Anti-Obese Activity of HPJ Extract on High Fat Diet-Induced Obese Mice

고지방 식이로 유도된 비만 쥐에서 HPJ 추출물의 항비만 효과

원해단·권해연·장아·김성집·신대희

*

^·임방호

*

^·정성현#

경희대학교 약학대학 약물학·임상약학교실, *(주)휴온스 중앙연구본부

(Received June 17, 2009; Revised July 27, 2009; Accepted August 10, 2009)

Anti-Obese Activity of HPJ Extract on High Fat Diet-Induced Obese Mice Hai-Dan Yuan, Hai-Yan Quan, Ya Zhang, Sung-Jib Kim, Dae-Hee Shin*, Bang-Ho Lim* and Sung-Hyun Chung

^#

Pharmacology and Clinical Pharmacy Lab, College of Pharmacy, Kyung Hee University, Seoul 130-701, Korea

*Research Complex, Sungkyunkwan University, Cheoncheon-dong, Jangan-gu, Suwon, Gyeonggi-do 440-746, Korea

Abstract

— In this study, we investigated the anti-obese activity of HPJ extract in C57BL/6J mice. The C57BL/6J mice were randomly divided into five groups: normal control group (Con), high fat diet control group (HFD), treatment groups with HPJ at 125 mg/kg (HPJ125), 250 mg/kg (HPJ250), or 500 mg/kg (HPJ500). To induce an obesity, mice were fed by a high fat diet for 6 weeks, and mice were administered with HPJ extract once a day for 8 weeks. At the end of treatment, we examined the effect of HPJ extract on body weight, plasma lipid, and lipogenic enzymes. HPJ extract was found to lower whole body and epididymal adipose tissue weights and lowered plasma levels of glucose, insulin, triglyceride (TG), total cho- lesterol (TC), non-esterified fatty acid (NEFA) and leptin, compared to those in HFD group. Histological analyses of the liver and fat tissues of mice treated with HPJ extract revealed significantly decreased number of lipid droplets and decreased size of adipocytes compared to the HFD group. In addition, HPJ extract preserved the morphological integrity of pancreatic islets. To elucidate an action mechanism of HPJ extract, Western blot and RT-PCR were performed using epididymal adipose tissues. HPJ extract up-regulated the levels of phosphorylated adenosine monophosphate-activated protein kinase (AMPK) and its substrate, acetyl-CoA carboxylasse (ACC). HPJ extract also attenuated lipogenic gene expressions of sterol reg- ulatory element-binding protein 1

^α

(SREBP1

^α

), fatty acid synthase (FAS), sterol-CoA desaturase 1 (SCD1) and glycerol- 3-phosphate acyltransferase (GPAT) in dose-dependent manners. In contrast, expressions of lipolytic genes such as per- oxisome proliferator-activated receptor-

^α

(PPAR-

^α

) and CD36, and fatty acid

^β

-oxidation gene, carnitine palmitoyltrans- ferase-1 (CPT-1) were increased. These results suggest that HPJ extract ameliorates obesity through inhibiting synthesis of lipogenic enzymes as well as stimulating fatty acid oxidation resulting from activation of AMPK, and HPJ extract could be developed as a potential therapeutic agent for obese patients.

Keywords □

HPJ extract, C57BL/6J mice, high fat diet, AMPK, lipogenic and lipolytic enzymes

비만

(Obesity)

은에너지섭취와소비의불균형으로인하여체

지방이과도하게축적되여생기는일종의대사성질환으로서생 활습관과밀접한관련이있다

.

^{1.2)비만은지방대사와당대사에}

불균형을초래하여고지혈증

,

고혈압

,

동맥경화

,

당뇨병

,

지방간

,

관절이상등다양한만성퇴행성질환들을유발하는원인이된다

.

^1.3)

현재비만을개선시키기위한치료전략으로과도한에너지 섭취의제한과적당한운동을권장하고있지만장기간꾸준히해 야한다는어려운점이있어최근에는식욕억제제

(

리덕틸

),

지방

흡수저해제

(

제니칼

)

와같은약물이임상에서사용되고있으나 불면증

,

혈압상승

,

지방변등부작용으로사용에제한을받고있 어이를대체할수있는새로운치료제의개발이요구되고있다

.

^4-7)

한편한의학적관점에서는비만을비습

(

지방과수분

)

과담음

(

체내수액의정체에의한노폐물

)

의과잉축적으로해석하기도 하며이러한대사성질환은예로부터방풍통성산

,

^{방기황기탕등}

의한방처방으로치료하여왔다

.

생약복합추출물인

HPJ

는기 존에시판된제품들과는전혀다른한방처방으로서예로부터사 용되어온비만치료처방의가감방이며택사

,

^시호

,

^저령

,

^대황

,

감초

,

건강

,

육계

,

작약

,

목단피

,

반하

,

승마등

11

종의생약재로 구성되어있다

.

따라서본연구에서는고지방식이로유도된

C57BL/6J

^비만쥐

#본논문에관한문의는저자에게로

(

^전화

) 02-961-0373 (

^팩스

) 02-957-0384

(E-mail) [email protected]

를이용하여

HPJ

^{추출물이체중}

,

^{혈중지질및지방대사에미}

치는영향등을검토하여

HPJ

^{의항비만효과와작용기전을살}

펴보았다

.

실험방법

실험재료

본연구에서사용한

HPJ

추출물은택사

,

시호

,

저령

,

대황

,

감 초등

11

종의생약으로구성되어있으며

(

주

)

휴온스로부터시료 를제공받아사용하였다

. HPJ

^{추출물은각구성생약을일정비}

율로혼합하여추출기에넣고물로

80~100

^o

C

에서

3~4

시간추 출한다음여과하고농축한다음동결건조하여얻은것을제공 받았다

.

^{시험물질은}

HPJ

^{추출물을물에용해하여혼화한것을}

사용하였다

.

실험동물및약물투여

6

주령의

C57BL/6J mouse

를

(

주

)

오리엔트사로부터구입하여

1

^{주일동안고형사료와물을섭취하면서실험실환경에적응시}

킨후본실험에사용하였다

.

^정상군

(Con),

^대조군

(high fat diet

군

, HFD),

실험군

HPJ 125 mg/kg

투여군

, HPJ 250 mg/kg

투여 군과

HPJ 500 mg/kg

^투여군등

5

^{개그룹으로나누었으며정상}

군마우스는

regular diet

^{를섭취시켰고나머지그룹은고지방식}

이를섭취시켰다

. HPJ

투여는고지방식이섭취

6

주후부터시

작하였다

. HPJ

^{는증류수에용해하여}

8

^{주간경구투여하였으며}

몸무게

,

^{식이섭취량및물섭취량은한주에두번씩측정하였다}

.

고지방식이의조성은

Table I

과같다

.

혈액지표분석

혈액지표분석을위한혈액채취는

12

^{시간절식시킨후실}

시하였다

.

⁸⁾채혈한혈액은

3,000 rpm

에서

10

분간원심분리한 혈청을분석에사용하였다

.

^{9)혈중포도당농도는}

glucose oxidase method(Trinder method)

를사용하여측정하였으며

,

흡광도측정 은

UV Spectrophotometer(U-3210, HITACHI

^TM

, Japan)

를사 용하였다

.

^{혈중 인슐린 농도는 마우스}

insulin ELISA kit (Shibayagi, Japan)

를 구입하여

ELISA reader(Labsystems, Finland)

로측정하였다

.

혈중총콜레스테롤

(total cholesterol),

혈 중중성지방

(triglyceride, TG)

^은시판

kit(Stanbio, USA)

^를구입

하여생화학분석기기

(SMARTLAB, USA)

를사용하여측정하였

고 혈중

leptin

은 마우스

leptin ELISA kit(LINCO research, USA)

^{를구입하여측정하였다}

.

Fat부위별무게(내장지방,부고환지방)측정

간

,

^{부고환지방}

, brown adipose tissue

^인

scapular

^부위도같

이적출하여부위별무게및형태를비교하였다

.

조직의형태학적관찰

쥐에서적출한간

,

췌장

,

부고환지방을

10% neutral buffered formalin

^{을사용하여고정}

.

^{이후탈수및포매과정을거쳐파라}

핀블럭을제작하고두께

5

^µ

m

^{의관상절편으로제작한후}

xylene

으로파라핀을제거시키고

, 100%, 95%, 90%, 80%, 70%

알코 올로친수화시킨후

hematoxylin

^과

eosin

^{으로염색하여광학현}

미경

(Olympus, Japan)

^{으로관찰하였다}

.

¹⁰⁾

AMPK인산화측정

부고환 지방에서

Western blot

^방법으로

phospho-AMPK, AMPK, phospho-ACC, ACC

를측정하였다

.

단백질분석을위 해부고환지방조직을

lysis buffer(50 mM Tris-HCL pH=7.5, 1 mM EDTA, 0.25% sucrose, 0.4 mg/m

l

digonin and 1.5 mM PMSF)

를이용하여균질화시켰다

.

¹¹⁾단백질정량은

Bio- Rad assay reagent(Bio-Rad, USA)

^{를이용하여측정하였다}

.

^정량

한단백질

40

µ

g

을

8% SDS-PAGE

로분리한후

gel

을

membrane (Milipore, Cat. No: IPVH00010)

에

transfer

하고

5% skim milk

^{로상온에서}

1

^시간

blocking

^한후

1 : 3000

^{비율로희석시}

킨

primary antibody(P-AMPK, AMPK, P-ACC, ACC)(Cell signaling Technology, Beverly, USA)

와

4

^o

C

에서

overnight

하 였다

.

^다음

Tris-buffered saline tween-20(TBST)

^으로

4

^번

washing

한후

1 : 5000

의비율로희석시킨

secondary antibody (Santa Cruz Biotechnology, Santa Cruz, USA)

와상온에서

1

시 간반응시켰다

.

^이후

TBST

^로

4

^번

washing

^하고

ECL solution (Amersham, Sweden)

을이용하여

X-ray

필름에

developing

하 였다

.

Table I −

Composition of the experimental diet

HFD 45%cal Regular diet

g% kcal% g% kcal%

Protein 24 20 20 21

Carbohydrate 41 35 66 68

Fat 24 45 5 12

kal/kg

^{4,776 3902}

Ingredient g kcal g kal

Casein (from milk) 200 800 200 800

Corn starch 155.036 620 150 600

Sucrose 50 200 500 2000

Dextrose 132 528 0 0

Cellulose 50 0 50 0

Soybean oil 25 225 0 0

Corn oil 175 1575 50 450

Mineral mixture 35 0 35 0

Vitamin mixture 10 40 10 40

TBHQ 0.014 0 0 0

DL-methionine - - 3 12

L-Cystine 3 12 0 0

Choline bitartrate 2.5 0 2.0 0

Total 837.6 4,000 1000 3,902

지방합성에관여하는유전자발현측정

부고환지방조직에서

total RNA

^분리는

guanidine thiocyanate- water saturated phenol/chroloform

분리방법을이용하였다

.

물

층에있는

total RNA

^{는이소프로판올을이용하여침전시켜분}

리한

RNA

는

260 nm

와

280 nm

의파장에서흡광도를측정하여 정량였고

,

총

RNA 10

µ

g

을

Moloney murine leukemia virus transcriptase

^와

Oligo(dT) 15 primer

^{를이용하여역전사하였다}

.

¹²⁾

Primer

의종류및서열은

Table VI

에표시한바와같다

. PCR

반 응조건은

95

^o

C

에서

30

초동안변성

, 30

초동안붙임

(

상응한붙 임온도는

Table VI

^에표시

), 72

^o

C

^에서

30

^{초동안연장을하여}

총

30 cycle

하였다

.

이후 반응 생성물을

0.5

µ

g/m

l

ethidium bromide

로염색된

1% agarose gel

을이용하여

100 V

에서전기

영동하였다

. CPN

^{은증폭된유전자들의대조군으로사용되었다}

.

에너지소모에관여하는유전자발현측정

부고환지방에서 에너지소모에관여하는 유전자인

CPT1

(carnitine palmitoyltransferase 1)

의발현을

RT-PCR

로측정하 였다

. Primer

^{의종류및서열은}

Table VI

^{에표시된바와같다}

. PCR

^{반응조건은}

95

^o

C

^에서

30

^{초동안변성}

, 55

^o

C

^에서

30

^초동

안붙임

, 72

^o

C

에서

30

초동안연장을하여총

30 cycle

하였다

.

이후반응생성물을

0.5

µ

g/m

l

ethidium bromide

로염색된

1%

agarose gel

^{을이용하여}

100 V

^{에서전기영동하였다}

. CPN

^은증

폭된유전자들의대조군으로사용하였다

.

자료분석및통계처리

모든실험결과는평균

±

표준오차로나타내었다

.

당뇨대조군

(DC)

과비교하여통계적유의성을

Student's

t

-test

로처리하였으 며 P

<0.05

^{이하인경우유의성있는차이가있는것으로판정}

하였다

.

실험결과 및 고찰

체중

Fig. 1

^{에서보듯이고지방식이대조군마우스는정상군마우스}

에비하여체중증가가뚜렷하였고개복했을때가장많은내장 지방을드러내었다

.

반면

HPJ

투여군마우스는대조군에비하여 덜뚱뚱하였고내장지방도적었다

.

^{부고환지방역시대조군마}

우스에서제일컸지만

HPJ

투여군에서는농도의존적으로작아

졌음을관찰할수있었다

.

^{하지만간의크기는각그룹에서차이}

가관찰되지않았다

. Table II

^는

8

^주동안

HPJ

^{추출물을경구투}

여한후체중변화에미치는영향을그룹간비교한결과이다

.

대 조군은정상군에비하여체중이

23.1%(

^p

<0.001)

^{유의적으로증}

Fig. 1 −

Gross appearance of whole body, abdomen, epididymal fat and liver.

가하였고

HPJ125, HPJ250, HPJ500

^{투여군은대조군에비하여}

농도의존적으로체중이

5.4%, 6.5%, 6.8%(

p

<0.05)

로유의적으 로감소하였다

.

^{체중증가량역시정상군에비하여대조군에서}

139.6%(

^p

<0.001)

^{유의적으로 증가하였고}

HPJ125, HPJ250, HPJ500

투여군은대조군에비하여

13.9%, 13.9%, 20.9%

감소 하였다

.

^{부고환지방무게는대조군에서정상군에비하여}

174.3%

(

^p

<0.01)

^{유의적으로증가하였고}

HPJ125, HPJ250, HPJ500

^투

여군에서는대조군에비하여

7.4%, 12.3%, 15.3%

농도의존적으 로감소하였다

.

^{갈색지방무게역시대조군에서정상군에비하}

여

64.7%(

^p

<0.01)

^{로유의적으로증가하였으나}

HPJ

^엑스투여

군에서는감소하는경향을보였다

.

이결과는

HPJ

추출물이

High fat diet

^{에의한체중증가를감소시키는효과가있음을나타내었다}

.

식이,음수량및식이효율

Table III

^{는그룹간식이}

,

^음수량

,

^{식이효율및에너지섭취량}

을나타내었다

.

^{대조군의경우정상군에비해식이섭취량은거}

의차이를나타내지않은반면

HPJ

추출물투여군은대조군에

비해용량의존적으로감소함을알수있었다

.

^{음용수섭취량은}

대조군에서정상군에비해

31.6%

^{감소하였고}

HPJ125, HPJ250, HPJ500

투여군에서는대조군에비해

10.4%, 14.1%, 16.1%

로 용량의존적으로상승함을알수있었다

.

^{식이효율은대조군에서}

정상군에비해

140%

증가하였고

HPJ

추출물투여군에서는대

조군에약간의감소는보이고있지만유의적으로차이가나지않 았다

.

^{하지만하루에너지섭취량은정상군에비해대조군에서}

평균

22.3%

증가하였고

HPJ125, HPJ250, HPJ500

투여군에서 는

5.1%, 5.8%, 7.5%

감소함을알수있었다

.

이결과는

HPJ

추

출물이식이섭취억제효과에기인된항비만작용이있을것 으로추측된다

.

혈중포도당,인슐린및인슐린저항성지수

Table IV

는

8

주동안시료를경구투여한후공복시혈당변 화를보여주고있다

.

^{대조군에비해}

HPJ125, HPJ250, HPJ500

투여군은 혈당이

19.1%(**

^p

<0.01), 24.2%(**

^p

<0.01), 26.0%

(**

p

<0.01)

로농도의존적으로감소하였다

.

인슐린수치역시

HPJ125, HPJ250, HPJ500

^{투여군에서 대조군에 비해 각각}

26.0%, 35.0%, 47.2%(

^p

<0.05)

^{감소하는경향을나타냈다}

.

^인슐

린저항성지수역시

HPJ125, HPJ250, HPJ500

투여군에서대조 군에비해각각

28.1%, 39.1%, 51.8%(

^p

<0.05)

^{감소하여농도}

의존적으로감소하는경향을보였다

.

혈액지표분석

비만은흔히고지혈증과동반되어있으며식이에의해유도된 흰쥐에서는혈중중성지방과콜레스테롤이증가된다고보고되고 있다

.

¹³⁾

Table V

^는

8

^{주동안시료를경구투여한후}

, 12

^시간절

식시켜공복상태의실험동물에서전혈을채취한후측정한중 성지방

(TG), total cholesterol(TC),

그리고지방세포에서분비되 는

adipokine

^인

leptin

^{의농도그리고}

NEFA

^{농도를나타낸것이}

다

.

혈중중성지방은대조군에서정상군에비해유의적으로높 았으며

(

p

<0.001) HPJ125, HPJ250, HPJ 500

투여군에서는대조 Table II −

Effects of HPJ extract on whole body, epididymal and brown fat weights

Group Initial Body weight (g) Final Weight gain (g) Epididymal fat weight(g) Brown fat weight (g)

Con 22.5±0.6 27.3±0.3 4.8±0.3 0.74±0.05 0.17±0.02

HFD 22.2±0.3

^###

33.6±0.8

^{### ###}

11.5±0.6

^{### ##}

2.03±0.11

^{## ##}

0.28±0.02

^##

HPJ125 21.7±0.3 *31.6±0.7* 9.9±0.8 1.88±0.13 0.28±0.04

HPJ250 21.5±0.4 *31.4±0.5* 9.9±0.7 1.78±0.16 0.25±0.02

HPJ500 22.2±0.3 *31.3±0.4* 9.1±0.6 1.72±0.03 0.26±0.01

Values represent the mean±SE (n=6).

^##

p <0.01,

^###

p <0.001 vs. Con; * p <0.05 vs. HFD

Table III −

Effects of HPJ extract on food and water intakes and feed efficiency ratio

Group Food intake (g/mouse) Water intake

(ml/mouse) Feed efficiency

ratio Energy intake (kcal/day)

Con 242.1 390.4 0.020±0.004

^###

09.64

HFD 241.2 267.2 0.048±0.006

^###

11.75

HPJ125 226.7 295.0 0.043±0.008

^###

11.05 HPJ250 222.3 311.0 0.042±0.003

^###

10.83 HPJ500 218.4 318.4 0.049±0.003

^###

10.64 Values represent the mean±SE (n=6).

^###

p <0.001 vs. Con

Table IV −

Effects of HPJ extract on fasting plasma glucose and insulin levels, and homeostasis model assessment values for insulin resistance (HOMA-IR)

Group Blood glucose (mg/d l ) Insulin

(mU/m l ) HOMA-IR

Con 100.8±5.0

^###

060.2±12.3 14.37±2.41 HFD 152.7±9.3

^###

235.6±34.1

^###

89.96±16.42

^###

HPJ125 123.5±8.9**0 174.4±39.1 52.57±11.42*

HPJ250 115.7±5.4**0 153.2±20.1 44.34±7.00**

HPJ500 113.0±5.3**0 124.5±20.6* 35.43±7.26**

Values represent the mean±SE (n=6). Homeostasis Model As- sessment was used to calculate an index of insulin resistance as insulin (mU/m l )×glucose (mM)/22.5.

^###

p <0.001 vs. Con, * p <

0.05, ** p <0.01 vs. HFD

군에비해각각

5.8%, 11.4%, 22.4%

^{낮아져서농도의존적으로}

낮아지는 경향을 보였다

.

총 콜레스테롤은

HFD

군에 비해

HPJ125, HPJ250, HPJ500

^{투여군에서 각각}

6.0%(

^p

<0.05), 9.0%(

^p

<0.01), 22.3%(

^p

<0.001)

^{로유의적으로낮아지는경향을}

보였다

. Leptin level

은대조군에비해

HPJ125, HPJ250, HPJ 500

^{투여군에서각각}

5.1%, 6.2%, 14.4%

^{로농도의존적으로낮}

아지는경향은보였지만유의적인경향은보이지않았다

. NEFA

level

은대조군에비해

HPJ125, HPJ250, HPJ 500

투여군에서 각각

9.2%, 9.5%, 20.2%(

^p

<0.01)

^{로농도의존적으로낮아지는}

경향을보였다

.

조직의형태학적관찰

Fig. 2

^는

HPJ

^{추출물이간}

,

^췌장

,

^{흰색지방인부고환지방의}

형태에미치는영향을살펴본결과이다

.

그림에서보는바와같 이간조직의염색결과에서대조군에서많은지방들이관찰되

었지만

HPJ

투여군에서는지방구가농도의존적으로현저하게

줄어든것을관찰할수있었다

.

췌장조직에서는

HPJ

투여군에

서췌장

islet

^{의모양이나구조가대조군에비해구조가뚜렷함}

Table V −

Effects of HPJ extract on plasma parameters

Group TG (mg/d l ) Cholesterol (mg/d l ) Leptin (ng/m l ) NEFA (mEq/ l )

Con 50.1±3.3 120.3±4.7 2.09±0.29 1151.2±63.0

HFD 87.1±3.7

^###

188.2±2.4

^###

13.74±1.55

^###

1700.2±62.9

^###

HPJ125 81.9±5.2 177.0±4.7* 13.04±1.47 1544.5±58.0

HPJ250 77.2±5.8 171.2±2.8** 12.89±0.94 1539.8±24.0

HPJ500 67.7±6.0* 146.3±3.2*** 11.76±1.23 1356.7±98.7**

Values represent the mean±SE (n=6). Plasma parameters were analyzed in plasma samples obtained from blood of 12 hr fasted mice.

###

p <0.001 vs. Con; * p <0.05, ** p <0.01, *** p <0.001 vs. HFD

Table VI −

Characteristics of specific primers used for RT-PCR analysis

Gene Forward primer (from 5' to 3') Reverse primer (from 5' to 3') Annealing temperature (

^o

C)

SREBP1 GCGCTACCGGTCTTCTATCA TGCTGCCAAAAGACAAGCG 57

FAS GATCCTGGAACGAGAACAC AGACTCGTGGAACACGGTGGT 57

SCD1 CGAGGGTTGGTTGTTGATCTGT ATAGCACTGTTGGCCCTGGA 57

GPAD GGTAGTGGATACTCTGTCGTCCA CATCAGCAACATCATTCGGT 66

PPAR-

^α

CCCTGAACATCGAGTGTCGA CTTGCCCAGAGATTTGAGGTCCT 57

CD36 TCCTCTGACATTTGCAGGTCTATC GTGAATCCAGTTATGGGTTCCAC 51

CPT1 CCTGGGCATGATTGCAAAG ACAGACTCCAGGTACCTGCTCAC 55

CPN ATGGTCAACCCCACCGTG TTAGAGTTGTCCACAGTCGGAGA 57

Fig. 2 −

Effects of HPJ extract on liver, pancreas, epididymal adipose tissue morphology. Magnification of histological section×200.

을관찰할수있었고부고환지방에서

HPJ

^{투여그룹의지방}

size

는대조군의지방

size

^{에비해작았다}

(Fig. 2).

^이결과는

HPJ

추출물이간에서지방의축적을억제하고췌장

islets

의파괴를

억제하며부고환지방의축적을감소시키는효과가있음을시사 하였다

.

Western blot

AMP-activated protein kinase(AMPK)

는

serine/threonine kinase

의일원으로세포내에너지상태를감지하는

"

에너지센 서

"

^{로알려져있는효소이다}

.

¹⁴⁾

AMPK

^{는세포내에너지가부}

족한상황

-

즉

ATP

에비해

AMP

가증가하는상황에서활성화 되어정상에너지균형을회복시키기위해

ATP

를소비하는과 정즉지방산

,

^{콜레스테롤등의합성을억제하고반대로}

ATP

^를

생산하는 과정즉지방산산화

,

해당과정을활성화시킨다

.

¹⁵⁾

AMPK

는지방세포로부터분비되는렙틴

(leptin)

과아디포넥틴

(adiponectin)

^{의 세포 내신호전달물질로서도 잘알려져 있}

다

.

^16,17)

Acetyl-CoA carboxylase(ACC)

는지방대사가증가할경

우에생성되는효소물질가운데하나이다

.

¹⁸⁾

AMPK

^는지방산

합성에서

ACC

^{등핵심효소를억제함으로써}

ATP

^{의추가적사}

용을억제한다

.

¹⁹⁾본연구에서는비만의표적단백질인

AMPK

및

ACC

^{의활성을보고자부고환지방조직에서}

AMPK

^를중심으

로활성화되는

marker

^{들의발현을}

Western blot

^{방법으로측}

정하였다

. Fig. 3

에서보는바와같이

HPJ

는용량의존적으로

AMPK

^{를현저하게인산화시켰으며}

ACC

^{역시현저하게인산}

화시켰다

.

^이결과

HPJ

^{투여로인해지방산산화가촉진되는것}

으로해석이가능하다

.

RT-PCR

HPJ

^의

AMPK

^{활성화결과를검증하기위해}

target gene

^들

(lipogenesis, lipolysis

관련유전자들

)

의발현을

RT-PCR

로측정 한결과

Fig. 4

^{에서보듯이}

HPJ

^{투여군에서}

lipogenesis

^관련

gene

들인

SREBP1

α

, FAS, SCD1, GPAT gene

들의발현은농 도의존적으로억제시킨반면에

lipolysis

관련

gene

들인

CD36,

PPAR-

^{α의발현은증가시켰고지방산산화에 관련된}

gene

^인

CPT1 gene

의발현역시증가시켰다

.

결 론

HPJ

추출물을

HFD

로비만을유도한

C57BL/6J

마우스에

8

주

간경구투여한결과대부분의지표에서대조군에비해

HPJ

^투

여군에서개선되는결과를보여주었다

.

이들은체중뿐만아니라 혈당수치에서도유의적인감소를나타내었으며체중감소작용 기전은흰색부고환지방에서

metabolic sensor

^{에해당하는단백}

질인

AMPK

의활성화와그로인한지방산산화촉진

,

그리고부

고환지방에서

SREBP1

^α

, FAS, SCD1, GPAT

^등

lipogenesis gene

^{들의발현을억제하여지방산의합성억제}

, CD36, PPAR-

^α

등

lipolysis gene

들의발현증가및지방산산화에관여하는단

백질인

CPT-1

^{발현증가등에기인하는것으로사료된다}

.

^결론

적으로

HPJ

^{추출물은고지방식이유도비만동물모델에서체중}

감소활성을나타내었고이로인해혈당치및지질프로파일이 개선되는활성을보여주어서향후비만치료제혹은예방목적 Fig. 3 −

Western blot analyses of phospho-AMPK, AMPK, phospho-

ACC, ACC levels in epididymal tissue. Lysate (40

^µ

g) was subjected to SDS-PAGE. Western blotting using antibodies against phospho-AMPK, AMPK, phospho-ACC, ACC, and actin was carried out as described in Materials and Methods. Actin was used as an control to evaluate relative expression of protein.

Fig. 4 −

Quantitative RT-PCR analyses of SREBP1-

^α

, FAS, SCD1,

GPAT, PPAR-

α,

CD36, UCP, CPT1, CPN genes in the

presence or absence of HPJ extrcat on epididymal adipose

tissue. Total RNA was subjected to RT-PCR as described in

Materials and Methods.

으로개발될수있을것으로기대된다

.

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