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 Z n2+-dependent endoproteases, have been instrumental in demonstrating t hat their respective substrates [synaptosomal-associated protein with M-r = 25 kDa (SNAP-25), synaptobrevin (Sbr), and syntaxin] are essenti al for regulated exocytosis from nerve terminals and neuroendocrine ce lls. 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-stimu lated glucose uptake, which results in part from enhanced fusion of th ese 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 result ing in a significant blockade of the insulin-stimulated translocation of GLUT4 to the plasmalemma. This establishes that these two toxin sub strates 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 adip ocytes, its functional homologue SNAP-23 is abundant and largely confi ned to the plasmalemma. SNAP-23 proved to be resistant to cleavage by BoNT/A. Consistent with these results, type A did not block insulin-in duced glucose uptake, precluding a demonstration of its likely importa nce in this process.