Sustained Stimulation of β(2)AR Inhibits Insulin Signaling in H9C2 Cardiomyoblast Cells Through the PKA-Dependent Signaling Pathway

INTRODUCTION: This study aimed to investigate the role of β(2) adrenergic receptor (β(2)AR) in insulin signaling transduction in H9C2 cardiomyoblast cells to understand the formation of the β(2)AR-insulin receptor (IR) protein complex and its role in insulin-induced Glut4 expression. METHODS: H9C2 cells were treated with various protein inhibitors (CGP, β(1)AR inhibitor CGP20712; ICI, β(2)AR 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 β(2)AR-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. RESULTS: 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 β(2)AR 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 β(2)AR, IR, insulin receptor substrate 1 (IRS1), Grb2-associated binding protein 1 (GAB1) and Grb2 existed in the same protein complex. CONCLUSION: The sustained stimulation of β(2)AR might inhibit insulin signaling transduction through the cAMP-PKA-JNK and MEK/JNK pathways in H9C2 cells.