Insulin enhances FSH-stimulated steroidogenesis by cultured rat granulosa cells

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Experimentally induced diabetes in female rats is associated with decreased ovarian functions. We have investigated the ovarian action of insulin using granulosa cells obtained from immature hypophysectomized, estrogen-treated rats. The cells were cultured for 2 days in a serum-free medium in the presence of follicle-stimulating hormone (FSH), with or without insulin. Medium steroids were determined by specific radioimmunoassay. Treatment with FSH caused a dose-dependent increase in the production of estrogen, progesterone, 20α-hydroxypregn-4-en-3-one (20α-OH-progesterone) and pregnenolone. Concomitant treatment with 100 ng/ml insulin increased the responsiveness of granulosa cells to FSH, decreasing the ED 50 values for FSH-stimulated estrogen and progestin production 2–2.75-fold. A lower dose of insulin, 10 ng/ml, also augmented FSH action. In contrast, treatment with insulin alone had no effect on steroid production. The insulin effect on progestin and estrogen biosynthesis was detected by 24 and 44 h after treatment, respectively. Also, the insulin action appeared to be specific since an insulin fragment, desoctapeptide insulin, exhibited no effect. Insulin also increased the FSH-stimulated increase in activity of 3β-hydroxysteroid dehydrogenase, the rate-limiting enzyme in the formation of progesterone, but was without effect on the activity of 20α-hydroxysteroid dehydrogenase, which converts progesterone to the inactive 20α-OH-progesterone. The effects of insulin on increasing FSH responsiveness could not be accounted for by changes in cell viability or total cell number. These results indicate that insulin exerts a specific action on granulosa cells to increase the FSH stimulation of estrogen and progestin production. Estrogen production appears to be enhanced by an increase in the activity of the aromatases, while enhancement of progestin production is mediated through increased production of pregnenolone and increased rate of conversion of pregnenolone to progesterone. Thus, decreases in ovarian functions during insulin-deficient states may be related to diminished steroidogenic responsiveness of the granulosa cell.