Activation of the PI3K/Akt/mTOR Pathway by Insulin Is Crucial for Amplification of FSH-Induced Differentiation, but Not Proliferation, of Ovarian Granulosa Cells.

Abstract

Follicle stimulating hormone (FSH) controls both proliferation and differentiation of granulosa cells. FSH induced differentiation and in particular steroidogenesis are synergized by insulin. Whether insulin potentiates the proliferative effect of FSH is not known. Moreover, the crosstalk between insulin and FSH intracellular signaling pathways in granulosa cells has not been examined. The aim of this study was to investigate whether insulin affects granulosa cell proliferation and to explore the mechanisms that are cooperatively regulated by insulin and FSH during granulosa cell proliferation and differentiation. Primary granulosa cells obtained from estradiol-treated immature rats were cultured in defined medium in the absence of serum. Proliferation was stimulated by treatment with FSH and activin, both at a 50 ng/ml final concentration. As expected, FSH/activin treatments stimulated granulosa cell proliferation as determined by MTT assays. However, FSH/activin-induced proliferation was not affected by co-treatment with increasing doses of insulin. Moreover, insulin itself did not stimulate granulosa cell proliferation. On the other hand, stimulation of P450aromatase (Cyp19) and P450 side chain cleavage (P450scc) expression by FSH was potentiated by the presence of insulin in the culture medium. Interestingly, dose-response experiments revealed a characteristic bell-shaped curve in which concentrations of 1, 10 and 50 μg/ml of insulin progressively potentiated the effect FSH on Cyp19 and P450scc. However, concentrations higher than 100 μg/ml led to a decrease of Cyp19 and P450scc stimulation. A similar response was observed in the stimulation of liver receptor homolog-1 by FSH. In the following experiments, we aimed to determine the intracellular signaling pathway mediating insulin effects, utilizing a dose of 10 μg/ml of insulin. For this purpose, granulosa cells were pretreated with PI3k inhibitors (wortmannin and LY294002), a MAPK inhibitor (UO126), or a SRC kinase inhibitor (PP2) prior to stimulation with FSH plus insulin. The potentiating effect of insulin on the FSH-induced expression of Cyp19 and P450scc was prevented by the two PI3k inhibitors. However, these inhibitors did not affect FSH-induced stimulation of gene expression. In contrast, the MAPK inhibitor decreased the expression of Cyp19 and P450scc expression to values significantly lower than those found in the presence of FSH alone. This suggests that MAPK inhibition prevented FSH stimulation, precluding any analysis of the effects of this pathway on insulin action. Finally, treatment with a SRC kinase inhibitor had no effect on the super-induction of Cyp19 or P450scc expression observed in the presence of FSH and insulin. To confirm these findings and to explore the downstream targets of PI3k/Akt, granulosa cells were treated with rapamycin, an inhibitor of mTOR kinase which is activated by Akt. Similar to the results observed by inhibiting PI3K, rapamycin treatment prevented the insulin-induced potentiation of FSH actions. Taken together, this evidence indicates that the effects of insulin in granulosa cell steroidogenesis are not only dose-related but they also follow a bell-shaped dose-response curve. These results also demonstrate that activation of the PI3K/Akt/mTOR pathway by insulin is crucial for the amplification of FSH-induced differentiation, but not proliferation, of ovarian granulosa cells. Supported by NIH R01HD057110 and R21HD066233. (poster)