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 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 immunofl...

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Main Authors:	Ploug, Thorkil, van Deurs, Bo, Ai, Hua, Cushman, Samuel W., Ralston, Evelyn
Format:	Online
Language:	English
Published:	The Rockefeller University Press 1998
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2141761/

id	pubmed-2141761
recordtype	oai_dc
spelling	pubmed-21417612008-05-01 Analysis of GLUT4 Distribution in Whole Skeletal Muscle Fibers: Identification of Distinct Storage Compartments That Are Recruited by Insulin and Muscle Contractions Ploug, Thorkil van Deurs, Bo Ai, Hua Cushman, Samuel W. Ralston, Evelyn Regular Articles 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). The Rockefeller University Press 1998-09-21 /pmc/articles/PMC2141761/ /pubmed/9744875 Text en This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Ploug, Thorkil van Deurs, Bo Ai, Hua Cushman, Samuel W. Ralston, Evelyn
spellingShingle	Ploug, Thorkil van Deurs, Bo Ai, Hua Cushman, Samuel W. Ralston, Evelyn Analysis of GLUT4 Distribution in Whole Skeletal Muscle Fibers: Identification of Distinct Storage Compartments That Are Recruited by Insulin and Muscle Contractions
author_facet	Ploug, Thorkil van Deurs, Bo Ai, Hua Cushman, Samuel W. Ralston, Evelyn
author_sort	Ploug, Thorkil
title	Analysis of GLUT4 Distribution in Whole Skeletal Muscle Fibers: Identification of Distinct Storage Compartments That Are Recruited by Insulin and Muscle Contractions
title_short	Analysis of GLUT4 Distribution in Whole Skeletal Muscle Fibers: Identification of Distinct Storage Compartments That Are Recruited by Insulin and Muscle Contractions
title_full	Analysis of GLUT4 Distribution in Whole Skeletal Muscle Fibers: Identification of Distinct Storage Compartments That Are Recruited by Insulin and Muscle Contractions
title_fullStr	Analysis of GLUT4 Distribution in Whole Skeletal Muscle Fibers: Identification of Distinct Storage Compartments That Are Recruited by Insulin and Muscle Contractions
title_full_unstemmed	Analysis of GLUT4 Distribution in Whole Skeletal Muscle Fibers: Identification of Distinct Storage Compartments That Are Recruited by Insulin and Muscle Contractions
title_sort	analysis of glut4 distribution in whole skeletal muscle fibers: identification of distinct storage compartments that are recruited by insulin and muscle contractions
description	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).
publisher	The Rockefeller University Press
publishDate	1998
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2141761/
_version_	1611422906369179648

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

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