Integrative regulation of lipid sensing by mTOR

摘要 甘油三酯占总摄入脂质的90%。饮食中TG摄入过多， 胆固醇衍生的脂蛋白是代谢性疾病的关键决定因素，代谢性疾病是糖尿病患者死亡的主要原因。 65岁或以上的人。循环和组织TG升高与年龄相关的代谢密切相关 疾病尽管我们了解脂质吸收，一个基本的问题仍然没有答案-如何 感觉到脂质？mTORC 1（雷帕霉素复合物1的机制靶点）是一种PI3 K样激酶， 酸和驱动蛋白质合成。我们令人兴奋的新数据表明，脂质的可用性激活mTORC1 信号-表明mTOR是脂质传感器。用BODIPY标记的棕榈酸管饲的实验 结果显示mTORC 1有很强的激活作用，这表明mTOR可能感知脂肪酸或脂肪酸 体内衍生物。mTOR如何感知脂质仍然未知。我们在培养细胞中的初步数据显示， 甘油二酯的可用性触发mTOR定位于溶酶体--这表明， 通过mTOR产生的甘油二酯发生在溶酶体膜上。由于mTOR随着年龄的增长而过度激活，我们建议 随着年龄的增长，溶酶体膜甘油二酯的增加导致mTORC 1过度活化。除了 通过脂质直接细胞自主调节mTORC 1信号，我们的新数据还揭示了一种新的细胞非- 外周mTORC1信号传导的自主调节。事实上，急性抑制mTORC1信号转导， 通过雷帕霉素的靶向立体定位递送下丘脑中基底，抑制脂质驱动的mTORC1 在外周组织中的信号传导。在此基础上，我们假设由于年龄的变化，mTOR的脂质感知发生了改变- 溶酶体膜脂质组成的相关变化是mTORC 1过度活化的机制。我们 进一步假设下丘脑中mTORC 1信号传导失调有助于过度激活 mTORC1信号在外周组织中的作用。为了验证我们的假设，我们提出了以下目标：目标1： 确定mTOR感知细胞甘油二酯的机制。在目标1中，我们将使用生物化学 和基于图像的方法以及蛋白质组学和溶酶体膜的无偏脂质组学， 表征mTORC1信号传导对年轻和老年小鼠中脂质可用性的响应。我们将确定新的 当脂质可用时与mTOR相互作用的调节蛋白。溶酶体膜靶向脂质组学 与哥伦比亚大学的Laura Beth McIntire博士合作，将揭示驱动细胞生长的特定脂质种类。 年轻和老年动物中mTORC 1过度激活。通过沉默候选靶点，我们将鉴定新的 在年轻和年老动物中介导mTOR的脂质感应功能的溶酶体中的候选蛋白。目的 2：通过CNS-外周mTORC1轴剖析全身脂质感受。在目标2中，我们将使用不同的集合 包括下丘脑神经元特异性GFP标记小鼠在内的工具，以确定脂质驱动的mTORC1 在外周组织中，mTOR 1活化先于mTOR 1活化，以及是否与年龄相关的mTOR过度活化 导致外周组织中mTOR过度激活。以确定何时何地 在年轻和老年小鼠中脂质激发后MBH中激活的mTOR，我们将使用脑切片（在时间点 当mTOR处于最大活性时），通过SYNAPT G2-Si四极杆-时间- 飞行（QToF）串联质谱仪，在ToF MS之前进行离子迁移率形状/结构预分离，使用 解吸电喷雾电离（DESI）作为正离子模式的离子源。然后我们将使用药物遗传学 抑制下丘脑mTOR信号传导的方法，以描绘脂质驱动的整合调节 年轻和老年小鼠中的mTOR信号传导。 意义：组织和循环脂质水平升高是心脏代谢疾病的关键决定因素。 衰老本身是代谢性疾病的一个主要危险因素，因此，促使研究人员了解 在衰老过程中改变细胞脂质代谢的因素。mTORC1信号转导的过度激活导致了 糖尿病和癌症的发展。我们的研究结果表明，改变脂质感受和 与年龄相关的mTORC 1过度激活。拟议中的研究将揭示导致 随着年龄的增长，mTORC1信号转导过度激活，因此可以设计新的治疗策略来预防 或逆转由过度激活的mTORC 1信号传导引起的慢性疾病。 1

Abstract Triglycerides constitute 90% of the total ingested lipid. Excessive intake of dietary TGs and elevated levels of intestine-derived lipoproteins are key determinants of metabolic disease—a leading cause of mortality in individuals 65 years or older. Increased circulating and tissue TGs strongly correlate with age-related metabolic disease. Despite our understanding of lipid absorption, a fundamental question remains unanswered—how are lipids sensed? mTORC1 (mechanistic target of rapamycin-complex 1) is a PI3K-like kinase that senses amino acids and drives protein synthesis. Our exciting new data show that availability of lipid activates mTORC1 signaling—suggesting that mTOR is a lipid sensor. Experiments with BODIPY-labelled palmitic acid gavaged into mice revealed robust mTORC1 activation—suggesting that mTOR likely senses fatty acid or a fatty acid derivative in vivo. How mTOR senses lipid remains unknown. Our preliminary data in cultured cells show that availability of diacylglycerol triggers the localization of mTOR to lysosomes—suggesting that sensing of diacylglycerols by mTOR occurs at lysosomal membranes. Since mTOR is hyperactivated with age, we propose that increases in lysosomal membrane diacylglycerol with age causes mTORC1 hyperactivation. In addition to the direct cell-autonomous regulation of mTORC1 signaling by lipid, our new data also reveal a novel cell non- autonomous regulation of peripheral mTORC1 signaling. Indeed, acutely suppressing mTORC1 signaling in the mediobasal hypothalamus via targeted stereotaxic delivery of rapamycin, dampens lipid-driven mTORC1 signaling in peripheral tissues. On this basis, we hypothesize that altered lipid sensing by mTOR due to age- related changes in lysosome membrane lipid composition is the mechanism for mTORC1 hyperactivation. We hypothesize further that dysregulation of mTORC1 signaling in hypothalamus contributes to the hyperactivation of mTORC1 signaling in peripheral tissues. To test our hypothesis, we present the following Aims: Aim 1: To determine the mechanism by which mTOR senses cellular diacylglycerols. In Aim 1, we will use biochemical and image-based approaches as well as proteomics and unbiased lipidomics of lysosomal membranes to characterize mTORC1 signaling in response to lipid availability in young and aged mice. We will identify novel regulatory proteins that interact with mTOR when lipid is available. Targeted lipidomics of lysosomal membranes with co-Investigator Dr. Laura Beth McIntire at Columbia University will reveal the specific lipid species that drive mTORC1 hyperactivation in young and old animals. By silencing candidate targets, we will identify novel candidate proteins at lysosomes that mediate the lipid sensing function of mTOR in young and old animals. Aim 2: To dissect systemic lipid sensing by a CNS-to-peripheral mTORC1 axis. In Aim 2, we will use a diverse set of tools including hypothalamic neuron-specific GFP-labeled mice to determine whether lipid-driven mTORC1 activation precedes mTORC1 activation in peripheral tissues, and whether age-related hyperactivation of mTOR in the hypothalamus leads to mTOR hyperactivation in peripheral tissues. To determine when and where is mTOR activated in MBH after a lipid challenge in young and aged mice, we will use brain sections (at timepoints when mTOR is maximally active) for mass spectrometry (MS) imaging via SYNAPT G2-Si quadrupole-time-of- flight (QToF) tandem mass spectrometer with ion mobility shape/structure pre-separation prior to ToF MS, using Desorption Electrospray Ionization (DESI) as the ion source in positive mode. We will then use pharmacogenetic approaches to dampen hypothalamic mTOR signaling to delineate the integrative regulation of lipid-driven mTOR signaling in young and aged mice. Significance: Increased tissue and circulating lipid levels are key determinants of cardiometabolic disease. Aging per se is a major risk factor for metabolic diseases, thus, prompting investigations to understand the factors that alter cellular lipid metabolism during aging. Hyperactivation of mTORC1 signaling leads to the development of diabetes and cancers. Our results indicate a mechanistic link between altered lipid sensing and age-related mTORC1 hyperactivation. The proposed studies will unravel novel mechanisms that lead to hyperactivation of mTORC1 signaling with age, such that new therapeutic strategies can be devised to prevent or reverse chronic conditions emanating from hyperactivated mTORC1 signaling. 1