Regulating SNARE mechanisms to remediate glucose homeostasis

描述(由申请人提供)：2型糖尿病(T2D)的发病率已达到流行病的程度，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问题。阻止糖尿病前期的发展和进展到T2D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功能t-SNARE(可溶性NSF附着蛋白受体)蛋白丰度的限制。与此相关的是，最近的一项人类胰岛研究表明，增加胞吐蛋白的表达，如t-SNARE蛋白Synaxin 4(Syn4)，可能会导致治疗糖尿病的再生方法的改进。在人类和啮齿动物的胰岛和骨骼肌中，Syn4丰度和/或活性的丧失与糖尿病有关，这些组织分别调节胰岛素的释放和胰岛素的敏感性。因此，长期目标是了解Syn4如何被操纵来治疗和预防糖尿病前期和T2D。发现靶向Syn4丰度/激活的方法来控制血糖失调和糖尿病前期，为疾病干预提供了一个诱人的机会。这项应用的目的是确定Syn4的丰富/激活如何在体内和分子水平上促进β细胞胰岛素分泌和骨骼肌胰岛素的作用，以及Syn4在这些组织中的丰度是如何调节的。初步数据显示，Syn4蛋白限制了人胰岛胰岛素的分泌；富含Syn4的胰岛更有效地降低了糖尿病小鼠的高血糖。此外，与糖尿病小鼠的胰岛和肌肉细胞一样，T2D人胰岛约40%缺乏Syn4蛋白。值得注意的是，将Syn4恢复到人的T2D胰岛可以完全挽救胰岛素的分泌。富含Syn4的小鼠也能抵抗年龄和高脂饮食诱导的胰岛素抵抗。中心假说是，糖尿病诱因刺激导致β细胞和骨骼肌细胞中Syn4缺乏，从而损害关键调控的胞吐事件，Syn4上调可以促进这些过程，以拯救/抵抗与糖尿病前期和T2D相关的应激。这项拟议研究的基本原理是，一旦知道Syn4的增加是如何促进和/或保护功能性β细胞团和外周胰岛素敏感性，以及Syn4的丰度是如何被调节的，那么Syn4就可以在糖尿病刺激下被操纵来避免疾病。为了验证这一点，我们开发了三个特定的目标：1)评估Syn4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Syn4在骨骼肌中的丰富/激活如何促进胰岛素敏感性；以及3)确定Syn4在糖尿病前期/组织中表达减弱的机制基础。目标将通过创新的可诱导的β细胞和骨骼肌特异的Syn4转基因小鼠来实现，这些转基因小鼠受到糖尿病刺激、内源性Syn4的多肽激活剂和活细胞成像生物传感器的挑战，并与使用人体组织的生化分析相结合。结果将对改善疾病的努力产生积极影响，因为已确定的机制极有可能提供新的治疗目标。

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.