Extract of Cassiae semen attenuates diabetic nephropathy via inhibition of advanced glycation end products accumulation in streptozotocin-induced diabetic rats

Chronic hyperglycemia leads to the formation of advanced glycation end products (AGEs), which accelerates the development of diabetic complications. Previous studies have shown that extract of Cassiae semen (CS), the seed of Cassia tora, has inhibitory activity on AGEs formation in vitro and reduces transforming growth factor-beta1 (TGF-β1) and extracellular matrix protein expression via inhibition of AGEs-mediated signaling in glomerular mesangial cells. In this study, to examine the preventive effects of CS extract on the development of diabetic nephropathy in vivo, streptozotocin (STZ)-injected diabetic rats were orally administered CS extract (200 mg/kg body weight/day) for 12 weeks. Serum glucose, triglycerides, and total cholesterol in diabetic rats were significantly higher compared to control rats. CS or aminoguanidine (AG) treatment significantly reduced these factors. Proteinuria and creatinine clearance were also significantly decreased in the CS-treated group compared with the untreated diabetic group. The CS-treated group had significantly inhibited COX-2 mRNA and protein, which mediates the symptoms of inflammation in the renal cortex of diabetic rats. Furthermore, histopathological studies of kidney tissue showed that in diabetic rats, AGEs, the receptor for AGEs, TGF-β1, and collagen IV were suppressed by CS treatment. Our data suggest that oral treatment of CS can inhibit the development of diabetic nephropathy via inhibition of AGEs accumulation in STZ-induced diabetic rats.     

Advanced glycation end product ligands for the receptor for advanced glycation end products: biochemical characterization and formation kinetics

Advanced glycation end products (AGEs) accumulate with age and at an accelerated rate in diabetes. AGEs bind cell-surface receptors including the receptor for advanced glycation end products (RAGE). The dependence of RAGE binding on specific biochemical characteristics of AGEs is currently unknown. Using standardized procedures and a variety of AGE measures, the present study aimed to characterize the AGEs that bind to RAGE and their formation kinetics in vitro. To produce AGEs with varying RAGE binding affinity, bovine serum albumin (BSA) AGEs were prepared with 0.5M glucose, fructose, or ribose at times of incubation from 0 to 12 weeks or for up to 3 days with glycolaldehyde or glyoxylic acid. The AGE-BSAs were characterized for RAGE binding affinity, fluorescence, absorbance, carbonyl content, reactive free amine content, molecular weight, pentosidine content, and N-epsilon-carboxymethyl lysine content. Ribose-AGEs bound RAGE with high affinity within 1 week of incubation in contrast to glucose- and fructose-AGE, which required 12 and 6 weeks, respectively, to generate equivalent RAGE ligands (IC50=0.66, 0.93, and 1.7 microM, respectively). Over time, all of the measured AGE characteristics increased. However, only free amine content robustly correlated with RAGE binding affinity. In addition, detailed protocols for the generation of AGEs that reproducibly bind RAGE with high affinity were developed, which will allow for further study of the RAGE-AGE interaction.     

Endoplasmic reticulum stress plays a role in the advanced glycation end product-induced inflammatory response in endothelial cells

Aims

Both advanced glycation end products (AGEs) and endoplasmic reticulum (ER) stress play important roles in the development of various diseases. This study aimed to clarify the consequence of AGE-induced ER stress and its underlying mechanisms in human umbilical venous endothelial cells (HUVECs).

Main methods

AGE-induced ER stress was assessed by the increased expression and activation of the ER stress marker proteins GRP78, IRE1α and JNK, which were detected using Western blot. NF-κB translocation was revealed using Western blot and immunofluorescent staining in IRE1α-knockdown HUVECs. The mechanism of AGE-induced ER stress was also explored by inhibiting the effect of reactive oxygen species (ROS) using NADPH oxidase 4 (Nox4) siRNA and the antioxidant reduced glutathione (GSH). The cellular ROS level was measured using flow cytometry.

Key findings

AGEs time- and dose-dependently enhanced the expression of GRP78 and increased the phosphorylation of IRE1α and its downstream signal JNK in HUVECs. siRNA-induced IRE1α down-regulation suppressed AGE-induced NF-κB p65 nuclear translocation. Inhibiting the ROS production using Nox4 siRNA or antagonizing ROS using GSH reduced cellular ROS level and attenuated AGE-induced GRP78 expression and IRE1α and JNK activation.

Significance

This study confirms that AGE-induced ER stress in HUVECs focuses on the ER stress-enhanced inflammatory response through JNK and NF-κB activation. It further reveals the involvement of ROS in the AGE-induced ER stress mechanism.     

Advanced glycation end productions and tendon stem/progenitor cells in pathogenesis of diabetic tendinopathy

Tendinopathy is a challenging complication observed in patients with diabetes mellitus. Tendinopathy usually leads to chronic pain, limited joint motion, and even ruptured tendons. Imaging and histological analyses have revealed pathological changes in various tendons of patients with diabetes, including disorganized arrangement of collagen fibers, microtears, calcium nodules, and advanced glycation end product (AGE) deposition. Tendon-derived stem/ progenitor cells (TSPCs) were found to maintain hemostasis and to participate in the reversal of tendinopathy. We also discovered the aberrant osteochondrogenesis of TSPCs in vitro. However, the relationship between AGEs and TSPCs in diabetic tendinopathy and the underlying mechanism remain unclear. In this review, we summarize the current findings in this field and hypothesize that AGEs could alter the properties of tendons in patients with diabetes by regulating the proliferation and differentiation of TSPCs in vivo.     

Advanced glycation end products impair function of late endothelial progenitor cells through effects on protein kinase Akt and cyclooxygenase-2

Endothelial progenitor cells (EPCs) exhibit impaired function in the context of diabetes, and advanced glycation end products (AGEs), which accumulate in diabetes, may contribute to this. In the present study, we investigated the mechanism by which AGEs impair late EPC function. EPCs from human umbilical cord blood were isolated, and incubated with AGE-modified albumin (AGE-albumin) at different concentrations found physiologically in plasma. Apoptosis, migration, and tube formation assays were used to evaluate EPC function including capacity for vasculogenesis, and expression of the receptor for AGEs (RAGE), Akt, endothelial nitric oxide synthase (eNOS), and cycloxygenase-2 (COX-2) were determined. Anti-RAGE antibody was used to block RAGE function. AGE-albumin concentration-dependently enhanced apoptosis and depressed migration and tube formation, but did not affect proliferation, of late EPCs. High AGE-albumin increased RAGE mRNA and protein expression, and decreased Akt and COX-2 protein expression, whilst having no effect on eNOS mRNA or protein in these cells. These effects were inhibited by co-incubation with anti-RAGE antibody. These results suggest that RAGE mediates the AGE-induced impairment of late EPC function, through down-regulation of Akt and COX-2 in these cells.     

Reduced cell replication and induction of apoptosis by advanced glycation end products in rat Schwann cells

We investigated the effects of advanced glycation end products (AGEs) derived from glucose, glyceraldehyde, and glycolaldehyde (designated as AGE-1, -2, and -3, respectively) on the viability, replication rate, and cytokine production of cultured Schwann cells. AGE-2 and -3, but not AGE-1, induced apoptosis, and significantly decreased the viability measured by MTT assay. The decrease was prevented completely by antioxidant alpha-lipoic acid and was prevented partially by p38 mitogen-activated protein kinase inhibitor SB202190. The decrease in mitochondrial membrane potential by AGE-2 and -3 was also observed. In addition, AGE-2 and -3 significantly suppressed the replication rate as shown by reduced bromodeoxyuridine uptake, whereas they enhanced the release of TNF-alpha and IL-1beta into the medium and activated nuclear factor-kappaB. The effects of AGE-1 on these measures were equivocal. The series of events elicited by AGE-2 and -3 may be responsible for some of the aspects of pathogenetic mechanisms in patients with diabetic neuropathy.     

Characterization of advanced glycation end products for biochemical studies: side chain modifications and fluorescence characteristics

Advanced glycation end products (AGEs) are known to be involved in the pathogenesis of several diseases and therefore effects of AGEs on cells are the objective of numerous investigations. Since AGEs used in biochemical studies are usually not chemically characterized, comparison of data is difficult if not impossible. To find a suitable characterization protocol, human serum albumin was reacted with different concentrations of glucose, methyl glyoxal, and glyoxylic acid. The obtained AGEs were characterized with respect to the extent of side chain modifications (lysine and arginine), the carboxymethyl lysine and carbonyl content, and the fibrillar state. Additionally, their fluorescence and absorbance characteristics were extensively studied. Although we found significant differences in the degree of modification and in AGE-specific fluorescence when using different modifiers, the results provide important information and allow comparing AGEs derived from different modifier concentrations. The results also suggest strong conformational changes within the modified proteins. In the present paper we propose a set of parameters that is sufficient to partially characterize AGEs used for biochemical studies.     

Plasma protein advanced glycation end products, carboxymethyl cysteine, and carboxyethyl cysteine, are elevated and related to nephropathy in patients with diabetes

In Diabetes Mellitus (DM), glucose and the aldehydes glyoxal and methylglyoxal modify free amino groups of lysine and arginine of proteins forming advanced glycation end products (AGEs). Elevated levels of these AGEs are implicated in diabetic complications including nephropathy. Our objective was to measure carboxymethyl cysteine (CMC) and carboxyethyl cysteine (CEC), AGEs formed by modification of free cysteine sulfhydryl groups of proteins by these aldehydes, in plasma proteins of patients with diabetes, and investigate their association with the albumin creatinine ratio (ACR, urine albumin (mg)/creatinine (mmol)), an indicator of nephropathy. Blood was collected from forty-two patients with type 1 and 2 diabetes (18–36 years) and eighteen individuals without diabetes (17–35 years). A liquid chromatography-mass spectrophotometric method was developed to measure plasma protein CMC and CEC levels. Values for ACR and hemoglobin A1C (HbA1C) were obtained. Mean plasma CMC (μg/l) and CEC (μg/l) were significantly higher in DM (55.73 ± 29.43, 521.47 ± 239.13, respectively) compared to controls (24.25 ± 10.26, 262.85 ± 132.02, respectively). In patients with diabetes CMC and CEC were positively correlated with ACR, as was HbA1C. Further, CMC or CEC in combination with HbA1C were better predictors of nephropathy than any one of these variables alone. These results suggest that glucose, glyoxal, and methylglyoxal may all be involved in the etiology of diabetic nephropathy.     

Characterization of advanced glycation end products: mass changes in correlation to side chain modifications

Advanced glycation end products (AGEs) that arise from the reaction of sugars with protein side chains are supposed to be involved in the pathogenesis of several diseases; therefore, the effects of AGEs on cells are the objective of numerous investigations. Because AGE modifications are an extremely heterogeneous group of side chain modifications, the exact characterization of an AGE-modified protein is impossible. To gain a deeper understanding about AGE formation kinetics and structures, AGEs can be characterized with respect to the degree of modification, specific side chain modifications, absorbance and fluorescence characteristics, and changes in the protein structure and molecular weight. For this study, human serum albumin (HSA)-AGEs derived from different concentrations of glucose, methyl glyoxal, and glyoxylic acid were used. The molecular mass of the obtained AGEs was determined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The mass data were compared with earlier results concerning the degree of lysine and arginine side chain modifications and AGE-specific fluorescence and absorbance data. The molecular masses were found to gradually increase with increasing concentrations of the individual modifier without reaching a plateau. The mass increase correlates very well with the AGE-specific absorbance at 360 nm and with the degree of side chain modifications. The mass spectrometric data prove, for the first time, that an increasing absorbance at 360 nm is directly correlated to a mass increase during the AGE formation process.     

Implications of advanced oxidation protein products (AOPPs), advanced glycation end products (AGEs) and other biomarkers in the development of cardiovascular diseases

Objective

To study the putative effects of Advanced Oxidation Protein Products (AOPPs) and Advanced Glycation End Products (AGEs) in the development and progression of cardiovascular disease (CVD).

Acetoacetate promotes the formation of fluorescent advanced glycation end products (AGEs)

Acetoacetate (AA) is an important ketone body, which produces reactive oxygen species (ROS). Advanced glycation end products (AGEs) are defined as final products of glycation process whose production is influenced by the levels of ROS. The accumulation of AGEs in the body contributes to pathogenesis of many diseases including complications of diabetes, and Alzheimer’s and Parkinson’s disease. Here, we evaluated the impact of AA on production of AGEs upon incubation of human serum albumin (HSA) with glucose. The effect of AA on the AGEs formation of HSA was studied under physiological conditions after incubation with glucose for 35 days. The physical techniques including circular dichroism (CD) and fluorescence spectroscopy were used to assess the impact of AA on formation and structural changes of glycated HSA (GHSA). Our results indicated that the secondary and tertiary structural changes of GHSA were increased in the presence of AA. The fluorescence intensity measurements of AGEs also showed an increase in AGEs formation. Acetoacetate has an activator effect in formation of AGEs through ROS production. The presence of AA may result in enhanced glycation in the presence of glucose and severity of complications associated with accumulation of AGEs.     

Quercetin inhibits advanced glycation end product formation via chelating metal ions,trapping methylglyoxal,and trapping reactive oxygen species

Physiological concentration of Mg²⁺, Cu²⁺, and Zn²⁺ accelerated AGE formation only in glucose-mediated conditions, which was effectively inhibited by chelating ligands. Only quercetin (10) inhibited MGO-mediated AGE formation as well as glucose- and ribose-mediated AGE formation among 10 polyphenols (1–10) tested. We performed an additional structure-activity relationship (SAR) study on flavanols (10, 11, 12, 13, and 14). Morin (12) and kaempherol (14) showed inhibitory activity against MGO-mediated AGE formation, whereas rutin (11) and fisetin (13) did not. These observations indicate that 3,5,7,4′-tetrahydroxy and 4-keto groups of 10 are important to yield newly revised mono-MGO adducts (16 and 17) and di-MGO adduct (18) having cyclic hemiacetals, while 3′-hydroxy group is not essential. We propose here a comprehensive inhibitory mechanism of 10 against AGE formation including chelation effect, trapping of MGO, and trapping of reactive oxygen species (ROS), which leads to oxidative degradation of 18 to 3,4-dihydroxybenzoic acid (15) and other fragments.     

Advanced glycation end products serve as ligands for lectin-like oxidized low-density lipoprotein receptor-1(LOX-1): biochemical and binding characterizations assay

Advanced glycation end products (AGEs) are a class of complex heterogeneous compounds which accumulate with age and is known to be involved in the pathogenesis of several diseases from diabetes to atherosclerosis. AGEs serve as ligands for multiple receptors including scavenger receptor (SR-A), CD36, and SR-BIota. Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) plays an important role in both atherosclerosis and is found to be an endothelial cell receptor for AGEs. To explore the binding characterization of AGEs to LOX-1, AGEs were prepared by three different reducing sugars (d-glucose, d-fructose, and d-ribose) and the biochemical characterization including, free amino groups, free amine content, fructosamine residues, carbonyl content, fluorescence, and absorbance were determined. The binding activity was determined by FITC labeled AGEs using Chinese hamster ovary-K1 cells stably transfected with human LOX-1 gene. The obtained AGEs showed significant differences in the extent of side chain modifications, carbonyl content, fluorescence, and absorption models. All of the AGEs showed specific and saturable binding to hLOX-1-CHO-K1 cells. Furthermore, dose-dependent binding processes were observed. However, the maximal cellular binding of AGEs differs between the sugars (glucose > ribose > fructose). In addition, oxidized low-density lipoprotein (ox-LDL) could significantly inhibit the binding of AGEs to LOX-1 with different inhibitory efficiency. LOX-1 serves as receptor for AGEs which may give some insight into the role of LOX-1 in the pathogenesis of diabetes and related disorders.     

Advanced glycoxidation and lipoxidation end products (AGEs and ALEs): an overview of their mechanisms of formation

Abstract

Advanced lipoxidation end products (ALEs) and advanced glycation end products (AGEs) have a pathogenetic role in the development and progression of different oxidative-based diseases including diabetes, atherosclerosis, and neurological disorders. AGEs and ALEs represent a quite complex class of compounds that are formed by different mechanisms, by heterogeneous precursors and that can be formed either exogenously or endogenously. There is a wide interest in AGEs and ALEs involving different aspects of research which are essentially focused on set-up and application of analytical strategies (1) to identify, characterize, and quantify AGEs and ALEs in different pathophysiological conditions; (2) to elucidate the molecular basis of their biological effects; and (3) to discover compounds able to inhibit AGEs/ALEs damaging effects not only as biological tools aimed at validating AGEs/ALEs as drug target, but also as promising drugs. All the above-mentioned research stages require a clear picture of the chemical formation of AGEs/ALEs but this is not simple, due to the complex and heterogeneous pathways, involving different precursors and mechanisms. In view of this intricate scenario, the aim of the present review is to group the main AGEs and ALEs and to describe, for each of them, the precursors and mechanisms of formation.     

Genotoxicity and immunogenicity of DNA-advanced glycation end products formed by methylglyoxal and lysine in presence of Cu

The highly reactive electrophile, methylglyoxal (MG), a break down product of carbohydrates, is a major environmental mutagen having potential genotoxic effects. Previous studies have suggested the reaction of MG with free amino groups of proteins forming advanced glycation end products (AGEs). This results in the generation of free radicals which play an important role in pathophysiology of aging and diabetic complications. MG also reacts with free amino group of nucleic acids resulting in the formation of DNA–AGEs. While the formation of nucleoside AGEs has been demonstrated previously, no extensive studies have been performed to assess the genotoxicity and immunogenicity of DNA–AGEs. In this study we report both the genotoxicity and immunogenicity of AGEs formed by MG–Lys–Cu²⁺ system. Genotoxicity of the experimentally generated AGEs was confirmed by comet-assay. Spectroscopical analysis and melting temperature studies suggest structural perturbations in the DNA as a result of modification. This might be due to generation of single-stranded regions and destabilization of hydrogen bonds. Immunogenicity of native and MG–Lys–Cu²⁺-DNA was probed in female rabbits. The modified DNA was highly immunogenic eliciting high titre immunogen specific antibodies, while the unmodified form was almost non-immunogenic. The results show structural perturbations in MG–Lys–Cu²⁺-DNA generating new epitopes that render the molecule immunogenic.     

Advanced glycation end products regulate anabolic and catabolic activities via NLRP3‐inflammasome activation in human nucleus pulposus cells

Intervertebral disc degeneration is widely recognized as a cause of lower back pain, neurological dysfunction and other musculoskeletal disorders. The major inflammatory cytokine IL‐1β is associated with intervertebral disc degeneration; however, the molecular mechanisms that drive IL‐1β production in the intervertebral disc, especially in nucleus pulposus (NP) cells, are unknown. In some tissues, advanced glycation end products (AGEs), which accumulate in NP tissues and promote its degeneration, increase oxidative stress and IL‐1β secretion, resulting in disorders, such as obesity, diabetes mellitus and ageing. It remains unclear whether AGEs exhibit similar effects in NP cells. In this study, we observed significant activation of the NLRP3 inflammasome in NP tissues obtained from patients with degenerative disc disease compared to that with idiopathic scoliosis according to results detected by Western blot and immunofluorescence. Using NP cells established from healthy tissues, our in vitro study revealed that AGEs induced an inflammatory response in NP cells and a degenerative phenotype in a NLRP3‐inflammasome‐dependent manner related to the receptor for AGEs (RAGE)/NF‐κB pathway and mitochondrial damage induced by mitochondrial reactive oxygen species (mtROS) generation, mitochondrial permeability transition pore (mPTP) activation and calcium mobilization. Among these signals, both RAGE and mitochondrial damage primed NLRP3 and pro‐IL‐1β activation as upstream signals of NF‐κB activity, whereas mitochondrial damage was critical for the assembly of inflammasome components. These results revealed that accumulation of AGEs in NP tissue may initiate inflammation‐related degeneration of the intervertebral disc via activation of the NLRP3 inflammasome.