br Materials and methods br Results

Materials and methods
Results
Discussion The incretin hormones potently stimulate insulin secretion, and it is generally assumed that this is mediated by an endocrine mechanism such that GIP and GLP-1 through the circulation reach the islet beta AR-13324 sale to stimulate their secretion. However, the rapid and extensive degradation of newly released GLP-1 and GIP (Deacon et al., 1995a, Deacon et al., 1995b, Vilsboll et al., 2006) has suggested that an indirect neural mechanism may contribute to the direct effect achieved by the circulating hormones (Ahrén, 2014). In fact, at the site of incretin hormone secretion and in the portal circulation there are alternative pathways for incretin hormone action (Bucinskaite et al., 2009). Previous studies provide evidence for the ability of incretin hormones to activate vagal nerve fibers (Balkan and Li, 2000, Ahrén, 2004), which would result in increased insulin secretion. Therefore, in this study we examined the role of vagal nerve signaling for incretin hormone action by blocking muscarinic receptors. The main finding was that blocking vagal nerve signaling reduced the insulin response to DPP-4 inhibition and to exogenous GLP-1 and GIP, whereas no such effect was observed in vitro in isolated islets and no effects of atropine was seen on intact GLP-1 and GIP levels. In order to study the effects of DPP-4 inhibition after atropine administration, it was necessary to initially develop an experimental model in which glucose was given directly in the duodenum, since atropine alters gastric emptying (Parkman et al., 1999) and such an effect is bypassed in this novel model. We therefore developed the DGGT model in anesthetized mice in which glucose was administered directly into the duodenum. For ethical reasons, the experiments were performed under anesthesia but the anesthesia used has been shown not to affect plasma glucose and insulin levels (Zuurbier et al., 2014). Therefore, although effects of the anesthesia on glucose or insulin levels or vagal activity can not be excluded we have minimized this limitation. The novel DGTT increased both glucose and insulin levels and, when compared to an isocaloric OGTT (Pacini et al., 2013), similar plasma glucose profiles were observed, although, the peak insulin response was slightly exaggerated and delayed with the DGTT. In response to duodenal glucose and in the presence of DPP-4 inhibition, mice that were given atropine had reduced insulin secretion. This indicates that part of the insulinotropic effect of DPP-4 inhibition, which acts to elevate endogenous intact incretin hormones, is dependent on activation of incretin hormone sensitive nerve fibers. In support of this, it was previously shown that the insulinotropic effects of a low dose sitagliptin, which causes elevations of local, but not circulating, intact incretin hormones, was abolished when antagonizing the GLP-1 receptor (Waget et al., 2011). Similarly, low doses of vildagliptin, insufficient to increase circulating incretin hormones, were shown to improve glycemia and insulin secretion (Omar and Ahrén, 2014). These current and past findings therefore suggest that incretin hormones, at the site of release and/or along the circulatory route to the pancreas, are able to increase insulin levels via triggering of vagal nerve signaling and this mechanism is operative during DPP-4 inhibition. The role for the vagus nerve in the secretion of incretin hormones is debated. It has been suggested that the vagus nerve is involved in the secretion of GLP-1 (Rocca and Brubaker, 1999, Deacon and Ahrén, 2011), whereas other studies have shown no effect of vagal nerve signaling on GLP-1 secretion (Ahrén and Holst, 2001, Hansen et al., 2004). Since the effects of DPP-4 inhibition is dependent on incretin hormone secretion, by blocking vagal nerve signaling, reduced levels of intact incretin hormones could therefore potentially explain the reduced insulin levels. However, atropine did not affect the increase in intact GLP-1 or intact GIP levels during duodenal feeding. Therefore, it could be excluded that reduction in insulin levels by atropine during DPP-4 inhibition was caused by reduced incretin hormone levels.