general, is usually slower, and requires lower calcium levels, although insulin granule exocytosis in b-cells and catecholamine release from chromaffin cells can also be triggered by high calcium concentrations [10,11]. Despite the apparent differences between SV and LDCV exocytosis, the exocytotic machinery in neuronal and endocrine cells shows remarkable similarities [4,12]. SNAREs operate during membrane fusion in both systems [12,13] and synaptotagmins also function as calcium sensors in excitable endocrine cells [4,14]. In our previous study, we identified synaptotagmin-7 as a high affinity calcium sensor in insulin secretion, and found that deletion of synaptotagmin-7 resulted in ,40% reduction in calcium- dependent insulin release [15], which indicates that other proteins must be responsible for regulating the remaining 60% of insulin secretion. Besides synaptotagmin-7, several other calcium-binding synaptotagmin isoforms are present in pancreatic islets [15]. Of these, mRNA level of the neuronal calcium sensor synaptotagmin- 9 is the highest after synaptotagmin-7 in pancreatic islets [15]. Since insulin release can be evoked by a wide range of calcium concentrations, from submicromolar to tens of mM [11], it is conceivable that the low affinity calcium sensor synaptotagmin-9 may regulate insulin secretion in response to high calcium concentrations, in collaboration with synaptotagmin-7, which is responsible for insulin release at low calcium concentrations. To investigate whether synaptotagmin-9 functions as a calcium sensor in glucose-stimulated insulin release, we generated pancreas-specific synaptotagmin-9 KO (p-S9X) mice, and tested the effects of synaptotagmin-9 deletion on glucose homeostasis, and on insulin secretion in vivo and in pancreatic islets and b-cells. Methods Animal Welfare All experiments involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of A*STAR (Agency for Science, Technology and Research) under IACUC #080351 and #090428. Generation of Pancreas-Specific Synaptotagmin-9 KO Mice Pancreas-specific synaptotagmin-9 KO mice (p-S9X) were generated by crossing synaptotagmin-9fl/fl mice containing Cre transgene under pdx1 promoter (Pdx1-Cre) with synaptotagmin- 9fl/fl mice. The Pdx1-Cre mouse line was a generous gift from Dr. Doug Melton (Harvard University). Floxed synaptotagmin-9 (synaptotagmin-9fl/fl) mice were described previously [9]. Litter- mates with genotypes of synaptotagmin-9fl/fl/Pdx1-Cre (p-S9X) and synaptotagmin-9fl/fl (control) were used in this study. Genotyping was performed on tail DNA by PCR using the following primers: 59-TGC TTC TGT CCG TTT GCC GGT-39 and 59-CTA AGT GCC TTC TCT ACA CCT-39 (PCR product of 500 bp indicates the presence of Cre transgene); 59- GCG TGA GAA AAC TCA CTG GAT G-39 and 59- GTC CAC TAG GGC TAG CCC AGG-39 (PCR fragment of 200 bp and 1100 bp indicate the presence of wild type and mutant allele, respectively); 59- GAG AGT GAT AGA GAA AGA CAC-39 and 59- GTT GAG ATG TAA TGT ATA CCT ATG C-39 (PCR product of 200 bp indicates the presence of mutant allele). For islet isolation, histological staining, electron microscopy and qPCR, mice were sacrificed by cervical dislocation. Altogether, 60 control and 53 p-S9X mice were used. All mice used in this study were bred and housed in our animal facility. Synaptotagmin-9 in Insulin Secretion Measurements of Body Weight and Body Composition Mice were fed ad libitum with free access to water. Body weight in p-S9X and control mice was monitored between 4 and 15 weeks of age. Body fat and lean mass was analyzed on 16 wk-old mice by using Echo MRI (Echo Medical Systems) as previously described [15]. Glucose Tolerance Tests and Blood Glucose Measurements Intraperitoneal glucose tolerance tests (IPGTT) was performed as previously described [15]. Briefly, 12–14 wk-old mice were fasted for 16–18 h with free access to water. Blood glucose levels were tested before and at 15, 30, 60, 90 and 120 min after i.p. injection of 20% D-glucose solution at 2 g/kg of body weight. ,4 ml of tail vein blood was taken at each time point, and blood glucose levels were determined by using Accu-Chek Advantage blood glucose meter (Roche Diagnostics). Glucose-Stimulated Insulin Secretion in vivo and Plasma Insulin Measurements Insulin secretion in vivo was induced by i.p. injection of 20% D- glucose solution at 2 g/kg of body weight on 12–14 wk-old mice after overnight fasting. ,20 ml blood samples were collected from tail vein at 3, 8, 15, 30 and 60 min after glucose injection. Blood samples were mixed with 2 ml of 0.5 M EDTA on ice and centrifuged at 10,0006 g for 5 min for plasma collection. Plasma insulin concentrations were determined by using Ultrasensitive Mouse Insulin ELISA Kit (Mercodia, Sweden). Histological Analysis and Electron Microscopy For histological analysis, pancreata from 4 mice of each group (p-S9X and control mice) were fixed in 10% formalin at 4uC for 24 h before paraffin embedding. Then 5 mm serial sections from each sample were processed for routine histological study (hematoxylin-eosin). For transmission electron microscopy, pan- creatic samples from 4 mice of each group (p-S9X and control mice) were fixed in 2.5% glutaraldehyde (Agar Scientific) and in 1% OsO4 in PBS (pH 7.4) at 4uC for 2 h each, dehydrated in ethanol series and embedded in araldite epoxy resin (EMS). Ultrathin (,90 nm) sections were stained with 2% uranyl acetate (Analar) for 10 min and examined by using a JEOL JEM-1220 electron microscope (JEOL Asia Pte). Islet Isolation and Insulin Measurements Islets from p-S9X and control mice were isolated by liberase digestion (0.25 mg/ml; Roche) and cultured overnight at 11.1 mM glucose in Advanced RPMI 1640 medium supplement- ed with 10% heat-inactivated fetal bovine serum, 1% penicillin/ streptomycin, 2 mM GlutaMax and 15 mM Hepes, pH 7.4 (Invitrogen). Subsequent experimental handling was performed with a Krebs-Ringer-Hepes medium (KRH) containing (in mM) 130 NaCl, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, and 2.56 CaCl2, supplemented with 1 mg/ml BSA and 3 mM D-glucose. The medium was buffered with 20 mM Hepes and NaOH to pH 7.4 and equilibrated with ambient air. For estimation of islet insulin content, islets were lysed by sonication in 200 ml of passive lysis buffer (Promega) after incubation in KRH medium containing 3 mM glucose for 2 h. Insulin content in the buffer was measured by using Mouse Insulin ELISA (Mercodia). For analysis of islet secretory responses, batches of 5 similar- sized islets from a single mouse were placed in a 40-mL flow chamber under continuous perifusion (0.3 ml/min) with KRH medium containing 3 mM glucose for 60 min at 37uC. The PLoS ONE | www.plosone.org 2 November 2010 | Volume 5 | Issue 11 | e15414