br Materials and methods br Results br Discussion Several st

Materials and methods
Results
Discussion Several studies highlighted an improvement of glycaemic control in diabetic patients receiving sunitinib, while the underlying mechanism remained still elusive [3], [4], [5], [6], [8]. The present study gives evidence that sunitinib directly and specifically stimulates insulin secretion. Firstly, sunitinib concentration-dependently stimulated GIIS without affecting basal release. Secondly, the effect was inhibited by adrenaline, a potent physiological inhibitor of insulin secretion [24]. These results allow the conclusion that the stimulatory effect of sunitinib on insulin secretion is direct and specific, via regulated signalling pathways, excluding an uncontrolled release of insulin due to a potentially toxic effect. Most interestingly, the stimulatory effect of sunitinib on insulin secretion was additive to the effects of FFAR1 activation and increase in cAMP by forskolin. The FFAR1 agonist TUG-469 was preferred to stimulate FFAR1, since the physiological FFAR1 agonists, i.e. long chain fatty acids, stimulate secretion not only via FFAR1 but also via metabolites [25], [26]. TUG-469 stimulates insulin secretion in a glucose-dependent manner in rodent and human islets and in INS-1E Dyphylline ([27] and Fig. 2). The stimulatory effect of sunitinib, i.e., an increase of insulin release of about 4% of the insulin content, was not affected by TUG-469. This suggests that sunitinib acts on an additional, FFAR1-independent signalling pathway which potentiates FFAR1-mediated phospholipase C activation, intracellular Ca release and DAG-dependent protein kinase C as well as protein kinase D1 stimulation [28], [29]. An important signalling pathway which augments insulin release in a glucose-dependent manner is the stimulation of adenylyl cyclase and the increase of cellular cAMP levels [30]. Thus, GLP-1R agonists, i.e. GLP-1 and exendin-4, do not stimulate basal secretion but potentiate GIIS in rodent and human islets [31], [32]. The INS-1E cell clone used in this study revealed a poor GLP-1R responsiveness although the cells express GLP-1R (unpublished observation). However, IBMX and forskolin, which specifically increase cellular cAMP levels, potentiated GIIS. Sunitinib had an additional effect on secretion in the presence of 1μM forskolin. In addition, the TKI did not increase cAMP levels in the presence of IBMX, indicating that sunitinib does not stimulate adenylyl cyclase. Since sunitinib further increased insulin secretion in the presence of IBMX it is also unlikely that its action implies an inhibition of phosphodiesterase. These results strongly suggest that sunitinib does not modulate cellular cAMP levels in insulin secreting cells. Furthermore, sunitinib did not alter phosphorylation of PKA at Thr197 although H89, a PKA inhibitor, abrogated sunitinib stimulation of insulin release but not the EPAC inhibitor ESI-05. The cAMP antagonist, Rp-8-bromo-cAMP, only attenuated the effect of sunitinib on secretion, probably due to its restricted membrane permeability [33]. So far, we have no evidence for a direct effect of the TKI on PKA or cellular cAMP levels. Our observations suggest a more distal effect, likely on PKA targets of the exocytotic machinery. Well studied targets of sunitinib are VEGFR and PDGFR [2]. Interestingly, upon activation by cAMP, PKA can be recruited to VEGFR2 via the scaffolding protein AKAP1 [34]. VEGFR1/2 is a tyrosine kinase reported to reduce islet inflammation and ameliorate β-cell function in rodent type-1 diabetic models [35]. There is a discrepancy between the improved insulin secretion in VEGF overexpressing mice which involves increased islet vascularisation and the beneficial effect of TKI on insulin secretion. Our study strongly suggests that sunitinib exerts a direct effect on β-cells, as the experiments were performed with a clonal β-cell line in the absence of any other cell type including endothelial cells. Previous studies report that sunitinib down-regulates expression of the insulin-like growth factor receptor-1 (IGF-1R), a tyrosine kinase receptor that sustains tumour growth but also β-cell function [20]. Insulin-dependent tyrosine phosphorylation of Munc18c, a regulator of SNARE-mediated vesicle fusion, has been reported to affect exocytosis and insulin secretion [36], [37]. The present results suggest that sunitinib does not impair IGF-1 action in β-cells, since it did not reduce IGF-1-mediated stimulation of PKB and ERK1/2 phosphorylation. Intact insulin and IGF-1 receptor signalling are essential for proper β-cell function and survival [22], [38], therefore a negative effect of sunitinib on IGF-1R is unlikely, as it would not improve but accentuate diabetes.