1
The polyphenol extract from Sechium edule shoots inhibits lipogenesis
2
and stimulates lipolysis via activation of AMPK signals in HepG2 cells
3
Cheng-Hsun Wua,b,c, Ting-Tsz Oud,＋, Chun-Hua Changd, Xiao-Zong Change,
4
Mon-Yuan Yangd, Chau-Jong Wangd, f, *
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6
a
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b
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c
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Taiwan.
Department of Anatomy, China Medical University, Taichung, Taiwan.
Department of Biochemistry, China Medical University, Taichung, Taiwan.
Department of Medical Research, China Medical University Hospital, Taichung,
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d
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Taichung, Taiwan.
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e
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f
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Taiwan.
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＋
Institute of Biochemistry and Biotechnology, Chung Shan Medical University,
Department of Medical Technology, Cishan Hospital, Kaohsiung, Taiwan.
Department of Medical Research, Chung Shan Medical University Hospital, Taichung,
These authors contributed equally to this work and therefore share first authorship
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*Corresponding author
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Chau-Jong Wang, Ph. D.
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Tel: +886-4-24730022ext11670, Professor of the Institute of Biochemistry and
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Biotechnology, Chung Shan Medical University.
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Fax: +886-4-2324-8167
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Address: No.110, Sec. 1, Jianguo N. Rd., South District, Taichung, Taiwan 402
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E-mail: wcj@csmu.edu.tw
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ABSTRACT
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Fatty liver may have implications about metabolic syndrome, such as obesity,
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hypertension and diabetes. Therefore, the development of pharmacological or natural
29
agents to reduce fat accumulation in the liver is an important effort. The Sechium
30
edule shoots have already been verified to decrease serum lipid, cholesterol and
31
prevent atherosclerosis. However, how Sechium edule shoots modulate hepatic lipid
32
metabolism is unclear. This study was designed to investigate the effects and
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mechanisms of polyphenol extracts (SPE) of Sechium edule shoots in reducing lipid
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accumulation in oleic acid- treated HepG2 cells. We found that water extracts (SWE)
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of Sechium edule shoots could decrease serum and hepatic lipid contents (e.g.
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triacylglycerol and cholesterol). Furthermore, SWE and SPE through the AMPK
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(AMP-activating protein kinase) signaling pathway could decrease lipogenic relative
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enzymes, such as FAS (fatty acid synthesis), HMGCoR (HMG-CoA reductase),
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SREBPs (sterol regulatory element binding proteins) and increase the expression of
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CPT-I (carnitine palmitoyltransferase I) and PPARα (peroxisome proliferators
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activated receptor α), which are critical regulators of hepatic lipid metabolism. These
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observations suggested that Sechium edule shoots have potential for developing health
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foods for preventing and remedying fatty liver.
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KEYWORDS: SPE (polyphenol extracts of Sechium edule shoots), SWE (water
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extracts of Sechium edule shoots), AMPK, FAS, HMGCoR, SREBPs
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2
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INTRODUCTION
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Excessive lipid may accumulate in liver,1 leading to obesity-associated fatty liver
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disease (FLD). 2 Fatty liver is closely associated with life-style-related diseases such as
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hyperlipidemia, hypertension, arteriosclerosis, type 2 diabetes mellitus and cancer.
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Fatty liver has a strong positive relationship to outbreaks of hepatitis, cirrhosis and
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cancer. 5 The fat that accumulates can cause inflammation and scarring in the liver. At
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its most severe, nonalcoholic fatty liver disease can progress to liver failure. 6 Therefore,
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prevention and treatment of lipid accumulation in liver are relevant to health
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promotion.
3, 4
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Previous research showed that the hepatic TG (triacylglycerol) content is
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significantly correlated with plasma TG levels and fat mass in humans. 7 As we know,
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the hepatic TG availability is controlled by the balance between FAS and oxidation in
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the liver. 8 The underlying cause of fat accumulation in NAFLD is mostly due to the
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synthesis of fatty acids and inhibition of fatty acid oxidation. 9 Several recent studies
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have demonstrated transcriptional regulation of the gene for the enzymes of synthesis
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of fatty acids, including FAS (fatty acid synthesis) and glycerol-3-phosphate
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acyltransferase (GPAT), by sterol regulatory SREBPs. 10, 11 Activation of FAS through
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modulation of SREBP-1 has been reported in human breast cancer. 12 The transcription
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factor PPAR is expressed at very low levels in the liver, and overexpression of this
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transcription factor in the liver leads to hepatic adipose accumulation with the
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expression of several adipogenic genes in the liver.
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enzymes of lipid production, such as FAS and GPAT, and can affect the formation of
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fatty acids and TG. In addition, SREBP-1 not only regulates the formation rate of TG
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but also determines whether TG can be released from the liver. 14, 15 SREBP-2 regulates
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the generation of cholesterol metabolism-related proteins such as HMGCoR and LDLR.
3
13
SREBP-1 can modulate the
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16, 17
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liver. PPARs are the sensors of in vivo lipids. They control the related genes, CPT, in
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lipid oxidation and thus play a role in regulating lipid metabolism, which controls
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almost all aspects in fatty acid metabolism. 18 AMPK phosphorylates and inactivates a
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number of metabolic enzymes involved in ATP-consuming cellular events including
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fatty acid and cholesterol synthesis, involving FAS
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of the AMPK pathway is necessary to prevent fat accumulation.
SREBP plays an important role in the process of controlling the formation of fatty
19
and HMGCoR. 20 The activation
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The Sechium edule shoots contain a lot of nutritional components including
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flavonoids which are known as a powerful polyphenol and antioxidant. 21 It is useful as
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a complementary treatment for artheriosclerosis and hypertension and as a diuretic and
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antiinflammatory remedy.
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cholesterol and prevent atherosclerosis. 22 However, how components of Sechium edule
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shoots modulate hepatic lipid metabolism is unclear.
22, 23
It has already been verified to decrease serum lipid,
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We examined the effect of the SWE and SPE on hepatic hypolipidemia. Human
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HepG2 cells were treated with indicated concentrations of SWE and SPE in the
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presence of OA for 24 h. We used this model to elucidate whether SWE and SPE
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prevents lipid accumulation in hepatic cells.
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MATERIALS AND METHODS
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Materials
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Leaves of fresh Sechium edule shoots were collected in Nantou County, located in
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central Taiwan. The 3-(4, 5-dimethylthiazol-zyl)-2, 5-diphenylterazolium bromide
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(MTT) and oleic acid were purchased from Sigma-Aldrich (St. Louis, Mo., U.S.A.).
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GPx, SOD and SREBP antibodies were obtained from Santa Cruz Biotechnology (CA,
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U.S.A.). Anti-pThr172-AMPK and anti-AMPK antibodies were purchased from Cell
4
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Signaling Technology (Beverly, MA, U.S.A). Anti-β-actin, Anti-FAS, Anti-SREBP-1,
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Anti-GPAT, Anti-HMGCoR, SREBP-2, Anti-LDLR and anti-catalase antibodies were
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purchased from Sigma-Aldrich.
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Preparation of SWE and SPE
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Fresh leaves were chopped and air dried under shade and milled to a coarse powder.
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The powder was used for the preparation of water extracts (SWE) and phenolic extracts
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(SPE). The powder (20 g) was then subjected to maceration with sufficient volume of
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distilled water (1000 ml) for 4℃ for 24 h. Then the aqueous extract was filtered and
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lypophilized to get the yield of 17.24 %. For preparation of the SPE, 100 g dried
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powder of Sechium edule was submerged in 300 mL of ethanol and heated at 50℃ for 3
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h. The extract was filtered and thereafter lyophilized under reduced pressure at room
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temperature. The powder was then resuspended in 500 mL of 50℃ distilled water,
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followed by extraction with 180 mL of ethyl acetate three times, redissolved in 250 mL
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of distilled water, stored at 70 ℃ overnight, and lyophilized. The presence and
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proportion of the main constituents of SPE were then analyzed by HPLC.
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HPLC (High Performance Liquid Chromatography) Analysis
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HPLC was performed with a Hitachi HPLC system (Hitachi, Danbury, CT, USA)
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which consisted of a pump (L-6200A), an ultraviolet detector (L-4250) and the Hitachi
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D-7000 HPLC System Manager program. A reported procedure was used for analyzing
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the phenolic acids, using a Mightysil RP-18 GP 250 column (Kanto, Tokyo, Japan) and
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two mobile phase solvent: solvent A, acetic acid/water (2:98, v/v), and solvent B, 0.5%
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acetic acid in water/acetonitrile (50:50, v/v). The flow rate was 1 mL/min. The gradient
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for the separation was 100% solvent A at 0 min, 70% solvent A and 30% solvent B at 5
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min, 65% solvent A and 35% solvent B at 50 min, 60% solvent A and 40% solvent B at
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55 min, 0% solvent A and 100% solvent B at 60 min, followed by a 5 min postrun with
5
123
HPLC grade water. Phenolic acids were detected at 260 nm.
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Cell Culture
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Human HepG2 cells obtained from the American Type Culture Collection were
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maintained in DMEM supplemented with 10% fetal calf serum, 100 U/ml penicillin,
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100 μg/ml streptomycin, and 2 mM L-glutamine and kept at 37℃ in a humidified
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atmosphere of 5 % CO2. Cells were grown to 70% confluence and then incubated in
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serum-free medium for 24 h before treatments. To induce FA (fatty acid) overloading,
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HepG2 cells at 70 % confluence were exposed to a long-chain oleic acid (OA).
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OA/BSA complex was prepared as reported previously.
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prepared in culture medium containing 1% BSA were diluted in culture medium to
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obtain the desired final concentrations. The OA/BSA complex solution was
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sterile-filtered through a 0.22 μm pore membrane filter and stored at -20 ℃.
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Cytotoxicity Assay
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HepG2 cells were seeded at a density of 1 x 106 cells/ ml in 24-well plates and incubated
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with oleic acid, SPE and SWE at various concentrations for 24 h. Thereafter, the
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medium was removed and 3-(4, 5-dimethylthiazol-zyl)-2, 5-diphenylterazolium
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bromide (MTT, 0.5 mg/ml) was added to incubate for 4 h. The viable cells were directly
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proportional to the production of formazan. Following dissolution in isopropanol, the
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absorbance was read at 563 nm with a spectrophotometer (Beckman DU640).
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Nile Red Stain
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HepG2 cells were seeded in 6-well plates (3x 106 cells /ml) and treated with 0.6 mM
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oleic acid and different concentrations of SPE and SWE for 24h. After the cells were
6
24
Stock solutions of 1M OA
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washed twice with PBS, they were fixed with 4% formaldehyde for 30 min and then
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stained with 40ug Nile red solution for 30 min at room temperature. Lipid-bounded
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Nile red fluorescence was observated using Inverted Fluorescence Microscopy.
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Preparation of Protein Extract of HepG2 Cells
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The proteins of cells were harvested in a cold RIPA (radioimmunoprecipitation assay)
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buffer [1% NP-40 (nonyl phenoxypolyethoxylethanol), 50 mM Tris–base, 0.1% SDS,
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0.5% deoxycholic acid, 150 mM NaCl, pH 7.5] containing leupeptin (17 μg/mL) and
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sodium orthovanadate (10 μg/mL). The cell mixture was vortexed at 4 °C for 4h. All
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mixtures were then centrifuged at 12,000 rpm at 4 °C for 10 min, and the protein
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contents of the supernatants were determined with the conmassie blue total protein
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reagent (Kenlor Industries, Inc, USA) using bovine serum albumin as the standard.
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Western Blot Analysis
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Equal amounts of protein samples (50 µg) were subjected to SDS-polyacrylamide gel
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electrophoresis and electrotransferred to nitrocellulose membranes (Millipore, Bedford,
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MA, USA). Membranes were blocked with 5% non-fat milk powder with 0.05% Tween
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20 in PBS and then incubated with the first antibody at 4 °C overnight. Thereafter,
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membranes were washed three times with 0.05% Tween 20 in PBS and incubated with
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the secondary antibody conjugated to anti-mouse horseradish peroxidase (GE
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Healthcare, Little Chalfont, Buckinghamshire, UK). Bands were detected and revealed
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by enhanced chemiluminescence using ECL Western blotting detection reagents and
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exposed ECL hyperfilm in FUJFILM Las-3000 (Tokyo, Japan). Protein quantitation
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was determined by densitometry using the FUJFILM-Multi Gauge V2.2 software.
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Statistical analysis
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Results are reported as the mean ± standard deviation of 3 independent experiments
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and statistical comparisons were evaluated by one-way analysis of variance (ANOVA).
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P values less than 0.05 was considered statistically significant.
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172
RESULTS
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SWE and SPE content assay
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The single-ring type of polyphenol compounds (gallic acid, GA) were used to
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determine the standard content of total polyphenol. The results show (Table 1) that in
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SWE, the single-ring polyphenol compounds were 4.41 + 0.02% polyphenol (using
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gallic acid as the standard), 3.32 + 0.17% flavonoids (using quercetin and naringenin as
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the standard), 26.73+ 2.18% carbohydrate, 4.67+ 1.46% protein and 3.25+1.611%
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lipid.
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The analysis of SPE revealed that it contained 17.74 + 0.05% total polyphenol (using
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gallic acid as the standard), 21.10+ 0.28% flavonoids (using quercetin and naringenin
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as the standard). The presence and proportion of the main constituents of SPE were then
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analyzed by HPLC (Fig. 1). For the standardization of SPE, the presence of
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protocatechuic acid (3.56 ± 0.14), gallocatechin gallate (11.06 ± 0.18), caffeic acid
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(4.42 ± 0.25), rutin (1.14 ± 0.13), quercetin (3.71 ± 0.32), naringenin (11.28 ± 1.12)
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contained in the SPE.
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The effect of SWE and SPE on cell viability of HepG2 cells
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By using different concentrations of SWE and SPE to treat HepG2 cells, after 24 h,
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we analyzed cell viability. Fig. 2A and 2B showed that from the result of MTT, the
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drug lethal dose (IC50) of SWE was more than 5 mg/ml and SPE was 2.32 mg/ml
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respectively. This experiment focuses on the premise that intracellular lipid
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accumulation will not cause any damage to cells. So the follow-up experiment used
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concentration of 1 and 5 mg/ml SWE and 0.5 and 1 mg/ml SPE for treatment of
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HepG2 cells.
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Inhibition of OA- induced lipid accumulation by SWE and SPE in HepG2 cells.
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The above results showed that the cell growth condition was good and cell survival
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rates remained at 100%. Our preliminary work has demonstrated the cell viability was
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unaffected at a concentration of 0.6 mM OA. Thus, we used 0.6 mM OA and SWE (1
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and 5 mg/ml) and SPE (0.5 and 1 mg/ml) to culture HepG2 cells in order to observe
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the fat accumulation. Fig. 3A is the result of using Nile red fluorescent staining to
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show that fat accumulation altered the red fluorescence with change in fat in
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cytoplasm in a dose dependent manner. Next, using Nile red staining and flow
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cytometric analysis to detect the intensity of fluorescence (Fig. 3B), we found that the
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intensity of fluorescence was proportional to fat content. Then, we quantified the
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intracellular fat content (Fig. 3C). The fat content of HepG2 was 2.1 times higher (*p
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＜0.05) than the control group after exposure to OA. SWE treatment of cells at doses
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of 1 or 5 mg/ml resulted in a reduction of lipid content (92.1% and 84.25%) as
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208
compared with the OA group (*p＜0.05). Treatment with SPE, also reduced lipid
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content about 88.1% and 83.21 (*p＜0.05) when compared with the OA group. These
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results showed that SWE and SPE had the effect of inhibiting intracellular fat
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accumulation. SPE was more efficient than SWE in causing this effect.
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Effect of SWE and SPE on the expression of TG synthesis related proteins.
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Fig. 4A shows that cells which were induced by OA had 1.24 times the expression of
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FAS. When compared with the control group, cells were exposed to 1 or 5 mg/ml
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SWE, the expression of FAS was 1.08 and 1.03 times respectively. Fig. 4A shows that
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cells which were induced by OA had 1.28 times the expression of SREBP-1. When
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compared with the control group, after exposure to 1 or 5 mg/ml SWE, the expression
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of SWE was 1.21 and 1.11 times respectively. GPAT is the rate-determining enzyme
219
of triglyceride synthesis. Fig. 4A also reveals that the expression of GPAT induced by
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OA was 1.25 times. When compared with the control group, after cells were exposure
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to 1 or 5 mg/ml SWE, the expression of GPAT was 1.22 and 1.03 times respectively.
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Fig. 4B shows that cells induced by OA had the expression of FAS 1.26 times. After
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exposure to 0.5 or 1.0 mg/ml SPE, the expression of FAS was 1.09 and 0.687 times
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respectively. SWE and SPE reduced the expression of FAS in HepG2 cells in a dose
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dependent manner after induction by OA.
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Fig. 4B shows that cells induced by OA had 1.31 times the expression of SREBP-1.
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After exposure to 0.5 or 1.0 mg/ml SPE, the expression of SREBP-1 was 1.17 and
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1.13 times respectively. SWE and SPE reduced the expression of FAS in HepG2 cells
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in a dose-dependent manner after induction by OA. Fig. 4B also reveals that the
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expression of GPAT was induced 1.29 times by OA. When compared with the control
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group, after cells were exposed to 0.5 or 1 mg/ml SPE, the expression of GPAT was
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0.98 and 0.91 times respective. Both SWE and SPE reduced the expression of GPAT
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in HepG2 cells after induced by OA, and the response was dose-dependent. From
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those data, we can validate that, through the inhibition of those transcription factors,
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SWE and SPE may regulate the synthesis of triglycerides.
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Effect of SWE and SPE on the expression of cholesterol synthesis related
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proteins
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HMGCoR is the rate-determining enzyme of cholesterol synthesis. To test whether
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the reduction of lipid accumulation in both SWE- and SPE- treated HepG2 cells is
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accompanied by changes the cholesterol biosynthesis, Western Blots were performed.
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As seen in Fig. 5A and B, the expression of HMGCoR, SREBP-2, and LDL-R were
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remarkably decreased by SWE (1 or 5 mg/ml) or SPE (0.5 or 1 mg/ml) treatment
243
compared with OA- treated group. From those data, we can validate that, through the
244
inhibition of those transcription factors and LDLR, SWE and SPE may regulate the
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synthesis of cholesterol (Fig. 5).
11
246
Effect of SWE and SPE on the expression of fatty acid oxidation related proteins
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CPT-1 is an enzyme in the body that helps change fat to energy. In this study, the
248
results have shown that SWE or SPE treatment increase the expression of PPARα
249
and CPT-A as compared with the OA group in HepG2 cells (fig. 6). Thus, through the
250
stimulation of those transcription factors, SWE and SPE were shown to increase the
251
oxidation of fatty acids.
252
Effect of SWE and SPE on the phosphorylation of AMPK
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AMPK is an important regulator in the metabolism mechanism for sugar and fat. In
254
Fig. 7A, cells were treated with 1 or 5 mg/ml SWE, the expression of p-AMPK was
255
significantly increased 1.33 and 1.40 fold respectively, as compared with the OA
256
group. We further observed the change ratio of AMPK /p-AMPK was increased (*p＜
257
0.05, **p ＜ 0.001), indicating that SWE can activate AMPK. In Fig. 7B, the
258
expression of p-AMPK was significantly increased 1.29 and 1.32 fold after treated
259
with 0.5 or 1 mg/ml SPE in HepG2 cells. We also observed the p-AMPK/AMPK ratio
260
had an upward trend. With those results, we prove that SWE and SPE can activate
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AMPK, and hence, reduce the lipid synthesis of cells.
262
DISCUSSION
263
264
Liver plays an essential role in lipid metabolism via regulating lipogenesis and
oxidative stress.
23
Excessive lipid accumulation in liver may progress to
12
265
steatohepatitis. 2 The mechanism study is well known in oleic acid-induced human
266
hepatoma HepG2 cells model. Here, we attempted to examine the hypolipidemia
267
effect and possible mechanism of SWE or SPE on hepatic lipid metabolism. Previous
268
reports indicated that a regulation of hepatic LDLR and HMGCoR activity could be
269
observed in HepG2 cells. 25, 26 The mechanism underlying fat accumulation of NAFLD
270
is mostly due to the synthesis of fatty acids and inhibition of fatty acid oxidation.
271
Generally, hepatic hypolipidemic mechanisms were highly associated with expression
272
of lipogenic enzyme, cholesterol biosynthesis, fatty acid β-oxidation, and TG
273
biosynthesis in HepG2 cells. In our present study, the hypolipidemic mechanisms of
274
SWE and SPE were related to expression of lipogenic enzyme (SREBP-1 and FAS),
275
cholesterol biosynthesis (HMGCoR, SREBP-2, and LDL-R), fatty acid β-oxidation
276
(PPAR-α and CPT-1), and TG biosynthesis (GPAT) in OA-induced HepG2 cells.
27
277
AMPK is a multisubunit enzyme recognized as a major regulator of lipid
278
biosynthetic pathways due to its role in the phosphorylation and inactivation of key
279
enzymes such as FAS. 28 Studies demonstrated that polyphenolic extracts from plants
280
can activate AMPK and inhibit FAS expression by preventing SREBP-1
281
transclocation to the nuclei.
282
Sechium edule shoots. 32 In this study, we found that both SWE and SPE contained
283
total polyohenols about 7.73 % and 38.84 %, respectively. These concentrations were
284
sufficient to lower lipid levels in the liver. 33 Therefore, both SWE and SPE have great
285
ability to activate AMPK and then reduce protein expression of SREBP-1, leading to
286
inhibit hepatic lipogenesis. Our data showed that AMPK plays a pivotal role in
287
hypolipid effect and both SWE and SPE can augment AMPK activation (Fig. 7).
288
SREBP-1 is a key lipogenic transcription factor regulating the gene expression of
289
lipogenic enzymes and is dedicated to the synthesis and uptake of fatty acids and
29-31
New paragraph polyphenols are widely found in
13
31, 34
290
triacylglycerol.
291
downstream factors, FAS and GAPT, were reduced in response to SWE or SPE
292
treatment in HepG2 cells (Fig. 4). Another recent study suggests that AMPK mediates
293
a decrease in SREBP-1 expression.
294
SWE and SPE to decrease FAS and GAPT expression may occur through AMPK
295
activation and SREBP-1 suppression.
296
Our data showed that the expression of SREBP-1 and its
29
Consequently, our data suggest the ability of
In addition to increase hepatic lipogenesis by activation of SREBP-1 may
35
297
contribute to the development of chemical-induced fatty liver.
298
phosphorylates and inhibits SREBP-2 activity to attenuate hepatic steatosis, 36 whereas
299
SREBP-2 primarily controls cholesterol homeostasis by activating genes required for
300
cholesterol synthesis and uptake.
301
Fig.7). Similarly, AMPK inhibits in vitro lipogenesis in hepatocytes through the
302
downregulation of the cleavage processing and transcriptional activity of SREBP.
303
PPAR-α is highly expressed in the liver where it activates genes involved in
304
β-oxidation of fatty acids.
305
increased expression of PPAR-α in OA-induced lipid accumulation cells.
38
37
AMPK
Our data corroborated these results (Fig.5 and
36
In our study, both SWE and SPE treatment result in an
306
In conclusion, the current study identifies that SWE and SPE can reduce lipid
307
accumulation. We also propose that AMPK is pivotal in closing the anabolic pathway
308
and promoting catabolism by down regulating the activity of key enzymes in lipid
309
metabolism, such as, HMGCoR and FAS. Both SWE and SPE can suppress fat
310
accumulation of the liver and could be developed as a potential therapeutic treatment in
311
order to reduce the formation of a fatty liver.
312
313
AKNOWLEDGMENTS:
314
This
work
was
supported
by
a
14
National
Science
Council
Grant
315
(NSC99-2321-B-040-001), Taiwan.
316
317
CONFLICTS OF INTEREST
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No potential conflicts of interest were disclosed.
319
320
REFERENCES:
321
1.
322
323
biochemical, metabolic, and clinical implications. Hepatology 2010, 51, 679-89.
2.
324
325
Fabbrini, E.; Sullivan, S.; Klein, S., Obesity and nonalcoholic fatty liver disease:
Evans, R. M.; Barish, G. D.; Wang, Y. X., PPARs and the complex journey to
obesity. Nature Medicine 2004, 10, 355-61.
3.
Brooks, S. C., 3rd; Brooks, J. S.; Lee, W. H.; Lee, M. G.; Kim, S. G.,
326
Therapeutic potential of dithiolethiones for hepatic diseases. Pharmacology &
327
Therapeutics 2009, 124, 31-43.
328
4.
Wu, C. H.; Lin, M. C.; Wang, H. C.; Yang, M. Y.; Jou, M. J.; Wang, C. J.,
329
Rutin inhibits oleic acid induced lipid accumulation via reducing lipogenesis
330
and oxidative stress in hepatocarcinoma cells. Journal of Food Science 2011, 76,
331
T65-72.
332
5.
Corrao, G.; Torchio, P.; Zambon, A.; D'Amicis, A.; Lepore, A. R.; di Orio, F.,
333
Alcohol consumption and micronutrient intake as risk factors for liver cirrhosis:
334
a case-control study. The Provincial Group for the study of Chronic Liver
335
Disease. Annals of Epidemiology 1998, 8, 154-9.
336
6.
Musso, G.; Gambino, R.; Cassader, M.; Pagano, G., A meta-analysis of
337
randomized trials for the treatment of nonalcoholic fatty liver disease.
338
Hepatology 2010, 52, 79-104.
339
7.
Hartz, A. J.; Rupley, D. C., Jr.; Kalkhoff, R. D.; Rimm, A. A., Relationship of
15
340
obesity to diabetes: influence of obesity level and body fat distribution.
341
Preventive Medicine 1983, 12, 351-7.
342
8.
Chan, D. C.; Watts, G. F.; Ng, T. W.; Hua, J.; Song, S.; Barrett, P. H.,
343
Measurement of liver fat by magnetic resonance imaging: Relationships with
344
body fat distribution, insulin sensitivity and plasma lipids in healthy men.
345
Diabetes, Obesity & Metabolism 2006, 8, 698-702.
346
9.
347
348
Duval, C.; Muller, M.; Kersten, S., PPARalpha and dyslipidemia. Biochimica et
Biophysica Acta 2007, 1771, 961-71.
10.
Heemers, H.; Maes, B.; Foufelle, F.; Heyns, W.; Verhoeven, G.; Swinnen, J. V.,
349
Androgens stimulate lipogenic gene expression in prostate cancer cells by
350
activation of the sterol regulatory element-binding protein cleavage activating
351
protein/sterol
352
Endocrinology 2001, 15, 1817-28.
353
11.
regulatory
element-binding
protein
pathway.
Molecular
Reddy, J. K.; Rao, M. S., Lipid metabolism and liver inflammation. II. Fatty
354
liver disease and fatty acid oxidation. American Journal of Physiology.
355
Gastrointestinal and Liver Physiology 2006, 290, G852-8.
356
12.
Magana, M. M.; Koo, S. H.; Towle, H. C.; Osborne, T. F., Different sterol
357
regulatory element-binding protein-1 isoforms utilize distinct co-regulatory
358
factors to activate the promoter for fatty acid synthase. The Journal of
359
Biological Chemistry 2000, 275, 4726-33.
360
13.
Magana, M. M.; Osborne, T. F., Two tandem binding sites for sterol regulatory
361
element binding proteins are required for sterol regulation of fatty-acid synthase
362
promoter. The Journal of Biological Chemistry 1996, 271, 32689-94.
363
14. Matsusue, K.; Haluzik, M.; Lambert, G.; Yim, S. H.; Gavrilova, O.; Ward, J. M.;
364
Brewer, B., Jr.; Reitman, M. L.; Gonzalez, F. J., Liver-specific disruption of
16
365
PPARgamma in leptin-deficient mice improves fatty liver but aggravates
366
diabetic phenotypes. The Journal of Clinical Investigation 2003, 111, 737-47.
367
15.
Shimano, H.; Horton, J. D.; Shimomura, I.; Hammer, R. E.; Brown, M. S.;
368
Goldstein, J. L., Isoform 1c of sterol regulatory element binding protein is less
369
active than isoform 1a in livers of transgenic mice and in cultured cells. The
370
Journal of Clinical Investigation 1997, 99, 846-54.
371
16.
Engelking, L. J.; Kuriyama, H.; Hammer, R. E.; Horton, J. D.; Brown, M. S.;
372
Goldstein, J. L.; Liang, G., Overexpression of Insig-1 in the livers of transgenic
373
mice inhibits SREBP processing and reduces insulin-stimulated lipogenesis. The
374
Journal of Clinical Investigation 2004, 113, 1168-75.
375
17.
Yahagi, N.; Shimano, H.; Hasty, A. H.; Matsuzaka, T.; Ide, T.; Yoshikawa, T.;
376
Amemiya-Kudo, M.; Tomita, S.; Okazaki, H.; Tamura, Y.; Iizuka, Y.; Ohashi,
377
K.; Osuga, J.; Harada, K.; Gotoda, T.; Nagai, R.; Ishibashi, S.; Yamada, N.,
378
Absence of sterol regulatory element-binding protein-1 (SREBP-1) ameliorates
379
fatty livers but not obesity or insulin resistance in Lep(ob)/Lep(ob) mice. The
380
Journal of Biological Chemistry 2002, 277, 19353-7.
381
18.
Briggs, M. R.; Yokoyama, C.; Wang, X.; Brown, M. S.; Goldstein, J. L.,
382
Nuclear protein that binds sterol regulatory element of low density lipoprotein
383
receptor promoter. I. Identification of the protein and delineation of its target
384
nucleotide sequence. The Journal of Biological Chemistry 1993, 268, 14490-6.
385
19.
Schoonjans, K.; Watanabe, M.; Suzuki, H.; Mahfoudi, A.; Krey, G.; Wahli, W.;
386
Grimaldi, P.; Staels, B.; Yamamoto, T.; Auwerx, J., Induction of the
387
acyl-coenzyme A synthetase gene by fibrates and fatty acids is mediated by a
388
peroxisome proliferator response element in the C promoter. The Journal of
389
Biological Chemistry 1995, 270, 19269-76.
17
390
20.
Zhou, G.; Myers, R.; Li, Y.; Chen, Y.; Shen, X.; Fenyk-Melody, J.; Wu, M.;
391
Ventre, J.; Doebber, T.; Fujii, N.; Musi, N.; Hirshman, M. F.; Goodyear, L. J.;
392
Moller, D. E., Role of AMP-activated protein kinase in mechanism of
393
metformin action. The Journal of Clinical Investigation 2001, 108, 1167-74.
394
21.
Oliaro-Bosso, S.; Calcio Gaudino, E.; Mantegna, S.; Giraudo, E.; Meda, C.;
395
Viola, F.; Cravotto, G., Regulation of HMGCoA reductase activity by
396
policosanol and octacosadienol, a new synthetic analogue of octacosanol. Lipids
397
2009, 44, 907-16.
398
22.
Chandrasekaran C. V. ; Vijayalakshmi M. A. ; Prakash K. ; Bansal V. S. ;
399
Meenakshi J.; A., A., Review Article: Herbal Approach for Obesity
400
Management. American Journal of Plant Sciences 2012, 3, 1003-14.
401
23.
402
403
Wellen, K. E.; Hotamisligil, G. S., Inflammation, stress, and diabetes. The
Journal of Clinical Investigation 2005, 115, 1111-9.
24.
Cousin, S. P.; Hugl, S. R.; Wrede, C. E.; Kajio, H.; Myers, M. G., Jr.; Rhodes,
404
C. J., Free fatty acid-induced inhibition of glucose and insulin-like growth
405
factor I-induced deoxyribonucleic acid synthesis in the pancreatic beta-cell line
406
INS-1. Endocrinology 2001, 142, 229-40.
407
25.
Kong, W.; Wei, J.; Abidi, P.; Lin, M.; Inaba, S.; Li, C.; Wang, Y.; Wang, Z.; Si,
408
S.; Pan, H.; Wang, S.; Wu, J.; Li, Z.; Liu, J.; Jiang, J. D., Berberine is a novel
409
cholesterol-lowering drug working through a unique mechanism distinct from
410
statins. Nature Medicine 2004, 10, 1344-51.
411
26.
Lu, N.; Li, Y.; Qin, H.; Zhang, Y. L.; Sun, C. H., Gossypin up-regulates LDL
412
receptor through activation of ERK pathway: a signaling mechanism for the
413
hypocholesterolemic effect. Journal of Agricultural and Food Chemistry 2008,
414
56, 11526-32.
18
415
27.
Zhang, S.; Zheng, L.; Dong, D.; Xu, L.; Yin, L.; Qi, Y.; Han, X.; Lin, Y.; Liu,
416
K.; Peng, J., Effects of flavonoids from Rosa laevigata Michx fruit against
417
high-fat diet-induced non-alcoholic fatty liver disease in rats. Food Chemistry
418
2013, 141, 2108-16.
419
28.
420
421
Carling, D., The AMP-activated protein kinase cascade--a unifying system for
energy control. Trends in Biochemical Sciences 2004, 29, 18-24.
29.
Kemp, B. E.; Stapleton, D.; Campbell, D. J.; Chen, Z. P.; Murthy, S.; Walter,
422
M.; Gupta, A.; Adams, J. J.; Katsis, F.; van Denderen, B.; Jennings, I. G.; Iseli,
423
T.; Michell, B. J.; Witters, L. A., AMP-activated protein kinase, super metabolic
424
regulator. Biochemical Society Transactions 2003, 31, 162-8.
425
30.
Hwang, J. T.; Park, I. J.; Shin, J. I.; Lee, Y. K.; Lee, S. K.; Baik, H. W.; Ha, J.;
426
Park, O. J., Genistein, EGCG, and capsaicin inhibit adipocyte differentiation
427
process via activating AMP-activated protein kinase. Biochemical and
428
Biophysical Research Communications 2005, 338, 694-9.
429
31.
Auger, C.; Teissedre, P. L.; Gerain, P.; Lequeux, N.; Bornet, A.; Serisier, S.;
430
Besancon, P.; Caporiccio, B.; Cristol, J. P.; Rouanet, J. M., Dietary wine
431
phenolics
432
hypercholesterolemic hamsters against aortic fatty streak accumulation. Journal
433
of Agricultural and Food Chemistry 2005, 53, 2015-21.
434
32.
435
436
catechin,
quercetin,
and
resveratrol
efficiently
protect
Flores, E. M., [The chayote, Sechium edule Swartz (Cucurbitaceae)]. Revista de
Biologia tropical 1989, 37 Suppl 1, 1-54.
33.
Ribeiro Rde, A.; de Barros, F.; de Melo, M. M.; Muniz, C.; Chieia, S.;
437
Wanderley, M. d. G.; Gomes, C.; Trolin, G., Acute diuretic effects in conscious
438
rats produced by some medicinal plants used in the state of Sao Paulo, Brasil.
439
Journal of Ethnopharmacology 1988, 24, 19-29.
19
440
34.
Winder, W. W.; Hardie, D. G., AMP-activated protein kinase, a metabolic
441
master switch: possible roles in type 2 diabetes. The American Journal of
442
Physiology 1999, 277, E1-10.
443
35.
You, M.; Fischer, M.; Deeg, M. A.; Crabb, D. W., Ethanol induces fatty acid
444
synthesis pathways by activation of sterol regulatory element-binding protein
445
(SREBP). The Journal of Biological Chemistry 2002, 277, 29342-7.
446
36.
Li, Y.; Xu, S.; Mihaylova, M. M.; Zheng, B.; Hou, X.; Jiang, B.; Park, O.; Luo,
447
Z.; Lefai, E.; Shyy, J. Y.; Gao, B.; Wierzbicki, M.; Verbeuren, T. J.; Shaw, R. J.;
448
Cohen, R. A.; Zang, M., AMPK phosphorylates and inhibits SREBP activity to
449
attenuate hepatic steatosis and atherosclerosis in diet-induced insulin-resistant
450
mice. Cell Metabolism 2011, 13, 376-88.
451
37.
Jung, E. J.; Kwon, S. W.; Jung, B. H.; Oh, S. H.; Lee, B. H., Role of the
452
AMPK/SREBP-1 pathway in the development of orotic acid-induced fatty liver.
453
Journal of Lipid Research 2011, 52, 1617-25.
454
455
38.
Rakhshandehroo, M.; Knoch, B.; Muller, M.; Kersten, S., Peroxisome
proliferator-activated receptor alpha target genes. PPAR Research 2010, 2010.
456
457
458
FIGURE CAPTION
459
Figure 1. The HPLC chromatogram of SPE (polyphenol extracts of Sechium
460
edule shoots). (A) HPLC chromatogram of free polyphenols from SPE (10 mg/mL,
461
10 μL). (B) HPLC chromatogram of eight kinds of standard polyphenols (1 mg/mL;
462
10 μL). Peaks: 1, gallic acid; 2, protocatechuic acid; 3, catechin; 4, gallocatechin
463
gallate; 5, caffeic acid; 6, rutin; 7, quercetin; 8, naringenin.
464
20
465
Figure 2. The cytotoxicity effects of SWE (water extracts of Sechium edule shoots)
466
and
467
hepatocarcinoma cell line. HepG2 cells were treated with various concentrations of
468
SWE (A) or SPE (B) for 24 hrs. Viability of HepG2 cells was determined by the MTT
469
assay. The results are presented as mean ± SD of two independent experiments.
SPE
(polyphenol
extracts
of Sechium
edule
shoots) on
human
470
471
Figure 3. Effects of SWE (water extracts of Sechium edule shoots) or SPE
472
(polyphenol extracts of Sechium edule shoots) on intracellular lipid accumulation
473
in HepG2 cells. Cells are cotreated with oleic acid (OA) 0.6 mM and various
474
concentrations of SWE or SPE for 24 hr. (A) After culturing, cells were fixed with
475
formalin and stained with nile red and (B) analyzed by flow cytometry. (C)
476
Quantitative assessment of the percentage of lipid accumulation and represents the
477
average of three independent experiments ± SD. SC, as an internal control of cell
478
stained with nile red. 0, as an induced control of cell treated with oleic acid only.
479
#p<0.05 compared with the SC group. *p<0.05 compared with the OA-induced group.
480
481
Figure 4. Treatment of SWE (water extracts of Sechium edule shoots) and SPE
482
(polyphenol extracts of Sechium edule shoots) decreased fatty acid biosynthesis
483
relative protein expression in OA (oleic acid)-induced HepG2 cell.
484
Cells were coexposed to OA (0.6 mM) and various doses of SWE (A) or SPW (B) for
485
24 hr. The FAS, SREBP-1 and GPAT protein levels were also examined under the
486
same conditions. The numbers below the panels represent quantification of the
487
immunoblot by densitometry. #p<0.05 compared with a control group. *p<0.05
488
compared with OA-induced group.
489
21
490
Figure 5. Treatment of SWE (water extracts of Sechium edule shoots) and SPE
491
(polyphenol extracts of Sechium edule shoots) decreased cholesterol biosynthesis
492
relative protein expression in OA (oleic acid)-induced HepG2 cell. Cells were
493
coexposed to OA (0.6 mM) and various doses of SWE (A) or SPE (B) for 24 hr. The
494
HMGCoR, SREBP-2 and LDLR protein levels were also examined under the same
495
conditions. The numbers below the panels represent quantification of the immunoblot
496
by densitometry. #p<0.05 compared with control group. *p<0.05 compared with
497
OA-induced group.
498
499
Figure 6. Treatment of SWE (water extracts of Sechium edule shoots) and SPE
500
(polyphenol extracts of Sechium edule shoots) increased fatty acid oxidation
501
relative protein expression in OA (oleic acid)-induced HepG2 cell. Cells were
502
coexposed to OA (0.6 mM) and various doses of SWE (A) or SPE (B) for 24 hr.
503
CPT-1 and PPARα were detected by Western blot analysis under the same conditions.
504
The numbers below the panels represent quantification of the immunoblot by
505
densitometry. #p<0.05 compared with control group. *p<0.05 compared with
506
OA-induced group.
507
508
509
Figure 7. Treatment of SWE (water extracts of Sechium edule shoots) and SPE
510
(polyphenol extracts of Sechium edule shoots) increased AMP-activated protein
511
kinase (AMPK) phosphorylation protein expression in OA (oleic acid)-induced
512
HepG2 cell. Cells were coexposed to OA (0.6 mM) and various doses of SWE (A) or
513
SPE (B) for 24 hr. AMPK phosphorylation (pThr172-AMPK) was detected by
514
Western blot analysis under the same conditions. The numbers below the panels
22
515
represent quantification of the immunoblot by densitometry. *p<0.05 compared with
516
OA-induced group.
23
517
Table 1. Components of SWE and SPE (polyphenol extracts of Sechium edule shoots).
SWE (%)
SPE (%)
Polyphenol (Gallic acid as STD)
4.41 ± 0.02
17.74 ± 0.05
Flavonoid
3.32 ± 0.17
21.10 ± 0.28
Flavone & Flavonol
1.54 ± 0.07
4.52 ± 0.07
Flavanone & Flavanonol
1.78 ± 0.10
16.58 ± 0.21
Carbohydrate
26.73 ± 2.18
Protein
4.67 ± 1.46
Lipid
3.25 ± 1.11
24
518
Figure 1
519
520
(A)
521
522
523
524
(B)
525
526
527
528
529
530
531
25
532
Figure 2
533
(A)
534
100
535
536
538
539
540
cell survival (%)
537
80
60
40
IC 50 > 5 mg/ml
541
20
542
0
543
0.0
0.5
544
545
1.0
2.0
3.0
5.0
2.0
3.0
SWE (mg/ml)
(B)
546
547
120
548
100
549
551
552
553
cell survival (%)
550
80
60
40
20
IC 50 = 2.32 mg/ml
554
0
555
556
0.0
0.1
0.5
1.0
SPE26(mg/ml)
557
Figure 3
558
(A)
559
560
561
562
563
564
565
566
567
568
569
570
571
572
573
574
575
576
577
578
579
580
581
582
27
583
(B)
584
585
586
587
588
589
590
591
592
593
594
595
596
597
(C)
598
599
600
601
28
602
Figure 4
603
(A)
604
605
1.6
SREBP-1 /  actin (fold of control)
606
607
608
609
#
1.4
*
1.2
1.0
0.8
0.6
0.4
0.2
610
0.0
C
0.0
611
1.0
5.0
OA + SWE (mg/ml)
612
613
616
617
618
*
1.2
1.0
0.8
0.6
0.4
0.2
1.2
1.0
0.8
0.6
0.4
0.2
0.0
619
#
1.4
GPAT /  actin (fold of control)
615
#
1.4
FAS /  actin (fold of control)
614
1.6
1.6
0.0
C
0.0
1.0
5.0
C
0.0
1.0
OA + SWE (mg/ml)
OA + SWE (mg/ml)
620
621
622
29
5.0
623
Figure 4
624
(B)
625
626
1.8
1.6
GPAT /  actin (fold of control)
627
628
629
630
#
1.4
*
1.2
*
1.0
0.8
0.6
0.4
0.2
0.0
C
0.0
631
632
1.8
636
1.4
*
1.2
*
1.0
0.8
0.6
0.4
0.2
*
1.4
*
1.2
1.0
0.8
0.6
0.4
0.2
0.0
637
#
1.6
#
SREBP-1 /  actin (fold of control)
635
FAS /  actin (fold of control)
634
1.0
1.8
1.6
633
0.5
OA + SPE (mg/ml)
0.0
C
0.0
0.5
1.0
C
OA + SPE (mg/ml)
0.0
0.5
OA + SPE (mg/ml)
638
639
640
641
30
1.0
642
Figure 5
643
(A)
644
645
1.8
646
LDLR /  actin (fold of control)
1.6
647
648
649
#
*
1.4
1.2
1.0
0.8
0.6
0.4
0.2
650
0.0
C
0.0
1.0
5.0
OA + SWE (mg/ml)
651
652
1.6
1.6
655
656
HMGCR /  actin (fold of control)
654
SREBP-2 /  actin (fold of control)
#
1.4
653
*
1.2
1.0
0.8
0.6
0.4
0.2
1.4
#
1.2
1.0
0.8
0.6
0.4
0.2
0.0
C
657
0.0
1.0
5.0
0.0
C
OA + SWE (mg/ml)
0.0
1.0
OA + SWE (mg/ml)
658
659
660
31
5.0
661
Figure 5
662
(B)
663
664
1.8
665
#
LDLR /  actin (fold of control)
1.6
666
667
668
*
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
669
C
0.0
0.5
1.0
OA + SPE (mg/ml)
670
671
1.8
1.6
674
675
1.2
*
1.0
0.8
0.6
0.4
0.2
#
1.4
*
1.2
*
1.0
0.8
0.6
0.4
0.2
0.0
C
676
SREBP-2 /  actin (fold of control)
673
HMGCR /  actin (fold of control)
672
1.6
#
1.4
0.0
0.5
0.0
1.0
C
OA + SPE (mg/ml)
0.0
0.5
OA + SPE (mg/ml)
677
678
32
1.0
679
Figure 6
680
(A)
681
682
683
684
685
686
687
688
1.8
689
692
693
*
1.4
PPAR /  actin (fold of control)
691
CPT-I /  actin (fold of control)
690
2.0
1.6
1.2
1.0
0.8
0.6
0.4
0.2
#
1.5
*
*
1.0
0.5
0.0
C
694
0.0
1.0
5.0
0.0
C
0.0
1.0
OA + SWE (mg/ml)
OA + SWE (mg/ml)
695
696
697
33
5.0
698
Figure 6
699
(B)
700
701
702
703
704
705
706
707
708
1.6
711
712
PPAR /  actin (fold of control)
710
CPT-I /  actin (fold of control)
709
3.0
*
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
C
713
0.0
0.5
*
2.5
2.0
#
1.5
1.0
0.5
0.0
1.0
C
OA + SPE (mg/ml)
0.0
0.5
OA + SPE (mg/ml)
714
715
716
717
718
34
1.0
719
Figure 7
720
(A)
721
722
723
724
725
726
727
728
729
731
732
733
1.6
pAMPK / AMPK (fold of control)
730
1.8
*
*
1.4
1.2
1.0
0.8
0.6
0.4
0.2
734
0.0
C
0.0
1.0
OA + SWE (mg/ml)
735
736
737
738
739
35
5.0
740
Figure 7
741
(B)
742
743
744
745
746
747
748
1.8
749
751
pAMPK / AMPK (fold of control)
750
1.6
*
*
0.5
1.0
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
C
0.0
OA + SPE (mg/ml)
36