The Regulating Effect Of Adiponectin On Lipid Metabolism In Chicken Primary Hepatocytes,Myoblasts,and Adipocytes

This study was conducted to investigate the role and mechanism of adiponectin on chicken primary hepatocytes,myoblasts,and adipocytes.Through the exogenous addition of recombinant adiponectin protein and adenovirus overexpression methods,the regulating effects of adiponectin on chicken lipid metabolism were explored,to provide new ideas for improving chicken production performance,improving feed conversion rate and treating metabolic diseases such as obesity.Experiment 1 The regulating effect of recombinant adiponectin on lipid metabolism in chicken primary hepatocytes was investigated.Firstly,after pretreatment with palmitic acid(saturated fatty acid)and oleic acid(unsaturated fatty acid),the effects of gradient addition of recombinant adiponectin on lipid metabolism of chicken primary hepatocytes were explored.As the results shown,5 ?g/mL recombinant adiponectin has a more significant lipid-lowering effect.Compared with the pretreatment of oleic acid,recombinant adiponectin has a more significant lipid-lowering effect under pretreatment of palmitic acid.Secondly,palmitic acid and the suitable recombinant adiponectin concentration(5 ?g/mL)were choosed for the regulating mechanism study.The results showed that recombinant adiponectin reduced(P<0.05)intracellular lipid content,while increased(P<0.05)triglyceride(TG)and very low density lipoprotein(VLDL)content in the medium of hepatocytes.Recombinant adiponectin significantly(P<0.01)increased the expression of lipid synthesis-related genes(FAS,ACC,PPAR? and SREBP1)after palmitic acid pretreatment.And there was a tendency that recombinant adiponectin increased the enzyme activity of FAS(P=0.07)and ME(P=0.08).In addition,the fatty acid oxidation-related gene liver-carnitine palmitoyl transferase 1(L-CPT1)was significantly(P<0.05)down-regulated,and adiponectin receptor 2(ADPNR2)and peroxisome proliferator-activated receptor ?(PPAR?)were significantly increased(P<0.05).Recombinant adiponectin also significantly(P<0.01)increased glucose and fatty acid uptake in hepatocytes.Overall,the proteins expression in AMPK/ACC/CPT1 signaling pathway and proteins expression of PGC1? and PGC1? had different changes at different time.The above results suggested that adiponectin was likely to combine with ADPNR2,promoting the uptake of fatty acids and glucose,and enhancing the transport of TG and VLDL from intracellular to extracellular,thereby reducing hepatocyte fat deposition.These results were inconsistent with the mechanism by which adiponectin plays a role in mammals and other domestic animals.Experiment 2 The regulating effect of recombinant adiponectin on lipid metabolism in chicken primary myoblast was investigated.Myoblasts were divided into four treatment groups: the control group,the recombinant adiponectin group,the palmitic acid treatment group,recombinant adiponectin and palmitic acid combined treatment group.The results showed that recombinant adiponectin reduced intracellular lipid content(TG and TCH)without affecting the viability of myoblasts.Adiponectin receptor(ADPNR1 and ADPNR2)mRNA levels were significantly(P<0.05)up-regulated,indicating that functions of adiponectin were mainly through binding to adiponectin receptors.In addition,recombinant adiponectin significantly(P<0.05)affected the levels of lipid metabolism-related genes FAS,ACC,PPAR?,PGC1?,PGC1?,PPAR?,L-CPT1,ATGL and FATP1,and promoted glucose and fatty acid uptake.In addition,recombinant adiponectin activated the AMPK/ACC/CPT1 signaling pathway,after pretreatment of palmitic acid,recombinant adiponectin activated the proteins expression of AMPK and ACC more significantly(P<0.05).Results of treatment with AMPK inhibitor(Compound C)showed that the addition of recombinant adiponectin significantly reduced TG(P<0.001)and TCH(P=0.08)content under palmitic acid pretreatment,but after Compound C pretreated for 1 h,this phenomenon was changed in which TG and TCH increased significantly(P<0.01).At the same time,AMPK and ACC phosphorylation levels and the protein expression of PGC1? returned to normal levels.In summary,recombinant adiponectin reduced intracellular lipid content,mainly by binding to adiponectin receptor,activating AMPK pathway,increasing cellular uptake of glucose and fatty acids,and promoting lipid oxidation.Experiment 3 The regulating effect of adiponectin on lipid metabolism in chicken primary adipocytes was investigated.Firstly,adipocytes were treated with recombinant adiponectin.The results showed that recombinant adiponectin significantly(P<0.001)promoted cellular glucose and fatty acid uptake and significantly(P<0.001)reduced intracellular lipid content.Oil red O staining and Bodipy staining showed that lipid droplets became smaller and FAS enzyme activity was significantly(P<0.05)decreased.In addition,recombinant adiponectin did not significantly affect the proteins expression of ACC,CPT1,PGC1?,and PGC1?,but at 1 h,AMPK phosphorylation was inhibited.The results suggested that recombinant adiponectin may promote glucose and fatty acid uptake,inhibit FAS enzyme activity thereby inhibiting lipid synthesis.In addition,recombinant adiponectin significantly(P<0.01)decreased adiponectin mRNA expression in adipocytes,indicating that a negative feedback mechanism of adiponectin existed in adipocytes.Secondly,adipocytes were treated with adenovirus overexpression of globular adiponectin,and the results showed that the adenovirus overexpression of globular adiponectin significantly(P<0.05)increased the intracellular and extracellular lipid content.The mRNA expression of adiponectin and its receptor in adipocytes and the adiponectin content in the cell medium were significantly(P<0.05)increased after adenovirus overexpression of globular adiponectin transfection.Adenovirus overexpression of globular adiponectin also significantly increased the mRNA expression of lipid metabolism-related genes of PPAR?,SREBP1,PPAR?(P<0.05)and the phosphorylated AMPK(P<0.01).The above results indicated that the adenovirus overexpression of globular adiponectin increased the lipid content inside and outside adipocytes and activated AMPK.In summary,the results showed that adiponectin played a lipid-lowering role in chicken primary hepatocytes and myoblasts,but their mechanism was not the same.For chicken primary adipocytes,the effect of exogenous addition of recombinant adiponectin and endogenous adenovirus over-expressing globular adiponectin on lipid metabolism were not consistent,but both affected the AMPK phosphorylation.