Sechium edule Shoot Extracts and Active Components

Apr 26, 2015 - Sechium edule Shoot Extracts and Active Components Improve. Obesity and a Fatty Liver That Involved Reducing Hepatic. Lipogenesis and ...
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Sechium edule Shoot Extracts and Active Components Improve Obesity and a Fatty Liver That Involved Reducing Hepatic Lipogenesis and Adipogenesis in High-Fat-Diet-Fed Rats Mon-Yuan Yang,† Kuei-Chuan Chan,‡,§ Yi-Ju Lee,† Xiao-Zong Chang,† Cheng-Hsun Wu,*,# and Chau-Jong Wang*,†,⊥ †

Institute of Biochemistry and Biotechnology, and §School of Medicine, Chung Shan Medical University, Taichung 40201, Taiwan ‡ Department of Internal Medicine, and ⊥Department of Medical Research, Chung Shan Medical University Hospital, Taichung 40201, Taiwan # Department of Anatomy, China Medical University, Taichung 40401, Taiwan ABSTRACT: Excess fat accumulation in the liver increases the risk of developing progressive liver injuries ranging from a fatty liver to hepatocarcinoma. In a previous study, we demonstrated that the polyphenol components of Sechium edule shoots attenuated hepatic lipid accumulation in vitro. Therefore, we investigated the effects and mechanisms of the extract of S. edule shoots (SWE) to modulate fat accumulation in a high-fat-diet (HFD)-induced animal model. In this study, we found that the SWE can reduce the body weight, adipose tissue fat, and regulate hepatic lipid contents (e.g., triglyceride and cholesterol). Additionally, treatment of caffeic acid (CA) and hesperetin (HPT), the main ingredients of SWE, also inhibited oleic acid (OA)induced lipid accumulation in HepG2 cells. SWE enhanced the activation of AMP-activating protein kinase (AMPK) and decreased numerous lipogenic-related enzymes, such as sterol regulator element-binding proteins (SREBPs), e.g., SREBP-1 and SREBP-2, and HMG-CoA reductase (HMGCoR) proteins, which are critical regulators of hepatic lipid metabolism. Taken together, the results demonstrated that SWE can prevent a fatty liver and attenuate adipose tissue fat by inhibiting lipogenic enzymes and stimulating lipolysis via upregulating AMPK. It was also demonstrated that the main activation components of SWE are both CA and HPT. KEYWORDS: fatty liver, caffeic acid, hesperetin, AMPK, obesity, lipogenesis, adipogenesis

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INTRODUCTION Obesity is characterized by an increase in fat mass and body weight.1 Excessive fat may accumulate in the liver, leading to a fatty liver,2,3 which is known to be a strong risk factor for nonalcoholic fatty liver disease (NAFLD) and cancer.4,5 Therefore, prevention and treatment of metabolic disorders via attenuating obesity, a fatty liver, and NAFLD are relevant to health promotion. Differentiated adipocytes store fatty acids in the form of triglycerides (TGs) in their cytoplasm with the involvement of various enzymes, such as fatty acid synthase (FAS).6 TGs are synthesized in the liver, secreted into the bloodstream, and transported to the peripheral organs, including adipose tissue.7,8 Several recent studies have demonstrated that hepatic TG availability is controlled by the balance between FAS and oxidation in the liver.9 The fat accumulation in NAFLD is mostly due to the synthesis of fatty acids and inhibition of fatty acid oxidation.10 Several studies have demonstrated transcriptional regulation of the gene for the enzymes of fatty acid synthesis by sterol regulatory element-binding proteins (SREBPs).11,12 Activation of FAS expression through modulation of SREBP-1 has been reported in human breast cancer.13 Inhibition of peroxisome proliferator-activated receptor (PPAR) function and stimulation of SREBP-1, the receptor molecules that control the enzymes responsible for the oxidation and synthesis of fatty acids, respectively, appear to contribute to the overall lipid load in the liver.14,15 © 2015 American Chemical Society

AMP-activating protein kinase (AMPK) phosphorylates and inactivates a number of metabolic enzymes involved in ATPconsuming cellular events, including fatty acid and cholesterol synthesis, involving FAS 16 and HMG-CoA reductase (HMGCoR).17 AMPK monitors intracellular energy status and regulates the uptake and metabolism of fatty acids as well as the synthesis and oxidation of fatty acids, cholesterol, glycogen, and protein to meet energy demand.18,19 Activation of the AMPK pathway is necessary to prevent fat accumulation. Recently, studies on obesity in the field of food science have focused on the search for herbal extracts that can suppress the accumulation of body fat.20 Sechium edule shoots (SWE) contain a lot of nutritional components, including flavonoids,21 which are known as powerful polyphenol antioxidants.17 They are useful as a complementary treatment for arteriosclerosis and hypertension and as a diuretic and anti-inflammatory remedy.4,22 They have been verified to decrease serum lipid and cholesterol and prevent atherosclerosis,22 but there are no reports on the effect of SWE on modulation of hepatic lipid metabolism. SWE have been shown to decrease serum lipid and cholesterol and to prevent atherosclerosis.22 Our previous Received: Revised: Accepted: Published: 4587

January 20, 2015 April 25, 2015 April 26, 2015 April 26, 2015 DOI: 10.1021/acs.jafc.5b00346 J. Agric. Food Chem. 2015, 63, 4587−4596

Article

Journal of Agricultural and Food Chemistry

stored at −80 °C for further investigations, which were completed within 2 weeks. All animal experimental protocols used in this study were approved by the Institutional Animal Care and Use Committee of the Chung Shan Medical University (IACUC, CSMU), Taichung, Taiwan (IACUC Approval Number 885). Hepatic Pathological Analysis. Liver tissues were fixed in 4% buffered paraformaldehyde overnight and embedded in paraffin. Consecutive paraffin sections were examined by hematoxylin and eosin (H&E) staining. Frozen sections were stained with Oil Red O and Sudan III reagents.30,31 Then, the samples were analyzed by light microscopy (Nikon Eclipse, Nikon, Japan). Blood Sample Analysis. Blood samples were collected into heparin tubes and immediately centrifuged at 3000g for 5 min at 4 °C. The serum was decanted and stored at 4 °C. Serum levels of total cholesterol, TGs, aspartate transaminase (AST), and alanine transaminase (ALT) were measured using clinical chemistry reagent kits (Randox Laboratories, Ltd., Antrim, U.K.). A free fatty acid (FFA) quantification kit (BioVision, Inc., Mountain View, CA) was performed to detect the FFA level of serum according to the protocol of the manufacturer. Determination of Total Cholesterol and TGs in Liver. Fresh liver was collected for the extraction of liver lipids. Liver lipids were refined as described previously. Briefly, liver was homogenized with chloroform/methanol (1:2, v/v), and then chloroform and distilled water (1:1, v/v) were added to the homogenate and mixed well. The lipid extract was centrifuged at 1500g for 10 min, and the lower clear organic phase solution was transferred into a glass tube and then lyophilized. The lyophilized powder was dissolved in chloroform/ methanol (1:2, v/v) as the liver lipid extract and stored at −20 °C. TGs and cholesterol in the lipid extracts were measured by enzymatic colorimetric methods using commercial kits (Human, Wiesbaden, Germany). Preparation of the Protein Extract. The protein extracts of cultured cells or liver tissues were harvested in a cold RIPA buffer [1% NP-40, 50 mM Tris−base, 0.1% sodium dodecyl sulfate (SDS), 0.5% deoxycholic acid, and 150 mM NaCl at pH 7.5] containing proteinase inhibitor (17 μg/mL leupeptin and 10 μg/mL sodium orthovanadate). The cell lysates were vortexed at 4 °C for 4 h, and the liver tissues were homogenized on ice for 3 min. All protein extracts were then centrifuged at 10 000 rpm at 4 °C for 20 min. The protein concentration was measured with Coomassie blue (Kenlor Industries, Inc., Santa Ana, CA) using bovine serum albumin as a standard. Western Blot Analysis. Protein samples (80 μg) were subjected to SDS−polyacrylamide gel electrophoresis and electrotransferred to nitrocellulose membranes (Millipore, Bedford, MA). Membranes were blocked with 5% non-fat milk powder/0.1% Tween-20 in Tris-buffered saline (TBS) and then hybridized with the primary antibody at 4 °C overnight. After washing with TBS 3 times, the secondary antibody was hybridized and conjugated to anti-mouse horseradish peroxidase (HRP) (GE Healthcare, Little Chalfont, U.K.) at room temperature for 1 h. HRP was activated by enhanced chemiluminescence (ECL) using ECL western blotting detection reagents and exposed ECL hyperfilm in Fujfilm LAS-3000 (Tokyo, Japan). Protein quantity was determined by densitometry using Fujfilm MultiGauge, version 2.2, software. Cytotoxicity Assay. HepG2 cells were seeded at a density of 5 × 105 cells/mL in 24 well plates and incubated with OA, SWE, CA, or HPT at various concentrations. After 24 h, the medium containing (3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) reagent (0.5 mg/mL) was replaced and incubated for 4 h. Formazan was produced by viable cells, which was dissolved with isopropanol. The absorbance was read at 563 nm by a spectrophotometer (Beckman DU640). Nile Red Stain. HepG2 cells were seeded in 6 well plates (2 × 106 cells/mL) and treated with 600 μM OA and the indicated concentration of SWE, CA, or HPT for 24 h. The cells were washed twice with phosphate-buffered saline (PBS) and fixed with 4% formaldehyde for 30 min. Cell were stained with 40 μg/mL Nile red at room temperature for 30 min. Lipid-bound Nile red fluorescence was

study showed that SWE reduced oliec acid (OA)-induced hepatic lipid accumulation in HepG2 cells.23 Therefore, in this study, we further investigated the anti-obesity effect of SWE in rats fed a high-fat diet (HFD). Adding caffeic acid (CA) and hesperetin (HPT), which are the main ingredients of SWE, also inhibit OA-induced lipid accumulation. In addition, animals fed with a HFD could serve as a model to attenuate hepatic lipid accumulation and evaluate the methods for healthcare foods for prevention of a fatty liver. The lipogenesis inhibitors could promote catabolism by downregulation of the activity of key enzymes of metabolism that account for the effect of healthcare foods.

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MATERIALS AND METHODS

Materials. Fresh S. edule shoots and leaves were collected in Nantou county, located in central Taiwan. GPx, SOD, SREBP-1, SREBP-2, HMGCoR, PPARα, and carnitine palmitoyltransferase-1 (CPT-I) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-pThr172-AMPK, anti-AMPK, and FAS antibodies were purchased from Cell Signaling Technology (Beverly, MA). Anti-β-actin and anti-catalase antibodies were purchased from Sigma-Aldrich. Preparation of SWE and Functional Polyphenol Extract from SWE. The fresh leaves were minced, air-dried under shade, and powdered for used. The extract prepared from 20 g of powder immersed in 1000 mL of distilled water under 4 °C for 24 h was filtered and lyophilized as the SWE with a yield of 17.24%. A total of 100 g of dried SWE powder was drenched with 300 mL of 50 °C methanol for 3 h. After refining 3 times, the extract was filtered and the solvent was removed from the combined extract using a vacuum rotary evaporator. The residue was dissolved in 500 mL of distilled water, removed the pigment with 200 mL of hexane, purified with 180 mL of ethyl acetate 3 times, and removed the solvent by a vacuum rotary evaporator. The residue was redissolved with distilled water and lyophilized to yield the powder as the polyphenol extracts of SWE (SWPE). The SWPE were analyzed for the presence and proportion of functional compounds by high-performance liquid chromatography (HPLC). HPLC Analysis. HPLC was performed with a Hitachi HPLC system (Hitachi, Danbury, CT), which consisted of a pump (L6200A), an ultraviolet detector (L-4250), and a Hitachi D-7000 HPLC system manager program. A systemic procedure for analyzing the phenolic acids contained a Mightysil RP-18 GP 250 column (Kanto, Tokyo, Japan), mobile phase 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, and 0% solvent A and 100% solvent B at 60 min, followed by a 5 min post-run with HPLC-grade water. Phenolic acids were detected at 260 nm. Animals and Treatment. The male Wistar rats (weight of 150 ± 10 g) obtained from the National Laboratory Animal Breeding and Research Center (Taipei, Taiwan) were housed and acclimated in laboratory conditions (23 ± 2 °C, 60 ± 5% relative humidity, and 12 h light/dark cycle) for at least 1 week before study. Different treatments of rats were divided into four groups (n = 10 per group) as follows: (A) normal meals (control), (B) HFD, (C) HFD + 0.5% SWE, and (D) HFD + 1.0% SWE. The composition of the HFD was 40% beef tallow, 26% casein, 15% corn starch, 9% sucrose, 5% cellulose, 4% mineral mixture, and 1% vitamin mixture.24−28 The ratio of mineral and vitamin mixtures was according to AIN-93.29 The metabolized energy of the control and HFD-fed rats, with or without SWE supplement, was 3.85 and 5.60 kcal/g, respectively. All rats, except the control group, were fed with the HFD for 12 weeks. The food intake of each rat was controlled as equal as possible. All rats were weighed every week. After the end of the experiment, animals were sacrificed to collect the blood sample and liver tissue. Serum was prepared and 4588

DOI: 10.1021/acs.jafc.5b00346 J. Agric. Food Chem. 2015, 63, 4587−4596

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Journal of Agricultural and Food Chemistry

Figure 1. (A) HPLC chromatogram of SWPE. HPLC chromatogram of 15 kinds of standard polyphenols. Peaks: 1, gallic acid (GA); 2, protocatechuic acid (PCA); 3, catechin (C); 4, gallocatechin gallate (GCG); 5, caffeic acid (CA); 6, epicatechin (EC); 7, p-coumaric acid (p-CA); 8, rutin (R); 9, ferulic acid (FA); 10, gossypin (G); 11, hesperetin (HPT); 12, resveratrol (RV); 13, quercetin (Q); 14, naringenin (N); and 15, hydroxyflavin (FlOH). (B) HPLC chromatogram of free polyphenols from SWPE. 4589

DOI: 10.1021/acs.jafc.5b00346 J. Agric. Food Chem. 2015, 63, 4587−4596

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Journal of Agricultural and Food Chemistry Table 1. Effects of SWE on the Relative Body Weight Change of Rats with Obesity Induced by a HFD NDa week

0 4 5 6 7 8 9 10 11 12

271.2 358.8 362.4 365.9 379.1 392.2 424.9 457.0 457.3 457.5

± ± ± ± ± ± ± ± ± ±

24.43 36.63b (32.80%) 35.93 (34.17%) 41.21 (35.53%) 28.48 (40.16%) 40.14 (44.78%) 23.90 (57.39%) 42.10 (69.98%) 31.81 (70.04%) 41.64 (70.10%)

HFD 274.1 448.6 459.3 470.1 491.7 511.1 518.2 523.3 537.2 551.0

± ± ± ± ± ± ± ± ± ±

13.73 36.42c 38.62c 51.35c 41.66c 59.21c 22.32c 45.63c 37.50c 51.73c

HFD + 0.5% SWE (63.58%) (67.47%) (71.34%) (79.11%) (86.88%) (89.02%) (91.15%) (96.19%) (101.23%)

269.9 441.2 446.0 450.8 470.4 493.9 503.3 512.7 507.6 502.5

± ± ± ± ± ± ± ± ± ±

13.25 38.43 (63.96%) 22.96 (65.73%) 37.81 (67.50%) 36.62 (75.65%) 66.31 (83.79%) 34.97 (87.34%) 71.23 (90.898%) 45.57d (88.56%) 49.84d (86.22%)

HFD + 1.0% SWE 266.0 433.5 435.6 437.6 455.6 481.5 486.9 492.2 485.6 479.0

± ± ± ± ± ± ± ± ± ±

25.28 20.83 (64.70%) 35.12 (65.38%) 61.42 (66.05%) 40.47 (74.83%) 39.97 (83.60%) 34.97 (85.33%) 56.73 (87.04%) 44.11d (84.32%) 55.86d (81.59%)

a

ND, normal diet group; HFD, rats fed a HFD-induced group; HFD + 0.5% SWE, rats fed a HFD with 0.5% SWE group; and HFD + 1.0% SWE, rats fed a HFD with 1.0% SWE group. bData = [(week x − week 0)/week 0] × 100%. Each value is expressed as the mean ± standard deviation (SD) (n = 10/group). Results were statistically analyzed with a one-way ANOVA. cp < 0.05 compared to the ND group. dp < 0.05 compared to the HFD group. measured and quantified the lipid content in cells by flow cytometry (Becton Dickinson, Mountain View, CA). Statistical Analysis. Overall differences among groups were analyzed by one-way analysis of variance (ANOVA). When overall analysis demonstrated the presence of significant differences among all of the different groups studied, the differences between specific groups were tested using Duncan’s multiple range test. All statistical analyses were performed using SigmaStat software (SPSS, Chicago, IL). p < 0.05 was considered statistically significant.

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RESULTS Functional Components of SWE. The presence and proportion of the main functional constituents of SWE were analyzed by HPLC. The HPLC results revealed that SWPE contained the presence of protocatechuic acid (PCA, 3.56 ± 0.11%), gallocatechin gallate (GCG, 1.18 ± 0.12%), caffeic acid (CA, 26.53 ± 0.28%), p-coumaric acid (p-CA, 7.32 ± 0.24%), hesperetin (HPT, 23.44 ± 0.18%), resveratrol (RV, 0.61 ± 0.04%), quercetin (Q, 4.18 ± 0.09%), and naringenin (N, 13.72 ± 0.14%) (Figure 1). Effect of SWE on Body Weight of Rats Fed with a HFD. The effect of SWE on lipid homeostasis was examined. By feeding rats with HFD and the indicated concentration of SWE for 12 weeks, we compared relative body weight changes. In Table 1 and Figure 2, the rats fed with a HFD increased in weight in the entire experimental duration. In the termination of this experiment, the weight increased to 101.23%. Rats fed with 0.5 or 1.0% SWE decelerated the weight gain significantly at weeks 11 and 12. The results showed that feeding with SWE decreased weight gain in HFD-fed rats. Effect of SWE on Body Fat Deposition. We further detected the lipid content of the liver in rats fed with a HFD or SWE (Table 2). First of all, we compared the weight of liver differences among different groups. The liver weight of rats fed a normal diet was 24.22 ± 3.08 g, and the liver weight of rats in the HFD group increased to 27.07 ± 2.09 g. After treating with 0.5 and 1.0% SWE, it was reduced to 23.54 ± 2.05 and 23.55 ± 3.21 g, respectively. Supplement of SWE significantly reversed the liver weight gain. The study also conducted weight variation of adipose tissue to evaluate whether SWE affected fat deposition. In Table 2, the results reveal that a HFD caused a significant increase in epididymal fat (23.27 ± 6.17 g), perirenal fat (40.01 ± 10.62 g), retroperitoneal fat (29.00 ± 10.70 g), and mesenteric fat (9.24 ± 2.53 g) in surrounding accumulation of adipose tissue compared to a ND, which means an increase in adipose tissue. However, 0.5 and 1.0%

Figure 2. Effects of SWE on the body weight change of rats with obesity induced by a HFD. Body weight change versus time in rats fed various diets. All values are the means ± SD (n = 10). ND, normal group (rats fed normal chow); HFD, rats fed with 40% beef tallow. (#) p < 0.05 compared to the ND group. (∗) p < 0.05 compared to the HFD group.

SWE showed a significant decrease in adipose tissue. The relative total fat was increased, while SWE reduced it even lower than the control. These results indicate that SWE inhibited adipose tissue fat deposition and obesity (Table 2). Effects of SWE on the Serum Lipid and Hepatic Lipids of Rat Induced by a HFD. Because liver plays a critical role in metabolic regulations, we analyzed the hepatic content of cholesterol and TGs. After feeding SWE with both a HFD and 0.5 and 1.0% SWE for 12 weeks, animals were sacrificed and then the serum content of cholesterol, TG, and FFA were analyzed. As shown in Table 3, serum glucose, cholesterol, and FFA levels were elevated by a HFD. SWE reversed the serum levels of glucose and lipids, especially FFA. Merely 0.5% SWE exerted a profound effect of lowering of FFAs elevated by a HFD. A HFD increased low-density lipoprotein-cholesterol (LDL-C) and decreased high-density lipoprotein-cholesterol (HDL-C) and, thus, enhanced the LDL-C/HDL-C ratio. Supplementation of SWE effectively reversed the lipoprotein profiles. Noticeably, the analysis of AST, ALT, blood urea nitrogen (BUN), creatinine (CRE), electrolyte, and ketone 4590

DOI: 10.1021/acs.jafc.5b00346 J. Agric. Food Chem. 2015, 63, 4587−4596

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Journal of Agricultural and Food Chemistry Table 2. Effects of SWE on the Weights of Organs and Adipose Tissue of Rats with Obesity Induced by a HFD relative tissue weights (mg/g of rat) liver weight epididymal fat perirenal fat retroperitoneal fat mesenteric fat total fat

ND 24.22 10.92 15.32 15.45 3.19 44.90

± ± ± ± ± ±

HFD

3.08a 1.84 6.29 7.02 1.35 14.21

27.07 23.27 40.01 29.00 9.24 101.54

± ± ± ± ± ±

HFD + 0.5% SWE

2.09b 6.17b 10.62b 10.70b 2.53b 25.75b

23.54 17.90 24.55 25.56 5.73 73.76

± ± ± ± ± ±

2.05c 4.08c 6.86c 9.63 1.08c 17.81c

HFD + 1.0% SWE 23.55 17.49 23.06 25.06 5.29 71.14

± ± ± ± ± ±

3.21c 4.35c 5.67c 11.88 2.27c 13.91c

a Each value is expressed as the mean ± SD (n = 10/group). Results were statistically analyzed with a one-way ANOVA. bp < 0.05 compared to the ND group. cp < 0.05 compared to the HFD group.

Table 3. Effects of SWE on the Serum Biochemical Parameters and Hepatic Lipids of Rats with Obesity Induced by a HFD ND total cholesterol (mg/dL) total TG (mg/dL) HDL-C (mg/dL) LDL-C (mg/dL) LDL-C/HDL-C glucose (mg/dL) AST (U/L) ALT (U/L) BUN (mg/dL) CRE (mg/dL) sodium (mmol/L) potassium (mmol/L) FFA (mmol/L) insulin (ng/mL) leptin (pg/mL) liver cholesterol (mg/dL) liver TG (mg/dL)

48.80 35.30 35.60 12.50 0.37 104.00 107.50 58.70 13.96 0.65 152.20 6.65 1.30 0.90 1.82 223.60 229.29

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

HFD a

9.97 12.45 6.34 6.46 0.25 10.08 17.86 10.30 1.18 0.05 4.95 0.32 0.26 0.03 0.26 18.10 23.04

63.40 85.80 29.41 18.80 0.68 132.60 131.70 60.50 12.06 0.62 155.80 6.47 1.64 0.99 1.90 661.24 622.86

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

HFD + 0.5% SWE b

12.73 29.08b 6.83 6.35b 0.37b 15.95b 24.91b 13.68 1.45 0.06 9.07 0.59 0.45 0.13 0.23 90.81b 18..97b

59.40 55.30 29.78 13.80 0.46 115.30 115.70 53.30 13.52 0.62 150.60 5.56 1.33 0.98 1.87 544.04 444.18

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

10.61 14.64c 6.43 5.32 0.14 2.64c 18.48 8.05 1.62 0.06 2.79 0.34 0.29 0.10 0.18 77.07c 20.01c

HFD + 1% SWE 56.80 45.00 32.09 12.60 0.41 107.50 107.50 47.10 13.60 0.63 155.30 5.47 1.30 0.98 1.83 471.48 429.27

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

12.52 11.01c 7.11 6.00c 0.24 12.96c 19.68c 6.10c 1.15 0.04 6.78 0.34 0.17c 0.14 0.33 54.19c 51.74c

a Each value is expressed as the mean ± SD (n = 10/group). Results were statistically analyzed with a one-way ANOVA. bp < 0.05 compared to the ND group. cp < 0.05 compared to the HFD group.

body showed that SWE did not burden the liver or affect kidney functions. Next, we analyzed the cholesterol content of liver tissues. The results (Table 3) showed that the cholesterol content of rats fed with a normal diet was 223.60 ± 18.10 mg/dL. The cholesterol content of rats fed with a HFD alone was 661.24 ± 90.81 mg/dL. The cholesterol content of rats fed with 0.5 and 1.0% SWE was 544.04 ± 77.07 and 471.48 ± 54.19 mg/dL, respectively. As shown in Table 3, the TG and cholesterol contents of liver in rats fed with SWE were lower than the TG and cholesterol contents of liver in rats fed with a HFD alone. We also observed the TG content in liver tissues. The results showed that the TG content of rats fed with a normal diet was 229.29 ± 23.04 mg/dL. The TG content of rats fed with a HFD alone was 622.86 ± 18.97 mg/dL, while the TG content of rats fed with 0.5 and 1.0% SWE was 444.18 ± 20.01 and 429.27 ± 51.74 mg/dL, respectively. It showed the reduction of fatty metamorphosis in livers of SWE-supplemented rats (Figure 3 and Table 3). SWE reduced the contents of cholesterol, TG, and FFA to inhibit lipid accumulation. Effect of SWE on the Hepatic-Lipid-MetabolismRelated Protein in HFD-Fed Rats. Previous studies showed that activation of AMPK-p could inhibit lipid accumulation and the activation of FAS and acetyl-CoA carboxylase (ACC).32,33 In an animal model, we examined the protein level in lipid metabolism. In panels A and B of Figure 4, the results showed that the rats fed with 0.5 or 1.0% SWE had lower lipid accumulation.

Figure 3. Histopathological examination of liver tissue in rats fed a control diet and HFD. Animals were fed a normal diet (control group), HFD, or HFD pair feeding with a 0.5 or 1% SWE diet for 12 weeks ad libitum (n = 10). Rats in all groups were sacrificed after 12 weeks of experiments. Liver tissues were obtained immediately after sacrifice and stained with H&E (100×, top), Oil Red O stain (400×, middle), and Sudan III stain (100×, bottom).

Effect of SWE on the Hepatic-Lipid-Oxidation-Related Protein in HFD-Fed Rats. In addition to the lipid synthesis, the hepatic lipid oxidation was also analyzed. PPARα was decreased in the HFD group, while SWE dose-dependently increased the PPARα (Figure 4C). SWE also increased the 4591

DOI: 10.1021/acs.jafc.5b00346 J. Agric. Food Chem. 2015, 63, 4587−4596

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Journal of Agricultural and Food Chemistry

Figure 4. SWE treatment reduced fatty-acid-synthesis-related protein expression in rats fed a HFD. Animals were fed a normal diet, HFD, or HFD pair feeding with a 0.5 or 1% SWE diet for 12 weeks ad libitum (n = 10). Protein extracts from rat liver were subjected to western blotting to detect FAS, SREBP-1, HMGCoR, SREBP-2, PPARα, CPT-I, phosphorylated AMPK, and AMPK. β-actin was used as a loading control. The numbers below the panels represent quantification of the immunoblot by densitometry. Data are presented as means ± SD from three independent experiments. (#) p < 0.05 compared to the ND group. (∗) p < 0.05 compared to the HFD group.

cytotoxicity in HepG2 cells.34,35 The cell cytotoxicity was not found under 4 mg/mL SWE, 200 μM CA, or HPT treatment (Figure 5A). SWE (1−2 mg/mL), CA (100−150 μM), and HPT (100−150 μM) can indeed reduce OA-induced lipid accumulation in HepG2 cells (panels B and C of Figure 5). Taken above, they showed that SWE, CA, and HPT have the ability to reduce the lipid accumulation in the liver.

expression of CPT-I and the activation of AMPK. In comparison to the control, 0.5 or 1.0% SWE significantly increased the AMPK phosphorylation (Figure 4D). Neither CPT-I nor AMPK was altered by a HFD. Hence, it was demonstrated that SWE inhibited lipid synthesis, stimulated lipid oxidation, and lowered hepatic lipid content. Effect of CA and HPT on the OA-Induced Lipid Accumulation by SWE in HepG2 Cells. The main polyphenols of SWE are CA and HPT. The effects of CA and HPT on the intracellular lipid accumulation of HepG2 cells were conducted by OA. This experiment was focused on the change of intracellular lipid accumulation but did not cause

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DISCUSSION The liver plays an important role in lipid homeostasis via regulation of lipogenesis and oxidative stress.36 Recent studies on obesity in the field of food science have focused on the 4592

DOI: 10.1021/acs.jafc.5b00346 J. Agric. Food Chem. 2015, 63, 4587−4596

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Journal of Agricultural and Food Chemistry

Figure 5. SWE, CA, and HPT attenuated OA-induced lipid accumulation in HepG2 cell. (A) SWE, CA, and HPT had no cytotoxicity effect on HepG2 cells. (B and C) Lipid accumulation was induced by 0.6 mM OA. Cells were co-treated with OA and the indicated concentrations of SWE, CA, or HPT for 24 h. Lipid-bound Nile red fluorescence was detected, and quantification of lipid in cells was immediately analyzed by flow cytometry. SWE-L, cells were treated with 1 mg/mL SWE as a low-dose SWE; SWE-H, cells were treated with 2 mg/mL SWE as a high-dose SWE; CA-L, 100 μM caffeic acid; CA-H, 150 μM caffeic acid; HPT-L, 100 μM hesperetin; and HPT-H, 150 μM hesperetin. 4593

DOI: 10.1021/acs.jafc.5b00346 J. Agric. Food Chem. 2015, 63, 4587−4596

Article

Journal of Agricultural and Food Chemistry

the nuclei49 and inhibits SREBP-2 activity to attenuate hepatic steatosis,50 whereas SREBP-2 primarily controls cholesterol homeostasis by activating genes required for cholesterol synthesis and uptake.51 In this study, we found that SWE can activate AMPK, reduce SREBP-1 expression (Figure 4), and lead to inhibition of hepatic lipogenesis, along with the other studies. Similarly, AMPK inhibits in vitro lipogenesis in hepatocytes through the downregulation of the cleavage processing and transcriptional activity of SREBP.50 In another study, the protein levels of CPT-I and PPARα were ascertainable participating in the process of lipolysis.52 In this study, we found that SWE had the same ability to activate PPARα and CPT-I expressions (Figure 4C). Others have also reported that activation of AMPK reduced fat storage while also increasing fatty acid oxidation.53 In our previous study, polyphenols were found in SWE at the level of about 7.73%.23 SWE and CA augmented AMPK activation and mediated a decrease in SREBP-1, FAS, and HMGCoR in HepG2 cells.23,34 In this study, we further demonstrated that SWE effectively improved various lipidrelated parameters (body weight, adipocyte tissue, and serumrelated parameters) in the HFD-induced rat model. It agrees with our previous study that CA exhibited potential as an antiobesity agent by suppressing lipogenesis enzymes44 or enhancing adipocyte glucose uptake, insulin secretion, and antioxidant capacity.54 HPT intake alone at a dietary level of 0.02% showed a decrease in the plasma level cholesterol and TGs and reduced the activity of relative lipogenesis enzymes in high-cholesterol-diet-fed rats.45 Meanwhile, in this study, we used CA and HPT, which are main ingredients of SWE, to show the effectiveness of reducing lipid accumulation in cells. Results in Figure 5 show that CA and HPT reduced the lipid accumulation in the liver. This is consistent with the CAenhanced activity of AMPK to accelerate lipolysis.34 In addition, CA inhibited OA-induced hepatic lipid accumulation and promoted the lipolysis via upregulation of AMPK. In conclusion, we showed that SWE can reduce lipid accumulation and elicit the fatty acid oxidation in the liver of HFD-induced rats. SWE would be developed as a potential therapeutic treatment to reduce the progression of a fatty liver and obesity.

search for functional food ingredients or herbal extracts that can mend the body weight and body fat.3,37−39 SWE suppressed OA-mediated lipid accumulation via regulating lipogenesis and lipolysis in an in vitro study.23 The present investigation verified that SWE improved HFD-induced obesity by decreasing cellular lipid accumulation and increasing lipolysis via activation of AMPK in rats. We first examined the effects of SWE on total cholesterol and TG contents in HFD-fed rats with respect to weight decrease (Figure 1) and serum cholesterol and TG reduction (Table 3). Our observations agree with other reports, which showed that polyphenols could significantly reduce the levels of total cholesterol and also availably diminish body weight in animals with a HFD.39 In addition, there are other polyphenols, such as PCA, p-CA, and naringenin, existing in SWE. In 2010, Liu et al. pointed out that CA and p-CA possess the potential to reduce TGs.40 Yoshida et al. showed that the administration of HPT and naringenin can reduce the FFA secretion in mouse adipocyte and improve lipolysis.41 The previous study also showed that naringenin can effectively decrease body fat.42 Hsu et al. revealed that PCA, p-CA, and CA have the potential to inhibit the differentiation of adipocytes.43 In a previous report, Hsu et al. also showed that the flavonoid and polyphenol can reduce body weight and fatty liver formation in beef-tallowinduced rat obesity. In our result, we found that the rat body weight increases under a similar induction and SWE decelerates the weight gain and a fatty liver via reducing the accumulation of liver fat and increasing the fatty acid oxidation.28 A previous study showed that CA (0.08%, w/w) or HPT (0.02%, w/w) exhibited the potential as an anti-obesity agent in an animal model.44,45 After the analysis of HPLC, SWE contained 26% CA and 23% HPT. Accordingly, it was evaluated that the usage of SWE in 0.5 or 1% would be efficacious to improve obesity and fatty liver formation in our model. The present results showed that SWE has the potential role to reduce obesity in rats. Actually, the duration of 12 weeks is not long enough to confirm the efficacy. In a future study, we will extend the experimental duration to observe the results. The synthesis of fatty acids and reduction of fatty acid oxidation are important events in regulating lipid accumulation of NAFLD.46,47 We suggested that SWE may inhibit the synthesis of fatty acid and cholesterol. FAS is the primary enzyme involved in lipogenesis and serves as a marker of the treatment effect in obesity.32 In the results, we found that SWE inhibited the expressions of FAS and HMGCoR in rats (panels A and B of Figure 4). AMPK has high attention-getting importance to modulate the homeostasis of fatty acids in the research of lipometabolism.48 Previous research indicated that polyphenolic components from many plants can activate AMPK.49 In fact, our previous in vitro study and other research works showed that AMPK can be activated by polyphenol extracts.23 We also investigated the effect of SWE on AMPK activity and the protein expression of SREBP-1. In this study, we found that SWE increased the activation of the AMPK pathway to regulate the lipid accumulation in vivo (Figure 4D). It is consistent that there are several reports demonstrating that AMPK plays an important role in regulating carbohydrate and lipid metabolism, serving as a metabolic master switch responsible for alterations in liver energy homeostasis.50 It also agrees with previous studies that polyphenol extracts from plenty of plants can activate AMPK.33 Activation of AMPK suppressed FAS expression because it prevents SREBP-1 translocation from

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AUTHOR INFORMATION

Corresponding Authors

*Telephone: +886-4-22053366, ext. 2113. Fax: +886-422038766. E-mail: [email protected]. *Telephone: +886-4-24730022, ext. 11670. Fax: +886-4-23248167. E-mail: [email protected]. Funding

This work was supported by the 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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