Extragustatory Functions of Bitter Taste Receptors

 描述(申请人提供)：味觉是通过激活口腔中的味觉感受器来区分营养物质和非营养物质所必需的。苦味已经演变成一种警告信号，提醒身体注意潜在的有害物质。多种苦味受体(T2Rs)在肠道上皮细胞中表达，它们很可能作为检测肠腔内容物的传感器。腔内T2R配体激活迷走神经传入，诱导胃肠激素释放，调节食物摄入和胃肠功能。有趣的是，在小鼠的结肠中，T2Rs的一部分，主要定位于肠内分泌(EEC)细胞，以及主要的T2R信号分子a-Gustducin，被高脂饮食上调，众所周知，这会导致肠道微生物区系失调、炎症、肥胖和代谢紊乱。T2R亚型也在超重/肥胖者的结肠中上调，在那里它们只定位于EEC细胞。此外，群体感应分子N-3-(氧代十二烷酰基)-高丝氨酸内酯(AHL)激活了EEC细胞株STC-1中与苦味转导相关的GPCR级联反应，并通过可能涉及T2Rs的途径诱导肠黏膜释放肠肽。群体感应分子对细菌交流至关重要，它们调节宿主的免疫反应。这一提议将检验这样的假设，即肠道T2Rs通过感知细菌产物和诱导肠道激素的释放来调节宿主对肠道微生物区系变化的功能反应。具体目标1将在KO小鼠中确定肠道微生物区系组成的变化是否调节T2Rs的表达，以及阻断T2R信号是否影响高脂饮食诱导的宿主反应。特定目的2将通过比较AHL和T2R配体对STC 1细胞和分离的粘膜细胞内钙和ERK磷酸化的影响，以及对正常小鼠或a-Gustducin基因缺失小鼠肠黏膜释放CCK、GLP-1和-2以及PYY的影响，来测试细菌产生的群体感应分子是否激活肠道T2Rs。这些研究将使用形态、成像和信号方法，用于高脂饮食的体内动物模型、肠道微生物区系的操纵和无菌小鼠，以及包括STC-1细胞、分离的粘膜细胞和肠道粘膜的器官型培养在内的体外模型系统。也将使用a-gustducin为空或a-gustducin启动子驱动GFP的小鼠品系。T2Rs或a-gustducin的siRNA敲除、信号通路的抑制剂和T2R抑制剂将用于控制细胞反应的特异性。肠道内宿主和微生物群之间的动态平衡是肥胖和代谢紊乱等疾病的重要因素。这一建议的影响是，更好地了解T2Rs在检测腔内细菌和调节宿主反应中的作用，将为治疗肥胖和其他涉及微生物组的疾病提供新的靶点。

 DESCRIPTION (provided by applicant): The sense of taste is essential for the discrimination between nutrients and non-nutrients through the activation of taste receptors in the oral cavity. Bitter taste has evolved as a warning signal by alerting the body to potentially harmful substances. Multiple bitter taste receptors (T2Rs) are expressed in gut epithelial cells and they are likely to serve as transducers for the detection of luminal contents. Intraluminal T2R ligands activate vagal afferents, induce release of gut hormones, and modulate food intake and gastrointestinal functions. Interestingly, in the mouse colon, a subset of T2Rs, which are predominantly localized to enteroendocrine (EEC) cells, and the major T2R signaling molecule, a-gustducin, are upregulated by high fat diet, which is known to induce dysbiosis of the gut microbiota, inflammation, obesity, and metabolic disorders. T2R subtypes are also upregulated in the colon of overweight/obese humans, where they are only localized to EEC cells. Moreover, the quorum-sensing molecule, N-3- (oxododecanoyl)-homoserine lactone (AHL) activates a GPCR cascade associated with bitter taste transduction in the EEC cell line, STC 1, and induce release of gut peptides from intestinal mucosa through a pathway likely involving T2Rs. Quorum-sensing molecules are critical for bacteria communication and they modulate host immune response. This proposal will test the hypothesis that intestinal T2Rs mediate host functional responses to changes in the gut microbiota by sensing bacterial products and inducing release of gut hormones. Specific Aim 1 will determine whether alterations of gut microbiota composition regulate T2Rs expression and whether blockade of T2R signaling affects the host response induced by high-fat diet using a-gustducin KO mice. Specific Aim 2 will test whether bacteria-produced quorum sensing molecules activate gut T2Rs by comparing the effects of AHL and T2R ligands on intracellular Ca2+ and ERK phosphorylation in STC 1 cells and dissociated mucosal cells, and on CCK, GLP-1 and -2, and PYY release from intestinal mucosa of normal mice or mice with a-gustducin gene deletion. These studies will use morphological, imaging and signaling approaches with in vivo animal models of high fat diet, manipulations of the gut microbiota, and germ free mice, and in vitro model systems including STC 1 cells, isolated mucosal cells, and organotypic cultures of the intestinal mucosa. Mouse lines null for a-gustducin or with the a-gustducin promoter driving GFP will also be used. siRNA knockdown of T2Rs or a-gustducin, inhibitors of signaling pathways, and a T2R inhibitor will be used to control for specificity of cellular responses. The homeostasis between host and microbiota in the gut lumen is an important factor in diseases such as obesity and metabolic disorders. The impact of this proposal is that a greater understanding of the roles of T2Rs in the detection of intraluminal bacteria and mediation of host response, will provide novel targets for the treatment of obesity and other disorders involving the microbiome.