Insulin-regulated Glut4 translocation: membrane protein trafficking with six distinctive steps.
The trafficking kinetics of Glut4, the transferrin (Tf) receptor, and LRP1 were quantified in adipocytes and undifferentiated fibroblasts. Six steps were identified that determine steady state cell surface Glut4: (i) endocytosis, (ii) degradation, (iii) sorting, (iv) sequestration, (v) release, and (vi) tethering/docking/fusion. Endocytosis of Glut4 is 3 times slower than the Tf receptor in fibroblasts (ken = 0.2 min(-1) versus 0.6 min(-1)). Differentiation decreases Glut4 ken 40% (ken = 0.12 min(-1)). Differentiation also decreases Glut4 degradation, increasing total and cell surface Glut4 3-fold. In fibroblasts, Glut4 is recycled from endosomes through a slow constitutive pathway (kex = 0.025-0.038 min(-1)), not through the fast Tf receptor pathway (kex = 0.2 min(-1)). The kex measured in adipocytes after insulin stimulation is similar (kex = 0.027 min(-1)). Differentiation decreases the rate constant for sorting into the Glut4 recycling pathway (ksort) 3-fold. In adipocytes, Glut4 is also sorted from endosomes into a second exocytic pathway through Glut4 storage vesicles (GSVs). Surprisingly, transfer from endosomes into GSVs is highly regulated; insulin increases the rate constant for sequestration (kseq) 8-fold. Release from sequestration in GSVs is rate-limiting for Glut4 exocytosis in basal adipocytes. AS160 regulates this step. Tethering/docking/fusion of GSVs to the plasma membrane is regulated through an AS160-independent process. Insulin increases the rate of release and fusion of GSVs (kfuseG) 40-fold. LRP1 cycles with the Tf receptor and Glut4 in fibroblasts but predominantly with Glut4 after differentiation. Surprisingly, AS160 knockdown accelerated LRP1 exocytosis in basal and insulin-stimulated adipocytes. These data indicate that AS160 may regulate trafficking into as well as release from GSVs.
From the Department of Biochemistry and Molecular Biology, University of Nevada School of Medicine, Reno, Nevada 89557.
Paul Duffield
PD
Brewer
the Section of Endocrinology and Metabolism, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520, and.
Estifanos N
EN
Habtemichael
From the Department of Biochemistry and Molecular Biology, University of Nevada School of Medicine, Reno, Nevada 89557.
Irina
I
Romenskaia
From the Department of Biochemistry and Molecular Biology, University of Nevada School of Medicine, Reno, Nevada 89557, cmastick@unr.edu.
Cynthia Corley
CC
Mastick
the School of Mathematics and Statistics, University of New South Wales, Sydney, New South Wales 2052, Australia.
Adelle C F
AC
Coster
D041721:3T3-L1 Cells; D017667:Adipocytes / Q000166:cytology / Q000378:metabolism*; D000818:Animals; D002454:Cell Differentiation / Q000502:physiology*; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D011992:Endosomes / Q000235:genetics / Q000378:metabolism; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism; D055785:Gene Knockdown Techniques; D051275:Glucose Transporter Type 4 / Q000235:genetics / Q000378:metabolism*; D007004:Hypoglycemic Agents / Q000378:metabolism / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism* / Q000494:pharmacology; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D051379:Mice; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D011973:Receptors, LDL / Q000235:genetics / Q000378:metabolism; D011990:Receptors, Transferrin; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism
D020690:GTPase-Activating Proteins; D051275:Glucose Transporter Type 4; D007004:Hypoglycemic Agents; D007328:Insulin; D026503:Low Density Lipoprotein Receptor-Related Protein-1; C486645:Lrp1 protein, mouse; D011973:Receptors, LDL; D011990:Receptors, Transferrin; C495693:Slc2a4 protein, mouse; C505647:Tbc1d4 protein, mouse; D025521:Tumor Suppressor Proteins
Adipocyte; Endocytosis; Exocytosis; Fibroblast; Glucose; Glucose Transport; Glucose Transporter Type 4 (Glut4); Insulin; Transferrin Receptor