Autophagy-facilitated secretion in metabolic maintenance

项目总结/摘要 这项提案的目的是证明一种新的非降解，非细胞自主，机制 自噬机制调节新陈代谢的机制。尽管新出现的证据表明自噬 异常与代谢功能障碍有关，代谢功能障碍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， 溶酶体我们最近发现了一种新的非降解功能，由ULK 1-Vps 34-Becn 1调控。 脂肪组织中的自噬机制，可能在系统性AMPK激活和胰岛素分泌中起关键作用。 致敏脂联素是一种脂肪源性激素（脂肪因子），其在循环中的减少是强烈的 与2型糖尿病（T2 D）和代谢综合征相关。由SNPs引起的低脂联素血症或 人类脂联素基因突变与胰岛素抵抗和T2 D相关， 脂联素敲除小鼠表型模仿人类表型并发展胰岛素抵抗。但尽管 脂联素的功能重要性，调节脂联素分泌的机制， 研究的关注和仍然模糊。我们发现，自噬过度活跃的突变小鼠， 自噬蛋白Becn 1的突变（Becn 1F 121 A基因敲入小鼠）改善了胰岛素敏感性 在应对高脂肪饮食喂养，这是在循环的因素造成的。为了鉴定Becn 1调节的， 胰岛素敏感性，循环因素，我们进行了激素筛选，并确定了较高水平的 自噬过度活跃的Becn 1F 121 A小鼠血清中的脂联素。我们发现一方面，脂肪- 特异性Becn 1F 121 A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，消耗Becn 1，或抑制两种上游自噬激酶，ULK 1和 vps 34降低小鼠循环脂联素。利用蛋白质组学和生物化学方法，我们发现 外囊组分Sec 6/Exoc 3是Becn 1的结合伴侣，并且优先结合过度活跃的 形式，Becn 1F 121 A。受初步数据的启发，我们提出了我们的总体假设，即ULK 1-Vps 34- Becn 1自噬轴通过Becn 1-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。我们将此途径称为“自噬促进分泌”。为了验证这一假设，我们提出了 以下目标，使用遗传学，成像，细胞生物学，生物化学和代谢方法的组合： 目的1是确定活性Becn 1是否通过脂联素激活AMPK并改善胰岛素敏感性 目的2是确定ULK 1-Vps 34-Becn 1自噬轴是否在脂肪组织中起作用， 非细胞自主调节全身胰岛素敏感性;目的3是证明分子 以Becn 1为中心的自噬机制通过串扰调节脂联素分泌 与外囊途径有关。总之，我们的研究将为Becn 1为中心机制建立一个新的范式 自噬途径在脂肪组织中预防T2 D，超越其自我降解的作用。

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.