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Volume 45 Issue 10
Oct.  2021
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PGC1α molecular characterization and its responsiveness to nutrient restriction, carbohydrate-enriched diets and glucose loadings in blunt snout bream (Megalobrama amblycephala)

  • Corresponding author: XU Chao, xuc1213@scau.edu.cn
  • Received Date: 2020-12-20
    Accepted Date: 2021-05-06
    Available Online: 2021-08-25
  • To explore the mechanism of peroxisome proliferator activated receptor γ coactivator 1α (PGC1α) in the glucose metabolism of blunt snout bream Megalobrama amblycephala, the partial cDNA of PGC1α was cloned, and the transcriptional response of this gene to nutrient restriction, carbohydrate-enriched diets and glucose loadings were investigated. The partial cDNA was 2566 bp with an open reading frame of 1 404 bp encoding 467 amino acids, and compared to Ctenopharyngodon idellus, it showed 96.79% homology. The mRNA levels of PGC1α in the brain and liver were significantly increased during 10 days of fasting, and then decreased to normal level after refeeding 1 h. The high-carbohydrate diet significantly decreased the mRNA levels of PGC1α in the brain and liver. In addition, the mRNA levels of PGC1α in the brain and liver both decreased significantly during the first 2 h, then returned to the basal value at 12 h. The results indicated that PGC1α plays an important role in glucose metabolism in M. amblycephala. The results obtained here will provide the theoretical foundation for completing the research of PGC1α functions involving the regulation of glucose homeostasis in fish.
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PGC1α molecular characterization and its responsiveness to nutrient restriction, carbohydrate-enriched diets and glucose loadings in blunt snout bream (Megalobrama amblycephala)

    Corresponding author: XU Chao, xuc1213@scau.edu.cn
  • 1. College of Marine Sciences of South China Agricultural University & Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou 510642, China
  • 2. Key Laboratory of Aquatic Nutrition and Feed Science of Jiangsu Province, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China

Abstract: To explore the mechanism of peroxisome proliferator activated receptor γ coactivator 1α (PGC1α) in the glucose metabolism of blunt snout bream Megalobrama amblycephala, the partial cDNA of PGC1α was cloned, and the transcriptional response of this gene to nutrient restriction, carbohydrate-enriched diets and glucose loadings were investigated. The partial cDNA was 2566 bp with an open reading frame of 1 404 bp encoding 467 amino acids, and compared to Ctenopharyngodon idellus, it showed 96.79% homology. The mRNA levels of PGC1α in the brain and liver were significantly increased during 10 days of fasting, and then decreased to normal level after refeeding 1 h. The high-carbohydrate diet significantly decreased the mRNA levels of PGC1α in the brain and liver. In addition, the mRNA levels of PGC1α in the brain and liver both decreased significantly during the first 2 h, then returned to the basal value at 12 h. The results indicated that PGC1α plays an important role in glucose metabolism in M. amblycephala. The results obtained here will provide the theoretical foundation for completing the research of PGC1α functions involving the regulation of glucose homeostasis in fish.

  • 糖类作为三大营养物质 (蛋白质、脂肪、糖) 中最廉价的能量源[1],水产饲料中添加适量糖水平可节约饲料中蛋白质原料的用量。然而,鱼类是先天性糖尿病体质,对糖类的利用率较低[2],当摄食高碳水化合物饲料后会出现血糖持续偏高,代谢负荷加重,进而抑制鱼体生长[2]。血糖调控机制是生理学研究的重要内容之一,糖稳态对生物有机体能量代谢平衡意义重大。过氧化物酶体增殖物激活受体γ辅助活化因子 1α (peroxisome proliferator activated receptor γ coactivator 1α,PGC1α)属于PGC1 转录辅助活化因子家族成员,其广泛参与机体的能量生成与利用过程[3]。糖代谢上的研究证实,PGC1α是调节机体糖异生的关键因子,其在肝细胞中过度表达可显著上调磷酸烯醇式丙酮酸羧激酶(PEPCK)、葡萄糖-6-磷酸酶(G6Pase)等糖异生关键酶的转录水平,导致肝糖输出增加,进而升高机体血糖水平[4]。糖尿病动物模型上的研究发现,干扰PGC1α表达会导致机体产生胰岛素抵抗和葡萄糖不耐受等现象[4]。然而,以上研究均来源于哺乳动物,水产动物上的研究尚较缺乏。目前,已克隆获得斑马鱼(Danio rerio)、金鱼(Carassius auratus)、草鱼(Ctenopharyngodon idella)、红鳍东方鲀(Takifugu rubripes)、齐口裂腹鱼(Schizothorax prenanti)等鱼类的PGC1α基因全长序列[5-6]。但对基因在鱼类糖代谢中的作用仍缺乏研究。此外,PGC1α分子克隆研究主要集中于杂食性和肉食性鱼类,对草食性鱼类上的研究较少。

    团头鲂(Megalobrama amblycephala)是我国重要的淡水草食性经济鱼类。相较肉食和杂食性鱼类,其具有较高的糖耐受能力,但过高的饲料糖水平仍会使其产生高血糖症状,进而抑制鱼体生长。因此,本实验旨在克隆团头鲂PGC1α基因的cDNA片段序列,并分析其分子特征;同时探究团头鲂PGC1α基因在饥饿、高糖饲料喂养及葡萄糖负荷下的表达量变化。本实验所获结果将为鱼类糖代谢的调控提供效应靶点,并有助于高糖低蛋白水产饲料的研发。

1.   材料与方法
  • 实验所用团头鲂购自于江苏省扬州市国家良种场。实验开始前,将鱼暂养于池塘网箱内2周,用含32%蛋白和31%糖水平的商品饲料(帅丰饲料有限公司,江苏省,中国)进行驯化。随后,挑选4条体质量量为 (43.72 ± 0.16) g的团头鲂进行麻醉(使用100 mg/L的MS-222),取其肝脏组织用液氮迅速冷冻,放置于−80 °C冰箱中用于PGC1α基因克隆。

  • 随后,选取54条鱼 [(62.96 ± 2.57) g]进行饥饿再投喂实验。实验分为3个组:连续10 d投喂组(fed 10 d group)、饥饿10 d组(fasted 10 d group)、饥饿10 d后投喂1 h组(re-fed 1 h group),每组3个重复。实验结束后,每缸(水体容量为200 L)随机选取4条鱼迅速使用100 mg/L的MS-222进行麻醉,取其脑和肝脏组织用液氮迅速冷冻,放置于−80 °C备用。

  • 饥饿实验后,随机选取160条健康的团头鲂幼鱼[(42.11 ± 0.64) g ]进行12周的养殖实验。实验分为2个组,每组4个重复。期间分别投喂基础饲料(control: 30%糖水平)和高糖饲料(high carbohydrate: 45%糖水平),每天2次 (08:00和17:00)。饲料配方如表1所示。养殖结束后,全部鱼饥饿24 h。随机选取6条鱼迅速使用100 mg/L的MS-222进行麻醉,取其脑和肝脏组织用液氮迅速冷冻,放置于−80 °C备用。

    对照组
    control
    高糖组
    HC
    原料/% ingredient
      鱼粉 fish meal 8.00 8.00
      豆粕 soybean meal 26.00 26.00
      菜粕 rapeseed meal 17.00 17.00
      棉粕 cottonseed meal 17.00 17.00
      鱼油 fish oil 2.00 2.00
      豆油 soybean oil 2.00 2.00
      玉米淀粉 corn starch 12.00 25.00
      微晶纤维素 microcrystalline cellulose 13.00 0.00
      磷酸二氢钙 Ca(H2PO4)2 1.80 1.80
      预混料 premix 1) 1.20 1.20
    营养成分 proximate composition (dry matter basis)
      水分/% moisture 6.96 6.93
      乙醚提取物/% ether extract 5.93 5.94
      粗灰分/% crude ash 8.46 8.18
      粗蛋白质/% crude protein 29.82 30.03
      粗纤维/% crude fiber 16.97 7.28
      可消化性糖/% digestible carbohydrate 2) 31.86 43.64
    总能/(MJ/kg) total energy 19.09 19.14
    注:① 预混料为每千克饲料提供以下矿物质(g/kg)和维生素(IU or mg/kg)包括 CuSO4·5H2O 2 g, FeSO4·7H2O 25 g, ZnSO4·7H2O 22 g, MnSO4·4H2O 7 g, Na2SeO3 0.04 g, KI 0.026 g, CoCl2·6H2O 0. 1 g, VA 900 000 IU, VD 200 000 IU, VE 4500 mg, VK3 220 mg, VB1 320 mg, VB2 1090 mg, VB5 2000 mg, VB6 500 mg, VB12 1.6 mg, VC 5000 mg, 泛酸 1000 mg, 叶酸 165 mg, 胆碱, 60 000 mg。② 可消化性糖含量 = 100- (水分+粗蛋白+乙醚提取物+粗灰分+粗纤维)
    Notes: ① premix supplied the following minerals (g/kg of diet) and vitamins (IU or mg/kg of diet): CuSO4·5H2O 2 g, FeSO4·7H2O 25 g, ZnSO4·7H2O 22 g, MnSO4·4H2O 7 g, Na2SeO3 0.04 g, KI 0.026 g, CoCl2·6H2O 0.1 g, vitamin A 900000 IU, vitamin D 200000 IU, vitamin E 4500 mg, vitamin K3 220 mg, vitamin B1 320 mg, vitamin B2 1090 mg, vitamin B5 2000 mg, vitamin B6 500 mg, vitamin B12 1.6 mg, vitamin C 5000 mg, pantothenate 1000 mg, folic acid 165 mg, choline 60 000 mg. ② digestible carbohydrate content = 100– (moisture+crude protein+ether extract+ash+crude fiber)

    Table 1.  Formulation and proximate composition of the different experimental diets

  • 对于葡糖糖负荷实验,将体质健壮的团头鲂幼鱼[(52.49 ± 1.02) g]随机分为2组,分别注射1.67 mL/kg体质量的生理盐水和1.67 g/kg体质量的葡萄糖溶液。将注射后的鱼随机放入6个循环养殖缸(水体容量为200 L),每缸4条鱼。随后,分别在0、1、2、4、8和12 h采集脑和肝脏组织用液氮迅速冷冻,放置于−80 °C备用。期间,每个时间点采集一个循环养殖缸,以降低反复采样对鱼体造成的应激。

  • 采用Trizol法(TaKaRa,宝生物公司,大连)对鱼体肝脏RNA进行提取。根据RNAiso Plus商业试剂盒说明书要求,将提取后的组织RNA分别进行结构完整性及浓度和纯度检测。随后,参照5′RACE System for Rapid Amplification of cDNA Ends,Version 2.0试剂盒(Invitrogen公司)操作说明,以总RNA为模板、Oligo(dT)16为反转录引物合成cDNA第一链。

  • 筛选NCBI网站中已发表的鱼类PGC1α基因的保守区域序列,使用Primer Premier 5.0设计合成简并引物(表2)并进行保守片段扩增。随后,根据SMARTer™ RACE cDNA Amplification (Clontech公司)试剂盒操作说明,分别使用引物PGC1α-F1,以团头鲂cDNA为模板并参照确定的扩增条件(94 °C 预变性 2 min;94 °C变性30 s,55 °C退火30 s,72 °C延伸2 min,进行34个循环;最后72 °C再延伸10 min),进行简并引物PCR的两轮扩增。第一轮扩增反应体系为ddH2O 31.5 μL,10×PCR buffer 5.0 μL,25 mmol/L MgCl2 3.0 μL,10 mmol/L dNTP mix 1.0 μL,PGC1α-R1 2.0 μL,Abridged Anchor Primer (10 μmol/L) 2.0 μL,dC-tailed cDNA 5.0 μL和Taq DNA 聚合酶(5 units/μL) 0.5 μL;第二轮扩增反应体系为第一轮扩增产物5.0 μL,ddH2O 33.5 μL,10×PCR buffer 5.0 μL,25 mmol/L MgCl2 3.0 μL,10 mmol/L dNTP mix 1.0 μL,PGC1α-R1 21.0 μL,AUAP or UAP (10 μmol/L) 1.0 μL和Taq DNA 聚合酶(5 units/μL) 0.5 μL。纯化后的PCR产物鉴定阳性克隆后进行测序并在NCBI网站验证核心片段。最后,使用DNA Star Package 5.01软件对PGC1α的正反序列进行组装,最终分别获得完整的团头鲂PGC1α基因cDNA片段序列。

    引物名称
    primer name
    序列(5→3)
    sequences (5→3)
    用途
    usage
    PGC1α-F1 TATGCCAACTCCTCCATCAACCCCA 中间片段扩增
    PGC1α-R1 AGGCAGGTCAGGGCAAAG 中间片段扩增
    PGC1α-F TGCCCTCGGTTCATTGTC qRT-PCR
    PGC1α-R GATTTCTGATTGGTCGCTGTA qRT-PCR
    EF1α-F CTTCTCAGGCTGACTGTGC qRT-PCR
    EF1α-R CCGCTAGCATTACCCTCC qRT-PCR
    注:PGC1α. 过氧化物酶体增殖物激活受体γ共激活因子1α;EF1α. 延长因子1α
    Notes:PGC1α. peroxisome proliferator-activated receptor γ coactivator 1α; EF1α. elongation factor 1α

    Table 2.  Primers used in the experiment

  • 根据NCBI的开放阅读框(ORF) Finder操作流程,寻找Pgc1α cDNA中的ORF。使用DNA MAN对Pgc1α氨基酸序列进行检测。ExPASy网站测定Pgc1α的等电点和分子量。NCBI网站(http://www.ncbi.nlm.nih.gov/gorf/gorf.html)检测Pgc1α的同源性基因。利用网站http://sable.cchmc.org/预测Pgc1α蛋白二级结构。MEGA 5.0对比Pgc1α相关物种的氨基酸序列。

  • 团头鲂脑和肝脏RNA提取及其目的基因PCR定量,均参照先前实验操作流程[7]。具体流程如下:RNA的提取过程严格参照TaKaRa® Plus RNA Purification (Invitrogen 货号:12 183-555)试剂盒说明书进行。同时,使用Prime Script® RT reageat Kit反转录试剂盒并按照说明书要求进行cDNA反转。随后,根据Takara SYBR® Premix Ex TaqTM试剂盒要求对目的基因进行荧光定量PCR检测,其20 μL反应体系分别包含cDNA稀释液 2 μL,SYBR® Premix Ex TaqTM 2 10 μL,ROX Reference Dye II 0.6 μL,上游引物 0.2 μL,下游引物 0.2 μL 和ddH2O 7 μL。最后,使用ABI Quantstudio™ DX实时定量PCR仪(Life Technologies 公司)进行荧光定量检测。期间,选择团头鲂内参引物EF1α (序列号:X77689.1)对目的基因(表2)的Ct值进行均一化处理,使用2−△△Ct[8]来计算组织样品中mRNA的相对表达量。

  • 利用SPSS 16.0软件并根据单因素方差分析要求对营养限制、高糖营养实验数据进行分析。同时,根据双因素方差分析要求对葡萄糖负荷实验数据进行分析;当存在显著差异(P<0.05)时,利用Turkey氏方法进行多重比较。

2.   结果
  • 实验获得2 566 bp的PGC1α基因片段序列,其中包含长度为1 404 bp的开放阅读框并其编码467个氨基酸(图1)。同时,Pgc1α蛋白分子量为53 375.53 U,等电点为6.56。转录因子结合位点分析结果显示,PGC1α序列包括MyOD、CREB和C/EBP等结合位点。氨基酸序列分析显示,N 端含有LXXLL 转录激活区,C端含有一个RNA识别区域和富含丝氨酸/精氨酸的结构域(SR)结构域。团头鲂Pgc1α蛋白结构中存在5个α-螺旋,6个β-折叠和12个转角(图2)。

    Figure 1.  Nucleotide sequence of PGC1α cDNA fragment and its encoding amino acid sequences in blunt snout bream

    Figure 2.  The secondary structure of the Pgc1α protein in blunt snout bream

  • 使用NCBI中的BLASTp 程序对团头鲂Pgc1α 氨基酸序列进行同源比对(图3),结果表明其与鲤科鱼类有很高的相似度。具体比对结果如下:PGC1α中草鱼96.79%、鲫(Carassius auratus) 87.58%、鲤 (Cyprinus carpio) 89.29%、黄颡鱼(Tachysurus fulvidraco) 71.97%、斑点叉尾鮰(Ictalurus punctatus) 71.95%、大西洋鲑(Salmo salar) 56.87%、大菱鲆(Scophthalmus maximus) 55.96%、银鲑(Oncorhynchus kisutch) 54.82%。使用MEGA 3.1软件将Pgc1α 氨基酸序列构建系统发育树,结果表明团头鲂Pgc1α序列与草鱼相似性最高并聚为一支,随后再与其他鲤科鱼类聚为一支。

    Figure 3.  Phylogenetic tree based on Pgc1α sequences by MEGA 3.1 software

  • 饥饿10 d组(10 d fasted group)的脑和肝脏中PGC1α基因表达量显著高于其他各组(P<0.05) (图4)。饥饿10 d后投喂1 h组(1 h-refed group)的脑和肝脏中PGC1α基因表达量与连续10 d投喂组(10 d-fed group) (P>0.05)无显著差异。

    Figure 4.  The mRNA levels of PGC1α in the brain (a) and liver (b) of blunt snout bream subjected to nutrient restriction

  • 经12周养殖实验后,高糖组(HC)鱼的脑和肝脏中PGC1α表达量均显著降低(P<0.05) (图5)。

    Figure 5.  The mRNA levels of PGC1α in the brain (a) and liver (b) of blunt snout bream subjected to different dietary carbohydrate levels

  • 葡萄糖负荷后,采样时间和注射剂量以及二者交互作用均显著影响脑和肝脏中PGC1α的表达(P<0.05) (图6)。葡萄糖负荷实验后,PGC1α表达量显著降低(P<0.05),最低值于1 h处出现。随后,它们的表达量均显著性上调(P<0.05),且在8 h回到基础值。生理盐水组,脑组织中PGC1α表达量随时间增加显著性升高(P<0.05)。就注射剂量而言,0 h处脑和肝脏中PGC1α的表达量及8 h肝脏中PGC1α的表达量均无显著差异(P>0.05)。除此之外,糖负荷组鱼脑和肝脏中PGC1α表达量均显著低于对照组(P<0.05)。

    Figure 6.  The mRNA levels of PGC1α in the brain (a) and liver (b) of blunt snout bream subjected to different glucose loadings

3.   讨论
  • PGC1α是过氧化物酶体增殖物激活受体γ辅激活因子1 基因家族成员之一,其在维持机体糖代谢稳态中发挥重要作用[3]。目前,关于PGC1α较为深入的研究主要集中于哺乳动物。本实验从团头鲂肝脏中克隆到PGC1α基因的cDNA片段序列。碱基及氨基酸序列比对结果显示,团头鲂PGC1α结构中包含多种与哺乳动物相同的结构位点,如N端LXXLL转录激活区、C端RNA识别区和SR结构域及MyOD、CREB、C/EBP等结合位点。在这些结构中,LXXLL转录激活区被认为是PGC1α与过氧化物酶体增殖物激活受体α (peroxisome proliferators-activated receptors α,PPARα)等细胞核受体转录因子相互作用的关键部位[9-10];SR结构域则包含多个磷酸化修饰位点可通过磷酸化和去磷酸化形式的转换,从而调控PGC1α蛋白与其下游蛋白间的相互作用[11]。然而,相较哺乳动物,团头鲂PGC1α基因结构上缺少与肌细胞增强因子2 C (myocyte enhancer factor 2 C,MEF2C)相互作用的结合位点,这与齐口裂腹鱼PGC1α基因上的研究结果相同[6]。这一改变被认为会降低PGC1α在调控鱼体糖代谢中的作用,由于PGC1α需通过结合并激活MEF2C来增加葡萄糖转运蛋白4 (glucose transporter type 4,GLUT4)的活性,进而增强机体的葡萄糖转运功能[12]。此外,同源性比对结果显示,团头鲂PGC1α基因与其他动物的该基因序列同源性较高,表明不同物种间PGC1α基因具有较高的保守性。

    营养限制实验中,饥饿10 d组(10 d fasted group)鱼的脑和肝脏中PGC1α基因表达量被显著性上调,但投喂1 h后它们呈现显著地下降趋势。先前的研究表明,饥饿条件下,小鼠(Mus musculus)下丘脑中PGC1α表达的升高,可以刺激刺鼠相关蛋白(agouti-related protein,AgRP)、神经肽Y (neuropeptide Y,NPY)等促食欲肽相关基因的表达,从而增强机体的摄食意识[13]。此外,游建华在HL-7702肝细胞株上的研究表明,过表达PGC1α可以激活肝细胞核因子4α (hepatocyte nuclear factor 4α,HNF4α),进而促进糖异生基因的表达和增加肝脏葡萄糖的输出,为机体提供能量[14]

    总的来讲,PGC1α与机体的糖脂代谢密切相关。研究表明,PGC1α是肝糖异生的关键调节因子,能够诱导糖异生关键酶的基因表达,导致肝糖异生增强,肝糖输出增加,进而升高空腹血糖水平[15]。此外,过表达小鼠肝脏中的PGC1可以激活过氧化物酶体增殖物激活受体α,增加脂肪酸的氧化,为机体提供能量[16]。本实验中,高糖组鱼的脑和肝脏中PGC1α基因表达量显著降低。这可能体现了团头鲂应对高糖营养的适应机制,由于PGC1α降低会抑制肝糖异生及脂肪酸氧化,从而有助于增强饲喂高糖鱼的代谢稳态。此外,饲喂高糖饲料致使鱼体血液中瘦素水平的升高,可能会进一步抑制脑和肝脏中PGC1α的表达[17]。然而,哺乳动物上的研究表明,PGC1α表达降低可能会抑制线粒体呼吸链酶及抗氧化酶的活性,导致活性氧(ROS)积聚,进而加剧细胞凋亡,降低代谢功能[18]

    本实验中,葡萄糖注射后团头鲂脑和肝脏PGC1α的表达量迅速发生变化,均在1~2 h降低至最小值。这与陈金虎等[19]在小鼠下丘脑腹内侧核神经元上的结果相似,研究显示糖负荷后腹内侧核PGC1α表达水平在2 h到达最低值。这可能归因于糖负荷后鱼体增加的胰岛素水平,由于胰岛素可以通过激活蛋白激酶B (protein kinase B,Pkb),致使Pgc1α磷酸化失活,进而降低 PGC1α表达水平[20-21]。随后,采样时间从2 h增至12 h,脑和肝脏中PGC1α表达量显著上调。这可能因为鱼体逐渐降低的胰岛素水平消弱了其对PGC1α表达的抑制能力。此外,上调的PGC1α表达水平进一步回应了2~12 h逐渐下降的鱼体血糖水平[22]。由于PGC1α能够通过激活Mef2c来诱导内源性GLUT4基因表达,增强外周组织摄取葡萄糖的能力,进而维持机体的血糖稳态[12]。此外,与葡萄糖注射组相比,生理盐水组鱼脑和肝脏中PGC1α的表达量随采样时间增加而逐渐升高。这可能是由于饥饿状态下,PGC1α的激活将会促进肝糖异生和脂肪酸氧化相关基因的表达,进而增加肝脏的葡萄糖输出及脂肪酸氧化能力,为机体短时间内提供能量[14,16]

4.   结论
  • 本研究从团头鲂肝脏中克隆得到了PGC1α基因片段序列,长2566 bp。营养限制、高糖营养实验发现,饥饿和饲喂高糖饲料均会显著性下调团头鲂脑和肝脏中PGC1α基因的表达量。此外,葡萄糖负荷后,脑和肝脏中PGC1α表达量显著降低,最低值出现在1~2 h处。以上结果可为研究PGC1α在鱼类糖代谢中的作用提供基础数据和理论依据。

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