Electroporated GLUT4-7myc-GFP detects in vivo glucose transporter 4 translocation in skeletal muscle without discernible changes in GFP patterns
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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 to directly measure the translocation of GLUT4 in adult skeletal muscle is needed. 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 following in vivo running exercise and pharmacological activation of AMP activated protein kinase (AMPK). Transfection led to expression of GLUT4-7myc-GFP that were dynamic in live flexor digitorum brevis fibres and which, upon insulin stimulation, exposed the myc-epitope extracellularly. Running exercise, as well as AMPK-activation by 5-Aminoimidazole-4-carboxamide ribonucleotide, induced ∼125% and ∼100% increase in extracellularly exposure of GLUT4 in the surface membrane of tibialis anterior muscle. Interestingly, the clear increase in surface-exposed GLUT4 content 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 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.
|Status||Udgivet - 2019|
CURIS 2019 NEXS 098
- Det Natur- og Biovidenskabelige Fakultet