Diurnal regulation of MTP and plasma lipids

描述(由申请人提供)：我们的目标是了解参与血浆脂类昼夜调节的分子机制。我们观察到血浆甘油三酯和载脂蛋白中的胆固醇呈现出昼夜节律。当动物受到食物夹带或暴露在持续的光照下时，这些节律就会改变。因此，食物和光诱导的机制都控制着血脂的变化。我们的目的是从生理、生化和分子水平上解释这两种机制。目的1：光诱导调节血脂和组织MTP水平的机制：转录因子异源二聚体CLOCK和BMal1正向调节光诱导下丘脑视交叉上核的昼夜节律。这些转录因子也在外周组织中表达。我们的假设是Clock/BMal1在光诱导的血脂和MTP表达的调节中起关键作用。为了评估这一点，我们将测量时钟突变(Clkmt/mt)、BMal1(BMal1-/-)基因敲除(KO)小鼠及其野生型(WT)兄弟姐妹的血浆甘油三酯、胆固醇和载脂蛋白B-脂蛋白。此外，还将对涉及脂肪代谢和昼夜节律的基因的蛋白质和mRNA水平进行量化。为了强调时钟和BMal1分别在肠道和肝脏动员外源性和内源性脂肪中的重要性，我们将研究这些组织的脂蛋白产生。然后，实验将重点了解这些转录因子如何调节MTP基因的表达。将确定MTP启动子中对昼夜节律调节至关重要的元件以及与这些顺式元件结合的转录因子。这些研究将阐明由CLOCK和BMal1调控的影响血脂昼夜节律的关键生理、生化和分子机制。此外，还将建立由CLOCK/BMal1调控MTP的分子联系。目的2：食物携带的血脂和脂蛋白波动的机制：我们假设Clock、BMal1和MTP在食物携带的血脂和肝脏和肠道中的脂代谢和Clock基因的表达中起关键作用。为了测试这一点，Clkmt/mt、BMal1-/-以及肠道和肝脏特异性MTP缺陷小鼠和对照组小鼠将暴露于不同的食物夹带计划中。此外，还将研究食物移除和禁食/再喂食对血脂和候选基因组织表达的影响。实验将被设计用来确定食物携带MTP表达的分子机制。时钟和BMal1在暴露于2小时喂养的细胞中循环MTP表达中的重要性将被确定。将确定MTP启动子中对食物夹带做出反应的元件以及与这些元件结合的转录因子。这些研究将提供有关肠道和肝脏MTP在食物携带外周时钟基因中的重要性的新信息。CLOCK和BMal1在食物携带调节血浆脂/脂蛋白中的关键作用将被确立。与公共健康相关：我们建议解开光和食物携带的导致每日血脂水平变化的机制。具体地说，时钟和BMal1以及肝脏和肠道特异的微粒体甘油三酯转移蛋白的作用将被阐明。

DESCRIPTION (provided by applicant): Our goal is to understand molecular mechanisms involved in the diurnal regulation of plasma lipids. We observed that plasma triglyceride and cholesterol in apoB-lipoproteins show diurnal rhythms. These rhythms are changed when animals are subjected to food entrainment or exposed to constant light. Therefore, both food and light induced mechanisms control variations in plasma lipids. Our aim is to explain both these mechanisms at physiological, biochemical and molecular levels. Aim 1: Light-entrained mechanisms controlling diurnal regulation of plasma lipids and tissue MTP levels: Heterodimers of transcription factors Clock and Bmal1 positively regulate circadian rhythms in the light-entrained suprachiasmatic nucleus of the hypothalamus. These transcription factors are also expressed in peripheral tissues. Our hypothesis is that Clock/Bmal1 are critical for light-induced regulation of plasma lipids and MTP expression. To evaluate this, we will measure plasma triglyceride, cholesterol and apoB-lipoproteins in Clock mutant (Clkmt/mt), Bmal1 (Bmal1-/-) knockout (KO) mice and their wild type (WT) siblings. In addition, protein and mRNA levels of genes involved in lipid metabolism and circadian rhythms will be quantified. To highlight the importance of Clock and Bmal1 in the mobilization of exogenous and endogenous lipids by the intestine and liver, respectively, we will study lipoprotein production by these tissues. Experiments will then focus to understand how these transcription factors regulate MTP gene expression. Elements in MTP promoter critical for circadian regulation and transcription factors that bind to these cis elements will be identified. These studies will elucidate key physiological, biochemical, and molecular mechanisms regulated by Clock and Bmal1 that contribute to diurnal rhythms in plasma lipids. In addition, molecular links in the regulation of MTP by Clock/Bmal1 will be established. Aim 2: Mechanisms involved in food-entrained oscillations in plasma lipids and lipoproteins: We hypothesize that Clock, Bmal1 and MTP play critical roles in food-entrainment of plasma lipids and expression of lipid metabolism and clock genes in the liver and intestine. To test this, Clkmt/mt, Bmal1-/-, as well as intestine and hepatic-specific MTP deficient and control mice will be exposed to different food entrainment schedules. Moreover, the effect of food removal and fasting/re-feeding on plasma lipids and tissue expression of candidate genes will be studied. Experiments will be designed to identify molecular mechanisms involved in food- entrainment of MTP expression. The importance of Clock and Bmal1 in the cyclic MTP expression in cells exposed to 2 h feeding will be ascertained. Elements in MTP promoter that respond to food entrainment and transcription factors that bind to these elements will be identified. These studies will provide novel information about the importance of intestinal and hepatic MTP in the entrainment of peripheral clock genes by food. Critical role of Clock and Bmal1 in food-entrained regulation of plasma lipids/lipoproteins will be established. PUBLIC HEALTH RELEVANCE: We propose to unravel light and food entrained mechanisms that contribute to daily changes in plasma lipid levels. Specifically, the role of Clock and Bmal1 as well as liver and intestine-specific microsomal triglyceride transfer protein will be elucidated.