Specific Advanced Glycation End-products Research Articles

Identification of Yellow Advanced Glycation End Products in Human Skin.

Skin yellowness is a hallmark of dull or unhealthy skin, particularly among Asians. Previous research has indicated a link between skin glycation and skin yellowness. However, the specific glycated chemicals contributing to yellowish skin appearance have not been identified yet. Using HPLC-PDA-HRMS coupled with native and artificially glycated human epidermal explant skin, we identified intensely yellow colored glycated chromophores "(1R, 8aR) and (1S, 8aR)-4-(2-furyl)-7-[(2-furyl)-methylidene]-2-hydroxy-2H,7H,8AH-pyrano-[2,3-B]-pyran-3-one" (abbreviated as AGEY) from human skin samples for the first time. The abundance of AGEY was strongly correlated with skin yellowness in the multiple skin explant tissues. We further confirmed the presence of AGEY in cultured human keratinocytes and 3D reconstructed human epidermal (RHE) models. Additionally, we demonstrated that a combination of four cosmetic compounds with anti-glycation properties can inhibit the formation of AGEY and reduce yellowness in the RHE models. In conclusion, we have identified specific advanced glycation end products with an intense yellow color, namely AGEY, in human skin tissues for the first time. The series of study results highlighted the significant contribution of AGEY to the yellow appearance of the skin. Furthermore, we have identified a potential cosmetic solution to mitigate AGEY formation, leading to a reduction in yellowness in the in vitro RHE models.

A 4-week high-AGE diet does not impair glucose metabolism and vascular function in obese individuals.

BACKGROUNDAccumulation of advanced glycation endproducts (AGEs) may contribute to the pathophysiology of type 2 diabetes and its vascular complications. AGEs are widely present in food, but whether restricting AGE intake improves risk factors for type 2 diabetes and vascular dysfunction is controversial.METHODSAbdominally obese but otherwise healthy individuals were randomly assigned to a specifically designed 4-week diet low or high in AGEs in a double-blind, parallel design. Insulin sensitivity, secretion, and clearance were assessed by a combined hyperinsulinemic-euglycemic and hyperglycemic clamp. Micro- and macrovascular function, inflammation, and lipid profiles were assessed by state-of-the-art in vivo measurements and biomarkers. Specific urinary and plasma AGEs Nε-(carboxymethyl)lysine (CML), Nε-(1-carboxyethyl)lysine (CEL), and Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H1) were assessed by mass spectrometry.RESULTSIn 73 individuals (22 males, mean ± SD age and BMI 52 ± 14 years, 30.6 ± 4.0 kg/m2), intake of CML, CEL, and MG-H1 differed 2.7-, 5.3-, and 3.7-fold between the low- and high-AGE diets, leading to corresponding changes of these AGEs in urine and plasma. Despite this, there was no difference in insulin sensitivity, secretion, or clearance; micro- and macrovascular function; overall inflammation; or lipid profile between the low and high dietary AGE groups (for all treatment effects, P > 0.05).CONCLUSIONThis comprehensive RCT demonstrates very limited biological consequences of a 4-week diet low or high in AGEs in abdominally obese individuals.TRIAL REGISTRATIONClinicaltrials.gov, NCT03866343; trialregister.nl, NTR7594.FUNDINGDiabetesfonds and ZonMw.

Deciphering Changes in the Structure and IgE-Binding Ability of Ovalbumin Glycated by α-Dicarbonyl Compounds under Simulated Heating.

As a complex reaction, biological consequences of the Maillard reaction (MR) on dietary proteins need to be deciphered. Despite previous studies on the structural and antigenic properties of ovalbumin (OVA) by MR, associated changes induced by specific MR intermediates and their downstream products are largely unknown. This study focused on the impacts of glycation by α-dicarbonyl compounds (α-DCs), intermediates of MR and precursors of advanced glycation end-products (AGEs), on the structural and IgE-binding properties of ovalbumin (OVA) under simulated heating. Methylglyoxal (MGO), glyoxal (GO), and butanedione (BU) were selected as typical α-DCs to generate glycated OVA with different AGE-modifications (AGE-Ms). The results showed that reactions between OVA and α-DCs generated OVA-AGE with various degrees of modification and conformational unfolding, and the reactivity of α-DCs followed the order GO > MGO > BU. Depending on the precursor type, the levels of 10 specific AGEs were verified, and the amounts of total AGEs increased with heating temperature and α-DC dosage. Compared to native OVA, glycated OVA showed reduced IgE-binding levels but with sRAGE-binding ligands, the extent of which was associated with the contents of total AGEs and Nε-carboxymethyllysine, and changes in certain protein conformational structures. High-resolution mass spectrometry further identified different AGE-Ms on the Lys and Arg residues of OVA, confirming variations in the glycation sites and their associations with the immunoreactive epitopes of OVA under different conditions.

The inhibitory effects of natural antioxidants on protein glycation as well as aggregation induced by methylglyoxal and underlying mechanisms

The non-enzymatic glycation of protein can result in the formation of advanced glycation end-products (AGEs), leading to the deposition of amyloid proteins, and it’s essential for the pathogenesis of diabetes complications and amyloid diseases. Reactive dicarbonyl compounds, such as methylglyoxal (MGO), are one of the most reactive glycating agents. Therefore, it’s crucially necessary to inhibit protein glycation and aggregation induced by MGO. In the present study, we aimed to systemically investigate the anti-glycation and anti-fibrillization activities of eight natural antioxidants, including apigenin, quercetin (Que), catechin, resveratrol (Res), and gallic acid (GA), L-ascorbic acid (L-AA), limonene, and β-carotene, during MGO-induced protein glycation and aggregation. Furthermore, the underlying mechanisms were clarified. The formation of AGEs and the degree of protein aggregation were characterized by optical detection, flow cytometry, and so on. The results demonstrated that eight selected natural antioxidants could inhibit glycation and protein aggregation induced by MGO via the synergy of scavenging free radicals, capturing MGO, and interacting with proteins, among which GA (300 μM) and Res (15 μM) had higher inhibition rates on both argpyrimidine (specific fluorescent AGEs, 17% and 22%, respectively) and protein amyloid aggregation (42% and 29%, respectively). These findings suggested that antioxidants could act as potential inhibitors of AGEs and glycation-induced protein aggregation, which were expected to become a new strategy for the prevention and treatment of diabetes and amyloid diseases. Besides, these inhibition mechanisms provided valuable insights into the design and development of candidate drugs for the prevention and treatment of AGEs and protein aggregation-related diseases.

Controlled Formation of Carboxymethyllysine in Bone Matrix through Designed Glycation Reaction.

ABSTRACTIt has been a challenge to establish a link between specific advanced glycation end products (AGEs) as causal agents of different pathologies and age‐related diseases, primarily because of the lack of suitable in vitro experimental strategies facilitating increased formation of a specific AGE, here carboxymethyllysine (CML), over other AGEs under controlled conditions. CML is of considerable importance to various oxidative stress–related diseases, because in vivo formation of this AGE is connected with cellular oxidative/carbonyl metabolism. The mechanistic implications of CML accumulation in bone remain to be elucidated. To facilitate such studies, we developed a new in vitro strategy that allows preferential generation of CML in bone matrix over other AGEs. Using bone samples from human donors of different age (young, middle‐age, and elderly), we show successful in vitro generation of the desired levels of CML and show that they mimic those observed in vivo in several bone disorders. Formation of such physiologically relevant CML levels was achieved by selecting two oxidative/carbonyl stress compounds naturally produced in the human body, glyoxal and glyoxylic acid. Kinetic studies using the two compounds revealed differences not only between their reaction rates but also in the progression and enhanced formation of CML over other AGEs (measured by their collective fluorescence as fluorescent AGEs [fAGEs]) Consequently, through the regulation of reaction time, the levels of CML and fAGEs could be controlled and separated. Given that the developed approach does not fully eliminate the formation of other uncharacterized glycation products, this could be considered as the study limitation. We expect that the concepts of our experimental approach can be used to develop diverse strategies facilitating production of the desired levels of selected AGEs in bone and other tissues, and thus, opens new avenues for investigating the role and mechanistic aspects of specific AGEs, here CML, in bone. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Chinese bayberry (Myrica rubra) phenolics mitigated protein glycoxidation and formation of advanced glycation end-products: A mechanistic investigation

Protein glycation and formation of advanced glycation end-products (AGEs) impose threats to the human health. This study firstly investigated the inhibition of Chinese bayberry (Myrica rubra) phenolics on AGEs formation through mechanistic analysis. Four common Chinese bayberry cultivars were selected to prepare phenolic-rich extracts (CBEs) and characterized for phenolic composition, and their anti-AGE properties were evaluated in multiple in vitro systems. Total sixteen phenolics were quantified in CBEs by UPLC-ESI-MS/MS. CBEs reduced total and specific fluorescent AGEs formation in various simulating models, and protected the protein from structural modification, oxidation, and cross-linking. Mechanistic analysis unveiled that scavenging of free radicals, inactivation of transition metals, interaction with protein to form complexes, and trapping of reactive α-dicarbonyls to form adducts underlain the mechanisms of the anti-glycative actions of CBEs. Chinese bayberry fruits, especially the cultivars Biqi and Wuzi, may be a promising dietary strategy to mitigate AGEs load in the human body.

Role of curd and yogurt in establishment and progression of diabetes through protein glycation and induction of inflammation

Curd and yogurt is rich in protein, calcium, magnesium and other vitamins and considered as beneficial for diabetics. The present study delves into another aspect of curd and yogurt; viz. its role in protein glycation and thereby initiation of inflammation. Curd contains glyoxal and methylglyoxal and thereby capable of glycating proteins. The present study has used curd set for 12 h, 24 h and yogurt. Rats were fed 1.5 g curd and yogurt twice a day for 45 days. General and specific Advanced Glycation End Products (AGEs) were measured from various tissues showing 6.5, 10.5 and 11.5 fold increase in general fluorescence in the liver tissue in 12 h curd, yogurt and 24 h curd fed rats respectively. The specific fluorescence was even higher in liver and kidney. Likewise, enhanced AGEs formation was also observed in intestines, heart, lungs, lens, soleus muscle and pancreas. Animals demonstrated lower SOD value and higher catalase and TBARS implying oxidative stress. The extent of stress was as 24 h > yogurt > 12 h fed rats. IL-6, IL-10 and TNF-α values were found to increase in a similar pattern while that of IL-4 and IFN-γ were reduced. OGTT revealed an impaired glucose tolerance in 24 h curd fed rats and yogurt. In view of lesser effect of 12 h set curd, its effect on diabetic animals was studied, demonstrating an increase in AGEs in all the tissues. Hence regular consumption of curds can lead to glycation of proteins, inflammation and oxidative stress that can cause progression of diabetes.

P 141 - Peripheral oxidative profile and specific advanced glycation end products can be a signature of cognitive decline in Alzheimer's disease

An early diagnosis of Alzheimer's disease (AD) is required for early and more effective treatment. Oxidative stress plays an early role in AD and could promote the formation of advanced glycation end products (AGEs) in AD brains. However, the peripheral association of specific AGEs and oxidative markers with cognitive decline remains to be defined in order to establish a clinical signature for an early diagnosis. Objectives: To measure the total antioxidant capacity (TAC), the levels of protein carbonyl (PC), AGEs precursors (methylglyoxal, MG; glyoxal, GO) and some specific AGEs (pentosidine; carboxylmethyl lysine, CML) and their association with clinical scores (MMSE and MoCA). Methods: These markers were measured in blood from patients with MCI (mild cognitive impairment) and AD at different stages and from age-matched controls by Western blot, ELISA or HPLC. Results: A decrease of TAC and an increase of AGEs precursor's levels (MG and GO) are observed in MCI patients. PC and CML levels raised later in AD groups. Pentosidine levels were not different. Interestingly, PC and CML levels are correlated with cognitive scores. Conclusion: Our results demonstrate that TAC decreases early in MCI stage while some oxidative markers and AGEs precursors increase and are correlated with cognitive decline. Overall, some oxidative stress markers have a potential as biomarkers for AD.

Advanced Glycation End Products, Oxidation Products, and the Extent of Atherosclerosis During the VA Diabetes Trial and Follow-up Study.

OBJECTIVETo determine whether plasma levels of advanced glycation end products and oxidation products play a role in the development of atherosclerosis in patients with type 2 diabetes (T2D) over nearly 10 years of the VA Diabetes Trial and Follow-up Study.RESEARCH DESIGN AND METHODSBaseline plasma levels of methylglyoxal hydroimidazolone, Nε-carboxymethyl lysine, Nε-carboxyethyl lysine (CEL), 3-deoxyglucosone hydroimidazolone and glyoxal hydroimidazolone (G-H1), 2-aminoadipic acid (2-AAA), and methionine sulfoxide were measured in a total of 411 participants, who underwent ultrasound assessment of carotid intima-media thickness (CIMT), and computed tomography scanning of coronary artery calcification (CAC) and abdominal aortic artery calcification (AAC) after an average of 10 years of follow-up.RESULTSIn risk factor–adjusted multivariable regression models, G-H1 was associated with the extent of CIMT and CAC. In addition, 2-AAA was strongly associated with the extent of CAC, and CEL was strongly associated with the extent of AAC. The combination of specific advanced glycation end products and oxidation products (G-H1 and 2-AAA) was strongly associated with all measures of subclinical atherosclerosis.CONCLUSIONSSpecific advanced glycation end products and metabolic oxidation products are associated with the severity of subclinical atherosclerosis over the long term and may play an important role in the “negative metabolic memory” of macrovascular complications in people with long-standing T2D.

Increased AGE-RAGE ratio in idiopathic pulmonary fibrosis.

BackgroundThe abnormal epithelial-mesenchymal restorative capacity in idiopathic pulmonary fibrosis (IPF) has been recently associated with an accelerated aging process as a key point for the altered wound healing. The advanced glycation end-products (AGEs) are the consequence of non-enzymatic reactions between lipid and protein with several oxidants in the aging process. The receptor for AGEs (RAGEs) has been implicated in the lung fibrotic process and the alveolar homeostasis. However, this AGE-RAGE aging pathway has been under-explored in IPF.MethodsLung samples from 16 IPF and 9 control patients were obtained through surgical lung biopsy. Differences in AGEs and RAGE expression between both groups were evaluated by RT-PCR, Western blot and immunohistochemistry. The effect of AGEs on cell viability of primary lung fibrotic fibroblasts and alveolar epithelial cells was assessed. Cell transformation of fibrotic fibroblasts cultured into glycated matrices was evaluated in different experimental conditions.ResultsOur study demonstrates an increase of AGEs together with a decrease of RAGEs in IPF lungs, compared with control samples. Two specific AGEs involved in aging, pentosidine and Nε-Carboxymethyl lysine, were significantly increased in IPF samples. The immunohistochemistry identified higher staining of AGEs related to extracellular matrix (ECM) proteins and the apical surface of the alveolar epithelial cells (AECs) surrounding fibroblast foci in fibrotic lungs. On the other hand, RAGE location was present at the cell membrane of AECs in control lungs, while it was almost missing in pulmonary fibrotic tissue. In addition, in vitro cultures showed that the effect of AGEs on cell viability was different for AECs and fibrotic fibroblasts. AGEs decreased cell viability in AECs, even at low concentration, while fibroblast viability was less affected. Furthermore, fibroblast to myofibroblast transformation could be enhanced by ECM glycation.ConclusionsAll of these findings suggest a possible role of the increased ratio AGEs-RAGEs in IPF, which could be a relevant accelerating aging tissue reaction in the abnormal wound healing of the lung fibrotic process.

Chemical Synthesis of Peptides Containing Site-Specific Advanced Glycation Endproducts.

In nature, proteins, lipids, and nucleic acids can nonenzymatically react with sugars and sugar degradation products to give rise to a diverse range of modifications, known as advanced glycation endproducts (AGEs). These AGEs typically occur at lysine and arginine residues of long-lived proteins, such as collagen, and can modify the structure and function of the native protein. AGEs accumulate during the normal aging process, and AGE formation is dramatically accelerated with diabetes. AGEs have also been implicated in a wide range of debilitating conditions including cardiovascular, renal failure, and neurodegenerative diseases. Thus, there is an ongoing interest in studying the role of AGEs in different aspects of these disorders. Typically, glycated proteins are prepared using nonspecific in vitro incubation techniques. However, this method results in a complex mixture of products which is then employed without further purification. In order to determine the effect of individual AGEs in a peptide sequence, in this Account, we highlight our synthetic methods for site-specifically introducing five frequently occurring AGEs, namely, Nε-(carboxymethyl)lysine (CML), Nε-(carboxyethyl)lysine (CEL), pyrraline, glyoxal-lysine dimer (GOLD), and methylglyoxal-lysine dimer (MOLD) into collagen peptides. Both a collagen model peptide (CMP) and the telopeptide region of human type I α1 collagen (CTP) were chosen due to being prone to glycation and cross-linking in vivo. For the preparation of the AGE-modified collagen peptides, we investigated both the initial preparation of AGE building blocks in solution followed by incorporation into Fmoc-SPPS, as well as an on-resin method whereby AGEs were selectively introduced by modification of the side-chain of an unprotected resin-bound lysine. Both of our synthetic methods enabled the site-specifically modified AGE-containing collagen peptides to be obtained in high purity and yield. In addition, the on-resin method had the added advantage of requiring fewer synthetic steps. We then evaluated the impact of the specific AGEs on the properties of the native protein and found that the AGE modifications protected against proteolytic digestion, enhanced copper binding at physiological pH, and, for the cross-linking AGEs, disrupted the triple helical structure of CMPs. Overall these synthetic methods offered a new strategy for preparing peptides site-specifically modified by AGEs, which can be applied to other peptidic systems, thereby enabling further insights into the biochemical consequences of AGEs.

Analysis of advanced glycation end products in the DHS Mind Study

AimsHuman studies of links between advanced glycation end-products (AGEs) and disease phenotypes are less common than studies of animal and cell models. Here, we examined the association of total AGEs with diabetes risk factors in a predominately type 2 diabetes (T2D) affected cohort. MethodsAGEs were measured using an enzyme linked immunosorbant assay in 816 individuals from the DHS Mind Study (n=709 T2D affected), and association analyses were completed. ResultsTotal AGEs were associated with estimated glomerular filtration rate (p=0.0054; β=−0.1291) and coronary artery calcification (p=0.0352; β=1.1489) in the entire cohort. No significant associations were observed when individuals with T2D were analyzed separately. In individuals without T2D, increased circulating AGEs were associated with increased BMI (p=0.02, β=0.138), low density lipoproteins (p=0.046, β=17.07) and triglycerides (p=0.0004, β=0.125), and decreased carotid artery calcification (p=0.0004, β=−1.2632) and estimated glomerular filtration rate (p=0.0018, β=−0.1405). Strong trends were also observed for an association between AGEs and poorer cognitive performance on the digit symbol substitution test (p=0.046, β=−6.64) and decreased grey matter volume (p=0.037, β=−14.87). ConclusionsAGEs may play an important role in a number of phenotypes and diseases, although not necessarily in interindividual variation in people with T2D. Further evaluation of specific AGE molecules may shed more light on these relationships.

Comprehensive analysis of maillard protein modifications in human lenses: effect of age and cataract.

In human lens proteins, advanced glycation endproducts (AGEs) originate from the reaction of glycating agents, e.g., vitamin C and glucose. AGEs have been considered to play a significant role in lens aging and cataract formation. Although several AGEs have been detected in the human lens, the contribution of individual glycating agents to their formation remains unclear. A highly sensitive liquid chromatography-tandem mass spectrometry multimethod was developed that allowed us to quantitate 21 protein modifications in normal and cataractous lenses, respectively. N(6)-Carboxymethyl lysine, N(6)-carboxyethyl lysine, N(7)-carboxyethyl arginine, methylglyoxal hydroimidazolone 1, and N(6)-lactoyl lysine were found to be the major Maillard protein modifications among these AGEs. The novel vitamin C specific amide AGEs, N(6)-xylonyl and N(6)-lyxonyl lysine, but also AGEs from glyoxal were detected, albeit in minor quantities. Among the 21 modifications, AGEs from the Amadori product (derived from the reaction of glucose and lysine) and methylglyoxal were dominant.

Biological Effects Induced by Specific Advanced Glycation End Products in the Reconstructed Skin Model of Aging.

Advanced glycation end products (AGEs) accumulate in the aging skin. To understand the biological effects of individual AGEs, skin reconstructed with collagen selectively enriched with Nɛ-(carboxymethyl)-lysine (CML), Nɛ-(carboxyethyl)-lysine (CEL), methylglyoxal hydroimidazolone (MG-H1), or pentosidine was studied. Immunohistochemistry revealed increased expression of α6 integrin at the dermal epidermal junction by CEL and CML (p<0.01). Laminin 5 was diminished by CEL and MG-H1 (p<0.05). Both CML and CEL induced a robust increase (p<0.01) in procollagen I. In the culture medium, IL-6, VEGF, and MMP1 secretion were significantly decreased (p<0.05) by MG-H1. While both CEL and CML decreased MMP3, only CEL decreased IL-6 and TIMP1, while CML stimulated TIMP1 synthesis significantly (p<0.05). mRNA expression studies using qPCR in the epidermis layer showed that CEL increased type 7 collagen (COL7A1), β1, and α6 integrin, while CML increased only COL7A1 (p<0.05). MG-H1-modified collagen had no effect. Importantly, in the dermis layer, MMP3 mRNA expression was increased by both CML and MG-H1. CML also significantly increased the mRNAs of MMP1, TIMP1, keratinocyte growth factor (KGF), IL-6, and monocyte chemoattractant protein 1 (MCP1) (p<0.05). Mixed effects were present in CEL-rich matrix. Minimally glycoxidized pentosidine-rich collagen suppressed most mRNAs of the genes studied (p<0.05) and decreased VEGF and increased MCP1 protein expression. Taken together, this model of the aging skin suggests that a combination of AGEs tends to counterbalance and thus minimizes the detrimental biological effects of individual AGEs.

Methylglyoxal in diabetes: link to treatment, glycaemic control and biomarkers of complications

Diabetic complications are major health problems worldwide, with the cost of caring for diabetes rising to US$245 billion in 2012 in the U.S.A. alone. It is widely recognized that non-enzymatic glycation in diabetes is a major cause of damage and dysfunction of key vascular cells. MG (methylglyoxal) is directly toxic to tissues, and is a major precursor of AGEs (advanced glycation end-products). Various propensities to diabetic complications are seen among individuals with diabetes, with accelerated rates occurring in some individuals with modest hyperglycaemia, while others never progress in spite of poor glycaemic control over many years. Since production and detoxification of MG is ultimately controlled by enzymatic mechanisms, both genetic and environmental factors could regulate tissue glycation and potentially account for these variable complication rates. Activation of pathways that determine MG levels occurs in susceptible patients, indicting an important role in pathogenesis. MG leads to formation of specific AGEs, which are likely to predict propensity to diabetic complications. We have shown recently that three specific plasma AGE biomarkers [MG-H1 (MG-derived hydroimidazolones), CEL (Nε-carboxyethyl-lysine) and CML (Nε-carboxymethyl-lysine)] predict biopsy-documented fast DN (diabetic nephropathy) progression. Since two of the predictive biomarkers are MG end-products, these outcomes support a role for MG in the development of DN. Our studies on MG and its end-products have also shown anti-complication effects of the drug metformin, which binds and inactivates MG, thus reducing MG-related AGEs. We have also shown that reducing post-meal glucose decreases MG levels, as well as levels of MG-related AGEs. Our clinical outcome studies have been based on the novel concept that the unique glycation products that we can measure reflect the activity of specific chemical pathways that are selectively activated by hyperglycaemia in patients that are inherently more susceptible to diabetic complications, and can be used to solve other diabetes-related medical questions.

Aegle marmelos Correa leaf extract prevents secondary complications in streptozotocin-induced diabetic rats and demonstration of limonene as a potent antiglycating agent

To study the antiglycating, antidiabetic and antioxidant properties of Aegle marmelos Correa leaf extract and identify the bioactive constituent. The effect of the chloroform extract of Aegle marmelos Correa was studied in streptozotocin-induced diabetic rats through evaluation of biochemical parameters. Antiglycation activity was assessed in vitro through measurement of total and specific advanced glycation end products, protein carbonyl formation and collagen solubility tests. Antioxidant potential was evaluated using the ferric-reducing antioxidant power assay and 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) assays. Identification of the bioactive component was attempted through silica gel column chromatography and GC-MS analysis. In-vivo studies for 60 days revealed that the extract prevented kidney damage and other secondary complications. The chloroform extract at 16 μg could inhibit protein glycation by 44.33% and pentosidine formation by 59.31%, and could effectively inhibit protein carbonyl formation. It could scavenge DPPH radicals up to 85.26% (IC50: 26 μg). Bio-guided fractionation revealed limonene as the bioactive component, which could account for the antiglycating activity shown by the chloroform extract. The chloroform extract of Aegle marmelos demonstrated antidiabetic antiglycating and antioxidant activity, effectively preventing kidney damage and establishment of cataracts. Limonene is reported for the first time as possessing potent antiglycating activity and is non-toxic at the concentration used.

Current therapeutic interventions in the glycation pathway: evidence from clinical studies

The increased formation of advanced glycation endproducts (AGEs) constitutes a potential mechanism of hyperglycaemia-induced micro- and macrovascular disease in diabetes. In vitro and animal experiments have shown that various interventions can inhibit formation and/or actions of AGEs, in particular the specific AGE inhibitor aminoguanidine and the AGEs crosslink breaker alagebrium, and the B vitamins pyridoxamine and thiamine, and the latter's synthetic derivative, benfotiamine. The potential clinical value of these interventions, however, remains to be established. The present review provides, from the clinical point of view, an overview of current evidence on interventions in the glycation pathway relating to (i) the clinical benefits of specific AGE inhibitors and AGE breakers and (ii) the potential AGE-inhibiting effects of therapies developed for purposes unrelated to the glycation pathway. We found that safety and/or efficacy in clinical studies with the specific AGE inhibitor, aminoguanidine and the AGE breaker, alagebrium, appeared to be a concern. The clinical evidence on the potential AGE-inhibiting effects of B vitamins is still limited. Finally, current evidence for AGE inhibition by therapies developed for purposes unrelated to glycation is limited due to a large heterogeneity in study designs and/or measurement techniques, which have often been sub-optimal. We conclude that, clinical evidence on interventions to inhibit formation and/or action of AGEs is currently weak and unconvincing.

A novel mechanism for antiglycative action of limonene through stabilization of protein conformation

Inhibition of protein glycation is known to ameliorate secondary complications in diabetes. In the present study antiglycative properties of limonene, a natural product, were evaluated using BSA as a model protein. AMG (aminoguanidine) was used as a positive control. Measurement of total AGEs (Advanced Glycation End-products) and specific AGEs revealed that limonene could inhibit protein glycation to the extent of 56.3% and 75.1% respectively at 50 μM concentration as against 54.4% and 82.2% by AMG at 1 mM. Congo red binding and CD (Circular Dichroism) analysis revealed inhibition of α-helix to β-sheet transition wherein 18.5% β-sheet structures were observed in glycated BSA (bovine serum albumin) as against 4.9% with limonene. Glycation of protein in the presence of urea was enhanced by 18%, while in the presence of limonene it was reduced by 23% revealing the stabilizing effect of limonene. Electrophoretic mobility was similar to the normal control and a zeta potential value of -12.1 mV as against -15.1 mV in diabetic control was observed. Inhibition of glycation in limonene treated samples was confirmed through LC-MS analysis wherein AGEs such as pentosidine, CML (N(ε)-(carboxymethyl)lysine), CEL (N(ε)-(carboxyethyl)lysine), MOLD (methylglyoxal-lysine dimer) and imidazolone observed in glycated samples were absent in limonene treated samples. PatchDock studies revealed that limonene could bind to the major glycation sites IB, IIA and IIB sub domains and AMG to the IIIA sub domain. Thus limonene is a potent protein glycation inhibitor that prevents protein glycation through a novel mechanism of stabilization of protein structure through hydrophobic interactions.

Beneficial Effects of Cinnamon Proanthocyanidins on the Formation of Specific Advanced Glycation Endproducts and Methylglyoxal-Induced Impairment on Glucose Consumption

Advanced glycation endproducts (AGEs) are a group of complex and heterogeneous compounds formed from nonenzymatic reactions. The accumulation of AGEs in vivo has been implicated as a major pathogenic process in diabetic complications and other health disorders, such as atherosclerosis and Alzheimer's disease, and normal aging. In this study, we investigate the inhibitory effects of cinnamon bark proanthocyanidins, catechin, epicatechin, and procyanidin B2 on the formation of specific AGE representatives including pentosidine, N(epsilon)-(carboxymethyl)lysine (CML), and methylglyoxal (MGO) derived AGEs. These compounds displayed obvious inhibitory effects on these specific AGEs, which are largely attributed to both their antioxidant activities and carbonyl scavenging capacities. Meanwhile, in terms of their potent MGO scavenging capacities, effects of these proanthocyanidins on insulin signaling pathways interfered by MGO were evaluated in 3T3-L1 adipocytes. According to the results, proanthocyanidins exerted protective effects on glucose consumption impaired by MGO in 3T3-L1 fat cells.

Kaempferol modulates pro-inflammatory NF-κB activation by suppressing advanced glycation endproducts-induced NADPH oxidase

Advanced glycation endproducts (AGE) are oxidative products formed from the reaction between carbohydrates and a free amino group of proteins that are provoked by reactive species (RS). It is also known that AGE enhance the generation of RS and that the binding of AGE to a specific AGE receptor (RAGE) induces the activation of the redox-sensitive, pro-inflammatory transcription factor, nuclear factor-kappa B (NF-kB). In this current study, we investigated the anti-oxidative effects of short-term kaempferol supplementation on the age-related formation of AGE and the binding activity of RAGE in aged rat kidney. We further investigated the suppressive action of kaempferol against AGE's ability to stimulate activation of pro-inflammatory NF-kB and its molecular mechanisms. For this study, we utilized young (6 months old), old (24 months old), and kaempferol-fed (2 and 4 mg/kg/day for 10 days) old rats. In addition, for the molecular work, the rat endothelial cell line, YPEN-1 was used. The results show that AGE and RAGE were increased during aging and that these increases were blunted by kaempferol. In addition, dietary kaempferol reduced age-related increases in NF-kappaB activity and NF-kB-dependant pro-inflammatory gene activity. The most significant new finding from this study is that kaempferol supplementation prevented age-related NF-kappaB activation by suppressing AGE-induced nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase). Taken together, our results demonstrated that dietary kaempferol exerts its anti-oxidative and anti-inflammatory actions by modulating the age-related NF-kappaB signaling cascade and its pro-inflammatory genes by suppressing AGE-induced NADPH oxidase activation. Based on these data, dietary kaempferol is proposed as a possible anti-AGE agent that may have the potential for use in anti-inflammation therapies.