Regulation of Glucagon Secretion from Pancreatic Islets

胰岛通过调节激素分泌在葡萄糖稳态中起关键作用。胰岛研究集中在分泌胰岛素的β细胞上，尽管另一种胰岛激素--α细胞的胰高血糖素的异常分泌会加剧糖尿病的病理。通过胰高血糖素受体拮抗剂使胰高血糖素作用正常化可以帮助糖尿病患者维持血糖正常并避免低血糖发作，但这种方法的进展因许多副作用而减慢。另一种方法是减少胰高血糖素分泌，但控制其胞吐作用的机制仍有争议。我们实验室和其他实验室的最新数据表明，长期以来对α细胞Ca 2+信号传导的关注可能是误导性的，胰高血糖素分泌的调节需要通过多种途径进行信号传导。这种复杂性推动了一种创新的研究策略，我们整合了专注于单个组件的机械实验数据，同时还测试了途径之间可能的串扰。在I型糖尿病中，β细胞被破坏后，胰高血糖素分泌的葡萄糖调节（GRGS）在体内丧失，这表明胰岛细胞类型之间的相互作用对α细胞功能至关重要。这导致了 旁分泌信号传导，其中来自胰岛β细胞和δ细胞的分泌因子限制α细胞功能。我们已经证明胰岛素和生长抑素协同作用，降低cAMP和PKA活性，从而降低胰高血糖素分泌，但其本身不能解释GRGS。初步数据表明，复合蛋白2在连接PKA活性与分泌中起关键作用。我们还表明，一种新的肾上腺素途径，EphA 4/7正向信号传导，被β细胞表面上的ephrinA 5配体激活。这种作用导致F-肌动蛋白聚合和胰高血糖素分泌减少，通过RhoA激活而增加，并且它似乎具有葡萄糖调节组分。因此，旁分泌和外分泌途径都驱动胰岛的GRGS，但用ephrinA 5处理的分散的α-细胞也表现出额外的GRGS机制，这似乎是α-细胞固有的。基于这些数据，我们假设，GRGS需要协同组合的旁分泌，adaptacrine，和细胞内在的信号传导途径。该假设将通过三个具体目的进行测试：1）确定旁分泌介导的PKA激活的复合蛋白2磷酸化在胰岛素和生长抑素介导的胰高血糖素分泌抑制中的作用; 2）确定RhoA激活在胰高血糖素分泌抑制中的作用; 3）确定RhoA激活在胰高血糖素EphA 4/7前向调节中的作用。 导致胰高血糖素分泌抑制的信号传导; 3）确定EphA 4/7正向信号传导在对葡萄糖的内在α细胞应答中的作用。我们发现的多种细胞内和细胞间信号传导机制将通过精确观察活细胞和胰岛中相关动力学的方法来阐明。该研究计划还将利用我们的发现，即GRGS的机制在小鼠和人类α细胞中是相似的，我们将尽可能在不同物种之间进行平行实验。这些实验将进一步加深我们对α细胞功能的理解，这是发现胰高血糖素调节和糖尿病治疗新的潜在靶点的关键一步。

The islet of Langerhans plays a key role in glucose homeostasis through regulated hormone secretion. Islet research has focused on the insulin-secreting β-cells, even though aberrant secretion of another islet hormone, glucagon from α-cells, exacerbates the pathology of diabetes. Normalization of glucagon action by glucagon receptor antagonism can help diabetic patients maintain euglycemia and avoid hypoglycemic episodes, but progress on this approach has been slowed by numerous side-effects. An alternate approach would be to reduce glucagon secretion, but the mechanism controlling its exocytosis remains controversial. Recent data from our lab and others suggest that the long-standing focus on α-cell Ca2+ signaling may have been misleading, and that regulation of glucagon secretion requires signaling through multiple pathways. This complexity drives an innovative research strategy where we integrate data from mechanistic experiments focused on individual components, while also testing for possible cross-talk between pathways. The loss of glucose-regulation of glucagon secretion (GRGS) in vivo after β-cells are destroyed in Type I diabetes suggests that interactions between islet cell types are critical to α-cell function. This has led to models of paracrine signaling where secreted factors from islet β- and δ-cells constrain α-cell function. We have shown that insulin and somatostatin work in concert to reduce cAMP and PKA activity, which lowers glucagon secretion, but does not by itself explain GRGS. Preliminary data point to a key role for complexin 2 in linking PKA activity to secretion. We have also shown that a novel juxtacrine pathway, EphA4/7 forward signaling, is activated by ephrinA5 ligands on the β-cell surface. This effect leads to F-actin polymerization and decreased glucagon secretion, putatively via RhoA activation, and it appears to have a glucose-regulated component. Thus, both paracrine and juxtracrine pathways drive GRGS from islets, but dispersed α-cells treated with ephrinA5 also exhibit an additional GRGS mechanism, which appears to be intrinsic to the α-cell. Based on these data, we hypothesize that GRGS requires a synergistic combination of paracrine, juxtacrine, and cell-intrinsic signaling pathways. This hypothesis will be tested via three specific aims: 1) Determine the role of paracrine-mediated PKA-activated phosphorylation of complexin 2 in insulin- and somatostatin-mediated inhibition of glucagon secretion; 2) Determine the role of RhoA activation in the juxtacrine EphA4/7 forward signaling that leads to inhibition of glucagon secretion; 3) Determine the role of EphA4/7 forward signaling in intrinsic α-cell response to glucose. The multiple intracellular and intercellular signaling mechanisms that we are uncovering will be elucidated by methods that allow precise observation of the pertinent dynamics in living cells and islets. The research plan will also leverage our findings that the mechanisms of GRGS are similar in mouse and human α-cells, and we will perform parallel experiments across species to the extent possible. These experiments will further our understanding of α-cell function, which is a critical step towards discovering new potential targets for the regulation of glucagon and treatment of diabetes.