Receptor Regulation of CCK Cell Function

CCK 细胞功能的受体调节

• 批准号: 8735944
• 负责人: Rodger A. Liddle
• 金额: $ 34.15万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-18 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8735944
• 关键词: Address; Animals; Apical; Attention; Binding; Blood; Calcium; Cell Line; Cell model; Cell physiology; Cells; Cholecystokinin; Cholecystokinin Receptor; Cholelithiasis; Data; Diet; Digestion; Disease; Distant; Eating; Endocrine; Enteroendocrine Cell; Exhibits; Family; Fatty Acids; Fatty acid glycerol esters; Feeding behaviors; Fluorescence; Fluorescence Microscopy; Fluorescence-Activated Cell Sorting; Food; Food Intake Regulation; Gall Bladder Diseases; Gallbladder; Gastrointestinal Hormones; Gastroparesis; Genetic; Hormonal; Hormones; Human; Immunoglobulin Domain; In Vitro; Individual; Ingestion; Intestines; Knockout Mice; Lead; Lipoprotein Binding; Lipoproteins; Measurement; Mediating; Methods; Modeling; Mus; Normal Cell; Nutrient; Obesity; Pancreatic enzyme; Pattern; Physiological; Property; Protein Family; Receptor Activation; Regulation; Role; Satiation; Second Messenger Systems; Signal Pathway; Signal Transduction; Stream; Study models; Surface; Techniques; Time; Tissues; Transgenic Mice; Transgenic Organisms; absorption; enhanced green fluorescent protein; feeding; gastrointestinal function; hormone regulation; in vivo; increased appetite; insight; interest; intestinal epithelium; lipolysis-stimulated receptor; lipoprotein-remnant receptor; member; mouse model; novel; public health relevance; receptor; receptor function; second messenger; tool

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.

描述(申请人提供)：CCK是一种主要的胃肠激素，由散布在肠道中的离散的肠内分泌细胞(称为I细胞)产生。CCK有几个对调节食物摄取和消化基本营养素很重要的作用。具体地说，CCK调节胆囊收缩，胰酶分泌，延缓胃排空，诱导饱腹感。与大多数胃肠道激素一样，CCK在进食后被分泌到血液中。摄入的脂肪是CCK分泌的最重要的饮食刺激物。营养物质如何与肠内分泌细胞相互作用，并刺激胃肠激素分泌，引起了人们的极大兴趣。尽管人们普遍认为营养物质直接刺激CCK的释放，但这一点最近受到了质疑，而且调控CCK细胞功能的细胞机制在很大程度上还不清楚。PI已经开发出一种分离和鉴定单个、活的、天然的肠道CCK细胞的方法，通过高度浓缩这些细胞，可以在体外研究CCK的分泌，识别这些细胞上的受体，并研究参与调节激素分泌的第二信使信号通路。结合这些方法，我们最近发现了CCK细胞上的一种新受体，称为免疫球蛋白样域包含受体(ILDR)。在肠道中，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会分泌CCK。ILDR是残基受体家族中的一个新成员，通过一种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，该受体对调节脂肪诱导的CCK分泌至关重要。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的分泌。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，(4)研究表达ILDR的细胞内信号的方法。新的模型将包括：(1)在CCK细胞中表达增强型绿色荧光蛋白的转基因小鼠(CCK-EGFP小鼠)，以实现CCK细胞的鉴定和分离；(2)ILDR基因敲除小鼠(ILDR-/-)；(3)双重CCK-EGFP转基因/ILDR基因敲除小鼠(CCK-EGFP/ILDR-/-)，以及条件性肠道CCK细胞特异性ILDR基因敲除小鼠。这些转基因小鼠将使我们能够研究完整小鼠的ILDR功能，以及来自正常和ILDR缺陷小鼠的分离CCK细胞。这一应用的中心假设是ILDR介导脂肪刺激的CCK分泌。这项建议的总体目的是了解脂肪和脂肪酸控制CCK分泌的机制。探讨CCK细胞ILDR的特征及其与激素分泌的关系：1.建立脂肪酸和脂蛋白调节ILDR功能的机制。2.研究ILDR对小鼠体内CCK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侧重于ILDR的调节，将其作为调节脂肪酸刺激的CCK分泌的关键步骤。在全球范围内，这些目标应该为胃肠道内分泌细胞被已知在激素分泌控制中起重要作用的营养物质调节的机制提供相当大的洞察力。