Abstract
Insulin stimulates the translocation of the glucose transporter GLUT4 from intracellular vesicles to the plasma membrane. In the present study we have conducted a comprehensive proteomic analysis of affinity-purified GLUT4 vesicles from 3T3-L1 adipocytes to discover potential regulators of GLUT4 trafficking. In addition to previously identified components of GLUT4 storage vesicles including the insulin-regulated aminopeptidase insulin-regulated aminopeptidase and the vesicle soluble N-ethylmaleimide factor attachment protein (v-SNARE) VAMP2, we have identified three new Rab proteins, Rab10, Rab11, and Rab14, on GLUT4 vesicles. We have also found that the putative Rab GTPase-activating protein AS160 (Akt substrate of 160 kDa) is associated with GLUT4 vesicles in the basal state and dissociates in response to insulin. This association is likely to be mediated by the cytosolic tail of insulin-regulated aminopeptidase, which interacted both in vitro and in vivo with AS160. Consistent with an inhibitory role of AS160 in the basal state, reduced expression of AS160 in adipocytes using short hairpin RNA increased plasma membrane levels of GLUT4 in an insulin-independent manner. These findings support an important role for AS160 in the insulin regulated trafficking of GLUT4.
Highlights
We wanted to determine which of these proteins might play a functional regulatory role in GLUT4 trafficking or determine which proteins may be targeted with GLUT4 into insulin-responsive vesicles
Summary of GLUT4 vesicle proteins identified by mass spectrometry from 3T3-L1 adipocytes Proteins shown were the consensus of three separate experiments
E Translocation of proteins to the plasma membrane of 3T3-L1 adipocytes treated with 100 nM insulin for 30 min
Summary
Materials—3T3-L1 murine fibroblasts, C2C12 fibroblasts, and Chinese hamster ovary (CHO) cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA). Immunoprecipitation and GST Pull-down of FLAG-tagged AS160— FLAG-AS160-expressing CHO cells were lysed in extraction buffer (1% Nonidet P-40, 137 mM sodium chloride, 10% glycerol, 25 mM Tris, pH 7.4), centrifuged at 18,000 ϫ g for 20 min, anti-FLAG antibody and protein G beads were added to the supernatant and incubated for 2 h at 4 °C with mixing. Immunoprecipitation of Endogenous AS160 from 3T3-L1 Adipocytes— The LDM fraction from basal or insulin-stimulated cells prepared as described above was resuspended in IP buffer (60 mM -octylglucoside, 1% Triton X-100, 137 mM sodium chloride, 10% glycerol, 25 mM Tris, pH 7.4), incubated for 30 min at 4 °C, and centrifuged at 200,000 ϫ g for 15 min. A region of interest was set around each cell, and the amount of fluorescence per unit area was determined for each channel using the Leica confocal software
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