Regulation of spatial organization and cell-cell communication in the islet of Langerhans

郎格汉斯胰岛中不同类型内分泌细胞的协调激素分泌是维持血糖稳态的关键。在肥胖、胰岛素抵抗和糖尿病中，空间胰岛组织和细胞-细胞通讯被破坏，导致协调激素调节的丧失。尽管胰岛组织和细胞-细胞通讯在胰岛功能中起着关键作用，但针对它们作为糖尿病潜在治疗方法的药物尚未开发出来。环状交叉口(ROBO)受体是胰岛表达的细胞黏附蛋白。在胰岛素分泌中，ROBO受体可以将细胞间的接触与细胞骨架的动态和囊泡的运输联系起来。在肥胖和糖尿病中，小鼠和人类胰岛中Robo的表达严重减少。我们最近发现，在小鼠的β细胞中，Robo1和Robo2的缺失会导致胰岛组织的丧失和糖耐量的降低。我们进一步证明，在β细胞选择性缺失Robo时看到的胰岛表型并不是由于β细胞向α细胞或三角洲细胞的转分化，也不是β细胞成熟丧失或β细胞死亡的结果。相反，我们的结果使我们假设，在β细胞中表达Robo有助于正确的胰岛组织，这是内分泌细胞-细胞交流和正确的葡萄糖反应所必需的。根据这一假说，肥胖时Robo的下调扰乱了胰岛的组织，导致细胞间通讯的丧失，从而导致糖反应功能障碍。了解Robo在β细胞中的表达如何调节胰岛组织和激素分泌，将为糖尿病的新药理学方法提供基础。我们将通过两个特定的目标来检验上述假设：在目标1中，我们将检验β细胞中Robo基因缺失会损害胰岛素和胰升糖素分泌的假说。我们将通过活体内观察同步的胰岛素分泌来进一步检验Robo基因的缺失破坏了功能上的β细胞-β细胞连接的假说。我们还将验证Robo通过调节内分泌细胞-细胞黏附来调节激素分泌的假设。在目标2中，我们将确定Robo在β细胞中的表达如何控制胰岛的组织，并确定Robo配体Sit在这一过程中的参与。我们将进一步确定β细胞中的信号通路和下游的Robo靶点，这可能是胰岛组织和糖耐量缺陷的原因。这些数据将在三个翻译方面对糖尿病产生重要影响：1)Robo信号可能被操纵以防止和恢复2型糖尿病患者胰岛组织和内分泌细胞-细胞通讯的恶化；2)Robo信号可用于在体外人类多能干细胞来源的胰岛样簇中提供正确的3D组织和细胞-细胞接触，以产生无限来源的真实胰岛用于移植；以及3)Robo受体及其细胞外配体Sit可用于生物工程方法，以开发能够支持临床胰岛移植的身体胰岛的结构完整性、生存和功能的基质支架。

Coordinated hormone secretion from the different endocrine cell types in the islets of Langerhans is critical to glucose homeostasis. Spatial islet organization and cell-cell communication are disrupted in obesity, insulin resistance and diabetes, leading to loss of coordinated hormone regulation. Despite the critical roles of islet organization and cell-cell communication in islet function, drugs that target them as potential therapies to diabetes have not yet been developed. Roundabout (Robo) receptors are cell adhesion proteins expressed in the islet. Robo receptors can link cell-cell contact to cytoskeleton dynamic and vesicle transport in insulin secretion. The expression Robo in the islets of mice and humans are severely diminished in obesity and diabetes. We have recently found that deletion of Robo1 and Robo2 in beta cells in mice results in loss of islet organization and impaired glucose tolerance. We further demonstrated that the islet phenotype seen upon beta cell-selective deletion of Robo is not due to transdifferentiation of beta cells to alpha or delta cells, nor is it the result of loss of beta cell maturation or beta cell death. Rather, our results led us to hypothesize that expression of Robo in beta cells facilitates correct islet organization which is required for endocrine cell-cell communication and correct glucose response. According to this hypothesis, the downregulation of Robo in obesity disrupts islet organization, leading to loss of cell-cell communication, thus contributing to dysfunctional glucose response. Understanding how expression of Robo in beta cells regulates islet organization and hormone secretion will provide the basis for new pharmacological approaches to diabetes. We will test the above hypothesis with two specific aims: in Aim 1, we will test the hypothesis that deletion of Robo in beta cells impairs insulin and glucagon secretion. We will further test the hypothesis that deletion of Robo disrupts functional beta cell-beta cell coupling using intravital visualization of synchronized insulin secretion. We will also test the hypothesis that Robo regulates hormone secretion through mediating endocrine cell-cell adhesion. In Aim 2, we will determine how expression of Robo in beta cells controls islet organization, and determine the involvement of the Robo ligand, Slit, in this process. We will further identify signaling pathways and downstream Robo targets in beta cells which could account for the defects in islet organization and glucose tolerance. These data will have important impact on diabetes in three translational aspects: 1) Robo signaling may be manipulated to prevent and restore the deterioration in islet organization and endocrine cell-cell communication in type-2 diabetics; 2) Robo signaling may be used to confer correct 3D organization and cell-cell contact in human pluripotent stem cells-derived islet-like clusters in vitro, to generate an unlimited source of bona fide islets for transplantation, and 3) Robo receptors and their extracellular ligand, Slit, may be used in bioengineering approaches for developing matrix scaffolds that could support structural integrity, survival and function in cadaver islets in clinical islet transplantation settings.