QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 14 March 2006
doi: 10.1242/jcs.02848

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Vankoningsloo, S. Articles by Arnould, T. Search for Related Content PubMed PubMed Citation Articles by Vankoningsloo, S. Articles by Arnould, T. Social Bookmarking                         What's this?

CREB activation induced by mitochondrial dysfunction triggers triglyceride accumulation in 3T3-L1 preadipocytes

¹ Laboratory of Biochemistry and Cellular Biology, University of Namur (F.U.N.D.P.), Rue de Bruxelles, 61, 5000 Namur, Belgium
² Eppendorf Array Technologies, Rue du Séminaire, 12, 5000 Namur, Belgium
³ Cardiovascular Research Unit of the Center for Experimental Surgery and Anesthesiology, Katholieke Universiteit Leuven (KUL), Belgium

View larger version (61K): [in a new window]   Fig. 1. Antimycin A and chloramphenicol induce triglyceride accumulation in 3T3-L1 preadipocytes. (A) Photomicrographs of 3T3-L1 cells (a) not treated or treated (b) for 8 days with 10 nM AA, (c) for 16 days with 100 µg/ml chloramphenicol and (d) for 8 days with an adipogenic cocktail. Cells were stained for the presence of neutral lipids with Oil Red O. (B-C) Quantitative determination of triglyceride accumulation in 3T3-L1 cells (B) incubated without a drug (controls, CTL) or incubated with the indicated concentrations for 8 days with antimycin A and (C) for 16 days with chloramphenicol. The absorbance of cell monolayers was spectrophotometrically determined at 490 nm after Oil Red O staining. Results are expressed in optical density (O.D.) as the mean ± s.d. for n=3 experiments. **P<0.01 and ***P<0.001, significantly different from control cells. Magnification, 150x.

View larger version (50K): [in a new window]   Fig. 2. Antimycin A and the adipogenic cocktail trigger CREB activation by phosphorylation on Ser133. (A) Western blot analysis for CREB and CREB phosphorylated on Ser133 (pCREB) performed with 20 µg of nuclear proteins extracted from 3T3-L1 cells incubated for 6, 24 and 48 hours, and 3 and 8 days without a drug (controls, CTL), with 10 nM AA or with the adipogenic cocktail (DIFF); TBP served as a loading control. (B) Immunostaining and confocal microscopic analysis of abundance and localization of pCREB in 3T3-L1 cells incubated for 24 hours (a) without, (b) with 10 nM AA or (c) with adipogenic cocktail. Arrows indicate nuclear localization of pCREB. (C) Quantitative detection of pCREB and total CREB that binds to a DNA consensus sequence in a colorimetric assay (TransAM kit). Assays were performed on 10 µg of nuclear protein extract from 3T3-L1 cells incubated for 24 hours without (CTL), with 10 nM AA or with adipogenic cocktail (DIFF). Phosphorylated and total forms of CREB binding of pCREB and total CREB to DNA was detected with anti-Ser133-pCREB or anti-CREB antibodies, respectively, followed by a colorimetric reaction in the presence of a HRP-conjugated secondary antibody. Absorbance values were measured at 450 nm for DNA-binding of pCREB and CREB, and signals obtained for pCREB were normalized against total CREB bound to the capture probe (n=3). (D) Effect of AA and the adipogenic cocktail on CREB transcriptional activity in 3T3-L1. Cells were transiently co-transfected with a plasmid encoding ß-galactosidase and a CREB-sensitive luciferase-reporter construct. The next day, cells were incubated for 24 hours without (CTL) or with 10 nM AA, or with the adipogenic cocktail (DIFF) and then processed for luciferase assay. Results were normalized against ß-galactosidase activity and expressed as fold-increase of controls as the mean ± s.d. for n=3 experiments. *P<0.05, **P<0.01, ***P<0.001 are significantly different from control cells.

View larger version (83K): [in a new window]   Fig. 3. Silencing of CREB by using specific siRNA prevents triglyceride accumulation in antimycin-A-treated 3T3-L1 cells. (A) Visualization of siRNA transfection efficiency in 3T3-L1 preadipocytes. Cells were transfected for 4 hours with fluorescein-labeled siRNA (20 nM) and processed for confocal microscopy after (a) 24 hours or (b) 48 hours. (B) Effect of a CREB-specific siRNA on CREB protein expression. 3T3-L1 preadipocytes were transfected for 4 hours with different concentrations of siRNA or incubated for 4 hours with the transfection reagent (JetSI). CREB abundance was determined by western blotting of 40 µg of clear cell lysate proteins prepared 48 hours after transfection. TBP served as a loading control. (C) Effect of a CREB-specific siRNA on AA-induced triglyceride accumulation. Cells were transfected for 4 hours with 100 nM siRNA and then incubated for 8 days without (controls, CTL) or with 10 nM AA. The absorbance of cell monolayers was spectrophotometrically determined at 490 nm after Oil Red O staining. Results are expressed in optical density (O.D.) as the mean ± s.d. for n=3 or the mean of n=2 experiments. *P<0.05, significantly different from control cells. (D) Photomicrographs of 3T3-L1 cells transfected or not for 4 hours with 100 nM siRNA and incubated or not (CTL) for 8 days with 10 nM AA before Oil Red O staining. Magnification, 75x.

View larger version (23K): [in a new window]   Fig. 4. Effect of the adipogenic cocktail on the transcription levels of leptin, IL-6, C/EBPß and C/EBP genes analyzed by RT-PCR. Cells were incubated for the indicated times with the adipogenic cocktail before RNA extraction, reverse-transcription and amplification in the presence of SYBR Green and specific primers. TBP was used as a house-keeping gene for data normalization.

View larger version (45K): [in a new window]   Fig. 5. (A) Comparison of the results obtained with RT-PCR and DNA microarray analyses for FAS, Clic4 and CPT-2 genes. Cells were incubated for the indicated times with the adipogenic cocktail (DIFF) or 10 nM antimycin A (AA) before RNA extraction, reversetranscription and amplification in the presence of SYBR Green and specific primers. TBP was used as a house keeping gene for data normalization. (B) Western blot analysis for GPDmit performed on 20 µg of clear cell lysate proteins prepared from 3T3-L1 cells incubated or not (controls, CTL) for 2, 4, 6 or 8 days with 10 nM AA. Equal protein loading was controlled by the immunodetection of PARP.

View larger version (39K): [in a new window]   Fig. 6. Comparison of gene expression profiles as quantified with the DNA microarray in differentiating 3T3-L1 adipocytes (white) and in 3T3-L1 preadipocytes incubated with 10 nM antimycin A (grey). Cells were differentiated or treated with 10 nM AA for (A) 2, (B) 4, (C) 6 and (D) 8 days; data are presented for some selected genes to illustrate major similarities and differences between both treatments. For the other genes, see Tables 1 and 3. Mean ratios indicate a fold-increase or decrease in gene expression. *, significant mean ratios, obtained from ratios falling out of the 95% confidence interval.

View larger version (26K): [in a new window]   Fig. 7. Representative time-course profiles of gene expression in 3T3-L1 preadipocytes incubated with 10 nM antimcyin A, determined by the DNA microarray is illustrated for (A) ATR I, (B) GPDmit, (C) CHOP-10 and (D) C/EBP. Similar profiles were obtained for the genes listed on the right in A-D. Mean ratios indicate a fold-increase or decrease in gene expression. *, significant mean ratios, obtained from ratios falling out of the 95% confidence interval.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter