2,4,5-Trimethoxybenzaldehyde, a Bitter Principle in Plants

Sep 15, 2014 - 2,4,5-Trimethoxybenzaldehyde (2,4,5-TMBA) is a bitter principle present in plant seeds, roots, and leaves. 2,4,5-TMBA isolated from car...
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2,4,5-Trimethoxybenzaldehyde, a Bitter Principle in Plants, Suppresses Adipogenesis through the Regulation of ERK1 Yu-Wen Wang and Chia-Feng Kuo* Department of Food Science, Nutrition, and Nutraceutical Biotechnology, Shih Chien University, 70 Ta-Chih Street, 104 Taipei, Taiwan ABSTRACT: Because of the prevalence of obesity, there is particular interest in finding potential therapeutic targets. In a previous study, we demonstrated that 2,4,5-trimethoxybenzaldehyde (2,4,5-TMBA), a bitter principle in plants and a natural cyclooxygenase II (COX-2) inhibitor, suppressed the differentiation of preadipocyts into adipocytes at the concentration of 0.5 mM. In this current study, we aimed to investigate the stage during adipogenesis that is critically affected by 2,4,5-TMBA and the effects of 2,4,5-TMBA on the time-course expression of signaling molecules MAP kinase kinase (MAPKK, represented by MEK) and extracellular signal-regulated kinase (ERK), transcription factors CCAAT/enhancer binding protein (C/EBP)α, β, and δ and peroxisome proliferator-activated receptor (PPAR)γ, lipogenic enzymes acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), and lipid droplet-coating protein perilipin A. When preadipocytes were co-cultured with 2,4,5-TMBA (0.5 mM) specifically at post-induction days 0−2, 2−4, 4−6, or 6−8 only, relative lipid accumulation was decreased by 67.93, 34.65, 49.56, and 34.32%, respectively. A time-course study showed that treatment of 2,4,5-TMBA suppressed the phosphorylation of ERK1 at the initial stage of adipogenesis but upregulated the phosphorylation at the late stage, which is opposite to the conditions required for the differentiation process. The overall expression of C/EBPα, β, and δ, PPARγ2, ACC, FAS, and perilipin A in preadipocytes was downregulated by the treatment of 2,4,5-TMBA. Taken together, our findings suggest that 2,4,5-TMBA suppresses adipogenesis through the regulation of ERK1 phosphorylation. Although results from in vitro studies cannot be directly extrapolated into clinical effects, our study will help to elucidate the anti-adipogenic potential of 2,4,5-TMBA. KEYWORDS: 2,4,5-TMBA, 3T3-L1 preadipocyte, adipogenesis, ERK

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INTRODUCTION With the prevalence of obesity-related diseases, there is great interest in defining potential therapeutic targets for obesity. Obesity is characterized by the growth of adipose tissue involving the increase in the size of adipocytes as well as the formation of new adipocytes from preadipocytes. In the presence of adipogenic factors, the preadipocytes undergo mitotic clonal expansion and further differentiate into lipidladen adipocytes, a process referred to as adipogenesis.1−3 Several transcription factors are involved in the process of adipogenesis. Among them, CCAAT/enhancer binding protein (C/EBP)α, β, and δ and peroxisome proliferator-activated receptor (PPAR)γ are considered to be crucial determinants.4 In the presence of adipogenic stimulation, the MAP kinase kinase/mitogen-activated protein kinases (MEK/MAPKs) signaling pathway is activated and followed by the expression of C/EBPβ and C/EBPδ. Activation of C/EBPβ and C/EBPδ further induces the expression of PPARγ and C/EBPα, which are essential for the expression of genes that produce the adipocyte phenotype.5,6 Extracellular signal-regulated kinase (ERK) is one of the MAPKs. The mechanisms that regulate the duration and extent of ERK activity play a pivotal role in regulating adipogenic process. ERK1 but not ERK2 is specifically required for in vivo and in vitro adipogenesis. The function of ERK1 needs to be timely regulated during adipogenesis. At the early stage, ERK1 is turned on by phosphorylation for the proliferative process. Later on, phosphorylation of ERK1 has to be suppressed to avoid the phosphorylation of PPARγ.7 © 2014 American Chemical Society

2,4,5-Trimethoxybenzaldehyde (2,4,5-TMBA) is a bitter principle present in plant seeds, roots, and leaves. 2,4,5TMBA isolated from carrot (Daucus carota L.) seeds inhibited the activity of cyclooxygenase II (COX-2) at the concentration of 0.5 mM.8 COX-2 not only plays a role in the inflammatory process but is also involved in the modulation of adipogenesis.9,10 In our previous study, 3T3-L1 preadipocytes and adipocytes were treated with 0.5 mM 2,4,5-TMBA to explore its effect on adipogenesis and lipolysis. The results showed that 2,4,5-TMBA suppressed differentiation of preadipocyts into adipocytes and enhanced lipolysis in mature adipocytes.11 In this study, we aimed to further investigate the stage during adipogenesis that is critically affected by 2,4,5-TMBA and the effects of 2,4,5-TMBA on the time-course expression of ERK1 and its downstream factors.

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MATERIALS AND METHODS

Chemicals. 2,4,5-TMBA, Dulbecco’s modified Eagle’s medium (DMEM), insulin, dexamethasone (DEX), and 3-isobutyl-1-methylxanthine (IBMX) were obtained from Sigma (St. Louis, MO). Fetal bovine serum (FBS) and penicillin−streptomycin were purchased from Biowest (Kancas, MO). Calf bovine serum (CBS) was obtained from Gibco (Grand Island, NY). Anti-C/EBPα, anti-MEK, antipMEK, and anti-fatty acid synthase (FAS) antibodies were purchased from Epitomics (Burlingame, CA). Anti-β-actin and anti-C/EBPδ Received: Revised: Accepted: Published: 9860

May 14, 2014 August 31, 2014 September 15, 2014 September 15, 2014 dx.doi.org/10.1021/jf503344v | J. Agric. Food Chem. 2014, 62, 9860−9867

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antibodies were obtained from Novus (Littleton, CO). Anti-ERK, antipERK, anti-acetyl-CoA carboxylase (ACC), and C/EBPβ were purchased from Cell Signaling (Danvers, MA). Anti-perilipin A and anti-PPARγ were obtained from Chemicon International (Billerica, MA) and Cayman (Ann Arbor, MI), respectively. Goat anti-rabbit peroxidase-conjugated antibody was purchased from Jackson ImmunoResearch (West Grove, PA). Cell Culture. 3T3-L1 preadipocytes (BCRC 60159) purchased from Bioresource Collection and Research Center (BCRC) of Food Industry Research and Development Institute (Hsinchu, Taiwan) were cultured in DMEM supplemented with 10% CBS at 37 °C in a humidified atmosphere containing 5% CO2. At 2 days after confluence, cells were cultured in FBS-containing DMEM (10%, v/v) with the addition of insulin (0.862 μM), DEX (1 μM), and IBMX (0.5 mM) to induce differentiation (day 0). On day 2, the medium was substituted by DMEM with 10% FBS and insulin (0.862 μM) for another 2 days. On day 4, the medium was changed to DMEM with 10% FBS only, and the same medium was provided again on day 6. 2,4,5-TMBA dissolved in DMSO was added in the medium (final concentration of 0.5 mM) at specific time periods. Control samples were prepared by the addition of isovolumetric dimethyl sulfoxide (DMSO) to the medium. Oil Red O Staining of Adipocytes and Quantification of Triglyceride Accumulation. The formation of lipid droplets in adipocytes was observed by Oil Red O staining. Cells were washed twice with phosphate-buffered saline (PBS) before the addition of 10% formaldehyde at room temperature. After 10 min, formaldehyde was removed. Cells were then fixed with fresh 10% formaldehyde for 1 h, followed by the washes with water twice and with 60% isopropanol for 5 min. The cells were completely dry at room temperature before they were stained with Oil Red O (three parts of 0.35% Oil Red O dye in isopropanol and two parts of water) for 15 min at room temperature with shaking and then washed 4 times with water before photographed under a microscope. For the measurement of lipid accumulated in adipocytes, the isopropanol-washed and completely dried cells were shaken with 100% isopropanol for 10 min. The dissolved lipid was quantified by measuring the absorbance at 500 nm. The results were expressed as the lipid accumulation relative to that of the control group. Measurement of PPARγ Activation. Activation of PPARγ in cells harvested at the second and eighth days of adipogenesis was measured by an enzyme-linked immunosorbent assay (ELISA) kit (Signosis, Sunnyvale, CA). Briefly, the nuclear extracts prepared from culture cells by an extraction kit (Signosis, Sunnyvale, CA) were added to the 96-well plate, which was pre-immobilized with PPARγ consensus sequencing oligo. The activated PPARγ was detected with the specific antibody against the PPARγ subunit and a horseradish peroxidase (HRP)-conjugated secondary antibody. The colorimetric detection was measured by a spectrophotometer at 450 nm. Western Blotting. Cells were washed with cold phosphatebuffered saline (PBS) twice, harvested, and centrifuged at 936g for 5 min. The pellets were resuspended in lysis buffer containing 150 mM sodium chloride, 1% Nonidet P 40, and 50 mM Tris-HCl at pH 7.5. The cell suspension was centrifuged at 16805g at 4 °C for 30 min. After centrifugation, the supernatant was collected for analysis and its protein content was determined by a DC Protein Assay Kit (Bio-Rad, Hercules, CA). Equal amounts of proteins from each sample were denatured and separated by gel electrophoresis before being transferred to polyvinylidene fluoride (PVDF) membranes (PerkinElmer, Waltham, MA). The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline containing Tween (TBST) (137 mM NaCl, 0.1% Tween-20, and 20 mM Tris-HCl at pH 8.3) for 1 h before sequentially incubated with primary antibody overnight in a cold room and HRP-conjugated secondary antibody for 1 h at room temperature. Immunoreactive proteins were detected using an enhanced chemiluminescence kit (ECL, PerkinElmer, Waltham, MA), and the film was analyzed using ImageQuant software (Molecular Dynamics, Sunnyvale, CA) to obtain densitometric values. For stripping and reprobing, the membrane was incubated with stripping buffer [0.2 M glycine, 3.5 mM sodium dodecyl sulfate (SDS), and 0.1% Tween-20 at

pH 2.2] at room temperature for 10 min. After the buffer was discarded, the membrane was incubated with fresh stripping buffer for another 10 min, followed by two 10 min washes by PBS buffer and two 5 min washes by TBST buffer. After striping, the blocking and probing were performed as mentioned above. The relative expression of each protein was calculated as the “densitometric value of protein/ densitometric value of β-actin collocated”. The ratio of phosphorylated proteins to total proteins was calculated by the comparison of relative expression. Statistical Analysis. Statistical analysis was carried out using Statistical Analysis System (SAS Institute, Cary, NC). Analysis of variance (ANOVA) and Student−Newmann−Keuls multiple range test were performed to evaluate the differences among groups (α = 0.05).

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RESULTS

Effect of the Incubation Period of 2,4,5-TMBA on Adipogenesis. To figure out the time period critically affected by 2,4,5-TMBA during adipogenesis, preadipocytes were cultured with 2,4,5-TMBA specifically at post-induction days 0−2, 2−4, 4−6, or 6−8 only (Figure 1A). At day 8, photos were taken for each group under a microscope and the relative accumulation of lipids was estimated. As shown in panels B and C of Figure 1, preadiopcytes were poorly differentiated without the presence of adipogenic factors (“−IDI”) but well-differentiated with the presence of adiogenic factors (“+IDI”). When preadipocytes were treated with 2,4,5-TMBA at different time periods in the presence of adipogenic factors, relative lipid accumulation was decreased by 67.93, 34.65, 49.56, and 34.32% for days 0−2, 2−4, 4−6, and 6−8 groups, respectively (Figure 1D). The most significant inhibitory effect was observed when 2,4,5-TMBA was present in the medium at days 0−2. Withdrawal of 2,4,5-TMBA from the medium did not restore adipogenesis. Effect of 2,4,5-TMBA on the Expression of MEK and ERK during Adipogenesis. Different from the study design for Figure 1 (Figure 1A), the study design for Figures 2−4 (Figure 2A) was to investigate the expression of signaling molecules and transcription factors at different stages when 2,4,5-TMBA was co-cultured with preadipocytes during the differentiation process. The cells were harvested at postinduction days 2, 4, 6, and 8 (Figure 2A). Phosphorylation of the MEK/ERK signaling pathway is crucial for the differentiation of preadipocytes. To study how MEK/ERK is affected during adipogenesis, preadipocytes cultured with 2,4,5-TMBA were harvested at post-induction days 2, 4, 6, and 8 (Figure 2A). 2,4,5-TMBA decreased the level of phosphorylated MEK at the early stage but increased the level at the late stage (Figure 2B). ERK1 but not ERK2 is specifically required for the differentiation of 3T3-L1. During adipogenesis, ERK1 has to be turned on by phosphorylation at the initial stage but shut off later on.7 Because activation of MEK leads to the phosphorylation of ERK1, the effect of 2,4,5TMBA on ERK1 phosphorylation was in accordance with the result of MEK phosphorylaiton; the expression of phosphorylated ERK1 was suppressed initially but enhanced at the terminal period (Figure 2C), which is opposite to the conditions required for adipogenesis. Effect of 2,4,5-TMBA on the Expression of C/EBPα, β, and δ and PPARγ during Adipogenesis. Activation of ERK is followed by the expression of C/EBPβ and C/EBPδ, which further induces the expression of PPARγ and C/EBPα.5,6 As the study design shown in Figure 2A, the expression of C/ EBPα, β, and δ and PPARγ was determined when 2,4,5-TMBA9861

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stage of adipogenesis decreases the transcription activity of PPARγ2 and suppresses differentiation of preadipocytes.13,14 Without the treatment of 2,4,5-TMBA, the ratio of PPARγ2 being phosphorylated was lower at the late stage than that at the early stage; however, culture with 2,4,5-TMBA increased the level of phosphorylated PPARγ2 at the late stage (Figure 3C), which decreased the transcription activity of PPARγ2, as shown in Figure 3D. The pattern of C/EBPα expression was similar to that of C/EBPβ and C/EBPδ. The level of C/EBPα gradually increased during adipogenesis, but the expression was suppressed in the presence of 2,4,5-TMBA (Figure 3E). Effect of 2,4,5-TMBA on the Expression of ACC, FAS, and Perilipin A. ACC is the rate-limiting enzyme in fatty acid de novo synthesis, providing malonyl-CoA substrate for the biosynthesis of fatty acids.15 FAS is a multifunctional protein with the function of catalyzing the synthesis of long-chain saturated fatty acids from acetyl-CoA and malonyl-CoA.16 As the study design shown in Figure 2A, the expression of ACC, FAS, and perilipin A was determined when 2,4,5-TMBA-treated preadipocytes were harvested at post-induction days 2, 4, 6, and 8, respectively. Without the presence of 2,4,5-TMBA, expression of ACC and FAS in preadipocytes was elevated along the process of adipogenesis. However, when preadipocytes were cultured with 2,4,5-TMBA, expression of ACC and FAS was suppressed at the terminal stage of adipogenesis, an important stage for lipid accumulation (panels A and B of Figure 4). At the late stage of lipid droplet formation and maturation, perilipin replaces the other droplet-coating proteins and becomes the only coating protein to reside on the large lipid droplets. Perilipin A is the predominant isoform of perilipin proteins found in mature adipocytes.17,18 Without the treatment of 2,4,5-TMBA, expression of perilipin A gradually increased during adipogenesis, reaching the maximum when the cells were fully differentiated (Figure 4C). Although a similar pattern was observed in 2,4,5-TMBA-treated cells, the overall expression of perilipin A was attenuated.

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DISCUSSION Previously, we showed that 2,4,5-TMBA suppresses the differentiation of preadipocytes.11 In this study, preadipocytes were co-cultured with 2,4,5-TMBA specifically at postinduction days 0−2, 2−4, 4−6, or 6−8 only to further figure out the stages that are critically affected by 2,4,5-TMBA during adipogenesis. Moreover, preadipocytes were co-cultured with 2,4,5-TMBA during the differentiation process and harvested at days 2, 4, 6, and 8 to study the expression of signaling molecules and transcription factors at different stages of adipogenesis (Figure 5). Although several signal transduction pathways are involved in adipogenesis, transient activation of the MEK/MAPKs signaling pathway is required for the differentiation of preadipocytes.19 Activation of MEK by adipogenic stimuli leads to the phosphorylation of MAPKs: ERK, c-Jun amino-terminal kinase (JNK), and p38 MAPK. ERK1 and ERK2 isoforms have distinct biological functions. ERK1 has been clearly linked to the regulation of preadipocyte differentiation and adiposity. Using ERK1−/− mice, Bost et al. demonstrated that ERK1−/− mice have fewer adipocytes and decreased adiposity than those of wild-type animals. In addition, ERK1−/− mice challenged with a high-fat diet are protected from obesity and insulin resistance.20 Bost et al. also showed that preadiocytes isolated from ERK1−/− mice exhibit impaired differentiation.7

Figure 1. Effect of the incubation period of 2,4,5-TMBA on adipogenesis. (A) Study design. “−IDI” refers to the absence of adipogenic factors (insulin, dexamethasone, and isobutylmethylxanthine) in DMEM. “+IDI” refers to the presence of adipogenic factors in culture medium. “D 0−2” to “D 6−8” groups refer to the specific post-induction period for the addition of 2,4,5-TMBA (0.5 mM) to medium containing adiopgenic factors. CBS, calf bovine serum; FBS, fetal bovine serum. (B) Photos taken under a microscope at day 8 (200×). (C) Photos taken under a microscope after Oil Red O staining at day 8 (200×). (D) Relative lipid accumulation at day 8. Values are means of three replicated cultures. Groups with the same letter are not significantly different (α = 0.05).

treated preadipocytes were harvested at post-induction days 2, 4, 6, and 8. When preadipocytes were cultured without the presence of 2,4,5-TMBA, the levels of C/EBPβ and C/EBPδ gradually increased throughout the intermediate to terminal differentiation periods. However, the treatment of 2,4,5-TMBA downregulated the overall expression of both proteins (panels A and B of Figure 3). PPARγ2 but not PPARγ1 is required for the activation of adipogenesis.12 Phosphorylation of PPARγ2 by ERK at the late 9862

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Figure 2. Effects of 2,4,5-TMBA on the expression of MEK and ERK during adipogenesis. (A) Study design. 2,4,5-TMBA (0.5 mM) was supplemented to culture medium during the adipogenic process. The cells were harvested at days 2, 4, 6, and 8. (B) Protein expression of phosphorylated MEK (pMEK) and MEK. (C) Protein expression of phosphorylated ERK (pERK) and ERK. Relative expression of proteins was calculated according to the reference band of β-actin. The ratio of phosphorylated proteins to total proteins was calculated by the comparison of relative expression. Values are means of three replicated cultures. Groups with the same letter are not significantly different (α = 0.05). MEK, MAP kinase kinase; ERK, extracellular signal-regulated kinase.

ylation at the early stage but elevated the phosphorylation during terminal differentiation, which is opposite to the conditions required for adipogenesis. The cytotoxic effect of 2,4,5-TMBA was estimated in our previous study.11 When preadiopcytes were co-cultured with 0.5 mM 2,4,5-TMBA during differentiation, the cell viability was decreased by 25.82% at the end of the 8 day differentiation period. On the basis of the results, the inhibition on adipogenesis observed in this study mostly resulted from the action of 2,4,5-TMBA instead of cytotoxicity. When the rats were orally administered with 100 mg/kg 2,4,5-TMBA, the serum concentration of 2,4,5-TMBA reached 5.44, 8.39, 9.09, and 5.64 μg/mL at 30, 60, 120, and 240 min, respectively (data not shown). Because rats did not show toxicity when gavaged with 2,4,5-TMBA at a dose of 2000 mg/kg,28 a serum concentration close to 0.5 mM (100 μg/mL) might be reached in vivo by administering a higher dose of 2,4,5-TMBA. Activation of MEK/MAPKs in preadipocyte is rapidly followed by the expression of C/EBPβ and C/EBPδ, which further induces the expression of PPARγ and C/EBPα, the two transcription factors that oversee the terminal differentiation

Phosphorylation at specific threonine and tyrosine residues by MEK leads to the activation of ERK1, and the activation needs to be timely regulated during adipogenesis.7,21 ERK1 has to be turned on at the early stage for a proliferative process but to be shut off later on to prevent the phosphorylation of PPARγ.7,20 PPARγ is a substrate of ERK, and its phosphorylation by ERK decreases its transcription activity.14,22 Prusty et al. observed a rapid induction of ERK1 phosphorylation following the exposure of preadipocyte to adipogenic stimuli, but the level of phosphorylation gradually declines during adipogenesis.23 Lii et al. showed that diallyl trisulfide, the second most abundant organosulfide in garlic oil, suppresses the differentiation of 3T3-L1 preadipocytes through prolonging ERK phosphorylation along adipogenesis.24 Turpin et al. reported carbamazepine, a dibenzazepine, strongly inhibited adipogenesis through activation of the ERK pathway.25 Moreover, both evodiamine (a major alkaloidal compound extracted from the fruit of Evodia fructus) and EGCG [(−)-epigallocatechin gallate] suppress preadipocyte differentiation through the ERK pathway.26,27 In this current study, treatment of 2,4,5-TMBA attenuated ERK phosphor9863

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Figure 3. Effects of 2,4,5-TMBA on the expression of transcription factors. The cells were cultured with 2,4,5-TMBA (0.5 mM) and harvested at days 2, 4, 6, and 8. Expression of (A) C/EBPβ, (B) C/EBPδ, (C) PPARγ2, and (E) C/EBPα. (D) Result of PPARγ activation. Relative expression of proteins was calculated according to the reference band of β-actin. The ratio of phosphorylated proteins to total proteins was calculated by the comparison of relative expression. Values are means of three replicated cultures. Groups with the same letter are not significantly different (α = 0.05). C/EBP, CCAAT/enhancer binding protein; PPAR, peroxisome proliferator-activated receptor.

process.29 C/EBPβ and C/EBPδ play a synergistic role in preadipocyte differentiation. Embryonic fibroblasts derived from mice lacking either C/EBPβ or C/EBPδ do not differentiate into mature adipocytes.30 PPARγ is the master regulator of adipogenesis.31 Activated PPARγ enhances the expression of C/EBPα, and then both factors synergistically induce the genes responsible for the maturation of adipocytes. PPARγ can promote adipogenesis in C/EBPα-deficient cells, but C/EBPα has no ability to promote adipogenesis in the

absence of PPARγ.32 PPARγ2 but not PPARγ1 is required for the activation of adipogenesis.12 PPARγ2−/− mouse embryonic fibroblasts showed a dramatically reduced capacity for adipogenesis compared to wild-type mouse embryonic fibroblasts.33 Phosphorylation of PPARγ2 by ERK decreases its transcription activity.14 In this study, co-culture with 2,4,5-TMBA downregulated the overall expression of C/EBPs and PPARγ2 but elevated the ratio of PPARγ2 being phosphorylated at the terminal stage (panels A−C and E of Figure 3), which results in 9864

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Figure 4. continued Expression of (A) ACC, (B) FAS, and (C) perilipin A. Relative expression of proteins was calculated according to the reference band of β-actin. Values are means of three replicated cultures. Groups with the same letter are not significantly different (α = 0.05). ACC, acetylCoA carboxylase; FAS, fatty acid synthase.

a decreased transcription activity of PPARγ2 (Figure 3D), leading to the attenuation of adipogenesis. This increased phosphorylation of PPARγ2 resulted from enhanced ERK1 phosphorylation by 2,4,5-TMBA at the terminal stage. The increase in size of adipocytes arises in part from progressive lipid accumulation during adipogenesis. Both ACC and FAS play crucial roles in de novo lipogenesis in mammals.15,16 The study by Wu et al. showed that husk extract, punicalagin, and ellagic acid exhibit FAS inhibitory activity. When 3T3-L1 adiocytes were incubated with these extracts and compounds, adipogenesis was inhibited.34 Lower levels of ACC and FAS were also observed in carbamazepinetreated preadipocytes, which showed impaired lipid accumulation.25 Loftus et al. have demonstrated that animals with lowered ACC and FAS activities showed significantly reduced adiposity.35 In a previous study, we showed that incubation with 2,4,5-TMBA decreased the amount of lipid accumulated in adipocytes.11 Results of this current study show that the inhibitory effect of 2,4,5-TMBA on the expression of ACC and FAS is mainly observed at the late period of adipogenesis, an important stage for lipid accumulation. One characteristic of mature adipocytes is the formation and enlargement of intracellular lipid droplets. The proteins that coat lipid droplets change during droplet biogenesis and play critical roles in the maintenance of the lipid droplet structure and regulation of lipid metabolism. At the late stage of lipid droplet formation and maturation, perilipins become the only coating protein and perilipin A is the predominant isoform found in mature adipocytes.18 In obese subjects, expression of perilipin A in adipose tissue is elevated.36 However, lipid accumulation in adipocytes is significantly decreased in perilipin null mice.37 In this study, we observed an inhibition on the expression of perilipin A when preadipocytes were cultured with 2,4,5-TMBA. Suppression of perilipin A could impair the formation of lipid droplets and maturation, leading to a decreased lipid accumulation as observed in 2,4,5-TMBAtreated cells. Taken together, our results showed that 2,4,5-TMBA may suppress adipogenesis through the regulation of ERK1 phosphorylation. 2,4,5-TMBA inhibited the phosphorylaton of ERK1 at the early stage of the adipogenic process but enhanced it at the late stage, which is opposite the conditions required for adipogenesis. Although all of the effects observed might be coincidental and not yet mechanistically interdependent, the opposite effects of 2,4,5-TMBA on ERK at the early and late stages of the 8 day adipogenic process may be mediated by the modulation of scaffolding protein AEBP-1 and phosphatases MKP-1 and MKP-3. AEBP-1 protects the ERK from specific phosphatase and is downregulated at the late stage of adipogenesis. Conversely, expression of MKP-1 and MKP-3 is upregulated in mature adipocytes.38−40 Prospectively, the interaction of 2,4,5-TMBA with AEBP-1, MKP-1, and MPL-3 will be worth studying. Moreover, application of microarray technology to monitor global changes in gene expression

Figure 4. Effects of 2,4,5-TMBA on the expression of lipogenic enzymes and lipid droplet-coating protein. The cells were cultured with 2,4,5-TMBA (0.5 mM) and harvested at days 2, 4, 6, and 8. 9865

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Figure 5. Proposed mechanism for the suppression of adipogenesis by 2,4,5-TMBA. expression during the differentiation of 3T3-L1 preadipocytes. J. Biol. Chem. 2002, 277, 46226−46232. (7) Bost, F.; Aouadi, M.; Caron, L.; Even, P.; Belmonte, N.; Prot, M.; Dani, C.; Hofman, P.; Pages, G.; Marchand-Brustel, Y. L.; Binetruy, B. The extracellular signal-regulated kinase isoform ERK1 is specifically required for in vitro and in vivo adipogenesis. Diabetes 2005, 54, 402− 411. (8) Momin, R. A.; De Witt, D. L.; Nair, M. G. Inhibition of cyclooxygenase (COX) enzymes by compounds from Daucus carota L. seeds. Phytother. Res. 2003, 17, 976−979. (9) Fajas, L.; Miard, S.; Briggs, M. R.; Auwerx, J. Selective cyclooxygenase-2 inhibitors impair adipocyte differentiation through inhibition of the clonal expansion phase. J. Lipid Res. 2003, 44, 1652−1659. (10) Ghoshal, S.; Trivedi, D. B.; Graf, G. A.; Loftin, C. D. Cyclooxygenase-2 deficiency attenuates adipose tissue differentiation and inflammation in mice. J. Biol. Chem. 2011, 286, 889−898. (11) Wu, M. R.; Hou, M. H.; Lin, Y. L.; Kuo, C. F. 2,4,5-TMBA, an inhibitor of cyclooxygenase-2, suppresses adipogenesis and enhances lipolysis in 3T3-L1 adipocytes. J. Agric. Food Chem. 2012, 60, 7262− 7269. (12) Ren, D.; Collingwood, T. N.; Rebar, E. J.; Wolffe, A. P.; Camp, H. PPARγ knockdown by engineered transcription factors: Exogenous PPARγ2 but not PPARγ1 reactivates adipogenesis. Cenes Dev. 2002, 16, 27−32. (13) Hu, E.; Kim, J. B.; Sarraf, P.; Spiegelman, B. M. Inhibition of adipogenesis through MAP kinas-mediated phosphorylation of PPARγ. Science 1996, 274, 2100−2103. (14) Camp, H. S.; Tafuri, S. R. Regulation of peroxisome proliferatoractivated receptor gamma activity by mitogen-activated protein kinase. J. Biol. Chem. 1997, 272, 10811−10816. (15) Lane, M. D.; Moss, J.; Plalkis, S. E. Acetyl coenzyme A carboxylase. Curr. Top. Cell. Regul. 1974, 8, 139−195. (16) Wakil, S. J.; Stoops, J. K.; Joshi, V. C. Fatty acid synthase and its regulation. Annu. Rev. Biochem. 1983, 52, 537−579. (17) Wolins, N. E.; Quaynor, B. K.; Skinner, J. R.; Schoenfish, M. J.; Tzekov, A.; Bicket, P. E. S3-12, adipophilin, and TIP47 package lipid in adipocytes. J. Biol. Chem. 2005, 280, 19146−19155. (18) Brasaemle, D. L. The perilipin family of structural lipid droplet proteins: Stabilization of lipid droplets and control of lipolysis. J. Lipid Res. 2007, 48, 2547−2559.

profiles during 3T3-L1 differentiation will help to elucidate the mode of regulation by 2,4,5-TMBA. Obesity results from the hypertropy of adipocytes and the recruitment of new adipocytes from preadipocytes. Both processes are significantly dependent upon the regulation of adipogenesis. Although results from in vitro studies cannot be directly extrapolated into clinical effects, our study will help to elucidate the anti-adipogenic potential of 2,4,5-TMBA.

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AUTHOR INFORMATION

Corresponding Author

*Telephone: 886-2-25381111, ext. 6214. Fax: 886-2-25334789. E-mail: [email protected]. Funding

This study was supported by the National Science Council (NSC 101-2313-B-158-001) and Shih Chien University (USC101-05-02001). Notes

The authors declare no competing financial interest.

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REFERENCES

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