Two Populations of Insulin Granules with Distinct Fusion Properties are Maintained by ABC Transporters ABCG1 and ABCA1

Abstract

Release of insulin from pancreatic beta cells is ultimately triggered by increased intracellular calcium. Insulin is stored in secretory granules whose membranes are enriched in cholesterol and contain synaptotagmins that serve as calcium sensors for SNARE-mediated exocytosis. Previously, we showed that efficient storage and secretion of insulin rely on cholesterol-regulatory ATP binding cassette proteins, ABCG1 and ABCA1. Now, using stable cell lines that express proinsulin tagged with GFP and are deficient in either ABCG1 or ABCA1, we have addressed further the roles of the two transporters in secretion. We assessed the fusion of purified insulin granules to planar supported bilayers in vitro and the exocytotic response to graded cell depolarization in situ. For granules purified from control cells, titration of calcium during in vitro fusion uncovered two distinct populations of granules differing in calcium sensitivity. Strikingly, granules purified from ABCG1-deficient cells selectively lacked the high-sensitivity population while granules from ABCA1-deficient cells lacked the low-sensitivity population. Addition of cholesterol to cells before purification restored the missing granule populations. Comparative examination of our results to in vitro fusion of dense core vesicles from PC12 cells, in which we manipulated expression of synaptotagmin isoforms, enabled us to relate our high- and low-sensitivity granule populations to the presence of synaptotagmin-7 and −9, respectively. Our parallel efforts examining depolarization-driven exocytosis in cell culture using a bulk secretion assay and single vesicle TIRF microscopy showed that deficiency of ABCG1 selectively inhibited GFP release elicited by mild depolarizations while deficiency of ABCA1 inhibited release elicited by stronger depolarizations. Collectively, our data argue that insulinoma cells use ABCG1 and ABCA1 to maintain two populations of insulin granules with distinct synaptotagmin isoforms and consequently distinct fusion properties.