Action of metformin on the insulin-signaling pathway and on glucose transport in human granulosa cells.

Abstract

Hyperinsulinemia in polycystic ovary syndrome is widely treated with the insulin sensitizer metformin, which, in addition to its systemic effects, directly affects the ovarian insulin-stimulated steroidogenesis pathway. Our aim was to investigate the interaction of metformin with the other insulin-stimulated ovarian pathway, namely that leading to glucose uptake. Human granulosa-luteal cells were cultured with metformin (10(-7) M), insulin (10 ng/ml) or metformin and insulin (met + ins) combined. Insulin receptor (IR) involvement was assessed by culture with an (anti)-insulin receptor (IR) antibody. The effect of metformin on insulin-receptor substrate proteins 1 and 2 (IRS-1 and -2) mRNA and protein expression was determined. The KGN granulosa-cell line was used to investigate the effect of insulin and metformin on Akt activation and glucose transporter-4 (Glut-4) expression. Glut-4 translocation from the cytosol to the membrane was determined in cytoplasmic and membrane-enriched fractions of protein lysates. IRS-1 mRNA and protein increased with all treatments. In contrast, basal IRS-2 mRNA levels were barely detectable, but transcription was up-regulated by metformin. The anti-IR antibody reduced treatment-stimulated IRS-1 to basal levels and IRS-2 expression to an even greater extent than IRS-1, showing greater dependence on the IR than IRS-1. Metformin in the presence of insulin activated Akt and this was dependent on phosphoinositide-3 kinase, as was translocation of Glut-4 to the membrane. Metformin was able to substantially enhance the insulin-stimulated translocation of Glut-4 transporters from the cytosol to the membrane. This net increase in Glut-4 transporters in the plasma membrane has the potential to increase glucose uptake and metabolism by granulosa cells of the insulin-resistant polycystic ovary, thereby facilitating follicle growth.