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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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AMPK activity does not affect GLUT4-EGFP basal localization and translocation and re-internalization kinetics in response to in situ contractions. A and B: t = 0 shows confocal images of basal GLUT4-EGFP–expressing muscle fibers immediately before in situ contractions in either a muscle-specific AMPKα2 inactive transgenic mouse (A) or a matched control mouse (B). Contractions were elicited using the high-voltage protocol for 2 × 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 fibers by diagonal arrows. Similar observations were made in fibers from five mice in each group. Numbers denote time in min during (t =) and after (t = +) contractions. Bars = 20 μm. C–E: Image quantifications of GLUT4-EGFP at the sarcolemma (C), the t-tubules (D), and the intracellular vesicle depots (E). Horizontal solid lines indicate contraction period. Horizontal dotted lines indicate the period after contractions. Data are means ± SE. n = 5.
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Figure 3: AMPK activity does not affect GLUT4-EGFP basal localization and translocation and re-internalization kinetics in response to in situ contractions. A and B: t = 0 shows confocal images of basal GLUT4-EGFP–expressing muscle fibers immediately before in situ contractions in either a muscle-specific AMPKα2 inactive transgenic mouse (A) or a matched control mouse (B). Contractions were elicited using the high-voltage protocol for 2 × 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 fibers by diagonal arrows. Similar observations were made in fibers from five mice in each group. Numbers denote time in min during (t =) and after (t = +) contractions. Bars = 20 μm. C–E: Image quantifications of GLUT4-EGFP at the sarcolemma (C), the t-tubules (D), and the intracellular vesicle depots (E). Horizontal solid lines indicate contraction period. Horizontal dotted lines indicate the period after contractions. Data are means ± SE. n = 5.

Mentions: 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.


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

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

AMPK activity does not affect GLUT4-EGFP basal localization and translocation and re-internalization kinetics in response to in situ contractions. A and B: t = 0 shows confocal images of basal GLUT4-EGFP–expressing muscle fibers immediately before in situ contractions in either a muscle-specific AMPKα2 inactive transgenic mouse (A) or a matched control mouse (B). Contractions were elicited using the high-voltage protocol for 2 × 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 fibers by diagonal arrows. Similar observations were made in fibers from five mice in each group. Numbers denote time in min during (t =) and after (t = +) contractions. Bars = 20 μm. C–E: Image quantifications of GLUT4-EGFP at the sarcolemma (C), the t-tubules (D), and the intracellular vesicle depots (E). Horizontal solid lines indicate contraction period. Horizontal dotted lines indicate the period after contractions. Data are means ± SE. n = 5.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 3: AMPK activity does not affect GLUT4-EGFP basal localization and translocation and re-internalization kinetics in response to in situ contractions. A and B: t = 0 shows confocal images of basal GLUT4-EGFP–expressing muscle fibers immediately before in situ contractions in either a muscle-specific AMPKα2 inactive transgenic mouse (A) or a matched control mouse (B). Contractions were elicited using the high-voltage protocol for 2 × 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 fibers by diagonal arrows. Similar observations were made in fibers from five mice in each group. Numbers denote time in min during (t =) and after (t = +) contractions. Bars = 20 μm. C–E: Image quantifications of GLUT4-EGFP at the sarcolemma (C), the t-tubules (D), and the intracellular vesicle depots (E). Horizontal solid lines indicate contraction period. Horizontal dotted lines indicate the period after contractions. Data are means ± SE. n = 5.
Mentions: 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.

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