Targeting PAK1 to improve functional beta-cell mass and insulin sensitivity

描述（由申请人提供）：随着糖尿病前期发展期间血糖升高，心血管后果（如中风、心肌梗死和死亡率）已经增加了2-4倍，但在理解糖尿病前期如何以及为什么发展方面仍然存在根本性差距。在具有类似糖尿病风险因素的人群中，无法预测哪些人群比其他人群更易患糖尿病，这是一个重要的问题，因为在解决这个问题之前，预防/治疗糖尿病前期/功能障碍的策略将在很大程度上仍然是站不住脚的。阻止功能障碍的策略需要多管齐下的方法，因为病理生理学涉及外周胰岛素抵抗和胰腺细胞功能障碍。与这两个过程中的失败有关的因素是治疗重点的需求;我们已经确定了p21激活激酶PAK 1，作为这样一个因素。此外，PAK 1丰度的丧失与人胰岛和人骨骼肌中的糖尿病和肥胖相关，这些组织分别是调节胰岛素释放和胰岛素敏感性的关键。因此，长期目标是了解如何操纵这些组织中的PAK 1通路来治疗/预防糖尿病前期，最终阻止进展为坦率的糖尿病。本申请的目的是在体内和分子水平上区分PAK 1在细胞胰岛素分泌和骨骼肌胰岛素作用中的功能（PAK 1在其他细胞类型中的信号传导和支架中发挥作用）。已知PAK 1途径在其他细胞类型中引起动态肌动蛋白细胞骨架变化，初步数据表明此类变化是胰岛素释放和葡萄糖清除机制的一部分。初步数据显示，经典的全身PAK 1基因敲除小鼠喂食42%的脂肪饮食仅10周，就会出现空腹高血糖、胰岛素不足和严重的葡萄糖耐受不良。此外，胰岛细胞中PAK 1丰度或信号的恢复恢复胰岛素分泌并减少细胞凋亡。这些发现引起了中心假设，即a）PAK 1是葡萄糖稳态的中心调节因子，通过肌动蛋白重塑调节细胞和骨骼肌细胞中的胞吐事件，以及B）PAK 1缺乏导致对血糖失调和糖尿病前期的易感性增加。这项研究的基本原理是，一旦知道PAK 1在细胞和骨骼肌细胞中的作用，就可以操纵PAK 1通路来避免糖尿病前期的功能障碍。这一假设将在两个具体目标中进行检验：1）描述PAK 1在细胞功能和存活中的作用机制，2）阐明PAK 1促进骨骼肌胰岛素敏感性的机制。目标将通过使用创新的可诱导细胞和骨骼肌PAK 1基因敲除小鼠和具有生化分析的活细胞成像生物传感器以及相关的人类胰岛和肌肉组织来实现。这些询问的结果有望对改善糖尿病前期的努力产生积极影响，因为所确定的效应物和机制极有可能提供新的治疗靶点。

DESCRIPTION (provided by applicant): As blood sugars rise during development of pre-diabetes, cardiovascular consequences such as stroke, myocardial infarction and mortality are already increasing by 2-4 fold, yet there remains a fundamental gap in understanding how and why pre-diabetes develops. The inability to predict, within a population with similar risk factors for diabetes, which are more susceptible than others represents an important problem because, until it is resolved, strategies to prevent/treat pre-diabetes/dysglycemia will remain largely untenable. Strategies to halt dysglycemia require a multi-pronged approach, since the pathophysiology involves both peripheral insulin resistance and pancreatic �ell dysfunction. Factors that are linked to failures in both processes are in-demand for therapeutic focus; we have identified the p21-activated kinase, PAK1, as such a factor. Further, losses of PAK1 abundance are associated with diabetes and obesity in human islets and human skeletal muscle, tissues that are key to regulating insulin release and insulin sensitivity, respectively. Thus, the long-term goal is to understand how the PAK1 pathways in these tissues can be manipulated to treat/prevent pre-diabetes, ultimately halting progression to frank diabetes. The objective of this particular application is to discriminate how PAK1 functions (PAK1 plays roles in signaling as well as scaffolding in other cell types) in �ell insulin secretion and skeletal muscle insulin action in vivo and at the molecular level. PAK1 pathways are known in other cell types to evoke dynamic actin cytoskeleton changes, and preliminary data suggest such changes to be part of insulin release and glucose clearance mechanisms. Preliminary data show that classic whole-body PAK1 knockout mice fed a 42% fat diet for just 10 weeks develop fasting hyperglycemia, insulin insufficiency and severe glucose intolerance. Additionally, restoration of PAK1 abundance or signaling in islet �ells restores insulin secretion and reduces �ell apoptosis. These findings give rise to the central hypotheses, that a) PAK1 is a central regulator of glucose homeostasis via functions in actin remodeling-regulated exocytosis events in both �ells and skeletal muscle cells, and b) that PAK1 deficiency culminates in heightened susceptibility to glycemic dysregulation and pre-diabetes. The rationale for the proposed research is that once it is known how PAK1 is needed in �ells versus skeletal muscle cells, that the PAK1 pathways can be manipulated to avert pre-diabetic dysglycemia. This hypothesis will be tested in two Specific Aims: 1) Delineate the mechanism(s) for PAK1 actions in �ell function and survival, 2) Elucidate the mechanism(s) by which PAK1 promotes skeletal muscle insulin sensitivity. Aims will be accomplished using innovative inducible �ell and skeletal muscle PAK1 knockout mice and live-cell imaging biosensors with biochemical assays, and relevant human islet and muscle tissues. Results of these interrogations are expected to positively impact efforts to ameliorate pre-diabetes, because the identified effectors and mechanisms are highly likely to provide new therapeutic targets.