Autophagy-facilitated secretion in metabolic maintenance

项目摘要/摘要 这项提议的目标是展示一种新的非降解、非细胞自主的机制。 自噬机制通过它来调节新陈代谢。尽管新出现的证据表明自噬 代谢异常与代谢功能障碍有关，这是自噬调节胰岛素的机制 敏感性在很大程度上是未知的。自噬通常被认为是一种破坏和降解过程，通过 溶酶体。我们最近发现了一种新的非降解性功能，由ULK1-Vps34-Becn1调节 脂肪组织中的自噬机制，它可能在全身AMPK激活和胰岛素中发挥关键作用 敏化。脂联素是一种脂肪衍生的激素(脂肪因子)，其在循环中的减少是强烈的 与2型糖尿病(T2D)和代谢综合征相关。SNPs或SNPs引起的低脂联素血症 人类脂联素基因突变与胰岛素抵抗和T2D有关，以及 脂联素基因敲除小鼠的表型与人类相似，并出现胰岛素抵抗。然而，尽管 脂联素是一种调节脂联素分泌的机制，但其功能重要性却鲜为人知 研究受到关注，仍然含糊其辞。我们发现自噬过度活跃的突变小鼠体内有一种活性 必需自噬蛋白Becn1(Becn1F121a敲入小鼠)的突变改善了胰岛素敏感性 应对高脂饮食喂养，这是由循环中的因素引起的。为了识别Becn1调控的， 胰岛素敏感型循环因子，我们进行了激素筛查，发现有较高水平的 自噬过度活跃的Becn1F121a小鼠血清中的脂联素。我们发现一方面，脂肪- 特定的Becn1F121a表达足以改善全身胰岛素敏感性并增加循环 脂联素水平；另一方面，耗尽Becn1，或抑制两个上游自噬蛋白激酶，ULK1和 Vps34，减少小鼠循环中的脂联素。利用蛋白质组学和生物化学方法，我们发现 外囊成分Sec6/Exoc3是Becn1的结合伙伴，并优先与超活性结合 表格，Becn1F121a。受初步数据的启发，我们提出了我们的总体假设，即ULK1-Vps34- Becn1自噬轴经Becn1-外囊结合促进脂联素分泌改善胰岛素敏感性 在脂肪细胞中。我们将这一途径称为“自噬促进的分泌”。为了检验这一假设，我们提出了 利用遗传学、成像学、细胞生物学、生物化学和代谢方法的组合，实现以下目标： 目的1是确定活性Becn1是否通过脂联素激活AMPK和改善胰岛素敏感性 目的2是确定ULK1-Vps34-Becn1自噬轴在脂肪组织中是否起作用 非细胞自主调节全身性胰岛素敏感性；目标3是证明分子 以Becn1为中心的自噬机制通过串扰调节脂联素分泌的机制 与排囊途径有关。总体而言，我们的研究将建立一个新的以贝克纳为中心的机制范式 脂肪组织中的自噬途径防止T2D，超越其在自我降解中的作用。

Project Summary/Abstract The goal of this proposal is to demonstrate a novel non-degradative, non-cell autonomous, mechanism by which the autophagy machinery regulates metabolism. Although emerging evidence suggests that autophagy abnormality is implicated in metabolic dysfunction, the mechanism by which autophagy regulates insulin sensitivity is largely unknown. Autophagy is generally considered as a destruction and degradation process via lysosomes. We recently discovered a new non-degradative function regulated by a ULK1-Vps34-Becn1 autophagy machinery in adipose tissue, which may play a pivotal role in systemic AMPK activation and insulin sensitization. Adiponectin is an adipose-derived hormone (adipokine), whose reduction in circulation is strongly associated with type 2 diabetes (T2D) and metabolic syndrome. Hypoadiponectinemia caused by SNPs or mutations in the Adiponectin gene in humans has been associated with insulin resistance and T2D, and adiponectin knockout mice phenocopy the human phenotypes and develop insulin resistance. However, despite the functional importance of adiponectin, the mechanism that regulates adiponectin secretion has received little research attention and remains obscure. We found that autophagy-hyperactive mutant mice harboring an active mutation in the essential autophagy protein Becn1 (Becn1F121A knock-in mice) have improved insulin sensitivity in response to high-fat diet feeding, which is caused by factors in the circulation. To identify the Becn1-regulated, insulin-sensitizing, circulating factors, we performed a hormonal screen and identified a higher level of adiponectin in the serum of autophagy-hyperactive Becn1F121A mice. We found that on one hand, adipose- specific Becn1F121A expression is sufficient to improve systemic insulin sensitivity and increase circulating adiponectin levels; on the other hand, depleting Becn1, or inhibiting two upstream autophagy kinases, ULK1 and Vps34, reduces circulating adiponectin in mice. Using proteomic and biochemical approaches, we discovered that the exocyst component Sec6/Exoc3 is a binding partner of Becn1, and preferentially binds to the hyperactive form, Becn1F121A. Motivated by the preliminary data, we propose our overall hypothesis that a ULK1-Vps34- Becn1 autophagy axis improves insulin sensitivity by promoting adiponectin secretion via Becn1-exocyst binding in adipocytes. We term this pathway as “autophagy-facilitated secretion”. To test the hypothesis, we propose the following aims, using a combination of genetic, imaging, cell biology, biochemistry, and metabolic approaches: Aim 1 is to determine whether active Becn1 activates AMPK and improves insulin sensitivity via adiponectin signaling; Aim 2 is to determine whether a ULK1-Vps34-Becn1 autophagy axis functions in adipose tissue to non-cell autonomously regulate systemic insulin sensitivity; and Aim 3 is to demonstrate the molecular mechanism by which the Becn1-centered autophagy machinery regulates adiponectin secretion via the crosstalk with the exocyst pathway. Overall, our study will establish a new mechanistic paradigm for the Becn1-centered autophagy pathway in adipose tissue to prevent T2D, beyond its role in self-degradation.