Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/98065
標題: 載脂蛋白apo VLDL-II 調節產蛋雞 VLDL粒徑-與三酸甘油脂轉移蛋白協同作用
Apolipoprotein VLDL-II mediates VLDL particle size in laying hens -synergism with microsomal triglyceride transfer protein functionality
作者: 葉宥麟
Yu-Lin Ye
關鍵字: 產蛋雞;極低密度脂蛋白;載脂蛋白Apo VLDL-II;微粒體三酸甘油酯轉移蛋白;粒徑大小;Laying hens;Very low density lipoprotein;Apolipoprotein VLDL-Il;Microsomal- triglyceride transfer protein (MTP);particle size.
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摘要: 
鳥類胚胎營養來源完全依賴蛋黃中脂質供應,因此母鳥於產蛋期間需提供更多脂質以形成蛋黃。產蛋母鳥之VLDL與未產蛋母鳥有以下幾點不同:1) 較小之粒徑以通過顆粒細胞基底膜,2) 較高的三酸甘油酯與磷脂比率,3) VLDL上鑲嵌著鳥類所獨有,受到雌激素誘導生成的載脂蛋白Apolipoprotein VLDL-II,此蛋白質已被證實可干擾VLDL脂蛋白分解酶作用(Lipoprotein lipase, LPL),保護VLDL於血液循環中運輸,完整的進入濾泡蓄積成蛋黃。但apo VLDL-II是否影響粒徑仍未有定論。微粒體三酸甘油酯轉移蛋白 (Microsomal Triglyceride Transfer protein, MTP)在肝臟VLDL形成過程中會促進三酸甘油脂轉移。然產蛋期apo VLDL-II出現與大量VLDL合成與分泌,其是否與MTP協同活性作用有關,以促進之三酸甘油酯運用組裝,進而影響VLDL合成、分泌與組成亦未有定論。本試驗中以小雞肝臟原代細胞和LMH-2A細胞株為模型,過度表達Apo VLDL-II或以雌激素刺激,並添加油酸 (Oleic acid, OA) 驅動VLDL之分泌能力,探討VLDL粒徑變化與MTP在此過程中之角色。電子顯微鏡下,原代肝細胞Control (C)組高峰為91-100 nm,Control+ OA (C+)組為111-120 nm,Apo VLDL-II transfection (T)組為31-40 nm,Apo VLDL-II transfection+OA (T+)組為21-30 nm。LMH-2A cells 之Control (C)為121-130 nm,Control+OA (C+)為81-90 nm,Transfection (T)為91-110 nm,Transfection +OA (T+)為51-60 nm,Estradiol induce (E)為111-120 nm,Estradiol induction (E+)為51-60 nm。單純添加OA於原代細胞中會導致細胞VLDL粒徑變大,但LMH-2A則是變小。兩種細胞過度表達Apo -II或以Estradiol induction產生Apo-II皆會使VLDL粒徑降低,OA與Apo-II overexpression 共處理則有加成效應使粒徑變小。以自製抗體做Western Blot 分析,顯示母雞肝臟MTP表現量並無大於公雞,而過度表達Apo VLDL-II與雌激素刺激之LMH-2A 細胞其MTP表現量亦無差異,但過度表達並添加OA處理會提高小雞原代肝細胞MTP表達。免疫沉澱分析確認MTP與Apo VLDL-II有交互作用,表示MTP活性可能受到ApoVLDL-II調節。推測Apo VLDL-II於小顆粒形成過程中協同MTP將脂質纏繞上Apo B,於富含TG之大顆粒形成時鑲嵌其上限制其粒徑並加速其分泌。綜上所述,過度表達與雌激素誘導產生Apo VLDL-II之肝臟細胞皆會降低VLDL粒徑,此現象應與Apo VLDL-II與MTP於VLDL分泌過程之交互作用調控有關。

Abstract

Plasma VLDL biology in laying hens differs distinctively from that of immature hens, including a smaller VLDL particle diameter, dramatic increases of actual and fractional of levels of VLDL-TG (triacylglycerol) and -PL (phospholipid) concentration, and the presence of an avian specific apolipoprotein; VLDL-II (Apo VLDL-II). These estrogen-induced alterations were suggested to provide TG-rich VLDL for yolk formation for embryo development, and thereby termed VLDLy. Precious studies have shown the peripheral role of Apo VLDL-II by hampering LPL (lipoprotein lipase) activity to escort intact VLDL to the ovary. Despite lacking direct evidences, due to the presence of Apo VLDL-II in VLDLy simultaneously with smaller particle size, Apo VLDL-II was postulated to mediate smaller particle diameter of VLDLy. . Microsomal triglyceride transfer protein (MTP) functions to prevent apo B from degradation and facilitate TG mobilization and traffic into vesicles for secretion. However, no studies have been tried to elucidate altered lipid compositions and massive secretion of VLDLy in relation to the role of MTP functionality in TG mobilization for VLDL secretion. In this study, chick primary hepatocytes and LMH-2A cell line in combination with Apo VLDL-II overexpression and estrogen induction and/or pulse stimulation by oleate to impose VLDL assembly and secretion were used to elucidate the role of Apo VLDL-II in mediating the change of VLDL size and its relationship with MTP in hepatic TG mobilization. Under scanning electron microscope (SEM), primary cells had VLDL particle size as Control (C); 91-100 nm, Control+ OA (oleic acid; C+); 111-120 nm, Transfection with apo VLDL-II expressing vector (T): 31-40 nm, T+OA(T+): 21-30 nm. In LMH-2A cells, VLDL particle was sized as Control (C): 121-130 nm, Control+ OA (C+): 81-90 nm, Transfection (T): 91-110 nm, Transfection +OA (T+): 51-60 nm, Estradiol induction (E): 111-120 nm, E+ OA(E+): 51-60 nm. Accordingly, oleate increased VLDL size in primary hepatocytes but decreased it in LMH-2A cells. Enforced expression of Apo VLDL-II and estrogen induction decreased the VLDL particle size in both chick primary hepatocytes and LMH-2A cells. No differences of hepatic MTP expression between laying hens and roosters, and so as by apo VLDL-II overexpression and estrogen induction in LMH-2A cells. However, Apo VLDL-II overexpression and oleate treatment increased MTP expression in chick primary hepatocytes. Immunoprecipitation analysis confirmed that Apo VLDL-II binds to MTP and apoB in laying hen hepatocytes, suggesting allosteric regulation of MTP activity by Apo VLDL-II along the assembly process of VLDL with Apo B and cargo lipids In summary, the presence of Apo VLDL-II indeed mediates the decrease of VLDLy particle size and intracellular it may interact with MTP and Apo B to facilitate VLDL assembly and secretion in the laying hens.
URI: http://hdl.handle.net/11455/98065
Rights: 同意授權瀏覽/列印電子全文服務,2021-01-16起公開。
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