P-643 The effect of hyperinsulinemia on FSH-mediated signalling pathways in granulosa cells - implications for follicle growth in women with PCOS
Authors: Rice S , Khalid A , Khan S , Lacey M , Homburg R 
Published: June 2023

Abstract

Study question
Does in vitro exposure of granulosa cells (GC) to an equivalent hyperinsulinemic (HI) dose of insulin alter key signalling pathways, potentially slowing follicle growth?

Summary answer
Exposure of GC to HI dose of insulin down-regulated FSH-mediated phosphorylation of AKT and ERK proteins, which was reversed by metformin but not myoinositol treatment

What is known already
Antral follicles utilise glycolytic methods of energy production releasing the metabolites pyruvate and lactate, which are then secreted into the follicular fluid or passed directly to the oocyte via gap junctions. Insulin increases GC glucose uptake and cross-talks with FSH-mediated intracellular pathways that determine follicle growth and glucose utilisation. We and others have shown that there is significant perturbation in glucose metabolism (as measured by reduced lactate production) in cultured luteinised GC (GLC) from women with anovulatory PCOS, compared to those with normal ovaries or polycystic ovarian morphology (PCOM), that represents both attenuated glucose uptake and reduced glycolytic activity.

Study design, size, duration
KGN-GC were exposed to 1000ng/ml insulin for 48h (mimicking prolonged HI in vivo) with/without metformin [10-7M] or myo-inositol [25mM]; followed by acute stimulation with FSH (10ng/ml at 15 and 30 mins) (n = 4-5). GLCs obtained from women with normal ovaries (n = 5), PCOM (n = 7) and PCOS (n = 2) were serum-starved and exposed acutely to insulin (10ng/ml for 30mins), glucose starved (20mins) and treated with a radiolabelled 2-deoxy-glucose mix. Fasting serum glucose and insulin were measured in PCOS women.

Participants/materials, setting, methods
Western blotting on proteins from KGN-GC was performed using antibodies against total and phosphorylated forms of AKT and ERK. Densitometry measurements of phospho:total forms were taken from individual treatments, normalised to loading controls (either β-actin/Vinculin) and then untreated controls. GLCs were lysed and uptake of 14C-2DG (non-metabolised) measured and expressed as percentage uptake of 14C-2DG in the insulin-treated normalised to untreated/control cells (100%). Insulin resistance (IR) was calculated from fasting glucose and insulin using HOMA2.

Main results and the role of chance
Chronic exposure of KGNs to high dose insulin significantly down-regulated acute FSH-mediated phosphorylation of AKT and ERK (ANOVA *p=0.03, multiple post-hoc t-tests p &lt; 0.05, two-tailed), without altering insulin receptor levels. Addition of metformin to FSH treatment, significantly reversed the HI-induced reduction in pAKT levels and enhanced it further (*p=0.02) but had no effect on pERK levels. Myoinositol had no effect on either pAKT or pERK levels. These findings demonstrated that HI directly inhibited FSH signalling events downstream of the insulin receptor in GC, but that metformin could counteract some of these detrimental effects. The 2-deoxy form of glucose is taken up into the cell but not metabolised further. Measurement of 14C-2DG showed that acute stimulation with post-prandial levels of insulin of GLCs taken from normal and PCOM women significantly increased glucose uptake (*p&lt;0.05, ratio of paired t-test, 2-tailed); whereas GLCs from women with PCOS and insulin resistance were unable to respond to insulin and take up glucose. Therefore, GLCs from insulin resistant women with PCOS retain their phenotype in vitro and this is not an artefact, since GLCs from normal and PCOM also subjected to the same stimulation regime were able to take up glucose in response to insulin stimulation.

Limitations, reasons for caution
A clear limitation is the small number of insulin resistant women with PCOS. In addition, human GLCs were collected after hormonal stimulation.

Wider implications of the findings
Women with IR, HI and PCOS undergoing ART may require extra gonadotrophin stimulation to grow and mature sufficient eggs. Additionally, the oocyte quality maybe compromised, as indicated by reduced glucose uptake. Treatment of women with metformin but not myoinositol may improve the dysregulated FSH-signalling pathways brought about by HI.

Trial registration number
not applicable