Regulation of Glucagon Secretion from Pancreatic Islets

郎格汉斯胰岛通过调节激素分泌，在血糖稳态中起着关键作用。胰岛研究一直集中在分泌胰岛素的β细胞上，尽管α细胞分泌的另一种胰岛激素--胰升糖素的异常分泌会加剧糖尿病的病理。用胰升糖素受体拮抗剂使胰升糖素作用正常化可以帮助糖尿病患者维持正常血糖，避免低血糖发作，但由于存在许多副作用，这一方法的进展缓慢。另一种方法是减少胰升糖素的分泌，但控制其胞吐的机制仍存在争议。来自我们实验室和其他实验室的最新数据表明，长期以来对α-细胞钙信号的关注可能具有误导性，并且对胰高血糖素分泌的调节需要通过多个途径来传递信号。这种复杂性推动了一种创新的研究策略，在这种策略中，我们整合了专注于单个组件的机械实验的数据，同时还测试了路径之间可能的串扰。I型糖尿病患者β细胞被破坏后，体内葡萄糖调节的胰升糖素分泌功能丧失，提示胰岛细胞类型之间的相互作用对α细胞的功能至关重要。这导致了模型的出现 胰岛β和δ细胞分泌因子的旁分泌信号限制了α细胞的功能。我们已经证明，胰岛素和生长抑素协同作用，减少cAMP和PKA活性，从而降低胰高血糖素的分泌，但本身并不能解释GRGS。初步数据表明，复合素2在将PKA活性与分泌联系起来的过程中发挥了关键作用。我们还发现了一个新的旁分泌途径，即EphA4/7正向信号转导，它被β-细胞表面的ephinA5配体激活。这种作用导致F-肌动蛋白聚合和胰升糖素分泌减少，推测是通过激活RhoA实现的，而且它似乎有一个受葡萄糖调节的成分。因此，旁分泌和旁分泌途径都驱动胰岛的GRG，但分散的α细胞也表现出额外的GRGS机制，这似乎是α细胞所固有的。基于这些数据，我们假设GRGS需要旁分泌、旁分泌和细胞内在信号通路的协同组合。这一假说将通过三个特定的目的来验证：1)确定旁分泌介导的PKA激活的复杂蛋白2磷酸化在胰岛素和生长抑素介导的抑制胰升糖素分泌中的作用；2)确定RhoA激活在旁分泌EphA4/7中的作用 3)确定EphA4/7正向信号在α细胞对葡萄糖的内在反应中的作用。我们正在揭示的多种细胞内和细胞间信号机制将通过允许精确观察活细胞和胰岛中的相关动态的方法来阐明。该研究计划还将利用我们的发现，即GRGS在小鼠和人类α细胞中的机制相似，我们将尽可能跨物种进行平行实验。这些实验将进一步加深我们对α细胞功能的理解，这是为调节胰高血糖素和治疗糖尿病发现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.