Doctor of Philosophy / Food Science Institute / J. Scott Smith / Advanced glycation endproducts (AGEs) are formed in many cooked meat products via Maillard browning reactions. Current research suggests consumption of these compounds may be a contributor to chronic diseases such as diabetes and heart diseases. Thus, information on the prevalence and inhibition of these compounds in food is desirable. The first objective was to determine the AGE content, as determined as N[superscript]ε-carboxymethyllysine (CML) level, in cooked meat and fish prepared by general cooking methods recommended by U.S. Department of Agriculture, Food Safety and Inspection Service (USDA-FSIS). We found AGE was detected in all the cooked samples, but the levels depended on the different cooking conditions. Broiling and frying at higher cooking temperatures produced higher levels of CML and broiled beef contained the highest CML content (21.84 μg/g). However, the baked salmon (8.59 μg/g) and baked tilapia (9.72 μg/g) contained less CML as compared to the other samples. In order to investigate the inhibitory effect of selected natural antioxidant on AGEs formation in cooked meat, four cereal brans, wheat (Jagger, JA), triticale (Spring Triticale, ST; Thundercale, TH), and Rye (RY) bran were added to beef patties before cooking. RY (42.0% inhibition), ST (27.5% inhibition), and TH (21.4% inhibition) brans significantly decreased CML formation compared with the control. The inhibition of CML was correlated to the water-holding activity (WHC) of the samples, and the radical scavenging activity of the brans. The effect of cereal bran extracts (JA, ST, TH, and RY), was studied in a bovine serum albumin and glucose (BSA-GLU) model system. The ST extract significantly (P <0.05) inhibited CML formation compared to the control group. ST particularly contained vanillic acid (VA), chlorogenic acid (CHA), gentisic acid (GEA), and ferulic acid (FA), where GEA and CHA mitigated CML with an average percentage decrease of 29.6% for CHA and 51.1% for GEA. It therefore may be useful in preventing AGEs formation by using ST bran as a food addictive, which contains abundant phenolic acids. In summary, current dietary AGEs database will provide important information for use in estimating AGEs exposure, and also these data demonstrate that a significantly reduced intake of dietary AGEs can be achieved by low heat AGE cooking methods such as baking, which can be used at home or in the meat industry. Cereal bran addition to meat products may reduce formation of AGEs that is a desired attribute for the processed meat products industry.

Methylglyoxal (MG), a metabolite of glucose, causes non-enzymatic glycation of proteins to form irreversible advanced glycation end products (AGEs). Increased MG production, which in turn gives rise to AGEs, has been linked to the development of complications in diabetes. However, the role of MG and AGEs in hypertension has not been investigated widely. The previous study from our laboratory showed that the cellular levels of MG and MG-induced AGE formation are significantly higher in cultured aortic smooth muscle cells from spontaneously hypertensive rats (SHR) than those from normotensive Wistar-Kyoto rats (WKY). Using immunofluorescence staining with specific monoclonal antibodies against MG-induced AGEs, the present studies show a strong association of MG and its AGE products (Nå-carboxyethyl-lysine and Nå-carboxymethyl-lysine) with hypertension in SHR. The blood pressure of SHR was not different from that of WKY rats at 5 wks of age. From 8 wks onwards, blood pressure was significantly elevated compared to age-matched WKY rats. Importantly, this increase in blood pressure coincided with an elevated MG level in plasma and aorta of SHR in an age-dependent fashion compared to age-matched WKY rats, although no difference was observed in blood glucose levels between these two strains. Our data showed an increased MG level in plasma and aorta, but not in kidney or heart, in SHR at an early age of 8 wks, suggesting, in addition to diabetes/hyperglycemic or hyperlipidemic conditions, the accumulation of MG in blood vessel walls plays an important role in the development of hypertension or its complications even in the absence of diabetes. Moreover, we observed increased blood pressure and vascular remodeling in Sprague Dawley rats which had been treated to increase endogenous MG and related AGEs. After inhibiting MG and MG-induced AGE generation in SHR, hypertension development in this genetic hypertension model was delayed and vascular remodeling was reversed. Our data indicate that increased MG and AGE formation may play an important role in the development of hypertension.

Methylglyoxal (MG), a metabolite of glucose, causes non-enzymatic glycation of proteins to form irreversible advanced glycation end products (AGEs). Increased MG production, which in turn gives rise to AGEs, has been linked to the development of complications in diabetes. However, the role of MG and AGEs in hypertension has not been investigated widely. The previous study from our laboratory showed that the cellular levels of MG and MG-induced AGE formation are significantly higher in cultured aortic smooth muscle cells from spontaneously hypertensive rats (SHR) than those from normotensive Wistar-Kyoto rats (WKY). Using immunofluorescence staining with specific monoclonal antibodies against MG-induced AGEs, the present studies show a strong association of MG and its AGE products (Nå-carboxyethyl-lysine and Nå-carboxymethyl-lysine) with hypertension in SHR. The blood pressure of SHR was not different from that of WKY rats at 5 wks of age. From 8 wks onwards, blood pressure was significantly elevated compared to age-matched WKY rats. Importantly, this increase in blood pressure coincided with an elevated MG level in plasma and aorta of SHR in an age-dependent fashion compared to age-matched WKY rats, although no difference was observed in blood glucose levels between these two strains. Our data showed an increased MG level in plasma and aorta, but not in kidney or heart, in SHR at an early age of 8 wks, suggesting, in addition to diabetes/hyperglycemic or hyperlipidemic conditions, the accumulation of MG in blood vessel walls plays an important role in the development of hypertension or its complications even in the absence of diabetes. Moreover, we observed increased blood pressure and vascular remodeling in Sprague Dawley rats which had been treated to increase endogenous MG and related AGEs. After inhibiting MG and MG-induced AGE generation in SHR, hypertension development in this genetic hypertension model was delayed and vascular remodeling was reversed. Our data indicate that increased MG and AGE formation may play an important role in the development of hypertension.

Methylglyoxal (MG) is a reactive metabolite presents in all biological systems. The accumulation of MG in diabetic patients and animals has been long recognized. Recently, studies have shown that MG levels are elevated in hypertensive rats. However, the pathological effects of MG in diabetes and related insulin resistance syndrome such as obesity are currently unknown. In the present study, the role of MG in the pathogenesis of insulin resistance was investigated.<p> First, it was observed that MG induced structural and functional changes of insulin. Incubation of human insulin with MG in vitro yielded MG-insulin adducts, as evidenced by additional peaks observed upon mass spectrometric (MS) analysis. Tandem MS analysis of insulin B-chain adducts confirmed attachment of MG at an arginine residue. [3H]-2-deoxyglucose uptake ([3H]-2-DOG) by 3T3-L1 adipocytes was significantly and concentration-dependently decreased after treatment with MG-insulin adducts, in comparison with the effect of native insulin at the same concentration. A significant decrease of glucose uptake induced by MG-insulin adducts was also observed in L8 skeletal muscle cells. Unlike native insulin, MG-insulin adducts did not inhibit insulin release from pancreatic â-cells. The degradation of MG-insulin by cultured liver cells was also decreased. In conclusion, MG modifies insulin by attaching to internal arginine residue in the â-chain of insulin. The formation of this MG-insulin adduct decreases insulin-mediated glucose uptake, impairs autocrine control of insulin secretion, and decreases insulin clearance. These structural and functional abnormalities of the insulin molecule may contribute to the pathogenesis of insulin resistance.<p> Second, the effects of MG on the insulin signaling pathway were investigated. After 9 weeks of fructose treatment, an insulin resistant state was developed in Sprague-Dawley (SD) rats, demonstrated as increased triglyceride and insulin levels, elevated blood pressure, and decreased insulin-stimulated glucose uptake by adipose tissue. A close correlation between insulin resistance and the elevated MG accumulation in adipose and skeletal muscle tissues was observed. The insulin resistant state and the elevated MG level were reversed by the MG scavenger, N-acetyl cysteine (NAC) and metformin. In cultured adipose cells, MG treatment impaired insulin signaling as measured by decreased tyrosine phosphorylation of insulin-receptor substrate-1 (IRS-1) and the decreased kinase activity of phosphatidylinositol 3-kinase (PI3K). The ability of NAC to block MG-impairment of PI3K activity and IRS-1 phosphorylation further confirmed the role of MG in the development of insulin resistance. In cultured skeletal muscle cells, MG treatment significantly reduced the expression of IRS-1 and PI3K at the mRNA level. Similar to adipose cells, MG also decreased tyrosine phosphorylation of IRS-1 and PI3K activity. We also examined the mechanism of metformin to inhibit AGEs. Using mass spectrometry, stable metformin-MG adducts were identified.<p> In addition, we investigated the causative effect of MG in the pathogenesis of obesity, another form of insulin resistance. This study revealed a previously unrecognized effect of MG in stimulating adipogenesis by up-regulating Akt signaling. In Zucker fatty rats, dramatically increased MG accumulations in serum and different tissues were identified. The serum MG level increased age. In 10 and 12 week-old obese rats, MG was 144±50% and 171±15% of the age-matched control Zucker rats; this value increased to 241±7 % and 329±10% by 14 and 16 weeks (P<0.05, n=4). Further study suggested that MG accumulation stimulates the phosphorylation of Akt and its effectors p21 and p27. The activated Akt pathway then increased the activity of Cdk2 and accelerates the cell cycle progression and proliferation of pre-adipocytes. The effects of MG were efficiently reversed by both alagebrium, and Akt inhibitor SH-6.<p> Overall, the current study investigated the effect of MG during the pathogenesis of insulin resistance syndrome. MG, as the most potent precursor of AGEs, impairs the activity of insulin signaling pathway by glycating the insulin molecule and other insulin signaling proteins. Moreover, this study observed a previously unrecognized causative effect of MG in the proliferation of adipocytes by up-regulating the Akt signaling pathway. The results from this study offer new mechanisms to explain the development of insulin resistance and to prevent the related diseases.

Methylglyoxal (MG) is a reactive metabolite presents in all biological systems. The accumulation of MG in diabetic patients and animals has been long recognized. Recently, studies have shown that MG levels are elevated in hypertensive rats. However, the pathological effects of MG in diabetes and related insulin resistance syndrome such as obesity are currently unknown. In the present study, the role of MG in the pathogenesis of insulin resistance was investigated.<p> First, it was observed that MG induced structural and functional changes of insulin. Incubation of human insulin with MG in vitro yielded MG-insulin adducts, as evidenced by additional peaks observed upon mass spectrometric (MS) analysis. Tandem MS analysis of insulin B-chain adducts confirmed attachment of MG at an arginine residue. [3H]-2-deoxyglucose uptake ([3H]-2-DOG) by 3T3-L1 adipocytes was significantly and concentration-dependently decreased after treatment with MG-insulin adducts, in comparison with the effect of native insulin at the same concentration. A significant decrease of glucose uptake induced by MG-insulin adducts was also observed in L8 skeletal muscle cells. Unlike native insulin, MG-insulin adducts did not inhibit insulin release from pancreatic â-cells. The degradation of MG-insulin by cultured liver cells was also decreased. In conclusion, MG modifies insulin by attaching to internal arginine residue in the â-chain of insulin. The formation of this MG-insulin adduct decreases insulin-mediated glucose uptake, impairs autocrine control of insulin secretion, and decreases insulin clearance. These structural and functional abnormalities of the insulin molecule may contribute to the pathogenesis of insulin resistance.<p> Second, the effects of MG on the insulin signaling pathway were investigated. After 9 weeks of fructose treatment, an insulin resistant state was developed in Sprague-Dawley (SD) rats, demonstrated as increased triglyceride and insulin levels, elevated blood pressure, and decreased insulin-stimulated glucose uptake by adipose tissue. A close correlation between insulin resistance and the elevated MG accumulation in adipose and skeletal muscle tissues was observed. The insulin resistant state and the elevated MG level were reversed by the MG scavenger, N-acetyl cysteine (NAC) and metformin. In cultured adipose cells, MG treatment impaired insulin signaling as measured by decreased tyrosine phosphorylation of insulin-receptor substrate-1 (IRS-1) and the decreased kinase activity of phosphatidylinositol 3-kinase (PI3K). The ability of NAC to block MG-impairment of PI3K activity and IRS-1 phosphorylation further confirmed the role of MG in the development of insulin resistance. In cultured skeletal muscle cells, MG treatment significantly reduced the expression of IRS-1 and PI3K at the mRNA level. Similar to adipose cells, MG also decreased tyrosine phosphorylation of IRS-1 and PI3K activity. We also examined the mechanism of metformin to inhibit AGEs. Using mass spectrometry, stable metformin-MG adducts were identified.<p> In addition, we investigated the causative effect of MG in the pathogenesis of obesity, another form of insulin resistance. This study revealed a previously unrecognized effect of MG in stimulating adipogenesis by up-regulating Akt signaling. In Zucker fatty rats, dramatically increased MG accumulations in serum and different tissues were identified. The serum MG level increased age. In 10 and 12 week-old obese rats, MG was 144±50% and 171±15% of the age-matched control Zucker rats; this value increased to 241±7 % and 329±10% by 14 and 16 weeks (P<0.05, n=4). Further study suggested that MG accumulation stimulates the phosphorylation of Akt and its effectors p21 and p27. The activated Akt pathway then increased the activity of Cdk2 and accelerates the cell cycle progression and proliferation of pre-adipocytes. The effects of MG were efficiently reversed by both alagebrium, and Akt inhibitor SH-6.<p> Overall, the current study investigated the effect of MG during the pathogenesis of insulin resistance syndrome. MG, as the most potent precursor of AGEs, impairs the activity of insulin signaling pathway by glycating the insulin molecule and other insulin signaling proteins. Moreover, this study observed a previously unrecognized causative effect of MG in the proliferation of adipocytes by up-regulating the Akt signaling pathway. The results from this study offer new mechanisms to explain the development of insulin resistance and to prevent the related diseases.

Diabetes is associated with increased fracture risk, which leads to increased morbidity and eventual mortality with a substantial financial burden. Type 2 Diabetics also have increased fracture risk, despite having the same or higher BMD as non-diabetics with a low fracture risk. One hypothesis for this is increased modifications made to the extra-cellular matrix via non-enzymatic glycation (NEG) that can occur in a hyperglycemic environment, such as with diabetes. The accumulation of NEG products, known as advanced glycation endproducts (AGEs) can possibly lead to microdamage and eventual weakening of the bone itself. We developed a time-response model in order to induce a wide range of AGEs in a manner that would sustain the mineral integrity of the bone and could be applied to a variety of bone sample types. This was performed on 65 rat tibias, distributed amongst 8 groups (3,7,10, & 14 days) for both ribose and control. Secondly, the protocol was performed on human cortical beam samples cut from 10 donor tibias with 3,5 and 7 day time points for ribose and control groups. All samples were incubated in a 0.6 M ribose solution or 0.0 M ribose control solution. There was a 7, 4, and 5-fold increase in AGEs at the 7, 10, and 14 day time points respectively over controls in the rat tibia study. There was no significant variation in cortical porosity, however TTMD was significantly less dense in the 14-day ribose treated groups. There was a trend toward higher AGEs with time in the human cortical beam specimens, but no significant increase. The AGEs values in the human cortical beam specimens were much lower than expected based on previous trials and reports in the literature. We were able to establish a time-response model for AGE accumulation in bone. However, the effects of AGEs on bone material properties remains inconclusive.

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Το Hypericum perforatum (Υπερικό το διάτρητο) είναι ένα από τα πιο παλιά φαρμακευτικά φυτά που χρησιμοποιούταν στη λαϊκή θεραπευτική πολλών διαφορετικών πολιτισμών, ως επουλωτικό, αντιφλεγμονώδες και αντικαταθλιπτικό. Αλκοολικά εκχυλίσματα αυτού συνιστούν σκευάσματα που κυκλοφορούν ως συμπληρώματα ή φάρμακα για διαταραχές της ήπιας και μέτριας κατάθλιψης. Φλαβονοειδή, φαινολικά οξέα, ναφθοδιανθρόνες και φλωρογλουκινόλες είναι οι κύριες ομάδες συστατικών που βρίσκονται σε μεγάλη περιεκτικότητα στα φυτικά εκχυλίσματα. Στην παρούσα μελέτη πραγματοποιήθηκε ανάπτυξη αναλυτικής μεθόδου για τον προσδιορισμό και την ποσοτικοποίηση κύριων βιοδραστικών συστατικών του Hypericum perforatum με τη χρήση υγρής χρωματογραφίας υψηλής απόδοσης με ανιχνευτή συστοιχίας φωτοδιόδων (Ηigh Performance Liquid Chromatography - Diode Array Detector, HPLC-DAD). Μετά από πιλοτικά πειράματα σε στήλη ανάστροφης φάσης Luna C-18, ο διαχωρισμός των συστατικών του μεθανολικού εκχυλίσματος έγινε με χρήση συστήματος βαθμιδωτής έκλουσης με τρεις διαλύτες: ρυθμιστικό διάλυμα 10 mM οξικού αμμωνίου, pH=4,5/ ακετονιτρίλιο/ μεθανόλη και ροή 1.0 mL/min. Η μέθοδος πιστοποιήθηκε με πρότυπα χλωρογενικού οξέος, ρουτίνης, υπεροζίτη, κερσετίνης, ισοκερσετίνης και υπερικίνης και τα ποιοτικά χαρακτηριστικά της μεθόδου είναι εντός των αποδεκτών ορίων που θεσπίζει ο ΕΜΕΑ. Η αναπτυχθείσα μέθοδος μπορεί να εφαρμοσθεί για τον ποιοτικό και ποσοτικό έλεγχο εκχυλισμάτων Hypericum, όπως και για τον χαρακτηρισμό της σύστασης διαφόρων taxa Hypericum τα οποία δεν έχουν μελετηθεί. Πρόσφατη μελέτη αναφέρθηκε στις νευροπροστατευτικές ιδιότητες του εκχυλίσματος σε διαβητικά πειραματόζωα και αυτό αποτέλεσε το έναυσμα διερεύνησης της επίδρασης αυτού στη μη ενζυμική γλυκοζυλίωση των πρωτεϊνών, που συμβαίνει σε συνθήκες υπεργλυκαιμίας στους ασθενείς με σακχαρώδη διαβήτη. Η πλεονάζουσα γλυκόζη αντιδρά μη ενζυμικά με πλήθος πρωτεϊνικών μορίων, επηρεάζοντας τη δομή και τη λειτουργία αυτών. Σε συνθήκες εμμένουσας υπεργλυκαιμίας και διάρκειας εβδομάδων η αντίδραση αυτή έχει ως αποτέλεσμα το σχηματισμό φθοριζουσών τελικών προϊόντων, μη-αναστρέψιμων (Advanced Glycation End-products, AGEs), τα οποία συνεισφέρουν στην ανάπτυξη των αγγειακών διαβητικών επιπλοκών. Ο έλεγχος της έντασης φθορισμού έγινε μετά από επώαση τριών ημερών αλβουμίνης βόειου ορού (10 mg/mL) και ριβόζης (0,5 M) σε όγκο 350 μL, παρουσία ή απουσία του φυτικού εκχυλίσματος και των συστατικών αυτού. Τα αποτελέσματα έδειξαν σημαντική ανασταλτική δράση του εκχυλίσματος (100 μg/mL) (70%) και των μεμονωμένων συστατικών του (100 μΜ), κύρια δε των φλαβονοειδών. Τα αποτελέσματα αυτά συνεισφέρουν στην επιστημονική αναζήτηση μη τοξικών αναστολέων σχηματισμού AGEs για την αντιμετώπιση των αγγειακών επιπλοκών του διαβήτη. Λέξεις-κλειδιά: Hypericum perforatum, HPLC ανάλυση, Τελικά προϊόντα προχωρημένης γλυκοζυλίωσης (AGEs) / Hypericum perforatum (Saint John’s wort) has been used since antiquity in folk medicine as a wound-healing, anti-inflammatory and antidepressant drug. Alcoholic extracts of this plant are the constituents of drug preparations or supplements that are used for mild to moderate depression. Flavonoids, phenolic acids, napthodianthrones and phloroglucinols are the main constituents of high content in herbal extracts. In the present study, a High Performance Liquid Chromatography (HPLC) analytical method was developed for the quality and quantity control of bioactive components of Hypericum perforatum methanolic extracts. After a great variety of elution gradients on a C-18 Luna column, the natural products were completely separated by a linear gradient of 10mM ammonium acetate (pH=4,5) -acetonitrile-methanol in one run within 60 min and flow 1.0 mL/min. The chromatographic method was validated using commercially available standards of chlorogenic acid, rutin, hyperoside, quercetin, isoquercitrin and hypericin and the quality values were within the acceptable limits of EMEA. The developed method can be applied towards the quality and quantity control of hypericum extracts and the characterization of the composition of Hypericum taxa, which have not been studied. A recent study mentioned the neuroprotective capacity of the extract in diabetic rats and that was the beginning of a research about its effect in non-enzymatic glycosylation of proteins that occurs in diabetic patients, in hyperglycaemia conditions. The redundant glycose react non-enzymatically with a number of proteins, affecting their structure and function. Under sustained hyperglycaemia conditions and in natural aging, further glycation of proteins causes the generation of AGEs (Advanced Glycation End-products).AGEs may fluoresce under ultraviolet light and contribute to the development of diabetic vascular complications. The measurement of fluorescence intensity was realized after a 3-days incubation of bovine serum albumin (10 mg/mL) with ribose (0,5 M) in the presence or absence of herbal extract and its constituents. The final volume of reaction was 350 μL. The results showed significant inhibitory effect of the extract (100 μg/mL) (70%) and its components (100 μΜ), mainly that of flavonoids. The present results contribute to scientific research of non-toxic inhibitors of AGEs formation in order to get faced the diabetic vascular complications.

Τα τελικά προϊόντα προχωρημένης γλυκοζυλίωσης (AGEs: Advanced Glycation Endproducts) παίζουν σημαντικό ρόλο στην παθογένεια των διαβητικών αγγειακών επιπλοκών. Το καλύτερα χαρακτηριζόμενο είναι η N-καρβοξυμεθυλ-λυσίνη (CML). Τα AGEs προκαλούν σημαντικές επιδράσεις στα αγγεία με την πρόσδεσή τους σε ειδικούς υποδοχείς της κυτταρικής επιφάνειας, όπως τον RAGE (Receptor for Advanced Glycation Endproducts). Διαλυτές μορφές του RAGE (sRAGE) εμφανίζονται στο ανθρώπινο αίμα και δρουν ως παγίδα αιχμαλωτίζοντας τους φλεγμονώδεις προσδέτες του RAGE εξωκυττάρια, προστατεύοντας με αυτό τον τρόπο τα κύτταρα από τη βλάβη που προάγεται από τα AGEs. Σκοπός αυτής της εργασίας ήταν να μελετηθούν τα επίπεδα του sRAGE, η πρωτεϊνική έκφραση του RAGE, καθώς και τα επίπεδα CML σε σχέση με διάφορες κλινικές και βιοχημικές παραμέτρους σε παιδιά, εφήβους και νεαρούς ενήλικες με ΣΔ1. Τα επίπεδα sRAGE και CML προσδιορίστηκαν με ELISA και η πρωτεϊνική έκφραση του RAGE στα μονοπύρηνα του περιφερικού αίματος με ανοσοαποτύπωση κατά Western σε 74 παιδιά, εφήβους και νεαρούς ενήλικες με ΣΔ1 (13± 4 χρονών) και 43 μάρτυρες αντίστοιχης ηλικίας, φύλου και σταδίου Tanner. Σ’ αυτή την εργασία τα αυξημένα επίπεδα sRAGE στα παιδιά με ΣΔ1 και πιο ειδικά, σ’ αυτά ηλικίας κάτω από 13 ετών και με διάρκεια διαβήτη κάτω από 5 έτη, μπορεί να είναι ένα προσωρινό προστατευτικό μέτρο ενάντια στην κυτταρική βλάβη και πιθανόν να είναι επαρκές για να εξουδετερώσει επαρκώς τα κυκλοφορούντα CML, εμποδίζοντας έτσι τις διαβητικές αγγειακές επιπλοκές. Επίσης, μια ήπια αύξηση της LDL θα μπορούσε να είναι ένα ερέθισμα για την αύξηση του sRAGE, οδηγώντας στη δέσμευση του CML και τελικά τη μείωση των επιπέδων CML στην κυκλοφορία. Τα μειωμένα επίπεδα της πρωτεϊνικής έκφρασης του RAGE 55 kd (υποδοχέα πλήρους μήκους) μπορεί να αντανακλούν την αυξημένη έκφραση του sRAGE στους ασθενείς με ΣΔ1 συνολικά λόγω της αποκοπής του RAGE με μεταλλοπρωτεϊνάσες. Με την παρουσία κάποιου παράγοντα κινδύνου, όπως αύξηση ηλικίας, περιμέτρου κοιλίας, BMI, συστολικής ή διαστολικής αρτηριακής πίεσης ή επιδείνωση λιπιδαιμικού προφίλ αυξάνεται η πρωτεϊνική έκφραση της ισομορφής αυτής, ενώ φαίνεται αντίστοιχα να μειώνονται τα επίπεδα του sRAGE. Φαίνεται τελικά ότι συνολικά στα παιδιά, τους εφήβους και τους νεαρούς ενήλικες με ΣΔ1 υπάρχει μια υποκλινική διαταραχή του άξονα sRAGE-RAGE-CML, η οποία δύναται να μετατραπεί σε κλινικά εμφανείς αγγειακές βλάβες, αν προστεθούν περαιτέρω επιβαρυντικοί παράγοντες. / The binding of Advanced Glycation Endproducts (AGEs) to their receptor (RAGE) plays a major role in the development of diabetic vascular complications. This work is based on the relation between circulating soluble RAGE (sRAGE) levels in children, adolescents and young adults with IDDM and RAGE protein expression in association with N-(carboxymethyl)lysine (CML), a major antigenic AGEs component. Circulating sRAGE and CML levels were determined by ELISA and RAGE protein expression was evaluated in peripheral blood mononuclear cells by western immunoblotting in 74 children, adolescents and young adults with IDDM (134 years old) and 43 age, sex and Tanner stage-matched controls. Serum sRAGE levels were significantly higher in IDDM than in controls, inversely correlated to diabetes duration and directly correlated to LDL levels. Furthermore, circulating CML levels were not significantly different between IDDM and controls. Also, the protein expression of the RAGE isoforms 55 kd (full-length), 64 kd and 100 kd, measured by western immunoblotting, was significantly lower in IDDM than in controls, whereas RAGE 37 kd levels were not significantly different between IDDM and controls. Finally, when there was a risk factor, such as increased age, poor lipid profile, increased BMI or waist circumference or increased systolic or diastolic pressure, then it seemed that isoforms RAGE 55, 64 and 100 kd were increased. Isoform RAGE 64 kd could be RAGE-v5, a splice variant which resulted in a change of amino acid sequence in the extracellular ligand-binding domain of RAGE. Isoform RAGE 37 kd seemed to be Δ8-RAGE, a soluble splice variant with probably protective function, which had been found increased in patients with increased HDL. Finally, isoform RAGE 100 kd seemed to be some other splice variant in peripheral mononuclear cells. In conclusion, increased serum levels of sRAGE seen in IDDM children may be a temporary protective measure against cell damage and may be sufficient to efficiently eliminate excessive circulating CML. Moreover, the lower protein expression of the full-length RAGE in IDDM may also reflect the increased sRAGE expression in patients due to RAGE cleavage by metalloproteases. Consequently, in IDDM children, adolescents and young adults there may be a subclinical perturbation of the sRAGE-RAGE-CML axis, which could lead to future clinical vascular damage if additional risk factors are added over time.

Ο Σακχαρώδης Διαβήτης (ΣΔ) είναι μια μεταβολική διαταραχή, που χαρακτηρίζεται από χρόνια υπεργλυκαιμία ως αποτέλεσμα διαταραχής στην έκκριση της ινσουλίνης ή τη δράση της ή και στα δύο αυτά χαρακτηριστικά. Οι μακροπρόθεσμες επιπτώσεις της χρόνιας υπεργλυκαιμίας στον ΣΔ διακρίνονται σε μικροαγγειακές και μακροαγγειακές επιπλοκές. Η μικροαγγειακή νόσος οδηγεί σε αμφιβληστροειδοπάθεια, νεφρική ανεπάρκεια και νευροπάθεια, ενώ η σχετιζόμενη με τον ΣΔ μακροαγγειακή νόσος προκαλεί αυξημένο κίνδυνο για έμφραγμα μυοκαρδίου, αγγειακά εγκεφαλικά επεισόδια και ακρωτηριασμούς των άκρων. Έχει βρεθεί ότι η υπεργλυκαιμία είναι η κύρια αιτία της μικροαγγειακής νόσου, ενώ στην παθογένεια της μακροαγγειακής νόσου συμμετέχει η υπεργλυκαιμία, αλλά και η αντίσταση στην ινσουλίνη. Ο σύνδεσμος ανάμεσα στην χρόνια υπεργλυκαιμία και την αγγειακή βλάβη έχει αποδοθεί σε τέσσερα ανεξάρτητα βιοχημικά μονοπάτια: 1. Αυξημένη δραστηριότητα του μονοπατιού της πολυόλης 2. Συσσώρευση τελικών προϊόντων προχωρημένης γλυκοζυλίωσης (Advanced Glycation Endproducts: AGEs) 3. Ενεργοποίηση της πρωτεϊνικής κινάσης C (PKC) και 4. Αυξημένη δραστηριότητα του μονοπατιού της εξοζαμίνης. Αυτά τα φαινομενικά μη σχετιζόμενα μεταξύ τους μοριακά μονοπάτια έχουν έναν υποκείμενο κοινό μηχανισμό : την υπερπαραγωγή ριζών υπεροξειδίου από τη μιτοχονδριακή αλυσίδα μεταφοράς ηλεκτρονίων. Οι μιτοχονδριακές ελεύθερες ρίζες οξυγόνου, μέσω ενεργοποίησης της πολυμεράσης της πολυ-ADP-ριβόζης, μερικώς αναστέλλουν το γλυκολυτικό ένζυμο αφυδρογονάση της 3-φωσφορικής γλυκεραλδεΰδης, με αποτέλεσμα τη συσσώρευση των γλυκολυτικών ενδιάμεσων προϊόντων, όπως της 3-φωσφορικής γλυκεραλδεΰδης και της 6-φωσφορικής φρουκτόζης, που αποτελούν υποστρώματα για τα τέσσερα παραπάνω βιοχημικά μονοπάτια. Το αποτέλεσμα της αντίστασης στην ινσουλίνη, όσον αφορά τις μακροαγγειακές επιπλοκές, είναι η αυξημένη ροή των ελεύθερων λιπαρών οξέων από τα λιποκύτταρα προς τα αρτηριακά ενδοθηλιακά κύτταρα. Η αυξημένη οξείδωση των ελεύθερων λιπαρών οξέων στα μιτοχόνδρια και η μιτοχονδριακή υπερπαραγωγή ελευθέρων ριζών οξυγόνου οδηγούν στην ενίσχυση των τεσσάρων μοριακών μονοπατιών με τον ίδιο ακριβώς μηχανισμό που έχει περιγραφεί παραπάνω για την υπεργλυκαιμία. Στην αντίσταση στην ινσουλίνη έχει παρατηρηθεί, επίσης, η μερική αναστολή του μονοπατιού της κινάσης της 3-φωσφατιδυλινοσιτόλης, που τελικά προάγει την ενίσχυση των αθηρογόνων και την καταστολή των αντι-αθηρογόνων ιδιοτήτων της ινσουλίνης. Ο κύριος στόχος αυτής της βιβλιογραφικής εργασίας είναι η περιγραφή των μονοπατιών που οδηγούν στη δημιουργία των, επαγόμενων από την υπεργλυκαιμία αλλά και την αντίσταση στην ινσουλίνη, διαβητικών αγγειακών επιπλοκών, καθώς και του κοινού μηχανισμού (παραγωγής ελευθέρων ριζών οξυγόνου) που βρίσκεται πίσω από αυτά τα μονοπάτια, παρέχοντας πλέον μια καινούρια βάση για μελλοντική έρευνα και ανακάλυψη φαρμάκων, προληπτικών και θεραπευτικών της διαβητικής αγγειοπάθειας. / Diabetes Mellitus is a metabolic disorder characterized by chronic hyperglycemia, due to decreased secretion of insulin and/ or decreased tissue sensitivity to insulin. The sequelae of chronic hyperglycemia in diabetes of all phenotypes are divided into microvascular and macrovascular complications. Microvascular disease causes blindness, renal failure, and neuropathy, and diabetes-accelerated macrovascular disease causes excessive risk for myocardial infarction, stroke, and lower limb amputation. Strict glycemic control has been shown to reduce both microvascular and macrovascular complications of diabetes. However, in contrast to diabetic microvascular disease, it is believed that hyperglycemia is not the major determinant of diabetic macrovascular disease : a large part of cardiovascular disease risk is due to insulin resistance. The link between chronic hyperglycemia and vascular damage has been established by four independent biochemical abnormalities : increased polyol pathway flux, increased formation of Advanced Glycation End-products (AGEs), activation of Protein Kinase C (PKC), and increased hexosamine pathway flux. These seemingly unrelated pathways have an underlying common denominator : overproduction of superoxide by the mitochondrial electron transport chain. Mitochondrial reactive oxygen species (ROS) partially inhibit the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, which diverts increased substrate flux from glycolysis to pathways of glucose overutilization. As for insulin resistance, it causes increased free fatty acid flux from adipocytes into endothelial cells and increased free fatty acid oxidation in macrovascular endothelial cells, resulting in mitochondrial overproduction of ROS by exactly the same mechanism described above about hyperglycemia. Furthermore, metabolic insulin resistance is characterized by pathway-specific impairment in phosphatidylinositol 3-kinase-dependent signaling, which also causes endothelial dysfunction. Preliminary experimental evidence in vivo suggests that these mechanisms leading to diabetic microvascular and macrovascular complications offer a novel basis for research and drug development, targeting to prevention and treatment of angiopathy in Diabetes Mellitus.