Protein Kinase B Stimulates the Translocation of GLUT4 but Not GLUT1 or Transferrin Receptors in 3T3-L1 Adipocytes by a Pathway Involving SNAP-23, Synaptobrevin-2, and/or Cellubrevin

doi:10.1074/jbc.274.40.28087

Protein Kinase B Stimulates the Translocation of GLUT4 but Not GLUT1 or Transferrin Receptors in 3T3-L1 Adipocytes by a Pathway Involving SNAP-23, Synaptobrevin-2, and/or Cellubrevin*

From the ^‡Department of Biochemistry, Imperial College, London SW7 2AY and the ^§Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, BS8 1TD United Kingdom

Abstract

An interaction of SNAP-23 and syntaxin 4 on the plasma membrane with vesicle-associated synaptobrevin-2 and/or cellubrevin, known as SNAP (solubleN-ethyl-maleimide-sensitive factorattachment protein) receptors or SNAREs, has been proposed to provide the targeting and/or fusion apparatus for insulin-stimulated translocation of the GLUT4 isoform of glucose transporter to the plasma membrane. By microinjecting 3T3-L1 adipocytes with the Clostridium botulinum toxin B or E, which proteolyzed synaptobrevin-2/cellubrevin and SNAP-23, respectively, we investigated the role of these SNAREs in GLUT4, GLUT1, and transferrin receptor trafficking. As expected, insulin stimulated the translocation of GLUT4, GLUT1, and transferrin receptors to the plasma membrane. By contrast, a constitutively active protein kinase B (PKB-DD) only stimulated a translocation of GLUT4 and not GLUT1 or the transferrin receptor. The GLUT4 response to PKB-DD was abolished by toxins B or E, whereas the insulin-evoked translocation of GLUT4 was inhibited by approximately 65%. These toxins had no significant effect on insulin-stimulated transferrin receptor appearance at the cell surface. Thus, insulin appears to induce GLUT4 translocation via two distinct routes, only one of which involves SNAP-23 and synaptobrevin-2/cellubrevin, and can be mobilized by PKB-DD. The PKB-, SNAP-23-, and synaptobrevin-2/cellubrevin-independent GLUT4 translocation pathway may involve movement through recycling endosomes, together with GLUT1 and transferrin receptors.

Footnotes

↵* This work was supported by grants from the Medical Research Council and the British Diabetic Association (to J. M. T.) and by Contract DAMD 17-97-C-7060 from the United States Army Medical Research and Material Command (to J. O. D.). The confocal microscopy was performed using the Bristol Cell Imaging Facility that was funded by an Infrastructure Grant from the Medical Research Council.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¶ British Diabetic Association Senior Research Fellow. To whom correspondence should be addressed. Tel.: 44-117-928-8273; Fax: 44-117-928-8274; E-mail, j.tavare@bristol.ac.uk.
↵2 P. G. P. Foran, L. M. Fletcher, P. B. Oatey, N. Mohammed, J. O. Dolly, and J. M. Tavaré, unpublished observations.
Abbreviations:

PI3-kinase

phosphatidylinositide (4,5)-bisphosphate kinase

BoNT

botulinum neurotoxin

Cbr

cellubrevin

GFP

green-fluorescent protein

IRAP

insulin-responsive amino peptidase

TfR

transferrin receptor

hTfR

human TfR

HA

hemagglutinin

PKB

protein kinase B

Sbr

synaptobrevin

TeTx

tetanus toxin

TRITC

tetramethylrhodamine β-isothiocyanate

GTPγS

guanosine 5′-3-O-(thio)triphosphate

PIPES

1,4-piperazinediethanesulfonic acid
- Received June 16, 1998.
- Revision received June 3, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.