Non-enzymatic Glycation Process Research Articles

Croton gratissimus Burch Herbal Tea Exhibits Anti-Hyperglycemic and Anti-Lipidemic Properties via Inhibition of Glycation and Digestive Enzyme Activities.

Over the years, the world has continued to be plagued by type 2 diabetes (T2D). As a lifestyle disease, obese individuals are at higher risk of developing the disease. Medicinal plants have increasingly been utilized as remedial agents for managing metabolic syndrome. The aim of the present study was to investigate the in vitro anti-hyperglycemic and anti-lipidemic potential of Croton gratissimus herbal tea infusion. The inhibitory activities of C. gratissimus on carbohydrate (α-glucosidase and α-amylase) and lipid (pancreatic lipase) hydrolyzing enzymes were determined, and the mode of inhibition of the carbohydrate digestive enzymes was analyzed and calculated via Lineweaver-Burk plots and Michaelis Menten's equation. Its effect on Advanced Glycation End Product (AGE) formation, glucose adsorption, and yeast glucose utilization were also determined. High-performance liquid chromatography (HPLC) was used to quantify the possible phenolic compounds present in the herbal tea infusion, and the compounds were docked with the digestive enzymes. C. gratissimus significantly (p < 0.05) inhibited α-glucosidase (IC50 = 60.56 ± 2.78 μg/mL), α-amylase (IC50 = 35.67 ± 0.07 μg/mL), as well as pancreatic lipase (IC50 = 50.27 ± 1.51 μg/mL) in a dose-dependent (15-240 µg/mL) trend. The infusion also inhibited the non-enzymatic glycation process, adsorbed glucose effectively, and enhanced glucose uptake in yeast cell solutions at increasing concentrations. Molecular docking analysis showed strong binding affinity between HPLC-quantified compounds (quercetin, caffeic acid, gallic acid, and catechin) of C. gratissimus herbal tea and the studied digestive enzymes. Moreover, the herbal tea product did not present cytotoxicity on 3T3-L1 cell lines. Results from this study suggest that C. gratissimus herbal tea could improve glucose homeostasis and support its local usage as a potential anti-hyperglycemic and anti-obesogenic agent. Further in vivo and molecular studies are required to bolster the results from this study.

Structure analysis and tyrosinase, melanogenesis, α-glucosidase, and nonenzymatic glycation inhibitory activities of polymeric proanthocyanidins from the pulp of Crataegus pinnatifida Bge.

Crataegus pinnatifida Bge. is a worthwhile industrial crop for its extensive application in Chinese herbal pharmaceuticals, food, and other industries. In this study, proanthocyanidins were extracted from the pulp of Crataegus pinnatifida Bge., and the structure, antityrosinase, antimelanogenesis, anti-α-glucosidase, and anti-glycation activities of these compounds were systematically elucidated. The combined utilization of high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS), matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), and nuclear magnetic resonance (NMR) demonstrated that the proanthocyanidins were B-type procyanidin polymers consisting of epicatechin . These polymers were efficient, reversible, and competitive inhibitors of both tyrosinase and α-glucosidase with IC 50 values of 0.10 ± 0.01 mg/mL (for monophenolase), 0.16 ± 0.02 mg/mL (for diphenolase), and 0.50 ± 0.02 µg/mL, respectively. Moreover, the process of nonenzymatic glycation was also delayed by proanthocyanidins. Further cell assays suggested that these polymers strongly inhibited intracellular tyrosinase activity and melanogenesis and induced apoptosis in mouse melanoma cells. The results of circular dichroism (CD), molecular docking , and molecular dynamics (MD) consistently revealed that the inhibition of procyanidins on the two enzymes was primarily driven by hydrogen bonding and hydrophobic interactions, and the binding of epicatechin caused structural stretching and conformational modification of tyrosinase. Consequently, this study confirms novel tyrosinase, melanogenesis, α-glucosidase, and nonenzymatic glycation inhibitors, which might offer scientific evidence for potential applications in the cosmetics and pharmaceutical industries. • Proanthocyanidins can be produced industrially for a yield of 11.08%. • Various biological activities of proanthocyanidins enabled their wide applications. • Hydrogen bond and hydrophobic interaction loosened tyrosinase secondary structures. • Proanthocyanidins can reduce melanogenesis by down-regulating tyrosinase proteins.

Contribution of Advanced Glycation End Products to PCOS Key Elements: A Narrative Review.

In the last decade, data has suggested that dietary advanced glycation end products (AGEs) play an important role in both reproductive and metabolic dysfunctions associated with polycystic ovary syndrome (PCOS). AGEs are highly reactive molecules that are formed by the non-enzymatic glycation process between reducing sugars and proteins, lipids, or nucleic acids. They can be formed endogenously under normal metabolic conditions or under abnormal situations such as diabetes, renal disease, and other inflammatory disorders. Bodily AGEs can also accumulate from exogenous dietary sources particularly when ingested food is cooked and processed under high-temperature conditions, such as frying, baking, or grilling. Women with PCOS have elevated levels of serum AGEs that are associated with insulin resistance and obesity and that leads to a high deposition of AGEs in the ovarian tissue causing anovulation and hyperandrogenism. This review will describe new data relevant to the role of AGEs in several key elements of PCOS phenotype and pathophysiology. Those elements include ovarian dysfunction, hyperandrogenemia, insulin resistance, and obesity. The literature findings to date suggest that targeting AGEs and their cellular actions could represent a novel approach to treating PCOS symptoms.

Birefringence effect studies of collagen formed by nonenzymatic glycation using dual-retarder Mueller polarimetry.

.Significance: Nonenzymatic glycation of collagen covalently attaches an addition of sugar molecules that initially were involved in a reversibly reaction with amino groups on the protein. Due to the ultimate formation of stable irreversible advanced glycation end products, the process of glycation leads to abnormal irreversible cross-linking, which ultimately accumulates with age and/or diabetes in the extracellular matrix, altering its organization.Aim: We report the use of dual-retarder Mueller polarimetry in conjunction with phase retardance to differentiate collagen cross-linking in a normal collagen gel matrix from that in tissues with nonenzymatic cross-linking.Approach: A dual-liquid crystal-based Mueller polarimetry system that involves electronic modulation of polarization state generators (PSGs) was employed to produce all types of polarization states without moving any part and enable detection of the signal directly using a Stokes polarimeter. The linear phase retardance response was obtained for the characterization of the solution and gel forms of collagen using differential Mueller matrix analysis.Results: We found that linear phase retardance measurements via differential Mueller matrix polarimetry successfully differentiated collagen gel matrices with different degrees of cross-linking formed by a nonenzymatic glycation process and demonstrated that this technology constitutes a quick and simple modality.Conclusions: This approach has high sensitivity for studying differences in fibrillar cross-linking in glycated collagen. Further, our work suggests that this method of structural analysis has potential clinical diagnostic value owing to its noninvasive and cost-efficient nature.

Glycyrrhizic Acid Scavenges Reactive Carbonyl Species and Attenuates Glycation-Induced Multiple Protein Modification: An In Vitro and In Silico Study.

The current study is aimed at studying the inhibitory effect of glycyrrhizic acid (GA) on D-ribose-mediated protein glycation via various physicochemical analyses and in silico approaches. Being a potent free radical scavenger and a triterpenoid saponin, GA plays a vital role in diminishing the oxidative stress and thus could be an effective inhibitor of the nonenzymatic glycation process. Our data showed that varying concentrations of GA inhibited the in vitro BSA-AGEs via inhibiting the formation of fructosamines, fluorescent AGEs, scavenging protein carbonyl and hydroxymethyl furfural (HMF) content, and protection against D-ribose-induced modification of BSA as evident by increased free Arg and Lys residues in GA-treated Gly-BSA samples. Moreover, GA also attenuated D-ribose-induced alterations in the secondary structure of BSA by protecting the α-helix and β-sheet conformers and amide-I band delocalization. In addition, GA attenuated the modification in β-cross amyloid structures of BSA and in silico molecular interaction study too showed strong binding of GA with higher number of Lys and Arg residues of BSA and binding energy (ΔG) of -8.8 Kcal/mol, when compared either to reference standard aminoguanidine (AG)-BSA complex (ΔG: -4.3 Kcal/mol) or D-ribose-BSA complex (ΔG: -5.2 Kcal/mol). Therefore, GA could be a new and favorable inhibitor of the nonenzymatic glycation process that ameliorates AGEs-related complications via attenuating the AGE formation and glycation-induced multiple protein modifications with a reduced risk of adverse effects on protein structure and functionality; hence, it could be investigated at further preclinical settings for the treatment and management of diabetes and age-associated complications.

Piperine exhibits preventive and curative effect on erythrocytes membrane modifications and oxidative stress against in vitro albumin glycation.

Inhibition of non-enzymatic glycation processes is an essential aspect of treating type 2 diabetes and related complications. In this study, piperine's preventative, simultaneous and curative effect in glucose-induced albumin glycation was examined by analyzing the structural and functional markers of albumin. The protective and antioxidant influence of piperine on erythrocytes was assessed by examining cellular membrane modifications with antioxidant status. Albumin glycation was performed in three different experimental sets of 21days at 37°C in dark conditions-using different piperine concentrations (250, 500, and 1,000μM) and time of addition of glucose (30mM)/piperine (1,000μM) in a respective solution at 10th day. Piperine with glycated albumin leads to decreased fructosamine, carbonyl group, and protein-bound glucose. It had protected free amino groups, thiol group, and reduced beta-amyloid, protein aggregates formation. The presence of piperine with glycated albumin prevented erythrocytes hemolysis, membrane modifications, and maintained the antioxidant status. Piperine showed the antiglycation effects in a dose-dependent manner, additionally, its pre-treatment exhibited maximum attenuation by manifesting its primarily preventive role. PRACTICAL APPLICATIONS: Piperine is a natural alkaloid compound found in pepper, has been reported to possess anti-cancer, anti-microbial, and anti-inflammatory properties. The present study evaluated the antiglycation potential of piperine in albumin's glycation and it displayed preventive action, protected erythrocytes from oxidative damage induced by glycated albumin. We concluded that the daily intake of piperine can be adequate to prevent glycation-induced diabetic complications development in hyperglycemic conditions.

The protective effects of bexarotene against advanced glycation end-product (AGE)-induced degradation of articular extracellular matrix (ECM)

Osteoarthritis (OA) is a common debilitating disease primarily characterised by excessive loss of the articular ECM, which is composed of up to 95% type II collagen. Among the factors that contribute to the pathogenesis of OA, the natural process of aging is regarded as the most significant risk factor. AGEs, which are extremely resilient to degradation, are produced in the body naturally as a result of the Maillard process of nonenzymatic glycation and are also introduced through diet and tobacco smoke. AGEs have a high affinity for collagen and therefore accumulate in joint tissues, where they induce increased expression of proinflammatory cytokines, chemokines, and degradative enzymes. Additionally, AGEs induce oxidative stress, which further exacerbates the degradative process. Type II collagen is targeted for degradation by matrix metalloproteinases (MMPs), particularly MMP-3 and MMP-13, and AGEs have been shown to trigger increased expression of these MMPs. The role of retinoid and rexinoid receptors as specific treatment targets has been receiving increasing attention. Bexarotene is a retinoid X receptor (RXR) agonist used for the treatment of T-cell lymphoma and other cancers which has displayed a favourable safety profile. Here, we examined the roles of RXR agonism using bexarotene on AGE-induced markers of OA, including oxidative stress, inflammatory response, and MMP-mediated degradation of type II collagen. Furthermore, we demonstrate that bexarotene inhibited phosphorylation of IκBα, thereby suppressing activation of the proinflammatory NF-κB cellular signalling pathway. These findings present a basis for selective targeting of RXR by bexarotene as a potential treatment of OA induced by AGEs.

Insights into the Progression of Labile Hb A1c to Stable Hb A1c via a Mechanistic Assessment of 2,3-Bisphosphoglycerate Facilitation of the Slow Nonenzymatic Glycation Process

Nonenzymatic glycation (NEG) of human hemoglobin (Hb A) consists of initial non covalent, reversible steps involving glucose and amino acid residues, which may also involve effector reagent(s) in the formation of labile Hb A1c (the conjugate acid of the Schiff base). Labile Hb A1c can then undergo slow, largely irreversible, formation of stable Hb A1c (the Amadori product). Stable Hb A1c is measured to assess diabetic progression after labile Hb A1c removal. This study aimed to increase the understanding of the distinctions between labile and stable Hb A1c from a mechanistic perspective in the presence of 2,3-bisphosphoglycerate (2,3-BPG). 2,3-Bisphosphoglycerate is an effector reagent that reversibly binds in the Hb A1c pocket and modestly enhances overall NEG rate. The deprotonation of C2 on labile Hb A1c in the formation of the Amadori product was previously proposed to be rate-limiting. Computational chemistry was used here to identify the mechanism(s) by which 2,3-BPG facilitates the deprotonation of C2 on labile Hb A1c. 2,3-Bisphosphoglycerate is capable of abstracting protons on C2 and the α-nitrogen of labile Hb A1c and can also deprotonate water and/or amino acid residues, therefore preparing these secondary reagents to deprotonate labile Hb A1c. Parallel reactions not leading to an Amadori product were found that include formation of the neutral Schiff base, dissociation of glucose from the protein, and cyclic glycosylamine formation. These heretofore under appreciated parallel reactions may help explain both the selective removal of labile from stable Hb A1c and the slow rate of NEG.

A Perspective on Reagent Diversity and Non-covalent Binding of Reactive Carbonyl Species (RCS) and Effector Reagents in Non-enzymatic Glycation (NEG): Mechanistic Considerations and Implications for Future Research.

This perspective focuses on illustrating the underappreciated connections between reactive carbonyl species (RCS), initial binding in the nonenzymatic glycation (NEG) process, and nonenzymatic covalent protein modification (here termed NECPM). While glucose is the central species involved in NEG, recent studies indicate that the initially-bound glucose species in the NEG of human hemoglobin (HbA) and human serum albumin (HSA) are non-RCS ring-closed isomers. The ring-opened glucose, an RCS structure that reacts in the NEG process, is most likely generated from previously-bound ring-closed isomers undergoing concerted acid/base reactions while bound to protein. The generation of the glucose RCS can involve concomitantly-bound physiological species (e.g., inorganic phosphate, water, etc.); here termed effector reagents. Extant NEG schemes do not account for these recent findings. In addition, effector reagent reactions with glucose in the serum and erythrocyte cytosol can generate RCS (e.g., glyoxal, glyceraldehyde, etc.). Recent research has shown that these RCS covalently modify proteins in vivo via NECPM mechanisms. A general scheme that reflects both the reagent and mechanistic diversity that can lead to NEG and NECPM is presented here. A perspective that accounts for the relationships between RCS, NEG, and NECPM can facilitate the understanding of site selectivity, may help explain overall glycation rates, and may have implications for the clinical assessment/control of diabetes mellitus. In view of this perspective, concentrations of ribose, fructose, Pi, bicarbonate, counter ions, and the resulting RCS generated within intracellular and extracellular compartments may be of importance and of clinical relevance. Future research is also proposed.

Refractive Index Tomograms and Dynamic Membrane Fluctuations of Red Blood Cells from Patients with Diabetes Mellitus

Background: Diabetic effects on blood rheology are important topics and have been extensively investigated. In particular, it has been known that diabetic RBCs exhibit significantly deteriorated deformability compared to healthy RBCs. Despite the importance of previously measured mechanical properties of diabetic RBCs, several problems remain to be elucidated. First, conventional techniques have difficulties in measuring intrinsic properties of individual cells. Detailed quantification of diabetic alteration in RBCs at the individual cell level is also hard to achieve. Accordingly, previous approaches do not allow the systematical correlative analyses to be performed on simultaneously measured various cell parameters. As a consequence, there are still no general agreements on the integrated effects of diabetic complications or hyperglycemia on individual human RBCs.Methods: To effectively settle the arisen problems, we quantitatively and non-invasively measured the individual RBCs from diabetic and healthy blood donors to figure out the characteristics of diabetic RBCs by employing the quantitative phase imaging (QPI) technique. From the reconstructed three-dimensional (3-D) refractive index (RI) tomogram and the time-series of 2-D phase map of individual RBCs, the morphological (volume, surface area, and sphericity), biochemical (Hb concentration and Hb content), and mechanical (membrane fluctuation) parameters were retrieved. Systematic correlative analyses among measured six RBC parameters and independently determined HbA1c level, defined as the ratio of HbA1c to total Hb, were also performed. In measuring red cells, only gently sedimented RBCs of discocytes were selected. After excluding erroneously measured RBCs, 40, 40, 40, 38, 39, 40 control RBCs of six healthy donors and 40, 40, 40, 40, 40, 40 diabetic RBCs of six diabetic patients were finally analyzed respectively for the study of diabetic effects on characteristics of individual human RBCs.Results: HbA1c level is defined as a percentage of HbA1c, the majority of glycated Hbs in RBC, to total Hb contents in blood. Because HbA1c is formed through 120-days long, non-enzymatic glycation process of Hb, HbA1c level has been perceived as an effective mean to estimate mean glucose concentration of individuals for the last three months. In other words, HbA1c reflects the degree of hyperglycemia. The most noticeable alterations in diabetic RBCs can be found when mean RBC membrane fluctuations of individuals are rearranged in the increasing order of the measured HbA1c levels. The mean values of membrane fluctuations for RBCs are 59.7 ¡¾ 6.2, 58.3 ¡¾ 4.9, 55.0 ¡¾ 5.4, 57.1 ¡¾ 7.4, 51.0 ¡¾ 6.8, and 49.6 ¡¾ 4.6 nm for healthy volunteers, and 45.1 ¡¾ 4.0, 46.9 ¡¾ 3.4, 48.8 ¡¾ 4.0, 47.6 ¡¾ 4.2, 43.0 ¡¾ 4.1, and 48.0 ¡¾ 5.0 nm for diabetic patients, in order of increasing HbA1c. As shown in Fig. 4, mean membrane fluctuation of non-diabetic RBCs tends to decrease as the HbA1c level increases (Pearson correlation coefficient of -0.87 with a p-value of 0.02). Beyond this level of HbA1c, RBCs from diabetic patients exhibit significantly decreased and settled membrane fluctuation. This observed negative correlation between RBC membrane fluctuation and HbA1 is also conceptually in accordance with the report of the previous study.Conclusions: We quantitatively and non-invasively characterize the morphological, biochemical, and mechanical alterations of individual RBCs by diabetes mellitus. HbA1c levels of subjects, which reflect one's mean blood glucose concentration of last three months, were also determined using hematology analyzers. Accordingly, correlative analyses among the retrieved individual RBC parameters and HbA1c level were performed to search unique features which cannot be addressed by conventional techniques with incapability of simultaneous measurement of those parameters. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

MICROHARDNESS EVALUATION OF NON ENZYMATICALLY GLYCATED BOVINE FEMUR CORTICAL BONE

Purpose: The aim of this study is to investigate deterioration in mechanical integrity of the collagen network of bovine bone formed by non-enzymatic glycation (NEG), a process that mimics aging by microindentation technique. Methods: Young and old bovine cortical bone specimens were rested in solutions for four weeks for the process of NEG and were grouped as ribosylated and non-ribosylated. A series of indentations were made on bone specimens weekly for each of 3-masses of 50 g, 100 g and 200 g for 10 s to detect the effect of indentation load and for each of five durations of 5 s, 10 s, 20 s, 30 s for 100 g to study the effect of indentation duration. The applied load was increased to 300 g, 500 g, 1000 g and 2000 g for 10 s to be able to make microcracks. Specimens were tested in the wet and dry state to study the effects of hydration on microhardness measurement. Results: Loads of 50 g, 100 g and 200 g for 10 s were able to differ ribosylated bone from non-ribosylated bone for the young and old bovine bones. Microhardness values increased with increasing incubation period. Microhardness of dry specimens were found to be statistically higher than that of wet specimens. Presence of extrinsic toughening mechanisms including crack bridging due to uncracked ligaments and collagen fibers were directly observed by scanning electron microscope (SEM). Ribosylated bone was found to have lower number of collagen bridging compared to non-ribosylated bovine bone. Conclusion: Microhardness test by these are able to discriminate non-modified collagen structure from modified collagen. On the other hand, it is found that microindentation was not able to discriminate the degree of NEG.

SAT0350 Glucose and Insulin Concentrations in Association with Hand Osteoarthritis: The Neo Study

BackgroundBody mass index (BMI) is an important risk factor for osteoarthritis (OA), however the underlying causal pathways are unclear. Earlier studies showed an association of diabetes mellitus and OA, suggesting...

Antiglycative effect of fruit and vegetable seed extracts: inhibition of AGE formation and carbonyl‐trapping abilities

Advanced glycation end-products (AGEs) are the final products derived from the non-enzymatic glycation process. AGEs are involved in the development of several health complications associated with diabetes and aging. Searching for anti- AGE extracts is necessary to mitigate the effects of age-related pathologies. The antioxidant and antiglycative activities of eight aqueous extracts of fruit and vegetable seeds were evaluated. All seed extracts (3.6 mg mL(-1)) exhibited anti-AGE activity in protein-glucose assay, ranging from 20 to 92% inhibition compared with aminoguanidine (4.87 mmol L(-1)). Green pepper extract exerted the highest anti-AGE activity. However, peach and pomegranate extracts exhibited the highest anti-AGE activity in protein-methylglyoxal assay, ranging from 0 to 79% inhibition. Hazelnut, almond and sesame extracts were not effective when methylglyoxal was the promoter. Apricot and peach extracts appeared to inhibit the formation of AGEs through their capacity for direct trapping of 1,2-dicarbonyls (IC50=0.14 mg mL(-1)). No relationship between antioxidant and phenolic compound content and antiglycative activity was found. Therefore other hydrophilic constituents in addition to phenolic acids must be involved in the antiglycative activity of the extracts. Aqueous extracts of fruits and vegetables can be considered in the prevention of glycation-associated complications of age-related pathologies.

Cellular and molecular players in the atherosclerotic plaque progression

Atherosclerosis initiation and progression is controlled by inflammatory molecular and cellular mediators. Cells of innate immunity, stimulated by various endogenous molecules that have undergone a transformation following an oxidative stress or nonenzymatic glycation processes, activate cells of the adaptive immunity, found at the borders of atheromas. In this way, an immune response against endogenous modified antigens takes place and gives rise to chronic low-level inflammation leading to the slow development of complex atherosclerotic plaques. These lesions will occasionally ulcerate, thus ending with fatal clinical events. Plaque macrophages represent the majority of leukocytes in the atherosclerotic lesions, and their secretory activity, including proinflammatory cytokines and matrix-degrading proteases, may be related to the fragilization of the fibrous cap and then to the rupture of the plaque. A considerable amount of work is currently focused on the identification of locally released proinflammatory factors that influence the evolution of the plaque to an unstable phenotype. A better understanding of these molecular processes may contribute to new treatment strategies. Mediators released by the immune system and associated with the development of carotid atherosclerosis are discussed.

Naturally occurring inhibitors against the formation of advanced glycation end-products

Advanced glycation end-products (AGEs) are the final products of the non-enzymatic reaction between reducing sugars and amino groups in proteins, lipids and nucleic acids. Recently, the accumulation of AGEs in vivo has been implicated as a major pathogenic process in diabetic complications, atherosclerosis, Alzheimer's disease and normal aging. The early recognition of AGEs can ascend to the late 1960s when a non-enzymatic glycation process was found in human body which is similar to the Maillard reaction. To some extent, AGEs can be regarded as products of the Maillard reaction. This review firstly introduces the Maillard reaction, the formation process of AGEs and harmful effects of AGEs to human health. As AGEs can cause undesirable diseases or disorders, it is necessary to investigate AGE inhibitors to offer a potential therapeutic approach for the prevention of diabetic or other pathogenic complications induced by AGEs. Typical effective AGE inhibitors with different inhibition mechanisms are also reviewed in this paper. Both synthetic compounds and natural products have been evaluated as inhibitors against the formation of AGEs. However, considering toxic or side effects of synthetic molecules present in clinical trials, natural products are more promising to be developed as potent AGE inhibitors.

Therapeutic Uses of Drug-Carrier Systems for Imidazole-Containing Dipeptide Compounds That Act as Pharmacological Chaperones and Have Significant Impact on the Treatment of Chronic Diseases Associated With Increased Oxidative Stress and the Formation of Advanced Glycation End Products

The purpose of this study was to determine how the naturally occurring molecules N-acetylcarnosine, L-carnosine, and carcinine, which are chemical or pharmacological chaperones, affect the cells and biomolecules of patients with skin diseases, cosmetic skin lesions, or underlying clinically significant visual impairment such as age-related cataracts, age-related retinal degeneration, and ocular complications of diabetes. We evaluated and characterized the effects of cited pharmacological chaperones on enzyme activity, protein structure in tissues, and other biomarkers of diseases in skin cells and tissues or in ocular tissues (human cataractous and normal lenses) derived from ophthalmic patients or age-matched donors. The samples were used to test imidazole-containing peptidomimetic chemical/pharmacological chaperones in relation to oxidative stress induced by reaction with lipid peroxides or advanced non-enzymatic glycation processes. Chaperone function is characterized by interaction with other proteins, mediating their folding, transport, and interaction with other molecules, lipid peroxidation products, and membranes. Although these therapies remain on hold pending further investigation, we present growing evidence demonstrating the ability of N-acetylcarnosine (lubricant eye drops) or carcinine pharmacological chaperone therapy to act as novel treatments for age-related cataracts, age-related macular degeneration, and ocular complications of diabetes. Finally, we examine strategies for identifying potential chaperone compounds and for experimentally demonstrating chaperone and transglycating (de-glycation) types of activity in in vitro and in vivo models of human age-related eye diseases, such as cataracts, and advanced glycation tissue protein-engineered systems.

Mapping of the ATP-binding domain of human fructosamine 3-kinase-related protein by affinity labelling with 5′-[p-(fluorosulfonyl)benzoyl]adenosine

The modification of proteins by reducing sugars through the process of non-enzymatic glycation is one of the principal mechanisms by which hyperglycaemia may precipitate the development of diabetic complications. Fn3K (fructosamine 3-kinase) and Fn3KRP (Fn3K-related protein) are two recently discovered enzymes that may play roles in metabolizing early glycation products. However, although the activity of these enzymes towards various glycated substrates has been established, very little is known about their structure-function relationships or their respective mechanisms of action. Furthermore, their only structural similarities noted to date with members of other kinase families has been with the bacterial aminoglycoside kinases. In the present study, we employed affinity labelling with the ATP analogue FSBA {5'-p-[(fluorosulfonyl)benzoyl]adenosine} to probe the active-site topology of Fn3KRP as an example of this enigmatic family of kinases. FSBA was found to modify Fn3KRP at five distinct sites; four of these were predicted to be localized in close proximity to its ATP-binding site, based on alignments with the aminoglycoside kinase APH(3')-IIIa, and examination of its published tertiary structure. The results of the present studies provide evidence that Fn3KRP possesses an ATP-binding domain that is structurally related to that of both the aminoglycoside kinases and eukaryotic protein kinases.

Glycotoxins: a possible threat to health?

AGEs are produced by a nonenzymatic glycation process called the Maillard reaction which involves the condensation of a reducing sugar or an aldehydic group and a protein amino group, with the formation of a series of reactive intermediates leading to stable, irreversible, seldom fluorescent, compounds known as advanced glycation end-products (AGEs). The reaction occurs both in food during heating and in animal and human tissues. During the heating of food containing sugars, lipids and proteins, nonenzymatic protein browning reaction occurs, resulting in the formation of a large series of compounds, which include the melanoidins and other AGEs. Accumulation of AGEs in the cells and in the extracellular matrix in animals is also observed; it is age-dependent, is related to cell or tissue turnover and is species-specific. These processes are accelerated in chronic diseases in which the production of pathogenetic AGEs and their accumulation rate are increased. The two research fields, the Maillard reaction in food chemistry and the glycation cascade in medicine, ran in parallel independently for a long time, but recently a new scientific society (the International Maillard Reaction Society, IMARS) has opened the way for an exciting fusion of knowledge. Pioneering studies resulted in concerns about the non-negligible bioavailability of dietary AGEs and related plasma concentrations in humans, and there are now claims that dietary AGEs may be toxic because of their bioreactivity in different chronic diseases. This has been confirmed by several studies in animals and in humans (AGEs-restricted diets resulted in a reduction of serum and tissue AGEs, related AGE receptors, inflammatory cells, and proatherosclerotic factors) and by in vitro studies on cells in culture. In view of the emerging evidence of a pathogenetic role of glycotoxins, an assessment of the benefits of a shift to a specific nutritional approach (the Mediterranean diet being the healthiest in terms of the low amount of AGEs and high amount of antioxidants) and an innovative therapeutic approach, aimed at directly breaking AGEs molecules or reversing their impaired gut absorption to favour their renal or faecal excretion, may be warranted. At any rate, a new scientific field is open for an interdisciplinary debate (involving chemists, geriatricians, and specialists in metabolic disorders, diabetes, and diet) paving the way for possible, remarkable clinical progress.

Glycated fetal calf serum affects the viability of an insulin-secreting cell line in vitro

The purpose of the present study was to evaluate the direct effects of advanced glycation end products (AGEs) on β-cells by their exposure to a glycated serum to estimate the cellular viability and the related insulin secretion. Glycation of fetal calf serum was obtained by incubation with 50 mol/L ribose at 37°C for 7 days; at the end of this incubation period, the pentosidine content ranged between 15 and 16 × 10 5 pmol/L. HIT-T15 cells, a pancreatic islet cell line, were grown and cultured for 5 days in Roswell Park Memorial Institute (RPMI) medium containing either not glycated (NGS) or glycated (GS) fetal calf serum. Cellular oxidative stress (ie, thiobarbituric acid-reactive substances) was assessed by high-performance liquid chromatography. Cellular viability was evaluated by detection of proliferation, cell necrosis, and cell apoptosis rate. The insulin secretion and the related intracellular content were evaluated by enzyme-linked immunosorbent assay. The present study reported, after 5 days of exposure to the glycation environment, a moderately reduced cellular proliferation (−20.44% ± 2.92%) with a corresponding increase of cell necrosis (+67.7% ± 1.56%) and cell apoptosis (+39.83% ± 2.92%) rate in comparison with the untreated cells. Oxidative intracellular stress was higher in GS conditions compared with the NGS ones (+293.3% ± 87.53%). Insulin release from GS-treated HIT-T15 cells was lower than that of NGS-treated cells both when cells were stimulated with low glucose concentration (2.8 mmol/L, −30.3% ± 4.91%) or when they were challenged with high glucose concentration (16.7 mmol/L, −29.2% ± 5.82%). Incubation of HIT-T15 cells with glycated serum also caused a significant decrease of insulin intracellular content (−44.47% ± 9.98%). Thus, AGEs were shown to exert toxic effects on insulin-secreting cells. Chronically high intracellular oxidative stress, due to accumulation of AGEs, affects the insulin secretion machinery. The present data suggest a pivotal role of the non-enzymatic glycation process in the onset and progression of diabetes during aging and a direct adverse effect of a glycated environment on the pancreatic islet cells.

Fructosamine-3-kinase-related-protein phosphorylates glucitolamines on the C-4 hydroxyl: Novel substrate specificity of an enigmatic enzyme

Fructosamine-3-kinase (FN3K) phosphorylates fructosamines to fructosamine-3-phosphates. Recent data from FN3K-knockout mouse indicate that this phosphorylation results in deglycation of proteins modified by non-enzymatic glycation process. A homolog of FN3K, the FN3K-related-protein (FN3KRP) displays 65% amino acid sequence identity with FN3K and is highly conserved in evolution. However, FN3KRP does not phosphorylate substrates of FN3K such as fructoselysine and its physiological function remains unknown. We observed that human erythrocytes that contain both enzymes phosphorylate N-methylglucamine (meglumine) to two products. One of these is meglumine-3-phosphate (Meg3P), an activity consistent with the known substrate specificity of FN3K. Here, we identify the second product as meglumine-4-phosphate (Meg4P) and show that it is produced specifically by FN3KRP. While it is unlikely that meglumine is the physiological target of FN3KRP, this novel specificity, along with FN3KRPs known phosphorylation of some ketosamines on the C-3 hydroxyl may prove useful in identifying the physiological substrates of this kinase.