A method to determine insulin responsiveness in synaptosomes isolated from frozen brain tissue
A method to determine insulin responsiveness in synaptosomes isolated from frozen brain tissue
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DOI：
10.1016/j.jneumeth.2016.01.006
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发表时间：
2016-03-01
影响因子：
3
通讯作者：
Taglialatela, Giulio
中科院分区：
文献类型：
--
作者：
Franklin, Whitney;Taglialatela, Giulio
背景资料：研究突触的胰岛素信号反应是了解疾病相关神经退行性病变分子机制的重要途径，新方法：我们建立了一种从新鲜或冷冻组织中分离功能性突触体，并在ATP存在下暴露于胰岛素的方法来研究突触水平的胰岛素反应结果：我们进行了ATP剂量-反应曲线、胰岛素剂量-反应曲线和胰岛素反应时间过程的优化，以检测胰岛素受体（IR）激活。我们还通过使用已知胰岛素抵抗的动物模型AtENPP 1-Tg小鼠证明了我们的方案反映了体内胰岛素反应性的程度。该方法优于检测全脑匀浆中IR的其他方法，这是由于能够检测IR响应而不混淆来自也表达IR的其他细胞区域和细胞类型的贡献。离体胰岛素刺激可以与从同一动物获得的基线突触体进行比较，这提高了可靠性和统计功效，同时减少了进行单独实验所需的动物数量。我们开发了一个可靠的，有效的方法来测量胰岛素驱动的突触体胰岛素受体的离体磷酸化，可以可靠地反映前，动物CNS中现有的胰岛素反应性状态。据我们所知，这是第一个用胰岛素刺激分离的突触体的证据，也是研究生理或疾病条件下突触CNS胰岛素反应性的有前途的新技术。(C)2016爱思唯尔B. V.保留所有权利。
Background: Studying the insulin signaling response at the synapse is an important approach to understand molecular mechanisms involved in disease-related neurodegenerative processes.New method: We developed a method for studying the insulin responsiveness at the synaptic level by isolating functional synaptosomes from fresh or frozen tissue and exposing them to insulin in the presence of ATP (a critical step) to detect insulin receptor (IR) activation.Results: We performed an ATP dose-response curve, insulin dose-response curve, and insulin response time course to optimize this method. We also demonstrated that our protocol reflects the degree of insulin responsiveness in vivo by using an animal model of known insulin resistance, AtENPP1-Tg mice.Comparison with existing method(s): This method is advantageous over other methods detecting IR in total brain homogenates due to the ability to detect IR response without confounding contributions from other cell areas and cell types also expressing IR. Furthermore, ex vivo insulin stimulation can be compared to baseline synaptosomes obtained from the same animal which improves reliability and statistical power while decreasing the number of animals required to perform individual experiments.Conclusions: We have developed a reliable, efficient method to measure insulin-driven ex vivo phosphorylation of the synaptosomal insulin receptor that can reliably reflect the pre-existing insulin responsiveness status in the CNS of the animal. To the best of our knowledge, this is the first evidence of stimulation of isolated synaptosomes with insulin and a promising new technique to study the synaptic CNS insulin responsiveness under physiological or disease conditions. (C) 2016 Elsevier B.V. All rights reserved.