| Intramuscular fat(IMF)content is closely related to meat quality,and study the regulation mechanism of IMF metabolism is arm to improve meat quality.C2C12 cells are a myoblast line with the ability to differentiate into adipocytes.Adipogenic differentiation of myoblasts can be used to accurately study the regulation mechanism by various factors on IMF metabolism in the cellular level.This experiments take adipogenic differentiation myocytes C2C12 as the research materious,and induce autophagy through serum-free and starvation method.Investigate the effects of autophagy on adipocyte differentiation,lipid droplets(LDs)remodeling,fat differentiation,lipid synthesis,fatty acid oxidation and other processes.And through the recovery of high glucose medium after fasting,to investigate the experiment of compensatory effect on the growth of LDs in adipocytes.Experiment results:1.Use 4.5g / L D-glouse highly glucose medium(containing 10% FBS,1% double antibody),and add 0.5 mg/L dexamethasone,0.5 mmol/L IBMX,10 μg/m L insulin,0.2mmol/L Indomethacin’s adipogenic induction solution,to culture and induce C2C12 myoblasts for 12 days successfully,which can show better cell lipophilic morphology.2.In this experiments,(1)Acquire adipogenic differentiation of C2C12.(2)mRNA expression of autophagy-related genes LC3 and p62 increased significantly with starvation treatment by serum-free and lower glucose(P <0.05);LC3Ⅱ / LC3Ⅰshearing increased significantly(P <0.05);the expression of p62 protein was significantly reduced(P <0.05).(3)And colocalization of LC3 and LDs were observed,indicating that autophagy and lipid droplets lipophagy by starvation in induced adipogenic differentiation myocytes was successful in this experiments.Adding 3-MA inhibitors,the expression of autophagy-related genes and the colocalization of LC3 and LDs were inhibitsed.(4)At the same time,it was observed that the TGs level decreased significantly after fasting treament in adipogenic differentiation myocytes(P <0.05);the size of LDs decreased(P <0.05);the numbers of LDs increased significantly(P <0.05).And surface structural proteins have the changes in LDs.The addition of 3-MA inhibitors can reduce the size of LDs,increase the number of LDs,and decrease the level of TGs.This indicates that starvation induces LDs remodeling and autophagy participates during the process of LDs remodeling.3.The method starvation of serum-free and lower-sugar,(1)significantly up-regulated fat differentiation initiation factors,lipid synthesis,lipid transport,and decomposition-related genes.Under fasting conditions,cells up-regulated lipid metabolic activity and promoted cell fat differentiation to ensure cell survival.(2)Inhibition of autophagy activity significantly down-regulated lipid metabolism,and inhibited the up-regulation of the neutral esterase gene ATGL expression.(3)As follow,suggesting that autophagy affect lipid metabolism from adipogenic differentiation myocytes under fasting,the autophagy pathway may promote neutral esterase hydrolysis.4.Under fasting condition,in order to respond to the lack of energy,(1)adipogenic myocytes can produce lipolysis,which leads to intracellular LDs remodeling,bigger LDs would degradate,smaller LDs would increase,and the contents of TGs decreased.(2)Using high glucose culture,small LDs is quickly filled with fat,the content of TGs increased significantly(P <0.01).At the same time,it was observed that the red fluorescence signal of LC3 did not decrease during the restoration of high glucose culture,and colocalization of LDs and LC3 still existed.(3)It is shown that the compensatory effect can promote rapidly filling fat in LDs,and autophagy-related protein LC3 participated in the process of LDs with filling fat,through adipogenic differentiated myocytes by high glucose recovery after starvation. |
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