Role of taste signaling elements in enteroendocrine cells

描述(申请人提供)：我们已经确定Gustducin、T1R3和其他几个支持味觉检测/转导的信号元件也在L和K亚型的肠道内分泌细胞中表达。在L细胞中，Gustducin和T1R3的一个功能是感觉肠腔内的葡萄糖或甜味剂。1-Gustducin基因敲除小鼠不会因肠腔内注射葡萄糖而从其肠内分泌L细胞分泌GLP-1。由于这一缺陷，这些小鼠的血浆胰岛素和血糖水平调节失调。对来自1-Gustducin基因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株和反义阻断Gustducin或药物抑制T1R3的研究表明，Gustducin和T1R3都是GLP-1释放所必需的，提示1-Gustducin基因敲除小鼠体内的缺陷是葡萄糖感觉水平的缺陷。本实验旨在进一步研究Gustducin、T1R3等味觉信号元件在肠道内分泌细胞中的功能。我们结合分子、细胞、转基因、生理学和药理学的方法：1.确定哪些关键的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它们的味觉信号元件的表达，并引起肠细胞糖和脂肪酸转运蛋白的上调；3.确定药物或遗传阻断肠内分泌细胞表达的味觉信号元件是否改变了肠内分泌细胞的激素释放；4.建立选择性缺乏肠道1-Gustducin、T1R3或Trpm5的条件性基因敲除小鼠；5.确定肠道中缺乏1-Gustducin、T1R3或Trpm5的条件性基因敲除小鼠是否对饮食诱导的肥胖具有抵抗力，在高脂肪饮食中表现出2细胞功能缺陷，并表现出能量消耗增加。我们将检验以下关键假设：1.特定类型的肠内分泌细胞表达多种味觉信号元件；2.大量营养素和味精通过激活肠内分泌细胞亚型中表达的味觉受体和味觉受体，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性基因敲除小鼠将在调节其血浆GLP-1、GIP和其他激素水平方面存在缺陷-导致：(A)无法上调某些肠道细胞转运蛋白，(B)葡萄糖平衡失调，(C)对饮食诱导的肥胖的抵抗力，(D)2细胞功能中断，(E)能量消耗增加。这种多学科的方法有望为肠道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中的大量营养素(例如葡萄糖和糖)和味觉物质(例如人工甜味剂)，以及这如何导致GLP-1和其他激素的释放以调节肠道功能。这项建议与食欲、饱腹感、肥胖症和糖尿病有医学上的相关性。 公共卫生相关性： 作为味觉检测/转导基础的许多相同的信号元件也在肠道内分泌细胞中表达。Gustducin和T1R3在肠内分泌的L细胞中表达，是葡萄糖或甜味剂在肠腔内释放GLP-1所必需的。这些实验旨在从功能上表征味觉蛋白和其他味觉信号元件在肠道内分泌细胞中的作用，这些实验与食欲、饱腹感、糖尿病和肥胖症有医学上的相关性。

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.