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First published online 9 December 2008
doi: 10.1242/jcs.027508

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Mild mitochondrial uncoupling induces 3T3-L1 adipocyte de-differentiation by a PPAR-independent mechanism, whereas TNF-induced de-differentiation is PPAR dependent

¹ Laboratory of Biochemistry and Cellular Biology, University of Namur (FUNDP), 61 Rue de Bruxelles, 5000 Namur, Belgium
² Eppendorf Array Technologies, 12 Rue du Séminaire, 5000 Namur, Belgium

View larger version (36K): [in this window] [in a new window]   Fig. 1. Effect of FCCP treatment on mitochondrial membrane potential and triglyceride content in differentiated adipocytes. (A) Determination of the m was assessed after differentiated (DIFF) adipocytes were not incubated or were incubated with 0.5 µM TMRE with or without 10 ng/ml TNF or 0.5 µM FCCP before FACS analysis. As positive control, differentiated cells were treated with 10 µM FCCP. (B,C) For TG determination, differentiated cells were incubated for 3 (white columns) or 6 days (black columns) with 10 ng/ml TNF or 0.5 µM FCCP. TG vesicles were then stained with ORO and the relative TG content in cell monolayers was (B) determined at 490 nm with a spectrophotometer and (C) visualized by phase-contrast microscopy (magnification 250x). (D) Glycerol release was measured in the 24-hour-old media using the INT kit, and (E) TG content was determined after lipid extraction in methanol-chloroform (using the same kit) in differentiated (DIFF) and undifferentiated (CTL) cells and then incubated for 1 (black columns), 3 (grey columns) or 6 (white columns) days with or without 10 ng/ml TNF, 0.5 µM FCCP or 10 µM isoproterenol (ISO). Representative results are expressed as percentages of differentiated cells. Values are means ± s.d. for n=3 experiments. ***P<0.001, significantly different from DIFF. ##P<0.01; ###P<0.001, significantly different from DIFF+ISO.

View larger version (37K): [in this window] [in a new window]   Fig. 2. Representative time-course gene expression profiles in 3T3-L1 cells. Differentiated (DIFF) cells incubated for 1, 3 or 6 days with or without (diamonds and black line) 10 ng/ml TNF (squares and short dashed line) or 0.5 µM FCCP (triangle and long dashed line). The gene expression profile as a function of time determined by cDNA microarray is illustrated for (A) AGT, (B) CHOP-10, (C) CPT 2 and (D) PPAR. Results are expressed as relative transcript abundance, transformed to log2. Set of genes displaying profiles similar to AGT and CTP 2 are listed between A and B and C and D, respectively. (E,F) Effect of FCCP or TNF on the DNA-binding activity of PPAR and C/EBP in adipocytes. DNA-binding activity for (E) PPAR and (F) C/EBP was determined in nuclear proteins prepared from differentiated (DIFF) and undifferentiated cells (CTL) and then incubated for an extra 24 (white columns) or 72 hours (black columns) with or without 10 ng/ml TNF, 0.5 µM FCCP or 10 µM isoproterenol using a Trans-AM assay. Results are expressed as percentages of DNA-binding activity found in differentiated cells as means ± s.d. for 3 experiments. *P<0.5, **P<0.01 and ***P<0.001, significantly different from DIFF cells. (G) Western blot analysis for PPAR was performed using the same conditions as above. Equal protein loading was controlled by the TBP immunodetection.

View larger version (34K): [in this window] [in a new window]   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.

View larger version (55K): [in this window] [in a new window]   Fig. 4. Effect of FCCP, TNF and isoproterenol in the expression of ATGL, HSL and perilipin A. (A) Quantitative gene expression analysis by real time RT-PCR of ATGL in differentiated 3T3-L1 (DIFF) incubated for 1 (white columns) or 3 (black columns) days with 10 ng/ml TNF, 0.5 µM FCCP or 10 µM isoproterenol. For data normalization, TBP was used as reference gene. Results are expressed in relative fold increase as means ± s.d. for n=3 experiments, ***P<0.001 and **P<0.01 significantly different from DIFF. (B) Western blot analysis for HSL, ATGL and perilipin A abundance was performed using the same conditions as above. (C) HSL phosphorylation (Ser563 and Ser660) status and abundance were analyzed after short incubation times using the same conditions as above. Equal protein loading was controlled by the immunodetection of -tubulin in all western blot experiments. Abundance and localization of HSL and perilipin A in adipocytes. (D,E) Differentiated cells (DIFF) were incubated on coverslips for 3 days with or without 10 ng/ml TNF, 0.5 µM FCCP or 10 µM isoproterenol before immunostaining and confocal microscopic analysis. At the end of the incubations cells were fixed, permeabilized and then incubated with the specific antibodies (green fluorescence) against HSL (D) and perilipin A (E). For nuclear staining TOPRO-3 was used (blue fluorescence).

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