Figure 3

Analysis of glucose metabolism and visualization of GLUT4 translocation. (A) Blood glucose levels of wt and mutant mice were assessed by oGTT (n ≥ 5 per genotype). Note elevated glucose levels in mdx mice, 30, 45, and 60 minutes after force-feeding of glucose (**P < 0.01). (B) Insulin levels were measured in blood during oGTT (n ≥ 5 per genotype, differences are not statistically significant). (C) Bar graph, representative immunoblot, and corresponding Coomassie-stained gel, showing GLUT4 protein levels in mutant relative to wt (100%) GC muscles (n = 4 per genotype). Differences are not statistically significant. Positions of molecular mass markers are indicated. (D) Immunolocalization and quantification of GLUT4 on cryosections of QF muscles from wt and mutant mice. Note increased immunoreactivity of GLUT4 at the periphery of myofibers in wt, cKO and dKO compared to mdx mice (***P < 0.001); details of quantification are described in the text. (E) Fluorescence microscopy of GFP-P1f-overexpressing (P1f-transfected) and control (Ctrl; GFP-P1f-untransfected) differentiated myotubes, both expressing a mCherry-HA-GLUT4 fusion protein; a topographic scheme of the mCherry-HA-GLUT4 fusion protein is shown below micrographs. Note reduced labeling of HA (yellow) in P1f-transfected compared to control myotubes. Bar, 10 μm. (F) Quantification of GLUT4 translocation. GLUT4 (mCherry) signals colocalizing with extracellular HA-tag signals were measured as described in the text. Note, surface GLUT4 labeling was reduced to 54% in differentiated myotubes overexpressing GFP-tagged P1f (P1f) compared to control cells (Ctrl) (n ≥ 13 per genotype; four independent experiments, ***P < 0.001). Bar, 10 μm. Data in (A-D), and F represent mean ± SEM.