Supplementary MaterialsESM Methods: (PDF 115 kb) 125_2014_3226_MOESM1_ESM. granule exocytic events. Compared with WT, islets showed far fewer exocytic events (an 83% decline at 15?mmol/l glucose). This decline was due to a 73% loss of responding cells and, in the remaining responsive cells, a 50% loss of exocytic responses per cell. An assay measuring granule re-acidification showed evidence for more recaptured granules in islets compared with WT. Conclusions/interpretation We showed that islets had a reduced calcium response to glucose and a reduction in syntaxin-1A. Within the islets, changes were manifest as both a reduction in responding cells and a reduction in fusing insulin granules per cell. Electronic supplementary material The online version of this article (doi:10.1007/s00125-014-3226-8) contains peer-reviewed but unedited supplementary material, which is available to authorised users. leptin receptor mutant mouse is usually a model for type 2 diabetes [1]. The animals gain weight and develop insulin intolerance and reduced insulin secretion [2]. The explanation for this reduction in secretion, as in type 2 diabetes, is still not clear, but may include: loss of islets; loss of beta cells [3]; loss of insulin content [2, 4, 5]; or reduced exocytic capacity. Direct comparison shows the proportionate loss of secretion parallels the loss of insulin content: 4.5- to sixfold for both [4, 5]. However, even though content is usually reduced, there is still more insulin present than that required to maintain normoglycaemia [5]. This suggests defects in insulin secretion are important in the disease; an idea supported by work in humans [6, 7] and in other models Rabbit Polyclonal to TBX2 of type 2 diabetes, such as the Goto-Kakizaki (GK) rat [8, 9]. The reduced insulin secretion could arise because of defects anywhere along the complex stimulus-secretion cascade. In mice there is evidence for reduced GLUT2 expression [10, 11], and reduced ATP production [12]. The next step in the cascade is the calcium response. In mice, both the size [12] and the temporal profile of the calcium response are altered [13]. Most recently, there is evidence, in other disease models, for any mis-positioning of calcium channels relative to the sites of insulin granule fusion [8, 14]. The final step of granule fusion is dependent around the soluble islets mRNA for synaptosomal-associated protein 25 (SNAP25) and vesicle-associated membrane protein 2 (VAMP2) increase GDC-0941 enzyme inhibitor and syntaxin-1A mRNA decreases [15]. This contrasts with the GK rat, in which all these SNAREs decrease GDC-0941 enzyme inhibitor [9, 16]. Functional studies in a reduction be showed with the GK rat in insulin granule fusion [17]. It’s been recommended that different settings of fusion may be widespread in types of severe disease that make use of high-glucose or palmitate publicity [18, 19]. Right here, transient granule fusion, termed kiss-and-run often, could preferentially discharge low molecular mass substances in support of discharge insulin [20] partially; if this is the widespread type of granule fusion in disease this may explain the decrease in insulin secretion. Nevertheless, the relevance of complete fusion or kiss-and-run fusion in islets is certainly questioned [21, feasible and 22] roles aren’t known in pet types of disease. Within the unchanged islet it really is known the fact that GDC-0941 enzyme inhibitor framework and physical romantic relationships between your cells are essential for secretion [23], and so are factors that transformation in disease [3, 5]. To comprehend beta cell secretory function in a islet we’ve created a live-cell two-photon assay to measure single-insulin-granule fusion occasions [24]. We’ve validated this technique on wild-type (WT) islets to verify it methods insulin granule fusion which the time training course and variety of fusion occasions fully take into account the measured quantity of insulin secretion [24]. As the technique does not have the temporal quality of capacitance dimension [25], its essential advantage is certainly it information replies from all cells inside the two-photon image slice. Using this method, we show here the secretory deficit in islets is definitely primarily due to a loss of responding cells and a reduction in granule (full) fusion in the remaining responsive cells. Methods Experimental solution.