Theingi Myint

Glycoxidation in aortic collagen from STZ-induced diabetic rats and its relevance to vascular damage

Glycoxidation reactions lead to the formation of permanent, irreversible chemical modifications and cross-links in protein, such as the glycoxidation products carboxymethyllysine (CML) and pentosidine. It has been implicated that CML as well as Amadori products play a role in the formation of superoxidative products, such as H2O2 and advanced glycosylation endproducts in trapping LDL. Therefore, a possible relationship between glycoxidation and lipoperoxidation might exist because oxidized lipoprotein, which has been directly linked to atheroma formation, could be produced by the superoxidative products released from the pathway of CML formation. Using a CML-specific monoclonal antibody (6D12) and a specific antiserum against hexitol-lysine (HL), an Amadori product, we studied the relationship between glycoxidation and lipoperoxidation by determining the aortic CML contents with ELISA and the fluorescence levels of lipoperoxidation side products, malondialdehyde (MDA) and hydroxynonenal (HNE) from STZ-induced diabetic rats and age-matched control rats. The immunohistochemical and ultrastructural changes relevant to glycoxidation and lipoperoxidation were also studied. The CML content measured by ELISA in DM rats was significantly higher than that in the control rats at 28 weeks (n = 11, P < 0.01). The levels of MDA-linked and HNE-linked fluorescence in the DM rats increased in a similar way and were significantly higher than the levels in control rats at 28 weeks (n = 11, both P < 0.01 at 28 weeks). The CML contents correlated with the fluorescence levels of both MDA-linked (n = 19, r = 0.638, P < 0.01) and HNE-linked fluorescence (n = 19, r = 0.629, P < 0.01) only in the DM rats, but not in the control rats. Our immunohistochemical study thus demonstrated that CML was initially formed in the aortic media of diabetic rats in the 16th week of diabetes, localized primarily in the extracellular matrix surrounding the aortic smooth muscle cells after HL occurred early in the 2nd week of diabetes. Consequently, a significant increase in the extracellular matrix and decrease in the area of the SMCs were observed in the aortic media in the DM rats by a morphometrical study. The in vivo results of this study provided the first evidence that CML correlated with fluorescence levels of MDA and HNE, and thus suggested the existence of a close relationship between glycoxidation and lipoperoxidation in vivo. This information is thus considered to shed some new light on the etiology of atherogenesis in diabetes.

Immunological detection of glycated proteins in normal and streptozotocin-induced diabetic rats using anti hexitol-lysine IgG

A polyclonal antibody specific for the Amadori compound, a product of an early stage of the Maillard reaction, was raised in rabbits by immunization with hexitol-lysine (1-glucitol-lysine or 1-mannitol-lysine) coupled with various carrier proteins. The affinity purified antibody has a high titre and preferentially recognizes the glucose adduct, in the presence of sodium borohydride, as judged on enzyme-linked immunosorbent assay as well as immunoblot analysis. The glycated proteins (Amadori products) in various tissues of normal and streptozotocin-induced diabetic rats were examined by immunoblot analysis. In diabetic conditions, kidney, liver, lens, brain and lung proteins are more susceptible to glycation than other tissue proteins. Heart, spleen, adrenal gland and muscle proteins exhibit similar extents of glycation in both normal and diabetic conditions. This is the first demonstration of a specific antibody against the Amadori compound being raised with a synthetic compound, and of the tissue distribution of glycated proteins in normal and diabetic conditions. The antibody was very useful for in vitro and in vivo experiments on the Maillard reaction.

Glycoxidation and lipid peroxidation of low-density lipoprotein can synergistically enhance atherogenesis

OBJECTIVE The purpose of this study was to clarify the role of glycoxidation and lipid peroxidation of low-density lipoprotein (LDL) in atherogenesis. METHODS AND RESULTS We examined the formation of N(epsilon)-(carboxymethyl) lysine (CML), a glycoxidation product, and malondialdehyde (MDA), a lipid peroxidation product, in vitro and their co-localization in human atherosclerotic lesions. Immunochemical analysis revealed that CML was formed in a time-dependent manner by human LDL incubated with copper ions and glucose, i.e. an in vitro model of glycoxidation of LDL. When LDL was exposed to copper ions alone, a small amount of CML was formed, however this was significantly less in oxidized LDL than glycoxidative LDL. In contrast, MDA formation was observed in both oxidation and glycoxidation of LDL, but not in glycation of LDL. Hexitol-lysine (HL), an Amadori product, was formed by both glycation and glycoxidation of LDL, but not by oxidation of LDL. Immunohistochemical analysis showed that CML and MDA accumulated mainly in macrophage/foam cells, while pyrraline, a non-oxidative product of glycation, and apolipoprotein B were localized in the extracellular matrix in atherosclerotic lesions. Atheromas were positive for CML and MDA, but negative for pyrraline. Macrophage/foam cells in atherosclerotic lesions exhibited co-localization of macrophage scavenger receptor-A with CML and MDA, but not with pyrraline. CONCLUSION Our results suggest that glycoxidation and lipid peroxidation of LDL synergistically promote the development of atherosclerotic lesions through interaction with macrophage scavenger receptor-A.

Characterization of Wild‐Type and Amyotrophic Lateral Sclerosis‐Related Mutant Cu,Zn‐Superoxide Dismutases Overproduced in Baculovirus‐Infected Insect Cells

Abstract: We describe the use of a baculovirus expression system to overproduce human Cu,Zn‐superoxide dismutase (SOD). Spodoptera frugiperda (Sf21) insect cells infected with a baculovirus carrying the Cu,Zn‐SOD cDNA synthesized a large amount of Cu,Zn‐SOD apoprotein in the conventional medium. The SOD activity of the apoprotein, which was initially very low, increased in a dose‐dependent manner when Cu2+ and Zn2+ were added to the medium. Cells grown in media supplemented with Cu2+ alone exhibited nearly maximal SOD activity. SOD activity reached 40% of the maximal level within 2 h after addition of Cu2+ to postinfected cells cultivated for 3 days in the conventional medium, and the activity gradually increased thereafter. The protein produced by the infected cells was purified by a simple procedure involving two chromatographic steps, DE52 ion exchange and ACA54 gel filtration. Identification of the recombinant Cu,Zn‐SOD with the human erythrocyte enzyme was confirmed by immunochemical reactivity to anti‐human Cu,Zn‐SOD antibody and by partial amino acid sequencing of peptides from purified protein (50 amino acid residues in total). We constructed three mutant enzymes, which have been found in familial amyotrophic lateral sclerosis and are overproduced in Sf21 cells, and purified them. Mutant enzymes Gly41Asp, His43Arg, and Gly85Arg exhibited 47, 66, and 99% of wild‐type SOD activity, respectively. The availability of this protein will facilitate investigation of the relationship between the structure and function of the mutant enzymes found in familial amyotrophic lateral sclerosis.

Glycation proceeds faster in mutated Cu, Zn-superoxide dismutases related to familial amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) involves the progressive degeneration of motor neurons in the spinal cord and motor cortex. It has been shown that 15–20% of patients with familial ALS (FALS) have defects in the Sod1 gene that encodes Cu, Zn‐superoxide dismutase (SOD). To elucidate the pathological role of mutated Cu, Zn‐SODs in FALS, the susceptibility of mutants to glycation was examined. Mutated Cu, Zn‐SODs (G37R, G93A, and I113T) related to FALS and wild type were produced in a baculovirus/insect cell expression system. Glycated and nonglycated proteins were separated on a boronate column, and the nonglycated fraction was then incubated with glucose. The mutated Cu, Zn‐SODs were found to be highly susceptible to glycation compared with the wild‐type enzyme as estimated by Western blot analysis using an anti‐hexitol lysine antibody. The mutated Cu, Zn‐SOD incubated with glucose generated higher levels of hydrogen peroxide than the wild‐type enzyme. Mutated Cu, Zn‐SODs were also shown to be highly susceptible to fructation, and the fructated mutant also produced higher levels of hydrogen peroxide than the wild type. These results suggest that high susceptibility of mutated Cu, Zn‐SODs to glycation could be the origin of the oxidative stress associated with neuronal dysfunction in FALS.

Reducing Sugars Induce Apoptosis in Pancreatic β-Cells by Provoking Oxidative Stress via a Glycation Reaction

Reducing sugars brought about apoptosis in isolated rat pancreatic islet cells as well as in a pancreatic β-cell-derived cell line, HIT. This apoptosis was characterized biochemically by internucleosomal DNA cleavage and morphologically by nuclear shrinkage, chromatic condensation, and apoptotic body formation. N-acetyl-L-cysteine and aminoguanidine inhibited the apoptosis. Proteins in β-cells were actually glycated by the binding with an antibody that can specifically recognize the protein glycated by fructose. The FACS analysis using dichlorofluorescin diacetate showed that reducing sugars increased intracellular peroxide levels preceding the induction of apoptosis. Levels of carbonyl and malondialdehyde were also increased. These results suggest that reducing sugars trigger oxidative modification and apoptosis in pancreatic β-cells by provoking oxidative stress mainly via a glycation reaction, which may explain the deterioration of β-cells under diabetic conditions.