Influence of insulin on the processes of autophagy and phagocytosis in bone marrow-derived macrophages

• Grant number: 21/04525-7
• Support Opportunities: Scholarships in Brazil - Master
• Start date: December 01, 2021
• End date: May 31, 2023
• Field of knowledge: Biological Sciences - Pharmacology - General Pharmacology
• Principal Investigator: Joilson de Oliveira Martins
• Grantee: Kamilla da Costa Pantoja
• Host Institution: Faculdade de Ciências Farmacêuticas (FCF). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Hyperglycemia is the main characteristic of diabetes mellitus (DM) and is associated with high mortality in affected individuals. In previous studies, we evaluated phosphatidylinositol 3 kinase (PI3K), protein kinase B (PKB, or Akt) and mitogen-activated protein kinase (MAPK) proteins in bone marrow-derived macrophages (BMDM) after lipopolysaccharide (LPS) stimulation, treated or not with insulin by measuring the inflammatory mediators: tumor necrosis factor (TNF) -±, interleukin (IL) -6 and IL-10. It was observed that insulin, by modulating the PI3K, AKT, ERK 1/2 and SAPK/JNK pathways, amplifies the secretion of TNF-± and IL-6 in BMDM under LPS stimulation. In addition, autophagy, a regulatory mechanism necessary for the maintenance of cellular homeostasis, has shown a relationship with the pathophysiology of DM. Thus, in this new study we aim to investigate how insulin influences the processes of autophagy and phagocytosis and the participation of the signaling pathways mentioned in BMDM from diabetic (streptozotocin, 65 mg / kg, ip, on five successive days), or non-diabetic mice, under LPS stimulation. For this, insulin treatment will happen simultaneously with LPS stimulation in 6 and 24 hours (Tessaro et al.2020), and we will quantify the cytokines (TNF-±, IL-6 and, IL-10) in the cell culture supernatant using ELISA method and NO using the Griess reaction method. The intracellular signaling pathways of PIK3, AKT, PKC alpha and PKCdelta, as well as autophagy pathway (BECLIN1, CLASS 3 PI3K, ULK1, LC3B) will be evaluated by Western blotting and RT-PCR techniques. Through phagocytic and microbicidal activity we will evaluate the functionality of BMDM and the role of insulin in these processes. (AU)