Trace Nutrients Research 33 : 43−48（2016）

Original Article

Dietary Oyster-Extract Decreases Cholesterol Content by Suppressing hepatic
HMG-CoA Reductase mRNA Expression Level and Enhancing Fecal Steroids
Excretions in Rats Fed High-Fat Diet
Anna Matsudo1）, Naoki Nishimura1）, Ryota Hosomi†, 1）, Shin Harumatsu2）, Suguru Fukuda2）,
Yoshikazu Matsuda2）, Munehiro Yoshida1） and Kenji Fukunaga1）
1）
Laboratory of Food and Nutritional Sciences, Faculty of Chemistry,
Materials and Bioengineering, Kansai University＊
2）
Central Research Institute, Japan Clinic Co., Ltd.＊＊

SummaryThis study demonstrated the effect of oyster-extract on hepatic lipid accumulation and cholesterol metabolism inrats fed high-fat diet. Male Wistar rats were divided into two groups and fed an AIN-93G modified high-fat diet(control diet) and 5% (w/w) oyster-extract containing diet (OE diet) for four weeks. Cholesterol metabolism was measured through serum and liver cholesterol contents, fecal neutral and acidic steroids excretions, and hepatic mRNA
expression levels of enzymes and nuclear receptors involved in cholesterol homeostasis. Dietary oyster-extract decreased hepatic cholesterol contents, perhaps through increasing fecal neutral and acidic steroids excretions and decreasing hepatic 3-hydroxy-3methylglutaryl-coenzyme A reductase (HMGCR) and acyl-coenzyme A:cholesterol acyltransferase-1 expression levels. In addition, the suppression of HMGCR expression in the liver could be through the
reduction of sterol regulatory element binding factor-2 pathway due to oyster-extract intake. This study found that
dietary administration of oyster-extract has hypocholesterolemic effects that may help prevent nonalcoholic fatty
liver disease.

Introduction

insulin resistance, hypertension, and dyslipidemia5）. The
progression of NAFLD is also partly linked to unhealthy

Nonalcoholic fatty liver disease (NAFLD) is one of the

dietary pattern6）. Therefore, dietary therapy is important

most common causes of liver disease worldwide and af-

and could be considered as the first choice treatment or at

fects 20% to 30% of the of the general population1）. The

least as important as medical therapy.

prevalence of NAFLD has grown proportionally with the

Oysters (Crassostrea gigas) contain many nutrients andrise in unfavorable food intake, patterns, sedentary life-

are widely consumed in Japan. The main components ofstyle, obesity, and metabolic syndrome. NAFLD is a condi-

oyster-extract include zinc, glycogen, and taurine, and oys-tion ranging from benign hepatic lipid accumulation toter-extract powder is currently being marketed as a sup-steatosis combined with inﬂammation. NAFLD can prog-

plement. The nutritional benefits of oyster-extract forress to non-alcoholic steatohepatitis (NASH), characterized

health maintenance and lifestyle-related diseases haveby inflammation, apoptosis, and ballooning degeneration.

been reported7, 8）. Our previous findings suggested that

NAFLD carries a 20%-50% risk for progressive fibrosis,

oyster-extract powder suppressed the initiator action of

30% risk for hepatic cirrhosis, and 5% risk for hepatic car-

carcinogens in mice and promoted the recovery of proxi-

cinoma2‒4）. NAFLD may be considered the hepatic event

mal tubular epithelial cell function in p-aminophenol-in-in the metabolic syndrome and is therefore linked withduced nephrotoxicity in rats9, 10）. However, few studies

common metabolic syndrome risk factors such as obesity,

have focused on the effects of dietary oyster-extract on


＊
＊＊
†

Address : 3-3-35, Yamate-cho, Suita, Osaka, 564-8680, Japan
Address : 10-1, Kainichi-cho, Uzumasa, Ukyo-ku, Kyoto, 616-8555, Japan orrespondence to: Ryota Hosomi, Faculty of Chemistry, Materials and Bioengineering, Kansai University, Suita,
COsaka, 564-8680, Japan.
Tel: +81-6-6368-1765, Fax: +81-6-6388-8609, E-mail: hryotan@kansai-u.ac.jp.
― 43 ―

the hepatic lipid accumulation in rats fed high-fat diet. The

Food consumption and body weight were recorded dai-

aim of this study was to investigate the effect of dietaryly. After feeding for 28 days, rats were weighed and sacri-oyster-extract on serum and liver lipid contents in rats fed

ficed under isoflurane (Intervet K.K.; Osaka, Japan) anes-

high-fat diet and examine the mechanisms underlying

thesia for 09:00-11:00. Blood was collected from thethese effects.

abdominal aorta without the use of anti-coagulant, andserum was obtained by centrifugation at 2,000×g for

Materials and Methods

15 min. The liver and abdominal white adipose tissue(WAT) from the epididymis, mesentery, perinephria, and

1. Materials

retroperitoneum were removed rapidly, then weighed,

Oyster-extract was obtained from Japan Clinic Co. Lt.

rinsed with saline, and frozen in liquid nitrogen, followed(Kyoto, Japan). Casein, dextrinized cornstarch, cornstarch,by storage at －80℃ until analysis. Feces were collectedsucrose, cellulose, American Institute of Nutrition (AIN)-

from each group every 24 hours for 7 days before being

93G mineral mixture, and AIN-93 vitamin mixture weresacrificed, dried to a constant weight, and ground to a finepurchased from Oriental Yeast Co. Ltd. (Tokyo, Japan). All

powder using a conventional mill. Rats were not deprived

other reagents were purchased from commercial sourcesof food prior to being sacrificed because food deprivation

and were reagent grade.

leads to a significant down regulation of the genes involved

2. Animals care and experimental diets

in

fatty

acid

synthesis

andcholesterol

metabolism12）.

The experimental protocol was reviewed and approvedby the Animal Ethics Committee of Kansai University and3. Analysis of lipid components

followed the “Guide for the Care and Use of Experimental

Serum trlacylglycerol (TAG), cholesterol, phospholipid

Animals” issued by the Prime Minister’s Office of Japan.

(PL),

Male 4-week-old Wistar rats obtained from Japan SLC,

non-HDL-cholesterol contents were determined using aInc. (Shizuoka, Japan) were kept in an air-conditioned

commercial service (Japan Medical Laboratory, Osaka,

room (temperature, 21-22℃; humidity, 55-65%; lights on,

Japan).

high-density

lipoprotein(HDL)-cholesterol,and

08:00-20:00), with free access to tap water and feed.

Total liver lipids were extracted using the chloroform/

Twelve rats were divided into the following 2 dietarymethanol/water method as described previously13）. Eachgroups of 6 rats each: control diet (AIN-93G modified high-total lipid sample was dilute to 10 mL with 2-propanol, andfat diet) and oyster-extract diet (OE diet). Table 1 presents

the TAG content was determined using an enzymatic as-

the composition of the experimental diets .

say kit (Triglyceride-E-Test Wako, Wako Pure Chemical

11）

Industries; Osaka, Japan). The cholesterol content in liver
was analyzed using a gas–liquid chromatograph (GC) sys-

Table 1 Composition of the experimental dietstem (GC-14B; Shimadzu Co.) with an SE-30 column

Groupscontrol
Dextrinized corn starchCorn starch
SucroseCellulose
Casein
L -Cystine
Choline bitartrate
AIN-93G mineral mixture
AIN-93 vitamin mixture
Soybean oil
Lard
Oyster-extract†tert-Butylhydroquinone

92.1
277.386
100
50
230
3
2.5
35
10
70
130
－
0.014

(Shinwa Chemical Industries, Kyoto, Japan) and 5α-choles-

OE
g/kg

tane as an internal standard14）. The phospholipid contents
were determined by phosphorus analysis15）.

92.1
241.286
100
50
216.8
3
2.5
35
10
70
129.3
50
0.014

Fecal neutral steroids content was determined by GC as
described above. The acidic steroids content in feces wasmeasured using an enzymatic assay commercial kit (Total
Bile Acid Test Kit; Wako Pure Chemical Industries Ltd.)according to the manufacturer’s method.
4. Analysis of mRNA expression level
Total RNA was extracted from liver using TRIzol®
RNA Isolation Reagents (Thermo Fisher Scientific K.K.,
Kanagawa, Japan). Then, cDNA was synthesized from to-

Oyster-extract provided by Japan Clinic Co., Ltd. (Kyoto,
Japan). The composition of oyster-extract was as follows (%w/w): carbohydrate, 61.0; protein, 22.4; fat, 1.2; moisture, 3.5;
and ash, 11.9.
AIN, American Institute of Nutrition.

†

tal RNA using a GoScript™ Reverse Transcription System(Promega KK., Tokyo, Japan). Real-time quantitative PCR
analysis was performed with an automated sequence detection system (Thermal Cycler Dice® Real Time System― 44 ―

Single; Takara Bio Inc., Shiga, Japan) using GoTaq® qPCR

5’-TGCCAGAGTGTTGTCCTCAG-3’; for SREBF-2, for-

Master mix (Promega KK.). The primer sequences used

ward: 5’-ATGACTCTACCCACGGCAAG-3’ and reverse:

for the detection of the ATP-binding cassette A1 (ABC)5’-TACTCAGCACCAGCATCACC-3’; for GAPDH, which

A1, ABCG5, ABCG8, acyl-coenzyme A:cholesterol acyl-

were designed using Pimer3Plus (http://primer3plus.

transferase-17α-hydroxylasecom/). Results were quantified using a comparative meth-(CYP7A1), 3-hydroxy-3-methylglutaryl coenzyme A reduc-od and were expressed as a relative level after normaliza-tase (HMGCR), low-density lipoprotein receptor (LDLR),

tion to the GAPDH expression level.

(ACAT-1),cholesterol

scavenger receptor class B type 1 (SR-B1), sterol regulatory element binding factor (SREBF)-2, and glyceraldehyde

5. Statistical analysis

3- phosphate dehydrogenase (GAPDH) were as follows:

Data represent means and standard error of mean

forward: 5’-CCCGGCGGAGTAGAAAGG-3’ and reverse:(SEM). The statistical significance of differences was eval-5’-AGGGCGATGCAAACAAAGAC-3’; for ABCA1, for-uated using Student’s t-test and means were considered

ward: 5’-ATGGCCTGTACCAGAAGTGG-3’ and reverse:

significantly different at p < 0.05 and p < 0.01. Analyses

5’-GGATACAAGCCCAGAGTCCA-3’; for ABCG5, for-were

ward: 5’-TTCTGCTGCAACGCTCTCTA-3’ and reverse:(GraphPad Software, California, USA).

performed

using

GraphPad

Prism6software5’-GGAGGAACGACATCTTGGAA-3’; for ABCG8, for-Results and Discussion

ward: 5’-AAGTACGCCATCGGCTCTTA-3’ and reverse:
5’-TCACCACCACGTCTGGTTTA-3’; for ACAT-1, forward: 5’-CCCAGACCCTTTGACTTTCA-3’ and reverse:

Oyster-extract has been reported the various beneficial

5’-GATCCGAAGGGCATGTAGAA-3’; for CYP7A1, for-effects7, 8）. Our previous studies suggested that dietaryward: 5’-AGAATATAGCGCGTGGGATG-3’ and reverse:

oyster-extract recovered proximal tubular epithelial cell

5’-GACATACAGCCAAAGCAGCA-3’; for HMGCR, for-function in p-aminophenol-induced nephrotoxicity and sup-ward: 5’-ACCGCCATGAGGTACGTAAG-3’ and reverse:pressed of the initiator action of the carcinogen in animal

5’-CGGCGCTGTAGATCTTTCTC-3’; for LDLR, forward:

experiment9, 10）. The present study examined the effects of5’-AAGAAGGCCTCTTACTCCGC-3’

dietary oyster-extract on hepatic cholesterol metabolism

andreverse:5’-GTCTAGCCCCACGTTGATGT-3’; for SRB1, forward:
5’-CACCTGTGGAGCAGTCTCAA-3’

andin normal rats fed high-fat diet.

reverse:Table 2 shows the growth parameters, organ weights,

Table 2 Growth parameters, organ weights, and serum lipid parameters in rats fed experimental diets for 4 weeks
control

OE

Growth parameters
Initial BW (g)Final BW (g)BW gain (g/day)Food intake (g/day)
Water intake (mL/day)Food efficiency (g/g)†

91.8 ± 2.2
305.4 ± 7.0
7.63 ± 0.29
17.0 ± 1.2
39.6 ± 5.6
0.450 ± 0.017

93.9 ± 1.4
312.1 ± 7.6
7.79 ± 0.28
17.4 ± 1.4
43.0 ± 5.2
0.448 ± 0.016

Relative organ weight (g/100g BW)
Liver
Epididymal WAT
Mesentery WAT
Perirenal and retroperitoneal WAT

4.16 ± 0.14
1.60 ± 0.05
1.31 ± 0.12
1.32 ± 0.07

4.01 ± 0.12
1.73 ± 0.09
1.61 ± 0.22
1.49 ± 0.10

Serum lipid parameters (mg/dL)
TAG
CholesterolPL
HDL-cholesterol
Non-HDL-cholesterol

95.3 ± 16.4
88.8 ± 5.6
159 ± 860.0 ± 4.5
28.8 ± 1.2

112.3 ± 11.0
103.3 ± 7.6
179 ± 1072.2 ± 6.6
31.2 ± 1.4

Data represent means±SEM (n = 7).
Food efficiency (g/g) = BW gain (g/day) / food intake (g/day); BW, body weight; HDL, high-density lipoprotein; OE, oyster-extract; PL, phospholipid; TAG, triacylglycerol, WAT, white adipose tissue.

†― 45 ―

and serum lipid contents in rats fed OE diet. The growthol contents between the two groups. Fig. 1 shows the he-parameters including the initial body weight, final bodypatic TAG, cholesterol, and PL contents in rats fed OE

weight, body weight gain, food intake, water intake, anddiet. The hepatic TAG and PL contents were not found tofood efficiency were not found to be significantly different

be significantly different between the experimental

between the experimental groups. There were also no sig-groups. Interestingly, OE diet significantly decreased thenificant differences in relative liver, epididymal, mesenter-hepatic cholesterol content compared with control diet.

ic, perirenal, and retroperitoneal WAT weights between

Dietary oyster-extract could decrease the hepatic choles-

the groups. The serum cholesterol, PL, and HDL-

terol content in rats fed high-fat diet. Two main mecha-

cholesterol contents in the rats fed OE diet tended to benisms have been proposed to explain the decreases inhigher, but not significantly, than that in the rats fed con-cholesterol levels in the serum and liver that are associat-

trol diet (p = 0.15, 0.14, and 0.15, respectively). Previous

ed with dietary food components. One is an increase instudy reported that basal diet contained taurine, supple-

excretion of steroids, including neutral and acidic steroids;

mented at level of 0.5% (w/w), enhanced the serum HDL-

the other is a change in cholesterol metabolism in the he-cholesterol content in normal rats . In the OE diet, tau-patic tissue, which is maintained through a balance of ex-

rine was contained about 0.3% (w/w) because oyster-extract

cretion, biosynthesis, storage, catabolism, and uptake ofcontained 5.6% (w/w) taurine17）. Therefore, the increasing

steroids. To test the first mechanism, we measured thetendency of HDL-cholesterol content in the OE groupfecal neutral and acidic excretions in rats fed OE diet. Fig.

might be caused by the taurine. No significant differences

2 shows the fecal neutral and acidic steroids contents inwere observed in the serum TAG and non-HDL-cholester-rats fed OE diet. The fecal neutral and acidic steroids con-Fig. 1 H epatic lipids contents in rats fed experimental diets for
4 weeks
Data represent means±SEM (n = 7). Values are statistically different compared with the control diet at*p < 0.05 using Student’s t-test.
PL, phospholipid; TAG, triacylglycerol.

Fig. 2 Fecal steroids of neutral and acidic excretions in rats fed
experimental diets for 4 weeks
Data represent means±SEM (n = 7). Values are statistically different compared with the control diet at*p < 0.05 and **p < 0.01 using Student’s t-test.

16）

Fig. 3 H epatic relative mRNA expression levels of genes encoding proteins involved cholesterol metabolism in rats fed experimental
diets for 4 weeks
Data represent means±SEM (n = 7). Values are statistically different compared with the control diet at *p < 0.05 and**p < 0.01 using Student’s t-test.
The mRNA expression levels were determined by real-time PCR analysis using the GAPDH mRNA expression level for normalization. The mRNA expression levels of genes are shown relative to those determined from the livers of rats fed the control diet (set at 100).
ABC, ATP-binding cassette; ACAT, acetyl-CoA acetyltransferase; CYP7A1, cholesterol 7α-hydroxylase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HMGCR, 3-hydroxy-3-methyl-glutaryl-CoA reductase; LDLR, low-density-lipoprotein receptor; SR-B1, scavenger receptor class B member 1; SREBF, sterol regulatory element-binding transcription factor― 46 ―

tents in the OE group were significantly higher than that

(Fig. 3). This result suggests that oyster-extract decreases

in the control diet. Previous study showed that diet con-

HMGCR expression level through the suppression of thetaining 3% (w/w) taurine decrease the serum and liver

SREBF-2 pathway.

cholesterol contents in part due to enhance the fecal bile

The major components of the oyster-extract were min-acid excretion18）. Taurine in the OE diet might affect theerals,

fecal steroids excretion, however, it was possible to consid-

Although taurine was a convincing component as a bioac-

er that a components other than taurine enhances the fe-tive component, it was not determined which components

cal steroids excretions because taurine was containedof the oyster-extract affected the cholesterol metabolism

about 0.3% (w/w) in OE diet. Further investigation is re-in rats. Therefore, future studies will determine whichquired to clarify which component in the oyster-extract iscomponents of the oyster-extract are responsible for theenhanced the fecal steroids excretions. Consequently, it

improvement of cholesterol metabolism.

carbohydrates,

protein,

and

amino

acids7‒10）.

has been suggested that the decreases in hepatic choles-

Conclusions

terol in rats fed OE diet could be attributable to the suppression of neutral and acidic steroids absorption in intestine through the enhancement of fecal neutral and acidic
steroids excretions.

Dietary oyster-extract led to decreased hepatic cholesterol content through the enhancement of fecal steroids

To test the second proposed mechanism, we analyzed

excretions and the suppression of HMGCR and ACAT-1

hepatic mRNA expression levels of genes encoding pro-

expression levels due to the suppression of the SREBF-2

teins involved in cholesterol metabolism. Fig. 3 shows thepathway. Therefore, the oyster-extract could be prevented

hepatic mRNA expression levels of genes related to cho-

the hepatic cholesterol accumulation under high-fat dietlesterol metabolism in rats fed OE diet. ABCA1 is a trans-conditions.

porter involved in the production of HDL, CYP7A1 is a

Acknowledgements

rate-limiting enzyme involved in the conversion of cholesterol to 7α-hydroxylated bile acid, ABCG5 and ABCD8 areheterodimer forms play major roles in the excretion of

We would like to thank Kazumasa Miyauchi of Kansaicholesterol into bile, SR-B1 is responsible for the selective

University for him help with animal care and lipid analy-

uptake of HDL, and LDLR is required for LDL uptake intoses.

cells . There were no differences in the expression levels
19）

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