【作者】 郑家浪；

【导师】 罗智；

【作者基本信息】 华中农业大学 ， 水产养殖， 2014， 博士

【摘要】 肉碱棕榈酰转移酶I (CPT I)是线粒体脂肪酸β-氧化的一种“限速酶”,它在脊椎动物中控制长链脂肪酸的β氧化,在降解脂肪过程中发挥着重要作用。近年来,哺乳动物中对于CPT I基因的结构和功能的研究取得了一定程度的进展。但是却很少有对鱼类CPT I基因研究和酶学性质的相关报道,也没有任何研究涉及外源营养素对鱼类CPT I基因表达和动力学性质的影响。锌是鱼类一种必需的微量元素,它的存在维系着鱼类生理发育的不同功能。迄今大量的学者研究了锌对鱼类生长、金属富集、氧化应激和组织病变等方面的影响,但是很少有学者关注锌对鱼类脂代谢的影响。本论文克隆了矛尾复虾虎鱼(Javelin goby, Synechogobius hasta)和黄颡鱼(yellow catfish, Pelteobagrus fulvidraco) CPT I的全长cDNA序列,并对其结构、进化、表达和动力学性质进行了研究。同时从CPT I的基因表达和动力学的角度探讨了水体和饲料锌水平对CPT I的影响机制。论文的主要研究结果和结论如下：1矛尾复虾虎鱼CPT I的克隆与动力学性质的研究在脊椎动物中,CPT I的调节在控制脂肪酸代谢方面起着重要的作用。在当前的研究中,我们克隆了虾虎鱼CPT I的cDNA全序列,分别为CPT Iα1a-1a, CPT I ala-1b、CPT Iala-1c、CPTIα1a-2、CPTIa2a、CPTIα2b1a、CPTIα2b1b和CPT Iβ。进化分析显示：CPT I基因的重复形成CPT I a=α和CPTIβ, CPT I α基因的重复形成CPT I α1和CPTIα2, CPTIα2基因进一步重复形成CPT Iα2a和CPT Iα2b, CPT Iα2b基因重复导致CPT Iα2b1a和CPT Iα2b1b的产生。CPT Iα1a基因的选择性剪切形成4种CPT I剪切突变体,分别为CPT Iαla-1a、CPT Iα1a-lb、CPT Iα1a-lc和CPT Iα1a-2。CPT I氨基酸序列比对和跨膜域分析表明虾虎鱼出现了关键氨基酸的替换和跨膜域的变化。关键氨基酸的替换和跨膜域的改变可能影响CPT I对丙二酸单酰辅酶A (M-CoA)的敏感性或者CPT I的催化活性,赋予鱼类CPT I新的酶学性质。为了深入了解虾虎鱼CPT I酶学性质,我们分离了肝脏、心脏、肌肉、肠道和脾脏的线粒体,然后采用酶动力学的方法测定了Michaelis-Menten常数Km和最大反应速率Vmax。结果显示：CPT I适宜的反应温度是34-40℃,最佳pH是7.4,适宜的孵育时间是5-25min,适宜的线粒体蛋白浓度为120μg/ml。虾虎鱼CPTI的动力学分析符合Michaelis-Menten规律。肉碱Km值在肌肉中最高,在其它组织中没有显著差异。棕榈酰辅酶A (P-CoA)的Km值在肝脏中最高,在肌肉中最低,在其它组织中没有显著差异。催化效率(Vmax/Km)在心脏中最高,在肝脏中最低。综上所述,我们的结果表明了不同组织中的CPT I动力学的差异可能反映了脂肪酸氧化的能力。虾虎鱼肝脏最低的CPT I催化效率可能是虾虎鱼易形成“脂肪肝“的一种重要原因。2黄颡鱼CPT I的克隆与表达的研究在当前的研究中,采用逆转录PCR和快速扩增cDNA末端(RACE)的方法,我们成功克隆得到了黄颡鱼CPT14种构型的cDNA全序列。根据进化关系,我们命名为CPT Iα1b、CPT Iα1a、CPT Iα2a和CPT Iβ。CPTI的进化分析暗示黄颡鱼CPT Iα1a和CPT Ia1b的产生可能是由于CPT Ia1基因的倍增。CPT Iα2仅存在鱼类中,可能是CPT Iα的一种亚家族。由于哺乳动物仅有一个单拷贝的CPT Iα基因,因此我们的结果暗示黄颡鱼多重的CPT I基因拷贝可能源于鱼类特异性的基因组倍增事件。另外,我们将黄颡鱼CPT I的氨基酸序列和其它物种进行比对,结果表明黄颡鱼出现了关键氨基酸的替换,例如Asp17, Val19, Ser24和Ala275。这些氨基酸同样出现在其它鱼类中,显示了和哺乳动物巨大差异。我们进一步采用Tmpred软件分析了黄颡鱼CPT I的跨膜域,结果显示CPT Iα1a和CPT Iαlb的氮端比哺乳动物CPT Iα的更短,但是两个跨膜域之间的环状结构比哺乳动物CPT la的更长。关键氨基酸的替换和跨膜域的改变可能影响CPT I对M-CoA的敏感性或者CPT I的催化活性,赋予鱼类CPT I新的酶学性质。同时,我们通过荧光定量的方法分析了黄颡鱼4种CPTI构型在肝脏、肌肉、心脏、肠道、鳃、脑、脾脏和肾脏中的mRNA表达水平。结果显示,4种构型均在上述组织中表达,但是有量的差异。CPT Iαla和CPTIβ倾向于在心脏和肌肉中表达,CPT Iαlb和CPT Iα2a的mRNA水平分别在肝脏和鳃中表达最高。我们进一步通过相同的方法检测了4种CPT I构型在黄颡鱼仔鱼,幼鱼和成鱼肝脏、心脏和肌肉中的表达水平。结果显示构型的转变贯穿于黄颡鱼的整个发育阶段。4种构型的共表达和发育的构型转换暗示了鱼类更加复杂的脂肪的利用,可能有助于不同组织中脂肪酸氧化的精准控制。3黄颡鱼CPT I动力学性质的研究为了深入了解黄颡鱼CPT I动力学性质,我们分离了肝脏、心脏、肌肉、肠道和脾脏的线粒体,然后采用酶动力学的方法测定了Michaelis-Menten常数Km和最大反应速率Vmax。结果显示：CPT I的最适的反应温度是36℃,最佳pH是8,适宜的孵育时间是3-20min,适宜的线粒体蛋白浓度为25-200μg/ml。黄颡鱼CPT Ⅰ的动力学分析符合Michaelis-Menten规律。肉碱Km值在肠道中最高,在脾脏中最低。反之,P-CoA的Km值在脾脏中最高,在肠道中最低。肝脏和心脏具有最大的Vmax值,肌肉和脾脏具有最低的Vmax值。相似地,催化效率(Vmax/Km)在肝脏和心脏中最高,在肌肉中最低。另外,不同组织中的CPT I酶活显示了和Vmax相似的趋势。综上所述,我们的结果表明不同组织中的CPT I动力学的差异可能反映了脂肪酸氧化的能力。在另一方面,与哺乳动物相比,黄颡鱼不同组织中Km差异显得很微弱,可能暗示物种的特异性。4水体慢性和急性锌暴露对黄颡鱼脂肪沉积、肉碱组成、CPT I的表达及其动力学性质的影响本实验是在Zn2+浓度分别为0.05mgl-1(对照)、0.35mgl-1和0.86mg l-1的水体中对黄颡鱼进行为期64d的慢性暴露实验,和在zn2+浓度分别为0.05mgl-1(对照)和4.71mgl-1的水体中对黄颡鱼进行为期96h的急性暴露实验,然后研究了水体慢性和急性锌暴露对黄颡鱼生长、脂肪沉积、肉碱组成、CPT Ⅰ的表达及其动力学性质的影响。慢性和急性锌暴露对黄颡鱼生长有抑制作用。慢性锌暴露显著增加了肝脏脂肪的含量(P<0.05),相反,急性锌暴露显著减少了肝脏脂肪的含量(P<0.05),无论是慢性还是急性锌暴露都显著减少了肌肉脂肪的含量(P<0.05)。慢性锌暴露减少了肝脏Michaelis-Menten常数Km和最大反应速率Vmax (P<0.05)但是在肌肉中两参数呈现相反趋势(Km减少,Vmax增加)。慢性锌暴露也显著影响了肝脏游离肉碱(FC),总肉碱(TC)和酰基肉碱的含量(AC),但并不影响肌肉各种肉碱组成的含量。慢性和急性锌暴露也影响了黄颡鱼肝脏和肌肉4种CPT I构型的表达,即CPT Ialb、CPT Ib、CPT Ia2a和CPT Ia。进一步分析,我们发现了构型的表达和Km,以及和Vmax呈现了很好的相关性。总之,慢性和急性锌暴露差异地影响了组织脂肪的沉积,CPT I的动力学性质及其表达水平。该研究提供了锌暴露影响脂代谢的一种新机制,也加深了我们对鱼类锌毒理的认识。5饲料锌缺乏和过量对黄颡鱼脂肪沉积、肉碱组成、CPT I的表达及其动力学性质的影响黄颡鱼分别投喂三种不同锌水平的饲料20mg kg11(对照),11.45mg kg-1(缺乏)和155mg kg-1(过量),实验周期为8周,然后研究了饲料锌缺乏和过量对黄颡鱼生长、脂肪沉积、肉碱组成、CPT I的表达及其动力学性质的影响。结果显示：锌水平不能显著影响黄颡鱼生长(P>0.05),锌缺乏倾向增加肝脏和肌肉脂肪的含量(P>0.05),但是过量锌显著减少了肝脏和肌肉的脂肪含量(P<0.05)。在肝脏中,锌缺乏显著增加了FC, AC和TC的含量(P<0.05),但是并不能显著影响FC/TC和AC/FC的比率(P>0.05)。锌过量也显著增加了TC和AC的含量,但是AC/FC值增加,FC的含量和FC/TC值减少。在肌肉中,锌缺乏增加了FC的含量,但是锌过量减少了FC的含量。相似地,锌缺乏和过量差异地影响了AC/FC和FC/TC的值。锌缺乏减少了肝脏和肌肉Km和Vmax值,但是锌过量增加Km和Vmax值。锌缺乏和过量也影响了黄颡鱼肝脏和肌肉4种CPT I构型的表达,即CPT Ialb, CPT Ib, CPT Ia2a和CPT Ia。进一步分析,我们发现构型的表达和Km,以及和Vmax呈现了很好的相关性。总之,锌缺乏和过量差异地影响了组织脂肪的沉积、CPT I的动力学性质及其表达水平,该研究为鱼类锌的营养和毒理提供了一种新的观点。更多还原

【Abstract】 Carnitine palmitoyltransferase I (CPT I) is frequently described as the’rate-limiting enzyme’of mitochondrial fatty acid β-oxidation. It controls the β-oxidation of long-chain fatty acid in vertebrate and plays an important part in the degradation of fat. The structure and function of CPT I have been studied in details in mammals. However, up to date, only limited information is available on CPT I gene and characterization in fish. Zinc is an essential micronutrient required for various biological processes. The impact of Zn exposure on growth, survival, histological changes in the gills and liver, metal bioaccumulation and the production of reactive oxygen species in fish has attracted wide attention. However, the underlying mechanism involved in the change of lipid metabolism as a response to Zn is poorly known in fish. In the present study, we describe the cloning, molecular characterization, phylogenetic analysis and the tissue expression profile of CPT I in Javelin goby Synechogobius hasta and yellow catfish Pelteobagrus fulvidraco, and investigate kinetic parameters of CPT I in various tissues. Moreover, we partially elucidate effects of waterborne and dietary Zn on CPT I expression and kinetic property.1Cloning and kinetics of CPT I from Javelin goby Synechogobius hastaThe regulation of CPT I is critical in the control of fatty acid metabolism in vertebrates. In the present study, we clone seven complete CPT I cDNA squences (CPT I ala-la, CPT I ala-lb, CPT I ala-lc, CPT I ala-2, CPT I a2a, CPT I a2bla, CPT I β) and a partial cDNA sequence (CPT I a2blb) from S. hasta. Phylogenetic analysis shows there are at least four CPT I duplications in S. hasta, CPT I duplication resulting in CPT I a and CPT Iβ, CPT I a duplication producing CPT I α1and CPT Iα2, CPT Iα2duplication generating CPT I a2a and CPT Iα2b, and CPT I α2b duplication creating CPT I a2bla and CPT Iα2b1b. Alternative splicing of CPT Iα1a results in the generation of four CPT I isoforms, CPT I ala-la, CPT I ala-lb, CPT I ala-lc and CPT Iα1a-2. CPT I ala-la and CPT I α1a-2both contain complete amino acid sequences and mutually exclusive alternative exons numbered8but CPT I α1a-1a terminates earlier than CPT I α-2in the3’UTR. Premature mRNA of CPT I α-1b and CPT I α1a-1c can be as a consequence of alternative polyadenylation signal and terminal exon. The key amino acid substitutions and modifications, and a functional motifs analysis reveal an intensely diversified CPT I gene family in fish, especially for CPT Ia2b isoform. These results may affect CPT I sensitivity to malonyl-CoA and/or catalytic activity in hasta compared to mammals.In order to get knowledge of kinetic properties of CPT I from yellow catfish, mitochondria from five different tissues (including liver, heart, spleen, intestine and muscle) were isolated. Then CPT I kinetic parameters (Michaelis-Menten constants, Km; maximal reaction rates, Vmax) were measured with substrate concentrations for carnitine varied from0.5mM to10mM, and for palmitoyl-CoA from0.02to0.6mM. Results showed that:CPT I optimum conditions were34-40℃and pH=7.4, the optical incubation time was5to25min, the optical mitochondria protein concentration was120ug/ml. Kinetic analysis revealed CPT I has a Michaelis-Menten behavior. The Km for carnitine was highest in the muscle, and no significant differences in the other tissues. The Km for palmitoyl-CoA was highest in the liver and lowest in the heart. The Vmax of CPT I was obtained in the heart, followed by the liver, the intestine and spleen, and the muscle had the lowest Vmax. The catalytic efficiency (Vmax/Km value) for carnitine and palmitoyl-CoA was highest in the heart, and the lowest in the liver. Thus, our results suggested the differences in CPT I kinetics parameters among various tissues may be a symbol of different capacities of fatty acid oxidation.2Cloning and expression of CPT I from yellow catfish Pelteobagrus fulvidracoIn the present study, we cloned the complete cDNA sequences of four CPT I gene isoforms from yellow catfish by RT-PCR and rapid amplification of cDNA ends (RACE) approaches, named as CPT Iα1b, CPT Iα1a, CPT Ia2a and CPT Iβ, respectively. Phylogenetic analysis indicated the generation of CPT Iα1a and CPT Iα1b may be due to duplication of CPT Ial. CPT Ia2exists only in fish and may be a subfamily of CPT Iα. Considering there is one copy of CPT Ia gene in mammals, our study indicated that genome duplication events had diversified the CPT I gene family in yellow catfish. Further, alignment of CPT I amino acid sequences from yellow catfish to those from mammals and other fish suggested some important substitutions occur at Asp17, Val19, Ser24and Ala275in yellow catfish. And TMpred analysis indicated the NH2terminus of CPT Ial is shorter and the loop between the first and second TMD is longer in yellow catfish CPT Ial isoform than those in mammalian CPT Iα and β. Both results may affect CPT I sensitivity to malonyl-CoA and/or catalytic activity in yellow catfish compared to mammals.The tissue-specific expression of four CPT I isoforms was determined via real-time qPCR in yellow catfish across liver, muscle, heart, intestine, gill, brain, spleen and kidney Four isoforms were present in all the tested tissues, but at varying levels. CPT Iα1a and CPT Iβ were expressed preferentially in heart and muscle. CPT Iαlb and CPT Iα2a mRNA expression levels were highest in liver and gill, respectively. Developmental expression of four CPT I isoforms was also detected across liver, muscle and heart in larva, juvenile and adult yellow catfish. The result suggested isoform switch occurred during development of yellow catfish. The co-expression of four CPT I isoforms and developmental isoform switch indicate more complex pathways of lipid utilization in fish than in mammals, allowing for precise control of lipid oxidation in individual tissue.3Study on kinetic property of CPT I from yellow catfish Pelteobagrus fulvidracoIn order to get knowledge of kinetic properties of CPT I from yellow catfish, mitochondria from five different tissues (including liver, heart, spleen, intestine and muscle) were isolated. Then CPT I kinetic parameters (Michaelis-Menten constants, Km; maximal reaction rates, Vmax) were measured with substrate concentrations for carnitine varied from0.5mM to10mM, and for palmitoyl-CoA from0.02to0.6mM. Results showed that: CPT I optimum conditions were36℃and pH=8.5, the optical incubation time was10to20min, the optical mitochondria protein concentration was44-176μg/ml. Kinetic analysis revealed CPT I has a Michaelis-Menten behavior. The Km for carnitine was highest in the intestine and lowest in the spleen. The Km for palmitoyl-CoA was highest in the spleen and lowest in the intestine. The Vmax of CPT I was obtained in the liver and heart, followed by the intestine, while muscle and spleen had the lowest Vmax. CPT I activities in yellow catfish in different tissues followed a similar trend with the Vmax. The catalytic efficiency (Vmax/Km value) for carnitine and palmitoyl-CoA was highest in the heart and liver, and lowest in the muscle. Thus, our results suggested the differences in CPT I kinetics parameters among various tissues may be a symbol of different capacities of fatty acid oxidation. On the other hand, these differences across tissues in yellow catfish were thinner than those in mammals, indicating a species specificity.4Effects of the chronic and acute zinc exposure on lipid content, carnitine composition, kinetics and expression of CPT I in yellow catfish Pelteobagrus fulvidracoThe present study is conducted to determine the effect of acute and chronic zinc exposure on carnitine concentration, CPT I kinetics, and expression levels of CPT I isoforms in liver and muscle of yellow catfish. To this end, yellow catfish are subjected to chronic waterborne Zn exposure (0.05mg Zn I-1,0.35mg Zn I-1and0.86mg Zn I-2, respectively) for8weeks and acute Zn exposure (0.05mg Zn I-1and4.71mg I-1Zn, respectively) for96h, respectively. Chronic Zn exposure increased remarkably lipid content in liver while acute Zn exposure decreased significantly hepatic lipid content (P<0.05). In muscle, lipid content declined significantly by both chronic and acute Zn exposure (P<0.05). In the chronic Zn exposure, Michaelis-Menten constants (Km) and maximal reaction rates (Vmax) values were reduced in liver while Km decreased and Vmax increased in muscle (P<0.05). Contrary to the chronic Zn exposure, the acute Zn exposure increased significantly Vmax in liver and Km in muscle (P<0.05), and did not affect Km in liver and Vmax in muscle (P>0.05). Chronic Zn exposure also significantly influences the contents of FC, TC and AC in liver, but not in muscle. The acute Zn exposure significantly increases FC, AC, TC contents in liver and muscle. The chronic and acute Zn exposure also influenced the mRNA levels of four CPT I isoforms (CPT Ialb, CPT Ib, CPT Ia2a and CPT Iala) in liver and muscle. Furthermore, correlations are observed in the mRNA levels between CPT I isoforms and Km, and between isoforms expression and CPT I Vmax. Thus, chronic and acute Zn exposure shows differential effects on carnitine content, CPT I kinetics and mRNA levels of four CPT I isoforms in yellow catfish, which provides new mechanism for Zn exposure on lipid metabolism and also novel insights into Zn toxicity in fish.5Effects of dietary Zn deficiency and excess on lipid content, carnitine composition, kinetics and expression of CPT I in yellow catfish Pelteobagrus fulvidracoThe present study was conducted to determine the effect of dietary Zn deficiency and excess on carnitine status, kinetics and expression of CPT I in the liver and muscle of yellow catfish. Yellow catfish were subjected to20(adequate Zn),11.45(Zn deficiency) and155(Zn excess) mg kg-1diet for8weeks. Zn deficiency tended to reduce lipid accumulation in liver and muscle (P>0.05) while Zn excess significantly induced lipid depletion in both tissues (P<0.05). In the liver, Zn deficiency increased FC, AC and TC contents (P<0.05), and did not significantly affect the ratios of FC/TC and AC/FC (P>0.05). Similarly, Zn excess also increased TC and AC contents, and AC/FC ratios, but reduced FC content and FC/TC ratio. In the muscle, FC content was promoted by Zn deficiency and inhibited by Zn excess. FC/TC ratio was stimulated by Zn deficiency and inhibited by Zn excess. In contrast, AC/FC ratio was reduced by Zn deficiency and induced by Zn excess. Zn deficiency also reduced Km and Vmax values while Zn excess increased them in the liver and muscle. Zn deficiency and excess influenced the expression levels of four CPTI isoforms, such as CPT Ialb, CPT Iβ, CPT Iα2a and CPT la la in the liver and muscle. Furthermore, some correlations were observed between the expression levels of CPT I isoforms and Km for carnitine, and between CPT I isoform expression and CPT I Vmax. Thus, for the first time, our study indicated that Zn deficiency and excess showed differential effects on carnitine status, kinetics and expression of CPT I in yellow catfish, which helped provide some novel insights into Zn nutrition and toxicology in fish.更多还原