Anti-adipogenic Effects of Corni Fructus in 3T3-L1 Preadipocytes

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1 Biotechnology and Bioprocess Engineering 19: (2014) DOI /s RESEARCH PAPER Anti-adipogenic Effects of Corni Fructus in 3T3-L1 Preadipocytes Chang-Ho Kang, Yoon-Jung Kwon, and Jae-Seong So Received: 8 July 2013 / Revised: 28 October 2013 / Accepted: 2 November 2013 The Korean Society for Biotechnology and Bioengineering and Springer 2014 Abstract Obesity is the most common health problem in developed countries and is considered a significant risk factor for many major human diseases. The goal of the present study was to evaluate the inhibitory effect of herbal extracts on adipocyte differentiation in 3T3-L1 preadipocytes. We screened the extracts of 30 plants, used in traditional medicine to test their effects against obesity. Among the tested extracts, Corni fructus ethanolic extract (CFEE), significantly inhibited adipocyte differentiation by more than 80% in 3T3-L1 preadipocytes in a dose-dependent manner. The level of adipogenesis in the 3T3-L1 cells was measured by Oil Red O staining and the triglyceride content assay. Furthermore, CFEE inhibited adipocyte differentiation by suppressing the expression of the adipogenic transcription factors CCAAT/enhancer binding protein (C/EBP) and peroxisome proliferator-activated receptor (PPAR), as confirmed by reverse transcription-polymerase chain reaction (RT-PCR) analysis. These results suggest that CFEE inhibits adipogenesis by suppressing the expression of adipogenic transcription factors. Keywords: adipogenesis, adipocyte, 3T3-L1, corni fructus 1. Introduction In spite of the current public awareness of obesity, its prevalence continues to increase. Obesity is the most common health problem in developed countries and is Chang-Ho Kang, Jae-Seong So * Department of Biological Engineering, Inha University, Incheon , Korea Tel: ; Fax: sjaeseon@inha.ac.kr Yoon-Jung Kwon Department of Organic and Nano System Engineering, Konkuk University, Seoul , Korea considered a significant risk factor for many major human diseases such as heart disease, cancer, arthritis, and diabetes [1]. The development of obesity is characterized by an increase in the number of fat cells and their lipid content as a result of adipocyte differentiation (adipogenesis). The primary role of the adipocytes involves the synthesis and storage of triglycerides during periods of caloric excess. As a result of adipogenesis, the size or number of fat cells increases and lipid drops accumulate [1]. Thus, preadipocyte cell lines are useful models for investigating the adipogenic process. An enhanced understanding of the process of adipogenesis would help prevent the initiation and progression of adipogenesis, which leads to obesity and obesity-related diseases in humans. Adipogenesis involves the induction and repression of a number of genes in a programmed manner [2]. The sequential expression of specific transcription factors, such as those of the CCAAT/enhancer-binding protein (C/EBP-α) and peroxisome proliferation activating receptor (PPAR-γ) families, plays a key role in the intracellular mechanisms involved in the early stage of adipogenesis. During adipogenesis, C/EBP-β expression is rapidly induced by hormonal stimulation, and it functions to regulate PPAR-γ transcription. PPAR-γ is a ligand-activated transcription factor and is a member of a nuclear receptor superfamily; its overexpression enhances adipogenesis in vitro. PPAR-γ is also known to promote the expression of an array of genes involved in the maturation of adipocytes [3]. The fructus of Cornus officinalis Sieb. et Zucc. (Corni fructus) has traditionally been used in medicinal practices in Korea. The major chemical constituents of Corni fructus include saponins, phenolic acids (gallic acid and tannic acid), and loganin [4]. Saponins and phenolic acids are known to have antioxidant activity [5]. Recently, it was reported that Corni fructus was beneficial in models of advanced glycation end product-mediated renal injury in streptozotocin (STZ)-treated diabetic rats [6] and db/db

2 Anti-adipogenic Effects of Corni Fructus in 3T3-L1 Preadipocytes 53 mice [7]. Therefore, in this study, 30 traditional herbs were selected through a literature review and evaluated for their antiadipogenic activity. Among these 30 herbs, Corni fructus was found to effectively inhibit adipocyte differentiation in terms of morphology and gene expression. 2. Materials and Methods 2.1. Chemicals Dulbecco s modified Eagle medium (DMEM), fetal bovine serum (FBS), and insulin were obtained from Gibco BRL (Grand Island; NY, USA). Dimethyl sulfoxide (DMSO), dexamethasone, isobutylmethylxanthine (IBMX), Oil Red O, free-glycerol reagent, (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT), TG assay kit, and TRIzol reagent were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The BrdU proliferation assay kit was purchased from Invitrogen (Carlsbad, CA, USA). All other chemicals were of analytical grade or equivalent and were purchased commercially Sample preparation Dried herbs were purchased from the Kyungdong Oriental Herbal Market in Korea. Each herb (50 g) was crushed and extracted 3 times with 95% ethanol at room temperature using a shaking incubator for 24 h (Table 1). The supernatant was filtered and evaporated in a vacuum below 45 C using a rotary evaporator (EYELA Ltd.; Kyoto, Japan). Each extract was suspended in DMSO and stored at 4 C until further use. Table 1. Inhibition of adipocyte differentiation by various herbal extracts Botanical origin Scientific name Used part Yield (%) Inhibition (%) * Corni fructus Cornus officinalis Sieb.et Zucc. fruit ± 1.35 Pinellia rhizome Pinellia ternata Thunberg Breit. root ± 1.50 Paeoniae Radix Paeonia lactiflora Pall. root ± 1.31 Pericarpium citri Citrus unshiu Markovich. pericarp ± 1.81 Schisandra chinensis Schisandra chinensis Baill. fruit ± 2.76 Atractylodes japonica Atractylodes japonica Koidz. root ± 1.90 Morus alba Morus alba L. bark ± 1.21 Polygoni multiflori caulis Polygonum multiflorum Thunb. root ± 1.23 Scutellaria radix Scutellaria baicalensis Georgi. root ± 1.21 Folium perillae Perillae folium leaves ± 1.31 Crataegi fructus Crataegus pinnatifida Bunge. fruit ± 1.31 Radix astragali Astragalus membranaceus Bunge root ± 2.71 Radix platycodi Platycodon grandiflorum A. DC. root ± 1.26 Siegesbeckiae herba Siegesbeckia glabrescens Makino leaves ± 1.46 Semen coicis Coix lachryma-jobi var. ma-yuen fruit ± 1.50 Carthamus tinctorius Carthamus tinctorius L. flower ± 1.51 Ephedra sinica Ephedra sinica Stapf. stem ± 1.27 Alismatis rhizoma Alisma plantago-aquatica var. orientale root ± 1.20 Rheum undulatum Rheum undulatum L. stem ± 1.36 Semen Phaseoli Phaseoli angularis Wight fruit ± 1.76 Mori fructus Morus alba L. fruit ± 1.38 Radix Glycyrrhizae Glycyrrhiza uralensis Fisch. root ± 1.44 Pinus densiflora Pinus densiflora Sieb. et Zuccarini leaves ± 1.15 Japanese persimmon Diospyros kaki Thunb. leaves ± 1.12 Artemisia capillaris Artemisia capillaris Thunberg. leaves ± 1.70 Flos Chrysanthemi Chrysanthemum morifolium leaves ± 1.46 Eriobotryae folium Eriobotrya japonica Lindl. leaves ± 1.25 Dendrobium nobile Dendrobium nobile Lindl. stem ± 1.16 Folium Phyllostachys Phyllostachys niger M. var. Henonis leaves ± 1.57 Semen Cassiae Cassia tora L. fruit ± 2.12 The inhibition data of the extracts from herbs are presented as the mean ± S.D., and inhibited adipocyte differentiation activity was examined at a concentration of 250 µg/ml. * The inhibitory rate (%) was defined as follows: [(sample OD control OD)/(MDI OD Control OD)] 100.

3 54 Biotechnology and Bioprocess Engineering 19: (2014) 2.3. Cell differentiation and sample treatment 3T3-L1 preadipocytes were purchased from American Type Culture Collection (ATCC) and cultured in DMEM supplemented with 10% (v/v) heat-inactivated FBS at 37 C in an atmosphere containing 5% CO 2. To induce adipocyte differentiation, 2-day post-confluent 3T3-L1 preadipocytes (day 0) were stimulated for 48 h (day 2) with a standard induction cocktail (0.5 mm 3-isobutyl-1-methylxanthine, 1 µm dexamethasone, and 1 µg/ml insulin; MDI) and herbal extracts (250 µg/ml), and then maintained for 4 days (day 6) in DMEM supplemented with 10% FBS and 1 µg/ml insulin. To examine the effects of the herbal extracts on adipocyte differentiation, the medium was treated every 2 day, until the end of the experiment on day 8. In the control experiments, cells were cultured in the same media containing a drug-free vehicle and baicalin (100 µm), which has been reported to be an adipogenesis inhibitor [8], which were used as a positive control Cell viability and Oil Red O staining Cell viability was assessed using the MTT assay [9]. 3T3- L1 preadipocytes were treated with Corni fructus ethanolic extracts (CFEE) at concentrations of 50, 100, and 200 µg/ml. After 24 h, 20 µl of MTT solution was added, and cells were incubated at 37 C for 4 h. The supernatant was then removed, and 200 µl of DMSO was added. Absorbance was measured on a microplate reader (Bio-Rad Model 550; Hercules, CA, USA) at 546 nm to obtain the percentage of viable cells. Eight days after inducing differentiation, the media was removed, and the cells were washed twice with phosphate-buffered saline (PBS) and fixed with 3.7% formalin for 15 min. To visualize the lipid droplets, the fixed cells were washed twice with 60% isopropanol (in PBS) and then stained with a 0.5% Oil Red O solution for 40 min at room temperature. The Oil Red O solution was then removed and the cells were washed twice with deionized water before being photographed. For quantification of Oil Red O uptake, cells were incubated with isopropanol, and the optical density of the solution was measured at 540 nm Measurement of triglyceride (TG) and glycerol The cellular TG content was measured using a commercial TG assay kit according to the manufacturer s instructions. In 6-well plates, cells were treated with plant extracts at a concentration of 100 µg/ml during adipocyte differentiation over 4 days. The cells were washed twice with PBS, suspended in 80 µl of a homogenizing solution (154 mm KCl, 1 mm ethylenediaminetetraacetic acid (EDTA), and 50 mm Tris; ph 7.4), and sonicated to homogenize the cell suspension. The residual cell lysate was centrifuged at 3,000 g for 5 min at 25 C to remove the fat layers. The supernatants were assayed to measure the TG and protein levels. TG was normalized to the protein concentration, which was determined by the Bradford assay using bovine serum albumin (BSA) as the standard. The results were expressed in milligrams of TG per milligram of cellular protein (TG (mg)/cellular protein (mg)). Lipolysis was assessed by determining the amount of glycerol released into the medium, according to the manufacturer s instructions. Specifically, differentiated 3T3-L1 adipocytes were treated with plant extracts for 24 h. After incubation, 80 µl of the medium was incubated with 120 µl of the free glycerol reagent for 15 min at room temperature. Glycerol levels were quantified by measuring the absorbance at 590 nm Reverse transcription-polymerase chain reaction (RT-PCR) analysis Total RNA was isolated from mature adipocytes (day 8) using TRIzol reagent. Reverse transcription was performed using a Superscript Preamplification System (Roche; Indianapolis, IN, USA) to synthesize first-strand cdna according to the manufacturer s instructions. Reverse transcription was performed at 45 C for 30 min and then at 94 C for 5 min to inactivate the reverse transcriptase. The following primers were used: PPARγ (forward): 5'-ACCACTCGC ATTCCTTTGAC-3', PPARγ (reverse): 5'-TCAGCGGGA AGGACTTTATG-3', C/EBPα (forward): 5'-ATGGAGTCG GCCGA CTTCTAC-3', C/EBPα (reverse): 5'-CAGGAA CTCGTCGTTGAAGGC-3', GAPDH (forward): 5'-AAC TTTGGCATTGTG GAAGGGC-3', GAPDH (reverse): 5'- GACACA TTGGGGGTAGGAACAC-3' (Bioneer, Korea). C/EBPα and PPARγ amplifications were performed by denaturing at 94 C for 30 sec, annealing at 55 C for 30 sec, extension at 72 C for 1 min for 25 cycles, followed by a final extension at 72 C for 5 min. The PCR products were loaded onto 1% agarose gels, stained with ethidium bromide, and visualized under UV light [10] BrdU proliferation assay Cell proliferation was assayed by BrdU staining. A cell proliferation assay kit was used to detect BrdU incorporation into the newly synthesized DNA of proliferating cells. Cells were fixed with 4% paraformaldehyde in PBS and cryoprotected with primary anti-brdu monoclonal antibody (1:200) in 10% normal goat serum overnight at 4 C. Goat anti-mouse Ig conjugated to fluoresce in isothiocyanate (No. F1010; Sigma; 1:500) were used as secondary antibodies. Propidium iodide (No. P4170; Sigma) staining was performed in PBS for 5 min. Images were taken using a Fluorescence Stereomicroscope (M165FC; Leica Ltd.) equipped with an Optronics CCD camera Statistical analysis The results are expressed as the means ± S.D. of three

4 Anti-adipogenic Effects of Corni Fructus in 3T3-L1 Preadipocytes 55 experiments. Statistical analysis was performed by the Student s t-test, and a p value of < 0.05 was considered to indicate a significant difference. 3. Results and Discussion 3.1. Inhibitory effect of 30 herbal extracts on adipocyte differentiation Adipocyte differentiation is a very complex process involving whole-cell changes, and is triggered by coordinated signaling by growth factors, cytokines, and hormones [2]. In this study, we screened the extracts of 30 herbs to test their potential anti-adipogenic activity. 3T3-L1 adipocytes were cultured and differentiated in DMEM containing 10% FBS for 6 ~ 8 day in the absence or presence of the extracts (at a final concentration of 250 µg/ml) according to the differentiation protocols. As shown in Table 1, CFEE had the highest inhibitory effect (87.11 ± 1.35%) on adipogenesis of 3T3-L1 preadipocytes, as assessed by intracellular triglyceride droplet staining with Oil Red O. In addition, extracts of Atractylodis japonica (87.02 ± 1.90%) and Rheum undulatum (82.42 ± 1.36%) displayed high inhibitory effects of more than 80%. These 3 extracts significantly suppressed lipid accumulation, and their inhibitory effect was higher than that of baicalin (100 µm). Conversely, extracts from Pinus densiflora ( ± 1.15%), Flos Chrysanthemi ( ± 1.46%), Mori Fructus ( ± 1.38%), Artemisia capillaries ( ± 1.70%), Pinellia rhizome ( 6.41 ± 1.50%), Pericarpium Citri ( 2.16 ± 1.81%), and Carthamus tinctorius ( 2.06 ± 1.51%) enhanced adipogenesis. As CFEE significantly inhibited adipocyte differentiation (by over 80%), we chose to further examine this herb extract. Previous research has shown that some flavonoids (including phloretin and nobiletin) enhance adipocyte differentiation in 3T3-L1 cells [11,12] Inhibitory effects of CFEE on cell viability and 3T3-L1 cell differentiation The effect of CFEE on cell viability in both preadipocytes and mature adipocytes was demonstrated by using the MTT assay (Fig. 1). CFEE displayed low toxicity in 3T3- L1 preadipocyte and mature adipocyte cultures, with the cell viability remaining at approximately 80 ~ 100%. In Fig. 2A, the effects of CFEE on lipid droplet formation in 3T3-L1 cells are presented by quantifying the amount of Oil Red O staining. Incubation of 3T3-L1 cells with 25, 50, 100, and 200 µg/ml CFEE decreased the lipid droplet amounts by 21, 72, 83, and 85%, respectively. Thus, CFEE Fig. 1. Effect of Corni fructus ethanolic extract (CFEE) on viability of 3T3-L1 cells. 3T3-L1 preadipocytes were differentiated into mature adipocytes as described in Materials and Methods. The cell viability was measured by using the MTT assay. (A) Preadipocytes; (B) mature adipocytes. Fig. 2. Effect of CFEE on Oil Red O staining in cultured 3T3-L1 cells. (A) Effect of CFEE on fat droplet formation in 3T3-L1 cells. The cells were stained with Oil Red O dye and examined under a light microscope. (B) Relative lipid content obtained by counting Oil Red O stained cells. Data are presented as mean ± S.D. (n = 3). * indicates p < 0.05.

5 56 Biotechnology and Bioprocess Engineering 19: (2014) Fig. 3. Effect of different concentrations of CFEE on lipid accumulation in differentiated 3T3-L1 cells. (A) Preadipocytes were treated with no additives (control) or in the presence of CFEE of different concentrations. # p < 0.05 compared with control; * p < 0.05 compared with MDI-treated group. was found to significantly reduce lipid accumulation in 3T3-L1 adipocytes, suggesting an anti-obesity effect. These results directly indicate that CFEE inhibits lipid deposition in a dose-dependent manner without causing cytotoxic effects. Adipose tissue is composed of adipocytes that store TG as a fuel for the body. Approximately 80 ~ 90% of the wet weight of adipocytes consists of TG. In order to understand adipocyte physiology, a mouse fibroblast cell line (3T3-L1 cells) has been widely used. Differentiation of 3T3-L1 cells is essential for optimal adipocyte formation [13,14]. As shown in Fig. 3, lipid accumulation was measured based on the TG content of 3T3-L1 cells differentiated in the presence of CFEE. The amount of TG in 3T3-L1 cells treated with 100 and 200 µg/ml CFEE was decreased by approximately 38 and 43%, respectively. Treatment of 3T3-L1 cells with CFEE was found to significantly decrease cell differentiation and TG accumulation in a dose-dependent manner CFEE inhibits the expression of C/EBP-α and PPAR-γ Several transcription factors that act in a sequential fashion to promote adipocyte differentiation have been identified [15]; these include members of the C/EBP and PPAR families [14,16]. For adipogenesis, the sequential expression of several genes must occur in a coordinated and temporal pattern. For example, C/EBP-β and C/EBP-γ mediate the transcriptional activation of PPAR-γ and C/EBP-α [17]. To investigate whether CFEE suppresses adipogenesis at a transcriptional level, we extracted total RNA from differentiated 3T3-L1 cells treated with increasing concentrations of CFEE (50, 100, and 200 µg/ml), and performed RT-PCR. As shown in Fig. 4A, PPAR-γ mrna expression was Fig. 4. Effect of CFEE on (A) mrna expression of CCAAT/ enhancer binding protein (C/EBP)-α and peroxisome proliferatoractivated receptor (PPAR)-γ and (B) cell proliferation. reduced by up to 41.2%, and C/EBP-α mrna expression was decreased by up to 75.9%. These observations suggest that CFEE can suppress adipogenesis at the mrna level. The results from cdna quantifications suggest that CFEE might stimulate cell proliferation. C/EBP proteins (C/EBP α, β, and γ) belong to the basic leucine zipper family of transcription factors, whereas PPAR-γ is a member of the nuclear receptor superfamily of transcription factors and is predominantly expressed in adipose tissue. These transcription factors appear to function as dominant activators of adipocyte differentiation [13]. PPAR-γ is induced prior to the transcriptional activation of most adipocyte-specific genes. The expression of PPAR-γ is sufficient to induce growth arrest and to initiate adipogenesis in exponentially growing fibroblast cell lines [18]. In addition, it was reported that the transcription factor C/EBP-α is a likely candidate for directly regulating adipocyte differentiation [19]. To confirm this, we used BrdU to monitor DNA replication (Fig. 4B). In the untreated cultures, a small percentage of cells were BrdU-positive. Treatment with CFEE resulted in a marked increase in the number of BrdU-positive cells. This result indicates that 3T3-L1 cell proliferation was increased in the CFEE-treated cultures more than in the untreated cultures. In conclusion, the results of this study show that CFEE has considerable anti-adipogenic activity, which is directly proportional to its concentration. CFEE suppresses the expression of specific genes associated with adipogenesis, and can be used as an alternative phytochemical or therapeutic

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