SUN-215 In-vitro Insulin Treatment of Human Granulosa-like Tumor Cells Results in Insulin Resistance, and May Serve as a Model for the Insulin Resistance Seen in Polycystic Ovary Syndrome (PCOS)

Abstract Disclosure: H. Han: None. K. Vann: None. A. Weidner: None. S.R. Hammes: None. O. Astapova: None. Background: Polycystic ovarian syndrome (PCOS) is characterized by hyperandrogenism and insulin resistance. Previous studies have demonstrated that four-day treatment with insulin increases androstenedione, testosterone, and dihydrotestosterone (DHT) in theca and stroma cells from patients with hyperandrogenism, insulin resistance, and acanthosis nigricans. However, there are no in vitro models of prolonged insulin treatment of granulosa cells, which express both insulin and the related insulin-like growth factor 1 (IGF1) receptors. Aim: We aim to create an in vitro model of insulin resistance by treating human granulosa-like tumor cells (KGN) with insulin for 24 hours to mimic chronic hyperinsulinemia, then characterize the phosphorylation cascade downstream of the insulin and IGF1 receptors. Methods: KGN cells were incubated in serum-starved media for two hours. Afterwards, cells were either treated with insulin (2.5 ug/uL) or phosphate buffered saline (PBS) for 24 hours (chronic insulin treatment). Insulin was then removed, and cells were incubated in serum-free media for two hours before treatment with or without 1ug/mL of insulin for 30 minutes (acute insulin treatment). We then performed western blot to analyze phosphorylated AKT, phosphorylated FOXO-3A, and insulin and IGF-1 receptors. Results: Cells pretreated with insulin for 24 hours resulted in significant downregulation of phosphorylated AKT and phosphorylated FOXO-3A, as well as reduction of insulin and IGF1 receptor levels, in comparison to cells pretreated with PBS. Conclusion: Incubating KGN cells with insulin for 24 hours creates a state of insulin resistance, with downregulation of both insulin and IGF1 receptors as well as their downstream signaling products. We propose that we may be able to utilize this in vitro model of pretreatment of KGN cells with 24 hours of insulin to simulate the hyperinsulinemia found in PCOS patients. Presentation: Sunday, July 13, 2025