Regulating SNARE mechanisms to remediate glucose homeostasis

描述（由申请人提供）：2型糖尿病（T2 D）的发病率已达到流行病的比例，约8.3%的美国人口被诊断为糖尿病前期，另有7900万人表现出糖尿病前期-这提醒人们迫切需要解决方案。阻止前驱糖尿病发展和进展为T2 D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β细胞功能障碍。这些过程中的每一个都受到胞吐t-SNARE（可溶性NSF附着蛋白受体）蛋白质丰度的限制。相关地，最近的人类胰岛研究表明，增加胞吐蛋白的表达，例如t-SNARE蛋白Syntaxin 4（Syn 4），可能导致改善治疗糖尿病的再生方法。Syn 4丰度和/或其活性的丧失与人类和啮齿动物胰岛和骨骼肌中的糖尿病相关，所述胰岛和骨骼肌分别是调节胰岛素释放和胰岛素敏感性的组织。因此，长期目标是了解如何操纵Syn 4来治疗和预防前驱糖尿病和T2 D。发现靶向Syn 4丰度/激活以控制血糖失调和前驱糖尿病的方法为疾病干预提供了诱人的机会。本申请的目的是确定Syn 4富集/活化如何在体内和分子水平上增强β细胞胰岛素分泌和骨骼肌胰岛素作用，以及这些组织中的Syn 4丰度如何调节。初步数据显示，Syn 4蛋白限制人胰岛胰岛素分泌;富含Syn 4的胰岛更有效地降低糖尿病小鼠的高血糖症。此外，T2 D人胰岛约40%缺乏Syn 4蛋白，糖尿病小鼠的胰岛和肌肉细胞也是如此。值得注意的是，Syn 4对人T2 D胰岛的恢复可以完全拯救胰岛素分泌。富含Syn 4的小鼠还抵抗年龄和高脂肪饮食诱导的胰岛素抵抗。中心假设是糖尿病致敏刺激是Syn 4缺陷的基础，以损害β细胞和骨骼肌细胞中的关键调节胞吐事件，并且Syn 4上调可以促进这些过程以拯救/抵抗与前驱糖尿病和T2 D相关的应激。所提出的研究的基本原理是，一旦知道Syn 4富集如何促进和/或保护功能性β细胞群和外周胰岛素敏感性，以及Syn 4丰度如何调节，就可以操纵Syn 4以避免面对致糖尿病刺激的疾病。开发了三个具体目的来测试这一点：1）评价Syn 4上调在预防/逆转糖尿病诱导的β细胞功能障碍中的作用，2）描述骨骼肌中Syn 4富集/活化如何促进胰岛素敏感性，以及3）确定前驱/糖尿病组织中减弱的Syn 4表达的机制基础。将使用创新的诱导型β细胞和骨骼肌特异性Syn 4转基因小鼠实现目标，这些小鼠用致糖尿病刺激物、内源性Syn 4的肽激活剂和与使用人体组织的生化测定配对的活细胞成像生物传感器进行挑战。结果将积极影响改善疾病的努力，因为所确定的机制很可能提供新的治疗靶点。

DESCRIPTION (provided by applicant): Incidences of type 2 diabetes (T2D) have reached epidemic proportions, with ~8.3% of the US population diagnosed and 79 million more exhibiting pre-diabetes - a reminder of the urgent need for solutions. Halting pre-diabetes development and progression to T2D requires a multi-pronged approach, since the pathophysiology involves both peripheral insulin resistance and pancreatic β cell dysfunction. Each of these processes is rate-limited by the abundance of exocytosis t-SNARE (Soluble NSF Attachment Protein Receptor) proteins. Relatedly, a recent human islet study suggests that increasing expression of exocytosis proteins, such as the t-SNARE protein Syntaxin 4 (Syn4), may lead to improved regenerative approaches to treat diabetes. Loss of Syn4 abundance and/or its activity is associated with diabetes in human and rodent islets and skeletal muscle, tissues which regulate insulin release and insulin sensitivity, respectively. Thus, the long-term goal is to understand how Syn4 can be manipulated for treatment and prevention of prediabetes and T2D. Discovery of ways to target Syn4 abundance/activation to control glycemic dysregulation and prediabetes offers a tantalizing opportunity for disease intervention. The objective of this application is to determine how Syn4 enrichment/activation functions to enhance β-cell insulin secretion and skeletal muscle insulin action both in vivo and at the molecular level, and how Syn4 abundance in these tissues is regulated. Preliminary data show that Syn4 protein is limiting for human islet insulin secretion; islets enriched with Syn4 more effectively reduce hyperglycemia in diabetic mice. Moreover, T2D human islets are ~40% deficient in Syn4 protein, as are islet and muscle cells of diabetic mice. Notably, restoration of Syn4 to human T2D islets can fully rescue insulin secretion. Syn4-enriched mice also resist age-and high-fat-diet induced insulin resistance. The central hypothesis is that diabetogenic stimuli underlie Syn4 deficiency to impair key regulated exocytosis events in β cells and skeletal muscle cells, and that Syn4 upregulation can boost these processes to rescue/resist stress associated with prediabetes and T2D. The rationale for the proposed research is that once it is known how Syn4 enrichment promotes and/or protects functional β cell mass and peripheral insulin sensitivity, and how Syn4 abundance is regulated, that Syn4 can be manipulated to avert disease in the face of diabetogenic stimuli. Three Specific Aims are developed to test this: 1) Evaluate Syn4 upregulation in prevention/reversal of diabetogenic-induced β-cell dysfunction, 2) Delineate how Syn4 enrichment/activation in skeletal muscle promotes insulin sensitivity, and 3) Determine the mechanistic basis for attenuated Syn4 expression in pre/diabetic tissues. Aims will be accomplished using innovative inducible β-cell- and skeletal muscle-specific Syn4 transgenic mice challenged with diabetogenic stimuli, peptide activators of endogenous Syn4, and live-cell imaging biosensors paired with biochemical assays using human tissues. Results will positively impact efforts to ameliorate disease as the identified mechanisms are highly likely to provide new therapeutic targets.