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Vol. 10, Issue 11, 3675-3688, November 1999
and
*Division of Biochemistry and Molecular Biology, University of
Glasgow, Glasgow G12 8QQ, Scotland; and Insulin and guanosine-5'-O-(3-thiotriphosphate)
(GTPCentre for
Molecular and Cellular Biology and Department of Physiology and
Pharmacology, University of Queensland, St. Lucia, Brisbane 4072, Queensland, Australia
S) both stimulate glucose transport and translocation of the
insulin-responsive glucose transporter 4 (GLUT4) to the plasma membrane
in adipocytes. Previous studies suggest that these effects may be
mediated by different mechanisms. In this study we have tested the
hypothesis that these agonists recruit GLUT4 by distinct trafficking
mechanisms, possibly involving mobilization of distinct intracellular
compartments. We show that ablation of the endosomal system using
transferrin-HRP causes a modest inhibition (~30%) of
insulin-stimulated GLUT4 translocation. In contrast, the GTPS
response was significantly attenuated (~85%) under the same
conditions. Introduction of a GST fusion protein encompassing the
cytosolic tail of the v-SNARE cellubrevin inhibited
GTPS-stimulated GLUT4 translocation by ~40% but had no effect on
the insulin response. Conversely, a fusion protein encompassing the
cytosolic tail of vesicle-associated membrane protein-2 had no
significant effect on GTPS-stimulated GLUT4 translocation but
inhibited the insulin response by ~40%. GTPS- and
insulin-stimulated GLUT1 translocation were both partially inhibited by
GST-cellubrevin (~50%) but not by GST-vesicle-associated membrane
protein-2. Incubation of streptolysin O-permeabilized 3T3-L1 adipocytes
with GTPS caused a marked accumulation of Rab4 and Rab5 at the cell
surface, whereas other Rab proteins (Rab7 and Rab11) were unaffected.
These data are consistent with the localization of GLUT4 to two
distinct intracellular compartments from which it can move to the cell
surface independently using distinct sets of trafficking molecules.
Present address: Department of Clinical
Biochemistry, Addenbrooks Hospital, University of Cambridge, Hills
Road, Cambridge, UK.
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