HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 16/2/249 01-0646fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by KARLSSON, M. Articles by GUSTAVSSON, J. Search for Related Content PubMed PubMed Citation Articles by KARLSSON, M. Articles by GUSTAVSSON, J.

Insulin induces translocation of glucose transporter GLUT4 to plasma membrane caveolae in adipocytes ¹

Department of Cell Biology, Faculty of Health Sciences, University of Linköping, S-58185 Linköping, Sweden

²Correspondence: Department of Cell Biology, Faculty of Health Sciences, S-58185 Linköping, Sweden. E-mail: peter.stralfors{at}mcb.liu.se.

SPECIFIC AIMS

The insulin receptor is localized to and signals in caveolae, and insulin signaling for metabolic control depends on intact caveolae. We earlier reported that in response to insulin, an initial rapid translocation of GLUT4 to the plasma membrane was followed by a slower transition of transporters to a detergent-insoluble plasma membrane fraction, coinciding with the increase in glucose uptake [Mol. Med., vol 2, p. 367 (1996)], which prompted us to examine the detailed localization of GLUT4 in the plasma membrane with a focus on caveolae.

PRINCIPAL FINDINGS

1. Cellular localization of GLUT4 in the plasma membrane in relation to caveolin as examined by immunofluorescence microscopy
We used double-immunofluorescence labeling to investigate the cellular localization of GLUT4 in relation to the caveolae marker protein caveolin in the plasma membrane of 3T3-L1 adipocytes. We sonicated the cells to expose the inner surface of the plasma membrane and fixed the membranes before antibody labeling. Fluorescence emitted for GLUT4 and caveolin indicated that a large fraction of plasma membrane GLUT4 and caveolin is colocalized in the plasma membrane. Evaluation of the fluorescence intensities from insulin-stimulated cells vs. control cells showed that insulin increased the amount of GLUT4 in the plasma membrane 2.2-fold: from 28 ± 1.4 (n=151 different membranes, four different preparations) to 61 ± 2.8 (n=134) arbitrary fluorescence units (P<0.01 by Student’s t test).

2. Caveolae localization of GLUT4 as determined by electron microscopy
To precisely localize GLUT4 in the plasma membrane, we immunogold labeled 3T3-L1 adipocyte plasma membranes for examination by transmission electron microscopy (Fig. 1 ). Caveolae were identified by their characteristic appearance and labeling with antibodies against caveolin (6 nm gold particles). A large fraction (85%) of GLUT4 (15 nm gold particles, arrows) was found in the caveolae structures. A small and significant fraction of GLUT4 was found outside caveolae (Fig. 1) . Counting of gold particles in the plasma membranes from insulin-stimulated cells vs. control cells showed that insulin increased the amount of GLUT4 labeling in the plasma membrane 2.2-fold: from 11.8 ± 3.0 (n=six different membranes, three different preparations) to 26.3 ± 2.2 (n=7) immunogold particles per µm² of plasma membrane (P<0.01 by Student’s t test) in accordance with immunofluorescence analysis. The fraction of GLUT4 labeling of the plasma membrane outside of caveolae was not affected by insulin: 12% in control cells vs. 13% in cells stimulated with insulin.

View larger version (81K): [in this window] [in a new window]   Figure 1. Immunogold electron microscopic localization of GLUT4 in 3T3-L1 adipocyte plasma membrane. 3T3-L1 adipocytes were incubated without (a) or with (b) insulin and plasma membranes were prepared. The inner surface of the plasma membrane was labeled with antibodies against GLUT4 (15 nm gold particles, arrows) and caveolin (6 nm gold particles).

3. Caveolae localization of GLUT4 as determined by biochemical and electron microscopical analysis of isolated caveolae
We confirmed the morphological localization of GLUT4 to caveolae by biochemical isolation (without detergent treatment) of caveolae fractions from purified plasma membranes of freshly isolated rat adipocytes (Fig. 2 a). GLUT4 was enriched 3.9 ± 0.7-fold (mean±SE, n=7 caveolae preparations) in control cells and 3.5 ± 0.6-fold in insulin-stimulated cells compared with total plasma membrane fractions. This can be compared to the enrichment of caveolin (sevenfold) and cholesterol (threefold). In response to insulin, the amount of GLUT4 tripled in the total plasma membrane and the caveolae fractions (Fig. 2b ).

View larger version (24K): [in this window] [in a new window]   Figure 2. GLUT4 in biochemically isolated caveolae. Rat adipocytes were incubated with (+) or without (minus) insulin. Plasma membrane (pm), caveolae (cav), and intracellular membrane (im) fractions were prepared. a) 3 µg protein were separated by SDS-PAGE and immunoblotted against GLUT4 or caveolin. b) Immunoblots for GLUT4 were quantified, mean ± SE; n = 7 separate experiments, P < 0.05 in comparison to controls, Student’s t test. c) Immunolabeling and electron microscopy of membranes in the caveolae fraction. GLUT4 (15 nm gold particles) and caveolin (6 nm gold particles). d) GLUT4 in immuno-isolated caveolae obtained by binding to anti-caveolin antibodies attached to magnetic beads. Equal aliquots of samples were separated by SDS-PAGE and immunoblotted with antibodies against GLUT4 or caveolin.

Electron microscopic examination of the caveolae fractions showed that 50–70% of the membrane fragments were labeled with antibodies against caveolin (Fig. 2c ). GLUT4 labeling was largely found in the same membranes as those labeled for caveolin (Fig. 2c ), demonstrating a caveolar localization of GLUT4 in biochemically isolated caveolae.

To unequivocally affirm that GLUT4 in the plasma membrane is localized in caveolae, we further purified the caveolae preparation by immunoisolation of caveolae using anti-caveolin antibodies attached to magnetic beads. Thus, highly purified caveolae membranes contained GLUT4 as determined by immunoblotting after SDS-PAGE (Fig. 2d ) (no caveolin or GLUT4 were detected in the immuno-isolate in the absence of anti-caveolin antibodies during the immunoisolation). The amount of GLUT4 increased (four- and twofold in two separate preparations) in the immunopurified caveolae in response to insulin treatment of the cells (Fig. 2d ).

CONCLUSIONS AND SIGNIFICANCE

Caveolae are small (50–100 nm diameter) invaginations, related to raft microdomains, of the plasma membrane. They are enriched in sphingolipids and cholesterol. Caveolae appear to form from lipid rafts in the presence of the principal structural protein caveolin, which serves as their marker. These specialized microdomains are common in endothelial, muscle, and adipose tissues and appear to be involved in uptake of solutes into the cytosol, in transcytosis, and in signal transduction.

We used a detergent-free method to prepare highly purified caveolae, as well as morphological examination of plasma membranes by immunofluorescence and immunogold electron microscopy, to establish the localization of GLUT4 within the plasma membrane. Our results unambiguously demonstrate that GLUT4 in the plasma membrane are located largely in caveolae; in response to insulin stimulation, a major portion of GLUT4 translocates to the caveolae. About 85% of GLUT4 in the plasma membrane were located in caveolae irrespective of insulin recruitment of the transporters, as determined by electron microscopy and immunogold labeling. This is compatible with the enrichment of GLUT4 in biochemically isolated caveolae from control and insulin-stimulated cells, as well as with the immunofluorescence images of GLUT4 in relation to caveolin. We earlier reported that GLUT4 translocate to a detergent-insoluble caveolae fraction of the plasma membrane of rat adipocytes and the appearance of GLUT4 in this fraction coincides with the insulin-stimulated increase in glucose uptake. Some reports, however, have not found GLUT4 in caveolae by immunogold labeling and electron microscopic examination. Differences in the preparation of membranes for microscopy, fixation, and immunolabeling protocols likely explain why GLUT4 has not been detected in caveolae. We have optimized our procedure to produce plasma membranes with large numbers of intact caveolae, which are then efficiently immunolabeled. The only tampering with the membrane/caveolae is a brief prefixation of the membranes in paraformaldehyde and a low concentration of glutaraldehyde. This is essential to preserve caveolae and to exclude antibody-induced antigen movement in the membrane. Overfixation, for example, using higher concentrations of glutaraldehyde precludes subsequent antibody binding and GLUT4 detection in caveolae. Lipid fixation with OsO₄ after the incubation with antibodies proved critical in order to retain caveolae structures for the electron microscopy. Another key step in the preparation of membranes for transmission electron microscopy was the combined lyophilization and metal sputtering (cryosputtering) using an in-house modified lyophilizer, a procedure that avoids structural damage artifacts caused by partial rehydration and contamination during transfer from lyophilization to sputtering. Moreover, sputtering with tungsten gives a very fine grain homogeneous coating, allowing high-resolution immunogold detection by transmission electron microscopy.

That 85% of the plasma membrane GLUT4 was located in caveolae independent of insulin indicates that insulin has no specific caveolae targeting effect on GLUT4, but merely enhances its delivery to the plasma membrane. This raises the question of what determines GLUT4 localization in the plasma membrane. Some proteins localized in caveolae have an amino acid sequence that binds the caveolin scaffolding domain, but such sequences have not been identified in the GLUT4 protein. On the other hand, it appears that for most caveolae proteins, a significant fraction is found outside the caveolae. Considering that caveolae constitute a substantial fraction of the adipocyte (and endothelial) plasma membrane, the partition coefficient of GLUT4 or any other protein may not have to be much in favor of caveolae in order to retain a large part of that protein population.

The finding that GLUT4 is induced to translocate to caveolae in response to insulin, together with our earlier findings that the insulin receptor signals in caveolae and that destruction of caveolae makes cells insulin resistant, demonstrates a critical role for caveolae in insulin control of its target cells (Fig. 3 ).

View larger version (121K): [in this window] [in a new window]   Figure 3. Schematic diagram depicting insulin receptor-stimulated glucose uptake through translocation of GLUT4 from intracellular vesicles to caveolae of the plasma membrane in an adipocyte. The nucleus is making a protrusion on the cell surface at the right side of the figure.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0646fje; to cite this article, use FASEB J. (December 14, 2001) 10.1096/fj.01-0646fje

This article has been cited by other articles:

				A. Ost, U. Ortegren, J. Gustavsson, F. H. Nystrom, and P. Stralfors Triacylglycerol Is Synthesized in a Specific Subclass of Caveolae in Primary Adipocytes J. Biol. Chem., January 7, 2005; 280(1): 5 - 8. [Abstract] [Full Text] [PDF]

				A. Kumar, Y.-P. Xiao, P. J. Laipis, B. S. Fletcher, and S. C. Frost Glucose deprivation enhances targeting of GLUT1 to lipid rafts in 3T3-L1 adipocytes Am J Physiol Endocrinol Metab, April 1, 2004; 286(4): E568 - E576. [Abstract] [Full Text] [PDF]

				D. Rubin and F. Ismail-Beigi Distribution of Glut1 in detergent-resistant membranes (DRMs) and non-DRM domains: effect of treatment with azide Am J Physiol Cell Physiol, August 1, 2003; 285(2): C377 - C383. [Abstract] [Full Text] [PDF]

				M. Kanzaki and J. E. Pessin Caveolin-associated Filamentous Actin (Cav-actin) Defines a Novel F-actin Structure in Adipocytes J. Biol. Chem., July 12, 2002; 277(29): 25867 - 25869. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 16/2/249 01-0646fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by KARLSSON, M. Articles by GUSTAVSSON, J. Search for Related Content PubMed PubMed Citation Articles by KARLSSON, M. Articles by GUSTAVSSON, J.