Stimulation of Lactate Production in Human Granulosa Cells by Metformin and Potential Involvement of Adenosine 5′ Monophosphate-Activated Protein Kinase

Context: Production of 3-carbon units (as lactate) by granulosa cells (GCs) is important in follicular and oocyte development and may be modulated by metformin. Objective: 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). Design: GCs were prepared from follicular aspirates. After exposure to metformin and other potential modulators of AMPK in culture, aspects of cellular function were examined. Setting: The study was conducted in a private fertility clinic/university academic center. Patients: Women undergoing routine in vitro fertilization participated in the study. Interventions: All agents were added in culture. Main Outcome Measures: 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. Results: Metformin led to a rapid increase in lactate production by GCs [minimum effective dose, 250 mu M; 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 mu M; maximum effect, 500 mu M (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 mu M; maximum effect, 250 mu M; P < 0.001). A level of metformin (125 mu M) insufficient for the stimulation of lactate output when used alone potentiated the effects of suboptimal doses of insulin on lactate production. Adiponectin (2.5 mu g/ml) had a small but significant effect on lactate output. Conclusions: 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. (J Clin Endocrinol Metab 94: 670-677, 2009)