唐卓懿 崔 坤 陳 強(qiáng) 譚 朋 廖 凱 麥康森 艾慶輝

(中國(guó)海洋大學(xué)教育部海水養(yǎng)殖重點(diǎn)實(shí)驗(yàn)室, 青島 266003)

飼料脂肪水平對(duì)大菱鲆幼魚TOR 信號(hào)通路的影響

唐卓懿 崔 坤 陳 強(qiáng) 譚 朋 廖 凱 麥康森 艾慶輝

(中國(guó)海洋大學(xué)教育部海水養(yǎng)殖重點(diǎn)實(shí)驗(yàn)室, 青島 266003)

研究旨在探究飼料脂肪水平對(duì)大菱鲆(Scophthalmus maximus)幼魚雷帕霉素受體(TOR)信號(hào)通路的影響。以大菱鲆幼魚[初始體重(8.6±0.01) g]為實(shí)驗(yàn)對(duì)象, 配制脂肪水平分別為11.69% (適宜脂肪組)和16.58% (高脂組)的2種等氮飼料在室內(nèi)循環(huán)水系統(tǒng)中進(jìn)行投喂實(shí)驗(yàn), 養(yǎng)殖周期為97d。結(jié)果顯示: (1)飼料脂肪水平升高對(duì)大菱鲆幼魚存活和特定生長(zhǎng)率(SGR)無顯著影響(P＞0.05)。(2)與適宜脂肪組相比, 肝臟中TOR和4EBP1 mRNA水平在高脂組上調(diào)(P＜0.05)而4EBP2 mRNA水平在高脂組下調(diào)(P＜0.05)。此外, 與適宜脂肪組相比, 肌肉中TOR和4EBP1 mRNA水平在高脂組下調(diào)(P＜0.05)而4EBP2 mRNA水平在高脂組無顯著變化。(3)飼料脂肪水平顯著影響AKT、TOR和4EBPs磷酸化水平。肝臟中p-AKT (T308和S473)/AKT、p-mTOR(S2448)/ mTOR和p-4EBP1 (T37/46)/4EBP1在高脂組顯著高于適宜脂肪組，而p-p70S6K (T389)/p70S6K在高脂組顯著低于適宜脂肪組。肌肉中p-AKT (S473)/AKT、p-mTOR (S2448)/mTOR和p-4EBP1 (T37/46)/4EBP1在高脂組顯著低于適宜脂肪組，而p-p70S6K (T389)/p70S6K在高脂組顯著高于適宜脂肪組。結(jié)果表明: 大菱鲆幼魚肝臟和肌肉TOR、4EBP1和4EBP2的mRNA表達(dá)量與AKT、TOR、4EBP1和p70 S6K磷酸化受到飼料脂肪水平調(diào)控, 飼料脂肪水平升高, 激活了肝臟TOR信號(hào)通路, 同時(shí)肌肉TOR信號(hào)通路受到部分抑制。

大菱鲆; 脂肪水平; 基因表達(dá); 磷酸化; TOR信號(hào)通路

雷帕霉素受體(TOR)信號(hào)通路是細(xì)胞感知營(yíng)養(yǎng)物質(zhì)并調(diào)控生長(zhǎng)的主要通路, 在進(jìn)化上極其保守。TOR是一種絲氨酸/蘇氨酸激酶, 它包含了兩個(gè)結(jié)構(gòu)功能不同的復(fù)合物——mTORC1和mTORC2。在mTORC1激活后, 其下游主要作用因子4EBPs和S6K發(fā)生磷酸化。S6K的磷酸化促進(jìn)了多種蛋白的轉(zhuǎn)錄激活和翻譯起始延伸。磷酸化的4EBPs與真核生物翻譯起始因子eIF-4E脫離, 激活后者, 使40S核糖體亞基與mRNA的5′端結(jié)合[1—3], 啟動(dòng)蛋白質(zhì)的翻譯。

在哺乳動(dòng)物中, TOR信號(hào)通路的研究已經(jīng)非常廣泛和深入[4]。在魚類上, 以斑馬魚(Danio rerio)為模式生物, 研究發(fā)現(xiàn)TOR信號(hào)通路調(diào)控腸道上皮細(xì)胞形成、細(xì)胞自噬[5,6]、心肌炎發(fā)生[7,8]和非酒精脂肪肝的發(fā)生[9]等。此外, 在大菱鲆(Scophthalmus maximus)[10]、建鯉(Cyprinus carpio var. Jian)[11—13]、軍曹魚(Rachycentron canadum)[14]、虹鱒(Oncorhynchus mykiss)[15—19]和草魚(Ctenopharyngodon idella)[20,21]中分別進(jìn)行了飼料中精氨酸、色氨酸、蛋氨酸、亮氨酸、油酸、膽堿水平以及豆粕替代魚粉和不同蛋能比對(duì)TOR信號(hào)通路的調(diào)控作用的研究。

然而, 目前關(guān)于飼料中脂肪水平對(duì)養(yǎng)殖魚類TOR信號(hào)通路的相關(guān)研究較少。大菱鲆肉質(zhì)鮮美, 營(yíng)養(yǎng)價(jià)值高, 經(jīng)濟(jì)價(jià)值高, 是我國(guó)重要養(yǎng)殖品種。在大菱鲆中, TOR信號(hào)通路受到飼料脂肪水平調(diào)控的研究目前尚未見報(bào)道。通過研究飼料脂肪水平對(duì)大菱鲆幼魚TOR信號(hào)通路的影響, 有助于深入了解飼料脂肪對(duì)大菱鲆蛋白質(zhì)合成調(diào)控的機(jī)理。

1 材料與方法

1.1 實(shí)驗(yàn)過程與樣品采集

配制粗蛋白含量為50%, 粗脂肪含量分別為11.69%和16.58%的兩種飼料(表 1)。大菱鲆幼魚購(gòu)自山東青島七好生物科技有限公司, 在青島鰲山衛(wèi)國(guó)家海洋科研中心實(shí)驗(yàn)基地暫養(yǎng)15d后, 饑餓24h。然后隨機(jī)選取體質(zhì)健壯、規(guī)格整齊、平均體重為(8.75±0.01) g的大菱鲆幼魚隨機(jī)分為2組, 每組6個(gè)重復(fù), 每個(gè)重復(fù)30尾。將2組飼料分別與6個(gè)養(yǎng)殖桶隨機(jī)匹配, 每日8:00和18:00各飽食投喂一次, 光照時(shí)間為12L：12D (7:30至19:30), 養(yǎng)殖周期97d。在實(shí)驗(yàn)結(jié)束后, 將實(shí)驗(yàn)魚禁食24h, 每桶隨機(jī)選6尾魚, 取肝臟、肌肉保存于–80℃, 用作分析實(shí)驗(yàn)。

表 1 實(shí)驗(yàn)飼料的配方以及常規(guī)營(yíng)養(yǎng)成分(%飼料干重)Tab. 1 Formulation and proximate composition of the experimental diet (% dry matter)

1.2 計(jì)算及統(tǒng)計(jì)方法

存活率(Survival rate, SR, %)=(初始實(shí)驗(yàn)魚數(shù)量–死亡實(shí)驗(yàn)魚數(shù)量)/初始實(shí)驗(yàn)魚數(shù)量×100

特定生長(zhǎng)率(Specific growth rate, SGR, %)=[Ln (實(shí)驗(yàn)魚終末體重)–Ln (實(shí)驗(yàn)魚初始體重)]/實(shí)驗(yàn)天數(shù)×100

1.3 總RNA提取及反轉(zhuǎn)錄

在液氮中研磨組織樣本, 使用TaKaRa RNAiso Reagent提取組織樣本總RNA, 用異丙醇法沉淀RNA, 凝膠電泳檢測(cè)RNA質(zhì)量。使用PrimeScript?RT reagent Kit With gDNA Eraser (Perfect Real Time)(TaKaRa, Japan)進(jìn)行反轉(zhuǎn)錄, 反轉(zhuǎn)錄結(jié)束后檢測(cè)cDNA濃度, 并稀釋到300 ng/μL, 置于–80℃冰箱中保存。

1.4 驗(yàn)證引物擴(kuò)增效率

熒光定量PCR引物參考張凱凱[22](表 2)。通過驗(yàn)證, 選用RPSD (DQ848899.1)為內(nèi)參基因。通過制作濃度標(biāo)準(zhǔn)曲線驗(yàn)證目標(biāo)序列與內(nèi)參序列的擴(kuò)增效率一致性[23]。

1.5 實(shí)時(shí)定量PCR

使用TransStart Top Green qPCR SuperMix (TRANSGEN, China)試劑盒進(jìn)行熒光定量反應(yīng)。定量程序設(shè)定為: 94攝氏度預(yù)熱30s; 設(shè)置循環(huán)為: 94攝氏度5s, 引物Tm值15s, 72攝氏度10s, 共45個(gè)循環(huán)。相對(duì)表達(dá)量采用2–ΔΔCt的方法計(jì)算。

1.6 Western Blot

稱取適量肝臟和肌肉樣品, 冰上勻漿。使用試劑盒(Beyotime Institute of Biotechnology, Jiangsu, China)測(cè)定蛋白濃度。使用裂解液稀釋所得蛋白,加入5×蛋白上樣緩沖液, 煮沸, 離心。配置聚丙烯酰胺凝膠(TOR使用6%, 其余蛋白使用12%), 150 V室溫電泳70min。室溫下100 V轉(zhuǎn)膜60min, 于5%脫脂奶粉TBST溶液中封閉, 洗膜。一抗4℃孵育過夜,洗膜。二抗37℃孵育60min, 洗膜。使用碧云天化學(xué)發(fā)光試劑(BeyoECL Plus kit)處理PVDF膜后于暗盒中壓片, 在暗室用X光片自動(dòng)洗片機(jī)(SGSB GROUP CO. LTD, Shanghai, China)中顯影, 得到相應(yīng)條帶。使用image J軟件進(jìn)行灰度分析。

2 結(jié)果

2.1 存活、生長(zhǎng)

大菱鲆幼魚存活率(均為100%)和特定生長(zhǎng)率(分別為2.34%/d和2.28%/d)在處理組間無顯著差異(P＞0.05, 表 3)。

2.2 肝臟和肌肉TOR信號(hào)通路相關(guān)基因mRNA表達(dá)量

在肝臟中, 高脂組TOR和4EBP1的mRNA水平顯著高于適宜脂肪組(P＜0.05), 而4EBP2表達(dá)量低于適宜脂肪組(P＜0.05); 在肌肉中, TOR和4EBP1表達(dá)量在高脂組顯著低于適宜脂肪組(P＜0.05), 而4EBP2表達(dá)量在兩組間無顯著差異(P＞0.05, 圖 1)。

2.3 肝臟和肌肉TOR信號(hào)通路相關(guān)蛋白磷酸化水平變化

在肝臟中, 與適宜脂肪組相比, p-AKT (Thr308)/ AKT、p-AKT (Ser473)/AKT、p-TOR (Ser2448)/TOR和p-4EBP1 (Thr37/46)/4EBP1蛋白水平在高脂組顯著上調(diào)(P＜0.05), 而p-p70 S6K (Thr389)/p70 S6K蛋白水平在高脂組顯著下調(diào)(P＜0.05)。在肌肉中, 與適宜脂肪組相比, p-AKT (Thr308)/AKT和p-p70 S6K (Thr389)/p70 S6K蛋白水平在高脂組顯著上調(diào),而p-AKT (Ser473)/AKT、p-TOR (Ser2448)/TOR和p-4EBP1 (Thr37/46)/4EBP1蛋白水平在高脂組顯著下調(diào)(P＜0.05)(圖 2)。

表 3 飼料脂肪水平對(duì)大菱鲆成活率(%)和特定生長(zhǎng)率(%/d)的影響(平均值±標(biāo)準(zhǔn)誤, n=6)Tab. 3 Effects of dietary lipid levels on the survival rate (%) and specific growth rate (SGR) (%/d) of juvenile Turbot(Scophthalmus maximus L.) (means±SEM, n=6)

3 討論

圖 1 大菱鲆幼魚不同組織TOR和4EBP1/2的mRNA表達(dá)量Fig. 1 Relative mRNA level of TOR and 4EBP1/2 in different tissuesA. 肝臟中相關(guān)基因表達(dá); B. 肌肉中相關(guān)基因表達(dá); 柱狀圖上標(biāo)星號(hào)表示有顯著差異(P＜0.05; 獨(dú)立樣本t檢驗(yàn))(n=3); TOR. 雷帕霉素受體; 4EBP1. 4E結(jié)合蛋白1; 4EBP2. 4E結(jié)合蛋白2A. Related genes expression in liver. B. Related genes expression in muscle. Asterisk indicates significant differences (P＜0.05; independent-samples t test) among treatments (n=3). TOR. Target of Rapamycin; 4EBP1. 4E binding protein 1; 4EBP2. 4E binding protein 2

圖 2 大菱鲆TOR信號(hào)通路相關(guān)蛋白的蛋白印跡Fig. 2 Western Blotting from tissues of turbotA. 肝臟和肌肉中相關(guān)蛋白的蛋白印跡的代表圖; B. 圖A數(shù)據(jù)的定量分析; 柱狀圖上標(biāo)星號(hào)表示有顯著差異(P＜0.05), 獨(dú)立樣本t檢驗(yàn))A. Representative images of western blot from liver, muscle; B. Quantitive analysis of data from A. Asterisk indicates significant differences (P＜0.05; independent-samples t test) among treatments (n=3)

目前, 魚類TOR和4EBPs的mRNA表達(dá)量受飼料脂肪水平調(diào)控的研究目前尚未見報(bào)道。本研究發(fā)現(xiàn)大菱鲆幼魚肝臟和肌肉TOR、4EBP1和4EBP2的mRNA表達(dá)量受到飼料脂肪水平調(diào)控。研究結(jié)果顯示: 在肝臟中, 高脂組TOR和4EBP1的mRNA水平顯著高于適宜脂肪組, 而4EBP2表達(dá)量低于適宜脂肪組; 在肌肉中, TOR和4EBP1的mRNA水平在高脂組低于適宜脂肪水平組, 4EBP2 mRNA水平在高脂組無顯著變化。與本研究類似, 在建鯉中的研究發(fā)現(xiàn), 飼料膽堿水平調(diào)控肝臟、肌肉、脾臟和頭腎的TOR和4EBP2 mRNA表達(dá)量[11,13], 許丹丹[24]的研究發(fā)現(xiàn)豆粕替代魚粉影響大菱鲆幼魚腸道4EBP1的mRNA表達(dá)而且4EBP1 mRNA表達(dá)量受攝食后取樣時(shí)間點(diǎn)的影響。以上研究表明建鯉和大菱鲆幼魚TOR和4EBPs的mRNA表達(dá)受到飼料營(yíng)養(yǎng)素的調(diào)控。而在張凱凱[22]的研究中, 支鏈氨基酸水平并未影響大菱鲆幼魚肌肉TOR、4EBP1和4EBP2的mRNA表達(dá); 與張凱凱的研究類似, Luo等[14]的研究表明軍曹魚幼魚TOR mRNA表達(dá)量不受飼料玉米蛋白粉水平調(diào)控, 這可能是由于飼料營(yíng)養(yǎng)素對(duì)TOR和4EBPs的調(diào)控存在種間差異。另外, 不同飼料營(yíng)養(yǎng)素對(duì)相同種類的魚體中TOR和4EBPs的作用機(jī)制可能也存在差異。飼料營(yíng)養(yǎng)素對(duì)魚類TOR和4EBPs的mRNA表達(dá)的作用機(jī)制還有待于進(jìn)一步研究。在人類的研究[25]表明, 4EBP1和4EBP2具有非常相似的基因序列和晶體結(jié)構(gòu), 4EBP2的氨基酸序列僅僅在60至63號(hào)位與4EBP1存在差異。4EBP1和4EBP2生理作用相同, 二者均能夠與真核翻譯啟動(dòng)子——eIFs結(jié)合并抑制后者活性。在魚類中, 有關(guān)4EBP1和4EBP2的比較研究相對(duì)較少, 二者受飼料營(yíng)養(yǎng)素調(diào)控的差異還有待于進(jìn)一步研究。

TOR是進(jìn)化上非常保守的絲氨酸/蘇氨酸激酶, TOR信號(hào)通路是細(xì)胞感知營(yíng)養(yǎng)素水平并調(diào)控生長(zhǎng)的重要通路。mTORC1是TOR信號(hào)通路中整合營(yíng)養(yǎng)素信息并調(diào)控下游作用因子——S6K和4EBPs的關(guān)鍵蛋白。胰島素和生長(zhǎng)因子通過PI3K-AKT和mTORC2-AKT途徑使AKT激活并且在Thr308和Ser473位點(diǎn)磷酸化, AKT激活mTORC1使后者在Ser2448位點(diǎn)磷酸化, mTORC1激活使TOR信號(hào)通路下游兩個(gè)重要作用因子——S6K和4EBPs磷酸化并啟動(dòng)蛋白質(zhì)的翻譯。在魚類中, AKT、TOR、4EBP1和p70 S6K磷酸化受到飼料脂肪水平調(diào)控的研究相對(duì)較少。本研究發(fā)現(xiàn)大菱鲆幼魚肝臟和肌肉中AKT、TOR、4EBP1和p70 S6K磷酸化受到飼料脂肪水平調(diào)控。研究結(jié)果顯示: 在肝臟中, 與適宜脂肪組相比, AKT (Thr308)、AKT (Ser473)、TOR (Ser2448)和p-4EBP1 (Thr37/46)磷酸化水平在高脂組顯著上調(diào); 而p70 S6K (Thr389)磷酸化水平在高脂組顯著下調(diào)。在肌肉中, 與適宜脂肪組相比, AKT (Thr308)和p70 S6K (Thr389)磷酸化水平在高脂組顯著上調(diào); 而AKT (Ser473)、TOR (Ser2448)和4EBP1 (Thr37/46)磷酸化水平在高脂組顯著下調(diào)。在塞內(nèi)加爾鰨(Solea senegalensis)的研究中[26], 肌肉AKT磷酸化在高脂組受到抑制, 此結(jié)果與本研究類似, 但肌肉S6K磷酸化水平在高脂組降低, 此結(jié)果與本研究結(jié)果相反。在人類的研究中, 2型糖尿病人肌肉普遍存在胰島素抵抗現(xiàn)象。Treins等[27]研究表明S6K敲除小鼠不會(huì)發(fā)生胰島素抵抗, 并且S6K的激活對(duì)PI3K-AKT通路存在抑制, 加劇胰島素抵抗。在本研究中, 肌肉AKT (Ser473)在高脂組降低, S6K磷酸化在高脂組升高, 與在哺乳動(dòng)物中對(duì)胰島素抵抗的研究結(jié)果一致。此外, 有研究表明4EBPs和S6K存在類似互補(bǔ)的作用, 當(dāng)4EBPs被敲除后, S6K磷酸化水平上升, 同時(shí)發(fā)生胰島素抵抗且p-AKT (Ser473)/ AKT下調(diào)[28], 在本研究中也發(fā)現(xiàn)了類似結(jié)果。

總之, 飼料脂肪水平升高, 肝臟TOR信號(hào)通路被激活, 肌肉TOR信號(hào)通路受到部分抑制。肝臟和肌肉TOR信號(hào)通路的變化規(guī)律不同, 可能與肌肉對(duì)胰島素的敏感性發(fā)生變化有關(guān)。

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EFFECTS OF DIETARY LIPID LEVELS ON TOR PATHWAY IN JUVENILE TURBOT (SCOPHTHALMUS MAXIMUS L.)

TANG Zhuo-Yi, CUI Kun, CHEN Qiang, TAN Peng, LIAO Kai, MAI Kang-Sen and AI Qing-Hui
(The Key Laboratory of Mariculture (Ministry Education of China), Ocean University of China, Qingdao 266003, China)

This study investigated the effects of dietary lipid levels on the TOR pathway in juvenile turbot using diets containing graded levels of lipid 11.69% (L11.69) and 16.58% (L16.58) (dry weight) to feed triple groups of juvenile turbot (8.6±0.01 g) for a 97d trial. The lipids level in diet did not impact survival rate and SGR. Compared those in L11.69 group, L16.58 group increased the gene expression of TOR, 4EBP1 but decreased 4EBP2 in liver, and it increased the phosphorylation of pAKT-S473, pAKT-T308, pmTOR-S2448, and p4EBP1-T37/46. In the muscle, L16.58 group had decreased TOR and 4EBP1 associated with pAKT-S473, pmTOR-S2448 and p4EBP1-T37/46 compared with those in L11.69 group. In conclusion, L16.58 diet can activate TOR pathway in liver while partially suppress TOR pathway in muscle of junvenile turbot compare with L11.69 diet.

Turbot; Dietary lipid level; Gene expression; Phosphorylation; TOR pathway

S963.1

A

1000-3207(2017)03-0491-06

10.7541/2017.63

2016-05-16;

2016-11-04

國(guó)家重點(diǎn)基礎(chǔ)研究發(fā)展計(jì)劃(973計(jì)劃)養(yǎng)殖魚類蛋白質(zhì)高效利用的調(diào)節(jié)機(jī)制(2014CB138600)資助 [Supported by Mechanisms of Protein Efficient Utilization in Farmed Fish, National Program on Key Basic Research Projects (973 Program) (2014CB138602)]

唐卓懿(1990—), 男, 廣東韶關(guān)人; 碩士研究生; 研究方向?yàn)樗a(chǎn)動(dòng)物營(yíng)養(yǎng)與飼料。E-mail: tzypublic@163.com

艾慶輝, 教授; 研究方向?yàn)樗a(chǎn)動(dòng)物營(yíng)養(yǎng)與飼料。E-mail: qhai@ouc.edu.cn