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

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.