L6 Myotubes or myoblasts were detached using trypsin/EDTA, transferred to Eppendorf tubes and centrifuged at 1000rpm for 3 min, resuspended in 20 µl SE Cell Line Nucleofector solution for L6 cells and P1 primary Nucleofector solution for SKMC cells (Lonza, Basel, Swiss) and 100 pmol of siRNA (L6 myotubes) or 0.8 ?g GLUT4mycGFP construct (L6 myoblasts and human SKMC) added. L6 cells were nucleofected by using the DS-137 program and SKMC cells with the DS-138 program in the Lonza 4D nucleofector. Cells were electroporated in 16-well microcuvette stripes (Lonza), pre-warmed RPMI1640 added (80µl), and then transferred to 1.5 ml microcentrifuge tubes or glass bottom slides containing DMEM with 10% FBS for 8h.
Summary of Content: Type 2 diabetes is an increasing worldwide epidemic that poses major health problems. In this paper, the authors describe for the first time that specific activation of ß2-adrenoceptors in skeletal muscle, but not white adipocytes, causes translocation of GLUT4 to increase glucose uptake, by a mechanism dependent on mTORC2. To confirm the involvement of GLUT4, siRNA directed against GLUT4 was nucleofected into L6 myotubes. This caused a markedly reduced ß -adrenoreceptor mediated glucose uptake compared to a scrambled siRNA. Direct demonstration of GLUT4 translocation following ß2-adrenoceptor-stimulated glucose uptake has been shown by transient and stable nucleofection of L6 myotubes and transient nucleofection of human primary skeletal muscle cells with different GLUT4 reporter constructs.