Changou Chun Austin, Ajoy Reni, Chou Szu-Yi
The Ph.D. Program for Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University; Integrated Laboratory, Center of Translational Medicine, Taipei Medical University; Core Facility, Taipei Medical University.
The Ph.D. Program for Neural Regenerative Medicine, College of Medical Science and Technology, Taipei Medical University; Graduate Institute of Neural Regenerative Medicine, College of Medical Science and Technology, Taipei Medical University.
J Vis Exp. 2017 Dec 7(130):56409. doi: 10.3791/56409.
Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNFα, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear. In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.
2型糖尿病（T2DM）是一场全球健康危机，其特征为胰岛素信号传导受损以及外周组织的慢性炎症。中枢神经系统（CNS）中的下丘脑是能量和胰岛素信号反应调节的控制中心。外周组织的慢性炎症以及某些趋化因子（如CCL5、TNFα和IL-6）的失衡会导致糖尿病和肥胖。然而，连接趋化因子与下丘脑胰岛素信号调节的功能机制仍不清楚。体外原代神经元培养模型是方便且简单的模型，可用于研究下丘脑神经元中的胰岛素信号调节。在本研究中，我们将与绿色荧光蛋白（GFP）偶联的外源性GLUT4蛋白（GFP-GLUT4）导入原代下丘脑神经元，以追踪胰岛素刺激后GLUT4的膜转位。通过去卷积显微镜记录GFP-GLUT4蛋白运输的延时图像，这使得用户在进行实验时能够生成高速、高分辨率的图像，同时不会对神经元造成显著损伤。在从CCR5基因敲除小鼠分离培养的CCR5缺陷型下丘脑神经元中观察到了CCR5在胰岛素调节的GLUT4转位中的作用。我们的结果表明，胰岛素刺激后，CCR5缺陷型下丘脑神经元中GLUT4的膜转位效率降低。
