Synaptotagmin-9 in Insulin Secretion Figure 2. Normal glucose tolerance and in vivo insulin secretion in p-S9X mice. (A) Intraperitoneal glucose tolerance test (IPGTT) in p-S9X and control mice. p-S9X mice (filled circles, N = 16) showed the same glucose increase and clearance as control mice (open circles, N = 15). (B) Insulin secretion in vivo induced by i.p. injection of glucose in p-S9X and control mice. Plasma insulin levels were determined in control (open circle, N = 21) and p-S9X mice (filled circle, N = 21) after i.p. injection of glucose. (C) Area under curve for the insulin secretion curves in (B). Data are presented as means 6 SEM. N = 21 for each genotype group. doi:10.1371/journal.pone.0015414.g002 S9X mouse islets, respectively; N = 23–26, NS) were the same for p-S9X and control mice. b-cells. These data exclude the involvement of synaptotagmin-9 in regulating insulin granule exocytosis. Insulin granule exocytosis in individual b-cells Considering that brain-specific deletion of synaptotagmin-9 resulted in specific loss of fast neurotransmitter release in neurons [9], and that synaptotagmin-9 may function as a low-affinity calcium sensor for fast insulin release, it is possible that we might have missed the insulin secretion defects in p-S9X mice in the perifusion experiments due to intrinsic limitation of low temporal resolution of this type of measurements. To better resolve calcium- dependent insulin release, we recorded capacitance changes in b- cells, as a measure of insulin granule exocytosis, in response to membrane depolarizations (Figure 5A). Isolated b-cells from p- S9X and control mice showed the same capacitance increases (Figure 5B), indicating that calcium-triggered insulin granule exocytosis was indistinguishable between p-S9X and control Discussion Insulin, along with several other hormones and neuropeptides, is responsible for the proper maintenance of glucose homeostasis. Due to its great physiological effects in regulating glucose clearance from the blood, insulin secretion is tightly regulated. Insulin granule exocytosis can be triggered by a wide range of calcium concentrations [11]. Therefore calcium sensing in b-cells is expected to be complex, and may involve more than one calcium sensors with appropriate calcium affinity and cooperativ- ity to respond to various intracellular calcium levels during a stimulus or under different stimuli. By analogy to neurotransmitter release, we hypothesized that insulin secretion is controlled by at least two types of calcium sensors: a low affinity sensor responsible Figure 3. Glucose-stimulated insulin secretion from p-S9X mouse islets showed the same pattern as in control mouse islets. (A) Insulin secretion from batches of 5 similar-sized islets in perifusion experiments in response to 20 mM glucose (arrow). N = 20 for control mouse islets (open circles), and 16 for p-S9X mouse islets (filled circles). (B) Insulin secretion during the first 15 minutes of stimulation (first phase) and over the entire stimulation period (total), calculated as sum of insulin levels in all fractions after baseline subtraction. (C) Insulin contents in isolated individual pancreatic islets from p-S9X and control mice. Data are presented as means 6 SEM. N = 14 for control (white bar) and 19 for p-S9X (grey bar). doi:10.1371/journal.pone.0015414.g003 PLoS ONE | www.plosone.org 5 November 2010 | Volume 5 | Issue 11 | e15414