METFORMIN ACTS DIRECTLY ON RAT GRANULOSA CELLS ACTIVATING THE AMPK AND REGULATING VEGF EXPRESSION.

DI PIETRO, MARIANA; SCOTTI LEOPOLDINA; PASCUALI NATALIA; OUBIÑA, GONZALO; VELAZQUEZ CANDELA; MATZKIN EUGENIA; RIVIERE EUGENIA; FRUNGIERI MONICA; PARBORELL FERNANDA; ABRAMOVICH DALHIA

Congreso; LXII REUNIÓN ANUAL DE LA SOCIEDAD ARGENTINA DE INVESTIGACIÓN CLÍNICA (SAIC); 2017

Polycystic ovary syndrome (PCOS) is a common disorder that affects women in reproductive age. Its symptoms are heterogeneous and range from chronic anovulation, oligo- or amenorrhea, and hyperandrogenism to obesity and insulin resistance. Metformin (MET) is an oral antihyperglycemic drug introduced in the treatment PCOS to manage hyperglycemia. MET has been shown to improve ovulation, pregnancy and live birth rates in patients with PCOS. The mechanism by which MET improves reproductive parameters are not fully understood. MET is a highly hydrophilic molecule so the organic cation transporters (OCTs) are actively involved in the cellular uptake of MET in different tissues. Its primary mechanism of action 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. The aims of the present work were to analyze a possible direct effect of MET on rat granulosa cells and to evaluate the presence of OCTs in this cell type. Materials and Methods: Sprague Dawley rats (21d) were injected subcutaneously with diethylstilbestrol (DES: 1mg/rat) daily for three days to stimulate the development of early antral follicles. Granulosa cells (GCs) were isolated by percoll gradient. RNA was isolated from the GCs and RT-PCR was performed for OCT 1-3. Another set of isolated GCs were stimulated with MET 0.01 or 0.1ng/ml with or without the OCT inhibitor cimetidine (CIM). Cells were harvested 48 h later and proteins extracted for OCTs, phospho-AMPK (P-AMPK) and VEGF measurement by western blot. Results: Expression of OCT 1, 2 and 3 was detected in GCs. P-AMPK was increased in rat GCs after metformin treatment. VEGF levels decreased after stimulation with MET. Inhibition of OCTs by CIM reversed these effects. Conclusion: We demonstrated that MET acts directly on rat GCs. Our findings suggest that MET enters GCs through OCTs. These results provide new evidence to explain the effect of MET on infertility treatments.