Regulating SNARE mechanisms to remediate glucose homeostasis

描述（由申请人提供）：2型糖尿病（T2D）的发病率已经达到流行病的程度，约8.3%的美国人口被诊断出患有糖尿病，7900多万人表现出糖尿病前期，这提醒我们迫切需要解决方案。阻止糖尿病前期发展和发展为T2D需要多管齐下的方法，因为病理生理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）蛋白丰度的限制。相关的，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Syntaxin 4 (Syn4)，可能导致改善再生治疗糖尿病的方法。在人类和啮齿动物的胰岛和骨骼肌中，Syn4丰度和/或其活性的丧失与糖尿病有关，这两种组织分别调节胰岛素释放和胰岛素敏感性。因此，长期目标是了解如何操纵Syn4来治疗和预防前驱糖尿病和T2D。针对Syn4丰度/激活来控制血糖失调和前驱糖尿病的方法的发现为疾病干预提供了一个诱人的机会。本应用程序的目的是确定Syn4富集/激活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Syn4丰度如何被调节。初步资料显示Syn4蛋白限制人胰岛胰岛素的分泌；富含Syn4的胰岛能更有效地降低糖尿病小鼠的高血糖。此外，糖尿病小鼠的胰岛细胞和肌肉细胞也存在约40%的T2D人胰岛Syn4蛋白缺失。值得注意的是，恢复人T2D胰岛的Syn4可以充分恢复胰岛素分泌。富含syn4的小鼠也能抵抗衰老和高脂肪饮食引起的胰岛素抵抗。核心假设是，糖尿病源性刺激导致Syn4缺乏，从而损害β细胞和骨骼肌细胞中关键的受调节的胞吐事件，而Syn4上调可以促进这些过程，以挽救/抵抗与前驱糖尿病和T2D相关的应激。该研究的基本原理是，一旦知道Syn4富集如何促进和/或保护功能性β细胞质量和外周胰岛素敏感性，以及Syn4丰度如何被调节，Syn4就可以在面对糖尿病刺激时被操纵以避免疾病。为了验证这一点，我们开发了三个特定目的：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)描述骨骼肌中Syn4的富集/激活如何促进胰岛素敏感性；3)确定糖尿病前期组织中Syn4表达减弱的机制基础。研究人员将利用创新的诱导型β细胞和骨骼肌特异性Syn4转基因小鼠，对其进行糖尿病刺激、内源性Syn4肽激活剂和活细胞成像生物传感器，以及使用人体组织进行生化检测。结果将积极影响改善疾病的努力，因为确定的机制极有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.