Monitoring FoxO1 Localization in Chemically Identified Neurons
Monitoring FoxO1 Localization in Chemically Identified Neurons
复制标题
DOI：
10.1523/jneurosci.4023-08.2008
复制
发表时间：
2008-12-10
影响因子：
5.3
通讯作者：
Elmquist, Joel K.
中科院分区：
文献类型：
--
作者：
Fukuda, Makoto;Jones, Juli E.;Elmquist, Joel K.
PI3K-Akt-FoxO1 通路有助于多种细胞类型（包括中枢神经系统神经元）中胰岛素和瘦素的作用。然而，事实证明，在化学鉴定的神经元中识别这些行为很困难。为了解决这个问题，我们开发了一种基于 FoxO1 核质穿梭的报告小鼠，用于监测特定神经元群体中的 PI3K-Akt 信号传导。报告基因 FoxO1 与绿色荧光蛋白 (FoxO1GFP) 融合，在普遍存在的启动子的控制下表达，该启动子被 loxP 侧翼转录阻断剂沉默。因此，报告基因在选定细胞中的表达取决于 Cre 重组酶的作用。使用该模型，我们发现胰岛素治疗导致 POMC 和 AgRP 神经元内 FoxO1GFP 以剂量和时间依赖性方式被核排除。体内给予胰岛素后，在 POMC 神经元中也观察到 FoxO1GFP 核排斥。此外，瘦素以剂量依赖性方式诱导 POMC 神经元中 FoxO1GFP 的瞬时核输出。最后，游离脂肪酸预处理会损害 POMC 神经元中胰岛素诱导的核输出，这是一种已知会诱导外周胰岛素靶组织胰岛素抵抗的范例。因此，我们的 FoxO1GFP 小鼠提供了一种在生理和病理生理条件下以细胞特异性方式监测 PI3K-Akt 信号传导状态的工具。
The PI3K-Akt-FoxO1 pathway contributes to the actions of insulin and leptin in several cell types, including neurons in the CNS. However, identifying these actions in chemically identified neurons has proven difficult. To address this problem, we have developed a reporter mouse for monitoring PI3K-Akt signaling in specific populations of neurons, based on FoxO1 nucleocytoplasmic shuttling. The reporter, FoxO1 fused to green fluorescent protein (FoxO1GFP), is expressed under the control of a ubiquitous promoter that is silenced by a loxP flanked transcriptional blocker. Thus, the expression of the reporter in selected cells is dependent on the action of Cre recombinase. Using this model, we found that insulin treatment resulted in the nuclear exclusion of FoxO1GFP within POMC and AgRP neurons in a dose- and time-dependent manner. FoxO1GFP nuclear exclusion was also observed in POMC neurons following in vivo administration of insulin. In addition, leptin induced transient nuclear export of FoxO1GFP in POMC neurons in a dose dependent manner. Finally, insulin-induced nuclear export was impaired in POMC neurons by pretreatment with free fatty acids, a paradigm known to induce insulin resistance in peripheral insulin target tissues. Thus, our FoxO1GFP mouse provides a tool for monitoring the status of PI3K-Akt signaling in a cell-specific manner under physiological and pathophysiological conditions.