A Bioactive Constituent of Ginger, 6‐Shogaol, Prevents Adipogenesis

JournalJournal of of Food Food Biochemistry Biochemistry ISSN ISSN 1745–4514 1745-4514

A BIOACTIVE CONSTITUENT OF GINGER, 6-SHOGAOL, PREVENTS ADIPOGENESIS AND STIMULATES LIPOLYSIS IN 3T3-L1 ADIPOCYTES SUJIN SUK1,2*, SANG GWON SEO1,3*, JAE GAK YU1, HEE YANG1, EUNSUN JEONG1, YOUNG JIN JANG1, SOONHAM SAMI YAGHMOOR4, YOUSSRI AHMED5, JEHAD MUSTAFA YOUSEF6, KHALID OMER ABUALNAJA7, ABDULRAHMAN LABEED AL-MALKI8, TAHA ABDULLAH KUMOSANI9, CHANG Y. LEE10,11,13, HYONG JOO LEE1,3,12,14 and KI WON LEE1,2,3,12,15

1WCU Biomodulation Major, Department of Agricultural Biotechnology, Center for Food and Bioconvergence, Seoul National University, Seoul 151-921, Korea 2Interdisciplinary Program in Agricultural Biotechnology Major, College of Agriculture and Life Sciences, Seoul National University, Seoul, Korea 3Advanced Institutes of Convergence Technology, Seoul National University, Suwon 443-270, Korea 4Experimental Biochemistry Unit, King Fahd Medical Research Center and Production of Bioproducts for Industrial Applications Research Group, King Abdulaziz University, Jeddah, Saudi Arabia 5Biochemistry Department, Faculty of Science and Production of Bioproducts for Industrial Applications Research Group, King Abdulaziz University, Jeddah, Saudi Arabia 6Biochemistry Department, Faculty of Science for Girl’s, Experimental Biochemistry Unit, King Fahd Medical Research Center and Production of Bioproducts for Industrial Applications Research Group, King Abdulaziz University, Jeddah, Saudi Arabia 7Biochemistry Department, Faculty of Science and Bioactive Natural Products Research Group, King Abdulaziz University, Jeddah, Saudi Arabia 8Biochemistry Department, Faculty of Science, Experimental Biochemistry Unit, King Fahd Medical Research Center and Bioactive Natural Products Research Group, King Abdulaziz University, Jeddah, Saudi Arabia 9Biochemistry Department, Faculty of Science, Experimental Biochemistry Unit, King Fahd Medical Research Center and Production of Bioproducts for Industrial Applications Research Group, King Abdulaziz University, Jeddah, Saudi Arabia 10Department of Food Science, Cornell University, Ithaca, NY 14850 11Production of Bio-products for Industrial Applications Research Group, King Abdulaziz University, Jeddah 22254, Saudi Arabia 12Research Institute of Bio Food Industry, Institute of Green Bio Science and Technology, Seoul National University, Pyeongchang 232-916, Korea

13Corresponding author. TEL: ABSTRACT TEL:+1-607-255-0114; +1-607-255-0114; FAX: FAX:+1-607-254-4868; +1-607-254-4868; EMAIL: [email protected] Zingiber ofﬁcinale Roscoe, one of the most widely used spices, has been reported to 14Corresponding author. TEL: have anti-obesity and anti-diabetes effects. In the present study, we investigated TEL:+82-2-880-4860; +82-2-880-4860; FAX: FAX:+82-2-873-5095; +82-2-873-5095; the effects of 6-shogaol, a bioactive compound present in ginger, on the EMAIL: [email protected] adipogenic process in 3T3-L1 preadipocytes. The anti-adipogenic effects of 15 Corresponding author. TEL: 6-shogaol was signiﬁcantly higher than the more widely investigated 6-gingerol, TEL:+82-2-880-4661; +82-2-880-4661; FAX: FAX:+82-2-873-5095; +82-2-873-5095; another major ginger constituent. We observed that 6-shogaol inhibited the EMAIL: [email protected] expression of two master regulators of adipogenesis, PPARγ and C/EBPα, and also Received for Publication January 9, 2015 stimulated lipolysis in mature 3T3-L1 adipocytes. Collectively, these results Accepted for Publication July 23, 2015 suggest that 6-shogaol, not 6-gingerol, is the major compound present in ginger responsible for its reported anti-adipogenic properties. doi:10.1111/jfbc.12191

*These authors contributed equally to this PRACTICAL APPLICATIONS work. Ginger is widely consumed all over the world, and has been associated with various health benefits. At least some of these benefits have been previously attrib- uted to 6-gingerol. In the present study, we observed that 6-shogaol has more potent anti-adipogenic effects than 6-gingerol in 3T3-L1 cells. This is the first study to investigate the anti-obesity effect of 6-shogaol in vitro, and provides a new perspective on future development of ginger-based anti-obesity strategies.

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INTRODUCTION improves insulin sensitivity by reducing blood glucose and elevating insulin levels (Goyal and Kadnur 2006). In addi- Obesity is a chronic condition characterized by an abnor- tion, ginger extract has been reported to enhance glucose mal increase in adipose tissue mass that results from uptake and inhibit adipocyte differentiation in vitro (Rani increases in adipocyte number (hyperplasia) and size et al. 2012). (hypertrophy) (Camp et al. 2002). The abnormal growth Ginger contains a plethora of bioactive compounds including of adipose tissue is associated with metabolic disorders gingerols (6-gingerol, 8-gingerol, and 10-gingerol), shogaols, including type 2 diabetes, atherosclerotic vascular diseases volatile oils, aryl alkenes, and diarylheptanoids (Chrubasik et al. and nonalcoholic fatty liver disease. The control of pro- 2005). Among these, the gingerols and shogaols have been iden- cesses responsible for obesity therefore has wide therapeutic tified as the major bioactive constituents present in fresh and implications (Hajer et al. 2008). 3T3-L1 cell lines can mimic dried ginger, respectively (Chrubasik et al. 2005). Previous adipogenic processes in vitro with appropriate cell culture studies have reported that fresh ginger contains about 2.0% of models (Tang et al. 2003). A number of studies have shown 6-gingerol, while 6-shogaol is scarcely detected in fresh ginger that specific phytochemicals may exert potential preventive (Govindarajan 1982). However, 6-shogaol is shown to be pro- effects against obesity due to their anti-adipogenic effects in duced during processing or storage of ginger, and dried ginger 3T3-L1 preadipocytes (Jung et al. 2013; Min et al. 2013; Seo contains about 0.2% of 6-shogaol (Govindarajan 1982; Jolad et al. 2013; Shin et al. 2014). et al. 2005). 6-Gingerol and 8-gingerol are relatively potent The 3T3-L1 preadipocyte cell line is commonly used agonists of transient receptor potential cation channel subfamily in modern adipogenesis studies. The cells differentiate into V member 1, and have been reported to contribute to the mature adipocytes when treated with an adipogenic cocktail medicinal properties of ginger (Dedov et al. 2002). Of particular (a mixture of isobutylmethyl xanthine (IBMX), dexametha- note, 6-gingerol has been reported to inhibit adipogenesis sone, and insulin; MDI) (Poulos et al. 2010). When MDI is in preadipocytes, as well as lipid accumulation in mature administered to confluent cells, the growth-arrested cells adipocytes (Tzeng and Liu 2013; Tzeng et al. 2014). However, the re-enter the cell cycle synchronously and undergo extra cell anti-adipogenic effects of 6-shogaol, another major component division, mimicking the first stage of adipogenesis called of ginger, has not been investigated. Therefore, in the present mitotic clonal expansion (MCE). Following MCE, when study, we investigated anti-adipogenic and pro-lipolytic activity insulin is added, the cells further differentiate into mature of 6-shogaol in vitro. adipocytes, acquiring the extensive ability to store lipids (Abdulrazaq et al. 2012). During this differentiation process, several adipogenic proteins such as peroxisome MATERIALS AND METHODS proliferator-activated receptor gamma (PPARγ), CCAAT/ enhancer binding protein alpha (C/EBPα), and fatty acid Materials synthase (FAS) are expressed, which regulate both the dif- 6-gingerol, 6-shogaol, IBMX, dexamethasone, insulin and ferentiation and lipid accumulation processes (Liu et al. oil red O powder were purchased from Sigma-Aldrich 2004; Farmer 2006; Rosen and MacDougald 2006). There- (St. Louis, MO). Dulbecco’s modified Eagle’s Medium fore, controlling the expression of these proteins is thought (DMEM), fetal bovine serum (FBS), bovine calf serum to be critical for the inhibition of adipogenesis. (BCS) and antibiotic-antimycotic were purchased from Zingiber officinale Roscoe (Z. officinale), commonly GIBCO® Life Technologies Corporation (Grand Island, known as ginger, has been widely used as an herbal medi- NY). Isopropyl alcohol was purchased from Junsei Chemical cine to treat a variety of ailments (Chrubasik et al. 2005). Co. (Tokyo, Japan). Primary antibodies against PPARγ,glyc- Ginger has been reported to have anti-obesity and anti- eraldehyde 3-phosphate dehydrogenase (GAPDH) and diabetic effects as well as anti-inflammatory, anti-oxidant horseradish peroxidase (HRP)-conjugated secondary anti- and anti-cancer properties (Kadnur and Goyal 2005; bodies were purchased from Santa Cruz Biotechnology Al-Suhaimi et al. 2011; Abdulrazaq et al. 2012; Skrovankova (Santa Cruz, CA). Primary antibodies against C/EBPα and et al. 2012). Previous studies have demonstrated that the FAS were obtained from Cell Signaling Biotechnology marked rise in body weight of rats fed on a high-fat diet (Beverly, MA). can be significantly reduced with ginger supplementation. Moreover, high-fat diet-fed rats consuming ginger exhibit significantly lower glucose and insulin levels, as well as Cell Culture reductions in lipid factors including total cholesterol, low- density lipoprotein cholesterol, triacylglycerol, free fatty 3T3-L1 preadipocytes were obtained from the ATCC acids and phospholipids in serum (Nammi et al. 2009). (Manassas, VA). Cells were maintained in DMEM supple- Another study has shown that treatment with ginger extract mented with 10% BCS and 1% antibiotic-antimycotic at

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37C under 5% CO2. Cells were subcultured every 2 or 3 days loaded onto sodium dodecyl sulfate-polyacrylamide at a density of about 1–2 × 105 cells per dish. gel electrophoresis and proteins were transferred to polyvinylidene fluoride membranes (GE Healthcare, Piscataway, NJ). Membranes were blocked with 5% skim Adipogenesis milk and incubated with specific primary antibodies fol- 3T3-L1 preadipocytes were seeded at 2.5 × 104 cells per lowed by HRP-conjugated secondary antibodies. Protein well in 24-well plates, and incubated in DMEM supple- bands were visualized using a chemiluminescence detection mented with 10% BCS until 100% confluence. After 2 days kit (Amersham Pharmacia Biotech, Piscataway, NJ). post-confluence, the media was changed to DMEM supplemented with 10% FBS, 0.5 mM IBMX, 1 μM dexamethasone and 5 μg/mL insulin. After 2 days, the media was Glycerol Release replaced with DMEM supplemented with 10% FBS and 3T3-L1 preadipocytes were differentiated as described 5 μg/mL insulin. After two further days, the media was above. Cells were then treated with 6-shogaol or 6-gingerol changed to DMEM supplemented with 10% FBS and cells for 4 hr. The media in each well were collected and stored at were incubated for 2 days. The total duration of adipogen- −20C. Glycerol concentrations in each media were deter- esis was 6 days from the induction of differentiation with mined using free glycerol reagent (Sigma-Aldrich), accord- hormonal cocktail. ing to the manufacturer’s instructions.

Oil Red O Staining Statistical Analysis 3T3-L1 preadipocytes were differentiated as described above in the presence or absence of 6-shogaol or 6-gingerol. Fully Data are expressed as mean ± SD. Significant differences differentiated cells were fixed with 3.7% (v/v) formaldehyde among multiple groups were evaluated by one-way analysis solution for 20 min and washed with phosphate buffered of variance followed by Tukey’s HSD tests. Data were ana- saline (PBS) three times. Lipid droplets in mature lyzed using SPSS Statistical Software (SPSS Inc., Chicago, adipocytes were then stained with oil red O solution for IL). Superscripts of different letters denote statistical signifi- 15 min and washed with PBS three times. The stained oil cance at P < 0.05. red O was then dissolved in isopropyl alcohol and absorbance was measured at 515 nm using a microplate reader (Molecular Devices, CA). RESULTS

Effect of 6-Shogaol on MDI-induced MTT Assay Adipogenesis of 3T3-L1 Preadipocytes 3T3-L1 preadipocytes were seeded at 2.5 × 104 cellsperwellin 6-gingerol and 6-shogaol are the major constituents in fresh 24-well plates, and incubated in DMEM supplemented with and dried ginger, respectively (structures are depicted in 10% BCS until 100% conﬂuence, before incubation with Fig. 1). Neither 6-shogaol nor 6-gingerol exhibited cytotox- DMEM supplemented with 10% FBS in the presence or absence icity (Fig. 2A). We next induced differentiation of 3T3-L1 of 6-gingerol or 6-shogaol. After 3 days, 1 mg/mL of 3-(4,5- preadipocytes into adipocytes either with or without Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) 6-shogaol or 6-gingerol. Differentiated cells exhibited sig- solution was added and cells were incubated for 1 hr. The dark niﬁcantly increased levels of intracellular lipid accumula- blue formazan cyrstals were dissolved in dimethyl sulphoxide tion, and 40 μM of 6-shogaol inhibited adipogenesis of (DMSO) and the absorbance at 570 nm was measured using a 3T3-L1 preadipocytes (Fig. 2B,C). These results together microplate reader (Molecular Devices). suggest that 6-shogaol, but not 6-gingerol, inhibits adipogenesis of 3T3-L1 preadipocytes. Western Blot Analysis 3T3-L1 preadipocytes were seeded in 6-cm dishes at a Effect of 6-Shogaol on Adipogenic/Lipogenic density of 1.5 × 105 cells per dish and differentiated as Protein Expression during MDI-induced described above in the presence or absence of 6-shogaol or Adipogenesis 6-gingerol. After full differentiation, cells were lysed with RIPA buffer obtained from Cell Signaling Technology We next investigated whether 6-shogaol could downregulate (Beverly, MA), and lysates were collected via centrifugation the expression of representative adipogenic/lipogenic (14,000 rpm, 4C, 10 min). The collected cell lysates were marker proteins during MDI-induced adipogenesis. MDI

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FIG. 1. THE CHEMICAL STRUCTURES OF (A) 6-GINGEROL AND (B) 6-SHOGAOL

treatment increased the protein expression of two master The anti-adipogenic effects of ginger have been previ- regulators of adipogenesis, PPARγ and C/EBPα, and this ously demonstrated in vitro (Rani et al. 2012). Among the expression was lowered by 6-shogaol at 40 μM (Fig. 3A). two major representative bioactive constituents of ginger, Moreover, expression of FAS (an enzyme involved in fatty 6-gingerol and 6-shogaol, only the anti-adipogenic effects of acid synthesis) was also increased by MDI treatment, and 6-gingerol have been reported (Tzeng and Liu 2013; Tzeng downregulated by 6-shogaol at 40 μM (Fig. 3B). These et al. 2014). However, previous studies on the pharmacoki- results suggest that 6-shogaol modulates the expression netics of ginger constituents have reported that only of adipogenic/lipogenic proteins, resulting in an anti- 6-shogaol and not 6-gingerol can be detected in free form adipogenic effect in 3T3-L1 preadipocytes. in human plasma (Yu et al. 2011). It was reported that 6-gingerol metabolites were detected in the forms of gluc- uronide or sulfate metabolites (Yu et al. 2011). We sought Effect of 6-Shogaol on Lipolysis in Mature to investigate the effect of 6-shogaol on adipogenesis of 3T3-L1 Adipocytes 3T3-L1 preadipocytes and observed that 6-shogaol, but not 6-gingerol, inhibits MDI-induced adipogenesis of 3T3-L1 Stimulating lipolysis is another important strategy for preadipocytes through the suppression of adipogenic/ obesity prevention/treatment. We therefore chose to investi- lipogenic marker proteins. We also observed that 6-shogaol gate whether 6-shogaol or 6-gingerol can stimulate lipolysis. more potently stimulates lipolysis than 6-gingerol in mature To measure the pro-lipolytic activity of 6-shogaol and 3T3-L1 adipocytes. Based on these data, we postulate that 6-gingerol, we first fully differentiated 3T3-L1 preadipocytes 6-shogaol has superior anti-obesity effects than 6-gingerol to mature adipocytes and treated the cells with 6-shogaol or in 3T3-L1 cells. 6-gingerol at the indicated concentrations. In comparison to Previous studies investigating the anti-adipogenic effects the nontreated group, 6-shogaol significantly increased of ginger constituents have widely focused on 6-gingerol. glycerol release and decreased intracellular lipid accumula- For example, Tzeng et al. reported that 6-gingerol inhibits tion, indicating the stimulation of lipid breakdown, a adipogenesis in 3T3-L1 cells (Tzeng and Liu 2013; Tzeng process called lipolysis (Fig. 4A,B). Neither 6-shogaol nor et al. 2014). In contrast to these previous results, we found 6-gingerol exhibited cytotoxicity (Fig. 4C), suggesting that that 6-gingerol did not show any detectable anti-adipogenic these observations were not due to a cytotoxic effect. These effect. This discrepancy could be due to different experi- results together suggest that 6-shogaol, but not 6-gingerol, mental conditions. Their study employed treatment with stimulates lipolysis in mature 3T3-L1 adipocytes. 50 μM of 6-gingerol for 8 days, whereas we treated 6-gingerol at 40 μM for 6 days. Their differentiation DISCUSSION regimen also included insulin, IBMX, indomethasone and dexamethasone for 8 days to induce differentiation, which is With global prevalence of obesity, numerous anti-obesity an atypical experimental condition. Since dexamethasone is drugs have been developed, which target central nervous a steroid that can induce various cellular responses includ- system or digestive system. However, they brought little ing cell proliferation and differentiation (McCulloch and success with various side effects such as cardiovascular dis- Tenenbaum 1986; Baud et al. 2005; Belanto et al. 2010), the eases. Phytochemicals from natural dietary plants and reported anti-adipogenic effects might be a combinational herbs, including ginger, have been consumed throughout effect of 6-gingerol and dexamethasone. long history, ensuring their safety. Recently, anti-obesity Previously, 6-shogaol was reported to exhibit anti- effects of natural phytochemicals are actively being oxidative and anti-inflammatory activities. Dugasani investigated. et al. reported that 6-shogaol is responsible for the most

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FIG. 3. EFFECTS OF 6-SHOGAOL ON MDI-INDUCED PPARΓ, C/EBPΑ AND FAS EXPRESSION (A) 6-shogaol inhibits MDI-induced PPARγ and C/EBPα protein expression. 3T3-L1 preadipocytes were differentiated into adipocytes in the presence of 6-shogaol at 40 μM. PPARγ and C/EBPα protein expressions were determined by Western blotting. GAPDH was used as a loading control. The data are representative of three independent FIG. 2. EFFECT OF 6-SHOGAOL AND 6-GINGEROL ON VIABILITY AND experiments. (B) 6-shogaol inhibits MDI-induced FAS protein MDI-INDUCED ADIPOGENESIS IN 3T3-L1 PREADIPOCYTES expression. 3T3-L1 preadipocytes were differentiated into adipocyte in (A) Neither 6-shogaol nor 6-gingerol exhibit cytotoxicity toward the presence of 6-shogaol at 40 μM. FAS protein expression was 3T3-L1 preadipocytes. Cells were cultured until confluence, before determined by Western blotting and GAPDH was used as a loading treatment with either 6-shogaol or 6-gingerol at the indicated control. The data are representative of three independent experiments. concentrations for 3 days. Cell viability is expressed as a percentage of control values, presented as mean values ± SD (n = 3). (B, C) 6-shogaol inhibits MDI-induced adipogenesis of 3T3-L1 preadipocytes more potently than 6-gingerol. Cells were differentiated in the presence of inflammation are major stimulants for metabolic disorders, 6-shogaol and 6-gingerol at the indicated concentrations. Intracellular these anti-oxidative and anti-inflammatory activities could lipids were stained by oil red O staining as described in the Materials be associated with its beneficial effects toward metabolic and Methods. (B) Intracellular lipid content is expressed as a disease. ± = percentage of control values, with mean values SD (n 3). The In the present study, we found that 6-shogaol more potently mean values marked with different superscript letters are significantly inhibits adipogenesis of 3T3-L1 preadipocytes and decreases different (P < 0.05). (C) Intracellular lipids were visualized by photograph. expression of various adipogenic/lipogenic marker proteins than 6-gingerol, and that 6-shogaol, but not 6-gingerol, increases lipolysis in mature adipocytes. These results together suggest that potent anti-oxidative and anti-inflammatory effects in 6-shogaol has promising potential to be developed as an anti- ginger (Dugasani et al. 2010), while Pan et al. showed that obesity agent. To that end, further studies focusing on the anti- 6-shogaol down-regulates inflammatory genes such as iNOS obesity effect of 6-shogaol and ginger in preclinical and clinical and COX-2 (Pan et al. 2008). Since oxidative stress and models is needed.

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ACKNOWLEDGMENTS This research was supported by the National Research Foundation grant (2012M3A9C4048818) funded by the Ministry of Science, ICT and Future Planning (MSIP), Bio- industry Technology Development Program (514004) funded by the Ministry of Agriculture, Food and Rural Affairs of the Republic of Korea, and by the Deanship of Scientiﬁc Research (DSR), King Abdulaziz University, Jeddah, Saudi Arabia, under grant No. 4-141-35-HiCi. The authors, therefore, acknowledge with thanks DSR technical and ﬁnancial support.

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