Integrative regulation of lipid sensing by mTOR

mTOR 对脂质传感的综合调节

• 批准号: 10674262
• 负责人: Rajat Singh
• 金额: $ 31.98万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10674262
• 关键词: Acute; Age; Aging; Amino Acids; Animals; BODIPY; Binding; Biochemical; Bolus Infusion; Brain; Caenorhabditis elegans; Cardiometabolic Disease; Cells; Cholesterol; Chronic; Chronic Disease; Complex; Corn Oil; Cultured Cells; Data; Denervation; Development; Diabetes Mellitus; Diet; Dietary Fats; Dietary Oils; Dietary intake; Diglycerides; Drosophila genus; Electrospray Ionization; Endothelial Cells; Enzymes; Exposure to; FRAP1 gene; Fatty Acids; Foundations; Geroscience; Grant; Growth; Hypothalamic structure; Image; Individual; Inflammation; Ingestion; Intestines; Investigation; Label; Lead; Leucine; Link; Lipids; Lipoproteins; Liver; Longevity; Lysosomes; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Membrane; Membrane Lipids; Metabolic Diseases; Metabolism; Molecular; Molecular Genetics; Mus; Nerve Degeneration; Neurons; Nonesterified Fatty Acids; Nutrient; Operative Surgical Procedures; Oral; Organism; Palmitic Acids; Peripheral; Pharmacogenetics; Phosphatidic Acid; Phosphotransferases; Physiological; Pro-Opiomelanocortin; Protein Biosynthesis; Proteins; Proteomics; Regulation; Research Personnel; Risk Factors; Role; Shapes; Signal Transduction; Sirolimus; Structure; Surface; Testing; Time; Tissues; Triglycerides; Universities; absorption; age related; aged; detection of nutrient; dietary; experimental study; genetic approach; genetic regulatory protein; healthspan; imaging approach; in vivo; ion mobility; ion source; lipid metabolism; lipidomics; lipoprotein lipase; lysosome membrane; mass spectrometer; mass spectrometric imaging; mortality; novel; novel therapeutic intervention; prevent; response; sensor; sugar; tool

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

摘要 甘油三酯占总摄入脂肪的90%。膳食中TGS摄入量过高和体内 肠源性脂蛋白是代谢性疾病的关键决定因素--这是 65岁或以上的个人。循环和组织TGS的增加与年龄相关的代谢密切相关 疾病。尽管我们了解了脂肪吸收，但一个根本的问题仍然没有答案--如何 感觉到血脂了吗？MTORC1(雷帕霉素-复合体1的机制靶点)是一种PI3K样激酶，它能感觉到氨基 酸性物质和促进蛋白质合成。我们令人兴奋的新数据表明，脂质的可获得性激活了mTORC1 信号传递--表明mTOR是一种脂质传感器。BODIPY标记棕榈酸灌胃实验 在小鼠身上发现了强大的mTORC1激活--这表明mTOR可能感觉到脂肪酸或脂肪酸 体内的衍生品。MTOR如何感知脂质仍是个未知数。我们在培养细胞中的初步数据显示 二酰甘油的可获得性触发mTOR对溶酶体的定位--提示感知 二酰甘油通过mTOR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，溶酶体膜上二酰甘油的增加会导致mTORC1的过度激活。除了……之外 通过脂质对mTORC1信号的直接细胞自主调节，我们的新数据还揭示了一种新的细胞非 自主调节外周mTORC1信号。事实上，尖锐地抑制mTORC1信号在 通过定向立体定向注射雷帕霉素抑制脂质驱动的mTORC1下丘脑内侧 外周组织中的信号。在此基础上，我们假设由于年龄的原因，mTOR改变了对血脂的感知- 溶酶体膜脂组成的相关变化是mTORC1高激活的机制。我们 进一步假设下丘脑中mTORC1信号的失调是导致过度激活的原因。 外周组织中mTORC1信号的表达。为了验证我们的假设，我们提出了以下目标：目标1： 确定mTOR感知细胞二酰甘油的机制。在目标1中，我们将使用生化 和基于图像的方法以及溶酶体膜的蛋白质组学和无偏脂组学来 表征mTORC1信号对年轻和老年小鼠脂肪供应的反应。我们将确定小说 当有脂类可用时与mTOR相互作用的调节蛋白。溶酶体膜的靶向脂质组学研究 哥伦比亚大学的劳拉·贝丝·麦金泰尔博士将与研究员劳拉·贝丝·麦金泰尔博士一起揭示导致 MTORC1在幼年和老年动物中的过度激活。通过让候选目标保持沉默，我们将确定新的 在幼年和老年动物中介导mTOR脂质感应功能的溶酶体候选蛋白。目标 2：通过中枢神经系统到外周的mTORC1轴来剖析全身性脂质感觉。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标记的小鼠在内的工具来确定脂质驱动的mTORC1 在外周组织中，激活先于mTORC1的激活，以及与年龄相关的mTOR的过度激活 导致外周组织中mTOR过度激活。确定何时何地是 在年轻和老年小鼠的脂质刺激后，mTOR在MBH中被激活，我们将使用脑切片(在时间点 当mTOR最活跃时)，用于通过Synapt G2-Si四极杆进行质谱学成像 飞行(QToF)串联质谱仪，具有离子迁移率形状/结构在TOF MS前预分离，使用 解吸电喷雾电离(DESI)作为正离子源。然后我们将使用药物遗传学 抑制下丘脑mTOR信号以描述脂质驱动的整合调节的方法 年轻和老年小鼠的mTOR信号。 意义：组织和循环中的脂质水平升高是心脏代谢性疾病的关键决定因素。 衰老本身是代谢性疾病的一个主要风险因素，因此，促使调查了解 在衰老过程中改变细胞脂代谢的因素。MTORC1信号的过度激活导致 糖尿病和癌症的发展。我们的研究结果表明，脂类感觉改变和 与年龄相关的mTORC1过度激活。拟议中的研究将揭开导致 随着年龄的增长，mTORC1信号的过度激活，从而可以设计新的治疗策略来预防 或逆转由过度激活的mTORC1信号引起的慢性疾病。 1