Receptor Regulation of CCK Cell Function

描述（由申请人提供）：胆囊收缩素（CCK）是一种主要的胃肠道激素，由分散在整个肠道中的离散肠内分泌细胞（称为I细胞）产生。CCK有几个行动是重要的调节食物摄入量和消化的必需营养素。具体而言，CCK调节胆囊收缩、胰腺酶分泌、延迟胃排空和诱导饱腹感。作为大多数胃肠道激素的典型，CCK在进食后分泌到血液中。脂肪是CCK分泌的最重要的食物刺激物。营养素如何与肠内分泌细胞相互作用并刺激胃肠激素分泌是一个值得关注的问题。尽管人们普遍认为营养素直接刺激CCK释放，但最近这一点受到了质疑，并且调节CCK细胞功能的细胞机制在很大程度上是未知的。PI开发了一种分离和表征单个、活的天然肠CCK细胞的方法，通过高度富集这些细胞，可以在体外研究CCK分泌，鉴定这些细胞上的受体，并研究参与调节激素分泌的第二信使信号通路。这些方法一起导致我们最近在CCK细胞上发现了一种新的受体，称为免疫球蛋白样结构域受体（ILDR）。在肠中，ILDR仅在CCK细胞中表达。PI已经产生了Ildr基因缺失的小鼠，并证明Ildr缺陷小鼠在脂肪喂养后不分泌CCK。ILDR是蛋白质残基受体家族的新成员，通过独特的作用机制被激活。这些初步数据表明，CCK细胞表达一种新的受体，这是至关重要的调节脂肪诱导的CCK分泌。PI将使用互补技术的组合来表征CCK细胞中的ILDR，并确定ILDR如何调节CCK分泌。这些方法包括：（1）天然CCK细胞的分离和鉴定，（2）体内和体外CCK分泌的测量，（3）细胞内钙荧光的定量，和（4）研究表达ILDR的细胞中细胞内信号传导的方法。新模式将包括：（1）在CCK细胞中表达增强的绿色荧光蛋白的转基因小鼠（CCK-eGFP小鼠），以能够鉴定和分离CCK细胞，（2）ILDR敲除小鼠（ILDR-/-），（3）双重CCK-eGFP转基因/ ILDR敲除小鼠（CCK-eGFP/ILDR-/-），和条件性肠CCK细胞特异性ILDR敲除小鼠。这些转基因小鼠将使我们能够研究完整小鼠和正常和ILDR缺陷小鼠分离的CCK细胞中的ILDR功能。本申请的中心假设是ILDR介导脂肪刺激的CCK分泌。本提案的总体目的是了解脂肪和脂肪酸控制CCK分泌的机制。ILDR的表征及其与CCK细胞中激素分泌的关系将通过以下具体目的来解决：1.建立脂肪酸和脂蛋白调节ILDR功能的机制。2.目的研究ILDR对小鼠胆囊收缩素分泌、胆囊功能和摄食行为的调节作用。这些目标中的每一个都将集中在ILDR的调节上，作为调节脂肪酸刺激的CCK分泌的关键步骤。更广泛地说，这些目标应该提供相当多的洞察GI内分泌细胞的调节机制，营养素已知是重要的激素分泌的控制。

DESCRIPTION (provided by applicant): Cholecystokinin (CCK) is a major gastrointestinal hormone that is produced by discrete enteroendocrine cells (known as I cells) that are scattered throughout the intestine. CCK has several actions that are important for the regulation of food intake and digestion of essential nutrients. Specifically CCK regulates gallbladder contraction, pancreatic enzyme secretion, delays gastric emptying, and induces satiety. As is typical of most GI hormones, CCK is secreted into the blood stream after ingestion of a meal. Ingested fat is the most important dietary stimulant of CCK secretion. There is considerable interest in how nutrients interact with enteroendocrine cells and stimulate gastrointestinal hormone secretion. Even though it is generally believed that nutrients stimulate CCK release directly this has recently come into question and the cellular mechanisms regulating CCK cell function are largely unknown. The PI has developed a method for isolating and characterizing individual, viable, native intestinal CCK cells and by highly enriching these cells it has been possible to study CCK secretion in vitro, identify receptors on these cells and investigate second messenger signaling pathways involved in regulated hormone secretion. Together these approaches led to our very recent discovery of a novel receptor on CCK cells, known as immunoglobulin-like domain containing receptor (ILDR). In the intestine, ILDR is expressed exclusively in CCK cells. The PI has generated mice with genetic deletion of Ildr and demonstrated that ILDR deficient mice do not secrete CCK following fat feeding. ILDR is a novel member of the remnant receptor family of proteins and is activated through a unique mechanism of action. These preliminary data indicate that CCK cells express a novel receptor that is critical for regulating fat-induced CCK secretion. The PI will use a combination of complementary techniques to characterize ILDR in CCK cells and establish how ILDR regulates CCK secretion. These methods include: (1) isolation and identification of native CCK cells, (2) measurements of CCK secretion in vivo and in vitro, (3) quantification of intracellular calcium fluorescence, and (4) approaches to study intracellular signaling in ILDR -expressing cells. Novel models will include: (1) transgenic mice expressing enhanced green fluorescent protein in CCK cells (CCK-eGFP mice) to enable the identification and isolation of CCK cells, (2) ILDR knockout mice (ILDR-/-), (3) dual CCK-eGFP transgenic / ILDR knockout mice (CCK-eGFP/ILDR-/-), and conditional, intestinal CCK cell specific ILDR knockout mice. These genetically modified mice will enable us to study ILDR function in intact mice and in isolated CCK cells from normal and ILDR deficient mice. The central hypothesis of this application is that ILDR mediates fat-stimulated CCK secretion. The overall purpose of this proposal is to understand the mechanism whereby fat and fatty acids control CCK secretion. Characterization of ILDR and its relationship to hormone secretion in CCK cells will be addressed by the following Specific Aims: 1. To establish the mechanism by which fatty acids and lipoproteins regulate ILDR function. 2. To characterize the role of ILDR in regulating CCK secretion, gallbladder function and feeding behavior in mice in vivo. Each of these aims will focus on regulation of ILDR as a critical step in the regulation of fatty acid-stimulated CCK secretion. More globally, these aims should provide considerable insight into the mechanisms by which GI endocrine cells are regulated by nutrients known to be important in the control of hormone secretion.