Metformin is commonly used to treat insulin resistance in women with polycystic ovary syndrome (PCOS) and also has direct effects on the ovary. The mechanism of this action is unknown, but could be via insulin-independent pathways, namely AMP activated protein kinase (AMPK). AMPK is an energy sensor, activated when ATP levels are depleted causing an inhibition of anabolic and an activation of catabolic pathways. We hypothesised that in the ovary, metformin acts via AMPK to mediate its effects. Our aim was to identify the catalytic subunits (α1 and α2) of AMPK in ovarian tissue and to determine whether metformin increased the phosphorylation of AMPK indicating activation. Cortex, stroma, theca and GC were collected from whole ovaries and luteinised cells (GLC) from IVF follicular flush. α1 and α2 mRNA and protein were determined by PCR and western blotting. GLC were cultured +/− metformin (10−4 M and 10−7 M) and AICAR (2 mM) for varying times between 30 mins and 48 hrs; cells lysed and total protein measured. Anti-phospho threonine (pThr) antibody was used to detect phosphorylated bands of AMPK (n=3 at each time point). α1 and α2 subunit activities were also measured by the 32P ATP method (n=3).
α1 and α2 were identified in all ovarian cells, with the highest expression in theca and stroma. α1 was higher than α2, except in cortex. There was an increase in α1 activity with metformin and AICAR after 1 hr (ANOVA P=<0.001), but no change in phosphorylation was detected by western blotting.
In summary, the catalytic subunits of AMPK were identified in all ovarian cell types with the highest levels being in theca and stroma. Metformin significantly increased AMPK activity in GLC. To conclude, as in other tissues, metformin activates AMPK in the ovary. This could be an important signalling step involved in mediating its effects.
06 - 07 Nov 2006
Society for Endocrinology