title: Insulin induces the translocation of GLUT4 from a unique intracellular organelle to transverse tubules in rat skeletal muscle.
abstract: Skeletal muscle surface membrane is constituted by the PM domain and its specialized deep invaginations known as TTs. We have shown previously that insulin induces a rapid translocation of GLUT4s from an IM pool to the PM in rat skeletal muscle (6). In this study, we have investigated the possibility that insulin also stimulates the translocation of GLUT4 proteins to TTs, which constitute the largest area of the cell surface envelope. PM, TTs, and IM components of control and insulinized skeletal muscle were isolated by subcellular fractionation. The TTs then were purified further by removing vesicles of SR origin by using a Ca-loading procedure. Ca-loading resulted in a five- to sevenfold increase in the purification of TTs in the unloaded fraction relative to the loaded fraction, assessed by immunoblotting with an anti-DHP-receptor monoclonal antibody. In contrast, estimation of the content of Ca(2+)-ATPase protein (a marker of SR) with a specific polyclonal antibody revealed that most, if not all, SR vesicles were recovered in the Ca-loaded fraction. Western blotting with an anti-COOH-terminal GLUT4 protein polyclonal antibody revealed that acute insulin injection in vivo (30 min) increased the content of GLUT4 (by 90%) in isolated PMs and markedly enhanced (by 180%) GLUT4 content in purified TTs. Importantly, these insulin-dependent changes in GLUT4 content of PM and purified TTs were seen in the absence of changes in the alpha 1-subunit of the Na(+)-K(+)-ATPase, a surface membrane marker.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Division of Cell Biology, Hospital for Sick Children, Toronto, Ontario, Canada.
authors.forename: A
authors.initials: A
authors.lastname: Marette
authors.forename: E
authors.initials: E
authors.lastname: Burdett
authors.forename: A
authors.initials: A
authors.lastname: Douen
authors.forename: M
authors.initials: M
authors.lastname: Vranic
authors.forename: A
authors.initials: A
authors.lastname: Klip
mesh_terms: D000818:Animals; D000252:Calcium-Transporting ATPases / Q000302:isolation & purification / Q000378:metabolism; D002458:Cell Fractionation; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D007328:Insulin / Q000494:pharmacology*; D008297:Male; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins / Q000302:isolation & purification / Q000378:metabolism*; D009132:Muscles / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D015388:Organelles / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011950:Receptors, Cholinergic / Q000302:isolation & purification / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel
chemical_list: D007328:Insulin; D009004:Monosaccharide Transport Proteins; D011506:Proteins; D011950:Receptors, Cholinergic; D019837:Ryanodine Receptor Calcium Release Channel; D000252:Calcium-Transporting ATPases