Effects of TZDs on expression and secretion of adiponectin in 3T3-L1 adipocytes. Dose effect of TGZ on the adionectin mRNA
level (A) and the secreted amount in media (B) is shown. After differentiation-induction on day 7, 3T3-L1 cells were treated with the indicated concentrations of TGZ,
0, 1, 3, and 10 μmol/l for 24 h. Five micrograms of total RNA was subjected to Northern blotting and quantified and normalized
relative to the 18-s rRNA signal. All mRNAs are plotted as percentage change relative to mRNA level by 0 μmol/l TGZ treatment.
The amount of adiponectin secreted into media was measured by quantitative Western blotting. The inset shows a representative
picture of Northern and Western blotting that was quantified. Time curve of the effect of 10 μmol/l TGZ on adiponectin mRNA
(C) is shown. After differentiation-induction on day 7, 3T3-L1 cells were treated with 10 μmol/l TGZ for the indicated time.
Northern blotting and quantification was performed as described in the legend of A. After differentiation-induction on day 7, 3T3-L1 cells were treated with 10 μmol/l TGZ, PGZ, RGZ, and Wy14643, respectively
(D). Northern blotting and quantification was performed as described for A. A 2.0-kb adiponectin promoter was subcloned into pGL3-luciferase vector (E). Adiponectin-luciferase and pRL-SV40 vector were cotransfected into day 7–differentiated 3T3-L1 cells. For each 12-well
dish, 1 μg adiponectin-luciferase and 10 ng pRL-SV40 were used. Three hours after transfection, the cells were switched to
medium containing vehicle (DMSO), or 10 μmol/l TGZ (T), PGZ (P), RGZ (R), or Wy14643 (W) for 24 h. Firefly and Renilla luciferase reporter activity was measured as described in research design and methods. Adiponectin-luciferase (firefly) activities were normalized by pRL-SV40 Renilla luciferase activity. Values are means ±
SE from three independent exeriments. *P < 0.01 compared with control values.