GLUT4 is the major glucose transporter in skeletal muscle. GLUT4 cycles to and from the plasma membrane and its exocytic rate is accelerated by insulin and muscle contraction to achieve a new steady state with more GLUT4 proteins at the muscle cell surface. To gain a better understanding of the molecular and cellular mechanisms that govern GLUT4 protein recycling, we developed an in vitro model in which myc-epitope-tagged GLUT4 or GLUT4-GFP is expressed in L6 skeletal muscle cells. The myc-epitope is inserted into an exofacial domain that is accessible to anti-myc antibodies from the outside of non-permeabilized cells, allowing one to count the number of transporters at the cell surface. This enables one to perform single-cell analysis using confocal fluorescence microscopy to quantify cell surface GLUT4myc or GLUT4myc-GFP in cells co-transfected with diverse cDNA constructs, treated with siRNAs, or co-stained with antibodies for other proteins of interest. Herein, we describe the methodology to perform these experimental approaches in insulin-stimulated L6 muscle cells.
Keywords: GLUT4 translocation; GLUT4-GFP; Glucose uptake; Insulin; L6 muscle cells; Skeletal muscle; Vesicle traffic.