Article pubs.acs.org/JAFC
Brown Alga Ecklonia cava Polyphenol Extract Ameliorates Hepatic Lipogenesis, Oxidative Stress, and Inflammation by Activation of AMPK and SIRT1 in High-Fat Diet-Induced Obese Mice Hyeyoon Eo,† You-jin Jeon,§ Myoungsook Lee,# and Yunsook Lim*,† †
Department of Food and Nutrition, Kyung Hee University, 26 Kyunghee-daero, Dongdaemun-gu, Seoul 130-701, Republic of Korea Department of Marine Life Sciences, Jeju National University, Jeju 690-756, Republic of Korea # Department of Food and Nutrition, Sungshin Women’s University and Research Institute of Obesity Sciences, Sungshin Women’s University, Seoul 136-742, Republic of Korea §
ABSTRACT: Obesity is considered to be a metaflammatory condition. Ecklonia cava, brown algae rich in polyphenols, has shown strong antioxidant activity in vitro. This study investigated the effect of E. cava polyphenol extract (ECPE) on the regulation of fat metabolism, inflammation, and the antioxidant defense system in high fat diet-induced obese mice. After obesity was induced by a high-fat diet (HFD), the mice were administered ECPE by gavage for 5 days/12 weeks. ECPE supplementation reduced body weight gain, adipose tissue mass, plasma lipid profiles, hepatic fat deposition, insulin resistance, and the plasma leptin/adiponectin ratio derived from HFD-induced obesity. Moreover, ECPE supplementation selectively ameliorated hepatic protein levels associated with lipogenesis, inflammation, and the antioxidant defense system as well as activation of AMPK and SIRT1. Collectively, ECPE supplement might have potential antiobesity effects via regulation of AMPK and SIRT1 in HFDinduced obesity. KEYWORDS: Ecklonia cava polyphenol extract, obesity, AMPK, SIRT1, hepatic lipid metabolism, inflammation
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INTRODUCTION Recently, obesity has been recognized as a chronic metabolic disorder.1 Under an obese condition, there are several abnormalities in lipid metabolism, the antioxidant defense system, and inflammatory responses. As the storage of triglyceride (TG) in adipose tissue exceeds its limit, it is likely to encourage the release of free fatty acids (FFA) into circulation, and released FFA could be brought into nonadipose tissues such as the liver.2 Obese subjects might undergo nonalcoholic fatty liver disease (NAFLD) characterized by an increase in fat accumulation in the liver caused by abnormalities, including reduced fatty acid (FA) oxidation or TG secretion and increased FA uptake or de novo lipogenesis.3 Moreover, the TG overload in liver could cause hepatic damage and increase reactive oxygen species (ROS), which induce lipid peroxidation and inflammation.4 At the molecular level, AMP-activated protein kinase (AMPK) and Sirtuin 1 (SIRT1) are regarded as key fuel sensors or metabolic modulators. It is well-documented in detail that AMPK plays a key role in hepatic lipid metabolism, through inactivation of several metabolic enzymes, in switching on catabolic pathways such as β-oxidation as well as switching off anabolic pathways involving lipid metabolism in the liver.5,6 SIRT1, an NAD+-dependent protein deacetylase, is involved in several metabolic pathways in various tissues.7 Especially, SIRT1 is involved in lipid and glucose metabolism, inflammatory response, and oxidative stress in hepatic tissue.8 Furthermore, it has been reported that SIRT1 modulates fatty acid β-oxidation and cholesterol/bile homeostasis through the peroxisome proliferator-activated receptor alpha (PPARα)/ peroxisome proliferator-activated receptor gamma coactivator 1 © 2014 American Chemical Society
(PGC1) pathway and liver X receptor (LXR)/farnesoid X receptor (FXR) signaling, respectively.9,10 SIRT1 might also regulate lipid metabolism by deacetylation of sterol regulatory element binding proteins (SREBPs), which are pivotal modulators of lipogenesis and cholesterolgenesis11,12 and inflammatory responses such as tumor necrosis factor alpha (TNFα) and interleukin 1 beta (IL-1β).9 Thereby, inactive or reduced levels of SIRT1 increased susceptibility to dyslipidemia, hepatic steatosis, inflammation, and endoplasmic reticulum stress.7,9 Recently, a partnership between AMPK and SIRT1 has been well-documented in a previous study.13 Briefly, when SIRT1 is stimulated by certain activators such as NAD+, SIRT1 regulates AMPK by deacetylation of liver kinase B1 (LKB1). According to previous studies, the mechanism provides a comprehensive explanation in hepatic lipid metabolism and NAFLD by suggesting an SIRT1/AMPK cycle.14 Therefore, partnership between AMPK and SIRT1 could be considered as a crucial target to regulate hepatic lipid metabolism related to NAFLD in obesity. Brown alga Ecklonia cava is produced in bountiful amounts on the coast of Jeju Island, Korea. E. cava contains copious polysaccharides such as fucoidans as well as polyphenols including dieckol, eckol, phloroglucinol, 6,6′-bieckol, and eckstolonol.15 In the previous study, E. cava ethanolic extracts suppressed the lipopolysaccharide (LPS)-induced cytokines such as TNFα and IL-1β and reduced the inducible nitric oxide Received: Revised: Accepted: Published: 349
June 17, 2014 November 14, 2014 December 5, 2014 December 5, 2014 dx.doi.org/10.1021/jf502830b | J. Agric. Food Chem. 2015, 63, 349−359
Journal of Agricultural and Food Chemistry
Article
99.9, 99.7, 99.5, 99.4, 97.6, 96.2, 95.8, 95.7, 95.3; ESI-MS [M − H]− at m/z 741.11. 2,7″-Phloroglucinol-6,6′-bieckol: amorphous powder; 1H NMR (400 MHz, methanol-d4) δ 5.57 (1H, s), 5.89 (1H, s), 5.74 (1H, m), 5.84 (1H, m), 5.74 (1H, m), 6.25 (1H, s), 6.14 (1H, s), 5.84 (1H, m), 5.89 (1H, m), 5.84 (1H, m), 6.52 (1H, s), 6.14 (1H, m), 6.44 (1H, m), 6.77 (1H, s), 6.72 (1H, s), 8.93 (1H, s), 8.93 (1H, s), 9.19 (1H, s), 9.19 (1H, s), 9.19 (1H, s), 9.04 (1H, s), 8.26 (1H, s), 9.94 (1H, s), 8.59 (1H, s), 9.88 (1H, s), 9.86 (1H, s), 9.25 (1H, s), 9.75 (1H, s), 9.21 (1H, s); 13C NMR (100 MHz, methanol-d6) δ 127.6, 143.0, 93.0, 137.1, 125.6, 147.2, 106.5, 152.2, 95.5, 152.4, 127.6, 137.1, 162.0, 98.7, 160.3, 95.5, 160.3, 98.8, 124.3, 147.2, 94.5, 144.1, 124.3, 147.2, 110.0, 144.1, 101.5, 151.8, 137.2, 144.1, 159.7, 96.7, 157.1, 95.5, 157.1, 96.7, 159.8, 97.8, 159.3, 95.2, 159.2, 97.9, 122.5, 153.9, 99.8, 156.8, 99.9, 152.8; ESI-MS [M − H]− at m/z 973.37. Pyrogallol-phloroglucinol-6,6′-bieckol: amorphous powder; 1H NMR (400 MHz, methanol-d4) δ 6.10 (1H, s), 5.99 (1H, s), 5.72 (1H, m), 5.75 (1H, m), 5.72 (1H, m), 6.25 (1H, s), 6.14 (1H, s), 5.88 (1H, δ, 2.21 Hz), 5.88 (1H, δ, 2.21 Hz), 5.85 (1H, δ, 2.21 Hz), 6.72 (1H, δ, 2.2 Hz), 6.08 (1H, δ, 2.2 Hz), 5.89 (1H, δ, 2.01 Hz), 5.54 (1H, δ, 2.01 Hz), 5.89 (1H, δ, 2.01 Hz), 9.116 (1H, s), 9.03 (1H, s), 8.92 (1H, s), 9.27 (1H, s), 9.20 (1H, s), 9.20 (1H, s), 9.18 (1H, s), 9.03 (1H, s), 8.92 (1H, s), 8.25 (1H, s), 9.94 (1H, s), 9.87 (1H, s), 9.87 (1H, s), 9.20 (1H, s), 9.20 (1H, s); 13C NMR (100 MHz, methanold4) δ 125.2, 145.9, 95.5, 145.9, 125.1, 147.9, 105.5, 148.4, 95.4, 151.9, 127.8, 135.3, 161.9, 97.9, 160.3, 95.5, 160.3, 97.8, 124.4, 144.4, 94.4, 144.4, 124.5, 147.9, 105.4, 148.4, 95.5, 151.4, 127.8, 138.3, 159.2, 100.2, 156.9, 99.5, 159.3, 96.3, 154.0, 96.6, 152.1, 122.5, 153.5, 96.6, 159.9, 99.0, 159.8, 94.6, 159.8, 98.6; ESI-MS [M − H]− at m/z 973.03. Phlorofucofuroeckol-A: amorphous powder; 1H NMR (400 MHz, methanol-d4) δ 6.63 (1H, s, H-7), 6.40 (1H, s, H-11), 6.26 (1H, s, H2), 5.97 (2H, δ, J = 2.1 Hz, H-200, 600), 5.94 (1H, t, J = 1.9 Hz, H40), 5.92 (1H, t, J = 2.0 Hz, H-400), 5.88 (2H, δ, J = 2.1 Hz, H-20, 60); 13C NMR (100 MHz, CD3OD) δ 162.7, 162.6, 161.0, 161.0, 154.0, 152.5, 152.0, 149.1, 149.0, 146.7, 144.7, 139.2, 136.2, 128.9, 125.9, 125.6, 123.2, 106.2, 106.1, 100.8, 100.2, 98.6, 98.5, 97.0, 96.2, 96.2; ESI-MS [M − H]− at m/z 601.36. Animals and Experimental Design. Male C57BL/6 mice (n = 40; 4 weeks old) were obtained from Orient Bio (Gyeonggi-do, South Korea) and were housed two or three per cage in a room (temperature, 21−23 °C; relative humidity, 45−55%; 12 h dark/ light cycle). After acclimation for 1 week, mice were then randomly divided into two groups: the normal control diet group (CD; n = 10) and the high-fat diet group (HFD; n = 30). CD was provided with regular diet (D12450B, 10% kcal fat, Research Diets, New Brunswick, NJ, USA), whereas HFD was provided with high-fat diet (D12451, 45% kcal fat, Research Diets) for 10 weeks. Food and distilled water were supplied ad libitum. After obesity was induced by the high-fat diet, the animals were divided into four groups, 10 mice in each group and supplemented as follows: CON, nonobese control group mice fed CD were gavaged with distilled water; HF, obese mice fed HFD were gavaged with distilled water; LE, obese mice fed HFD were gavaged ECPE at a (low) dose of 100 mg/kg/day; HE, obese mice fed HFD were gavaged with ECPE at a (high) dose of 500 mg/kg/day. The identical volume of gavage with either distilled water or ECPE was administrated by oral gavage 5 times a week for 12 weeks. ECPE was freshly suspended in distilled water. The body weight and food intake were measured twice a week. Mice were fasted for 15 h after the last treatment and then euthanized by inhalation with isoflurane (Baxter, Deerfield, IL, USA). Blood was collected by cardiac puncture using a heparin (SigmaAldrich, St. Louis, MO, USA) coated syringe. Plasma was obtained by centrifugation at 3000 rpm for 15 min. Tissues such as the liver and adipose pads were removed after cardiac puncture, washed of blood or foreign materials by saline, weighed, and immediately frozen in liquid nitrogen. All of the samples were stored at −80 °C until they were used for experiments. Experimental protocol was approved by the Institutional Animal Care and Use Committee of the Kyung Hee University.
synthase (iNOS) and the cyclooxygenase-2 (COX-2) expression by blocking transcription factors of the nuclear factor kappa B (NFκB) and the mitogen-activated protein kinase (MAPK) activation in BV2 microglia.16 Among the polyphenols of E. cava, dieckol is the most ample compound in E. cava, and it has been reported for having some beneficial potential such as antioxidative effects and antithrombotic activity.17,18 A recent study reported that dieckol, which was isolated from E. cava, inhibited adipogenesis by activating AMPK and suppressing adipocyte differentiation in 3T3-L1 preadipocytes.19 However, little research has focused on the molecular mechanisms by which E. cava polyphenol extract (ECPE) regulates hepatic lipid metabolism in an obese animal model. We investigated how ECPE ameliorates hepatic lipid metabolism, oxidative stress, and inflammation through modulation of AMPK and SIRT-1 in high-fat diet (HFD)induced obese mice.
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MATERIALS AND METHODS
Preparation of ECPE. ECPE used in this study was provided from Aqua Green Tech., Jeju, Korea. The preparation of dieckol-rich polyphenols from E. cava was described previously.20 In brief, the E. cava was collected from the coast of Jeju Island, Korea. The powdery E. cava (500 g) was suspended in 10 L of 70% ethanol and stirred for 24 h at room temperature. The solution was filtered and then concentrated under reduced pressure to give the oily extract. Then, the organic soluble fraction was taken by treatment with ethyl acetate (47.85 g), and the extract was evaporated under vacuum. Quantification of Total Phenol and Major Compounds in ECPE. The total phenol content in the polyphenol extract was measured by using the protocol described by Kim et al.21 The quantitative analysis in the major compounds in the polyphenol extract from E. cava was performed by their HPLC analysis and quantitative standard curve. The quantitative standard curves of the main compounds were calculated by their area (%) in high-performance liquid chromatography (HPLC) under concentrations between 25 and 400 μg/mL. Identification of Major Compounds in ECPE and Chemical Structure of Dieckol. The structural identification of the major compounds in the polyphenol extract from E. cava was performed by using the method described in a previous paper.22 The major compounds in the extract were confirmed by LC-DAD-ESI/MS and 1D NMR spectrum. To operate LC-DAD-ESI/MS, 10 μL of 5 mg/mL sample solution was directly injected on an Atlantis T3 column (3 μm 3.0 × 150 mm column) (Waters, Milford, MA, USA). The mobile phase comprised acetonitrile/water in gradient mode as follows: acetonitrile with 0.1% formic acid/water with 0.1% formic acid (0−40 min, 10:90−40:60 v/v; 40−50 min, ∼50:50 v/v; and 50−60 min, ∼100:0 v/v). The flow rate was 0.2 mL/min, and the UV absorbance was detected at 290 nm. The source voltage was set to 4.5 kV and the capillary temperature to 250 °C. The other conditions were as follows: capillary voltage, −36.5 V; interoctapole lens voltage, 10 V; sheath gas, 80 psi (551.6 kPa); auxiliary gas, 20 psi (137.9 kPa). The major compounds were confirmed as dieckol, 2,7″-phloroglucinol-6,6′bieckol, pyrogallol-phloroglucinol-6,6′-bieckol, and phlorofucofuroeckol-A, respectively. After purification of the compounds from the E. cava extract, their 1H NMR and 13C NMR spectra were measured with a JEOL JNM-LA 300 spectrometer (JEOL Ltd., Tokyo, Japan), and the mass spectra (FAB-MS and EIMS) were recorded on a JEOL JMS 700 spectrometer. Dieckol: amorphous powder; 1H NMR (400 MHz, methanol-d4) δ 6.15 (1H, s), 6.13 (1H, s), 6.09 (1H, δ, 2.9 Hz), 6.06 (1H, δ, 2.9 Hz), 6.05 (1H, δ, 2.9 Hz), 5.98 (1H, δ, 2.8 Hz), 5.95 (1H, δ, 2.8 Hz), 5.92 (3H, m); 13C NMR (100 MHz, methanol-d4) δ 161.8, 160.1, 157.8, 155.9, 154.5, 152.4, 147.3, 147.2, 147.1, 146.9, 144.3, 144.1, 143.4, 143.3, 138.6, 138.5, 126.5, 126.2, 125.6, 125.5, 124.9, 124.6, 124.5, 350
dx.doi.org/10.1021/jf502830b | J. Agric. Food Chem. 2015, 63, 349−359
Journal of Agricultural and Food Chemistry
Article
Figure 1. Quantification and identification of E. cava polyphenol extract (ECPE): (A) polyphenol contents in ECPE quantified by HPLC analysis; (B) electrospray ionization mass spectrometry (ESI/MS) of ECPE and chemical structure of dieckol. Intraperitoneal Glucose Tolerance Test (IPGTT). A week before the end point, the animals were fasted overnight for 16 h prior to the IPGTT test. The 50% glucose solution (2 g/kg) was injected intraperitoneally. Blood glucose level was measured with a glucometer (NOCODING1, Seoul, Korea) at 0 (as basal fasting blood glucose level, FBG), 15, 30, 60, 90, and 120 min after injection of glucose solution. The area under the curve (AUC) was calculated using the trapezoidal rule as follows: AUCi= [(blood glucose)i + (blood glucose)i−1] × [(time)i − (time)i−1]/2. Plasma Biomarker Analysis. The contents of plasma triglyceride (TG), total cholesterol (TC), and high-density lipoprotein cholesterol (HDL-C) were determined using commercial kits (Bio-Clinical
System, Gyeonggi-do, South Korea). The atherogenic index (AI) was estimated as follows: AI = ([TC] − [HDL-C])/[HDL-C]. Plasma glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) were determined by enzymatic assays with commercial kits (Bio-Clinical System). The plasma concentrations of leptin, adiponectin, and insulin were determined using enzyme-linked immuosorbent assay (ELISA) commercial kits (RayBiotech, Inc., Norcross, GA, USA). The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated as follows: fasting insulin (μU/ mL) × fasting glucose (mmol/L)/22.5. All of the commercial kits were performed according to the manufacturer’s instructions, respectively. 351
dx.doi.org/10.1021/jf502830b | J. Agric. Food Chem. 2015, 63, 349−359
Journal of Agricultural and Food Chemistry
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Table 1. Effects of ECPE on Body Weight, Food Intake, Liver Weight, and Adipose Tissue Weighta groupb body weight (g) before supplementation after supplementation gain food intake (g/day) food efficiency ratio (%) liver (g) total adipose tissue (g) visceral fat (g) epididymal mesenteric retroperitoneal subcutaneous fat (g) inguinal brown
CON
HF
LE
HE
30.35 ± 0.58 a 37.31 ± 0.80 a 6.95 ± 0.44 a 3.17 ± 0.06 c 2.84 ± 0.16 a 1.23 ± 0.07 ab 3.88 ± 0.28 a 2.71 ± 0.19 a 1.43 ± 0.10 a 0.66 ± 0.07 a 0.62 ± 0.03 a 1.17 ± 0.11 a 0.98 ± 0.07 a 0.20 ± 0.04 a
38.56 ± 1.09 b 48.51 ± 0.73 c 9.96 ± 0.79 b 2.84 ± 0.05 ab 4.59 ± 0.39 c 1.30 ± 0.13 ab 8.88 ± 0.48 c 5.65 ± 0.26 c 2.08 ± 0.09 c 1.94 ± 0.17 c 1.63 ± 0.08 c 3.24 ± 0.26 b 2.83 ± 0.25 b 0.41 ± 0.05 c
39.23 ± 0.74 b 48.07 ± 0.70 c 8.84 ± 0.65 ab 2.97 ± 0.05 b 3.87 ± 0.28 bc 1.48 ± 0.15 b 8.31 ± 0.35 bc 4.91 ± 0.13 b 1.77 ± 0.11 b 1.55 ± 0.11 b 1.59 ± 0.08 c 3.39 ± 0.29 b 3.04 ± 0.28 b 0.35 ± 0.04 bc
38.01 ± 0.61 b 44.96 ± 0.78 b 6.94 ± 0.62 a 2.78 ± 0.05 a 3.23 ± 0.26 ab 1.08 ± 0.05 a 7.67 ± 0.32 b 5.03 ± 0.15 b 2.34 ± 0.10 c 1.32 ± 0.10 b 1.37 ± 0.05 b 2.64 ± 0.23 b 2.36 ± 0.23 b 0.28 ± 0.03 ab
Values are means ± SEM. Mean values with unlike letters are significantly different (p < 0.05). bCON, mice fed the control diet (negative control); HF, mice fed the high-fat diet (positive control); LE, mice supplemented with low dose (100 mg/kg/day) of ECPE; HE, mice supplemented with high dose (500 mg/kg/day) of ECPE. a
acid−0.8% TBA mixture were heated in a water bath at 95 °C for 60 min. After cooling with tap water, distilled water and a mixture of nbutanol and pyridine (15:1 v/v) were added and then centrifuged at 4000 rpm for 10 min to obtain a clear supernatant. The absorbance of the supernatant was measured at 532 nm. Statistical Analysis. The results are expressed as means ± standard error of mean (SEM). Significant differences among the groups were determined by one-way ANOVA using SPSS (version 20.0 for Windows, SPSS Inc., Chicago, IL, USA). Duncan’s multiplerange tests was followed when differences were identified among the groups at p values of
Brown Alga Ecklonia cava Polyphenol Extract Ameliorates Hepatic Lipogenesis, Oxidative Stress, and Inflammation by Activation of AMPK and SIRT1 in High-Fat Diet-Induced Obese Mice Hyeyoon Eo,† You-jin Jeon,§ Myoungsook Lee,# and Yunsook Lim*,† †
Department of Food and Nutrition, Kyung Hee University, 26 Kyunghee-daero, Dongdaemun-gu, Seoul 130-701, Republic of Korea Department of Marine Life Sciences, Jeju National University, Jeju 690-756, Republic of Korea # Department of Food and Nutrition, Sungshin Women’s University and Research Institute of Obesity Sciences, Sungshin Women’s University, Seoul 136-742, Republic of Korea §
ABSTRACT: Obesity is considered to be a metaflammatory condition. Ecklonia cava, brown algae rich in polyphenols, has shown strong antioxidant activity in vitro. This study investigated the effect of E. cava polyphenol extract (ECPE) on the regulation of fat metabolism, inflammation, and the antioxidant defense system in high fat diet-induced obese mice. After obesity was induced by a high-fat diet (HFD), the mice were administered ECPE by gavage for 5 days/12 weeks. ECPE supplementation reduced body weight gain, adipose tissue mass, plasma lipid profiles, hepatic fat deposition, insulin resistance, and the plasma leptin/adiponectin ratio derived from HFD-induced obesity. Moreover, ECPE supplementation selectively ameliorated hepatic protein levels associated with lipogenesis, inflammation, and the antioxidant defense system as well as activation of AMPK and SIRT1. Collectively, ECPE supplement might have potential antiobesity effects via regulation of AMPK and SIRT1 in HFDinduced obesity. KEYWORDS: Ecklonia cava polyphenol extract, obesity, AMPK, SIRT1, hepatic lipid metabolism, inflammation
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INTRODUCTION Recently, obesity has been recognized as a chronic metabolic disorder.1 Under an obese condition, there are several abnormalities in lipid metabolism, the antioxidant defense system, and inflammatory responses. As the storage of triglyceride (TG) in adipose tissue exceeds its limit, it is likely to encourage the release of free fatty acids (FFA) into circulation, and released FFA could be brought into nonadipose tissues such as the liver.2 Obese subjects might undergo nonalcoholic fatty liver disease (NAFLD) characterized by an increase in fat accumulation in the liver caused by abnormalities, including reduced fatty acid (FA) oxidation or TG secretion and increased FA uptake or de novo lipogenesis.3 Moreover, the TG overload in liver could cause hepatic damage and increase reactive oxygen species (ROS), which induce lipid peroxidation and inflammation.4 At the molecular level, AMP-activated protein kinase (AMPK) and Sirtuin 1 (SIRT1) are regarded as key fuel sensors or metabolic modulators. It is well-documented in detail that AMPK plays a key role in hepatic lipid metabolism, through inactivation of several metabolic enzymes, in switching on catabolic pathways such as β-oxidation as well as switching off anabolic pathways involving lipid metabolism in the liver.5,6 SIRT1, an NAD+-dependent protein deacetylase, is involved in several metabolic pathways in various tissues.7 Especially, SIRT1 is involved in lipid and glucose metabolism, inflammatory response, and oxidative stress in hepatic tissue.8 Furthermore, it has been reported that SIRT1 modulates fatty acid β-oxidation and cholesterol/bile homeostasis through the peroxisome proliferator-activated receptor alpha (PPARα)/ peroxisome proliferator-activated receptor gamma coactivator 1 © 2014 American Chemical Society
(PGC1) pathway and liver X receptor (LXR)/farnesoid X receptor (FXR) signaling, respectively.9,10 SIRT1 might also regulate lipid metabolism by deacetylation of sterol regulatory element binding proteins (SREBPs), which are pivotal modulators of lipogenesis and cholesterolgenesis11,12 and inflammatory responses such as tumor necrosis factor alpha (TNFα) and interleukin 1 beta (IL-1β).9 Thereby, inactive or reduced levels of SIRT1 increased susceptibility to dyslipidemia, hepatic steatosis, inflammation, and endoplasmic reticulum stress.7,9 Recently, a partnership between AMPK and SIRT1 has been well-documented in a previous study.13 Briefly, when SIRT1 is stimulated by certain activators such as NAD+, SIRT1 regulates AMPK by deacetylation of liver kinase B1 (LKB1). According to previous studies, the mechanism provides a comprehensive explanation in hepatic lipid metabolism and NAFLD by suggesting an SIRT1/AMPK cycle.14 Therefore, partnership between AMPK and SIRT1 could be considered as a crucial target to regulate hepatic lipid metabolism related to NAFLD in obesity. Brown alga Ecklonia cava is produced in bountiful amounts on the coast of Jeju Island, Korea. E. cava contains copious polysaccharides such as fucoidans as well as polyphenols including dieckol, eckol, phloroglucinol, 6,6′-bieckol, and eckstolonol.15 In the previous study, E. cava ethanolic extracts suppressed the lipopolysaccharide (LPS)-induced cytokines such as TNFα and IL-1β and reduced the inducible nitric oxide Received: Revised: Accepted: Published: 349
June 17, 2014 November 14, 2014 December 5, 2014 December 5, 2014 dx.doi.org/10.1021/jf502830b | J. Agric. Food Chem. 2015, 63, 349−359
Journal of Agricultural and Food Chemistry
Article
99.9, 99.7, 99.5, 99.4, 97.6, 96.2, 95.8, 95.7, 95.3; ESI-MS [M − H]− at m/z 741.11. 2,7″-Phloroglucinol-6,6′-bieckol: amorphous powder; 1H NMR (400 MHz, methanol-d4) δ 5.57 (1H, s), 5.89 (1H, s), 5.74 (1H, m), 5.84 (1H, m), 5.74 (1H, m), 6.25 (1H, s), 6.14 (1H, s), 5.84 (1H, m), 5.89 (1H, m), 5.84 (1H, m), 6.52 (1H, s), 6.14 (1H, m), 6.44 (1H, m), 6.77 (1H, s), 6.72 (1H, s), 8.93 (1H, s), 8.93 (1H, s), 9.19 (1H, s), 9.19 (1H, s), 9.19 (1H, s), 9.04 (1H, s), 8.26 (1H, s), 9.94 (1H, s), 8.59 (1H, s), 9.88 (1H, s), 9.86 (1H, s), 9.25 (1H, s), 9.75 (1H, s), 9.21 (1H, s); 13C NMR (100 MHz, methanol-d6) δ 127.6, 143.0, 93.0, 137.1, 125.6, 147.2, 106.5, 152.2, 95.5, 152.4, 127.6, 137.1, 162.0, 98.7, 160.3, 95.5, 160.3, 98.8, 124.3, 147.2, 94.5, 144.1, 124.3, 147.2, 110.0, 144.1, 101.5, 151.8, 137.2, 144.1, 159.7, 96.7, 157.1, 95.5, 157.1, 96.7, 159.8, 97.8, 159.3, 95.2, 159.2, 97.9, 122.5, 153.9, 99.8, 156.8, 99.9, 152.8; ESI-MS [M − H]− at m/z 973.37. Pyrogallol-phloroglucinol-6,6′-bieckol: amorphous powder; 1H NMR (400 MHz, methanol-d4) δ 6.10 (1H, s), 5.99 (1H, s), 5.72 (1H, m), 5.75 (1H, m), 5.72 (1H, m), 6.25 (1H, s), 6.14 (1H, s), 5.88 (1H, δ, 2.21 Hz), 5.88 (1H, δ, 2.21 Hz), 5.85 (1H, δ, 2.21 Hz), 6.72 (1H, δ, 2.2 Hz), 6.08 (1H, δ, 2.2 Hz), 5.89 (1H, δ, 2.01 Hz), 5.54 (1H, δ, 2.01 Hz), 5.89 (1H, δ, 2.01 Hz), 9.116 (1H, s), 9.03 (1H, s), 8.92 (1H, s), 9.27 (1H, s), 9.20 (1H, s), 9.20 (1H, s), 9.18 (1H, s), 9.03 (1H, s), 8.92 (1H, s), 8.25 (1H, s), 9.94 (1H, s), 9.87 (1H, s), 9.87 (1H, s), 9.20 (1H, s), 9.20 (1H, s); 13C NMR (100 MHz, methanold4) δ 125.2, 145.9, 95.5, 145.9, 125.1, 147.9, 105.5, 148.4, 95.4, 151.9, 127.8, 135.3, 161.9, 97.9, 160.3, 95.5, 160.3, 97.8, 124.4, 144.4, 94.4, 144.4, 124.5, 147.9, 105.4, 148.4, 95.5, 151.4, 127.8, 138.3, 159.2, 100.2, 156.9, 99.5, 159.3, 96.3, 154.0, 96.6, 152.1, 122.5, 153.5, 96.6, 159.9, 99.0, 159.8, 94.6, 159.8, 98.6; ESI-MS [M − H]− at m/z 973.03. Phlorofucofuroeckol-A: amorphous powder; 1H NMR (400 MHz, methanol-d4) δ 6.63 (1H, s, H-7), 6.40 (1H, s, H-11), 6.26 (1H, s, H2), 5.97 (2H, δ, J = 2.1 Hz, H-200, 600), 5.94 (1H, t, J = 1.9 Hz, H40), 5.92 (1H, t, J = 2.0 Hz, H-400), 5.88 (2H, δ, J = 2.1 Hz, H-20, 60); 13C NMR (100 MHz, CD3OD) δ 162.7, 162.6, 161.0, 161.0, 154.0, 152.5, 152.0, 149.1, 149.0, 146.7, 144.7, 139.2, 136.2, 128.9, 125.9, 125.6, 123.2, 106.2, 106.1, 100.8, 100.2, 98.6, 98.5, 97.0, 96.2, 96.2; ESI-MS [M − H]− at m/z 601.36. Animals and Experimental Design. Male C57BL/6 mice (n = 40; 4 weeks old) were obtained from Orient Bio (Gyeonggi-do, South Korea) and were housed two or three per cage in a room (temperature, 21−23 °C; relative humidity, 45−55%; 12 h dark/ light cycle). After acclimation for 1 week, mice were then randomly divided into two groups: the normal control diet group (CD; n = 10) and the high-fat diet group (HFD; n = 30). CD was provided with regular diet (D12450B, 10% kcal fat, Research Diets, New Brunswick, NJ, USA), whereas HFD was provided with high-fat diet (D12451, 45% kcal fat, Research Diets) for 10 weeks. Food and distilled water were supplied ad libitum. After obesity was induced by the high-fat diet, the animals were divided into four groups, 10 mice in each group and supplemented as follows: CON, nonobese control group mice fed CD were gavaged with distilled water; HF, obese mice fed HFD were gavaged with distilled water; LE, obese mice fed HFD were gavaged ECPE at a (low) dose of 100 mg/kg/day; HE, obese mice fed HFD were gavaged with ECPE at a (high) dose of 500 mg/kg/day. The identical volume of gavage with either distilled water or ECPE was administrated by oral gavage 5 times a week for 12 weeks. ECPE was freshly suspended in distilled water. The body weight and food intake were measured twice a week. Mice were fasted for 15 h after the last treatment and then euthanized by inhalation with isoflurane (Baxter, Deerfield, IL, USA). Blood was collected by cardiac puncture using a heparin (SigmaAldrich, St. Louis, MO, USA) coated syringe. Plasma was obtained by centrifugation at 3000 rpm for 15 min. Tissues such as the liver and adipose pads were removed after cardiac puncture, washed of blood or foreign materials by saline, weighed, and immediately frozen in liquid nitrogen. All of the samples were stored at −80 °C until they were used for experiments. Experimental protocol was approved by the Institutional Animal Care and Use Committee of the Kyung Hee University.
synthase (iNOS) and the cyclooxygenase-2 (COX-2) expression by blocking transcription factors of the nuclear factor kappa B (NFκB) and the mitogen-activated protein kinase (MAPK) activation in BV2 microglia.16 Among the polyphenols of E. cava, dieckol is the most ample compound in E. cava, and it has been reported for having some beneficial potential such as antioxidative effects and antithrombotic activity.17,18 A recent study reported that dieckol, which was isolated from E. cava, inhibited adipogenesis by activating AMPK and suppressing adipocyte differentiation in 3T3-L1 preadipocytes.19 However, little research has focused on the molecular mechanisms by which E. cava polyphenol extract (ECPE) regulates hepatic lipid metabolism in an obese animal model. We investigated how ECPE ameliorates hepatic lipid metabolism, oxidative stress, and inflammation through modulation of AMPK and SIRT-1 in high-fat diet (HFD)induced obese mice.
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MATERIALS AND METHODS
Preparation of ECPE. ECPE used in this study was provided from Aqua Green Tech., Jeju, Korea. The preparation of dieckol-rich polyphenols from E. cava was described previously.20 In brief, the E. cava was collected from the coast of Jeju Island, Korea. The powdery E. cava (500 g) was suspended in 10 L of 70% ethanol and stirred for 24 h at room temperature. The solution was filtered and then concentrated under reduced pressure to give the oily extract. Then, the organic soluble fraction was taken by treatment with ethyl acetate (47.85 g), and the extract was evaporated under vacuum. Quantification of Total Phenol and Major Compounds in ECPE. The total phenol content in the polyphenol extract was measured by using the protocol described by Kim et al.21 The quantitative analysis in the major compounds in the polyphenol extract from E. cava was performed by their HPLC analysis and quantitative standard curve. The quantitative standard curves of the main compounds were calculated by their area (%) in high-performance liquid chromatography (HPLC) under concentrations between 25 and 400 μg/mL. Identification of Major Compounds in ECPE and Chemical Structure of Dieckol. The structural identification of the major compounds in the polyphenol extract from E. cava was performed by using the method described in a previous paper.22 The major compounds in the extract were confirmed by LC-DAD-ESI/MS and 1D NMR spectrum. To operate LC-DAD-ESI/MS, 10 μL of 5 mg/mL sample solution was directly injected on an Atlantis T3 column (3 μm 3.0 × 150 mm column) (Waters, Milford, MA, USA). The mobile phase comprised acetonitrile/water in gradient mode as follows: acetonitrile with 0.1% formic acid/water with 0.1% formic acid (0−40 min, 10:90−40:60 v/v; 40−50 min, ∼50:50 v/v; and 50−60 min, ∼100:0 v/v). The flow rate was 0.2 mL/min, and the UV absorbance was detected at 290 nm. The source voltage was set to 4.5 kV and the capillary temperature to 250 °C. The other conditions were as follows: capillary voltage, −36.5 V; interoctapole lens voltage, 10 V; sheath gas, 80 psi (551.6 kPa); auxiliary gas, 20 psi (137.9 kPa). The major compounds were confirmed as dieckol, 2,7″-phloroglucinol-6,6′bieckol, pyrogallol-phloroglucinol-6,6′-bieckol, and phlorofucofuroeckol-A, respectively. After purification of the compounds from the E. cava extract, their 1H NMR and 13C NMR spectra were measured with a JEOL JNM-LA 300 spectrometer (JEOL Ltd., Tokyo, Japan), and the mass spectra (FAB-MS and EIMS) were recorded on a JEOL JMS 700 spectrometer. Dieckol: amorphous powder; 1H NMR (400 MHz, methanol-d4) δ 6.15 (1H, s), 6.13 (1H, s), 6.09 (1H, δ, 2.9 Hz), 6.06 (1H, δ, 2.9 Hz), 6.05 (1H, δ, 2.9 Hz), 5.98 (1H, δ, 2.8 Hz), 5.95 (1H, δ, 2.8 Hz), 5.92 (3H, m); 13C NMR (100 MHz, methanol-d4) δ 161.8, 160.1, 157.8, 155.9, 154.5, 152.4, 147.3, 147.2, 147.1, 146.9, 144.3, 144.1, 143.4, 143.3, 138.6, 138.5, 126.5, 126.2, 125.6, 125.5, 124.9, 124.6, 124.5, 350
dx.doi.org/10.1021/jf502830b | J. Agric. Food Chem. 2015, 63, 349−359
Journal of Agricultural and Food Chemistry
Article
Figure 1. Quantification and identification of E. cava polyphenol extract (ECPE): (A) polyphenol contents in ECPE quantified by HPLC analysis; (B) electrospray ionization mass spectrometry (ESI/MS) of ECPE and chemical structure of dieckol. Intraperitoneal Glucose Tolerance Test (IPGTT). A week before the end point, the animals were fasted overnight for 16 h prior to the IPGTT test. The 50% glucose solution (2 g/kg) was injected intraperitoneally. Blood glucose level was measured with a glucometer (NOCODING1, Seoul, Korea) at 0 (as basal fasting blood glucose level, FBG), 15, 30, 60, 90, and 120 min after injection of glucose solution. The area under the curve (AUC) was calculated using the trapezoidal rule as follows: AUCi= [(blood glucose)i + (blood glucose)i−1] × [(time)i − (time)i−1]/2. Plasma Biomarker Analysis. The contents of plasma triglyceride (TG), total cholesterol (TC), and high-density lipoprotein cholesterol (HDL-C) were determined using commercial kits (Bio-Clinical
System, Gyeonggi-do, South Korea). The atherogenic index (AI) was estimated as follows: AI = ([TC] − [HDL-C])/[HDL-C]. Plasma glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) were determined by enzymatic assays with commercial kits (Bio-Clinical System). The plasma concentrations of leptin, adiponectin, and insulin were determined using enzyme-linked immuosorbent assay (ELISA) commercial kits (RayBiotech, Inc., Norcross, GA, USA). The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated as follows: fasting insulin (μU/ mL) × fasting glucose (mmol/L)/22.5. All of the commercial kits were performed according to the manufacturer’s instructions, respectively. 351
dx.doi.org/10.1021/jf502830b | J. Agric. Food Chem. 2015, 63, 349−359
Journal of Agricultural and Food Chemistry
Article
Table 1. Effects of ECPE on Body Weight, Food Intake, Liver Weight, and Adipose Tissue Weighta groupb body weight (g) before supplementation after supplementation gain food intake (g/day) food efficiency ratio (%) liver (g) total adipose tissue (g) visceral fat (g) epididymal mesenteric retroperitoneal subcutaneous fat (g) inguinal brown
CON
HF
LE
HE
30.35 ± 0.58 a 37.31 ± 0.80 a 6.95 ± 0.44 a 3.17 ± 0.06 c 2.84 ± 0.16 a 1.23 ± 0.07 ab 3.88 ± 0.28 a 2.71 ± 0.19 a 1.43 ± 0.10 a 0.66 ± 0.07 a 0.62 ± 0.03 a 1.17 ± 0.11 a 0.98 ± 0.07 a 0.20 ± 0.04 a
38.56 ± 1.09 b 48.51 ± 0.73 c 9.96 ± 0.79 b 2.84 ± 0.05 ab 4.59 ± 0.39 c 1.30 ± 0.13 ab 8.88 ± 0.48 c 5.65 ± 0.26 c 2.08 ± 0.09 c 1.94 ± 0.17 c 1.63 ± 0.08 c 3.24 ± 0.26 b 2.83 ± 0.25 b 0.41 ± 0.05 c
39.23 ± 0.74 b 48.07 ± 0.70 c 8.84 ± 0.65 ab 2.97 ± 0.05 b 3.87 ± 0.28 bc 1.48 ± 0.15 b 8.31 ± 0.35 bc 4.91 ± 0.13 b 1.77 ± 0.11 b 1.55 ± 0.11 b 1.59 ± 0.08 c 3.39 ± 0.29 b 3.04 ± 0.28 b 0.35 ± 0.04 bc
38.01 ± 0.61 b 44.96 ± 0.78 b 6.94 ± 0.62 a 2.78 ± 0.05 a 3.23 ± 0.26 ab 1.08 ± 0.05 a 7.67 ± 0.32 b 5.03 ± 0.15 b 2.34 ± 0.10 c 1.32 ± 0.10 b 1.37 ± 0.05 b 2.64 ± 0.23 b 2.36 ± 0.23 b 0.28 ± 0.03 ab
Values are means ± SEM. Mean values with unlike letters are significantly different (p < 0.05). bCON, mice fed the control diet (negative control); HF, mice fed the high-fat diet (positive control); LE, mice supplemented with low dose (100 mg/kg/day) of ECPE; HE, mice supplemented with high dose (500 mg/kg/day) of ECPE. a
acid−0.8% TBA mixture were heated in a water bath at 95 °C for 60 min. After cooling with tap water, distilled water and a mixture of nbutanol and pyridine (15:1 v/v) were added and then centrifuged at 4000 rpm for 10 min to obtain a clear supernatant. The absorbance of the supernatant was measured at 532 nm. Statistical Analysis. The results are expressed as means ± standard error of mean (SEM). Significant differences among the groups were determined by one-way ANOVA using SPSS (version 20.0 for Windows, SPSS Inc., Chicago, IL, USA). Duncan’s multiplerange tests was followed when differences were identified among the groups at p values of
