Sumoylation And Its Regulatory Mechanism In Carbohydrate Influencing Lipid Metabolism Of Yellow Catfish Pelteobagrus Fulvidraco

SUMOylation(small ubiquitin-related modifier,SUMO)is a kind of important post-translational modification.It is characterized by SUMO proteins which covalently binds to the ?-amino group of lysine residues in intracellular target proteins in an ATP-dependent process.SUMOylation plays a central role in cell homeostasis by regulating a large number of key biological processes such as signal transduction,nucleoplasm transport,transcription regulation,DNA repair and cell cycle.Generally speaking,compared to mammals,fish show the capacity of utilization for carbohydrate at a low level.However,appropriate dietary carbohydrate partially replaced the protein utilization for fish growth.Yet,the relation between SUMOylation and dietary carbohydrate remains unknown in teleost.Carbohydrate-mediated the SUMOylation of lipid metabolism-related genes regulate hepatic lipid metabolism in fish remained unknown.What is the potential mechanism? Using yellow catfish as experimental animal model,we first isolated and identified the c DNA sequences of 10 SUMOylation-related genes involved in SUMOylation and de SUMOylation.Then examine their molecular characterization,tissue distribution,and first understanding the effect of different dietary carbohydrate to SUMOylation in yellow catfish in vivo and in vitro.Finally,based on the SUMOylation of SREBP1,the mechanism of glucose-mediated SUMOylation of SREBP1 to regulate hepatic lipid metabolism was explored.Results are as follows:1 Molecular characterization and function analysis for the SUMOylation-related genes from yellow catfishThe c DNA sequences of 10 SUMOylation-related genes involved in SUMOylation and de SUMOylation(sumo1? sumo2? sumo3? sae1? uba2? ubc9? pias1? senp1?senp2?senp3)were successfully obtained by RT-PCR and RACE methods from P.fulvidraco.In this study,as a preliminary step to unravel the physiological role of the SUMOylation-related genes,we determined the tissue distribution patterns of these genes.Their m RNAs were widely expressed in various tissues,but at variable levels,indicating expression of the SUMOylation-related genes had tissue specificity and species specificity.All of these members shared similar domain structure to their orthologous genes of vertebrates.Phylogenetic analysis further identified these genes,and confirmed their classification.These results provide evidence that the SUMOylation process can also be fully reconstituted in fish,which opens the possibility to further explore the physiological function of SUMOylation and the mechanism of induction,regulation and execution of SUMOylation in fish.2 The effects of carbohydrate addition on the transcriptional responses to SUMOylation-related genes in yellow catfishThe effects of carbohydrate addition on the transcriptional responses to SUMOylation-related genes were investigated.The experiment was divided into two parts.In Exp.1 was conducted to determinate their transcriptional responses to different levels(15%,21%,27%)of dietary carbohydrate.Exp.2 was taken to determine the effect of glucose incubation(control,10 m M,20 m M)on m RNA expression of these genes in primary hepatocytes from yellow catfish.Our study indicated that dietary carbohydrate levels differentially influenced their m RNA expression in a dose-dependent manner,and glucose incubation differentially influence their m RNA expression in dose-ways.It is understandable that the carbohydrate-induced changes of these gene expression will in turn influence the utilization and metabolism of carbohydrate in fish,which would contribute to our understanding of the molecular basis for SUMOylation functions of carbohydrate.3 Glucose-mediated SUMOylation of SREBP1 regulates hepatic lipid metabolism in the liver of yellow catfishOur previous study indicated that dietary carbohydrate levels differentially influenced the SUMOylation-related genes m RNA expression in a dose-dependent manner in the liver of yellow catfish.In mammals,carbohydrates are widely involved in the regulation of lipid metabolism-related transcription factors and genes,and many transcription factors are substrate proteins which modified by SUMOylation.Does carbohydrate-mediated the SUMOylation of lipid metabolism-related transcription factors regulate the hepatic lipid metabolism of yellow catfish? Glucose incubation(control,15 m M,30 m M)primary hepatocytes from yellow catfish for 48 hours,then we examined the accumulation of TG,the expression lipid metabolism related genes(chrebp,pparr,lxr?,srebp1,acc?,fas,scd1,lpl),the enzyme activities of synthesis-related enzymes(G6PD,6PGD,ICDH,ME,FAS),the protein levels of lipid metabolism related transcription factors or genes,and the SUMOylation level of lipid metabolism related transcription factors.The results show that glucose promoted the accumulation of TG in primary hepatocytes of yellow catfish,and this process mainly achieved by up-regulated the enzyme activities of synthesis-related enzymes and some other genes expressions.The SUMOylation level of SREBP1 was detected,and glucose reduced the SUMOylation level of SREBP1 and promoted the protein expression of SREBP1 and its target gene SCD1.We elucidated the roles and mechanisms of SUMOylation in the regulation of carbohydrate on hepatic fat deposition and lipid metabolism.These results extend our understanding into nutrition metabolism and physiological function of carbohydrate,and provides novel insights on the mechanism and prevention of fatty liver disease in fish.