Effect of dexamethasone on peroxisome proliferator activated receptor-gamma mRNA expression in 3T3-L1 adipocytes with the human recombinant adiponectin

Background The fat derived protein adiponectin plays an important role in the regulation of glucose metabolism.Theaim of this study was to provide the experimental basis for further investigating on adiponectin （ADPN） function.Itseukaryotic recombinant was constructed and expressed in precursor cells of 3T3-L1 adipocytes.The effects ofdexamethasone on peroxisome proliferator activated receptor-gamma （PPAR-γ） mRNA expression in 3T3-L1 cells withhuman recombinant adiponectin were assessed.Methods The recombinant plasmid pMD18-T-hADPN and eukaryotic expression vector pcDNA3.1were digested bytwo restrictive endonucleases and adiponectin and linear pcDNA3.1were obtained.Then,they were ligated andtranslated into JM109.The recombinant pcDNA3.1-hADPN so obtained was identified by digestion by restrictiveendonuclease and nucleotide sequencing.The 3T3-L1 precursor cells were transfected using SuperFect TransfectionReagent （Qiagen）.Furthermore,3T3-L1 cells with human recombinant adiponectin incubated with dexamethasone （0.5mmol/L） for 24 hours,cells were collected and total RNA was extracted.The PPAR-γ mRNA expression was quantifiedby semiquantitative reverse transcription-polymerase chain reaction （RT-PCR）.Results After eukaryotic recombinant was digested by Hind Ⅲ and EcoR Ⅰ,fragments of 800 bp and 5.4 kb wereidentified by nucleotide sequence scanning and consistent with theoretical values.Electrophoretogram of RT-PCR in3T3-L1 precursors showed only one band in front of 250 bp,which was consistent with theoretical value 234 bp.In the3T3-L1 cells,3T3-L1 cells with plasmid and 3T3-L1 cells human recombinant adiponectin,treatment withdexamethasone （0.5 mmol/L） decreased PPAR-γ mRNA expression compared to untreated controls （P<0.01）.Effect ofdexamethasone on PPAR-γ mRNA expression in 3T3-L1 cells was reversed by stably transfected human recombinantadiponectin.Conclusion The 3T3-L1 cells stably transfected human recombinant adiponectin had increased PPAR-γ mRNAexpression.Dexamethasone suppressed PPAR-γ mRNA expression in the 3T3-L1 cells.Effect of dexamethasone onPPAR-γ mRNA expression in 3T3-L1 cells was reversed by stably transfected human recombinant adiponectin.