胰島素在Hep3B肝癌細胞中對nocturnin基因表現調節之研究

Abstract

生理時鐘在生物體的許多生理層面上扮演重要角色，其包含代謝與行為表徵。近期有越來越多研究指出作息不正常的人與代謝異常症有高度關聯，暗示生理時鐘與代謝有著緊密的連結。Nocturnin (Noc) 起初在非洲蟾蜍(Xenopus)及小鼠的許多組織中發現，特別是在肝臟組織，其mRNA在夜間表現量會上升。Noc是一個生理時鐘所調控的去腺嘧啶酶 (deadenylase)，當小鼠noc基因有缺陷時，會減少高油脂飲食造成的肥胖與脂肪肝，此外葡萄糖耐受性與胰島素感受性都有變異，進而得知Noc在肝臟執行正常的代謝功能上十分重要;肝臟是維持體內能量平衡與代謝的最重要器官，最主要是受到胰島素的調節，當胰島素感受性受損或其功能失調時，往往造成代謝異常，然而胰島素與Noc之間的關聯在過去的研究中尚未有明確的探討。本篇論文針對胰島素如何調節hNoc基因表現的分子機制進行研究，首先我們發現胰島素會促進肝癌細胞Hep3B中hNoc基因的表現，而且胰島素並不影響hNoc的mRNA及protein穩定性，表示胰島素對於hNoc的影響可能是在轉錄調節的層次；我們進一步針對hNoc啟動子進行研究，證實位在其中的強化子(Enhancer-box; E-box)對於胰島素促進hNoc基因表現調節是很重要的；另外利用ChIP (chromatin immunoprecipitation)實驗，發現轉錄因子USF1，USF2，Clock以及Bmal1在胰島素促進下與E-boxes形成複合體；在hNoc轉錄起始點附近的Histone3的第9及14位離胺酸乙醯化之程度有顯著增加，因此我們認為胰島素對於hNoc基因表現主要是透過轉錄的調節。此外，我們研究胰島素所涉及的訊息傳遞路徑，我們發現胰島素影響hNoc轉錄調控是經由PI3K/AKT路徑而非MAPK路徑。透過本篇論文的研究，我們對於胰島素調節hNoc活化的分子機制有更明確的闡述，也強化了Nocturnin在胰島素於肝臟執行代謝功能的重要性。

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.