IRS-1 as a key point in the regulation of insulin-sensitivity in granulosa cells from PCOS women

Background: Polycystic Ovary Syndrome (PCOS) is a common endocrine disorder among women and is characterised by chronic low-grade inflammation, ovulatory dysfunction, hyperandrogenism, and often by insulin-resistance. However, little is really known on insulin/IGF signalling in PCOS ovaries. This study aim ed to investigate the amount of Insulin (IR) and IGF-I receptor type I (IGF-IR1) and their intracellular mediators in granulosa cells from PCOS women with respect to controls. Methods: 28 women with PCOS [age: 34.8±4.2 yr; BMI: 25.4±5.5 kg/m2] diagnosed following the Rotterdam criteria, and 44 healthy controls [age: 37.7±4.3 yr; BMI: 23.4±4.3 kg/m2] were enrolled. Granulosa cells were isolated from follicular fluids and were lysed. Total protein content was quantified using a spectrophotometer. Total and phosphorylated insulin receptor (INSR) [pY1162/pY1163], total IGF1-receptor (IGF1R), total and phosphorylated AKT [pS473], total and phosphorylated ERK1 [pT202/pY204]- ERK2 [pT185/pY187], and phosphorylated IRS-1 [pS312] were measured by using specific ELISA kits. Moreover, due to the low amount of protein content obtained from each sample, a multiplex bead-based assay was performed in other 14 PCOS women and 14 controls in order to evaluate total and phosphorylated GSK3β [pS9], IRS1 [pS636], AKT [pS473], mTOR [pS2448], P70S6K [pT412], IR [pY1162/pT1163], PTEN [pS380], GSK3α [pS21], TSC2 [pS939], RPS6 [pS235/pS236], IGF1R [pY1135/pY1136], belonging to the Akt/mTOR pathway which is pivotal for insulin and IGF1R signaling. Student’s T-test and Fisher’s exact test were used to analyze the data. Results: Phosphorylated IRS-1 was measurable in 9/21 PCOS patients and was undetectable in controls (P = 0.0002). Total and phosphorylated INSR, total IGF1R, total and phosphorylated AKT and total and phosphorylated ERK1-2 were similar in PCOS and controls. The multiplex analysis showed, however, that phosphorylated IGF1R (P = 0.02), IRS1 (P = 0.04), mTOR (P = 0.05), p70S6K (P <0.0001), INSR (P = 0.01), GSK3α (P = 0.02), and TSC2 (P = 0.01) were higher in PCOS with respect to controls. No differences were found in phosphorylated GSK3β, AKT, PTEN, RPS6 between PCOS and controls. Conclusion: The phosphorylated fraction of IRS-1 observed in PCOS with both methodologies, methods suggests that IRS1 has a key role in the regulation of insulin-sensitivity in PCOS. Indeed, the increased phosphorylation at the Ser312 and Ser636 sites of IRS-1 is compatible with reduced insulin sensitivity. Acknowledgments: This work was funded by the Italian Ministry of Health (grant number GR-2018-12367635 “Bando di Ricerca Finalizzata-Giovani Ricercatori 2018”).