The relevance of depolarizing and non-depolarizing stimuli for the biphasic kinetics of insulin secretion.The role of the metabolic amplification.

在这个项目中，我们打算进一步深入了解胰岛素分泌的双相动力学的基础机制，这是一个可以在人类胰岛和啮齿动物胰岛中观察到的特征。已知去极化诱导的Ca2+内流对于刺激的胰岛素分泌是必需的，然而，刺激分泌的程度由另外的、目前不完全理解的信号决定。这些信号被概括为"放大途径"或"代谢放大"。胰腺β-细胞中的刺激-分泌-偶联具有一个特定的功能：只有那些同时也是β-细胞营养物的刺激（"燃料促分泌素"）才能够诱导分泌的持续增加。营养促分泌素必须代谢才能刺激胰岛素分泌，这一事实导致放大途径中存在大量令人困惑的潜在信号化合物。用新鲜分离的胰岛进行的实验使我们假设乙酰乙酸从线粒体到胞质溶胶的cataplerotic出口和随后的胞质乙酰辅酶A浓度的增加在代谢放大的过程中可能具有信号传导功能。胞质乙酰辅酶A如何精确地导致分泌增加是一个悬而未决的问题，但某些蛋白质的可逆乙酰化似乎是一个合理的机制。这种可逆的乙酰化作用已经在许多蛋白质中得到描述。有趣的是，肌动蛋白细胞骨架的调节因子可以在这些蛋白质中找到。这就引出了我们在这个项目中试图回答的以下问题：1.乙酰乙酸输出的信号作用和由此产生的细胞溶质乙酰辅酶A的增加能否被证实？2.细胞骨架的功能是否受到某些调节蛋白可逆乙酰化的影响？3.最后，这种乙酰化与胰岛素颗粒的流动性和融合频率之间是否存在联系？

In this project we intend to gain further insight into the mechanisms which underlie the biphasic kinetics of insulin secretion, a feature which can be observed with human islets as well as with rodent islets. The depolarization-induced influx of Ca2+ is known to be necessary for the stimulated insulin secretion, however, the extent to which secretion is stimulated is determined by additional, currently incompletely understood signals. These signals are summarized as “amplifying pathway” or “metabolic amplification”. The stimulus-secretion-coupling in the pancreatic beta-cell has one specific feature: only those stimuli which are also nutrients for the beta-cell (“fuel secretagogues”) are able to induce a lasting increase of secretion. The fact that nutrient secretagogues have to be metabolized in order to stimulate insulin secretion has led to a bewildering multitude of potential signaling compounds in the amplifying pathway. Experiments with freshly isolated islets have led us to the hypothesis that the cataplerotic export of acetoacetate from the mitochondria to the cytosol and the ensuing increase of the cytosolic acetylCoA concentration could have a signaling function in the course of the metabolic amplification. How precisely cytosolic acetylCoA leads to increased secretion is an open question, but the reversible acetylation of certain proteins appears as a plausible mechanism. Such a reversible acetylation has been described for a number of proteins. Interestingly, regulators of the actin cytoskeleton can be found among these proteins. This leads to the following questions which we seek to answer in this project: 1. Can a signaling role of the export of acetoacetate and the resulting increase of cytosolic acetylCoA be demonstrated? 2. Is the function of the cytoskeleton affected by the reversible acetylation of certain regulatory proteins? 3. Finally, is there a relation between this acetylation and the mobility and fusion frequency of insulin granules?