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.
Gastrointestinal hormones and CCK, in particular, are important regulators of gastrointestinal function. Globally, they coordinate the ingestion, digestion, and absorption of food and essential nutrients. However, because of difficulties in isolating and purifying enteroendocrine cells, we know very little about the manner in which nutrients regulate hormone secretion. In this proposal the PI has developed a method for highly purifying CCK cells and has identified a novel receptor on CCK cells that is responsible for fat-stimulated CCK secretion. The studies described in this proposal will not only extensively characterize the unique interaction of fatty acids and lipoproteins with this receptor but will als, for the first time, allow detailed analysis of the intracellular signaling properties resulting fro fatty acid activation in native CCK cells. Finally, we will assess the physiological importance of CCK cells in vivo with a particular emphasis on gallstone formation and feeding behavior. It is possible that this approach could lead to novel ways to regulate GI hormone function, independent of eating a meal. If successful, this proposal could have implications for treating diseases related to CCK action including obesity and gallbladder disease.
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