Role And Mechanism Of 9-PAHSA In White Adipocyte Browning

ObjectiveWhite adipose tissue(WAT)has the capacity to acquire browning adipose tissue(BAT)-like properties via the so-called “browning” process.Browning of white adipose tissue is a novel mechanism to counteract obesity.Activation of G-protein-coupled receptor 120(GPR120)can promote the browning of white fat.9-PAHSA,an endogenous mammalian lipid,which is acting as the ligand of GPR120 to enhance glucose uptake and exert anti-inflammatory effect.However,the effects of 9-PAHSA on lipid metabolism have not been evaluated.In the study,we would like to investigate the biological effects of9-PAHSA on adipocyte browning.MethodsTo investigated the roles of 9-PAHSA on 3T3-L1 adipocytes browning,we established the in vitro adipogenic differentiation models of 3T3-L1 adipocytes.9-PAHSA induced browning of 3T3-L1 adipocytes via enhanced expression of brown fat specific genes tested by western blotting(WB)and real-time fluorescent quantitative PCR(q PCR).Oil Red O staining was used to detect the lipid droplets size and cells size in 9-PAHSA-treated 3T3-L1 adipocytes.To test the effect of 9-PAHSA on browning of adipocytes in vivo,we used male C57 BL ob/ob transgenic mice(ob/ob)as obesity models and we used wild-type C57 mice as control.Mice were randomly grouped into 4: ob/ob control group;ob/ob+9-PAHSA intervention group(50 mg/kg per day);WT control group;WT+9-PAHSA intervention group(50 mg/kg per day)(n=5 mice for each group).9-PAHSA was given to mice by gavage once a day for one month.The control groups were given with the same volume of vehicle(50% PEG400,0.5% Tween-80,49.5% H2O)at the corresponding time points.Subcutaneous WAT(s WAT)in WT and ob/ob mice were harvested.H&E staining was used to detect lipid droplets size and adipocytes size.9-PAHSA-induced browning in white adipocytes of WT mice and ob/ob mice was investigated by determining expression levels of brown adipocyte-specific genes/proteins by q PCR,WB and immunochemical staining.The effects of 9-PAHSA on brown fat markers in 3T3-L1 cells were tested when GPR120 gene was silenced.To investigate the molecular mechanism of 9-PAHSA on adipocyte browning,lipopolysaccharide(LPS)-induced inflammatory model was conducted.The influence of 9-PAHSA and GPR120 on LPS-induced inflammation and the potential metabolism underlying it were tested.Results1.9-PAHSA treatment enhanced expression of brown fat-specific genes uncoupling protein 1(UCP1),peroxisome proliferator-activated receptor ? coactivator 1?(PGC 1?),CCTTA/enhancer binding protein ?(C/EBP?)and PR domain-containing 16(PRDM16)on day 8 of the differentiation process in a dose-dependent manner.In addition,Oil Red O staining indicated that 9-PAHSA treatment induced smaller,multilocular lipid droplets accumulation and smaller adipocytes size in 3T3-L1 adipocytes as compared to control,which exhibited the characteristics of brown adipocytes.2.With treatment of 9-PAHSA,the expression of brown fat-specific genes(UCP1,PGC-1?,C/EBP? and PRDM16)were dramatically up-regulated at day 4 and stayed high at day 8.3.9-PAHSA treatment increased m RNA expression of browning markers including UCP1,PGC1?,PRDM16 and cell death inducing factor DFFA like effecter a(Cidea)and significantly increased UCP1 and PGC1? protein levels,in both WT mice and ob/ob mice as compared with control.By H&E staining,we found that the fat cells in s WAT were smaller and multilocular in 9-PAHSA-treated WT mice and ob/ob mice than that in their control group,which were typically associated with the browning process.4.Knockdown of GPR120 almost completely reversed the enhanced effects of 9-PAHSA on browning related genes(PRDM16,C/EBP?,PGC1? and UCP1).5.LPS stimulated phosphorylation of nuclear factor-?B(NF-?B)and inhibitor of NF-?B(I?B),promoted monocyte chemotactic protein 1(MCP1)and Interleukin 6(IL-6)secretion and inhibited expression of browning markers including UCP1,PGC1?,PRDM16 and Cidea.6.9-PAHSA inhibited LPS stimulated phosphorylation of I?B and NF-?B,and inhibited MCP and IL-6 expression.These effects of 9-PAHSA were mediated by GPR120.Conclusion1.9-PAHSA promotes browning of 3T3-L1 adipocytes in vitro.9-PAHSA exerts its influence on induction of 3T3-L1 adipocytes browning at the terminal stage of 3T3-L1 cells differentiation.2.9-PAHSA induces browning of s WAT in WT mice and ob/ob mice in vivo.3.9-PAHSA promotes adipocyte browning through GPR120.4.LPS activates NF-?B signaling pathway and promotes inflammation in 3T3-L1 adipocytes,then inhibits adipocyte browning.5.9-PAHSA inhibits LPS-induced inflammation in 3T3-L1 adipocytes through GPR120.6.9-PAHSA induces adipocyte browning via GPR120-mediated anti-inflammatory role.