Molecular regulation of gut lipid metabolism by mTOR and autophagy proteins

摘要 饮食中甘油三酯(TGS)的吸收失调会导致肥胖或脂肪营养不良，其中每一种都可以 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以维持 质量控制。我们已经证明，自噬通过在多种组织中降解胞浆脂滴(LDS) 吞脂性。肠道中的自噬是否有助于食物中TGS的吸收尚不清楚。膳食 甘油三酯(TGS)以游离脂肪酸的形式被肠细胞吸收，在 内质网(ER)膜由不同的TG合成酶组成。新生产的TGS Enter 内质网管腔形成乳糜粒以供分泌，或以胞质LDs的形式暂时储存。在此应用程序中， 以培养的肠细胞和小鼠为模型，我们将研究营养传感器之间的相互作用。 MTORC1、自噬蛋白Lc3和TG合成酶调节摄入的脂质的命运。我们 假设肠上皮细胞中游离脂肪酸可激活mTOR并将其定位于内质网 在那里它使当地的LC3池磷酸化。磷酸化(P)-Lc3从典型的自噬中解偶联 作为与甘油三酯合成酶相互作用的支架，调节甘油三酯的生物发生--这是关键的一步 推动乳糜粒的形成和分泌。我们进一步提出，在生理状态下，脂噬作用 有助于清除暂时储存的胞质低密度脂蛋白，从而限制可用于 分泌物。为了检验这些假设，我们提出了以下具体目标：S.A.1：描述 Lc3在内质网膜上的磷酸化和相互作用体。在这个目标中，我们将确定磷酸化 在脂质可利用性过程中，内质网细胞膜上Lc3的特征和相互作用伙伴。我们将确定 MTORC1或其下游靶标ULK1对LC3磷酸化的贡献。我们将使用站点定向 突变研究每个已识别的磷酸化对内质网中TG合成的作用， 肠细胞分泌甘油三酯。我们将确定新确定的LC3互动合作伙伴中的哪一个 调节甘油三酯的合成和分泌。S.A.2：剖析mTOR、Lc3和吞脂性之间的相互作用 饮食中的脂肪吸收。在S.A.中，我们将在培养的Caco 2肠道细胞中使用定点突变，并 新的小鼠模型，以剖析mTOR，LC3和ER定位的TG合成酶之间的串扰 调节TG合成和TG进入内质网。S.A.3：确定肠道mTOR的贡献 肥胖期间代谢综合征发生的信号和吞脂作用。MTORC1信号和 自噬在肥胖中都受到抑制，这本身就与饮食中TGS的吸收增加有关。 因此，我们将使用高脂肪喂养的小鼠模型的功能获得的mTOR信号或丢失 自噬的作用探讨肠道特异性mTOR和自噬在肠易激综合征发生中的作用 代谢综合征。我们将探讨雷帕霉素对mTOR信号的战略性定时抑制 会减少肠道甘油三酯的吸收，预防代谢综合征。我们将建立范式转换的角色 对于mTOR、LC_3和自噬在饮食甘油三酯吸收的分子调控中的作用。我们的发现将创造一个 考虑mTOR和LC3如何与ER的新互动伙伴进行沟通以进行监管的框架 脂类新陈代谢的基本方面。 意义：代谢综合征是一个重大的全球健康问题，影响着超过44%的 美国人口年龄超过50岁。代谢综合征通过以下作用影响健康寿命 心脑血管健康、运动、视力、认知和肿瘤发展。目前的提案将 描绘了mTOR和ATG蛋白在调节肠道甘油三酯代谢中的新的串扰，为进一步研究奠定了基础 用于治疗调节肠道中的mTOR信号，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.