Soleus muscles of fed rats were fixed by vascular perfusion with paraformaldehyde; individual fibers were teased and immunostained with a polyclonal antibody against the COOH-terminal of GLUT4. The binding sites were visualized by a horseradish peroxidase–coupled secondary antibody and diaminobenzidine. The fibers were embedded in epoxy resin and studied by electron microscopy. Strong immunoreactivity was found in subsarcolemmal clusters of vesicles and cisternae, Golgilike structures, and triadic junctions. Clusters of vesicles between myofibrils were occasionally stained. The plasma membrane was unlabeled. However, the plasma membrane was labeled when the rats had been injected with insulin (40 U/kg body wt) 15 min before perfusion fixation. In non–insulin-injected rats, the plasma membrane might show spotty staining close to clusters of intensely labeled subsarcolemmal vesicles. This may have been due to diffusion but may also indicate that there are domains of GLUT4 in the plasma membrane of nonstimulated fibers or that the endogenous insulin activity to some extent had translocated GLUT4 from the intracellular pool into the plasma membrane. Coated vesicles that were also labeled were found adjacent to subsarcolemmal vesicles and cisternae; it is possible that coated vesicles play a role during insulin- or contraction-induced translocation of GLUT4 between subsarcolemmal pool and plasma membrane. It has been proposed that glucose uptake into skeletal muscle fibers takes place across the t-tubule membrane rather than across the plasma membrane. This would explain the presence of GLUT4 at triadic junctions. Alternatively, we suggest that GLUT4 in t-tubules represents a second intracellular pool.

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