The Effects And Molecular Mechanisms Of LRP16Gene On Insulin Resistance In3T3-L1Adipocytes

Objective: To investigate the effects and possible molecular mechanisms ofLRP16gene on insulin resistance in murine3T3-L1adipocytes.Methods:(1) The LRP16gene over-expressing adipocyte cell line3T3-L1-LRP16and its control cell line3T3-L1-3.1were constructed usinglipidosome transfection technology. They were cultured and induced fordifferentiating into adipocytes by regular method. Before the followingexperiments, the adipocytes were pretreated with or without100μM NF-κBinhibitor PDTC for24hours, then were incubated with100nM insulin for30minutes at37℃. The insulin-stimulated glucose uptake rates were determined by2-deoxy-[3H]-D-glucose assay. Expressions of key proteins in IRS-1signalingpathway such as pIRS-1(Ser307), pIRS-1(Tyr1179), PI3-K(p85), pAkt(Ser473) andpAkt(Thr308), were detected by Western blot respectively.(2) Full-differentiated3T3-L1-LRP16and3T3-L1-3.1adipocytes, with or without pretreatment of100μM PDTC for24hours, were incubated with100nM insulin for30minutes.The protein contents of both nuclear NF-κB p65and total NF-κB p65weredetermined by Western blot assay. The intracellular distributions of NF-κB p65proteins in3T3-L1-3.1and3T3-L1-LRP16adipocytes were detected byimmunofluorescence analysis. The contents of active NF-κB in the nuclearextracts prepared from3T3-L1-LRP16and3T3-L1-3.1adipocytes weredetermined by non-radioactive electrophoretic mobility shift assay (EMSA) andsupershift assay to evaluate the DNA binding activity of NF-κB p65protein. Helacells were transiently co-transfected with the structural expression vectorsincluding pNF-κB-Luc, pcDNA-3.1-LRP16, pcDNA-3.1, and the internal controlvector pRL-TK, followed by treatment of100μM PDTC. Then the NF-κB relativeluciferase activity which reflected the transcriptional activity of NF-κB was detected using a Dual-luciferase Reporter Gene Assay system.(3) The mRNAexpressions of inflammatory cytokines in3T3-L1-3.1and3T3-L1-LRP16adipocytes such as TNF-α, IL-6, IL-1β were measured by real-time quantitativePCR. The concentrations of TNF-α, IL-6, and IL-1β in the supernatants ofcultured3T3-L1-3.1and3T3-L1-LRP16adipocytes were measured using ELISAkits.Results:(1) Over-expression of LRP16gene significantly inhibited theinsulin-stimulated glucose uptake in3T3-L1adipocytes: the levels of glucoseuptake in3T3-L1-LRP16adipocytes were significantly reduced to45.7%of thatin3T3-L1-3.1adipocytes (P<0.05). LRP16over-expression in3T3-L1adipocytesalso increased the protein expression of pIRS-1(Ser307), but decreased theexpressions of pIRS-1(Tyr1179), PI3-K(p85), pAkt(Ser473) and pAkt(Thr308).PDTC treatment abolished the above inhibitory effects of LRP16oninsulin-stimulated glucose uptake and IRS-1signaling.(2) Over-expression ofLRP16gene enhanced the DNA binding activity of NF-κB p65,EMSA showedthe DNA binding activity of NF-κB p65in3T3-L1-LRP16adipocytes was2.6-fold of that in3T3-L1-3.1adipocytes. LRP16also promoted the nucleartranslocation of NF-κB p65,and increased the expression of nuclear NF-κB p65protein in3T3-L1adipocytes. LRP16enhanced the transcriptional activity ofNF-κB in a dose-dependent manner in Hela cells, which could be abolished bytreatment with a NF-κB inhibitor of PDTC.(3) LRP16gene enhanced theexpressions of inflammatory cytokines: the relative levels of IL-6, IL-1β andTNF-α mRNA transcripts in3T3-L1-LRP16adipocytes were4.53,7.17and9.58-fold of that in3T3-L1-3.1adipocytes, respectively (P<0.05). Theconcentrations of IL-6, IL-1β and TNF-α in the supernatants of cultured3T3-L1-LRP16adipocytes were1.54,1.78and2.13-fold of that in3T3-L1-3.1adipocytes, respectively (P<0.05).Conclusions: LRP16gene enhanced the transcription of NF-κB, promotedthe nuclear translocation and DNA binding of NF-κBp65, upregulated the expressions of inflammatory cytokines, subsequently impaired the IRS-1signaling pathway and induced insulin resistance in3T3-L1adipocytes.