Oxidative modification of glycated low density lipoprotein in the presence of iron
Abstract
This is the first observation for contributing to the glycation of low density lipoprotein (LDL) to oxidative modification of its own lipids and protein. Human plasma LDL was glycated by incubation with glucose (G-LDL). Glucose incorporated into apoprotein B was approximately 10 mol/mol of apoprotein (2.8 % modification of lysine residues) and 84 % of G-LDL was adsorbed on phenylboronate affinity column. G-LDL incubated with Fe3+ for 4 h caused a significantly higher level of lipid peroxidation than U-LDL (untreated with glucose), and a higher molecular weight protein was observed in apoprotein B on SDS-polyacrylamide gel electrophoresis (SDS-PAGE), increasing with incubation period. Corresponding to change on SDS-PAGE, G-LDL exposed to Fe3+ moved faster than G-LDL per se or U-LDL to anode on agarose gel electrophoresis. The higher the Fe3+ concentration, the more lipid peroxidation was caused. α-tocopherol or probucol suppressed the lipid peroxidation of G-LDL exposed to Fe3+.
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Cited by (52)
Glycoxidized low-density lipoprotein enhances monocyte chemoattractant protein-1 mRNA expression in human umbilical vein endothelial cells: Relation to lysophosphatidylcholine contents and inhibition by nitric oxide donor
2002, Metabolism: Clinical and ExperimentalLow-density lipoprotein (LDL) may undergo more glycation or oxidation in patients with diabetes mellitus than in nondiabetic subjects. We investigated whether glycoxidized LDL (goLDL) induces monocyte chemoattractant protein-1 (MCP-1) mRNA expression through activation of nuclear factor-kappaB (NF[kappa ]B), and determined the effect of nitric oxide (NO) on MCP-1 mRNA expression in human umbilical vein endothelial cells (HUVEC). Oxidized (oxLDL) or goLDL enhanced MCP-1 mRNA expression in HUVEC, and preincubation with NOR3, a NO donor, abrogated such stimulation. goLDL increased NF[kappa ]B-DNA binding activity in HUVEC and this effect was also suppressed by NOR3. We measured lysophosphatidylcholine (lyso-PC) contents in modified LDL using electrospray ionization liquid chromatography/mass spectrometry (LC/MS) to identify its molecular species. MCP-1 mRNA expression and NF[kappa ]B activation correlated significantly with palmitoyl- and stearoyl-lyso-PC contents in LDL. Our results suggest that LDL modified by glycation and oxidation may contribute to the development of accelerated atherosclerosis in the presence of diabetes, a process that may be prevented by increased vascular NO availability.
Remnant-like lipoprotein particles in type 2 diabetic patients with apolipoprotein E3/3 and apolipoprotein E2 genotypes
2002, Metabolism: Clinical and ExperimentalApolipoprotein (apo) E2 and diabetes mellitus are known to be associated with an accumulation of remnant lipoproteins in plasma. In this study, effects of type 2 diabetes mellitus and/or apo E2 genotypes on remnant-like lipoprotein particles (RLP) were assessed. Thirty-three subjects were divided into 6 groups: 7 apo E3/3 nondiabetic subjects, 6 apo E3/3 diabetic patients, 5 apo E3/2 nondiabetic subjects, 6 apo E3/2 diabetic patients, 5 apo E2/2 nondiabetic subjects, and 4 apo E2/2 diabetic patients. First, the effect of diabetes mellitus on RLP were estimated by comparing the apo E3/3 nondiabetic group with the apo E3/3 diabetic group. Plasma levels of RLP-cholesterol (chol) in the apo E3/3 diabetic group and the uptake of RLP from the apo E3/3 diabetic group by macrophages were significantly greater compared with the apo E3/3 nondiabetic group. Second, the effect of apo E2 on RLP was estimated in nondiabetic subjects. Apo E2/2 nondiabetic subjects had type III hyperlipoproteinemia (HLP). Plasma levels of RLP-chol in the apo E2/2 nondiabetic group and the uptake of RLP from the apo E2/2 nondiabetic group by macrophages were significantly greater compared with the apo E3/3 and apo E3/2 nondiabetic groups. Third, the effects of both apo E2 and diabetes on RLP were estimated. Plasma levels of RLP-chol in the apo E2 (E3/2 and E2/2) diabetic groups and the uptake of RLP from apo E2 (E3/2 and E2/2) diabetic groups by macrophages were significantly greater compared with apo E3/3 nondiabetic and diabetic groups or the apo E3/2 nondiabetic group. In diabetes, a gene dose effect of apo E2 on plasma levels of RLP-chol and uptake of RLP by macrophages was present (apo E3/3 [lt ] apo E3/2 [lt ] apo E2/2). The apo E2/2 diabetic group had type III HLP. Furthermore, uptake of RLP from the apo E2/2 diabetic group with type III HLP was significantly greater compared with the apo E2/2 nondiabetic group with type III HLP. In conclusion, type 2 diabetes was associated with increased RLP-chol in plasma and atherogenic RLP. In nondiabetes, apo E2/2 contributes to increased plasma RLP-chol and atherogenic RLP. In diabetes, additional effects of apo E2 to increase RLP-chol in plasma and to enhance the uptake of RLP by macrophages are present. RLP from apo E2/2 diabetes with type III HLP are more atherogenic than those from apo E2/2 nondiabetes with type III HLP.
In vivo and in vitro evidence for the glycoxidation of low density lipoprotein in human atherosclerotic plaques
2000, AtherosclerosisAlthough there have been suggestions that the glycation and oxidation of low density lipoprotein (LDL) might increase its atherogenic potential, little is known about the presence of glycoxidative LDL in human atherosclerotic lesions. We developed specific antibodies against different immunological epitopes of AGE structures, including Nε-(carboxymethyl)lysine-protein adduct (CML), a glycoxidation product, and structure(s) other than CML (nonCML), and a monoclonal antibody against oxidized phosphatidylcholine (oxPC), as an epitope of oxidized LDL. Immunohistochemical analysis demonstrated that the CML- and oxPC-epitopes were accumulated mainly in macrophage-derived foam cells in atherosclerotic lesions, including fatty streaks and atherosclerotic plaques. On the other hand, the nonCML-epitope and apolipoprotein B were localized mainly in extracellular matrices of atherosclerotic lesions. The CML- and oxPC-epitopes were characterized by a model antigen-generating system using the copper ion-induced peroxidation and/or glucose-induced glycation of LDL. The glycoxidation of LDL caused the formation of CML-epitope with increasing concentrations of copper ion and glucose. It was also formed to some extent in LDL incubated with high concentrations (500 mM) of glucose. However, no CML-epitope was observed in oxidized LDL induced by copper ion alone. On the other hand, the formation of oxPC-epitope in LDL was dependent on copper ion-induced peroxidation, but independent of glucose-induced glycation. The addition of chelators, ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid, reduced the increase in electrophoretic mobility and TBARS caused by the peroxidation and glycoxidation of LDL, but had no effects on the formation of fructosamine caused by the glycation and glycoxidation of LDL. Chelators as well as aminoguanidine protected the formation of CML-epitope in glycated or glycoxidative LDL. Although the formation of oxPC-epitope was completely inhibited by the addition of chelators, it was partially protected by aminoguanidine. These in vitro results suggest that the glycoxidative modification of LDL may occur in the arterial intima, and may contribute to the development of human atherosclerotic lesions.
Glycated phosphatidylethanolamine promotes macrophage uptake of low density lipoprotein and accumulation of cholesteryl esters and triacylglycerols
1999, Journal of Biological ChemistryNon-enzymatic glycation of low density lipoprotein (LDL) has been suggested to be responsible for the increase in susceptibility to atherogenesis of diabetic individuals. Although the association of lipid glycation with this process has been investigated, the effect of specific lipid glycation products on LDL metabolism has not been addressed. This study reports that glucosylated phosphatidylethanolamine (Glc-PtdEtn), the major LDL lipid glycation product, promotes LDL uptake and cholesteryl ester (CE) and triacylglycerol (TG) accumulation by THP-1 macrophages. Incubation of THP-1 macrophages at a concentration of 100 μg/ml protein LDL specifically enriched (10 nmol/mg LDL protein) with synthetically prepared Glc-PtdEtn resulted in a significant increase in CE and TG accumulation when compared with LDL enriched in non-glucosylated PtdEtn. After a 24-h incubation with LDL containing Glc-PtdEtn, the macrophages contained 2-fold higher CE (10.11 ± 1.54 μg/mg cell protein) and TG (285.32 ± 4.38 μg/mg cell protein) compared with LDL specifically enriched in non-glucosylated PtdEtn (CE, 3.97 ± 0.95, p < 0.01 and TG, 185.57 ± 3.58 μg/mg cell protein, p < 0.01). The corresponding values obtained with LDL containing glycated protein and lipid were similar to those of LDL containing Glc-PtdEtn (CE, 11.9 ± 1.35 and TG, 280.78 ± 3.98 μg/mg cell protein). The accumulation of both neutral lipids was further significantly increased by incubating the macrophages with Glc-PtdEtn LDL exposed to copper oxidation. By utilizing the fluorescent probe, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI), a 1.6-fold increase was seen in Glc-PtdEtn + LDL uptake when compared with control LDL. Competition studies revealed that acetylated LDL is not a good competitor for DiI Glc-PtdEtn LDL (5–6% inhibition), whereas glycated LDL gave an 80% inhibition, and LDL + Glc-PtdEtn gave 93% inhibition of uptake by macrophages. These results indicate that glucosylation of PtdEtn in LDL accounts for the entire effect of LDL glycation on macrophage uptake and CE and TG accumulation and, therefore, the increased atherogenic potential of LDL in hyperglycemia.
Oxidative damage of vascular smooth muscle cells by the glycated protein-cupric ion system
1998, AtherosclerosisTo clarify the mechanism of cellular injury through the nonenzymatic reaction of glucose with proteins, we studied the cytotoxic effect of glycated bovine serum albumin on cultured smooth muscle cells in the presence of cupric ion. Glycated proteins were prepared by incubating bovine serum albumin with 0.5 M d-glucose in 0.3 M sodium phosphate buffer at 37°C for 2, 4 and 16 weeks (g-BSA-2, g-BSA-4 and g-BSA-16, respectively). Early glycation products, such as fructosamine, were formed more than two weeks after incubation. However, the immunoreactivity of glycated proteins to anti-AGE antibody was 12-fold higher in g-BSA-16 than in g-BSA-2. Both g-BSA-2 and g-BSA-16 showed a concentration-dependent cytotoxicity in smooth muscle cells in the presence of 80 μM cupric ion by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) dye reduction assay and dye exclusion test. Flow cytometry and spectrofluorophotometry using dihydrorhodamine 123 showed that the extracellular generation of oxidants was dose-dependently enhanced with increasing concentrations of g-BSA-2 or g-BSA-16 in the presence of cupric ion. However, no difference was observed in the intracellular generation of oxidants between the presence and absence of glycated proteins by flow cytometry using 2′, 7′-dichlorofluorescein diacetate. Cytotoxicity and oxidant generation were prevented by catalase and tiron, but not by superoxide dismutase or mannitol, a hydroxyl radical scavenger. These results indicate that smooth muscle cells may be damaged by reactive oxygen species which are produced extracellularly by the interaction with the early glycation products and cupric ion, and suggest that hydrogen peroxide may be a candidate for reactive oxygen species which contribute to such oxidative damage of smooth muscle cells.
Do E-series prostaglandins and their metabolites influence oxidation of native and glycated low-density lipoproteins?
1998, Prostaglandins and Other Lipid MediatorsOxidation of lipoproteins, and, in particular, low-density lipoproteins (LDL), has been shown to play a significant role in the pathogenesis of atherosclerosis. Oxidized LDL are endocytosed via scavenger receptors to form lipid-laden foam cells. The non-enzymatic reaction of glucose with proteins and lipoproteins results in a modified LDL involved in the pathogenesis of late complications in diabetes mellitus. In the present paper, the influence of various E-series prostaglandins (PGE1; 13,14-dihydro PGE1; 13,14-dihydro 15-keto PGE1; and PGE2) on oxidation of native and glycated LDL was investigated. The effect of these agents in the concentration range from 1 pg/mL to 1.6 μg/mL on copper-induced oxidation of native and glycated LDL was tested. The concentration of each agent causing the maximal effect on oxidation of native LDL, as measured by the formation of thiobarbituric acid-reacting substances, was chosen to estimate the effect on 2, 4, 8, and 24 h oxidation of glycated LDL. The study was performed with LDL isolated by sequential ultracentrifugation from normolipidemic individuals. LDL (0.25 mg protein/mL) was oxidatively modified with 5 μm CuSO4. The glycosylation of LDL was performed by incubation of LDL with 500 mM glucose for varying periods of time ranging from 10 to 31 days. Our results show that only 13,14-dihydro PGE1 significantly inhibits copper-induced oxidation of native LDL, while the other examined E-series prostaglandins in vitro are ineffective as reducing agents in LDL-oxidation.