Dissertations / Theses on the topic 'Glycation'

Forty-two non-diabetics whose glycated haemoglobin levels could not be explained by their levels of fasting glycaemia or glucose tolerance were identified as part of the Islington Diabetes Survey. Mean glycated haemoglobin levels that were high relative to their blood glucose levels (HIGH GLYCATORS) were found in 22 subjects whilst 20 subjects had low relative levels (LOW GLYCATORS). A repeat glucose tolerance test in 13 of the 42 subjects (7 high and 6 low glycators) showed that they remained categorised in the two groups four years after the discrepancy was identified. Mean blood glucose levels calculated from two 5-point diurnal profiles also failed to explain the glycated haemoglobin levels in the two groups. Further biochemical and haematological tests revealed that glycated albumin showed a better relationship with blood glucose levels than did glycated haemoglobin. Intra-erythrocyte glucose concentrations were significantly lower than the plasma glucose in the low glycators, whilst the levels of erythrocyte 2,3-diphosphoglycerate (a known catalyst of glycation) were significantly higher in the high glycators. These findings may partially explain the original discovery of low and high glycators. The effects of high fibre/low glucose and high glucose/low fibre diets on glycated proteins were studied in normal subjects. Only glycated albumin levels changed significantly, probably due to small changes in plasma glucose occurring on each diet, which were not mirrored by similar changes in the erythrocyte. When another group of normal subjects received 1 g vitamin C daily for three months, significant reductions in glycated albumin and glycated haemoglobin were observed, with the former being reduced to a greater extent. This suggests that vitamin C has the potential of being used therapeutically to reduce glycation. In conclusion, for normal subjects, glycated albumin is a more reliable indicator of blood glucose control than glycated haemoglobin.

RAGE is a multi-ligand pattern recognition receptor. RAGE can bind several damage associated molecular pattern proteins. RAGE- ligand interaction is pathophysiologically relevant to several major diseases including diabetes and certain cancers. RAGE inhibition has been reported to reduce morbidity in these disease states. However, to design better RAGE inhibitors it is necessary to understand the structural basis behind the RAGE-ligand interaction and currently this is not well understood. This thesis focuses on understanding the interaction of RAGE with two of its ligands; AGEs and S100B. AGEs are highly heterogeneous and are formed as a result of non-enzymatic glycation. A panel of AGEs were characterized in terms of their side chain modifications, thermal stability, secondary structure, aggregation and surface charge. These glycation induced changes were then correlated to RAGE binding. Building on these results the role of AGE-RAGE interaction in pancreatic cancer cell proliferation and migration was determined. Ribose modified BSA induced ROS formation, which then triggered NF-?B upregulation via RAGE induced ROS signaling. Ribose BSA increased pancreatic cell proliferation and migration. Anti-RAGE antibodies and RAGE inhibitors prevented AGE induced cellular effects. The role of ribose modified BSA was also determined in macrophage activation and pro-inflammatory cytokine release. Rapid internalization was observed of the ribose-BSA and confocal imaging revealed the internalization of the AGE compound into the lysosomes which lead to the ROS production, NF-?B activation and pro-inflammatory cytokine release in a RAGE independent signaling mechanism. Finally, the role of tryptophan residues of the V domain in domain stability and S100B binding was determined. We have generated single, double and triple tryptophan mutants of the V domain by site directed mutagenesis. The effect of Trp residues in the domain stability could not elucidated as no change was observed in the secondary structure of the mutants when compared to the wild type suggesting the plasticity of the V-domain. The fluorescence emission and life time properties of each Trp residue was determined. Our binding assays of the Trp Ala mutants indicate tighter binding of the S100B to the mutants. The S100-RAGE peptide structures suggest multi modal interaction of S100B-RAGE interaction.

Background: In the past 60 years, the median age of the entire world population has increased and ageing and chronic diseases are now the main medical concerns in the developed world. The identification of early signs of disease pathogenesis is vital for prevention and targeting populations at risk in order to reduce morbidity and mortality. Glycation is well-established as an index of control, or otherwise, and a predictor of end-organ damage, for people with type 2 diabetes. At the beginning of the work for this thesis (2010), evidence was beginning to be raised to suggest that since glycation levels vary considerably, in normoglycaemic, non-diabetic individuals, glycation cannot be solely related to glucose levels and early reports allowed for speculations about a relationship between early glycation and oxidative stress. The aim of this thesis was to establish the relationship between early glycation and oxidative stress in normoglycaemia using techniques from the full breadth of Human Nutrition Research. Methods: In study 1, existing epidemiological data were used to identify relationships between proxies for redox status and early glycation in non-diabetic individuals. One-way ANOVA, Chi-squared and multivariate linear regressions, adjusted for all known confounders were used to explore associations of HbA1c with self-reported smoking status and fruit & vegetables consumptions in the Scottish Health Surveys 2003-2010, among individuals without known diabetes and HbA1c<6.5%. In studies 2 and 3, the associations from epidemiology were explored in mechanistic laboratory studies with high physiological relevance (using physiological concentrations and conditions) to better characterise the effect of oxidative stress and antioxidants on early glycation. In study 2, bovine serum albumin (BSA), reduced BSA (mercaptalbumin) (both 40g/L), and human plasma were incubated with glucose concentrations 0-30 mM for 4 weeks at 37oC. All were tested pre-oxidized for 8 hours prior to glycation with 10nM H202, or continuously exposed to 10nM H2O2 throughout the incubation period. Fructosamine was measured (nitroblue tetrazolium method) at two and four weeks. In study 3, Bovine Serum Albumin (BSA) was pre-treated prior to in vitro glycation: either no treatment (native), pre-oxidised (incubated with 10nM H2O2, for 8 hours) or incubated with a mixture of phenolic acids at physiologically relevant concentrations, for 8 hours). In-vitro glycation was carried out in presence of i) glucose only (0, 5 or 10mM), ii) glucose (0, 5 or 10mM) plus H2O2 (10nM), or iii) glucose (0, 5 or 10mM) plus phenolic acids (10-160nM). Fructosamine was again measured using the nitroblue tetrazolium method. Prior to the experimental study we carried out a systematic literature review of dietary interventions reporting plasma concentrations polyphenol metabolites, to inform the design of a physiologically relevant in-vitro study. In study 4, clinical trial data and biological samples were analysed from a randomised controlled dietary advice trial in obese pregnant women, a group at risk from higher glycation and oxidative stress. Samples and data from the UPBEAT study trial (n=117) were analysed. Plasma fructosamine, plasma sRAGE, urinary Ferric Reducing Ability of Plasma (FRAP), urinary Total Phenol (TP) and urinary Advanced Oxidised Protein Products (AOPPs) were measured at 16-18+6 and 27-28+6 weeks gestation. Dietary recalls were used to calculate fruit and vegetable and polyphenol intake at the same timepoints. Data were analysed to identify associations between dietary variables and biochemical markers, as well as their relationships with diagnosis of complications. Associations between maternal variables and neonatal anthropometry were also investigated. Results: In study 1, HbA1c was higher in smokers by 0.25 SDs (0.08%), and 0.38 SDs higher (0.14%) in heavy smokers (>20cigarettes/day) than non-smokers (p<0.001 both). Smokers were twice as likely to have HbA1c in the ‘pre-diabetic’ range (5.7-6.4%) (p<0.001, adj.model). Pre-diabetes and low grade inflammation did not affect the associations. For every extra 80g vegetable portion consumed, HbA1c was 0.03 SDs (0.01%) lower (p=0.02), but fruit consumption did not impact on HbA1c, within the low range of consumptions in this population. In study 2, oxidized BSA (both pre-oxidised and continuously exposed to H2O2) was more readily glycated than native BSA at all glucose concentrations (p=0.03). Moreover, only oxidized BSA was glycated at physiological glucose concentration (5mM) compared to glucose-free control (glycation increased by 35% compared to native albumin p<0.05). Both 5mM and 10mM glucose led to higher glycation when mercaptalbumin was oxidised than un-oxidised (p<0.05). Fructosamine concentration in human plasma was also significantly higher when oxidized and exposed to 5mM glucose, compared to non-oxidised plasma (p=0.03). The interaction between glucose concentration and oxidation was found to be significant in all protein models (p<0.05). In study 3, the presence of six phenolic acids with BSA during in-vitro glycation did not lower fructosamine formation. However, when BSA was pre-incubated with phenolic acids, significantly lower concentration of fructosamine was detected under glycoxidative conditions (glucose 5 or 10mM plus H2O2 10nM) (p<0.001 vs. native BSA). In study 4, women in the lowest quartile of total polyphenol intake had 8% greater fructosamine levels compared to those in the top quartile. Total polyphenol intake was negatively correlated with sRAGE levels. Diagnosis of severe preeclampsia was associated with elevated AOPPs. Maternal polyphenol intake was positively correlated with birth weight, while maternal glycoxidation showed the opposite relationship. Conclusions: Study 1 added evidence to relate smoking (an oxidative stress proxy) to protein glycation in normoglycaemic subjects. This association has implications for individuals exposed to ROS and for epidemiological interpretation of HbA1c and its clinical usefulness. Study 2 offered a mechanistic background to the previously shown epidemiological association. This study demonstrated for the first time albumin glycation in-vitro, using physiological concentrations of albumin, glucose and hydrogen peroxide. These results identified low-grade oxidative stress as a key element early in the glycation process, especially in glucose concentrations relative to normoglycaemia. Furthering those findings, study 3 showed that protein-phenolic acid interactions are important regulators of protein glycation. Together those studies highlighted that protein pre-treatment, either with oxidants or phenolic acids, is an important regulator of subsequent glycation in a physiologically relevant system. An important outcome of those studies is that high quality in-vitro studies under conditions closer to physiology are feasible and should be employed more frequently. Finally, study 4 demonstrated an association between polyphenol intake and glycation during pregnancy, with an impact on neonatal outcome measures. Maternal glycoxidation is a promising marker of pre-eclampsia and neonatal anthropometry and could be modulated by maternal lifestyle and dietary habits. Overall, the results of this thesis implicate that drivers of redox status have the capacity to modulate protein glycation in normoglycaemia. These results challenge the assumption that glycation levels are solely dependent on circulating glucose levels and suggest a useful application of glycation outside the field of diabetes.

Introduction: Glycation is a non-enzymatic reaction between a sugar and a free-amino group contained in molecules such as proteins, amino acids, DNA, RNA, and lipids. In the initial phase, the carbonyl group of the reducing carbohydrate condenses with the free-amino group on the biomolecule to form a reversible glycosylamine, which is then converted to a more stable Amadori product. Once formed, these products can with time undergo to dehydration, cyclization, oxidation, and rearrangements forming a polymorphic group of compounds collectively referred as Advanced Glycation End-products (AGEs). The accumulation of AGEs in vivo is considered the most important link between diabetes and oxidative disease. The rate of glycation could account for the long-term diabetic complications. Nevertheless, the role of AGEs is yet to be determined and it is, still nowadays, an important and challenging field of study. Little is known about their presence and their consequences on adipose tissue; in particular, the role of AGEs on adipogenesis and fat cell functionality remains fully unexplored. Methods: In this project, 3T3-L1 cell line and stromal vascular fraction cells isolated from human adipose tissue were used. The purpose was to set up an in vitro model for investigating the effects of a chronic exposure to a glycation-inducing environment on the adipogenetic process. Morphological analyses, E.L.I.S.A. assay for AGEs, expression analysis of lipid and glucose metabolism genes, glucose uptake, and immunofluorescence for RAGE, GLP1r, GLUT4 and AGEs, were performed on mature adipocytes. Moreover, the gene expression profile obtained in vitro, was compared to that of subcutaneous and visceral adipose tissues obtained from 64 obese subjects with type 2 diabetes or with normal glucose tolerance underwent to bariatric surgery. In collaboration with the Mass Spectrometry Service (CNR – IENI Padova), the use of mass spectrometry to characterize post-translation modifications induced by glycation was evaluated. Results and conclusions: Following an exposition to a glycation-inducing environment, an increase of intracellular AGEs both by immunofluorescence and E.L.I.S.A. was observed in our in vitro model. Preliminary analysis on cell cultures suggested that a glycation-inducing environment importantly impacts on gene expression profile. In glycating conditions, an increased expression of Glut4 and, on the contrary, an insulin-induced glucose uptake reduction was observed. These results allow us to hypothesize an influence of AGEs on GLUT4 translocation to the plasma-membrane and/or on its functionality. Finally, although mass spectrometry represents a high potential technique was shown that it has several disadvantages and technical challenges. In particular, several post-translational modifications can affect simultaneously the same protein, thus the total mass increase will reflect the sum of all modifications. Furthermore, spectra analysis of a whole cellular or tissue lysates resulted excessively complex to underline small differences. Multiple steps in which proteins of a specific molecular weight range would be isolated, concentrated and separately assayed are therefore required in order to simplify the analysis by mass spectrometry.
Introduzione: La glicazione è una reazione non enzimatica tra uno zucchero e un gruppo amminico contenuto in molecole come proteine, aminoacidi, DNA, RNA e lipidi. Nella fase iniziale, il gruppo carbonilico del carboidrato riducente condensa con il gruppo amminico libero delle biomolecole formando una glicosil-ammina reversibile, la quale verrà poi convertita in un prodotto più stabile, detto prodotto di Amadori. Nel tempo, tali prodotti possono subire processi di disidratazione, ciclizzazione, ossidazione e riarrangiamenti formando un gruppo polimorfico di composti indicati come prodotti di glicazione avanzata (AGEs). L'accumulo degli AGEs in vivo è considerato il più importante collegamento tra il diabete e la malattia ossidativa. In particolare, il tasso di glicazione potrebbe spiegare le complicanze diabetiche a lungo termine. Tuttavia, la loro presenza a livello del tessuto adiposo ed il loro ruolo nell’adipogenesi e nella funzionalità dell’adipocita, non sono ancora stati del tutto determinati e sono a tutt’oggi un importante e stimolante campo di ricerca. Metodi: In questo progetto, sono state utilizzate cellule 3T3-L1 e cellule della frazione vasculo- stromale isolate dal tessuto adiposo umano allo scopo di costruire un modello in vitro per indagare gli effetti indotti sul differenziamento adipogenico dovuti da una esposizione cronica ad un ambiente inducente la glicazione. Sugli adipociti maturi ottenuti in vitro sono stati eseguite: analisi morfologiche, saggi E.L.I.S.A. per gli AGEs, analisi di espressione di geni coinvolti nel metabolismo lipidico e glucidico, misurazione della capacità di captazione del glucosio ed analisi di immunofluorescenza per RAGE, GLP1r, GLUT4 ed AGEs. È stata inoltre confrontata l'espressione genica ottenuta in vitro, con quella misurata nelle biopsie di tessuto adiposo viscerale e sottocutaneo di 64 soggetti obesi con diabete di tipo 2 o con normale tolleranza al glucosio sottoposti ad intervento di chirurgia bariatrica. In collaborazione con il servizio di Spettrometria di Massa (CNR – IENI, Padova), è stato valutato l'utilizzo della spettrometria per la caratterizzazione delle eventuali modifiche post-traduzionali indotte dalla glicazione. Risultati e conclusioni: È stato osservato, sia mediante immunofluorescenza, sia mediante test E.L.I.S.A., che un ambiente inducente alla glicazione porta effettivamente ad un aumento degli AGEs intracellulari. Analisi preliminari delle colture cellulari, inducono ad ipotizzare che possano esistere alcuni effetti indotti dall’ambiente glicante sull'espressione genica. In condizioni glicanti, è stata osservata un’aumentata espressione di Glut4 e una ridotta captazione di glucosio che suggerisce una probabile influenza degli AGEs sulla traslocazione e/o sulla funzionalità del trasportatore del glucosio. Tale osservazione suggerisce che possa esistere una probabile influenza degli AGEs sulla traslocazione /o sulla funzionalità del trasportatore del glucosio. Nonostante la spettrometria di massa sia una metodica molto sensibile ed abbia alte potenzialità, ha anche alcuni svantaggi: modifiche post traduzionali, siano esse enzimatiche o non-enzimatiche, possono essere contemporaneamente presenti sulla stessa proteina; pertanto, l’incremento di massa totale rifletterà la somma di tutte le modifiche. Lo spettro di un lisato intero, si è dimostrato eccessivamente complesso per poter apprezzare piccole differenze. Risulta pertanto necessaria un’analisi composta da più passaggi in cui, in prima istanza verrà isolata e concentrata una frazione di proteine caratterizzate da uno specifico range di peso molecolare che solo in seguito verrà analizzata mediante spettrometria di massa; suddividendo in questo modo l'intero spettro in piccole parti.

Aging is an irreversible degenerative process characterized by a general decline in cellular metabolic activity accompanied with progressive deterioration of cellular components resulting in enhanced mortality. Although, average life expectancy has increased, it is associated with different aging related diseases such as cardiovascular diseases, cancer, diabetes and so on. Hence, there is demand of active research on aging process and aging associated diseases to make added years of older people healthy. Short lived organisms, such as yeast are excellent model for aging research, as longevity-defining genes and signaling pathways discovered in yeast have been shown to define cellular and organismal longevity in eukaryotes across phyla. Yeast displays two distinct lifespan, namely, replicative lifespan (RLS) and chronological lifespan (CLS), which serve as models for proliferating (mitotic) and non-proliferating (post-mitotic) tissues in higher eukaryotes, respectively. RLS is defined as the number of daughter cells produced by a mother cell before cell division ceases; whereas CLS is the duration of cell survival in the stationary phase. Nutrients play important role in chronological aging of yeast. Calorie restriction is associated with lifespan extension in yeast and other higher organism including C elegans, drosophila, and mouse. While, non calorie restriction in terms of glucose (2%) leads to activation of various signaling pathways such as TOR/Sch9 and RAS/AC/PKA, which stimulate cellular senescence in yeast. In addition to these factors, non calorie restriction in terms of glucose can promote formation of advanced glycation reaction products (AGEs). AGEs are formed as a result of series of non-enzymatic reactions between protein and reducing sugars. AGEs are known to affect functions of many intracellular proteins and also AGE modification of proteins results in protease resistance, which affect the protein homeostasis. Glycated proteins and aggregates are responsible for several age associated diseases like Alzheimer‘s disease, Parkinson‘s disease, familial amyloidotic polyneuropathy, amyloidotic lateral sclerosis and diabetic complications. Thus, targeting AGE formation could be a rational approach to extend the lifespan and delay aging. Therefore, thesis deals with the development of yeast as a model system to study glycation induced aging and targeting AGEs by glycation inhibitors. For that, we have studied the effect of glycation and glycation inhibitors on yeast chronological lifespan. Further, we explored the effect of presence of glycation inhibitors on AGE modification and on global proteome through proteomics approach. Lastly, we have investigated the effect of glycation inhibitors on aging responsive genes using microarray and real time PCR.
AcSIR

Evidence suggests a role for glycation and glycoxidative changes in damaged tissues and plasma components. LDL modified as a result of glucoxidation stress, is potentially atherogenic, and is implicated in the accelerated atherosclerosis associated with diabetic complications. The purpose of this thesis is to investigate and establish the importance of glycoxidative changes to LDL and its role in the chemical complications associated with diabetes. LDL was chemically modified in vitro with glyoxal (dicarbonyl aldehyde) to produce a crosslinked structure (glyoxalated) LDL. The complex produced (glyoxalated LDL) was used for the production of specific antibodies. Anti-glyoxalated LDL antibody was employed in specific and sensitive ELISA assays to measure the levels of glyoxalated LDL in human plasma. Levels of glyoxalated LDL measured in diabetics were generally up to 20% higher (n = 182, P <0.001) when compared to non-diabetics, reference and hyperlipidemic groups. An anti-glyoxalated LDL autoantibody ELISA assay was developed to assess the immunological importance of glyoxalated LDL and similar crosslinked structures in human plasma. Diabetics with poor glycaemic control demonstrated a 22% increase in anti-glyoxalated LDL autoantibodies (n = 39, P <0.0001) compared to reference groups, where less than a 3% increase was detected in hyperlipidemic non-diabetic subjects (n = 33, P <0.08). Unlike glycated haemoglobin, no significant age relationship was detected. Although glyoxal is a common product of glucose and lipoprotein autoxidation, levels of glyoxalated LDL and anti-glyoxalated LDL autoantibodies measured in diabetics, hyperlipidemic and reference groups, suggest a direct relationship between hyperglycaemia and the presence of glyoxal mediated crosslinks on human LDL. Glyoxalated LDL measured with these specific immunochemical assays may predict the possible complications associated with diabetes better than glycated haemoglobin because it appears not to have a direct relationship with age at high concentrations observed in diabetics.

Diabetes and cancer are major health problems because of their high incidences worldwide. A growing body of epidemiological evidence indicats a molecular link between diabetes and breast cancer. Patients with diabetes mellitus have an increased likelihood of developing various types of cancers including breast cancer through the formation of advanced glycation endproducts (AGEs) which lead to cellular and bio-molecular dysfunction. However, the effects of AGEs have been poorly investigated on breast cancer cells. This current study examined for the first time the biological effects of various concentrations of BSA-derived AGEs on the invasive and hormone–independent breast cancer cell line MDA-MB-231 and on non- invasive hormone-dependent human breast cancer cell line MCF-7. Bovine serum albumin was glycated-using methylglyoxal for three days. Crosslinked AGEs were assessed using sodium dodecyl sulphate polyacrylamide gel electrophoresis followed by Coomassie blue staining. Different assays including cell proliferation, migration and invasion through the MatrigelTM with assessment of matrix metalloproteinase (MMP) activity by using Zymogaraphy were performed to investigate the effect of different concentrations of BSA-AGE on both cell lines in vitro. Furthermore, signalling pathways were investigated by Western blotting and using kinexusTM phspho-protein microarray. The expression of the main receptor for AGEs (RAGE) involved on the MDA-MB231 and MCF-7 breast cancer cell lines was assessed by Western blotting and Calibur Flow Cytometer System. The results of this study demonstrated that BSA-AGEs increased MDA-MB-cell, proliferation, migration and invasion through the MatrigelTM associated with an enhancement of matrix metalloproteinase (MMP)-9 activities, in a dose-dependent manner, up-regulated the expression of the receptor for AGEs (RAGE) and of the key signalling protein, phospho-extracellular-signal regulated kinase (p-ERK)-1/2. In addition, the blockade of BSA-AGE/RAGE interactions using anti-neutralizing RAGE antibody reduced the expression of p-ERK1/2. Furthermore, in BSA-AGE-treated cells, phospho-protein micro-array analysis revealed the main enhancement of the over-phosphorylation of (ERK1/2), (p70S6K1), (STAT)-3 and (MAPK) p38, involved in cell survival, cell growth cell cycle and protein synthesis. In contrast, MCF-7 showed stimulatory effects of BSA-AGEs, on cell proliferation and migration, as compared to non-modified BSA. However, BSA-AGEs did not change the weak invasive capacity of MCF-7 cells to cross a reconstituted basement membrane. In addition, BSA-AGEs induced over-phosphorylation of RAGE in MCF-7 cells. The investigation of signalling pathways suggests that BSA-AGEs might contribute to breast cancer development through activation of MAPK pathway and activation of CREB1 transcription factor in MCF-7 cells. Throughout the study, the non-modified BSA had a negligible effect. In conclusion, BSA-AGEs might contribute to breast cancer development and progression of breast cancer. In addition, the up-regulation of RAGE and key phosphor-protein signalling expression induced by BSA-AGEs might be a promising target for therapy to prevent the development of breast cancer in diabetic patients.

Apolipoproteins have important roles in the transport of lipids and the regulation of lipoprotein metabolism as cofactors for enzymes and ligands for receptor-binding. Their function and metabolism are closely related to the development of many diseases. This dissertation describes the investigation of the distribution and metabolism of apoE and glycated apoE in diabetes, obesity and hyperlipidaemia in comparison with healthy people. In order to carry out the research, I developed several robust laboratory methods and techniques for the isolation and measurement of apoE and glycated apoE. These included (1) a modified in-house ultracentrifugation for isolation of lipoprotein fractions (2) high sensitivity sandwich enzyme-linked immunosorbent assay (ELISA) for apoE and (3) m-aminophenylboronate affinity chromatography for the separation of glycated and non- glycated apoE.In healthy people the apoE concentration in different lipoprotein fractions is influenced by age, gender and apoE genotype. The effect of atorvastatin on serum apoE concentration in patients with type 2 diabetes with nephropathy was dependent on the dose of atorvastatin and apoE genotype and was strongly correlated with the reduction in triglycerides (TG) in very low density lipoprotein (VLDL).The effect of bariatric surgery on obese patients with and without diabetes demonstrated that after bariatric surgery, VLDL-apoE increased and apoE in low density lipoprotein (LDL), high density lipoprotein (HDL) and d>1.21g/ml fractions decreased; both glycated LDL-apoE and glycated HDL-apoE decreased. Total apoE and glycated apoE concentrations in plasma decreased to levels comparable to those of healthy controls. However, the distribution within the lipoprotein fractions was very different. The effect of niacin/laropiprant (LRPT) on lipoproteins in hyperlipidaemia patients was assessed in a blind crossover trial. Niacin/LRPT slightly decreased VLDL-apoE and LDL-apoE. It had no effect on apoE in HDL. Glycated apoE did not change in hyperlipidaemia. These results show that, compared with healthy people, the apoE distribution in obese and hyperlipidaemia patients is abnormal despite no change in total apoE concentration in some cases. The results also demonstrate that glycated apoE originates preferentially from VLDL. Various mechanisms for these results and relationships with other lipids are discussed. Furthermore, I suggest several potential directions, especially in vitro, for further research on apoE function and metabolism.

Methylglyoxal (MG) is a reactive dicarbonyl derived as a by-product of glycolysis. If MG is not metabolized by glyoxalase-1 (Glo1), it glycates macromolecules producing advanced glycation end products (AGEs); these have been linked to larger infarct sizes and poorer cardiac function after myocardial infarction (MI). Proteins of the extracellular matrix (ECM) are prime targets for glycation by MG, but it is unknown if MG modification of the ECM may be a mechanism that contributes to the poor repair and function of the post-MI heart. This study sought to examine if MG-induced modifications of ECM proteins negatively affect fibroblast and endothelial cell function. Analysis with an MG-derived hydroimidazolone 1 (MG-H1) antibody confirmed MG modification of laminin and collagen type (Col) 1, 3, and 4. MG modifications decreased endothelial cell (EC) adhesion on Col3, Col4, and laminin and angiogenesis on ECMatrix. Furthermore, alpha smooth muscle actin staining indicated increased myofibroblast differentiation of fibroblasts on MG-modified proteins. Following induction of MI, extracted mouse hearts were decellularized and compared to healthy controls. Perhaps a result of technical challenges, both western blot and immunohistochemistry contrasted previous data by displaying a marked decrease in MG-H1 modifications post-MI. Overall, these results indicate that MG modifications of the ECM negatively influence EC and fibroblast function, requiring more research on their impact in cardiovascular disease progression.

Tubulointerstitial disease is a prominent phenomenon that underlies diabetic nephropathy and correlates closely with declining renal function. The formation of myofibroblasts represents a pivotal process in the development of tubulointerstitial fibrosis. Although the origin of these cells remains unclear, the cytokine transforming growth factor-p (TGF-P) has been shown to mediate the in vitro phenotypic transformation of tubular epithelial cells into myofibroblasts, a process known as transdifferentiation. The pathogenic link between chronic hyperglycaemia and the development of tubulointerstitial injury has not been fully elucidated, however diabetes mellitus is associated with the increased accumulation of a group of heterogeneous protein modifications termed advanced glycation endproducts (AGEs). Interactions between AGEs and specific receptors such as RAGE induce pleiotropic cellular effects including the increased expression of cytokines. This thesis explored the role of AGEs in inducing epithelial to myofibroblast transformation. Specific binding of I AGE-BSA to cell membranes prepared from a rat proximal tubule cell-line, was observed. Binding characteristics, immunoblotting and real time PCR experiments established that this binding site was RAGE. Whole cells exposed to AGEs including a physiological AGE-moiety, carboxymethy-lysine, demonstrated time and dose-dependent epithelial-myofibroblast transdifferentiation as determined by morphological changes and de-novo oc-smooth muscle actin expression. Reciprocal loss of expression of the epithelial antigens E-cadherin and zonula occludens was also seen. The addition of neutralising antibodies to RAGE or to TGF-6 abrogated AGE-mediated effects and prevented an observed AGE-RAGE augmentation of TGF-6 protein production. Furthermore, there was evidence of proximal tubular transdifferentiation in long-term diabetic rats and in a renal biopsy from a patient with type I diabetes. Administration of an AGE cross-link breaker, phenyl-4,5-dimethylthiazloium (ALT 711) reduced AGE accumulation, TGF-6 expression and transdifferentiation in diabetic rats. These studies provide a novel mechanism to explain the development of tubulointerstitial disease manifested in diabetic nephropathy and provide a new treatment target.

Glycation is a series of chemical interactions occurring in food and biological systems between reducing sugars and proteins leading to the formation of Advanced Glycation End products (AGEs). Ingestion of dietary AGEs and/or their formation in-vivo are mainly associated with cardiovascular and other age-related diseases and complications of long term diabetes. Potential strategies to prevent AGE formation can help to reduce risk factors associated with thermal processing of many foods. The overall objective of this research was focused on the identification of potential AGE inhibitors and investigation of their activity in glucose and ribose-based model systems containing lysozyme. The carboxylic acid functional group was chosen as a potential candidate based on their ability to interact with Schiff bases in addition to their ability to form amide bonds and carboxylate salts with the lysine side chains of proteins. The model systems were incubated with and without selected carboxylic acids (maleic, acetic, oxalic and citraconic) at 50°C for 12, 24 and 48h at pH 6.5. The effect of carboxylic acids on the glycation of lysozyme was studied by electrospray ionization mass spectrometry (ESI-MS). The experimental results showed that none of the carboxylic acids were able to form amide linkages with lysozyme under the experimental conditions and only maleic acid was able to form carboxylate salts, however, oxalic acid was the only acid able to interact with the Schiff base and form 1,3-oxazolidine-4,5-dione intermediate and thus hinder its rearrangement into Amadori product and consequently inhibit glycation. As a result the percentage of free or unreacted lysozyme was the highest in oxalic acid model systems (9.4% in the case of glucose, 7.1% in the case of ribose system) and was even higher than the control systems (6.0% in the case of glucose, 1.2% in the case of ribose system) of both glucose and ribose. In addition, all carboxylic acids were able to modify the relative distribution of different glycoforms generated during the incubation period however oxalic acid was the most efficient in shifting the distribution of glycoforms to lower molecular weight clusters which can additionally contribute to its anti-glycation activity.

The receptor for advanced glycation endproducts (RAGE) is a 35-kDa polypeptide of the immunogloblin superfamily that has been implicated as a mediator of both acute and chronic vascular inflammation. RAGE has also recently been implicated in the pathology of pulmonary hypertension (PH): a rare, progressive disease of the small pulmonary arteries characterised by pulmonary vascular remodelling, thrombosis, vasoconstriction and increased pulmonary vascular resistance. A ligand for RAGE, the calcium binding protein MTS1/S100A4, is expressed in occlusive vascular lesions of patients with advanced PH. MTS1/S100A4 is upregulated and secreted by pulmonary arterial smooth muscle cells (PASMCs) in vitro on activation of the 5HT1b receptor and 5HT transporter (5HTT). Additionally, the proliferative effect of 5HT on these cells, which is mediated by 5HT1b and 5HTT, may be inhibited by antagonism of RAGE or reduced bioavailability of MTS1/S100A4. These data suggest that MTS1/S100A4, through its action at RAGE, is a key mediator of 5HT-induced hPASMC proliferation. Transgenic mice overexpressing MTS1/S100A4 are observed to develop obliterative pulmonary vascular disease and possess increased right ventricular pressure at baseline and after hypoxia when compared to wildtype mice (WT). These increases occur in the absence of an increase in pulmonary vascular remodelling suggesting that MTS1/S100A4 overexpression is associated with some other structural or functional change in the pulmonary circulation. We sought to further our understanding of the role of RAGE in pulmonary hypertension through treatment with a small molecule inhibitor of monocyte chemoattractant protein 1(MCP-1), a marker of downstream of RAGE rage activation; through further characterisation of the MTS1/S100A4 mouse in a chronic hypoxic model of PAH; and through treatment with soluble RAGE (sRAGE) to reduce RAGE ligand bioavailability in vivo. In each case systolic right ventricular pressure (sRVP), right ventricular hypertrophy (RVH) and pulmonary vascular remodelling were measured in normoxic conditions or after a two week chronic hypoxia challenge to induce PH. These in vivo experiments were supplemented with functional studies in isolated intrapulmonary arteries to assess vascular reactivity and vascular elastance as well as studies of pulmonary fibroblast proliferation in vitro. Treatment with the MCP-1 synthesis inhibitor Bindarit produced no detectable effects upon the pulmonary response of mice to chronic hypoxia, though this study may have been hampered by difficulties with the methylcellulose vehicle. MCP-1 produced no degree of proliferation in pulmonary fibroblasts and neither augmented nor inhibited proliferation induced by 5HT. We found little evidence for the exacerbation of PH in MTS1/S100A4 mice in normoxia, hypoxia or after 4 weeks of normoxic recovery. Mean RVP was elevated above that in WT mice exposed to hypoxia. However, MTS1/S100A4 mice appeared protected against hypoxia-induced vascular remodelling and decreases in vascular elastance. No other significant differences in sRVP, RVH or remodelling were observed between strains. Vessels isolated from MTS1/S100A4 mice tended towards an enhanced contractile response to 5HT in normoxia compared with vessels in WT mice but were also more sensitive to the nitric oxide donor SNP. These differences in vasoreactivity were largely abolished by exposure to hypoxia. Treatment with soluble RAGE (sRAGE) to reduce RAGE ligand bioavailability produced a significant reduction in sRVP after hypoxia in comparison to vehicle-dosed mice -possibly associated with the prevention of a hypoxia-induced decrease in proximal vascular elastance. However, no benefit upon the development of remodelling or the extent of RVH was observed. Vessels isolated from mice treated with sRAGE and challenged with hypoxia showed a marked increase in contractility. Further work demonstrated that sRAGE produces a small, slowly developing contraction in isolated vessels and that the maximal force of contraction to 5HT was markedly augmented in the presence of sRAGE. Finally, treatment with sRAGE did not inhibit fibroblast proliferation in vitro as induced by 5HT but was observed to cause a small degree of proliferation alone and to augment hypoxia-induced proliferation. In summary, we have reported a number of seemingly contradictory findings associated with RAGE in pulmonary hypertension. Treatment with sRAGE produced a beneficial reduction in hypoxia-induced PH associated with protection against decreased proximal vascular elastance but produced no change in hypoxia-induced RVH or remodelling as well as greatly increasing vascular contractility. MTS1/S100A4 mice show some evidence of deleterious changes to the pulmonary circulation, but these may be offset by beneficial compensatory mechanisms such as increased sensitivity to nitric oxide and protection against vascular remodelling. MTS1/S100A4 stimulates smooth muscle cell proliferation suggesting that it may involved pulmonary vascular remodelling. However, inhibition of RAGE was observed to enhance fibroblast proliferation in response to hypoxia here. Fibroblasts are important regulators of SMC proliferation in vivo. These findings therefore suggest a more complicated relationship between RAGE, its ligands and the remodelling process. Since both MTS1/S100A4 overexpression and sRAGE treatment in vivo produced findings which are difficult to reconcile using the currently employed techniques, it is clear that furthering our understanding of RAGE will require study with greater focus upon the interaction of different cell types in the pulmonary vasculature and the manner in which the disturbance of this may lead to alterations in the physical and physiological properties of the pulmonary circulation.

Le diabète est associé à divers types de complications au niveau vasculaire affectant la micro et la macro-vasculature, ce qui contribue à l'augmentation de l'incidence d'infarctus du myocarde, d'ACV, de néphropathie et de rétinopathie. Parmi les mécanismes expliquant l'apparition de ces complications, la glycation des protéines joue un rôle important. En effet, il est connu que la glycation des protéines de la matrice extracellulaire (élastine, collagène) affecte les propriétés mécaniques des tissus constitués de celles-ci. Nous pensons que la glycation des protéines du cytosquelette peut également affecter les propriétés mécaniques de cellules présentes au niveau de la vasculature, telles que la cellule du muscle lisse vasculaire ou la cellule endothéliale, et ainsi affecter les fonctions cellulaires dépendantes d'une réorganisation du cytosquelette, telles que la contraction cellulaire. Le glyoxal (GO) est un composé hautement réactif de la famille des oxoaldéhydes, considéré comme un puissant agent de glycation au niveau cellulaire, puisqu'il réagit rapidement avec les groupements amines des protéines de façon à former des produits de glycation avancés (PGA). L'étude présentée dans cette thèse démontre d'une part que l'exposition à cet agent de glycation entraîne une augmentation de la rigidité cellulaire ainsi qu'une augmentation de la réponse contractile cellulaire générée par la machinerie actomyosine, en réponse à l'AngII. À la lumière de ces résultats, nous proposons qu'une exposition au GO peut induire des modifications post-traductionnelles de type non-enzymatique des protéines impliquées dans la machinerie contractile et ainsi altérer la fonction contractile cellulaire. C'est pourquoi nous avons en second lieu évalué la glycation de trois protéines impliquées dans la machinerie contractile (actine, ROCK, gelsoline) par un essai basé sur la réaction d'une sonde fluorescente et perméable à la membrane cellulaire, soit le carboxyfluorescéine diacetate succinimidyl ester (CFDA-SE), avec les amines primaires des protéines. Par cet essai, nous avons observé une augmentation de la glycation de l'actine et de ROCK, de même qu'une augmentation de l'interaction entre l'actine et la GSN. Cette thèse montre également l'implication de l'activité kinase de ROCK dans l'amplification de la réponse contractile, suggérant que la glycation de ROCK pourrait moduler son activité. En conclusion, la modification des protéines cellulaires par le GO pourrait affecter leurs fonctions et propriétés mécaniques, notamment par la modulation d'importantes interactions protéine-protéine impliquées dans la contraction cellulaire et dans l'organisation du cytosquelette.

Des études récentes ont montré que les produits de glycation avancée (AGE) pouvaient jouer un rôle dans la pathogénèse des troubles vasculaires en insuffisance rénale. Nous avons alors émis l’hypothèse que l’augmentation de la concentration des AGE observé dans le cas d’insuffisance rénale joue un rôle important dans la pathogenèse de l’hypertension artérielle, entre autres, en aggravant la dysfonction endothéliale et les dommages vasculaires. De plus, nous croyions que les effets des AGE sur la dysfonction endothéliale sont causés par l’activation des récepteurs RAGE sur les cellules vasculaires. Pour vérifier ces hypothèses nous avons étudié dans un premier temps l’effet du S100b, un agoniste des récepteurs des AGE (RAGE), sur la réponse vasodilatatrice dépendante de l’endothélium au carbachol et la réponse vasoconstrictrice à la phényléphrine et l’ET-1 sur des segments d’aorte de rats in vitro montés dans des bains à organe, et, dans un deuxième temps, l’effet du S100b sur la fonction endothéliale, notamment l’expression de la NO synthase endothéliale (eNOS) et la production des radicaux libres de l’oxygène (ROS). Les résultats obtenus indiquent que le S100b induit une diminution de la réponse vasorelaxante dépendante du NO au carbachol. Par contre, le S100b entraine une augmentation de la réponse vasoconstrictrice à la phényléphrine. Ce dernier effet n’est pas modifié dans les vaisseaux dénudés de l’endothélium, ni l’inhibition de la production de NO dans les vaisseaux intacts. L’effet du S100b est néanmoins atténué en présence d’indométacine, un inhibiteur de la cyclo-oxygénase, suggérant que l’activation des RAGE module la production de certaines eicosanoides. Enfin, le S100b induit une diminution de la réponse vasoconstrictrice à l’ET-1 qui est bloquée en présence de L-NAME, un inhibiteur de la production de NO. Ces derniers résultats indiquent que l’effet du S100b sur la réponse vasoconstrictrice à l’ET-1 dépend de la relâche de NO. Par ailleurs les résultats obtenus sur des segments d’aorte de rats et des cellules endothéliales en culture indiquent que le S100b n’affecte pas la production de ROS dans les segments d’aorte, bien qu’il stimule leur production dans les cellules endothéliales isolées. Par contre, le S100b tend à augmenter l’expression de la eNOS dans l’endothélium des vaisseaux en culture. En conclusion, la stimulation des RAGE avec le S100b affecte la réponse aux agents vasoconstricteurs et vasodilatateurs sur des segments d’aorte de rats normaux de façon dépendante et, possiblement, indépendante de l’endothélium. Ces effets seraient causés, en partie, par la modulation de la relâche de NO et de l’expression de la eNOS, ainsi que par la production d’eicosanoides et de ROS et, possiblement, l’activation du système endothélinergique. Ainsi, les AGE peuvent affecter le tonus vasculaire en modifiant la fonction endothéliale.
Des études récentes ont montré que les produits de glycation avancée (AGE) pouvaient jouer un rôle dans la pathogénèse des troubles vasculaires en insuffisance rénale. Nous avons alors émis l’hypothèse que l’augmentation de la concentration des AGE observé dans le cas d’insuffisance rénale joue un rôle important dans la pathogenèse de l’hypertension artérielle, entre autres, en aggravant la dysfonction endothéliale et les dommages vasculaires. De plus, nous croyions que les effets des AGE sur la dysfonction endothéliale sont causés par l’activation des récepteurs RAGE sur les cellules vasculaires. Pour vérifier ces hypothèses nous avons étudié dans un premier temps l’effet du S100b, un agoniste des récepteurs des AGE (RAGE), sur la réponse vasodilatatrice dépendante de l’endothélium au carbachol et la réponse vasoconstrictrice à la phényléphrine et l’ET-1 sur des segments d’aorte de rats in vitro montés dans des bains à organe, et, dans un deuxième temps, l’effet du S100b sur la fonction endothéliale, notamment l’expression de la NO synthase endothéliale (eNOS) et la production des radicaux libres de l’oxygène (ROS). Les résultats obtenus indiquent que le S100b induit une diminution de la réponse vasorelaxante dépendante du NO au carbachol. Par contre, le S100b entraine une augmentation de la réponse vasoconstrictrice à la phényléphrine. Ce dernier effet n’est pas modifié dans les vaisseaux dénudés de l’endothélium, ni l’inhibition de la production de NO dans les vaisseaux intacts. L’effet du S100b est néanmoins atténué en présence d’indométacine, un inhibiteur de la cyclo-oxygénase, suggérant que l’activation des RAGE module la production de certaines eicosanoides. Enfin, le S100b induit une diminution de la réponse vasoconstrictrice à l’ET-1 qui est bloquée en présence de L-NAME, un inhibiteur de la production de NO. Ces derniers résultats indiquent que l’effet du S100b sur la réponse vasoconstrictrice à l’ET-1 dépend de la relâche de NO. Par ailleurs les résultats obtenus sur des segments d’aorte de rats et des cellules endothéliales en culture indiquent que le S100b n’affecte pas la production de ROS dans les segments d’aorte, bien qu’il stimule leur production dans les cellules endothéliales isolées. Par contre, le S100b tend à augmenter l’expression de la eNOS dans l’endothélium des vaisseaux en culture. En conclusion, la stimulation des RAGE avec le S100b affecte la réponse aux agents vasoconstricteurs et vasodilatateurs sur des segments d’aorte de rats normaux de façon dépendante et, possiblement, indépendante de l’endothélium. Ces effets seraient causés, en partie, par la modulation de la relâche de NO et de l’expression de la eNOS, ainsi que par la production d’eicosanoides et de ROS et, possiblement, l’activation du système endothélinergique. Ainsi, les AGE peuvent affecter le tonus vasculaire en modifiant la fonction endothéliale.
Recent studies suggest that advanced glycation end products (AGEs) could play an important role in vascular pathogenesis in kidney disease. We hypothesized that increased AGEs concentrations in kidney disease play an important role in the pathogenesis of arterial hypertension, by increasing endothelial dysfunction and vascular damages. Furthermore, we believe that the effects of AGEs on endothelial dysfunction are caused by the activation of AGEs receptors (RAGE) on vascular cells. To test this hypothesis, we studied first the effect of S100b, an AGEs receptor agonist, on the endothelium dependent vasodilatation response to carbachol and the vasoconstriction response to phenylephrine and ET-1 on rats thoracic aorta segments in vitro placed in organ bath, and, secondly, the effect of S100b on the endothelial function, especially the expression of endothelial NO synthase (eNOS) and the production of reactive oxygen species (ROS). Our results indicate that S100b induces a decrease in the NO dependent vasodilatation response to carbachol, but an increase in the vasoconstriction response to phenylephrine. This effect of S100b on phenylephrine response is not modified in vessels without endothelium, nor following the inhibition of the NO production in intact vessels. The effect of S100b is however decreased with indometacine, a cyclooxygenase inhibitor, suggesting that RAGE activation modulates the production of eicosanoids. Finally, S100b induces a decrease in the vasoconstriction response to ET-1 that is blocked in presence of L-NAME, a NO production inhibitor. These results indicate that the effect of S100b on the vasoconstriction response to ET-1 is dependent on NO release. In addition, results obtained on cultured rat aorta segments and endothelial cells indicate that S100b does not affect the production of ROS althouch it stimulates their production in isolated endothelial cells. Otherwise, S100b tend to increase the expression of eNOS in the endothelium of cultured vessels. In conclusion, the stimulation of RAGE by S100b affects the response to vasoconstrictors and vasodilators agents on intact rat thoracic aorta segments in an endothelium dependant and, possibly, independent manner. These effects may be related, in part, to the modulation of NO release and eNOS expression, the production of eicosanoids and ROS, and, possibly, the activation of the endothelinergic system. Thus, AGEs could affect vascular tone through the modulation of the endothelial function.
Recent studies suggest that advanced glycation end products (AGEs) could play an important role in vascular pathogenesis in kidney disease. We hypothesized that increased AGEs concentrations in kidney disease play an important role in the pathogenesis of arterial hypertension, by increasing endothelial dysfunction and vascular damages. Furthermore, we believe that the effects of AGEs on endothelial dysfunction are caused by the activation of AGEs receptors (RAGE) on vascular cells. To test this hypothesis, we studied first the effect of S100b, an AGEs receptor agonist, on the endothelium dependent vasodilatation response to carbachol and the vasoconstriction response to phenylephrine and ET-1 on rats thoracic aorta segments in vitro placed in organ bath, and, secondly, the effect of S100b on the endothelial function, especially the expression of endothelial NO synthase (eNOS) and the production of reactive oxygen species (ROS). Our results indicate that S100b induces a decrease in the NO dependent vasodilatation response to carbachol, but an increase in the vasoconstriction response to phenylephrine. This effect of S100b on phenylephrine response is not modified in vessels without endothelium, nor following the inhibition of the NO production in intact vessels. The effect of S100b is however decreased with indometacine, a cyclooxygenase inhibitor, suggesting that RAGE activation modulates the production of eicosanoids. Finally, S100b induces a decrease in the vasoconstriction response to ET-1 that is blocked in presence of L-NAME, a NO production inhibitor. These results indicate that the effect of S100b on the vasoconstriction response to ET-1 is dependent on NO release. In addition, results obtained on cultured rat aorta segments and endothelial cells indicate that S100b does not affect the production of ROS althouch it stimulates their production in isolated endothelial cells. Otherwise, S100b tend to increase the expression of eNOS in the endothelium of cultured vessels. In conclusion, the stimulation of RAGE by S100b affects the response to vasoconstrictors and vasodilators agents on intact rat thoracic aorta segments in an endothelium dependant and, possibly, independent manner. These effects may be related, in part, to the modulation of NO release and eNOS expression, the production of eicosanoids and ROS, and, possibly, the activation of the endothelinergic system. Thus, AGEs could affect vascular tone through the modulation of the endothelial function.

Le vieillissement de la peau est la résultante du chrono et du photo-vieillissement qui partagent des changements moléculaires incluant les modifications des protéines telles que la glycation. La glycation se produit lorsque le glucose ou ses dérivés dicarbonylés, le glyoxal et de méthylglyoxal, réagissent avec les protéines conduisant à la formation de produits de glycation avancée (AGE). Le but de ces travaux est de mieux comprendre le rôle des glyoxalases dans la détoxification des dicarbonyles et dans la protection des protéines au cours du vieillissement de la peau. Afin de comprendre la régulation du systeme dans la peau humaine pendant le chono et le photo-vieillissement, des coupes de peau de donneurs jeunes et âgées ont été analysées par immunohistochimie. Le système des glyoxalase, glyoxalase 1 (GLO1) et glyoxalase 2 (Glo2), et AGE ont été localisés et détectés dans les échantillons de peau. Pour comprendre le rôle des GLO en réponse au stress dicarbonyle, des kéartinocytes HaCaT ont été soumise à un stress dicarbonyle de 24h et l'expression et les activités des glyoxalases ont été déterminées. Enfin, des études protéomiques ont été réalisées sur des cellules HaCaT surexprimant Glo2 ou inhibant GLO1 et nous sommes en train d'identifier des protéines cibles des carboxymethyllysines. Nos résultats montrent que le système des glyoxalases est présent dans la peau en particulier dans les cellules basales et est capable de protéger les protéines cellulaires contre les dommages oxydatifs. Nous espérons que notre étude peut contribuer à comprendre le rôle des glyoxalases dans la maintenance protéique qui est un élément clé de l'homéostasie cellulaire au cours du vieillissement
Skin aging is the result of intrinsic and photo-aging, due to UV exposure, that both share important molecular features including alterations of proteins such as glycation. Glycation occurs when glucose or its derivates the dicarbonyl compounds glyoxal and methylglyoxal react with proteins leading to the formation of advanced glycation endproducts (AGE). The aim of this work is to better understand the role of glyoxalases in the detoxification of dicarbonyls and in the protection of keratinocyte’s proteins during skin aging.In order to understand glyoxalases regulation in human skin during intrinsic and photo-aging, skin sections from young and old donors were analysed by immunohistochemistry. The glyoxalase system, glyoxalase 1 (Glo1) and glyoxalase 2 (Glo2), and AGE were localized and detected in the skin samples. To understand the role of glyoxalases in response to dicarbonyl stress, HaCaT keratinocytes were subjected to a 24h dicarbonyl stress and the expression and activities of glyoxalases were determined. HaCaT cells silenced for Glo1 or overexpressing Glo2 were developed and were used in proteomic studies to identify target proteins preferentially glycated by dicarbonyl compounds.Finally, proteomic studies were performed on cellular clones overexpressing Glo2 or inhibiting Glo1 and we are currently identifying proteins targets for carboxymethyllysine modifications.Our results show that glyoxalase system is present in skin especially in the basal cells and is able to protect cellular proteins against oxidative damages. We expect that our study may contribute to decipher the role of glyoxalases in protein maintenance, which is a key element of cellular homeostasis during aging

Advanced Glycation Endproducts (AGEs), resulting from the non-enzymatic reaction of reducing sugars with proteins, accumulate in patients with diabetes mellitus and with advancing age and are implicated in the pathogenesis of vascular disease. Oxidative stress also participates in vascular pathology and has also been reported in the context of diabetes and ageing. This study set out to explore the contribution of AGEs to oxidant stress generation, particularly by examining their effects on the respiratory burst of neutrophils and lymphoblasts. Using chemiluminescence to detect reactive oxygen species (ROS), AGEs did not stimulate the neutrophil respiratory burst directly, but caused a dose-dependent enhancement of the neutrophil respiratory burst in response to a mechanical stimulus (up to 265% +/- 42%, p=0.022) or chemical stimulation with fMLP (formylleucylphenylalanine) 100nM (up to 218% +/- 19%, p<0.001). This phenomenon was immediate and reversible, and depended on the simultaneous presence of AGEs with the additional stimulus; hence AGEs appear to act as neutrophil 'co-agonists'. The in vivo correlates of mechanical and chemical stimulation may be vascular stress and microbial exposure respectively, especially since some acute vascular events have been correlated with infective episodes. The 'co-agonist' effect of AGEs on the neutrophil respiratory burst appears to involve upregulation of the NADPH oxidase enzyme, as evidenced by a DPI-dependent suppression of basal and augmented ROS output. This in turn is dependent upon the generation of arachidonic acid (which may potentiate NADPH oxidase subunit function), via cytosolic phospholipase A2 (cPLA2) activation. The whole process is sensitive to adjustments of the intracellular redox status, implying a role for upstream redox signalling.

Although the risk of malformations in the offspring of diabetic mothers remains the leading cause of perinatal mortality, the pathogenesis has not been elucidated. The hypothesis of this study was that protein glycation and oxygen free radicals play a role in hyperglycemia-induced malformations. CD-1 mouse embryos (0-2 somites) were cultured under hyperglycemic conditions for 48 hours with the exogenous addition of anti-glycating agents and oxygen free radical scavengers. The exogenous addition of aspirin (ASA) and D-lysine reduced significantly the malformations and embryonic glycated protein levels. Salicylate, arachidonic acid (AA), and to a lesser extent, indomethacin also exerted protective effects, but with no effect on glycated protein levels. We hypothesize that ASA, salicylate and indomethacin are protective by exerting free radical scavenging action; and ASA and D-lysine are acting as potent anti-glycating agents. Moreover, we suggest that AA may have inhibited hyperglycemia-induced malformations through the protective action of prostaglandins against free radical damage. Serum media lipid peroxidation (LPO) was reduced in the ASA and indomethacin groups possibly due to either a direct free radical scavenging action and/or the inhibitory effects of these agents on cyclooxygenase activity thereby decreasing the oxygen free radicals produced by this enzyme system. On the other hand, AA was associated with an increased level of LPO in the serum media. As the evidence has shown, the cause of hyperglycemia-induced malformations appears to be multifactorial and no one agent can completely eliminate the problem, although protective action can be exerted at different levels of the glycation-free radical cascade of tissue damage.

Background and Methods: The involvement of iron in the risk for, and complications of, type 2 diabetes has generated substantial interest over the past 15 years, initially sparked by an association with raised serum ferritin, and the observation that people with iron overload diseases frequently develop diabetes. Considerable advances have since been made in understanding the effect glucose has on molecules, cells, and tissues; and the role that oxidative stress plays in the development of the pathologies of long-term diabetes. Poorly liganded iron is potentially both a contributor to, and consequence of, these complications. In vitro experiments with glucose-incubated transferrin by earlier workers have demonstrated loss of function with increasing glycation, leading to the suggestion that the failure of this key iron-binding protein may contribute to diabetic pathology, via the presence of redox active non-transferrin-bound iron (NTBI). In vitro glycated transferrin is examined here by ultrafiltration, to assess loss of function and possible oxidative fragmentation. Mass spectrometry is used to identify a range of amino acid glycation sites on in vitro glycated transferrin for the first time. Finally, several groups have previously measured NTBI in people with diabetes, finding little agreement in results. NTBI is measured here in a cohort of people with type 2 diabetes, using a new adaptation of earlier NTBI assays. NTBI is also assessed in pre-dialysis chronic kidney disease (CKD) stages I to III for the first time. Results and Conclusions: Experiments with glycated transferrin in vitro demonstrate oxidative fragmentation, explaining the loss of function reported by earlier groups. In vitro glycated transferrin examined by mass spectrometry reveals a substantial number and range of amino acids subject to glycation. Comparison with in vivo glycated transferrin suggests that many of the in vitro glycation sites are not glycated in vivo, and that there are many oxidized methionine residues which are potential artefacts, or likely to be repaired by methionine sulphoxide reductases in vivo. A study of people with type 2 diabetes finds no direct association between NTBI and protein glycation. Unexpected correlations between NTBI and LDL, and LDL and haemoglobin with increasing protein glycation, are reported for the first time. NTBI is suggested to be iron sourced from haemoglobin or haem, from erythrocyte haemolysis prior to sample collection. In people with pre-dialysis CKD stages I to III no significant difference in NTBI level compared to controls is seen, or correlations with markers of renal function. No link between NTBI and kidney function at this stage of disease is indicated.

Impaired wound healing is associated with hyperglycaemia in patients suffering from diabetes mellitus. Dicarbonyl compounds such as methylglyoxal (MG) are formed at an increased rate under hyperglycaemic conditions in diabetes. They take part in glycation of proteins and serve as precursors of advanced glycation end products (AGEs). The mechanism behind glycation and possible dysfunction of growth factors involved in wound healing is investigated in this study. Platelet derived growth factor BB (pDGF-BB) is a potent mitogenic and chemotactic factor. It plays essential roles in the recruitment and proliferation of cells involved in wound healing and tissue repair. PDGF-BB may represent a key target for sugar induced molecular modifications during hyperglycaemic conditions of diabetes. PDGF-BB was glycated using glucose-6-phosphate and MG. Matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) and sodium dodecyl polyacrylamide gel ecletrophoresis analysis were employed to confirm PDGF-BB glycation. A time and dose dependent glycation of PDGF-BB was achieved for the first time in this study. Peptide mass mapping was used following enzymatic hydrolysis and subsequent MALDI-TOF-MS analysis to localise methylglyoxal-hydroimidazolone adduct formation on arginine residues in PDGF-BB incubated with MG. The proliferation and migration of human dermal fibroblasts were significantly reduced in glycated PDGF-BB treated cells compared to native PDGF-BB treated cells. Similarly reduced wound recovery was observed in the presence of glycated PDGF-BB compared to native PDGF-BB in artificially wounded cell monolayer. These results may help to understand pathological mechanisms underlying impaired wound healing in diabetic patients. Glycation inhibitors could prevent the formation of AGEs and dysfunction ofPDGF-BB. Two new inhibitors i.e. aged garlic extract and S-allyl cysteine (SAC) were identified to inhibit formation of glucose and MG derived AGEs. Both inhibitors showed promising multi-functional and multi-stage inhibitory properties when compared to pyridoxamine and aminoguanidine, two well established glycation inhibitors. Antiglycation properties of aged garlic extract and SAC combined with their already well established antioxidant, metal chelating and carbonyl scavengmg properties may limit glycation induced complications. VI

Methylglyoxal is a physiological dicarbonyl metabolite and potent argininedirected glycating agent. It often modifies proteins at functional sites producing loss of positive charge, structural distortion and inactivation. Plasma methylglyoxal is increased in hyperglycaemia associated with diabetes and is linked to the development of vascular complications of diabetes – particularly nephropathy, retinopathy and neuropathy. The effects of dicarbonyl glycation on beta cells and involvement in early stage dysfunction and development of type 2 diabetes mellitus are not known. The aim of this project was to investigate the effect of dicarbonyl protein glycation on beta cell function and related involvement in the development of diabetes. Studies were performed in an in vitro model of beta cell dysfunction - MIN6 insulinoma cells incubated under low and high glucose concentrations, and in a pre-clinical in vivo model of decline of glucose tolerance preceding development of type 2 diabetes - high fat diet-induced insulin resistant mice. Dicarbonyl metabolism and protein damage by glycation and oxidation were studied by stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry. Localisation of methylglyoxal glycation adducts within the pancreas were visualised by immunostaining. Interactions between the extracellular matrix protein, collagen IV, and MIN6 cells in vitro were investigated and impairments in adhesion were assessed following glycation with methylglyoxal. Impairments in adhesion of MIN6 cells to methylglyoxal-glycated collagen IV were assessed using atomic force microscopy force spectroscopy. The results show that MIN6 cells were resistant to accumulation of methylglyoxal when incubated in high glucose concentration although the flux of methylglyoxal was increased 41%. Glycation of collagen IV by methylglyoxal impairs binding to MIN6 cells in vitro resulting in a 91% decrease in the energy necessary to detach cells from the extracellular matrix protein. In high fat diet fed mice the concentration of methylglyoxal in the pancreas was increased. Visualisation of MG-H1 adduct residues in the pancreas showed they were predominantly on the extracellular matrix. In conclusion, protein glycation by methylglyoxal occurs in MIN6 cells in vitro and in the mouse pancreas in vivo. Although the methylglyoxal concentration in the pancreas of high fat diet fed, insulin resistant mice was increased, the lack of a concurrent increase in methylglyoxal protein glycation adducts suggests there may be increased turnover of methylglyoxal-modified proteins. Impairment of beta cell attachment to the extracellular matrix protein, collagen IV, by methylglyoxal and increased protein turnover stimulated by an increased rate of methylglyoxal glycation may impair beta cell function in pre-diabetes in vivo. Glycation by methylglyoxal may contribute to beta cell glucotoxicity and dysfunction with progression to type 2 diabetes mellitus.