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The role of carnosine on Ca2+ handling, control of oxidative stress and protection against protein glycation: advances and applications of the study "life without carnosine"

Grant number: 19/25032-9
Support type:Regular Research Grants
Duration: June 01, 2020 - May 31, 2022
Field of knowledge:Health Sciences - Physical Education
Principal Investigator:Guilherme Giannini Artioli
Grantee:Guilherme Giannini Artioli
Home Institution: Escola de Educação Física e Esporte (EEFE). Universidade de São Paulo (USP). São Paulo , SP, Brazil
Assoc. researchers:Antonio Carlos Seguro ; Bruno Gualano ; Craig Sale ; Lívia de Souza Gonçalves ; Maria Heloisa Massola Shimizu ; Marisa Helena Gennari de Medeiros

Abstract

This project aims to assess, using in vivo experimental models to challenge homeostasis (acute physical exercise and diabetes mellitus) along with biochemical and functional markers, whether carnosine plays the following physiological roles: protection against oxygen and nitrogen reactive species and toxic by-products of the metabolism; protection against protein glycation and carbonylation; metabolic control, inflammatory profile and glucose uptake in experimental model of diabetes; regulation of Ca2+ motility during striated muscle contraction. Three independent and complementary studies are proposed, all of which using CARNS1 (gene encoding the sole enzyme responsible for endogenous synthesis of carnosine - the strain has already been developed and is available to our research team) knockout Wistar rats (KO) that will be compared with their wild type (WT) control counterparts. Study 1: samples of skeletal and cardiac muscle obtained at rest (no challenge to homeostasis) will be submitted to SERCA activity and SERCA-Ca2+ affinity assays as well as Ca2+ transport using the Time-Resolved Fluorescence Resonance Energy Transfer assay (TR-FRET). Study 2: WT and KO rats will be treated with streptozotocin to induce diabetes (DB), or only buffer with no active principle (SHAM) to serve as non-diabetic controls (CON), therefore forming 4 groups: WT-DB, WT-CON, KO-DB, KO-CON. Following diabetes confirmation, the animals will be continuously monitored (body mass, fasting glycemia, water and food intake) for 8 weeks, when the experiment will be terminated. In the last week of follow-up, the following functional assessments will be carried out: echocardiography and euglycaemic-hiperinsulinemic clamp (subgroup 1), insulin tolerance test (ITT) and kidney function via inulin clearance (subgroup 2), and skeletal muscle contractile properties and mitochondrial respiration in heart tissue homogenates (subgroup 3). Blood and selected tissues (soleus, heart and kidneys) will be collected at the end of the experiments in all subgroups for the determination of the following parameters: in blood, plasma or serum: fasting glycaemia, insulinemia, glycated haemoglobin, lipid profile (LDL, HDL, VLDL and TG), inflammatory profile (TNF-±, IL-6, IL-1², IL-10), reactive aldehyde (HNE and HHE) and their carnosine-adducts; in tissue: malondialdehyde (TBARS), 3-nitrotyrosine, 8-isoprostane, enzymatic activity (SOD, CAT and glutathione peroxidase), total and oxidised glutathione, carbonylated protein, glycated protein, advanced glycation-end products and fluorescent dihidroetidine (DHE) probe for visualisation of peroxide radicals in histological sections. Study 3: WT and KO rats will be euthanised at rest or immediately after an acute 100-min swimming exercise session with extra load corresponding to 5% of the body mass - a protocol that has been shown to acutely increase markers of oxidative stress. Blood and tissues (soleus, heart and kidneys) will be collected and submitted to the same analyses described in study 2. (AU)