Microtubule Regulation of Pancreatic Beta Cell Function and Diabetes

在胰岛β细胞中，胰岛素颗粒(IG)形成于细胞质深处的高尔基复合体。它们需要主动地从生产现场运输到质膜下，以调节分泌。以前的假设是MT依赖的运输定向地将IGs沿着笔直的微管(MT)轨迹运送到细胞外周，从而预测MT在胰岛素分泌中的积极作用。然而，我们在前一个捐赠周期的数据显示，MT依赖的转运限制了预先存在的IGs的分泌，而IGs通常存在于过多的数量中。我们还发现，这种负调控功能归因于β-cell MT的独特结构。在许多其他分泌细胞中，MT是由 中心体，并组织成径向轨道，允许定向货物移动。相反，大多数β细胞MT在高尔基体(高尔基体衍生MT，GDMT)处成核，在细胞内部以致密的非方向性网络组织，细胞外围有稳定的亚膜MT束。实验测试和数学模型表明，这种配置导致将IGs捕获在细胞质中，将它们储存起来，以便在β细胞长期功能期间持续释放胰岛素，以避免胰岛素不足引发的糖尿病。这一功能还可以防止胰岛素在每种刺激下过度分泌，以避免高胰岛素血症引起的低血糖。重要的是，我们发现葡萄糖刺激触发MT网络的重新配置：它通过cAMP/Epac2介导的信号诱导新的GDMT成核，这是新的IG生物合成所必需的。它还通过磷酸化微管相关蛋白(MAP)tau来诱导β细胞外周MT的分解，从而增强胰岛素的分泌。然而，负责β细胞MT组织及其作用于葡萄糖下游的很大一部分细胞内机制仍然难以捉摸。有趣的是，我们的初步数据表明，葡萄糖诱导的MT重塑依赖于胰岛微环境。在这个提议中，我们将检验一个假设，即受细胞内分子机制和细胞间旁分泌信号调控的β-细胞MT网络的最佳动态结构对于β-细胞功能和葡萄糖稳态是必不可少的。通过我们的具体目标，我们将：(1)研究β细胞中独特的MT网络的建立和调控；(2)确定MT对β细胞中IG转运和定位的依赖调节；(3)确定MT在胰岛α-β细胞旁分泌通讯中的作用。

In pancreatic islet β cells, insulin granules (IG) are formed at the Golgi complex deep inside the cytoplasm. They need to be actively transported from the site of production to underneath the plasma membrane for regulated secretion. It was previously assumed that MT-dependent transport directionally delivers IGs to the cell periphery along the straight microtubule (MT) tracks, predicting a positive role of MTs in insulin secretion. However, our data over the previous grant cycle show that MT-dependent transport restricts secretion of pre-existing IGs, which are normally present in excessive numbers. We have also found that this negative regulatory function was ascribed to the unique configuration of β-cell MTs. In many other secretory cells, MTs are assembled from the centrosome and organized as radial tracks that allow directional cargo movement. In contrast, most β-cell MTs are nucleated at the Golgi (Golgi-derived MTs, GDMTs) and are organized as a dense, non-directional meshwork in the cell interior, with addition of stable sub-membrane MT bundles at the cell periphery. Experimental tests and mathematical modeling indicate that this configuration leads to trapping of IGs within the cytoplasm, storing them for sustainable insulin release during long-term β-cell function to avoid insulin-insufficiency-induced diabetes. This function also prevents insulin over-secretion at each stimulus to avoid hyperinsulinemia-induced hypoglycemia. Importantly, we show that glucose stimulus triggers reconfiguration of the MT networks: it induces new GDMT nucleation via the cAMP/EPAC2-mediated signals, which is essential for new IG biosynthesis. It also induces MT disassembly in β-cell periphery to enhance insulin secretion by phosphorylating tau, a well-established microtubule associated protein (MAP). However, a big part of intracellular mechanisms that are responsible for β-cell MT organization and its action downstream of glucose remains elusive. Intriguingly, our preliminary data suggest that glucose-induced MT remodeling depends on the islet microenvironment. In this proposal, we will test a hypothesis that optimal dynamic architecture of the β-cell MT networks, modulated by intracellular molecular machinery and intercellular paracrine signals, is essential for β-cell function and glucose homeostasis. By addressing our Specific Aims, we will: (1) investigate building and regulating the unique MT networks in β cells, (2) determine MT-dependent regulation of IG transport and positioning in β cells, and (3) determine roles of MTs in paracrine α-β cell communication in islets.