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| Author(s): |
Raphael de Souza Pinto
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Faculdade de Medicina (FM/SBD) |
| Defense date: | 2011-08-10 |
| Examining board members: |
Marisa Passarelli;
Everardo Magalhães Carneiro;
Maria Lucia Cardillo Correa Giannella;
Nadja Cristhina de Souza Pinto Lardner;
Edna Regina Nakandakare
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| Advisor: | Marisa Passarelli |
| Abstract | |
Advanced glycation end products (AGE) impair reverse cholesterol transport, by decreasing the HDL-mediated cholesterol efflux from macrophages. We evaluated the role of advanced glycated albumin (AGE-albumin) on the generation of reactive oxygen species (ROS) by mitochondria and NADPH oxidase, and its implication on the HDL receptor (ABCA-1) level in macrophages. AGE-albumin was prepared by incubation with glyoxal (GO), methylglyoxal (MGO) or glycolaldehyde (GAD) and control albumin (C-albumin) with phosphate buffered saline alone. C and AGE-albumin were incubated along time with J774 macrophages in order to determine ROS production and ABCA-1 protein level by flow citometry. Macrophages treated with GO, MGO and GAD-albumin presented, respectively, 24%, 25% and 24% increased ROS production compared to cells treated with C-albumin. The increase in ROS production was prevented by cell treatment with a NADPH oxidase inhibitor or mitochondrial uncoupler, demonstrating a role of NADPH oxidase and mitochondria in AGE-albumin-induced ROS generation. Compared to cells treated with C-albumin, basal mitochondrial respiration, determined by oxygraphy, was 35% and 46% reduced in cells exposed, respectively, to GO and GAD-albumin and was not restored after cell treatment with mitochondrial uncoupling. Intracellular carbonyl content increased 41% in macrophages treated with GAD-albumin as compared to C-albumin. In macrophages treated with GAD-albumin, the reduction in ABCA-1 content observed after 8 hours of treatment was accompained by the increase of ROS production. Aminoguanidine that prevented ROS generation was able to restore ABCA-1 levels. On the other hand, benfotiamine failed to restore ABCA-1 protein levels which was ascribed to a lesser reduction in ROS generation by this compund. These results point to a role of AGE-albumin on the reduction of cellular cholesterol efflux, notably by diminishing ABCA-1 protein level in macrophages which is associated with intracellular oxidative stress. Inhibition of oxidative stress induced by AGE-albumin prevents disturbances in reverse cholesterol transport by curbing the reduction of ABCA-1 elicited by advanced glycation in macrophages and therefore may contribute to the prevention of atherosclerosis in diabetes mellitus (AU) | |