Role of taste signaling elements in enteroendocrine cells

描述（由申请人提供）：我们已经确定，味觉调节蛋白、T1 R3和几种其他的作为味觉检测/转导基础的信号传导元件也在肠内分泌细胞的L和K亚型中表达。L细胞中gustducin和T1 R3的一个功能是感知肠道内腔内的葡萄糖或甜味剂。1-Gustducin基因敲除小鼠不从其肠内分泌L细胞分泌GLP-1以响应葡萄糖输注到肠腔中。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。对来自1-gustducin敲除小鼠的切碎的十二指肠组织或分离的十二指肠绒毛的研究表明，GLP-1分泌的缺陷与神经支配无关。人和小鼠肠内分泌细胞系的研究和味蛋白的反义阻断或T1 R3的药理学抑制表明，味蛋白和T1 R3都是GLP-1的肠内分泌细胞释放所需的，这表明1-味蛋白敲除小鼠体内的缺陷是葡萄糖感觉水平。 本文提出的实验旨在进一步表征肠内分泌细胞中味蛋白、T1 R3和其他味觉信号元件的功能。我们结合了分子、细胞、转基因、生理和药理学的方法：1。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依赖于它们的味觉信号元件的表达以及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学阻断是否改变了肠内分泌细胞的激素释放; 4.产生选择性缺乏肠道中1-gustducin、T1 r3或Trpm 5的条件性敲除小鼠; 5.确定在肠道中缺乏1- gustducin、T1 r3或Trpm 5的条件性基因敲除小鼠是否对饮食诱导的肥胖有抵抗力，在高脂肪饮食中是否表现出2细胞功能缺陷，并表现出能量消耗增加。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.大量营养素和促味剂通过激活肠内分泌细胞亚型中表达的味觉调节蛋白和味觉受体，引起GLP-1和其他激素从肠内分泌细胞释放; 3.缺乏肠内分泌细胞表达的味觉信号传导元件的条件性敲除小鼠将在调节其GLP-1、GIP和其他激素的血浆水平方面具有缺陷-导致：（a）不能上调某些肠细胞转运蛋白，（B）葡萄糖稳态失调，（c）对饮食诱导的肥胖症的抗性，（d）2细胞功能的破坏，（e）增加的能量消耗。 这种多学科的方法有希望为肠内分泌细胞功能的性质提供重要的新见解。特别是，肠内分泌细胞如何感知肠腔中的大量营养素（例如葡萄糖和糖）和促味剂（例如人工甜味剂），以及这如何刺激GLP-1和其他激素的释放以调节肠道功能。该建议与食欲、饱腹感、肥胖和糖尿病具有医学相关性。 公共卫生相关性： 许多与味觉检测/转导相关的信号传导元件也在肠内分泌细胞中表达。在肠内分泌L细胞中表达的Gustducin和T1 R3是响应于小肠腔内的葡萄糖或甜味剂而释放GLP-1所必需的。提出的功能性表征肠内分泌细胞中的味蛋白和其他味觉信号传导元件的作用的实验与食欲、饱腹感、糖尿病和肥胖症具有医学相关性。

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.