Figure 7.

Disruption of flotillin-1-containing domains prevents activation of C3G, movement of GLUT4 to the sarcolemma, and glucose uptake after insulin treatment. A) Sucrose density gradient centrifugation. Skeletal muscle cells were differentiated for 5 days and treated with or without 10 mM methyl-meta-cyclodextrin (MbetaCD) for 1 h before insulin stimulation (160 nM for 10 min) in the presence or absence of MbetaCD. Detergent-resistant microdomains (DRMs fractions) were separated from the bulk of cellular membranes and cytosolic proteins (Non-DRMs fractions) by equilibrium sucrose density gradient centrifugation. Expression of flotillin-1 and GLUT4 was examined by imunoblotting analysis using specific antibody probes. B, C) Coimmunoprecipitation. Skeletal muscle cells were differentiated for 5 days and treated with or without 10 mM methyl-meta-cyclodextrin (MbetaCD) for 1 h before insulin stimulation (160 nM for 5 min) in the presence or absence of MbetaCD. Cell lysates were immunoprecipitated with anti-Ins-Rβ IgGs (B) and C3G IgGs (C). Immunoprecipitates were then subjected to Western blotting analysis with anti-flotillin-1 IgGs (B, C) and anti-phosphotyrosine IgGs (C). MbetaCD did not change total InsR-β, flotillin-1, and C3G protein expression (data not shown). D) Immunofluorescence. Skeletal muscle cells were differentiated for 5 days and treated with or without 10 mM methyl-meta-cyclodextrin (MbetaCD) for 1 h before insulin stimulation (160 nM for 10 min) in the presence or absence of MbetaCD. Cells were then incubated with flotillin-1 and GLUT4 IgGs. Expression of flotillin-1 and GLUT4 was evaluated using fluorescent secondary antibodies. Green: flotillin-1; red: GLUT4. E) Glucose uptake. Control skeletal muscle cells were differentiated for 5 days and treated with or without 10 mM methyl-meta-cyclodextrin (MbetaCD) for 1 h before insulin stimulation (160 nM for 10 min) in the presence or absence of MbetaCD. ³H-2-deoxy-glucose (1 μCi/ml) was added during the last 5 min of incubation. Cells were subsequently solubilized, and ³H content was determined by scintillation counting. Values are mean ± SE; n = 9; *P ≤ 0.005 (CTL+Ins+MbetaCD vs. CTL+Ins).