ATF3 in Beta cell signaling, expression & destruction

描述(申请人提供)：目标和意义：糖尿病是美国的一个主要健康问题。多年来的研究表明，β细胞破坏在糖尿病的发病机制和并发症中起着重要作用。这项建议的目的是阐明激活转录因子3(ATF3)在应激诱导的信号转导、β细胞功能障碍和破坏中的作用。这项拟议的研究将检验以下假设。 (1)目的1：验证应激信号至少部分通过MAPK和NFkB通路在β细胞中诱导ATF3的假说。阻断这些通路激活的显性负性分子将被用来确定它们是否可以抑制应激信号(IL-1β、高血糖和高脂血症)对β细胞中ATF3的诱导。此外，激活这些通路的结构性活性突变体将被用来确定它们是否可以在没有外源信号的情况下诱导ATF3的表达。 (2)目的2：验证ATF3表达导致胰岛β细胞功能障碍、破坏和糖尿病发生的假说。米非司酮诱导系统将用于以可诱导的方式在胰岛产生表达ATF3的转基因小鼠。这些小鼠的特征是形态、免疫组织化学和生理参数，以及胰岛细胞死亡和细胞增殖。 (3)目的3：验证ATF3在细胞因子诱导的β细胞功能障碍和破坏中起重要作用的假说。胰岛细胞将从野生型(ATF3/)、杂合子(ATF3/-)和纯合子(ATF3-/-)ATF3基因敲除小鼠中分离出来。将细胞置于促炎细胞因子、一氧化氮供体S-亚硝基谷胱甘肽(GSNO)或培养液(对照组)中。细胞死亡将在不同的时间点进行分析，以确定ATF3是否是应激信号诱导有效的β细胞死亡所必需的。此外，还将分析胰岛功能，以确定ATF3是否是细胞因子诱导β细胞功能障碍所必需的。在体内实验中，野生型和基因敲除小鼠将被注射链脲佐菌素(STZ)以诱导糖尿病。将分析血糖水平和糖尿病发病率，以确定ATF3基因敲除小鼠是否比野生型小鼠对STZ不那么敏感。

DESCRIPTION (provided by applicant): Goal and Significance: Diabetes is a major health problem in the U.S. Many years of research indicate that beta cell destruction plays an important role in the pathogenesis and complication of diabetes. The goal of this proposal is to elucidate the roles of Activating Transcription Factor 3 (ATF3), a stress-inducible gene, in stress-induced signal transduction, beta cell dysfunction and destruction. The proposed research will test the following hypotheses. (1) Aim 1: To test the hypothesis that ATF3 is induced in beta cells by stress signals, at least in part, through the MAPK and NFkB pathways. Dominant negative molecules that block the activation of these pathways will be used to determine whether they could inhibit the induction of ATF3 in beta cells by stress signals (IL-1beta, hyperglycemia, and hyperlipidemia). In addition, constitutively active mutants that activate these pathways will be used to determine whether they could induce the expression of ATF3 in the absence of exogenously applied signals. (2) Aim 2: To test the hypothesis that expression of ATF3 leads to beta cell dysfunction, destruction and the development of diabetes. The mifepristone-inducible system will be used to generate transgenic mice expressing ATF3 in the islets in an inducible manner. These mice will be characterized for morphological, immunohistochemical and physiological parameters, and for islet cell death and cell proliferation. (3) Aim 3: To test the hypothesis that ATF3 plays an essential role in cytokine-induced beta cell dysfunction and destruction. Islet cells will be isolated from wild type (ATF3+/+), heterozygous (ATF3+/-) and homozygous (ATF3-/-) ATF3 knockout mice. Cells will be subjected to pro-inflammatory cytokines, nitric oxide donor S-nitroso glutathione (GSNO), or medium (control). Cell death will be analyzed at various time points to determine whether ATF3 is "necessary" for stress signals to induce efficient beta cell death. In addition, islet functions will be analyzed to determine whether ATF3 is necessary for cytokines to induce beta cell dysfunction. For in vivo experiments, wild type and knockout mice will be injected with streptozotocin (STZ) to induce diabetes. Blood glucose levels and diabetes incidence will be analyzed to determine whether ATF3 knockout mice are less sensitive to STZ than wild type mice.