medium was then switched to KRH containing 20 mM glucose for 30 min. Fractions of the medium were collected every 3 min, starting at 6 min before stimulation. Insulin concentration in each fraction was measured by using Mouse Insulin ELISA (Mercodia). Ca2+ Measurements Calcium measurements were performed as described previously [15]. Briefly, isolated and overnight-cultured islets were loaded with 3 mM Fluo-4 in KRH buffer containing 3 mM glucose for 1 hour, then placed in a perifusion chamber on the stage of an inverted microscope (Nikon Eclipse TE2000-U) and continuously superfused with KRH buffer containing 3 mM glucose at 37uC. Fluo-4 was excited at 488 nm by using a 175-watt xenon arc lamp, and emitted signal was projected onto a CCD camera (CoolSNAP HQ2, Photometrics) after passing through a 535DF35 band pass optical filter (Omega Optical). Images were collected every 2 sec, and fluorescence signals from individual cells were measured as a function of time using Metafluor software (Molecular devices). Fluo-4 images were acquired for 2 minutes in KRH buffer containing 3 mM glucose, and a further 10 minutes after the superfusion was switched to KRH buffer containing 20 mM glucose. Lag-time for [Ca2+]i rise was defined as the time from the buffer switch (3 mM to 20 mM glucose) to the first value above baseline average, which was calculated during the 2 minutes before stimulation. Calcium rise was calculated as the difference between the average basal value and highest peak value. RNA Extraction and Quantitative RT-PCR Total RNA was extracted from islets of p-S9X and control mice with TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. RNA samples were first treated with DNase I (Roche), and then reverse-transcribed with RevertAid (Fermentas). Quantitative real time PCR was performed by using SYBR Green chemistry and gene-specific primers on an Applied Biosystems StepOnePlus real-time PCR system. Primers used for detecting mouse synaptotagmin-9 (mus musculus synaptotagmin V) gene expression were designed by using PerkinElmer’s Primer Express Software (59-TAA GAC ACC TCC AGA CTC CA-39 and 59- TCA CTT CCT GAA GAT GGA CT-39, accession number: NM_016908). Cyclophilin was used as the internal standard to determine relative mRNA levels between samples (59-TGG AGA GCA CCA AGA CAG ACA-39 and 59-TGC CGG AGT CGA CAA TGA T-39, accession number: NM_011149). Electrophysiology Measurements Electrophysiology measurements were carried out essentially as previously described [16]. Briefly, Single b-cells were obtained by digestion of isolated pancreatic islets with 0.025 mg/ml trypsin [17], distributed on polylysine-coated cover glasses and cultured for 24 hours as described for islets. Membrane capacitance was recorded from single b-cells using standard whole-cell patch-clamp technique [18]. Exocytosis was elicited by a 500 ms depolarizing pulse from 270 to 0 mV. Single b-cells were identified by the absence of the transient Na+-current and the outward TEA- resistant K+-current when the cells were given a depolarization pulse from 270 to 0 mV. Pipette resistance ranged between 3 and 5 MV when pipettes were filled with intracellular solution containing (in mM): 135 KCl, 10 NaCl, 1 MgCl2, and 3 MgATP, 5 Hepes (pH 7.2 with KOH). Extracellular solution contained (in mM): 118 NaCl, 20 TEA-Cl (tetraethylammonium chloride), 5.6 KCl, 2.6 CaCl2, 1.2 MgCl2, 5 D-glucose, and 5 Hepes (pH 7.4, with NaOH). Cells were stimulated at low frequency (,0.05 Hz) to allow full recovery of exocytotic capacity between pulses. Measurements were performed by using EPC10 plus patch clamp Synaptotagmin-9 in Insulin Secretion amplifier and analyzed by using Patchmaster software. Exocytosis was detected as changes in cell membrane capacitance (Cm), which was estimated by the Lindau-Neher technique implement- ing the ‘Sine+DC’ feature of the lock-in module [19]. The amplitude of the sine wave was 10 mV and the frequency was set at 1 kHz. All Cm measurements were performed at 30–32uC. Statistical Analysis Data are presented as means 6 SEM. Comparisons of data from p-S9X and control mice were made by using Student’s two- tailed t-test for independent data. The significance limit was set at P,0.05. Results Generation of pancreas-specific synaptotagmin-9 KO (p- S9X) mice To investigate whether synaptotagmin-9 was involved in the regulation of glucose homeostasis and insulin secretion, we generated pancreas-specific synaptotagmin-9 KO mice by crossing synaptotagmin-9fl/fl with and without Pdx1-Cre transgene (Figure 1A). P-S9X and littermate synaptotagmin-9fl/fl (control) mice were used in the experiments. Pdx1 promoter drove specific expression of Cre recombinase in the pancreas, with no detectable expression in other tissues, as further confirmed by the lack of Cre recombination activity outside the pancreas in p-S9X mice (Figure 1B). Under the Pdx1 promoter, Cre expression in the pancreatic epithelium started at early embryonic stages, which ensured efficient synaptotagmin-9 deletion in the pancreas (Figure 1C). Body weight, glucose and insulin levels P-S9X mice were viable and fertile, and showed no differences from synaptotagmin-9fl/fl in their postnatal development, includ- ing body weight gain and body composition (Figure S1A and S1B). We compared synaptotagmin-9fl/fl with wild type mice, and found no difference in body weight, glucose tolerance and glucose levels (data not shown), therefore, we used synaptotagmin-9fl/fl as control to p-S9X mice in subsequent experiments. P-S9X mice and their control showed similar fasting glucose levels (4.460.1 and 4.460.1 mmol/l, N = 27 and 25 for control and p-S9X, respectively; NS) and fed glucose levels (9.060.3 and 9.960.5 mmol/l, N = 13 and 15 for control and p-S9X, respectively; NS). Fasting insulin levels were the same (0.2360.003 and 0.2360.003 ng/ml, N = 21 and 23 for control and p-S9X, respectively; NS) and fed insulin levels were also similar in control and p-S9X mice (0.7860.14 and 0.6460.14 ng/ ml, N = 8 and 7 for control and p-S9X, respectively; NS). These results indicate that pancreas-specific deletion of synaptotagmin-9 had no effect on glucose homeostasis in p-S9X mice. Glucose tolerance test and acute insulin response Deletion of synaptotagmin-7, a calcium sensor in insulin secretion, resulted in glucose intolerance and impaired insulin secretion in synaptotagmin-7 KO mice, even though the KO mice exhibited normal resting and fed glucose levels [15]. To examine whether p-S9X mice were also defective in response to glucose overload, we performed glucose tolerance tests and measured glucose-induced insulin secretion in vivo in p-S9X and control mice. After i.p. glucose injection, blood glucose increase and clearance were similar in p-S9X and control mice (Figure 2A). Plasma insulin levels were determined at 3, 8, 15, 30 and 60 minutes after glucose injection. No difference was observed in glucose-induced insulin secretion in vivo (Figure 2B), and the area PLoS ONE | www.plosone.org 3 November 2010 | Volume 5 | Issue 11 | e15414