Identification of dietary lipid cues that regulate metabolism in Drosophila.

膳食脂肪是定义我们饮食中卡路里含量的一个主要因素。此外，最近的研究发现，脂质是能够诱导细胞和全身代谢反应的信号。野生果蝇以腐烂的水果为食，这是一种含有植物和真菌成分的富含卡路里的食物。真菌脂不同于植物脂；真菌脂肪酸更短、更饱和，并且含有真菌类固醇而不是植物类固醇。饮食中的甾醇对果蝇的细胞膜和激素的产生至关重要，因为果蝇是固醇营养缺乏症患者。为了研究不同的脂肪成分如何影响果蝇的新陈代谢，我创造了两种新的食谱，植物和酵母食物。这两种饮食都有非常相似的高卡路里含量，并且蛋白质/糖/脂比例几乎相同。我发现酵母脂可以提升全身胰岛素信号。高胰岛素信号水平导致果蝇快速发育和高繁殖率，但会缩短成虫的平均寿命。我发现脂蛋白颗粒需要将膳食酵母脂肪信号传递给产生胰岛素的神经元。为了到达他们的神经元目标，一种名为LTP的特定脂蛋白颗粒需要越过血脑屏障，该屏障控制着进入大脑的所有流量。我已经证明，营养酵母脂类提高血脑屏障细胞内钙水平，并且靶向受体介导的特定脂蛋白颗粒LTP(脂转移蛋白)的转位需要高细胞内钙水平。在大脑中，LTP丰富了与胰岛素产生细胞直接相连的不同神经元。我的数据表明，这些LTP阳性神经元的活动触发了胰岛素的释放进入循环。我现在想了解这些脂质是如何激活BBB细胞的，以及随后允许LTP通过BBB传输到特定神经元和大脑的机制。为此，我提出了以下目标：1.确定激活血脑屏障细胞的饮食脂质，以及它们的作用机制(在苍蝇和脊椎动物细胞培养模型中)。2.绘制连接血脑屏障细胞和特定神经元的神经胶质网络图。识别通过血脑屏障传递LTP的膜运输途径(作为对代谢变化的响应)。我的拨款提案在这里概述了分离和鉴定激活血脑屏障细胞中钙信号的饮食脂质的详细方案，并描述了区分结构性脂类(调节质膜组成以诱导细胞反应)或脂信号线索的策略。它描述了一种尖端的遗传学方法来确定血脑屏障细胞和特定神经元之间连接的模式和可塑性。这项提案还概述了我将如何使用内源性YFP标记的Rab蛋白文库来确定跨越LTP使用的BBB的膜运输路线。

Dietary lipids are one major element that defines the caloric content of our diet. In addition, recent studies identified lipids as cues capable to induce a cellular and systemic metabolic response. In the wild fruit flies feed on rotting fruits, a diet rich in calories with plant and fungal components. Fungal lipids are different from plant lipids; fungal fatty acids are shorter and more saturated, and contain fungal sterols rather than plant sterols. Dietary sterols are critical for Drosophila membrane and hormone production because flies are sterol auxotrophs. To study how different lipid compositions affect metabolism in flies I have created two new recipes, plant and yeast food. Both diets share a very similar high caloric richness and have almost identical protein/sugar/lipid ratios. I have discovered that yeast lipids elevate systemic Insulin signaling. The high Insulin signaling levels result in fast development and high reproductive rates, but decrease the average lifespan of adult fruit flies. I found that lipoprotein particles are required to convey the dietary yeast lipid signal to Insulin producing neurons. To reach their neuronal targets, a specific lipoprotein particle called LTP needs to cross the Blood Brain Barrier, which controls all traffic into the brain. I have shown that nutritional yeast lipids elevate intracellular Calcium levels in Blood Brain Barrier cells and that high intracellular Calcium levels are required for the targeted receptor mediated translocation of the specific lipoprotein particle LTP (Lipid transport protein). In the brain, LTP enriches on distinct neurons that are directly connected to Insulin producing cells. My data suggest that the activity of these LTP positive neurons triggers the release of Insulin into circulation.I would now like to understand how these lipids activate BBB cells, and the mechanisms that then allow the transport of LTP across the BBB to specific neurons with the brain. To do so, I propose the following aims:1. To identify dietary lipids that activate Blood Brain Barrier cells, and the mechanisms by which they act (in flies and in a vertebrate cell culture model). 2. To map the glio-neuronal network that connects Blood Brain Barrier cells with specific neurons.3. To identify membrane traffic routes that deliver LTP across the Blood Brain Barrier (in response metabolic changes). My grant proposal outlines here detailed protocols to isolate and identify dietary lipids that activate Calcium signaling in BBB cells, and describes strategies to discriminate between structural lipids (that modulate the composition of plasma membranes to induce cell reactivity) or lipid signal cues. It describes a cutting edge genetic approach to identify the pattern and plasticity of connections between BBB cells and specific neurons. This proposal also outlines how I will use a library of endogenously YFP-tagged Rab proteins to identify membrane trafficking routes across the BBB used by LTP.