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First published online July 13, 2004
doi: 10.1242/10.1242/jcs.01207

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Inhibition of adipocyte differentiation by mechanical stretching through ERK-mediated downregulation of PPAR₂

Department of Cellular and Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka-city, 422-8526, Japan

View larger version (65K): [in a new window]   Fig. 1. Effect of cyclic stretching on adipocyte differentiation of 3T3-L1 cells. (A) A protocol of cyclic stretching during the differentiation of 3T3-L1 cells and phase-contrast microscopic images of 3T3-L1 cells undergoing differentiation into adipocytes with or without stretching. Confluent cultures of 3T3-L1 cells grown on a collagen-coated elastic silicon membrane were induced to undergo differentiation with or without cyclic stretching. (a) Undifferentiated 3T3-L1 cells at confluence. (b) 3T3-L1 cells induced without cyclic stretching for 45 hours after incubation in induction medium containing DEX, MIX, INS and FBS (DEX/MIX/INS). (c) Oil-Red-O-stained image of post-maturation-period cells that were not subjected to cyclic stretching during the induction period. (d) 3T3-L1 cells induced with cyclic stretching to 130% of the original size at a frequency of 1Hz for 45 hours after incubation in the induction medium. (e) Oil-Red-O-stained image of post-maturation-period cells that were subjected to cyclic stretching during the induction period. The arrows in (d,e) represent the direction of the stretching. (B,C) Relative GPDH activity (B) and triglyceride content (C) during the post-maturation period in 3T3-L1 cells with (grey) or without (black) various amount of cyclic stretching (rest, 110%, 120%, 130% and 175%; 1 Hz each) during the induction period (n=4; **P<0.01).

View larger version (44K): [in a new window]   Fig. 2. Effect of cyclic stretching on the expression of mRNA for C/EBPs and PPARs during the induction period. Cells were grown in induction medium containing DEX, MIX, INS and FBS (DEX/MIX/INS) with or without cyclic stretching. (A) The representative results of hybridization experiments showing quantitatively amplified PCR products. The total RNA of 3T3-L1 cells at different times during the respective induction periods was subjected to quantitative RT-PCR and blots of the PCR products were hybridized with DIG-labelled cDNA probes corresponding to each target mRNA.(B-F) Summarized data showing the expression of C/EBP (B), C/EBPß (C), C/EBPd (D), PPAR1 (E), and PPAR2 (F) by RT-PCR analysis. Each value is expressed as relative amount of mRNA for target gene compared with that expressed in undifferentiated cells (i.e. expression before induction, Pre=1), which was calculated using a standard curve of a serial dilution of cDNA form the undifferentiated 3T3-L1 cells. The white bar represents the cells before induction (Pre), the black bar represents the cells not subjected to stretching (Rest) and the grey bar represents the cells subjected to cyclic stretching (Stretch; at 130%, 1 Hz) at different time points during induction (n=3 each). *P<0.05 vs control (0 hours); **P<0.01 vs control (0 hours); P<0.05 vs without cyclic stretching (rest), corresponding to each time point; P<0.01 vs without cyclic stretching (rest), corresponding to each time point.

View larger version (42K): [in a new window]   Fig. 3. Effects of cyclic stretching at different time points during the induction period on adipocyte differentiation of 3T3-L1 cells. (A) The protocols of cyclic stretching at different time points during the induction period. Briefly, the 3T3-L1 cells, treated with induction medium containing DEX, MIX, INS and FBS (DEX/MIX/INS), were subjected to cyclic stretching (at 130%, 1 Hz) for the entire period (0-45 hours; S45), for the first 15-hour period (early phase, 0-15 hours; SE15) and for the final 15-hour period (late phase, 30-45 hours; SL15) of the induction period of 45 hours. R45, without stretching for the entire period. (B) Relative GPDH activity. (C) Triglyceride content of post-maturation 3T3-L1 cells with (grey) or without (black) cyclic stretching at different time points during the induction period (n=4; *P<0.05; **P<0.01). (D) The total RNA of 3T3-L1 cells at the end of the induction period was subjected to quantitative RT-PCR and blots of the PCR products were hybridized with DIG-labelled cDNA probes corresponding to each target mRNA. (n=5; **P<0.01).

View larger version (23K): [in a new window]   Fig. 4. Western-blot analysis of the activation of the ERK/MAPK pathway. (A) Representative western-blot analyses using anti-ERK and anti-phosphorylated-ERK antibodies. (B,C) Summarized results of the densitometric analyses of total ERKs (B) and phosphorylated ERKs (C) (n=3 each). (D) Summarized results of the effect of PD98,059 on the activation of ERKs (n=8-10 each). The 3T3-L1 cells were incubated in induction medium (DEX/MIX/INS) containing either 20 µM PD98,059 (designated PD) or 0.01% DMSO (Vehi) for 45 hours with or without cyclic stretching. The white bar represents the cells before induction (Pre), the black bar represents the cells without stretching (Rest) and the grey bar represents the cells with cyclic stretching (Stretch; at 130%, 1 Hz) during the induction period. *P<0.05; **P<0.01.

View larger version (37K): [in a new window]   Fig. 5. Effect of PD98,059 on the stretch-induced attenuation of mRNA expression of C/EBP and PPAR. The 3T3-L1 cells were incubated in induction medium (DEX/MIX/INS) containing either 20 µM PD98,059 (designated PD) or 0.01% DMSO (Vehi) for 3 hours and 45 hours with or without cyclic stretching. The total RNA of 3T3-L1 cells at different times during the respective induction periods was subjected to quantitative RT-PCR and blots of the PCR products were hybridized with DIG-labelled cDNA probes corresponding to each target mRNA. (A) Representative results of the hybridization experiment for quantitatively amplified PCR products. The results of C/EBP at 3 hours as well as PPAR2 and PPAR1 at 45 hours after the onset of the induction period are shown. (B-D) Summarized results of the quantitative RT-PCR analysis of the effect of PD98,059 on the expression of C/EBP (B), PPAR2 (C) and PPAR1 (D). The white bar represents the cells before induction (Pre), the black bar represents the cells without stretching (Rest) and the grey bar represents the cells with cyclic stretching (Stretch; at 130%, 1 Hz) at 3 hours or 45 hours of induction (n=4 each). Abbreviations: Pre, before induction; Vehi, 0.01% DMSO; PD, 20 µM PD98,059. *P<0.05; **P<0.01.

View larger version (41K): [in a new window]   Fig. 6. Western-blot analysis of the PPAR protein. (A) Specificity of western-blot analyses using anti-PPAR antibody. Total cellular proteins (20 µg) from undifferentiated (Pre) and differentiated (Mat) 3T3-L1 cells were analysed by western blot using anti-PPAR antibody. (B) Representative western blot and (C) summarized densitometric analyses of PPAR expressed in the differentiating 3T3-L1 cells with or without the stretching in the presence or absence of PD98,059. The 3T3-L1 cells were incubated in induction medium (DEX/MIX/INS) containing either 20 µM PD98,059 (designated PD) or 0.01% DMSO (Vehi) for 45 hours with or without cyclic stretching. The black bar represents the cells without stretching (Rest) and the grey bar represents the cells with cyclic stretching (Stretch; at 130%, 1 Hz) during the induction period (n=4 each). *P<0.05; **P<0.01.

View larger version (84K): [in a new window]   Fig. 7. Effect of PD98,059 on the stretch-induced inhibition of adipocyte differentiation of 3T3-L1 cells. (A) Phase-contrast microscopic images of 3T3-L1 cells at 45 hours after incubation in induction medium containing either20 µM PD98.059 or 0.01% DMSO with or without cyclic stretching (a-d), and the images of Oil-Red-O-stained 3T3-L1 cells after a maturation period of 9 days (e-h). (a,e) Unstretched cells incubated with 0.01% DMSO during the induction period. (b,f) Stretched cells incubated with 0.01% DMSO during the induction period. (c,g) Unstretched cells incubated with 20 µM PD98,059 during the induction period. (d,h) Stretched cells incubated with 20 µM PD98,059 during the induction period. (B) GPDH activity. (C) Intracellular triglyceride accumulation after maturation period of differentiated 3T3-L1 cells with (grey) or without (black) cyclic stretching (130%, 1 Hz), in the presence or absence of 20 µM PD98,059 during the induction period (n=4 each). Abbreviations: PD, 20 µM PD98,059; Vehi, 0.01% DMSO; DEX/MIX/INS, induction medium containing DEX, MIX, INS and FBS; *P<0.05; **P<0.01.

View larger version (54K): [in a new window]   Fig. 8. Effect of troglitazone on the stretch-induced inhibition of adipocyte differentiation of 3T3-L1 cells. (A) Phase-contrast microscopic images Oil-Red-O-stained 3T3-L1 cells after a maturation period of 9 days. (a) 3T3-L1 cells without induction (Pre). (b) Unstretched cells (Rest) incubated with 0.01% DMSO (Vehi) during the induction period. (c) Unstretched cells incubated with 30 µM troglitazone (Tro) during the induction period. (d) Stretched cells (Stretch) incubated with 0.01% DMSO (Vehi) during the induction period. (e) Stretched cells incubated with 30 µM troglitazone during the induction period. (B) GPDH activity and (C) intracellular triglyceride accumulation after maturation period of differentiated 3T3-L1 cells with (grey) or without (black) cyclic stretching (130%, 1 Hz), and in the presence or absence of troglitazone (30 µM) during the induction period (n=5 each). Abbreviations: Tro, 30 µM troglitazone; Vehi, 0.01% DMSO; DEX/MIX/INS, induction medium containing DEX, MIX, INS and FBS; *P<0.05; **P<0.01.

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