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1 Original article 운동과학, 2015 년, 제 24 권제 3 호. Exercise Science, 2015, Vol.24, No.3. pissn eissn 운동중골격근내 PGC-1α 농도는 PPARβ/δ 를통한후전사기전에의하여조절된다 : PPARβ/δ silence 가 ubiquitin 을통한 PGC-1α 안정성에미치는영향 정수련 1), 김기진 1), 고진호 2)* 1) 계명대학교 2) Washington University School of Medicine Su-Ryun Jung, Ki-Jin Kim, Jin-Ho Koh (2015). PGC-1α concentration in skeletal muscle by exercise is controlled by PPARβ/δ through post-transcriptional mechanism: the effects of PPARβ/δ silence on PGC-1α ubiquitination and stability. Exercise Science, 24(3): PURPOSE: The aim of this study was to identified which PPARβ/δ control PGC-1α protein stability in rat skeletal muscle by long-term endurance exercise. METHODS: shpparβ/δ was over-expressed in rat epitrochlearis (Epi) muscle using electrical pulse-mediated gen transfer (electroporation; EPO) method. Then animal underwent 2 wk long swimming exercise. To evaluate PPARβ/δ, PGC-1α ubiquitination, PGC-1α and mitochondrial enzyme expression in rat skeletal muscle, Epi muscles were dissected 18 h post final bout of swimming exercise. RESULTS: PPARβ/δ, PGC-1α, COX I and NADH protein expression in shpparβ/δ over-expression sedentary (Sed) skeletal muscle were significantly decreased over 55%, but PGC-1α ubiquitination was significantly increased 2.6 fold when compared to scramble (Scr) treated Sed muscle. PPARβ/δ, PGC-1α, COX I and NADH protein expression in 2 wks swimming exercise with Scr gene treated muscle were significantly increased over 2 fold, but PGC-1α ubiquitination was significantly decreased 66% when compared to Scr gene treated Sed muscle. PPARβ/δ, PGC-1α, COX I and NADH protein expression in 2 wks swimming exercise with shpparβ/δ gene treated muscle were not increased, but PGC-1α ubiquitination was significantly increased 1.96 fold when compared to Scr gene treated Sed muscle. CONCLUSIONS: Our results indicate that PPARβ/δ controls PGC-1α protein stability in rat skeletal muscle following long-term endurance exercise. Key words: long-term endurance exercise, PARβ/δ, PGC-1α, ubiquitin, mitochondrial enzymes 주요어 : 장기간지구성운동, PPARβ/δ, PGC-1α, ubiquitin, 미토콘드리아효소 Ⅰ. 서론 근지구력은탄수화물과지방등의에너지원을이용하여 ATP 를생산하는능력에의하여좌우된다 [11]. 미토콘드리아는에너지원을산화시켜 ATP를생산하는근육의발전소로서운동부하가지속적으로주어질때더많은에너지를산화할수있도록그수와양이증가된다 [10]. Peroxisome proliferator-activated receptor-gamma coactivator-1 alpha(pgc-1α) 는미토콘드리아기능, 생합성및세포에너지대사의전사조절과관련된기전의통합자로간주되고있는데 [12,24], 최근리간드 (ligand) 에의하여활성화되는핵수용체 peroxisome proliferator-activated receptor beta/delta(pparβ/δ) 가 PGC-1α의안정성과미토콘드리아생합성에영향을미치는것으로보고되어 [5,8,14], 지구성운동의골격근 적응반응으로유도되는미토콘드리아생합성과두인자간에어떠한관련성이있는지확인할필요가있다. 지구성운동은 PGC-1α 단백질발현을빠르게증가시키지만자극이주어지지않으면생산이중단되며단백질도빠르게분해된다 [2,6,9,13,14,26,27,31]. 골격근내미토콘드리아생합성이 PGC-1α 에의하여조절됨으로서미토콘드리아생합성을유지또는증가시키기위한골격근내 PGC-1α 단백질농도의수준은매우중요하다. 이러한관점에서 PPARβ/δ 의과발현이 PGC-1α 단백질의안정성을증가시킨다는선행연구의결과 [14] 는매우흥미롭다. PPARβ/δ 과발현이 1회지구성운동후 PGC-1α의빠른감소를지연시켜미토콘드리아효소의농도를높은수준으로유지시킬수있는것으로나타났다 [14]. 이는 PPARβ/δ가 PGC-1α 교신저자 : 고진호, spogion@gmail.com Tel: , Fax: 이논문은 2014 년정부 ( 교육부 ) 의재원으로한국연구재단의지원을받아수행된연구임 (NRF-2014S1A5B5A ) Received 29 Jun. 2015, Revised 4 Aug. 2015, Accepted 21 Aug. 2015

2 운동과학, 2015 년, 제 24 권제 3 호, 단백질의안정성을증가시켜골격근미토콘드리아기능을증가시킬수있다는중요한정보이지만, 실제로지구성운동에의하여증가된 PARβ/δ가 PGC-1α의단백질안정성을증가시키는지확인하기위해서는장기간지구성운동에의한 PPARβ/δ의역할을살펴보는것이중요하다. 이를증명하기위한가장효과적인방법은장기간지구성운동으로골격근 PPARβ/δ가증가 ( 과발현 ) 되도록하는동시에다른근육에서는 PPARβ/δ 발현이억제되도록유도하여이를비교하는방법일것이다. Knock down 모델을이용하여 6세대이상골격근 PPARβ/δ 발현을억제시키는것은미토콘드리아기능감소에의한운동능력저하로대조군보다수축활성이감소될수있으므로, 본연구에서는약 50% 수준으로 PPARβ/δ의발현을억제시키기위해 shpparβ/δ를 epitrochlearis(epi) 에과발현시키는방법을이용하였다. Epi는삼두근에붙어있는 0.2 mm 정도의매우가늘고작은근육으로서 [19] 운동자극에대한반응이크며, electroporation(epo) 효율이높다 [15]. 본연구는흰쥐의왼쪽 Epi 근육에서 PPARβ/δ의발현이정상근육보다낮게발현되도록 shpparβ/δ(shot-hairpin PPARβ/δ RNA-silencing constructs) 를과발현시킨후 2주간지구성운동을실시하였다. 세포의핵으로전이된 shrna는 hairpin(double strand structure) 구조를형성하여목표 mrna를파괴함으로서단백질발현을감소시키게된다. 따라서본연구의목적은골격근 PPARβ/δ 발현억제가장기간지구성운동에의한 PGC-1α 단백질안정성에어떠한영향을미치는지확인하는것이며, 이는 PPARβ/δ 가장기간지구성운동에의한골격근미토콘드리아생합성에어떠한역할을수행하는지확인할수있을것이다. Ⅱ. 연구방법 1. 실험동물 실험동물은 3 주령 ( 약 50 g) 의수컷 Wistar rat( 흰쥐 ) 16마리를이용하였다. 흰쥐는좌업그룹 (Sedentary, Sed) 과 2주간수영운동그룹 (Exercise, Ex) 으로각각 8마리씩무작위분류하였다. 모든그룹의흰쥐골격근에전기자극을이용한유전자전이법 (electorporation, EPO) 으로 shpparβ/δ 와 Scramble gene(scr, short hairpin이형성되지만유전자에영향을미치지않도록 NCBI blast search를통해확인한후유전자를제작 ) 을각각좌 우측 Epi 근육에과발현시켰다. 흰쥐는일반사료 (Purina, USA) 를물과함께자유롭게섭취하도록하였다. 사육실온도는 21 로유지하였으 며, 명기 (06:00-18:00) 와암기는각각 12시간으로조절하였다 [15]. 본연구는 Animal Studies Committee of Washington University School of Medicine(USA) 에의해승인되었다. 2. 운동프로그램 수영은 Ploug et al [23] 과 Terada et al [26,27] 의방법을수정하여사용하였다. 수영운동은하루 3시간씩 2회분할하여총 6시간, 2주간실시하였으며, 1회실시후 45분간휴식과함께물과사료를자유롭게섭취토록하였다. 원통형수조 (80 cm 120 cm, 수심 50~60 cm) 내수온은 35 로일정하게유지하였다. 운동종료후사료섭취는자유롭게이루어질수있도록하였다. 본연구에사용된수영트레이닝의운동강도는속도 13 m/min, 경사도 0% 의장시간트레드밀달리기와유사하도록설정되었다 [23,26,27]. 3. shpparβ/δ 및 Scr Plasmid 제작과정 shrna 는흰쥐 PPARβ/δ mrna 의 silence 가능부위에해당하는 5 -CCCTTCATCATCCACGAC ATATTTTCAAGAGAaatatgtcgaggatgatgaaggg ( 밑줄 ; hairpin loop, 소문자 ; target complementary sequence) 를 prnat-h1.1(genescript, USA) 의 multi cloning site에삽입하였다 [15]. shpparβ/δ가세포내로형질이입된후 H1 promoter 에의하여 hairpin 모양을한 double strand silence RNA가생성되도록설계하였다 [15]. Cloning 산물은공급업체의프로토콜에따라서 E.coli(One Shot TOP10 Competent Cell, Invitrogen, USA) 에형질전환한후 SOC media에넣어 1시간동안배양 (37 ) 하였다. 배양된 E.coli는 aga-plate에서이튿날까지배양 (37 ) 하였으며, 하나의 colony 를 ampicillin을첨가된 LB broth 에넣어이튿날까지배양하였다. 배양된 E.coli는 plasmid mini-prep kit(qiagen, USA) 으로정제하였으며 [14], HEK293A 세포에이입하여 PPARβ/δ 단백질의발현유무를확인한후실험에이용하였다. 4. 전기자극유전자전이 (Electroporation) Isoflurane gas로생쥐를마취시킨후 20 μg의 shpparβ/δ와 Scr plasmid 를각각좌측과우측 Epi 근육에주사기를이용하여주입하였다. 주입직후근육에전기자극 (100-ms duration, 1Hz frequency, 200 volts/cm amplitude) 을주어 plasmid DNA 가근육의핵속으로유입되도록하였다 [1]. 290 정수련, 김기진, 고진호

3 5. Western blotting 막자사발을이용하여동결된근육을분말로만들고 Radioimmunoprecipitation assay(ripa) buffer를이용하여균질화하였으며, 동결과해동과정을 3회반복한후 Lowry et al [18] 의방법으로단백질농도는측정하였다 [14]. 균질화된시료는 laemmli sample buffer(lsb) 를첨가한후 5분간 100 물에서용해하였다. 샘플은전기영동후단백질을 nitrocellulose membrane 에전이 (transfer) 하여다음의일차항체와함께배양하였다 [14]. PPARβ/δ(Santacruz, USA), PGC-1α(Calbiochem, USA), ubiquitin(affymetrix Bioscience, USA), cytochrome c(cyto C, BD Biosciences, USA), cytochrome oxidase subunit I(COX I, Life technologies, USA), β-actin(sigma, USA). Horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG(PPARβ/δ, PGC-1α), donkey anti-mouse IgG(COX I, CYTO C, β-actin) 그리고 HRP-streptavidin(ubiquitin) 과같은이차항체는 Jackson ImmunoResearch Laboratories(USA) 에서구매하였다. ECL kit(sigma, USA) 으로발광시킨후 C-DiGit blot scanner(li-cor, USA) 를이용하여 antibody가결합된단백질밴드를시각화하였다. 단백질밴드의상대적강도는 C-DiGit blot scanner의소프트웨어인 Image Studio Digits(Li-COR, USA) 을이용하였다 [14]. 6. 자료처리 수집된자료는 SigmaPlot 12.0 통계패키지를이용하여측정항목별평균및표준편차를산출하였다. 각인자간에상관관계를분석하기위하여 pearson s 상관관계분석을실시하였다. 처치그룹및시기간상호작용을분석하기위하여 two-way repeated measures ANOVA 를실시하였으며, 사후검정은 Tukey를실시하였다. 모든통계적유의수준 (α) 은 p<0.05 미만으로설정하였다. Ⅲ. 연구결과 1. 지구성운동과 shpparβ/δ 과발현이 PPARβ/δ 및 PGC-1α 발현에미치는영향 PPARβ/δ 발현을억제하고 2주간수영운동을실시한흰쥐의 Epi 근육에서 PPARβ/δ 와 PGC-1α의발현을분석한결과는 <Fig. 1A,1B,1C> 와같다. shpparβ/δ 를처치한좌업그룹의근육은 Scr을처치한근육보다 PPARβ/δ 와 PGC-1α 발현이각각 70%(p<0.001) 및 58%(p<0.01) 감소하였다. Scr을처치하고 2주간수영운동을실시한그룹의 PPARβ/δ와 PGC-1α의발현은좌업그룹의 Scr 처치근육보다각각 2.7배 (p<0.001) 및 2.5배 (p<0.001) 증가하였다. 그러나 2주간지구성운동을실시하였음에도불구하고 shpparβ/δ 처치한그룹의 PPARβ/δ 및 PGC-1α 발현은좌업그룹의 Scr 처치근육보다증가되지않았으며, 2주간운동과 Scr을처치한그룹보다는 PPARβ/δ 및 PGC-1α 발현이각각 84%(p<0.001) 및 75%(p<0.001) 감소한것으로나타났다. 2주간지구성운동및 shpparβ/δ 를처치한그룹의 PPARβ/δ가좌업그룹의 shpparβ/δ를처치한그룹보다약 2.1 배 (p<0.001) 증가한것은 PPARβ/δ의기능이완전히억제되지않고 2주간지구성운동에의하여부분적으로증가되었음을의미한다. 이는 knock out 모델과같이완전한기능의억제로나타날수있는보상작용에의한부작용 (side effects) 을감소시킬수있으며, shpparβ/δ 과발현을통한 PPARβ/δ 발현억제가효과적으로이루어졌음을의미한다 (Fig. 1A,1B). 또한 2주간지구성운동및 shpparβ/δ 를처치한그룹의 PGC-1α가좌업그룹의 shpparβ/ δ를처치한그룹보다약 2배 (p<0.001) 증가한것은 PPARβ/δ 의발현이완전히억제되지않고 2주간지구성운동에의하여약 2.1배증가하였기때문이다. PGC-1α 의발현양상은 <Fig. 1B> 의 PPARβ/δ 발현양상과일치하며, 정적상관관계가있는것으로나타났는데 (Fig. 1E), 이는장기간지구성운동에의하여증가되는 PPARβ/δ가 PGC-1α 단백질의안정성을증가시킨다는것을의미한다 (Fig. 1). 2. 지구성운동과 shpparβ/δ 과발현이 PGC-1α ubiquitination 에미치는영향 PARβ/δ가 PGC-1α 의빠른분해를지연시켜단백질안정성을증가시키는지확인하기위하여 PGC-1α ubiquitination을분석하였다 (Fig. 1A,1D). shpparβ/δ를처치한좌업그룹의 PGC-1α ubiquitination은 Scr 을처치한근육보다 2.6배 (p<0.001) 증가하였다. Scr을처치하고 2주간수영운동을실시한그룹의 PGC-1α ubiquitination은좌업그룹의 Scr 처치근육보다 66%(p<0.001) 감소하였다. 그러나 2주간지구성운동을실시하였음에도 shpparβ/δ 처치한그룹의 PGC-1α ubiquitination은좌업그룹의 Scr 처치근육보다 1.96배 (p<0.01) 증가하였으며, 2주간운동과 Scr을처치한그룹보다는 4.5배 (p<0.001) 증가한것으로나타났다 (Fig. 1A,1D). PGC-1α ubiquitination과 PPARβ/δ 는부적관련성이있으며 (Fig. 1F), 이러한결과들은지구성운동에의하여증가된 PPARβ/δ가 PGC-1α 단백질의안정성을증가시킨다는것을보여준다. 운동중골격근내 PGC-1α 농도는 PPARβ/δ 를통한후전사기전에의하여조절된다 : PPARβ/δ silence 가 ubiquitin 을통한 PGC-1α 안정성에미치는영향 291

4 운동과학, 2015 년, 제 24 권제 3 호, Fig. 1. The effects of PPARβ/δ silencing with 2 wks swimming exercise on PGC-1αprotein stability in skeletal muscle. (A) Protein band image by western blot. (B) PPARβ/δ expression, (C) PGC-1α expression, (D) PGC-1α ubiquitination by 2 wks endurance and shpparβ/δ overexpression. (E) and (F) Pearson's correlation. Sed; sedentary, Scr; scramble, * p<0.01 significantly different between group. # p<0.01 significantly different within group. Fig. 2. The effects of PPARβ/δ silencing with 2 wks swimming exercise on mitochondrial protein in skeletal muscle. (A) Protein band image by western blot. (B) CYTO C expression, (C) COX I expression by 2 wks endurance and shpparβ/δ overexpression. Sed, sedentary; Scr, scramble; CYTO C, cytochrome c; COX I, cytochrome oxidase subunit I; * p<0.01 significantly different between group. # p<0.01 significantly different within group. 3. 지구성운동과 shpparβ/δ 과발현이골격근미토콘드리아효소발현에미치는영향 PPARβ/δ 발현억제에의한 PGC-1α 단백질안정성감소가 PPARβ/δ의조절을받는 CYTO C와 PGC-1α의조절을받는 COX I의발현에어떠한영향을미치는지분석한결과는 <Fig. 2> 에나타난바와같다. shpparβ/δ를처치한좌업그룹의 CYTO C와 COX I의발현은 Scr을처치한근육보다각각 73%(p<0.001) 및 68%(p<0.001) 감소 292 정수련, 김기진, 고진호

5 하였다. Scr을처치하고 2주간수영운동을실시한그룹의 CYTO C와 COX I의발현은좌업그룹의 Scr 처치근육보다발현이각각 2.7배 (p<0.001) 및 2.1배 (p<0.001) 증가하였다. 그러나 2주간지구성운동을실시하였음에도 shpparβ/δ 처치한그룹의 CYTO C와 COX I의발현은좌업그룹의 Scr 처치근육보다증가되지않았으며, 2주간운동과 Scr을처치한그룹보다 CYTO C와 COX I 발현이각각 61%(p<0.01) 및 74%(p<0.001) 감소한것으로나타났다. 2주간지구성운동및 shpparβ/δ 를처치한그룹의 CYTO C와 COX I 발현이좌업그룹의 shpparβ/δ 를처치한그룹보다각각 3.7배 (p<0.001) 및 2.5배증가한것은 PPARβ/δ의발현이완전히억제되지않고 2주간지구성운동에의하여 PPARβ/δ와 PGC-1α가각각약 2.1배및 2배증가되었기때문이다 (Fig. 2). Ⅳ. 논의 PPARβ/δ 는지방산이화작용과에너지항상성의강력한조절자역할을수행하는데 [3,22], 이러한기능들은 PGC-1α와매우유사한것으로나타났다 [24,32]. 세포와조직연구에서도 PPARβ/δ 와 PGC-1α 의관련성은매우높은것으로나타났으며 [29], 이는 PGC-1α의많은대사적효과가 PPARβ/δ를통하여매개될가능성이높다는것을의미한다. 실제로골격근 PPARβ/δ의과발현은지방산산화와에너지소비의증가 [20], 그리고미토콘드리아생합성증가에의한러닝지구력증가등 [30] 과같은 PGC-1α 과발현에의한결과와유사한것으로나타났다 [16]. 이러한관점에서골격근에 PPARβ/δ 과발현이후전사기전을통한 PGC-1α 단백질의안정성을증가시킨다는결과 [14] 는매우흥미롭다. 왜냐하면이는 PPARβ/δ가결핍되거나감소되면 PGC-1α 단백질안정성감소에따른미토콘드리아효소의발현이감소되거나장애가나타날수있기때문이다. 특히지구성운동은 PGC-1α와 PPARβ/δ의발현을증가시키는데 [4,7,21], PPAR β/δ의발현이차단된다면지구성운동에의하여 PGC-1α가생산되더라도단백질이빠르게감소되어미토콘드리아생합성에장애가나타날수있다. 본연구는이러한관련성을증명하기위하여 2주간지구성운동을실시하는흰쥐의왼쪽 Epi에 PPARβ/δ 의단백질이증가되지않도록 shpparβ/δ를과발현시켜 Scr을처치한오른쪽 Epi와비교하였다. 장기간지구성운동에의한 PPARβ/δ 의발현증가는 PGC-1α ubiquitination을감소시켜 PGC-1α 안정성을증가시켰 으며, 이는 PGC-1α 와미토콘드리아효소를증가시키는결과로 나타났다 (Fig. 1,2). 그러나 PPARβ/δ 의발현이억제된근육은장기간지구성운동을실시했음에도 PGC-1α ubiquitination이증가되었으며, 이는 PGC-1α 단백질감소와함께미토콘드리아효소의발현도감소시켰다 (Fig. 1,2). 선행연구는 Ubiquitin이 PGC-1α N-terminus에결합하며 [28], PPARβ/δ도 PGC-1α의 N-terminus 에결합하여상호작용한다고하였다 [33]. 이는지구성운동에의한 PPARβ/δ의증가가 PGC-1α의 N-terminus 에결합하여 ubiquitin이 PGC-1α의 N-terminus 에결합하는것을방해할가능성을보여주는것인데, 본연구는장기간지구성운동에의하여증가된 PPARβ/δ 가 PGC-1α의 ubiquitination을감소시켜 PGC-1α의단백질안정성을증가시킨다는것을보여주었다. 또한후전사기전에의한 PGC-1α 안정성증가는미토콘드리아생합성증가를유도한다는것을확인하였다. PGC-1α 의조절장애는인슐린저항성및당뇨병의발병요인이될수있으며 [17], 근기능감소에직접적인영향을미칠수있다 [25]. 이러한결과들은골격근의 PGC-1α 농도를일정한수준이상으로유지하는것이건강에유익하다는것을보여준다. 규칙적인지구성운동은골격근 PGC-1α를증가시키고유지하는가장좋은방법이나규칙적이고지속적으로지구성운동을실시할수없는노인, 근기능감소자등과같은대상자에게는현실적인대안이요구된다. PPARβ/δ 는지구성운동후 PGC-1α의빠른감소를지연시킬수있으므로 PPARβ/δ를활용한약품이그대안이될것으로생각된다. 왜냐하면 PPARβ/δ는리간드에의하여활성화되는핵수용체성전사인자이므로대상자에게비교적안전하게처치될수있기때문이다. PGC-1α 단백질안정성에대한 PPARβ/δ 과발현연구 [14] 와지구성운동중 PPARβ/δ 발현억제를시도한본연구는골격근미토콘드리아생합성에대한 PGC-1α의역할이 PPARβ/δ에의한후전사기전에의존적으로이루어진다는것을보여주었지만, PGC-1α 전사와관련된추가연구가요구된다. PPARβ/δ의과발현이 PGC-1α mrna 전사를증가시키지않으므로 [8,30], 지구성운동에의하여증가된 PPARβ/δ가 PGC-1α mrna 를증가시킨것은아니다. 그러나장기간지구성운동이 PPARβ/δ 와관련이없는다른기전을경유하여 PGC-1α mrna 전사를증가시켰을가능성을배재할수는없다. 따라서장기간지구성운동에의한 PGC-1α mrna의발현이분석된다면지구성운동에의하여증가되는 PPARβ/δ와관련된미토콘드리아생합성기전을보다명확하게설명할수있을것으로생각된다. 운동중골격근내 PGC-1α 농도는 PPARβ/δ 를통한후전사기전에의하여조절된다 : PPARβ/δ silence 가 ubiquitin 을통한 PGC-1α 안정성에미치는영향 293

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<303620C1A4BCF6B7C320BFDC5FC3D6C1BE2E687770> 운동과학, 2014년, 제23권제4호 Exercise Science, 2014, Vol. 23, No. 4 원문 PGC-1α 단백질이생쥐근육세포의글리코겐분해및해당효소의발현을조절한다. 고진호 1) ㆍ김기진 2) ㆍ김상현 2) ㆍ정수련 2) 1) Washington University 2) 계명대학교 Abstract Koh, Jin-Ho, Kim, Ki-Jin,