The Impacts Of Testosterone On Inflammatory Factors And Insulin Sensitivity In 3T3-L1 Adipocytes And Their Related Molecular Mechanisms

PartⅠThe impacts of testosterone on productions of inflammatory factors in 3T3-L1 adipocytes and screeening the key moleculars of signalling pathway related of the productions of inflammatory factorsObjectives:The metabolic inflammtion(low-grade chronic inflammtion) is common in PCOS patients,which are characterized by hyperandrogenism or hyperandrogenemia.The clinical researches have proved that the hyperandrogenemia is closely correlated with the metabolic inflammation,but the mechanism is unclear.We conduct this study to ellucidate the time and dose effects of androgen on pruduction of some inflammation factors in 3T3-L1 adipocytes,and to screen the key moleculars of signalling pathway related of the productions of inflammatory factors.Methods:(1)Without lipopolysaccharide(LPS) treatment,namely in basic state,matured 3T3-L1 adipocytes were treated with testosterone at a concentration of 10^-9 to 10^-5 mol·L^-1 for either short(10 or 30 minutes) or long(12 hours,24 hours and 48 hours) treatment course,none-testosterone treatment groups were the controls.Secretion of inflammatory factors,i.e.interleukin-6(IL-6) and monocyte chemoattractant protein-1(MCP-1),in the culture medium were determined by enzyme-linked immunosorbent assay(ELISA).mRNA expression of IL-6 and MCP-1 were determined by reverse transcriptase PCR. Phosphorylation and total proteins of NF-κB,p38MAPK and ERK1/2 were analyzed by western blot withβ-actin as the reference gene.(2)In LPS-stimulated state,adipocytes were treated with LPS for 6 hours.Before the LPS treatment,testosterone at a concentration of 10-9 to 10-5 mol/L for either short or long period was given.Cells treated with LPS served as controls.Likely,secretion of IL-6 and MCP-1,in the culture medium were determined by ELISA.mRNA expression of IL-6 and MCP-1 were determined by RT-PCR.Phosphorylation and total proteins of NF-κB,p38MAPK and ERK1/2 were analyzed by western blot withβ-actin as the reference gene. Results:(1)In basic state,①the secretion of IL-6 and MCP-1 in the culture medium were higher in the testosterone-treated groups than in the control groups(P＜0.05).Compared to other testosterone-treated groups,the concentration of IL-6 and MCP-1 were higher in the group treated with 10^-5M testosterone for 24 hours(P＜0.05).②Compared to the control groups,the mRNA expression of IL-6 and MCP-1 were higher in the groups treated with testosterone for 12 hours,especially with testosterone of 10^-5M and 10^-9 M(P＜0.05).③With a short treatment course of testosterone,more NF-κB and ERK1/2 were phosphated than in control,especially with testosterone of 10^-5M.More NF-κB and ERK1/2 were phosphated following the testosterone treatment(10-9,10-7,10-5M) for 12h,especially with a testosterone concentration of 10^-9 and 10^-5M(p＜0.05.While no more p38MAPK were phosphated in testosterone-treated groups than in controls.(2)In LPS-stimulated-state,the secretion of IL-6 and MCP-1 in the culture medium were higher in the 10^-5M testosterone-pretreated groups than in the only-LPS-treated groups (P＜0.05),especially treated for 48 hours(p＜0.05);While the secretion of IL-6 and MCP-1 in the culture medium were lower in the 10^-7M testosterone-treated groups than in the only-LPS-treated groups(p＜0.05);②The mRNA expression of IL-6 and MCP-1 were higher in 10^-5M testosterone-pretreated 12h groups than that in only-LPS-treated groups(p＜0.05), And the mRNA expression of IL-6 and MCP-1 were lower in 10^-7M testosterone-pretreated groups than that in only-LPS-treated groups(p＜0.05).③More NF-κB and ERK1/2 were phosphated in 10^-5M testosterone pretreated groups than in only-LPS-treated groups especially in 12h(p＜0.05).Less NF-κB and ERK1/2 were phosphated in 10^-7M testosterone pretreated groups than in only-LPS-treated groups(p＜0.05).And no more p38MAPK were phosphated in testosterone-pretreated groups than in only-LPS-treated groups(p＞0.05).Conclusion:1.Testosterone could increase the expression and secretion of inflammatory factors in 3T3-L1 adipocytes;and high dose testosterone could promote the LPS-stimulated inflammatory factors production.2.Testosterone could activate the lower signaling molecular NF-kB and ERK1/2. PartⅡTo testify the hypothesis that testosterone increases the expression of inflammatory factors in adipocytes through the activation of NF-κB and ERK1/2.Objectives:To further testify the hypothesis that testosterone increases the expression of inflammatory factors in adipocytes through the activation of NF-κB and ERK1/2,namely NF -kB and ERK1/2 are the "cross-talk" between androgen signal pathway and inflammatory signal pathway.Methods:(1)In basic state,matured adipocytes were pretreated with PDTC(inhibitor of NF-κB) or PD98059(inhibitor of ERK1/2)for 2 hours,then treated with 10^-5mol/L testosterone for 24h,and the secretion of IL-6 and MCP-1 in the culture medium were determined by ELISA.And matured adipocytes were pretreated with PDTC(inhibitor of NF-κB) or PD98059(inhibitor of ERK1/2)for 2 hours,then treated with 10^-5mol/L testosterone for 24h,the mRNA expression of IL-6 and MCP-1 were determined by RT-PCR. Phosphorylation and total proteins of NF-κB and ERK1/2 were analyzed by western blot withβ-actin as the reference proteins.And the ability of NF-κB binding to DNA was determined by EMSA.(2)In LPS-stimulated state,adipocytes were treated with LPS for 6 hours.Before the LPS treatment,testosterone was given at a concentration of 10^-5 mol/L for 12hours or 48 hours.Cells treated with LPS served as controls.In another experiment,adipocytes were manipulated following the same protocol except that cells were pretreated with PDTC (inhibitor of NF-κB) or PD98059(inhibitor of ERK1/2) for 2 hours.Concentrations and mRNA expression of IL-6 and MCP-1,and phosphorylation of NF-κB and ERK1/2 were measured in each experiment.And the ability of NF-κB binding to DNA was determined by EMSA.Results:In both conditions,PD98059 could partially reverse the impacts of testosterone on pruductions of IL-6 and MCP-1 in 3T3-L1 adipocytes;while PDTC could completely reverse impacts of testosterone on pruductions of IL-6 and MCP-1 in 3T3-L1 adipocytes.Conclusions:In basic state and in LPS-stimulated state,higher concentration of testosterone could promote the production and secretion of IL-6 and MCP-1 via activating the pathway ERK 1/2/NF-κB. PartⅢTo explore the molecular mechanisms testosterone on insulin sensitivity in 3T3-L1 adipocytesObjectives:To explore the molecular mechanisms of testosterone on insulin sensitivity in 3T3-L1 adipocytes in vitro.Methods:In basic state,matured 3T3-L1 adipocytes were treated with 10^-5mol/L testosterone for 12 hours.While in LPS-stimulated state,3T3-L1 adipocytes were treated with LPS for 6 hours.Before the LPS treatment,testosterone was given at a concentration of 10^-5 mol/L for 12 hours.In another experiment,adipocytes were manipulated following the same protocol except that cells were pretreated with PDTC(inhibitor of NF-κB) or PD98059(inhibitor of ERK1/2) for 2 hours.The productions of IRS-1,Tyr941p IRS-1,GLUT-4 and the membrane proteins of GLUT-4 stimulated by insulin were analysed by western blot.Results:(1) In basic state,3T3-L1 adipocytes treated with 10^-5M testosterone within 12h obviously increased pruduction of IRS-1 and GLUT-4(P＜0.05),while decreased the insulin-stimulated Tyr941p IRS-1 and membrane protein GLUT-4 production(P＜0.05).While pretreated with PDTC(inhibitor of NF-κB) or PD98059(inhibitor of ERK1/2),the decreased production of insulin-stimulated Tyr941p IRS-1 and membrane protein GLUT-4 could be partially reversed.(2) In LPS-stimulated state,pretreated with 10^-5mol·1^-1 testosterone for 12 hours,the protein of IRS-1 were obviously increased(P＜0.05)and GLUT-4 were obviously decreased (P＜0.05),while the insulin-stimulated Tyr941p IRS-1 and membrane protein GLUT-4 production were obviously decreased,too(P＜0.05).Pretreated with PDTC(inhibitor of NF-κB) or PD98059(inhibitor of ERK1/2),the decreased production of insulin-stimulated Tyr941p IRS-1 and membrane protein GLUT-4 could be partially reversed,too.Conclusion:Testosterone could induce insunlin resistance via ERK1/2/NF-κB pathway.