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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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Depletion of AMPKα2 activity markedly reduces AICAR-mediated GLUT4-EGFP translocation to both the sarcolemma and t-tubules. A and B: t = 0 shows confocal images of a basal GLUT4-EGFP–expressing muscle fiber in situ in either an AMPKα2 inactive transgenic mouse (A) or a matched control FVB mouse (B). Immediately after t = 0, AICAR was administrated intravenously via the tail vein. 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 made in fibers from five to six mice in each group. Numbers denote time in min. Bars = 20 μm. C–E: Image quantifications of GLUT4-EGFP at the sarcolemma (C), the t-tubules (D), and the intracellular vesicle depots (E). Area under curves were calculated. *Statistical difference between groups (P < 0.05). Data are means ± SE. n = 5–6.
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Figure 5: Depletion of AMPKα2 activity markedly reduces AICAR-mediated GLUT4-EGFP translocation to both the sarcolemma and t-tubules. A and B: t = 0 shows confocal images of a basal GLUT4-EGFP–expressing muscle fiber in situ in either an AMPKα2 inactive transgenic mouse (A) or a matched control FVB mouse (B). Immediately after t = 0, AICAR was administrated intravenously via the tail vein. 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 made in fibers from five to six mice in each group. Numbers denote time in min. Bars = 20 μm. C–E: Image quantifications of GLUT4-EGFP at the sarcolemma (C), the t-tubules (D), and the intracellular vesicle depots (E). Area under curves were calculated. *Statistical difference between groups (P < 0.05). Data are means ± SE. n = 5–6.

Mentions: 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.


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

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

Depletion of AMPKα2 activity markedly reduces AICAR-mediated GLUT4-EGFP translocation to both the sarcolemma and t-tubules. A and B: t = 0 shows confocal images of a basal GLUT4-EGFP–expressing muscle fiber in situ in either an AMPKα2 inactive transgenic mouse (A) or a matched control FVB mouse (B). Immediately after t = 0, AICAR was administrated intravenously via the tail vein. 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 made in fibers from five to six mice in each group. Numbers denote time in min. Bars = 20 μm. C–E: Image quantifications of GLUT4-EGFP at the sarcolemma (C), the t-tubules (D), and the intracellular vesicle depots (E). Area under curves were calculated. *Statistical difference between groups (P < 0.05). Data are means ± SE. n = 5–6.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2927934&req=5

Figure 5: Depletion of AMPKα2 activity markedly reduces AICAR-mediated GLUT4-EGFP translocation to both the sarcolemma and t-tubules. A and B: t = 0 shows confocal images of a basal GLUT4-EGFP–expressing muscle fiber in situ in either an AMPKα2 inactive transgenic mouse (A) or a matched control FVB mouse (B). Immediately after t = 0, AICAR was administrated intravenously via the tail vein. 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 made in fibers from five to six mice in each group. Numbers denote time in min. Bars = 20 μm. C–E: Image quantifications of GLUT4-EGFP at the sarcolemma (C), the t-tubules (D), and the intracellular vesicle depots (E). Area under curves were calculated. *Statistical difference between groups (P < 0.05). Data are means ± SE. n = 5–6.
Mentions: 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.

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