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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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In situ contractions induce GLUT4-EGFP translocation to and re-internalization from both sarcolemma and t-tubules with similar kinetics. A: t=0 shows confocal image of a basal GLUT4-EGFP–expressing muscle fiber just before in situ contractions in an ICR mouse. Contractions were elicited using the high-voltage protocol for 3 × 5 min separated by 90 sec of rest. 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. t = denotes accumulated contraction time; t = + denotes time during recovery after contractions. Bar = 20 μm. B-D: Image quantifications of GLUT4-EGFP at the sarcolemma (B), the t-tubules (C), and the intracellular vesicle depots (D). Horizontal solid lines indicate contraction period. Horizontal dotted lines indicate the period after contractions. Data are means ± SE. n = 5–8.
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Figure 2: In situ contractions induce GLUT4-EGFP translocation to and re-internalization from both sarcolemma and t-tubules with similar kinetics. A: t=0 shows confocal image of a basal GLUT4-EGFP–expressing muscle fiber just before in situ contractions in an ICR mouse. Contractions were elicited using the high-voltage protocol for 3 × 5 min separated by 90 sec of rest. 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. t = denotes accumulated contraction time; t = + denotes time during recovery after contractions. Bar = 20 μm. B-D: Image quantifications of GLUT4-EGFP at the sarcolemma (B), the t-tubules (C), and the intracellular vesicle depots (D). Horizontal solid lines indicate contraction period. Horizontal dotted lines indicate the period after contractions. Data are means ± SE. n = 5–8.

Mentions: 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).


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

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

In situ contractions induce GLUT4-EGFP translocation to and re-internalization from both sarcolemma and t-tubules with similar kinetics. A: t=0 shows confocal image of a basal GLUT4-EGFP–expressing muscle fiber just before in situ contractions in an ICR mouse. Contractions were elicited using the high-voltage protocol for 3 × 5 min separated by 90 sec of rest. 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. t = denotes accumulated contraction time; t = + denotes time during recovery after contractions. Bar = 20 μm. B-D: Image quantifications of GLUT4-EGFP at the sarcolemma (B), the t-tubules (C), and the intracellular vesicle depots (D). Horizontal solid lines indicate contraction period. Horizontal dotted lines indicate the period after contractions. Data are means ± SE. n = 5–8.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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Figure 2: In situ contractions induce GLUT4-EGFP translocation to and re-internalization from both sarcolemma and t-tubules with similar kinetics. A: t=0 shows confocal image of a basal GLUT4-EGFP–expressing muscle fiber just before in situ contractions in an ICR mouse. Contractions were elicited using the high-voltage protocol for 3 × 5 min separated by 90 sec of rest. 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. t = denotes accumulated contraction time; t = + denotes time during recovery after contractions. Bar = 20 μm. B-D: Image quantifications of GLUT4-EGFP at the sarcolemma (B), the t-tubules (C), and the intracellular vesicle depots (D). Horizontal solid lines indicate contraction period. Horizontal dotted lines indicate the period after contractions. Data are means ± SE. n = 5–8.
Mentions: 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).

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