Investigating how apolipoprotein E genotypes modify fatty acid metabolism

本项目的总体目标：阐明载脂蛋白E(ApoE)基因型对长链多不饱和脂肪酸(LC-PUFA)代谢的影响(S)。 背景：血浆LC-PUFA池是将LC-PUFA带入大脑的关键，它是动态的，不断地与器官和组织交换FA。构成脑膜的三分之一的脂肪酸是花生四烯酸(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-肝脏-脑FA轴。 APOE基因在人类中存在三种亚型(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、血浆和脑中存在APOE基因特异性的LC-PUFA动态平衡失调。使用脑内原位灌注，我和我的合作者Frederic Calon和我显示，4个月和13个月大的hAPOE4小鼠的大脑摄取14C-DHA的能力低于喂食n-3LC-PUFA不足的hAPOE2小鼠。这一耐人寻味的结果表明，在hAPOE4小鼠的生命早期，LC-PUFA动态平衡的解除调控就开始了。 因此，NSERC-DG计划的假设是，在很小的时候，APOE基因与饮食中的脂肪酸相互作用，调节脂肪酸的分布、它们的运输蛋白以及它们在脂肪-肝脏-脑轴的基因表达水平。 今后五年的具体奋斗目标： 1.通过APOE基因分型和膳食脂肪摄入量研究脂肪-肝-脑轴的脂肪酸动态平衡。 2.用原位脑血流灌注法评价不同apoE基因型和不同饮食对脑内ARA和DHA的摄取。 实验方法 在目前的研究计划中，我们建议使用表达人APOE2、APOE3或APOE4的转基因小鼠和C57BL6/129SV小鼠作为对照(与转基因小鼠的遗传背景相同)。三种不同的饮食将被用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-3LC-PUFA的饮食。小鼠将被喂食这些食物16周。我们将对ARA和DHA、脂肪酸转运蛋白基因及其各自在每个器官和组织中的蛋白表达进行量化。我们将用我们新的LC-MS-MS仪器开发血脂学方法学。大脑对ARA和DHA的摄取将通过APOE基因和饮食脂肪摄入量进行评估，使用与拉瓦尔大学的Frederic Calon合作的原位脑灌注。 新颖性和预期意义：研究脂肪-肝脏-脑轴是一件新奇的事情，因为我们使用了一种综合的方法，而不是针对器官的方法。考虑到LC-PUFA对器官和组织的调节作用以及APOE在脂质运输和稳态中的作用，研究与APOE基因相关的多器官LC-PUFA动态平衡是重要的。

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.