Association of Metformin's Effect to Increase Insulin-Stimulated Glucose Transport With Potentiation of Insulin-Induced Translocation of Glucose Transporters From Intracellular Pool to Plasma Membrane in Rat Adipocytes

To examine the cellular mechanism of the antihyperglycemic action of metformin, we studied its effect on various functional and molecular parameters involved in the pathogenesis of insulin resistance. Isolated rat adipocytes were incubated with or without metformin (1–100 μg/ml) for 2 h at 37°C followed by an incubation with or without insulin (1.72 nM). Metformin treatment had no significant effect on basal 3-O-methylglucose uptake. In contrast, metformin increased insulin-stimulated glucose transport in a dose-dependent manner up to 43 ± 7%. This effect was neither associated with a significant effect of metformin on trace insulin binding (1.74 ± 0.20% without metformin vs. 1.89 ± 0.30% with metformin; P > 0.05) nor with an effect of metformin on insulin-receptor kinase activity as measured by 32P incorporation into the 95,000-M_r β-subunit of the insulin receptor and an exogenous substrate, histone 2B. Determination of glucose-transporter numbers in subcellular membrane fractions, plasma membranes (PMs), and low-density microsomes (LDMs) with cytochalasin B binding revealed that metformin's effect to increase insulin-stimulated glucose transport is associated with a potentiation (38 ± 5%) of insulininduced translocation of glucose transporter from the LDM to the PM, whereas the basal state was not significantly affected (basal LDM 61.3 ± 4.2 vs. basal metformin LDM 63.6 ± 7.1, P < 0.05; basal PM 6.4 ± 2.1 vs. basal metformin PM 7.2 ± 2.8, P > 0.05; insulin LDM 32.7 ± 5.4 vs. insulin-metformin LDM 15.8 ± 3.7, P < 0.01; insulin PM 28.6 ± 4.6 vs. insulin-metformin PM 45.3 ± 4.9 pmol/mg membrane protein, P < 0.01). Immunodetection of glucose-transporter isoforms HepG2 erythrocyte (GLUT1) and muscle adipose tissue (GLUT4) in adipocyte membrane fractions separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and blotted to nitrocellulose revealed that insulin-induced translocation of GLUT1 and GLUT4 is potentiated by metformin. Determination of specific GLUT1 and GLUT4 mRNA levels revealed that this metformin effect is not associated with an increase in glucose-transporter gene expression. We conclude that 1) metformin increases insulinstimulated 3-O-methylglucose transport by potentiating insulin-induced translocation of GLUT1 and GLUT4 glucose transporter from LDM to the PM without affecting glucose-transporter gene expression, and 2) because neither insulin binding nor activation of insulin-receptor kinase in intact cells was significantly altered by metformin, these results suggest that metformin acts at the level of glucose transport.