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

描述(申请人提供)：随着糖尿病前期发展过程中血糖的升高，中风、心肌梗塞和死亡率等心血管后果已经增加了2-4倍，但对糖尿病前期的发展方式和原因的理解仍然存在根本差距。在一个有类似糖尿病风险因素的人群中，无法预测比其他人更容易患糖尿病的风险因素，这是一个重要的问题，因为在问题得到解决之前，预防/治疗糖尿病前期/血糖紊乱的战略将在很大程度上站不住脚。阻止血糖异常的策略需要多管齐下，因为病理生理学涉及外周胰岛素抵抗和胰腺β细胞功能障碍。与这两个过程中的失败相关的因素是治疗重点的需求；我们已经确定了p21激活的激酶，PAK1，就是这样一个因素。此外，PAK1丰度的丧失与人类胰岛和人类骨骼肌中的糖尿病和肥胖症有关，这两种组织分别是调节胰岛素释放和胰岛素敏感性的关键组织。因此，长期目标是了解如何操纵这些组织中的PAK1通路来治疗/预防糖尿病前期，最终阻止进展为糖尿病。这一特殊应用的目的是在体内和分子水平上区分PAK1在β细胞胰岛素分泌和骨骼肌胰岛素作用中的功能(在其他细胞类型中，PAK1在信号和支架中发挥作用)。在其他类型的细胞中，已知PAK1通路可以引起肌动蛋白细胞骨架的动态变化，初步数据表明，这种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，典型的全身PAK1基因敲除小鼠只喂食42%脂肪饲料10周，就会出现空腹高血糖、胰岛素不足和严重的糖耐量异常。此外，恢复胰岛β细胞中PAK1的丰度或信号可以恢复胰岛素的分泌，减少β细胞的凋亡。这些发现提出了如下中心假设：a)PAK1通过参与β细胞和骨骼肌细胞中肌动蛋白重塑调节的胞吐活动，是葡萄糖动态平衡的中枢调节因子；b)PAK1缺乏导致对血糖调节失调和糖尿病前期的易感性增加。这项拟议研究的基本原理是，一旦知道β细胞和骨骼肌细胞中需要PAK1的方式，就可以操纵PAK1通路来避免糖尿病前期的血糖紊乱。这一假说将在两个特定的目标下进行检验：1)描绘PAK1在β细胞功能和存活中的作用机制(S)；2)阐明PAK1促进骨骼肌胰岛素敏感性的机制(S)。目标将使用创新的可诱导β细胞和骨骼肌PAK1基因敲除小鼠和带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。这些审讯的结果预计将对改善糖尿病前期的努力产生积极影响，因为已确定的效应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 β cell 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 β cell 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 β cells restores insulin secretion and reduces β cell 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 β cells 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 β cells 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 β cell function and survival, 2) Elucidate the mechanism(s) by which PAK1 promotes skeletal muscle insulin sensitivity. Aims will be accomplished using innovative inducible β cell 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.