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ROLE OF O-GLUCONACILATION IN THE ACTIVITY OF THE FOXO1 TRANSCRIPTION FACTOR: VASCULAR ENDOTHELIAL CELL PHYSIOLOGY IN HYPERGLYCEMIA

Grant number: 21/04378-4
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): May 01, 2021
Effective date (End): April 30, 2022
Field of knowledge:Biological Sciences - Pharmacology - Cardiorenal Pharmacology
Principal researcher:Rita de Cassia Aleixo Tostes Passaglia
Grantee:Carina Amarante Pedersoli
Home Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
Associated research grant:13/08216-2 - CRID - Center for Research in Inflammatory Diseases, AP.CEPID

Abstract

The chronic increase in the content of intracellular O-GlucoNAcilated proteins (O-GlcNAc) is one of the changes promoted by diabetes mellitus (DM). DM is one of the main risk factors for hypertension, atherosclerosis, stroke, coronary artery disease and heart failure, which are largely responsible for the high rate of morbidity and mortality of patients with this metabolic disorder. The hyperglycemic state negatively modulates several regulatory factors for the activity of vascular endothelial cells, causing dysfunction in their ability to respond to humoral and mechanical stimuli. The changes produced by hyperglycemia in the release of dilating and constricting factors by endothelial cells compromise the fine adjustment of vascular tone, with a consequent increase in peripheral vascular resistance, contributing to lesions in organs such as the kidneys, heart, eyes and brain. In addition, it alters the metabolism of the endothelial cell, in order to displace glucose to other pathways, such as the hexosamine biosynthesis (VBH) pathway. Hyperglycemia, by generating excessive activation of this pathway, causes a chronic increase in the content of O-GlucoNAcilated proteins, resulting in changes in the activity, location and recycling of important proteins, such as nitric oxide endothelial synthase (eNOS). Previous data from the laboratory show that the eNOS modified by O-GlcNAc has its function of producing impaired nitric oxide (NO), configuring itself as a sign of endothelial dysfunction. In addition, modification by O-GlcNAc increases the expression, translocation and activity of the FoxO1 transcription factor. FoxO1 activates genes associated with both resistance responses to oxidative stress and endothelial cell dysfunction. Therefore, the objective of this work is to use human endothelial cells and subject them to stress due to high glucose concentration to investigate the hypothesis that the increase in proteins modified by O-GlcNAc modifies FoxO1 activity, increasing oxidative stress and reducing the bioavailability of NO in the vascular endothelium. Initially, the rate of proteins modified by O-GlcNAc and the presence of FoxO1 in the cytosol and in the nucleus will be determined by western blot. An additional block of experiments will be carried out by increasing or preventing modification by O-GlcNAc using thiamet G and L-DON, OGA and GFAT inhibitors, respectively. The formation of oxidizing species will be measured using the DCFDA fluorescent probe. To investigate FoxO1's direct O-GlucoNAcillation, HEK293T cells will be enriched with the plasmid encoding this transcription factor and, after stimulation with thiamet G, the FoxO1-O-GlcNAc complex will be immunoprecipitated. To test this hypothesis in an animal model, preparations of arteries isolated from spontaneously hyperglycemic animals, db / db mice, will be mounted to measure the relaxation promoted by acetylcholine. These animals will also be treated with AS 1842856, an inhibitor of FoxO1 activity, to investigate the recovery of endothelium-dependent relaxation.

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