The initial picture of preadipocyte response to a nitrooxidative environment

FUNK MELANIA; BRUZZONE ARIANA; SALVADOR GABRIELA; URANGA ROMINA,

Salta

Congreso; Joint LV annual SAIB meeting and XIV PABMB congress, 5 al 9 de noviembre de 2019, Salta, Argentina.; 2019

SAIB

Funk, MI; Bruzzone, A; Salvador, GA; Uranga, RM. . Libro de resúmenes: página 51. LV SAIB/PABMB Congress, 5 al 8 de noviembre de 2019, Salta, Argentina.92CB-P47THE INITIAL PICTURE OF PREADIPOCYTE RESPONSE TO A NITRO-OXIDATIVE ENVIRONMENTFunk MI1, Bruzzone A1,2, Salvador GA1,2, Uranga RM1,2.1INIBIBB-CONICET, 2DBByF -UNS, Bahía Blanca, Argentina. E-mail: ruranga@criba.edu.arCumulative evidence indicates that obesity-related metabolic dysfunction is more associated with the existence of hypertrophic dysfunctional adipocytes rather than to numerous small adipocytes. For this reason, the study of adipocyte development as a major cause of obesity has become a relevant area of research. We have previously shown that menadione-induced oxidative stress inhibits adipogenesis and increases triglyceride content in the mature adipocyte, with the PI3K/Akt pathway playing a crucial role. Here, we tested the effect of nitro-oxidative stress (NS) induced by sodium nitroprusside (SNP) on 3T3-L1 preadipocytes. For this purpose, we exposed 3T3-L1 preadipocytes to 0.0?1.0 mM SNP for 24 h, and we characterized the level of cellular injury triggered by NS. Cell mortality was close to 100% after 24-h incubation in the presence of concentrations of SNP over 0.2 mM. Low SNP concentrations (0.1?0.2 mM) altered neither mitochondrial functionality (MF) nor increased reactive oxygen species (ROS). However, lipid peroxides were found to be increased, and the permeability of the cell membrane (measured as LDH leakage) was significantly altered. These findings indicate that SNP triggers the generation of ROS that are early scavenged, and only the remaining lipid peroxidation products and damage to cell membrane are detected after 24 h. We also evaluated the short-term effect of the exposure to SNP by incubating 3T3-L1 cells with 0.0?10.0 mM SNP for 2 h. It was found that MF was not affected by 0.5 mM SNP, but significantly diminished by concentrations of SNP over that (20%, 40%, and 60% decrease by 1 mM, 5 mM, and 10 mM, respectively). Moreover, 0.5 mM tripled ROS levels, doubled the levels of lipid peroxides and significantly altered membrane permeability (4-fold increased LDH leakage). We then evaluated the phosphorylation levels of Akt, ERK1/2, and the stress-activated protein kinase/Jun-amino-terminal kinase (SAPK/JNK), four well known kinases involved in cellular redox signaling, after the incubation with 0.5 mM SNP (increased markers of NS without affecting MF) and 5 mM SNP (increased markers of NS with decreased MF). The four kinases were found to increase their phosphorylation level, thus, to be activated by both concentrations of SNP. Immunocytochemistry analysis revealed a different subcellular distribution of the kinases upon SNP treatment: the active form of Akt showed a preferential nuclear localization, regardless of the concentration of SNP; p-ERK1/2 increased their nuclear localization only in the condition where MF was not affected; p-SAPK/JNK increased all over the cell. Together, our findings show the response-time distribution that arises from the SNP treatment of preadipocytes. Our next goal is to evaluate the differentiation capacity of these preadipocytes acutely exposed to SNP.