In vitro and ex vivo effects of 17b-estradiol and metformin on glucose uptake, activation of FOXO3a and insulin receptor signaling pathways in ovarian cells

Abstract

The estrogens are responsible for many biologic process in ovaries. Their production and secretion contribute significantly for maintenance of women physiology. The 17 ²-estradiol (E2) is the more active form of natural estrogen. To perform its biological functions depends on estrogen receptor activation (ER± ou ²). Additionally, E2 controls the PI3K/AKT signaling pathway due to increased interaction with ER±, stimulating glucose metabolism. The E2 antidiabetogenic potential has clinical significance in the insulin resistance (RI) treatment, as commonly observed in Polycystic Ovarian Syndrome (PCOS). Although there has been a great advance in the characterization and understanding of energy change in RI, the number of substances used in the treatment of IR-related morbidities is still very limited. The treatment of IR through insulin sensitizing agents, such as metformin, is still controversial, as some researchers advocate its use only in patients with proven glucose intolerance. Additionally, as mentioned before, E2 controls the PI3K/AKT pathway which, in response to insulin, induces FOXO3a transcription factor phosphorylation. This, in turn, can be inhibited by metformin, preventing protein translocation to the cytoplasm. Thus, there would be decreased expression of their target genes, which could induce IR. Our project aims to verify the effects of 17²-estradiol and metformin treatment, isolated and combined, on glucose uptake and their role in FOXO3a and insulin receptor (InsR) signaling pathways using ovarian cells in vitro and ex vivo. For that, CHO 1-15 cells (ATCC CRL9606) and primary cells will be used. The CHO cells will be treated with low and high glucose concentration (LowGlic = 1,000 mg/L and HighGlic = 1,800 mg/L, respectively) and subdivided into 8 groups, as follow: 1) Control (no treatment); 2) 17²-estradiol; 3) metformin; 4) insulin; 5) 17²-estradiol and metformin; 6) 17²-estradiol and insulin; 7) metformin and insulin, and 8) 17²-estradiol, metformin and insulin combined. Glucose uptake will be evaluated by spectrophotometry and the expression of molecules related to FOXO3a and InsR signaling pathways will be evaluated by Real Time PCR and Western Blot analyses. The effects of treatments on cell behavior will be evaluated by proliferation and apoptosis assays. All results will be submitted to the relevant statistical analyzes. (AU)