Pei Jinli, Xiao Zhengpan, Guo Ziyi, Pei Yechun, Wei Shuangshuang, Wu Hao, Wang Dayong
Key Laboratory of Ministry of Education for Tropical Bioresources, Hainan University, Haikou, Hainan 570228, People's Republic of China.
Laboratory of Biotechnology and Molecular Pharmacology, School of Life and Pharmaceutical Sciences, Hainan University, Haikou, Hainan 570228, People's Republic of China.
Diabetes Metab Syndr Obes. 2020 Oct 21;13:3887-3898. doi: 10.2147/DMSO.S268028. eCollection 2020.
This study aimed to investigate the role of β adrenergic receptor (βAR) in insulin signaling transduction in H9C2 cardiomyoblast cells to understand the formation of the βAR-insulin receptor (IR) protein complex and its role in insulin-induced expression.
H9C2 cells were treated with various protein inhibitors (CGP, βAR inhibitor CGP20712; ICI, βAR inhibitor ICI 118,551; PKI, PKA inhibitor myristoylated PKI; PD 0325901, MEK inhibitor; SP600125, JNK inhibitor) with or without insulin or isoproterenol (ISO) before RNA-sequencing (RNA-Seq) and quantitative-PCR (Q-PCR). Yeast two-hybrid, co-immunoprecipitation and His-tag pull-down assay were carried out to investigate the formation of the βAR-IR protein complex. The intracellular concentrations of cAMP in H9C2 cells were tested by high performance liquid chromatography (HPLC) and the phosphorylation of JNK was tested by Western blot.
Gene Ontology (GO) analysis revealed that the most significantly enriched processes in the domain of molecular function (MF) were catalytic activity and binding, whereas in the domain of biological processes (BP) were metabolic process and cellular process. Furthermore, the enriched processes in the domain of cellular components (CC) were cell and cell parts. The Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis showed that the most significant pathways that have been altered included the PI3K-Akt and MAPK signaling pathways. Q-PCR, which was performed to verify the gene expression levels exhibited consistent results. In evaluating the signaling pathways, the sustained stimulation of βAR by ISO inhibited insulin signalling, and the effect was primarily through the cAMP-PKA-JNK pathway and MEK/JNK signaling pathway. Yeast two-hybrid, co-immunoprecipitation and His-tag pull-down assay revealed that βAR, IR, insulin receptor substrate 1 (IRS1), Grb2-associated binding protein 1 (GAB1) and Grb2 existed in the same protein complex.
The sustained stimulation of βAR might inhibit insulin signaling transduction through the cAMP-PKA-JNK and MEK/JNK pathways in H9C2 cells.
本研究旨在探讨β肾上腺素能受体（βAR）在H9C2心肌成纤维细胞胰岛素信号转导中的作用，以了解βAR-胰岛素受体（IR）蛋白复合物的形成及其在胰岛素诱导表达中的作用。
在进行RNA测序（RNA-Seq）和定量聚合酶链反应（Q-PCR）之前，用各种蛋白抑制剂（CGP，βAR抑制剂CGP20712；ICI，βAR抑制剂ICI 118,551；PKI，蛋白激酶A抑制剂肉豆蔻酰化PKI；PD 0325901，丝裂原活化蛋白激酶/细胞外信号调节激酶（MEK）抑制剂；SP600125，应激活化蛋白激酶（JNK）抑制剂）处理H9C2细胞，同时加入或不加入胰岛素或异丙肾上腺素（ISO）。进行酵母双杂交、免疫共沉淀和His标签下拉实验，以研究βAR-IR蛋白复合物的形成。通过高效液相色谱法（HPLC）检测H9C2细胞中细胞内环磷酸腺苷（cAMP）的浓度，通过蛋白质免疫印迹法检测JNK的磷酸化。
基因本体论（GO）分析显示，在分子功能（MF）领域中最显著富集的过程是催化活性和结合，而在生物学过程（BP）领域中是代谢过程和细胞过程。此外，在细胞成分（CC）领域中富集的过程是细胞和细胞部分。京都基因与基因组百科全书（KEGG）通路分析表明，改变最显著的通路包括磷脂酰肌醇-3激酶（PI3K）-蛋白激酶B（Akt）和丝裂原活化蛋白激酶（MAPK）信号通路。用于验证基因表达水平的Q-PCR结果一致。在评估信号通路时，ISO对βAR的持续刺激抑制了胰岛素信号传导，其作用主要通过cAMP-蛋白激酶A（PKA）-JNK通路和MEK/JNK信号通路。酵母双杂交、免疫共沉淀和His标签下拉实验表明，βAR、IR、胰岛素受体底物1（IRS1）、生长因子受体结合蛋白2相关结合蛋白1（GAB1）和生长因子受体结合蛋白2（Grb2）存在于同一蛋白复合物中。
βAR的持续刺激可能通过cAMP-PKA-JNK和MEK/JNK通路抑制H9C2细胞中的胰岛素信号转导。
