Role of interaction between reactive oxygen species and ferroptosis pathway in methylglyoxal-induced injury in mouse embryonic osteoblasts / 南方医科大学学报

OBJECTIVE@#To explore the interaction between reactive oxygen species (ROS) and ferroptosis in methylglyoxalinduced injury of mouse embryonic osteoblasts (MC3T3-E1 cells).@*METHODS@#MC3T3-E1 cells were treated with methylglyoxal to establish a cell model of diabetic osteoporosis. CCK-8 assay was used to detect the viability of MC3T3-E1 cells. Rhodamine 123 staining followed by photofluorography was used to examine mitochondrial membrane potential (MMP). The intracellular ROS level was detected by 2', 7'-dichlorodihydrofluorescein diacetate staining with photofluorograph. Alkaline phosphatase (ALP) activity in the cells was detected using an ALP kit, the number of mineralized nodules was determined with alizarin red S staining, and the level of iron ions was detected using a detection kit. The expression level of glutathione peroxidase 4 (GPX4, a marker protein that inhibits ferroptosis) in the osteoblasts was determined using Western blotting.@*RESULTS@#Treatment of MC3T3-E1 cells with 0.6 mmol/L methylglyoxal for 24 h significantly inhibited the expression level of GPX4 (P < 0.001), increased intracellular iron ion concentration, decreased the cell viability, increased the loss of MMP and intracellular ROS level, decreased both ALP activity and the number of mineralized nodules in the cells (P < 0.001). Co-treatment of MC3T3-E1 cells with 2 mmol/L N-acetylcysteine (NAC, a ROS scavenger) and methylglyoxal significantly increased the expression level of GPX4 (P < 0.01); co-treatment with 4 mmo/L FER-1 (a ferroptosis inhibitor) and methylglyoxal obviously decreased the intracellular ROS level (P < 0.001). Co-treatment of the cells either with NAC and methylglyoxal or with FER-1 and methylglyoxal attenuated methylglyoxal-induced injuries in the osteoblasts (P < 0.001).@*CONCLUSION@#The interaction between ROS and ferroptosis pathway plays an important role in methylglyoxal-induced injury of mouse embryonic osteoblasts.

Advances in regulation of glyoxalase 1 expression in diseases / 中国药理学通报

Glyoxalase 1 (Glo 1) is an MG detoxification enzyme widely present in humans. It is highly conserved among different species and is closely related to the development of cancer, diabetes and nervous system diseases. In this paper, the expression and regulation of Glo lby the Glo 1 copy number variation (CNV), single nucleotide polymorphism (SNP), and transcription factor and non-coding RNA in the above diseases were summarized, aiming to deeply understand the role and the regulation of expression change rules of Glo 1 in the occurrence and development of diseases, so as to provide potential targets for disease prevention and treatment.

Butein protects from PC12 cells apoptosis induced by methylglyoxal via inhibiting p53 signaling pathway / 中国药理学通报

Aim To study the effect of butein on apop-tosis of PC12 cells induced by methylglyoxal (MG) and its mechanism. Methods Being pretreated with different concentrations of butein, PC12 cells were damaged by 1.5 mmol·L-1MG. Cell viability and cell toxicity were evaluated by MTT and LDH assay. Cell apoptosis and death were analyzed by PI and Ho-echst 33342. The antioxidant gene and proapoptotic gene expressions were determined by RT-PCR. The protein expression of p53 was detected by Western blot. Results Being pretreated with 2.5~10 μmol· L-1butein for 1 h significantly increased the cell via-bility,decreased LDH release,and protected from cell nuclei shrinkage, condensation and cleavage by MG. Meanwhile, butein increased the gene expression of SOD2, decreased the gene expression of proapoptotic genes p53 and caspase-9, and lowered the protein ex-pression of p53. Conclusion Butein can protect ap-optosis of PC12 cells from MG in a dose-dependent manner,which is linked with antioxidation and inhibi-ting p53 and caspase-9 gene expression.

Effects of methylglyoxal on oxidative stress and NLRP3 inflammasome signaling in HK-2 cells / 医学研究生学报

Objective Methylglyoxal can cause the injury of human proximal tubular epithelial cell line(HK-2 cells),but the exact mechanism is still unclear. The present study aimed to explore the influence of oxidative stress and the expression of NLRP3 inflammasome in HK-2 cells induced by methylglyoxal. Methods HK-2 cells at logarithmic phase were divided into six groups:control group and 100,200,400,800,1600 μmol/L methylglyoxal groups (cells were cultured in 100,200,400,800,1600 μmol/L methylg-lyoxal concentration for 24 h). Superoxide dismutase(SOD)levels were assayed by thibabituric acid method. Release of lactate dehydro-genase(LDH)activity was detected by assay kit.ROS production was measured by DCFH-DA staining. The expression levels of NLRP3,caspase-1,IL-1β and NF-κB were evaluated by western blot. Results Compared with control group,different methylglyoxal concen-trations could enhance ROS level and LDH activity in HK-2 cells(P<0.05)and reduce SOD level significantly(P<0.05). The results of western blot showed the protein levels of NLRP3,caspase-1,IL-1β and NF-κB were significant up-regulated after the addition of methylglyoxal(P<0.05). Conclusion Methylglyoxal may induce the injury of HK-2 cells by oxidant stress and activating of NLRP3 inflammasome signaling.

Correlation of serum methylglyoxal and brain-derived neurotrophic factor with cognitive function in elderly patients with type 2 diabetes mellitus / 中华内分泌代谢杂志

Objective To investigate the association of serum brain-derived neurotrophic factor (BDNF) and methylglyoxal (MG) levels with cognitive function in elderly patients with type 2 diabetes mellitus (T2DM). Methods The normal population and elderly patients with T2DM were frequency-matched by age, sex, and educational level. BDNF was detected by ELISA assay, MG by HPLC assay, and cognitive function by sets of repetitive mental state examination (RBANS) in the two groups. Results (1) Compared with control group, serum BDNF level in T2DM group was significantly decreased [ (4.97±3.05 vs 11.77±7.92)ng /ml, P<0.01]while serum MG level was elevated [(67.91 vs 43.86) nmol /L, P<0.05]. The increasing of serum MG was related to the decreasing of serum BDNF. (2) Compared with control group, the scores for standardized tests of cognitive scale, visual breadth, immediate memory, delayed memory, and attention areas in T2DM group were significantly decreased (all P<0.05). After the influencing factors were adjusted by multiple regression, the associations of serum BDNF level with cognitive scale standardized score, the delay associated with memory and attention functions were still evident, and serum MG level in T2DM group was still related with the levels of delayed memory, immediate memory, total scale standardization (all P<0.05). (3) Serum BDNF level was negatively correlated with serum MG level (P=0.031). Conclusions Cognitive function of elderly patients with T2DM is related with serum MG and BDNF levels. The increased serum MG as well as the decreasd serum BDNF levels maybe involved in the pathogenesis of impaired cognitive function.

Protective effect of astragalosides IV on retinal pigmentepithelium injury induced by methylglyoxal / 中国药理学通报

Aim To investigate the protective effect of astragaloside IV (AS-Ⅳ) on human retinal pigment epithelium injury induced by methylglyoxal (MGO), and explore its molecular mechanism.Methods The injury of ARPE-19 cells was induced by MGO and the cell viability was measured by CCK-8 method.The morphology of cell nucleus was analyzed by Hoechst 33342 staining and the cell apoptosis was analyzed by flow cytometry to detect labbled Annexin V-FITC/PI.JC-1 staining and fluorescence probe DCFH-DA were employed to evaluate the change of mitochondrial membrane potential and reactive oxygen species (ROS).The levels of SOD, MDA, caspase-9 and caspase-3 were determined by respective kits.Western blot was used to analyse the expression of Bcl-2, Bax and PARP.Results AS-Ⅳ could significantly inhibit the decrease of cell viability induced by MGO, improve the morphology of cell nucleus, reduce the ARPE-19 cell apoptosis rate and the level of ROS and MDA, and increase the activity of SOD.Furthermore, AS-Ⅳ could enhance mitochondrial membrane potential, the ratio of Bcl-2/Bax and the expression of PARP, and inhibit the activation of caspase-9 and caspase-3.Conclusion AS-Ⅳ may protect ARPE-19 cells from the injury induced by MGO by increasing the antioxidant ability of ARPE-19 cells and inhibiting cell apoptosis.

Rapid Identification of Methylglyoxal Trapping Constituents from Onion Peels by Pre-column Incubation Method

The methylglyoxal (MGO) trapping constituents from onion (Allium cepa L.) peels were investigated using pre-column incubation of MGO and crude extract followed by HPLC analysis. The peak areas of MGO trapping compounds decreased, and their chemical structures were identified by HPLC-ESI/MS. Among major constituents in outer scale of onion, 2-(3,4-dihydroxybenzoyl)-2,4,6-trihydroxy-3(2H)-benzofuranone (2) was more effective MGO scavenger than quercetin (6) and its 4′-glucoside, spiraeoside (3). After 1 h incubation, compound 2 trapped over 90% MGO at a concentration of 0.5 mM under physiological conditions, but compounds 3 and 6 scavenged 45%, 16% MGO, respectively. HPLC-ESI/MS showed that compound 2 trapped two molecules of MGO to form a di-MGO adduct and compounds 3 and 6 captured one molecule of MGO to form mono-MGO adducts, and the positions 6 and 8 of the A ring of flavonoids were major active sites for trapping MGO.

Hypericin, a Naphthodianthrone Derivative, Prevents Methylglyoxal-Induced Human Endothelial Cell Dysfunction

Methylglyoxal (MGO) is a highly reactive metabolite of glucose which is known to cause damage and induce apoptosis in endothelial cells. Endothelial cell damage is implicated in the progression of diabetes-associated complications and atherosclerosis. Hypericin, a naphthodianthrone isolated from Hypericum perforatum L. (St. John’s Wort), is a potent and selective inhibitor of protein kinase C and is reported to reduce neuropathic pain. In this work, we investigated the protective effect of hypericin on MGO-induced apoptosis in human umbilical vein endothelial cells (HUVECs). Hypericin showed significant anti-apoptotic activity in MGO-treated HUVECs. Pretreatment with hypericin significantly inhibited MGO-induced changes in cell morphology, cell death, and production of intracellular reactive oxygen species. Hypericin prevented MGO-induced apoptosis in HUVECs by increasing Bcl-2 expression and decreasing Bax expression. MGO was found to activate mitogen-activated protein kinases (MAPKs). Pretreatment with hypericin strongly inhibited the activation of MAPKs, including P38, JNK, and ERK1/2. Interestingly, hypericin also inhibited the formation of AGEs. These findings suggest that hypericin may be an effective regulator of MGO-induced apoptosis. In conclusion, hypericin downregulated the formation of AGEs and ameliorated MGO-induced dysfunction in human endothelial cells.

N-acetylcysteine protects H9c2 cells against injuries induced by methyl-glyoxal / 中国病理生理杂志

AIM:To investigate the protective effect of N-acetylcysteine (NAC) on H9c2 cells from injuries induced by methylglyoxal (MG) and the potential mechanism.METHODS:H9c2 cells were divided into control group, MG treatment group, NAC +MG treatment group, SP600125 pretreatment +MG group, NAC group and SP600125 group.The viability of the H9c2 cells was measured by CCK-8 assay.The protein levels of p-JNK and t-JNK were tested by Western blot .The changes of intracellular reactive oxygen species ( ROS) were evaluated by 2′, 7′-dichlorofluorescein di-acetate (DCFH-DA) staining.Mitochondrial membrane potential (MMP) was measured by rhodamine 123 (Rh123) stai-ning.The morphological changes in apoptotic cardiomyocytes were detected by Hoechst 33258 staining.RESULTS: Du-ring 100~800 μmol/L concentration range , MG caused significantly reduced viability of the H 9c2 cells in a dose-depend-ent manner.NAC had a protective effect on H9c2 cells against the injuries induced by MG during 500~1 500μmol/L con-centration range through raising cell viability , inhibiting cellular oxidative stress and improving MMP ( P <0.01 ) . SP600125,an inhibitor of JNK, showed the protective effect similar to NAC on H9c2 cells against MG-induced injuries, in-cluding attenuating oxidative stress , improving MMP and suppressing apoptosis .CONCLUSION: N-acetylcysteine offers obvious protective effect on H9c2 cells against the injuries induced by methylglyoxal .The underlying mechanisms may be associated with decreasing the production of ROS , ameliorating MMP , inhibiting the activation of JNK and suppressing ap-optosis.

No short-term effects of calorie-controlled Mediterranean or fast food dietary interventions on established biomarkers of vascular or metabolic risk in healthy individuals

BACKGROUND/OBJECTIVES: This study addressed the question whether the composition of supposedly 'healthy' or 'unhealthy' dietary regimes has a calorie-independent short-term effect on biomarkers of metabolic stress and vascular risk in healthy individuals. SUBJECTS/METHODS: Healthy male volunteers (age 29.5 +/- 5.9 years, n = 39) were given a standardized baseline diet for two weeks before randomization into three groups of different dietary regimes: fast food, Mediterranean and German cooking style. Importantly, the amount of calories consumed per day was identical in all three groups. Blood samples were analyzed for biomarkers of cardiovascular risk and metabolic stress after two weeks of the baseline diet and after two weeks of the assigned dietary regime. RESULTS: No dietary intervention affected the metabolic or cardiovascular risk profile when compared in-between groups or compared to baseline. Subjects applied to the Mediterranean diet showed a statistically significant increase of uric acid compared to baseline and compared to the German diet group. Plasma concentrations of urea were significantly higher in both the fast food group and the Mediterranean group, when compared to baseline and compared to the German diet group. No significant differences were detected for the levels of vitamins, trace elements or metabolic stress markers (8-hydroxy-2-deoxyguanosine, malondialdehyde and methylglyoxal, a potent glycating agent). Established parameters of vascular risk (e.g. LDL-cholesterol, lipoprotein(a), homocysteine) were not significantly changed in-between groups or compared to baseline during the intervention period. CONCLUSIONS: The calorie-controlled dietary intervention caused neither protective nor harmful short-term effects regarding established biomarkers of vascular or metabolic risk. When avoiding the noxious effects of overfeeding, healthy individuals can possess the metabolic capacity to compensate for a potentially disadvantageous composition of a certain diet.

No short-term effects of calorie-controlled Mediterranean or fast food dietary interventions on established biomarkers of vascular or metabolic risk in healthy individuals

BACKGROUND/OBJECTIVES: This study addressed the question whether the composition of supposedly 'healthy' or 'unhealthy' dietary regimes has a calorie-independent short-term effect on biomarkers of metabolic stress and vascular risk in healthy individuals. SUBJECTS/METHODS: Healthy male volunteers (age 29.5 +/- 5.9 years, n = 39) were given a standardized baseline diet for two weeks before randomization into three groups of different dietary regimes: fast food, Mediterranean and German cooking style. Importantly, the amount of calories consumed per day was identical in all three groups. Blood samples were analyzed for biomarkers of cardiovascular risk and metabolic stress after two weeks of the baseline diet and after two weeks of the assigned dietary regime. RESULTS: No dietary intervention affected the metabolic or cardiovascular risk profile when compared in-between groups or compared to baseline. Subjects applied to the Mediterranean diet showed a statistically significant increase of uric acid compared to baseline and compared to the German diet group. Plasma concentrations of urea were significantly higher in both the fast food group and the Mediterranean group, when compared to baseline and compared to the German diet group. No significant differences were detected for the levels of vitamins, trace elements or metabolic stress markers (8-hydroxy-2-deoxyguanosine, malondialdehyde and methylglyoxal, a potent glycating agent). Established parameters of vascular risk (e.g. LDL-cholesterol, lipoprotein(a), homocysteine) were not significantly changed in-between groups or compared to baseline during the intervention period. CONCLUSIONS: The calorie-controlled dietary intervention caused neither protective nor harmful short-term effects regarding established biomarkers of vascular or metabolic risk. When avoiding the noxious effects of overfeeding, healthy individuals can possess the metabolic capacity to compensate for a potentially disadvantageous composition of a certain diet.

Acetilação radicalar de amino ácidos, peptídeos e nucleobases pelos sistemas biacetilo/peroxinitrito e metilglioxal/peroxinitrito / Radical acetylation of aminoacids, peptides, and nucleobases by the biacetyl or methylglyoxal/peroxynitrite systems

Biacetilo (2,3-butanediona) é um contaminante de comida e cigarro, também implicado na hepatoxicidade do álcool e em doenças pulmonares. O metilglioxal (MG), um α-oxoaldeído reativo frequentemente associado ao diabetes e envelhecimento, é produto da fragmentação oxidativa de trioses fosfato, acetona e aminoacetona. Por sua vez, peroxinitrito - um potente oxidante, agente nitrante e nucleófilo formado in vivo pela reação controlada por difusão do ânion radical superóxido com o radical óxido nítrico (k ~1010 M-1s-1) é capaz de se adicionar a CO2 e compostos carbonílicos, gerando produtos potencialmente tóxicos ou sinalizadores celulares. Aminoácidos, peptídeos e nucleobases podem ser acetilados nos grupos amina e na porção desoxiribose. Relativamente ao tratamento com peroxinitrito isolado, níveis superiores de 3-nitrotirosina foram detectados quando tirosina foi tratada com peroxinitrito/biacetilo ou metilglioxal. Ambos os grupos amina de lisina (Lys) ou um deles de derivados de lisina bloqueados e um deles (Ac-Lys-OMe, Z-Lys-OMe) foram acetilados pelo sistema biacetilo ou metilglioxal/peroxinitrito. Em tetrapeptídeos sintéticos contendo lisina como aminoácido amino-terminal (H-KALA-OH, Ac-KALA-OH and H-K(Boc)ALA-OH), a lisina foi acetilada pelo sistemas dicarbonilico/peroxinitrito no grupo α-amina (em maior extensão) e/ou no ε-amina (em menor extensão). No conjunto, estes resultados podem ser interpretados à luz do mecanismo proposto para a reação de compostos α-dicarbonílicos com peroxinitrito, o qual envolve sequencialmente: (i) adição nucleofílica de peroxinitrito à carbonila; (ii) homólise do aduto peroxinitroso formado, liberando •NO2 e um radical oxila do reagente carbonílico; (iii) β-clivagem do radical oxila a um ácido carboxílico (ácido acético no caso de biacetilo e ácido fórmico, a partir de metilglioxal) e radical acetila; (iv) captação do radical acetila pelo oxigênio molecular dissolvido dando acetato, ou por aminoácido ou nucleobase...

Diacetyl (2,3-butanedione) is a food and cigarette contaminant recently implicated in alcohol hepatotoxicity and lung disease. In turn, methylglyoxal (MG) is an α-oxoaldehyde frequently associated with diabetes and aging that is putatively formed by the oxidative fragmentation of trioses phosphate, acetone and aminoacetone. Peroxynitrite - a potent oxidant, nitrating agent and nucleophile formed in vivo by the diffusion-controlled reaction of superoxide radical with nitric oxide (k ~1010 M-1s-1) - is able to form adducts with carbon dioxide and carbonyl compounds. When initially present in the reaction mixtures before addition of ONOO-, amino acids, peptides and nucleobases undergo acetylation at the amino group and purine moieties in the presence of biacetyl or methylglyoxal. Higher levels of 3-nitrotyrosine nitration were measured when peroxynitrite/biacetyl or metilglioxal was added to tyrosine, in comparison with peroxynitrite alone. Both amino groups of L-lysine or one of the amino groups of L-lysine derivatives (Z-Lys-OH and Ac-Lys-OH) were acetylated by biacetyl and methylglyoxal/peroxynitrite system. Using tetrapeptides containing lysine at the terminal amino acid (H-KALA-OH, Ac-KALA-OH and H-K(Boc)ALA-OH), the lysine residue was acetylated at both or either α-amino (major adduct) and ε-amino group (minor adduct). Altogether these data can be interpreted by the mechanism proposed to describe the reaction of α-dicarbonyls with peroxynitrite as follows: (i) nucleophilic addition of peroxynitrite to the carbonyl group of the reagent; (ii) homolysis of the formed peroxynitroso carbonyl adduct to •NO2 and a carbonyloxyl radical; (iii) β-cleavage of the oxyl radical to acetyl radical plus acetic acid (from diacetyl) or formic acid (from methylglyoxal); (iv) competitive scavenging of the acetyl radical by dissolved molecular oxygen and by added amino acid, peptide or nucleobase, ultimately yielding acetate or acetylated biomolecule. If occurring in vivo...

Preferential recognition of methylglyoxal-modified calf thymus DNA by circulating antibodies in cancer patients.

Methylglyoxal (MG) has been implicated in mutagenesis and cancer. Present study probes the antigenicity of MG damaged DNA in cancer patients. Purified calf thymus DNA was damaged by the synergistic action of MG, lysine (Lys) and CuSO4 for 24 h at 37oC. DNA modifications produced single-strand breaks, hyperchromicity in UV spectrum and increased fluorescence intensity. Binding characteristics of auto-antibodies in cancer patients were assessed by direct binding and inhibition ELISA. These antibodies exhibited enhanced binding with the modified DNA, as compared to the native form. The effect was more pronounced when affinity-purified IgG was used in place of the serum. In conclusion, MG-modified DNA presents unique epitopes which are recognized as non-self by the immune system and may, therefore, be one of the factors for the autoantibody induction in cancer patients.

Distinct synergistic action of piperacillin and methylglyoxal against Pseudomonas aeruginosa.

The dicarbonyl compound methylglyoxal is a natural constituent of Manuka honey produced from Manuka flowers in New Zealand. It is known to possess both anticancer and antibacterial activity. Such observations prompted to investigate the ability of methylglyoxal as a potent drug against multidrug resistant Pseudomonas aeruginosa. A total of 12 test P. aeruginosa strains isolated from various hospitals were tested for their resistances against many antibiotics, most of which are applied in the treatment of P. aeruginosa infections. Results revealed that the strains were resistant to many drugs at high levels, only piperacillin, carbenicillin, amikacin and ciprofloxacin showed resistances at comparatively lower levels. Following multiple experimentations it was observed that methylglyoxal was also antimicrobic against all the strains at comparable levels. Distinct and statistically significant synergism was observed between methylglyoxal and piperacillin by disc diffusion tests when compared with their individual effects. The fractional inhibitory concentration index of this combination evaluated by checkerboard analysis, was 0.5, which confirmed synergism between the pair. Synergism was also noted when methylglyoxal was combined with carbenicillin and amikacin.

Mecanismo de oxidação aeróbica de acetoacetato e 2-metilacetoacetato catalisada por mioglobina: implicações em desordens cetogênicas / Mechanism of the aerobic oxidation of acetoacetate and 2- methylacetoacetate catalyzed by Mb: implications for ketogenic disorders

Acetoacetato (AA) e 2-metilacetoacetato (MAA) são compostos β-cetoácidos acumulados em diversas desordens metabólicas como no diabetes e na isoleucinemia, respectivamente. Examinamos o mecanismo de oxidação aeróbica de AA e MAA iniciada por intermediários reativos de mioglobina de coração de cavalo (Mb) gerados pela adição de H2O2. Uma rota quimioluminescente que envolve um intermediário dioxetânico cuja termólise gera espécies α-dicarbonílicas (metilglioxal e biacetilo) foi proposta e estudada. Emissão de luz ultra fraca acompanha a reação, e sua intensidade aumenta linearmente pelo aumento da concentração tanto de Mb (10-500 µM) quando AA (10-100 mM). Estudos de consumo de oxigênio mostraram que MAA é, como esperado, quase uma ordem de grandeza mais reativo que AA. Estudos de EPR com captação de spin, utilizando MNP, possibilitaram detectar adutos de MAA atribuíveis a um radical centrado no Cα (aN = 1.55 mT) e ao radical acetila (aN = 0.83 mT). O sinal do radical acetila é totalmente suprimido por sorbato, um conhecido e eficiente supressor de espécies tripletes, o que é consistente com uma rota reacional envolvendo um intermediário dioxetânico. Clivagem-α da ligação carbonila-carbonila do produto biacetilo triplete produziria, de fato, radicais acetila. Além disso, utilizando AA como substrato para Mb/H2O2, um sinal de EPR atribuível ao aduto MNP-AA• (aN = 1.46 mT e aH = 0.34 mT) foi observado e confirmado por efeito isotópico. O consumo de oxigênio e o rendimento de compostos α-dicarbonílicos foram dose-dependentes à concentração de AA ou MAA (1-50 mM) bem como à concentração de H2O2 adicionado às misturas de reação contendo Mb (até 1:10 quando medido o consumo de oxigênio, e até 1:25 quando medido o rendimento de compostos α-dicarbonílicos) e tert-butilhidroperóxido (até 1:200). Os perfis de pH (5,8-7,8) para consumo de oxigênio e rendimento de compostos α-dicarbonílicos mostraram maiores rendimentos para baixos valores de pH, indicativo de ferrilMb...

Acetoacetate (AA) and 2-methylacetoacetate (MAA) are β-ketoacids accumulated in several metabolic disorders such as diabetes and isoleucinemia, respectively. Here we examine the mechanism of AA and MAA aerobic oxidation initiated by the reactive enzyme intermediates formed by the reaction of muscle horse myoglobin (Mb) with H2O2. A chemiluminescent route involving a dioxetane intermediate whose thermolysis yields triplet α-dicarbonyl species (methylglyoxal and diacetyl) is envisaged. Accordingly, the ultraweak light emission that accompanies the reaction increases linearly by raising the concentration of both Mb (10-500 µM) and AA (10- 100 mM). Oxygen uptake studies revealed that MAA is, expectedly, almost one order of magnitude more reactive than AA. EPR spin-trapping studies with MNP detected spin adducts from MAA attributable to an α-carbon-centered radical (aN = 1.55 mT) and to an acetyl radical (aN = 0.83 mT). As the acetyl radical signal is totally suppressed by sorbate, a well-known efficient triplet species quencher, the dioxetane hypothesis seems to be reliable. The α-cleavage of the carbonyl-carbonyl bond of a putative excited triplet diacetyl product would, in fact, leads to an acetyl radical. Furthermore, using AA as substrate for Mb/H2O2, an EPR signal assignable to a MNP-AA• adduct (aN = 1.46 mT and aH = 0.34 mT) was observed and confirmed by isotope effect. Oxygen consumption and α-dicarbonyl yield were also dependent on AA or MAA concentrations (1-50 mM) as well as on the concentration of peroxide added to the Mb-containing reaction mixtures: H2O2 (up to 1:10 when measuring oxygen uptake and up to 1:25 when measuring the α-dicarbonyl yield) and t-butOOH (up to 1:200). The pH profiles (5.8-7.8) of oxygen consumption and α-dicarbonyl yield show higher reaction rates at lower pHs, indicative of a ferrylMb intermediate. Evaluating Mb lesion, both β-ketoacids reduced disorganization of the secondary and tertiary protein structure elicited by H2O2...

The relationship between glyoxalaseⅠand diabetic complications / 中国药理学通报

GlyoxalaseⅠthat presents in all human tissues detoxifies α-oxoaldehydes and prevents the formation of advanced glycation end products(AGEs).AGEs and their main precursor namely methylglyoxal(MG)produce cytotoxicity and have extensive relationship with diabetic complications.Therefore,to elevate glyoxalaseⅠactivity may be a new path to release such complications.

The cytotoxic effect of methylglyoxal on BDNF and TrkB expression in rat hippocampal neurons / 中华行为医学与脑科学杂志

Objective To investigate the mechanisms of methylglyoxal(MG)-induced injury of hippocam-pal neurons. Methods Primary cultured of hippocampal neurons from 1-day-old Sprague-Dawley rats were incuba-ted with MG for different time and dose period. Cells proliferation were assayed by methyl thiazolyl tetrazolium (MTT),and apoptosis was quantified by flow cytometer using annexin V-FITC and propidium iodide (PI) stai-ning. The protein and mRNA levels of brain-derived neurotrophie factor (BDNF) and tyrosine kinase B(TrkB) were assayed with Western Blotting and real-time PCR. Results Treatment with MG resulted in a concentration-dependent (r=0.946, P < 0.01) and time-dependent (r=0.993, P < 0.01) decreasing neurons viability. Com-pared with Oh group(1. 633±0. 153)%, 100 μM MG treatment for 2h,6h, 12h and 24h,the cellular apeptosis rate were significantly increased ((2. 833±0. 153)%, (3. 367±0. 153)%, (4. 433±0. 404)% and (8. 833± 0. 306)% respectivdy,all P<0.01). MG also increased the BDNF mRNA and protein expression after 12h treat-ment (P<0.05 or P<0.01),but decreased the TrkB mRNA and protein expression in the cells after 6h treatment (P<0.05 or P < 0.01). Conclusion MG has direct toxic effect on hippocampal neurons and can impaire the BD-NF-TrkB signal pathway by inhibiting the expression of TrkB,and increasing the apoptosis of hippocampal neurons.

Toxicidade de aminoacetona e células produtoras de insulina / Cytotoxity of aminoacetone on insulin-producing cells

Danos induzidos por hiperglicemia em tecidos no diabetes são caracterizados por quatro mecanismos conectados: aumento do fluxo metabólico através da via do poliol, ativação da proteína quinase C (PKC), aumento da atividade da via das hexosaminas e aumento da produção intracelular dos precursores dos produtos finais de glicação avançada (AGEs). Entre eles, os derivados de metilglioxal, um potente agente de modificação de proteínas e DNA, têm sido associados a complicações microvasculares no diabetes: nefropatia, retinopatia e neuropatia. O metilglioxal é produzido a partir das trioses fosfato, acetona e aminoacetona, um catabólito de treonina e glicina, gerado na matriz mitocondrial. A aminoacetona sofre oxidação enzimática, catalisada por aminoxidase sensível a semicarbazida (SSAO), ou química, catalisada por íons de cobre e ferro, produzindo metilglioxal, H2O2 e NH4 +. Sabendo que metilglioxal e H2O2 são capazes de induzir apoptose e/ou necrose em células produtoras de insulina (RINm5f) propomos uma possível atividade pró-oxidante da aminoacetona sobre células beta do pâncreas. O tratamento destas linhagens com aminoacetona/Cu(II) aumentou a morte celular, fluxo de Ca2+ intracelular, produção de NO, fragmentação do DNA, depleção dos níveis de glutationa reduzida (GSH), expressão gênica da proteína apoptótica Bax, enzimas antioxidantes - glutationa peroxidase (GPx), glutationa redutase (GRd), catalase e isoformas de superóxido dismutases (CuZnSOD e MnSOD) - e óxido nítrico sintase induzida (iNOS). Embora as concentrações normais e patológicas da aminoacetona, provavelmente seja muito menores que as usadas nos experimentos, sugerimos que, em tecidos de diabéticos, um acúmulo da aminoacetona em longo prazo pode conduzir a danos oxidativos e eventualmente morte das células beta do pâncreas.

Tissue damages induced by hyperglycemia in diabetics are characterized by four linked mechanisms: increased flux through the polyol pathway, protein kinase C (PKC) activation, increased hexosamine pathway activity and intracellular production of advanced glycation end product (AGE) precursors. The production of AGEs by modifying proteins and DNA agent, such as methylglyoxal, has been implicated in microvascular complications in diabetes: nephropathy, retinopathy and neuropathy. Methylglyoxal is putatively produced in vivo from trioses phosphate, acetone and aminoacetone, a catabolite of threonine and glycine synthesized in the mitochondrial matrix. Aminoacetone has been reported to undergo semicarbazide sensitive amine oxidase- catalyzed and copper- and iron-catalyzed oxidations by molecular oxygen to methylglyoxal, NH4 + ion and H2O2. Considering that methylglyoxal and H2O2 have been found to promote apoptosis/necrosis to insulin-producing cells (RINm5f), we propose a possible pro-oxidant role of aminoacetone in pancreatic beta-cells. Treatment of RINm5f cells with aminoacetone plus Cu(II) ion promotes an increase of non-viable cells, influx of Ca2+ ions, NO production, DNA fragmentation, depletion of reduced glutathione (GSH) levels, and increased mRNA expression of pro-apoptotic protein (Bax), antioxidant enzymes - glutathione peroxidase (GPx), glutathione reductase (GRd), MnSOD, CuZnSOD and catalase - and inducible nitric oxide synthase (iNOS). Although both normal and pathological concentrations of aminoacetone are probably much lower than those used here, it is tempting to propose that excess aminoacetone in diabetic patients, at long term, may drive oxidative damage and eventually death of pancreatic beta-cells.

Effect of Methylglyoxal on the Oxidative Stress in Trabecular Meshwork Cells

PURPOSE: To investigate the effect of methylglyoxal (MG), intermediate metabolite of advanced glycation end products(AGE), on the induction of oxidative stress in human trabecular meshwork cells (HTMC). METHODS: Primarily cultured HTMC were exposed to at concentrations of 0, 30, 100, and 300 micrometer of MG for 18 hours, with or without co-exposure to N-acetyl-cysteine. Cellular survival and apoptosis were assessed by MTT assay and flow cytometry using annexin-PI double staining. Production of nitric oxide (NO), superoxide, and reactive oxygen species (ROS) was assessed by Griess assay, cytochrome c assay, and dichlorofluorescein diacetate assay, respectively. RESULTS: MG did not affect cellular survival at concentrations under 100 micrometer, but induced apoptosis of HTMC at concentrations over 100 micrometer. MG decreased NO production, accompanied with increased superoxide production. In addition, MG increased ROS, which were abolished by N-acetylcysteine. CONCLUSIONS: MG induced oxidative stress by decreasing NO production, accompanied by increasing superoxide and ROS productions in HTMC. AGE could induce trabecular meshwork dysfunction.

Methylglyoxal Induces Apoptosis Mediated by Reactive Oxygen Species in Bovine Retinal Pericytes

One of the histopathologic hallmarks of early diabetic retinopathy is the loss of pericytes. Evidences suggest that the pericyte loss in vivo is mediated by apoptosis. However, the underlying cause of pericyte apoptosis is not fully understood. This study investigated the influence of methylglyoxal (MGO), a reactive -dicarbonyl compound of glucose metabolism, on apoptotic cell death in bovine retinal pericytes. Analysis of internucleosomal DNA fragmentation by ELISA showed that MGO (200 to 800 micrometer) induced apoptosis in a concentration-dependent manner. Intracellular reactive oxygen species were generated earlier and the antioxidant, N-acetyl cysteine, inhibited the MGO-induced apoptosis. NF-kB activation and increased caspase- 3 activity were detected. Apoptosis was also inhibited by the caspase-3 inhibitor, Z-DEVD-fmk, or the NF- kB inhibitor, pyrrolidine dithiocarbamate. These data suggest that elevated MGO levels observed in diabetes may cause apoptosis in bovine retinal pericytes through an oxidative stress mechanism and suggests that the nuclear activation of NF-kB are involved in the apoptotic process.