Cloning, Expression, Kinetic Properties Of CPT I And Response To Zinc In Synechogobius Hasta And Pelteobagrus Fulvidraco

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.