ANALYSIS OF GLUT4 DISTRIBUTION IN WHOLE SKELETAL-MUSCLE FIBERS - IDENTIFICATION OF DISTINCT STORAGE COMPARTMENTS THAT ARE RECRUITED BY INSULIN AND MUSCLE CONTRACTIONS

The effects of insulin stimulation and muscle contractions on the subc ellular distribution of GLUT4 in skeletal muscle have been studied on a preparation of single whole fibers from the rat soleus. The fibers w ere labeled for GLUT4 by a preembedding technique and observed as whol e mounts by immunofluorescence microscopy, or after sectioning, by imm unogold electron microscopy. The advantage of this preparation for cel ls of the size of muscle fibers is that it provides global views of th e staining from one end of a fiber to the other and from one side to t he other through the core of the fiber. In addition, the labeling effi ciency is much higher than can be obtained with ultracryosections. In nonstimulated fibers, GLUT4 is excluded from the plasma membrane and T tubules. It is distributed throughout the muscle fibers with similar to 23% associated with large structures including multivesicular endos omes located in the TGN region, and 77% with small tubulovesicular str uctures. The two stimuli cause translocation of GLUT4 to both plasma m embrane and T tubules. Quantitation of the immunogold electron microsc opy shows that the effects of insulin and contraction are additive and that each stimulus recruits GLUT4 from both large and small depots. I mmunofluorescence double labeling for GLUT4 and transferrin receptor ( TfR) shows that the small depots can be further subdivided into TfR-po sitive and TfR-negative elements. Interestingly, we observe that coloc alization of TfR and GLUT4 is increased by insulin and decreased by co ntractions. These results, supported by subcellular fractionation expe riments, suggest that TfR-positive depots are only recruited by contra ctions. We do not find evidence for stimulation-induced unmasking of r esident surface membrane GLUT4 transporters or for dilation of the T t ubule system (Wang, W., P.A. Hansen, B.A. Marshall, J.O. Holloszy, and M. Mueckler. 1996. J. Cell Biol, 135:415-430).