Mechanisms underlying the biological function of fatty acids

背景资料。单种脂肪酸(FA)形式多样，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)确定主要FA的个体效应，c)它们对细胞功能的影响。在过去的6年里，我积累了有关FA生理水平的知识，这将极大地促进未来的研究。此外，我们使用新的delta6去饱和酶基因敲除小鼠模型定义了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。这些结果还导致了一条新的研究路线，以了解去饱和酶在FA信号中的调制作用。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Delta9去饱和酶，影响反式母牛油酸中共轭亚油酸(CLA)的水平。我们以前已经证明，CLA通过改变小窝的膜成分和蛋白质定位来影响小窝介导的信号转导。这些观察表明，从头合成通过控制FA供应来影响细胞信号传递。*目标和训练。在迄今进展的基础上，总体目标是继续研究FA对小窝介导的信号转导的个体影响以及FA去饱和酶的调节作用。使用分子、饮食和生理方法，短期目标是使用体外和体内模型系统来研究：*1：个体FA对小窝膜脂组成的影响*2：个体FA对小窝驻留蛋白定位和下游信号的影响*3：FA去饱和度对小窝膜脂组成和信号的影响*需要资金支持1名博士、4名硕士和5名本科生。HQP将接受研究方法、统计分析和沟通技能方面的培训。研究将通过新闻稿、同行评议出版物和演示文稿进行传播。*实验方法。单独的FA将被添加到乳腺、前列腺、结肠和肝脏细胞系的培养液中。用超速离心法将小窝分离出来。膜FA的组成将用气相色谱分析。信号将通过免疫印迹确定，基因表达将通过RT-PCR确定。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，精选FA将用于SCD1和D6D基因敲除小鼠的喂养研究，以确定FA的单独作用以及去饱和酶对小凹细胞膜成分和细胞信号转导的影响。这项研究将促进我们对个体FA在细胞信号和功能中的生物学作用的基础知识，并支持知识经济下的HQP培训。**

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. **