Metformin acts directly on rat granulosa cells activating the AMPK and regulating VEGF expression.

SCOTTI LEOPOLDINA; ABRAMOVICH DALHIA; PARBORELL FERNANDA; DI PIETRO MARIANA

Simposio; Frontiers in Reproduction; 2017

Polycysticovary syndrome (PCOS) is a common disorder that affects between 5% and 8% ofwomen in reproductive age. Its symptoms are heterogeneous and range fromchronic anovulation, oligo- or amenorrhea, and hyperandrogenism to obesity andinsulin resistance. Metformin(1,1-dimethylbiguanide hydrochloride, MET) is an oral antihyperglycemic drugwidely used for the treatment of type 2 diabetes. Its primary mechanism ofaction is through the activation of the AMP-activated protein kinase (AMPK),which acts as an energy sensor by monitoring the AMP/ATP status of the cell.Activation of AMPK produces the pleiotropic beneficial effects of this drug,such as the suppression of endogenous glucose production and the improvement inlipid metabolism. MET has been introduced in the treatment for PCOS to manageinsulin resistance and hyperglycemia. However, the effects of this drug on PCOSare wide and exceed those related to glucose metabolism. In this regard, METhas been shown to improve ovulation, pregnancy and live birth rates in PCOSpatients. However, the mechanism by which MET improves reproductive parametersare not fully understood. MET is a highly hydrophilic molecule that ispartially able to enter the cell by passive diffusion through the membrane. Inaddition, organic cation transporters (OCTs) are actively involved in thecellular uptake of MET in different tissues. The aim of the present work was toanalyze a possible direct effect of MET on rat granulosa cells and to evaluatethe involvement of OCTs in these effects. Materialand Methods: Immature Sprague Dawley rats were injected with DES for three daysand then sacrificed. The ovaries were obtained and granulosa cells (GCs) wereisolated by percoll gradient. RNA was isolated and RT-PCR was performed for OCT1-3. Another set of isolated granulosa cells were cultured in DMEM:F12 medium andstimulated with MET 0.01 or 0.1 ng/ml. Cell were harvested 48 h later andprotein extracted for OCTs, AMPKP, AMPK and VEGF measurement by western blot. Results:Phosphorylation of AMPK was increased after metformin treatment. Moreover, VEGFlevels were lower in GCs stimulated with metformin than basal GCs. Inhibitionof OCTs by cimetidine reversed these effects. Conclusion:These results suggest a possible direct effect of MET on rat GCs. MET is ableto activate the AMPK cascade thus regulating cell metabolism. In addition, VEGFproduction, which is increased in PCOS patients, decreases after MET treatment.Moreover, this is the first time that organic cation transporters 1-3 aredescribed to be expressed in GCs. As it has already been demonstrated that OCTstransport MET into different types of cells, they are likely involved in thesedirect effects of MET on GCs. These results provide new evidence to explain theeffect of MET on infertility treatments besides its effects on systemic metabolism,in patients that suffer mainly from insulin resistance and infertility, such asPCOS patients.