Molecular cloning and expression pattern of 11 genes involved in lipid metabolism in yellow catfish Pelteobagrus fulvidraco

11 genes involved in lipid metabolism were cloned from liver of yellow catfish Pelteobagrus fulvidraco, including CPT 1A, CPT 1B, PPARα, PPARγ, SREBP-1, G6PD, 6PGD, FAS, acetyl-CoA ACCa, ACCb, and LPL. Phylogenetic analysis further identified these genes, and confirmed the classification and evolutionary status of yellow catfish. mRNA of all eleven genes was present in liver, muscle, mesenteric adipose, ovary and heart, but at varying levels. The present study will facilitate further studies on the regulation of lipid metabolism at the molecular level for the fish species.     

黄颡鱼溶酶体酸性脂肪酶基因的cDNA序列、启动子及其转录调控分析

通过分子结构、组织表达及启动子结构功能分析,探讨黄颡鱼(Pelteobagrus fulvidraco)溶酶体酸性脂肪酶(Lysosmal acid lipase,lal)基因的分子特征及转录调控,为lal在鱼类脂质代谢和转录水平的调控提供新的视野.实验采用RT-PCR和RACE克隆lal基因的cDNA全长序列,其长度...     

Dietary Lipid Levels Influence Lipid Deposition in the Liver of Large Yellow Croaker (Larimichthys crocea) by Regulating Lipoprotein Receptors,Fatty Acid Uptake and Triacylglycerol Synthesis and Catabolism at the Transcriptional Level

Ectopic lipid accumulation has been observed in fish fed a high-lipid diet. However, no information is available on the mechanism by which dietary lipid levels comprehensively regulate lipid transport, uptake, synthesis and catabolism in fish. Therefore, the present study aimed to gain further insight into how dietary lipids affect lipid deposition in the liver of large yellow croaker(Larimichthys crocea). Fish (150.00±4.95 g) were fed a diet with a low (6%), moderate (12%, the control diet) or high (18%) crude lipid content for 10 weeks. Growth performance, plasma biochemical indexes, lipid contents and gene expression related to lipid deposition, including lipoprotein assembly and clearance, fatty acid uptake and triacylglycerol synthesis and catabolism, were assessed. Growth performance was not significantly affected. However, the hepato-somatic and viscera-somatic indexes as well as plasma triacylglycerol, non-esterified fatty acids and LDL-cholesterol levels were significantly increased in fish fed the high-lipid diet. In the livers of fish fed the high-lipid diet, the expression of genes related to lipoprotein clearance (LDLR) and fatty acid uptake (FABP11) was significantly up-regulated, whereas the expression of genes involved in lipoprotein assembly (apoB100), triacylglycerol synthesis and catabolism (DGAT2, CPT I) was significantly down-regulated compared with fish fed the control diet, and hepatic lipid deposition increased. In fish fed the low-lipid diet, the expression of genes associated with lipoprotein assembly and clearance (apoB100, LDLR, LRP-1), fatty acid uptake (CD36, FATP1, FABP3) and triacylglycerol synthesis (FAS) was significantly increased, whereas the expression of triacylglycerol catabolism related genes (ATGL, CPT I) was reduced compared with fish fed the control diet. However, hepatic lipid content in fish fed the low-lipid diet decreased mainly due to low dietary lipid intake. In summary, findings of this study provide molecular insight into the role of lipid deposition in the liver in response to different dietary lipid contents.     

黄颡鱼对四种动物性蛋白原料在不同制粒工艺下的表观消化率研究

实验以三氧化二铬(Cr₂O₃)为外源指示剂,采用“70%基础饲料+30%实验原料”的方法配制实验饲料,测定了初始体质量为(28.68±0.49) g的黄颡鱼(Peltobagrus fulvidraco)对国产鱼粉、进口鱼粉、进口鸡肉粉和脱脂黄粉虫粉在膨化制粒和非膨化制粒两种工艺下的干物质、粗蛋白、粗脂肪和氨基酸表观消化率。结果显示,在非膨化制粒工艺下,黄颡鱼对进口鸡肉粉的干物质表观消化率显著高于另外3种原料(P<0.05),黄颡鱼对黄粉虫粉蛋白消化率最低(P<0.05),黄颡鱼对进口鸡肉粉脂肪表观消化率显著高于国产鱼粉和进口鱼粉(P<0.05); 在膨化制粒工艺下,进口鸡肉粉的干物质表观消化率显著低于另外3种原料(P<0.05),国产鱼粉和进口鱼粉的粗蛋白质消化率达94%以上,显著高于另外两种原料(P<0.05),但进口鸡肉粉的脂肪表观消化率显著低于其他原料(P<0.05)。在非膨化制粒工艺和膨化制粒工艺下国产鱼粉、进口鱼粉和黄粉虫粉的干物质表观消化率无显著性差异,但在非膨化制粒工艺下进口鸡肉粉的干物质表观消化率显著高于膨化制粒工艺(P<0.05)。对于国产鱼粉、进口鱼粉和黄粉虫粉而言,非膨化制粒工艺的蛋白消化率显著低于膨化制粒工艺(P<0.05),而鸡肉粉则相反。膨化加工工艺进口鱼粉的脂肪表观消化率显著高于非膨化加工工艺(P<0.05),而膨化加工工艺的黄粉虫脂肪表观消化率显著低于非膨化加工工艺(P<0.05)。氨基酸的消化率结果与粗蛋白的表观消化率变化趋势基本一致。由此可知,对于黄颡鱼饲料,国产鱼粉和进口鱼粉是最佳的蛋白质来源,进口鸡肉粉和黄粉虫亦可以作为其优质的蛋白质来源。对于进口鱼粉、国产鱼粉和黄粉虫粉,膨化制粒工艺更有利于黄颡鱼对其干物质、蛋白、脂肪和氨基酸等营养元素的消化利用,而对于进口鸡肉粉,非膨化制粒工艺更有利于黄颡鱼对营养元素的消化利用。     

氨氮胁迫下不同食性鱼类仔鱼抗氧化和非特异性免疫的差异响应

为探究氨氮胁迫对不同食性鱼类的影响, 研究选取不同食性的4种鱼类(鲢Hypophthalmichthys molitrix、草鱼Ctenopharyngodon idella、团头鲂Megalobrama amblycephala和黄颡鱼Pelteobagrus fulvidraco)仔鱼为研究对象, 探讨不同浓度氨氮(0、1、2和3 mg/L)短期胁迫(96h)对其生长、抗氧化和非特异性免疫响应的影响及其分子机制。结果显示: (1)氨氮胁迫导致4种仔鱼的体长生长速度呈现剂量依赖性减缓; (2)不同浓度氨氮导致4种仔鱼体内T-AOC、CAT和GPx含量显著降低(P<0.05), 2和3 mg/L氨氮暴露显著降低了草鱼、团头鲂和黄颡鱼仔鱼体内SOD活力(P<0.05), 仅检测到黄颡鱼gpx及鲢sod转录水平出现显著性下调(P<0.05); (3)在不同浓度氨氮胁迫下, 4种仔鱼相关免疫基因转录水平均呈现一定的上调, 仅鲢il1β转录水平显著下降(P<0.05), 相对地草鱼仔鱼LYZ含量显著性下降, 黄颡鱼仔鱼LYZ含量在2 mg/L氨氮组显著性上升(P<0.05)。双因素方差分析显示, 氨氮对所有抗氧化酶、免疫指标及免疫相关基因有显著影响, 不同种仔鱼之间T-AOC、CAT、GPx和C3及基因cuznsod、gpx、il1β和c3之间差异显著(P<0.05), 但鱼种和氨氮互作效应仅对C3和基因gpx、tnfα、il1β、c3影响显著(P<0.05)。研究表明, 高浓度氨氮急性胁迫引起仔鱼生长迟缓和氧化应激, 降低了其抗氧化性能, 削弱了其非特异免疫防御机能。相比较而言, 肉食性的黄颡鱼仔鱼对氨氮的耐受性较其他几种仔鱼弱, 草食性的团头鲂和草鱼仔鱼居中, 而鲢仔鱼对氨氮的耐受性较强。     

Genome-wide association study reveals the genetic basis of growth trait in yellow catfish with sexual size dimorphism

Sexual size dimorphism has been widely observed in a large number of animals including fish species. Genome-wide association study (GWAS) is a powerful tool to dissect the genetic basis of complex traits, whereas the sex-differences in the genomics of animal complex traits have been ignored in the GWAS analysis. Yellow catfish (Pelteobagrus fulvidraco) is an important aquaculture fish in China with significant sexual size dimorphism. In this study, GWAS was conducted to identify candidate SNPs and genes related to body length (BL) and body weight (BW) in 125 female yellow catfish from a breeding population. In total, one BL-related SNP and three BW-related SNPs were identified to be significantly associated with the traits. Besides, one of these SNPs (Chr15:19195072) was shared in both the BW and BL traits in female yellow catfish, which was further validated in 185 male individuals and located on the exon of stat5b gene. Transgenic yellow catfish and zebrafish that expressed yellow catfish stat5b showed increased growth rate and reduction of sexual size dimorphism. These results not only reveal the genetic basis of growth trait and sexual size dimorphism in fish species, but also provide useful information for the marker-assisted breeding in yellow catfish.     

Upstream regulators of apoptosis mediates methionine-induced changes of lipid metabolism

Although the role of methionine (Met), as precursor for l-carnitine synthesis, in the regulation of lipid metabolism has been explored. Met seems to have tissue- and species-specific regulatory effect on lipid metabolism, implying that the mechanisms in Met regulation of lipid metabolism is complex and may involve the upstream regulatory pathway of lipid metabolism. The present study was performed to determine the mechanism of apoptosis signaling pathways mediating Met-induced changes of hepatic lipid deposition and metabolism in fish, and compare the differences of the mechanisms between the fish and mammals. By iTRAQ-based quantitative proteome analyses, we found that both dietary Met deficiency and excess evoked apoptosis signaling pathways, increased hepatic lipid deposition and caused aberrant hepatic lipid metabolism of yellow catfish Pelteobagrus fulvidraco. Using primary hepatocytes from P. fulvidraco, inhibition of caspase by Z-VAD-FMK blocked the apoptotic signaling pathways with a concomitant reversal of Met deficiency- and excess-induced increase of lipid deposition, indicating that apoptosis involved the Met-mediated changes of hepatic lipid metabolism. Moreover, we explored the roles of three upstream apoptotic signaling pathways (PI3K/AKT-TOR pathway, cAMP/PKA/CREB pathway and LKB1/AMPK-FOXO pathway) influencing hepatic lipid metabolism of P. fulvidraco. The three upstream pathways participated in apoptosis mediating Met-induced changes of lipid metabolism in P. fulvidraco. At last, HepG2 cell line was used to compare the similarities of mechanisms in apoptosis mediating Met-induced changes of lipid metabolism between fish and mammals. Although several slight differences existed, apoptosis mediated the Met-induced changes of lipid metabolism between fish and mammals. The present study reveals novel apoptosis-relevant signal transduction axis which mediates the Met-induced changes of lipid metabolism, which will help understand the mechanistic link between apoptosis and lipid metabolism, and highlight the importance of the evolutionary conservative apoptosis signaling axis in regulating Met–induced changes of hepatic lipid metabolism.     

黄颡鱼母本的肠系膜脂肪沉积对繁育性能的影响

为探索肠系膜脂肪堆积对黄颡鱼母本繁育性能的影响, 选择了4个母本群体来评价肠系膜脂肪沉积与黄颡鱼繁育性能的关联: group 1, 野生黄颡鱼群体[肠系膜脂肪指数(0.56±0.17)%]; group 2, 肠系膜脂肪较少的黄颡鱼养殖群体[肠系膜脂肪指数(1.97±0.40) %]; group 3和4, 肠系膜脂肪较多的2个黄颡鱼养殖群体[肠系膜脂肪指数(5.92±1.85)%和(9.62±1.01)%]。研究结果显示肠系膜脂肪体重比与性腺指数总体上呈负相关。人工繁殖结果表明, group 1和group 2的产卵率、受精率和单尾母鱼出苗量无显著性差异, 但显著高于group 3和group 4; group 1和2的苗种畸形率显著低于group 3和4。黄颡鱼雌鱼血清中促黄体生成素(LH)和卵黄蛋白原(VTG)的水平随着肠系膜脂肪沉积量的增大而降低; 相反, 肝脏和卵子中的油脂和糖原含量随着肠系膜脂肪沉积量的增大而升高。研究发现, 肠系膜脂肪过度沉积的黄颡鱼母本的生理指标和繁殖性能明显下降, 生产出来的苗种质量欠佳; 建立完善的亲本培育模式并减少肠系膜脂肪沉积能够显著改善繁殖性能, 文章为提高鱼类苗种质量提供了思路。     

New insights into the mitochondrial carnitine palmitoyltransferase enzyme system

Dissection of the mitochondrial carnitine palmitoyltransferase (CPT) enzyme system in terms of its structure/function relationships has proved to be a formidable task. Although no one formulation has gained universal agreement we believe that the weight of evidence supports a model with the following features: a) in any given tissue CPT I and CPT II are distinct proteins; b) CPT I, unlike CPT II, is detergent labile; c) within a species CPT II is expressed body wide, whereas CPT I exists as tissue specific isoforms; d) malonyl-CoA and other CPT I inhibitors probably interact at the catalytic center of the enzyme, not with a regulatory subunit. The amino acid sequences of rat and human CPT II (deduced from cDNA clones) show them to be similar proteins (greater than 80% identity) but encoded by mRNAs of significantly different sizes. Efforts to clone and sequence the cDNA for rat liver CPT I are presently underway.     

Characterization and tissue expression of twelve selenoproteins in yellow catfish Pelteobagrus fulvidraco fed diets varying in oxidized fish oil and selenium levels

BackgroundSelenium (Se) functions through selenoproteins and is essential to growth and metabolism of vertebrates. The present study was conducted to identify twelve selenoproteins genes (selenoe, selenof, selenoh, selneoi, selenom, selenok, selneon, selenoo, selenot, selenos, selenou and msrb1) from yellow catfish. Their mRNA expression patterns, as well as their response to dietary oxidized fish oils and Se addition were explored.MethodsWe use 3′and 5′ RACE PCR to clone full-length cDNA sequence of twelve selenoprotein genes from yellow catfish. Their mRNA expression patterns were assessed via quantitative real-time PCR. Yellow catfish were fed diet adequate Se+ fresh fish oil, adequate Se+ oxidized fish oil, high Se+ fresh fish oil and high Se+ oxidized fish oil, respectively, for 10 weeks. Their kidney, heart, brain and testis were used to assess the mRNA expression of twelve selenoprotein.ResultsTwelve selenoprotein genes had similar domains with mammals and the other fish. Their mRNAs were expressed widely in eleven tissues but varied with the tissues. Dietary oxidized fish oils and Se addition influenced their mRNA abundances of twelve selenoproteins in a tissue-dependent manner.ConclusionOur study demonstrated the characterization and expression of twelve selenoproteins, and elucidated their responses in yellow catfish fed diets varying in oxidized fish oils and Se addition, which increased our knowledge into the biological function and regulatory mechanism of Se and selenoproteins in fish.     

灌喂氧化鱼油后黄颡鱼胃肠道黏膜黑色素细胞分化和黑色素合成途径基因的差异表达

以黄颡鱼(Pelteobagrus fulvidraco)为实验对象, 灌喂氧化鱼油、鱼油7d后, 提取胃肠道黏膜总RNA, 采用RNA-seq测序并做转录组分析, 分析了黑色素生物合成途径关键酶(酪氨酸酶)及其相关蛋白基因、黑素体运动的3个蛋白基因、α黑素细胞刺激激素途径和WNT/β-catenin、EDN3和EDNRB、KIT及其配体KITL3个信号通路的主要蛋白基因的差异表达活性。结果显示, 黄颡鱼胃肠道黏膜中存在黑色素细胞分化和发育过程、黑色素合成及其调控途径的代谢网络, 通过绘制代谢网络得到了关键性酶或蛋白质的基因信息。在灌喂氧化鱼油后, 控制黑色素合成途径主要基因的表达活性显著下调, 可能导致黑色素合成量的不足; α-MSH激素途径主要基因差异表达上调, 具备促进黑色素细胞分化和发育的调控基础; 而调控黑色素细胞分化和发育的3个信号通路主要基因也有差异表达。因此, 黄颡鱼受灌喂氧化鱼油的影响, 黑色素细胞分化和发育过程受到较大影响, 会影响到鱼体成熟的黑色素细胞的数量, 同时, 黑色素的生物合成量不足将导致引起黄颡鱼体色的变化。     

Molecular cloning and mRNA tissue expression of thyroid hormone receptors in yellow catfish Pelteobagrus fulvidraco and Javelin goby Synechogobius hasta

Thyroid hormones (THs) play a pivotal role in many physiological functions in vertebrates, including fish. Their effects are mediated by thyroid hormone receptors (TRs), which are members of the nuclear hormone receptor superfamily. In this study, full-length cDNA sequences of TRs from yellow catfish Pelteobagrus fulvidraco and Javelin goby Synechogobius hasta were cloned and their mRNA tissue expression profiles were determined. In P. fulvidraco, the validated cDNAs encoding for TRα and TRβ were 1789 and 1848 bp in length, encoding peptides of 401 and 378 amino acid residues, respectively. In addition, a TRβ spliced variant (named P. fulvidraco-TRβv), containing a 60-bp insertion, was detected. In S. hasta, cDNAs encoding for TRαA, TRαB and TRβ were 1827, 2295 and 2258 bp in length, encoding peptides of 401, 409 and 393 amino acid residues, respectively. The phylogenetic analysis revealed that TRα and TRβ cDNAs grouped into two separate clusters with other vertebrate counterparts and two TRα sequences grouped separately, suggesting that the two TRαs derived from paralogous genes that might arise during a teleost-specific genome duplication event. All TR mRNAs were detected in various tissues sampled. The mRNA levels of both TRα and TRβ from P. fulvidraco were the highest in brain, followed by liver, and lowest in heart, intestine, muscle, gill and spleen. However, in S. hasta, TRαA, TRαB and TRβ showed the highest mRNA levels in brain and lowest in muscle. Identification and mRNA tissue expression of TR genes from P. fulvidraco and S. hasta provide an initial step towards understanding their biological roles in the two fish species.     

Nutrient digestibility and intestinal enzyme activity of Clarias batrachus (Linn.) juveniles fed on dried fish and chicken viscera incorporated diets

A feeding trial was conducted for 56 days to study the effect of replacement of fish meal by dried fish and chicken viscera, and a combination of oil cakes, in the diet of Clarias batrachus juveniles. The nutritional values of these by-products were studied through a digestibility experiment. No significant difference in nutrient digestibility was observed in different diets. Even 19.59% lipid in the diet of catfish did not affect the nutrient digestibility. Both amylolytic and proteolytic enzymes in the intestine of juveniles were studied. A decreased protease activity due to replacement of animal protein by plant protein and a decreased (P < 0.01) aspartate aminotransferase (ASAT) activity could be observed after inclusion of 22% of dried fish viscera in the diet of the catfish. Though body lipid content increased in fish fed a high level of lipid, fat-free body composition did not vary among the fish fed on different diets.     

Growth and reproductive performance of the Asian catfish Clarias macrocephalus (Gunther) fed artificial diets

Four natural ingredient diets similar in nutrient composition (crude protein = 42–44%; P/E ratio = 115–120 mg/kcal) but different in protein sources, were formulated and fed to hatchery-reared catfish to measure the relative performance of the catfish fed alternative broodstock diets. The control feed was a combination of fish-by-catch and commercial fish pellets. In trial I, growth of the catfish was slow over a 36-week period, but some fish became gravid. Diets 1, 2, and 3 and the control feed were tested in trial II. Growth of fish did not differ significantly (P > 0.05) and female fish in all treatments became gravid. For fish induced to spawn from April to August (1994), hatching rate showed significant differences among treatments (P < 0.05). Relative fecundity, fertilization and hatching rates, and production of 3-day-old larvae were significantly different among fish induced to spawn in November (1994) when another incubation setup was used. Among the diets, diets 2 and 3 best enhanced reproductive performance of the catfish.     

Effects of 2-methylisoborneol (MIB), and ethanol on the expression and activity of cytochrome P450s from the channel catfish

Injection of 1 mg kg⁻¹ of 2-methylisoborneol (MIB) caused an increase in a CYP1A-like protein of approximately 56 kDa from the kidneys of juvenile channel catfish as determined by western blot analysis using antibodies raised against trout CYP1A1. Ethoxyresorufin O-deethylase activity from kidneys of MIB-injected fish was significantly elevated as compared to ethanol-injected controls. Static exposure of juvenile channel catfish to 10 ppm MIB caused a statistically significant induction of a CYP2K-like protein of approximately 53 kDa from the livers of treated catfish as determined by western blot analysis using antibodies raised against trout CYP2K1. Treatment of juvenile channel catfish with ethanol (1 ml kg⁻¹) reduced the expression of one kidney and two constitutive liver P450s, while increasing another kidney form. There was no difference in carbon tetrachloride-induced lipid peroxidation in livers after ethanol treatment. Thus, MIB and ethanol affect the expression of at least three P450 isoforms in channel catfish tissues.     

Porcine α1,3-galactosyltransferase: tissue-specific and regulated expression of splicing isoforms

Expression of the Galα_1,3 Gal epitope on membrane glycolipids and glycoproteins is known to vary widely from one tissue to another. In the course of studying the mechanisms underlying this variability, we have isolated from pig cDNA four sequences corresponding to four isoforms of α_1,3-galactosyltransferase (α_1,3GT), the Golgi enzyme that links galactose in α_1,3 on the galactose residue of N-acetyllactosamine. The isoforms differ from each other in the alternative presence of two nucleotide stretches of 36 and 63 base pairs in a segment encoding the stem region of the protein. Stable expression experiments show that all four isoenzymes can confer α-galactosyltransferase activity to HeLa cells, and that they are all located within the Golgi compartment, indicating that variations in length in the stem region do not affect enzyme activity or cellular localization. Analysis of RNA from different pig organs and cells shows quantitative differences between tissues in levels of α_1,3GT, as well as qualitative differences, the four isoforms being unequally represented in different tissues.     

Inter-tissue and inter-species characteristics of the mitochondrial carnitine palmitoyltransferase enzyme system

Properties of the carnitine palmitoyltransferase (EC 2.3.1.21) (CPT) enzyme system were compared in isolated mitochondria from a range of tissues in rodents, monkey, and man. Common features were as follows: (a) while membrane-bound, CPT I, but not CPT II, was inhibited reversibly by malonyl-coenzyme A (CoA) and irreversibly by CoA esters of certain oxirane carboxylic acids; (b) the detergent, Tween-20, readily solubilized CPT II in active form while leaving CPT I membrane associated and catalytically functional; (c) octyl glucoside and Triton X-100 released active CPT II but caused essentially complete loss of CPT I activity. Use of [3H]tetradecylglycidyl-CoA, a covalent ligand for CPT I, yielded estimates of the enzyme's monomeric molecular size: approximately 86 kDa in non-hepatic tissues and approximately 90-94 kDa in liver, depending upon species. A polyclonal antibody to purified rat liver CPT II recognized a single protein in each tissue; its apparent molecular mass was approximately 70 kDa in all rat tissues and approximately 68 kDa in all mouse tissues as well as monkey and human liver. On Northern blot analysis a rat liver CPT II cDNA probe detected a single approximately 2.5-kilobase mRNA in all rat and mouse tissues examined. The following points are emphasized. First, CPT I and II are different proteins. Second, within a species CPT II, but not CPT I, is probably conserved across tissue lines. Third, slight variations in size of both enzymes were found in different species, although, at least in the case of CPT II, significant amino acid identity exists among the various isoforms. Fourth, CPT I, unlike CPT II, requires membrane integrity for catalytic function. Finally, the strategic use of detergents provides a simple means of discriminating between the two enzyme activities.