Nutritional Regulation Of Lipolysis In Oreochromis Niloticus

Excessive fat accumulation has been observed in some farmed fish,and this has caused by high energy density(fat or carbohydrate)in the diet,unbalanced nutrient composition,and improper dietary sources.studies on fish have indicated that excessive fat accumulation is one of the phenotypes of metabolic syndromes with low growth and feed efficiency,poor immunity and anti-stress ability,and energy homeostasis disorder.Excessive fat accumulation was also found in in Oreochromis niloticus farming.Lipolysis describes the process that triglycerdies are hydrolyzed to produce fatty acids and glycerol.Many drugs and nutritional additives are developed for obesity treatment and weight control based on lipolysis regulation in mammals.However,the study on fish lipolysis,especially its mechanism and the nutrient regulation of lipolysis during O.niloticus farming,is still limited.This study focused on the mechanism of O.niloticus lipolysis and its application on reduction of lipid accumulation and promotion of lipid utilization during O.niloticus farming.The techniques of molecular biology,cell biology and nutrition were used to determin the agonist for O.niloticus lipolysis.Meanwhile,2 additives were used in the feed of O.niloticus in order to study their mechanism and effect on stimulating lipolysis and lipid usage during O.niloticus farming.This study can provide reference for the study on mechanism of lipolysis and nutriten regulation of lipid utilization in O.niloticus.The main results and conclusion of this study are as follows:1.Characteristics of O.niloticus adipose triglyceride lipase expressionAdipose triglyceide lipase(ATGL)is the rate-limiting enzyme of the first step in trigleceride(TG)hydrolysis.Many lipolysis agonists can regulate the activity of ATGL in mammals.However,fish ATGL,especially the genetic structure and expression of O.niloticus are less understood.In this study,full-length gene of O.niloticus ATGL was colned,analyzed and blasted on line.The ORF length of ATGL is 1539 bp,encoding512 amino acids.The polypeptide has a theoretical molecular weight of 56.96 k Da and an isoelectric point of 5.81,which is mainly located in the cytoplasm.The functional domain was analyzed and confirmed that O.niloticus ATGL belonged to the Patatin-like phospholipase domain-containing protein(PNPLA)family and the domain was between 1-250 amino acids.The gene composistion of O.niloticus ATGL showed a highly similarity with large yellow croaker(Larimichthys crocea)(78%),followed by Epinephelus fuscoguttatus x Epinephelus lanceolatus(76%)and the similarity to Cynoglossussemilaevis,Boleophthalmuspectinirostris,Pangasianodon hypophthalmus was 70%.Experssion in different tissues showed that this lipase had the highest expression in liver and adipose tissues followed by kidney,brain,heart,red muscle,spleen and head kidney tissue,and the lowest expression was gill.Fatsing treatment was used for ATGL study during lipolysis promoted.On the first day of starvation,the expression levels of ATGL in muscle,liver and adipose tissues were significantly increased.The expression of ATGL in the liver tissue was 15 times higher than that in the control group and falled to the same as control on Day 3.The expression of ATGL in the muscle gradually decreased during the fasting trail,and decreased to the lowest on Day 5.Adipose tissue maintained a high level of ATGL expression throughout the fasting trail.The results indicated that ATGL is sensitive on lipolysis during fasting.These results provided a throretical basis for the further study on lipolysis agoinst and the lipid utilization in O.niloticus.2.Optimization of O.niloticus adipocytes and hepatocytes culturing and lipolysis agonist selectionCell culture has the advantages of convenient operation,homogeneity and could directly exposure to drugs,thus becoming one of the important tools for physiological research.This study firstly optimized the method of O.niloticus adipocyte and hepatocyte culturing by referring other fish species and the O.niloticus physiology.The results showd that adipocytes and hepatocytes in O.niloticus needed to culturing at 28~0C and the centrifugation conditions should be 400g at room temperature.Three lipolysis agonists which are commenlly used in mammal cells were choiced in this study to select the appropriate agonists for O.niloticus lipolysis.The results showed that isoprenaline(ISO),thyroid-stimulating hormone(TH)couldn't stimulated lipolysis in O.niloticus as they did in mammals,or even showed an inhibtion effect.However,forskolin(FSK)could significantly up-regulate the lipolysis of O.niloticus adipocyte and hepatocyte.Therefore,FSK was choosed as a tool for studing the lipolysis of O.niloticus.For further study on the lipid composition and content in O.niloticus adipocyte and hepatocyte,thin layer chromatography(TLC)was used to analyze the cells.This study firstly screened the TLC solvent,results showed that the solvent heptane:diethy;ether:acetic acid=55:45:1 was appropriate for all the lipid stander:TG,sn1,2/1,3-DG and OA.Afterwards,the linear equation is constructed by using the TG and its absorbance after iodine dyeing.The linera relationship is:TG concertration=0.0023*absorbance value-3.2534,R~2=0.9988.This result indicated that TG could be analyzed quantitatively by TLC,which provided the technology for the subsequent experimnents.The adipocyte and hepatocyte after being stimulated by FSK were analyzed by TLC,result showed that TG content was significantly decreased while DG accumulated,indicating that DG played an important role in O.niloticus lipolysis.3.Effect of forskolin on O.niloticus lipolysis and lipid utilizationPrevious study optimized the method for O.niloticus cell culturing and selected the lipolysis agonist.This study further focused on the the regulation of lipolysis in O.niloticus adipocyte and hepatocyte.Different doses of FSK were fistly selected by their performance on O.niloticus lipolysis.The result showed that 50?M FSK could stimulate lipolysis both in adipocyte and hepatocyte.Following by deteming the gene expression during FSK stimulation,the results showed that FSK significantly up-regulate the ATGL and PKA gene as well as?-oxidation genes expression,PPAR?and CPT1,thus indicating that FSK stimulated lipolysis by upregulating lipase and fatty acid oxidation.We further studied the effect of FSK on lipid utilization in vivo.The results showed that,despite no difference on growth performance and protein content in whole fish and muscle,feed coefficient rate,hepatosomatic and mesenteric fat index as well as the lipid droplet area in liver were singnificantly down-regulated by FSK,indicating that FSK could induce lipid utilization in O.niloticus.The results of gene expression indicated that lipase related genes,ATGL,HSL,MGL and PKA as well as?oxidation genes,PPAR?,FABP1,CPT1 and ACO were significantly up-regulated.These results declared that FSK could induce O.niloticus lipid utilization in vivo by up-regulating lipolysis and?oxidation,indicating the application prospects of FSK in O.niloticus farming.4.Effect of diglyceride on O.niloticus lipolysis and lipid utilizationPrevious study showed that FSK could stimuled O.niloticus lipolysis while caused the accumulation of diaglyceride(DG),indicating that DG play an important role in O.niloticus lipolysis.Study on mammals have shown that DG could stimulate lipolysis.Consider the safety as food additive,DG might be more suitable for O.niloticus farming.Therefore,this study investigated the effects of DG on O.niloticus lipolysis in vivo and in vitro.The results showed that DG could stimulate lipolysis of O.niloticus adipocyte and hepatocyte as well as the gene expression of lipase and FA metabolism related genes.Using different doses of DG as lipid source of O.niloticus feed.The results indicated that DG could improve the growth performance and feed coefficient ration of O.niloticus.The crude lipid,TG content of whole fish.Liver and adipose tissues ase well as the lipid droplet area in liver tissue were significantly down-regulated by DG diet,indicating that DG diet could improve the lipid utilization during O.niloticus farming.The gene expression showed that DG could up-regulate the lipase(ATGL,HSL,MGL),?oxidation(FAS,LPL,SREBP,ACO,CPT1,PPAR?)and protein metabolism related genes,indicating that DG diet could reduce fat accumulation and induce protein content by up-regulating lipolysis,FA oxidation and protein synthesize.However,high dose of DG diet should negative effect during O.niloticus farming by derceased the survival rate and increase the expression of stress-related genes.Therefore,further research is needed to determine the DG diet application during O.niloticus farming.In summary,results of this paper indicate that O.niloticus ATGL regulates the lipolysis in vitro and can be induced by FSK and DG.Meanwhile,FSK and DG can also decrease the lipid accumulation,increase the lipid utilization and protein biosynthesize in O.niloticus.These results provide a reference for redcing excessive lipid accumulation and promoting proetein saparing effect during O.niloticus farming.