PANCREATIC ISLETS DYNAMICS REGULATING GLUCAGON SECRETION

朗格汉斯胰岛通过调节激素分泌在葡萄糖稳态中起关键作用。胰岛的研究主要集中在分泌胰岛素的β-细胞上，尽管另一种胰岛激素——来自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的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的分泌，但控制其胞吐的机制仍有争议。最近来自我们实验室和其他人的数据表明，长期以来对α-细胞Ca²+信号的关注可能被误导了，胰高血糖素分泌的调节需要通过多种途径发出信号。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，同时也测试了路径之间可能的串扰。1型糖尿病患者β-细胞被破坏后体内胰高血糖素分泌抑制功能（GIGS）的丧失表明，胰岛细胞类型之间的相互作用对α-细胞功能至关重要。这导致了胰岛β和δ细胞分泌因子约束α细胞功能的旁分泌信号模型。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的分泌，但本身并不能解释GIGS。初步数据表明，复杂蛋白2在连接PKA活性和分泌方面起着关键作用。我们还发现了一种新的近胞嘧啶途径，EphA4/7正向信号通路，可被β细胞表面的ephrinA5配体激活。这导致f -肌动蛋白聚合和胰高血糖素分泌减少，推测是通过RhoA激活。因此，旁分泌和近分泌途径都能驱动胰岛的GIGS，但经ephrinA5处理的分散α-细胞也表现出额外的GIGS机制，这似乎是α-细胞固有的。基于这些数据，我们假设GIGS需要旁分泌、近分泌和细胞内在信号通路的协同组合。这一假设将通过三个特定目的进行验证：1)确定旁分泌介导的paca激活的复合物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激活在近分泌EphA4/7正向信号通路抑制胰高血糖素分泌中的作用；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在活细胞和胰岛中精确观察相关动力学的方法来阐明。研究计划还将利用我们在小鼠和人类α-细胞中GIGS机制相似的发现，我们将尽可能在物种间进行平行实验。这些实验将进一步加深我们对α-细胞功能的理解，这是发现调节胰高血糖素和治疗糖尿病的新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-inhibition of glucagon secretion (GIGS) in vivo after β-cells are destroyed in Type 1 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 GIGS. 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 leads to F-actin polymerization and decreased glucagon secretion, putatively via RhoA activation. Thus, both paracrine and juxtracrine pathways drive GIGS from islets, but dispersed α-cells treated with ephrinA5 also exhibit an additional GIGS mechanism, which appears to be intrinsic to the α-cell. Based on these data, we hypothesize that GIGS 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 GIGS 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.