title: Separation of IRS-1 and PI3-kinase from GLUT4 vesicles in rat skeletal muscle.
abstract: In fat and muscle tissues, insulin stimulates cellular glucose uptake by initiating a phosphorylation cascade which ultimately results in the translocation of the GLUT4 glucose transporter isoform from an intracellular vesicular storage pool(s) to the plasma membrane in fat and to t-tubules in skeletal muscle. Insulin receptor substrate-1 (IRS-1) and phosphatidylinositol 3-kinase (PI3-kinase) are known to be involved in cellular responses to insulin such as GLUT4 translocation, but the biochemical mechanism(s) connecting IRS-1 and PI3-kinase to GLUT4-containing intracellular membranes remains unclear. Here, in control and insulin-stimulated rat skeletal muscle, the intracellular localization of these two proteins was compared to that of GLUT4 using subcellular fractionation by sucrose velocity gradients followed by immunoblotting. Our data show that insulin-sensitive GLUT4-containing vesicles are present in fractions 1 through 10, whereas IRS-1 and PI3-kinase are found in fractions 16 through 24. These results indicate that in intracellular fractions derived from skeletal muscle, IRS-1 and PI3-kinase are excluded from membranes harboring GLUT4.
authors.affiliation: Department of Biochemistry, Boston University School of Medicine, Massachusetts 02118, USA.
authors.forename: J L
authors.initials: JL
authors.lastname: Kummer
authors.forename: M R
authors.initials: MR
authors.lastname: Calera
authors.forename: P F
authors.initials: PF
authors.lastname: Pilch
mesh_terms: D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D004789:Enzyme Activation; D005947:Glucose / Q000378:metabolism; D051275:Glucose Transporter Type 4; D015151:Immunoblotting; D007328:Insulin / Q000378:metabolism / Q000494:pharmacology; D055504:Insulin Receptor Substrate Proteins; D008297:Male; D009004:Monosaccharide Transport Proteins / Q000302:isolation & purification* / Q000378:metabolism; D009124:Muscle Proteins; D018482:Muscle, Skeletal / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000302:isolation & purification* / Q000378:metabolism; D010750:Phosphoproteins / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011972:Receptor, Insulin / Q000302:isolation & purification / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D051275:Glucose Transporter Type 4; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; C526296:Irs1 protein, rat; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D010750:Phosphoproteins; C495694:Slc2a4 protein, rat; D019869:Phosphatidylinositol 3-Kinases; D011972:Receptor, Insulin; D005947:Glucose