Metformin prevents methylglyoxal-induced apoptosis by suppressing oxidative stress in vitro and in vivo.

Methylglyoxal (MGO) is an active metabolite of glucose and plays a prominent role in the pathogenesis of diabetic vascular complications, including endothelial cell apoptosis induced by oxidative stress. Metformin (MET), a widely prescribed antidiabetic agent, appears to reduce excessive reactive oxygen species (ROS) generation and limit cell apoptosis. However, the molecular mechanisms underlying this process are still not fully elucidated. We reported here that MET prevents MGO-induced apoptosis by suppressing oxidative stress in vitro and in vivo. Protein expression and protein phosphorylation were investigated using western blotting, ELISA, and immunohistochemical staining, respectively. Cell viability and apoptosis were assessed by the MTT assay, TUNEL staining, and Annexin V-FITC and propidium iodide double staining. ROS generation and mitochondrial membrane potential (MMP) were measured with fluorescent probes. Our results revealed that MET prevented MGO-induced HUVEC apoptosis, inhibited apoptosis-associated biochemical changes such as loss of MMP, the elevation of the Bax/Bcl-2 ratio, and activation of cleaved caspase-3, and attenuated MGO-induced mitochondrial morphological alterations in a dose-dependent manner. MET pretreatment also significantly suppressed MGO-stimulated ROS production, increased signaling through the ROS-mediated PI3K/Akt and Nrf2/HO-1 pathways, and markedly elevated the levels of its downstream antioxidants. Finally, similar results were obtained in vivo, and we demonstrated that MET prevented MGO-induced oxidative damage, apoptosis, and inflammation. As expected, MET reversed MGO-induced downregulation of Nrf2 and p-Akt. In addition, a PI3K inhibitor (LY-294002) and a Nrf2 inhibitor (ML385) observably attenuated the protective effects of MET on MGO-induced apoptosis and ROS generation by inhibiting the Nrf2/HO-1 pathways, while a ROS scavenger (NAC) and a permeability transition pores inhibitor (CsA) completely reversed these effects. Collectively, these findings broaden our understanding of the mechanism by which MET regulates apoptosis induced by MGO under oxidative stress conditions, with important implications regarding the potential application of MET for the treatment of diabetic vascular complications.

Metformin abrogates the voiding dysfunction induced by prolonged methylglyoxal intake.

Methylglyoxal (MGO) is a reactive carbonyl species found at high levels in blood of diabetic patients. The anti-hyperglycemic drug metformin can scavenger MGO and reduce the formation of advanced glycation end products (AGEs). Here, we aimed to investigate if MGO-induced bladder dysfunction can be reversed by metformin. Male C57/BL6 mice received 0.5% MGO in drinking water for 12 weeks, and metformin (300 mg/kg, daily gavage) was given in the last two weeks. The bladder functions were evaluated by performing voiding behavior assays, cystometry and in vitro bladder contractions. MGO intake markedly elevated the levels of MGO and fluorescent AGEs in serum and reduced the mRNA expression and activity of glyoxalase (Glo1) in bladder tissues. Glucose levels were unaffected among groups. MGO intake also increased the urothelium thickness and collagen content of the bladder. Void spot assays in conscious mice revealed an increased void volume in MGO group. The cystometric assays in anesthetized mice revealed increases of basal pressure, non-voiding contractions frequency, bladder capacity, inter-micturition pressure and residual volume, which were accompanied by reduced voiding efficiency in MGO group. In vitro bladder contractions to carbachol, α,ß-methylene ATP and electrical-field stimulation were significantly greater in MGO group. Metformin normalized the changes of MGO and AGEs levels, Glo1 expression and activity, urothelium thickness and collagen content. The MGO-induced voiding dysfunction were all restored by metformin treatment. Our findings strongly suggest that the amelioration of MGO-induced voiding dysfunction by metformin relies on its ability to scavenger MGO, preventing its accumulation in blood.

Coadministration of liposomal methylglyoxal increases the activity of amphotericin B against Candida albicans in leukopoenic mice.

In this study, we investigated the therapeutic efficacy of a combination of liposomal amphotericin B (Lip-Amp B) and Methylglyoxal (Lip-MG) against Candida albicans in the leukopoenic mice. The antifungal efficacy of Lip-Amp B or Lip-MG or a combination of Lip-Amp B and Lip-MG was evaluated by the analysis of the survival rate and the fungal load in the treated mice. The immune-stimulatory effect of Lip-MG on macrophages was evaluated by analysing the secretion of proinflammatory cytokines. C. albicans infected mice treated at the doses of 1 and 2 mg/kg of Lip-Amp B showed 20% and 50% survival rates, respectively. Whereas the mice treated with free Amp B at the same doses died within 40 days of treatment. Interestingly, C. albicans infected mice treated with a combination of Lip-Amp B and Lip-MG had 70% survival rate on day 40 postinfection. Moreover, treatment of macrophages with Lip-MG increased their fungicidal activity and the secretion of proinflammatory cytokines, including TNF-α and IL-1ß. These findings suggested that co-treatment with Lip-Amp B and Lip-MG had a synergistic effect and could be effective against C. albicans in immunocompromised subjects.

Methylglyoxal-Mediated Dopamine Depletion, Working Memory Deficit, and Depression-Like Behavior Are Prevented by a Dopamine/Noradrenaline Reuptake Inhibitor.

Methylglyoxal (MGO) is an endogenous toxin, mainly produced as a by-product of glycolysis that has been associated to aging, Alzheimer's disease, and inflammation. Cell culture studies reported that MGO could impair the glyoxalase, thioredoxin, and glutathione systems. Thus, we investigated the effect of in vivo MGO administration on these systems, but no major changes were observed in the glyoxalase, thioredoxin, and glutathione systems, as evaluated in the prefrontal cortex and the hippocampus of mice. A previous study from our group indicated that MGO administration produced learning/memory deficits and depression-like behavior. Confirming these findings, the tail suspension test indicated that MGO treatment for 7 days leads to depression-like behavior in three different mice strains. MGO treatment for 12 days induced working memory impairment, as evaluated in the Y maze spontaneous alternation test, which was paralleled by low dopamine and serotonin levels in the cerebral cortex. Increased DARPP32 Thr75/Thr34 phosphorylation ratio was observed, suggesting a suppression of phosphatase 1 inhibition, which may be involved in behavioral responses to MGO. Co-treatment with a dopamine/noradrenaline reuptake inhibitor (bupropion, 10 mg/kg, p.o.) reversed the depression-like behavior and working memory impairment and restored the serotonin and dopamine levels in the cerebral cortex. Overall, the cerebral cortex monoaminergic system appears to be a preferential target of MGO toxicity, a new potential therapeutic target that remains to be addressed.

Short-Term Alterations in Behavior and Astroglial Function After Intracerebroventricular Infusion of Methylglyoxal in Rats.

Methylglyoxal (MG) is a by-product of glycolysis. In pathological conditions, particularly diabetes mellitus, this molecule is unbalanced, causing widespread protein glycation. In addition to protein glycation, other effects resulting from high levels of MG in the central nervous system may involve the direct modulation of GABAergic and glutamatergic neurotransmission, with evidence suggesting that the effects of MG may be related to behavioral changes and glial dysfunction. In order to evaluate the direct influence of MG on behavioral and biochemical parameters, we used a high intracerebroventricular final concentration (3 µM/µL) to assess acute effects on memory and locomotor behavior in rats, as well as the underlying alterations in glutamatergic and astroglial parameters. MG induced, 12 h after injection, a decrease in locomotor activity in the Open field and anxiolytic effects in rats submitted to elevated plus-maze. Subsequently, 36 h after surgery, MG injection also induced cognitive impairment in both short and long-term memory, as evaluated by novel object recognition task, and in short-term spatial memory, as evaluated by the Y-maze test. In addition, hippocampal glutamate uptake decreased and glutamine synthetase activity and glutathione levels diminished during seventy-two hours after infusion of MG. Interestingly, the astrocytic protein, S100B, was increased in the cerebrospinal fluid, accompanied by decreased hippocampal S100B mRNA expression, without any change in protein content. Taken together, these results may improve our understanding of how this product of glucose metabolism can induce the brain dysfunction observed in diabetic patients, as well as in other neurodegenerative conditions, and further defines the role of astrocytes in disease and therapeutics.

Long-term intake of the reactive metabolite methylglyoxal is not toxic in mice.

Reactive carbonyls, including methylglyoxal (MG), are considered toxic compounds in foodstuffs because they irreversibly modify proteins and produce advanced glycation end products (AGEs). Therefore, we studied the long-term effect of increased MG intake in mature adult mice. Six-month-old C57BL/6N mice received MG by drinking water (2.5 mg/ml; i.e., 200-300 mg/kg BW/d) until death. This treatment caused an immediate strong increase in urine MG and a delayed moderate increase in plasma MG. At 24 months of age, mice administered MG showed no changes in the blood and tissue activity of glyoxalase-1 (Glo1), an intracellular MG-detoxifying enzyme; no signs of renal insufficiency and diabetes, including unchanged AGE modifications of plasma and vessel proteins; reduced tumour incidence; and slightly increased survival. Mice simultaneously deficient in the receptor for AGEs (RAGE) and overexpressing Glo1 exhibited higher basal plasma MG levels and did generally not respond to long-term MG intake. In vitro experiments supported the minor relevance of Glo1 in the detoxification of circulating MG but the important role of plasma albumin as an MG scavenger. In conclusion, the detoxification of dietary MG through renal excretion and further mechanisms largely prevents the toxicity of MG and possibly other food-derived reactive carbonyls in mature adults.

Psoralea corylifolia L. Seed Extract Attenuates Methylglyoxal-Induced Insulin Resistance by Inhibition of Advanced Glycation End Product Formation.

Accumulation of advanced glycation end products (AGEs) in the body has been implicated in the pathogenesis of metabolic conditions, such as diabetes mellitus. Methylglyoxal (MGO), a major precursor of AGEs, has been reported to induce insulin resistance in both in vitro and in vivo studies. Psoralea corylifolia seeds (PCS) have been used as a traditional medicine for several diseases, but their potential application in treating insulin resistance has not yet been evaluated. This study is aimed at investigating whether PCS extract could attenuate insulin resistance induced by MGO. Male C57BL/6N mice (6 weeks old) were administered 1% MGO in their drinking water for 18 weeks, and the PCS extract (200 or 500 mg/kg) was orally administered daily from the first day of the MGO administration. We observed that both 200 and 500 mg/kg PCS extract treatment significantly improved glucose tolerance and insulin sensitivity and markedly restored p-Akt and p-IRS1/2 expression in the livers of the MGO-administered mice. Additionally, the PCS extract significantly increased the phosphorylation of Akt and IRS-1/2 and glucose uptake in MGO-treated HepG2 cells. Further studies showed that the PCS extract inhibited MGO-induced AGE formation in the HepG2 cells and in the sera of MGO-administered mice. PCS extract also increased the expression of glyoxalase 1 (GLO1) in the liver tissue of MGO-administered mice. The PCS extract significantly decreased the phosphorylation of ERK, p38, and NF-κB and suppressed the mRNA expression of proinflammatory molecules including TNF-α and IL-1ß and iNOS in MGO-administered mice. Additionally, we demonstrated that the PCS extract attenuated oxidative stress, as evidenced by the reduced ROS production in the MGO-treated cells and the enhanced expression of antioxidant enzymes in the liver of MGO-administered mice. Thus, PCS extract ameliorated the MGO-induced insulin resistance in HepG2 cells and in mice by reducing oxidative stress via the inhibition of AGE formation. These findings suggest the potential of PCS extract as a candidate for the prevention and treatment of insulin resistance.

Diabetes with heart failure increases methylglyoxal modifications in the sarcomere, which inhibit function.

Patients with diabetes are at significantly higher risk of developing heart failure. Increases in advanced glycation end products are a proposed pathophysiological link, but their impact and mechanism remain incompletely understood. Methylglyoxal (MG) is a glycolysis byproduct, elevated in diabetes, and modifies arginine and lysine residues. We show that left ventricular myofilament from patients with diabetes and heart failure (dbHF) exhibited increased MG modifications compared with nonfailing controls (NF) or heart failure patients without diabetes. In skinned NF human and mouse cardiomyocytes, acute MG treatment depressed both calcium sensitivity and maximal calcium-activated force in a dose-dependent manner. Importantly, dbHF myocytes were resistant to myofilament functional changes from MG treatment, indicating that myofilaments from dbHF patients already had depressed function arising from MG modifications. In human dbHF and MG-treated mice, mass spectrometry identified increased MG modifications on actin and myosin. Cosedimentation and in vitro motility assays indicate that MG modifications on actin and myosin independently depress calcium sensitivity, and mechanistically, the functional consequence requires actin/myosin interaction with thin-filament regulatory proteins. MG modification of the myofilament may represent a critical mechanism by which diabetes induces heart failure, as well as a therapeutic target to avoid the development of or ameliorate heart failure in these patients.

Methylglyoxal at metronomic doses sensitizes breast cancer cells to doxorubicin and cisplatin causing synergistic induction of programmed cell death and inhibition of stemness.

Potent anticancer activity coupled with absence of toxicity at therapeutic dose established the glycolytic metabolite, methylglyoxal, as a promising candidate against malignant neoplasia. In this preclinical study we illustrate the applicability of methylglyoxal in formulating an optimally designed combination regimen with chemotherapeutic drugs against breast cancer. Results demonstrated a synergistic augmentation in doxorubicin and cisplatin mediated cytotoxicity in human breast cancer cell lines MDA MB 231 & MCF 7 with methylglyoxal co-treatment at metronomic concentrations. The cell death due to combination treatment was significantly prevented by N-Acetylcysteine and the synergistic effects were attenuated in presence of inhibitors for apoptosis and necroptosis, in MDA MB 231 and MCF 7 cells, respectively. Additionally, acridine orange staining and immunoblotting with LC3B antibody indicated the suppression of doxorubicin induced autophagy flux with methylglyoxal co-treatment. This report documents for the first time the preferential targeting of breast cancer stem cells by methylglyoxal. Combination treatment with doxorubicin or cisplatin hindered mammosphere forming efficiency and inclusively eliminated both cancer stem as well as non-stem cancer cells. The synergistic effect was validated in Ehrlich mammary carcinoma cell induced murine ascites model and the combination advantage in vivo was achieved without any additional deleterious effect to liver and kidney. Our present study evidences the implications of methylglyoxal inclusion in adjuvant multimodal chemotherapeutics against breast cancer and offers noteworthy insights into the possible outcome.

Inhibition of Methylglyoxal-Induced Histone H1 Nε-Carboxymethyllysine Formation by (+)-Catechin.

Reactive dicarbonyl species (RCS) such as methylglyoxal (MGO) and glyoxal (GO) are common intermediates in protein damage, leading to the formation of advanced glycation end products (AGEs) through nonenzymatic glycation. (+)-Catechin, a natural plant extract from tea, has been evaluated for its ability in trapping GO and MGO. However, (+)-catechin is also reported to have both antioxidant ability and pro-oxidant properties. Until now, whether (+)-catechin can inhibit the formation of nonenzymatic glycation and the mechanism of the inhibition in nucleoprotein nonenzymatic glycation is still unclear. In the present study, histone H1 and MGO were used to establish an in vitro (100 mM phosphate buffer solution (PBS), pH 7.4, 37 °C) protein glycation model to study the trapping ability of (+)-catechin. Our data show that MGO caused dose-dependent protein damage, and the content of MGO-induced Schiff base formation was inhibited by (+)-catechin when the molecular ratio of catechin:MGO was 1:6. The formation of Nε-carboxymethyllysine (CML) was reduced significantly when the ratio of (+)-catechin and MGO was 1:1, which was similar to the inhibition effect of aminoguanidine (AG). The formation of CML under in vitro conditions can be inhibited by low concentration (12.5-100 µM) of (+)-catechin but not with high concentration (200-800 µM) of (+)-catechin. The reason is that the high concentration of (+)-catechin did not inhibit CML formations due to H2O2 produced by (+)-catechin. In the presence of catalase, catechin can inhibit MGO-induced CML formation. In conclusion, the trapping ability of (+)-catechin may be more effective at the early stage of nonenzymatic glycation. However, a high concentration (200-800 µM) of (+)-catechin may not inhibit the formation of CML because it induced the increase of H2O2 formation.

Methylglyoxal Impairs ß2-Adrenoceptor-Mediated Vasodilatory Mechanisms in Rat Retinal Arterioles.

Methylglyoxal, a highly reactive dicarbonyl compound, is formed as a by-product of glycolysis and plays an important role in the pathogenesis of diabetic complications, including diabetic retinopathy. However, it remains to be determined how methylglyoxal affects the regulatory mechanisms of retinal blood flow. In this study, we examined the effects of methylglyoxal on ß2-adrenoceptor-mediated vasodilatory mechanisms in rat retinal arterioles. The retinal vasodilator responses were assessed by measuring the diameter of retinal arterioles in the fundus images. Intravitreal injection of methylglyoxal significantly diminished the vasodilation of retinal arterioles induced by the ß2-adrenoceptor agonist salbutamol. The vasodilator effect of BMS-191011, a large-conductance Ca2+-activated K+ (BKCa) channel opener, on retinal arterioles was also attenuated by methylglyoxal. In contrast, methylglyoxal had no significant effect on retinal vasodilator response to forskolin. Methylglyoxal attenuated retinal vasodilator response to salbutamol under blockade of BKCa channels with iberiotoxin, an inhibitor of the channels. These results suggest that methylglyoxal attenuates ß2-adrenoceptor-mediated retinal vasodilation by impairing the coupling of the ß2-adrenoceptor to the guanine nucleotide-binding protein (Gs protein) and the function of the BKCa channel. Increased methylglyoxal in the eyes may contribute to the impairment of regulatory mechanisms of retinal blood flow in patients with diabetic retinopathy.

Methylglyoxal treatment in lactating mothers leads to type 2 diabetes phenotype in male rat offspring at adulthood.

PURPOSE: Environmental and nutritional disorders during perinatal period cause metabolic dysfunction in the progeny and impair human health. Advanced glycation end products (AGEs) are primarily produced during metabolism of excess blood glucose, which is observed in diabetes. Methylglyoxal (MG) is a precursor for the generation of endogenous AGEs, which disturbs the metabolism. This work aimed to investigate whether the maternal MG treatment during lactation programs the progeny to metabolic dysfunction later in life. METHODS: Female Wistar rats were divided into two groups: control group (C) treated with saline and MG group treated with MG (60 mg/kg/day) by gavage throughout the lactation period. Both mothers and offspring were fed a standard chow. At weaning, breast milk composition was analyzed and mothers euthanized for blood and tissue sample collections. At 90 days of age, offspring were submitted to glucose tolerance test (ivGTT) and euthanized for blood and tissue samples collection. RESULTS: MG mothers showed increase in glucose and fructosamine levels; however, they showed low insulin levels and failure in ß-cell function (p < 0.05). MG mothers also showed dyslipidemia (p < 0.05). Moreover, breast milk had elevated levels of glucose, triglycerides, cholesterol and fructosamine and low insulin (p < 0.05). Interestingly, MG offspring had increased body weight and adipose tissue at adulthood, and they also showed glucose intolerance and failure in ß-cell function (p < 0.05). Besides, MG offspring showed dyslipidemia (p < 0.05) increasing cardiovascular diseases risk. CONCLUSIONS: Maternal MG treatment negatively affects the male rat offspring, leading to type 2 diabetes and dyslipidemia in later life, possibly by changes in breast milk composition.

Methylglyoxal displays colorectal cancer-promoting properties in the murine models of azoxymethane and CT26 isografts.

Methylglyoxal (MG), a highly reactive carbonyl species (RCS) with pro-oxidant and proinflammatory properties, may be a colon tumor-promoting factor in food and biological systems. In the present study, we found that consumption of MG significantly deteriorated azoxymethane (AOM)-induced colonic preneoplastic lesions in ICR mice, in which biomarkers of oxidative stress and inflammation within the body and feces induced by MG-fueled carbonyl stress may have played important roles. Interestingly, exposure to MG also led to increases in the serum low-density lipoprotein (LDL)/high-density lipoprotein (HDL) ratio and fecal bile acid levels in mice, which may be critical factors involved in MG-induced colonic lesions. Additionally, MG treatment (50mg/kg body weight (BW); intraperitoneally) promoted tumor growth of CT26 isografts in mice partly by carbonyl stress-evoked protumorigenic responses, including low-grade inflammation and oxidative stress. Furthermore, primary tumor cells isolated from mice with MG-induced CT26 isografts had greater proliferative and migratory activities as well as stem-like properties compared to those isolated from the vehicle controls. Excitingly, enhanced expression or activation of proteins that modulate cell survival, proliferation, or migration/invasion was also observed in those cells. In conclusion, it is conceivable that MG-induced carbonyl stress may be the pivotal promoter involved in colon cancer progression.

Lactobacillus demonstrate thiol-independent metabolism of methylglyoxal: Implications toward browning prevention in Parmesan cheese.

Endogenous production of α-dicarbonyls by lactic acid bacteria can influence the quality and consistency of fermented foods and beverages. Methylglyoxal (MG) in Parmesan cheese can contribute toward undesired browning during low temperature ripening and storage conditions, leading to the economic depreciation of affected cheeses. We demonstrate the effects of exogenously added MG on browning and volatile formation using a Parmesan cheese extract (PCE). To determine the influence of Lactobacillus on α-dicarbonyls, strains were screened for their ability to modulate concentrations of MG, glyoxal, and diacetyl in PCE. It was found that a major metabolic pathway of MG in Lactobacillus is a thiol-independent reduction, whereby MG is partially or fully reduced to acetol and 1,2-propanediol, respectively. The majority of lactobacilli grown in PCE accumulated the intermediate acetol, whereas Lactobacillus brevis 367 formed exclusively 1,2-propanediol and Lactobacillus fermentum 14931 formed both metabolites. In addition, we determined the inherent tolerance to bacteriostatic concentrations of MG among lactobacilli grown in rich media. It was found that L. brevis 367 reduces MG exclusively to 1,2-propanediol, which correlates to both its ability to significantly decrease MG concentrations in PCE, as well as its significantly higher tolerance to MG, in comparison to other lactobacilli screened. These findings have broader implications toward lactobacilli as a viable solution for reducing MG-mediated browning of Parmesan cheese.

Intracellular methylglyoxal induces oxidative damage to pancreatic beta cell line INS-1 cell through Ire1α-JNK and mitochondrial apoptotic pathway.

An increased intracellular methylglyoxal (MGO) under hyperglycemia led to pancreatic beta cell death. However, its mechanism in which way with MGO induced beta cell death remains unknown. We investigated both high glucose and MGO treatment significantly inclined intracellular MGO concentration and inhibited cell viability in vitro. MGO treatment also triggered intracellular advanced glycation end products (AGEs) formation, declined mitochondrial membrane potential (MMP), increased oxidative stress and the expression of ER stress mediators Grp78/Bip and p-PERK; activated mitochondrial apoptotic pathway, which could mimic by Glo1 knockdown. Aminoguanidine (AG), a MGO scavenger, however, prevented AGEs formation and MGO-induced cell death by inhibiting oxidative stress and ER stress. Furthermore, both antioxidant N-acetylcysteine (NAC) and ER stress inhibitor 4-phenylbutyrate (4-PBA) could attenuate MGO-induced cell death through ameliorating ER stress. MGO treatment down-regulated Ire1α, a key ER stress mediator, increased JNK phosphorylation and activated mitochondrial apoptosis; down-regulated Bcl-2 expression which could be attenuated by the JNK inhibitor SP600125 and further inhibited cytochrome c leakage from mitochondria and blocked the conversion of pro caspase 3 into cleaved caspase 3, all these might contribute to the inhibition of INS-1 cell apoptosis. Ire1α down-regulation by Ire1α siRNAs mimicked MGO-induced cytotoxicity by activating the JNK phosphorylation and mitochondrial apoptotic pathway. In summary, we demonstrated that increased intracellular MGO induced cytotoxicity in INS-1 cells primarily by activating oxidative stress and further triggering mitochondrial apoptotic pathway, and ER stress-mediated Ire1α-JNK pathway. These findings may have implication on new mechanism of glucotoxicity-mediated pancreatic beta-cell dysfunction.

Methyglyoxal administration induces modification of hemoglobin in experimental rats: An in vivo study.

Methylglyoxal, a highly reactive α-oxoaldehyde, increases in diabetic condition and reacts with proteins to form advanced glycation end products (AGEs) following Maillard-like reaction. In the present study, the effect of methylglyoxal on experimental rat hemoglobin in vivo has been investigated with respect to structural alterations and amino acid modifications, after external administration of the α-dicarbonyl compound in animals. Different techniques, mostly biophysical, were used to characterize and compare methylglyoxal-treated rat hemoglobin with that of control, untreated rat hemoglobin. In comparison with methylglyoxal-untreated, control rat hemoglobin, hemoglobin of methylglyoxal-treated rats (32mg/kgbodywt.dose) exhibited slightly decreased absorbance around 280nm, reduced intrinsic fluorescence and lower surface hydrophobicity. The secondary structures of hemoglobin of control and methylglyoxal-treated rats were more or less identical with the latter exhibiting slightly increased α-helicity compared to the former. Compared to control rat hemoglobin, methylglyoxal-treated rat hemoglobin showed higher stability. Peptide mass fingerprinting analysis revealed modifications of Arg-31α, Arg-92α and Arg-104ß of methylglyoxal-treated rat hemoglobin to hydroimidazolone adducts. The modifications thus appear to be associated with the observed structural alterations of the heme protein. Considering the increased level of methylglyoxal in diabetes mellitus as well as its high reactivity, AGE-induced modifications may have physiological significance.

CHOP deficiency inhibits methylglyoxal-induced endothelial dysfunction.

Epidemiological studies suggested that diabetic patients are susceptible to develop cardiovascular complications along with having endothelial dysfunction. It has been suggested that methylglyoxal (MGO), a glycolytic metabolite, has more detrimental effects on endothelial dysfunction rather than glucose itself. Here, we investigated the molecular mechanism by which MGO induces endothelial dysfunction via the regulation of ER stress. Biochemical data showed that 4-PBA significantly inhibited MGO-induced protein cleavages of PARP-1 and caspase-3. In addition, it was found that high glucose-induced endothelial apoptosis was enhanced in the presence of GLO1 inhibitor, suggesting the role of endogenous MGO in high glucose-induced endothelial dysfunction. MGO-induced endothelial apoptosis was significantly diminished by the depletion of CHOP with si-RNA against human CHOP, but not by SP600125, a specific inhibitor of JNK. The physiological relevance of this signaling pathway was demonstrated in CHOP deficiency mouse model, in which instillation of osmotic pump containing MGO led to aortic endothelial dysfunction. Notably, the aortic endothelial dysfunction response to MGO infusion was significantly improved in CHOP deficiency mice compared to littermate control. Taken together, these findings indicate that MGO specifically induces endothelial dysfunction in a CHOP-dependent manner, suggesting the therapeutic potential of CHOP inhibition in diabetic cardiovascular complications.

Glyoxalase I drives epithelial-to-mesenchymal transition via argpyrimidine-modified Hsp70, miR-21 and SMAD signalling in human bronchial cells BEAS-2B chronically exposed to crystalline silica Min-U-Sil 5: Transformation into a neoplastic-like phenotype.

Glyoxalase I (Glo1) is the main scavenging enzyme of methylglyoxal (MG), a potent precursor of advanced glycation end products (AGEs). AGEs are known to control multiple biological processes, including epithelial to mesenchymal transition (EMT), a multistep phenomenon associated with cell transformation, playing a major role in a variety of diseases, including cancer. Crystalline silica is a well-known occupational health hazard, responsible for a great number of human pulmonary diseases, such as silicosis. There is still much debate concerning the carcinogenic role of crystalline silica, mainly due to the lack of a causal demonstration between silica exposure and carcinogenesis. It has been suggested that EMT might play a role in crystalline silica-induced lung neoplastic transformation. The aim of this study was to investigate whether, and by means of which mechanism, the antiglycation defence Glo1 is involved in Min-U-Sil 5 (MS5) crystalline silica-induced EMT in BEAS-2B human bronchial epithelial cells chronically exposed, and whether this is associated with the beginning of a neoplastic-like transformation process. By using gene silencing/overexpression and scavenging/inhibitory agents, we demonstrated that MS5 induced hydrogen peroxide-mediated c-Jun-dependent Glo1 up-regulation which resulted in a decrease in the Argpyrimidine-modified Hsp70 protein level which triggered EMT in a novel mechanism involving miR-21 and SMAD signalling. The observed EMT was associated with a neoplastic-like phenotype. The results obtained provide a causal in vitro demonstration of the MS5 pro-carcinogenic transforming role and more importantly they provide new insights into the mechanisms involved in this process, thus opening new paths in research concerning the in vivo study of the carcinogenic potential of crystalline silica.

Neuronal overexpression of Glo1 or amygdalar microinjection of methylglyoxal is sufficient to regulate anxiety-like behavior in mice.

GLO1 (Glyoxalase1) is a ubiquitous cellular enzyme that detoxifies methylglyoxal (MG), which is a byproduct of glycolysis. Previously, we showed that ubiquitous overexpression of Glo1 reduced concentrations of MG and increased anxiety-like behavior, whereas systemic injection of MG reduced anxiety-like behavior. We further showed that MG is a competitive partial agonist at GABA-A receptors. Based on those data we hypothesized that modulation of GABAergic signaling by MG underlies Glo1 and MG's effects on anxiety-like behavior. As previous studies used ubiquitous overexpression, we sought to determine whether neuronal Glo1 overexpression was sufficient to increase anxiety-like behavior. We generated ROSA26 knock-in mice with a floxed-stop codon upstream from human Glo1 (FLOXGlo1KI) and bred them with mice expressing CRE recombinase under the direction of the Synapsin 1 promoter (Syn-CRE) to limit overexpression of Glo1 specifically to neurons. Furthermore, since previous administration of MG had been systemic, we sought to determine if direct microinjection of MG into the basolateral amygdala (BLA) was sufficient to reduce anxiety-like behavior. Thus, we performed bilateral microinjections of saline, MG (12µM or 24µM), or the positive control midazolam (4mM) directly into the BLA. FLOXGlo1KIxSyn-CRE mice showed significantly increased anxiety-like behavior compared to their FLOXGLO1xWT littermates. In addition, bilateral microinjection of MG and midazolam significantly decreased anxiety-like behavior compared to saline treated mice. These studies suggest that anatomically specific manipulations of Glo1 and MG are sufficient to induce changes in anxiety-like behavior.

Effects of methylglyoxal and glyoxalase I inhibition on breast cancer cells proliferation, invasion, and apoptosis through modulation of MAPKs, MMP9, and Bcl-2.

Emerging evidence indicates that methylglyoxal (MG) can inhibit tumorigenesis. Glyoxalase I (GLOI), a MG degradation enzyme, is implicated in the progression of human malignancies. However, little is known about the roles of MG and GLOI in breast cancer. Our purpose was to investigate the anticancer effects of MG and inhibition of GLOI on breast cancer cells and the underlying mechanisms of these effects. Our findings demonstrate that cell viability, migration, invasion, colony formation, and tubule formation were significantly restrained by addition of MG or inhibition of GLOI, while apoptosis was significantly increased. Furthermore, the expression of p-JNK, p-ERK, and p-p38 was markedly upregulated by addition of MG or inhibition of GLOI, whereas MMP-9 and Bcl-2 expression levels were dramatically decreased. These effects were augmented by combined treatment with MG and inhibition of GLOI. Collectively, these data indicate that MG or inhibition of GLOI induces anticancer effects in breast cancer cells and that these effects are potentiated by combination of the 2. These effects were modulated by activation of the MAPK family and downregulation of Bcl-2 and MMP-9. These findings may provide a new approach for the treatment of breast cancer.