The Effects Of TNF-α And Pioglitazone On Expression Of Adiponutrin And PPARγ In 3T3-L1 Adipocytes

Objective To investigate the effects of TNF-αand pioglitazone on adiponutrin mRNA level in 3T3-L1 adipocytes and its time-effect.Methods 3T3-L1 cells during differentiation (2d,4d,6d,8d) and full differentiation (6h,12h,24h,48h,72h) were incubated with Pioglitazone(100μmol/l) or TNF-α(100ng/mL) in their optimal concentration. Total RNA or protein was extracted from homogenized cells in various time of cell differentiation. Every condition was repeated three times. Semi-quantitative RT-PCR used to test adiponutrin gene expression and western blotting used to test the varies of PPARγprotein levels .Results (1) The expression of adiponutrin mRNA and PPARγprotein was lower in the undifferentiated 3T3-L1 adipocytes.Adiponutrin expression increased dramatically in 3T3-L1 cells. The level of PPARγwas gradually increased with cells differentiation(P <0.05). When cells were fully differentiated, PPARγexpression similarly increased and got its peak at full differentiation 3T3-L1 adipocytes for 48 hours. (2) The effects of TNF-α(100ng/mL) and Pioglitazone(100umol/l )on Adiponutrin mRNA and PPARγexpression in 3T3-L1 adipocytes for different time were investigated. By treated with TNF-α, the adiponutrin mRNA expression was increased in 3T3-L1 cells. In contrast, pioglitazone dramatically downregulated adiponutrin mRNA (P <0.05) in a time-dependent manner. Contrast to the control, TNF-αstrongly downregulated PPARγexpression during adipose conversion and fully differentiated 3T3-L1 cells.When cells were incubated with pioglitazone,PPARγprotein were all elevated in differentiating cells.While pioglitazone had no influence on the expression of PPARγin differentiated 3T3-L1 adipocytes.(3)The differentiated 3T3-L1 adipocytes were treated with 100ng/mL TNF-αand pioglitazone together for 48 hours. Adiponutrin mRNA expression was downregulated by 16.7% compared with the control. (4) Adiponutrin mRNA had no correlation with PPARγ.Conclusions Taken together; the adiponutrin mRNA expression in the differentiated 3T3-L1 adipocytes is higher than that in 3T3-L1 preadipocytes. Adiponutrin mRNA expression were modulated by TNF-αand pioglitazone in the 3T3-L1 cells. Pioglitazone can suppress adiponutrin mRNA expression in the 3T3-L1 adipocytes.On the contrary, TNF-αcan increase it. And pioglitazone can also suppress the effects of TNF-αon expression of adiponutrin mRNA in the 3T3-L1 adipocytes. pioglitazone treatment resulted in a 42.3% decrease in the level of adiponutrin mRNA expression (P<0.001); TNF-αtreatment was associated with a 31.5% increase(P <0.001). The PPARγlevels were with a significant trend toward decreased expression in TNF-αtreatment. Nevertheless pioglitazone treatment was associated with increasein PPARγexpression.