Apigenin Promotes Browning Of White Adipose Tissue Through Activating PI3K-Akt-mTOR Pathway To Suppress Autophagy

Objective: With the improvement of people’s living conditions and the prevalence of sedentary lifestyles of office workers,the number of obese people has been increasing exponentially year by year.It has been proven that browning of white adipose tissue has a positive significance in the treatment of obesity and related metabolic diseases,and the effect of compounds with flavonoids structure on browning of white adipose tissue has gradually come into the field of vision of researchers.In this study,we investigated the effects of apigenin(AP)on browning of white adipose tissue in vivo(obese C57BL/6 mice)and in vitro experiment(3T3-L1 and primary adipocytes of obese SD rats)and its possible potential mechanism,providing an important basis for clinical treatment of obesity and related diseases.Methods: 1.In vivo experiment: thirty 6 week-old healthy male C57BL/6 mice were randomly divided into two groups.one group(20 mice)was fed with high-fat diet(HFD),and the other(10 mice)was fed with normal diet as a control(Ctrl).After 12 weeks of feeding,the weight of the HFD group was greater than 20% of that of the Ctrl group,showing the obesity model was successfully established.After the successful establishment of the obesity model,the HFD group was randomly divided into two sub-groups.One sub-group was fed with high-fat diet and the other was gavaged with AP,with vehicle(PBS)as a HFD,and the control group was fed with normal diet.The experiment was divided into the Ctrl group,the HFD group,and the AP group.After 4 weeks,the following indicators were measured:(1)The Weight,Length,Lee’ s Index and FI;(2)Glucose tolerance test(GTT),insulin tolerance test(ITT);(3)Weight of epididymal WAT(eWAT),dorsal interscapular BAT(BAT),subcutaneous inguinal WAT(iWAT),perirenal WAT(pWAT),omental WAT(oWAT),heart,liver,spleen,kidney,and lung weights;(4)The biochemical indicators including TG(triglyceride),INS(insulin),TC(cholesterol),FPG(fasting plasma glucose),insulin resistance index(HOMA-IR)and serum Resistin;(5)Cold tolerance test;(6)The expression levels of PGC-1α,PRDM16,UCP-1,LC3Ⅰ/Ⅱ,Beclin-1 and p62 in iWAT were detected by the method of western blotting.2.In vitro experiment: First,primary SD rat preadipocytes of iWAT,BAT and oWAT were extracted and induced by a cocktail induction method and so did 3T3-L1 preadipocytes.After 8 days of induction,it was successfully induced into mature adipocytes.Then,the mature adipocytes from various adipose depots and 3T3-L1 were treated with AP concentrations indicating 5,25,50,100,200 μM and PBS.Subsequently,the accumulation of triglycerides was detected.After obtaining the optimal concentration of AP,the experiment was divided into Control group and AP group to determine the expression of autophagy-related proteins.After determining the effect of AP on autophagy,the pharmacologic autophagy activator rapamycin was used to synchronize the action.In this experiment,the cells were sub-divided into Ctrl group,RP group,AP group and AP+RP group and were explored the influence of AP on the browning process after reversing the effect of AP on autophagy during browning.At the end of the experiment,we used pharmacological inhibitors to explore the autophagy classic pathway PI3K-Akt-mTOR pathway and to explore the role of AP in promoting browning.Indicators measured in vitro:(1)MTS method was used to detect cell viability;(2)Oil red O staining was used to observe the accumulation of lipid droplets;(3)Immunofluorescence was used to investigate the expression of UCP-1 in 3T3-L1 cells;(4)qRT-PCR was used to detect the browning-related genes including UCP-1,PGC-1α,PRDM16,PPAR-γ,CEBP/β,COX-8P and ATP5B;(5)MDC staining to observe the number of autophagosomes;(6)Western blotting to detect UCP-1,PRDM16,PGC-1α,LC3Ⅰ/Ⅱ,Beclin-1,p62,p-PI3 K,PI3K,p-Akt,Akt,p-mTOR and mTOR protein expression levels.Results: 1.In vivo:(1)After AP treatment,the size and shape of the four white adipose depots of the mice were smaller than those in the HFD group,while the BAT morphological size was slightly larger than in the HFD group;(2)After AP treatment,the body weight,Lee’s index and FI of the mice decreased significantly compared with that of the HFD group;(3)Cold tolerance experiments showed that after AP treatment,the body temperature of the mice was more stable at 4 ℃;(4)After AP treatment,the weight of all visceral adiposes in the AP treatment group was decreased compared with that in the HFD group;(5)The weights of the white adipose tissues were decreased compared with that in the HFD group,while the weight of BAT increased;(6)After AP treatment,various biochemical indicators(TG,INS,TC,FPG,HOMA-IR and Resistin)of the mice were lower than that of the HFD group;(7)After AP treatment,the expression level of browning marker proteins(PGC-1α,PRDM16 and UCP-1)in subcutaneous iWAT was increased,and the expression of autophagy marker proteins(LC3Ⅰ/Ⅱ,Beclin-1,p62)was decreased;(8)The GTT and ITT results showed that AP could repair the glucose tolerance and insulin tolerance damage caused by HFD and it was benefit for glucose homeostasis.2.In vitro:(1)Oil Red O staining results showed that a large amount of lipids were accumulated in four kinds of adipocytes,and fat cells were induced to mature after 8 days of induction culture;(2)The optimal concentration was selected according to the effect of MTS on cell viability and browning effect after AP treatment.The optimal concentration of AP for primary adipocytes was 100 μM,and the optimal concentration for 3T3-L1 was 50 μM.The treatment time was chosen to be 48 h;(3)Western blotting and RT-PCR results show that AP promoted the browning of white fat;(4)MDC experiments and western blotting results showed that AP inhibited autophagy during induction;(5)Western blotting results show that after rapamycin reverses AP’s autophagy inhibition,the browning effect was also reversed,suggesting that the role of AP in promoting browning may be exerted by inhibiting autophagy;(6)Western blotting results showed that the mechanism of AP in promoting browning may be by activating the PI3K-Akt-mTOR signaling pathway.Conclusion: Our experimental results show that apigenin promotes the browning of white fat,both in vivo and in vitro experiment.We have concluded at the cellular level through methods such as autophagy activator and PI3K-Akt-mTOR pathway inhibitor that the mechanism may be through the activation of the classic autophagy pathway PI3K-Akt-mTOR to inhibit autophagy and achieve the purpose of promoting browning of white adipose tissue.