Extragustatory Functions of Bitter Taste Receptors

 描述（由申请人提供）：味觉对于通过激活口腔中的味觉受体来区分营养物和非营养物是必不可少的。苦味已经演变成一种警告信号，通过警告身体潜在的有害物质。多种苦味受体（T2 R）在肠上皮细胞中表达，并且它们可能充当用于检测管腔内容物的换能器。管腔内T2 R配体激活迷走神经传入，诱导肠道激素的释放，并调节食物摄入和胃肠道功能。有趣的是，在小鼠结肠中，主要定位于肠内分泌（EEC）细胞的T2 R亚组和主要的T2 R信号传导分子α-味蛋白被高脂肪饮食上调，已知高脂肪饮食诱导肠道微生物群的生态失调、炎症、肥胖和代谢紊乱。T2 R亚型也在超重/肥胖人的结肠中上调，其中它们仅定位于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（阿勒）激活EEC细胞系STC 1中与苦味转导相关的GPCR级联，并通过可能涉及T2 R的途径诱导肠肽从肠粘膜释放。群体感应分子是细菌通讯的关键，它们调节宿主的免疫反应。该提案将测试这样的假设：肠道T2 R通过感应细菌产物并诱导肠道激素的释放来介导宿主对肠道微生物群变化的功能反应。具体目标1将确定肠道微生物群组成的改变是否调节T2 R表达，以及T2 R信号传导的阻断是否影响使用α-gustducin KO小鼠由高脂肪饮食诱导的宿主应答。具体目标2将通过比较阿勒和T2 R配体对STC 1细胞和解离的粘膜细胞中的细胞内Ca 2+和ERK磷酸化的影响，以及对正常小鼠或具有α-gustducin基因缺失的小鼠的肠粘膜中CCK、GLP-1和-2和PYY释放的影响，来测试细菌产生的群体感应分子是否激活肠道T2 R。这些研究将使用形态学、成像和信号传导方法，包括高脂饮食、肠道微生物群操作和无菌小鼠的体内动物模型，以及包括STC 1细胞、分离的粘膜细胞和肠粘膜器官型培养物的体外模型系统。还将使用α-味蛋白无效或具有驱动GFP的α-味蛋白启动子的小鼠系。T2 R或α-味觉蛋白的siRNA敲低、信号传导途径的抑制剂和T2 R抑制剂将用于控制细胞应答的特异性。宿主和肠道内微生物群之间的稳态是肥胖和代谢紊乱等疾病的重要因素。这一提议的影响是，更好地理解T2 R在检测管腔内细菌和介导宿主反应中的作用，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.