consumption of sericin reduces serum lipids, ameliorates glucose tolerance and elevates serum...

Consumption of Sericin Reduces Serum Lipids, Ameliorates Glucose Toleranceand Elevates Serum Adiponectin in Rats Fed a High-Fat Diet

Yukako OKAZAKI,1;y Shoko KAKEHI,2 Yonghui XU,2 Kazuhisa TSUJIMOTO,3 Masahiro SASAKI,3

Hiroshi OGAWA,4 and Norihisa KATO2

1Faculty of Human Life Sciences, Fuji Women’s University, Ishikari 061-3204, Japan2Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima 739-8528, Japan3Seiren Co., Ltd., Fukui 918-8560, Japan4Tezukayamagakuin University, Osaka 590-0113, Japan

Received January 26, 2010; Accepted April 27, 2010; Online Publication, August 7, 2010

[doi:10.1271/bbb.100065]

The effect was examined of dietary sericin on the lipidand carbohydrate metabolism in rats fed with a high-fatdiet. The rats were fed with a 20% beef tallow diet withor without sericin at the level of 4% for 5 weeks. Thefinal body weight and white adipose tissue weight wereunaffected by dietary manipulation. The consumptionof sericin significantly reduced the serum levels oftriglyceride, cholesterol, phospholipids and free fattyacids. Serum very-low-density lipoprotein (VLDL)-triglyceride, VLDL-cholesterol, low-density lipoprotein(LDL)-cholesterol and LDL-phospholipids were alsosignificantly reduced by the sericin intake. Livertriglyceride and the activities of glucose 6-phosphatedehydrogenase and malic enzyme, the lipogenic en-zymes, were also reduced by the sericin intake. Dietarysericin caused a marked elevation in serum adiponectin.The consumption of sericin suppressed the increases inplasma glucose and insulin levels after an intraperito-neal glucose injection. These results imply the usefulnessof sericin for improving the lipid and carbohydratemetabolism in rats fed on a high-fat diet.

Key words: sericin; serum lipid; adiponectin; glucosetolerance; rat

The silk protein, sericin, is the main constituent ofcocoon proteins (20–30% of the total cocoon weight)specially synthesized in the middle silk gland of thesilkworm, Bombyx mori. Sericin is mostly removed fromthe cocoon and disposed of without any use when thecocoon is used for making silk textiles. We havepreviously found that sericin had several physiologicalfunctions, including anti-oxidative, anti-colon tumor andanti-skin tumor activities, which appear to be beneficialfor health.1–6) These characteristic effects of sericinmake this protein a valuable natural ingredient for thecosmetic and food industries. We have previouslyspeculated that sericin had dietary fiber-like activity byvirtue of its protease-resistant property.6) Consistentwith this hypothesis, we have demonstrated the stronganti-constipation activity of dietary sericin in ratsreceiving atropine.7) Our recent study also suggests that

undigested sericin in the colon of rats fed with sericinsuppressed colonic oxidative stress and carcinogenesisin 2,4-dimethylhydrazine-treated rats.8) The consump-tion of dietary fiber is known to prevent hyperlipidemiaand improve glucose tolerance.9,10) These facts led us topostulate that a sericin intake would have a favorableeffect on rats fed a high-fat diet. The present study wasconducted to test this possibility by examining the effectof dietary sericin on the lipid and glucose metabolism inrats fed on a high-fat diet.

Materials and Methods

Animals and diets.Male Sprague Dawley rats (5 weeks of age) were

purchased from Hiroshima Laboratory Animal Center (Hiroshima,

Japan) and maintained according to the ‘‘Guide for the Care and Use of

Laboratory Animals’’ established by Hiroshima University and

approved by the ethics committee of the same university. The animals

were individually housed in an air-conditioned room at 23–24 �C with

a 12-h light cycle (light, 8:00–20:00). After being fed on a stock diet

(MF, Oriental Yeast Co., Tokyo, Japan) for 7 d, the rats were assigned

to two groups of 12–13 rats each (the control group and sericin group).

The composition of the basal diet (% w/w) was beef tallow, 20; casein,

24; L-cystine, 0.2; cellulose, 5; sucrose, 20; corn starch, 26.3; vitamin

mixture, 1; and salt mixture, 3.5.11) One group of rats was fed with the

basal diet, while the other group was fed with the 4% (w/w) sericin

diet. The method for preparing sericin was as described elsewhere.5)

The amino acid composition of casein and sericin was determined by

an LC 10A amino acid analyzer (Shimazu, Kyoto, Japan) (Table 1).

The level of dietary protein was adjusted by reducing dietary casein.

The same amount of each experimental diet was incorporated daily

into food cups at 7:00 p.m. (9 g for days 1–4, 11 g for days 5–7, 15 g for

days 8–13, 18 g for days 14–21, 19 g for 22–27, 20 g for 28–32, and

21 g for days 33–35) to prevent any difference of food intake by the

two groups. All of the diet was consumed each day until the next day’s

diet was served. Feces were collected for the final 3 d. At the end of the

feeding period, the rats were sacrificed by decapitation under

anesthesia by diethyl ether. Blood was collected and the serum was

separated by centrifugation at 2;000� g for 20min. The liver and

epididymal and perirenal adipose tissues were quickly removed,

weighed and stored at �80 �C prior to being analyzed.

Measurements. Hepatic lipids and the hepatic activities of glucose

6-phosphate dehydrogenase (G6PD, EC, 1.1.1.49), malic enzyme (ME,

EC, 1.1.1.40) and carnitine palmitoyltransferase I (CPT I, EC,

2.3.1.21) were determined as previously described.12) Serum lip-

oproteins were isolated by ultracentrifugation by the method of Ogawa

y To whom correspondence should be addressed. Fax: +81-133-74-8344; E-mail: yokazaki@fujijoshi.ac.jpAbbreviations: VLDL, very-low-density lipoprotein; LDL, low-density lipoprotein; HDL, high-density lipoprotein; TBARS, thiobarbituric acid-

reactive substances; G6PD, glucose 6-phosphate dehydrogenase; ME, malic enzyme; CPT I, carnitine palmitoyltransferase I

Biosci. Biotechnol. Biochem., 74 (8), 1534–1538, 2010

et al.13) Serum triglyceride, cholesterol and phospholipids, and the

levels of these lipids in the individual lipoprotein fractions were

determined by using enzymatic kits (Wako Pure Chemicals, Osaka,

Japan). Fecal total lipids were gravimetrically determined after the

fecal lipids had been extracted by the method of Folch et al.14) The

apparent digestibility of dietary fat was calculated as (intake of dietary

fat� fecal total lipids)/intake of dietary fat� 100. The serum

concentrations of adiponectin, leptin and resistin were determined by

using ELISA kits (one kit for adiponectin, manufactured by Otsuka

Pharmaceutical Co., Tokyo, Japan; and kits for leptin and resistin,

manufactured by B-Bridge International, USA). Lipid peroxidation in

the serum and liver was estimated by measuring thiobarbituric acid

reactive-substances (TBARS).15,16) An intraperitoneal glucose toler-

ance test was performed on day 24 (13:00–16:00) of the experiment

after 6 h of food deprivation. A D-glucose solution was injected

intraperitoneally at a dose of 2 g/kg of body weight. Blood samples

were taken from the tail vein at zero time, and 30, 60 and 120min after

the injection. Plasma glucose and insulin were measured by using

commercial kits (Wako Pure Chemicals, Osaka, Japan, and Shibayagi,

Gunma, Japan, respectively).

Statistical analysis. Data are expressed as the mean� SE. The

statistical significance of the difference between means was analyzed

by Student’s t-test. Results are considered significant at p < 0:05.

Results

There was no difference in final body weight betweenthe control group and sericin group (Table 2). The food

intake was also unaffected by the dietary treatment.Dietary sericin significantly reduced the serum levelsof triglyceride (�33%), cholesterol (�16%), phospho-lipids (�18%) and free fatty acids (�27%) (Table 2).Serum VLDL-triglyceride (Fig. 1A), VLDL-cholesterol,LDL-cholesterol (Fig. 1B) and LDL-phospholipids(Fig. 1C) were also significantly reduced by the sericinintake. The reduction in serum triglyceride appeared tobe in large part ascribable to the reduction in VLDL-triglyceride. Serum TBARS was unaffected by dietarysericin.The hepatic level of triglyceride was markedly

reduced (�43%) by the sericin intake (Table 3), whilethe levels of cholesterol and phospholipids were un-affected by dietary manipulation. There was a significantcorrelation between the hepatic triglyceride concentra-tion and serum triglyceride (r ¼ 0:93, p < 0:05). Thehepatic activities of G6PD and ME, which are lipogenicenzymes, were suppressed by sericin consumption. Thehepatic triglyceride concentration was correlated withthe activities of G6PD and ME (G6PD: r ¼ 0:94,

Table 1. Amino Acid Composition of Sericin

Amino acidCasein Sericin

Molar percent

Asp 6.4 19.1

Thr 4.9 6.0

Ser 5.3 30.4

Glu 19.7 4.1

Pro 11.6 0.8

Gly 3.3 12.2

Ala 4.6 4.6

Cys 0.2 <0:05

Val 7.1 2.6

Met 2.6 <0:05

Ile 5.6 1.4

Leu 8.7 0.6

Tyr 4.1 3.8

Phe 4.2 0.4

His 2.6 0.9

Lys 6.4 10.2

Arg 2.6 2.8

Values are means by triplicate analyses.

Table 2. Effect of Dietary Sericin on Serum Lipids in Rats Fed on aHigh-Fat Diet

Control Sericin

Final body weight (g) 336� 4 327� 3

Food intake (g/35 d) 580� 0 580� 0

Serum

Triglyceride (mg/100ml) 384� 42 259� 28�

Cholesterol (mg/100ml) 97� 4 81� 3�

Phospholipids (mg/100ml) 232� 11 191� 7�

Free fatty acids (mmol/100ml) 79� 6 58� 8�

TBARS (mmol/100ml) 3:90� 0:37 3:95� 0:42

Each value is the mean� SE (n ¼ 12{13).�Significantly different from the control group by Student’s t-test

(p < 0:05).

TBARS, thiobarbituric acid-reactive substances

A

B

C

0

50

100

150

200

250

300

350

VLDL LDL HDL

Control

Sericin

Tri

glyc

erid

e(m

g/10

0 m

l)

*

0

510

15

2025

30

3540

45

VLDL LDL HDLC

hole

ster

ol(m

g/10

0 m

l)

* *

0

10

20

30

40

50

60

70

80

90

VLDL LDL HDL

*Pho

spho

lipid

s(m

g/10

0 m

l)

Fig. 1. Effect of Dietary Sericin on Serum Lipoprotein Lipids(A, Triglyceride; B, Cholesterol; C, Phospholipids) in Rats Fed ona High-Fat Diet.

Each value is the mean� SE (n ¼ 12{13). �Significantly differentfrom the control group by Student’s t-test (p < 0:05).

Effect of Sericin on Lipid and Glucose Metabolism 1535

p < 0:001; ME: r ¼ 0:93, p < 0:001). The hepaticCPT I activity was not affected by the sericin intake,and liver TBARS was unaffected by dietary sericin(Table 3). Dietary sericin caused an elevation in fecaldry weight (p < 0:05), but there was no difference infecal total lipids between the control and sericin groups.The apparent digestibility of dietary fat was alsounaffected by dietary manipulation. There was nodifference in adipose tissue weights (epididymal andperirenal adipose tissues) between the two groups.

Dietary sericin caused a 64% elevation in theserum adiponectin concentration (p < 0:05), but withoutaffecting leptin and resistin (Fig. 2A, B, and C,respectively). The serum adiponectin concentrationwas inversely correlated with the triglyceride concen-tration in the serum and liver (serum: r ¼ �0:78,p < 0:05; liver: r ¼ �0:76, p < 0:05).The plasma glucose (Fig. 3A) and insulin (Fig. 3B)

levels 30min after the glucose injection were signifi-cantly lower in the rats fed on the sericin diet than inthose fed on the control diet. There was no difference inthese parameters at zero time after the injection.

Discussion

This study demonstrated for the first time that dietarysericin lowered the levels of serum triglyceride andcholesterol in rats fed on a high-fat diet. The suppressionof serum triglyceride was mainly ascribed to a reductionin VLDL-triglyceride. In addition, the sericin intakeresulted in lower concentrations of serum cholesterol,VLDL-cholesterol and LDL-cholesterol, but withoutaffecting HDL-cholesterol. Since blood VLDL-trigly-ceride and LDL-cholesterol are considered to be riskfactors for arteriosclerosis, sericin intake may bebeneficial for preventing arteriosclerosis by reducingVLDL and LDL lipids.

The results of this study further indicate a significantcorrelation between the hepatic triglyceride concentra-tion and serum triglyceride. It is therefore postulated thatthe preventive effect of dietary sericin on hepatic lipid

accumulation may contribute to the decrease in hepatictriglyceride secretion to the serum. The fecal excretionand apparent digestibility of lipids did not differ betweenthe control and sericin groups. The triglyceride-loweringeffect of dietary sericin could therefore not be accountedfor by a mechanism involving the digestibility of dietaryfat. The serum level of free fatty acids and the hepaticactivities of ME and G6PD in the present study weresignificantly reduced by the sericin intake, whereas theactivity of CPT I, the rate-limiting enzyme of fatty acidoxidation, was not affected by dietary sericin. Theelevation of hepatic lipogenesis and release of free fattyacids from adipose tissue to the liver lead to theformation of fatty liver that is accelerated by increasedesterification to triglyceride.17) These results suggest thatdietary sericin suppressed high-fat feeding-mediatedfatty liver by a mechanism involving the suppressionof hepatic lipogenesis and a lower level of serum freefatty acids.Our previous studies have reported that a dietary

addition of 3% sericin significantly suppressed thecolonic oxidative stress markers in 1,2-dimethylhydra-

Table 3. Effect of Dietary Sericin on Liver Lipids, the EnzymesRelating to Lipid Metabolism and Digestion of Dietary Fat in Rats Fedon a High-Fat Diet

Control Sericin

Liver

Weight (g) 13:8� 0:3 13:1� 0:2

Triglyceride (mg/g of tissue) 23:5� 2:0 15:7� 1:3�

Cholesterol (mg/g of tissue) 1:74� 0:19 1:82� 0:23

Phospholipids (mg/g of tissue) 22:5� 0:5 22:7� 0:4

TBARS (nmol/g of tissue) 155� 2 162� 6

G6PD activity (mmol/min�g of tissue) 8:96� 0:53 7:20� 0:47�

ME activity (mmol/min�g of tissue) 5:78� 0:28 4:80� 0:28�

CPT I activity (mmol/min�g of tissue) 0:15� 0:02 0:15� 0:02

Feces

Dry weight (g/3 d) 3:4� 0:1 4:1� 0:1�

Total lipids (mg/3 d) 237� 10 234� 6

Apparent digestibility of dietary fat (%) 98:1� 0:1 98:1� 0:1

Adipose tissue (epididymalþ perirenal)

Weight (g) 14:2� 0:7 12:8� 0:9

Each value is the mean� SE (n ¼ 12{13).�Significantly different from the control group by Student’s t-test

(p < 0:05).

TBARS, thiobarbituric acid-reactive substances

A

B

C

0

100

200

300

400

500

600

Control Sericin

*

Seru

m a

dipo

nect

in (

ng /

100

ml)

0

10

20

30

40

50

Control Sericin

Seru

m le

ptin

(ng

/ 10

0 m

l)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Control Sericin

Seru

m r

esis

tin

(µg

/ 10

0 m

l)

Fig. 2. Effect of Dietary Sericin on Serum Adipocytokines (A,Adiponectin; B, Leptin; C, Resistin) in Rats Fed on a High-Fat Diet.

Each value is the mean� SE (n ¼ 12{13). �Significantly differentfrom the control group by Student’s t-test (p < 0:05).

1536 Y. OKAZAKI et al.

zine-treated animals.2,8) This antioxidative activity ofsericin appears to be partly mediated by its chelationwith metal ions through its hydroxyl (serine) andcarboxyl (aspartic acid) groups. Since oxidative stresshas been considered to be associated with hyperlipide-mia, hepatic steatosis and insulin resistance,18) weexamined whether dietary sericin could reduce the indexof oxidative stress (TBARS) in the blood and liver ofrats fed on a high-fat diet. The results indicate noinfluence of dietary sericin on serum and liver TBARS.

Of interest was the finding that dietary sericinmarkedly elevated the serum level of adiponectin.Adiponectin has favorable functions for regulatingglucose and lipid metabolism in both skeletal muscleand liver. It prompts fatty acid oxidation and glucoseutilization in skeletal muscle by activating AMP-activated protein kinase (AMPK).19) Adiponectin hasalso been reported to down-regulate the expression ofsterol regulatory element-binding protein (SREBP) 1c,the master regulator which up-regulates hepatic lipo-genic enzymes, and to improve insulin sensitivity.19–21)

Although it is necessary to test if dietary sericin canaffect the serum adiponectin concentration in a time-dependent manner and influence the non-fasting plasmainsulin level, our results imply that the increase in serumadiponectin by sericin intake may, at lease in part,contribute to the protective effect against hyperlipide-mia, fatty liver and glucose intolerance in rats fed on ahigh-fat diet.

The results of this study demonstrate that supplemen-tal sericin ameliorated glucose tolerance and suppressed

insulin secretion in rats fed on a high-fat diet. Theelevation of serum free fatty acids induced by a high-fatdiet has been suggested to be frequently associated withinsulin resistance.22–24) This elevation has been reportedto suppress the insulin action of insulin receptorsubstrate 1 (IRS1)-associated phosphatidylinositol 3-kinase (PI3K) activity, and to decrease glucose utiliza-tion in peripheral tissue.25) Taken together, the suppres-sive effect of sericin on the serum level of free fattyacids may be associated with an increase in glucoseuptake by the peripheral tissue, resulting in improvedglucose tolerance. PI3K activates serine/threonine pro-tein kinase (Akt) which is involved in the translocationof glucose transporter 4 (GLUT4) to the cell membrane.Further studies are in progress to investigate whethersupplemental sericin can increase glucose transport inthe tissue by enhancing PI3K Akt-mediated GLUT4translocation. We have recently studied the response ofplasma glucose for 120min in rats after an oraladministration of a glucose solution with or withoutvarious concentrations of sericin (Okazaki et al.,unpublished data). The results indicated no influenceof sericin on the plasma glucose response. The possi-bility that sericin can directly suppress intestinal glucoseabsorption thus appears to be negated.In conclusion, our study has provided the first

evidence for dietary sericin reducing serum and hepaticlipids, improving glucose tolerance and elevating serumadiponectin concentration in rats fed on a high-fat diet.These results imply a beneficial effect of sericin on high-fat diet-induced metabolic syndrome. The protein sericinhas a high content of serine (30% of total amino acids)and very low content of methionine (Table 1). A recentstudy has reported that dietary supplementation withserine attenuated the plasma homocysteine-raising effectof dietary methionine.26) An elevated level of homo-cysteine is known to promote insulin resistance.27,28) It isthus of interest to test whether the promoting effect ofsericin on insulin sensitivity would be partly mediatedby its high content of serine relative to methionine.Further studies are required to examine if the specificamino acid pattern and the protease-resistant property ofsericin are related to the mechanism for the effects onlipid and carbohydrate metabolism.

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A

B

0

50

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350

400

0 30 60 90 120

Time (min)

Pla

sma

gluc

ose

(mg

/ 100

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