Stimulation of lactate production in human granulosa cells by metformin and potential involvement of adenosine 5' monophosphate-activated protein kinase.

Abstract

Production of 3-carbon units (as lactate) by granulosa cells (GCs) is important in follicular and oocyte development and may be modulated by metformin. The aim of the study was to examine the action of metformin on GC lactate production and potential mediation via AMP-activated protein kinase (AMPK). GCs were prepared from follicular aspirates. After exposure to metformin and other potential modulators of AMPK in culture, aspects of cellular function were examined. The study was conducted in a private fertility clinic/university academic center. Women undergoing routine in vitro fertilization participated in the study. All agents were added in culture. Lactate output of GCs was measured. Cell extracts were prepared after culture, and phosphorylated forms of AMPK and acetyl CoA carboxylase (ACC) were assayed using Western analysis. Metformin led to a rapid increase in lactate production by GCs [minimum effective dose, 250 microm; maximum dose studied, 1 mm (1.22-fold; P < 0.01)]. This dose range of metformin was similar to that required for stimulation of phospho-AMPK in GCs [minimum effective dose, 250 microm; maximum effect, 500 microm (2.01-fold; P < 0.001)]. Increasing phospho-ACC, as a representative downstream target regulated by AMPK, was apparent over a lower range (minimum effective dose, 31 microm; maximum effect, 250 microm; P < 0.001). A level of metformin (125 microm) insufficient for the stimulation of lactate output when used alone potentiated the effects of suboptimal doses of insulin on lactate production. Adiponectin (2.5 microg/ml) had a small but significant effect on lactate output. Metformin activates AMPK in GCs, stimulating lactate production and increasing phospho-ACC. Metformin also enhances the action of suboptimal insulin concentrations to stimulate lactate production.