Evaluation of the action of triiodothyronine (T3) in the purinergic system, with the participation of extranuclear pathways, in human adipose tissue

Abstract

White Adipose Tissue (TAB) is an important endocrine organ, essential for metabolic regulation of the organism, through adipokine production and secretion, such as adiponectin and leptin. It is known that TAB expresses receptors of the purinergic system, which are important in lipid metabolism, and can be activated by adenosine triphosphate (ATP) and its derivatives. In addition, TAB is modulated by thyroid hormones (HT), mainly triiodothyronine (T3), through extranuclear pathways, among them MAPK/ERK and PI3K/Akt. There is an important relationship between HT and purinergic signaling in different physiological and pathological systems, however, this relationship in adipose tissue is poorly understood in the literature. In previous results, we identified the expression of purinergic receptors in human subcutaneous adipocytes, from global transcriptome data, which determined the choice of P2X7, P2Y1 and P2Y11 receptors, to be investigated in the present study. Thus, the objective is to validate the results of the action of T3 on the expression of P2X7, P2Y1 and P2Y11 receptors, as well as to determine if this action is mediated via non-canonical mechanisms, by activating the MAPK/ERK and PI3K pathways, and if there are the translocation of these receptors that are located on the cell membrane, in addition to measuring the release of ATP and expression of adipokines. For this, human pre-adipocytes (HPAd-S802s-05A), after differentiation, will be submitted to treatment with T3 (10nM), for 24h, in the presence or absence of inhibitors of the MAPK/ERK pathways (PD98059 - 50uM) and PI3K ( LY294002 - 50 ¼M). The following parameters will be analyzed: cell viability (MTT), confirmation of inhibition of the pathways (western blot), determination of lipid accumulation (oil red staining), basal lipolysis (NEFA and Glycerol assay), oxidative stress (malonaldehyde and carbonylation measurements), protein expression of P2 purinergic receptors and adipokines (immunofluorescence and western blot), translocation of P2 receptors (immunofluorescence), quantification of extracellular ATP (HS II luciferin- luciferase bioluminescence assay), quantification of NTPDAses (immunofluorescence). The present work will allow to determine, if the action of T3 in the modulation of purinergic P2 receptors, involves the activation of non-nuclear pathways, and what is the reflex of the action of the hormone and inhibition of the pathways, in the release of extracellular ATP, in the subcutaneous adipose tissue. human. The understanding of this involvement, of T3 on components of purinergic signaling, can lead us to identify effects observed in disorders, such as Obesity. (AU)