Natural Product Sciences 16(4) : 291-294 (2010)
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Inhibitory Activity of Three Varieties of Adlay (Coix Seed) on Adipocyte Differentiation in 3T3-L1 cells
Mi Kyeong Lee1,*, Eunjin Shin1,QingLiu1, BangYeon Hwang1, Jungbok Lee2, Sun Yeou Kim3, and Jae-Hak Lee4
1College of Pharmacy, Chungbuk National University, Cheongju 361-763, Korea
2Research Institute, Sonimedi, Gangwon-do 220-702, Korea
3Graduate School of East-West Medical Science, Kyung Hee University Global Campus, Gyeonggi-do 446-701, Korea
4Korea Plant Resource Institute, attached Well Root, Gyeonggi-do 413-832, Korea
Abstract −In obesity, adipocytes undergo abnormal growth characterized by increased cell numbers and differentiation. Thus, inhibition of mitogenesis of preadipocytes and their differentiation to adipocytes would be beneficial for the prevention and progression of obesity. In the present study, we attempted to evaluate anti- adipogenic activity of adlay (Coix seed, the seed of Coix lacryma-jobi L. var. ma-yuen Stapf) employing preadipocytes cell line, 3T3-L1 as an in vitro assay system. Because several varieties of adlay are in use in Korea, anti-adipogenic activity of three varieties of adlay such as Sang-Gang, Jo-Hyun and Yulmu-Ilho was evaluated.
All the three varieties of adlay showed significant inhibitory activity on adipocyte differentiation as assessed by measuring fat accumulation using Oil Red O staining. Adlay, however, showed little effects on adipocyte proliferation. Further studies with interval treatment demonstrated that adlay exerted inhibitory activity on adipocyte differentiation via acting on early stage of adipogenesis. Taken together, adlay might be useful in the prevention of obesity.
Keywords −Adlay, Variety, 3T3-L1 cells, adipocyte differentiation, Coix seed, obesity.
Introduction
Obesity is no longer considered only a cosmetic problem but associated with several pathological disorders, including diabetes, hypertension, atherosclerosis and cancer. Obesity is a major public health problem in industrialized counties and has become the leading metabolic disease in the world (Kopelman, 2000).
Adipose tissue, as a metabolic and endocrine organ, plays an essential role in regulating energy balance.
Adipocyte is the primary site for energy storage and accumulates triglycerides during nutritional excess. As an endocrine organ, adipocytes express and secrete molecules known as adipokines, which regulate physiologic processes such as glucose metabolism, appetite and inflammatory responses (Rosen and Spiegelman, 2006; Bastard et al., 2006).
Adlay, also called Coix seeds, is an annual crop which is widely distributed in Asian country including Korea.
Adlay has been served as meals or beverages. In traditional medicine, adlay was also called Coicis Semen,
the seeds of Coix lacryma-jobi L. var. ma-yuen Stapf (Gramineae), and has been used as diuretic and as an anti- inflammatory, antitumor and analgesic agent (Bae, 2000).
Recent studies also demonstrated the antiallergic and antimicrobial effects of adlay (Yu et al., 2008; Huang et al., 2009). In addition, adlay has been suggested to be effective for the treatment of obesity (Kim et al., 2004;
Yokoi et al., 2009; Ha et al., 2010).
Because of importance in use, varieties of adlay have been developed to improve crops, nutritional composition, resistance to plant disease and harmful insects, and etc.
Sang-Gang, Jo-Hyun and Yulmu-Ilho are three varieties of adlay, which are widely consumed in Korea. They showed improvement in various aspects. Yulmu-Ilho is known to possess higher protein and starch content whereas Sang- Gang is more resistant to plant diseases and temperature.
Although three varieties of adlay have been known to improve in several aspects, their detailed characteristics, especially biological effects have not been evaluated yet.
In the present study, we attempted to investigate the anti- adipogenic activity of these three varieties of adlay employing the mouse preadipocyte cell line, 3T3-L1 as an in vitro assay system by measuring fat accumulation.
*Author for correspondence
Tel: +82-43-261-2818; E-mail: mklee@chungbuk.ac.kr
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Methods and materials
Plant material−Three varieties of adlay such as Sang- Gang (CS-A), Jo-Hyun (CS-B) and Yulmu-Ilho (CS-C) were provided from Korea Plant Resource Institute in June 2009.
Sample preparation−The dried seeds of three varieties of adlay (each 100 g) were extracted with water in ultrasonic apparatus for 3 h at 40oC. Removal of the solvent in vacuo yielded total extract of Sang-Gang (2.2 g), Jo-Hyun (1.8 g) and Yulmu-Ilho (3.0 g), respectively.
The extracts were dissolved in water and filtered in membrane filter (0.2µm) before treatment.
Culture of 3T3-L1 cells−3T3-L1 mouse embryo fibroblasts were obtained from the American Type Culture Collection (Manassas, VA). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supple- mented with 10% fetal bovine serum (FBS) until confluence. Two days after confluence (day 0), cells were stimulated to differentiate with differentiation medium containing DMEM with 10% FBS, 0.5 mM 3-isobutyl-1- methyl-xanthine, 1µM insulin and 1µM dexamethasone for 2 days (day 2). Cells were then maintained in DMEM supplemented with 10% FBS and 1µM insulin for another 2 days (day 4), followed by culturing with DMEM with 10% FBS for an additional 4 days (day 8). All media contained 100 IU/mL penicillin and 100µg/mL strepto- mycin. Cells were maintained at 37oC in a humidified atmosphere of 95% air-5% CO2. The cultures were treated with test samples for whole culture period (day 0- 8) for general experiment. In some experiment, test samples were treated after differentiation was induced (fully differentiated cells) for three day. For the evaluation of time dependent activity, test samples were treated for indicated time-periods.
Oil Red O staining−Lipid droplets in cells were stained with Oil Red O. Eight days after differentiation induction, cells were washed three times with PBS and fixed with 10% formalin at room temperature for 1 hr.
After fixation, cells were washed once with PBS and stained with freshly diluted Oil Red O solution (3 parts of 0.6% Oil Red O in isopropyl alcohol and 2 parts of water) for 15 min. Cells were then washed twice with water and visualized. For quantitative analysis, Oil Red O staining was dissolved with isopropyl alcohol and optical density was measured at 520 nm by ELISA plate reader.
Measurement of cell viability−3T3-L1 cells were treated with vehicle or samples to be tested for 72 hr. Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)- 2.5-diphenyltetrazolium bromide (MTT) assay. 3T3-L1
cells were incubated with 0.5 mg/mL of MTT in the last 2 hr of the culture period tested. Reduction of MTT to formazan was assessed in an ELISA plate reader.
Statistical analysis−The evaluation of statistical significance was determined by one-way Anova test with a value of p < 0.05 or less considered to be statistically significant.
Results and discussion
Obesity arises from abnormal growth of adipocyes leading to increased total fat mass. Abnormal deposit of fat mass is occurred by increased numbers of adipocytes containing lipids due to processes of mitogenesis and differentiation. Although adipocyte differentiation is an important process for cellular function in normal condition, pathological adipocyte differentiation confers abnormal expression of adipocytokines, which are implicated in metabolic disorders. Thus, inhibition of adipocyte differen- tiation has been suggested an important therapeutic approach for prevention and treatment of obesity.
Adlay is widely distributed in Korea and served as meals or beverages. In addition, adlay, also called Coicis Semen, has been used as diuretic and as an anti- inflammatory, antitumor and analgesic agent in traditional medicines. Recently, adlay has been suggested to be effective for the treatment of obesity (Kim et al., 2004;
Yokoi et al., 2009). Because of several varieties of adlay are consumed in Korea, we attempted to investigate the anti-adipogenic activity of three widely consumed varieties of adlay, such as Sang-Gang, Jo-Hyun and Yulmu- Ilho.
Adipocyte differentiation was induced by the differen- tiation mixture including IBMX, insulin and dexame- thasone. Differentiated adipocytes showed accumulation of lipid droplets in their cytoplasm and easily distinguished by microscopic observation after Oil Red O staining (Fig. 1). When adlay extracts were treated to 3T3-L1 cells during whole differentiation period, fat accumulation was significantly reduced in differentiated 3T3-L1 cells (Fig. 2A). At a concentration of 300µg/ml, adlay extract inhibited fat accumulation up to 50% of fully differentiated cells. Microscopic observation also showed the decreased fat droplet in 3T3-L1 cells by the treatment of adlay extract (Fig. 1). Interestingly, although three varieties of adlay are known to differ in ingredient content and cultivation method, all the three varieties of adlay exerted similar activity in our assay system.
However, adlay extract showed little activity when treated to fully differentiated adipocytes (Fig. 2B). These results
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suggest that adlay might be effective in the prevention of adipogenesis but not in lipolysis.
Adipogenesis, the development of mature fat cells from preadipocytes, is an intensely studied model of cellular differentiation. The process of adipogenesis includes alteration of cell shape, growth arrest, clonal expansion, and a complex sequence of changes in gene expression and storage of lipid, which occurs in several stages (Gregoire et al., 1998; Lefterova and Lazar, 2009). For differentiation, preadipocytes enter growth arrest and continue to subsequent differentiation process by appro- priate mitogenic and adipogenic signals. During the initial stage of differentiation, a dramatic decrease of preadipo- cyte factor-1 (Pref-1) expression accompanies a rapid
Fig. 1. Effects of CS-A, CS-B and CS-C on the morphology of 3T3-L1 cells.
Cultures were induced to differentiate for 8 days. Cells were treated with samples for whole differentiation process (days 0 - 8). On day 8, cultures were then subjected to Oil Red O staining. Figures are representatives of three independent experiments with similar results.
Fig. 2. Effects of three varieties of adlay, CS-A, CS-B and CS-C on adipocyte differentiation in 3T3-L1 cells.
Cultures were induced to differentiate for 8 days. Cells were treated with samples for whole differentiation process (days 0 - 8) (A) or treated after differentiation was induced (fully differentiated adipocytes) for three days (B). Cultures were then subjected to Oil Red O staining. Relative fat accumulation (%) was calculated as 100×[(absorbance of sample-treated−absorbance of undifferen- tiated control) / (absorbance of differentiated control−absorbance of undifferentiated control)]. Results are expressed as mean ± SD of three independent experiments, each performed using triplicate wells. *p < 0.05; **p < 0.01 compared with differentiated control.
Fig. 3. Effects of CS-A on adipocyte differentiation during differentiation process.
Cultures were induced to differentiate and CS-A was added at different periods of differentiation, namely, during days 0 - 2, 2 -4, 4 - 8, 0 - 4, 2 - 8 and 0 - 8. On day 8, cells were stained with Oil Red O. Relative fat accumulation (%) was calculated as 100× [(absorbance of sample-treated−absorbance of undifferentiated control) / (absorbance of differentiated control−absorbance of undifferentiated control)]. Values are expressed as means±SD of triplicate experiments. *p < 0.05; **p < 0.01 compared with differen- tiated control.
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increase in the expression of PPARγ and CCAAT/
enhancer-binding proteins (C/EBPs). During the terminal stage of differentiation, enzymes involved in triacylglycerol metabolism such as fatty acid synthase and glycerol-3- phosphate dehydrogenase increase to a great extent (Farmer, 2006; White and Stephens, 2010). Therefore, we further examined which stage of adipogenic differen- tiation was effectively inhibited by adlay. During differen- tiation, 3T3-L1 cells were incubated with 300µg/ml Sang- Gang at different periods of differentiation, namely, during days 0-2, 2-4, 4-8, 0-4, 2-8 and 0-8. On day 8, cells were stained with Oil Red O and quantitated. As shown in Fig. 3, adlay inhibited adipocyte differentiation when treated during days 0-2, 2-4, 0-4 and 0-8. However, there was no significant inhibitory effect when treated during days 4-8. These results suggest that adlay could effectively inhibit the early stage of adipocyte differen- tiation.
Adipocyte mass can be reduced not only by inhibiting adipogenesis but also by decreasing adipocyte number (Rayalam et al., 2008). Therefore, we investigated the effect of adlay on adipocyte number. As shown in Fig. 4, adlay showed weak inhibitory effect on mature adipocyte number.
In conclusion, although three varieties of adlay differ in some aspects, all the three varieties of adlay significantly reduced fat accumulation in 3T3-L1 cells. Our study also showed that adlay was more effective in the prevention of adipogenesis by acting early stage of adipogenesis.
Therefore, adlay might be useful in the prevention of obesity.
Acknowledgement
This work was supported by the research grant of the Rural Development Administration in 2009 (PJ006985:
Mass Production Technique and Selection of Best Genetic Resource with Regulatory Activity for Metabolic Disease) and Korea Research Foundation Grant funded by the Korea government (MEST) (No. 2010-0015470).
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Received November 11, 2010 Revised December 10, 2010 Accepted December 13, 2010
Fig. 4. Effects of CS-A, CS-B and CS-C on cell viability of 3T3- L1 cells.
CS-A, CS-B and CS-C were treated to confluent 3T3-L1 cells for 3 days. Cell viability was measured by MTT assay. Relative cell viability (%) was calculated as 100×(absorbance of sample- treated / absorbance of control). Results are expressed as the mean
±S.D. of three independent experiments, each performed using triplicate wells.
