Iron is necessary for life, but the simultaneous iron-catalyzed formation of reactive oxygen species (ROS) is involved in pathogenesis of many diseases. One of them is diabetes mellitus, a widespread disease with severe long-term complications, including neuropathy, retinopathy, and nephropathy. Much evidence points to methylglyoxal, a potent glycating agent, as the key mediator of diabetic complications. In diabetes, there is also a peculiar dysregulation of iron homeostasis, leading to an expansion of redox-active iron. This in vitro study focuses on the interaction of methylglyoxal with ferritin, which is the main cellular protein for iron storage. Methylglyoxal effectively liberates iron from horse spleen ferritin, as well as synthetic iron cores; in both cases, it is partially mediated by superoxide. The interaction of methylglyoxal with ferritin increases the production of hydrogen peroxide, much above the generation of peroxide by methylglyoxal alone, in an iron-dependent manner. Glycation with methylglyoxal results in structural changes in ferritin. All of these findings can be demonstrated with pathophysiologically relevant (submillimolar) methylglyoxal concentrations. However, the rate of iron release by ascorbate, the ferroxidase activity, or the diameter of gated pores even in intensely glycated ferritin is not altered. In conclusion, although the functional features of ferritin resist alterations due to glycation, the interaction of methylglyoxal with ferritin liberates iron and markedly increases ROS production, both of which could enhance oxidative stress in vivo. Our findings may have implications for the pathogenesis of long-term diabetic complications, as well as for the use of ferritin as a nanocarrier in chemotherapy.
Ferritin with methylglyoxal generates both Fenton reagents, ferrous iron, and hydrogen peroxide.Methylglyoxal produces structural modifications in ferritin that are detectable even in excess of another protein.Even heavily glycated ferritin has been found to resist functional alteration.
- Klíčová slova
- Glycation, diabetes mellitus, labile iron pool, protein cross-links, resistance to oxidative stress,
- MeSH
- ferritiny * metabolismus MeSH
- koně MeSH
- lidé MeSH
- pyruvaldehyd * metabolismus MeSH
- reaktivní formy kyslíku * metabolismus MeSH
- železo metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- ferritiny * MeSH
- pyruvaldehyd * MeSH
- reaktivní formy kyslíku * MeSH
- železo MeSH
A side effect of diabetes is formation of glycated proteins and, from them, production of advanced early glycation end products that could determine aberrant immune responses at the systemic level. We investigated a relevant aberrant post-translational modification (PTM) in diabetes based on synthetic peptides modified on the lysine side chain residues with 1-deoxyfructopyranosyl moiety as a possible modification related to glycation. The PTM peptides were used as molecular probes for detection of possible specific autoantibodies developed by diabetic patients. The PDC-E2(167-186) sequence from the pyruvate dehydrogenase complex was selected and tested as a candidate peptide for antibody detection. The structure-based designed type I' β-turn CSF114 peptide was also used as a synthetic scaffold. Twenty-seven consecutive type 1 diabetic patients and 29 healthy controls were recruited for the study. In principle, the 'chemical reverse approach', based on the use of patient sera to screen the synthetic modified peptides, leads to the identification of specific probes able to characterize highly specific autoantibodies as disease biomarkers of autoimmune disorders. Quite surprisingly, both peptides modified with the (1-deoxyfructosyl)-lysine did not lead to significant results. Both IgG and IgM differences between the two populations were not significant. These data can be rationalized considering that i) IgGs in diabetic subjects exhibit a high degree of glycation, leading to decreased functionality; ii) IgGs in diabetic subjects exhibit a privileged response vs proteins containing advanced glycation products (e.g., methylglyoxal, glyoxal, glucosone, hydroimidazolone, dihydroxyimidazolidine) and only a minor one with respect to (1-deoxyfructosyl)-lysine.
- MeSH
- diabetes mellitus 1. typu metabolismus MeSH
- glykosylace MeSH
- glyoxal metabolismus MeSH
- imidazoly metabolismus MeSH
- imunoanalýza MeSH
- ketosy metabolismus MeSH
- lidé MeSH
- lysin chemie metabolismus MeSH
- peptidy chemie metabolismus MeSH
- posttranslační úpravy proteinů MeSH
- produkty pokročilé glykace metabolismus MeSH
- pyruvaldehyd metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- glucosone MeSH Prohlížeč
- glyoxal MeSH
- imidazolone MeSH Prohlížeč
- imidazoly MeSH
- ketosy MeSH
- lysin MeSH
- peptidy MeSH
- produkty pokročilé glykace MeSH
- pyruvaldehyd MeSH
The great research interest in the quantification of reactive carbonyl compounds (RCCs), such as methylglyoxal (MGO) in biological and environmental samples, is reflected by the fact that several publications have described specific strategies to perform this task. Thus, many reagents have also been reported for the derivatization of RCCs to effectively detect and quantify the resulting compounds using sensitive techniques such as liquid chromatography coupled with mass spectrometry (LC-MS). However, the choice of the derivatization protocol is not always clear, and a comparative evaluation is not feasible because detection limits from separate reports and determined with different instruments are hardly comparable. Consequently, for a systematic comparison, we tested 21 agents in one experimental setup for derivatization of RCCs prior to LC-MS analysis. This consisted of seven commonly employed reagents and 14 similar reagents, three of which were designed and synthesized by us. All reagents were probed for analytical responsiveness of the derivatives and stability of the reaction mixtures. The results showed that derivatives of 4-methoxyphenylenediamine and 3-methoxyphenylhydrazine-reported here for the first time for derivatization of RCCs-provided a particularly high responsiveness with ESI-MS detection. We applied the protocol to investigate MGO contamination of laboratory water and show successful quantification in a lipoxidation experiment. In summary, our results provide valuable information for scientists in establishing accurate analysis of RCCs.
- Klíčová slova
- carbonyl derivatization, hydroxylamine, lipoxidation, phenylenediamine, phenylhydrazine, water analysis,
- MeSH
- chemické látky znečišťující vodu analýza MeSH
- chromatografie kapalinová metody MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací metody MeSH
- laboratoře normy MeSH
- limita detekce MeSH
- pyruvaldehyd analýza chemie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- chemické látky znečišťující vodu MeSH
- pyruvaldehyd MeSH
Methylglyoxal production is increased in diabetes. Methylglyoxal is efficiently detoxified by enzyme glyoxalase 1 (GLO1). The aim was to study the effect of diabetic and CKD milieu on (a) GLO1 gene expression in peripheral blood mononuclear cells; (b) GLO1 protein levels in whole blood; and (c) GLO1 activity in RBCs in vivo in diabetic vs. non-diabetic subjects with normal or slightly reduced vs. considerably reduced renal function (CKD1-2 vs. CKD3-4). A total of 83 subjects were included in the study. Gene expression was measured using real-time PCR, and protein levels were quantified using Western blotting. Erythrocyte GLO1 activity was measured spectrophotometrically. GLO1 gene expression was significantly higher in subjects with CKD1-2 compared to CKD3-4. GLO1 protein level was lower in diabetics than in non-diabetics. GLO1 activity in RBCs differed between the four groups being significantly higher in diabetics with CKD1-2 vs. healthy subjects and vs. nondiabeticsfig with CKD3-4. GLO1 activity was significantly higher in diabetics compared to nondiabetics. In conclusion, both diabetes and CKD affects the glyoxalase system. It appears that CKD in advanced stages has prevailing and suppressive effects compared to hyperglycaemia. CKD decreases GLO1 gene expression and protein levels (together with diabetes) without concomitant changes of GLO1 activity.
- Klíčová slova
- chronic kidney disease, diabetes, diabetic nephropathy, glyoxalase,
- MeSH
- chronická renální insuficience krev patologie MeSH
- diabetes mellitus krev patologie MeSH
- diabetické nefropatie krev patologie MeSH
- laktoylglutathionlyasa krev MeSH
- lidé středního věku MeSH
- lidé MeSH
- pyruvaldehyd krev MeSH
- senioři MeSH
- studie případů a kontrol MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- GLO1 protein, human MeSH Prohlížeč
- laktoylglutathionlyasa MeSH
- pyruvaldehyd MeSH
Neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), are increasing in prevalence. Currently, there are no effective and specific treatments for these disorders. Recently, positive effects of the orexigenic hormone ghrelin on memory and learning were demonstrated in mouse models of AD and PD. In this study, we tested the potential neuroprotective properties of a stable and long-lasting ghrelin analog, Dpr(3)ghrelin (Dpr(3)ghr), in SH-SY5Y neuroblastoma cells stressed with 1.2 mM methylglyoxal (MG), a toxic endogenous by-product of glycolysis, and we examined the impact of Dpr(3)ghr on apoptosis. Pre-treatment with both 10(-5) and 10(-7) M Dpr(3)ghr resulted in increased viability in SH-SY5Y cells (determined by MTT staining), as well as reduced cytotoxicity of MG in these cells (determined by LDH assay). Dpr(3)ghr increased viability by altering pro-apoptotic and viability markers: Bax was decreased, Bcl-2 was increased, and the Bax/Bcl-2 ratio was attenuated. The ghrelin receptor GHS-R1 and Dpr(3)ghr-induced activation of PBK/Akt were immuno-detected in SH-SY5Y cells to demonstrate the presence of GHS-R1 and GHS-R1 activation, respectively. We demonstrated that Dpr(3)ghr protected SH-SY5Y cells against MG-induced neurotoxicity and apoptosis. Our data suggest that stable ghrelin analogs may be candidates for the effective treatment of neurodegenerative disorders.
- MeSH
- apoptóza účinky léků MeSH
- ghrelin analogy a deriváty farmakologie MeSH
- glykolýza účinky léků MeSH
- L-laktátdehydrogenasa metabolismus MeSH
- lidé MeSH
- MAP kinasový signální systém účinky léků MeSH
- membránový potenciál mitochondrií účinky léků MeSH
- nádorové buněčné linie MeSH
- neuroprotektivní látky farmakologie MeSH
- neurotoxické syndromy prevence a kontrola MeSH
- proteiny regulující apoptózu biosyntéza genetika MeSH
- pyruvaldehyd toxicita MeSH
- receptory ghrelinu biosyntéza MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- ghrelin MeSH
- Ghsr1a protein, human MeSH Prohlížeč
- L-laktátdehydrogenasa MeSH
- neuroprotektivní látky MeSH
- proteiny regulující apoptózu MeSH
- pyruvaldehyd MeSH
- receptory ghrelinu MeSH
Reactive dicarbonyls stimulate production of advanced glycation endproducts, increase oxidative stress and inflammation and contribute to the development of vascular complications. We measured concentrations of dicarbonyls - methylglyoxal (MG), glyoxal (GL) and 3-deoxyglucosone (3-DG) - in the heart and kidney of a model of metabolic syndrome - hereditary hypertriglyceridemic rats (HHTg) and explored its modulation by metformin. Adult HHTg rats were fed a standard diet with or without metformin (300 mg/kg b.w.) and dicarbonyl levels and metabolic parameters were measured. HHTg rats had markedly elevated serum levels of triacylglycerols (p<0.001), FFA (p<0.01) and hepatic triacylglycerols (p<0.001) along with increased concentrations of reactive dicarbonyls in myocardium (MG: p<0.001; GL: p<0.01; 3-DG: p<0.01) and kidney cortex (MG: p<0.01). Metformin treatment significantly reduced reactive dicarbonyls in the myocardium (MG: p<0.05, GL: p<0.05, 3-DG: p<0.01) along with increase of myocardial concentrations of reduced glutathione (p<0.01) and glyoxalase 1 mRNA expression (p<0.05). Metformin did not have any significant effect on dicarbonyls, glutathione or on glyoxalase 1 expression in kidney cortex. Chronically elevated hypertriglyceridemia was associated with increased levels of dicarbonyls in heart and kidney. Beneficial effects of metformin on reactive dicarbonyls and glyoxalase in the heart could contribute to its cardioprotective effects.
- MeSH
- deoxyglukosa analogy a deriváty metabolismus MeSH
- dieta MeSH
- fyziologický stres MeSH
- glutathion metabolismus MeSH
- glyoxal metabolismus MeSH
- hypertriglyceridemie farmakoterapie genetika patofyziologie MeSH
- hypoglykemika terapeutické užití MeSH
- krysa rodu Rattus MeSH
- laktoylglutathionlyasa metabolismus MeSH
- metformin terapeutické užití MeSH
- myokard metabolismus MeSH
- potkani Wistar MeSH
- pyruvaldehyd metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- 3-deoxyglucosone MeSH Prohlížeč
- deoxyglukosa MeSH
- glutathion MeSH
- glyoxal MeSH
- hypoglykemika MeSH
- laktoylglutathionlyasa MeSH
- metformin MeSH
- pyruvaldehyd MeSH
Proteins are subject to oxidative modification and the formation of adducts with a broad spectrum of reactive species via enzymatic and non-enzymatic mechanisms. Here we report that in vitro non-enzymatic methylglyoxal (MGO) binding causes the inhibition and formation of MGO advanced glycation end-products (MAGEs) in Na+/K+-ATPase (NKA). Concretely, MGO adducts with NKA amino acid residues (mainly Arg) and Nε-(carboxymethyl)lysine (CML) formation were found. MGO is not only an inhibitor for solubilized NKA (IC50=91±16μM), but also for reconstituted NKA in the lipid bilayer environment, which was clearly demonstrated using a DPPC/DPPE liposome model in the presence or absence of the NKA-selective inhibitor ouabain. High-resolution mass spectrometric analysis of a tryptic digest of NKA isolated from pig (Sus scrofa) kidney indicates that the intracellular α-subunit is naturally (post-translationally) modified by MGO in vivo. In contrast to this, the β-subunit could only be modified by MGO artificially, and the transmembrane part of the protein did not undergo MGO binding under the experimental setup used. As with bovine serum albumin, serving as the water-soluble model, we also demonstrated a high binding capacity of MGO to water-poorly soluble NKA using a multi-spectral methodology based on electroanalytical, immunochemical and fluorimetric tools. In addition, a partial suppression of the MGO-mediated inhibitory effect could be observed in the presence of aminoguanidine (pimagedine), a glycation suppressor and MGO-scavenger. All the results here were obtained with the X-ray structure of NKA in the E1 conformation (3WGV) and could be used in the further interpretation of the functionality of this key enzyme in the presence of highly-reactive metabolic side-products, glycation agents and generally under oxidative stress conditions.
- Klíčová slova
- Aminoguanidine, Enzyme inhibition, Mass spectrometry, Methylglyoxal, Oxidative post-translational modification, Reactivity, Sodium pump,
- MeSH
- guanidiny farmakologie MeSH
- hmotnostní spektrometrie MeSH
- konformace proteinů MeSH
- krystalografie rentgenová MeSH
- ledviny metabolismus MeSH
- ouabain farmakologie MeSH
- oxidační stres MeSH
- produkty pokročilé glykace chemie metabolismus MeSH
- pyruvaldehyd chemie metabolismus MeSH
- sérový albumin hovězí metabolismus MeSH
- skot MeSH
- sodíko-draslíková ATPasa antagonisté a inhibitory chemie metabolismus MeSH
- Sus scrofa MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- guanidiny MeSH
- ouabain MeSH
- pimagedine MeSH Prohlížeč
- produkty pokročilé glykace MeSH
- pyruvaldehyd MeSH
- sérový albumin hovězí MeSH
- sodíko-draslíková ATPasa MeSH
An electrophoretic apparatus with a flow-gating interface has been developed, enabling hydrodynamic sequence injection of the sample into the separation capillary from the liquid flow by underpressure generated in the outlet electrophoretic vessel. The properties of the apparatus were tested on an artificial sample of an equimolar mixture of 100μM potassium and sodium ions and arginine. The repeatability of the injection of the tested ions expressed as RSD (in%) for the peak area, peak height and migration time was in the range 0.76-2.08, 0.18-0.68 and 0.28-0.48, respectively. Under optimum conditions, the apparatus was used for sequence monitoring of the reaction between the antidiabetic drug phenyl biguanide and the glycation agent methyl glyoxal. The reaction solution was continuously sampled by a microdialysis probe from a thermostated external vessel using a syringe pump at a flow rate of 3μLmin-1 and was injected into a separation capillary at certain time intervals. The electrophoretic separation progressed in a capillary with an internal diameter of 50μm with a length of 11.5cm and was monitored using a contactless conductivity detector.
- Klíčová slova
- Antidiabetic drug, Flow-gating interface, Hydrodynamic injection, Microdialysis, Sequence analysis, Short capillary separation,
- MeSH
- arginin MeSH
- biguanidy chemie MeSH
- časové faktory MeSH
- draslík MeSH
- elektroforéza kapilární přístrojové vybavení metody MeSH
- hydrodynamika * MeSH
- hypoglykemika chemie MeSH
- mikrodialýza MeSH
- pyruvaldehyd chemie MeSH
- roztoky MeSH
- sodík MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- arginin MeSH
- biguanidy MeSH
- draslík MeSH
- hypoglykemika MeSH
- phenyl biguanide MeSH Prohlížeč
- pyruvaldehyd MeSH
- roztoky MeSH
- sodík MeSH
Inflammation and oxidative and dicarbonyl stress play important roles in the pathogenesis of type 2 diabetes. Metformin is the first-line drug of choice for the treatment of type 2 diabetes because it effectively suppresses gluconeogenesis in the liver. However, its "pleiotropic" effects remain controversial. In the current study, we tested the effects of metformin on inflammation, oxidative and dicarbonyl stress in an animal model of inflammation and metabolic syndrome, using spontaneously hypertensive rats that transgenically express human C-reactive protein (SHR-CRP). We treated 8-month-old male transgenic SHR-CRP rats with metformin (5 mg/kg/day) mixed as part of a standard diet for 4 weeks. A corresponding untreated control group of male transgenic SHR-CRP rats were fed a standard diet without metformin. In a similar fashion, we studied a group of nontransgenic SHR treated with metformin and an untreated group of nontransgenic SHR controls. In each group, we studied 6 animals. Parameters of glucose and lipid metabolism and oxidative and dicarbonyl stress were measured using standard methods. Gene expression profiles were determined using Affymetrix GeneChip Arrays. Statistical significance was evaluated by two-way ANOVA. In the SHR-CRP transgenic strain, we found that metformin treatment decreased circulating levels of inflammatory response marker IL-6, TNFα and MCP-1 while levels of human CRP remained unchanged. Metformin significantly reduced oxidative stress (levels of conjugated dienes and TBARS) and dicarbonyl stress (levels of methylglyoxal) in left ventricles, but not in kidneys. No significant effects of metformin on oxidative and dicarbonyl stress were observed in SHR controls. In addition, metformin treatment reduced adipose tissue lipolysis associated with human CRP. Possible molecular mechanisms of metformin action-studied by gene expression profiling in the liver-revealed deregulated genes from inflammatory and insulin signaling, AMP-activated protein kinase (AMPK) signaling and gluconeogenesis pathways. It can be concluded that in the presence of high levels of human CRP, metformin protects against inflammation and oxidative and dicarbonyl stress in the heart, but not in the kidney. Accordingly, these cardioprotective effects of metformin might be especially effective in diabetic patients with high levels of CRP.
- MeSH
- C-reaktivní protein biosyntéza genetika MeSH
- cytokiny metabolismus MeSH
- exprese genu MeSH
- glukosa metabolismus MeSH
- krysa rodu Rattus MeSH
- lidé MeSH
- lipolýza účinky léků genetika MeSH
- metformin farmakologie MeSH
- myokard metabolismus MeSH
- oxidační stres účinky léků genetika MeSH
- potkani inbrední SHR MeSH
- potkani transgenní MeSH
- proteinkinasy aktivované AMP genetika metabolismus MeSH
- pyruvaldehyd metabolismus MeSH
- srdeční komory metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- lidé MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- C-reaktivní protein MeSH
- cytokiny MeSH
- glukosa MeSH
- metformin MeSH
- proteinkinasy aktivované AMP MeSH
- pyruvaldehyd MeSH
Protein glycation is a complex process that plays an important role in diabetes mellitus, aging, and the regulation of protein function in general. As a result, current methodological research on proteins is focused on the development of novel approaches for investigating glycation and the possibility of monitoring its modulation and selective inhibition. In this paper, a first sensing strategy for protein glycation is proposed, based on protein electroactivity measurement. Concretely, the label-free method proposed is based on the application of a constant-current chronopotentiometric stripping (CPS) analysis at Hg-containing electrodes. The glycation process was monitored as the decrease in the electrocatalytic protein signal, peak H, observed at highly negative potentials at around -1.8 V (vs Ag/AgCl3 M KCl), which was previously ascribed to a catalytic hydrogen evolution reaction (CHER). Using this method, a model protein bovine serum albumin was investigated over 3 days of incubation with the glycation agent methylglyoxal in the absence or presence of the glycation inhibitor aminoguanidine (pimagedine). The electrochemical methodology presented here could open up new possibilities in research on protein glycation and oxidative modification. The methodology developed also provides a new option for the analysis of protein intermolecular interactions using electrochemical sensors, which was demonstrated by the application of a silver solid amalgam electrode (AgSAE) for monitoring the glycation process in samples of bovine serum albumin, human serum albumin, and lysozyme.
- MeSH
- biotest * MeSH
- elektrochemie MeSH
- elektrody * MeSH
- glykosylace účinky léků MeSH
- glykovaný sérový albumin MeSH
- guanidiny farmakologie MeSH
- inhibitory enzymů farmakologie MeSH
- katalýza MeSH
- konformace proteinů MeSH
- lidé MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- muramidasa analýza chemie MeSH
- produkty pokročilé glykace MeSH
- pyruvaldehyd farmakologie MeSH
- sekvence aminokyselin MeSH
- sérový albumin hovězí analýza chemie MeSH
- sérový albumin analýza chemie MeSH
- skot MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- glykovaný sérový albumin MeSH
- guanidiny MeSH
- inhibitory enzymů MeSH
- muramidasa MeSH
- pimagedine MeSH Prohlížeč
- produkty pokročilé glykace MeSH
- pyruvaldehyd MeSH
- sérový albumin hovězí MeSH
- sérový albumin MeSH
