Department of Cellular and Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka-city, 422-8526, Japan
* Author for correspondence (e-mail: nakyamk{at}ys7.u-shizuoka-ken.ac.jp)
Accepted 9 March 2004
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Summary |
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Key words: Adipocyte differentiation, Stretching, PPAR, ERK, Mechanotransduction
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Introduction |
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Adipocytes are the major cellular component in fat tissue and excessive growth, differentiation and hypertrophy of adipocytes are fundamental processes of obesity. Maturation of adipocytes can occur among cells from a pre-existing pool of adipocyte progenitor cells (preadipocytes) that are present irrespective of age (Gregore et al., 1998). Therefore, from a pathophysiological point of view, both the proliferation and differentiation of preadipocytes into mature adipocytes remain important issues in this context.
A range of mammalian non-sensory cells such as fibroblasts (Kanda and Matsuda, 1993; MacKenna et al., 1998
), cardiomyocytes (Sadoshima and Izumo, 1997
), vascular endothelial cells (Azuma et al., 2000
; Davies and Tripathi, 1993
; Kanda and Matsuda, 1993
; Saito et al., 2003
; Tanabe et al., 2000
), smooth-muscle cells (Kanda and Matsuda, 1993
; Li and Xu, 2000
; Oldenhof et al., 2002
; Tanabe et al., 2000
), osteoblasts (Duncan, 1995
) and skeletal muscle cells (Csukly et al., 2002
) can respond to mechanical stimuli, including stretching. Interestingly, most of these cells, as well as adipocytes, are considered to be of mesodermal origin. To the best of our knowledge, no study has yet been conducted on the direct effects of mechanical stimulation on adipocyte differentiation. Here, we demonstrate that cyclic mechanical stretching inhibited the differentiation of mouse 3T3-L1 cells into adipocytes; this effect was mostly attributable to the reduced expression of the peroxisome proliferator-activated receptor (PPAR)
2, an adipocyte-specific nuclear hormone receptor/adipogenic transcription factor (Tontonoz et al., 1994a
; Tontonoz et al., 1994b
). This reduced expression was mediated via the activation of an extracellular-signal-regulated-protein kinase/mitogen-activated-protein kinase (ERK/MAPK) pathway. The modulation of adipocyte differentiation in response to stretching might provide further insight into the physiological significance of the local application of mechanical stress to fat tissues with respect to the inhibition of differentiation and the renewal of adipocytes.
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Materials and Methods |
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Measurement of glycerol-3-phosphate dehydrogenase activity and triglyceride content
3T3-L1 cells under the various conditions were rinsed twice with PBS(), resuspended in 600 µl 25 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM phenylmethylsulfonylfluoride (PMSF), 1 mg ml1 pepstatin, and 1 mg ml1 leupeptin, and then lysed by sonication at 4°C. An aliquot of the homogenate of the cells was cleared by centrifugation at 12,800 g for 5 minutes at 4°C, and the supernatant was subjected to glycerol-3-phosphate dehydrogenase (GPDH) assay according to a previously reported method (Wise and Green, 1979); the other aliquot of the homogenate was mixed with an equal volume of chloroform/methanol mix (2:1), mixed vigorously for 10 minutes and centrifuged at 12,800 g for 10 minutes at 4°C. The resulting chloroform layer was dried, resuspended in 1% Triton X-100 and subjected to a triglyceride assay using a commercially available kit (Triglyceride G-test WAKO, Wako Pure Chemicals).
Western-blot analysis of ERK1/2 and PPAR
3T3-L1 cells of various conditions were rinsed twice with ice-cold PBS(), resuspended, and lysed in a lysis buffer consisting of 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% sodium dodecyl sulfate (SDS), 1% NP-40, 1 mM PMSF, 1 mg ml1 pepstatin, 1 mg ml1 leupeptin at 4°C. Cell extract containing an equal amount of protein (20 µg) was resolved by 10% SDS-polyacrylamide-gel electrophoresis (SDS-PAGE) under reducing conditions. Proteins was transferred to a Biotrace PVDF membrane (Pall Gelmann Laboratory, Ann Arbor, MI) and the membrane was blocked with 5% w/v bovine serum albumin (BSA) in 20 mM Tris-HCl, pH 7.4, 500 mM NaCl, 0.1% Tween-20 (TBST-500). Western-blot analysis was then carried out using polyclonal anti-ERK1/2 antibody (sc-94, Santa Cruz Biotechnology, Santa Cruz, CA, USA), monoclonal anti-phosphorylated-ERK antibody (sc-7383, Santa Cruz Biotechnology) or polyclonal anti-PPAR antibody (sc-7196, Santa Cruz Biotechnology). Control analyses were also carried out using mouse and rabbit IgG (Inter-Cell Technologies, Hopewell, NJ, USA). Each primary antibody (1:5000-1:10,000) and horseradish-peroxidase-conjugated secondary antibody (1:5000) were diluted with TBST-500 containing 5% BSA. Immunoreactive signals were visualized using the ECLplus system (Amersham Pharmacia Biotech, Buckinghamshire, UK).
Reverse transcription polymerase chain reaction (RT-PCR)
Total RNA (2 µg) extracted from 3T3-L1 cells was reverse-transcribed with oligo dT12-18 (Amersham Biosciences, Uppsala, Sweden) and M-MLV reverse transcriptase (Wako Pure Chemicals) at 37°C for 90 minutes. The resultant cDNA cocktail was heat inactivated and diluted to 400 µl with water. The reaction cocktail (10 µl) consisted of 1 µl of the diluted cDNA (corresponding to 5 ng of input total RNA), 12.5 pmol each of the following sense and antisense oligonucleotide pairs (PPAR1: 5'-AGAAGTCACACTCTGACAGG-3' and 5'-CAATCGGATGGTTCTTCGGA-3'; PPAR
2: 5'-ACTGCCTATGAGCACTTCAC-3' and 5'-CAATCGGATGGTTCTTCGGA-3'; C/EBP
: 5'-TGGACAAGAACAGCAACGAG-3' and 5'-AATCTCCTAGTCCTGGCTTG-3'; C/EBPß: 5'-ACTACGGTTACGTGAGCCTC-3' and 5'-CAGCTGCTTGAACAAGTTCC-3'; C/EBP
: 5'-ACCTCTTCAACAGCAACCAC-3' and 5'-TTCTGCTGCATCTCCTGGTT-3'; ß-actin: 5'-GAGACCTTCAACACCCCAGC-3' and 5'-CACGGAGTACTTGCGCTCAG-3'), 2 nmol of dNTP, and 0.25 U of Taq DNA polymerase in a buffer supplied by the manufacturer. In addition, dimethylsulfoxide (DMSO) was also included in the reaction cocktail to a 10% (v/v) concentration for amplification of C/EBPß and C/EBP
cDNAs. The protocols for the temperature cycling of the polymerase chain reaction (PCR) were 30 seconds at 94°C, 30 seconds at 58°C, and 1 minute at 72°C for 22 cycles (ß-actin), 24 cycles (C/EBPß, C/EBP
) or 27 (C/EBP
, PPAR
1, PPAR
2) cycles. The number of cycles was determined to ensure quantitative amplification. Serially diluted (four times) cDNA cocktail, corresponding 20 ng, 5 ng, 1.25 ng and 0.3125 ng of input total RNA of reverse transcription reaction, of both differentiated and undifferentiated 3T3-L1 cells was used to generate a standard curve in each PCR experiment. The resulting PCR products were electrophoresed through 1.5% agarose, blotted onto a Biodyne B membrane (Pall Gelmann), and then hybridized with digoxygenin (DIG)-labelled cDNA probes for each target. During the revision of this work, real-time PCR was also performed on ABI GeneAmp 5700 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) using QuantiTect SYBR Green PCR kit (Qiagen, Valencia, CA, USA) to confirm the quantitative results of PPAR
2, PPAR
1 and ß-actin mRNAs.
Quantification of the chemiluminescent signal
Chemiluminescent images of western- or Southern-blot analyses on X-ray film were acquired using a flatbed scanner with a transparent film adapter unit (CanoScan FB1200S with FAU-S10, Canon, Tokyo, Japan) and measured by NIH Image software (version 1.62, National Institutes of Health, Bethesda, MD, USA).
Statistical analysis
The data are expressed as the mean±s.e.m. Statistical analyses were performed with Fisher's protected least significant difference test or Scheffe's F-test after analysis of variance using StatView, version 4.5 (Abacus Concepts, Berkeley, CA, USA). Differences were considered to be significant when P was less than 0.05.
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Results |
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The effects of cyclic stretching on differentiation markers for adipocytes (Wise and Green, 1979) were also examined. Both the expression of cytosolic GPDH activity and the accumulation of intracellular triglycerides were significantly reduced when the 3T3-L1 cells were stretched at a frequency of 1 Hz within a range 110-175% of the original length during induction (Fig. 1B,C). Because the maximal inhibitory effect of cyclic stretching on the expression of these differentiation markers was obtained when the cells reached 130% of their original length, an optimal cyclic stretching percentage of 130% at 1 Hz was applied to the cells in the following experiments.
Cyclic stretching attenuates the induction of several transcription factors for adipocyte differentiation
Three members of the C/EBP family (C/EBP, C/EBPß and C/EBP
) and
-isoforms of the PPAR family (PPAR
1 and PPAR
2) play important roles in the regulation of adipocyte differentiation (Cao et al., 1991
; Tontonoz et al., 1994a
; Tontonoz et al., 1994b
; Yeh et al., 1995
). Thus, the effect of cyclic stretching on the expression of these transcription factors was assessed in the present study by quantitative reverse-transcription PCR (RT-PCR).
During the induction period, C/EPBß, C/EPB and PPAR
1 were rapidly induced as early as 30 minutes (Fig. 2A,C-E); C/EBP
and PPAR
2 (Fig. 2A,B,F) were also induced but much later during the induction period (
45 hours). The cyclic stretching (130%, 1 Hz) significantly reduced the expression of C/EBP
, PPAR
1 and PPAR
2 (Fig. 2D-F). By contrast, cyclic stretching showed no appreciable effect on the expression of C/EBPß mRNA (Fig. 2C) and only a slightly facilitative effect on the expression of C/EBP
mRNA (Fig. 2B).
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Cascade and/or cross-regulation has been reported between adipogenic transcription factors, including C/EBPs and PPARs (Cao et al., 1991
; Tanaka et al., 1997
; Tontonoz et al., 1994a
; Tontonoz et al., 1994b
; Wu et al., 1995
; Wu et al., 1999
; Yeh et al., 1995
). It is possible that the reduced expression of PPAR
2 mRNA was due to an earlier suppression of a transient burst of C/EBP
mRNA expression in response to cyclic stretching. To address the effects of cyclic stretching on the sequential expression of C/EBPs and PPAR
s, the cells that underwent the commitment of differentiation were stretched during the early phase (SE15; 0-15 hours) or the late phase (SL15; 30-45 hours) of the induction period (Fig. 3A). After 9 days of maturation, cytosolic GPDH activity and triglyceride accumulation were measured and compared with those observed under the resting (R45; 0-45 hours) and stretching (S45; 0-45 hours) conditions throughout the induction period.
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The GPDH activity and triglyceride content was significantly reduced when the cells were stretched throughout the induction period (S45) and when they were stretched during the late phase of induction (SL15). By contrast, neither GPDH activity nor triglyceride accumulation was affected by cyclic stretching during the early phase of induction (SE15) (Fig. 3B,C) when the expression of C/EBP mRNA peaked in resting cells 3 hours after the onset of induction.
The effect of cyclic stretching in the late phase of the induction period on the expression of C/EBPs and PPAR mRNAs was also examined. The expression of PPAR
1 and PPAR
2 mRNA decreased in response to the stretching during the late phase of the induction period, and no appreciable change on the expression of C/EBP
, C/EBPß and C/EBP
isoforms was observed (Fig. 3D). These results indicated that only the application of cyclic stretching during the late phase of induction could inhibit the adipocyte differentiation of 3T3-L1 cells, which was accompanied by the downregulation of PPAR
1 and PPAR
2 without any changes in the expression of C/EBP mRNA.
Cyclic stretching activates the ERK/MAPK pathway
Several lines of evidence have suggested that ERK1/2 is involved in the signal transduction pathway in a range of cell types, including vascular endothelial cells, in response to mechanical stimuli (MacKenna et al., 1998; Li and Xu, 2000
; Oldenhof et al., 2002
). In order to clarify whether cyclic stretching activates ERK1/2 in differentiating 3T3-L1 cells, we performed western-blot analysis using anti-phosphorylated-ERK antibody and anti-ERK antibody to examine the effects of stretching on the phosphorylation of ERK1/2 during the induction period (Fig. 4A). No significant differences were observed in the total ERK levels among the cells, regardless of the status of differentiation or of the mechanical stimulus (Fig. 4B). Nevertheless, the phosphorylation of ERK1/2 in these cells had increased significantly by the end of the induction period in response to cyclic stretching (Fig. 4C). The stretch-induced phosphorylation of ERK1/2 was abolished by the presence of PD98,059 (Fig. 4D), a selective inhibitor of MEK1 (an isoform of MAPK/ERK kinase), at 20 µM, a concentration previously shown to be highly specific to the MEK-ERK pathway (Oldenhof et al., 2002
).
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MEK inhibitor restores the stretch-induced reduction of adipogenic transcription factors and the stretch-induced inhibition of adipocyte differentiation of 3T3-L1 cells
We next investigated whether the activation of ERKs was involved in the stretch-induced decrease in the expression of mRNA for C/EPB, PPAR
1 and PPAR
2. PD98,059 was applied to 3T3-L1 cells during the induction period with and without cyclic stretching, and the expression levels of mRNA for C/EBP
, PPAR
1 and PPAR
2 were examined by RT-PCR (Fig. 5A). The expression of both C/EBP
and PPAR
2, which had been reduced in response to cyclic stretching, was restored by PD98,059 (Fig. 5B,C). However, the expression of PPAR
1, which had been lowered in response to cyclic stretching, was not restored by PD98,059 (Fig. 5D).
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The expression of PPAR at the protein level was also examined. The PPAR
protein was significantly increased during the differentiation (Fig. 6A). The cyclic stretching significantly reduced PPAR
expression at the protein level, which was also restored by PD98,059 (Fig. 6B,C). These results indicated that the stretch-induced downregulation of PPAR
mRNA was consistent with the decrease in PPAR
protein.
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We also studied the effect of PD98,059 on the stretch-induced inhibition of the differentiation of 3T3-L1 cells. PD98,059 (20 µM) was administered to cells with and without cyclic stretching during the 3T3-L1 cell induction period. The unidirectionally orienting response of the cells was abolished by PD98,059 when it was administered during the induction period (Fig. 7Ab,d). The cells were then washed and cultured for 9 days of maturation without being subjected to stretching. The number of differentiated adipocytes carrying droplets of cytoplasmic triglycerides in the cells that had been subjected to the stretching in the presence of PD98,059 was similar to that of cells without the stretching (Fig. 7Ag,h). Likewise, the reduced expression of cytoplasmic GPDH activity and decreased intracellular accumulation of triglyceride observed after the maturation period was restored to the control level by PD98,059 (Fig. 7B,C). These results suggest that the inhibitory effect of cyclic stretching on the adipocyte differentiation of 3T3-L1 cells is mediated by the activation of the MEK-ERK pathway.
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To examine whether the inhibitory effect of cyclic stretching on the differentiation of 3T3-L1 cells can be restored by the activation of PPAR, the cells were treated with troglitazone, a synthetic PPAR
ligand. The addition of troglitazone at 30 µM, a concentration previously shown to act as a full agonist of PPAR
in 3T3-L1 cells (Camp et al., 2000
), offset the decreased number of lipid-droplet-laden differentiated adipocytes in response to the stretching (Fig. 8Ad,e). The reduced cytoplasmic GPDH activity (Fig. 8B) and the decreased intracellular accumulation of triglyceride (Fig. 8C) were overcome by troglitazone. Thus, the inhibitory effect of the cyclic stretching on the differentiation of 3T3-L1 cells could be restored through activation of PPAR
by the synthetic ligand. Administration of troglitazone together with DEX-MIX-INS cocktail during induction period significantly increased the intracellular triglyceride in resting cells, whereas the cyclic stretching counteracted the action of troglitazone (Fig. 8C).
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Discussion |
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It has already been shown that the cascade-like expression of three members of the C/EBP family (C/EBP, C/EBPß, C/EBP
) and PPAR
(
1 and
2 isoforms of PPAR) plays an important role in the terminal differentiation of preadipocytes to triglyceride-laden adipocytes (Cao et al., 1991
; Tanaka et al., 1997
; Tontonoz et al., 1994a
; Tontonoz et al., 1994b
; Wu et al., 1995
; Wu et al., 1999
; Yeh et al., 1995
). In the present study, the application of cyclic stretching during the entire induction period significantly reduced the expression of C/EBP
, PPAR
1 and PPAR
2 mRNAs (Fig. 2D-F). However, when the stretching condition was applied during the late stage of the induction period only, the expression of C/EBP
mRNA was not inhibited, although this condition did inhibit the expression of both PPAR
2 and PPAR
1 mRNAs (Fig. 3D). Furthermore, when the stretching condition was applied only during the early stage of the induction period (a condition under which the expression of C/EBP
mRNA would be expected to be downregulated), no effect was observed on the terminal differentiation of 3T3-L1 cells (Fig. 3B,C). Regardless of the protocol, neither C/EPB
nor C/EPBß mRNA showed any notable changes in response to the stretching condition (Fig. 2B,C, Fig. 3D). Adipocyte differentiation of the cells was also inhibited at a similar level by the `delayed' stretching protocol, which suggests that the downregulation of PPAR
s was the most likely reason for the inhibition of adipocyte differentiation under the stretching condition. Consistent with the finding of the downregulation of PPAR
s by the stretching, the adipocyte differentiation was restored by troglitazone, an activator of PPAR
during the induction period (Fig. 8). However, there seems to be an alternative possibility that the mechanical deformation of the cells might affect either generation or availability of endogenous PPAR
ligands. It should be noted that administration of troglitazone during induction period significantly augmented accumulation of triglyceride in resting cells, whereas the stretching restrained this action of troglitazone (Fig. 8C). Therefore, the stretching might counteract the stimulating effect of troglitazone on differentiation.
An earlier study using ectopic overexpression experiments in uncommitted NIH3T3 fibroblasts suggested that the expression of C/EBPß alone could induce endogenous PPAR mRNA and stimulate adipogenesis (Wu et al., 1995
). By contrast, the ectopic overexpression of C/EBP
alone was not found to induce PPAR
and adipocyte differentiation. Moreover, it was shown that the dual overexpression of both C/EBP
and C/EBPß enhanced PPAR
mRNA expression and differentiation (in this context, it should be noted that DEX was still required to achieve a fully induced level of PPAR
mRNA) (Wu et al., 1996
). The synergistic role of C/EBPß and C/EBP
in adipocyte differentiation was also indicated using C/EBPß/ and C/EPB
/ mice both in vivo and in vitro (Tanaka et al., 1997
). These results, taken together, suggest that the coordinated expression of C/EBPß and C/EBP
is a prerequisite for the efficient expression of PPAR
, leading to fully differentiated adipocytes. Therefore, the downregulation of C/EBP
mRNA by itself as a result of the application of stretching during the early stage of induction period showed minor role in the inhibition of adipocyte differentiation.
The stretch-induced blockade of adipocyte differentiation was reversed by the inhibition of the ERK/MAPK pathway (Fig. 7), with concomitant restoration of the expression of PPAR2 at the mRNA (Fig. 5C) and protein (Fig. 6B,C) levels. By contrast, the expression of PPAR
1 mRNA, which was reduced in response to the stretching condition, was not restored by the inhibition of the ERK/MAPK pathway (Fig. 5D). Therefore, the reduced expression of PPAR
2 was responsible for the inhibition of the differentiation of 3T3-L1 cells in response to cyclic stretching, whereas the reduction in expression of PPAR
1 mRNAs played a minor role in the inhibition of cell differentiation. In addition, it has been reported that PPAR
activity is negatively regulated by phosphorylation with ERK (Hu et al., 1996
; Camp and Tafuri, 1997
; Adams et al., 1997
). It is possible that, once PPAR
is produced during the induction period of 3T3-L1 cells, its transcriptional activity is inhibited by phosphorylation with the stretch-induced MAPK. Thus, the inhibitory phosphorylation of PPAR
by the MEK-ERK signalling pathway might also be responsible for the stretch-induced inhibition of the differentiation of 3T3-L1 cells.
A range of mechanosensor molecules, thought to transduce mechanical forces into intracellular signals, have been proposed [e.g. mechanically gated ion channels (Hamill and Martinac, 2001), membrane-integrated growth factor receptors (Li and Xu, 2000
) and integrins (Geiger and Bershadsky, 2002
)]. At present, it remains unclear how 3T3-L1 cells receive the mechanical force that leads to the activation of ERK (followed by the attenuation of adipocyte differentiation); in other words, the means by which a negative signal is propagated for the expression of adipogenic transcription factors remains unknown. In this regard, it has been shown that ERK are activated by integrin-mediated cell adhesion to the extracellular matrix (ECM) in rat cardiac fibroblasts (MacKenna et al., 1998
). During the process of differentiation to adipocytes, drastic changes are known to take place in cell morphology, cytoskeletal components and the level and type of ECM components secreted. In turn, all of these factors are recognized to exert significant influence on adipocyte differentiation (Gregore et al., 1998
). Significant morphological changes were commonly observed in our experiments, even during the induction period (Fig. 1Aa,b) (i.e. in the early phase of adipocyte differentiation of 3T3-L1 cells). It is possible that, in the present cases, the appropriate cytoskeletal rearrangement was disturbed; thus, the correct arrangement has been suggested to be a prerequisite for terminal differentiation (Lieber and Evans, 1996
; Gregore et al., 1998
; Kawaguchi et al., 2003
). This conclusion is plausible, because exposure to cyclic stretching during the induction period induced in these cells the tendency to orient in a unidirectional manner (Fig. 1Ad). Interestingly, the stretch-induced unidirectionally oriented response of the cells disappeared with the administration of PD98,059 (Fig. 7Ab,d), suggesting a link between this orienting response and the inhibition of adipocyte differentiation through the activation of the ERK/MAPK pathway. In addition, Spiegelman and Ginty indicated that fibronectin matrices decrease lipogenic gene expression and lead to decreased triglyceride accumulation (Spiegelman and Ginty, 1983
). In this context, the interaction between the cell matrix and nuclear events has been suggested to play an important role in the cell differentiation (Gregore et al., 1998
). Collectively, physical deformation caused by the stretching of membrane components and/or cytoskeletal components (both of which interact directly or indirectly with the ECM) might be involved in the inhibitory mechanism of adipocyte differentiation in response to cyclic stretching.
Finally, the physiological significance of the present study should be briefly addressed. The mechanosensitivity of preadipocytes suggest that gymnastic exercise and/or massage act directly on preadipocytes as a mechanical stimulus; such stimulus might activate the ERK/MAPK system, which in turn could lead to the prevention of adipocyte differentiation and renewal. This line of reasoning further implies that local massage (vibration or passive stretching applied to the body) might ameliorate obesity-associated physical conditions in terms of the prevention of adipocyte differentiation.
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Acknowledgments |
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