Xu Yingke, Nan Di, Fan Jiannan, Bogan Jonathan S, Toomre Derek
Department of Biomedical Engineering, MOE Key Laboratory of Biomedical Engineering, Zhejiang Provincial Key Laboratory of Cardio-Cerebral Vascular Detection Technology and Medicinal Effectiveness Appraisal, Zhejiang University, Hangzhou 310027, China Department of Cell Biology, Yale University School of Medicine, New Haven, 06510, USA
Department of Biomedical Engineering, MOE Key Laboratory of Biomedical Engineering, Zhejiang Provincial Key Laboratory of Cardio-Cerebral Vascular Detection Technology and Medicinal Effectiveness Appraisal, Zhejiang University, Hangzhou 310027, China.
J Cell Sci. 2016 May 15;129(10):2085-95. doi: 10.1242/jcs.174805. Epub 2016 Apr 13.
Glucose transporter 4 (GLUT4; also known as SLC2A4) resides on intracellular vesicles in muscle and adipose cells, and translocates to the plasma membrane in response to insulin. The phosphoinositide 3-kinase (PI3K)-Akt signaling pathway plays a major role in GLUT4 translocation; however, a challenge has been to unravel the potentially distinct contributions of PI3K and Akt (of which there are three isoforms, Akt1-Akt3) to overall insulin action. Here, we describe new optogenetic tools based on CRY2 and the N-terminus of CIB1 (CIBN). We used these 'Opto' modules to activate PI3K and Akt selectively in time and space in 3T3-L1 adipocytes. We validated these tools using biochemical assays and performed live-cell kinetic analyses of IRAP-pHluorin translocation (IRAP is also known as LNPEP and acts as a surrogate marker for GLUT4 here). Strikingly, Opto-PIP3 largely mimicked the maximal effects of insulin stimulation, whereas Opto-Akt only partially triggered translocation. Conversely, drug-mediated inhibition of Akt only partially dampened the translocation response of Opto-PIP3 In spatial optogenetic studies, focal targeting of Akt to a region of the cell marked the sites where IRAP-pHluorin vesicles fused, supporting the idea that local Akt-mediated signaling regulates exocytosis. Taken together, these results indicate that PI3K and Akt play distinct roles, and that PI3K stimulates Akt-independent pathways that are important for GLUT4 translocation.
葡萄糖转运蛋白4（GLUT4；也称为SLC2A4）存在于肌肉和脂肪细胞的细胞内囊泡中，并在胰岛素的作用下转位至质膜。磷脂酰肌醇3激酶（PI3K）-Akt信号通路在GLUT4转位中起主要作用；然而，一个挑战是要弄清楚PI3K和Akt（有三种异构体，Akt1-Akt3）对整体胰岛素作用的潜在不同贡献。在这里，我们描述了基于CRY2和CIB1（CIBN）N端的新型光遗传学工具。我们使用这些“Opto”模块在3T3-L1脂肪细胞中在时间和空间上选择性激活PI3K和Akt。我们使用生化分析验证了这些工具，并对IRAP-pHluorin转位进行了活细胞动力学分析（IRAP也称为LNPEP，在此作为GLUT4的替代标志物）。令人惊讶的是，Opto-PIP3在很大程度上模拟了胰岛素刺激的最大效应，而Opto-Akt仅部分触发转位。相反，药物介导的Akt抑制仅部分减弱了Opto-PIP3的转位反应。在空间光遗传学研究中，将Akt聚焦靶向细胞的一个区域标记了IRAP-pHluorin囊泡融合的位点，支持局部Akt介导的信号调节胞吐作用的观点。综上所述，这些结果表明PI3K和Akt发挥不同的作用，并且PI3K刺激对GLUT4转位重要的Akt非依赖性途径。
