Defining insulin signalling pathways in granulosa-lutein (GL) cells in women with polycystic ovary syndrome (PCOS)
Jalini Joharatnam1, Giuseppina Lamanna2, Geoffrey Trew2, Stuart Lavery2, Kate Hardy1 & Stephen Franks1,2
Aim: PCOS is associated with peripheral insulin resistance and we have previously shown that glucose uptake and metabolism are impaired in granulosa lutein (GL) cells of anovulatory women with PCOS (anovPCO). The aim of this study was to delineate insulin-signalling pathways in GL cells from women with anovPCO and to compare the response to insulin with that in GL cells from ovulatory women with (ovPCO) and without polycystic ovaries (controls).
Methods: Primary culture of GL cells obtained at time of egg collection from 3 groups of patients undergoing routine IVF: women with normal ovaries and regular cycles (n=12), women with polycystic ovaries and regular cycles (n=8) and women with polycystic ovaries and oligomenorrhoea (n=11). Primary cultures of GL cells were incubated with insulin (1, 10, 100 ng/ml) and analysed for glucose uptake, lactate accumulation and progesterone production. Cell lysates were prepared for identification of insulin signalling pathways, specifically those involving PI3-kinase (P13-K) and MAPK, using western immunoblotting.
Results: As previously shown by there was impairment of glucose metabolism in cells from anovPCO women with conservation of insulin stimulated progesterone formation. Using phospho-specific antibodies to Akt Ser 473 and GSK 3β Ser 9, no significant impairment insulin stimulated PI3K signalling was observed. In the MAPK pathways, p42 ERK phosphorylation was significantly greater in controls compared to anovulatory and ovulatory PCOS patients in response to 100ng/ml insulin.
Conclusions: We have confirmed selective resistance to insulin in glucose metabolism in granulosa lutein cells in women with anovulatory PCOS. The absence of any obvious impairment in the insulin-stimulated PI3K signalling pathway in response to insulin in women with anovulatory PCOS is unexpected. We did however see impairment in MARK signalling and the significance of this finding with respect to impaired glucose metabolism in granulosa cells remains to be determined.