Regulatory Mechanism Of Autophagy In Dedifferentiation Of 3T3-L1 Adipocytes

Insulin is a key inducing factor during preadipocytes adipogenic differentiation.Insulin signaling is mainly initiated through the Insuin-AKT-mTOR-PPAR? pathway and continues to regulate preadipocytes differentiation,which is inversely related to mature adipocytes dedifferentiation in humans and mouse.Earlier studies in our laboratory showed that the removal of exogenous insulin during the mouse primary preadipocytes differentiation in vitro could cause the differentiation process to stop and dedifferentiated.As characteristics of adipocytes dedifferentiation,downregulation of adipogenic markers,upregulation of stemness markers and loss of intracellular lipids.These dedifferentiated cells regained the potentials of the stem cell-like characteristics.Autophagy is involved in lipid degradation in adipocytes and liver cells.Based on the fact that mTOR is a key molecule in the autophagy signaling pathway,we speculated that autophagy plays an important role in the mature adipocytes dedifferentiation.This article mainly used molecular biology methods to study the mechanism of autophagy in mouse mature adipocytes dedifferentiation.We used the “classic cocktail method” to induce 3T3-L1 mouse preadipocytes differentiation in vitro for 10 days.At the end of differentiation(D10),added insulin signal or autophagy signal intervening agents to promote mature adipocytes dedifferentiation,and set to DD0,that is,D10 and DD0 were at the same time.In our previous experiments,we only explored the characteristics of 3T3-L1 adipocytes dedifferentiation when insulin signal was simply inhibited and autophagy signal was promoted.It is still insufficient to reveal the mechanism of autophagy in mature adipocytes.Therefore,this experiment used six different intervention methods to further explore the role of autophagy during mature adipocytes dedifferentiation.Afterthe dedifferentiation,the medium was changed every two days and the cells were continuously treated for 12 days(DD12).The group was set as follows.1.Non-intervention control group,using complete growth medium(CGM)to continuously culture 3T3-L1 mature adipocytes;2.Insulin signal inhibition group,using CGM but additionally supplemented OSI-906 to inhibit insulin signal of 3T3-L1 adipocytes;3.Autophagy signal induction group,using CGM but additionally supplemented Rapa to induce autophagy in 3T3-L1 adipocytes;4.Autophagy signal inhibition group,using CGM but additionally supplemented Baf A1 to inhibit autophagy in 3T3-L1 adipocytes;5.Insulin signal inhibition and autophagy signal induction group,using CGM but additionally supplemented OSI-906 and Rapa to inhibit insulin signal and induce autophagy in 3T3-L1 adipocytes;6.Simultaneously inhibit insulin signal and autophagy group,using CGM but additionally supplemented OSI-906 and Baf A1 to inhibit insulin signal and autophagy in 3T3-L1 adipocytes.In addition,in order to test whether the dedifferentiated cells can regain stem cell-like ability,the cells were re-induced for adipogenic redifferentiation(RD)using CGM containing 1.7 ?M insulin that were counted as DD10/RD0 culture for 10 days(RD10).The experimental results of this paper are as follows.1.Dedifferentiation and autophagy control group(insulin signal nonintervention group): 3T3-L1 adipocytes continued to be cultured in CGM for 12 days(D10/DD0 to DD12).Lipid droplets continued to increase and continued to fuse.This phenomenon is consistent with our previous experimental results.In addition,the level of phosphorylation of AKT,a key molecule in insulin signaling,and the levels of adipogenic molecules C/EBP? and PPAR? were at high levels throughout the process.At DD12,the translation level of P62 was significantly up-regulated(p <0.01)compared to the beginning of dedifferentiation(DD0),and the transcription and translation levels of LC3 were significantly down-regulated(p <0.01)compared to thebeginning of dedifferentiation(DD0).This shows that during this process,autophagy levels are generally down-regulated.2.During the process of dedifferentiation of 3T3-L1 adipocytes(D10/DD0)to(DD12),continued simple inhibition of insulin signal can cause the adipogenic differentiation process to stop and lipid droplets to gradually disappear.At DD12,the cell adipogenesis rate decreased from 85.12±5.20% to 15.42±5.10%,and most of the original mature adipocytes showed fibroblast-like morphology.The target molecule transcription and translation levels showed that phosphorylation of AKT level was significantly down-regulated(p <0.05).The inhibition rate of insulin signal by OSI-906 was 76.20±3.36%,and the adipogenic molecule PPAR? and C/EBP? transcription and translation levels were significantly down-regulated(both p <0.01).These are consistent with previous laboratory results.The transcription level of stem cell marker CD105 was significantly up-regulated(p<0.01).In addition,the lipid droplet fusion-related molecule FSP27 was significantly down-regulated(p <0.05).During this process,the level of transcription and translation of the autophagy marker molecule LC3 was down-regulated in the early and middle stages(DD0 to DD6),and up-regulated in the late stage(DD6 to DD12).The level of transcription and translation of P62 was up-regulated in the early and middle stages(DD0 to DD6),and down-regulated in the late stage(DD6 to DD12).The above results show that 76.20±3.36% inhibition of insulin signal can lead to dedifferentiation of mature adipocytes,indicating that the insulin signal is negatively correlated with 3T3-L1 adipocytes dedifferentiation,which is consistent with the results of previous work we have done with mouse primary cells.3.Continued simple promotion of autophagy signaling from D10/DD0 to DD12 can cause some lipid droplets to be lost.At DD12,only a few fibroblast-like cells appeared.The cell adipogenesis rate decreased from 85.12±5.20% to 50.26±2.05%.The target molecule transcription and translation level results showed that the phosphorylation of AKT level was significantly down-regulated(p <0.01),so the inhibitory rate of insulin signal by Rapa was 54.33±2.26%.The levels of transcription and translation of PPAR? and C/EBP? were significantly down-regulated(both p<0.05),consistent with previous experimental results.In addition,the transcription and translation levels of FSP27 were also down-regulated(p <0.05).The above results show that the promotion of autophagy can partially inhibit the insulin signaling pathway and promote the mature adipocytes dedifferentiation.However,compared with the group that simply inhibits insulin signaling,its inhibitory rate on insulin signal is lower and the dedifferentiation process is slower.4.From D10/DD0 to DD12,continue to simply inhibit autophagy signaling.The cell adipogenesis rate decreased from 85.12±5.20% to 65.27±2.19%.In addition,the target molecule transcription and translation levels showed that inhibition of autophagy had little effect on insulin signaling and expression of key adipogenic molecules(p > 0.05).This shows that after inhibiting autophagy signals,insulin signal transmission cannot be inhibited,further explaining the role of autophagy in mature adipocytes dedifferentiation caused by lack of insulin signal.5.Continuous inhibition of insulin signal and promoting autophagy signal from D10/DD0 to DD12.At DD12,most mature adipocytes showed fibroblast-like morphology and adipogenesis rate decreased from 85.12±5.20% to 10.21±3.01%.The target molecule transcription and translation level results showed that the phosphorylation of AKT was significantly down-regulated(p <0.01)compared to the group that inhibited insulin signal alone.Therefore,the combined inhibition rate of insulin signal by OSI-906 and Rapa was 82.15±2.39%.In addition,the transcription level of C/EBP? was significantly down-regulated(both p <0.01),but did not significantly inhibit the transcription and translation level of PPAR?,and the transcription level of CD105 was up-regulated.The above results indicate that simultaneous inhibition of insulin signal and promotion of autophagy can further inhibit insulin signal.The above results show that the two effects can synergistically inhibit insulin signal and promote mature adipocytes dedifferentiation.6.Continuous inhibition of insulin and autophagy signal from D10/DD0 to DD12.Most of the lipid droplets disappeared at DD12,and the cell adipogenesis rate decreased from 85.12±5.20% to 15.42±2.17%.However,the number of cells containing lipid droplets was slightly more than the group that inhibits insulin signaland promotes autophagy,and the number of fibroblast-like cells was less than the group that inhibited insulin signal alone.The results of the target molecule transcription and translation levels showed that compared with the inhibition of insulin signal group alone,the levels of AKT phosphorylation,PPAR? and C/EBP? in the simultaneous inhibition of insulin and autophagy signal groups were not further suppressed.The results showed that simultaneous inhibition of insulin and autophagy signal could not further inhibit insulin signal,nor could it further promote mature adipocytes dedifferentiation.In addition,the cells were re-induced for adipogenic redifferentiation(RD)using CGM containing 1.7 ?M insulin that were counted as DD10/RD0 culture for 10 d(RD10).We have got adipocytes and the adipogenesis rate of re-differentiation was70.25±3.08%.The results show that the addition of insulin alone is sufficient to the adipogenic redifferentiation of dedifferentiated adipocytes,that is,insulin plays a key role in white adipogenic redifferentiation.In summary,our experimental results show that the insulin-mTOR-autophagy signaling pathway plays an important role in mature adipocytes dedifferentiation.Among them,autophagy can feedbackly inhibit insulin signal,inhibit AKT phosphorylation,and then inhibit the expression of adipogenic molecules PPAR? and C/EBP?,thereby promoting mature adipocytes dedifferentiation.In addition,dedifferentiated adipocytes can be redifferentiated into mature adipocytes,indicating that dedifferentiated adipocytes have similar characteristics to adipose stem cells.