Molecular regulation of gut lipid metabolism by mTOR and autophagy proteins

摘要 膳食甘油三酯（TG）吸收失调导致肥胖或脂肪营养不良，每一种都可以 导致胰岛素不敏感。自噬降解溶酶体中不需要的细胞质内容物， 质量控制我们已经证明，自噬通过以下途径降解多种组织中的胞质脂滴（LD）： 脂肪吞噬肠道自噬是否有助于膳食TG的吸收仍不清楚。膳食 甘油三酯（TG）作为游离脂肪酸被肠上皮细胞吸收，其在消化道内再酯化为TG。 内质网（ER）膜的TG合成酶。新生产的TG进入 内质网腔形成乳糜微粒用于分泌或作为细胞溶质LD暂时储存。在本申请中， 以培养的肠上皮细胞和小鼠为模型，我们将研究营养传感器之间的相互作用， mTORC 1、自噬蛋白LC 3和TG合成酶调节摄入脂质的命运。我们 假设肠上皮细胞中游离脂肪酸可激活mTOR并将其定位于ER膜 在那里它磷酸化LC 3的局部池。磷酸化（P）-LC 3与典型自噬解偶联 并作为支架与TG合成酶相互作用以调节TG生物合成-这是关键步骤 驱动乳糜微粒的形成和分泌。我们进一步提出，在生理状态下， 有助于清除暂时储存的细胞溶质LD，从而限制了可用于治疗的TG的量。 分泌物为了检验这些假设，我们提出了以下具体目标：1：描述 磷酸化和相互作用组的LC 3在ER膜。在这个目标中，我们将确定磷酸化 在脂质可用性期间，LC 3在ER膜上的签名和相互作用伴侣。康贝特人将以 mTORC 1或其下游靶标ULK 1对LC 3磷酸化的贡献。我们将使用网站导向 诱变以研究每种鉴定的磷酸化对ER中TG合成的功能， 以及肠上皮细胞的TG分泌。我们将确定哪些新发现的LC 3相互作用伙伴 调节TG的合成和分泌。S.A. 2：为了剖析mTOR、LC 3和脂肪吞噬之间的相互作用， 膳食脂质吸收在S.A. 2，我们将在培养的Caco 2肠细胞中使用定点诱变， 新的小鼠模型，以剖析mTOR，LC 3和ER定位的TG合成酶之间的串扰， 调节TG合成和TG进入ER。S.A. 3：确定肠道mTOR的贡献 信号传导和脂肪吞噬对肥胖期间代谢综合征的发展的影响。mTORC 1信号传导和 自噬在肥胖中各自受到抑制，其本身与膳食TG吸收增加有关。 因此，我们将使用高脂肪喂养mTOR信号传导功能获得或mTOR信号传导功能丧失的小鼠模型。 自噬的功能，以探索肠道特异性mTOR和自噬对发展的贡献。 代谢综合征我们将探索雷帕霉素对mTOR信号的策略性定时抑制是否 将减少肠道TG吸收，预防代谢综合征。我们将建立范式转换的角色 mTOR、LC 3和自噬在饮食TG吸收的分子调节中的作用。我们的发现将创造一个 考虑mTOR和LC 3如何与ER中的新型相互作用伙伴进行通信以调节 脂质代谢的基本方面。 重要性：代谢综合征是一个重要的全球性健康问题，影响超过44%的人， 美国50岁以上的人口。代谢综合征影响健康寿命通过影响 心/脑血管健康、运动、视觉、认知和肿瘤发展。目前的提案将 描述了一种新的mTOR和ATG蛋白在调节肠道TG代谢中的相互作用，为研究mTOR和ATG蛋白在调节肠道TG代谢中的相互作用奠定了基础。 用于治疗性调节肠道中的mTOR信号传导以预防或治疗代谢综合征。

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.