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Kinetics of contraction-induced GLUT4 translocation in skeletal muscle fibers from living mice.

Lauritzen HP, Galbo H, Toyoda T, Goodyear LJ - Diabetes (2010)

Bottom Line: AICAR increased GLUT4-EGFP translocation to both sarcolemma and t-tubules with similar kinetics.Ablation of AMPKalpha2 activity in AMPKalpha2 inactive transgenic mice did not change GLUT4-EGFP's basal localization, contraction-stimulated intracellular GLUT4-EGFP vesicle depletion, translocation, or re-internalization, but diminished AICAR-induced translocation.We have developed a novel imaging system to study contraction-stimulated GLUT4 translocation in living mice.

View Article: PubMed Central - PubMed

Affiliation: Research Division, Joslin Diabetes Center and Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT

Objective: Exercise is an important strategy for the treatment of type 2 diabetes. This is due in part to an increase in glucose transport that occurs in the working skeletal muscles. Glucose transport is regulated by GLUT4 translocation in muscle, but the molecular machinery mediating this process is poorly understood. The purpose of this study was to 1) use a novel imaging system to elucidate the kinetics of contraction-induced GLUT4 translocation in skeletal muscle and 2) determine the function of AMP-activated protein kinase alpha2 (AMPKalpha2) in this process.

Research design and methods: Confocal imaging was used to visualize GLUT4-enhanced green fluorescent protein (EGFP) in transfected quadriceps muscle fibers in living mice subjected to contractions or the AMPK-activator AICAR.

Results: Contraction increased GLUT4-EGFP translocation from intracellular vesicle depots to both the sarcolemma and t-tubules with similar kinetics, although translocation was greater with contractions elicited by higher voltage. Re-internalization of GLUT4 did not begin until 10 min after contractions ceased and was not complete until 130 min after contractions. AICAR increased GLUT4-EGFP translocation to both sarcolemma and t-tubules with similar kinetics. Ablation of AMPKalpha2 activity in AMPKalpha2 inactive transgenic mice did not change GLUT4-EGFP's basal localization, contraction-stimulated intracellular GLUT4-EGFP vesicle depletion, translocation, or re-internalization, but diminished AICAR-induced translocation.

Conclusions: We have developed a novel imaging system to study contraction-stimulated GLUT4 translocation in living mice. Contractions increase GLUT4 translocation to the sarcolemma and t-tubules with similar kinetics and do not require AMPKalpha2 activity.

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Related in: MedlinePlus

Contraction-mediated GLUT4-EGFP translocation to both sarcolemma and t-tubules. A and B: Examples of the regions of interest (ROIs) used for image quantifications. A: Fluorescence values of GLUT4-EGFP perinuclear and nonperinuclear depots were measured within the same ROI. Quantification software settings were set to measure vesicles >2 μm in size inside the ROI and to discriminate between perinuclear and nonperinuclear depots. B: The level of GLUT4-EGFP translocation in the muscle fibers at the different time points was measured in ROIs along each sarcolemma side and in four randomly chosen ROIs in the t-tubules region. Translocation ROIs excluded vesicles structures above 2 μm in size. C: In vivo staining of t-tubules after intravenous injection of the water soluble dye sulforhodamine B, showing a high fluorescence staining in the capillaries running along the sarcolemma together with a more weakly fluorescent striated pattern in the t-tubules region. D and E: t = 0 shows confocal images of basal GLUT4-EGFP–expressing ICR mouse muscle fibers just before in situ contractions. Repeated single contractions (2 Hz) were elicited using either “high” (1.1–3 V) (D) or “low” (0.1–0.9 V) (E) voltage. Muscle was stimulated for 3 × 5 min (D) or 3 × 10 min (E) periods separated by 90 sec of rest. Images are shown from one muscle fiber subjected to either high- (D) or low-voltage (E) contractions. Horizontal arrows indicate sarcolemma, while GLUT4-EGFP depots are indicated near the nuclei by vertical arrows and inside the fiber by diagonal arrows. Similar observations were done in fibers from five to eight mice. Numbers denote time in min. Bars = 20 μm. F-H: Image quantifications of GLUT4-EGFP at the sarcolemma (F), the t-tubules (G), and the intracellular vesicle depots (H). *Statistical difference between groups (P < 0.05) in values obtained at the end of identical contraction bouts. Arbitrary units shown are the actual grey value of ROI fluorescence divided by ROI fluorescence value at t = 0 (f/f0). Data are means ± SE. n = 5–8.
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Figure 1: Contraction-mediated GLUT4-EGFP translocation to both sarcolemma and t-tubules. A and B: Examples of the regions of interest (ROIs) used for image quantifications. A: Fluorescence values of GLUT4-EGFP perinuclear and nonperinuclear depots were measured within the same ROI. Quantification software settings were set to measure vesicles >2 μm in size inside the ROI and to discriminate between perinuclear and nonperinuclear depots. B: The level of GLUT4-EGFP translocation in the muscle fibers at the different time points was measured in ROIs along each sarcolemma side and in four randomly chosen ROIs in the t-tubules region. Translocation ROIs excluded vesicles structures above 2 μm in size. C: In vivo staining of t-tubules after intravenous injection of the water soluble dye sulforhodamine B, showing a high fluorescence staining in the capillaries running along the sarcolemma together with a more weakly fluorescent striated pattern in the t-tubules region. D and E: t = 0 shows confocal images of basal GLUT4-EGFP–expressing ICR mouse muscle fibers just before in situ contractions. Repeated single contractions (2 Hz) were elicited using either “high” (1.1–3 V) (D) or “low” (0.1–0.9 V) (E) voltage. Muscle was stimulated for 3 × 5 min (D) or 3 × 10 min (E) periods separated by 90 sec of rest. Images are shown from one muscle fiber subjected to either high- (D) or low-voltage (E) contractions. Horizontal arrows indicate sarcolemma, while GLUT4-EGFP depots are indicated near the nuclei by vertical arrows and inside the fiber by diagonal arrows. Similar observations were done in fibers from five to eight mice. Numbers denote time in min. Bars = 20 μm. F-H: Image quantifications of GLUT4-EGFP at the sarcolemma (F), the t-tubules (G), and the intracellular vesicle depots (H). *Statistical difference between groups (P < 0.05) in values obtained at the end of identical contraction bouts. Arbitrary units shown are the actual grey value of ROI fluorescence divided by ROI fluorescence value at t = 0 (f/f0). Data are means ± SE. n = 5–8.

Mentions: 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).


Kinetics of contraction-induced GLUT4 translocation in skeletal muscle fibers from living mice.

Lauritzen HP, Galbo H, Toyoda T, Goodyear LJ - Diabetes (2010)

Contraction-mediated GLUT4-EGFP translocation to both sarcolemma and t-tubules. A and B: Examples of the regions of interest (ROIs) used for image quantifications. A: Fluorescence values of GLUT4-EGFP perinuclear and nonperinuclear depots were measured within the same ROI. Quantification software settings were set to measure vesicles >2 μm in size inside the ROI and to discriminate between perinuclear and nonperinuclear depots. B: The level of GLUT4-EGFP translocation in the muscle fibers at the different time points was measured in ROIs along each sarcolemma side and in four randomly chosen ROIs in the t-tubules region. Translocation ROIs excluded vesicles structures above 2 μm in size. C: In vivo staining of t-tubules after intravenous injection of the water soluble dye sulforhodamine B, showing a high fluorescence staining in the capillaries running along the sarcolemma together with a more weakly fluorescent striated pattern in the t-tubules region. D and E: t = 0 shows confocal images of basal GLUT4-EGFP–expressing ICR mouse muscle fibers just before in situ contractions. Repeated single contractions (2 Hz) were elicited using either “high” (1.1–3 V) (D) or “low” (0.1–0.9 V) (E) voltage. Muscle was stimulated for 3 × 5 min (D) or 3 × 10 min (E) periods separated by 90 sec of rest. Images are shown from one muscle fiber subjected to either high- (D) or low-voltage (E) contractions. Horizontal arrows indicate sarcolemma, while GLUT4-EGFP depots are indicated near the nuclei by vertical arrows and inside the fiber by diagonal arrows. Similar observations were done in fibers from five to eight mice. Numbers denote time in min. Bars = 20 μm. F-H: Image quantifications of GLUT4-EGFP at the sarcolemma (F), the t-tubules (G), and the intracellular vesicle depots (H). *Statistical difference between groups (P < 0.05) in values obtained at the end of identical contraction bouts. Arbitrary units shown are the actual grey value of ROI fluorescence divided by ROI fluorescence value at t = 0 (f/f0). Data are means ± SE. n = 5–8.
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Related In: Results  -  Collection

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Figure 1: Contraction-mediated GLUT4-EGFP translocation to both sarcolemma and t-tubules. A and B: Examples of the regions of interest (ROIs) used for image quantifications. A: Fluorescence values of GLUT4-EGFP perinuclear and nonperinuclear depots were measured within the same ROI. Quantification software settings were set to measure vesicles >2 μm in size inside the ROI and to discriminate between perinuclear and nonperinuclear depots. B: The level of GLUT4-EGFP translocation in the muscle fibers at the different time points was measured in ROIs along each sarcolemma side and in four randomly chosen ROIs in the t-tubules region. Translocation ROIs excluded vesicles structures above 2 μm in size. C: In vivo staining of t-tubules after intravenous injection of the water soluble dye sulforhodamine B, showing a high fluorescence staining in the capillaries running along the sarcolemma together with a more weakly fluorescent striated pattern in the t-tubules region. D and E: t = 0 shows confocal images of basal GLUT4-EGFP–expressing ICR mouse muscle fibers just before in situ contractions. Repeated single contractions (2 Hz) were elicited using either “high” (1.1–3 V) (D) or “low” (0.1–0.9 V) (E) voltage. Muscle was stimulated for 3 × 5 min (D) or 3 × 10 min (E) periods separated by 90 sec of rest. Images are shown from one muscle fiber subjected to either high- (D) or low-voltage (E) contractions. Horizontal arrows indicate sarcolemma, while GLUT4-EGFP depots are indicated near the nuclei by vertical arrows and inside the fiber by diagonal arrows. Similar observations were done in fibers from five to eight mice. Numbers denote time in min. Bars = 20 μm. F-H: Image quantifications of GLUT4-EGFP at the sarcolemma (F), the t-tubules (G), and the intracellular vesicle depots (H). *Statistical difference between groups (P < 0.05) in values obtained at the end of identical contraction bouts. Arbitrary units shown are the actual grey value of ROI fluorescence divided by ROI fluorescence value at t = 0 (f/f0). Data are means ± SE. n = 5–8.
Mentions: 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).

Bottom Line: AICAR increased GLUT4-EGFP translocation to both sarcolemma and t-tubules with similar kinetics.Ablation of AMPKalpha2 activity in AMPKalpha2 inactive transgenic mice did not change GLUT4-EGFP's basal localization, contraction-stimulated intracellular GLUT4-EGFP vesicle depletion, translocation, or re-internalization, but diminished AICAR-induced translocation.We have developed a novel imaging system to study contraction-stimulated GLUT4 translocation in living mice.

View Article: PubMed Central - PubMed

Affiliation: Research Division, Joslin Diabetes Center and Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT

Objective: Exercise is an important strategy for the treatment of type 2 diabetes. This is due in part to an increase in glucose transport that occurs in the working skeletal muscles. Glucose transport is regulated by GLUT4 translocation in muscle, but the molecular machinery mediating this process is poorly understood. The purpose of this study was to 1) use a novel imaging system to elucidate the kinetics of contraction-induced GLUT4 translocation in skeletal muscle and 2) determine the function of AMP-activated protein kinase alpha2 (AMPKalpha2) in this process.

Research design and methods: Confocal imaging was used to visualize GLUT4-enhanced green fluorescent protein (EGFP) in transfected quadriceps muscle fibers in living mice subjected to contractions or the AMPK-activator AICAR.

Results: Contraction increased GLUT4-EGFP translocation from intracellular vesicle depots to both the sarcolemma and t-tubules with similar kinetics, although translocation was greater with contractions elicited by higher voltage. Re-internalization of GLUT4 did not begin until 10 min after contractions ceased and was not complete until 130 min after contractions. AICAR increased GLUT4-EGFP translocation to both sarcolemma and t-tubules with similar kinetics. Ablation of AMPKalpha2 activity in AMPKalpha2 inactive transgenic mice did not change GLUT4-EGFP's basal localization, contraction-stimulated intracellular GLUT4-EGFP vesicle depletion, translocation, or re-internalization, but diminished AICAR-induced translocation.

Conclusions: We have developed a novel imaging system to study contraction-stimulated GLUT4 translocation in living mice. Contractions increase GLUT4 translocation to the sarcolemma and t-tubules with similar kinetics and do not require AMPKalpha2 activity.

Show MeSH
Related in: MedlinePlus