Molecular regulation of gut lipid metabolism by mTOR and autophagy proteins

mTOR 和自噬蛋白对肠道脂质代谢的分子调节

• 批准号: 10079452
• 负责人: Rajat Singh
• 金额: $ 37.76万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-17 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10079452
• 关键词: Adipose tissue; Affect; Age-Years; Automobile Driving; Autophagocytosis; Biogenesis; Blood Circulation; Caco-2 Cells; Cells; Chylomicrons; Cognition; Data; Development; Diet; Dietary Fats; Endoplasmic Reticulum; Enterocytes; Enzymes; FRAP1 gene; Fatty Acids; Fatty acid glycerol esters; Growth; Health; Hypertriglyceridemia; Hypothalamic structure; Ingestion; Insulin; Intestines; Knockout Mice; Lipids; Lipodystrophy; Liver; Locomotion; Longevity; Lysosomes; Membrane; Metabolic Diseases; Metabolic syndrome; Modeling; Molecular; Mus; Mutagenesis; Neurons; Nonesterified Fatty Acids; Nutrient; Obesity; Pathway interactions; Pharmacology; Phosphorylation; Physiological; Population; Predictive Factor; Production; Proteins; Proteomics; Quality Control; Regulation; Role; Signal Transduction; Sirolimus; Site-Directed Mutagenesis; Source; Testing; Therapeutic; Thinness; Tissues; Triglyceride Metabolism; Triglycerides; Vision; Work; absorption; aged; base; cerebrovascular health; feeding; gain of function; global health; healthspan; inhibitor/antagonist; lipid metabolism; loss of function; mouse model; novel; phosphoproteomics; prevent; scaffold; sensor; stem; tumor; uptake

Abstract Dysregulation of absorption of dietary triglycerides (TGs) leads to adiposity or lipodystrophy, each of which can cause insulin insensitivity. Autophagy degrades unwanted cytoplasmic contents in lysosomes to maintain quality control. We have shown that autophagy degrades cytosolic lipid droplets (LDs) in multiple tissues via lipophagy. Whether autophagy in gut contributes to absorption of dietary TGs remains unknown. Dietary triglycerides (TGs) are absorbed by enterocytes as free fatty acids, which are re-esterified to TGs at the endoplasmic reticulum (ER) membrane by distinct TG synthesizing enzymes. Nascently produced TGs enter the ER lumen to form chylomicrons for secretion or are stored transiently as cytosolic LDs. In this application, using cultured enterocytes and mice as models, we will investigate how interplay between the nutrient sensor mTORC1, autophagy protein LC3, and TG synthesis enzymes regulates the fate of ingested lipid. We hypothesize that free fatty acid availability in enterocytes activates and localizes mTOR to ER membranes where it phosphorylates local pools of LC3. Phosphorylated (P)-LC3 is uncoupled from canonical autophagy and serves as scaffolds that interact with TG synthesis enzymes to regulate TG biogenesis – a key step driving chylomicron formation and secretion. We propose further that in the physiological state lipophagy contributes to clearance of transiently-stored cytosolic LDs, thus limiting the amount of TGs available for secretion. To test these hypotheses, we propose the following specific aims: S.A. 1: To characterize the phosphorylations and interactome of LC3 at the ER membrane. In this aim, we will identify the phosphorylation signature and interacting partners of LC3 at the ER membrane during lipid availability. We will determine the contribution of mTORC1 or its down-stream target ULK1 to LC3 phosphorylation. We will use site-directed mutagenesis to study the function of each of the identified phosphorylations towards TG synthesis in the ER, and TG secretion from the enterocyte. We will determine which of the newly-identified LC3 interacting partners regulate TG synthesis and secretion. S.A. 2: To dissect the interplay between mTOR, LC3, and lipophagy in dietary lipid absorption. In S.A. 2, we will use site-directed mutagenesis in cultured Caco2 intestinal cells, and novel mice models, to dissect the crosstalk between mTOR, LC3 and ER-localized TG synthesis enzymes in regulation of TG synthesis and TG entry into the ER. S.A. 3: To determine the contribution of gut mTOR signaling and lipophagy to development of metabolic syndrome during obesity. mTORC1 signaling and autophagy are each suppressed in obesity, which per se associates with increased absorption of dietary TGs. Consequently, we will use high fat feeding of mouse models of gain-of-function of mTOR signaling or loss-of- function of autophagy to explore the contribution of gut-specific mTOR and autophagy to development of metabolic syndrome. We will explore whether strategically-timed inhibition of mTOR signaling by rapamycin will reduce intestinal TG absorption and prevent metabolic syndrome. We will establish paradigm-shifting roles for mTOR, LC3, and autophagy in the molecular regulation of dietary TG absorption. Our findings will create a framework to consider how mTOR and LC3 communicate with novel interacting partners at the ER to regulate fundamental aspects of lipid metabolism. Significance: The metabolic syndrome is a significant global health problem affecting greater than 44% of the U.S. population aged more than 50 years. The metabolic syndrome affects health-span through effects on cardio/cerebrovascular health, locomotion, vision, cognition, and tumor development. The current proposal will delineate a novel crosstalk of mTOR and ATG protein in the regulation of gut TG metabolism, setting the basis for therapeutic modulation of mTOR signaling in the gut to prevent or treat the metabolic syndrome.

抽象的 饮食甘油三酸酯（TGS）苦难的失调导致肥胖或脂肪营养不良，每种都可以 引起胰岛素不敏感。自噬降解溶酶体中不需要的细胞质含量以维持 质量控制。我们已经表明，自噬在多个时间安排中降解了胞质脂质液滴（LDS） 脂肪。肠道中的自噬是否有助于饮食TG的苦难仍然未知。饮食 甘油三酸酯（TGS）被肠肠上皮细胞作为游离脂肪酸吸收，这些脂肪酸被重新置于TGS。 内质网（ER）膜通过不同的TG合成酶。无效产生的TG进入 ER管腔形成乳糜微粒进行分泌或瞬时存储为胞质LDS。在此应用程序中 使用培养的肠球细胞和小鼠作为型号，我们将研究营养传感器之间的相互作用 MTORC1，自噬蛋白LC3和TG合成酶调节摄入的脂质的命运。我们 假设肠上皮细胞中的游离脂肪酸可用性激活并将MTOR定位到ER膜 它磷酸化LC3的局部池。磷酸化（P）-LC3与规范自噬取消耦合 并用作与TG合成酶相互作用以调节TG生物发生的支架 - 一个关键步骤 驱动乳糜微粒形成和分泌。我们进一步提出，在物理状态下 有助于清除暂时存储的胞质LDS，从而限制了可用的TG量 分泌。为了检验这些假设，我们提出以下具体目的：S.A. 1：表征 LC3在ER膜上的磷酸化和相互作用组。在此目标中，我们将确定磷酸化 脂质可用性期间，在ER膜上的LC3的签名和相互作用的伴侣。我们将确定 MTORC1或其下游目标ULK1对LC3磷酸化的贡献。我们将使用网站定向 诱变以研究每个已鉴定的磷酸化对ER中TG合成的功能， 和肠肠细胞的TG分泌。我们将确定哪些新识别的LC3互动合作伙伴 调节TG合成和分泌。 S.A. 2：剖析MTOR，LC3和脂肪的相互作用 饮食脂质滥用。在S.A. 2中，我们将在培养的CACO2肠细胞中使用位置定向的诱变，以及 新的小鼠模型，以在MTOR，LC3和ER - 定位的TG合成酶之间剖析串扰 TG合成和TG进入ER的调节。 S.A. 3：确定肠道MTOR的贡献 肥胖期间代谢综合征发展的信号传导和寄生虫。 mtorc1信号和 自噬在肥胖症中均受到抑制，这本身本身与饮食TG的滥用增加有关。 因此，我们将使用MTOR信号的功能获得的小鼠模型的高脂肪进食或丧失。 自噬的功能，探索肠道特异性mTOR和自噬对发展的贡献 代谢综合征。我们将探讨雷帕霉素对MTOR信号传导的策略性抑制 将减少肠道TG抽象并预防代谢综合征。我们将建立范式移动角色 用于MTOR，LC3和自噬在饮食TG滥用的分子调节中。我们的发现将创建一个 框架以考虑MTOR和LC3如何与ER的新型互动合作伙伴进行沟通以调节 脂质代谢的基本方面。 意义：代谢综合征是一个重大的全球健康问题，影响超过44％ 美国人口年龄超过50岁。代谢综合征通过对 心脏/脑血管健康，运动，视力，认知和肿瘤发育。当前的建议将 在调节肠道TG代谢中，描绘了MTOR和ATG蛋白的新型串扰，树立了基础 用于肠道中MTOR信号传导的热调节，以预防或治疗代谢综合征。