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. The aim of this study was to provide the experimental basis for further investigating on adiponectin (ADPN) function. Its eukaryotic recombinant was constructed and expressed in precursor cells of 3T3-L1 adipocytes. The effects of dexamethasone on peroxisome proliferator activated receptor-gamma (PPAR-gamma) mRNA expression in 3T3-L1 cells with human recombinant adiponectin were assessed.


METHODS
The recombinant plasmid pMD18-T-hADPN and eukaryotic expression vector pcDNA3.1(+) were digested by two restrictive endonucleases and adiponectin and linear pcDNA3.1(+) were obtained. Then, they were ligated and translated into JM109. The recombinant pcDNA3.1(+)-hADPN so obtained was identified by digestion by restrictive endonuclease and nucleotide sequencing. The 3T3-L1 precursor cells were transfected using SuperFect Transfection Reagent (Qiagen). Furthermore, 3T3-L1 cells with human recombinant adiponectin incubated with dexamethasone (0.5 mmol/L) for 24 hours, cells were collected and total RNA was extracted. The PPAR-gamma mRNA expression was quantified by 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 were identified by nucleotide sequence scanning and consistent with theoretical values. Electrophoretogram of RT-PCR in 3T3-L1 precursors showed only one band in front of 250 bp, which was consistent with theoretical value 234 bp. In the 3T3-L1 cells, 3T3-L1 cells with plasmid and 3T3-L1 cells human recombinant adiponectin, treatment with dexamethasone (0.5 mmol/L) decreased PPAR-gamma mRNA expression compared to untreated controls (P < 0.01). Effect of dexamethasone on PPAR-gamma mRNA expression in 3T3-L1 cells was reversed by stably transfected human recombinant adiponectin.


CONCLUSIONS
The 3T3-L1 cells stably transfected human recombinant adiponectin had increased PPAR-gamma mRNA expression. Dexamethasone suppressed PPAR-gamma mRNA expression in the 3T3-L1 cells. Effect of dexamethasone on PPAR-gamma mRNA expression in 3T3-L1 cells was reversed by stably transfected human recombinant adiponectin.
She
Qi-mei
QM
Department of Endocrinology, Shijingshan Hospital of Beijing, Beijing 100043, China.
Zhao
Jing
J
Wang
Xia-lian
XL
Zhou
Chang-man
CM
Shi
Xian-zhong
XZ
D041721:3T3-L1 Cells; D052242:Adiponectin / Q000502:physiology*; D000818:Animals; D003907:Dexamethasone / Q000494:pharmacology*; D007333:Insulin Resistance; D051379:Mice; D047495:PPAR gamma / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D052242:Adiponectin; D047495:PPAR gamma; D012333:RNA, Messenger; D003907:Dexamethasone