synaptotagmin-9 functions as a low-affinity calcium sensor in insulin granule exocytosis. An earlier study showed insulin secretion was reduced by ,30% in isolated rat islets with synaptotagmin-9 knockdown by adenoviral infection [20]. The apparent discrepancy between this and our present study may be due to our choice of the experimental subjects, as the pattern of glucose-induced insulin secretion is not identical in rat and mouse [21,22]. It is possible that distinct patterns of calcium response in rat and mouse may result in different calcium levels, and thus requiring different calcium sensing proteins. The discrepancy could also be due to our choice in deleting synaptotagmin-9: genetic knockout vs. adeno- virus-mediated RNAi, which may have undesired off-target effects [23]. The lack of effect by synaptotagmin-9 deletion in the pancreas could be the result of functional compensation by another synaptotagmin. We first generated synaptotagmin-7 and -9 double KO (DKO) mice, and compared insulin secretion in the DKO, synaptotagmin-7 KO and wild type control mice (refer to Materials and Methods S1). Although both DKO and synapto- tagmin-7 KO mice showed impaired insulin secretion when compared with control mice, there was no further impairment in insulin secretion in DKO mice than in synaptotagmin-7 KO mice (Figure S3). In addition to synaptotagmin-7 and -9, which are expressed at high levels in pancreatic islets, two other calcium- binding synaptotagmins, synaptotagmin-3 and -5, are expressed at moderate levels in the islets [15]. We evaluated the expression of synaptotagmin-3, -5 and -7, but did not detect an upregulation of these synaptotagmins in p-S9X mice (data not shown). These results demonstrate that the lack of effect in insulin secretion by synaptotagmin-9 deletion was unlikely due to functional compen- sation, leading to the conclusion that synaptotagmin-9 is not a regulator of insulin secretion. Besides synaptotagmins, other C2-containing proteins may participate in the regulation of insulin secretion. Previous studies have shown a potential role of the calpain family in insulin secretion [24,25], possibly through partial proteolysis of SNAP-25 for the first phase [5] and ICA512 proteolysis for the second phase [26]. In conclusion, our results show that synaptotagmin-9 does not regulate glucose homeostasis or glucose-stimulated insulin secre- tion in mice. Our current efforts are focused on identifying the References 1. Henquin JC, Nenquin M, Ravier MA, Szollosi A (2009) Shortcomings of current models of glucose-induced insulin secretion. Diabetes Obes Metab 11 (Suppl 4): 168–179. 2. Ashcroft FM, Proks P, Smith PA, Ammala C, Bokvist K, et al. 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(EPS) Figure S2 Normal calcium response in p-S9X mouse islets. Representative calcium responses induced by a rise in glucose concentration from 3 to 20 mM in control (red trace) and p-S9X (black trace) mouse islets (N = 23 and 26, respectively). P- S9X and control islets showed similar calcium response pattern with regard to timing of response (lag time) and amplitude of calcium rise (see RESULTS section for mean values and SEM). (EPS) Figure S3 Comparison of insulin secretion in control, synaptotagmin-7 KO and synaptotagmin-7/-9 DKO mouse islets. Insulin secretion in response to 20 mM glucose was measured in batches of 5 similar-sized islets during the first 15 minutes (A, first phase) and the second 15 minutes (B, second phase). N = 18, 39 and 28 for control (Ctrl., white bar), synaptotagmin-7 KO (Syt72/2, grey bar) and synaptotagmin-7/ -9 DKO (DKO, black bar), respectively. Data are presented as means 6 SEM. Statistics is indicated on the graph. NS: not significant. 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