Evidence That Insulin Stimulates 5alpha-Reductase (Type 1) mRNA and Protein Expression in Ovarian Granulosa Cells: A Possible Mechanism of Ovulatory Dysfunction Under Hyperinsulinaemic State.

Abstract

Ovulatory dysfunction seen in women under pathophysiological conditions is often associated with hyperandrogenism and hyperinsulinaemia. Increased levels of 5α-reductase, an enzyme that reduces androgens to their 5α-reduced metabolites, have been reported in the follicular fluid collected from women with polycystic ovarian syndrome (PCOS). Previous reports from our laboratory have demonstrated that dihydrotestosterone (DHT), a 5α-reduced metabolite of testosterone, has an inhibitory effect on ovarian granulosa cell mitogenesis by reducing FSH mediated cyclin D2 mRNA expression both in vivo and in vitro, leading to cell cycle arrest. Since androgen-induced disruption of ovulation occurs under hyperinsulinaemic conditions, in the present study we have tested the hypothesis that the disruptive effects of androgens on follicular development might be augmented by increased production of 5α-reduced metabolites through the stimulation of 5α-reductase by insulin. The human granulosa-like tumor cell line, KGN, was used as a model system. To test whether insulin exerts an effect on 5α-reductase, 12 hour serum starved KGN cells were treated with 1.0µg/ml insulin for 3 time intervals (0, 4, and 6 hours). The media were then removed and total RNA was isolated using TRIzol reagent. The 5α-reductase mRNA expression (both type 1 and 2) was measured by real time PCR using probes purchased from Applied Biosystems (Foster City, CA). The results show that while there was no appreciable amount of 5α-reductase type 2 mRNA expression in these cells, type 1 enzyme showed a two-fold increase in response to insulin by 6 hours when 5α-reductase protein also showed a significant increase in expression at 12 hour compared to control. These results were confirmed by using primary cultures of rat granulosa cells. Granulosa cells harvested from immature rats were allowed to attach overnight in phenol red free DMEM-F12 and then treated with insulin (0, 1.0 and 10.0µg/ ml) for 12 hours. Real time PCR showed a two-fold increase in the 5α-reductase type 1 mRNA expression in response to insulin dose of 1.0 and 10.0µg/ml compared to control. Thus, the stimulatory effect of insulin on 5α-reductase can be demonstrated both in primary cultures of granulosa cells and a human granulosa cell line. These results suggest that under pathophysiological conditions such as PCOS, insulin not only augments production of androgens, but also stimulates the conversion of these androgens to their 5α-reduced metabolites by increasing 5α-reductase activity. These excess 5α-reduced metabolites might contribute to impaired granulosa cell mitogenesis contributing to anovulation. (Supported by NIH Grant-HD-38424) (poster)