Searchable abstracts of presentations at key conferences in endocrinology
Endocrine Abstracts (2009) 19 P283

SFEBES2009 Poster Presentations Reproduction (22 abstracts)

Metabolic effects of insulin and modification by androgen in immortalised mouse granulosa cells

J Joharatnam , K Hardy & S Franks

Imperial College, London, UK.

Polycystic ovary syndrome (PCOS) is characterised by hyperandrogenism and insulin resistance. It is thought that androgen excess contributes to the mechanism of insulin resistance. Granulosa cells in PCOS demonstrate impaired insulin-induced glucose uptake and lactate accumulation1. The aim of this study was to characterise insulin signalling (via the PI3-Kinase (PI3-K) pathway) and glucose metabolism in an immortalised mouse granulosa cell line (KK1)2, and to investigate the effect of short-term exposure to androgens.

Materials and methods: For protein expression studies, KK1 cells were cultured in serum-supplemented medium 199 for 24 h; medium was removed, the cells washed, then incubated in serum free medium for 8 h. Cell lysates were subjected to PAGE and Western immunoblotting was performed for key proteins in the insulin-signalling pathway. Glucose uptake and lactate production after 48 h were measured in conditioned medium using a COBAS bioanalyser.

Results: An increase in phosphorylated AKT was observed within 5 min of insulin stimulation with a steady increase up to 1 h. This was reversed by pre-incubation with the PI3-K inhibitor, LY294002. In cultured cells, insulin stimulated glucose uptake at 24 and 48 h and an increase in lactate production at the same time points. Again, these effects were reversed by LY294002. Androgen pre-incubation for 48 h, (either 10−8 molar testosterone (T) or dihydrotestosterone (DHT)), increased expression of insulin-stimulated phospho-AKT compared to insulin alone. Insulin-stimulated glucose uptake (P=0.012) and lactate production (P=0.008) were significantly enhanced following pre-incubation with T and a similar effect was observed for DHT.

Conclusion: We have characterised insulin action in a mouse granulosa cell line. The surprising result was the augmentation by androgens, rather than the expected inhibition, of insulin-stimulated glucose uptake and lactate production. This suggests that the insulin resistance observed in granulosa cells from women with PCOS cannot be explained by short-term exposure to androgen.

References: 1. Rice et al. Human Reproduction 2005 20 (2) 373–381.

2. Kananen et al. Molecular Endocrinology 1995 9 616–627.

Acknowledgments: Wellbeing of Women.

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