argpyrimidine and Diabetes-Mellitus

Decay accelerating factor (DAF or CD55) is a cell associated C3 and C5 convertase regulator originally described in terms of protection of self-cells from systemic complement but now known to modulate adaptive T cell responses. It is expressed on all cell types. We investigated whether nonenzymatic glycation could impair its function and potentially be relevant to complications of diabetes mellitus and other conditions that result in nonenzymatic glycation including cancer, Alzheimer's disease, and aging. Immunoblots of affinity-purified DAF from erythrocytes of patients with diabetes showed pentosidine, glyoxal-AGEs, carboxymethyllysine, and argpyrimidine. HPLC/MS analyses of glucose modified DAF localized the sites of AGE modifications to K

Topics: Amino Acids; Arginine; Catalytic Domain; CD55 Antigens; Complement Activation; Diabetes Mellitus; Erythrocytes; Glucose; Glycation End Products, Advanced; Humans; Lymphocyte Activation; Lysine; Models, Molecular; Ornithine; Protein Conformation; Pyrimidines; Ribose

Diabetes mellitus represents a major metabolic disorder affecting millions of people all over the world. Currently available therapeutic treatments are not good enough to control the long-term complications of diabetes. Active targeting via inclusion of a specific ligand on the nanoparticles provides effective therapeutic approach in different diseases. However, such specific drug delivery systems have not been explored much in diabetes due to lack of suitable biological targets in this disorder. Our objective is to synthesize a ligand-tagged drug-loaded nanoparticle for delivery of the drug at specific sites to enhance its therapeutic efficiency in diabetic condition. The nanoparticles have been prepared by using biocompatible ethylene glycol-bis (succinic acid N-hydroxysuccinimide ester) dimers. Although advanced glycation end products (AGEs) are the root causes of diabetic complications, argpyrimidine, an AGE, possesses antioxidant and reducing activities. AGE interacts selectively with its cell surface receptors (RAGE), which are significantly increased in diabetic condition. We have selected RAGE as the target of argpyrimidine, which is tagged on the nanoparticles as a ligand. Rutin, having anti-hyperglycemic and anti-glycating activities, has been used for nanoencapsulation. Rutin-loaded argpyrimidine-tagged nanoparticles have been synthesized and characterized. We have demonstrated the drug releasing capacity and target specificity of the synthesised drug delivery system under ex vivo and in vivo conditions.

Topics: Animals; Antioxidants; Biocompatible Materials; Cells, Cultured; Diabetes Mellitus; Drug Delivery Systems; Ethylene Glycol; Male; Nanoparticles; Ornithine; Polymers; Pyrimidines; Rats; Rats, Wistar; Rutin; Succinimides

Chronic high glucose levels lead to the formation of advanced glycation end-products (AGEs) as well as AGE precursors, such as methylglyoxal (MG) and glyoxal, via non-enzymatic glycation reactions in patients with diabetic mellitus. Glyoxalase 1 (GLO-1) detoxifies reactive dicarbonyls that form AGEs. To investigate the interaction between AGEs and GLO-1 in mesangial cells (MCs) under diabetic conditions, AGE levels and markers of oxidative stress were measured in GLO-1-overexpressing MCs (GLO-1-MCs) cultured in high glucose. Furthermore, we also examined levels of high glucose-induced apoptosis in GLO-1-MCs. In glomerular MCs, high glucose levels increased the formation of both MG and argpyrimidine (an MG-derived adduct) as well as GLO-1 expression. GLO-1-MCs had lower intracellular levels of MG accumulation, 8-hydroxy-deoxyguanosine (an oxidative DNA damage marker), 4-hydroxyl-2-nonenal (a lipid peroxidation product), and nitrosylated protein (a marker of oxidative-nitrosative stress) compared to control cells. Expression of mitochondrial oxidative phosphorylation complexes I, II, and III was also decreased in GLO-1-MCs. Furthermore, fewer GLO-1-MCs showed evidence of apoptosis as determined by terminal deoxynucleotidyl transferase-mediated dUTP nick labeling assay, and activation of both poly (ADP-ribose) polymerase 1 cleavage and caspase-3 was lower in GLO-1-MCs than in control cells cultured in high glucose. These results suggest that GLO-1 plays a role in high glucose-mediated signaling by reducing MG accumulation and oxidative stress in diabetes mellitus.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Aldehydes; Animals; Apoptosis; Caspase 3; Cell Line; Cells, Cultured; Deoxyguanosine; Diabetes Mellitus; Glycation End Products, Advanced; Hyperglycemia; Lactoylglutathione Lyase; Lipid Peroxidation; Mesangial Cells; Mice; Mitochondria; Ornithine; Oxidative Stress; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Pyrimidines; Pyruvaldehyde

Methylglyoxal (MG), an alpha-dicarbonyl compound, can be produced in vivo by several metabolic pathways and the Maillard reaction. It reacts rapidly with proteins to form advanced glycation end products or AGEs. We previously isolated and characterized a blue fluorescent product of the reaction between MG and arginine, which we named argpyrimidine. We found that argpyrimidine was stable to acid hydrolysis, which allowed us to hydrolyze tissue proteins with 6 N HCl and quantify argpyrimidine by high-performance liquid chromatography. Here we report argpyrimidine concentrations in human lens and serum proteins as determined by HPLC. We have also measured pentosidine, a fluorescent AGE derived from pentose sugars, and compared the concentrations of pentosidine and argpyrimidine. We found two- to threefold higher argpyrimidine concentrations in diabetic serum proteins than in nondiabetic controls (9.3 +/- 6.7 vs 4.4 +/- 3.4 pmol/mg). We found a significant correlation (P = 0.0001) between serum protein argpyrimidine and glycosylated hemoglobin. Argpyrimidine concentrations were approximately seven times greater in brunescent cataractous lenses than in aged noncataractous lenses. Pentosidine concentrations in serum and lens proteins were much lower than argpyrimidine concentrations; in general, argpyrimidine levels were 10--25 times higher than pentosidine. Results from our study confirm that MG-mediated arginine modifications occur in vivo and provide a method for assessing protein-arginine modification by MG in aging and diabetes.

Topics: Adolescent; Adult; Aged; Analysis of Variance; Arginine; Blood Proteins; Diabetes Mellitus; Humans; Lens, Crystalline; Lysine; Middle Aged; Ornithine; Pyrimidines; Pyruvaldehyde

Methylglyoxal (MG), an endogenous metabolite that increases in diabetes and is a common intermediate in the Maillard reaction (glycation), reacts with proteins and forms advanced glycation end products. In the present study, we identify a novel MG-arginine adduct and also characterize the structure of a major fluorescent adduct. In addition, we describe the immunochemical study on the MG-arginine adducts using monoclonal antibody directed to MG-modified protein. Upon incubation of Nalpha-acetyl-L-arginine with MG at 37 degrees C, two nonfluorescent products and one fluorescent product were detected as the major products. The nonfluorescent products were identified as the Ndelta-(5-hydro-5-methyl-4-imidazolon-2-yl)-L-ornithine derivatives (5-hydro-5-methylimidazolone) and a novel MG-arginine adduct having a tetrahydropyrimidine moiety (Ndelta-(4-carboxy-4,6-dimethyl-5, 6-dihydroxy-1,4,5,6-tetrahydropyrimidine-2-yl)-L-ornithine). On the basis of the following chemical and spectroscopic evidence, the major fluorescent product, putatively identified as Ndelta-(5-methylimidazolon-2-yl)-L-ornithine (5-methylimidazolone), was found to be identical to Ndelta-(5-hydroxy-4, 6-dimethylpyrimidine-2-yl)-L-ornithine (argpyrimidine): (i) the low and high resolution fast atom bombardment-mass spectrometry gave a molecular ion peak at m/z of 297 (M+H) and a molecular formula of C10H25O6N4, respectively, which coincided with argpyrimidine; (ii) the 1H NMR spectrum of this product in d6-Me2SO showed a singlet at 2.10 ppm corresponding to six protons; (iii) the peak corresponding to the 5-methylimidazolone derivative was not detected by the liquid chromatography-mass spectrometry with the mode of selected ion monitoring; (iv) incubation of 5-hydro-5-methylimidazolone, a putative precursor of 5-methylimidazolone, at 37 degrees C for 14 days scarcely generated 5-methylimidazolone. On the other hand, as an immunochemical approach to the detection of these MG adducts, we raised the monoclonal antibodies (mAb3C and mAb6B) directed to the MG-modified protein and found that they specifically recognized the major fluorescent product, argpyrimidine, as the dominant epitope. The immunohistochemical analysis of the kidneys from diabetic patients revealed the localization of argpyrimidine in intima and media of small artery walls. Furthermore, the accumulation of argpyrimidine was also observed in some arterial walls of the rat brain after middle cerebral artery occlusion followed by re

Topics: Animals; Antibodies, Monoclonal; Arginine; Brain Chemistry; Chromatography, High Pressure Liquid; Diabetes Mellitus; Enzyme-Linked Immunosorbent Assay; Female; Humans; Kidney; Magnetic Resonance Spectroscopy; Mice; Mice, Inbred BALB C; Models, Chemical; Ornithine; Proteins; Pyrimidines; Pyruvaldehyde; Rats