Insulin-regulated cellular glucose uptake changes the amount of glucose transporter-4 (GLUT4) in the plasma membrane (PM), primarily regulated by the tethering and fusion of GLUT4-containing vesicles to PM; however, the molecular mechanisms remain unknown. Here we utilized time-lapse total internal reflection fluorescence microscopy (TIRFM) to test the involvement of the exocyst complex in these processes in living mature rat adipose cells, as the exocyst complex is important for the exocytosis of GLUT4 in 3T3-L1 developing adipocytes. We compared the mobility of GLUT4 vesicles in cells expressing the wild-type Exo70 subunit of the exocyst complex or a dominant-negative mutant Exo70-N. Cells expressing the Exo70 mutant exhibited a bimodal distribution of vesicle mobility. However, inhibition of GLUT4 vesicle mobility with Exo70-N mutant did not affect the insulin-stimulated fusion of these immobilized vesicles as indicated by exposure of GLUT4 on PM: insulin augments the amount of PM GLUT4 in response to insulin in Exo70-N mutant cells by the same extent as in wild-type cells. Exo70 was reported to play an essential role in the translocation of GLUT4 in 3T3-L1 adipocytes, which are fibroblasts stimulated to develop into adipocytes. In mature adipose cells, Exo70 is not required for the successful fusion of GLUT4-containing vesicles to PM, and both exocyst and GLUT4 translocate to the PM after insulin stimulation. Thus in these mature rat adipose cells, the exocyst complex is not important in GLUT4 vesicle trafficking and its disruption does not abolish insulin-dependent GLUT4 translocation to the PM. We suggest a developmental role of the exocyst complex to set up sites for GLUT4 vesicle exocytosis? ? Tankyrase is a poly(ADP-ribose) polymerase which was described as a telomere-associated protein that was suggested to regulate telomere length. However, in 3T3-L1 adipocytes, tankyrase is found on peripheral membranes, is associated with the Golgi, and colocalizes with GLUT4 storage vesicles. It has been shown that tankyrase binds insulin-responsive amino peptidase (IRAP), a GLUT4 vesicle-associated protein. Recently, it was reported that genetic inactivation of tankyrase 2 (Tnks 2) had no effect on telomere length but that body weight was decreased in Tank 2 knockout mice, suggesting that tankyrase might have telomere-independent roles in pathways such as those involved in glucose metabolism and/or glucose homeostasis. In the present study, we utilized the Tnks 2 KO mouse model to test this hypothesis in vivo and in vitro. Our results show that Tnks 2 deficiency increases glucose uptake in white adipose cells (WAT) without causing a major change in the whole body glucose metabolism.

Project Start
Project End
Budget Start
Budget End
Support Year
1
Fiscal Year
2007
Total Cost
$317,250
Indirect Cost
City
State
Country
United States
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Lizunov, Vladimir A; Lisinski, Ivonne; Stenkula, Karin et al. (2009) Insulin regulates fusion of GLUT4 vesicles independent of Exo70-mediated tethering. J Biol Chem 284:7914-9