探討HLJ1透過影響生理時鐘調控代謝及粒線體形態變化所扮演之角色

Abstract

生理時鐘為調控代謝的重要機制，分為中央及器官周遭生理時鐘。在肝臟中，若週期紊亂會造成脂肪肝等代謝疾病。粒線體同為維持生理運作的中樞，過去研究指出粒線體形態的動態平衡會受到生理時鐘的調節。實驗室過去在人類肝臟熱休克蛋白40相似蛋白(HLJ1)的研究中發現HLJ1基因剔除(HLJ1-/-)小鼠肝臟有脂肪生成和代謝異常的現象，且在正常(WT)及HLJ1-/-小鼠肝臟RNA微陣列結果分析發現生理時鐘相關基因路徑有顯著差異。故本篇研究欲探討HLJ1在生理時鐘調控脂肪代謝的關係及對粒線體形態的影響。RNA微陣列路徑分析顯示HLJ1與生理時鐘高度相關。欲進一步探討兩者的相關性，我們使用小鼠胚胎成纖維細胞（MEF）及小鼠的肝細胞(hepatocyte)，並利用地塞米松(dexamethasone)同步細胞生理時鐘。細胞實驗顯示生理時鐘相關基因確實呈現週期震盪，但WT及HLJ1-/-組別只有部分時間點有差異，整體沒有顯著差異，在小鼠肝臟實驗也獲得相同結果。在研究脂肪代謝的實驗中，結果顯示小鼠血清中三酸甘油酯與總膽固醇也會隨時間產生週期，且兩組在相位與量上都有差異。但在脂肪代謝相關基因與蛋白表現量中，兩組並無差異。我們發現HLJ1在細胞中週期不規律但在肝臟中不管基因還是蛋白層面都有明顯週期的變化。接著，我們利用西方墨點法及免疫螢光染色探討影響線粒體形態，結果可見融合/裂變蛋白表達及和螢光圖中的粒線體形態會受到羰基氰化物間氯苯腙(CCCP)而形成分裂態，但在WT及HLJ1-/- MEF沒有顯著差異。最後，為了更清楚的觀察粒線體形態，我們建立一株粒線體會自發螢光的NIH3T3細胞，可用於進一步研究粒線體形態。

Circadian clock is the organizer of body biological functions, the disruption of clock in liver causes metabolic diseases such as fatty liver. Mitochondrial morphological change is also a critical mechanism that involves in many metabolic processes, researches have indicated that the dynamic balance of mitochondrial morphology is regulated by circadian clocks. Study of HLJ1 in our lab has found that HLJ1 knockout (HLJ1-/-) mice had abnormalities in lipogenesis and metabolism in liver. We wonder to know the relationship between HLJ1, circadian rhythms and mitochondria dynamic. We utilized mouse embryonic fibroblast (MEF) and hepatocyte from wild-type (WT) and HLJ1-/- mice for experiments. The results displayed the expression levels of circadian related genes were oscillated, but there were no significant difference between those two groups. The same results were seen in liver of WT and HLJ1-/- mice. The results of lipid metabolism of mice showed that triglyceride and total cholesterol in serum also produced rhythmicity by time, the phase and amount were different in two groups. However, there was no difference between the two groups in lipid metabolism related genes and proteins in liver. In addition, the duration pattern of HLJ1 was significant in mRNA and protein level in liver compared to the cell experiments. In the study of mitochondrial morphology, there was no difference in fusion/fission proteins expression and images of immunofluorescence staining of WT and HLJ1-/- MEF. Finally, we generated auto-fluorescent mitochondria in NIH3T3 that could be used for further investigation of mitochondrial morphology.