Perilipin A Regulates Lipolysis Stimulated By Chronic High Insulin And IL-6 Level And The Molecular Mechanisms

The enzymatic hydrolysis of stored neutral lipid in adipocytes is an exquisitely regulatedprocess that maintains whole body energy homeostasis and is vital to metabolic health.Perilipin A is a important structural and functional protein,which locates on the surface oflipid droplets in adipocytes and acts as one of terminal targets among several lipolyticpathway,so it plays a key regulatory role during adipocytes lipolysis.Hormones andcytokines are important factors affecting lipolysis.However,up to now,the regulatorymechanisms of perilipin A during chronic high insulin and IL-6 stimulate lipolysis remainsunclear.Moreover,Porcine has more abundant body fat than rodents,and has many similarphysiological characters to human.Thus,porcine is regarded as an important animal modelfor researching human obesity and metabolic diseases.While at present,there are no reportsabout perilipin A regulating porcine lipolysis,and the effect of chronic high insulin and IL-6level on porcine adipocytes lipolysis also remains unclear.In this study,porcine preadipocytes were isolated from 1-7 days old piglets and culturedto induce differentiation.Then the differentiated porcine adipocytes were incubated withdifferent insulin and IL-6 concentrations for various times(24h or 48h).After that,theconcentration of glycerol and free fatty acids(FFAs)in the media was measured as anindicator of the lipolysis and FFA consumption.Lipid accumulation morphology wasvisualized by light microscopy.Further,gene expression ofperilipin A,PPARγ2,HSL,ATGL,TNFα,IL-6,PGC-1α,CPT-1 and UCP2 were determined with semi-quantitative RT-PCR andperilipin A protein expression was detected by western blot.Meanwhile,PKA and ERKinhibitors were used to determine the involved lipolytic pathway,and rosiglitazone was usedto analyze the regulatory role of perilipin A in IL-6 treatment lipolysis.The results are asfollows:1.Chronic high insulin dose significantly promoted lipolysis in porcine adipocytes.Theexpression of perilipin A protein were significantly reduced as well as the mRNA expressionof perilipin A and PPARγ2,however,the mRNA expression of TNFαand IL-6 wassignificantly increased. 2.The inhibitors of both PKA(H89)and ERK(PD98059)could repress the lipolysisstimulated by chronic high insulin concentration partially,and the combination of them couldrepress the lipolysis completely.Moreover,PD98059 could inhibit the down regulation ofperilipin A induced by insulin,but H89 did not present this kind of role.3.IL-6 receptor system was expressed in porcine adipocytes.Chronic high IL-6 dosesignificantly promoted lipolysis in porcine adipocytes.Furthermore,the mRNA expression ofperilipin A and PPARγ2 were significantly reduced,but the expression of PGC-1α,CPT-1 andUCP2 mRNA was significantly increased.4.The inhibitor of PKA(H89)didn't affect the lipolysis stimulated by IL-6(100ng/mL).However,the inhibitor of ERK(PD98059)had significant inhibitory role in this kind oflipolysis,and the down regulation ofperilipin A induced by IL-6 was significantly repressed.5.Rosiglitazone significantly repressed the lipolysis stimulated by IL-6 in porcine adipocytes,and lipolytic response of the adipocytes to isoprenaline was enhanced.Meanwhile,rosiglitazone significantly increased the mRNA expression of perilipin A,PPARγ2,FAS andPGC-1α的mRNA,but reduced the expression oflL-6 mRNA.Above all,Chronic high insulin dose significantly promoted lipolysis in porcineadipocytes,and decreased the expression of perilipin A protein and mRNA,this process couldbe associated with the up regulation of IL-6 and TNFα.Both PKA and ERK pathwaymediated the lipolysis stimulated by chronic high insulin dose,but the down regulation ofperilipin A expression was only involved in ERK pathway,rather than PKA pathway,so weinfered that the means of PKA lipolytic pathway during insulin treatment lipolysis wasinvolved in the phosphorylation of perilipin A and activation of HSL and ATGL.Chronichigh IL-6 dose directly stimulated lipolysis in porcine adipocytes by repressing perilipin Aand promoting gene expression of PGC-1α,CPT-1 and UCP2.This process was onlymediated by ERK pathway.Rosiglitazone repressed the lipolysis stimulated by IL-6 inporcine adipocytes by increasing perilipin A expression,which maybe related to the upregulation of PPARγ2 and FAS as well as the down regulation of IL-6.