Zanthoxylum Piperitum DC Ethanol Extract Suppresses Fat Accumulation in Adipocytes and High Fat Diet-Induced Obese Mice by Regulating Adipogenesis
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J Nutr Sci Vitaminol, 58, 393–401, 2012 Zanthoxylum piperitum DC Ethanol Extract Suppresses Fat Accumulation in Adipocytes and High Fat Diet-Induced Obese Mice by Regulating Adipogenesis So Young GWON1, Ji Yun AHN1, Tae Wan KIM2 and Tae Youl HA1,* 1 Division of Metabolism and Functionality Research, Korea Food Research Institute, Seongnam 463–746, Republic of Korea 2 Department of Food Science and Biotechnology, Andong National University, Andong 760–749, Republic of Korea (Received May 2, 2012) Summary This study was conducted to determine the anti-obesity effects of Zanthoxylum piperitum DC fruit ethanol extract (ZPE) in 3T3-L1 adipocytes and obese mice fed a high-fat diet. We evaluated the influence of the addition of ZPE to a high-fat diet on body weight, adipose tissue weight, serum and hepatic lipids in C57BL/6 mice. In addition, adipogenic gene expression was determined by Western blot and real-time reverse transcription-PCR analysis. We assessed the effect of ZPE on 3T3-L1 preadipocyte differentiation. ZPE reduced weight gain, white adipose tissue mass, and serum triglyceride and cholesterol levels (p,0.05) in high-fat diet-fed C57BL/6 mice. ZPE decreased lipid accumulation and PPARg, C/EBPa, SREBP-1, and FAS protein and mRNA levels in the liver. ZPE inhibited in vitro adi- pocyte differentiation in a dose-dependent manner and significantly attenuated adipogenic transcription factors, such as PPARg, C/EBPa, and SREBP-1 in 3T3L1 cells. These findings suggest that Z. piperitum DC exerts an anti-obesity effect by inhibiting adipogenesis through the downregulation of genes involved in the adipogenesis pathway. Key Words 3T3-L1 adipocyte, high-fat diet, anti-obesity, Zanthoxylum piperitum DC fruit Obesity is the most common metabolic disease in peroxisome proliferator-activated receptor g (PPARg) developed countries and has become a global epidemic (10). These factors modulate adipogenesis-related gene in recent years (1). It is associated with numerous expression and lipid storage in adipocytes (11). Ste- chronic diseases including type 2 diabetes, dyslipidemia, rol regulatory element-binding protein (SREBP-1) also atherosclerosis, hypertension, cardiovascular diseases, plays a role by upregulating many lipogenic genes, such stroke, and certain forms of cancer (2–5). Obesity can as fatty acid synthase (FAS). be defined as an increased fat mass due to an increase in Zanthoxylum piperitum DC (ZPDC) has been used in the number and size of adipocytes. Adipogenesis, which Korea as a spice and as a traditional medicine for vom- is the process by which undifferentiated preadipocytes iting, diarrhea, and abdominal pain (12). The fruit and are converted to differentiated adipocytes, is closely leaves of Z. piperitum contain aliphatic acid amides (13, related to the etiologies of obesity and obesity-related 14), terpenoids (15, 16), flavonoids (17), an alkaloid metabolic disorders (6). Controlling adipogenesis is a (18), and other phenolics (19). Previous studies reported potential strategy for obesity prevention because adipo- that a glycoprotein isolated from ZPDC possesses anti- cyte differentiation plays a key role in fat mass growth inflammatory properties (20), and ZPDC fruits and (7–9). The master adipogenic transcription regulators leaves exhibit antioxidant and hepatoprotective effects are CCAAT/enhancer binding protein a (C/EBPa) and (21). However, anti-obesity effects of Z. piperitum DC fruit have not yet been demonstrated. We hypothesized * To whom correspondence should be addressed. that ZPDC contains functional compounds that pos- E-mail: [email protected] sess anti-obesity properties. Therefore, we evaluated the Abbreviations: C/EBPa, CCAAT/enhancer binding protein effects of an ethanol extract of Z. piperitum DC fruit in a; DMEM, Dulbecco’s modified Eagle’s medium; ELISA, en- a 3T3-L1 cell culture system and investigated its effects zyme-linked immunosorbent assay; FAS, fatty acid synthase; on body weight gain, adiposity, liver steatosis, and gene FBS, fetal bovine serum; GAE, gallic acid equivalents; H&E, expression in high-fat diet-induced obese mice. hematoxylin and eosin; HDLC, high-density lipoprotein cho- lesterol; HFD, high-fat diet control group; HFD1ZPE, 0.5% MATERIALS AND METHODS ZPE with high-fat diet; IBMX, isobutyl-3-methylxanthine; ND, normal diet group; PPARg, peroxisome proliferator-activated Chemicals and regents. Isobutyl-3-methylxanthine receptor g; QE, quercetin equivalents; SREBP-1, sterol regula- (IBMX), dexamethasone, insulin, and Oil Red O were tory element-binding protein; TC, total cholesterol; TG, triglyc- purchased from Sigma-Aldrich Chemical Co. (St. Louis, eride; ZPDC, Zanthoxylum piperitum DC; ZPE, ethanol extract MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), from fruit of Z. piperitum DC. fetal bovine serum (FBS), bovine calf serum, and antibi- 393 394 GWON SY et al. otics (penicillin and streptomycin) were purchased from complete. Life Technologies (Burlington, ON, Canada). Antibod- Oil Red O staining and cell quantification. After differ- ies against PPARg, SREBP-1, C/EBPa, and b-actin were entiation was induced, cells were stained with Oil Red obtained from Santa Cruz Biotechnology (Santa Cruz, O (0.2% Oil Red O in 60% isopropanol). The cells were CA, USA), and an antibody against FAS was purchased washed twice with phosphate buffered saline (PBS), from Cell Signaling Technology (Danvers, MA, USA). fixed with 10% formalin for 1 h, dried, and stained with Preparation of ethanol extract from fruit of Z. piperitum Oil Red O for 10 min. The cells were washed with 70% DC (ZPE). The fruits of Z. piperitum DC originating ethanol and water, and then dried. The lipid content of from Korea were purchased from Khung-dong Oriental stained cells was visualized by microscopy (Olympus medicine market (Seoul, Korea) and identified by Pro- IX71, Tokyo, Japan). The stained lipid droplets were fessor Y. M. Park, Department of Life Science, Cheongju dissolved in isopropanol and quantified by measuring University. Voucher specimens (KFRI-ZPDC090705) absorbance at 500 nm. were deposited in the Korea Food Research Institute. The Animal experiments. C57BL/6 male mice (4 wk old, dried fruits (1.0 kg) were soaked in 70% ethanol (10 L) n530) were purchased from Central Laboratory Animal at room temperature for 12 h. The ethanol extract was (Seoul, Korea). After acclimation to the commercial chow filtered through filter paper (Whatman Grade No. 2, for a week, they were divided into three groups accord- New Jersey, USA), and concentrated under a vacuum at ing to diet: normal diet (ND), high-fat diet (HFD), or 40˚C. The concentrated extracts were then freeze-dried. high-fat diet supplemented with 0.5% ZPE (HFD1ZPE). Finally, the dried extract (216.8 g) obtained from fruits All mice were housed individually during the study. The of Z. piperitum DC (1,000 g) were stored at 220˚C until experimental diets were based on the AIN-76 diet, and use. The total polyphenol content in the extract was the high-fat diet contained 20% fat (lard 50 g/kg, coco- determined using the Folin-Ciocalteous method (22) nut butter 70 g/kg, cocoa oil 30 g/kg, corn oil 50 g/kg) and total flavonoid content was determined using the and 0.5% cholesterol (w/w). The energy content of the diethylene glycol method (23). Results were expressed high-fat diet was 19 MJ/kg (4,625 kcal), whereas that of in terms of gallic acid equivalents (GAE), and flavonoid the normal diet was 16 MJ/kg (3,850 kcal). The animal content was expressed in terms of quercetin equiva- room was maintained at 2361˚C and 5362% humidity lents (QE). The main compounds in ZPE were analyzed with a 12-h light/dark cycle. The mice had free access to by HPLC analysis. Samples were separated on a JASCO water. The diet was given to the mice at 10:00 every day. HPLC system (JASCO Corporation, Tokyo, Japan) and The body weight was measured once a week, and food XterraTM RP18 column (4.63250 mm, Waters Corpo- intake was measured every day by subtraction of pre- ration, Milford, MA, USA). The mobile phase consisted and post-weights of food jars for 6 wk. Energy efficiency of solvents A (50 mM sodium phosphate, 10% metha- was calculated as the energy intake divided by the body nol, pH 3.3) and B (70% methanol). The gradient elutes weight gain. were filtered through a 0.45 mm Millipore filter and All animal experiments were approved by the Korea degassed prior to use. The linear gradient profile was Food Research Institutional Animal Care and Use from 70% A to 30% B in 7 min, 65% A to 35% B in Committee. 18 min, 60% A to 40% B in 20 min, 50% A to 50% B in Biochemical analysis. All mice were sacrificed after a 5 min, 100% B in 35 min and then 100% A in 25 min, 12-h fast. Blood was collected by orbital venipuncture followed by re-equilibration of the column to its initial and centrifuged at 3,000 rpm for 15 min to separate the conditions. The constituent was detected with UV wave- serum, which was stored at 270˚C until analysis. Tri- length at 280 nm. glyceride (TG), total cholesterol (TC), and high-density Cell culture and differentiation. The 3T3-L1 mouse lipoprotein cholesterol (HDLC) were enzymatically ana- fibroblast cells were purchased from the American Type lyzed with a commercial kit (Shinyang Chemical, Seoul, Culture Collection (Manassas, VA, USA). Cells were cul- Korea). Low-density lipoprotein cholesterol (LDLC) was tured in Dulbecco’s modified Eagle’s medium contain- calculated from TG, TC, and HDLC concentrations using ing 10% calf serum, 100 U/mL penicillin, 100 mg/mL the following equation: LDLC5TC2HDLC2(TG/5) (24). streptomycin, and 2 mM L-glutamine (Invitrogen, Carls- The hepatic lipids were extracted using the Folch method bad, CA, USA) at 37˚C under 5% CO2.