Discovery and characterization of signaling molecules from tetracosahexaenoic acid

二十四碳六烯酸信号分子的发现和表征

• 批准号: RGPIN-2017-06465
• 负责人: Bazinet, Richard
• 金额: $ 2.04万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2017
• 资助国家: 加拿大
• 起止时间: 2017-01-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=630610
• 关键词: Discovery; characterization; signaling; molecules; tetracosahexaenoic

Docosahexaenoic acid (22:6n-3; DHA) is an omega-3 polyunsaturated fatty acid that is highly abundant in the brain, where it is thought to regulate many important processes. Through a series of desaturations and elongations, largely occurring in the liver, alpha-linolenic acid (18:3n-3) ALA is converted to DHA. One especially interesting, and sometimes confusing, biochemical step is that ALA, upon a few steps, is converted to tetracosahexaenoic acid (24:6n-3;THA) before one round of beta-oxidation removes two carbons producing DHA. We, and others, have been a bit perplexed by the apparent complexity of this pathway and think that some of the issues may arise by not examining the pathway kinetically. Thus, the overall objective of this grant proposal is to understand if THA is a short lived and committed precursor to DHA, as is commonly accepted, or if THA is a product of DHA and/or is possibly itself a signaling molecule. Our focus will be on the liver for synthesis studies as that is thought to be the major site of synthesis, although other tissues will be examined, while we will focus on the brain for signaling studies. The first specific objective of this grant is to quantify and compare the rate of synthesis of DHA to THA with the rate of synthesis of THA to DHA in vivo using our established and newly developed kinetic models in rodents. The first model requires a 5 minute intravenous infusion of labelled THA and DHA (which we have sourced) in the unanaesthetized, free-living rat. The second approach to this question is similar, but labelled THA and DHA are infused for 3 hours and the appearance of their “products” are measured in the blood and applied in a kinetic model that determines their synthesis/secretion rate. Comparing these two kinetic models will not only achieve these objectives, but will give us insight into why tissue levels of THA are low by testing if THA is extensively secreted from the liver. The second part of this grant is to identify and quantify THA in the brain and to test if it is enzymatically converted into molecules that could be involved in signaling. Our second objective will focus on the brain and will determine if THA is present in common fatty acid signaling pathways. To do this, we have developed sensitive LC- and GC-MS methods to measure THA and its candidate signaling derivatives. We will now apply these methods to detect if THA is released from neuronal membranes upon ischemia into the unesterified fatty acid pool, a phenomenon common among signaling molecules. We will also use these techniques to identify if THA is enzymatically converted to molecules that act as signaling molecules similar to DHA and arachidonic acid (ARA, 20:4n-6). We will also perform these experiments in wildtype mice and in mice lacking critical enzymes for the synthesis of the signaling molecules.

二十二碳六烯酸(22：6N-3；DHA)是一种omega-3多不饱和脂肪酸，在大脑中含量丰富，被认为调节许多重要的过程。通过一系列的脱饱和度和伸长，主要发生在肝脏，α-亚麻酸(18：3n-3)丙氨酸转化为DHA。一个特别有趣但有时令人困惑的生化步骤是，丙氨酸在几个步骤后转化为四碳六烯酸(24：6N-3；THA)，然后一轮β-氧化去除两个产生DHA的碳。我们和其他人对这一途径的明显复杂性感到有点困惑，并认为一些问题可能是因为没有从动力学上研究这一途径而产生的。因此，这项赠款提案的总体目标是了解THA是否如普遍接受的那样是DHA的短暂和承诺的前体，或者THA是否是DHA的产物和/或本身可能是信号分子。我们的重点将放在肝脏上进行合成研究，因为它被认为是主要的合成部位，尽管其他组织将被检查，而我们将专注于大脑的信号研究。这项资助的第一个具体目标是使用我们建立的和新开发的啮齿动物动力学模型来量化和比较体内DHA到THA的合成速率和THA到DHA的合成速率。第一个模型需要在未麻醉的自由生活的大鼠中静脉输注标记的THA和DHA(我们已经获得)5分钟。这个问题的第二种方法与此类似，但将标记为THA和DHA的药物输注3小时，在血液中测量其“产物”的外观，并应用于确定其合成/分泌速率的动力学模型。比较这两个动力学模型不仅可以实现这些目标，还可以通过测试肝脏是否广泛分泌THA来深入了解组织中THA水平低的原因。这笔拨款的第二部分是识别和量化大脑中的THA，并测试它是否被酶转化为可能参与信号传递的分子。我们的第二个目标将集中在大脑上，并将确定THA是否存在于常见的脂肪酸信号通路中。为此，我们开发了灵敏的LC-MS和GC-MS方法来测定THA及其候选信号衍生物。我们现在将应用这些方法来检测THA是否在缺血时从神经细胞膜释放到未酯化的脂肪酸池中，这是信号分子中常见的现象。我们还将使用这些技术来鉴定THA是否被酶促转化为类似于DHA和花生四烯酸(ARA，20：4N-6)的信号分子。我们还将在野生型小鼠和缺乏合成信号分子的关键酶的小鼠身上进行这些实验。