl species such as glyoxal and methylglyoxal are ubiquitous by-products of cell metabolism that potently induce the formation of AGEs by nonenzymatic protein glycation and may achieve plasma concentrations of 0.3–1.5 μmol/L. In this in vitro study histone H1 glycation by glyoxal, methylglyoxal, or ADP-ribose was used to model nonoxidative protein glycation, permitting us to distinguish specific AGE inhibition from general antioxidant action. Rutin derivatives were tested as AGE inhibitors because rutin, a common dietary flavonoid that is consumed in fruits, vegetables, and plant-derived beverages, is metabolized by gut microflora to a range of phenolic compounds that are devoid of significant antioxidant activity and achieve blood concentrations in the μmol/L range. Our data show that in a 1:1 stoichiometry with glyoxal or methylglyoxal, 3,4-dihydroxyphenylacetic acid (DHPAA) and 3,4-dihydroxytoluene (DHT) are powerful inhibitors of CML and argpyrimidine histone H1 adduct formation, respectively. Furthermore, when DHPAA and DHT were tested as inhibitors of histone H1 glycation by the powerful glycating agent ADP-ribose, they inhibited glycation as effectively as aminoguanidine. These results suggest that dietary flavonoids may serve as effective AGE inhibitors and suggest mechanisms whereby fruit- and vegetable-rich diets contribute to the prevention of processes resulting in aging and diabetes complications.     

Glycation and post-translational processing of human interferon-gamma expressed in Escherichia coli

Until recently, nonenzymatic glycosylation (glycation) was thought to affect the proteins of long living eukaryotes only. However, in a recent study (Mironova, R., Niwa, T., Hayashi, H., Dimitrova, R., and Ivanov, I. (2001) Mol. Microbiol. 39, 1061-1068), we have shown that glycation takes place in Escherichia coli as well. In the present study, we demonstrate that the post-translational processing (proteolysis and covalent dimerization) observed with cysteineless recombinant human interferon-gamma (rhIFN-gamma) is tightly associated with its in vivo glycation. Our results show that, at the time of isolation, rhIFN-gamma contained early (but not advanced) glycation products. Using reverse phase high performance liquid chromatography in conjunction with fluorescence measurements, enzyme-linked immunosorbent assay, and mass spectrometry, we found that advanced glycation end products arose in rhIFN-gamma during storage. The latter were identified mainly in the Arg/Lys-rich C terminus of the protein, which was also the main target of proteolysis. Mass spectral analysis and N-terminal sequencing revealed four major (Arg140/Arg141, Phe137/Arg138, Met135/Leu136, and Lys131/Arg132) and two minor (Lys109/Ala110 and Arg90/Asp91) cleavage sites in this region. Tryptic peptide mapping indicated that the covalent dimers of rhIFN-gamma originating during storage were formed mainly by lateral cross-linking of the monomer subunits. Antiviral assay showed that proteolysis lowered the antiviral activity of rhIFN-gamma, whereas covalent dimerization completely abolished it.     

Effect of pyridoxamine on chemical modification of proteins by carbonyls in diabetic rats: characterization of a major product from the reaction of pyridoxamine and methylglyoxal

Advanced glycation end products (AGEs) from the Maillard reaction contribute to protein aging and the pathogenesis of age- and diabetes-associated complications. The alpha-dicarbonyl compound methylglyoxal (MG) is an important intermediate in AGE synthesis. Recent studies suggest that pyridoxamine inhibits formation of advanced glycation and lipoxidation products. We wanted to determine if pyridoxamine could inhibit MG-mediated Maillard reactions and thereby prevent AGE formation. When lens proteins were incubated with MG at 37 degrees C, pH 7.4, we found that pyridoxamine inhibits formation of methylglyoxal-derived AGEs concentration dependently. Pyridoxamine reduces MG levels in red blood cells and plasma and blocks formation of methylglyoxal-lysine dimer in plasma proteins from diabetic rats and it prevents pentosidine (an AGE derived from sugars) from forming in plasma proteins. Pyridoxamine also decreases formation of protein carbonyls and thiobarbituric-acid-reactive substances in plasma proteins from diabetic rats. Pyridoxamine treatment did not restore erythrocyte glutathione (which was reduced by almost half) in diabetic animals, but it enhanced erythrocyte glyoxalase I activity. We isolated a major product of the reaction between MG and pyridoxamine and identified it as methylglyoxal-pyridoxamine dimer. Our studies show that pyridoxamine reduces oxidative stress and AGE formation. We suspect that a direct interaction of pyridoxamine with MG partly accounts for AGE inhibition.     

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.     

AGEs in human lens capsule promote the TGFβ2‐mediated EMT of lens epithelial cells: implications for age‐associated fibrosis

Proteins in basement membrane (BM) are long‐lived and accumulate chemical modifications during aging; advanced glycation endproduct (AGE) formation is one such modification. The human lens capsule is a BM secreted by lens epithelial cells. In this study, we have investigated the effect of aging and cataracts on the AGE levels in the human lens capsule and determined their role in the epithelial‐to‐mesenchymal transition (EMT) of lens epithelial cells. EMT occurs during posterior capsule opacification (PCO), also known as secondary cataract formation. We found age‐dependent increases in several AGEs and significantly higher levels in cataractous lens capsules than in normal lens capsules measured by LC‐MS/MS. The TGFβ2‐mediated upregulation of the mRNA levels (by qPCR) of EMT‐associated proteins was significantly enhanced in cells cultured on AGE‐modified BM and human lens capsule compared with those on unmodified proteins. Such responses were also observed for TGFβ1. In the human capsular bag model of PCO, the AGE content of the capsule proteins was correlated with the synthesis of TGFβ2‐mediated α‐smooth muscle actin (αSMA). Taken together, our data imply that AGEs in the lens capsule promote the TGFβ2‐mediated fibrosis of lens epithelial cells during PCO and suggest that AGEs in BMs could have a broader role in aging and diabetes‐associated fibrosis.     

The pathogenic role of Maillard reaction in the aging eye

The proteins of the human eye are highly susceptible to the formation of advanced glycation end products (AGEs) from the reaction of sugars and carbonyl compounds. AGEs progressively accumulate in the aging lens and retina and accumulate at a higher rate in diseases that adversely affect vision such as, cataract, diabetic retinopathy and age-related macular degeneration. In the lens AGEs induce irreversible changes in structural proteins, which lead to lens protein aggregation and formation of high-molecular-weight aggregates that scatter light and impede vision. In the retina AGEs modify intra- and extracellular proteins that lead to an increase in oxidative stress and formation of pro-inflammatory cytokines, which promote vascular dysfunction. This review outlines recent advances in AGE research focusing on the mechanisms of their formation and their role in cataract and pathologies of the retina. The therapeutic action and pharmacological strategies of anti-AGE agents that can inhibit or prevent AGE formation in the eye are also discussed.     

Glycation of H1 Histone by 3-Deoxyglucosone: Effects on Protein Structure and Generation of Different Advanced Glycation End Products

Advanced glycation end products (AGEs) culminate from the non-enzymatic reaction between a free carbonyl group of a reducing sugar and free amino group of proteins. 3-deoxyglucosone (3-DG) is one of the dicarbonyl species that rapidly forms several protein-AGE complexes that are believed to be involved in the pathogenesis of several diseases, particularly diabetic complications. In this study, the generation of AGEs (N^ε-carboxymethyl lysine and pentosidine) by 3-DG in H1 histone protein was characterized by evaluating extent of side chain modification (lysine and arginine) and formation of Amadori products as well as carbonyl contents using several physicochemical techniques. Results strongly suggested that 3-DG is a potent glycating agent that forms various intermediates and AGEs during glycation reactions and affects the secondary structure of the H1 protein. Structural changes and AGE formation may influence the function of H1 histone and compromise chromatin structures in cases of secondary diabetic complications.     

UVA Light-excited Kynurenines Oxidize Ascorbate and Modify Lens Proteins through the Formation of Advanced Glycation End Products: IMPLICATIONS FOR HUMAN LENS AGING AND CATARACT FORMATION*

Advanced glycation end products (AGEs) contribute to lens protein pigmentation and cross-linking during aging and cataract formation. In vitro experiments have shown that ascorbate (ASC) oxidation products can form AGEs in proteins. However, the mechanisms of ASC oxidation and AGE formation in the human lens are poorly understood. Kynurenines are tryptophan oxidation products produced from the indoleamine 2,3-dioxygenase (IDO)-mediated kynurenine pathway and are present in the human lens. This study investigated the ability of UVA light-excited kynurenines to photooxidize ASC and to form AGEs in lens proteins. UVA light-excited kynurenines in both free and protein-bound forms rapidly oxidized ASC, and such oxidation occurred even in the absence of oxygen. High levels of GSH inhibited but did not completely block ASC oxidation. Upon UVA irradiation, pigmented proteins from human cataractous lenses also oxidized ASC. When exposed to UVA light (320–400 nm, 100 milliwatts/cm², 45 min to 2 h), young human lenses (20–36 years), which contain high levels of free kynurenines, lost a significant portion of their ASC content and accumulated AGEs. A similar formation of AGEs was observed in UVA-irradiated lenses from human IDO/human sodium-dependent vitamin C transporter-2 mice, which contain high levels of kynurenines and ASC. Our data suggest that kynurenine-mediated ASC oxidation followed by AGE formation may be an important mechanism for lens aging and the development of senile cataracts in humans.     

Transthyretin is up-regulated by sex hormones in mice liver

The chaperone function of alpha-crystallin is significantly affected in diabetes. Increased formation of advanced glycation end products (AGEs) is the likely cause. This study was aimed to investigate the effect of AGE crosslinks on the chaperone activity of alpha-crystallin and to show the effect of an AGEs crosslink breaker, phenacyl-4,5-dimethylthiazolium bromide (DMPTB). Recombinant alphaA-crystallin was prepared by expressing it in Escherichia coli and purified by size exclusion chromatography. Glycation of alphaA-crystallin was performed with 1-100 mM glucose-6-phosphate (G6P) as the glycating agent for a period of 1-15 days. To break AGE crosslinks, pre-glycated alphaA-crystallin was treated with 0.1-20 mM DMPTB for 3 days. Excess G6P and DMPTB were removed by gel filtration before performing additional experiments. AGEs and crosslinked proteins were estimated by measuring non-tryptophan fluorescence and by SDS-PAGE. Chaperone activity was determined with alcohol dehydrogenase as the target protein. With increasing duration of glycation and G6P concentration, chaperone activity of alpha-crystallin decreased. When pre-glycated alphaA-crystallin was treated with 5-20 mM DMPTB, a DMPTB concentration-dependent recovery of chaperone activity was seen. Lower concentrations, 0.1, 0.5, and 1.0 mM, of DMPTB also showed significant recovery of the chaperone activity. SDS-PAGE analysis after DMPTB treatment showed 40% decrease in crosslinked proteins and fluorescence scan indicated 30% decrease in AGEs. DMPTB is expected to regain alpha-crystallin chaperone activity and provide structural stability to other eye lens proteins that are in aggregation mode which emphasizes the clinical importance of the present finding.     

The in vitro inhibition effect of 2 nm gold nanoparticles on non-enzymatic glycation of human serum albumin

The reaction of amino groups of protein and the carbonyl groups of reducing sugar molecules, non-enzymatically induce a series of chemical reactions that form a heterogeneous group of compounds known as advanced glycation end products (AGEs). The accumulation of AGEs is associated with various disease conditions that include complications in diabetes, Alzheimer's disease and aging. The current study monitored the extent of non-enzymatic glycation of human serum albumin (HSA) in order to estimate the formation of HSA related AGEs in the presence of 2 nm gold nanoparticles. The rate of glycation was evaluated using several analytical methods. Physiological concentrations of HSA and glyceraldehyde mixtures, incubated with various concentrations of negatively charged 2 nm gold nanoparticles, resulted in a lower reaction rate than mixtures without 2GNP. Moreover, increasing concentrations of gold nanoparticles exhibited a pronounced reduction in AGE formation. High performance liquid chromatography, UV-visible spectroscopy and circular dichroism analytical methods provide reliable techniques for evaluating AGE formation of HSA adducts.     

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.     

Histone H1 as a Reporter Protein to Investigate Glycation in Bacteria

Nonenzymatic glycosylation (glycation) of proteins is a multistage chemical process starting as a condensation reaction between reducing sugars and primary amino groups (mainly from the side chains of Lis and Arg) and ending up with formation of complex heterocyclic compounds called advanced glycation end products (AGEs). For a long time, glycation has been attributed to the long-lived eukaryotes (including in humans) only. In a recent study, we showed that glycation also occurs in bacteria. The present study aims to prove that bacterial cytoplasm contains soluble glycating compounds. To this end, Lis/Arg-rich histone H1 isolated from rat liver was treated with deproteinized Escherichia coli cytoplasm through a dialysis membrane. This treatment leads to accumulation of AGEs as well as to a remarkable degradation of the reporter protein on storage at 4°C. Our results indicate also that glycation can be inhibited by acetylsalicylic acid (aspirin), thiamine (vitamin B1), and pyridoxine (vitamin B6).     

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.