Control of liver autophagy by phosphatidic acid signaling

描述(由申请人提供)：适当储存和利用脂类对于维持细胞能量平衡是必不可少的，这种平衡的干扰会导致各种人类疾病，包括代谢综合征。细胞脂质通常以甘油三酯的形式以脂滴的形式储存，并在营养缺乏期间被水解为脂肪酸以提供能量。近年来，巨自噬(简称自噬)已成为肝细胞和其他多种细胞中甘油三酯分解代谢和脂滴清除的主要途径。自噬确保了在营养匮乏的条件下大分子的循环，以及消除有缺陷的细胞器、长寿的蛋白质和细胞内蛋白质聚集体。由于其在消除细胞“废物”和消耗能源方面的显著效率，自噬越来越多地被视为一种可用于治疗各种人类疾病的途径。最近发现的大脂吞噬提出了一些基本问题，包括是否有专门针对这种选择性类型的自噬的分子途径，以及这一过程的有效性是否受到饮食和衰老的影响。在这项建议中，我们探讨了脂酶磷脂酶D_1(PLD_1)是大脂吞噬的正向调节因子的可能性。磷脂酰胆碱能将磷脂酰胆碱水解成生物活性的脂磷脂酸，从而调节信号转导和膜转运过程。我们发表的数据表明，由于突变肝细胞中自噬小体的数量减少和体积变小，缺乏PLD1的小鼠在肝脏中表现出自噬缺陷。值得注意的是，这种表型是由饥饿条件诱导的，与蛋白质聚集体的积累无关，而在缺乏ATG5或ATG7等基因的突变小鼠中观察到的典型情况是，ATG5或ATG7对基础和饥饿诱导的自噬都是必不可少的。在机制水平上，我们的结果表明，PLD1衍生的磷脂酸的一个关键功能是促进自噬小体的成熟，促进这些细胞器与内体/溶酶体的融合。再加上初步观察显示，在饥饿小鼠的Pld1基因敲除的肝脏以及基因敲除的成纤维细胞中，脂滴更加突出，我们的结果强烈表明PLD1与饥饿诱导的巨脂噬有关。在这项建议中，我们计划用肝细胞中选择性缺乏PLD1的小鼠直接测试这一假说。我们还评估了“应激源”，如高脂饮食和年龄，对高脂吞噬疗效的影响，预测它们可能进一步揭示脂肪积累和脂代谢的特定基因型差异。我们期待我们的研究将阐明大脂吞噬在控制脂代谢中的作用，并为未来的工作提供基础，测试刺激PLD1途径是否可以在人类条件下促进大脂吞噬，如脂肪肝疾病。

DESCRIPTION (provided by applicant): Proper storage and utilization of lipids is essential to maintain cellular energy homeostasis and perturbation of this balance can lead to a variety of human disorders, including metabolic syndrome. Cellular lipids are generally stored as triglycerides in the form of lipid droplets and are hydrolyzed into fatty acids for energy supply during nutrient deprivation. Recently, macroautophagy (simply called autophagy) has emerged as a major cellular pathway mediating the catabolism of triglycerides and the clearance of lipid droplets (i.e., "macrolipophagy") in hepatocytes and many other cell types. Autophagy ensures the recycling of macromolecules under conditions of nutritional scarcity, as well as the elimination of defective organelles, long- lived proteins, and intracellular protein aggregates. Because of its remarkable efficiency at eliminating cellular "waste" and consuming energy sources, autophagy is increasingly viewed as a pathway that can be exploited for the therapy of various human conditions. The recent discovery of macrolipophagy has raised a number of fundamental questions, including whether there are molecular pathways dedicated to this selective type of autophagy and whether the efficacy of this process is affected by diet and aging. In this proposal, we explore the possibility that the lipid enzyme phospholipase D1 (PLD1) is a positive modulator of macrolipophagy. PLD1 hydrolyzes phosphatidylcholine into the bioactive lipid phosphatidic acid, which regulates signaling and membrane trafficking processes. Our published data indicate that mice lacking PLD1, which are viable, exhibit defects in autophagy in the liver, based on the occurrence of both fewer and smaller autophagosomes in mutant hepatocytes. Remarkably, this phenotype is induced by starvation conditions and is not associated with the accumulation of protein aggregates, as typically observed in mutant mice lacking genes such as Atg5 or Atg7 which are essential for both basal and starvation-induced autophagy. At the mechanistic level, our results suggest that a key function of PLD1-derived phosphatidic acid is to promote the maturation of autophagosomes by facilitating the fusion of these organelles with endosomes/lysosomes. Together with preliminary observations showing that lipid droplets are more prominent in Pld1 knockout liver from starved mice as well as in knockout fibroblasts, our results strongly suggest that PLD1 is implicated in starvation- induced macrolipophagy. In this proposal, we plan to directly test this hypothesis using mice that lack PLD1 selectively in hepatocytes. We also assess the impact of "stressors", such as high fat diet and age, on the efficacy of macrolipophagy, with the prediction that they may further unmask genotype-specific differences in fat accumulation and lipid metabolism. We anticipate our studies will clarify the role of macrolipophagy in the control of lipid metabolism and provide the basis for future work testing whether stimulating the PLD1 pathway can be exploited to promote macrolipophagy in human conditions, such as fatty liver diseases.