Glut4 is sorted from a Rab10 GTPase-independent constitutive recycling pathway into a highly insulinresponsive Rab10 GTPase-dependent sequestration pathway after adipocyte differentiation

Background: AS160 regulates insulin-sensitive glucose transport in adipocytes. Results: Knockdown of the AS160 substrate Rab10 differentially affected Glut4 and LRP1 in adipocytes and fibroblasts. Conclusion: Glut4 and LRP1 are sorted from a Rab10-independent constitutive trafficking pathway into a highly regulated Rab10-dependent pathway in adipocytes. Significance: Rab10 is a marker for the specialized insulin-sensitive pathway in adipocytes and an AS160 substrate that limits exocytosis through this pathway

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% (k en = 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 (k ex = 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 (k seq) 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. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc

Rab14 limits the sorting of Glut4 from endosomes into insulin-sensitive regulated secretory compartments in adipocytes

Insulin increases glucose uptake by increasing the rate of exocytosis of the facilitative glucose transporter isoform 4 (Glut4) relative to its endocytosis. Insulin also releases Glut4 from highly insulin-regulated secretory compartments (GSVs or Glut4 storage vesicles) into constitutively cycling endosomes. Previously it was shown that both overexpression and knockdown of the small GTP-binding protein Rab14 decreased Glut4 translocation to the plasma membrane (PM). To determine the mechanism of this perturbation, we measured the effects of Rab14 knockdown on the trafficking kinetics of Glut4 relative to two proteins that partially co-localize with Glut4, the transferrin (Tf) receptor and low-density-lipoprotein-receptorrelated protein 1 (LRP1). Our data support the hypothesis that Rab14 limits sorting of proteins from sorting (or 'early') endosomes into the specialized GSV pathway, possibly through regulation of endosomal maturation. This hypothesis is consistent with known Rab14 effectors. Interestingly, the insulin-sensitive Rab GTPase-activating protein Akt substrate of 160 kDa (AS160) affects both sorting into and exocytosis from GSVs. It has previously been shown that exocytosis of GSVs is rate-limited by Rab10, and both Rab10 and Rab14 are in vitro substrates of AS160. Regulation of both entry into and exit from GSVs by AS160 through sequential Rab substrates would provide a mechanism for the finely tuned 'quantal' increases in cycling Glut4 observed in response to increasing concentrations of insulin