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.

The purpose of this study was to investigate the metabolic pathways of methylglyoxal in S. quadricauda.

Methylglyoxal synthetase, which catalyzes the formation of methylglyoxal and inorganic phosphate from dihydroxyacetone phosphate, was found in extracts of Proteus vulgaris. An efficient purification procedure utilizing ion exchange column chromatography and isoelectric focusing has been developed. Homogeneity of the enzyme preparation was confirmed by polyacrylamide gel electrophoresis and rechromatography.Two components of methylglyoxal synthetase were obtained upon isoelectric focusing. A comparison of the chemical and physical properties of the two components was carried out. The enzyme is a dimer. In the presence of inorganic phosphate, the hyperbolic saturation kinetics with dihydroxyacetone phosphate are shifted to sigmoidal.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第24914号 / 農博第2577号 / 新制||農||1103(附属図書館) / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 井上 和生, 教授 佐々木 努, 准教授 後藤 剛 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Methylglyoxal (MG) is a reactive dicarbonyl compound mainly formed during glucose and fructose metabolism. Diabetic patients have increased plasma levels of MG. Our laboratory has shown that treatment with MG induces insulin resistance and type II diabetes in male Sprague-Dawley rats. However, the increases in endogenous MG level attained in different organs and its contribution to the pathogenesis of diabetes following the administration of either high glucose or high fructose diet have not been addressed. The present study aims to investigate whether the harmful effects induced by increased consumption of glucose and/or fructose is linked to increased MG generation. In vitro studies have suggested that L-arginine is an effective MG scavenger. Accordingly, another goal is to determine whether L-arginine pretreatment would scavenge MG under in vivo setting and reduce the harmful effects of hyperglycemia. MG and reduced glutathione (GSH) levels were determined in plasma and urine and in different organs of male Sprague-Dawley rats after 12 weeks of treatment with either high fructose or high glucose diet. GSH plays an important role in the degradation of MG and bears an inverse relationship with the levels of MG. The key results obtained suggest that both diets significantly increased blood pressure and plasma MG levels. A high fructose but not a high glucose diet, increased the plasma total cholesterol, triglycerides levels and total cholesterol/HDL ratio in parallel with the increases in MG and GSH levels in the liver. Increased MG levels seen in both aorta and mesenteric artery induced by high glucose or fructose diet was attenuated by pretreatment with L-arginine. These findings suggest that elevated MG level induced by treatment with high carbohydrate diets in both conduit (aorta) and resistance type (mesneteric artery) vessels may be linked to endothelial dysfunction seen in hyerglycemic/diabetic states. High glucose but not high fructose diet significantly increased MG levels in the pancreas. This observation is consistent with the well-known glucotoxicity caused by hyperglycemia in the pancreas. Taken together, these data provide the first evidence that elevated MG levels in certain organs/tissues following consumption of high fructose and/or glucose diet(s) may play a critical role in contributing to the metabolic abnormalities and the endothelial dysfunction that precedes the onset of macro and microvascular complications in either hyperglycemic and/or type II diabetic states. Interestingly, quenching of elevated MG levels in tissues by pretreamtent with L-arginine overcomes MG-induced vascular damage and endothelial dysfunction caused by high fructose and high glucose diet regimens.

The glyoxalase system, which has been known since 1913, is widely distributed in nature. The system consists of two enzymes, glyoxalase I and glyoxalase II. Methylglyoxal is very unstable and undergoes oxidation and polymerization reactions. One of the purposes of this study was to find a simple, convenient and reproducible method of methylglyoxal preparation. Another objective was the purification of both glyoxalase enzymes employing affinity chromatography as a major step. The purified enzymes were to be characterized by chemical, physical and kinetic properties as an approach to the understanding of the biological function of the system.

Das Vorkommen von alpha-Dicarbonylverbindungen werden in 2 Schwerpunkten dokumentiert. Zum einen werden mehrere Honige auf Ihren Gehalt an 3-DG, GO und MGO neben HMF analysiert und die Bildung wärend einer Lagerung beobachtet. Zum anderen wird die Entstehung von alpha-Dicarbonylen und anderen cytotoxischen Glucoseabbauprodukten bei der Behandlung von Lösungen der Peritonealdialyse mit hohen Druck beobachtet.

Glyco-oxidation is linked to the pathophysiology of diabetes and diabetic complications. The process of glyco-oxidation generates reactive dicarbonyls, which form adducts on arginine residues in distributions throughout the proteome that are site-specific depending on the protein microenvironment. Dicarbonyl adducts are thus markers for glyco-oxidative stress. Various approaches using mass spectrometry permits the identification, localization, and quantification of these dicarbonyl adducts. Using MG as a model dicarbonyl, a shotgun proteomics approach identified the sites for modification of major plasma proteins. Thirty five sites on seven abundant plasma proteins were found, and investigation into the microenvironment surrounding the target arginine sites revealed a neighboring charged residue motif where adjacent residues were either negatively or positively charged. One of the sites identified was R257 in HSA, which is located in the important drug binding site I. We validated drug site I as a target for MG modification by the adaptation of two assays to monitor the effect of MG modification. MG significantly decreases the rate of hydrolysis of PGE2 in drug site I, and induces the displacement of prodan from drug site I. Molecular modeling of warfarin docking at drug site I with the MG-modified R257 resulted in significantly decreased binding and change in binding orientation. The oxidation products of susceptible residues methionine, tryptophan, and cysteine were evaluated using MRM of oxidized HSA peptides. Oxidation of methionine gave the M+16 single oxidized product, and M329 in HSA was the most responsive site. Oxidation of the sole W214 tryptophan produced the W+32 double oxidation product, and oxidation of C34 produced the C+48 triple oxidation product. MG, 3DG, and glucosone were evaluated for propensity to modify 12 HSA sites based on MRM of dicarbonyl modified HSA. Dicarbonyl modification was independent of arginine solvent accessibility. In a clinical study using nephropathy as an endpoint, sites of oxidation and modification of HSA by MG, 3DG, and glucosone were quantified by MRM. The most important variable among diabetic subjects was metformin use, and subjects taking metformin had significantly reduced markers for glyco-oxidation. These findings may be useful in the development of new diabetes therapies that aim to ameliorate glyco-oxidative stress.

Methylglyoxal (MG) is a reactive á-oxoaldehyde and a glucose metabolite. Previous studies in our laboratory have shown that MG induces the production of reactive oxygen species (ROS), such as superoxide (O2.-), nitric oxide (NO) and peroxynitrite (ONOO-), in vascular smooth muscle cells (VSMCs, A-10 cells). However, the effect of endogenous MG and mechanisms of MG-induced oxidative stress have not been thoroughly explored. The present study investigated fructose (a precursor of MG)- induced ONOO- formation in A-10 cells and whether this process was mediated via endogenous MG formation; roles of MG in regulating mitochondrial ROS (mtROS) production and mitochondrial functions in A-10 cells; and effect of MG on neutrophils in patients with type 2 diabetes mellitus (T2DM). Fructose induced intracellular production of MG in a concentration- and time- dependent manner. A significant increase in the production of NO, O2.−, and ONOO− was observed in the cells exposed to fructose or MG. Fructose- or MG-induced ONOO− generation was significantly inhibited by MG scavengers and by O2.− or NO inhibitors. The data showed that fructose treatment increased the formation of ONOO− via increased NO and O2.− production in A-10 cells, and this effect was directly mediated by an elevated intracellular concentration of MG. By inhibiting complex III and manganese superoxide dismutase activities, MG induced mitochondrial overproduction of O2.-, and mitochondrial ONOO- further. MG also reduced mitochondrial ATP synthesis, indicating the dysfunction of mitochondria. In addition, MG increased plasma NO levels in patients with T2DM, which reflected the oxidative status in those patients. MG-induced oxidative stress in patients with T2DM significantly enhanced levels of cytokines released from neutrophils. Moreover, the neutrophils from T2DM patients showed a greater proclivity for apoptosis, which was further increased by in vitro MG treatment. Our data demonstrate that MG-induced oxidative damage, particularly ONOO- production, contributes to the pathogenesis of T2DM and its vascular complications.

Diabetes mellitus is characterized by hyperglycemia, oxidative stress and excessive production of intermediary metabolites including methylglyoxal (MGO), a reactive carbonyl. MGO can readily interact with proteins, lipids and DNA, and cause an imbalance of the cellular antioxidant system leading to carbonyl stress. The effects of MGO can be devastating if the targeted molecules are responsible for the maintenance of membrane potentials and ionic homeostasis. Here we show that MGO disrupts the vascular isoform of ATP-sensitive K+ (KATP) channels by acting on the mRNAs of Kir6.1 and SUR2B subunits thereby regulating vascular tone. Our results show that the 3’ untranslated region (UTR) of Kir6.1 mRNA and the coding region of SUR2B mRNA are targeted by MGO causing a disruption of vascular KATP channels. The destabilization of the mRNAs of KATP channel can in turn affect K+ homeostasis of vascular smooth muscles as well as vascular responses to circulating vasodilators and vasoconstrictors.