Viscothionin Isolated from Korean Mistletoe Improves Nonalcoholic

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Viscothionin isolated from Korean mistletoe improves nonalcoholic fatty liver disease via the activation of adenosine monophosphate-activated protein kinase Sokho Kim, Dongho Lee, Jae-Kyung Kim, Jae-Hun Kim, Jong-Heum Park, Ju-Woon Lee, and Jungkee Kwon J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf503535s • Publication Date (Web): 10 Nov 2014 Downloaded from http://pubs.acs.org on November 19, 2014

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Journal of Agricultural and Food Chemistry

Viscothionin isolated from Korean mistletoe improves nonalcoholic fatty liver disease via the activation of adenosine monophosphate-activated protein kinase

Sokho Kim,†,∥ Dongho Lee,†,‡,∥ Jae-Kyung Kim,§ Jae-Hun Kim,§ Jong-Heum Park,§ JuWoon Lee,§ and Jungkee Kwon*,†

†

Department of Laboratory Animal Medicine, College of Veterinary Medicine, Chonbuk

National University, Jeonju, Jeonbuk 561-156, Republic of Korea ‡

Gwangju Regional Food and Drug Administration, Ministry of Food and Drug Safety,

Gwangju, Jeonnam 500-480, Republic of Korea §

Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute,

Jeongeup, Jeonbuk 580-185, Republic of Korea

*To whom correspondence should be addressed: Jungkee Kwon Department of Laboratory Animal Medicine, College of Veterinary Medicine, Chonbuk National University, 664-14, Duckjin-Dong, Jeonju, Jeonbuk, 561-156, Republic of Korea. Tel: +82-63-270-3884; Fax: +82-63-270-3780; E-mail: [email protected]

Author Contributions ∥

These authors contributed equally to this study.

Notes 1

ACS Paragon Plus Environment


The authors declare no competing financial interest.

Funding This work was supported by a grant from the National Research Foundation of Korea, funded by the Korean Government (NRF-2013R1A1A2065158). This work was also supported by the Nuclear Research & Development Program of the Korea Science and Engineering Foundation, through a grant funded by the Government of the Republic of Korea.

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Abstract

In the present study, we investigated the effects of viscothionin, a compound isolated from Korean mistletoe (Viscum album coloratum) on nonalcoholic fatty liver disease (NAFLD) in both in vitro and in vivo models. We discovered a connection between viscothionin and the adenosine monophosphate-activated protein kinase (AMPK) signaling pathway, which is involved in lipid metabolism. Viscothionin was shown to significantly attenuate lipid accumulation in HepG2 cells treated with oleic acid, which induces lipid accumulation. Moreover, the phosphorylation of AMPK and acetyl-coenzyme A carboxylase in HepG2 cells were increased by viscothionin treatment. Viscothionin was orally administered to high fat diet-induced obese mice and subsequently evaluated histopathological analysis associated with AMPK signaling pathways. A significant reduction in the extent of hepatic steatosis was revealed in viscothionin-treated obese mice. Thus, viscothionin mediates its beneficial effects on NAFLD via AMPK signaling pathways, suggesting that it may be a potential target for novel NAFLD treatments.

KEYWORDS: viscothionin, Korean mistletoe, lipid accumulation, NAFLD, AMPK

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Introduction

Nonalcoholic fatty liver disease (NAFLD) is defined by the accumulation of fat in the liver in the absence of significant ethanol consumption. NAFLD comprises various liver diseases, including hepatic steatosis and steatohepatitis, which progresses to hepatic cirrhosis in 1025% of all cases within 8 years.1 The liver plays a vital role in lipid metabolism by tightly regulating both adipogenesis, glycogen synthesis, and lipid peroxidation.2 Several studies have suggested that excessive intake of fat leading to visceral obesity and diabetes-linked insulin resistance places a heavy burden on the liver, making these major risk factors in the development of NAFLD.3 NAFLD is also independently regarded as a risk factor for cardiovascular disease.4 Currently, the incidence of NAFLD in adults and children is increasing rapidly due to the progression of obesity and type 2 diabetes epidemics.5 Therefore, both the prevention and treatment of NAFLD are important public health concerns. As described above, consumption of dietary fat is one of the major causes of NAFLD in society. Recent studies of fatty livers have focused on alternative medicine that is aimed at identifying effective food ingredients or crude herbal extracts that can attenuate the accumulation of hepatic lipids. Adenosine monophosphate-activated protein kinase (AMPK) is a metabolic regulator that mediates adaptation to various cellular stressors from both the environment and nutrition.6 AMPK consists of a serine/threonine kinase and controls lipid metabolism by acetylcoenzyme A carboxylase (ACC) phosphorylation. Once activated, AMPK stimulates fatty acid oxidation to inhibit hepatic lipid accumulation and glycogen synthesis. Thus, the AMPK signaling pathway has been proposed as a possible target for treating NAFLD.7 Fatty acid

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synthase (FAS) is involved in lipid anabolism by mediating the synthesis of long-chain fatty acids from acetyl-CoA.8, 9 The transcription of FAS has been shown to be upregulated by sterol regulatory element binding protein-1 (SREBP-1),10, 11 which regulates the expression of various genes whose products are required for de novo lipogenesis.12 Mistletoe is the popular name given to various species of semi-parasitic plants that grow on a number of different species of trees worldwide. Mistletoes have been shown to have a number of pharmacological activities, including nervine, hypertensive, cardiac depressant, vasodilator, or relaxant effects. Moreover, mistletoe can slow and/or steady an excessive heart rate and has been shown to exert diuretic and stimulant effects.13-15 Korean mistletoe (Viscum album coloratum) is a traditional herb that has more recently been used for the treatment of cancer, cardiovascular disease, and arthrosis. Many bioactive components have been identified in Korean mistletoe, including viscothionin, lectin, alkaloids, steroids, triterpenes, flavonoids, and polysaccharides.16 Although extracts from Korean mistletoe have previously been reported to have an anti-obesity effect,17 as of yet no studies have evaluated whether Korean mistletoe-derived viscothionin possesses hypolipidemic activity and, if so, what is the underlying mechanisms of this activity. Thus, in the present study, we extracted and identified viscothionin from Korean mistletoe, and investigated its effects on AMPK-mediated lipid metabolism as it relates to NAFLD using in vitro and in vivo models.

Materials and Methods

Preparation of Korean mistletoe. Korean mistletoe, growing on oak trees, was collected in

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February 2013 in the Kangwon province of South Korea. Mistletoe plants were washed twice with distilled water before removing the excess water by drying in the shade for 2 days, after which time the plants were freeze-dried. Mistletoe leaves and branches were ground in a homogenizer (Hanil, Seoul, Korea) and strained through a sieve with a mesh size of 500 µm. The resultant mistletoe powder was stored at -80°C until use.

Isolation of viscothionin from Korean mistletoe. Isolation of viscothionin from mistletoe was performed as described previously with slight modification.18 Briefly, 50 g of powdered mistletoe was mixed with 750 ml of 2% acetic acid and stirred overnight at 4°C. The homogenate was centrifuged at 6,000 rpm for 30 min, and the resulting supernatant was lyophilized. The dried matter was then dialyzed using membrane tubing (molecular weight cutoff 3.5 kDa) against 25 mM sodium acetate buffer (pH 4.8) at 4°C. The dialysate was loaded on a CM Sepharose Fast Flow cation exchange column (20 × 4.6 cm) and washed with the same acetate buffer. Retained compounds were then eluted with a NaCl gradient (0 – 0.5 M) and 15 ml fractions were collected using a DC-1200 fraction collector (Eyela, Sunnyvale, CA, USA). The eluted fractions were monitored by determining their absorbance at 280 nm (Figure 1A), and the appropriate fractions were pooled for eventual lyophilization. Specifically, the pooled fractions were first dialyzed against distilled water using the same membrane tubing as described above. Next, the NaCl-free dialysate was relyophilized, dissolved in 1.5 ml of distilled water, and passed through a Sephadex G-50 gel filtration column (70 × 1.4 cm). Among the collected fractions, the fractions in the second peak, which showed cytotoxic activity, (Figure 1B) were pooled and concentrated by centrifugation using a centrifugal filtration unit with a molecular weight cutoff of 9 kDa (Thermo Scientific,

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Rockford, IL, USA). The preparation of isolated viscothionin resulted in a single polypeptide band, approximately 6 kDa in size, as determined by SDS-PAGE analysis (Figure 1C).

Polyacrylamide

gel

electrophoresis

and

immunoblotting.

Polyacrylamide

gel

electrophoresis of isolated viscothionin was carried out using precast 12% NuPAGE Bis-Tris gels (Invitrogen, San Diego, CA, USA) at 100 V for 1 h in NuPAGE 2-(N-morpholino) ethanesulfonic acid (MES) sodium dodecyl sulfate running buffer (Invitrogen, San Diego, CA) according to the manufacturer’s instructions. A SeeBlue Plus2 prestained standard protein marker (Invitrogen) was used to determine the apparent molecular masses of the extracted and concentrated mistletoe proteins. Gels were stained with Coomassie Brilliant Blue R-250 for visualization.

Chemicals. DMSO, oleic acid (OA), and compound C were purchased from Sigma (St. Louis, MO, USA). Primary antibodies for AMPK, phosphorylated AMPK (pAMPK), ACC, phosphorylated ACC (pACC), and β-actin were purchased from Cell Signaling (Beverly, MA, USA). Secondary antibodies (horseradish peroxidase-conjugated anti-rabbit, anti-goat, and anti-mouse IgG antibodies) were obtained from Millipore (Temecula, CA, USA). Cell culture medium and other in vitro ingredients were purchased from Hyclone (Logan, UT, USA).

Cell culture. HepG2 cells (American Type Culture Collection, Manassas, VA, USA) were cultured at 37˚C under a humidified atmosphere supplemented with 5% CO2. Cells were routinely sub-cultured twice per week and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin,

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100 µg/ml streptomycin, and 5.5 mM D-glucose. Upon reaching 70% confluence, media on HUVEC cells was replaced with serum-free DMEM overnight and cells were used as described previously.19

Cell

viability

assay.

Cell

survival

was

determined

using

a

3-(4,5-

dimethylthiazoldimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay kit from Sigma. HepG2 cells were seeded in 48-well plates at a density of 2.0×104 cells per well, incubated for 48 h, and treated with various concentrations (0.5 to 10 µM) of viscothionin for 24 h. After the incubation period, 20 µl of the kit solution was added to each well. Plates were then incubated for 3 h at 37°C in 5% CO2. The resultant formazan crystals were subsequently dissolved in MTT solubilization solution, and the absorbance of each well at 540 nm was determined using a PowerWave2multiplate reader spectrophotometer (Bio-Tek Instruments, Winooski, VT, USA).

Lipid accumulation analysis. Lipid accumulation was analyzed using an Oil Red O staining kit according to the manufacturer’s suggested protocol (Lifeline cell technology, Carlsbad, CA, USA). Oil Red O-stained cells were imaged with an Observer A1 microscope (Carl Zeiss, Germany) at 100 X magnification. After image acquisition, accumulated intracellular Oil Red O dye was eluted with isopropanol and quantified by determining its optical absorbance at 540 nm using a PowerWave2multiplate reader spectrophotometer (Bio-Tek Instruments, Winooski, VT, USA).

Animals and in vivo experimental design. Forty male C57BL/6J mice, approximately six

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weeks of age, were purchased from Dae-han Biolink (Daejeon, Korea). All mice were verified to be free of murine viruses, bacteria, and parasites and were maintained in microisolator cages under pathogen-free conditions on a 12 hour light/dark schedule for one week. Mice were randomly divided into two groups and fed either a normal diet (n=8) or a high fat diet (HFD, n=32) containing 45% of its calories from fat. HFD mice were fed for eight weeks in order to induce obesity. After eight weeks, mice receiving the normal diet were orally administered 0.1 ml saline daily, while HFD-fed mice were randomly divided into three subgroups. Each subgroup was orally administered either 0.1 ml saline (n=8), 1 mg/kg viscothionin in 0.1 ml saline (n=8), 10 mg/kg viscothionin in 0.1 ml saline (n=8), or 10 mg/kg simvastatin in 0.1 ml saline daily for 4 weeks. Treatments were maintained while the mice were fed a HFD from weeks zero to four. All mice had free access to food and water. Body weight for each mouse was measured at 1 and 4 weeks after beginning the treatments. At the end of the experimental period, food was withheld for 12 h and the mice were anesthetized with ether. Subsequently, cardiac punctures were performed and the mice were perfused with cold physiological saline. The livers were then harvested, weighed, photographed, and processed. Livers were processed for histology, real-time RT-PCR, immunoblotting, and other analyses; samples were stored at −80°C until needed for analysis. All animals were cared for in accordance with the Chonbuk National University institutional guidelines for the care and use of experimental animals.

Histopathology. Trimmed liver tissues were fixed in 10% normal buffered formalin for 24 h. All of the fixed samples were embedded in paraffin and cut into 4–5 micron sections. The resulting sections were deparaffinized with xylene, rehydrated with a graded alcohol series,

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and stained with hematoxylin and eosin (H & E). Tissue histopathology was then evaluated using a conventional light microscope (magnification 200 X; Carl Zeiss, Jena, Germany). H & E-stained sample sections were imaged and the areas of the stained regions, in pixels, were quantified using the Image Pro analysis program.

Measurement of intracellular triglyceride contents. Intracellular triglyceride contents were measured using a commercial triglyceride assay kit (BioAssay Systems, Hayward, CA, USA) according to the manufacturer’s instructions. Briefly, HepG2 cells were pretreated with viscothionin at various concentrations (0, 1, or 5 µM) for 1 h before treatment with 10 mM OA in conditioned medium for 48 h. Additionally, cells were pretreated either with or without compound C (20 µM) or viscothionin (5 µM) for 1 h and then treated with OA for 48 h. After treatment, cells were washed twice with phosphate-buffered saline (PBS) prior to the addition of 75 µl of homogenizing solution (154 mM KCl, 1 mM EDTA, 50 mM Tris, pH 7.4) to lyse the cells. The resultant cell lysate was centrifuged at 3000×g for five min at 4°C to remove cellular lipids. The supernatants were then assayed for triglyceride and protein contents. Triglyceride contents were normalized to protein concentrations, which were determined from a standard BSA curve. Results were expressed as mg of triglyceride per mg of cellular protein. To evaluate the triglyceride contents in liver tissues, the tissues were homogenized in 1 ml of PBS and the protein concentrations of the resultant lysates were determined. Homogenates were extracted with 5 ml of a chloroform/methanol mixture, vortexed vigorously, allowed to separate into 2 phases, and centrifuged at 2500 rpm for 15 minutes at 4°C. The total hepatic triglyceride concentrations were normalized to the respective protein concentrations and are presented as mg of lipid per g of tissue protein.

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Immunoblotting. Total proteins from HepG2 cell lysates and liver tissue lysates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis using 12% to 15% gels. Proteins were then electrophoretically transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA, USA). Membranes were blocked in 5% skim milk in PBS and then incubated with primary antibodies, which were diluted 1:500 in 1% skim milk in PBS overnight at 4°C. Membranes were then incubated with peroxidaseconjugated goat anti-rabbit IgG antibodies (1:5,000; Millipore, Bedford, MA, USA) for 1 h. Immunoreactive bands were visualized with SuperSignal West Dura Extended Duration Substrate (Thermo Scientific, San Jose, CA, USA) and analyzed using a chemiImager analyzer system (Alpha Innotech, San Leandro, CA, USA).

RNA preparation and real-time RT-PCR. Total RNA was isolated from cells and precipitated with Ribo EX (Geneall, Daejeon, Korea) according to the manufacturer’s instructions. mRNA was reverse transcribed to cDNA using a Maxime RT PreMix kit (Intron, Seongnam, Korea) according to the manufacturer’s instructions. For real-time RT-PCR, cDNA was amplified using a Mastercycler Gradient 5331 Thermal Cycler (Eppendorf, Germany). Real-time PCR runs were monitored by measuring the fluorescence signal after each cycle with an ABI Step One Plus Sequence Detection System (Applied Biosystems, Singapore). Specific primers for each gene were designed using Primer Express software (Applied Biosystems). The following sense and antisense primers were used for real-time RTPCR

quantification

in

HepG2

GGAGCCATGGATTGCACTTT-3’

and

cells

(forward

and

reverse):

5’-TCAAATAGGCCAGGGAAGTCA-3’

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5’for


SREBP-1,

5’-CGTCCTCTTCCCCGCCACT-3’

GCACCACATCCTCAAACACCACA-3’

for

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and FAS,

5’and

5’-

TCTCCTCTGACTTCAACAGCGAC-3’ and 5’-CCCTGTTGCTGTAGCCAAATTC-3’ for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which was used as a housekeeping gene. The following sense and antisense primers were used in real-time RT-PCR for quantification in liver tissue (forward and reverse): 5’-GCAGCCACCATCTAGCCTG-3’ and 5’-

CAGCAGTGAGTCTGCCTTGAT-3’

for

SREBP-1,

5’-

CCTGGATAGCATTCCGAACCT-3’ and 5’-AGCACATCTCGAAGGCTACACA-3’ for FAS, and 5’-GCATGGCCTTCCGTGTTC-3’ and 5’- GATGTCATCATACTTGGCAGGTTT-3’ for GAPDH, which was used as a housekeeping gene. All experiments were performed at least three times.

Statistical analysis. Results are presented as the mean ± standard error. Data were analyzed using Student`s t-test (for two groups), one-way ANOVA, and Tukey’s test (for more than two groups). P values

Viscothionin Isolated from Korean Mistletoe Improves Nonalcoholic

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