The Polyphenol Extract from Sechium edule Shoots Inhibits

Article pubs.acs.org/JAFC

The Polyphenol Extract from Sechium edule Shoots Inhibits Lipogenesis and Stimulates Lipolysis via Activation of AMPK Signals in HepG2 Cells Cheng-Hsun Wu,†,‡,§,+ Ting-Tsz Ou,∥,+ Chun-Hua Chang,∥ Xiao-Zong Chang,⊥ Mon-Yuan Yang,∥ and Chau-Jong Wang*,∥,# †

Department of Anatomy, China Medical University, Taichung 40402, Taiwan Department of Biochemistry, China Medical University, Taichung 40402, Taiwan § Department of Medical Research, China Medical University Hospital, Taichung 40402, Taiwan ∥ Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung 40201, Taiwan ⊥ Department of Medical Technology, Cishan Hospital, Kaohsiung 84247, Taiwan # Department of Medical Research, Chung Shan Medical University Hospital, Taichung 40201, Taiwan ‡

ABSTRACT: Fatty liver may have implications for metabolic syndrome, such as obesity, hypertension, and diabetes. Therefore, the development of pharmacological or natural agents to reduce fat accumulation in the liver is an important effort. The Sechium edule shoots have already been verified to decrease serum lipids and cholesterol and prevent atherosclerosis. However, how Sechium edule shoots modulate hepatic lipid metabolism is unclear. This study was designed to investigate the effects and mechanisms of polyphenol extracts (SPE) of Sechium edule shoots in reducing lipid accumulation in oleic acid-treated HepG2 cells. We found that water extracts (SWE) of Sechium edule shoots could decrease serum and hepatic lipid contents (e.g., triacylglycerol and cholesterol). Furthermore, SWE and SPE through the AMPK (AMP-activating protein kinase) signaling pathway could decrease lipogenic relative enzymes, such as FAS (fatty acid synthase), HMGCoR (HMG-CoA reductase), and SREBPs (sterol regulatory element binding proteins), and increase the expression of CPT-I (carnitine palmitoyltransferase I) and PPARα (peroxisome proliferators activated receptor α), which are critical regulators of hepatic lipid metabolism. These observations suggested that Sechium edule shoots have potential for developing health foods for preventing and remedying fatty liver. KEYWORDS: SPE (polyphenol extracts of Sechium edule shoots), SWE (water extracts of Sechium edule shoots), AMPK, FAS, HMGCoR, SREBPs

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INTRODUCTION Excessive lipid may accumulate in liver,1 leading to obesityassociated fatty liver disease (FLD).2 Fatty liver is closely associated with life-style-related diseases such as hyperlipidemia, hypertension, arteriosclerosis, type 2 diabetes mellitus, and cancer.3,4 Fatty liver has a strong positive relationship to outbreaks of hepatitis, cirrhosis, and cancer.5 The fat that accumulates can cause inflammation and scarring in the liver. At its most severe, nonalcoholic fatty liver disease (NAFLD) can progress to liver failure.6 Therefore, prevention and treatment of lipid accumulation in liver are relevant to health promotion. Previous research showed that the hepatic TG (triacylglycerol) content is significantly correlated with plasma TG levels and fat mass in humans.7 As we know, the hepatic TG availability is controlled by the balance between FAS and oxidation in the liver.8 The underlying cause of fat accumulation in NAFLD is mostly due to the synthesis of fatty acids and inhibition of fatty acid oxidation.9 Several recent studies have demonstrated transcriptional regulation of the gene for the enzymes of synthesis of fatty acids, including FAS (fatty acid synthase) and glycerol-3-phosphate acyltransferase (GPAT), by sterol regulatory element binding proteins (SREBPs).10,11 Activation of © 2013 American Chemical Society

FAS through modulation of SREBP-1 has been reported in human breast cancer.12 The transcription factor peroxisome proliferators activated receptor (PPAR) is expressed at very low levels in the liver, and overexpression of this transcription factor in the liver leads to hepatic adipose accumulation with the expression of several adipogenic genes in the liver.13 SREBP-1 can modulate the enzymes of lipid production, such as FAS and GPAT, and can affect the formation of fatty acids and TG. In addition, SREBP-1 not only regulates the formation rate of TG but also determines whether TG can be released from the liver.14,15 SREBP-2 regulates the generation of cholesterol metabolism-related proteins such as HMG-CoA reductase (HMGCoR) and low density lipoprotein receptor (LDLR).16,17 SREBP plays an important role in the process of controlling the formation of fatty liver. PPARs are the sensors of in vivo lipids. They control the related gene, carnitine palmitoyltransferase (CPT), in lipid oxidation and thus play a role in regulating lipid metabolism, which controls Received: Revised: Accepted: Published: 750

October 14, 2013 December 25, 2013 December 30, 2013 December 30, 2013 dx.doi.org/10.1021/jf404611a | J. Agric. Food Chem. 2014, 62, 750−759

Journal of Agricultural and Food Chemistry

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almost all aspects in fatty acid metabolism.18 AMP-activating protein kinase (AMPK) phosphorylates and inactivates a number of metabolic enzymes involved in ATP-consuming cellular events including fatty acid and cholesterol synthesis, involving FAS19 and HMGCoR.20 The activation of the AMPK pathway is necessary to prevent fat accumulation. The Sechium edule shoots contain many nutritional components including flavonoids, which are known as a powerful polyphenol and antioxidant.21 It is useful as a complementary treatment for arteriosclerosis and hypertension and as a diuretic and anti-inflammatory remedy.22,23 It has already been verified to decrease serum lipid and cholesterol and prevent atherosclerosis.22 However, how components of Sechium edule shoots modulate hepatic lipid metabolism is unclear. We examined the effect of the SWE and SPE on hepatic hypolipidemia. Human HepG2 cells were treated with indicated concentrations of SWE and SPE in the presence of OA for 24 h. We used this model to elucidate whether SWE and SPE prevents lipid accumulation in hepatic cells.

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treatments. To induce FA (fatty acid) overloading, HepG2 cells at 70% confluence were exposed to a long-chain oleic acid (OA). OA/BSA complex was prepared as reported previously.24 Stock solutions of 1 M OA prepared in culture medium containing 1% BSA were diluted in culture medium to obtain the desired final concentrations. The OA/ BSA complex solution was sterile-filtered through a 0.22 μm pore membrane filter and stored at −20 °C. Cytotoxicity Assay. HepG2 cells were seeded at a density of 1 × 106 cells/mL in 24-well plates and incubated with oleic acid, SPE, and SWE at various concentrations for 24 h. Thereafter, the medium was removed and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 0.5 mg/mL) was added to incubate for 4 h. The viable cells were directly proportional to the production of formazan. Following dissolution in isopropanol, the absorbance was read at 563 nm with a spectrophotometer (Beckman DU640). Nile Red Stain. HepG2 cells were seeded in 6-well plates (3 × 106 cells/mL) and treated with 0.6 mM oleic acid and different concentrations of SPE and SWE for 24 h. After the cells were washed twice with PBS, they were fixed with 4% formaldehyde for 30 min and then stained with 40 μg of Nile red solution for 30 min at room temperature. Lipid-bound Nile red fluorescence was observated using inverted fluorescence microscopy. Preparation of Protein Extract of HepG2 Cells. The proteins of cells were harvested in cold RIPA (radioimmunoprecipitation assay) buffer [1% NP-40 (nonyl phenoxypolyethoxylethanol), 50 mM Trisbase, 0.1% SDS, 0.5% deoxycholic acid, 150 mM NaCl, pH 7.5] containing leupeptin (17 μg/mL) and sodium orthovanadate (10 μg/ mL). The cell mixture was vortexed at 4 °C for 4 h. All mixtures were then centrifuged at 12 000 rpm at 4 °C for 10 min, and the protein contents of the supernatants were determined with the Coomassie blue total protein reagent (Kenlor Industries, Inc., USA) using bovine serum albumin as the standard. Western Blot Analysis. Equal amounts of protein samples (50 μg) were subjected to SDS−polyacrylamide gel electrophoresis and electrotransferred to nitrocellulose membranes (Millipore, Bedford, MA, USA). Membranes were blocked with 5% nonfat milk powder with 0.05% Tween 20 in PBS and then incubated with the first antibody at 4 °C overnight. Thereafter, membranes were washed three times with 0.05% Tween 20 in PBS and incubated with the anti-mouse secondary antibody conjugated to horseradish peroxidase (GE Healthcare, Little Chalfont, Buckinghamshire, U.K.). Bands were detected and revealed by enhanced chemiluminescence using ECL Western blotting detection reagents and exposed ECL hyperfilm in FUJFILM Las-3000 (Tokyo, Japan). Protein quantitation was determined by densitometry using the FUJFILM-Multi Gauge V2.2 software. Statistical Analysis. Results are reported as the mean ± standard deviation of three independent experiments and statistical comparisons were evaluated by one-way analysis of variance (ANOVA). P values less than 0.05 were considered statistically significant.

MATERIALS AND METHODS

Materials. Leaves of fresh Sechium edule shoots were collected in Nantou County, located in central Taiwan. The 3-(4, 5-dimethylthiazol-zyl)-2, 5-diphenylterazolium bromide (MTT) and oleic acid were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). GPx, SOD, and SREBP antibodies were obtained from Santa Cruz Biotechnology (CA, U.S.A.). Anti-pThr172-AMPK and anti-AMPK antibodies were purchased from Cell Signaling Technology (Beverly, MA, U.S.A). Anti-β-actin, anti-FAS, anti-SREBP-1, anti-GPAT, anti-HMGCoR, SREBP-2, anti-LDLR, and anti-catalase antibodies were purchased from Sigma-Aldrich. Preparation of SWE and SPE. Fresh leaves were chopped and airdried under shade and milled to a coarse powder. The powder was used for the preparation of water extracts (SWE) and phenolic extracts (SPE). The powder (20 g) was then subjected to maceration with sufficient volume of distilled water (1000 mL) for 4 °C for 24 h. Then the aqueous extract was filtered and lypophilized to get the yield of 17.24%. For preparation of the SPE, 100 g of dried powder of Sechium edule was submerged in 300 mL of ethanol and heated at 50 °C for 3 h. The extract was filtered and thereafter lyophilized under reduced pressure at room temperature. The powder was then resuspended in 500 mL of 50 °C distilled water, followed by extraction with 180 mL of ethyl acetate three times, redissolved in 250 mL of distilled water, stored at 70 °C overnight, and lyophilized. The presence and proportion of the main constituents of SPE were then analyzed by HPLC. HPLC (High Performance Liquid Chromatography) Analysis. HPLC was performed with a Hitachi HPLC system (Hitachi, Danbury, CT, USA), which consisted of a pump (L-6200A), an ultraviolet detector (L-4250), and the Hitachi D-7000 HPLC System Manager program. A reported procedure was used for analyzing the phenolic acids, using a Mightysil RP-18 GP 250 column (Kanto, Tokyo, Japan) and two mobile phase solvent: solvent A, acetic acid/ water (2:98, v/v), and solvent B, 0.5% acetic acid in water/acetonitrile (50:50, v/v). The flow rate was 1 mL/min. The gradient for the separation was 100% solvent A at 0 min, 70% solvent A and 30% solvent B at 5 min, 65% solvent A and 35% solvent B at 50 min, 60% solvent A and 40% solvent B at 55 min, 0% solvent A and 100% solvent B at 60 min, followed by a 5 min postrun with HPLC grade water. Phenolic acids were detected at 260 nm. Cell Culture. Human HepG2 cells obtained from the American Type Culture Collection were maintained in DMEM supplemented with 10% fetal calf serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine and kept at 37 °C in a humidified atmosphere of 5% CO2. Cells were grown to 70% confluence and then incubated in serum-free medium for 24 h before

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RESULTS SWE and SPE Content Assay. The single-ring type of polyphenol compounds (gallic acid, GA) were used to determine the standard content of total polyphenol. The results show (Table 1) that in SWE, the single-ring polyphenol compounds were 4.41% ± 0.02% polyphenol (using gallic acid as the standard), 3.32% ± 0.17% flavonoids (using quercetin and naringenin as the standard), 26.73% ± 2.18% carbohydrate, 4.67% ± 1.46% protein, and 3.25% ± 1.611% lipid. The analysis of SPE revealed that it contained 17.74% ± 0.05% total polyphenol (using gallic acid as the standard) and 21.10% ± 0.28% flavonoids (using quercetin and naringenin as the standard). The presence and proportion of the main constituents of SPE were then analyzed by HPLC (Figure 1). For the standardization of SPE, the presence of protocatechuic acid (3.56% ± 0.14%), gallocatechin gallate (11.06% ± 0.18%), caffeic acid (4.42% ± 0.25%), rutin (1.14% ± 0.13%), quercetin 751

dx.doi.org/10.1021/jf404611a | J. Agric. Food Chem. 2014, 62, 750−759


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Inhibition of OA-Induced Lipid Accumulation by SWE and SPE in HepG2 Cells. The above results showed that the cell growth condition was good, and cell survival rates remained at 100%. Our preliminary work has demonstrated that cell viability is unaffected at a concentration of 0.6 mM OA. Thus, we used 0.6 mM OA and SWE (1 and 5 mg/mL) and SPE (0.5 and 1 mg/mL) to culture HepG2 cells in order to observe the fat accumulation. Figure 3A is the result of using Nile red fluorescent staining to show that fat accumulation altered the red fluorescence with change in fat in cytoplasm in a dose dependent manner. Next, using Nile red staining and flow cytometric analysis to detect the intensity of fluorescence (Figure 3B), we found that the intensity of fluorescence was proportional to fat content. Then, we quantified the intracellular fat content (Figure 3C). The fat content of HepG2 was 2.1 times higher (*p < 0.05) than the control group after exposure to OA. SWE treatment of cells at doses of 1 or 5 mg/ mL resulted in a reduction of lipid content (92.1% and 84.25%) compared with the OA group (*p < 0.05). Treatment with SPE, also reduced lipid content about 88.1% and 83.21% (*p < 0.05) compared with the OA group. These results showed that SWE and SPE had the effect of inhibiting intracellular fat accumulation. SPE was more efficient than SWE in causing this effect.

Table 1. Components of SWE and SPE (polyphenol extracts) of Sechium edule Shoots SWE (%) polyphenol (gallic acid as STD) flavonoid flavone and flavonol flavanone and flavanonol carbohydrate protein lipid

4.41 3.32 1.54 1.78 26.73 4.67 3.25

± ± ± ± ± ± ±

0.02 0.17 0.07 0.10 2.18 1.46 1.11

SPE (%) 17.74 21.10 4.52 16.58

± ± ± ±

0.05 0.28 0.07 0.21

(3.71% ± 0.32%), and naringenin (11.28% ± 1.12%) was confirmed in the SPE. The Effect of SWE and SPE on Cell Viability of HepG2 Cells. Using different concentrations of SWE and SPE to treat HepG2 cells, after 24 h, we analyzed cell viability. Figure 2A,B showed that from the result of MTT, the drug lethal dose (IC50) of SWE was more than 5 mg/mL and that of SPE was 2.32 mg/mL respectively. This experiment focuses on the premise that intracellular lipid accumulation will not cause any damage to cells. So the follow-up experiment used concentrations of 1 and 5 mg/mL SWE and 0.5 and 1 mg/mL SPE for treatment of HepG2 cells.

Figure 1. The HPLC chromatogram of SPE (polyphenol extracts of Sechium edule shoots). (A) HPLC chromatogram of free polyphenols from SPE (10 mg/mL, 10 μL). (B) HPLC chromatogram of eight kinds of standard polyphenols (1 mg/mL; 10 μL). Peaks: (1) gallic acid; (2) protocatechuic acid; (3) catechin; (4) gallocatechin gallate; (5) caffeic acid; (6) rutin; (7) quercetin; (8) naringenin. 752



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Figure 4B shows that cells induced by OA had 1.31 times the expression of SREBP-1. After exposure to 0.5 or 1.0 mg/mL SPE, the expression of SREBP-1 was 1.17 and 1.13 times, respectively. SWE and SPE reduced the expression of FAS in HepG2 cells in a dose-dependent manner after induction by OA. Figure 4B also reveals that the expression of GPAT was induced 1.29 times by OA. When compared with the control group, after cells were exposed to 0.5 or 1 mg/mL SPE, the expression of GPAT was 0.98 and 0.91 times respective. Both SWE and SPE reduced the expression of GPAT in HepG2 cells after induced by OA, and the response was dose-dependent. From those data, we can validate that, through the inhibition of those transcription factors, SWE and SPE may regulate the synthesis of triglycerides. Effect of SWE and SPE on the Expression of Cholesterol Synthesis Related Proteins. HMGCoR is the rate-determining enzyme of cholesterol synthesis. To test whether the reduction of lipid accumulation in both SWE- and SPE-treated HepG2 cells is accompanied by changes in cholesterol biosynthesis, Western Blots were performed. As seen in Figure 5A,B, the expression of HMGCoR, SREBP-2, and LDL-R were remarkably decreased by SWE (1 or 5 mg/ mL) or SPE (0.5 or 1 mg/mL) treatment compared with the OA-treated group. From those data, we can validate that, through the inhibition of those transcription factors and LDLR, SWE and SPE may regulate the synthesis of cholesterol (Figure 5). Effect of SWE and SPE on the Expression of Fatty Acid Oxidation Related Proteins. CPT-1 is an enzyme in the body that helps change fat to energy. In this study, the results have shown that SWE or SPE treatment increases the expression of PPARα and CPT-1 compared with that in the OA group in HepG2 cells (Figure 6). Thus, through the stimulation of those transcription factors, SWE and SPE were shown to increase the oxidation of fatty acids. Effect of SWE and SPE on the Phosphorylation of AMPK. AMPK is an important regulator in the metabolism mechanism for sugar and fat. In Figure 7A, when cells were treated with 1 or 5 mg/mL SWE, the expression of p-AMPK was significantly increased 1.33- and 1.40-fold respectively, compared with that in the OA group. We further observed that the ratio of AMPK/p-AMPK was increased (*p < 0.05, **p < 0.001), indicating that SWE can activate AMPK. In Figure 7B, the expression of p-AMPK was significantly increased 1.29- and 1.32-fold after treatment with 0.5 or 1 mg/mL SPE in HepG2 cells. We also observed that the p-AMPK/AMPK ratio had an upward trend. With those results, we prove that SWE and SPE can activate AMPK and, hence, reduce lipid synthesis in cells.

Figure 2. The cytotoxicity effects of SWE (water extracts of Sechium edule shoots) and SPE (polyphenol extracts of Sechium edule shoots) on human hepatocarcinoma cell line. HepG2 cells were treated with various concentrations of SWE (A) or SPE (B) for 24 h. Viability of HepG2 cells was determined by the MTT assay. The results are presented as mean ± SD of two independent experiments.

Effect of SWE and SPE on the Expression of TG Synthesis Related Proteins. Figure 4A shows that cells that were induced by OA had 1.24 times the expression of FAS. When compared with the control group, in cells that were exposed to 1 or 5 mg/mL SWE, the expression of FAS was 1.08 and 1.03 times, respectively. Figure 4A shows that cells that were induced by OA had 1.28 times the expression of SREBP-1. When compared with the control group, after exposure to 1 or 5 mg/mL SWE, the expression of SREBP-1 was 1.21 and 1.11 times, respectively. GPAT is the rate-determining enzyme of triglyceride synthesis. Figure 4A also reveals that the expression of GPAT induced by OA was 1.25 times. When compared with the control group, after cells were exposure to 1 or 5 mg/mL SWE, the expression of GPAT was 1.22 and 1.03 times, respectively. Figure 4B shows that cells induced by OA had the expression of FAS 1.26 times. After exposure to 0.5 or 1.0 mg/ mL SPE, the expression of FAS was 1.09 and 0.687 times, respectively. SWE and SPE reduced the expression of FAS in HepG2 cells in a dose dependent manner after induction by OA.

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DISCUSSION The liver plays an essential role in lipid metabolism via regulating lipogenesis and oxidative stress.23 Excessive lipid accumulation in liver may progress to steatohepatitis.2 The mechanism study is well-known in oleic acid-induced human hepatoma HepG2 cells model. Here, we attempted to examine the hypolipidemia effect and possible mechanism of SWE or SPE on hepatic lipid metabolism. Previous reports indicated that a regulation of hepatic LDLR and HMGCoR activity could be observed in HepG2 cells.25,26 The fat accumulation in NAFLD is mostly due to the synthesis of fatty acids and inhibition of fatty acid oxidation.27 Generally, hepatic hypolipidemic mechanisms are highly associated with expression of lipogenic enzymes, cholesterol biosynthesis, fatty 753



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Figure 3. Effects of SWE (water extracts of Sechium edule shoots) or SPE (polyphenol extracts of Sechium edule shoots) on intracellular lipid accumulation in HepG2 cells. Cells are cotreated with oleic acid (OA) 0.6 mM and various concentrations of SWE or SPE for 24 h. (A) After culturing, cells were fixed with formalin and stained with Nile red and (B) analyzed by flow cytometry. (C) Quantitative assessment of the percentage of lipid accumulation; data represent the average of three independent experiments ± SD. SC indicates an internal control of cell stained with Nile red; 0 indicates an induced control of cells treated with oleic acid only. #p < 0.05 compared with the SC group; *p < 0.05 compared with the OA-induced group.

acid β-oxidation, and TG biosynthesis in HepG2 cells. In our present study, the hypolipidemic mechanisms of SWE and SPE were related to expression of lipogenic enzymes (SREBP-1 and FAS), cholesterol biosynthesis (HMGCoR, SREBP-2, and LDL-R), fatty acid β-oxidation (PPAR-α and CPT-1), and TG biosynthesis (GPAT) in OA-induced HepG2 cells. AMPK is a multisubunit enzyme recognized as a major regulator of lipid biosynthetic pathways due to its role in the phosphorylation and inactivation of key enzymes such as FAS.28 Studies demonstrated that polyphenolic extracts from plants can activate AMPK and inhibit FAS expression by preventing SREBP-1 translocation to the nuclei.29−31

Polyphenols are widely found in Sechium edule shoots.32 In this study, we found that both SWE and SPE contained total polyphenols about 7.73% and 38.84%, respectively. These concentrations were sufficient to lower lipid levels in the liver.33 Therefore, both SWE and SPE have great ability to activate AMPK and then reduce protein expression of SREBP-1, leading to inhibition of hepatic lipogenesis. Our data showed that AMPK plays a pivotal role in the hypolipid effect, and both SWE and SPE can augment AMPK activation (Figure 7). SREBP-1 is a key lipogenic transcription factor regulating the gene expression of lipogenic enzymes and is dedicated to the synthesis and uptake of fatty acids and triacylglycerol.31,34 Our data showed that the expression of SREBP-1 and its 754



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Figure 4. Treatment of SWE (water extracts of Sechium edule shoots) and SPE (polyphenol extracts of Sechium edule shoots) decreased fatty acid biosynthesis relative protein expression in OA (oleic acid)-induced HepG2 cell. Cells were coexposed to OA (0.6 mM) and various doses of SWE (A) or SPW (B) for 24 h. The FAS, SREBP-1, and GPAT protein levels were also examined under the same conditions. The numbers below the panels represent quantification of the immunoblot by densitometry. #p < 0.05 compared with a control group; *p < 0.05 compared with OA-induced group. 755



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Figure 5. Treatment of SWE (water extracts of Sechium edule shoots) and SPE (polyphenol extracts of Sechium edule shoots) decreased cholesterol biosynthesis related protein expression in OA (oleic acid)-induced HepG2 cell. Cells were coexposed to OA (0.6 mM) and various doses of SWE (A) or SPE (B) for 24 h. The HMGCoR, SREBP-2, and LDLR protein levels were also examined under the same conditions. The numbers below the panels represent quantification of the immunoblot by densitometry. #p < 0.05 compared with control group. *p < 0.05 compared with OAinduced group. 756



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Figure 6. Treatment with SWE (water extracts of Sechium edule shoots) and SPE (polyphenol extracts of Sechium edule shoots) increased fatty acid oxidation related protein expression in OA (oleic acid)-induced HepG2 cell. Cells were coexposed to OA (0.6 mM) and various doses of SWE (A) or SPE (B) for 24 h. CPT-1 and PPARα were detected by Western blot analysis under the same conditions. The numbers below the panels represent quantification of the immunoblot by densitometry. #p < 0.05 compared with control group; *p < 0.05 compared with OA-induced group. 757



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Figure 7. Treatment of SWE (water extracts of Sechium edule shoots) and SPE (polyphenol extracts of Sechium edule shoots) increased AMPactivated protein kinase (AMPK) phosphorylation protein expression in OA (oleic acid)-induced HepG2 cell. Cells were coexposed to OA (0.6 mM) and various doses of SWE (A) or SPE (B) for 24 h. AMPK phosphorylation (pThr172-AMPK) was detected by Western blot analysis under the same conditions. The numbers below the panels represent quantification of the immunoblot by densitometry. *p < 0.05 compared with OAinduced group.

developed as a potential therapeutic treatment in order to reduce the formation of a fatty liver.

downstream factors, FAS and GAPT, were reduced in response to SWE or SPE treatment in HepG2 cells (Figure 4). Another recent study suggests that AMPK mediates a decrease in SREBP-1 expression.29 Consequently, our data suggest that the ability of SWE and SPE to decrease FAS and GAPT expression may occur through AMPK activation and SREBP-1 suppression. In addition increase of hepatic lipogenesis by activation of SREBP-1 may contribute to the development of chemically induced fatty liver.35 AMPK phosphorylates and inhibits SREBP-1 activity to attenuate hepatic steatosis,36 whereas SREBP-2 primarily controls cholesterol homeostasis by activating genes required for cholesterol synthesis and uptake.37 Our data corroborated these results (Figure 5 and Figure 7). Similarly, AMPK inhibits in vitro lipogenesis in hepatocytes through the downregulation of the cleavage processing and transcriptional activity of SREBP.36 PPAR-α is highly expressed in the liver where it activates genes involved in β-oxidation of fatty acids.38 In our study, both SWE and SPE treatment result in an increased expression of PPAR-α in OA-induced lipid accumulation cells. In conclusion, the current study identifies that SWE and SPE can reduce lipid accumulation. We also propose that AMPK is pivotal in closing the anabolic pathway and promoting catabolism by down regulating the activity of key enzymes in lipid metabolism, such as HMGCoR and FAS. Both SWE and SPE can suppress fat accumulation of the liver and could be

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AUTHOR INFORMATION

Corresponding Author

*Tel: +886-4-24730022, ext. 11670. Fax: +886-4-2324-8167. Mailing address: No.110, Sec. 1, Jianguo N. Rd., South District, Taichung, Taiwan 402. E-mail: [email protected]. Author Contributions +

C.-H.W. and T.T.O. contributed equally to this work and therefore share first authorship. Funding

This work was supported by a National Science Council Grant NSC99-2321-B-040-001), Taiwan. Notes

The authors declare no competing financial interest.

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REFERENCES

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The Polyphenol Extract from Sechium edule Shoots Inhibits

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