Does insulin or glucose regulate alpha-cell function?

描述（由申请人提供）：血糖保持在一个狭窄的范围内，主要是由于胰岛素和胰高血糖素，它们分别由朗格汉斯岛的β细胞和α细胞分泌。虽然胰岛素分泌已被研究得很好，但调节胰高血糖素分泌的信号仍有激烈的争论。低血糖是I型糖尿病患者死亡的重要原因，也是胰岛素治疗高血糖的主要限制。因此，了解α细胞如何感知和响应循环葡萄糖水平是至关重要的。目前尚不清楚是直接检测到葡萄糖，还是α细胞受到邻近β细胞的控制，最有可能是通过胰岛素分泌。这项研究将开发两种方法，通过这些方法我们可以将原代大鼠α细胞与邻近β细胞的影响分离开来。第一种方法是使用限制于α细胞的绿色荧光蛋白（GFP）表达获得纯α细胞群，并标记这些细胞，通过荧光激活细胞分选（FACS）进行纯化。然后将开发技术来优化培养这些纯化的α细胞的条件。将检查细胞聚集和细胞单层的使用，以确定哪种方法能更严格地调节胰高血糖素的分泌。另一种方法是下调α细胞中胰岛素受体的表达。本研究的第二个方面的目的是确定胰高血糖素基因表达的步骤（RNA水平，RNA稳定性和胰高血糖素翻译）在葡萄糖和/或胰岛素的反应中被调节。这些步骤的识别将为研究α细胞对营养物质的反应提供新的解读。此外，基因表达在mRNA稳定性和翻译方面的调控几乎总是通过rna -蛋白相互作用进行的，这种相互作用的鉴定将为解剖α细胞的营养作用提供有价值的工具。本提案中描述的研究将提供新的技术和新的读数，通过这些技术和读数可以研究α细胞对其营养环境的反应。

DESCRIPTION (provided by applicant): Blood glucose remains within a narrow range, primarily due to insulin and glucagon, which are secreted from the beta and alpha cells of the islets of Langerhans, respectively. Although insulin secretion has been well studied the signals that regulate glucagon secretion are still hotly debated. Hypoglycemia is a significant cause of death amongst Type I diabetics and the major limitation upon insulin treatment of hyperglycemia. Thus, it is crucial to understand how alpha cells sense and respond to circulating glucose levels. It is unclear whether glucose is detected directly or whether the alpha cell is under the control of neighboring beta cells, most likely through insulin secretion. This study will develop 2 methods by which we can separate primary rat alpha cells from the effects of neighboring beta cells. The first approach is to obtain pure alpha cell populations using green fluorescent protein (GFP) expression restricted to the alpha cells to tag these cells for purification by fluorescence activated cell sorting (FACS). Techniques will then be developed to optimize the conditions for culturing these purified alpha cells. The use of cell aggregation and cell monolayers will be examined to determine which method gives the tighter regulation of glucagon secretion. The other method is to down-regulate insulin receptor expression in the alpha cells. The aim of the second aspect of this study is to identify steps of proglucagon gene expression (RNA levels, RNA stability and proglucagon translation) that are regulated in response to glucose and/or insulin. The identification of such steps would provide novel read-outs by which to study alpha cell responses to nutrients. Furthermore, the regulation of gene expression at mRNA stability and translation is almost always via RNA-protein interactions, the identification of which would provide valuable tools in the dissection of nutrient effects upon the alpha cell. The research described in this proposal will provide new techniques and new read-outs by which the study of the alpha cell responses to its nutrient environment can be studied.