Prolactina aumenta a expressão de enzimas antioxidantes e ativa o receptor ativado por proliferador de peroxissoma (ppar) em células INS- 1E

Abstract

Diabetes mellitus (types 1 and 2) is characterized by pancreatic beta cells death induced, among other factors, by oxidative stress (OE). It is known that prolactin (PRL) improves the survival of beta cells against this condition, however the mechanism by which this occurs is still unknown. Since PRL modulates the gene expression of antioxidant enzymes in different cell types, our objective was to investigate whether PRL protects pancreatic beta cells from oxidative stress by increasing the production of antioxidant enzymes and what signaling pathways would be involved in this effect. For this, we used beta cell line from rats insulinoma, INS-1E. Cells were pretreated for 24 h with vehicle (0.3 mmol/L NaHCO3 + 0.3% BSA) or PRL (0.5 μg/mL), followed by induction of oxidative stress with hydrogen peroxide (H2O2) for evaluation of cell viability, mitochondrial production of reactive oxygen species (ROS) and protein content of antioxidant enzymes catalase (CAT), glutathione peroxidase 1 (GPx1), superoxide dismutase 1 (SOD1) and superoxide dismutase 2 (SOD2). The protein content of these enzymes was also evaluated in the time-course experiment, in which the cells were treated with PRL (0.5 μg/mL) at different times. Physiologically, during pregnancy there is marked pituitary secretion of PRL. Therefore, in order to indicate possible signaling pathways to be investigated, our next step was to perform bioinformatics analyzes with the genes upregulated in islets of pregnant rats previously identified by our research group from macroarray. The findings of these analyzes led us to carry out the reporter promoter assay using different concentrations of PRL for 24h. Finally, to test whether the effects of PRL are mediated by the signaling pathway suggested by bioinformatics analyzes, we again evaluate cell viability in the presence of siRNA for target gene knockdown. Statistical analyzes were performed by t-test or ANOVA followed by the Bonferroni posttest. PRL increased the survival of pancreatic beta cells exposed to H2O2, as well as the protein content of SOD2 and CAT. We also observed decrease in mitochondrial H2O2 concentration in cells pretreated with the hormone. Bioinformatics analyzes revealed enrichment of the peroxisome proliferator-activated receptor (PPAR) pathway, which is involved in the transcription of antioxidant enzymes. Using the reporter promoter assay we observed that PRL activates PPAR. Among the existing PPAR isoforms, we evaluated whether PRL would be dependent on PPARγ to prevent cell death caused by EO, since PPARγ expression is increased by PRL in preadipocyte cell line and in adipose tissue of rats. However, the knockdown of this transcription factor did not alter the efficiency of PRL in protecting beta cells from death caused by H2O2. We conclude that PRL increases the expression of antioxidant enzymes and this action seems to be independent of PPARγ. Future investigations are needed to identify the signaling pathway that mediates this effect.