Rac1 and AMPK Account for the Majority of Muscle Glucose Uptake Stimulated by Ex Vivo Contraction but Not In Vivo Exercise

Sylow, Lykke; Møller, Lisbeth L. V.; Kleinert, Maximilian; D’Hulst, Gommaar; Groote, Estelle De; Schjerling, Peter; Steinberg, Gregory R.; Jensen, Thomas E.; Richter, Erik A.

doi:10.2337/db16-1138

Cited by 51 publications

(50 citation statements)

References 55 publications

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“…Therefore, the use of rodent models of exercise and muscle contraction combined with genetic or pharmacological manipulations is essential to comprehensively explore signal transduction and mechanisms underlying the benefits of exercise. Two commonly used models to simulate the human exercise response are rodent treadmill running and in situ electrical stimulation of skeletal muscle with an intact nerve and blood supply (Gehrig et al , ; Sylow et al , ). For example, phosphoproteomic analysis of mouse skeletal muscle has been utilized to investigate the role of AMP‐activated protein kinase (AMPK) in muscle contraction‐stimulated fatty acid oxidation (Dzamko et al , ), the role of mTORC2 in treadmill exercise‐induced signal transduction (Kleinert et al , ), and signal transduction during the recovery phase following muscle contraction (Potts et al , ).…”

Section: Introductionmentioning

confidence: 99%

Phosphoproteomics reveals conserved exercise‐stimulated signaling and AMPK regulation of store‐operated calcium entry

et al. 2019

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Exercise stimulates cellular and physiological adaptations that are associated with widespread health benefits. To uncover conserved protein phosphorylation events underlying this adaptive response, we performed mass spectrometry‐based phosphoproteomic analyses of skeletal muscle from two widely used rodent models: treadmill running in mice and in situ muscle contraction in rats. We overlaid these phosphoproteomic signatures with cycling in humans to identify common cross‐species phosphosite responses, as well as unique model‐specific regulation. We identified > 22,000 phosphosites, revealing orthologous protein phosphorylation and overlapping signaling pathways regulated by exercise. This included two conserved phosphosites on stromal interaction molecule 1 (STIM1), which we validate as AMPK substrates. Furthermore, we demonstrate that AMPK‐mediated phosphorylation of STIM1 negatively regulates store‐operated calcium entry, and this is beneficial for exercise in Drosophila. This integrated cross‐species resource of exercise‐regulated signaling in human, mouse, and rat skeletal muscle has uncovered conserved networks and unraveled crosstalk between AMPK and intracellular calcium flux.

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Section: Introductionmentioning

confidence: 99%

Phosphoproteomics reveals conserved exercise‐stimulated signaling and AMPK regulation of store‐operated calcium entry

et al. 2019

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“…Exercise induces a complex array of signalling pathways (Hoffman et al, 2015), which cannot be recapitulated faithfully in reductionistic ex vivo/in vitro models (Sylow et al, 2017). To test the performance of the GLUT4 translocation assay in vivo, we subjected GLUT4 F I G U R E 2 In vivo exercise stimulated GLUT4 translocation in tibialis anterior (TA) muscle.…”

Section: In Vivo Exercise-stimulated Glut4 Translocationmentioning

confidence: 99%

Electroporated GLUT4‐7myc‐GFP detects in vivo glucose transporter 4 translocation in skeletal muscle without discernible changes in GFP patterns

Knudsen

Henríquez‐Olguín

et al. 2019

Experimental Physiology

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New Findings What is the central question of this study?Resolving the mechanism(s) leading to glucose transporter 4 (GLUT4) translocation to the muscle surface membrane has great therapeutic potential. However, the measurement of GLUT4 translocation is technically challenging. Here, we asked whether electroporation of GLUT4‐7myc‐GFP into skeletal muscle could be used as a tool to study GLUT4 translocation in vivo. What is the main finding and its importance?By acutely inducing GLUT4‐7myc‐GFP expression in skeletal muscle, we verified that in vivo exercise and AICAR stimulation increased the GLUT4 presence in the sarcolemma measured as myc signal. Importantly, the increased myc signal in the sarcolemma was not accompanied by major visual changes in the distribution of the GFP signal. Abstract Insulin and exercise lead to translocation of the glucose transporter 4 (GLUT4) to the surface membrane of skeletal muscle fibres. This process is pivotal for facilitating glucose uptake into skeletal muscle. To study this, a robust assay is needed to measure the translocation of GLUT4 in adult skeletal muscle directly. Here, we aimed to validate a simple GLUT4 translocation assay using a genetically encoded biosensor in mouse skeletal muscle. We transfected GLUT4‐7myc‐GFP into mouse muscle to study live GLUT4 movement and to evaluate GLUT4 insertion in the muscle surface membrane after in vivo running exercise and pharmacological activation of AMP‐activated protein kinase (AMPK). Transfection led to expression of GLUT4‐7myc‐GFP that was dynamic in live flexor digitorum brevis fibres and which, upon insulin stimulation, exposed the myc epitope extracellularly. Running exercise, in addition to AMPK activation by 5‐aminoimidazole‐4‐carboxamide ribonucleotide, induced ∼125 and ∼100% increase, respectively, in extracellularly exposure of GLUT4 in the surface membrane of tibialis anterior muscle. Interestingly, the clear increase in surface‐exposed GLUT4 content induced by insulin, exercise or AMPK activation was not accompanied by any discernible reorganization of the GLUT4‐GFP signal. In conclusion, we provide a detailed description of an easy‐to‐use translocation assay to study GLUT4 accumulation at the surface membrane induced by exercise and exercise‐mimicking stimuli. Notably, our analyses revealed that increased GLUT4 surface membrane accumulation was not accompanied by a discernible change in the GLUT4 localization pattern.

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“…Hypoxia and contractile activity also activate AMPK. In addition to the potential requirement for AMPK activity, normal regulation of contraction-and exercise-stimulated glucose uptake also requires the Rho GTPase Rac1 [20,21], which is activated by insulin and which induces actin cytoskeleton remodeling at the plasma membrane in skeletal muscle cells [22]. Rac1 mediates this process by inducing cortical F-actin remodeling, which involves the recruitment of actin regulatory proteins such as cofilin and Arp2/3 to the actin filaments [23].…”

Section: Introductionmentioning

confidence: 99%

The TRPC6-AMPK Pathway is Involved in Insulin-Dependent Cytoskeleton Reorganization and Glucose Uptake in Cultured Rat Podocytes

Rachubik¹,

Szrejder²,

Rogacka³

et al. 2018

Cell Physiol Biochem

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Background/Aims: Podocytes are dynamic polarized cells on the surface of glomerular capillaries that are an essential part of the glomerular filtration barrier. AMP-activated protein kinase (AMPK), a key regulator of glucose and fatty acid metabolism, plays a major role in obesity and type 2 diabetes. Accumulating evidence suggests that TRPC6 channels are crucial mediators of calcium transport in podocytes and are involved in regulating glomerular filtration. Here we investigated whether the AMPK-TRPC6 pathway is involved in insulin-dependent cytoskeleton reorganization and glucose uptake in cultured rat podocytes. Methods: Western blot and immunofluorescence analysis confirmed AMPKα and TRPC6 expression, the phosphorylation of proteins associated with actin cytoskeleton reorganization (PAK, rac1, and cofilin), and the expression of insulin signaling proteins (Akt, Insulin receptor). Coimmunoprecipitation and immunofluorescence results demonstrated AMPKα/TRPC6 interaction. To ask whether TRPC6 is involved in the insulin regulation of glucose transport, we measured insulin-dependent (1, 2-³H)-deoxy-D-glucose uptake into podocytes after reducing TRPC6 activity pharmacologically and biochemically (TRPC6 siRNA). Results: The results suggested a key role for the TRPC6 channel in the mediation of insulin-dependent activation of AMPKα2 and glucose uptake. Moreover, AMPK and TRPC6 activation were required to stimulate the Rac1 signaling pathway. Conclusion: These results suggest a potentially important new mechanism that regulates glucose transport in podocytes and that could be injurious during diabetes.

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Rac1 and AMPK Account for the Majority of Muscle Glucose Uptake Stimulated by Ex Vivo Contraction but Not In Vivo Exercise

Cited by 51 publications

References 55 publications

Phosphoproteomics reveals conserved exercise‐stimulated signaling and AMPK regulation of store‐operated calcium entry

Phosphoproteomics reveals conserved exercise‐stimulated signaling and AMPK regulation of store‐operated calcium entry

Electroporated GLUT4‐7myc‐GFP detects in vivo glucose transporter 4 translocation in skeletal muscle without discernible changes in GFP patterns

The TRPC6-AMPK Pathway is Involved in Insulin-Dependent Cytoskeleton Reorganization and Glucose Uptake in Cultured Rat Podocytes

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