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© The Rockefeller University Press, 0021-9525/1998//1429 $5.00
The Journal of Cell Biology, Volume 142, Number 6, , 1998 1429-1446

Regular Articles

Analysis of GLUT4 Distribution in Whole Skeletal Muscle Fibers: Identification of Distinct Storage Compartments That Are Recruited by Insulin and Muscle Contractions



Thorkil Ploug*,{ddagger},||, Bo van Deurs§, Hua Ai*,{ddagger}, Samuel W. Cushman||, and Evelyn Ralston

* Copenhagen Muscle Research Centre, Rigshospitalet; {ddagger} 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

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).

Key Words: membrane trafficking • glucose transport • endosomes • exercise • diabetes

Abbreviations used in this paper: LM, light microscopy; SR, sarcoplasmic reticulum; TfR, transferrin receptor.

T. Ploug was supported by a fellowship from the Weimann Foundation, (Copenhagen, Denmark) and by a grant (504-14) from the Danish National Research Foundation. E. Ralston and T. Ploug acknowledge a North Atlantic Treaty Organization collaborative research grant. (1643–95).

Address all correspondence to T. Ploug, Department of Medical Physiology, The Panum Institute, University of Copenhagen, Blegdamsvej 3C, DK-2200 Copenhagen N, Denmark. Tel.: (45) 35-32-74-35. Fax: (45) 35-32-74-20. E-mail: t.ploug{at}mfi.ku.dk

2. In the text, we refer to fibers prepared from basal, nonstimulated soleus muscle as basal fibers, and to fibers from insulin-stimulated muscle as insulin-stimulated fibers, etc.


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