Production Of Advanced Glycation End-products Research Articles

Therapeutic Strategies in the Treatment of Diabetic Nephropathy - a Translational Medicine Approach

Type 2 Diabetes (T2D) is an important cause of renal dysfunction and the most common cause of end-stage renal disease (ESRD). Diabetic nephropathy is also associated with an increased risk of vascular disease and patient mortality. Aggressive management of hypertension to reduce microalbuminuria, together with tight glycaemic control are important therapeutic strategies for renal and vascular disease prevention in T2D. The main pathophysiological mechanisms associated with diabetic nephropathy result from activation of the renin-angiotensin-aldosterone system (RAAS), protein kinase C pathway, pro-inflammatory cytokines and various growth factors. Angiotensin II and transforming growth factor-beta (TGF-beta) are two important molecular mediators. The production of advanced glycation end-products (AGEs) and increased oxidative stress further exacerbates renal injury. These molecular changes within the renal tissue result in mesangial expansion, increased extracellular matrix deposition and an alteration in podocyte structure and function. Therapeutic targeting of these molecular pathways is an important area of translational research in diabetes. The elucidation of new genetic associations and proteomic biomarkers of diabetic kidney disease will also assist in the identification and treatment of high-risk patients. This review article will discuss both the molecular and clinical aspects of diabetic nephropathy, providing a bench-to-bedside research perspective to potential new therapeutic strategies.

Seasonal Phytochemical Variation of Anti-Glycation Principles in Lowbush Blueberry (Vaccinium angustifolium)

Diabetic hyperglycaemia promotes the production of advanced glycation end-products (AGEs), which play a significant role in the development of complications associated with type 2 diabetes mellitus. Vaccinium angustifolium, a medicinal plant used for the treatment of diabetes, produces a variety of phenolic metabolites with putative anti-diabetic activities. To assess optimal cultivation time, seasonal changes in the concentration of six phenolic compounds in leaves and twelve compounds in stems were examined using HPLC-DAD and examined in relation to seasonal changes in AGE inhibition activity, assessed with a fluorescence-based assay. A seasonal decline occurred in the concentration of chlorogenic acid, rutin, and quercetin 3-arabinoside in leaves and chlorogenic acid in stems. The concentration of (+)-catechin, and (-)-epicatechin in stems declined within two weeks before rising and fluctuating insignificantly. AGE inhibition activity of leaves was significantly greater at the final compared to the initial collection date whereas the activity of stems did not change significantly. Relative to the leaf extract, the stem was a more potent inhibitor of AGE formation, which could be a result of the unique phytochemistry of stems. Together, these results revealed significant seasonal variation in the phenolic profile and anti-glycation effects of V. angustifolium extracts and indicated late summer as the collection time yielding optimal activity.

Effects of methylglyoxal on amyloid‐beta peptide

Diabetics exhibit elevated levels of carbohydrates and their oxidized byproducts such as methylglyoxal (MGO). With the excess production and accumulation of the amyloid‐beta peptide (ABP) in AD, the linkage between diabetes and AD may involve ABP and MGO. We studied the effects of MGO‐induced ABP modification on conformational properties. Human isoform ABP fragment 1–42 was used in our study. Fluorescence and absorbance spectroscopy assessed MGO‐induced production of advanced glycation endproducts (AGEs), size exclusion chromatography monitored conformational changes, and thioflavin T fluorescence tracked fibrillation. MGO decreased the amount of high molecular weight (HMW) ABP aggregate and increased the low molecular weight (LMW) species relative to the control. Products obtained from incubation of MGO with free arginine, which absorbed in the 220nm range, also appear in HMW ABP when incubated with MGO. The LMW ABP species contained an MGO‐derived adduct that absorbed in the 280nm range, suggesting that conformational differences in the structures may promote reactivity to different amino acid residues. MGO affected the rates of ABP aggregation as compared with control, indicating that AGEs may influence ABP packing densities. These observations suggest a plausible molecular explanation for the relationship between diabetes and AD, providing novel avenues for therapeutic intervention.

TPI deficiency and neurodegeneration in Drosophila: an AGE‐dependent link to a common mechanism?

We have conducted forward screens in Drosophila to identify genes required for maintenance of neuronal viability. wasted away (wstd), is defined by a hypomorphic, autosomal recessive mutation associated with progressive motor impairment, severely reduced lifespan, and vacuolar neuropathology. Molecular analysis demonstrates that wstd encodes the glycolytic enzyme, triosphosphate isomerase (TPI). Mutations causing TPI deficiency have also been identified in humans resulting in progressive neurological dysfunction, neurodegeneration and early death. However, the mechanistic basis of the human syndrome remains puzzling. Decreased ATP levels do not appear to cause the observed phenotypes in TPI deficient flies or humans because ATP levels remain normal. Moreover, we find no genetic evidence that the mutant TPI is misfolded or involved in aberrant protein‐protein associations. We propose instead that the enzymatic block in wstd mutants leads to an accumulation of methylglyoxal and the consequent enhanced production of advanced glycation endproducts (AGEs), which are ultimately responsible for death and dysfunction of TPI deficient neurons. Evidence in support of this interpretation will be described. Our results favor the idea that TPI serves an essential neuroprotective role and support the possibility that AGEs may also contribute to the pathogenesis of other human neurological disorders.

RAGE modulates myocardial injury consequent to LAD infarction via impact on JNK and STAT signaling in a murine model

The receptor for advanced glycation end-products (RAGE) has been implicated in the pathogenesis of ischemia-reperfusion (I/R) injury in the isolated perfused heart. To test the hypothesis that RAGE-dependent mechanisms modulated responses to I/R in a murine model of transient occlusion and reperfusion of the left anterior descending coronary artery (LAD), we subjected male homozygous RAGE(-/-) mice and their wild-type age-matched littermates to 30 min of occlusion of the LAD followed by reperfusion. At 48 h of reperfusion, hematoxylin and eosin staining revealed significantly larger infarct size in wild-type versus RAGE(-/-) mice. Contractile function, as evaluated by echocardiography 48 h after reperfusion, revealed that fractional shortening was significantly higher in RAGE(-/-) versus wild-type mice. Plasma levels of creatine kinase were markedly decreased in RAGE(-/-) versus wild-type animals. Integral to the impact of RAGE deletion on diminished myocardial damage after infarction was significantly decreased apoptosis in the heart, as assessed by TUNEL staining, release of cytochrome c, and caspase-3 activity. Experiments investigating the impact of RAGE on early signaling pathways influencing myocardial ischemic injury revealed attenuation of JNK and STAT5 phosphorylation in RAGE(-/-) mouse hearts versus robust activation observed in wild-type mice upon ischemia and reperfusion. Solidifying the link to RAGE, these experiments revealed that infarction stimulated the rapid production of advanced glycation end-products in the heart. Thus, we tested the effect of ligand decoy soluble RAGE (sRAGE). Administration of sRAGE protected the myocardium from ischemic damage, similar to the effects observed in RAGE(-/-) mouse hearts. Taken together, these data implicate RAGE and its ligands in the pathogenesis of I/R injury and identify JNK and STAT signal transduction as central downstream effector pathways of the ligand-RAGE axis in the heart subjected to I/R injury.

Adjuvant strategies for prevention of glomerulosclerosis

The glomerulosclerosis which frequently complicates diabetes and severe hypertension is mediated primarily by increased mesangial production and activation of transforming growth factor-beta (TGF-beta), which acts on mesangial cells to boost their production of matrix proteins while suppressing extracellular proteolytic activity. Hyperglycemia and glomerular hypertension work in various complementary ways to stimulate superoxide production via NADPH oxidase in mesangial cells; the resulting oxidant stress results in the induction and activation of TFG-beta. Nitric oxide, generated by glomerular capillaries and by mesangial cells themselves, functions physiologically to oppose mesangial TGF-beta overproduction; however, NO bioactivity is compromised by oxidant stress. In addition to low-protein diets and drugs that suppress angiotensin II activity, a variety of other agents and measures may have potential for impeding the process of glomerulosclerosis. These include vitamin E, which blunts the rise in mesangial diacylglycerol levels induced by hyperglycemia; statins and (possibly) policosanol, which down-regulate NADPH oxidase activity by diminishing isoprenylation of Rac1; lipoic acid, whose potent antioxidant activity antagonizes the impact of oxidant stress on TGF-beta expression; pyridoxamine, which inhibits production of advanced glycation endproducts; arginine, high-dose folate, vitamin C, and salt restriction, which may support glomerular production of nitric oxide; and estrogen and soy isoflavones, which may induce nitric oxide synthase in glomerular capillaries while also interfering with TGF-beta signaling. Further research along these lines may enable the development of complex nutraceuticals which have important clinical utility for controlling and preventing glomerulosclerosis and renal failure. Most of these measures may likewise reduce risk for left ventricular hypertrophy in hypertensives, inasmuch as the signaling mechanisms which mediate this disorder appear similar to those involved in glomerulosclerosis.

Stiffening Our Resolve Against Adult Weight Gain

Increasing adiposity with a clinical endpoint of obesity is associated with a progressive increase in risk of cardiovascular diseases (CVD). However, we are only beginning to understand the physiological and pathophysiological mechanisms by which excessive body fat storage causes CVD. In this issue of Hypertension , the findings of Wildman et al1 extend and strengthen previous observations2–5 by showing that changes in arterial stiffness, as measured by aortic pulse-wave velocity (PWV), are related to changes in body weight over a 2-year follow-up period in healthy men and women aged 20 to 40 years. Weight gain was associated with an increase, whereas weight loss was associated with a decrease in aortic PWV, independent of changes in blood pressure. The greatest increases in aortic PWV were observed in those subjects who gained the most weight (≥4.5 kg). Importantly, the changes in aortic PWV were related to changes in body mass index (BMI), indicating that the associations with body weight were mediated by changes in adiposity (rather than fat-free mass). Changes in aortic PWV over time also were related to baseline body weight, BMI, and waist circumference (an indirect measure of abdominal adiposity), providing evidence that higher initial levels of total and abdominal body fatness are associated with greater future increases in arterial stiffness. The study included subjects with a broad range of BMI, suggesting that the findings are applicable to both nonobese and obese adults in the general population. An important finding was that black adults demonstrated greater increases in weight gain-adjusted and blood pressure-adjusted aortic PWV over time, suggesting that they undergo greater increases in arterial stiffness in response to the same age-associated weight gain compared with whites. These observations provide further support for the emerging concept that adiposity-driven weight gain during adulthood contributes significantly to the adverse changes …

Biochemical pathways for microvascular complications of diabetes mellitus.

To review the current biochemical theories on how diabetes contributes to microvascular disease. MEDLINE search (1980-June 2003) and bibliographies of articles obtained on this topic. Articles identified from the data sources were evaluated and those deemed relevant to this review were incorporated. The prevailing biochemical theories on how diabetes leads to microvascular disease include increased polyol (sorbitol/aldose reductase) pathway flux, production of advanced glycation end-products, generation of reactive oxygen species, and activation of diacylglycerol and protein kinase C isoforms. These pathways contribute to endothelial damage and dysfunction and may alter gene functioning. Each pathway, via varied and often overlapping mechanisms, contributes to altered microvascular function that leads to the development of retinopathy, neuropathy, and nephropathy, the major microvascular complications associated with diabetes.

Cytotoxicity test and cytogenetic analysis of effects of aminoguanidine in vitro

The potential genotoxic activity of chemical substances in vitro is usually assessed by the micronucleus test and by karyological analysis. Use of the fluorescent plus Giemsa (FPG) technique is also recommended in the event that positive results are found in the micronucleus test, or if there is an increased rate of structural and numerical chromosome aberrations compared with controls. The tested substance, aminoguanidine (AG), has a marked ability to inhibit the toxic effects of carbonyl products (carbonyl stress) that arise during the end-phases of non-enzymatic protein glycation both in vitro and in vivo. The importance of this ability follows the finding that the production of advanced glycation end-products (AGE) is a part of the molecular mechanism of the pathogenesis of chronic diabetic complications. The aim of this study was to test the cytotoxic and clastogenic effects of AG on cells of the diploid cell line B-HEF-2, derived from a three-month-old male fetus. The results of the test did not reveal any induction of micronucleus production in the analyzed cells at AG concentrations ranging between 1 x 10(-2) and 1 x 10(-4) mol.L-1. Karyological analysis showed no clastogenic effect of the tested substance nor any increased rate of structural chromosome aberrations. The positive properties of AG and to its potential use as a glycoxidation inhibitor and AGE production are somewhat dimmed by its ionic nature, which hampers hydrophobic interaction with the nonpolar components of biological membranes. For this reason, the authors will further study the cytotoxicity and cytogenetic analysis of Schiff bases of AG synthesis on the basis of natural aldehydes (resorcine aldehyde, pyridoxal, etc.) in which antiglycation activity has been detected.

Glucose and reactive oxygen species.

This review aims at presenting new concepts of glucose-induced damage in diabetes via an increased production of oxygen free radicals. Reactive oxygen species modulate various biological functions by stimulating transduction signals, some of which are involved in diabetes pathogenesis and complications. Diabetes is characterized by high glucose concentrations that lead, via several mechanisms (glucose autoxidation, stimulation of the polyol pathway, activation of the reduced form of nicotinamide adenine dinucleotide phosphate oxidase, and production of advanced glycation endproducts), to an increased production of reactive oxygen species. The resulting oxidative stress (the imbalance between reactive oxygen species production and the antioxidant defences) can play a key role in diabetes pathogenesis. Superoxide radicals generated by the reduced form of nicotinamide adenine dinucleotide phosphate oxidase may thus contribute to impaired endothelium-dependent vascular relaxation by the inactivation of nitric oxide, and more generally to vascular dysfunction, thereby contributing to accelerated atherosclerosis in diabetic patients. The increased production of reactive oxygen species induced by hyperglycaemia has also been suggested to be involved in platelet dysfunction, in tissue remodelling (via metalloproteinases), and in redox regulation of glucose transport in skeletal muscle. Beyond the classic treatments for diabetes, new therapeutic strategies involving antioxidants or anti-advanced glycation endproduct molecules are proposed. Future methods could take into account the signalling pathways and genes that are regulated by reactive oxygen species.

An immunochemical study on tau glycation in paired helical filaments

Glycation is a non-enzymatic posttranslational modification that involves a covalent linkage between a sugar and an amino group of protein molecule forming ketoamine. Subsequent oxidation, fragmentation and/or crosslinking of ketoamine leads to the production of advanced glycation endproducts (AGEs). Formation of AGEs causes detrimental effects on the structure and function of affected proteins. Accumulation of AGEs has been implicated in normal aging and in the pathogenesis of diabetes-associated complications and Alzheimer's disease (AD). Of all AGEs, N ε-(carboxymethyl)lysine (CML) is a major glycoxidation product known to be stable and accumulate progressively in vivo. In order to determine if tau is glycated in AD, we raised a rabbit antibody to CML that demonstrated its usefulness in detecting glycation of different proteins in vitro, including BSA, ribonuclease, lysozyme and recombinant tau. Immunochemical analyses indicated that ribose and glucose-6-phosphate are more effective than glucose in generating CML formation in these proteins. We used this antibody to probe for glycation in the following human tau preparations: tau of normal brains and preparations of soluble PHF-tau as well as insoluble PHF from AD brains. All three principal tau components resolved from PHF-tau on Western blots showed CML immunoreactivity indicating that tau is glycated in PHF-tau; and insoluble PHF exhibited prominent CML immunoreactivity on top of the stacking gel. Moreover, immunoelectron microscopic analyses indicate that the anti-CML antibody labels predominantly PHF in aggregates. Taken together, these results suggest that tau becomes glycated in PHF-tau and glycation may play a role in stabilizing PHF aggregation leading to tangle formation in AD.

12-Lipoxygenase products increase monocyte:endothelial interactions.

In summary, we suggest that hyperglycemia causes upregulation of 12-lipoxygenase activity. The increased production of 12-LO products, 12(S) and 15(S)-HETE, activates monocyte integrins which result in enhanced adhesion of monocytes to endothelium. The binding of monocytes to endothelium is a key early event in development of atherosclerosis. Upregulation of this process by vascular cells exposed to chronic elevations in glucose may be one explanation for the accelerated atherosclerosis observed in patients with Type 2 diabetes.