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

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Abstract
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.

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