Kinetics of Contraction-Induced GLUT4 Translocation in Skeletal Muscle Fibers From Living Mice

Plasmid and transfection procedures.

The construction of GLUT4-EGFP has been described previously (16,29). Mice were transfected as previously described (14,29,30). Mice were briefly anesthetized with 90 mg/kg pentobarbital i.p., and the skin was opened to expose the quadriceps muscle to the bombardment of DNA/gold particles using the Helios gene gun system. Each muscle was shot twice with 200 psi pressure with 1 μg DNA/0.5 mg gold (0.6 μm gold particle size). After transfection, the skin was closed using sutures and the animals were allowed to recover.

Time-lapse microscopy.

Five days after transfection, mice were anesthetized and mounted in dental cement (5,29). For contraction experiments, immediately prior to mounting the mice, microelectrodes were placed into the superficial muscle fiber layer near the groin and knee region of the quadriceps muscle, producing single twitch contractions only in the superficial layers of the quadriceps muscle fibers without losing imaging focus. Such stimulation activates muscle fibers via stimulation of the motor nerve fibers and motor endplates. Stimulation was not applied directly to the nerve because it resulted in either no visually detectable contractions or entire muscle belly contractions resulting in loss of optical focus during contraction.

For AICAR stimulation, the mouse was mounted, a catheter was placed in a tail vein, and a single confocal image was taken. This was followed by an additional 30-min stabilization period followed by another basal image (t = 0). There was no significant difference between the two basal images.

For the contraction protocol, immediately after the second basal image (t = 0), direct electrical stimulation to the muscle fibers was initiated. Two contraction treatments of different intensities were used: 1) contractions elicited by lower voltage and the parameters were frequency 2 Hz, duration 60 milliseconds, voltage 0.1–0.9 V, consisting of three bouts of 10 min, separated by 90 s of rest and 2) contractions elicited by higher voltage and the parameters were frequency 2 Hz, duration 60 milliseconds, voltage 1.1–3.0 V, consisting of three bouts of 5 min, separated by 90 s of rest. Both protocols elicited repeated single twitch contractions. Protocols were selected based on empirical observations of contraction force taken together with what was possible without losing optical focus of the imaged muscle fiber. We did not use an increasing contraction frequency because it made it very difficult to continually monitor fiber contraction. In our experience, the voltage field intensity generated by a higher voltage induced visually greater fiber contraction amplitude. This difference in contraction intensity is what we refer to as high- or low-voltage contractions throughout the study.

Contractions of the imaged fibers were continually monitored during the contraction periods through the oculars of the microscope, and voltage was adjusted to maintain force. Images were collected at the end of each contraction period and every 10-min post-contraction up to 130 min. The imaging of a high-voltage in situ contraction period is shown in supplementary video 1 (available online at http://diabetes.diabetesjournals.org/cgi/content/full/dc10-0233/DC1), illustrating that clear images are not obtainable when the fibers are contracting.

For AICAR stimulation, AICAR was given as an intravenous bolus injection (1 g/kg) through the tail vein, and images were collected every 10 min for 240 min. Images were collected with a 63×, 1.2 NA Zeiss C-Apochromat objective on a Zeiss-LSM-410 confocal microscope with ArKr-488 nanometer (nm) laser line for excitation of EGFP, while green emission light was collected between 500 and 530 nm using the standard fluorescein isothiocyanate filter. Sulforhodamine B images were collected as described (5).

Image analysis.

The tagged image file format (TIFF) images obtained with the Zeiss confocal software were imported into Metamorf Software (version 6.1; Universal Imaging). Image stacks were created, and brightness and contrast were adjusted. Since it has previously been reported that GLUT4 translocation and re-internalization in muscle are products of endocytosis and exocytosis movements, we measured the net amount of GLUT4-EGFP at the various time points (31). The net amount of GLUT4-EGFP at membrane surfaces was quantified using Metamorf Software by drawing thin regions of interest (ROIs) at the sarcolemma and t-tubule regions, excluding vesicle structures, as previously described (5,15,16). Briefly, intracellular vesicle depots were quantified by measuring changes in gray value intensity in GLUT4-EGFP vesicles larger than ∼2 μm in the visible fiber area inside an ROI covering both sarcolemma and t-tubules (Fig. 1A). The threshold and classifying settings in the Metamorf Software were used to discriminate depots above 2 μm in size from the background and emerging staining of sarcolemma and t-tubules. These Metamorf settings were also used to further discriminate between perinuclear and nonperinuclear GLUT4-EGFP depots. For sarcolemma, measurements from both sarcolemma edges of the confocal picture were averaged, the perinuclear areas were excluded, and measurements were normalized to the area of the sarcolemma ROI (Fig. 1B). For t-tubules, measurements from four randomly chosen areas were averaged (Fig. 1B). To compensate for variation in GLUT4-EGFP expression among fibers, the arbitrary units shown are the actual gray value of ROI fluorescence divided by ROI fluorescence value at t = 0 (f/f₀).

To verify the striated appearance and orientation of the t-tubules, we used sulforhodamine B, a water soluble dye that is not taken up by muscle fibers but will fill and stain the lumen of the t-tubules (32–34) (Fig. 1C). Sulforhodamine B was injected into the tail vein, and a confocal picture of quadriceps fibers was obtained 3 min later when the dye had fully loaded the t-tubules. Staining resulted in two very strong vertical florescent lines representing the capillaries and interstitial space near the sarcolemma and fine striated lines indicating the lumen of the t-tubules. The difference in the intensity of the sulforhodamine B staining is due to the much smaller diameter of the t-tubule lumen (∼100 nm) compared with the flanking capillaries (∼40 μm) and interstitial space. This striated pattern for t-tubules is consistent with previous findings (4,5,15,35).

Statistical analysis.

Gray value means from ROIs were imported into Sigma plot 10.0. Values obtained at the end of the various contraction bouts (Fig. 1) and calculated areas under the curve (Fig. 5) were compared between groups by t test. Data are means ± SE, *P < 0.05.

Contraction stimulates GLUT4-EGFP translocation to both the sarcolemma and t-tubules.

In the basal state, GLUT4-EGFP was found in larger and smaller vesicle structures (Fig. 1D and E) (t = 0, diagonal arrow) and in the perinuclear region (t = 0, vertical arrow), as has been previously reported (4,5,16,29,36). There was no continuous presence at the sarcolemma (Fig. 1D and E) (t = 0, horizontal arrow) and no regular striated pattern throughout the fiber. Following basal imaging, the superficial fibers of the quadriceps were contracted with high or low voltage. In mice subjected to high voltage contractions, there was marked staining along the sarcolemma by the 5 min time point (Fig. 1D) (t = 5), quantified as a 60% increase above basal (Fig. 1F). A striated pattern emerged inside the muscle fibers by 5 min showing translocation to the t-tubules (Fig. 1D) (t = 5). Compared with the basal state, there was a 90% increase in t-tubule GLUT4-EGFP after 5 min of contraction (Fig. 1G). In parallel, there was a marked depletion of GLUT4-EGFP vesicles in intracellular GLUT4-EGFP vesicle depots, which also included the perinuclear region (Fig. 1D [t = 5] and H). Two additional 5-min contraction periods further increased GLUT4-EGFP staining of sarcolemma and t-tubules (>200% increase from basal) simultaneously with a further reduction in GLUT4-EGFP vesicle depots (70% decrease from basal) (Fig. 1D [t = 10, t = 15] and H). The depot depletion corresponded to a 75% maximal reduction in nonperinuclear depots and a 65% maximal reduction in perinuclear depots.

With contractions elicited by low voltage, there was a significant but modest GLUT4-EGFP translocation to the sarcolemma by 10 min, which appeared in the form of continuous GLUT4-EGFP staining (Fig. 1E [t = 10] and F). There was also the emergence of t-tubule region striations (Fig. 1E [t = 10] and G). The increases in GLUT4-EGFP at the sarcolemma and t-tubules were accompanied by a 30% decrease in intracellular GLUT4 vesicle depots (Fig. 1E and H). During two additional 10-min periods of contractions, translocation to the sarcolemma and t-tubules steadily increased, and GLUT4-EGFP vesicle depots were further reduced (Fig. 1E [t = 20, t = 30, diagonal arrow] and F–H). Peak GLUT4-EGFP staining of the sarcolemma (Fig. 1E) (t = 30, horizontal arrow) and t-tubules (Fig. 1E) (t = 30, striated pattern) occurred at the same time, indicating that GLUT4 translocation to these distinct cell surface membranes followed similar kinetics (Fig. 1F–H). This is in contrast to what we have previously reported for insulin-mediated GLUT4-EGFP translocation (5). Comparison of high- and low-voltage contraction revealed that the high-voltage protocol resulted in greater levels of GLUT4-EGFP translocation to the sarcolemma and t-tubules and a greater decrease in GLUT4 vesicle depots (Fig. 1F–H).

Kinetics of GLUT4-EGFP re-internalization.

We next investigated the kinetics of GLUT4-EGFP re-internalization in the period following muscle contractions, using the 3 × 5 min high-voltage contraction protocol that resulted in maximal translocation. Similar to the previous experiment (Fig. 1), GLUT4-EGFP translocation to the sarcolemma and t-tubules increased gradually throughout the contractions, and there was a concomitant decrease in intracellular depots (Fig. 2). Interestingly, peak GLUT4-EGFP depot depletion had occurred by 10 min (Fig. 2A [t = 10] and D), whereas GLUT4-EGFP translocation to the sarcolemma and t-tubules continued to increase during the first 10 min following the cessation of contractions (Fig. 2A [t = +10] and B,C). This indicates a delay in the arrival of GLUT4-EGFP at the sarcolemma and t-tubules. Following peak translocation to the sarcolemma and t-tubules, GLUT4-EGFP underwent gradual net re-internalization and was almost fully re-internalized by 130 min after contractions (Fig. 2A–C). In parallel, GLUT4-EGFP re-emerged in the depots (Fig. 2A and D). The time course of GLUT4-EGFP re-internalization was similar for the sarcolemma and t-tubules (Fig. 2A–C).

Contraction-stimulated GLUT4-EGFP translocation and re-internalization are normal in AMPKα2 inactive transgenic mice.

AMPK has been proposed to regulate contraction-stimulated glucose transport in skeletal muscle, although there is now considerable data suggesting that AMPK is not necessary for this process (24,27,28). The role of AMPK in regulating GLUT4 translocation kinetics in skeletal muscle has not been studied. Here, contraction-regulated GLUT4-EGFP kinetics were compared in AMPKα2 inactive transgenic mice and their wild-type littermates. In the basal state, there was a normal distribution of GLUT4-EGFP in the intracellular depots and the perinuclear area (Fig. 3A and B) (t = 0, diagonal and vertical arrow), with no continuous presence at the sarcolemma and no regular continuous striated pattern (Fig. 3A and B [t = 0]). Thus, ablation of AMPKα2 activity did not affect GLUT4-EGFP localization in the basal state.

AMPKα2 inactive mice and their matched controls were subjected to 2 × 5 min of high-voltage stimulation. After contractions, GLUT4-EGFP was redistributed from the basal depots (Fig. 3A) (t = 0, t = 10, diagonal and vertical arrows) to the sarcolemma (Fig. 3A) (t = 10, horizontal arrow) and t-tubules (Fig. 3A) (t = 10, striated pattern). During 60 min of recovery, basal GLUT4-EGFP distribution was reestablished (Fig. 3A) (t = 60). When compared with controls, there was no difference in GLUT4-EGFP translocation and re-internalization (Fig. 3B). Image quantification showed that maximal translocation was reached at t = 10 min for both the sarcolemma (∼260% above basal) (Fig. 3C) and t-tubules (∼275% above basal) (Fig. 3D). This was accompanied by a 65% reduction in GLUT4-EGFP, corresponding to 65% maximal reduction in nonperinuclear depots and a 55% maximal reduction in perinuclear GLUT4-EGFP depots (Fig. 3E). Re-internalization of GLUT4-EGFP occurred immediately after contractions with similar rates for the sarcolemma and t-tubules indicating similar kinetics (Fig. 3C and D). Ablation of AMPKα2 activity did not influence GLUT4-EGFP kinetics in response to contractions (Fig. 3C–E). Thus, AMPKα2 activity is not necessary for normal GLUT4 trafficking kinetics before, during, and after in situ contractions in living animals.

AICAR-stimulated GLUT4-EGFP translocation and re-internalization.

A previous study suggested that the AMPK activator AICAR only increases GLUT4 translocation to the sarcolemma and not to the t-tubules (23). However, this finding is not consistent with the central role of the t-tubules in GLUT4 translocation (4,5,35) and glucose transport (15,35) and the finding that AICAR stimulates glucose transport to the same extent as insulin and contraction (20). Therefore, we investigated the effects of AICAR on GLUT4-EGFP translocation kinetics. AICAR was injected as a bolus into the tail vein immediately after t = 0, and the first post-injection image was obtained 10 min later. AICAR resulted in a gradual but significant increase in staining along the sarcolemma by 20–40 min after AICAR injection (Fig. 4A [t = 20–40] and B [supplementary video 2]). Furthermore, a clear striated pattern appeared by 20–40 min after AICAR injection demonstrating GLUT4-EGFP translocation to the t-tubules (Fig. 4A [t = 20–40] and C [supplementary video 2]). Maximal translocation occurred 90–100 min after AICAR injection with a ∼300% increase above basal for both the sarcolemma and t-tubules. Increased GLUT4-EGFP at the sarcolemma and t-tubules was accompanied by a gradual redistribution of GLUT4-EGFP from depots (Fig. 4). The depot depletion corresponded to a 70% maximal reduction in nonperinuclear depots and a 60% maximal reduction in perinuclear GLUT4-EGFP depots. GLUT4-EGFP vesicles were depleted 20 min after AICAR injection (Fig. 4) (t = 20, supplementary video 2), and depletion further progressed at 80 min (Fig. 4D). Re-internalization of GLUT4-EGFP from the sarcolemma and t-tubules followed an identical time course (Fig. 4). Maximal translocation did not differ in response to AICAR (Fig. 4) and contractions (Figs. 1 and 2), and re-internalization kinetics were also similar in comparing the two stimuli (Figs. 1, 2, and 4). Thus, AICAR stimulates GLUT4-EGFP translocation to both the sarcolemma and t-tubules in a similar manner to muscle contraction.

AICAR-stimulated GLUT4-EGFP translocation is impaired in AMPKα2 inactive transgenic mice.

We determined if AMPKα2 was necessary for AICAR-stimulated GLUT4-EGFP translocation. Compared with control mice, the AMPKα2 inactive transgenic mice had minor staining along the sarcolemma (Fig. 5A and C) and very weak striations in the t-tubule region following AICAR injection (Fig. 5B and D). Furthermore, there was only limited depletion of GLUT4-EGFP vesicle depots (Fig. 5B and E). It is interesting that the changes in the GLUT4-EGFP depots did not fully match sarcolemma and t-tubule GLUT4-EGFP, as there was a modest depletion of GLUT4-EGFP depots without an accompanying increase at the cell surface membranes (Fig. 5C–E). Based on our experience with this method, we believe that there is also an intermediate step in the GLUT4 translocation and re-internalization processes that appear as a light background “haze” upon imaging, and this haze can be observed in Fig. 5A.

The minimal GLUT4-EGFP translocation that is present in these mice may be due to very low levels of AMPKα1 subunit in the muscle fibers and/or from nonspecific systemic effects of AICAR. In fact, we observed that injection of AICAR caused minor trembling of the mice (this can be seen as the muscle fiber vibrating in first half of supplementary video 2). Thus, the majority of the AICAR-induced GLUT4-EGFP translocation is abolished in AMPKα2 inactive transgenic mice.