Detection of insulin-regulated GLUT4-translocation by the insertion of a protein C epitope in L6 myoblasts.

Abstract Insulin stimulates glucose transport by translocation of the membrane glucose transporter GLUT4 from intracellular vesicles to the plasma membrane. GLUT4 is highly expressed in adipose tissue and skeletal muscle. We have constructed a cDNA containing the human GLUT4 inserted by a 12 amino acid protein C epitope in the first extracellular (exofacial) domain of the human GLUT4 (GLUT4-PC). Stable expression of GLUT4-PC in L6 myoblasts (L6-GLUT4-PC) was confirmed in immunofluorescence using monoclonal antibodies against protein C. The protein C staining yielded labeling in perinuclear vesicles strongly co-localizing with GLUT4 detected with antibodies directed against the endofacial part of GLUT4. The L6-GLUT4-PC cells were further characterized in a direct cell-based enzyme-linked immunosorbent assay by the use of β-galactosidase. Cell surface binding of monoclonal protein C antibodies was detected with β-galactosidase-conjugated secondary antibodies and chlorophenolred-β- d -galactopyranoside (CPRG) as substrate in 2% paraformaldehyde fixed cells. In this assay, stimulation with insulin created a rapidly detectable recruitment of GLUT4-PC to the cell surface. This cell-based enzyme-linked immunosorbent GLUT4 assay was shown to be comparable with that of previously reported radioactive assays.