Indomethacin Promotes Browning Of White Adipocytes And Study On Its Mechanism

Background and Objective: Obesity is a worldwide health problem.With the improvement of people’s living standard and the change of life style,the number of obesity is increasing year by year.Browning of white fat can promote the body’s energy consumption and effectively improve the body’s glucose and lipid metabolism.Previous studies have confirmed that browning of white fat has become a new idea for the study of obesity and its related metabolic diseases and a new target for the development of weight-loss drugs.The purpose of this study was to explore the effect of indomethacin(IDM)on browning of white adipocytes and its mechanism,so as to provide an important experimental basis for IDM in the clinical treatment of obesity and related diseases.Methods: In this study,3T3-L1 preadipocytes were used as experimental subjects and induced by the classical cocktail induction method;After 8 days of induction,western blotting and oil-red o staining results showed successful induction of mature fat cells;Then MTS method was used to screen the optimal concentration and time of IDM(10,25,50,100,200 μM)to treat mature 3T3-L1 preadipocytes;After the optimal concentration was obtained,the experiment was divided into Ctrl group,IDM high,medium and low concentration groups,rosiglitazone group and β3receptor agonist group to detect the effect of IDM on triglyceride accumulation in mature 3T3-L1 adipocytes,and the effect of IDM treatment on glucose uptake in3T3-L1 preadipocytes;western blotting and q RT-PCR were used to determine the expression of protein and m RNA(UCP-1,PRDM16,PGC-1α)related to browning.Next,we investigated the possible mechanism of IDM inducing browning of white adipocytes,and whether IDM induced browning of white adipocytes through activation of PPARγ.We divided the experiment into Ctrl group,IDM optimal dose group,PPARγ inhibitor group and IDM optimal dose + PPARγ inhibitor group.First,we observed the effect of IDM on lipid droplet aggregation and glucose uptake in3T3-L1 adipocytes following PPARγ inhibition by oil-red o staining and glucose uptake assay.Next,we investigated the influence of IDM and PPARγ transcriptional activity through luciferase reporter gene.Then,we verified the interaction between PPARγ and PRDM16 by immunoprecipitation assay.Finally,the expression levels of browning proteins such as UCP-1,PRDM16 and PGC-1α were detected by western blotting.Results:(1)Oil-red o staining results showed that after 8 days of differentiation induction,large amounts of lipids were observed in 3T3-L1 preadipocytes,suggesting that we successfully induced the 3T3-L1 preadipocytes to mature adipocytes with lipid accumulation function;(2)MTS method was used to detect cell viability and promote browning level.In this experiment,the optimal concentration of 3T3-L1 was 50 μm,and the treatment time was 48 h;(3)Western blotting and q RT-PCR results show that IDM increased m RNA and protein expression of UCP-1,PRDM16,and PGC-1 in a concentration-dependent manner;(4)The results of luciferase reporter gene showed that IDM enhanced the transcriptional activity of PPARγ;(5)The results of immunoprecipitation showed that IDM promoted the interaction of PPARγ with PRDM16;(6)Western blotting showed that PPARγ inhibitor reversed the browning effect of IDM on white adipocytes.Conclusion: Indometacin can promote the browning of white adipocytes,and the mechanism may be related to the activation of PPARγ transcriptional activity to induce the expression of browning protein in white adipocytes.