Abstract
We report a rapid and sensitive colorimetric approach to quantitate the amount of glucose transporters exposed at the surface of intact cells, using L6 muscle cells expressing GLUT4 containing an exofacial myc epitope. Unstimulated cells exposed to the surface 5 fmol GLUT4myc per mg protein. This value increased to 10 fmol/mg protein in response to insulin as 2-deoxyglucose (10 microM) uptake doubled. The results are substantiated by immunofluorescent detection of GLUT4myc in unpermeabilized cells and by subcellular fractionation. We further show that wortmannin and the cytoskeleton disruptors cytochalasin D and latrunculin B completely blocked these insulin effects. The rapid quantitative assay described here could be of high value to study insulin signals and to screen for potential anti-diabetic drugs.
Publication types
-
Research Support, Non-U.S. Gov't
MeSH terms
-
Androstadienes / pharmacology
-
Animals
-
Biological Transport
-
Bridged Bicyclo Compounds, Heterocyclic / pharmacology
-
Cell Fractionation
-
Cell Line
-
Colorimetry / methods
-
Cytochalasin D / pharmacology
-
Deoxyglucose / metabolism
-
Enzyme Inhibitors / pharmacology
-
Glucose Transporter Type 4
-
Immunoenzyme Techniques*
-
Insulin / pharmacology*
-
Monosaccharide Transport Proteins / analysis*
-
Muscle Proteins*
-
Muscle, Skeletal / chemistry*
-
Muscle, Skeletal / cytology
-
Muscle, Skeletal / metabolism
-
Nucleic Acid Synthesis Inhibitors / pharmacology
-
Phosphoinositide-3 Kinase Inhibitors
-
Recombinant Fusion Proteins / metabolism
-
Thiazoles / pharmacology
-
Thiazolidines
-
Wortmannin
Substances
-
Androstadienes
-
Bridged Bicyclo Compounds, Heterocyclic
-
Enzyme Inhibitors
-
Glucose Transporter Type 4
-
Insulin
-
Monosaccharide Transport Proteins
-
Muscle Proteins
-
Nucleic Acid Synthesis Inhibitors
-
Phosphoinositide-3 Kinase Inhibitors
-
Recombinant Fusion Proteins
-
Thiazoles
-
Thiazolidines
-
Cytochalasin D
-
Deoxyglucose
-
latrunculin B
-
Wortmannin