Black Rice with Giant Embryo Attenuates Obesity-Associated

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Black Rice with Giant Embryo Attenuates ObesityAssociated Metabolic Disorders in ob/ob Mice Yoon-Mi Lee, Sang-Ik Han, Yu-Jin Won, Eunji Lee, Eunju Park, Seock-Yeon Hwang, and Kyung-Jin Yeum J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05361 • Publication Date (Web): 13 Dec 2015 Downloaded from http://pubs.acs.org on December 13, 2015

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Journal of Agricultural and Food Chemistry

Black Rice with Giant Embryo Attenuates Obesity-Associated Metabolic Disorders in ob/ob Mice

Yoon-Mi Lee1, Sank-Ik Han2, Yu-Jin Won3, Eunji Lee4, Eunju Park4 , Seock-Yeon Hwang5, Kyung-Jin Yeum1,*

1

Division of Food Biosciences, College of Biomedical and Health Sciences, Konkuk

University, Chungju-si, South Korea 2

National Institute of Crop Science, Rural Development Administration, Miryang-si, South

Korea 3

Department of Biomedical Chemistry, College of Biomedical and Health Sciences, Konkuk

University, Chungju-si, South Korea 4

Department of Food and Nutrition, Kyungnam University, Changwon, South Korea

5

Department of Biomedical Laboratory Science, College of Health and Medical Science,

Daejeon University, Daejeon-si, South Korea

*

Corresponding author: Kyung-Jin Yeum

Division of Food Bioscience, college of Biomedical and Health Sciences, Konkuk University, Chungju-si, Chungcheongbuk-do, Korea, 380-701 Telephone: 82-43-840-3586 Fax: 82-43-840-3585 E-mail: [email protected] 1

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ABSTRACT

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Obesity is closely associated with metabolic disorders such as hyperglycemia and

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dyslipidemia. Leptin-deficient ob/ob mice (C57BL/6J-ob/ob) and C57BL/6J were randomly

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assigned to a diet of black rice with giant embryo (BR), white rice (WR) or AIN-93G

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(control), and pair-fed for 14 weeks. Although there was not a significant difference in body

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weight, BR-fed ob/ob mice had 1) significantly lower body fat mass than WR- and control-

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fed ob/ob mice determined by dual-energy X-ray absorptiometry 2) significantly lower blood

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glucose, serum insulin, and triacylglycerol levels than control-fed ob/ob mice, and 3)

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significantly lower liver weight, hepatic triacylglycerol and hepatic lipid droplets than both

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WR- and control-fed ob/ob mice. Furthermore, DNA damage in the liver determined by

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phosphorylated H2AX protein and in the kidney, determined by single cell gel

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electrophoresis was significantly lower in BR-fed than WR- and control-fed ob/ob mice. This

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study indicates that BR ameliorates obesity and its related metabolic disorders.

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Keywords

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Obesity, metabolic disorders, black rice with giant embryo, fatty liver, DNA damage

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Introduction

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Obesity is becoming a serious health problem in developed and developing countries. In

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particular, obesity is responsible for the incidence of metabolic disorders such as

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hyperglycemia along with insulin resistance, dyslipidemia, and hypertension, eventually

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progressing to type II diabetes, cardiovascular disease and other age-related diseases.1 In

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addition, dysregulation of fatty acid metabolism leads to fat accumulation in the liver

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contributes to non-alcoholic fatty liver disease (NAFLD). The frequency of occurrence of

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NAFLD is approximately 40-90% in obese people.2-4 NAFLD encompasses a wide degree of

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fatty liver diseases beginning with steatosis, proceeding to non-alcoholic steatohepatitis

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(NASH) with fibrosis and inflammation in the liver, and eventually to liver cirrhosis and

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hepatocellular carcinoma.5

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Fat accumulation may increase oxidative stress due to an imbalance of reactive oxygen

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species (ROS), which in turn induces dysregulation of metabolic-related genes.6 Fat

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accumulation can also impair antioxidant defense systems making obese people susceptible

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to oxidative stress.7 The imbalance in redox status causes oxidation of polyunsaturated fatty

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acids and sugars which result in the production of reactive carbonyl species, that can further

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react with the nucleophilic sites of proteins and DNA, leading to cellular dysfunction.8 The

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link between obesity and oxidative stress poses a new strategy of reducing oxidative stress in

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order to prevent obesity and its related metabolic disorders.9, 10

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Recent studies have been focused on natural products for their antioxidant activity including

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the sequestering action of reactive carbonyl species as anti-obesity agents.8, 11, 12 Increasing

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evidence suggests that the consumption of grains ameliorates obesity and its related 3



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metabolic disorders.13-15 In those studies, the bioactive compounds were identified in the bran

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layer and inside pigmented grains. Previously, we have reported a high amount of fat-soluble

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bioactive components such as carotenoids, vitamin E and γ-oryzanol in black rice with giant

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embryo.16 In addition, it was reported that black rice with giant embryo contains high

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amounts of γ-aminobutyric acid (GABA) and anthocyanins.17 Although black rice extracts

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have been reported to mitigate fatty liver in high-fat diet-fed mice, the effect of whole black

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rice with giant embryo on obesity and its related metabolic disorders has not yet been

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tested.14 Thus, we examined the impact of black rice with giant embryo on obesity and its

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related metabolic disorders in leptin deficient ob/ob mice.

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Methods and Materials

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Reagents

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All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise

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indicated. Antibody against phospho-H2AX (Ser 19) and β-actin were purchased from Cell

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signaling (Danvers, MA).

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Preparation of animal diets

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Black rice with giant embryo and white rice were provided by the National Institute of Crop

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Science, Rural Development Administration, Republic of Korea. A detailed method for the

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development and culture of black rice with giant embryo has been previously reported.17 The

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animal diets were prepared as pellets (Feedlab corporation, Guri, Republic of Korea). AIN4


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93G, which was used for the control diet in this study, has been modified with white rice

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(WR) or black rice with giant embryo (BR). Compositions of each diet are listed in Table 1.

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Animal study

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All procedures were approved by the Institutional Animal Care and Use Committee of

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Konkuk University (KU14060). Five-week-old male mice (C57BL/6J, n=45; ob/ob,

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C57BL/6J-ob/ob, n=55) were purchased from Japan SLC Inc. Japan, and were kept for 1

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week to acclimate to experimental housing conditions (23~25℃, 40~60% humidity, 12 h

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light/dark cycle). Next, C57BL/6J and ob/ob mice were randomly assigned to AIN93G-

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modified with black rice with giant embryo (BR), AIN93G-modified with white rice (WR)

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and AIN93G (control), and pair-fed for 14 weeks. To prevent excessive eating of specific

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diets because of different taste or flavor, C57BL/6J and ob/ob mice were each pair-fed the

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same amount of 2.5~3 g for C57BL/6J and 4.5~5g for ob/ob mice daily. Based on their

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previous day intake for C57BL/6J and C57BL/6J-ob/ob mice, the lesser intake was used in

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each group of mice. Water was given ad libitum.

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Initial glucose levels were determined by a glucometer (SD biosensor, Suwon, Korea) with

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blood obtained from the tail vein, and body weight was measured every 3 days per week

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throughout the feeding period. In the final week of the feeding period, blood pressure was

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assessed using a non-invasive tail-cuff method (Muromachi MK-1030, Tokyo, Japan) and

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body fat was measured using dual-energy X-ray absorptiometry (GE Lunar PIXImus,

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Fitchburg, WI). All animals underwent a fasting period for 16 h before sacrifice. On the day

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of sacrifice, liver, white adipose tissue, and kidneys were immediately extracted for weight 5



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measurement and frozen in liquid nitrogen, and a part of the liver (right lobe) and right

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kidney were fixed in 10% formalin for histology analysis. Blood was collected and

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centrifuged at 3000 rpm for 15 minutes at 4℃, serum samples were aliquotted into cryotubes,

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and all were stored at -80℃ until analysis.

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Immunohistochemistry

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After extraction, the tissue was fixed in 10% formalin for 24 h, embedded in paraffin, and cut

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into sections 4-6 µm thick. Sections were than stained with hematoxylin and eosin (H&E).

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Liver tissue was observed at x200 magnification, and kidney tissue at x400 magnification.

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The score of lipid droplets in the H&E stained liver section was assessed as 0 (non-

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remarkable), 1 (minimal), 2 (mild), 3 (moderate) or 4 (marked) as per the pathologist’s

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opinions. Three sections and 3 fields per tissue were observed (n=5 in each group).

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Analysis of blood serum

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Serum triacylglycerol and cholesterol levels were determined by colorimetric assay kit

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according to the manufacturer’s instructions (Cayman chemical, Ann Arbor, MI). Insulin was

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measured by an ultra sensitive mouse ELISA assay (Crystral Chem, Downers Grove, IL). All

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color changes were read by a microplate reader (BioTek, Winooski, VT).

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Western blot 6


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Frozen tissues were weighed and lysed with an RIPA buffer (25 mM Tris-HCl, pH 7.6, 150

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mM NaCl, 1% NP-40, 1% sodium deoxycholate and 0.1% SDS with protease inhibitors).

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After tissues were lysed, they were centrifuged at 12000 rpm for 10 min, and the protein

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contents in the supernatant were normalized using the Bradford method (BCA protein assay,

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BioRad, Hercules, CA). The normalized protein was mixed with a 2X sample buffer (65.8

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mM Tris-HCl, pH 6.8, 2.1% SDS, 26.3% glycerol, 0.01% bromophenol blue, 710 mM β-

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mercaptoethanol), and loaded onto SDS-PAGE gels. After eletrophoresis, the gel was

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transferred on a PVDF membrane (Millipore, Billerica, MA), and incubated with primary

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antibody (p-H2AX, β-actin) overnight at 4℃. Horse radish peroxidase-conjugated secondary

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antibody was then added into the membrane. The protein was visualized by enhanced

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chemiluminescence (GE Healthcare, Piscataway, NJ).

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Alkaline comet assay

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Kidney tissues were mixed with 75 µl of 0.7% low melting agarose (LMA) and applied on to

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a 0.5% LMA-covered slide. The samples on the slide were lysed with buffer (2.5 M sodium

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chloride, 100 mM EDTA, 10 mM Tris, 1% sodium laurylasarcosine (pH 10), 1% Triton X-

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100, and DMSO was added before use) for 1 h under cold conditions followed by immersion

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with an alkaline buffer (300 mM sodium hydroxide, 10 mM Na2EDTA, pH 13.0) at 4℃ for

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40 min. The samples were then electrophoresed for 20 min at 4℃ with a voltage of 25V. The

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slides were neutralized with a buffer (0.4 M Tris, pH 7.5) for 5 min three times, and treated

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with ethanol for 5 min. After addition of an interchelating agent (50 µl of ethidium bromide

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in each slide), the results were observed under fluorescence microscopy. Measurement of tail 7



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extent moment was conducted by the Komet 5.0 kinetic imaging analyzer (LiverPool, UK).

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Statistics

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Data obtained from at least three independent experiments are expressed as mean ± S.D. The

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difference between two samples was analyzed by two-tailed unpaired Student’s t-test. For

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another multiple comparison, Duncan and Tukey were used. All n values are described in the

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figure legends. A p value under 0.05 is regarded as significant.

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Results and Discussion

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Intake of black rice with giant embryo attenuated total body fat accumulation in ob/ob

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mice

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C57BL/6J and ob/ob mice (C57BL/6J-ob/ob) were pair-fed. As expected, the body weights of

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ob/ob mice increased at a much higher rate than those of their C57BL/6J counterpart

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throughout the experimental period. Final body weights of mice were not affected by the

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different animal diets among the three groups of ob/ob mice or C57BL/6J (Fig. 1A). It should

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be pointed out that there was a possibility of caloric restriction due to pair feeding and a

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reduction of dietary intake from long periods of living inside at cage toward the end of the

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study. Interestingly, the total body fat of BR-fed mice was significantly lower than those of

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WR- and control-fed mice (P

Black Rice with Giant Embryo Attenuates Obesity-Associated

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