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J. Cell Biol.,
Volume 142, Number 6, September 21, 1998 1429-1446
* Copenhagen Muscle Research Centre, Rigshospitalet; The effects of insulin stimulation and muscle
contractions on the subcellular distribution of GLUT4
in skeletal muscle have been studied on a preparation
of single whole fibers from the rat soleus. The fibers
were labeled for GLUT4 by a preembedding technique
and observed as whole mounts by immunofluorescence
microscopy, or after sectioning, by immunogold electron microscopy. The advantage of this preparation for
cells of the size of muscle fibers is that it provides global
views of the staining from one end of a fiber to the
other and from one side to the other through the core
of the fiber. In addition, the labeling efficiency 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 ~23% associated with large structures including multivesicular endosomes located in the TGN region, and 77% with small
tubulovesicular structures. The two stimuli cause translocation of GLUT4 to both plasma membrane and T
tubules. Quantitation of the immunogold electron microscopy shows that the effects of insulin and contraction are additive and that each stimulus recruits
GLUT4 from both large and small depots. Immunofluorescence double labeling for GLUT4 and transferrin receptor (TfR) shows that the small depots can be further subdivided into TfR-positive and TfR-negative elements. Interestingly, we observe that colocalization of
TfR and GLUT4 is increased by insulin and decreased
by contractions. These results, supported by subcellular fractionation experiments, suggest that TfR-positive
depots are only recruited by contractions. We do not
find evidence for stimulation-induced unmasking of
resident surface membrane GLUT4 transporters or for dilation of the T tubule system (Wang, W., P.A.
Hansen, B.A. Marshall, J.O. Holloszy, and M. Mueckler. 1996. J. Cell Biol. 135:415-430).
Department of Medical Physiology and § Structural Cell Biology Unit,
Department of Medical Anatomy, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen N, Denmark; Experimental Diabetes, Metabolism, and Nutrition Section, Diabetes Branch, National Institute of Diabetes and Digestive and
Kidney Diseases; and ¶ Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes
of Health, Bethesda, Maryland 20892-4062
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