Caffeic Acid Phenethyl Ester, a Major Component of Propolis

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Caffeic Acid Phenethyl Ester, a Major Component of Propolis, Suppresses High Fat Diet-Induced Obesity through Inhibiting Adipogenesis at the Mitotic Clonal Expansion Stage Seung Ho Shin,†,# Sang Gwon Seo,†,‡,§,# Soyun Min,† Hee Yang,† Eunjung Lee,†,‡ Joe Eun Son,† Jung Yeon Kwon,∥ Shuhua Yue,⊥ Min-Yu Chung,† Kee-Hong Kim,∥ Ji-Xin Cheng,⊥,¶ Hyong Joo Lee,*,√ and Ki Won Lee*,‡,§,√ †

Department of Agricultural Biotechnology and Center for Agricultural Biomaterials, ‡WCU Biomodulation Major, Department of Agricultural Biotechnology and Center for Food and Bioconvergence, Seoul National University, Seoul 151-921, Republic of Korea § Advanced Institutes of Convergence Technology, Seoul National University, Suwon 443-270, Republic of Korea √ Research Institute of Bio Food Industry, Institute of Green Bio Science and Technology, Seoul National University, Pyeongchang 232-916, Republic of Korea ∥ Department of Food Science, ⊥Weldon School of Biomedical Engineering, ¶Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, United States S Supporting Information *

ABSTRACT: In the present study, we aimed to investigate the antiobesity effect of CAPE in vivo, and the mechanism by which CAPE regulates body weight in vitro. To confirm the antiobesity effect of CAPE in vivo, mice were fed with a high fat diet (HFD) with different concentrations of CAPE for 5 weeks. CAPE significantly reduced body weight gain and epididymal fat mass in obese mice fed a HFD. In accordance with in vivo results, Oil red O staining results showed that CAPE significantly suppressed MDI-induced adipogenesis of 3T3-L1 preadipocytes. FACS analysis results showed that CAPE delayed MDIstimulated cell cycle progression, thereby contributing to inhibit mitotic clonal expansion (MCE), which is a prerequisite step for adipogenesis. Also, CAPE regulated the expression of cyclin D1 and the phosphorylation of ERK and Akt, which are upstream of cyclin D1. These results suggest that CAPE exerts an antiobesity effect in vivo, presumably through inhibiting adipogenesis at an early stage of adipogenesis. KEYWORDS: caffeic acid phenethyl ester, diet-induced obesity, 3T3-L1 preadipocytes, adipogenesis, mitotic clonal expansion

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INTRODUCTION Obesity is a major health problem worldwide that is associated with the increased risk of type 2 diabetes and other chronic diseases.1,2 Obesity is characterized by an increase in adipose tissue mass, which is associated with an increased fat cell number (hyperplasia) and cell size (hypertrophy).3 It is the murine 3T3-L1 cell line that can mimic in vivo fat cell hyperplasia and hypertrophy. The 3T3-L1 cell line is a wellestablished in vitro model for adipogenesis, a differentiation program of preadipocytes to adipocytes.4−6 Adipogenesis of 3T3-L1 preadipocytes involves growth arrest, mitotic clonal expansion (MCE), and terminal differentiation.6−8 In particular, MCE is a prerequisite step for transcriptional activation and terminal differentiation of adipocytes.9 Thus, suppression of MCE could be an effective strategy for the prevention of adipogenesis during the development of obesity. MCE is associated with elevated mRNA levels of adipogenic transcription factors, such as CCAAT/enhancer-binding protein (C/EBP) α, C/EBPβ, and peroxisome proliferatoractivated receptor (PPAR) γ.9 One of the major events that occur during MCE is increased levels of cell cycle regulators, including cyclins and cyclin-dependent kinases (CDKs).10 During the G1 phase of cell cycling, activated cyclin D and E bind with their catalytic partners to form active kinase © XXXX American Chemical Society

complexes. While cyclin D binds to CDK4 or CDK6 and cyclin E binds to CDK2, cyclin A and B interact with CDK2 and CDK1, leading to the initiation of cell cycle progression through S phase. These kinase complexes inactivate retinoblastoma (Rb) proteins and/or induce hyperphosphorylation of Rb,11 resulting in transcriptional activation of the S phaseassociated genes,12 which allows the preadipocytes synchronously to pass through the G1/S checkpoint.9 MCE is also associated with a decrease of p27, a CDK inhibitor,13 which inhibits the phosphorylation of Rb through kinase complexes, such as CDK4/cyclin D1.14 ERK and PI3K/Akt-mediated signaling pathways are also involved in the initiation of cell cycle progression, contributing to adipogenesis.15,16 Therefore, blockade or a delay of the cell cycle progression by inactivating cell cycle regulators, upregulation of CDK inhibitors, and/or inhibiting MAPK and PI3K/Akt-mediated signaling pathways during MCE would effectively contribute to suppressing adipogenesis. Received: November 19, 2013 Revised: March 9, 2014 Accepted: March 10, 2014

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difference between the pump laser and Stokes laser was tuned to 2840 cm−1, which matches the Raman shift of the symmetric CH2 stretch vibration in lipid molecules. The forward detected CARS signal was collected by an air condenser (numerical aperture = 0.55), transmitted through a 600/65-nm bandpass filter and detected by a photomultiplier tube (PMT, H7422-40, Hamamatsu, Japan). For TPEF imaging of intracellular fluorescent CAPE, the back-reflected signal was collected by the objective, spectrally separated from the excitation source, transmitted through a 520/40-nm bandpass filter and detected by a PMT (H7422-40, Hamamatsu, Japan) mounted at the back port of the microscope. Acquisition time for each image was 1.12 s. Images were analyzed using Fluo-View software (Olympus, PA, USA). Western Blot Assay. Western blot analysis was performed as previously described.22 In brief, 3T3-L1 preadipocytes were seeded in a 6 cm dish at a density of 1.5 × 105 cells per dish and cultured for two days with DMEM supplemented with 10% BCS. Confluent cells were subjected to adipogenesis with MDI in the presence or absence of CAPE (10, 20, and 40 μM). Cells were lysed, loaded onto 10% sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE), and transferred to nitrocellulose membranes (GE healthcare, Piscataway, NJ, USA). The membrane was blocked with 5% skim milk and incubated with the indicated primary antibodies. The membrane was then incubated with HRP-conjugated secondary antibodies. Protein bands were visualized by a chemiluminescence detection kit (Amersham Pharmacia Biotech; Piscataway, NJ, USA), and the density of the protein bands was quantified using the NIH Image J analysis program. Trypan Blue Assay. 3T3-L1 preadipocytes were seeded in 24-well plates at a density of 2.5 × 104 cells per well and incubated in DMEM supplemented with 10% FBS with or without CAPE (10, 20, and 40 μM). Cells were trypsinized and stained with 0.4% trypan blue for 5 min. The stained cells were loaded onto a hematocytometer, and the viable cells were counted. Flow Cytometry Using a Fluorescence-Activated Cell Sorter (FACS) Analysis. 3T3-L1 preadipocytes were seeded in a 6 cm dish at a density of 1.5 × 105 cells per dish. Confluent cells were differentiated with MDI cocktail in the presence or absence of CAPE (40 μM). After centrifugation (1,000 rpm for 3 min), cells were suspended in 70% ethanol (v/v) and fixed overnight at 4 °C. The fixed cells were then centrifuged (1,500 rpm for 3 min), resuspended in PBS containing propidium iodide (PI) solution (Sigma, St. Louis, MO, USA) and RNase (Amresco, Solon, OH, USA), and incubated at 37 °C in the dark (10 min). Cell fluorescence was measured using a FACS calibur flow cytometer (Becton-Kickinson; San Jose, CA, USA). Ten thousands cells in each sample were analyzed. Real-Time Quantitative PCR. Messenger RNA (mRNA) was extracted from mouse epididymal fat tissue using RNA-Bee (TELTEST; Pearland, TX, USA) and reverse transcribed into cDNA using Promega product (Fitchburg, WI, USA). Real-time quantitative PCR was performed as described previously23 using the SYBR Green RTPCR kit from Fermentas (Glen Burnie, MD, USA). Each reaction yielded amplicons of 101 bp. Results are expressed relative to β-actin. Statistical Analysis. In vitro data were expressed as the means ± SD, and Student’s t test was used for comparisons. A probability value of p < 0.05 or p < 0.01 was used as the criterion for statistical significance. For in vivo study, data were expressed as the means ± SEM. One-way ANOVA with Newman−Keuls test was used to evaluate mean differences between groups. Data were analyzed using Graph Pad Prism (version 4.03; Graph Pad Software, Inc., San Diego, CA, USA). Means with superscripts of different letters were significantly different at p < 0.05.

Caffeic acid phenethyl ester (CAPE) is a polyphenol abundantly found in propolis, a resinous mixture that honey bees collect from tree buds, sap flows, and other botanical sources. Approximately 12−17 mg/g of CAPE is obtained from propolis ethyl acetate extract.17,18 So far, there has been no study about the antiobesity effect of CAPE in vivo. Although studies conducted by Juman et al.19,20 demonstrated that CAPE reduced the production and secretion of adipokines as well as transcription factors associated with adipogenesis in 3T3-L1 preadipocytes, the molecular mechanism by which CAPE regulates adipogenesis is not studied. Hence, we investigated the antiobesity effect of CAPE in the diet-induced obesity (DIO) animal model and the mechanism by which CAPE regulates body weight using 3T3-L1 preadipocytes.

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MATERIALS AND METHODS

Chemicals. CAPE (of which purity is 99.4%) was obtained from Chromadex Inc. (Santa Ana, CA, USA). Antibodies against p-Akt, Akt, and C/EBPα were purchased from Cell Signaling Biotechnology (Beverly, MA, USA), and antibodies against p-ERK, ERK, cyclin D1, cyclin A1, and PPARγ were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody against β-actin was purchased from Sigma (St. Louis, MO, USA). Dexamethasone, 3-isobutyl-1-methylxanthine (IBMX), and insulin were purchased from Sigma. Animals and Diets. Male C57BL/6N mice (5-wk old) were purchased from Koatech (Pyeongtaek, Korea). After 1 week of acclimation, mice were housed in climate-controlled quarters (23 ± 3 °C at 50 ± 10% humidity) with a 12-h light/12-h dark cycle. All experimental procedures were approved by Hallym University (Gangwon-do, Korea). Mice were divided into five dietary groups (n = 10−11 per each group): a normal diet (10% fat from calorie), a high fat diet (HFD; 60% fat from calorie), and a HFD supplemented with 0.02, 0.1, or 0.5% CAPE (w/w). The standard diet and HFD were purchased from Research Diets, Inc. (New Brunswick; NJ, USA) and were given in the form of pellets for 5 weeks. Body weight was recorded every week, and food intake was monitored every two days. Food was removed 12 h before the animals were sacrificed. The mice were anesthetized, and adipose tissue, the liver, and kidney were removed, weighed, and stored at −70 °C until analysis. Cell Culture and Adipocytes Differentiation. 3T3-L1 mouse preadipocyte cell line was kindly provided by Dr. J. B. Kim (Seoul National University, Seoul, Korea). 3T3-L1 preadipocytes were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% bovine calf serum (BCS) at 5% CO2 and 37 °C until confluence. Confluent cells were incubated in DMEM supplemented with 10% fetal bovine serum (FBS) and MDI cocktail [1 μM dexamethasone, 0.5 mM IBMX, and 5 μg/mL insulin]. After 2 days, the medium was replaced with DMEM containing 10% FBS and insulin (5 μg/mL). Medium was then replaced every two days with DMEM containing 10% FBS. Oil Red O Staining. 3T3-L1 preadipocytes were seeded in 24-well plates at a density of 2.5 × 104 cells/well and incubated until confluence. The cells were then incubated with 10% FBS DMEM supplemented with MDI cocktail with or without CAPE (10, 20, and 40 μM). The medium was then changed to 10% FBS DMEM supplemented with 5 μg/mL of insulin, and replaced every two days. After the induction of cell differentiation, the medium was removed, and the mature 3T3-L1 adipocytes were fixed in 10% formalin. After fixing with 10% formalin, the cells were washed with 1,2-propanediol (Sigma, St. Louis, MO, USA) and stained with Oil red O solution. Stained cells were then washed with phosphate buffered saline (PBS), and the lipid droplets stained with Oil red O were eluted with isopropyl alcohol and quantified by measuring the absorbance at 515 nm by spectrophotometer. Multimodal Coherent Anti-Stokes Raman Scattering (CARS) Microscope. CARS and two-photon excitation fluorescence (TPEF) imaging analysis were performed as described previously.21 For CARS/TPEF imaging of lipid droplets in adipocytes, the wavenumber

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RESULTS AND DISCUSSION CAPE Suppresses HFD-Induced Body Weight Gain in C57BL/6N Mice. To investigate the antiobesity activity of CAPE in vivo, mice (5 wk old) were fed with a HFD supplemented with different concentrations of CAPE (0, 0.02, 0.1, or 0.5%) for 5 weeks. In regard to the antiobesity effect of CAPE in DIO animal model, Bezerra et al. reported that CAPE B

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induced body weight gain change was much more significant than food intake change. To normalize weight gain change to food intake, we estimated food efficiency ratio (FER) which was calculated by dividing the weight increase by the food intake. Compared to control mice, HFD-fed mice had 3.3 times greater FER, which was significantly decreased by the supplementation of CAPE at 0.02, 0.1, and 0.5% (p < 0.05) (Figure 1D). To investigate which organ was most affected by HFD, we weighed several organs including epididymal fat and liver. HFD increased epididymal fat mass by 3.5 times and CAPE significantly decreased epididymal fat mass (p < 0.05) without affecting the weights of other major organs, such as kidney and liver (Figure 1E). Indeed, dietary supplementation of CAPE at 0.5% decreased adipose tissue mass to control levels (p < 0.05) (Figure 1E). In particular, high concentration of CAPE (0.5%) nearly normalized it to the extent observed in the mice fed a control diet. We next examined the effect of CAPE on the expression of genes associated with adiposity. CAPE inhibited mRNA expression of adipogenic and lipogenic genes such as PPARγ, C/EBPα, and fatty acid synthase (FAS) (Figure S1, Supporting Information), and induced mRNA expression of lipolytic and thermogenic genes such as adipose triacylglycerol lipase (ATGL), peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), and long chain acyl CoA synthetase (ACSL) (Figure S2, Supporting Information). These data collectively suggest that the supplementation of CAPE attenuates HFD-induced body weight gain, which is attributable to fat mass reduction possibly by inhibiting adipogenesis and increasing energy expenditure. CAPE Inhibits MDI-Induced Adipogenesis in 3T3-L1 Preadipocytes. To elucidate the underlying molecular mechanism of the antiobesity effect of CAPE, we used 3T3L1 preadipocytes. Since Juman et al. reported that CAPE inhibits the differentiation of 3T3-L1 preadipocytes,19,20 we confirmed the antiadipogenic activity of CAPE. Consistent with a previous report by Juman et al., CAPE dose-dependently inhibited the adipogenesis of 3T3-L1 preadipocytes (Figure 2A,B). We attempted to compare the antiadipogenic activity of CAPE with genistein, which is widely known as a suppressor of adipogenesis 25−27 during the differentiation of 3T3-L1 preadipocytes. Interestingly, a lower level of CAPE (40 μM) more effectively reduced cellular lipid contents than genistein (80 μM) (Figure 2B). CARS/TPEF microscopy has recently been developed as a novel noninvasive and label-free imaging technique to visualize lipid-rich biological structures and naturally fluorescent intracellular molecules.21 In an attempt to visualize lipid accumulation in differentiated adipocytes, CARS/TPEF microscopic analysis was performed. We observed that CAPE treatment dramatically reduces the number and size of lipid droplets of fully differentiated 3T3-L1 cells with a complete inhibition of adipogenesis at 40 μM (Figure 2C). Since CAPE is a naturally fluorescent molecule, CARS/TPEF microscopy also allowed us to visualize intracellular accumulation of CAPE, which is mostly found in the cytoplasm of differentiated 3T3L1 preadipocytes (Figure 2C). To confirm the antiadipogenic effect of CAPE at the molecular level, we checked the protein level of two well-known adipocyte marker genes, PPARγ and C/EBPα. Figure 2D showed that CAPE attenuated the protein level of PPARγ and C/EBPα (Figure 2D). We next performed the trypan blue assay to check whether these antiadipogenic activities of CAPE come from its cytotoxicity. The trypan blue assay showed that CAPE,

improves glucose sensitivity and reduces hepatic steatosis without altering body weight.24 However, in their study, they fed HFD to mice without CAPE for 8 weeks and started CAPE administration after the mice already became obese. Unlike the previous study, we started HFD and CAPE administration at the same time. The result shows that HFD-fed mice showed greater level of adiposity (Figure 1A). However, HFD-fed mice

Figure 1. Effects of CAPE on HFD-induced obesity in C57BL/6N mice (n = 11). (A and B) CAPE treatment for 5 weeks alleviated HFD-induced weight gain in C57BL/6N mice. (C) Food intake was slightly reduced by CAPE (0.1 and 0.5%). (D) Food efficiency ratio was attenuated by CAPE treatment. (E) Epididymal fat tissue mass was significantly reduced by CAPE treatment, but liver and kidney tissue masses were unaffected by CAPE treatment. Values are expressed as the means ± SEM. The means marked with superscript letters are significantly different (p < 0.05).

supplemented with CAPE showed decreased level of adiposity in a dose-dependent manner (Figure 1A). Indeed, HFD-fed mice displayed significantly increased body weight (p < 0.01) (Figure 1B). CAPE decreased HFD-induced body weight gain completely to control levels at 0.5% (p < 0.01) (Figure 1B). Although food intake was affected slightly by HFD or CAPE as the same tendency as weight gain (Figure 1C), HFD or CAPEC

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that CAPE suppresses MDI-induced adipogenesis of 3T3-L1 preadipocytes dose-dependently. CAPE Exerts Its Antiadipogenic Activity at the Early Stage of Differentiation. A previous study conducted by Juman et al.19 demonstrated that 50 μM CAPE significantly decreases total lipid contents of differentiated 3T3-L1 preadipocytes by ∼17% compared to that of MDI-treated control cells.19 However, our result showed that 40 μM CAPE completely inhibited adipogenesis of 3T3-L1 preadipocytes. We hypothesized that this discrepancy might be due to different CAPE-treated period, because they treated CAPE during days 3−7,19 whereas we treated CAPE for the whole adipogenesis process. To clarify this discrepancy and to gain insight into the molecular mechanism underlying CAPE-inhibited adipogenesis, we examined the effect of CAPE (40 μM) on the adipogenesis of 3T3-L1 preadipocytes at different stages of cell differentiation (Figure 3A). Oil red O staining results showed that

Figure 3. Effects of CAPE on 3T3-L1 preadipocytes during different stages of MDI-induced adipogenesis. (A) A time schedule for CAPE treatment during cellular differentiation. (B) CAPE reduced lipid accumulation when treated during days 0−2. Data are expressed as the mean ± SEM from three separate experiments. ## indicate a significant difference between the control group and the group treated with MDI alone (p < 0.01), and ** indicate a significant difference between groups treated with MDI alone and treated with MDI plus CAPE (p < 0.01).

Figure 2. Effects of CAPE on MDI-induced adipogenesis in 3T3-L1 preadipocytes. (A,B) CAPE effectively inhibited MDI-induced adipogenesis of 3T3-L1 preadipocytes. Antiadipogenic effect of CAPE was better than that of genistein. Data are expressed as the means ± SEM from three separate experiments. (C) CAPE inhibited intracellular lipid accumulation of adipocytes, as evidenced by multimodal CARS/ TPEF microscopy. (D) CAPE suppressed the protein expressions of MDI-induced PPARγ and C/EBPα. Data are representative of three independent experiments that showed the same tendency. (E) CAPE did not show cytotoxicity in 3T3-L1 preadipocytes up to 40 μM. Data are expressed as the means ± SEM from three separate experiments. ## indicate a significant difference between the control group and the group treated with MDI alone (p < 0.01), and ** indicate a significant difference between groups treated with MDI alone and treated with MDI plus CAPE (p < 0.01).

CAPE treatment between days 0−2 and 0−4 significantly suppressed MDI-induced adipogenesis (p < 0.01) (Figure 3B). In particular, the treatment of CAPE at 40 μM during day 0−4 almost blocked lipid accumulation similar to the extent observed in undifferentiated control cells. Notably, CAPE exerted little effect on the adipogenesis of 3T3-L1 preadipocytes when it was treated at days 2−4, 2−6, 4−6, and 4−8. Since adipogenesis of 3T3-L1 preadipocytes consist of MCE in the early phase of differentiation and terminal differentiation,6−8 these results indicate that CAPE inhibits adipogenesis by suppressing cellular differentiation during MCE, an early stage of adipogenesis. Also, CAPE treatment during days 2−4

up to 40 μM, did not show any toxicity in 3T3-L1 preadipocytes (Figure 2E). Collectively, these findings indicate D

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regulated in MDI-treated 3T3-L1 cells and that CAPE (40 μM) suppressed MDI-mediated upregulation of cyclin D1 (Figure 5A). We checked the mRNA expression level of cyclin D1 to

in our study showed antiadipogenic activity similar to the previous result by Juman et al. Since they treated CAPE after MDI treatment, their result corresponds to the CAPE treatment during days 2−4 in our study. CAPE Delays Cell Cycle Progression during the Early Stage of Cellular Differentiation. We further attempted to elucidate the effect of CAPE on MCE during adipogenesis. The inhibitory effect of CAPE on cell cycle progression was examined (Figure 4). FACS analysis data showed that MDI-

Figure 5. Effects of CAPE on cyclin D1 expression and its upstream signaling pathway in 3T3-L1 preadipocytes. (A) CAPE suppressed MDI-induced cyclin D1 protein expression. Data are representative of three independent experiments that showed the same tendency. (B) CAPE suppressed MDI-induced cyclin D1 mRNA expression. Data are expressed as the mean ± SEM from three separate experiments. ## indicate a significant difference between the control group and the group treated with MDI alone (p < 0.01), * indicates a significant difference between groups treated with MDI alone and treated with MDI plus CAPE (p < 0.05), and **, indicate a significant difference between groups treated with MDI alone and those treated with MDI plus CAPE (p < 0.01). (C) CAPE suppressed MDI-induced ERK and Akt phosphorylation. Data are representative of three independent experiments that showed the same tendency.

Figure 4. Effects of CAPE on MDI-induced cell cycle progression in 3T3-L1 preadipocytes. (A) CAPE inhibited the MDI-induced cell cycle progression of 3T3-L1 preadipocytes. Data are representative of three independent experiments that showed the same tendency. (B) The population of cells in each stage of the cell cycle was quantified.

induced cell cycle progression was likely delayed by 40 μM of CAPE treatment (Figure 4A). The cell population in each stage of the cell cycle was quantified (Figure 4B). Untreated 3T3-L1 preadipocytes showed no remarkable changes in cell cycle progression during 36 h of culture, mostly staying in the G1/S checkpoint (basal state). However, cells passed the G1/S checkpoint at 16 h of MDI treatment and passed the S to G2/ M phase at 20 h of MDI treatment. The cells exited the M phase and reentered into G1 phase from 24 to 36 h of MDI treatment. Meanwhile, cells treated with CAPE at 40 μM stayed at the G1/S checkpoint until 20 h of MDI treatment, but they entered S phase at 24 h. Moreover, we observed that cells treated with CAPE did not exit the G2/M phase and were arrested at the G2/M phase at 36 h (Figure 4B). These collectively indicate that CAPE delays cell cycle progression during MCE, thereby inhibiting adipogenesis of 3T3-L1 preadipocytes stimulated by MDI. CAPE Suppresses the Expression of Cyclin D1 during MCE. Since G1/S progression was significantly attenuated by CAPE treatment, we measured the protein expression level of cyclin D1, which has been reported to be implicated in G1/S transition. We found that the protein level of cyclin D1 was up-

investigate whether CAPE regulates cyclin D1 expression at the transcriptional level. We found that the mRNA level of cyclin D1 was also suppressed by 40 μM CAPE treatment (Figure 5B), indicating that CAPE inhibits cyclin D1 expression at the transcriptional level. The result that CAPE decreased both the mRNA and protein levels of cyclin D1 prompted us to investigate the upstream signaling pathway of cyclin D1 expression. We estimated the effect of CAPE on MDI-induced ERK and Akt phosphorylation, which have been reported as upstream of cyclin D1. We found that MDI-induced phosphorylation of ERK and Akt was decreased by CAPE (40 μM) (Figure 5C). Collectively, CAPE suppressed mitotic clonal expansion of 3T3-L1 by downregulating cyclin D1 by blocking MDI-mediated phosphorylation of ERK and Akt during MCE. E

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ASSOCIATED CONTENT

S Supporting Information *

Effects of CAPE on HFD-induced mRNA expression of adipogenic and lipogenic genes in C57BL/6N mice (N = 55); effects of CAPE on mRNA expression of lipolytic and thermogenic genes in C57BL/6N mice (N = 55). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*(H.J.L.) Tel: 82-2-880-4853. Fax: 82-2-873-5095. E-mail: [email protected]. *(K.W.L.) Tel: 82-2-880-4661. Fax: 82-2-878-6178. E-mail: [email protected]. Author Contributions #

S.H.S. and S.G.S. contributed equally to this work.

Funding

This research was supported by grant from National Research Foundation (NRF) of Korea (No. 2012M3A9C4048818) funded by the ministry of Science, ICT and Future Planning. This research was also supported by the grant from National Leap Research Program (No. 2010-0029233) through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology of Korea, Republic of Korea. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Kang, M. J., Suk, S. J., and Jung, E. S. for critical reading of this manuscript and Lee, D. E. and Song, N. R. for discussions.

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ABBREVIATIONS USED ACSL, long chain acyl CoA synthetase; ATGL, adipose triacylglycerol lipase; BCS, bovine calf serum; CAPE, caffeic acid phenethyl ester; CARS, coherent anti-Stokes Raman scattering; CDK, cyclin-dependent kinase; C/EBP, CCAATenhancer-binding protein; DIO, diet-induced obesity; ERK, extracellular signal-regulated kinase; FACS, fluorescence activated cell sorting; FAS, fatty acid synthase; FBS, fetal bovine serum; FER, food efficiency ratio; HFD, high fat diet; HRP, horseradish peroxidase; IBMX, isobutylmethylxanthine; MAPK, mitogen-activated protein kinase; MCE, mitotic clonal expansion; MDI, mixture of isobutylmethylxanthine, dexamethasone and insulin; PI, propidium iodide; PI3K, phosphatidylinositol 3-kinase; PGC-1α, peroxisome proliferator-activated receptor gamma coactivator 1α; PPAR, peroxisome proliferator-activated receptor; Rb, retinoblastoma; TPEF, twophoton excitation fluorescence

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