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Effects of leptin on lymphocyte metabolism and differentiation

Grant number: 17/07818-0
Support type:Scholarships in Brazil - Doctorate (Direct)
Effective date (Start): October 01, 2017
Effective date (End): September 30, 2021
Field of knowledge:Biological Sciences - Physiology - General Physiology
Principal Investigator:Renata Gorjao
Grantee:Vinicius Leonardo Sousa Diniz
Home Institution: Pró-Reitoria de Pós-Graduação e Pesquisa. Universidade Cruzeiro do Sul (UNICSUL). São Paulo , SP, Brazil

Abstract

Leptin is an adipokine that has an important role in metabolism and modulates the immune response. Leptin acts on lymphocytes stimulating Th1 cells response and inhibiting regulatory T cells (Treg), contributing to the development of the inflammatory process. The activation of lymphocytes is associated with specific metabolic pathways that are important for optimizing their function. In addition, the predominance of the glycolytic pathway may promote differentiation of Th0 cells into Th1 and Th17, which have proinflammatory features, suppressing the polarization to Th2 phenotype. Inhibition of the glycolytic pathway blocks this process and promotes differentiation into Treg cells. Treg lymphocytes use fatty acids and mitochondrial metabolism during differentiation. The regulation of the metabolic pathways associated with lymphocyte proliferation as well as the metabolic changes resulting from the effects of leptin on lymphocytes have not been described. The aim of this study is to evaluate the effect of leptin on the metabolic changes associated with the process of lymphocyte differentiation as well as the alterations of the signaling pathways involved with this process. Lymphocytes will be isolated from venous blood of healthy men aged between 18 to 50 years old. Subsequently, cells will be treated with 10, 25, 40, 50 and 100 ng/mL leptin for 24 hours, followed by stimulation with Phorbol Myristate Acetate (PMA) and ionomycin for 12 hours or phytohemagglutinin (PHA) for 48 hours. After this period, the following parameters will be evaluated in lymphocytes: Th1, Th2 and Th17 profile and expression of GLUT1 and leptin receptor in cell membrane by flow cytometry; proliferative capacity by incorporation of BRDU and flow cytometric analysis; phosphorylation and total expression of proteins involved with the signaling of leptin (JAK2, STAT3, ERK1/2, Akt and p38) and analysis of electron transport chain (I to IV) protein complexes by western blotting; mRNA expression of beta oxidation enzymes by real-time PCR; lactate production and activity of hexokinase, citrate synthase, glucose 6-phosphate dehydrogenase (G6PDH) and pyruvate dehydrogenase by spectrometry; mitochondrial transmembrane potential; oxidation assay of [U-14C] palmitate to CO2; O2 consumption kinetics through the oxygraphy. The same analyzes will be performed in the presence of leptin signaling pathway antagonists: JAK2/STAT3, p38MAPK and ERK1/2. The present study will allow a better comprehension of the metabolic effects promoted by leptin and involved in the process of lymphocyte differentiation that may be related to changes in immune response control observed in obese individuals and with diseases related to immunodeficiency. (AU)