The Effect Of PPARγ On Differentiation Of 3T3-L1 Preadipocytes And The Relationship With 11β-HSD1

Objective:The proliferation and differentiation of adipocytes result inadipogenesis and energy metabolism disequilibrium. Obesity and kinds ofdiseases related with obesity occur. Peroxisome proliferator-activatedreceptors (PPARs) are members of nuclear receptor superfamily and onekind of transcription factors. The activation of PPARs affects the pathwayof adipocyte differentiation. 11β-hydroxysteroid dehydrogenase type 1(11β-HSD1) is one of microsome enzymes, which converts biologicallyinactive cortisone to active cortisol. Glucocorticoids have been shown topotentiate the proliferation, differentiation and apoptosis of adipocytes. Inthe present study, 3T3-L1 preadipocytes were first induced todifferentiate into mature adipocytes, followed by incubation inPPARγagonist-pioglitazone and PPARγantagonist-GW9662. Themorphology of adipocytes and mRNA levels of differentiation markergenes were observed.Methods:1. Confluent 3T3-L1 preadipocytes were treated with 0.5mM IMX, 2uM dexamethasone and 10ug/ml insulin together for 48h, followed bystandard medium DMEM culture with 10ug/ml insulin for another 48h,then refeeding with standard medium. During the course of differentiation,PPARγagonist-pioglitazone was added to the medium. We observed theadipocytes on day0, day4 and day8 under microscope, respectively andreal-time PCR was used to study the expression of differentiation markergenes.2. Western blot and real-time PCR were used to observe the effect ofdifferent levels of PPARTagonist and antagonist on mRNA expression of11β-HSD1 and GR. During the course of adipocyte differentiation,mRNA expression of 11B-HSD1 and GR was also detected.Results:1. After differentiation, the 3T3-L1 preadipocytes turned to be largerand round rather than of spindle shape, and contained large droplets oftriglycerides. Over 80% of the preadipocytes appeared to be differentiatedinto mature adipocytes on day8. Over 60% of the cells treated withpioglitazone appeared to be differentiated into mature adipocyte on day4and over 95% of the cells on day8. The mRNA expression of markergenes such as C/EBPα,LPL,FAS,aP2 and GLUT4 upregulated duringthe differentiation day by day and in the cells stimulated withpioglitazone the expression increased more than the control cellsdramatically (P＜0.01). 2. 11β-HSD1 protein expression was up-regulated during the courseof adipocyte differentiation, but compared with those found in untreatedcells, the protein levels were always lower. When cells treated withGW9662, the mRNA expression of 11β-HSD1 increased as opposed tothe effect of pioglitazone (P＜0.01). Gene expression of 11β-HSD1decreased when cells treated with pioglitazone and increased whentreated with GW9662 in a dose- and time- dependent manner (P＜0.01).Conclusion:1. The differentiation program in the present study turned to be higheffectively suitable for 3T3-L1 preadipocytes and more than 80% of thepreadipocytes appeared to be differentiated into mature adipocytes onday8 when the oil droplets were obvious in the cytoplasm. PPARγagonist-pioglitazone promote the differentiation of 3T3-L1 preadipocytes.2. Gene expression of 11β-HSD1 decreased when cells treated withpioglitazone and increased when treated with GW9662 in a dose- andtime- dependent manner, but the expression of GR displayed diversetrend. The effect of PPARγagonist and 11β-HSD1 on adipogenesis mayhave crosslink and the interaction may exist between PPARγand GR.