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

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

CCN-2 is up-regulated by and mediates effects of matrix bound advanced glycated end-products in human renal mesangial cells

CCN-2, also known as connective tissue growth factor (CCN-2/CTGF) is a cysteine rich, extracellular matrix protein that acts as a pro-fibrotic cytokine in tissues in many diseases, including in diabetic nephropathy. We have published that soluble advanced glycation end products (AGEs), that are present in increased amounts in diabetes, induce CCN-2. However in vivo AGEs are known to be heavily tissue bound and whether matrix bound AGEs regulate CCN-2 has not been investigated. In this study we determined in human renal mesangial cells if CCN-2 is induced by matrix associated AGEs and if CCN-2 may then secondarily mediate effects of matrix AGEs on extracellular matrix expansion. Data generated show that CCN-2 mRNA and protein expression are induced by matrix bound AGEs, and in contrast, this was not the case for TGF-β1 mRNA regulation. Using CCN-2 adenoviral anti-sense it was found that CCN-2 mediated the up-regulation of fibronectin and the tissue inhibitor of matrix metalloproteinase, TIMP-1, that was caused by matrix bound AGEs. In conclusion, CCN-2 is induced by non-enzymatically glycated matrix and it mediates downstream fibronectin and TIMP-1 increases, thus through this mechanism potentially contributing to ECM accumulation in the renal glomerulus in diabetes.     

Advanced glycation endproducts: from precursors to RAGE: round and round we go

The formation of advanced glycation endproducts (AGEs) occurs in diverse settings such as diabetes, aging, renal failure, inflammation and hypoxia. The chief cellular receptor for AGEs, RAGE, transduces the effects of AGEs via signal transduction, at least in part via processes requiring the RAGE cytoplasmic domain binding partner, diaphanous-1 or mDia1. Data suggest that RAGE perpetuates the inflammatory signals initiated by AGEs via multiple mechanisms. AGE–RAGE interaction stimulates generation of reactive oxygen species and inflammation—mechanisms which enhance AGE formation. Further, recent data in type 1 diabetic kidney reveal that deletion of RAGE prevents methylglyoxal accumulation, at least in part via RAGE-dependent regulation of glyoxalase-1, a major enzyme involved in methylglyoxal detoxification. Taken together, these considerations place RAGE in the center of biochemical and molecular stresses that characterize the complications of diabetes and chronic disease. Stopping RAGE-dependent signaling may hold the key to interrupting cycles of cellular perturbation and tissue damage in these disorders.     

Modulation of insulin action by advanced glycation endproducts: a new player in the field

Insulin resistance is characterized by an impaired responsiveness to the action of insulin at its multiple target organs. The accumulation of advanced glycation endproducts (AGEs) has been demonstrated in clinical settings of insulin resistance such as in diabetes, hypertension, and obesity. In this review we have focused on advanced glycation as a modulator of insulin resistance. Structural and functional abnormalities of the insulin molecule by glycation and methylglyoxal may contribute to the pathogenesis of insulin resistance. In addition, it is likely that AGEs interfere in the complex molecular pathways of insulin signaling and as such in insulin resistance.     

Cathepsins D and L reduce the toxicity of advanced glycation end products

Advanced glycation end product-modified proteins are known for accumulating during aging and in several pathological conditions such as diabetes, renal failure, and neurodegenerative disorders. There is little information about the intracellular fate of endocytosed advanced glycation end products (AGEs) and their influence on proteolytic systems. However, it is known that the lysosomal system is impaired during aging. Therefore, undegraded material may accumulate and play a considerable role in the development of diverse diseases. To investigate if AGEs can be degraded and to test whether they accumulate because of impaired lysosomal proteases we studied the effects of advanced glycation end products on the endosomal-lysosomal system. Five different types of AGEs were generated by bovine serum albumin incubation with glyoxal, methylglyoxal, glucose, fructose, and ribose. The first experiments revealed the uptake of AGEs by the macrophage cell line RAW 264.7. Further investigations demonstrated an increase in cathepsin D and L activity and an increase in mature cathepsins D and L. Increased activities were accompanied by the presence of more lysosomes, measured by staining with LysoTracker blue. To specify the roles of cathepsins D and L we used knockout cells to test the roles of both cathepsins on the toxicity of advanced glycation end products. In summary we conclude that both cathepsins are required for a reduction in advanced glycation end product-induced cytotoxicity.     

Diabetic kidney disease: a role for advanced glycation end-product receptor 1 (AGE-R1)?

Diabetic patients are postulated to be in a perpetual state of oxidative stress and inflammation at sites where chronic complications occur. The accumulation of AGEs derived from both endogenous and exogenous sources (such as the diet) have been implicated in the development and progression of diabetic complications, particularly nephropathy. There has been some interest in investigating the potential for reducing the AGE burden in chronic disease, through the action of AGE “clearance” receptors, such as the advanced glycation end-product receptor 1 (AGE-R1). Reducing the burden of AGEs has been linked to attenuation of inflammation, slower progression of diabetic complications (in particular vascular and renal complications) and has been shown to extend lifespan. To date, however, there have been no direct investigations into whether AGE-R1 has any role in modulating normal kidney function, or specifically during the development and progression of diabetes. This mini-review will focus on the recent advances in knowledge around the mechanistic function of AGE-R1 and the implications of this for the pathogenesis of diabetic kidney disease.     

Aldose reductase (AKR1B3) regulates the accumulation of advanced glycosylation end products (AGEs) and the expression of AGE receptor (RAGE)

Diabetes results in enhanced chemical modification of proteins by advanced lipoxidation end products (ALEs) and advanced glycation end products (AGEs) precursors. These modifications have been linked to the development of several secondary diabetic complications. Our previous studies showed that aldose reductase (AR; AKR1B3) catalyzes the reduction of ALEs and AGEs precursors; however, the in vivo significance of this metabolic pathway during diabetes and obesity has not been fully assessed. Therefore we examined the role of AR in regulating ALEs and AGEs formation in murine models of diet-induced obesity and streptozotocin-induced diabetes. In comparison with wild-type (WT) and AR-null mice fed normal chow, mice fed a high-fat (HF) diet (42% kcal fat) showed increased accumulation of AGEs and protein-acrolein adducts in the plasma. AGEs and acrolein adducts were also increased in the epididymal fat of WT and AR-null mice fed a HF diet. Deletion of AR increased the accumulation of 4-hydroxy-trans-2-nonenal (HNE) protein adduct in the plasma and increased the expression of the AGE receptor (RAGE) in HF fed mice. No change in AGEs formation was observed in the kidneys of HF-fed mice. In comparison, renal tissue from AR-null mice treated with streptozotocin showed greater AGE accumulation than streptozotocin-treated WT mice. These data indicated that AR regulated the accumulation of lipid peroxidation derived aldehydes and AGEs under conditions of severe, but not mild, hyperglycemia and that deletion of AR increased RAGE-induction via mechanisms that were independent of AGEs accumulation.     

Non-enzymatic glycation of proteins: From diabetes to cancer

Incubation of proteins with glucose leads to their non-enzymatic glycation and formation of Amadori products known as an early glycation product. Oxidative cleavage of Amadori products is considered as a major route to advanced glycation endproducts (AGEs) formation in vivo. Non-enzymatic glycation of proteins or Maillard reaction is increased in diabetes mellitus due to hyperglycemia and leads to several complications such as blindness, heart disease, nerve damage, and kidney failure. The early and advanced glycation products are accumulated in plasma and tissues of diabetic patients and cause production of autoantibodies against corresponding products. The advanced glycation products are also associated with other diseases like cancer. This review summarizes current knowledge of these stage specific glycated products as common and early diagnostic biomarkers for the associated diseases and the complications with the aim of a novel therapeutic target for the diseases.     

In vitro glycation of a tissue-engineered wound healing model to mimic diabetic ulcers

Diabetic foot ulcers are a major complication of diabetes that occurs following minor trauma. Diabetes-induced hyperglycemia is a leading factor inducing ulcer formation and manifests notably through the accumulation of advanced glycation end-products (AGEs) such as N-carboxymethyl-lysin. AGEs have a negative impact on angiogenesis, innervation, and reepithelialization causing minor wounds to evolve into chronic ulcers which increases the risks of lower limb amputation. However, the impact of AGEs on wound healing is difficult to model (both in vitro on cells, and in vivo in animals) because it involves a long-term toxic effect. We have developed a tissue-engineered wound healing model made of human keratinocytes, fibroblasts, and endothelial cells cultured in a collagen sponge biomaterial. To mimic the deleterious effects induced by glycation on skin wound healing, the model was treated with 300 µM of glyoxal for 15 days to promote AGEs formation. Glyoxal treatment induced carboxymethyl-lysin accumulation and delayed wound closure in the skin mimicking diabetic ulcers. Moreover, this effect was reversed by the addition of aminoguanidine, an inhibitor of AGEs formation. This in vitro diabetic wound healing model could be a great tool for the screening of new molecules to improve the treatment of diabetic ulcers by preventing glycation.     

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.     

Non-enzymatic glycation of proteins: a cause for complications in diabetes

Diabetes mellitus is one of the most common non-communicable diseases, and is the fifth leading cause of death in most of the developed countries. It can affect nearly every organ and system in the body and may result in blindness, end stage renal disease, lower extremity amputation and increase risk of stroke, ischaemic heart diseases and peripheral vascular disease. Hyperglycemia in diabetes causes non-enzymatic glycation of free amino groups of proteins (of lysine residues) and leads to their structural and functional changes, resulting in complications of the diabetes. Glycation of proteins starts with formation of Shiff's base, followed by intermolecular rearrangement and conversion into Amadori products. When large amounts of Amadori products are formed, they undergo cross linkage to form a heterogeneous group of protein-bound moieties, termed as advanced glycated end products (AGEs). Rate of these reactions are quite slow and only proteins with large amounts of lysine residues undergo glycation with significant amounts of AGEs. The formation of AGEs is a irreversible process, causing structural and functional changes in protein leading to various complications in diabetes like nephropathy, retinopathy, neuropathy and angiopathy. The present review discusses about role of glycation in various complications of diabetes.     

RAGE and glyoxalase in kidney disease

Glycation is an important reaction in the regulation of physiological state. When poorly controlled, however, glycation can also result in the accumulation of glycated proteins (advanced glycation endproducts; AGEs) in the body. This AGE accumulation is termed glycative stress, and is an established pathological factor: to date, glycative stress has been closely associated with not only kidney diseases, but also kidney aging. Accumulating evidence demonstrates that the progression of renal tubular damage and tubular aging are often correlated with activation of the receptor for the AGE (RAGE)-AGE pathway or decreased activity of glyoxalase 1, which is an anti-glycation enzyme to lower glycative stress. Further, glycative stress exacerbates the derangement of protein homeostasis: the posttranslationally modified proteins by glycation often lose or gain their functions. Such deranged protein homeostasis leads to endoplasmic reticulum (ER) stress, a state of ER dysfunction in which the quality control of proteins is defective, as well as to induction of its stress signal, the unfolded protein response (UPR), in the kidney. The lowering of glycative stress via modulation of RAGE-AGE axis or glyoxalase 1 activity is beneficial for tubular homeostasis and the subsequent prevention and treatment of kidney disease, suggesting the possibility of novel therapeutic approaches which target glycative stress. In this review, we focused on the impact of glycative stress in the kidney, especially the role of RAGE and glyoxalase 1. Further we also discuss the crosstalk between glycative stress and ER stress in their effect on protein homeostasis.     

Substituted benzenediol Schiff bases as promising new anti-glycation agents

A feature of diabetes is that the rate of protein glycation and the formation of advanced glycation endproducts (AGEs) increases spontaneously due to the abnormally elevated levels of sugar in the blood. The glycation of proteins is associated with a large number of late diabetic complications (retinopathy, neuropathy, atherosclerosis, end stage renal diseases, rheumatoid arthritis and neurodegenerative diseases). The increase in diabetic complications is a major cause of morbidity and mortality, which has increased significantly in the last two decades. Therefore, there is a considerable recent interest in the identification of lead molecules, which can inhibit the glycation process or slow it down considerably. A new class of anti-glycation agents has been identified, based on the spectrofluorimetric analysis of fluorescent advanced glycation endproducts (AGEs), benzenediol Schiff bases, and their structure-activity relationships have been studied. Some of these compounds have shown a promising anti-glycation potential in vitro.     

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

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

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

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

Advanced glycation endproducts and their pathogenic roles in neurological disorders

Glycation is implicated in neurological disorders. In some cases it plays a key role in the pathogenesis, in others it plays a co-adjuvant role or it appears as a consequence of degenerative changes and protein accumulation stemming from other pathways. In this work, we attempt to provide a concise, updated review of the major recent findings concerning glycation in neurological diseases. After a short introduction covering advanced glycation endproducts (AGEs) and the receptor for AGEs (RAGE), we will discuss the impact of glycation in central nervous system disorders including Alzheimer’s, Parkinson’s and Creutzfeldt–Jakob disease, as well as peripheral diabetic polyneuropathies. Therapies directed at lowering the concentrations of RAGE ligands including AGEs, blocking RAGE signaling, preventing oxidative stress or lowering methylglyoxal (MGO) levels may significantly decrease the development of AGE-related pathologies in patients with neurological disorders. Many drugs are on the pipeline and the future clinical trials will reveal if the promising results translate into clinical application.     

Effect of advanced glycation end products on lens epithelial cells in vitro

The extended exposure of proteins to reducing sugars leads to nonenzymatic glycation with the accumulation of advanced glycation end products (AGEs). Long-lived proteins, such as collagen and crystallins, are subjected to this modification, and are implicated as causal factors in several diseases including diabetic complications, cataracts, and arteriosclerosis. One means through which AGEs modulate cellular interactions is via binding to specific receptors. In the current study, the existence of AGEs in human anterior polar lens capsules of cataracts was confirmed using a combination of dot-immunoblot and fluorescent detection. Human lens epithelial cells (LECs) attached to anterior lens capsules expressed mRNA for the receptor for AGEs (RAGE). The interaction of LECs with AGEs using bovine lens epithelial explants demonstrated that AGEs induced mRNAs and proteins of fibronectin, collagen type I, aberrant extracellular matrix proteins, and alpha-SMA, a specific marker for myofibroblastic cells. These findings suggest that AGEs may alter cellular functions which induce mRNAs and proteins associated with fibrosis in LECs.     

Rutin metabolites: Novel inhibitors of nonoxidative advanced glycation end products

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

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

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

Targeting advanced glycation with pharmaceutical agents: where are we now?

Advanced glycation end products (AGEs) are the final products of the Maillard reaction, a complex process that has been studied by food chemists for a century. Over the past 30 years, the biological significance of advanced glycation has also been discovered. There is mounting evidence that advanced glycation plays a homeostatic role within the body and that food-related Maillard products, intermediates such as reactive α-dicarbonyl compounds and AGEs, may influence this process. It remains to be understood, at what point AGEs and their intermediates become pathogenic and contribute to the pathogenesis of chronic diseases that inflict current society. Diabetes and its complications have been a major focus of AGE biology due to the abundance of excess sugar and α-dicarbonyls in this family of diseases. While further temporal information is required, a number of pharmacological agents that inhibit components of the advanced glycation pathway have already showed promising results in preclinical models. These therapies appear to have a wide range of mechanistic actions to reduce AGE load. Some of these agents including Alagebrium, have translated successfully to clinical trials, while others such as aminoguanidine, have had undesirable side-effect profiles. This review will discuss different pharmacological agents that have been used to reduce AGE burden in preclinical models of disease with a focus on diabetes and its complications, compare outcomes of those therapies that have reached clinical trials, and provide further rationale for the use of inhibitors of the glycation pathway in chronic diseases.