Phosphorylation and Activation of AMP-activated Protein Kinase (AMPK) by Metformin in the Human Ovary Requires Insulin

Metformin is commonly used to treat women with polycystic ovary syndrome, but its precise mechanism of action is unclear, and it even appears to have direct ovarian effects. At the cellular level, it may act either via an insulin-dependent pathway or an independent pathway by activating AMP-activated protein kinase (AMPK). In the ovary, metformin directly decreased estradiol and progesterone production by human granulosa cells, and inhibition of progesterone production by metformin in rat granulosa cells caused an increase in phosphorylated AMPK (pAMPK). We investigated whether metformin activates AMPK in the human ovary by looking for changes in phosphorylation of AMPK and its downstream target acetyl CoA carboxylase (ACC). mRNA and protein for α1 and α2 AMPK subunits were present in all human ovarian tissue. Neither 100 nm nor 2 mm of metformin affected subunit expression. After 1 or 4 h, neither dose of metformin increased pAMPK or pACC, although after 1 h, the addition of insulin significantly enhanced pAMPK, whereas insulin alone had no effect on phosphorylation of either AMPK or ACC. The addition of compound C, an inhibitor of AMPK, negated the effect of metformin in the presence of insulin on pAMPK. This effect on AMPK was not due to a change in the ADP/ATP ratio measured by HPLC. In summary, the presence of insulin was required to cause a metformin-induced increase in pAMPK in these human ovarian cells. Although previous data suggest that metformin may act via an insulin-independent pathway, our results therefore imply that insulin may be required to initiate an effect.