Kaushik M. Desai

Oxidative stress and aging: is methylglyoxal the hidden enemy?

Aging is a multifactorial process that involves changes at the cellular, tissue, organ and the whole body levels resulting in decreased functioning, development of diseases, and ultimately death. Oxidative stress is believed to be a very important factor in causing aging and age-related diseases. Oxidative stress is caused by an imbalance between oxidants such as reactive oxygen species (ROS) and antioxidants. ROS are produced from the mitochondrial electron transport chain and many oxidative reactions. Methylglyoxal (MG) is a highly reactive dicarbonyl metabolite formed during glucose, protein and fatty acid metabolism. MG levels are elevated in hyperglycemia and other conditions. An excess of MG formation can increase ROS production and cause oxidative stress. MG reacts with proteins, DNA and other biomolecules, and is a major precursor of advanced glycation end products (AGEs). AGEs are also associated with the aging process and age-related diseases such as cardiovascular complications of diabetes, neurodegenerative diseases and connective tissue disorders. AGEs also increase oxidative stress. In this review we discuss the potential role of MG in the aging process through increasing oxidative stress besides causing AGEs formation. Specific and effective scavengers and crosslink breakers of MG and AGEs are being developed and can become potential treatments to slow the aging process and prevent many diseases.

Vascular methylglyoxal metabolism and the development of hypertension

Objectives The pathogenic process of diabetes mellitus is associated with increased methylglyoxal (MG). MG causes non-enzymic glycation of proteins to form irreversible advanced glycation endproducts (AGEs). However, the correlation between MG and essential hypertension is unknown. The aim of the present study was to investigate whether MG, MG-induced AGEs, and oxidative stress were increased in the aorta of spontaneously hypertensive rats (SHR) and whether an increased formation of MG and related AGEs was correlated with the development of high blood pressure in these rats. Methods High-performance liquid chromatography (HPLC) was used to determine MG and reduced glutathione levels in plasma and aorta. MG-induced AGEs, Nϵ-carboxyethyl-lysine (CEL) and Nϵ-carboxymethyl-lysine (CML), in aorta were determined using immunohistochemistry. Hydrogen peroxide and superoxide levels in aorta and glutathione peroxidase and reductase activities were also determined. Results Aortic and plasma MG levels were significantly elevated in SHR, but not in Wistar–Kyoto (WKY) rats, at 8, 13 and 20 weeks of age, in parallel with blood pressure increase. Immunohistochemistry revealed more intense staining for CML and CEL in aorta from SHR than those of WKY rats from 8 weeks onwards. Most of the staining was localized to endothelial cells. Superoxide and hydrogen peroxide levels were significantly elevated in aorta of SHR at 13 weeks, whereas reduced glutathione levels, glutathione peroxidase and glutathione reductase activities were significantly decreased compared to WKY rats. Conclusions Increased aortic MG, AGE formation and oxidative stress were associated with blood pressure increase in SHR, which may cause endothelial dysfunction and altered vascular reactivity.

F2-isoprostane evidence of oxidant stress in the insulin resistant, obese Zucker rat: effects of vitamin E

We have concurrently investigated oxidant stress, glucose tolerance and glucose-stimulated insulin responses in the obese Zucker rat, a widely used model of insulin resistance. The plasma level of the lipid peroxidation product 8-epi-prostaglandin F2alpha, a sensitive in vivo marker of oxidant stress, was elevated approximately 5-fold in 13-week old obese relative to age-matched, insulin-sensitive lean Zucker rats. Supplementation of the diet with vitamin E (as (+)-alpha-tocopherol acetate, 0.5% w/w) for 4 weeks, reduced plasma 8-epi-prostaglandin F2alpha and concomitantly reversed glucose-stimulated hyperinsulinaemia in the obese Zucker rat without worsening glucose tolerance. We therefore provide evidence of oxidant stress, measured as elevated plasma 8-epi-prostaglandin F2alpha, for the first time in the obese Zucker rat which now provides a rationale for the beneficial effects of antioxidants on insulin action previously reported in this model of insulin resistance.

Chronic Methylglyoxal Infusion by Minipump Causes Pancreatic β-Cell Dysfunction and Induces Type 2 Diabetes in Sprague-Dawley Rats

OBJECTIVE The incidence of high dietary carbohydrate-induced type 2 diabetes is increasing worldwide. Methylglyoxal (MG) is a reactive glucose metabolite and a major precursor of advanced glycation end products (AGEs). MG levels are elevated in diabetic patients. We investigated the effects of chronic administration of MG on glucose tolerance and β-cell insulin secreting mechanism in 12-week-old male Sprague-Dawley rats. RESEARCH DESIGN AND METHODS MG (60 mg/kg/day) or 0.9% saline was administered by continuous infusion with a minipump for 28 days. We performed glucose and insulin tolerance tests and measured adipose tissue glucose uptake and insulin secretion from isolated pancreatic islets. We also used cultured INS-1E cells, a pancreatic β-cell line, for molecular studies. Western blotting, quantitative PCR, immunohistochemistry, and transferase-mediated dUTP nick-end labeling (TUNEL) assay were performed. RESULTS In rats treated with MG and MG + l-buthionine sulfoximine (BSO), MG levels were significantly elevated in plasma, pancreas, adipose tissue, and skeletal muscle; fasting plasma glucose was elevated, whereas insulin and glutathione were reduced. These two groups also had impaired glucose tolerance, reduced GLUT-4, phosphoinositide-3-kinase activity, and insulin-stimulated glucose uptake in adipose tissue. In the pancreatic β-cells, MG and MG + BSO reduced insulin secretion, pancreatic duodenal homeobox-1, MafA, GLUT-2, and glucokinase expression; increased C/EBPβ, nuclear factor-κB, MG-induced AGE, Nε-carboxymeythyllysine, and receptor for AGEs expression; and caused apoptosis. Alagebrium, an MG scavenger and an AGE-breaking compound, attenuated the effects of MG. CONCLUSIONS Chronic MG induces biochemical and molecular abnormalities characteristic of type 2 diabetes and is a possible mediator of high carbohydrate-induced type 2 diabetes.

Attenuation of hypertension development by scavenging methylglyoxal in fructose-treated rats.

Objectives Methylglyoxal is a reactive dicarbonyl intermediate of metabolism produced in the body. It reacts with certain proteins and forms damaging advanced glycation endproducts (AGEs) such as Nϵ-carboxyethyl-lysine (CEL) and Nϵ-carboxymethyl-lysine (CML). Increased methylglyoxal levels are found in diabetes mellitus and associated with hypertension development in the spontaneously hypertensive rats (SHR). The purpose of this study was to investigate whether increased endogenous formation of methylglyoxal and methylglyoxal-induced AGEs caused hypertension development in normotensive Sprague Dawley rats. Methods The rats were fed chronically for 16 weeks with fructose, a known precursor of methylglyoxal formation. One group of rats was cotreated with fructose and metformin, an AGEs formation inhibitor. Methylglyoxal and reduced glutathione (GSH) were measured by high performance liquid chromatography, whereas hydrogen peroxide was measured by a dicholorofluorescin assay. Immunohistochemistry was performed for endothelial nitric oxide synthase (eNOS), CEL and CML. Results Fructose-fed rats had elevated blood pressure, serum methylglyoxal and triglycerides and reduced serum levels of GSH. Methylglyoxal, hydrogen peroxide and CEL were increased in the aorta, whereas eNOS was reduced. CEL and CML were also increased in the mesenteric artery endothelium along with media/lumen ratio, signifying structural remodelling. All the harmful changes in fructose-fed rats were attenuated in metformin and fructose cotreated rats. Conclusion Increased methylglyoxal, AGEs, oxidative stress and reduced eNOS along with structural remodeling of the vessel wall in the aorta and mesenteric artery likely play a role in the pathogenesis of hypertension.

Methylglyoxal scavengers attenuate endothelial dysfunction induced by methylglyoxal and high concentrations of glucose.

BACKGROUND AND PURPOSE Endothelial dysfunction is a feature of hypertension and diabetes. Methylglyoxal (MG) is a reactive dicarbonyl metabolite of glucose and its levels are elevated in spontaneously hypertensive rats and in diabetic patients. We investigated if MG induces endothelial dysfunction and whether MG scavengers can prevent endothelial dysfunction induced by MG and high glucose concentrations.

Alagebrium attenuates acute methylglyoxal-induced glucose intolerance in Sprague-Dawley rats

Background and purpose:  Alagebrium is a breaker of cross‐links in advanced glycation endproducts. However, the acute effects of alagebrium on methylglyoxal (MG), a major precursor of advanced glycation endproducts have not been reported. MG is a highly reactive endogenous metabolite, and its levels are elevated in diabetic patients. We investigated whether alagebrium attenuated the acute effects of exogenous MG on plasma MG levels, glucose tolerance and distribution of administered MG in different organs in Sprague‐Dawley rats.

Free radical generation by methylglyoxal in tissues.

Methylglyoxal (MG) is a reactive dicarbonyl intermediate of the glycolytic pathway. Increased oxidative stress is associated with conditions of increased MG, such as diabetes mellitus. Increased oxidative stress is due to an increase in highly reactive by-products of metabolic pathways, the so-called reactive oxygen species, such as superoxide anion, hydroxyl radical, hydrogen peroxide, nitric oxide and peroxynitrite. These reactive species react with a variety of proteins, enzymes, lipids, DNA and other molecules and disrupt their normal function. Oxidative stress causes many pathological changes that lead to vascular complications of diabetes mellitus, hypertension, neurodegenerative diseases and aging. In this review we summarize the correlation of elevated MG and various reactive oxygen species, and the enzymes that produce them or take part in their disposal, such as antioxidant enzymes and cofactors. The findings reported in various studies reviewed have started filling in gaps in our knowledge that will ultimately provide us with a clear picture of how the whole process that causes cellular dysfunction is initiated.

Methylglyoxal, protein binding and biological samples: are we getting the true measure?

Methylglyoxal (MG), a reactive metabolic byproduct and a precursor of advanced glycation endproducts (AGEs), is elevated in diabetes. In the body MG is free or reversibly or irreversibly bound (mostly with proteins). Variable plasma MG values have been reported. MG is commonly measured using high performance liquid chromatography. We tested several protocols on different biological samples, which resulted in significant differences in MG values measured in a given sample. The different values do not appear due to the release and detection of bound MG under assay conditions. Protocols that provide consistent values of MG in biological samples are recommended.

Nitric oxide synthase inhibition exaggerates the hypotensive response to ghrelin : role of calcium-activated potassium channels

Objective To investigate the mechanism underlying the observation that infusion of the growth hormone secretagogue peptide, ghrelin, produces a decrease in mean arterial pressure (MAP) with no change in heart rate. Method The effect of a single bolus infusion of ghrelin (12 nmol/kg intravenously) on the changes in MAP and heart rate was determined in 12-week-old male anaesthetized Sprague–Dawley rats subjected to pretreatment with either the nitric oxide synthase (NOS) inhibitor, Nω-nitro-L-arginine methyl ester (L-NAME; 0.7 mg/ml by mouth for 5 days), or vehicle (control). Results Ghrelin produced a significant decrease in MAP at 20 min (P < 0.05) after infusion in the control group, without any change in heart rate. The MAP recovered partially over 1 h. The ghrelin-evoked decrease in MAP was much greater (P < 0.01) and was sustained for 1 h in rats subjected to NOS inhibition. Pretreatment with the cyclo-oxygenase inhibitor, indomethacin, failed to affect the responses in either group. Intravenous infusion of 50 μg/kg each of apamin and charybdotoxin (ChTX), a combination that is known to block Ca2+-activated K+ channels or the endothelium-derived hyperpolarization process, attenuated the decrease in MAP evoked by ghrelin in both control and NOS-inhibited rats. A sodium nitroprusside-induced decrease in MAP was unaffected in the presence of apamin–ChTX, but acetylcholine-evoked hypotension was significantly reduced in both groups. Conclusion These data suggest that the Ca2+-activated, K+-channel-mediated, ghrelin-evoked decrease in MAP may be significant in states of endothelial dysfunction associated with reduced nitric oxide availability.