Preventive Effects of Taurine on Development of Hepatic Steatosis

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J. Agric. Food Chem. 2011, 59, 450–457 DOI:10.1021/jf103167u

Preventive Effects of Taurine on Development of Hepatic Steatosis Induced by a High-Fat/Cholesterol Dietary Habit )

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YUAN-YEN CHANG,†,‡,O CHUNG-HSI CHOU,§,O CHIH-HSIEN CHIU, KUO-TAI YANG,^ YI-LING LIN,# WEI-LIEN WENG,r AND YI-CHEN CHEN*,

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† Department of Microbiology and Immunology, and Institute of Microbiology and Immunology, School of Medicine, #Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung 402, Taiwan, ‡Clinical Laboratory, Chung Shan Medical University Hospital, Taichung 402, Taiwan, §Zoonoses Research Center and School of Veterinary Medicine, and Department of Animal Science and Technology, National Taiwan University, Taipei 106, Taiwan, ^Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan, and rInstitute of Biochemistry, National Yang-Ming University, Taipei 112, Taiwan. O Y.-Y.C. and C.-H.C. contributed equally as first authors.

Nonalcoholic fatty liver (NAFL) is also called hepatic steatosis and has become an emergent liver disease in developed and developing nations. This study was to exam the preventive effects of taurine (Tau) on the development of hepatic steatosis via a hamster model. Although hepatic steatosis of hamsters was induced by feeding a high-fat/cholesterol diet, drinking water containing 0.35 and 0.7% Tau improved (p < 0.05) the serum lipid profile. Meanwhile, the smaller (p < 0.05) liver sizes and lower (p < 0.05) hepatic lipids in high-fat/cholesterol dietary hamsters drinking Tau may be partially due to higher (p < 0.05) fecal cholesterol, triacylglycerol, and bile acid outputs. In the regulation of lipid homeostasis, drinking a Tau solution upregulated (p < 0.05) low-density lipoprotein receptor and CYP7A1 gene expressions in high-fat/cholesterol dietary hamsters, which result in increased fecal cholesterol and bile acid outputs. Drinking a Tau solution also upregulated (p < 0.05) peroxisome proliferator-activated receptor-R (PPAR-R) and uncoupling protein 2 (UPC2) gene expressions in high-fat/cholesterol dietary hamsters, thus increasing energy expenditure. Besides, Tau also enhanced (p < 0.05) liver antioxidant capacities (GSH, TEAC, SOD, and CAT) and decreased (p < 0.05) lipid peroxidation (MDA), which alleviated liver damage in the high-fat/cholesterol dietary hamsters. Therefore, Tau shows preventive effects on the development of hepatic steatosis induced by a high-fat/cholesterol dietary habit. KEYWORDS: Antioxidant capacity/enzyme; hepatic/fecal lipids; hepatic steatosis; lipid homeostasis; serum lipids; taurine

INTRODUCTION

An imbalanced energy dietary habit, that is, hypercaloric food or saturated fat, results in increased body weights, serum lipids, and hepatic lipids. Nonalcoholic fatty liver (NAFL) is also called hepatic steatosis, which is a collection of excessive amounts of triacylglycerol (TAG) and other fats inside liver cells. NAFL has become an emergent liver disease since its prevalence is estimated as 20-30% in the general population of western countries (1, 2). NAFL is associated with other diseases that influence fat metabolism including obesity, diabetes, and hyperlipidemia (3). As we know, TAG is the major component of lipid accumulation in livers, which may cause lipid peroxidation in livers. Hence, the clinical implications of NAFL are mostly due to its potential to cause a chronic inflammation and then progress to cirrhosis, liver failure, and hepatocellular carcinoma. Therefore, food scientists and nutritionists strive to reduce or attenuate the occurrence of NAFL by decreasing hepatic lipid accumulation through a dietary modification. *To whom correspondence should be addressed. Tel: 8862-33664180. Fax: 886-2-27324070. E-mail: [email protected].

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Published on Web 12/02/2010

Taurine (Tau) (2-amino ethanesulfonic acid) is one of the major and free intracellular amino acids in many mammalian tissues, such as brain, retina, myocardium, skeletal muscle, liver, platelets, and leukocytes (4). Its biophysiological functions include detoxification, antioxidant, membrane stabilization and osmoregulation, neuromodulation, and brain and retina developments (4). Recently, two studies reported that supplementing Tau can alleviate cardiac and liver abnormalities in NZB/W F1 mice, a well-known and popular utilized lupus-prone mice strain, fed with a high-cholesterol diet (5, 6). Currently, Tau is a legal additive in food products, that is, milk, beverage, and functional foods. Additionally, a hypocholesterolemic effect of Tau accounts for increasing cholesterol 7-R hydroxylase (CYP7A1) expressions and fecal cholesterol and bile acid outputs, as well as decreasing levels of liver lipid (7). As we know, increased serum lipids always couple with the symptom of hepatic steatosis. Hence, normalizing serum lipids is also one of the ways to hinder the occurrence of hepatic steatosis. The sources of body lipids are mainly from diets and de novo synthesis in the body. Hepatic TAG homeostasis in mammal animals is regulated by TAG biosynthesis (i.e., sterol regulatory element-binding protein-1c, SREBP-1c; fatty acid synthase, FAS)

© 2010 American Chemical Society

Article and energy expenditure (peroxisome proliferator-activated receptor-R, PPAR-R; uncoupling protein-2; UCP2). Although it was mentioned that Tau upregulates CYP7A1 and 3-hydroxy3-methylglutaryl-CoA reductase (HMG-CoA reductase) in hepatic cells (8), the information about Tau on other regulators of lipid homeostasis is quite limited to our knowledge. A high-fat dietary habit results in hepatic lipid peroxidation when hepatic malondialdehyde (MDA) contents are increased and, thus, increases the probabilities of hepatic inflammation and steatosis (9, 10). Glutathione (GSH) derived from three kinds of amino acids (cysteine, glutamate, and glycine) has been found as the major endogenous antioxidant in hepatocytes (11). In addition, there are also three major antioxidant enzymes, that is, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px), which are capable to counteract reactive oxygen species and hydrogen peroxide (H2O2) in the biological system (12). Briefly, overproduction of reactive oxygen species (O2-•) due to lipid peroxidation in the body can be converted into H2O2 and oxygen (O2) by SOD, and H2O2 can be further detoxified by either CAT or GSH-Px to form water and O2. Therefore, the objectives of the present study were to investigate whether (1) Tau could improve antioxidant capacities and enzymes of high-fat/cholesterol dietary hamsters; (2) Tau could normalize gene expressions related to hepatic lipid homeostasis, such as low-density lipoprotein (LDL) receptor, HMG-CoA reductase, CYP7A1, SREBP-1c, FAS, PPAR-R, and UCP2 in high-fat/cholesterol dietary hamsters; and (3) Tau could attenuate the hepatic damage induced by a high-fat/cholesterol dietary habit. MATERIALS AND METHODS Animal and Diets. The animal usage and protocol were reviewed and approved by Chung Shan Medical University Animal Care Committee (IACUC No.: 565). Thirty-two male Golden Syrian hamsters that were 5 weeks of age were purchased from the National Laboratory Animal Center (National Science Council, Taipei, Taiwan). Two hamsters were housed in each cage in an animal room at 22 ( 2 C with a 12/12 h light-dark cycle. Chow diets containing 48.7% (w/w) carbohydrate, 23.9% (w/w) protein, 5.0% (w/w) fat, 5.1% (w/w) fiber, and 7.0% ash (Laboratory Rodent Diet 5001, PMI Nutrition International/Purina Mills LLC, United States) and water were provided for 1 week of acclimation. According to the previous study from our research group, feeding hamsters with a high-fat/cholesterol diet (HFCD) formulated with saturated fat (butter) significantly increased hepatic lipids and oxidative stress, thus leading to hepatic damage (10). Hence, a chow diet was regarded as a low-fat/cholesterol diet (LFCD), while a HFCD was formulated as 92.8% (w/w) chow diets supplemented with 7% (w/w) butter and 0.2% (w/w) cholesterol (Table 1). Butter and cholesterol were purchased from ICN Biomedicals, Inc. (Irvine, United States). Tau with a purity of 99.6% was kindly provided from Forever Chemical Co. Ltd. (Taiwan). In the beginning of the experiment, hamsters with two hamsters per cage were randomly assigned to one of the following groups: (1) LFCD with normal distilled water (LFCD þ NDW), (2) HFCD with normal distilled water (HFCD þ NDW), (3) HFCD with 0.35% (w/v) Tau solution (HFCD þ 0.35% Tau), and (4) HFCD with 0.7% (w/v) Tau solution (HFCD þ 0.7% Tau). All hamsters were fed the assigned diets and drinking water ad libitum. The experimental period lasted for 4 weeks. The body weights of hamsters were individually recorded every week. Feed and water were changed every day. The daily feed and water per cage were determined and further divided by two for obtaining daily feed (g) and water intakes (mL) on a per hamster daily basis. Collection of Liver, Visceral Fat, Serum, and Feces. At the end of the experiment, all feed was removed 14 h before euthanization. After the hamsters were euthanized by CO2, liver and visceral adipose tissue in the abdominal cavity from each hamster were removed and weighted. Liver was stored at -80 C for further analyses. Blood samples were also collected by an intracardiac puncture. Serum was separated from blood

J. Agric. Food Chem., Vol. 59, No. 1, 2011

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Table 1. Compositions and Nutrients of Experimental Diets feed composition (%) ingredients

LFCD

HFCD

chow dieta butter cholesterol

100 0 0

92.8 7 0.2 nutrient composition (%)

nutrients

LFCD

HFCD

protein fat nitrogen-free extract fiber cholesterol calorie (kcal/100 g)

23.9 5.0 48.7 5.1 0.0 335.0

22.18 11.64 45.19 4.73 0.2 376.0

a Chow diet was purchased from PMI Nutrition International/Purina Mills LLC (Laboratory Rodent Diet 5001) (United States).

samples by centrifugation at 3000g for 10 min and then stored at -80 C for further analyses. Feces were collected from each cage 24 h before the end of experiment and stored at -80 C for further analyses. Determination of Serum Lipid Parameters and Liver Damage Indices. Serum total cholesterol (TC), TAG, and high-lipoprotein cholesterol (HDL-C) were measured using commercial kits (Randox Laboratories Ltd., Antrim, United Kingdom). In the HDL-C analysis, LDL, very low-density lipoprotein (VLDL), and chylomicrometer in serum were precipitated by the addition of phosphotungstic acid in the presence of magnesium ions. After centrifugation (3000g for 10 min), the cholesterol concentration in the HDL fraction was determined using the TC commercial kit (Randox Laboratories Ltd.). Those methods were based on detection of colored end products at 500 nm. The atherogenic index (AI) was calculated by the formulation of non-HDL-C/HDL-C. The serum liver damage indices [glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) values] were determined using commercial enzymatic kits (Ortho-Clinical Diagnostics, Inc., Rochester, NY) with an EKTACHEM DT 60 II/DTSC (Estman Kodak Co., Rochester, NY). Determination of Hepatic/Fecal Cholesterol, TAG, and Bile Acids. Hepatic and fecal lipid extractions were measured according to the procedures of Tzang et al. (13). Briefly, hepatic and fecal lipids were extracted by chloroform and methanol (2:1, v/v). The extracts were dried under N2 and resuspended in isopropanol via an ultrasonic cleaner (model: DC150H, Taiwan Delta New Instrument Co. Ltd., TW) for a sufficient dissolution. Cholesterol and TAG concentrations were measured using commercial kits (Randox Laboratories Ltd.). 3R-Hydroxy bile acids comprised about 60% of the fecal bile acids in the hamster (14), and 3R-hydroxy bile acids can be determined using an enzymatic method (Randox Laboratories Ltd.). Preparation of Liver Homogenate. A 0.5 g amount of liver was homogenized on ice in 4.5 mL of phosphate buffer saline (PBS, pH 7.0, containing 0.25 M sucrose) and centrifuged at 12000g for 30 min. The supernatant was collected for further analyses. The protein content in the supernatant was measured according to the procedures of a Bio-Rad protein assay kit (catalog #500-0006, Bio-Rad Laboratories, Inc., Hercules, CA) using bovine serine albumin as a standard. Determination of Hepatic MDA and GSH Contents and Trolox Equivalent Antioxidant Capacity (TEAC). The hepatic MDA and GSH contents were performed according to procedures as described by Yang et al. (10). A 0.5 mL amount of liver homogenate was mixed with 0.75 mL of TBA solution in a Teflon tube, and then, 4.25 mL of trichloroacetic acid-HCl (TCA-HCl) reagent was added. The tube was flushed with nitrogen and closed. A blank was prepared in the same manner, but PBS (pH 7.0) replaced the liver homogenate. The tubes were boiled for 30 min and then cooled. The colored solution was centrifuged at 4000g for 15 min. A clear and colored supernatant was transferred to a cuvette, and the absorbance was measured at 535 nm using an Implen NanoPhotometer (model 1443, Implen GmbH, Munich, Germany). The hepatic MDA content was calculated by taking the extinction coefficient

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of MDA to be 1.56  10 M cm at 535 nm. Because of the unique thiol compound in GSH, 2,2-dithiobisnitrobenzoic acid (DTNB) is commonly used for thiol assay. In the analysis of GSH contents, equal volumes of liver homogenate and 10% TCA solution were mixed well and placed in an ice box for 5 min and then centrifuged. The clear layer, Tris base (0.25 M)ETDA (20 mM) buffer (pH 8.2) and DTNB (10 mM) solution were mixed well. After 5 min, the absorbance was measured at 412 nm using an Implen NanoPhotometer (model 1443, Implen GmbH). The hepatic GSH content was calculated by taking the extinction coefficient of 2-nitro-5-thiobenzoic acid (NTB) to be 1.36  104 M-1 cm-1. Hepatic TEAC was analyzed according to a method described by Hung et al. (15). A free radical, ABTSþ, can be generated by mixing ABTS (100 μM) with H2O2 (50 μM) and peroxidase (4.4 U/mL). The TEAC value was expressed as a scavenging capacity against ABTSþ. Briefly, a 0.25 mL mixture of ABTS, H2O2, and peroxidase and 1.5 mL of dd H2O were mixed well and placed under a dark room. After 30 min, 0.25 mL of diluted liver homogenate (1%, v/v) was then added. The absorbance was measured at 734 nm after the interaction of sample solution for 10 min. The decrease in absorption at 734 nm after the addition of reactant was used to calculate the TEAC value. A standard curve was plotted for Trolox on scavenging ABTSþ capacity and was used for calculation for TEAC of samples. The higher TEAC value of a sample results in the stronger antioxidant activity. Determination of Hepatic SOD, CAT, and GSH-Px Activities. Hepatic SOD was measured by the inhibitory effect of SOD on pyrogallol autoxidation (16). Briefly, 100 μL of liver homogenate was mixed well with 650 μL of PBS (pH 7.0). After centrifugation at 6000g for 10 min (4 C), 10 μL of supernatant was mixed with 3 mL of Tri-HCl buffer (50 mM, pH 8.2) and 15 μL of pyrogallol (0.2 mM). The absorbance change caused by the formation of the yellow pyrogallol oxidation product, purpurogallin, was recorded at 420 nm in 3 min. One unit of SOD activity was defined as the amount of enzyme that inhibited the autoxidation of pyrogallol by 50%. The hepatic SOD activity was expressed by unit/mg protein. The hepatic CAT activity was performed according to the procedure as described by Hong and Lee (17) with a slight modification. Briefly, 450 μL of liver homogenate was mixed well with 50 μL of triton X-100 (10% v/v). After centrifugation at 6000g for 10 min (4 C), a mixture of 10 μL of supernatant and 9990 μL of PBS (pH 7.0) was reacted with 0.5 mL of H2O2 (30 mM). The difference in absorbance between 0 and 3 min was measured at 240 nm. The hepatic CAT activity was calculated by taking the extinction coefficient of H2O2 to be 39.5 M-1 cm-1. One unit of CAT was expressed as the amount of enzyme that decomposes 1 mol H2O2 per min at 25 C. The hepatic CAT activity was expressed by unit/mg protein. The hepatic GSH-Px activity was measured according to the procedure as described by Hong and Lee (17) with a slight modification. Briefly, 2 μL of liver homogenate was mixed with 988 μL of reaction solution containing 100 mM PBS (pH 7.0), 10 mM EDTA, 10 mM NaN3, 2 mM NAPDH, 10 mM GSH, and 1 U/mL GSH reductase. After 5 min, 100 μL of H2O2 (2.5 mM) was then added. The difference of absorbance between 0 and 3 min was measured at 340 nm. Hepatic GSH-Px activity was calculated by taking the extinction coefficient of NADPH to be 6.22  106 nM-1 cm-1 and expressed by nmol NADPH oxidized/min/mg protein. Determination of Hepatic Tumor Necrosis Factor-r (TNF-r) and Interleukin-1β (IL-1β) Levels. The hepatic TNF-R and IL-1β levels were measured by enzyme-linked immunosorbent assay (ELISA) and were performed according to the commercial manufacturer’s instructions (TNF-R and IL-1β kits, eBioscience, Inc., San Diego, CA). In the assays, an aliquot of liver (100-200 ng of protein) was extracted in the foregoing liver homogenate. First, the capture antibody (anti-TNF-R and anti-IL1β) was diluted with a coating buffer as a working solution. Each well of flat-bottom 96-well ELISA plates was coated with 100 μL of working solution and incubated at 4 C overnight, and then, the plates were rinsed three times with washing buffer (1 PBS with 0.05% Tween-20). The blocking reaction was performed by adding 200 μL of assay diluent reagent for 1 h at room temperature, and then, the plates were rinsed three times with washing buffer. Prepared samples were added, followed by incubation for 2 h at room temperature, and then, the plates were rinsed three times with washing buffer. A 100 μL amount of diluted detection antibody with assay diluent reagent was added to plates and incubated for 2 h at room temperature, and then, the plates were rinsed five times with washing buffer. A 100 μL amount of diluted streptavidin-HRP with assay diluent reagent was added and then incubated for 20 min at room 5

Chang et al. temperature, and then, the plates were rinsed seven times with washing buffer. A 100 μL amount of substrate solution was added and incubated for 30 min at room temperature, and 50 μL of stop solution (2 N H2SO4) was added. Immediately, the optical density value of each well was read at 450 nm in an ELISA reader (Dynex Technologies, United Kingdom). Hepatic TNF-R and IL-1β levels were both expressed by pg/mg protein. Hepatic mRNA Expressions of LDL Receptor, HMG-CoA Reductase, CYP7A1, SREBP-1c, FAS, PPAR-r, and UCP2, and Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH). Total RNA was isolated from the stored frozen liver tissues by using the protocol described by E. Z. N. A. Tissue RNA Kit (Omega Bio-Tek, Inc., Norcross, GA). Reverse transcription was carried out with 2 μg of total RNA, 8 μL of reaction buffer, 2 μL of dNTPs, 4.8 μL of MgCl2, 4 μL of Oligo-dT (10 μmol/L), and 200 U RTase (Promega, Madison, WI) with diethyl pyrocarbonate (DEPC) H2O in a final volume of 40 μL at 42 C for 1 h. After a heat inactivation, 1 μL of cDNA product was used for a PCR amplification. The appropriate primers of target genes were designed for hamster’s LDL receptor (GenBank no.: M94387), HMG-CoA reductase (GenBank no.: M12705), CYP7A1 (GenBank no.: L04690), SREBP-1c (GenBank no.: U09103), FAS (GeneBank no.: AF356086), PPAR-R (GenBank no.: NM001113418.1), UCP2 (GenBank no.: AF096288.1), and GAPDH (GenBank no.: DQ403053.1) as follows: LDL receptor sense, 50 -ACAGATTCAGTTCCAGGCAG-30 ; antisense, 50 -TGGGGACAAGAGGTTTTCAG-30 ; HMG-CoA reductase sense, 50 -AACTGAGAGCACAAGCAGAG-30 ; antisense, 50 -ATCACAAGCACGAGGAAGAC-30 ; CYP7A1 sense, 50 -TTTGGACACAGAAGCATT-30 ; antisense, 50 -TCCATGTCATCAAAGGTA-30 ; SREBP-1c sense, 50 -GGTGGGCACTGAGGCAAAGC-30 ; antisense, 50 -CGCACACAGGGCTAGGCGGG-30 ; FAS sense, 50 -AGCCCCTCAAGTGCACAGTG-30 ; antisense, 50 -CACGTGTATGCCCTGGCGCC-30 ; PPAR-R sense, 50 -GGACAAGGCCTCAGGGTACC-30 ; antisense, 50 -CCACCATCTTGGCCACAAGC-30 ; UCP2 sense, 50 -TCAGCCTCGATGTTCCCAGC-30 ; antisense, 50 -AGCCAGGGTCTAGGGGAAGA-30 ; GAPDH sense, 50 -GACCCCTTCATTGACCTCAAC-30 ; antisense, 50 -GGAGATGATGACCCTTTTGGC-30 . The sizes of reaction products are as follows: LDL receptor, 477 bp; HMG-CoA reductase, 583 bp; CYP7A1, 497 bp; SREBP-1c, 412 bp; FAS, 347 bp; PPAR-R, 421 bp; UCP2, 403 bp; and GAPDH, 264 bp. GAPDH was used as an internal control in all reactions. The PCR amplification was performed using a DNA thermal cycler (ASTEC PC-818, ASTEC Co., Ltd., Fukuka, Japan) under the following conditions: LDL receptor and CYP7A1: 30 cycles at 94 C for 1 min, 51 C for 1 min, and 72 C for 2 min followed by 10 min at 72 C; HMG-CoA reductase: 30 cycles at 94 C for 1 min, 52 C for 1 min, and 72 C for 2 min followed by 10 min at 72 C; SREBP-1c and FAS: 35 cycles at 94 C for 1 min, 55 C for 1 min, and 72 C for 2 min followed by 10 min at 72 C; PPAR-R: 25 cycles at 94 C for 1 min, 55 C for 1 min, and 72 C for 2 min followed by 10 min at 72 C; UCP2: 40 cycles at 94 C for 1 min, 52 C for 1 min, and 72 C for 2 min followed by 10 min at 72 C; GAPDH: 25 cycles at 94 C for 1 min, 52 C for 1 min, and 72 C for 2 min followed by 10 min at 72 C. The final products were subjected to electrophoresis on a 2% agarose gel and detected by ethidium bromide staining via a UV light. The relative expression levels of the mRNAs of the target genes were normalized using the GAPDH internal standard. Histopathological Analysis. For a histopathological study, liver tissues were placed in formalin for no more than 24 h and were fixed in neutral-buffered formalin solution, dehydrated in graded alcohol, cleared in xylene, and embedded in paraffin. These blockers were later sectioned using a microtome, dehydrated in graded alcohol, embedded in paraffin section, and stained with hematoxylin and eosin (H&E) (18). Statistical Analysis. The experiment was conducted using a completely random design. Data were analyzed using analysis of variance. Significant differences were determined at 0.05 probability level, and differences between treatments were tested using the least significant difference test. All statistical analyses of data were performed using SAS 9.0 (SAS Institute, Inc., 2002). RESULTS

Effects of Tau on Body Weight, Weight Gain, Feed and Water Intake, and Sizes of Visceral Fat and Liver. Feed intakes of highfat/cholesterol dietary hamsters (HFCD þ NDW, HFCD þ 0.35% Tau, and HFCD þ 0.7% Tau groups) were higher (p < 0.05) than

Article

J. Agric. Food Chem., Vol. 59, No. 1, 2011

Table 2. Body Weight, Feed Intake, Water Intake, and Sizes of Visceral Fat and Liver of the Experimental Hamsters

453

a

groups

LFCD þ NDWb

HFCD þ NDWb

HFCD þ 0.35% Taub

HFCD þ 0.7% Taub

initial body weight (g) final body weight (g) weight gain (g) feed intake (g/hamster/day) water intake (mL/hamster/day) visceral fat (g/100 g body weight) liver (g/100 g body weight)

85.91 ( 1.76 a 101.75 ( 1.62 b 15.84 ( 0.86 b 6.76 ( 0.14 b 10.41 ( 0.10 a 1.96 ( 0.12 b 3.34 ( 0.05 c

85.78 ( 2.39 a 109.38 ( 2.07 a 23.60 ( 1.56 a 7.09 ( 0.06 a 10.57 ( 0.14 a 2.48 ( 0.08 a 4.44 ( 0.23 a

82.88 ( 1.90 a 105.69 ( 1.79 ab 22.81 ( 1.55 a 7.13 ( 0.08 a 10.60 ( 0.19 a 2.23 ( 0.08 ab 3.82 ( 0.11 b

86.56 ( 3.72 a 102.47 ( 2.90 b 15.90 ( 1.75 b 7.15 ( 0.10 a 10.58 ( 0.19 a 2.13 ( 0.14 b 3.80 ( 0.07 b

a Data are given as means ( SEMs (n = 8, except feed intake and water intake, n = 4). Mean values with different letters in each testing parameter were significantly different (p < 0.05). b LFCD þ NDW, low-fat/cholesterol dietary þ normal distilled water group; HFCD þ NDW, high-fat/cholesterol dietary þ normal distilled water group; HFCD þ 0.35% Tau, high-fat/cholesterol dietary þ 0.35% (w/v) Tau solution group; and HFCD þ 0.7% Tau, high-fat/cholesterol dietary þ 0.7% (w/v) Tau solution group.

Table 3. Serum, Liver, and Fecal Lipid Profiles and Fecal Bile Acid Contents of the Experimental Hamstersa groups

LFCD þ NDWb

HFCD þ NDWb

HFCD þ 0.35% Taub

HFCD þ 0.7% Taub

268.57 ( 25.79 b 251.08 ( 3.74 b 120.89 ( 6.21 a 130.19 ( 12.91 b 1.09 ( 0.11 b

256.13 ( 20.49 b 243.25 ( 12.05 b 119.95 ( 5.41 a 123.30 ( 9.74 b 1.04 ( 0.99 b

15.10 ( 0.94 b 21.13 ( 1.12 b

15.11 ( 0.86 b 22.71 ( 0.66 b

serum TAG (mg/dL serum) TC (mg/dL serum) HDL-C (mg/dL serum) non HDL-C (mg/dL serum) AI

182.79 ( 13.61 c 112.88 ( 4.00 c 73.75 ( 2.70 b 39.12 ( 3.92 c 0.54 ( 0.07 c

329.01 ( 17.65 a 283.43 ( 7.32 a 119.88 ( 4.03 a 163.55 ( 8.36 a 1.38 ( 0.10 a liver

TAG (mg/g tissue) TC (mg/g tissue)

11.32 ( 0.60 c 5.30 ( 0.38 c

19.98 ( 0.91 a 26.55 ( 1.37 a feces

TAG (mg/g dry feces) TC (mg/g dry feces) bile acid (nmol/g dry feces)

1.98 ( 0.10 a 0.71 ( 0.02 b 0.47 ( 0.07 c

2.07 ( 0.14 a 0.71 ( 0.03 b 0.51 ( 0.02 bc

2.14 ( 0.09 a 0.87 ( 0.04 a 0.64 ( 0.03 ab

2.14 ( 0.07 a 0.85 ( 0.04 a 0.69 ( 0.05 a

a

Data are given as means ( SEMs (serum and liver, n = 8; feces, n = 4). Mean values with different letters in each testing parameter were significantly different (p < 0.05). LFCD þ NDW, low-fat/cholesterol dietary þ normal distilled water group; HFCD þ NDW, high-fat/cholesterol dietary þ normal distilled water group; HFCD þ 0.35% Tau, highfat/cholesterol dietary þ 0.35% (w/v) Tau solution group; and HFCD þ 0.7% Tau, high-fat/cholesterol dietary þ 0.7% (w/v) Tau solution group.

b

that of the low-fat/cholesterol dietary group (LFCD þ NDW group) during the experimental period, but water intakes in all of the groups were not ( p >0.05) different (Table 2). However, drinking 0.7% Tau decreased body weights in the high-fat/cholesterol dietary habit and, meanwhile, even showed no ( p > 0.05) difference on final body weight and weight gain as compared to the LFCD þ NDW group after 4 weeks of feeding (Table 2). These results imply that Tau could hinder body weight gain in a high-fat/ cholesterol dietary habit. After the hamsters were sacrificed at the end of experiment, the visceral fat and liver were collected, and their sizes relative to body weight were also calculated (Table 2). Among all groups, the largest ( p