Analysis of GLUT4 distribution in whole skeletal muscle fibers: identification of distinct storage compartments that are recruited by insulin and muscle contractions

Abstract

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 approximately 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).

Figure 2

High magnification view of GLUT4 immunofluorescence in basal muscle fibers. Single fibers were stained as described in Fig. 1. A series of confocal images, centered on a nucleus, were recorded from the surface of the fiber (0.0 μm) and at depths of 0.7, 1.4, and 2.1 μm. GLUT4 is present in numerous small tubulovesicular structures and in larger perinuclear elements that form a belt around the nucleus. Bar, 5 μm.

Figure 3

The large, but not the small depots of GLUT4, are close to the TGN at the surface and in the core of basal fibers. Single basal fibers were stained simultaneously with an antibody to TGN38 (tgn) followed by a Texas red–conjugated secondary antibody and to GLUT4 (gt) followed by a fluorescein-conjugated secondary antibody. Single confocal images were recorded, separately for each fluorophore, from the surface (left panels) or the core (right panels) of the fibers. The inside views were rotated by 90° compared with the surface views, explaining why the striations are horizontal. When the green and red images are superimposed and viewed in color, their overlap, shown in yellow, is only partial. The smaller depots of GLUT4 have no corresponding TGN staining and an occasional TGN has no visible GLUT4 (small arrows, orphan structures). However, in both areas, each of the large depots of GLUT4 has a TGN counterpart (arrowheads, some of the corresponding elements). Bars, 5 μm.

Figure 10

Partial association of GLUT4 and transferrin receptor-containing structures at the surface and in the core of basal fibers. Single basal fibers were stained simultaneously with an antibody to the transferrin receptor (tfr) followed by a Texas red– conjugated secondary antibody, and to GLUT4 (gt) followed by a fluorescein-conjugated secondary antibody. Single confocal images were recorded, separately for each fluorophore, from the surface (left panels) or the core (right panels) of the fibers. The core views were rotated by 90° compared with surface views. In the large depots of the perinuclear belt, there is a large degree of overlapping (yellow) which is exaggerated by saturation of the image. In the smaller depots that are encircled by this belt, there is much less overlap. The small depots are shown, enlarged, in the bottom panel which displays their computer-drawn contours. Most of the depots are small and round. Only a small fraction of these overlap (arrows). Some depots are larger and irregularly shaped. They show a large degree of overlap (arrowheads). Notice, however, that the green and red contours are differently shaped. Bars, 5 μm.

Figure 11

Association of GLUT4 and TfR is increased by insulin but decreased by contractions. Single fibers from insulin- or contraction-stimulated muscle were stained and observed as described in the legend for Fig. 10. Note the increased overlap of the two stainings in insulin-stimulated fibers and the decreased overlap in contraction-stimulated fibers compared with basal fibers (refer to Fig. 10), suggesting that GLUT4⁺/TfR⁻ elements are recruited by insulin and GLUT4⁺/TfR⁺ elements are recruited by contractions. Bars, 5 μm.

Figure 6

Confocal images reveal translocation of GLUT4 to the plasma membrane and T tubules of stimulated fibers.Teased single fibers from basal (bas), insulin- (ins), contraction- (ex), and insulin- and contraction-stimulated muscles (ins+ex) were permeabilized and stained with rabbit anti-GLUT4 followed by biotin-conjugated anti-rabbit IgG and FITC-labeled streptavidin. Confocal images (12 for each condition) were collected from a plane close to the center of the fibers, as described in the Methods section. Notice the appearance of a continuous plasma membrane staining and of cross-striations, especially noticeable in insulin- and contraction-stimulated fibers (ins+ex), and the progressive disappearance of the strong dotted pattern when going from basal fibers to fibers stimulated with either or both stimuli. Insets, an enlarged view of the fiber surface. Bar, 5 μm.

Figure 9

GLUT4 is recruited from both large and small depots after stimulation. z series of four confocal images starting at the surface of the fibers, with successive images separated by 0.7 μm were recorded from 10–15 different fibers for each of the four conditions (bas, ins, ex, and ins+ex). Projections of the series were calculated. Shown here is one representative projection for each condition. Note that the intensity of the perinuclear staining and the density of the small depots both decrease with stimulation, with single stimuli causing a decrease intermediate to that caused by combined stimulation. Bar, 5 μm.

Figure 1

Immunofluorescence localization of GLUT4 in single fibers from basal soleus muscle. Single fibers were stained with anti-GLUT4 antiserum followed by biotin-conjugated anti-rabbit IgG and FITC-labeled streptavidin. (a) At the surface of the fibers, in some areas, myonuclei (arrowheads) are aligned with the axis of the fiber and joined by regular rows of GLUT4 aggregates (arrows). (b) In other areas, nuclei are not aligned with the axis of the fiber and the staining pattern appears less orderly. Dark channels (asterisks) correspond to capillaries. (c) In the core of the fibers, the staining consists of dotted lines (arrows) and weak cross-striations. Bar, 10 μm.

Figure 4

Localization of GLUT4 in basal fibers observed by preembedding immunogold electron microscopy. Single fibers were stained and embedded in epoxy resin as described in Materials and Methods. (a and b) Low magnification views of two areas close to the plasma membrane, one with a nucleus (a), the other with myofibrils extending very close to the plasma membrane (b). Both show small (arrowheads) and large (arrow) clusters of GLUT4. In a they surround a nucleus (N). The large clusters are next to a Golgi complex (G) and to multivesicular bodies (e; see enlarged area in i). Other recognizable elements are mitochondria (m) and myofibrils that fill the core of the fiber, with the characteristic A and I band alternation. Note the almost complete absence of single grains on the plasma membrane. (c– e) Distribution of GLUT4 in the core of the fibers. In basal conditions, most GLUT4 staining is in clusters of grains (arrowheads) in the I band, near the A–I band intersection, or more rarely in the A band. T tubules at the triad junctions (small arrows) are unlabeled but there is labeling of terminal cisternae of the SR (d and e) and of what appears to be the fenestrated SR in the A band (arrowheads). (f–j) Details of GLUT4 localization showing association with Golgi complex and endosomes. (f–g) Next to nuclei (N) that are close to the fiber surface, stacks of Golgi cisternae (arrowheads) can be recognized. GLUT4 is concentrated in the cisternae farthest away from the nucleus and in tubulovesicular structures just beyond or lateral to the cisternae. In h, GLUT4 is associated with a Golgi complex which is in the myofibrillar core and is not associated with a nucleus. In i, the gold grains are next to Golgi cisternae and to mutivesicular bodies, presumably endosomes (e), whereas j shows an abundantly labeled endosome in the myofibrillar core. Bars: (a–c) 0.5 μm; (d–j) 0.2 μm.

Figure 4

Localization of GLUT4 in basal fibers observed by preembedding immunogold electron microscopy. Single fibers were stained and embedded in epoxy resin as described in Materials and Methods. (a and b) Low magnification views of two areas close to the plasma membrane, one with a nucleus (a), the other with myofibrils extending very close to the plasma membrane (b). Both show small (arrowheads) and large (arrow) clusters of GLUT4. In a they surround a nucleus (N). The large clusters are next to a Golgi complex (G) and to multivesicular bodies (e; see enlarged area in i). Other recognizable elements are mitochondria (m) and myofibrils that fill the core of the fiber, with the characteristic A and I band alternation. Note the almost complete absence of single grains on the plasma membrane. (c– e) Distribution of GLUT4 in the core of the fibers. In basal conditions, most GLUT4 staining is in clusters of grains (arrowheads) in the I band, near the A–I band intersection, or more rarely in the A band. T tubules at the triad junctions (small arrows) are unlabeled but there is labeling of terminal cisternae of the SR (d and e) and of what appears to be the fenestrated SR in the A band (arrowheads). (f–j) Details of GLUT4 localization showing association with Golgi complex and endosomes. (f–g) Next to nuclei (N) that are close to the fiber surface, stacks of Golgi cisternae (arrowheads) can be recognized. GLUT4 is concentrated in the cisternae farthest away from the nucleus and in tubulovesicular structures just beyond or lateral to the cisternae. In h, GLUT4 is associated with a Golgi complex which is in the myofibrillar core and is not associated with a nucleus. In i, the gold grains are next to Golgi cisternae and to mutivesicular bodies, presumably endosomes (e), whereas j shows an abundantly labeled endosome in the myofibrillar core. Bars: (a–c) 0.5 μm; (d–j) 0.2 μm.

Figure 12

Immunogold EM localization of the TfR in basal fibers. Single fibers were stained for TfR and embedded in epoxy resin as described in Materials and Methods. (a) Low magnification overview showing an area around a nucleus near the plasma membrane. TfR is present in large aggregates in the area of the Golgi complex (arrow) and in smaller aggregates all around the nucleus (arrowheads) in a pattern that resembles that seen for GLUT4 in Fig. 4. (b) A stack of Golgi cisternae (arrow) showing TfR labeling, both in the cisternae and in vesicles of different sizes around the cisternae (arrowheads). (c and d) Labeling is often seen associated with multivesicular bodies (arrowheads) of different sizes. In c, the labeling seems excluded from the internal structures whereas in d they are labeled. Note also a network of labeled, tubulovesicular structures (arrows) around the multivesicular bodies. Bars: (a) 0.5 μm; (b–d) 0.2 μm.

Figure 5

Insulin and muscle contractions do not affect accessibility of GLUT4 NH₂- or COOH-terminal epitopes. Teased single fibers, each ∼9-mm long, were prepared from basal (Bas), insulin- (Ins), contraction- (Ex) and insulin- and contraction-stimulated muscles (ExI). They were permeabilized and stained with antibodies to the COOH- or NH₂-terminal part of GLUT4 or to caveolin-3. Primary antibody binding was quantitated by measuring the amount of ¹²⁵I-labeled secondary antibody binding. Amount of bound GLUT4 antibodies is expressed as percent of caveolin-3 binding to fibers from the same muscle in order to correct for possible variations in sarcomere length during fixation. Results are presented as mean ± SEM of two or three separate experiments with 10–15 fibers per experiment. Analysis of variance (ANOVA) did not detect any significant difference between stimulation states within each antibody group (P > 0.05). Omission of the primary antibodies resulted in less than 2% of the labeling obtained with primary antibodies against GLUT4.

Figure 7

Translocation of GLUT4 in stimulated fibers observed by preembedding immunogold electron microscopy. (a) Overview of a fiber that has been maximally stimulated with both insulin and contractions, showing two nuclei (N) and several mitochondria (m). The plasma membrane contains numerous single grains (black arrows) and the T tubules of several triad junctions also appear labeled (white arrowheads). GLUT4 labeling is also found at the pole of one of the nuclei, in the Golgi complex region (G). (b) Despite the heavy labeling of the plasma membrane (black arrows) in a fiber from contraction-stimulated muscle, the Golgi stack (G) still has fairly dense polarized GLUT4 labeling. (c) Overview of a fiber from an insulin-stimulated muscle showing dense labeling of the plasma membrane (black arrows). (d–h) Labeling of junctional T tubules (white arrows) in fibers from exercise- (d and f) or insulin and exercise-stimulated (e and g) muscle. In e, the nonjunctional part of a T tubule leaving a triad can be seen (white arrowheads) coursing between the myofibrils. The labeling of apparently fenestrated SR (f and g, white arrowheads) is mostly in the form of single grains and not of clusters of grains as seen in nonstimulated fibers. (h) Labeling of the T tubule in a cross-sectioned triad in a fiber from insulin-stimulated muscle. (i) Labeling of an endosome in the myofibrillar core of an insulin-stimulated fiber. (j) The labeling of the TGN area in an insulin and exercise-stimulated fiber appears lighter than in basal fibers. Bars: (a–c) 0.5 μm; (d–g, i, and j) 0.2 μm; (h) 0.05 μm.

Figure 7

Translocation of GLUT4 in stimulated fibers observed by preembedding immunogold electron microscopy. (a) Overview of a fiber that has been maximally stimulated with both insulin and contractions, showing two nuclei (N) and several mitochondria (m). The plasma membrane contains numerous single grains (black arrows) and the T tubules of several triad junctions also appear labeled (white arrowheads). GLUT4 labeling is also found at the pole of one of the nuclei, in the Golgi complex region (G). (b) Despite the heavy labeling of the plasma membrane (black arrows) in a fiber from contraction-stimulated muscle, the Golgi stack (G) still has fairly dense polarized GLUT4 labeling. (c) Overview of a fiber from an insulin-stimulated muscle showing dense labeling of the plasma membrane (black arrows). (d–h) Labeling of junctional T tubules (white arrows) in fibers from exercise- (d and f) or insulin and exercise-stimulated (e and g) muscle. In e, the nonjunctional part of a T tubule leaving a triad can be seen (white arrowheads) coursing between the myofibrils. The labeling of apparently fenestrated SR (f and g, white arrowheads) is mostly in the form of single grains and not of clusters of grains as seen in nonstimulated fibers. (h) Labeling of the T tubule in a cross-sectioned triad in a fiber from insulin-stimulated muscle. (i) Labeling of an endosome in the myofibrillar core of an insulin-stimulated fiber. (j) The labeling of the TGN area in an insulin and exercise-stimulated fiber appears lighter than in basal fibers. Bars: (a–c) 0.5 μm; (d–g, i, and j) 0.2 μm; (h) 0.05 μm.

Figure 8

Stimulation does not affect the diameter of T tubules. Muscles from basal and stimulated rats were fixed by perfusion with 3% formaldehyde + 0.5% glutaraldehyde (top) or with 2% formaldehyde alone (bottom), embedded and sectioned (refer to Materials and Methods). Under the microscope, the A–I junction was followed until a longitudinally sectioned triad junction was encountered. A photograph was taken, the grid was then shifted by a few sarcomeres, and then the search resumed. After five photographs another fiber was selected, etc. The diameter of the T tubules was measured from photographs taken at a primary magnification of 25,000, with a 7× magnifying glass equipped with a measuring scale. The photographs show a typical T tubule for each condition. For each of the fixation procedures, T tubules were sampled from two independent experiments, each including the four stimulation states. Two-way ANOVA did not detect any interaction between fixation conditions and T tubule diameter, nor any effect of stimulation on T tubule diameter (P > 0.05). Therefore, measurements obtained by the two fixation procedures were pooled. The average diameter (mean ± SEM) as well as the total number of T tubules measured (n) are shown for each condition. Bar, 0.2 μm.

Figure 13

Subcellular membrane fractionation shows that TfR only translocates in response to contractions. Four membrane fractions (F1–F4) were prepared from rat hindleg muscles from four group of rats (Bas, Ins, Ex, and ExI). 5 μg of protein from each fraction were separated by SDS-PAGE and analysed by Western blotting using antibodies against GLUT4 and TfR followed by ¹²⁵I-labeled secondary antibody. The bands were quantitated with a PhosphorImager and the contribution of each band to the sum of bands in the four fractions is expressed in percentages. Values are means ± SEM of 3–5 independent experiments. Insulin and contractions result in an additive translocation of GLUT4 to F1 (enriched in plasma membranes and T tubules), from F3 and F4 (enriched in intracellular membranes). In contrast, only contractions result in translocation of TfR from F2 and F3 to F1. TfR-positive vesicles recruited from F2 are probably GLUT4 negative, since the GLUT4 content in F2 is unaffected by stimulation, whereas those recruited from F3 are probably GLUT4 positive.