Investigating how apolipoprotein E genotypes modify fatty acid metabolism

本组织的总体目标：阐明载脂蛋白E（APOE）基因型对长链多不饱和脂肪酸（LC-PUFA）代谢的作用。 背景：血浆LC-PUFA库是将LC-PUFA带入脑的关键，它是动态的，不断地与器官和组织交换FA。构成大脑膜的脂肪酸中有三分之一是花生四烯酸（ARA）和二十二碳六烯酸（DHA）：这两种LC-PUFA几乎完全由饮食提供。以往的研究表明存在脂肪-肝-脑FA轴。 APOE基因在人类中有三种亚型（E2、E3和E4）。在我们之前的NSERC发现基金（DG）中，我们已经证明在肝脏，脂肪组织，血浆和大脑中的LC-PUFA稳态中存在APOE基因型特异性失调。使用原位脑内灌注，我的合作者Frederic Calon和我发现，与喂食n-3 LC-PUFA缺乏饮食的hAPOE 2小鼠相比，4个月和13个月大的hAPOE 4小鼠的14 C-DHA脑摄取量较低。这一有趣的结果表明，LC-PUFA稳态失调在hAPOE 4小鼠的生命早期开始。 因此，该NSERC-DG计划的假设是，在年轻时，APOE基因型与膳食脂肪酸相互作用，以调节脂肪酸谱、其转运蛋白及其在脂肪-肝-脑轴中的基因表达水平。 今后五年的具体目标： 1.通过APOE基因型和膳食脂肪摄入量研究脂肪-肝-脑轴的脂肪酸稳态。 2.使用原位脑灌注评价不同载脂蛋白E基因型和饮食对脑ARA和DHA摄取的影响。 实验方法 在目前的研究计划中，我们建议使用表达人APOE 2、APOE 3或APOE 4的转基因小鼠品系和C57 BL 6/129 SV小鼠作为对照（与转基因小鼠相同的遗传背景）。将使用三种不同的饮食来改变组织和器官的脂肪酸谱：A）对照饮食，B）富含DHA的饮食和C）缺乏n-3 LC-PUFA的饮食。小鼠将被喂食这些饮食16周。我们将量化ARA和DHA，脂肪酸转运蛋白基因的表达以及它们各自在每个器官和组织中的蛋白质。我们将用我们新的LC-MS-MS仪器开发生物化学方法学。将与拉瓦尔大学的Frederic Calon合作，使用原位脑灌注，通过APOE基因型和膳食脂肪摄入量评价ARA和DHA的脑摄取。 新奇和预期意义：研究脂肪-肝脏-大脑轴是一种新颖的方法，因为我们使用的是综合方法，而不是器官靶向方法。考虑到LC-PUFA对器官和组织的调节作用以及apoe在脂质转运和稳态中的作用，研究与APOE基因型相关的多器官LC-PUFA稳态是重要的。

OVERALL AIM OF THE PROGRAM: To elucidate the role(s) of apolipoprotein E (APOE) genotypes on long chain polyunsaturated fatty acids (LC-PUFA) metabolism. BACKGROUND: The plasma LC-PUFA pool is critical to bring LC-PUFA to the brain and it is dynamic, constantly exchanging FA with organs and tissues. One third of the fatty acids composing brain membranes are arachidonic acid (ARA) and docosahexaenoic acid (DHA): two LC-PUFAs provided almost exclusively by the diet. Previous studies suggested that there is an adipose-liver-brain FA axis. The APOE gene has three existing isoforms in human (E2, E3 and E4). In our previous NSERC-discovery grant (DG), we have shown that there are APOE-genotype specific dysregulation in LC-PUFA homeostasis in the liver, the adipose tissue, the plasma and the brain. Using in situ intracerebral perfusion, my collaborator Frederic Calon and I showed lower brain uptake of 14C-DHA in 4- and 13-month-old hAPOE4 mice compared to uptake in hAPOE2 mice fed a deficient diet in n-3 LC-PUFA. This intriguing result suggests that LC-PUFA homeostasis deregulation starts early in life in hAPOE4 mice. Therefore, the hypothesis for this NSERC-DG program is that, at a young age, APOE genotype interacts with dietary fatty acids to modulate the fatty acid profile, their transport proteins and their levels of gene expression in the adipose-liver-brain axis. SPECIFIC OBJECTIVES FOR THE NEXT FIVE YEARS: 1. To investigate the fatty acid homeostasis of the adipose-liver-brain axis by APOE genotype and dietary fat intake. 2. To evaluate brain ARA and DHA uptake with respect to the different APOE genotypes and diets using in situ brain perfusion. EXPERIMENTAL APPROACH In the current research program, we propose to use transgenic mouse strains expressing either humans APOE2, APOE3 or APOE4 and C57BL6 / 129SV mice as control (same genetic background than the transgenic mice). Three different diets will be used to alter the fatty acid profile of tissues and organs: A) a control diet, B) a diet enriched with DHA and C) a diet deprived in n-3 LC-PUFA. Mice will be fed these diets for 16 weeks. We will quantify ARA and DHA, the expression of fatty acid transporters genes as well as their respective proteins in each organs and tissues. We will develop lipididomics methodology with our new LC-MS-MS instrument. The brain uptake of ARA and DHA will be evaluated by APOE genotype and dietary fat intake using in situ cerebral perfusion with the collaboration of Frederic Calon at Université Laval. NOVELTY AND EXPECTED SIGNIFICANCE: Studying the adipose-liver-brain axis is something novel as we use an integrative approach instead of an organ targeted approach. Investigating multi-organ LC-PUFA homeostasis in relation to APOE genotype is important considering the regulatory role of LC-PUFA for organs and tissues and the role of apoe in lipid transport and homeostasis.