Mechanisms underlying the biological function of fatty acids

背景。由于形式的多样性，单个脂肪酸（FA）具有独特的生物学效应。因此，我的研究计划的长期目标是a)确定FA调节信号通路的机制，b)定义主要FA的个体效应，c)它们对细胞功能的影响。在过去的6年里，我对FA的生理水平有了一定的了解，这对未来的研究有很大的帮助。此外，我们使用新的δ 6去饱和酶敲除小鼠模型确定了α -亚麻酸（ALA）的抗炎作用。这一发现揭穿了ALA没有生物活性的教条。这些结果也导致了一条新的研究路线，以了解去饱和酶对FA信号传导的调节作用。我们对硬脂酰辅酶a去饱和酶（SCD1）的研究进一步支持了这一机制。我们发现SCD1，一种δ 9去饱和酶，影响反式异丙酸中共轭亚油酸（CLA）的水平。我们之前已经表明，CLA通过改变小泡膜组成和蛋白质定位影响小泡介导的信号传导。同时，这些观察结果表明，从头合成通过控制FA供应影响细胞信号传导。***目标和培训。基于迄今为止的进展，总体目标是继续研究FA对小泡介导的信号传导的个体影响以及FA去饱和酶的调节作用。利用分子、饮食和生理学方法，通过体外和体内模型系统研究短期目标：***1：单个FA对小囊膜脂组成的影响***2：单个FA对小囊蛋白定位和下游信号的影响***3:FA去饱和酶对小囊膜脂组成和信号的影响***拟资助1名博士、4名硕士和5名本科生。HQP将接受研究方法、统计分析和沟通技巧方面的培训。研究成果将通过新闻稿、同行评议的出版物和报告传播。* * *实验方法。单个FA将添加到乳腺、前列腺、结肠和肝细胞系的培养基中。小囊泡将通过超离心分离出来。膜FA组成将通过气相色谱分析。信号将通过免疫印迹和RT-PCR检测基因表达。siRNA将被用来通过抑制SCD1和D6D的活性来分离单个FA的作用。同样，将选择FA用于SCD1和D6D敲除小鼠的饲养研究，以确定FA的个体作用以及去饱和酶对小泡膜组成和细胞信号传导的影响。***意义这项研究将推进我们关于个体FA在细胞信号传导和功能中的生物学作用的基础知识，并支持HQP培训以知识为基础的经济。**

Background. By virtue of a diversity of form, individual fatty acids (FA) have unique biological effects. Therefore, the long term objective of objective of my research program is to a) determine mechanisms by which FA regulate signalling pathways, b) define individual effects of major FA and c) their impact on cellular function. In the past 6 years, I have produced knowledge regarding physiological levels of FA, which will greatly inform future studies. Also, we defined an anti-inflammatory role for alpha-linolenic acid (ALA) using the novel delta 6 desaturase knock out mouse model. This finding debunks dogma that ALA has no biological activity. These results also led to a new line of research to understand the modulatory role of desaturases on FA signalling. Further support for this mechanism was provided in our work on stearoyl CoA desaturase (SCD1). We show that SCD1, a delta 9 desaturase enzyme, influences levels of conjugated linoleic acid (CLA) from trans vaccenic acid. We have previously shown that CLA influences caveolae-mediated signalling by altering caveolae membrane composition and protein localization. In tandem, these observations suggest that de novo synthesis influences cell signalling by controlling FA supply.***Objectives and Training. Building on progress to date, the overall objective is to continue examining the individual effects of FA on caveolae mediated signaling and the modulatory role of FA desaturases. Using molecular, dietary and physiological approaches the short term objectives are to investigate using in vitro and in vivo model systems the:***1: effect of individual FA on caveolae membrane lipid composition***2: effect of individual FA on caveolae-resident protein localization and downstream signalling***3: effect of FA desaturases on caveolae membrane lipid composition and signalling***Funds are requested to support 1 PhD, 4 MSc and 5 undergraduates. HQP will be trained in research methodologies, statistical analyses, and communication skills. Research will be disseminated via press releases, peer reviewed publications, and presentations.***Experimental Approach. Individual FA will be added to the media of breast, prostate, colon, and liver cell lines. Caveolae will be isolated from by ultracentrifugation. Membrane FA composition will be analyzed by gas chromatography. Signalling will be determined by western immunoblotting and gene expression by RT-PCR. siRNA will be used to isolate effects of individual FA by inhibiting the activity of SCD1 and D6D. Similarly, select FA will be used in feeding studies with SCD1 and D6D knock out mice to determine the individual role of FA and the influence of desaturases on caveolae membrane composition and cell signalling.***Significance. This research will advance our foundational knowledge regarding the biological role of individual FA in cell signalling and function and support HQP training for a knowledge based economy. **