| Grant number: | 18/07283-1 |
| Support Opportunities: | Regular Research Grants |
| Start date: | February 01, 2020 |
| End date: | May 31, 2022 |
| Field of knowledge: | Biological Sciences - Physiology - General Physiology |
| Principal Investigator: | Renata Gorjao |
| Grantee: | Renata Gorjao |
| Host Institution: | Pró-Reitoria de Pós-Graduação e Pesquisa. Universidade Cruzeiro do Sul (UNICSUL). São Paulo , SP, Brazil |
| Associated researchers: | Adriana Cristina Levada-Pires ; Sandro Massao Hirabara |
Abstract
In Obesity, there is increased plasma leptin and lymphocyte polarization to Th1 cells accompanied by inhibition of regulatory T cells (Treg). However, the relationship between leptin concentrations and lymphocyte differentiation in humans is not established. The flow of metabolites through the glycolytic pathway correlates with the differentiation of Th0 cells to Th1 and Th17, which have inflammatory activity. Inhibition of the glycolytic pathway blocks this process and promotes differentiation for Treg cells. Our hypothesis is that, in concentrations found in plasma of obese patients, leptin stimulates lymphocyte polarization to the Th1 and Th17 profile, a process accompanied by increased activity of the glycolytic pathway. The effect of different leptin concentrations (5, 10, 25, 40, 50, 100 and 200 ng/mL) will be evaluated in the process of lymphocyte differentiation and metabolism obtained from eutrophic volunteers (protocol 1). Experiments will also be performed on lymphocytes obtained from obese individuals and data compared with those from eutrophic subjects (protocol 2). In protocol 1, lymphocytes will be isolated from venous blood from healthy and eutrophic men aged 18 to 40 years old. Subsequently, lymphocytes will be treated with 5 to 200 ng/mL of leptin for 24 hours, and then stimulated with phorbol myristate acetate (PMA) and ionomycin for 12 hours or phytohemagglutinin (PHA) for 48 hours. In parallel, we will evaluate the same parameters in lymphocytes obtained from the blood of obese and eutrophic individuals without treatment with leptin (protocol 2). The results obtained in protocol 2 will be correlated with the volunteers' plasma leptin concentration and the data obtained in protocol 1 with the dose response curve obtained. The following parameters will be evaluated: membrane integrity and DNA fragmentation by propidium iodide incorporation; Th1, Th2 and Th17 profile; GLUT1 and leptin receptor expression on the plasma membrane; proliferative capacity by BRDU incorporation by flow cytometry; phosphorylation and total expression of proteins involved in leptin signaling (JAK2, STAT3, ERK1/2, Akt and p38) and analysis of the expression of electron transport chain protein complexes (I to IV) by western blotting; mRNA expression of enzymes involved with beta-oxidation by real-time PCR; lactate production and hexokinase activity, citrate synthase, glucose 6-phosphate dehydrogenase (G6PDH) and pyruvate dehydrogenase by spectrophotometry; mitochondrial transmembrane potential by flow cytometry; [U-14C] palmitate oxidation test to CO2 and oxygen uptake kinetics of O2. The same analyzes will be performed in the presence of leptin-activated signaling pathway antagonists: JAK2/STAT3, p38MAPK and ERK1/2. The present study will allow to determine the effects of different leptin concentrations on the lymphocyte differentiation process that may be related to the inflammatory lymphocyte profile observed in obese individuals. (AU)
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