Role of taste signaling elements in enteroendocrine cells

描述（由申请人提供）：我们已经确定味觉检测/转导的gustducin， T1R3和其他几种信号元件也在肠内分泌细胞的L和K亚型中表达。L细胞中的gustducin和T1R3的一个功能是感知肠道内的葡萄糖或甜味剂。1-Gustducin敲除小鼠的肠内分泌L细胞不分泌GLP-1以响应葡萄糖输注到肠腔。由于这种缺陷，这些小鼠的血浆胰岛素和葡萄糖水平调节功能失调。对1-gustducin敲除小鼠切碎的十二指肠组织或分离的十二指肠绒毛进行的研究表明，GLP-1分泌的缺陷与神经支配无关。对人和小鼠肠内分泌细胞系的研究以及gustducin的反义阻断或T1R3的药理抑制表明，gustducin和T1R3都是肠内分泌细胞释放GLP-1所必需的，这表明1-gustducin敲除小鼠体内的缺陷在葡萄糖感觉水平。本实验旨在进一步表征gustducin、T1R3等味觉信号元件在肠内分泌细胞中的功能。我们将分子、细胞、转基因、生理和药理等方法结合起来：确定哪些关键味觉信号分子在哪些肠内分泌细胞中表达；2. 确定肠内分泌细胞的激素释放是否取决于其味觉信号元件的表达，并引起肠细胞中糖和脂肪酸转运蛋白的上调；3. 确定肠内分泌细胞表达的味觉信号元件的药理学或遗传阻断是否会改变肠内分泌细胞的激素释放；4. 在肠道中选择性缺失1-gustducin、T1r3或Trpm5的条件敲除小鼠；5. 确定在肠道中缺乏1- gustducin， T1r3或Trpm5的条件敲除小鼠是否对饮食诱导的肥胖有抵抗力，在高脂肪饮食中表现出细胞功能缺陷，并表现出增加的能量消耗。我们将测试以下关键假设：1。特定类型的肠内分泌细胞表达多种味觉信号元件；2. 大量营养素和味觉剂通过激活肠内分泌细胞亚型中表达的味觉素和味觉受体，诱导肠内分泌细胞释放GLP-1和其他激素；3. 缺乏肠内分泌细胞表达的味觉信号元件的条件敲除小鼠将在调节血浆GLP-1、GIP和其他激素水平方面存在缺陷，从而导致：(a)无法上调某些肠细胞转运蛋白，(b)葡萄糖稳态失调，(c)对饮食引起的肥胖产生抵抗，(d) 2种细胞功能破坏，(e)能量消耗增加。这种多学科方法有望为肠内分泌细胞功能的本质提供重要的新见解。特别是肠内分泌细胞如何感知肠腔内的大量营养物质（如葡萄糖和糖）和味觉物质（如人工甜味剂），以及这如何导致刺激GLP-1和其他激素的释放来调节肠道功能。这一建议与食欲、饱腹感、肥胖和糖尿病有医学相关性。

DESCRIPTION (provided by applicant): We have determined that gustducin, T1R3 and several other signaling elements that underlie taste detection/transduction also are expressed in L and K subtypes of enteroendocrine cells. One function of gustducin and T1R3 in L cells is to sense glucose or sweeteners within the lumen of the gut. 1-Gustducin knockout mice do not secrete GLP-1 from their enteroendocrine L cells in response to infusion of glucose into the gut lumen. Because of this defect these mice have dysfunctional regulation of their plasma insulin and glucose levels. Studies with minced duodenal tissue or isolated duodenal villi from 1-gustducin knockout mice show that the defect in GLP-1 secretion is independent of innervation. Studies with human and mouse enteroendocrine cell lines and antisense blockade of gustducin or pharmacological inhibition of T1R3 indicate that both gustducin and T1R3 are required for enteroendocrine cell release of GLP-1, suggesting that the defect in 1-gustducin knockout mice in vivo is at the level of glucose sensation. The experiments proposed here aim to further characterize the functions of gustducin, T1R3 and other taste signaling elements in enteroendocrine cells. We have combined molecular, cellular, transgenic, physiological and pharmacological methods to: 1. Determine which key taste signaling molecules are expressed in which enteroendocrine cells; 2. Determine if hormone release from enteroendocrine cells depends on their expression of taste signaling elements and elicits upregulation of sugar and fatty acid transporters in enterocytes; 3. Determine if pharmacologic or genetic block of enteroendocrine cell-expressed taste signaling elements alters hormone release from enteroendocrine cells; 4. Generate conditional knockout mice selectively lacking 1-gustducin, T1r3 or Trpm5 in gut; 5. Determine if conditional knockout mice lacking 1- gustducin, T1r3 or Trpm5 in gut are resistant to diet-induced obesity, show defective 2 cell function on a high fat diet, and exhibit increased energy expenditure. We will be testing the following key hypotheses: 1. Specific types of enteroendocrine cell type express multiple taste signaling elements; 2. Macronutrients and tastants elicit release of GLP-1 and other hormones from enteroendocrine cell by activating gustducin and taste receptors expressed in subtypes of enteroendocrine cells; 3. Conditional Knockout mice lacking enteroendocrine cell-expressed taste signaling elements will have defects in regulating their plasma levels of GLP-1, GIP and other hormones - leading to: (a) failure to upregulate certain enterocyte transporters, (b) dysregulation of glucose homeostasis, (c) resistance to diet-induced obesity, (d) disruption of 2 cell functions, (e) increased energy expenditure. This multidisciplinary approach has promise for providing significant new insights into the nature of enteroendocrine cell function. In particular, how enteroendocrine cells sense macronutrients (e.g. glucose and sugars) and tastants (e.g. artificial sweeteners) in the gut lumen, and how this leads to stimulation of release of GLP-1 and other hormones to regulate gut functions. This proposal has medical relevance to appetite, satiety, obesity, and diabetes. PUBLIC HEALTH RELEVANCE: Many of the same signaling elements that underlie taste detection/transduction also are expressed in enteroendocrine cells. Gustducin and T1R3, expressed in enteroendocrine L cells, are required for the release of GLP-1 in response to glucose or sweeteners within the lumen of the small intestine. The experiments proposed to functionally characterize the roles of gustducin and other taste signaling elements in enteroendocrine cells have medical relevance to appetite, satiety, diabetes and obesity.