| Due to the shortage of fish oil,plant oil source has been widely used in aquatic animal feed in recent years.At the same time of improving economic benefits and feed production,plant oil sources,due to the unbalanced nutrition of fatty acids,especially the lack of long-chain fatty acids and n-3 fatty acids,lead to the decline of fish growth performance and the occurrence of nutritional metabolic diseases,affecting the healthy and sustainable development of the aquaculture industry.Therefore,lipid replacement needs more reliable research foundation of fish lipid metabolism physiology.However,due to the diversity of fish species,metabolic patterns are different,and related research is very limited.In fact,there are many representative fish with relatively clear nutritional background but large difference in fatty acid demand,such as grass carp and nile tilapia,which are the typical representatives of n-3 type and n-6 type fish with essential fatty acid demand,but there are few studies on relevant lipid metabolism.Based on the existing research,this paper will discuss the following scientific issues: 1)what are the differences and similarities between n-3 fatty acids and n-6 fatty acids in demand-type fish? 2)Are the metabolic patterns of n-3 and n-6 types of fatty acids identical in different types of cells?3)Is the metabolism of long-chain saturated fatty acids the same in fish with different demand for fatty acids? In view of the above problem,this article through the establishment of grass carp and tilapia liver/muscle primary cell model,the use of radioactive marker fatty acids to track its fatty acid intake,FA beta oxidation,lipids esterification and other key lipids metabolic process and accurate quantitative.The expression levels of lipid metabolism key genes and regulatory factor genes/proteins were detected to comprehensively describe the similarities and differences of lipid metabolism patterns of LCFA in different fish cells.The main research results and conclusions of this paper are as follows:1.Establishment of cell modelThe cell model is stable and reproducible,which is a good choice for studying the lipid metabolism of specific tissues and organs in fish.In this chapter,the appropriate extraction and culture methods of primary hepatocytes and mucsle cells of grass carp and tilapia were first explored.Through the exploration of the concentration and time of co-incubation of fatty acids,the conditions suitable for studying the metabolism of fatty acids in fish cells were determined.The primary hepatocytes of grass carp and tilapia were obtained by collagenase digestion,and the suitable conditions for culture were: DEME high-glucose medium containing 10%FBS+1%P/S;Primary muscle cells of grass carp and tilapia were obtained by tissue mass culture method,and FBS and glucose levels were explored.The appropriate medium for passage was DEME low glucose(1g/L)medium containing 20%FBS+1%P/S.Then palmitic acid was used to explore the appropriate concentration and time of fatty acid co-incubation with four kinds of cells.The results showed that within 24 hours,the concentration of fatty acid was 100 μmol/L,which was suitable for exploring the influence of LCFA on lipid metabolism in fish cells.2 Study on the cellular metabolic mechanism of long chain fatty acids in the hepatocytes of grass carp and tilapiaAfter the establishment of primary cell models of grass carp and Nile tilapia liver,this part of the experiment used labeled fatty acid tracing and other techniques to conduct quantitative detection of the important processes of long-chain fatty acid assimilation and dissimilation metabolism of liver cells,and also,the expression levels of key genes and regulatory factors of lipid metabolism were detected,and explored the n-3 and n-6 fatty acid demand-type fish lipid metabolism models and their key points of difference.The results showed that the effect of LNA on FA fatty acid accumulation in grass carps was stronger than that of LA.However,LA is stronger than LNA in the process of LCFA uptake and oxidation of in tilapia,indicating that LA has a stronger assimilation and alienation effect in tilapia liver,and does not show a deposition effect on the differentiation of the two PUFA.Compared with PUFA,saturated fatty acids only occupy a dominant position in the intake of fatty acids,and their involvement in important intracellular metabolic processes,including fatty acid oxidized lipid deposition,is low,but it also has an influence on the secretion and regulation of lipid metabolism of grass carp to a certain extent.Except the process of LCFA absorption,PUFA dominates most processes of lipid metabolism in both grass carp and tilapia.To sum up,the key metabolic differences of lipid metabolism in hepatocytes of grass carp and tilapia are mainly reflected in the process of fatty acid uptake and ester deposition,and ultimately affect the deposition of total lipid in hepatocytes.Grass carp prefer LNA uptake and deposition,and there is no significant difference between the final deposition of lipid and LNA in hepatocytes of tilapia by improving the assimilation and foreignization of LA metabolism.3 Study on the cellular metabolic mechanism of long chain fatty acids in muscle cells of grass carps and tilapiaAfter the establishment of primary muscle cell models of grass carp and Nile tilapia,this part of the experiment used labeled fatty acid tracing and other techniques to conduct quantitative detection of the important processes of long-chain fatty acid assimilation and dissimilation metabolism of muscle cells,and also,the expression levels of key genes and regulatory factors of lipid metabolism were detected,and explored the n-3 and n-6 fatty acid demand-type fish lipid metabolism models and their key points of difference.The results showed that the two kinds of fish muscle cells had a stronger ability to absorb PUFA,but a weaker ability to consume PUFA than PA,which eventually led to the deposition of PUFA in muscle cells,especially tilapia muscle cells.However,the metabolic difference between LA and LNA in muscle cells was more interesting.There was no difference in the final fatty acid deposition between grass carp muscle cells and LA group under the condition of high intake of LNA and low oxidation,and the same result was found in tilapia muscle cells.In summary,compared with PUFA,PA dominates the process of lipid metabolism in tilapia hepatocytes,but has no significant effect on grass carp.The key difference of PUFA metabolism between grass carp and tilapia muscle cells is mainly reflected in the strength of the activation of PPARa--mainly activated by LNA in grass carp muscle cells,while LA is slightly stronger than LNA in tilapia muscle cells.Combining the effects of three fatty acids on lipid metabolism of liver and muscle cells,we elucidate the metabolic characteristics of different types of LCFA in two different types of fish cells:1)n-3pufa has good metabolism and accumulation ability in grass carps liver cells,and can activate the dissimilar metabolism of fatty acids in muscle cells;2)n-6pufa has better metabolism and accumulation ability in tilapia liver cells,and has an activation effect on muscle cell fatty acid catabolism;3)both types of fish have similar metabolic capacity for saturated fatty acids,which are good energy supply substrates for muscles.However,they are also easy to accumulate in liver and cause excessive accumulation,which has adverse effects on fish health. |
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