Diurnal regulation of MTP and plasma lipids

描述（由申请人提供）：我们的目标是了解涉及血浆脂质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B-脂蛋白胆固醇的昼夜节律。当动物受到食物夹带或暴露在恒定的光照下时，这些节奏就会改变。因此，食物和光诱导的机制控制血浆脂质的变化。我们的目标是在生理、生化和分子水平上解释这两种机制。目标1：控制血脂和组织MTP水平的昼夜调节的光诱导机制：转录因子Clock和Bmal 1的异源二聚体积极调节下丘脑光诱导的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/Bmal 1对光诱导的血脂和MTP表达的调节至关重要。为了评估这一点，我们将测量Clock突变（Clkmt/mt）、Bmal 1（Bmal 1-/-）敲除（KO）小鼠及其野生型（WT）同胞的血浆甘油三酯、胆固醇和载脂蛋白B-脂蛋白。此外，还将对参与脂质代谢和昼夜节律的基因的蛋白质和mRNA水平进行定量。为了突出Clock和Bmal 1在肠和肝分别动员外源性和内源性脂质中的重要性，我们将研究这些组织的脂蛋白产生。然后，实验将集中于了解这些转录因子如何调节MTP基因表达。将鉴定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Clock和Bmal 1调控的关键生理、生化和分子机制，这些机制有助于血浆脂质的昼夜节律。此外，还将建立Clock/Bmal 1调节MTP的分子联系。目标二：在血浆脂质和脂蛋白的食物夹带振荡参与的机制：我们假设，时钟，Bmal 1和MTP在血浆脂质的食物夹带和脂质代谢和时钟基因的表达在肝脏和肠道中发挥关键作用。为了测试这一点，将Clkmt/mt、Bmal 1-/-以及肠和肝特异性MTP缺陷和对照小鼠暴露于不同的食物夹带时间表。此外，将研究食物去除和禁食/再喂养对血浆脂质和候选基因的组织表达的影响。实验将被设计来鉴定涉及食物夹带MTP表达的分子机制。Clock和Bmal 1在暴露于2小时喂养的细胞中的循环MTP表达中的重要性将被确定。MTP启动子中响应食物夹带的元件和与这些元件结合的转录因子将被鉴定。这些研究将提供新的信息肠道和肝脏MTP的重要性，在夹带外周时钟基因的食物。Clock和Bmal 1在血浆脂质/脂蛋白的食物夹带调节中的关键作用将被建立。公共卫生相关性：我们建议解开导致血脂水平每日变化的光和食物夹带机制。具体而言，时钟和Bmal 1以及肝脏和胆固醇特异性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.