Application of the Snare Hypothesis to Insulin Exocytosis

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Several of the proteins which, it has been suggested, regulate neurotransmitter release have recently been identified in the (3-cell, and for a number of them a function in insulin exocytosis has been established. The ubiquitous fusion factor NSF is present in the insulin-secreting cell line HIT-T15. In addition, aSNAP, also present in these cells, appears to be one of the factors in cytosol required for Ca2+-triggered insulin exocytosis (Kiraly-Borri et at., 1996). The v-SNARE VAMP-2 was localized to insulin secretory granules (Jacobsson et a/., 1994). It is also expressed on the SLMVs in the (3-cell, as demonstrated by its colocalization with synaptophysin, a synaptic vesicle membrane protein (Regazzi eta/., 1995). Clostridial neurotoxins have been used to investigate the functional importance of several of the SNARE proteins. As insulin-secreting cells do not express the ganglioside receptors for the clostridial neurotoxins on the cell surface, the toxins were introduced into the cells following cell permeabilization (Regazzi eta/., 1995, Boyd et a/., 1995). Short treatment of the permeabilized cells with tetanus toxin (TeTx) light chain resulted in a dose-related cleavage of VAMP-2 and of cellubrevin which is also expressed both on secretory granules and SLMVs. Both TeTx and preactivated botulinum toxin B (BoNT/B) caused inhibition of Ca2+-stimulated exocytosis in insulin-secreting cell lines and rat islet cells (Regazzi et a/., 1995). At high concentrations of the toxins (50 nM), stimulated exocytosis was abolished. This suggests that VAMP-2 and/or cellubrevin are required for the docking/fusion process of the secretory granules.

The t-SNARE SNAP-25 is also expressed in the (3-cell and has been localized mainly to the plasma membrane (Sadoul eta/., 1995). SNAP-25 was cleaved by treatment of SLO-permeabilized cells with botulinum toxins (BoNT) A or E. The two neurotoxins inhibited Ca2+-induced insulin exocytosis but failed to abolish the process completely (Sadoul et a/., 1995). This could be due to the failure of the toxins to cleave SNAP-25 already complexed to other fusion proteins (escaping detection by Western blotting) or to the requirement for SNAP-25 at a penultimate step in insulin exocytosis.

The other t-SNARE candidate protein, syntaxin, is also expressed in islet cells (Jacobsson et a/., 1994). Treatment of digitonin-

permeabilized mouse islets with an antibody against syntaxin has been reported to attenuate Ca2+-stimulated exocytosis (Martin et al., 1995). In recent studies, we observed complete inhibition of Ca2+-mediated insulin exocytosis with BoNT/Cl, associated with syntaxin cleavage (Lang, Niemann and Wollheim, unpublished observations). Taken together, these results suggest that at least some of the proteins implicated in the regulation of synaptic vesicle fusion in nerve terminals also regulate exocytosis of secretory granules in the |3-cell.

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