title: GLUT4 is internalized by a cholesterol-dependent nystatin-sensitive mechanism inhibited by insulin.
abstract: Insulin slows GLUT4 internalization by an unknown mechanism. Here we show that in unstimulated adipocytes, GLUT4 is internalized by two mechanisms. Approximately 80% of GLUT4 is internalized by a mechanism that is sensitive to the cholesterol-aggregating drug nystatin, and is independent of AP-2 clathrin adaptor and two putative GLUT4 endocytic motifs. The remaining GLUT4 is internalized by an AP-2-dependent, nystatin-resistant pathway that requires the FQQI GLUT4 motif. Insulin inhibits GLUT4 uptake by the nystatin-sensitive pathway and, consequently, GLUT4 is internalized by the AP-2-dependent pathway in stimulated adipocytes. The phenylalanine-based FQQI GLUT4 motif promotes AP-2-dependent internalization less rapidly than a tyrosine-based motif, the classic form of aromatic-based motifs. Thus, both a change in the predominant endocytosis pathway and the specific use of a suboptimal internalization motif contribute to the slowing of GLUT4 internalization in insulin-stimulated adipocytes. Insulin also inhibits the uptake of cholera-toxin B, indicating that insulin broadly regulates cholesterol-dependent uptake mechanisms rather than specially targeting GLUT4. Our work thus identifies cholesterol-dependent uptake as a novel target of insulin action in adipocytes.
authors.affiliation: Department of Biochemistry, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA.
authors.forename: Vincent
authors.initials: V
authors.lastname: Blot
authors.forename: Timothy E
authors.initials: TE
authors.lastname: McGraw
mesh_terms: D041721:3T3-L1 Cells; D033962:Adaptor Protein Complex 2 / Q000378:metabolism; D017667:Adipocytes / Q000166:cytology / Q000187:drug effects; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002784:Cholesterol / Q000378:metabolism*; D004705:Endocytosis / Q000187:drug effects*; D051275:Glucose Transporter Type 4 / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D051379:Mice; D008954:Models, Biological; D008969:Molecular Sequence Data; D016296:Mutagenesis; D050505:Mutant Proteins / Q000378:metabolism; D009761:Nystatin / Q000494:pharmacology*; D021381:Protein Transport / Q000187:drug effects; D014168:Transferrin / Q000378:metabolism; D022161:Transport Vesicles / Q000187:drug effects
chemical_list: D033962:Adaptor Protein Complex 2; D051275:Glucose Transporter Type 4; D007328:Insulin; D050505:Mutant Proteins; D014168:Transferrin; D009761:Nystatin; D002784:Cholesterol