Mechanism Of Autophagy In Insulin-mediated Dedifferentiation Of 3T3-L1 Adipocytes

Obesity is a disease caused by abnormal accumulation of lipids in white mature adipocytes(hereinafter referred to as adipocytes),which is manifested as abnormal hypertrophy of adipocyte volume and abnormal increase in number.In recent years,studies have shown that adipocytes can remove lipid droplet(LD)from the cytoplasm and differentiate into fibroblast-like cells(dedifferentiated fat cells,DFATCs).Compared with adipocytes,DFATCs showed up-regulated stem cell marker SOX2 and down-regulated adipocyte marker PPAR?.Under specific induction conditions,DFATCs can be redifferentiated into fat cells,bone cells,smooth muscle cells,vascular endothelial cells and nerve cells.Autophagy is a common regulatory mechanism in eukaryotes.In the case of nutrient deficiency or stress,cells digest,degrade organelles,proteins and other components that are senescent or non-function in the body to maintain energy balance.Relevant studies have shown that starvation can induce autophagy in mammalian cells.The autophagic lysosomes were produced during autophagy which can target,select,wrap and degrade lipid droplets to provide energy for the life activities of cells.Factors such as insulin and high fat stress can activate mammalian target of rapamycin(mTOR),which inhibiting autophagy.Previous studies in our laboratory have shown that inhibiting insulin signaling can promote the dedifferentiation of white adipocytes.So how does autophagy play a role in insulin-mediated adipocyte dedifferentiation?3T3-L1 mouse preadipocytes were used as the materials in this experiment,the "classic cocktail method" was first used to induce their white lipogenic differentiation in vitro.Subsequently,the dedifferentiation of adipocytes was facilitated by the addition of insulin signal-specific inhibitor(OSI-906,linstinib)and autophagy was induced in the process.The role of autophagy in the dedifferentiation of insulin-mediated white adipocytes was analyzed through the morphological changes of dedifferentiated adipocytes and the transcription and translation levels of key molecules of insulin signaling and lipogenesis.The experimental results are as follows:1.The terminal concentration of 1?M OSI-906 was used to promote the dedifferentiation of adipocytes,and the time-series expression characteristics of lipid-related molecules and autophagy related molecules were observed by cell morphology detection.The results showed that the lipid drops in the cytoplasm of adipocytes gradually decreased after blocking the insulin signaling pathway,and the lipid formation rate decreased from 80%±5.5% to 10%±3.5%.Besides,the transcription and translation levels of the lipid-related molecule PPAR were significantly down-regulated,but the expression levels of the autophagy related molecule LC3 and the stem cell marker molecule SOX2 were significantly up-regulated.2.After the lipogenic differentiation of 3T3-L1 preadipocytes,100 nM Rapa was added to observe whether it could promote the process of dedifferentiation,the change characteristics of cellular morphology and the time-series expression of lipid-related molecules and autophagy related molecules were analyzed.The results showed that after the induction of autophagy,the large lipid droplets in the cytoplasm of adipocytes further fused and became larger,and the number of small lipid droplets significantly decreased,but the number of cells containing large lipid droplets showed no significant change and the process of dedifferentiation was slow.The expression of lipid-related molecule PPAR was down-regulated,and the expression of autophagy related molecule LC3 and stem cell marker molecule SOX2 were up-regulated.In summary,the results demonstrated that autophagy positively regulated the dedifferentiation of white adipocytes through the insulin signaling pathway.After the induction of autophagy in white fat cells,autophagy can regulate the signal through negative feedback and block the insulin signaling pathway,thus promoting the process of adipocyte dedifferentiation.Combined with the previous experimental results of our laboratory,this work will provide useful reference basis for the study of the correlation between the dedifferentiation process of white fat cells and autophagy,it also can supplement people's understanding of white fat cells in biology and may provides new ideas for the prevention and treatment of obesity.