A potential link between insulin signaling and GLUT4 translocation: Association of Rab10-GTP with the exocyst subunit Exoc6/6b

Abstract

Insulin increases glucose transport in fat and muscle cells by stimulating the exocytosis of specialized vesicles containing the glucose transporter GLUT4. This process, which is referred to as GLUT4 translocation, increases the amount of GLUT4 at the cell surface. Previous studies have provided evidence that insulin signaling increases the amount of Rab10-GTP in the GLUT4 vesicles and that GLUT4 translocation requires the exocyst, a complex that functions in the tethering of vesicles to the plasma membrane, leading to exocytosis. In the present study we show that Rab10 in its GTP form binds to Exoc6 and Exoc6b, which are the two highly homologous isotypes of an exocyst subunit, that both isotypes are found in 3T3-L1 adipocytes, and that knockdown of Exoc6, Exoc6b, or both inhibits GLUT4 translocation in 3T3-L1 adipocytes. These results suggest that the association of Rab10-GTP with Exoc6/6b is a molecular link between insulin signaling and the exocytic machinery in GLUT4 translocation.

Keywords: Exocyst; GLUT4; Insulin; Rab10.

Fig 1

Association of Rab10 with Exoc 6 and 6b. Growth on selective medium of several dilutions (10⁻¹ to 10⁻⁴) of the various diploid yeast cells, from the same number of cells in the stocks, in the absence and presence of 3-aminotriazole (3AT) is shown. Part A. The diploid cells (lanes 1 – 7) contained the following plasmid pairs: 1, empty bait vector and Gal activation domain (GAD)-Exoc6 carboxy terminus (CT); 2, empty bait vector and GAD-Exoc6b CT; 3, LexA-Rab10 Q68L and empty prey vector; 4 and 6, LexA-Rab10 Q68L and GAD-Exoc6b CT; 5 and 7 LexA-Rab10 Q68L and GAD-Exoc6 CT. Part B. The diploid cells contained the GAD-Exoc6b CT prey plasmid together with LexA-Rab10 Q68L (lanes 1, 4, 6), LexA-Rab10 wild-type (lanes 2, 5, 7), or LexA-Rab10 T23N (lane 3). In both parts A and B growth on 1, 5, 10, and 50 mM 3AT was determined, but for ease of presentation the results with the higher concentrations are not shown. 50 mM 3AT was required to inhibit completely the growth of cells containing LexA-Rab10 Q68L and GAD-Exoc6b CT.

Fig 2

siRNA knockdown of Exoc 6 and 6b. The immunoblot for Exoc6/6b was performed with SDS samples of 3T3-L1 adipocytes containing control siRNA alone (Con), siRNA for Exoc6 or Exoc6b plus Con, or a mixture of siRNAs for Exoc 6 and 6b. The 1× load was 20 μg protein. The standards (Stds) were purified recombinant 3×Flag-tagged Exoc 6 or 6b. The 1 load for the standards was 1 ng. The 3×Flag tag on the standards decreased their mobility slightly. A replicate of this immunoblot with SDS samples from a separate experiment gave similar results.

Fig 3

Effects of Exoc 6 and 6b knockdown on GLUT4 at the cell surface. 3T3-L1 adipocytes containing control siRNA (Con) or siRNA for Exoc6 or Exoc6b plus Con or the mixture of siRNAs for Exoc 6 and 6b were analyzed for HA-GLUT4-GFP at the cell surface in the basal (B) or insulin (I) state, as described in the Experimental Procedures. For each experiment the values were normalized to 1.0 for the control siRNA in the insulin state. The results are the means +/− the standard deviation for seven separate experiments. * designates p<0.05 for the comparison with the control insulin value.

Fig 4

Effect of Exoc6/6b knockdown on insulin signaling. 3T3-L1 adipocytes containing control siRNA (Con) or the mixture of siRNAs for Exoc6 and Exoc6b were treated with insulin (I) or left in the basal state (B). SDS samples of these were immunoblotted for the phosphothreonine 642 on TBC1D4 or phosphoserine 473 on Akt. The 1 load was 20 μg protein. A replicate of this experiment gave the same results.