Botulinum neurotoxin B inhibits insulin-stimulated glucose uptake into 3T3-L1 adipocytes and cleaves cellubrevin unlike type A toxin which failed to proteolyze the SNAP-23 present.
Types A, B, and C1 botulinum neurotoxin (BoNT), a group of selective Zn2+-dependent endoproteases, have been instrumental in demonstrating that their respective substrates [synaptosomal-associated protein with Mr = 25 kDa (SNAP-25), synaptobrevin (Sbr), and syntaxin] are essential for regulated exocytosis from nerve terminals and neuroendocrine cells. The colocalization of Sbr, or its homologue cellubrevin (Cbr), in the majority of the glucose transporter-isotype 4 (GLUT4)-containing vesicles from adipocytes implicates their involvement in insulin-stimulated glucose uptake, which results in part from enhanced fusion of these vesicles with the plasmalemma. In this study, exposure of cultured 3T3-L1 adipocytes to BoNT/B in a low-ionic strength medium was found to block insulin-evoked glucose uptake by up to 64%. BoNT/B was shown by immunoblotting to cause extensive proteolysis of Cbr and Sbr resulting in a significant blockade of the insulin-stimulated translocation of GLUT4 to the plasmalemma. This establishes that these two toxin substrates contribute to the insulin-regulated fusion of GLUT4-containing vesicles with the plasmalemma, at least in this differentiated 3T3-L1 clone. Although SNAP-25 was not detectable in the differentiated adipocytes, its functional homologue SNAP-23 is abundant and largely confined to the plasmalemma. SNAP-23 proved to be resistant to cleavage by BoNT/A. Consistent with these results, type A did not block insulin-induced glucose uptake, precluding a demonstration of its likely importance in this process.
Chen
F
F
Department of Biochemistry, Imperial College, London, U.K.
Foran
P
P
Shone
C C
CC
Foster
K A
KA
Melling
J
J
Dolly
J O
JO
D016475:3T3 Cells; D017667:Adipocytes / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D000954:Antigens, Surface / Q000378:metabolism; D001905:Botulinum Toxins / Q000494:pharmacology*; D019274:Botulinum Toxins, Type A / Q000494:pharmacology*; D002352:Carrier Proteins / Q000187:drug effects / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry; D010450:Endopeptidases; D005947:Glucose / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D006868:Hydrolysis; D007328:Insulin / Q000494:pharmacology*; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051379:Mice; D009004:Monosaccharide Transport Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D009124:Muscle Proteins; D009419:Nerve Tissue Proteins / Q000378:metabolism; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D050683:R-SNARE Proteins; D013347:Subcellular Fractions / Q000378:metabolism; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D051040:Vesicle-Associated Membrane Protein 3
D000954:Antigens, Surface; D002352:Carrier Proteins; D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D007328:Insulin; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D009419:Nerve Tissue Proteins; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D050683:R-SNARE Proteins; C100493:SNAP23 protein, human; C495693:Slc2a4 protein, mouse; C107017:Snap23 protein, mouse; C494209:Snap25 protein, mouse; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D051040:Vesicle-Associated Membrane Protein 3; C096323:rimabotulinumtoxinB; D010450:Endopeptidases; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; C096324:botulinum toxin type C; D005947:Glucose