FIGURE 1.
Effect of metformin on glucose uptake and components of the insulin signaling pathway in L6 myotubes. L6 myotubes were incubated in the absence or presence of metformin (1 mm) for 16 h prior to incubation with insulin (100 nm) and/or PI3K inhibitors (100 nm wortmannin (WM) or 50 μm LY 294002) for the penultimate 30-min period for insulin and 45-min period for PI3K inhibitors. At the end of the incubation period, muscle cells were used for assay of 2-deoxyglucose (2DG) uptake (A). Data from A was used calculate net changes in 2-deoxyglucose uptake so as to take account of any changes in basal uptake induced by the PI3K inhibitor (B). Alternatively, muscle cells were lysed at the end of indicated treatments and used for immunoprecipitation (IP) of IRS-1 and immune pellets subjected to SDS-PAGE and immunoblotting (IB) with antibodies against phosphotyrosine, IRS-1, or p85-PI3K (C), for lipid extraction to measure PIP3 abundance as reported under “Experimental Procedures” (D), or for immunoblotting with antibodies against phospho-PKB Ser473, phospho-GSK3α/β Ser21/9, and native PKB and GSK3 (E). Bar graph results are expressed as a mean ± S.E. for between 3 and 10 independent experiments. The asterisk represents a significant change (p < 0.05) relative to the untreated control, and the double asterisk indicates a significant change (p < 0.05) between the indicated bars. Phosphoblots are representative of three separate experiments.