Functional studies in 3T3L1 cells support a role for SNARE proteins in insulin stimulation of GLUT4 translocation.
Insulin stimulation of glucose transport in the major insulin-responsive tissues results predominantly from the translocation to the cell surface of a particular glucose transporter isoform, GLUT4, residing normally under basal conditions in intracellular vesicular structures. Recent studies have identified the presence of vesicle-associated membrane protein (VAMP) 2, a protein involved in vesicular trafficking in secretory cell types, in the vesicles of insulin-sensitive cells that contain GLUT4. The plasma membranes of insulin-responsive cells have also been shown to contain syntaxin 4 and the 25 kDa synaptosome-associated protein (SNAP-25), two proteins that form a complex with VAMP 2. The potential functional involvement of VAMP 2, SNAP-25 and syntaxin 4 in the trafficking of GLUT4 was assessed in the present study by determining the effect on GLUT4 translocation of microinjection of toxins that specifically cleave VAMPs or SNAP-25, or microinjection of specific peptides from VAMP 2 and syntaxin 4. Microinjection of tetanus toxin light chain or botulinum D toxin light chain resulted in an 80 and 61% inhibition respectively of insulin stimulation of GLUT4 translocation in 3T3L1 cells assessed using the plasma-membrane lawn assay. Botulinum A toxin light chain, which cleaves SNAP-25, was without effect. Microinjection of an N-terminal VAMP 2 peptide (residues 1-26) inhibited insulin stimulation of GLUT4 translocation by 54%. A syntaxin 4 peptide (residues 106-122) inhibited insulin stimulation of GLUT4 translocation by 40% whereas a syntaxin 1c peptide (residues 226-260) was without effect. These data taken together strongly suggest a role for VAMP 2 in GLUT4 trafficking and also for syntaxin 4. They further indicate that the isoforms of SNAP-25 isolated to date that are sensitive to cleavage by botulinum A toxin light chain do not appear to be involved in GLUT4 translocation.
Macaulay
S L
SL
CSIRO, Division of Biomolecular Engineering, 343 Royal Parade, Parkville 3052, Victoria, Australia.
Hewish
D R
DR
Gough
K H
KH
Stoichevska
V
V
MacPherson
S F
SF
Jagadish
M
M
Ward
C W
CW
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001905:Botulinum Toxins / Q000008:administration & dosage / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D051275:Glucose Transporter Type 4; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000008:administration & dosage / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008845:Microinjections; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D009124:Muscle Proteins; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010446:Peptide Fragments / Q000008:administration & dosage / Q000138:chemical synthesis / Q000494:pharmacology; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D013744:Tetanus Toxin / Q000008:administration & dosage / Q000494:pharmacology; D033921:Vesicular Transport Proteins
D051275:Glucose Transporter Type 4; D007328:Insulin; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D050600:SNARE Proteins; C495693:Slc2a4 protein, mouse; C494209:Snap25 protein, mouse; D050825:Synaptosomal-Associated Protein 25; D013744:Tetanus Toxin; D033921:Vesicular Transport Proteins; C096325:botulinum toxin type D; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D001905:Botulinum Toxins