日本鵪鶉卵黃囊膜內胚層上皮細胞藉由cyclicAMP調控第一型固醇轉醯酶增加膽固醇酯化之機制

Sterol O-acyltransferase 1 enhances cholesterol esterification via cyclic AMP-dependent pathway in the yolk sac membrane endodermal epithelial cells of quails

王琇慧(Siou-Huei Wang)

指導教授 ： 丁詩同

國立臺灣大學/生物資源暨農學院/動物科學技術學研究所/碩士(2015年)

https://doi.org/10.6342/NTU.2015.01735

摘要

營養分之吸收利用於胚胎發育過程中扮演重要角色，禽胚隨著胚胎發育衍生出胚外組織卵黃囊膜 (yolk sac membrane, YSM)，覆蓋整個卵黃以吸取蛋中之營養分，YSM由外胚層、中胚層、及內胚層三個胚層細胞所構成，其中內胚層上皮細胞 (endodermal epithelial cells, EECs) 為主要吸收營養分之細胞。禽胚於孵化前一週為快速生長發育期，卵黃內約 68% 的脂質會於此時期大量被吸收利用，此時期死亡的禽胚未能有效利用脂質，孵出之雛禽仍有 YSM 滯留於體外、未吸收入腹腔之現象，因此增加胚胎發育時期脂質之利用可改善胚胎發育及孵化率，然而目前對於YSM如何利用卵黃之機制尚未釐清，因此本試驗之目的為探討卵黃囊膜 EECs 利用卵黃中脂質之機制，以提升胚胎孵化率。
　　卵黃中游離之膽固醇，在孵化後期大量於 EECs 中被酯化成膽固醇酯，固醇轉醯酶 (sterol O-acyltransferase, SOAT) 能將膽固醇與長鏈脂肪酸酯化，在哺乳動物中 SOAT 有兩種同功酶，SOAT1 及 SOAT2，然而在家禽胚胎 YSM 中 SOAT1 為主要之作用酵素，我們推論 SOAT1 與胚胎利用卵黃中脂質之機制有關。為了解 SOAT1 之調控機制，進而增加禽胚對脂質之利用，我們先選殖 SOAT1 之啟動子，預測啟動子上可能存在的轉錄因子結合位，並利用鵪鶉 EECs 及肝臟細胞培養系統進行試驗，加入具有潛力影響 SOAT1 表現之內泌素及營養分，結果顯示，在經高濃度之升糖素、磷酸二酯酶抑制劑 (isobutylmethylxanthine, IBMX) 及低濃度之葡萄糖刺激時，SOAT1 mRNA 表現量皆顯著上升於兩種細胞中。此結果代表 SOAT1 可能受 cAMP-dependent pathway 所調控，因此更進一步使用腺苷酸環化酶促進劑 (forskolin) 和 cAMP 的類似物 (dibutyryl-cAMP) 進行處理，發現兩種細胞中 SOAT1 mRNA 表現量也都顯著上升。我們推測 cAMP 會影響PKA進而調節下游因子，將細胞處理 PKA 之抑制劑－H89 後， SOAT1 mRNA 之表現會因為 PKA 被抑制後表現量下降，H89 亦會抑制由 IBMX 所誘導之 SOAT1 mRNA 表現量，更進一步測試 PKA 活性，可發現在 IBMX 刺激下 PKA 活性顯著增加，並於共處理H89時其活性被抑制。為證實 cAMP-dependent pathway 刺激之下 SOAT1 酯化膽固醇之活性亦增加，我們利用帶有螢光之膽固醇 (NBD-cholesterol) 培養於 EECs 中，追蹤細胞內膽固醇位置及膽固醇累積情形，發現經 dibutyryl-cAMP 所刺激之 EECs 其綠螢光表現較對照組高，顯示經 dibutyryl-cAMP 的刺激可能會促進SOAT1之表現，並增加酯化之膽固醇以油滴的形式儲存於細胞內。最後我們藉由連續截切序列及點突變啟動子之實驗，找出啟動子 -349 bp ~ -341 bp 之 cAMP-responsive element 為調節 SOAT1 表現之重要轉錄因子結合位。
　　綜合上述，我們成功找到影響 SOAT1 表現之調節機制，cAMP 經由 PKA 對 SOAT1 基因表現正向調控，進而增加膽固醇酯以油滴的形式儲存於細胞內，此發現對於調控 YSM 利用蛋黃內脂質有了新方向，期望未來可以找到相關調節因子添加於飼糧或直接注射入蛋內之方式，促進禽類胚胎發育對於脂質之利用，最終能有效改善孵化率。

關鍵字

第一型固醇轉醯酶 ； 膽固醇酯 ； cAMP-dependent pathway ； 卵黃囊膜 ； 胚胎發育

並列摘要

Nutrient utilization is a critical factor for embryonic development. The developing embryo obtains nutrients from albumen and yolk. Yolk sac membrane (YSM) is derived from three germ layers, endoderm, mesoderm and ectoderm. Nutrients are mainly uptaken by endodermal epithelial cells (EECs). Avian embryos grow dramatically before hatching. Approximately 68% of the lipids in yolk are absorbed during this stage. However, a clear mechanism of lipids utilization in yolk remains elusive. In this current study, we examined the mechanism of lipids utilization during embryonic development in an attempt to ameliorate avian hatchability.
　　Cholesterol is stored as free cholesterol in the yolk, and can be esterified into cholesteryl esters after transporting into EECs. SOAT1 and SOAT2 are genes encoded the isozymes, sterol O-acyltransferase (SOAT) which is an intracellular enzyme that esterifies long-chain fatty acid and cholesterol to form cholesterol ester. Activity of SOAT1 is higher than SOAT2 in YSM during the final week of embryonic development. We hypothesized that SOAT1 play a role to mediate uptake of free fatty acid and yolk cholesterol in EECs and is responsible for absorption and transportation of yolk lipid into developing embryos. First, we cloned avian SOAT1 promoter from chickens and analyzed its sequence for potential regulatory elements. Then, we established the EECs culture system to elucidate the cellular functions of SOAT1. We evaluated the effects of hormones and nutrients on SOAT1 gene expression in Japanese quail EECs and hepatocytes. Results showed that treatment of glucagon, isobutylmethylxanthine (IBMX) and glucose increased SOAT1 mRNA expression in EEC and hepatocytes, suggesting that SOAT1 is regulated by a cAMP-dependent pathway. Expression of SOAT1 mRNA was increased by adenylate cyclase activator (forskolin) and cAMP analog (dibutyryl-cAMP) treatments in EECs and hepatocytes. Furthermore, we showed protein kinase A (PKA) activity was increased by IBMX treatment, whereas co-treatment of PKA inhibitor, H89, negated the increase in PKA activity. To investigate SOAT1 enzyme activity, we incubated EECs with their substrates. Results suggested that cAMP induced higher activity of cholesterol esterification and thereby more intracellular lipids in EECs. We also found that a cAMP-responsive element (CRE) is located in the SOAT1 promoter region between -349 and -341 bp. By promoter deletion and point-mutation, we demonstrated that CRE played a critical function in mediating the expression of SOAT1.
　　In conclusion, expression of SOAT1 is regulated via cAMP-dependent pathway. In the future, we will determine the factors that regulate SOAT1 activity to improve the utilization of lipids in the EECs and increase embryonic growth to promote hatchability of fertilized eggs.

並列關鍵字

Sterol O-acyltransferase 1 ； cholesteryl ester ； cAMP-dependent pathway ； yolk sac membrane ； embryonic development

參考文獻

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