Neuronal Calcium Sensor Synaptotagmin-9 Is Not Involved in the Regulation of Glucose Homeostasis or Insulin Secretion Natalia Gustavsson 1 * . , Xiaorui Wang1., Yue Wang 1 , Tingting Seah1, Jun Xu 2 , George K. Radda1, Thomas C. Su¨ dhof2, Weiping Han1,3* 1 Laboratory of Metabolic Medicine, Singapore Bioimaging Consortium, Agency for Science Technology and Research (A*STAR), Singapore, Singapore, 2 Howard Hughes Medical Institute and Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Palo Alto, California, United States of America, 3 Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore Abstract Background: Insulin secretion is a complex and highly regulated process. It is well established that cytoplasmic calcium is a key regulator of insulin secretion, but how elevated intracellular calcium triggers insulin granule exocytosis remains unclear, and we have only begun to define the identities of proteins that are responsible for sensing calcium changes and for transmitting the calcium signal to release machineries. Synaptotagmins are primarily expressed in brain and endocrine cells and exhibit diverse calcium binding properties. Synaptotagmin-1, -2 and -9 are calcium sensors for fast neurotransmitter release in respective brain regions, while synaptotagmin-7 is a positive regulator of calcium-dependent insulin release. Unlike the three neuronal calcium sensors, whose deletion abolished fast neurotransmitter release, synaptotagmin-7 deletion resulted in only partial loss of calcium-dependent insulin secretion, thus suggesting that other calcium-sensors must participate in the regulation of insulin secretion. Of the other synaptotagmin isoforms that are present in pancreatic islets, the neuronal calcium sensor synaptotagmin-9 is expressed at the highest level after synaptotagmin-7. Methodology/Principal Findings: In this study we tested whether synaptotagmin-9 participates in the regulation of glucose-stimulated insulin release by using pancreas-specific synaptotagmin-9 knockout (p-S9X) mice. Deletion of synaptotagmin-9 in the pancreas resulted in no changes in glucose homeostasis or body weight. Glucose tolerance, and insulin secretion in vivo and from isolated islets were not affected in the p-S9X mice. Single-cell capacitance measurements showed no difference in insulin granule exocytosis between p-S9X and control mice. Conclusions: Thus, synaptotagmin-9, although a major calcium sensor in the brain, is not involved in the regulation of glucose-stimulated insulin release from pancreatic b-cells. Citation: Gustavsson N, Wang X, Wang Y, Seah T, Xu J, et al. (2010) Neuronal Calcium Sensor Synaptotagmin-9 Is Not Involved in the Regulation of Glucose Homeostasis or Insulin Secretion. PLoS ONE 5(11): e15414. doi:10.1371/journal.pone.0015414 Editor: Kathrin Maedler, University of Bremen, Germany Received July 31, 2010; Accepted September 16, 2010; Published November 9, 2010 Copyright: 2010 Gustavsson et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by intramural funding from A*STAR (Agency for Science, Technology and Research, Singapore) Biomedical Research Council (W.H.). T.C.S. is a Howard Hughes Medical Institute investigator. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: weiping_han@sbic.a-star.edu.sg (WH); natalia_gustavsson@sbic.a-star.edu.sg (NG) . These authors contributed equally to this work. Introduction Maintenance of glucose homeostasis requires adequate amount and precise pattern of insulin secretion, which is tightly controlled and regulated in response to blood glucose levels and neuroen- docrine cues [1,2]. A failure in these mechanisms leads to hyperglycemia and type 2 diabetes, or life-threatening hypoglyce- mic conditions [1]. The basic cellular events in the excitation- secretion coupling of pancreatic b-cells are relatively well described, such as glucose uptake into b-cells and consequent changes in ATP/ADP ratio, membrane ion channel dynamics, and elevated cytoplasmic calcium as the trigger for insulin granule exocytosis [1,2]. However, the precise mechanism and temporal events from calcium elevation to insulin granule exocytosis remain elusive, and we have just begun to define the proteins involved in calcium sensing in insulin secretion regulation [3,4,5]. Synaptotagmins, a family of at least 15 proteins, are primarily expressed in neurons and endocrine cells [4,6]. Due to sequence variations at the Ca2+-coordinating sites and the flanking residues, only eight synaptotagmins bind Ca2+, and they exhibit diverse Ca2+ binding properties [4,6]. Synaptotagmin-1, -2 and -9 regulate fast neurotransmitter release, and function as calcium sensors for neurotransmission of their respective brain regions [7,8,9]. These synaptotagmins have low calcium affinity and therefore can respond within milliseconds at the active zone where cytoplasmic calcium concentration is high (,10–40 mM) to trigger synaptic vesicle (SV) exocytosis [4]. In contrast, exocytotic rate of insulin granules and of large-dense core vesicles (LDCV) in PLoS ONE | www.plosone.org 1 November 2010 | Volume 5 | Issue 11 | e15414