Figure 1. Generation of pancreas-specific synaptotagmin-9 KO (p-S9X) mice. (A) Homologous recombination and cre excision strategy for generating pancreas-specific synaptotagmin-9 KO mice. Syt9fl/fl (synaptotagmin-9fl/fl) showing targeted exon 2 and flanking loxP sites, p-S9X showing synaptotagmin-9 gene structure after exon 2 deletion by cre recombination. (B) Representative genotyping patterns for p-S9X and Syt9fl/fl mice. Note that deletion of exon 2 was only observed in the pancreas of p-S9X. (C) Synaptotagmin-9 mRNA levels were analyzed by quantitative real time PCR on total RNA extracted from p-S9X and Syt9fl/fl (ctrl.) mouse islets. Data are presented as means 6 SEM. N = 4 mice for each genotype. P,0.01. doi:10.1371/journal.pone.0015414.g001 under curve for the first phase of insulin secretion or for the entire measurement period was the same in p-S9X and control mice (Figure 2C). These data indicate that glucose-stimulated insulin Synaptotagmin-9 in Insulin Secretion secretion in p-S9X mice was adequate to regulate glucose homeostasis under glucose overload, similar to that in control mice, and further support the notion that synaptotagmin-9 was not involved in the regulation of glucose homeostasis. Glucose-induced insulin release from isolated islets The fact that p-S9X mice showed normal glucose tolerance and glucose-induced insulin secretion in vivo argues against synapto- tagmin-9 as a calcium sensor in insulin granule exocytosis. Since synaptotagmin-9 was deleted during embryonic development, p- S9X mice might have developed mechanisms to compensate for defects in calcium-dependent insulin granule exocytosis, e.g. increased insulin content or increased number of islets in b-cells, and as such, we failed to detect impaired insulin secretion in vivo as measured in tail vein blood. To directly examine insulin secretion, we performed perifusion experiments on isolated pancreatic islets, and measured insulin secretion in response to glucose stimulation (Figure 3A). Basal insulin secretion at 3 mM glucose was the same in p-S9X and control mouse islets. High glucose challenge induced more than 15-fold enhancement in insulin secretion at 6 minutes in both p-S9X and control islets, representing the peak amplitude of the first phase of glucose-stimulated insulin secretion. Insulin levels in the fractions collected between 9 and 15 minutes gradually decreased, but continued to show sustained elevation above basal levels until the end of stimulation, corresponding to the second phase of insulin secretion. There was no difference in peak amplitude or time course of insulin release between p-S9X and control mouse islets. Consequently, net insulin secretion during the first 15 minutes of stimulation (first phase) and over the entire stimulation period was similar between p-S9X and control islets (Figure 3B). Islet insulin content was the same in control and p-S9X mouse islets (Figure 3C). These results showed that deletion of synaptotagmin-9 had no effects on the amount or kinetics of glucose-induced insulin secretion in p-S9X mice. Morphological and ultrastructural characterization To examine the involvement of synaptotagmin-9 in the regulation of islet development and maintenance, we tested whether deletion of synaptotagmin-9 in the pancreas led to changes in islet architecture. Histological analysis of the pancreas showed no pathological signs in p-S9X mouse islets, and no differences with regard to the number, size or shape of islets between p-S9X and control mice (Figure 4A). Vascularization of islets and cell morphology were similar as well. We then examined pancreatic b-cell ultrastructure. Electron microscopy of pancreatic islets showed no differences between p-S9X and control mice in terms of the number and appearance of insulin granules and b-cell ultrastructure in general (Figure 4B). These data demonstrate that islet development and maintenance were not dependent on synaptotagmin-9, and that there were no compensatory changes in islet architecture or b-cell ultrastructure. Glucose-induced calcium responses As calcium is the triggering signal for insulin granule exocytosis, alterations in glucose-induced calcium responses might have compensated for the deletion of synaptotagmin-9. To test this, we recorded intracellular calcium changes to high glucose stimulation in isolated islets from p-S9X and control mice. Glucose-induced Ca2+ responses showed a similar pattern in p- S9X and control mouse islets (Figure S2). Calcium rise (188639 vs. 249636 a.u. for control and p-S9X mouse islets, respectively; N = 23–26, NS) and lag time (8867 vs. 7467 s for control and p- PLoS ONE | www.plosone.org 4 November 2010 | Volume 5 | Issue 11 | e15414