Monitoring chemosensory responses of GLP-1 enteroendocrine cells.

描述(由申请人提供)：本提案有三个目标。首先是检测两个现存的肠内分泌细胞STC-1和GluTag细胞对营养物质的动员反应。二是获得新型可逆转化的肠内分泌细胞系，并研究其钙离子对营养物质的反应。第三，在一只转基因小鼠中，原位检测肠内分泌细胞对营养物质的钙反应，转基因小鼠被设计表达钙传感器GFP-aequorin。被检测的营养素包括葡萄糖、脂肪、蛋白粉、氨基酸和一些苦味化合物。 为了实现第一个目标，我们将在STC-1和GluTag细胞中引入非侵入性生物发光钙传感器GFP-aequorin。然后，我们将研究出使用这种钙离子传感器监测肠内分泌细胞对营养物质的反应的技术细节。为了第二个和第三个目标，我们将建立一个同时表达GFP-aequorin和温度敏感的SV40T抗原(TsTag)的转基因小鼠系，该基因来自原胰高血糖素启动子，该启动子在L类型的肠内分泌细胞中具有活性，可以产生胰高血糖素样肽-1(GLP-1)。将从这些转基因小鼠的小肠和大肠中分离出新型转化的L型肠道内分泌细胞系。在有限的温度下生长会使tsTag失活，并导致这些细胞株恢复到更类似于天然肠道内分泌细胞的未转化表型。在这些“天然”的L细胞中，钙离子对营养物质的反应将在体外进行检测。为了最终的目标，我们将在原位监测这些转基因动物的小肠和大肠中肠内分泌L细胞的反应。 预计这项工作的结果将验证使用GFP-aequorin在体外和原位监测肠内分泌细胞反应的技术方法。这种方法应该提供有关肠内分泌细胞对营养物质反应的重要新信息和初步数据。基于目前的工作和开发的试剂，未来的R01建议将更详细地在机制水平上检查L型肠内分泌细胞在体外和原位的反应。 这一建议与胃肠道新陈代谢、食欲和饱腹感有关。从这项提议中获得的知识可能会导致对肥胖、厌食症、恶病质和糖尿病等疾病状态的有效干预。

DESCRIPTION (provided by applicant): This proposal has three goals. First is to examine Ca++ mobilization responses to nutrients of two existing enteroendocrine cells, STC-1 and GluTag cells. Second is to derive novel reversibly transformed enteroendocrine cell lines and characterize their Ca++ responses to nutrients. Third is to examine in situ Ca++ responses to nutrients by enteroendocrine cells in a transgenic mouse engineered to express the calcium sensor GFP-aequorin. The nutrients to be tested include glucose, fats, peptones, amino acids and some bitter compounds. To accomplish the first goal we will introduce a non-intrusive bioluminescent Ca++ sensor, GFP-aequorin, into STC-1 and GluTag cells. We will then work out the technical details of using this Ca++ sensor to monitor enteroendocrine cell responses to nutrients. For the purposes of the second and third goals we will generate a transgenic mouse line expressing both GFP-aequorin and temperature sensitive SV40 T antigen (tsTag) from the proglucagon promoter that is active in L type enteroendocrine cells that produce glucagon like peptide-1 (GLP-1). Novel transformed L type enteroendocrine cell lines will be isolated from the small and large intestines of these transgenic mice. Growth at the restrictive temperature will inactivate tsTag and cause these cell lines to revert to a non-transformed phenotype more akin to that of native enteroendocrine cells. Ca++ responses to nutrients will be examined in vitro in these "native" L cells. For the final goal we will monitor responses of enteroendocrine L cells in situ in the small and large intestines of these transgenic animals. It is anticipated that results from this work will validate the technical approach of using GFP-aequorin to monitor enteroendocrine cell responses in vitro and in situ. This approach should provide important new information and preliminary data on enteroendocrine cell responses to nutrients. Future R01 proposals based on the present work and the reagents developed here will examine in greater detail and at mechanistic levels the responses of L type enteroendocrine cells in vitro and in situ. This proposal has relevance to GI metabolism, appetite and satiety. The knowledge gained from this proposal may lead to effective interventions for disease states including obesity, anorexia, cachexia and diabetes.