| As the biggest energy storage organ, adipose tissue plays a major role in nutrient homeostasis. In animal, adipose tissue development imbalance may lead to many problems, such as inducing disease and lowering the quality of animal products, which will affect the breeding benefit. Adipocytes are the major components of adipose tissue. The development of adipocytes directly decides the biological characteristics and function of adipose tissues, which will affect fat deposition in body. Therefore, illustrating the development mechanism of adipocytes will provide important theoretical references for the work which ensures the metabolism balance and improves fat deposition in animal. Some in vivo researches on adipose tissue development in red sea bream and zebrafish have been studied. At the same time, seven primary preadipocyte culture systems in fish, including grass carp, have been established to explore the developmental characteristics. But, the time and position of grass carp adipocytes appeared in vivo during the ontogenetic development process, the molecular event and the changes of organelle such as mitochondria during preadipocytes differentiation process in vitro are still not clear. Highly unsaturated fatty acid(HUFA), especially DHA and EPA, could impact adipocytes development through suppressing fatty acid synthesis, increasing lipidolysis and fatty acid β-oxidation, and reduce lipid accumulation ultimately. In fish, it has been suggested that HUFA have a role in preventing the development of adiposity, but the mechanisms are still largely unknown.Objectives of this study were to 1) study the fundamental laws of grass carp adipocytes in vivo and in vitro and to 2) explore the influence and mechanism of HUFA on adipocytes differentiation and lipolysis. In present study, firstly, histological technique including paraffin and frozen sections were taken to observe the time and position of adipocytes appeared in vivo during the ontogenetic development of grass carp by different staining methods, including hematoxline and eosin(HE), oil red O and BODIPY. The expression of lipogenesis related genes including peroxisome proliferator-activated receptors γ(PPAR γ) and CCAAT-enhancer binding proteinα(C/EBP α) and so on at different time points were detected by q PCR. In addition, the expression sites and time of PPAR γ was detected by immunohistochemistry. Then, the molecular events and mitochondria development process were studied in grass carp adipocytes developed in vitro. Ultrastructure observation and mitochondria fluorescent staining were used to study the change of mitochondria during adipocytes differentiation. Mitochondrial protein content was also detected. The expression of lipogenesis related genes and mitochondrial relevant genes were detected by q PCR. Next, in order to explore the influence of HUFA on adipocytes differentiation, high throughput sequencing technology was applied to analyze the differentially expressed genes after treating with DHA, we found that several signaling pathway were affected by DHA in adipocyte. Then, to explore the role of Wnt/β-catenin signaling pathway in regulation of DHA on adipocyte, an inhibitor(FH535) of β-catenin signaling pathway was used to co-treating adipocytes with DHA, the lipid content, genes and protein expression change of cell were analyzed. Finally, we explored the influence of HUFA(DHA and EPA) on grass carp mature adipocytes lipolysis in vivo and in vitro. Mature adipocyte were isolated and treated with HUFA, the release of glycerol and genes were detected. In addition, a 95-day feeding trial was conducted with two diets formulated with either lard oil(as control) or fish oil(HUFA as treatment) as the main lipid source. The glycerol and free fatty acid(FFA) released from the isolated adipocytes of both groups were detected. The expression of lipolysis-related genes in adipose tissue was also analyzed.The main results obtained as follows:1. In vivo research showed that grass carp visceral adipocytes appeared around the hepatopancreas on 16 days post fertilization, then they developed and transferred gradually to mesenterium and formed adipose tissue; the early developmental stage of adipose tissue in grass carp was caused by active recruitment of adipocytes as opposed to hypertrophy of the cell;2. Cellular lipid content, droplets number, mitochondria number and mitochondrial protein content, the expression level of lipogenesis related genes and mitochondria related genes increased during the differentiation of grass carp adipocytes in vitro, these resultes demonstrated that mitochondria may play a impiont role in adipocyte differentiation.3. During the adipocytes differentiation process, DHA inhibited lipid accumulation in cells with 7days treatment, while DHA promoted lipid accumulation in cells with 2 days treatment. At the same time, the change of the expression level of lipogenesis related genes was consistent with the change of lipid content. High throughput sequencing results further showed that DHA treated for 7 days could down-regulated lipogenesis related genes and up-regulated β-catenin, DHA treated for 2 days up-regulated lipogenesis related genes in adipocytes. These results showed that Wnt signaling pathways may be plays an important role in DHA regulating adipocyte differentiation4. DHA could activate the expression of β-catenin and its downstream genes during grass carp adipocytes differentiation. At the same time, when β-catenin was inhibited by FH535 in the existence with DHA, the inhibition effect of DHA on adipocyes lipogenesis was relieved, implying that DHA could inhibit adipocytes differentiation through activating Wnt/β-catenin signaling pathway;5. The release of glycerol in grass carp mature adipocytes was significantly increased after treating with DHA and EPA, and the lipolysis-related genes were also elevated, implying that HUFA could promote grass carp adipocyte lipolysis. In addition, feeding with HUFA could promote the hydrolysis ability of grass carp mature adipocytes and elevate the expression of lipolysis-related genes. |
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