7,8-Dihydroxyflavone inhibits adipocyte differentiation via antioxidant activity and induces apoptosis in 3T3-L1 preadipocyte cells

Life Sciences 144 (2016) 103–112

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7,8-Dihydroxyflavone inhibits adipocyte differentiation via antioxidant activity and induces apoptosis in 3T3-L1 preadipocyte cells Ji Won Choi a, Chang Won Lee a, Jisun Lee a, Doo Jin Choi a, Jae Kyung Sohng b, Yong Il Park a,⁎ a b

Department of Biotechnology, The CUK Agromedical Research Center, The Catholic University of Korea, Bucheon, Gyeonggi-do 420-743, Republic of Korea Department of Pharmaceutical Engineering, Institute of Biomolecule Reconstruction, Sun Moon University, Asansi, Chungnam 336-708, Republic of Korea

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Article history: Received 21 May 2015 Received in revised form 29 September 2015 Accepted 25 November 2015 Available online 02 December 2015 Keywords: 7,8-Dihydroxyflavone Adipocyte Differentiation Antioxidant Anti-obesity

a b s t r a c t Aims: Anti-obesity effects of a natural plant flavonoid 7,8-dihydroxyflavone (7,8-DHF) were evaluated using 3T3L1 preadipocyte cells. Main methods: The cell viability was determined using MTT assay. Effects of 7,8-DHF on intracellular lipid droplets and intracellular reactive oxygen species (ROS) were measured using a 2,7-dichlorofluorescein diacetate (DCFDA) assay and Oil Red O staining method, respectively. Apoptotic cell death was monitored by annexin V-FITC/ PI double staining and by a TUNEL assay. Antioxidant enzyme mRNA levels and protein expression of adipogenic transcription factors were determined by real-time PCR and Western blotting, respectively. Key findings: Whereas the cell viability of 3T3-L1 preadipocytes was not affected by lower concentrations of 7,8DHF (b20 μM), higher concentrations of 7,8-DHF (N 20 μM) induced apoptotic cell death. 7,8-DHF (b20 μM) significantly reduced the intracellular lipid droplets and the expression of major adipogenic transcription factors, such as CCAAT/enhancer-binding protein-α (C/EBP-α), C/EBP-β, and peroxisome proliferator activated receptor-γ (PPAR-γ). 7,8-DHF treatment also dose-dependently reduced the intracellular ROS level, attenuated MAPK pathway activation, and increased the expression of antioxidant enzymes, such as Mn-superoxide dismutase (Mn-SOD), catalase (CAT), and heme oxygenase-1 (HO-1). Significance: The results of this study indicated that 7,8-DHF inhibits the adipogenesis of 3T3-L1 preadipocyte cells by down-regulating the expression of adipogenic transcription factors, reduces lipid accumulation, and attenuates ROS accumulation by inducing antioxidant enzymes in differentiated 3T3-L1 cells, suggesting for the first time that 7,8-DHF has an anti-obesity effect in vitro via its anti-oxidant activity. © 2015 Elsevier Inc. All rights reserved.

1. Introduction Obesity induces serious health problems independently or in association with other diseases, including coronary heart disease, certain forms of cancer and osteoarthritis of large and small joints [1]. The development of obesity is characterized by an increased number of fat cells and increased lipids in those fat cells as a result of the mitogenesis and differentiation processes of adipocytes [2]. Adipocyte differentiation requires the activation of several adipogenic transcription factors such as CCAAT/enhancer-binding protein-α, β (C/EBP-α, β) and peroxisome proliferator activated receptor-γ (PPAR-γ). At the terminal phase of differentiation, adipocytes synthesize adipose tissue-specific products including an adipocyte-specific fatty acid binding protein (aP2) [3]. The increase in fat-derived reactive oxygen species (fat ROS) during the development of obesity has been reported to be a main causative factor of the aberrant regulation of adipocytokines and the decreased expression of antioxidative enzymes in adipose tissue [4,5]. The ⁎ Corresponding author at: Department of Biotechnology, The Catholic University of Korea, 43 Jibong-ro, Wonmi-gu, Bucheon, Gyeonggi-do 420-743, Republic of Korea. E-mail address: [email protected] (Y.I. Park).

http://dx.doi.org/10.1016/j.lfs.2015.11.028 0024-3205/© 2015 Elsevier Inc. All rights reserved.

increased ROS level is also known to trigger the activation of the mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK), p38, and Jun N-terminal kinase (JNK), which are involved in the initiation of the differentiation of preadipocytes, and activated MAPKs trigger the proliferation of adipocytes [6,7]. Indeed, oxidative stress induces lipid accumulation in HepG2 cells and facilitates adipose differentiation by accelerating mitotic clonal expansion [8,9]. Several reports have described that antioxidant compounds could suppress adipogenesis [10,11], and thus any compound eliminating ROS is a potential target for attenuating obesity. Many antioxidants have been reported to exhibit potent anti-obesity effects [12–14]. Recently, flavonoids, which are abundant in plant foods, have attracted significant public attention due to their various biological activities [15,16]. 7,8-Dihydroxyflavone (7,8-DHF, 7,8-dihydroxy-2phenyl-4H-chromen-4-one) is a naturally occurring flavone found in plants including primula tree [17]. 7,8-DHF has been reported to exert beneficial health effects, such as anti-inflammatory, vasorelaxing and antihypertensive effects [18,19]. Several studies have demonstrated the antioxidant activity of 7,8-DHF, including a protective effect against hydrogen peroxide (H2O2)-induced DNA damage, a neuroprotective

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effect against glutamate-induced toxicity, and protective effects against 6-hydroxydopamine-induced cytotoxicity on PC12 cells and oxidative stress-induced cell damage on human keratinocytes [20–23]. Interestingly, several agonists of TrkB such as BDNF, neurotrophon-3,4, and 7,8-DHF have been reported to increase brown adipose tissue which dissipates energy directly, unlike white adipose tissue which accumulates excess energy in in vivo models [24]. Therefore, 7,8-DHF, which is a well-known antioxidant against several stimuli, may exhibit a direct anti-obesity effect. However, no specific studies have addressed the direct association between 7,8-DHF and obesity. We hypothesized that 7,8-DHF could regulate the adipogenic differentiation of preadipocytes and lipid metabolism during differentiation. To test this hypothesis, the effects of 7,8-DHF on the proliferation of preadipocytes, lipid accumulation during cell differentiation, and the expression of adipogenesis-specific transcription factor proteins were investigated in vitro using the 3T3-L1 preadipocyte cell line [3,25]. Considering that oxidative stress induces lipid accumulation and facilitates adipose differentiation [8,9], the effects of 7,8-DHF on intracellular ROS generation during cell differentiation and the underlying mechanisms of its anti-obesity effect via antioxidant activity were also examined. 2. Methods and materials 2.1. Materials and reagents Dulbecco's modified eagle's media (DMEM) and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific (Waltham, MA, USA), and antibiotics and PBS were obtained from GIBCO-BRL (Grand Island, NY, USA). The murine 3T3-L1 pre-adipocyte cell line (ATCC® CL173™) was purchased from the American Type Culture Collection (Manassas, VA, USA), and 7,8-dihydroxyflavone (7,8-DHF), Oil-Red O stock solution, IBMX (3-isobutyl-1-methylxanthine), dexamethasone, insulin, and a triglyceride (TG) assay kit were purchased from SigmaAldrich (St. Louis, MO, USA). Antibodies specific to C/EBP-α, C/EBP-β, PPAR-γ, aP2, total extracellular signal-regulated kinases (t-ERK), phospho-ERK (p-ERK), total-p38 MAPK (t-p38), phospho-p38 MAPK (p-p38), manganese superoxide dismutase (Mn-SOD), catalase (CAT), heme oxygenase-1 (HO-1), cleaved-caspase 8, cleaved-caspase 9, cleaved-caspase 3 and goat anti-rabbit IgG-HRP were obtained from Cell Signaling Technology (Danvers, MA, USA). 3-(4,5dimethylthiazol-yl)-diphenyl tetrazoliumbromide (MTT) was provided by DUCHEPA Biochemie (Haarlem, Netherlands) and 1,1-diphenyl-2picrylhydrazyl (DPPH) was purchased from Wako Pure Chemical Industries (Chuo-Ku, Osaka, JPN). 2.2. Cell culture 3T3-L1 cells (Mouse embryonic fibroblast-adipose like cell line) were grown in DMEM supplemented with 10% FBS, 2 mM L-glutamine, and 100 U/ml penicillin/streptomycin and maintained at 37 °C in a 5% CO2 incubator. Upon reaching confluence (Day 0), cells were stimulated with 0.5 μM IBMX, 1 μM dexamethasone and 10 μg/ml insulin in DMEM containing 10% FBS (MDI differentiation medium) for 2 days (Day 2). Cells were then maintained in DMEM containing 10% FBS and 10 μg/ ml insulin for another 2 days (Day 4), followed by DMEM containing 10% FBS for 4 days (Day 8), at which point over 90% of cells became mature adipocytes with lipid-filled droplets. 2.3. Measurement of cell viability The cytotoxicity of 7′8′-DHF to 3T3-L1 cells was determined by measuring the cell viability using the MTT assay. Precultured cells (2 × 104 cells/well) in DMEM were seeded on a 96-well microplate and exposed to different concentrations (0, 1, 10, and 20 μM) of 7′8′-DHF for 24 h. MTT solution (100 μg/well) was then added and the plates were

incubated for 4 h. After removal of the media, 200 μl DMSO was added into each well to solubilize the formazan crystals, and the absorbance was read at 570 nm on a microplate reader (Molecular Devices, CA, USA). 2.4. Oil Red O staining and measurement of triglyceride content To determine the lipid content, 3T3-L1 preadipocytes (7 × 104 cells/ ml) were incubated in MDI differentiation medium with 7,8-DHF (0– 20 μM) for 8 days in 6-well plates and stained with Oil Red O dye. After incubation, cells were washed gently with PBS, fixed with 4% paraformaldehyde at room temperature for 1 h and then stained with Oil Red O staining solution for 30 min. After staining, excess dye was removed and the plates were rinsed with PBS and dried. The stained lipid droplets were first visualized under the microscope (×100, Olympus, Tokyo, Japan), and then the stained lipid droplets were solubilized by adding isopropanol (Duksan, Korea) to each well to enable quantitative measurement of the lipid content. The absorbance was read at 500 nm using a microplate reader. Cellular triglyceride (TG) contents were measured using a commercial triglyceride assay kit (Sigma-Aldrich, MO, USA) according to the manufacturer's instructions. Differentiated adipocytes (Day 8) were treated with different concentrations of 7,8-DHF (0–20 μM) in 6-well plates. Cells were washed with PBS, scraped into 200 μL PBS, and homogenized by sonication for 1 min. The lysates were assayed for total TG. 2.5. Western blot analysis The cells grown for 8 days in differentiation medium with 7,8-DHF (0–20 μM) were collected by scraping in lysis buffer (20 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, pH 7.8) containing protease and phosphatase inhibitor cocktails (Roche, Germany). After 1 h on ice, the cell lysates were centrifuged at 14,000 rpm for 30 min at 4 °C. The supernatant was used to determine the protein concentration by the Bradford assay. After SDS-PAGE on 10 and 12% gels, proteins were transferred to nitrocellulose membranes and incubated with the indicated primary antibodies overnight at 4 °C, and washed three times. The protein bands were then detected using horseradish peroxidase (HRP)-conjugated antibodies for 2 h at room temperature and viewed using the enhanced chemiluminescence method (AbClon, Seoul, Korea). The relative protein levels were quantified using Image J software from the NIH (Bethesda, MD, USA). 2.6. DPPH radical-scavenging assay The effect of 7,8-DHF on the scavenging of DPPH radicals was examined by a modified method described earlier by Yamaguchi et al. [26]. Briefly, 1.8 ml of DPPH solution (0.1 mM, in methanol) was incubated with varying concentrations of 7,8-DHF (0.2 ml). The reaction mixture was incubated for 10 min at room temperature, and the absorbance of the resulting solution was read at 518 nm on a microplate reader. The radical scavenging activity was determined as a decrease in the absorbance of DPPH and calculated using the following equation: Scavenging effect ð%Þ ¼ ½ð1−A518 ðsampleÞ=A518 ðcontrolÞ  100:

2.7. Measurement of intracellular ROS The fluorescent dye DCF-DA was used to detect the level of intracellular ROS. 3T3-L1 preadipocytes (7 × 104 cells/ml) were incubated in MDI differentiation medium with 7,8-DHF (0–20 μM) for 8 days in 6well plates. Cells were harvested and stained with 10 μM DCF-DA (Thermo Fisher Scientific, MA, USA) for 45 min at 37 °C. The fluorescence intensity was monitored using a fluorescence spectrophotometer with excitation and emission wavelengths of 485 nm and 530 nm,

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respectively. Cells were seeded on an 8-well cell culture slide (SPL Life Sciences, Gyeonggi-Do, Korea) at 2 × 105 cells/well. Upon reaching confluence, cells were incubated in MDI differentiation medium with 7,8DHF (20, 30 μM) for 8 days and then washed gently with PBS, fixed with 1% paraformaldehyde for 15 min at 4 °C and stained with rhodamine (Thermo Fisher Scientific, MA, USA) for 3 h at 37 °C. After cytosolic staining, the cells were washed gently with PBS three times and stained with 10 μM DCF-DA for 30 min at 37 °C. After washing with PBS, the stained cells were mounted on a microscope slide in mounting medium (Vector Laboratories, CA, USA) and visualized using fluorescence microscopy (×100).

and 3 were examined, as these proteins are known to play key roles in the activation of apoptotic cell death (Fig. 1C). After the cells were treated with 7,8-DHF (0–100 μM) for 24 h, the protein expression levels of cleaved caspase 8, cleaved caspase 9, and cleaved caspase 3 in 3T3-L1 cells were measured by Western blot analysis. 7,8-DHF significantly increased the levels of all of these proteins in a dose-dependent manner compared to the untreated control (Con) cells, suggesting that 7,8DHF induces apoptotic cell death in 3T3-L1 preadipocytes.

2.8. Statistical analysis

To investigate the effect of 7,8-DHF on lipid accumulation during the differentiation of 3T3-L1 preadipocytes to mature adipocytes, the cells were treated with various concentrations of 7,8-DHF every 2 days for 8 days and intracellular oil droplets were stained with Oil Red O dye. As shown in Fig. 2A, the Oil Red O staining demonstrated that the intensity of stained regions (pink-colored) dramatically increased in a dosedependent manner as the concentration (0, 1, 10, 20 μM) of 7,8-DHF was increased, suggesting that 7,8-DHF significantly suppressed lipid accumulation. Consistent with this observation, after solubilizing the stained lipid droplets with isopropanol, the spectrophotometric measurement of the lipid content exhibited a dose-dependent decrease (Fig. 2B). In the control cells (Con, differentiated but 7,8-DHF-untreated cells), the intracellular lipids accumulated at high levels compared to the undifferentiated preadipocyte cells (UC), indicating that the preadipocyte cells were fully differentiated into mature adipocytes, actively synthesizing lipids. However, this elevated level of lipids was markedly reduced by 25, 45, and 59%, respectively, upon treatment with 7,8-DHF (1, 10, 20 μM), demonstrating that 7,8-DHF inhibited lipid accumulation during the differentiation of adipocyte cells. In addition, the increased TG contents in differentiated adipocytes (Con) also decreased to 9, 31, and 54% upon 7,8-DHF (1, 10, and 20 μM) treatment (Fig. 2C).

Data are reported as the mean ± SD. Comparisons between two groups were performed by unpaired Student's t-tests. A p-value of less than 0.05 was considered statistically significant. 3. Results 3.1. Effect of 7,8-DHF on cell viability and apoptotic proteins in 3T3-L1 cells To determine the effects of 7,8-DHF on the growth of proliferating preadipocytes, the cell viability was measured by an MTT assay after treatment with 7,8-DHF for 24 h. As shown in Fig. 1B, the cell viability decreased in a dose-dependent manner as the 7,8-DHF concentration increased, with 104%, 93%, 88.6%, 57.6% and 51.8% viabilities recorded at 7,8-DHF concentrations of 1, 10, 20, 50 and 100 μM, respectively, compared to untreated control cells (Con). At relatively higher concentrations (N 50 μM), 7,8-DHF exhibited significant cytotoxicity to the preadipocyte cells, while the cytotoxicity was not significant at lower concentrations (b20 μM). To clarify whether the cytotoxicity observed over 50 μM 7,8-DHF is associated with apoptotic cell death, the effects of 7,8-DHF on the cleavage of apoptotic proteins such as caspase 8, 9,

3.2. Effects of 7,8-DHF on lipid accumulation and intracellular TG content

Fig. 1. Effects of 7,8-DHF on the viability of 3T3 L1 cells and the expression of apoptotic proteins. (A) Structure of 7,8-DHF. (B) Cell viability was assessed using the MTT assay after 24 h exposure to increasing concentrations of 7,8-DHF. The data are expressed as a percentage normalized to untreated control cells. (C) Cells were collected 24 h after 7,8-DHF treatment and processed for Western blotting analysis of cleaved-caspase 8, cleaved-caspase 9, and cleaved-caspase 3. Tubulin was used as a loading control. Data = mean ± SD, n = 3. *p b 0.05; **, p b 0.01; ***, p b 0.001, Student's t-test compared to control (Con).

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Fig. 2. Effects of 7,8-DHF on the lipid accumulation (A, B) and TG levels (C) of 3T3-L1 cells. (A) Cells were cultured with or without 7,8-DHF (0–20 μM) for 8 days. Cells were then stained with Oil Red O staining solution, and Oil Red O positive regions in the adipocytes were visualized under a light microscope (×100, Olympus). (B) The stained lipid droplets were solubilized with isopropanol and the absorbance was read at 500 nm using a microplate reader. (C) Cellular TG content was measured using a commercial TG assay kit. Data = mean ± SD, n = 3. *p b 0.05; **, p b 0.01; ***, p b 0.001, Student's t-test compared to control (0 μg/ml). (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)

3.3. 7,8-DHF suppresses the expression of adipogenic proteins during adipocyte differentiation Various transcription factors and adipocyte-specific proteins such as C/EBP-α, C/EBP-β, PPAR-γ and aP2 are known to be expressed throughout the progression of adipogenesis [27]. To examine the influence of 7,8-DHF on the expression of these adipogenesis-specific proteins, 3T3-L1 cells were treated with 7,8-DHF (1, 10, and 20 μM) during differentiation. Western blot analysis revealed that 7,8-DHF induced a significant and dose-dependent reduction in the expression of both PPAR-γ, a major transcription factor regulating adipogenesis (Fig. 3A), and aP2, an important marker protein of mature adipocyte cells (Fig. 3B), compared to cells in the differentiated control group (Con). In addition, treatment with 20 μM 7,8-DHF during differentiation also significantly reduced the expression of both C/EBP-β (Fig. 3C), the first transcription factor, and C/ EBP-α (Fig. 3D), which is a C/EBP-β mediated protein, by 63% and 61%, respectively, compared to the control group (Con, differentiated cells not treated with 7,8-DHF). These results indicate that 7,8-DHF significantly inhibits the differentiation of preadipocytes to mature adipocytes by down-regulating the expression of various adipocyte-specific transcription factors and proteins involved in lipid metabolism.

3.4. 7,8-DHF inhibits intracellular ROS accumulation during adipocyte differentiation ROS have been reported to accumulate in adipocytes in parallel with lipids during adipogenesis [4]. To determine whether 7,8-DHF inhibits the generation of ROS in adipocytes, the differentiating cells were cultured with 7,8-DHF and then stained with 10 μM DCF-DA, and the fluorescence was measured using a fluorescence spectrophotometer. As shown in Fig. 4A, while ROS production markedly increased in

differentiated 3T3-L1 adipocytes (Con) compared to the undifferentiated control cells (UC), the increased intracellular ROS level in differentiated adipocytes decreased in response to treatment with 7,8-DHF (1, 10, and 20 μM) by 11, 15, and 37%, respectively, compared with untreated control cells (Con, 100%). Changes in the ROS levels in the cytosol of 3T3-L1 cells were monitored by image analysis after staining the cells with DCF-DA, and the stained cells were visualized using fluorescence microscopy. Rhodamine was used as a counter stain. As shown in Fig. 4B, many differentiated adipocyte control cells (Con) were heavily stained with DCF-DA, indicating that the differentiating cells accumulate substantial amounts of ROS in their cytosol. However, the number of DCF-DA-positive cells decreased significantly in a dose-dependent manner due to the treatment of cells with 7,8-DHF (20 and 30 μM), suggesting that 7,8-DHF inhibited ROS production during adipocyte differentiation (Fig. 4B). This result suggests that 7,8-DHF may exert antioxidant activity in adipocyte cells.

3.5. 7,8-DHF up-regulates the expression of antioxidant enzymes and scavenges DPPH radicals To confirm the antioxidant activity of 7,8-DHF, the effects of 7,8-DHF on the expression of several intracellular antioxidant enzymes and in vitro DPPH radical scavenging activity were examined. The 3T3-L1 cells were cultured in differentiation medium in the presence (1, 10, and 20 μM) or absence of 7,8-DHF, and the levels of the antioxidant proteins Mn-SOD, CAT, and HO-1 were determined by Western blot analysis. As shown in Fig. 5, the protein levels of Mn-SOD, CAT, and HO-1 were markedly increased in the differentiated control cells (Con) compared to the undifferentiated control cells (UC). These elevated levels of antioxidant enzymes during differentiation were further increased

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Fig. 3. Effects of 7,8-DHF on the expression of adipogenic transcription factors in 3T3 L1 cells. Cells were differentiated with 1, 10, and 20 μM of 7,8-DHF for 8 days. Cells were collected and processed for Western blot analysis of the expression levels of adipogenic proteins such as (A) C/EBP-α, (B) C/EBP-β, (C) PPAR-γ, and (D) aP2. Actin was used as a loading control. The data are expressed as the fold change normalized to control cells. Data = mean ± SD, n = 3. *p b 0.05; **, p b 0.01; ***, p b 0.001, Student's t-test compared to control (0 μg/ml).

in a dose-dependent manner by treatment with 7,8-DHF (1, 10, and 20 μM) (Fig. 5A, B, and C). On the other hand, 7,8-DHF also exhibited DPPH scavenging activity comparable to that of ascorbic acid, used as a positive control (Fig. 5D). The radical scavenging activity of DPPH was 2, 6, 15, 32.5, and 66% at 18.75, 37.5, 75, 150, and 300 μM 7,8-DHF, respectively. These results on DPPH radical scavenging activity indicate that 7,8-DHF can also directly remove the ROS. 3.6. Effect of 7,8-DHF on the MAPK signaling pathway during adipocyte differentiation To study the molecular mechanism of the antioxidant action of 7,8DHF during adipocyte differentiation, the effects of 7,8-DHF on the activation of mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinase (ERK) and p38, were examined by

Western blot analysis. MAPK proteins are known to be activated by ROS and are necessary to accelerate the differentiation of preadipocyte cells [7,28]. As shown in Fig. 6, the protein levels of both phospho-p38 (p-p38) and phospho-ERK (p-ERK) in the differentiated control cells (Con) significantly increased compared to those in undifferentiated control cells (UC), indicating that these MAPKs were activated by phosphorylation during the differentiation of preadipocytes. This observation was consistent with the elevated ROS level in differentiated cells (Con) as shown in Fig. 6A and B. However, treatment of differentiating adipocyte cells with 7,8-DHF (1, 10, and 20 μM) significantly reversed these increases in a dose-dependent manner (Fig. 6). Treatment with 20 μM 7,8-DHF inhibited the phosphorylation of p38 (Fig. 6A) and ERK (Fig. 6B) by 2.3-fold and 2.1-fold, respectively, compared to control cells (Con) that were differentiated but not treated with 7,8-DHF. These results suggest that 7,8-DHF down-regulates the MAPK signaling pathway during differentiation.

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Fig. 4. Inhibitory effect of 7,8-DHF on intracellular ROS production by 3T3-LT1 cells during adipocyte differentiation. 3T3-L1 preadipocytes were incubated for 2 days in MDI differentiation medium with 7,8-DHF (0, 1, 10, 20 μM) and then cultured for 6 days in DMEM medium. Cells were harvested and stained with 10 μM DCF-DA. After incubation, fluorescence intensity was monitored using a fluorescence spectrophotometer (A). The data are expressed as a percentage change normalized to control cells. (B) The change in ROS in the cytosol was determined by staining with DCF-DA and rhodamine and visualizing stained cells by fluorescence microscopy (×100). Data = mean ± SD, n = 3. *p b 0.05; **, p b 0.01; ***, p b 0.001, Student's t-test compared to control (0 μg/ml).

4. Discussion Several studies suggested that oxidative stress induces lipid accumulation and facilitates adipose differentiation [8,9]. 7,8-DHF has been reported for its antioxidant activity [20–23]. However, no previous studies explored the direct association between 7,8-DHF and an antiobesity effect, especially in terms of reducing the adipose tissue mass. In this study, we examined the effects of 7,8-DHF on the proliferation of preadipocytes, lipid accumulation during cell differentiation, intracellular ROS generation during cell differentiation, and adipogenesisrelated signaling pathways in association with ROS generation. Many plant flavonoids such as quercetin, α-mangostin, oroxylin A, and resveratrol have been reported to inhibit cell proliferation by inducing apoptotic cell death in 3T3-L1 adipocytes [29–31]. Several studies

showed that pro-apoptotic proteins can reduce body weight and body fat, and Kim et al. suggested that reductions in adipose mass are usually induced by apoptosis [32]. Consistent with these reports, the results of our study showed that treatment with 7,8-DHF (N 20 μM) reduces the number of viable 3T3-L1 preadipocytes, suggesting that 7,8-DHF inhibits the cell proliferation of preadipocytes. Decreasing the cell numbers of either pre-adipocytes or mature adipocytes is an effective strategy to reduce adipose tissue mass [33,34]. The decreased viability of pre-adipocytes by 7,8-DHF treatment was shown to be associated with the induction of apoptotic cell death. The caspase protein family, including caspase-3, 8, and 9, play a pivotal role in apoptosis induced by a variety of stimuli [35,36]. Western blot analysis showed that 7,8DHF resulted in a dose-dependent cleavage of caspase-3, 8, and 9. This finding indicates that 7,8-DHF induces apoptotic cell death in 3T3-L1

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Fig. 5. 7,8-DHF up-regulates the expression of antioxidant enzymes and scavenges DPPH radicals. Cells were differentiated with 7,8-DHF for 8 days, and the expression levels of anti-oxidant enzymes such as (A) catalase, (B) HO-1, and (C) Mn-SOD were measured by Western blot analysis. (D) The radical scavenging activity of 7,8-DHF was measured by the DPPH assay. The absorbance was read at 518 nm against a blank using a microplate reader. The data are expressed as a decrease in the absorbance of DPPH and calculated using the following equation: Scavenging effect (%) = (1 − A sample 518 / A control 518) × 100. The data are expressed as the fold change normalized to control cells. Data = mean ± SD, n = 3. *p b 0.05; **, p b 0.01; ***, p b 0.001, Student's t-test compared to control (0 μg/ml).

pre-adipocyte cells by activating pro-apoptotic factors such as caspase family proteins, suggesting that 7,8-DHF may ultimately reduce the number of fat cells in adipose tissue by inhibiting the proliferation of the pre-adipocyte cells. To investigate the effect of 7,8-DHF on lipid accumulation during the differentiation of 3T3-L1 preadipocytes to mature adipocytes, the cells were treated with relatively lower concentrations of 7,8-DHF (b 20 μM), at which the cell viability was not significantly affected. The Oil Red O staining showed that 7,8-DHF induces a dramatic, dosedependent decrease in the level of intracellular oil droplets, suggesting that 7,8-DHF significantly suppresses lipid synthesis during differentiation of adipocyte cells. Adipogenesis, a process involving lipid accumulation, is sequentially and cooperatively regulated by various transcription factors and adipocyte-specific genes, such as C/EBP-β, C/EBP-α, PPAR-γ, and aP2 [37,38]. C/EBP-α and C/EBP-β are the representative transcription

factors of adipocyte differentiation, playing critical roles in mediating adipogenesis. C/EBP-β expression is induced in the early stage of differentiation, and the expression of C/EBP-α and PPAR-γ is associated with the late stage differentiation. These transcription factors act together to differentiate pre-adipocytes into mature adipocytes by transactivating adipocyte-specific genes [39]. In the present study, we found that treatment of 3T3-L1 adipocyte cells with 7,8-DHF significantly diminished the C/EBP-α and C/EBP-β expression during adipocyte differentiation. Furthermore, 7,8-DHF also suppressed the expression of PPAR-γ and adipocyte-specific fatty acid binding protein (aP2) on 3T3-L1 cells. PPAR-γ is well-known as a master regulator of adipogenesis [40]. aP2 is a terminal adipocyte differentiation marker gene that triggers the appearance of lipid droplets in the cytoplasm of differentiating 3T3-L1 cells, and throughout this process, lipid droplets increase in size and coalesce [37]. The results of our study suggested that the 7,8-DHF-induced reduction in lipid accumulation and TG content in 3T3-L1 adipocytes is

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Fig. 6. Effects of 7,8-DHF on the MAPK signaling pathway during adipocyte differentiation. Cells were differentiated with 7,8-DHF for 8 days, and the phosphorylation levels of (A) p38 and (B) ERK were measured by Western blot analysis. The data are expressed as the fold change normalized to control cells. Data = mean ± SD, n = 3. *p b 0.05; **, p b 0.01; ***, p b 0.001, Student's t-test compared to control (0 μg/ml).

mediated by the down-regulation of C/EBP-α, β, PPAR-γ, and aP2 expression during adipogenesis. Similarly, some other flavonoids such as resveratrol, luteolin, and quercetin have also been reported to decrease the expression of PPAR-γ and C/EBP-α, resulting in decreased fat accumulation [41,42]. Additionally, in agreement with the findings of other research groups [20–23], our study also showed that 7,8-DHF exerts an antioxidant activity by scavenging intracellular ROS from differentiating 3T3-L1 cells and by DPPH radical scavenging. Interestingly, ROS production during obesity development has been reported to arise from several sources, including oxidation of the lipid molecule, activation of NADPH oxidase, and upregulation of C/EBP-α and PPAR-γ [4,43], and the ROS level markedly increases during adipocyte differentiation. Indeed, ROS facilitate the dimerization of C/EBP-β, leading to the upregulation of downstream adipogenic signals including C/EBP-α, and ROS have been found to accelerate adipose differentiation by facilitating mitotic clonal expansion [9,44]. In this regard, several reports have described that antioxidant compounds could suppress adipogenesis [10, 11], and many antioxidants have been reported to exhibit potent antiobesity effects [12–14]. Indeed, Lee et al. demonstrated that antioxidant treatment prevented the localization of C/EBP-β to centromeres, initiating adipogenesis, and arrested the cell cycle, preventing mitotic clonal expansion, resulting in adipocyte differentiation [9]. Moreover, wellknown antioxidants such as α-lipoic acid and NAC (N-acetylcysteine) inhibit adipocyte differentiation on 3T3-L1 cells and suppress the development of obesity in rats. Interestingly, however, other antioxidants such as GSH, ascorbic acid, and retinoic acid have been shown to have no significant anti-obesity effects [10,45,46], suggesting that there may be no direct correlation between a compound's antioxidant activity and its anti-obesity effects. As only some antioxidants show antiobesity activity, we tried to identify the molecular events induced by 7,8-DHF in 3T3-L1 cells to clearly determine whether the observed antioxidant activity of 7,8-DHF can contribute to the amelioration of obesity. ROS have been reported to accumulate in parallel with lipid accumulation in adipocytes during adipogenesis [4]. To confirm the antioxidant activity of 7,8-DHF, we examined its effects on the expression of several intracellular antioxidant enzymes. Western blot analysis showed that the levels of the antioxidant proteins manganese superoxide dismutase (Mn-SOD), catalase (CAT), and heme oxygenase-1 (HO-1) markedly increased in the differentiated cells (Con), and these elevated protein levels during differentiation were further increased by treatment with 7,8-DHF. Mn-SOD converts the superoxide anion to H2O2 in the

mitochondria during the eukaryotic TCA cycle reaction, and then H2O2 can be converted to water by CAT [47]. HO-1 is known to be induced by oxidative stress to provide protection against oxidative insult, and increased HO-1 prevents cell death and dysfunction [48]. Several studies have reported that HO-1 induction suppressed the development of obesity and oxidative stress-induced dysfunction could be reversed by increased expression of anti-oxidant enzymes including HO-1 [49,50]. Therefore, the results of our study suggested that 7,8-DHF can inhibit the intracellular ROS accumulation during adipocyte differentiation through its antioxidant activity, not only by up-regulating the expression of intracellular antioxidant enzymes, such as Mn-SOD, CAT, and HO-1, but also by directly scavenging ROS. In turn, ROS scavenging by 7,8-DHF suppresses the expression of adipogenic transcription factors such as C/EBPα, C/EBP-β, PPAR-γ, and aP2, and may ultimately confer 7,8-DHF the capability to suppress the adipogenic differentiation of adipocytes. 7,8-DHF was also found to reduce the phosphorylation of ERK and p38 in differentiated 3T3-L1 cells. ERK and p38 are protein kinases from the family of mitogen-activated protein kinases (MAPKs) including JNK. MAPKs are well known as key proteins in diverse signaling events from the cell membrane to the nucleus via cascades of phosphorylation, and MAPK signaling regulates various intracellular events, ranging from cell survival to cell death [51]. In addition, there have been many reports about the role of MAPKs in adipocyte differentiation and obesity; MAPK proteins are activated by ROS and are necessary to initiate the differentiation of preadipocyte cells [6,7]. In addition, ERK activity is known to be necessary for the expression of the crucial adipogenic regulators, including C/EBP-α, C/EBP-β, C/EBP-δ, and PPAR-γ, either in 3T3-L1 cells or in another cellular model [52,53]. Our results showed that treatment of differentiating adipocyte cells with 7,8-DHF significantly inhibited the phosphorylation of p38 and ERK, suggesting that 7,8-DHF attenuates the activation of the MAPK signaling pathway during differentiation. Inhibition of p38 activation decreased the phosphorylation and activation of C/EBP-β, and decreased BMP-2 (bone morphogenic protein-2)-induced adipocyte formation and PPAR-γ activity [54,55]. Bost et al. noted that ERK and p38 are required during mitotic clonal expansion (MCE) of preadipocytes, and suggested that specific drugs could be developed by modulating the MAPK pathway to suppress the development of obesity [56]. Consequently, these results support the potential use of 7,8-DHF as an anti-obesity drug candidate, as 7,8-DHF inhibits the expression of adipogenic transcription factors and the production of oil droplets and ROS by reducing the phosphorylation of p38 and ERK.

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Interestingly, in contrast to our observation, Chan et al. reported that 1 μM of 7,8-DHF treatment did not inhibit adipogenesis in 3T3-L1 cells [57]. One of the possible explanations for this different observation would be the differences between Chan's system and our system used for adipocyte differentiation. To differentiate 3T3-L1 preadipocyte cells in differentiation media, Chan et al. additionally treated their cells with ciglitazone that is selective and potent PPARγ ligand and strongly stimulates adipogenesis and ROS generation in 3T3-L1 cells [58]. Ciglitazone is generally known as an insulin sensitizing agent that enhances the response of target cells and tissues to insulin. Ciglitazone stimulates glucose uptake into the cells by activating insulin signaling pathways, resulting in enhancement of adipogenesis. Therefore, in case of those preadipocyte cells treated with insulin sensitizing agents such as ciglitazone in the differentiating media, the progression of differentiation would be much faster than ciglitazone-untreated cells as in the case of our study. This may have caused the cells become less sensitive to any anti-adipogenic compounds such as 7,8-DHF and thus Chan et al. could observe no effect on 3T3-L1 adipogenesis with 1 μM 7,8-DHF treatment. In the presence of ciglitazone in differentiating media, higher concentration of 7,8-DHF would have been required to inhibit the differentiation of preadipocytes. The results of present in vitro study clearly demonstrated that the 7,8-DHF has a potential anti-adipogenic and anti-obesity effect via its antioxidant activity. 7,8-DHF was shown to reduce fat cell numbers and adipose mass by inducing the apoptosis of preadipocyte cells and inhibit the differentiation of preadipocyte cells by the downregulation of adipogenic transcription factors through its antioxidant action, which is mediated by the inhibition of intracellular ROS accumulation via the induction of antioxidant enzymes and by the attenuation of MAPK activation. However, in vivo and clinical studies on protective efficacy of 7,8-DHF against obesity related diseases need to be conducted for future application of 7,8-DHF as an active ingredient of health beneficial foods or new therapeutic agent to treat obesity. There have been several reports that 7,8-DHF shows potent biological activity both in vivo and in vitro. Especially Chan et al. indicated that 7,8-DHF enhanced systemic energy expenditure which could reduce insulin resistance and adipocity in vivo [57,59]. Wu et al. assessed the safety of 7,8DHF in mice and reported that there were no significant among-group differences in serum levels of BUN, CRE, indicators of renal function and ALT, an indicator of hepatic function and no significant change in body weight as well [60]. These results suggested that 7,8-DHF could be largely non-toxic to human within the certain level of dosages although further details on safety and clinical efficacy need to be obtained through clinical tests in the future. Nevertheless, these in vivo findings on the effectiveness and safety of 7,8-DHF and our current in vitro results together may support the strong possibility for 7,8-DHF being potent candidate for the development of clinically effective anti-obesity agents in the future. 5. Conclusions In conclusion, we report, for the first time, that 7,8-DHF is a potential anti-adipogenic agent that could alleviate obesity. The potent antiobesity effect of 7,8-DHF was mediated by its inhibition of the adipogenic differentiation of 3T3-L1 preadipocyte cells, lipid accumulation, and the expression of adipogenic transcription factors such as PPAR-γ, C/EBP-α, C/EBP-β, and aP2. 7,8-DHF was also shown to induce the apoptotic death of 3T3-L1 preadipocyte cells, inhibit intracellular ROS accumulation by stimulating the expression of antioxidant enzymes such as Mn-SOD, CAT, and HO-1, and attenuate the activation of the MAPK signaling pathway during differentiation. Accordingly, we suggest that 7,8-DHF could be beneficial to treat and prevent obesity and obesity-associated metabolic syndrome. Conflict of interest statement The authors declare no conflicts of interest.

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