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Fig. 3. Effect of PPAR and RXR ligands (Lig) in 3T3-L1 adipocytes responding to TNF or FCCP. (A) Differentiated cells (DIFF) were transiently cotransfected with a PPAR-sensitive luciferase reporter construct and with a β-galactosidase expression vector. After transfection, cells were incubated for 24 (white columns) or 72 hours (black columns) with or without 10 ng/ml TNF, 0.5 µM FCCP or with rosiglitazone (10 µM) and 9-cis retinoic acid (10 µM) before luciferase assay. Results were normalized for β-galactosidase activity and expressed as fold increase; values are means ± s.d. for 3 experiments, ^#P<0.05, ^##P<0.01 and ^###P<0.001, significantly different from DIFF, and ^**P<0.01 and ^***P<0.001, significantly different from the corresponding condition without ligands. (B) Differentiated or undifferentiated (CTL) cells incubated for an extra 6 days with or without 10 ng/ml TNF or 0.5 µM FCCP (black columns), in the presence or absence of 10 µM rosiglitazone and 10 µM 9-cis retinoic acid (grey columns), were stained with ORO and the TG content in cells was then quantified by measuring the absorbance of cell monolayers with a spectrophotometer at 490 nm. Results are expressed in optical density values as means ± s.d. for n=3 experiments. ^##P<0.01, ^###P<0.001, significantly different from DIFF and ^***P<0.001, significantly different from DIFF+TNF cells. NS (not significant) versus DIFF+FCCP. (C) Western blot analysis for PPAR abundance, using the same conditions as above. Equal protein loading was controlled by the immunodetection of TBP. Effect of TNF or FCCP on FFA β-oxidation. (D) After 1 (white columns) or 3 days (black columns) of incubation with or without 10 ng/ml TNF or 0.5 µM FCCP, preadipocytes, adipocytes and de-differentiated adipocytes were labelled with [¹⁴C]oleate. Released CO₂ was harvested on filter papers and the radioactivity counted. Results are expressed in cpm ¹⁴CO₂ normalized for cpm of the [¹⁴C]oleate uptake and for protein content. Results are presented as percentages of controls (preadipocytes); values are means ± s.d. for n=3 experiments. ^*P<0.05, ^**P<0.01 and ^***P<0.001 are significantly different from the corresponding control. NS (not significant) versus differentiated cells for both times. Effect of TNF or FCCP on lipid synthesis. (E) [³H]acetate was added during the last 24 hours of adipocyte incubation with or without 10 ng/ml TNF or 0.5 µM FCCP during 1 (white columns) or 3 days (black columns) of treatment. Incorporation of [³H]acetate into lipids was quantified after lipid extraction (cpm) and normalized for protein content. Values are mean ± s.d. for n=3 and n=4 experiments respectively. ^**P<0.01, ^***P<0.001, significantly different from DIFF.

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