Action of Metformin on the Insulin-Signaling Pathway and on Glucose Transport in Human Granulosa Cells

• Published in: The journal of clinical endocrinology and metabolism 2011-03, Vol.96 (3), p.E427-E435
• Main Authors: Rice, Suman, Pellatt, Laura Jane, Bryan, Stacey Joanna, Whitehead, Saffron Ann, Mason, Helen Diane
• Format: Article
• Language: English
• Subjects: Biological Transport, Active - drug effects; Blotting, Western; Cell Line; Enzyme Activation; Female; Glucose - metabolism; Glucose Transporter Type 4 - metabolism; Granulosa Cells - drug effects; Granulosa Cells - metabolism; Humans; Hypoglycemic Agents - pharmacology; Insulin - physiology; Insulin Receptor Substrate Proteins - metabolism; Metformin - pharmacology; Oncogene Protein v-akt - physiology; Phosphatidylinositol 3-Kinases - metabolism; Phosphorylation; Protein Transport; Receptors, Cell Surface - metabolism; Reverse Transcriptase Polymerase Chain Reaction; RNA - biosynthesis; RNA - isolation & purification; Signal Transduction - drug effects
• ISSN: 0021-972X; 1945-7197
• DOI: 10.1210/jc.2010-2060

We demonstrate a direct interaction of metformin with key components of the classical insulin-signaling pathway in human ovarian granulosa cells. Context: 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. Objective: Our aim was to investigate the interaction of metformin with the other insulin-stimulated ovarian pathway, namely that leading to glucose uptake. Design: 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. Main outcome measures: 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. Results: 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. Conclusion: 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.