The effects of insulin on nocturnin expression regulation in Hep3B cells

Circadian clocks temporally organize many aspects of physiology, including metabolic processes and behaviors. Recently, more and more studies indicate that the circadian clocks are intimately interlocked with metabolism. Nocturnin (Noc), a circadian-controlled deadenylase, originally was discovered as a nocturnally expressed deadenylase in many tissues of Xenopus and mouse, especially in liver. The nocturnin knock-out mice has striking physiological features which are characterized as resistant to diet-induced obesity and hepatic steatosis, and the altered glucose tolerance and insulin sensitivity. These suggest that Nocturnin play roles in regulation of the metabolic functions in liver. On the other hand, liver is the major metabolic organ of insulin actions. However, so far, the relationship between insulin and nocturnin is poorly understood. In this study, we confirm that insulin can up-regulate hNoc in mRNA and protein level in Hep3B cells, and the presence of insulin does not affect the mRNA and protein stability of hNoc. We propose the major regulation step is the transcriptional regulation. Moreover, we learn from the promoter assay that the canonical enhancer-boxes (E-boxes) located at the promoter of hNoc are responsible for the hNoc upregulation by insulin. The basic helix-loop-helix (bHLH) family transcription factors USF1, USF2, Clock and Bmal1 were demonstrated to form a dynamic complex on the E-boxes and contribute to the hNoc transcription activation after insulin treatment. Furthermore, the pathway of insulin-induced hNoc upregulation is demonstrated to be phosphoinositide 3’-kinase (PI3K)/AKT pathway but not mitogen-activated protein kinase (MAPK) pathway. Conclusively, hNoc activation by insulin is mediated by the PI3K/AKT pathway and is mainly regulated at the transcriptional regulation level. These findings unveil the detail molecular mechanism of hNoc activation by insulin and enforce the importance of Nocturnin in the anabolic roles of insulin in liver.