Physiology and regulation of T2R bitter taste receptors in enteroendocrine cells

描述（由申请人提供）：哺乳动物通过单个小“苦味”分子激动剂刺激单个细胞表面受体来识别食物中的苦味成分。这些味觉2受体（T2 R）由单外显子g蛋白偶联受体（GPCR）基因家族编码，并且主要在舌头表面和周围口腔组织上的味觉受体细胞（TRC）中表达。存在参与大部分甜味感测的单独GPCR，其是味觉1受体（T1 R）单体T1 R2和T1 R3的异源二聚体。单一甜味和整个苦味T2受体家族也在口腔外的区域表达，包括肺、胰腺和小肠。它们在舌头中的功能显然是允许在潜在的食物来源之间进行味觉辨别，然而，口腔外的味觉感受器的生理作用并不直观。在最近的出版物中，我们表明，肠道T2 R信号的激活导致肠内分泌细胞分泌多肽激素胆囊收缩素（CCK）和胰高血糖素样肽-1（GLP-1）的增加，CCK的两个主要作用是限制食物摄入和减缓胃运动。这些结果表明，肠道T2 R可能限制 吸收苦味和潜在的有毒分子，尽管在口中的味道厌恶。我们还表明，肠道的表达和活性的T2受体是由固醇调节元件结合蛋白-2（SREBP-2）转录因子，这是诱导细胞胆固醇水平低，并激活积累新的细胞胆固醇所需的基因。富含植物的饮食胆固醇含量低，相对于富含胆固醇的动物肉饮食，苦味和潜在有毒物质的比例更高。因此，通过SREBP-2对低胆固醇植物饮食的T2 R活性的诱导提供了一种机制，使肠道对潜在毒性分子的存在“敏感”，以通过CCK依赖性抑制肠道运动和食物摄入来限制它们的吸收。应该注意的是，CCK和肠肽激素胃泌素具有相同的羧基末端，我们在早期研究中使用的ELISA检测两种激素具有相似的灵敏度。因此，胃泌素可能是肠道中T2 R信号诱导的相关激素，我们目前的项目旨在解决这一关键问题。我们还发现CCK/胃泌素增加ABCB 1外排转运蛋白的表达和活性，已知ABCB 1外排转运蛋白可阻止细胞摄取多种有毒有机小分子。这提供了一种更积极的机制，以限制在用餐期间或意外消耗的有毒分子的吸收。总之，这些研究提供了一个连接营养感测与保护免受毒素摄入的分子机制。目前的提议结合了在选择的小鼠模型中的生理学研究，这些小鼠模型在编码营养感测和毒素外排途径的关键蛋白质的基因中具有突变，以严格测试该机制。我们还建议采用一种新的离体小肠原位检测直接监测T2 R信号和毒素流出之间的联系。

DESCRIPTION (provided by applicant): Mammals recognize bitter constituents in food through individual small "bitter" molecule agonists stimulation of individual cell surface receptors. These taste 2 receptors (T2Rs) are encoded by a family of single-exon g- protein coupled receptor (GPCR) genes and are predominantly expressed in taste receptor cells (TRCs) on the surface of the tongue and surrounding oral tissue. There is a lone GPCR involved in the majority of sweet taste sensing which is a heterodimer of Taste 1 Receptor (T1R) monomers T1R2 and T1R3. The single sweet and the entire family of bitter T2 receptors are also expressed in areas outside of the oral cavity including the lung, pancreas, and small intestine. Their function in the tongue is obviously to allow taste discrimination between differen potential food sources, however, the physiologic role for taste receptors outside of the oral cavity is not intuitively obvious. In recent publications, we showed that activation of intestinal T2R signaling leads to an increase in secretion of polypeptide hormones cholecystokinin (CCK) and glucagon-like-peptide-1 (GLP-1) from enteroendocrine cells and two major actions of CCK are to limit food intake and slow gastric motility. These results suggest intestinal T2Rs may limit absorption of bitter and potentially toxic molecules that are ingested despite the taste aversion in the mouth. We also showed that intestinal expression and activity of the T2Rs is regulated by the sterol regulatory element binding protein -2 (SREBP-2) transcription factor, which is induced when cellular cholesterol levels are low and activates genes required for accumulating new cell cholesterol. Plant enriched diets are both low in cholesterol and have a higher proportion of bitter tasting and potentially toxic substances relative to diets rich in cholesterol-laden animal flesh. Thus, induction of T2R activity through SREBP-2 on low-cholesterol plant diets provides a mechanism to "sensitize" the gut to the presence of potentially toxic molecules to limit their absorption through CCK dependent suppression of both gut motility and food in take. It should be noted that CCK and the gut peptide hormone gastrin have identical carboxyl termini and the ELISA we used in our early studies detects both hormones with similar sensitivity. Thus, gastrin may be the relevant hormone that is induced by T2R signaling in the gut and our current project is designed to address this key issue. We also showed CCK/gastrin increases expression and activity of the ABCB1 efflux transporter, which is known to prevent cellular uptake of a wide range of toxic organic small molecules. This provides a more active mechanism to limit absorption of toxic molecules consumed during a meal or by accident. Taken together, these studies provide a molecular mechanism connecting nutrient sensing with protection from toxin ingestion. The current proposal combines physiologic studies in select mouse models that have mutations in genes encoding critical proteins of the nutrient sensing and toxin efflux pathways to rigorously test the mechanism. We also propose to adopt a novel in situ assay with isolated small intestine to monitor the connection between T2R signaling and toxin efflux directly.