Circadian Expression of Procolipase in the Liver: A Metabolic Signal

描述（由申请人提供）：越来越多的证据表明，昼夜节律的改变会导致人类肥胖。尽管生物钟的分子机制已经被描述得很清楚，但从生物钟到新陈代谢和行为的输出途径却不太清楚。最近对小鼠在持续黑暗条件下肝脏和其他组织中异位原胶原酶和胰脂肪酶相关蛋白2 （PLRP2）表达的研究，提出了这些蛋白参与环境应激下摄食行为和脂质代谢调节的可能性。重要的是，5'-AMP，一个在葡萄糖和脂质代谢中起中心作用的分子，调节了这些蛋白质的肝脏表达。由于原胶原酶在翻译后被加工释放一种有效的饱腹感调节剂肠抑素，以及脂解的辅助因子，而PLRP2是一种脂肪酶，这些发现对能量稳态的昼夜调节具有重要意义。在此，我们提出肝脏和其他外周器官中的procolipase和PLRP2表达代表了一种新的输出，可以根据环境触发调节摄食行为或脂质代谢。为了确定procolipase和PLRP2在响应环境信号中的作用，并开始揭示肝脏中procolipase基因表达背后的分子机制，我们提出了以下目标：1)通过表征procolipase缺陷小鼠、肠抑素缺陷小鼠、PLRP2缺陷小鼠和colipase转基因小鼠，确定procolipase和PLRP2在响应环境基因表达调节因子中的作用；2)确定ampk介导的通路是否在代谢刺激下调节procolipase和PLRP2的异位表达；3)利用HepG2细胞鉴定调节procolipase异位表达的顺式作用元件。在Aim 1中，我们将监测野生型、原胶原酶缺乏型和肠抑素缺乏型小鼠的体温、体重、活动、摄食行为以及食物和水的摄取量。我们将比较血液和肝脏中甘油三酯和脂肪酸的水平与procolipase和PLRP2的昼夜表达。本实验将提供证据，证明procolipase和PLRP2的异位表达具有功能影响。在Aim 2中，我们使用HepG2细胞和AMPK缺陷小鼠测试了amp活化激酶（AMPK）在调节procolipase异位表达中的作用。在Aim 3中，我们将定位含有介导对AMPK激活剂反应的顺式元件的原胶原酶基因区域。我们将在HepG2中进行启动子分析。这些研究将为进一步的研究提供依据，以确定从生物钟到5'- AMP到异位组织中原胶原酶表达的新途径中的相关转录因子。有了这些知识，就有可能开发出针对肥胖的新型时间生物学疗法。公共卫生相关性：肥胖已成为发达国家最常见的营养失调，肥胖的合并症显著增加了全世界的死亡率和医疗费用。生物或昼夜节律对能量稳态有深远的影响，这些节律的变化会导致代谢综合征或与肥胖相关的饮食模式紊乱，如暴饮暴食或夜食。拟议的研究有可能通过调节环境对生物钟的影响来开辟治疗肥胖的新途径。

DESCRIPTION (provided by applicant): There is emerging evidence that alterations in circadian rhythms contribute to obesity in humans. Although the molecular mechanisms of the circadian clock are mostly described, the output pathways from the clock to metabolism and behavior are less well understood. The recent description of ectopic procolipase and pancreatic lipase related protein 2 (PLRP2) expression in the liver and other tissues when mice are kept in constant darkness raises the possibility that these proteins contribute to the regulation of feeding behavior and lipid metabolism during environmental stress. Importantly, 5'-AMP, a molecule with a central role in glucose and lipid metabolism, regulated the liver expression of these proteins. Since procolipase is processed post- translationally to release a potent regulator of satiety, enterostatin, and a co-factor for lipolysis, colipase, and PLRP2 is a lipase, these findings have important implications for the circadian regulation of energy homeostasis. Herein, we propose that procolipase and PLRP2 expression in the liver and other peripheral organs represents a novel output that modulates feeding behavior or lipid metabolism in response to environmental triggers. To determine the role of procolipase and PLRP2 in response to environmental signals and to begin unraveling the molecular mechanisms behind procolipase gene expression in the liver, we propose the following Aims: 1) to determine the role of procolipase and PLRP2 in the response to environmental modulators of gene expression by characterizing procolipase-deficient, enterostatin-deficient, PLRP2-deficient and colipase transgene mice; 2) to determine if AMPK-mediated pathways regulate the ectopic expression of procolipase and PLRP2 in response to metabolic stimuli and 3) to identify the cis-acting elements that regulate the ectopic expression of procolipase using HepG2 cells. For Aim 1, we will monitor the body temperature, body weight, activity, feeding behavior and food and water intake of wild-type, procolipase- deficient and enterostatin-deficient mice kept in constant darkness or entrained to restricted-feeding. We will compare the blood and liver levels of triglycerides and fatty acids with the circadian expression of procolipase and PLRP2. The proposed experiments will provide evidence that the ectopic expression of procolipase and PLRP2 has functional consequences. In Aim 2, we test the role of AMP-activated kinase (AMPK) in regulating the ectopic expression of procolipase using HepG2 cells and AMPK-deficient mice. In Aim 3, we will locate the regions of the procolipase gene that contain the cis-elements that mediate the response to AMPK activators. We will perform the promoter analysis in HepG2. These studies will provide the justification for additional studies to identify the responsible transcription factors in a novel pathway from the circadian clock through 5'- AMP to procolipase expression in ectopic tissues. With this knowledge comes the potential to develop novel chronobiological therapies for obesity. PUBLIC HEALTH RELEVANCE: Obesity has become the most common nutritional disorder in the developed world and the co-morbidities of obesity contribute significantly to mortality and medical costs throughout the world. Biological or circadian rhythms have profound effects on energy homeostasis and changes in these rhythms can result in metabolic syndrome or in disordered eating patterns associated with obesity like binge eating or night eating. The proposed studies have the potential to open new avenues in the treatment of obesity through modulating the effects of the environment on biological clocks.