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 alpha-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 microM) 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-kappaB activation and increased caspase-3 activity were detected. Apoptosis was also inhibited by the caspase-3 inhibitor, Z-DEVD-fmk, or the NF-kappaB 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-kappaB are involved in the apoptotic process.     

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

Abstract

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-1β. 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.     

A copper chelating agent suppresses carbonyl stress in diabetic rat lenses

To clarify whether transition metals are involved in carbonyl stress in diabetic tissues, we observed the effects of a metal chelating agent, trientine (TE) hydrochloride on the levels of methylglyoxal (MG), 3-deoxyglucosone (3-DG), advanced glycation end products, 8-hydroxy-2′-deoxyguanosine (8-OHdG), and polyol pathway metabolites along with semicarbazide-sensitive amine oxidase (SSAO) enzyme activity in lenses from streptozotocin-induced diabetic rats. Lens MG and 3-DG levels were significantly higher in diabetic rats than nondiabetic controls, and TE significantly restored the increase of these compounds. Lens argpyrimidine was also increased in diabetic rats as compared with controls and was significantly reduced by TE. Lens SSAO activity and 8-OHdG were also significantly elevated in diabetic rats, and TE suppressed both of them, whereas TE showed no effect on the polyol pathway metabolites. The results indicate that transition metals play a significant role in the formation of MG and 3-DG via oxidative stress and SSAO activity.     

NAD+ Availability and Proteotoxicity

It has been shown that NAD⁺ availability is important for neuronal survival following ischemia (Liu et al., Neuromolecular Med 11:28–42, 2009). It is proposed here that NAD⁺ may also control proteotoxicity by influencing both formation and catabolism of altered proteins. It is suggested that low NAD⁺ availability promotes synthesis of methylglyoxal (MG) which can induce formation of glycated proteins, ROS, and dysfunctional mitochondria. That glyoxalase overexpression and carnosine are both protective against MG and ischemic injury support this proposal. Recognition and elimination of altered proteins is enhanced by NAD⁺ through effects on stress protein expression and autophagy.     

Concentrations ofd-lactate and its related metabolic intermediates in liver,blood, and muscle of diabetic and starved rats

This is a report investigating the methylglyoxal (MG) bypass in animals, by whichd-lactate is produced from triosephosphate via MG. Rats were made diabetic using streptozotocin or starved for 72 h.d-Lactate and various metabolites related to it, such asl-lactate, pyruvate, methylglyoxal, glucose, and inorganic phosphate, were measured in the blood plasma, liver, and skeletal muscle of the rats. Diabetic and starved rats had significantly higher levels ofd-lactate in plasma, liver, and skeletal muscle compared with the control group. In contrast, pyruvate levels in plasma, liver, and skeletal muscle was markedly lower than normal in diabetic and starved rats.l-Lactate level lowered markedly in plasma, liver, and skeletal muscle of starved rats and elevated in liver of diabetic rats. Differences between plasmal-lactate level for diabetes and control were not significant. MG level was significantly elevated in plasma and depressed in livers and muscles of starved rats as well as livers of diabetic rats. Hepatic glycerol content was markedly increased in those states. Enzyme activities related tod- andl-lactate, such as pyruvate kinase, phosphofructokinase, aldolase, and glyoxalase I, were measured in the livers of these rats. Pyruvate kinase activity decreased in these states, but other enzyme activities showed no significant changes.d-Lactate was much more excreted thanl-lactate in the urine of diabetic and fasted rats compared with normal rats.     

Preparation and quantification of methylglyoxal in human plasma using reverse-phase high-performance liquid chromatography

OBJECTIVES: To detect methylglyoxal (MG), a highly reactive alpha-oxoaldehyde found widespread throughout biological life, in human plasma using reverse-phase high-performance liquid chromatography method (RP HPLC) with UV detection. DESIGN AND METHODS: The processing of human plasma required protein precipitation with trifluoroacetic acid (TFA), incubation of the supernatant (2 h) with 1,2-diamino-4,5-dimethoxybenzene (DDB) to convert MG to 6,7-dimethoxy-2-methylquinoxaline (DMQ), freeze-drying, and RP HPLC analysis using 6,7-dimethoxy-2,3-dimethylquinoxaline (DMDQ) as an internal standard (IS). Simplified methods for the synthesis of MG and DDB are also described. RESULTS: Calibration curves were linear in the range of 200-1000 nM. The limit of detection was 30.6 and 45.9 pmol, at 215 and 352 nm, respectively. The intraday coefficients of variation were 6.9-12.6% for 215 nm and 3.5-12.6% for 352 nm. The interday coefficients of variation were 9.6-12.8% for 215 nm and 7.2-14.7% for 352 nm. Sample storage conditions together with statistical evaluation are also described. CONCLUSIONS: Here we present a rapid and inexpensive method for the determination of methylglyoxal in human plasma using RP HPLC with UV detection. The simplicity of the reported RP HPLC method makes it suitable for the detection of methylglyoxal in many human plasma samples.     

Energy metabolism, altered proteins, sirtuins and ageing: converging mechanisms?

The predominant molecular symptom of ageing is the accumulation of altered gene products. Nutritional studies show that ageing in animals can be significantly influenced by dietary restriction. Genetics has revealed that ageing may be controlled by changes in intracellular NAD/NADH ratio regulating sirtuin activity. Physiological and other approaches indicate that mitochondria may also regulate ageing. A mechanism is proposed which links diet, exercise and mitochondria-dependent changes in NAD/NADH ratio to intracellular generation of altered proteins. It is suggested that ad libitum feeding conditions decrease NAD availability which also decreases metabolism of the triose phosphate glycolytic intermediates, glyceraldehyde-3-phosphate and dihydroxyacetone-phosphate, which can spontaneously decompose into methylglyoxal (MG). MG is a highly toxic glycating agent and a major source of protein advanced-glycosylation end-products (AGEs). MG and AGEs can induce mitochondrial dysfunction and formation of reactive oxygen species (ROS), as well as affect gene expression and intracellular signalling. In dietary restriction–induced fasting, NADH would be oxidised and NAD regenerated via mitochondrial action. This would not only activate sirtuins and extend lifespan but also suppress MG formation. This proposal can also explain the apparent paradox whereby increased aerobic activity suppresses formation of glycoxidized proteins and extends lifespan. Variation in mitochondrial DNA composition and consequent mutation rate, arising from dietary-controlled differences in DNA precursor ratios, could also contribute to tissue differences in age-related mitochondrial dysfunction.     

Attenuation of hypertension development by aminoguanidine in spontaneously hypertensive rats: role of methylglyoxal

BACKGROUND: Methylglyoxal (MG), a metabolite of glucose, and MG-induced advanced glycation endproducts (AGEs) are causatively associated with vascular complications of diabetes mellitus. We have previously reported elevated levels of MG and MG-induced AGEs in spontaneously hypertensive rats (SHR). The purpose of this study was to investigate the causative role of MG and MG-induced AGEs in the pathogenesis of hypertension in SHR. METHODS: Young SHR were treated with an AGE inhibitor, aminoguanidine, for 9 weeks. HPLC was used to determine plasma and aortic MG and reduced glutathione levels. The MG-induced AGEs, N epsilon-carboxyethyl-lysine (CEL) and argpyramidine, in the aorta were determined by immunohistochemistry. Vascular relaxation of small mesenteric arteries was measured using myograph. RESULTS: Chronic treatment with aminoguanidine attenuated age-dependent blood pressure (BP) increase in SHR. Plasma and aortic MG levels, and aortic levels of MG-induced AGEs, were significantly reduced after aminoguanidine treatment, which were comparable to those from age-matched Wistar Kyoto rats. Free radical level was significantly lowered, whereas reduced glutathione level was significantly increased by aminoguanidine treatment in the aortic tissues from SHR. Moreover, aminoguanidine therapy prevented the morphologic damage of vascular tissues in SHR and restored the endothelium-dependent relaxation to acetylcholine. Chronic aminoguanidine treatment also increased aortic endothelial nitric oxide synthase expression and reduced inducible nitric oxide synthase expression. CONCLUSIONS: The MG and MG-induced AGEs contribute to the pathogenesis of hypertension by altering the redox balance, causing vascular eutrophic inward remodeling, and inducing endothelial dysfunction in SHR.     

Two α-dicarbonyls downregulate migration,invasion, and adhesion of liver cancer cells in a p53-dependent manner

Background

Hepatocellular carcinoma accounts for more than 600,000 deaths per year due to it being a highly invasive tumor. The α-dicarbonyl, methylglyoxal demonstrates efficacy at reducing tumor burden, however the anti-cancerous activities of 3-deoxyglucosone, have never been studied.

Aims

To determine the anti-cancerous potential of methylglyoxal and 3-deoxyglucosone on liver tumor cells.

Methods

The in vitro effects of methylglyoxal and 3-deoxyglucosone were studied by investigating migration, invasion, and adhesion of Huh-7, HepG2, and Hep3B cells.

Results

3-Deoxyglucosone inhibited migration of Huh-7 and HepG2 cells. Methylglyoxal decreased migration of HepG2 cells. Additionally, 3-deoxyglucosone and methylglyoxal impaired invasion, and adhesion of Huh-7 and HepG2 cells. In Hep3B cells, a p53 null cell line, 3-deoxyglucosone and methylglyoxal had no effect on migration, invasion, or adhesion. However, both compounds inhibited invasion of wild-type p53 transfected Hep3B cells. Silencing of p53 in Huh-7 and HepG2 cells abrogated the effects of the α-dicarbonyls on cell invasion. 3DG and MG did not alter p53 total protein but promoted nuclear translocation of p53.

Conclusions

These studies suggest that 3-deoxyglucosone and methylglyoxal impair invasion, migration, and adhesion of hepatocellular carcinoma. The effects of both compounds on cell invasion are dependent on p53 and imply that α-dicarbonyls could be efficacious in the treatment of p53-expressing invasive liver tumors.     

焦亚硫酸钠与盐酸-L-半胱氨酸在延缓抗坏血酸注射液变色中的作用

本文探讨了焦亚硫酸钠与盐酸-L-半胱氨酸这两种稳定剂在延缓抗坏血酸注射液变色中的作用。盐酸-L-半胱氨酸能延缓2-酮-L-古洛糖酸的降解速率,也能与丙酮醛发生化学反应,延缓丙酮醛的变色速率;焦亚硫酸钠可能改变了丙酮醛的聚合方式而延缓丙酮醛的变色速率。从而在一定程度上解释了这两种稳定剂延缓抗坏血酸注射液变色的原因。