Academic literature on the topic 'Advanced glycation endproducts (AGE)'

Abstract The balance between protein synthesis and degradation regulates the amount of expressed proteins. This protein turnover is usually quantified as the protein half-life time. Several studies suggest that protein degradation decreases with age and leads to increased deposits of damaged and non-functional proteins. Glycation is an age-dependent, non-enzymatic process leading to posttranslational modifications, so-called advanced glycation endproducts (AGE), which usually damage proteins and lead to protein aggregation. AGE are formed by the Maillard reaction, where carbonyls of carbohydrates or metabolites react with amino groups of proteins. In this study, we quantified the half-life time of two important receptors of the immunoglobulin superfamily, the neural cell adhesion molecule (NCAM) and the receptor for advanced glycation end products (RAGE) before and after glycation. We found, that in two rat PC12 cell lines glycation leads to increased turnover, meaning that glycated, AGE-modified proteins are degraded faster than non-glycated proteins. NCAM is the most prominent carrier of a unique enzymatic posttranslational modification, the polysialylation. Using two PC12 cell lines (a non-polysialylated and a polysialylated one), we could additionally demonstrate, that polysialylation of NCAM has an impact on its turnover and that it significantly increases its half-life time.

Abstract Advanced glycation endproducts (AGEs) are a diverse group of molecular adducts formed in environments high in reducing sugars that accumulate with aging and in diabetes. This study tests the hypothesis that AGEs inhibit the stabile osseointegration of dental implants through tissue interactions that interfere with bone turnover and compromise the biomechanical properties at the bone-implant interface. Maxillary first molars were extracted from 32 rats and allowed to heal for 4 weeks. Titanium implants (1 mm × 3 mm) were placed in the healed sockets of 2 groups of 16 rats consisting of 8 rats injected 3 times/wk for 1 month with AGE (prepared from glucose and lysine) and 8 rats injected with vehicle as a control. AGE injections continued for an additional 14 or 28 days before sacrifice. X-ray images, blood, and tissues were collected to examine bone/implant contact ratio, serum pyridinoline ([PYD] a collagen breakdown marker), osteocalcin ([OSC] a bone formation marker), and for immunohistochemistry with antibodies to AGE and the bone turnover-marker protein matrix metalloproteinase1. Compared with the AGE-treated groups, the controls showed significantly higher bone/implant contact at both 14- and 28-day time points. PYD (P < .05) and OSC (trend) levels from controls showed decreases at 28 days when compared with AGE-treated groups. Immunohistochemistry with AGE-specific and bone turnover marker antibodies showed stronger staining associated with the implant/tissue interface in AGE-treated rats. Our studies indicate an association between AGE and inhibition of bone turnover, suggesting that the formation of AGE in high glycemic conditions, such as diabetes, may contribute to a slower rate of osseointegration that negatively affects implant stability.

There is increasing evidence for the role of intestinal permeability as a contributing factor in the pathogenesis of diabetes; however, the molecular mechanisms are poorly understood. Advanced glycation endproducts, of both exogenous and endogenous origin, have been shown to play a role in diabetes pathophysiology, in part by their ligation to the receptor for advanced glycation endproducts (RAGE), leading to a proinflammatory signalling cascade. RAGE signalling has been demonstrated to play a role in the development of intestinal inflammation and permeability in Crohn’s disease and ulcerative colitis. In this review, we explore the role of AGE-RAGE signalling and intestinal permeability and explore whether activation of RAGE on the intestinal epithelium may be a downstream event contributing to the pathogenesis of diabetes complications.

Glucocorticoids are very effective anti-inflammatory drugs and widely used for inflammatory bowel disease (IBD) patients. However, approximately 20% of IBD patients do not respond to glucocorticoids and the reason for this is largely unknown. Dietary advanced glycation endproducts (AGEs) are formed via the Maillard reaction during the thermal processing of food products and can induce a pro-inflammatory reaction in human cells. To investigate whether this pro-inflammatory response could be mitigated by glucocorticoids, human macrophage-like cells were exposed to both LPS and AGEs to induce interleukin-8 (IL8) secretion. This pro-inflammatory response was then modulated by adding pharmacological compounds interfering in different steps of the anti-inflammatory mechanism of glucocorticoids: rapamycin, quercetin, and theophylline. Additionally, intracellular reactive oxygen species (ROS) were measured and the glucocorticoid receptor phosphorylation state was assessed. The results show that AGEs induced glucocorticoid resistance, which could be mitigated by quercetin and rapamycin. No change in the phosphorylation state of the glucocorticoid receptor was observed. Additionally, intracellular ROS formation was induced by AGEs, which was mitigated by quercetin. This suggests that AGE-induced ROS is an underlying mechanism to AGE-induced glucocorticoid resistance. This study shows for the first time the phenomenon of dietary AGE-induced glucocorticoid resistance due to the formation of ROS. Our findings indicate that food products with a high inflammatory potential can induce glucocorticoid resistance; these results may be of great importance to IBD patients suffering from glucocorticoid resistance.

Nonenzymatic glycosylation or glycation of macromolecules, especially proteins leading to their oxidation, play an important role in diseases. Glycation of proteins primarily results in the formation of an early stage and stable Amadori-lysine product which undergo further irreversible chemical reactions to form advanced glycation endproducts (AGEs). This review focuses these products in lysine rich proteins such as collagen and human serum albumin for their role in aging and age-related diseases. Antigenic characteristics of glycated lysine residues in proteins together with the presence of serum autoantibodies to the glycated lysine products and lysine-rich proteins in diabetes and arthritis patients indicates that these modified lysine residues may be a novel biomarker for protein glycation in aging and age-related diseases.

Recent investigations have indicated that reactive metabolites and AGE-RAGE-mediated inflammation might play an important role in the pathogenesis of ischemia-reperfusion injury in liver transplantation. In this observational clinical study, 150 patients were enrolled following liver transplantation from deceased donors. The occurrence of short-term complications within 10 days of transplantation was documented. Blood samples were collected prior to transplantation, immediately after transplantation, and at consecutive time points, for a total of seven days after transplantation. Plasma levels of methylglyoxal were determined using HPLC, whereas plasma levels of L-arginine, asymmetric dimethylarginine, advanced glycation endproducts-carboxylmethyllysine, soluble receptor for advanced glycation endproducts, and total antioxidant capacity were measured by ELISA. Patients following liver transplantation were shown to suffer from increased RAGE-associated inflammation with an AGE load mainly dependent upon reactive carbonyl species-derived AGEs. In contrast, carboxylmethyllysine-derived AGEs were of a minor importance. As assessed by the ratio of L-arginine/asymmetric dimethylarginine, the bioavailability of nitric oxide was shown to be reduced in hepatic IRI, especially in those patients suffering from perfusion disorders following liver transplantation. For the early identification of patients at high risk of perfusion disorders, the implementation of asymmetric dimethylarginine measurements in routine diagnostics following liver transplantation from deceased donors should be taken into consideration.

Preeclampsia/hypertensive disorders of pregnancy (PE/HDP) is a serious and potentially life-threatening disease. Recently, PE/HDP has been considered to cause adipose tissue inflammation, but the detailed mechanism remains unknown. We exposed human primary cultured adipocytes with serum from PE/HDP and healthy controls for 24 h, and analyzed mRNA expression of several adipokines, cytokines, and ligands of the receptor for advanced glycation endproducts (RAGE). We found that the mRNA levels of interleukin-6 (IL-6), C-C motif chemokine ligand 2 (CCL2), high mobility group box 1 (HMGB1), and RAGE were significantly increased by the addition of PE/HDP serum. Among RAGE ligands, advanced glycation endproducts (AGE) and HMGB1 increased mRNA levels of IL-6 and CCL2 in SW872 human adipocytes and mouse 3T3-L1 cells. The introduction of small interfering RNA for RAGE (siRAGE) into SW872 cells abolished the AGE- and HMGB1-induced up-regulation of IL-6 and CCL2. In addition, lipopolysaccharide (LPS), a ligand of RAGE, increased the expression of IL-6 and CCL2 and siRAGE attenuated the LPS-induced expression of IL-6 and CCL2. These results strongly suggest that the elevated AGE, HMGB1, and LPS in pregnant women up-regulate the expression of IL-6 and CCL2 via the RAGE system, leading to systemic inflammation such as PE/HDP.

Background:- Diabetes mellitus (OM) is a metabolic disorder commonly associated with advanced glycation endproducts (AGEs) and the specific receptor for AGE (RAGE). Cardiovascular disease is the main cause of mortality in patients with OM. Accordingly, accelerated atherosclerosis and AGEs are. common pathological features of OM. Chronic AGE exposure and AGE-activated intracellular signalling mechanisms were examined in coronary arteries as a causative link for the phenotypic modulation of VSMC associated with the severe atherosclerotic complications seen in diabetic patients. Methods and Results:- Cultured porcjfle coronary artery vascular smooth .muscle (CVSM) cells exposed to AGE for 96 hours were used as a relevant diabetic model. Calcium imaging studies and electrophysiology studies revealed chronic AGE treatment lead to significantly enhanced intracellular Ca2+ release and sustained activation of Ca2+-sensitive membrane ion channels, respectively, using agonists to evoke intracellular Ca2+ mobilization. Intracellular Ca2+ release from caffeinesensitive intracellular stores was enhanced in CVSM cells with AGE treatment, suggesting an upregulation of RyRs, or distinct RyR subtype expression. Brd U proliferation assay results confirmed proliferation was significantly increased in AGEtreated CVSM cells, which was abolished with cyclosporin A (CsA). Calcineurin activity, nuclear factor of activated T-cells (NFAT)-binding and RAGE expression were all significantly increased in CVSM cells after AGE exposure for 96 hC?urs. Conclusions:- This study concludes that the activation of the Ca2+-dependent calcineurin/NFAT pathway is responsible for the increased proliferative response in CVSM cells chronically exposed to AGE. An upregulation of the RAGE receptor after 96 hours AGE treatment mediates the phenotypic modulation of porcine coronary VSMC from a fully differentiated contractile phenotype to a dedifferentiated synthetic proliferative phenotype. The results of this study suggest that the uncontrollable proliferation of VSMCs associated with the accelerated atherosclerosis seen in diabetic coronary vasculature is partly due to the irreversible formation and deposition of AGEs.

Study of lysozyme in the context of diabetic nephropathy Introduction Lysozyme (LZ), enzyme known for its antimicrobial and immunomodulant activity, has recently shown to be effective in the context of diabetic nephropathy, one of the major complications associated to diabetes and one of the major cause of end stage kidney disease in Western countries. Experimental evidences strictly link the development and progression of the diabetic nephropathy to the enormous production and accumulation of Advanced Glycation Endproducts (AGEs). AGEs are a chemically heterogeneous group of macromolecules which formation is dramatically increased in diabetic patient mainly due to chronic hyperglicaemia. Nevertheless, several factor, such oxidative and carbonyl stress and reduced renal clereance seem to be related to the increase of the production and accumulation of AGEs. The first step of AGEs formation starts in presence of reducing sugars, as glucose, which are able to interact with free amino group (Maillard reaction), forming Schiff base. The rearrangements from the Schiff base leads to the formation of Amadori products, that are eventually oxidazed, leading to the formation of AGEs. AGEs binding to structural proteins and deposition in tissues alter functions, leading to stiffening, enhanced local cytokine production and is a marker of cumulative metabolic stress. At cellular level, AGEs-induced events, depend mainly by their interaction with specific receptor, among which RAGE, is the most well characterized. AGE-RAGE interactions determine a number of events, such as an increased production of reactive oxygen species (ROS), induction of pro-inflammatory mediators, upregulation of RAGE receptor. In the last decades, it has been shown that hen egg white LZ is able to act effectively as AGEs scavenger (Kd= 50 nM). In in vivo models it was demonstrated that orally administered microencapsulated LZ, can act prevent the insurgence of a number of relevant early manifestations of diabetic nephropathy, such as microalbuminuria and glomerular hypertrophy. Aim The aim of the present work is to develop an in vitro model for studying the molecular mechanisms responsible for the nephroprotective activity of LZ, through the identification and validation of an adequate cell line and a marker. In order contribute to understand the higher in vivo effectiveness of microencapsulated LZ compared to an equidose of “free” LZ, a sophisticated model of incannulated rat will be developed. Results and discussion An adequate cell model was selected among three different cell lines: a primary culture of endothelial cells (ADMEC), due to the implication of vascular tissue in diabetes and two proximal tubular cell lines, LLC-PK1 (porcine cells) and HK-2 (human cells) due to the tubular involvement in the context of diabetic nephropathy. HK-2 cell line was selected on the basis of its dose-dependent sensibility to the AGE treatments, quantified, in terms of viability, by means of MTT test. One of the most relevant AGE-induced effect is the induction of inflammatory response. A pivotal event in the context of inflammation is represented by macrophage recruitment. For this reason, it was performed a migration assay, using monocyte U937 differentiated in macrophage, toward a stimulus represented by supernatants obtained treating HK-2 cells with AGE e LZ. AGE treatment induced a significant increased (+50%) of the migrated macrophage. The co-treatment significantly prevented the migration induction. LZ treatment, as expected, did not modify migratory ratio. It is known that cytokine play a crucial role in macrophage recruitment. Interleukin-6 (IL-6), for what diabetic nephropathy is concerned, has a pivotal role. On the basis of this evidence, the IL-6 mRNA levels after AGE and LZ treatments were measured. According with expectations, AGE induced a significant IL-6 mRNA increase after 24 h of treatment. LZ exposition did not induced any modifications, whereas the co-treatment with AGE and LZ showed that LZ prevented IL-6 mRNA increase. In order to confirm the effectiveness of LZ toward AGE-induced IL-6 increase, an ELISA assay was performed. LZ was able to significantly and dose-dependently reduce the AGE-induced release of IL-6 in the supernatants. Several mechanisms have been reported to be capable to induce IL-6 release. The mechanisms that determine IL-6 release are several. On the basis of the recognized crucial role attributed to RAGE receptor, it was tested the capacity of LZ to influence RAGE mRNA levels, by means of RT-PCR technique. The results showed no variations of RAGE levels after exposition to AGE and LZ for 24 up to 96 h. The induction of ROS represents one of the events attributable to AGEs. Nevertheless, on the cellular model employed, this parameter did not show any significant increase. Considering that LZ activity is associated also to its AGE scavenging action, it was evaluated by means of confocal microscopy, if the presence of LZ could influence the AGE capacity to enter inside the cell. The images obtained by means of this technique showed that AGEs and LZ are both able to enter in the cells. In order to investigate the co-treatment effect a cytometry analysis was performed. The results suggest that LZ is not able, in this cellular model, to prevent the entrance on AGEs inside the cells, excluding that LZ scavenging action could be the cause of preventive action of in vitro IL-6 production. Subsequent studies were performed on pathways potentially involved in the action of LZ toward AGEs, such as MAPK cascade (p38) and lysosomial degradation. Preliminary data showed a possible involvement of p38. In fact, AGEs showed the capacity to increase phosphorylation level of this protein, effect reduced by the co-treatment with an equimolar dose of LZ. Promising are also the first results obtained from the lysosomial localization study of AGE, in presence of LZ. In fact, the qualitative analysis of the obtained figures confirms the hypothesis that LZ is able to bring AGEs in lysosomes, supporting their degradation. Taken together these data do not exclude stochastically that LZ action is mainly antidotic. However, the LZ capacity to act toward pivotal mediators of AGE-induced effects, such as IL-6 and p38, open new work perspectives for what LZ action is concerned, allowing to speculate its use as cooperating drug in the control of inflammation in the context of diabetic nephropathy. In order to investigate if the higher effectiveness of orally administered microencapsulated LZ was due to the capacity of microparticles to deliver more efficiently the LZ in the blood stream an in vivo test was performed. The results showed that microparticles can contribute significantly to deliver LZ more efficiently to serum
Studio del lisozima nel contesto della nefropatia diabetica Introduzione Il lisozima (LZ), enzima noto per la sua attività antibatterica ed immunomodulante, è stato recentemente identificato come principio attivo con potenzialità terapeutica nel contesto della nefropatia diabetica, una delle principali complicanze associate al diabete e principale causa di patologie renali terminali nell’Occidente sviluppato. Evidenze sperimentali correlano strettamente l’insorgenza e la progressione della nefropatia diabetica all’abnorme formazione ed accumulo di molecole note come prodotti finali della glicosilazione avanzata (Advanced Glycation Endproducts, AGE). Gli AGE sono un gruppo chimicamente eterogeneo di macromolecole la cui formazione risulta drammaticamente incrementata nel paziente diabetico a causa dell’iperglicemia cronicizzata. La formazione degli AGE ha inizio in presenza di zuccheri riducenti, come il glucosio, i quali nella loro forma aperta sono in grado di interagire con gruppo amminici liberi (reazione di Maillard) formando le basi di Schiff. Tali composti si riarrangiano in prodotti di Amadori, dai quali derivano direttamente gli AGE. Gli effetti patologici AGE-indotti derivano dal loro deposito tissutale e dal legame con proteine a lento turnover, con conseguente riduzione della funzionalità e/o dell’elasticità degli stessi. Gli eventi AGE-indotti, a livello cellulare, dipendono principalmente dalla loro interazione con i propri recettori specifici, tra i quali il meglio caratterizzato è RAGE. L’interazione AGE-RAGE è alla base di numerosi eventi tra i quali un ruolo centrale spetta all’aumento di specie reattive dell’ossigeno (ROS), all’induzione del rilascio di stimoli pro-infiammatori e all’upregolazione del recettore RAGE stesso. Recentemente, è stato dimostrato che il LZ da bianco d’uovo è in grado di fungere efficacemente da scavenger degli AGE (Kd= 50 nM). In modelli in vivo con diabete indotto, è stato dimostrato che il LZ microincapsulato, somministrato oralmente, è in grado di prevenire l’insorgenza di alcuni dei principali sintomi precoci della nefropatia diabetica, quali microalbuminuria ed ipertrofia glomerulare. Scopo L’obiettivo di questo lavoro di dottorato è quello di sviluppare un modello in vitro per lo studio dei meccanismi molecolari alla base dell’attività nefroprotettiva del LZ attraverso l’individuazione e la validazione di una linea cellulare e di un marker adeguati. Per chiarire alcuni aspetti di ordine farmacocinetico è stata anche quantificata la concentrazione plasmatica di LZ, in seguito a somministrazione orale in forma microincapsulata, rispetto alla somministrazione in forma libera. Risultati e discussione La selezione di un modello cellulare adeguato ai successivi studi in vitro è avvenuta nell’ambito di tre linee cellulari potenzialmente adatte, ovvero: una coltura primaria di cellule endoteliali del microcircolo dermico (ADMEC), per via dell’implicazione del sistema vascolare nel diabete e due linee cellulare di tubulo prossimale renale, LLC-PK1 (di origine suina) e HK-2 (di origine umana), per via del coinvolgimento tubulare nel contesto della nefropatia diabetica. Una serie di esperimenti ha permesso di far ricadere la scelta sulle cellule HK-2, le quali hanno mostrato la maggior sensibilità ai trattamenti con AGE (decremento dose-dipendente e tempo-indipendente) in termini di vitalità misurata mediante MTT test e per questioni di ordine pratico e logistico. Uno dei principali effetti degli AGE nell’organismo è l’induzione della risposta infiammatoria. Un evento cruciale dell’infiammazione è rappresentato dal reclutamento macrofagico. Sulla base di questi presupposti è stato effettuato un saggio di migrazione dei monociti U937, differenziati a macrofagi, verso uno stimolo rappresentato da sovranatanti ottenuti trattando le cellule HK-2 con AGE e LZ. Il trattamento con AGE ha indotto un incremento significativo (+ 50%) dei macrofagi migrati. Il trattamento con il LZ, come atteso, non ha modificato il tasso migratorio mentre, in presenza di AGE, è stato in grado di prevenire significativamente l’induzione della migrazione. È noto che le citochine svolgono un ruolo fondamentale nel reclutamento macrofagico. Nell’ambito della nefropatia diabetica, l’interleuchina-6 (IL-6) risulta rivestire un ruolo rilevante. Si è quindi deciso di misurare i livelli di mRNA di IL-6 in seguito a trattamenti con AGE e LZ. In conformità alle nostre aspettative, gli AGE hanno indotto un incremento statisticamente significativo dei livelli di mRNA di IL-6 dopo 24 h di trattamento. L’esposizione al LZ non ha indotto modificazione, mentre il co-trattamento con AGE e LZ ha evidenziato la capacità del LZ di prevenire l’incremento dei livelli di IL-6, mantenendoli a valori comparabili a quelli dei controlli. Per confermare l’attività del LZ nei confronti di IL-6 AGE-indotto, è stato condotto un saggio ELISA che ha rafforzato i dati ottenuti mediante RT-PCR: il LZ è in grado di ridurre, in maniera dose-dipendente e statisticamente significativa, la quantità di IL-6 rilasciata AGE-indotta. I meccanismi che portano al rilascio di IL-6 sono molteplici. Nell’arco di questa ricerca, ci si è focalizzati su alcuni eventi direttamente correlati all’incremento del rilascio di IL-6. Visto il ruolo chiave attribuito al recettore RAGE negli effetti AGE-indotti, si è deciso di testare la capacità del LZ di influenzare i livelli di mRNA di RAGE, mediante RT-PCR. I risultati non hanno evidenziato variazioni dei livelli di RAGE dopo esposizione al LZ a 24 e 96 h. L’induzione delle ROS rappresenta uno degli eventi notoriamente attribuibili ad un’azione AGE-indotta. Tuttavia, nel modello cellulare da noi utilizzato, questo parametro non ha mostrato innalzamenti rispetto al controllo negativo, pur mostrandosi sensibile al trattamento con ABAP (induttore di ROS di riferimento). Dal momento che l’attività del LZ è associata anche alla sua azione di scavenger degli AGE si è deciso di investigare, mediante microscopia confocale, se la presenza del LZ potesse influenzare la capacità degli AGE di penetrare all’interno della cellula. Le immagini acquisite con questa tecnica hanno permesso di stabilire che gli AGE e il LZ sono in grado di penetrare attraverso la membrana cellulare. Per quel che concerne il co-trattamento sono stati effettuati alcuni studi di citometria a flusso. I risultati suggeriscono che il LZ non è in grado, in questo modello cellulare, di ridurre l’ingresso degli AGE nella cellula, escludendo che l’azione di scavenger sia alla base della prevenzione della produzione di IL-6 in vitro. Successivi studi, sono stati effettuati su due possibile vie coinvolte negli effetti AGE-indotti, quali la cascata delle MAPk, mediante p38, e la via di degradazione lisosomiale. I dati preliminari ottenuti hanno evidenziato un possibile ruolo i p38. Gli AGE si sono dimostrati capaci di incrementare i livelli di fosforilazione di questa proteina, effetto ridotto dalla contemporanea presenza di una dose equimolare di LZ. Promettenti sono anche i primi risultati ottenuti dallo studio di co-localizzazione degli AGE, in presenza di LZ, nei lisosomi. Le immagini acquisite confermano, infatti, l’ipotesi che il LZ sia in grado di veicolare gli AGE nei lisosomi, favorendone la degradazione. Nel loro insieme i risultati ottenuti non escludono che l’azione del LZ nei confronti degli AGE e dei suoi effetti sia prettamente di tipo antidotico e di scavenging. Tuttavia, la capacità del LZ di agire su un mediatore chiave degli effetti AGE-indotti come IL-6 e p38, aprono una nuova finestra di studio per quel che concerne il LZ, permettendo di ipotizzare un suo possibile impiego come co-adiuvante per il controllo dell’infiammazione nella nefropatia diabetica. Al fine di investigare se la maggior efficacia del lisozima microincapsulato sia dovuta alla capacità dei microsistemi di veicolare il LZ, nel torrente circolatorio di quanto non avvenga con la forma libera è stato messo a punto un modello in vivo nell’ambito del quale il LZ è stato somministrato oralmente microincapsulato oppure libero. Nel corso di tale prova è stato dimostrato che i microsistemi sono capaci di veicolare il lisozima a livelli serici superiori di quanto non avvenga dopo somministrazione di un’equidose di LZ in forma libera. Questi risultati dimostrano l’efficacia dei microsistemi orali per la veicolazione in circolo di principi biologicamente attivi

Kohlenhydrate und Proteine gehören neben Wasser und Fetten zu den quantitativ bedeutendsten Grundbestandteilen biologischer Systeme und der Lebensmittel. Unter milden Bedingungen in lebenden Organismen oder unter thermischer Belastung bei der Lebensmittelverarbeitung können reduzierende Kohlenhydrate amin-katalysiert durch die Abspaltung von Wasser und Fragmentierungen des Kohlenstoffgerüsts abgebaut werden, wobei die noch reaktiveren 1,2-Dicarbonylverbindungen entstehen. Aus der Reaktion der N-α-Aminogruppe und funktioneller Gruppen der Seitenketten von Aminosäuren mit Kohlenhydraten bzw. 1,2-Dicarbonylverbindungen können stabile Endprodukte entstehen. In vivo können proteingebundene Maillard-Produkte (MRPs) aus der Reaktion mit Glucose (Amadori-Produkte) oder 1,2-Dicarbonylverbindungen (Advanced Glycation Endproducts: AGEs) entstehen. Beispielsweise ist das „N-terminale“ N-α-Fructosylderivat der β-Kette des Hämoglobins ein etablierter Parameter zur Diagnose von Diabetes mellitus (HbA1c-Wert). Diese nicht-enzymatische, posttranslationale Modifizierung von Proteinen wird allgemein als Glykierung bezeichnet und kann die Funktionalität von Proteinen beeinträchtigen. Deshalb wird untersucht, ob die Trübung der Augenlinsen, die Versteifung von Blutgefäßen oder Schädigungen von Nervenzellen durch eine erhöhte Glykierung verursacht werden. Diese Veränderungen treten im Alter und bei Stoffwechselkrankheiten wie Diabetes mellitus und Urämie auf, die durch eine erhöhte Glucosekonzentration bzw. die Anreicherung von 1,2-Dicarbonylverbindungen im Blut gekennzeichnet sind. Zwar gibt es Publikationen zum Vorkommen N-terminaler Amadori-Produkte an Hämoglobin und in Lebensmitteln, aber die Bildung N-terminaler AGEs wurde bisher nur in wenigen Studien untersucht. Deshalb waren die Bildung und das Vorkommen N-terminaler AGEs im physiologischen Modell, in Hämoglobin und in Backwaren Gegenstand der vorliegenden Arbeit. In der vorliegenden Arbeit wurde erstmals systematisch die Sequenzabhängigkeit der Bildung der Fructosylderivate bzw. der CM-Derivate in Konkurrenz zu den Glyoxal-2(1H)-Pyrazinonen am N-Terminus von Peptiden unter physiologischen und backtechnologischen Bedingungen untersucht. Dabei wurde nachgewiesen, dass die Variation der C-terminalen Aminosäure in Dipeptiden den Glykierungsgrad und das Produktspektrum erheblich beeinflusst. Mit dem konsequenten Nachweis der N-terminalen von Glyoxal und Methylglyoxal ableitbaren Carboxyalkylderivate und 2(1H)-Pyrazinone in humanen Hämoglobin wurde die Relevanz der N-terminalen Glykierung in vivo untermauert. Damit wird eine umfassendere Beurteilung des Dicarbonylstresses und der Glykierung insbesondere bei Urämikern und Diabetikern ermöglicht. Am Beispiel von Backwaren wurde für Lebensmittel gezeigt, dass unter trockenen Reaktionsbedingungen die 2(1H)-Pyrazinone und in wasserhaltigen Systemen die Carboxyalkylderivate bevorzugt zu erwarten sind.

Advances in our understanding of diabetes in human patients and experimental models indicate that a number of mechanisms may contribute to sensory nerve damage in diabetic polyneuropathy (DPN). In addition to oxidative stress, hyperglycemia and hyperlipidemia, recent research in pain, advanced glycation endproduct (AGE), and proteomics specify a contributory role for altered neuronal calcium homeostasis in DPN. Technology advances indicate neuronal energy balance and mitochondrial biogenesis, fission, and fusion are additional potential mechanisms. The effects of dysregulation or loss of insulin signaling and the effects of glucagon-like peptide-1 (GLP-1) and its receptor (GLP-1R) are also implicated.

Background and aims: Diabetic kidney disease occurs in ca. 40% patients with diabetes. Approximately 1 in 5 patients with type 1 diabetes mellitus (T1DM) and 1 in 3 patients with type 2 diabetes mellitus (T2DM) develop early decline in renal function (EDRF), requiring renal dialysis after 5 - 20 years. Currently, at the time of normoalbuminuria or new onset microalbuminuria (incipient diabetic nephropathy), it is uncertain which patients are at risk of EDRF. With Joslin Kidney Study investigators, we found patients with T1DM who later developed EDRF (Decliners) have higher fractional excretion (FE) of 6 glycated amino acids - fructosyl-lysine and 5 advanced glycation endproducts (AGEs), compared to patients with stable renal function (Non-decliners). However, FE of any single glycated amino acid could not classify Decliners or Non-decliners. The aim of this study was to apply artificial intelligence machine learning to develop diagnostic algorithms to classify Decliners and Non-decliners by optimum combination of levels of glycated and oxidized amino acids in plasma and urine, related FEs and conventional clinical chemistry variables. Materials and methods: Patients with T1DM with stable renal function (n = 63) and EDRF (n = 22) were recruited for this study. Data on levels of 14 glycated and oxidized amino acids in plasma, urine, related FEs, glycated hemoglobin A1C, log(urinary albumin creatinine ratio, ACR), age, gender and duration of diabetes at the time of new onset microalbuminuria were included as features in algorithm development. Algorithms were trained and tested on 90%/10% data split, repeated 1000 times, using the Extreme Gradient Boosting method. Results: The algorithm gave an optimal classification of Decliners and Non-decliners. Optimum with features: A1C, log[ACR], FE(Nꞷ-carboxymethylarginine, CMA), FE(glyoxal-derived hydroimidazolone, G-H1) and plasma concentration of Nε-carboxymethyl-lysine (CML) free adduct; For The diagnostic performance for risk prediction of future EDRF was (mean ± SD): sensitivity 74 ± 9%, specificity 91 ± 45 and accuracy 87 ± 4%. The positive likelihood ratio LR+ was 11.0, indicating that this method gives strong, often conclusive evidence of future EDRF in patients with T1DM. In contrast, algorithms with A1C and logACR only as features gave LR+ 2.6, providing small evidence of risk of future EDRF. Conclusion: With conclude that FEs of glycated amino acids are novel risk predictors of EDRF, likely linked to reporting of early-decline of cationic amino acid transporter function in the renal tubular epithelium. Genetic polymorphism of these amino acid transporters has been linked to rapid decline in renal function in genome-wide association studies. Measurement of only 3 glycated amino acids, CMA, G-H1 and CML, produced an algorithm with optimum risk prediction of EDRF. With further validation, including in patients with T2DM and with chronic kidney disease without diabetes, this method may markedly improve clinical risk prediction of EDRF.