268 THE EFFECTS OF METFORMIN ON THE INSULIN SIGNALING PATHWAY ON PORCINE GRANULOSA LUTEIN CELLS IN A PRIMARY CULTURE SYSTEM

Abstract

Metformin (N,N22-dimethylbiguanide) is an oral antihyperglycaemic drug which increases insulin-stimulated glucose uptake as an insulin sensitizing agent (ISA) and has direct effects on ovarian steroidogenesis in human. To investigate the effects of metformin on the insulin signaling pathway on porcine granulosa lutein cells in a primary culture system, we examined mRNA expressions of porcine insulin receptor, insulin-like growth factor-1 receptor, and insulin receptor substrate-1; expressions of downstream targets (Raf, MEK1/2, ERK, PDK1, mTOR, p70, and nuclear factor kB) of the insulin receptor signaling pathway; the luciferase activity of transcription factors activator protein 1; and nuclear factor kB. Granulosa cells were plated in DMEM and 10% fetal bovine serum at a density of 3.5 × 10⁷ in a T-25 tissue culture flask and cultured for 48 h at 37°C in a humidified atmosphere of 5% CO₂ and 95% air. Then, porcine granulosa lutein cells (pGLs) were cultured in a serum-free DMEM as a control group or supplemented with 10^-5 M metformin, 100 ng mL^-1 of insulin, or both for 24 h. The monolayer was collected for RT-PCR and western blot analysis. For transient transfections, porcine granulosa cells were plated in DMEM and 10% FBS at a concentration of 2.5 × 10⁶/well in 24-well culture dishes for 16 h and then transfected with plasmid constructs using FuGene6 Transfection Reagent. Reporter vectors used in these studies were AP-1-luc and NF-κ B-luc. Cells were treated post-transfection with 10^-5 M metformin, 100 ng mL^-1 insulin, or both for 48 h. The luciferase activity was assayed using the dual luciferase assay kit with a Microlumat LB 96 P luminometer. Metformin with insulin significantly increased mRNA expressions of insulin receptor, insulin-like growth factor-1 receptor, and insulin receptor substrate-1, whereas metformin alone had no significant effect on the expression of genes. Metformin alone and with insulin significantly increased expressions of downstream targets of the insulin receptor signaling pathway except MEK2. Metformin with insulin significantly elicited an induction of luciferase activity in the transfection of activator protein 1 and nuclear factor kB reporter, whereas metformin alone did not. Metformin induced expressions of target molecules in the insulin signaling pathway but had no effect on the luciferase activity of activator protein 1 and nuclear factor kB in porcine granulosa lutein cells. Metformin with insulin increased the luciferase activity of activator protein 1. These results suggest that metformin with insulin could change the function of ovarian granulosa cells induced by activator protein 1 activity.