TSPO INTERFERENCE ON THE PATHWAY OF ACTIVATION OF 3T3-L1 ADIPOCYTES LINEAGE

Abstract

Obesity is associated to low grade chronic inflammation and represents one of the risk factor for the development a series of co-morbidities, such as insulin resistance and type 2 diabetes mellitus. TSPO (translocator protein 18 kDa) is involved in several cellular functions, including steroid biosynthesis and transport, porphyrins transport, apoptosis, heme biosynthesis, ion transport, cellular burst, oxidative processes and immunomodulation. However, the presence and functions of TSPO in adipose tissue and obesity is not well established. Our research group has investigated the immunomodulatory role of TSPO, and the present project will evaluate the expression and function of TSPO receptor in adipocytes 3T3-L1, and the action of TSPO in Raw 264.7 macrophage polarization. For this, 3T3-L1 and Raw 264.7 are treated or not with Diazepan and/or Ro5-4864 agonists and/or PK11195 antagonist, and also stimulated or not with LPS. Will be investigated TSPO gene expression in Raw 264.7 lineage and the differentiation process of 3T3-L1 lineage by RT-PCR real time, as well as the glucose metabolism, and GLUT-4 and TSPO protein expression by Western blot. The viability, proliferation, fragmentation of DNA and cell cycle of 3T3-L1 pre-adipocytes, and the ability of TSPO ligands-induced polarization in Raw 264.7 co-cultured of macrophages and adipocytes will be analyzed by flow cytometer. The mediators inflammatory secretion (TNF, MCP-1, adiponectin, resistin, leptin, IL-6, IL-10 and IL-12) will be quantified in cultures and co-cultures supernatants by enzyme immunoassays; the 3T3-L1 metabolic activity will be analyzed by MTT and lipolysis and triacylglycerol accumulation will be analyzed by colorimetric assay. Thus, we can better characterize and understand the role of TSPO receptor in inflammatory and metabolic pathways of adipose tissue and macrophages polarization, beyond the main role of macrophages in inflammatory and metabolic mechanisms of the adipose tissue.