Measuring GLUT4 translocation in mature muscle fibers

Lauritzen, Hans P.M.M.; Schertzer, Jonathan D.

doi:10.1152/ajpendo.00066.2010

Cited by 35 publications

(37 citation statements)

References 64 publications

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“…It is now well established that insulin stimulates glucose transport in adipose and muscle cells through the translocation of glucose transporter 4 (GLUT4) from intracellular sites to the plasma membrane (5,13,15). However, whereas the molecular mechanism of GLUT4 translocation has been extensively studied in primary adipose cells and cultured adipocytes during the past years, relatively few studies have focused on GLUT4 trafficking in primary skeletal muscle cells (18,20). In part, this is due to the presence of abundant microfibrillar protein and large amounts of nuclei such that most fractionation protocols suffer from poor resolution for the analysis of the subcellular distribution of GLUT4 in skeletal muscle.…”

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confidence: 99%

Insulin stimulates fusion, but not tethering, of GLUT4 vesicles in skeletal muscle of HA-GLUT4-GFP transgenic mice

Lizunov

Stenkula

Lisinski

et al. 2012

American Journal of Physiology-Endocrinology and Metabolism

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Insulin regulates glucose uptake into fat and muscle by modulating the subcellular distribution of GLUT4 between the cell surface and intracellular compartments. However, quantification of these translocation processes in muscle by classical subcellular fractionation techniques is confounded by contaminating microfibrillar protein; dynamic studies at the molecular level are almost impossible. In this study, we introduce a musclespecific transgenic mouse model in which HA-GLUT4-GFP is expressed under the control of the MCK promoter. HA-GLUT4-GFP was found to translocate to the plasma membrane and T-tubules after insulin stimulation, thus mimicking endogenous GLUT4. To investigate the dynamics of GLUT4 trafficking in skeletal muscle, we quantified vesicles containing HA-GLUT4-GFP near the sarcolemma and T-tubules and analyzed insulin-stimulated exocytosis at the single vesicle level by total internal reflection fluorescence and confocal microscopy. We found that only 10% of the intracellular GLUT4 pool comprised mobile vesicles, whereas most of the GLUT4 structures remained stationary or tethered at the sarcolemma or T-tubules. In fact, most of the insulin-stimulated exocytosis emanated from pretethered vesicles, whereas the small pool of mobile GLUT4 vesicles was not significantly affected by insulin. Our data strongly suggest that the mobile pool of GLUT4 vesicles is not a major site of insulin action but rather locally distributed. Most likely, pretethered GLUT4 structures are responsible for the initial phase of insulin-stimulated exocytosis.hemagglutinin; glucose transporter 4; green fluorescent protein; insulin; fusion MUSCLE, ESPECIALLY SKELETAL MUSCLE, is a major direct contributor to mammalian systemic glucose homeostasis (1, 10). It is now well established that insulin stimulates glucose transport in adipose and muscle cells through the translocation of glucose transporter 4 (GLUT4) from intracellular sites to the plasma membrane (5, 13, 15). However, whereas the molecular mechanism of GLUT4 translocation has been extensively studied in primary adipose cells and cultured adipocytes during the past years, relatively few studies have focused on GLUT4 trafficking in primary skeletal muscle cells (18,20). In part, this is due to the presence of abundant microfibrillar protein and large amounts of nuclei such that most fractionation protocols suffer from poor resolution for the analysis of the subcellular distribution of GLUT4 in skeletal muscle. Likewise, because of technical limitations, morphological analyses of GLUT4 in skeletal muscle by photolabeling techniques, immunofluorescence, and electron microscopy have not provided sufficient information about the kinetics and dynamics of GLUT4 recycling through the multiple intracellular compartments.Recent alternative approaches involving ectopic expression of tagged glucose transporters in culture cells offer novel opportunities for the molecular analysis of GLUT4 translocation (9,11,35). In these studies, recombinant GLUT4 reporters carrying extracellular ep...

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confidence: 99%

Insulin stimulates fusion, but not tethering, of GLUT4 vesicles in skeletal muscle of HA-GLUT4-GFP transgenic mice

Lizunov

Stenkula

Lisinski

et al. 2012

American Journal of Physiology-Endocrinology and Metabolism

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“…Reduced energy turnover may be exacerbated by reduced insulin sensitivity in COPD [6]; insulin driving cellular glucose uptake via the GLUT-4 receptor [7]. Understanding mechanisms underlying these changes may lead to treatments to improve exercise capacity in patients with COPD.…”

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confidence: 99%

Pathways associated with reduced quadriceps oxidative fibres and endurance in COPD

et al. 2012

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Reduced quadriceps endurance in chronic obstructive pulmonary disease (COPD) is associated with a predominance of type II glycolytic fibres over type I oxidative fibres (fibre shift) and reduced muscle energy stores. The molecular mechanisms responsible for this remain unknown. We hypothesised that expression of known regulators of type I fibres and energy production in quadriceps muscle would differ in COPD patients with and without fibre shift.We measured lung function, physical activity, exercise performance, quadriceps strength and endurance (nonvolitionally) in 38 Global Initiative for Chronic Obstructive Lung Disease stage I-IV COPD patients and 23 healthy age-matched controls. Participants underwent a quadriceps biopsy: type I and II fibre proportions were determined using immunohistochemistry and fibre shift defined using published reference ranges. Calcineurin A, phosphorylated AMP kinase (phospho-AMPK)-a, protein kinase A-a catalytic subunits, modulators of calcineurin activity and calmodulin, 14-3-3 proteins were measured by Western blotting, and myocyte-enriched calcineurin-interacting protein-1 mRNA measured by quantitative PCR. Downstream, nuclear myocyte enhancer factor-2 capable of DNA binding was quantified by transcription factor ELISA.Unexpectedly, calcineurin expression was higher, while phospho-AMPK was lower, in COPD patients with fibre shift compared to COPD patients without fibre shift. Phospho-AMPK levels correlated with quadriceps endurance in patients.Reduced phospho-AMPK may contribute to reduced quadriceps oxidative capacity and endurance in COPD.KEYWORDS: AMP kinase, calcineurin, myocyte enhancer factor-2, protein kinase A R educed quadriceps endurance is associated with exercise limitation in chronic obstructive pulmonary disease (COPD) [1]. Underlying the loss of endurance is a reduction in type I myosin and oxidative enzymes [2], the hallmarks of the reduced oxidative type I to glycolytic type II fibre ratio observed in the quadriceps of COPD patients, which we will refer to in this study as the presence of fibre shift [3]. Type I fibres rely exclusively on oxidative metabolism to generate ATP, type IIx fibres depend on glycolysis, and type IIa fibres utilise both oxidative and glycolytic metabolism [4]. Since oxidative metabolism generates several times more ATP than glycolysis per molecule of glucose [5], type I fibres are fatigue-resistant compared to type II fibres.COPD patients, with their fewer oxidative fibres, have fewer muscle energy stores and exhibit metabolic stress from ATP depletion at rest and at low workloads, unlike controls. Reduced energy turnover may be exacerbated by reduced insulin sensitivity in COPD [6]; insulin driving cellular glucose uptake via the GLUT-4 receptor [7]. Understanding mechanisms underlying these changes may lead to treatments to improve exercise capacity in patients with COPD.Pathways influencing muscle type I fibre differentiation during development, muscle oxidative enzymes and energy production/glucose uptake have been descri...

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“…We expressed IL-6-EGFP in the superficial portion of the quadriceps muscle in living mice using gene gun transfection (26–28,31–33). Five days later, mice were anesthetized and the transfected fibers were subjected to intravital imaging (34).…”

Section: Resultsmentioning

confidence: 99%

Contraction and AICAR Stimulate IL-6 Vesicle Depletion From Skeletal Muscle Fibers In Vivo

et al. 2013

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Recent studies suggest that interleukin 6 (IL-6) is released from contracting skeletal muscles; however, the cellular origin, secretion kinetics, and signaling mechanisms regulating IL-6 secretion are unknown. To address these questions, we developed imaging methodology to study IL-6 in fixed mouse muscle fibers and in live animals in vivo. Using confocal imaging to visualize endogenous IL-6 protein in fixed muscle fibers, we found IL-6 in small vesicle structures distributed throughout the fibers under basal (resting) conditions. To determine the kinetics of IL-6 secretion, intact quadriceps muscles were transfected with enhanced green fluorescent protein (EGFP)-tagged IL-6 (IL-6-EGFP), and 5 days later anesthetized mice were imaged before and after muscle contractions in situ. Contractions decreased IL-6-EGFP–containing vesicles and protein by 62% (P < 0.05), occurring rapidly and progressively over 25 min of contraction. However, contraction-mediated IL-6-EGFP reduction was normal in muscle-specific AMP-activated protein kinase (AMPK) α2-inactive transgenic mice. In contrast, the AMPK activator AICAR decreased IL-6-EGFP vesicles, an effect that was inhibited in the transgenic mice. In conclusion, resting skeletal muscles contain IL-6–positive vesicles that are expressed throughout myofibers. Contractions stimulate the rapid reduction of IL-6 in myofibers, occurring through an AMPKα2-independent mechanism. This novel imaging methodology clearly establishes IL-6 as a contraction-stimulated myokine and can be used to characterize the secretion kinetics of other putative myokines.

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Measuring GLUT4 translocation in mature muscle fibers

Cited by 35 publications

References 64 publications

Insulin stimulates fusion, but not tethering, of GLUT4 vesicles in skeletal muscle of HA-GLUT4-GFP transgenic mice

Insulin stimulates fusion, but not tethering, of GLUT4 vesicles in skeletal muscle of HA-GLUT4-GFP transgenic mice

Pathways associated with reduced quadriceps oxidative fibres and endurance in COPD

Contraction and AICAR Stimulate IL-6 Vesicle Depletion From Skeletal Muscle Fibers In Vivo

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