DOI : 10.4093/kdj.2009.33.5.375 ORIGINAL ARTICLES 스트렙토조토신으로유발된당뇨병모델에서과식증에대한시상하부 FoxO1의역할 울산대학교의과대학울산대학교병원내과 1, 울산대학교의과대학생의과학연구소 2, 울산대학교생명과학부 3 남궁일성 1 김재근 2 김세진 1 허성재 1 이진우 1 김은숙 1 윤창호 3 이병주 3 김영일 1 The Role of Hypothalamic FoxO1 on Hyperphagia in Streptozotocin-Induced Diabetic Mice Il Seong Nam-Goong 1, Jae Geun Kim 2, Se Jin Kim 1, Seong Jae Hur 1, Jin Woo Lee 1, Eun Sook Kim 1, Chang Ho Yun 3, Byung Ju Lee 3, Young Il Kim 1 1 Department of Internal Medicine, University of Ulsan College of Medicine, Ulsan University Hospital, 2 Biomedical Research Center, University of Ulsan College of Medicine, 3 Department of Biological Sciences, University of Ulsan, Ulsan, Korea Abstract Background: Streptozotocin-induced diabetic animals are characterized by hyperphagia due to deficiencies of insulin and leptin. Forkhead box-containing protein of the O subfamily-1 (FoxO1) regulates energy homeostasis by regulating energy expenditure and food intake as well as mediating insulin and leptin signals in the hypothalamus. To identify the mediator of diabetic hyperphagia, we examined the effects of insulin or leptin on hypothalamic FoxO1 expression in a diabetic animal model. Methods: Diabetes was induced in mice (C57BL/6) by intraperitoneal administration of streptozotocin (200 mg/kg). Stainless steel cannula was implanted into the lateral ventricle of the brain in each mouse. After three weeks, the mice were administered saline, insulin or leptin via intracerebroventricular (ICV) route. The medial hypothalamus was isolated to evaluate the mrna expressions of FoxO1 and neuropeptides. Results: Streptozotocin-induced diabetic mice exhibited significant elevations of blood glucose and food intake and significantly low levels of serum insulin and leptin. The levels of hypothalamic FoxO1 mrna were significantly increased in diabetic mice. The hypothalamic expression of neuropeptide Y (NPY) mrna was increased, but the expression of preproopiomelanocortin (POMC) mrna was decreased in diabetic mice. ICV administration of insulin or leptin attenuated the upregulation of hypothalamic FoxO1 mrna, and resulted in downregulation of NPY mrna and upregulation of POMC mrna in diabetic mice. Conclusion: We observed that the expression of hypothalamic FoxO1 mrna was increased in streptozotocin-induced diabetic mice, and that it was significantly attenuated by central administration of insulin or leptin. These results suggest that hypothalamic FoxO1 is the direct mediator of diabetic hyperphagia. (Korean Diabetes J 33:375-381, 2009) Key words: Diabetes Mellitus, Forkhead Transcription Factors, Hyperphagia, Hypothalamus, Insulin, Leptin 접수일자 : 2009 년 5 월 4 일, 통과일자 : 2009 년 10 월 5 일교신저자 : 김영일, 울산대학교병원내분비내과, E-mail: yikuls@uuh.ulsan.kr 본연구는대한당뇨병학회의 2005 년제 12 회세르비에연구비와울산대학교병원생의과학연구소학술연구비 ( 과제번호 : 2006-14) 의지원을받아수행되었음. 375
서론당뇨병은인슐린의상대적또는절대적인결핍으로인해에너지대사의장애를초래하며조절되지않은당뇨병에서는흔히고혈당, 체중감소와더불어심한과식증이동반된다 1). 위와같은상황에서는세포내에너지결핍을보상하기위해시상하부의식욕조절중추에서신경펩타이드의조절을통하여식사섭취의증가가유발되는것이발생기전의하나로제시되고있다 2,3). 한편, 인슐린과렙틴은식사섭취를조절하는대표적인호르몬으로서시상하부식욕조절중추에식욕억제신호를전달한다. 혈중인슐린과렙틴은스트렙토조토신으로유발된당뇨병실험동물모델에서급격하게저하되어있으며, 시상하부에서식욕을증가시키는신경펩타이드인 neuropeptide Y (NPY) 와 agouti gene-related protein (AgRP) 의합성이증가되고식욕을감소시키는신경펩타이드인 preproopiomelanocortin (POMC) 의합성이감소되어있다고알려져있다 2-7). Forkhead box-containing protein of the O superfamily-1 (FoxO1) 은인슐린신호전달을매개하는전사조절인자로세포의분화와대사에관여하는것으로알려져있으며최근의연구에서는 FoxO1이시상하부에서인슐린뿐만아니라렙틴에의한식욕조절신호를매개하고식욕조절신경펩타이드의전사를조절하여식사섭취및에너지대사를조절한다고하였다 8-10). 그러나현재까지당뇨병에서인슐린과렙틴에의한식사섭취의변화를매개하는전사조절인자에대한구체적인기전은잘알려져있지않다. 본연구에서는인슐린과렙틴이결핍된당뇨병동물모델에서시상하부의 FoxO1이인슐린또는렙틴에어떤영향받는지확인하여당뇨병성과식증의기전에대해알아보고자하였다. 대상및방법 1. 실험동물사용한실험동물은 7주령의 C57BL/6 strain의수컷생쥐로대한실험동물센터에서구입한후울산대학교생명과학부실험동물사육장에서섭씨 23~25 의온도와 50% 습도및 12시간의일조주기하에서 2주이상을적응시키며정상적인물과먹이를공급하여사육하였다. 2. 당뇨병의유발당뇨병을유발하기위하여스트렙토조토신 (Sigma, St Louis, MO) 200 mg/kg를생쥐의복강으로투여하였고, 대조군은동량의 0.05 M sodium citrate buffer (ph 4.5) 를복강으로투여하였다. 스트렙토조토신을주입한지약 3일후혈당측정기 ( 라이프스캔원터치울트라 TM, 존슨앤존슨메디칼 ) 를이용하여혈당을측정하여 2회이상공복혈당이 16.7 mmol/l 이상일경우당뇨병이유발된것으로확인하였다. 실험기간중당뇨가유발된실험동물과대조군의체중및먹이섭취량을매일관찰하였으며실험군과대조군각각의시상하부와혈액을분리하여실험에사용하였다. 3. 뇌실내삽관및약물의투여생쥐의뇌실에인슐린, 렙틴또는 0.9% 생리식염수를주입하기위하여 pentobarbital (7.5 mg/kg B.W.) 과 ketamine hydrochloride (25 mg/kg B.W.) 을이용하여마취를한후에정위뇌수술장치를이용하여스테인리스금속관 (25G) 을측뇌실에삽입하였다. 실험동물을일주일동안회복시킨후 Hamilton 주사기를이용하여측뇌실에 500 nmol의인슐린과 1 μg/ 2 μl의렙틴및동량의 0.9% 생리식염수를주입하고 1 시간후 FoxO1 mrna의합성을확인하기위하여시상하부를적출하여단백질을분리하였다. 4. RNA 추출과 cdna 합성분리된조직의 RNA (ribonucleic acid) 는 Tri reagent (Sigma) 를이용하여추출하였다. 떼어낸시상하부를 500 μ L의 Tri reagent 속에서분쇄한후상온에서 5분간두었다가 chloroform 100 μl를첨가한후 vortexing했다. 그후얼음에 3분두었다가 14,000 rpm로 4 에서 15분간원심분리하였다. 원심분리한후상층액을새로운튜브로옮기고동량의차가운 isopropanol을첨가해서 vortexing했다. 영하 20 에서하룻밤동안보관후 14,000 rpm로 4 에서 15분간원심분리하여침전물을 75% 에탄올로 2~3회씻어주고다시원심분리한침전물을말린후 diethylpyrocarbonate (DEPC) 에침전물을녹였다. RNA 정량은분광광도계로 260 nm에서수행하였으며, A280에대한 A260의흡광도비율은약 1.6~2.0이었다. 흡광도측정후영하 80 에보관하였다. 5. Real Time PCR 분석정량을마친 total RNA는다음과같은조건하에서역전사를수행하였다. DEPC에녹인 RNA 2 μg을 cdna 합성전까지얼음위에두고 oligo-dt (50 pmol, Invitrogen Co.) 와 DEPC를섞어 65 에서 3분간변성시켰다. Reverse transcription cocktail (5X reverse transcription buffer, 0.1 376
남궁일성외 8 인 : 스트렙토조토신으로유발된당뇨병모델에서과식증에대한시상하부 FoxO1 의역할 M DTT, 10 mm dntps, RNasin, reverse transcriptase) 을첨가하여총 20 μl의부피로맞추고 37 에서 2시간동안반응시킨후합성된 cdna를 gel loading을통해확인하였다. Gel loading을통해서확인된 cdna 1 μl에 FoxO1 sense primer (5'-ATG CTC AAT CCA GAG GGA GG-3') 와 antisense primer (5'-AAG TCA TCG TTG CTG TGG GA-3'), NPY sense primer (5'-GCT AGG TAA CAA ACG AAT GG-3') 와 antisense primer (5'-GCA TTT TCT GTG CTT TCT CT-3'), POMC sense primer (5'-GCT AGG TAA CAA ACG AAT GG-3') 와 antisense primer (5'-GCA TTT TCT GTG CTT TCT CT-3'), GAPDH sense primer와 antisense primer를각각 1 μl씩첨가한후에 SYBR green (Quigen) 을사용하여 real time PCR을수행하였다. PCR (polymerase chain reaction) 조건은 FoxO1은 95 에서 5분간 pre- denaturation, 94 에서 40초간 denaturation, 56 에서 40초간 annealing, 72 에서 1분간 elongation의조건으로 45회를반복하고 72 에서 10분간 post-elongation 하였으며, NPY와 POMC은 94 에서 5분간 pre-denaturation, 94 에서 30초간 denaturation, 55 에서 30초간 annealing, 72 에서 35초간 elongation의조건으로 45회를반복하고 72 에서 6분간 post-elongation하였다. 6. 혈액의면역학적검사당뇨병이유발된실험동물과식사제한을한실험동물에서혈중인슐린과렙틴의농도를확인하기위하여각각실험군과대조군의심장으로부터혈액을수집한후원심분리를통해혈청만을분리하였다. Millipore사의 multiplex ELISA kit를사용하여면역효소방법으로혈중인슐린과렙틴의농도를측정하였다. 7. 통계학적분석실험결과는평균 ± 표준편차로나타내었고 GraphPad Prism (Version 4.0, GraphPad Software, Inc.) 프로그램을사용하여각군간의유의성은 Student s t test로검증하였 고 P < 0.05 이하일때유의성이있는것으로판정하였다. 결과 1. 스트렙토조토신으로유발된당뇨병생쥐의먹이섭취량및혈중호르몬의변화당뇨병을유발하기위하여스트렙토조토신을생쥐의복강으로주입하였고 3일후혈당을측정하여당뇨병의유발을확인하였다 ( 대조군, 6.8 ± 0.8 mmol/l; 실험군, 25.5 ± 2.8 mmol/l, P < 0.001, Table 1). 당뇨병이유발된생쥐는대조군에비해일일먹이섭취량이현저하게증가하였다 ( 대조군, 4.2 ± 0.4 g/day; 실험군, 7.4 ± 0.9 g/day, P < 0.001, Table 1). 혈액에서인슐린과렙틴의농도를면역효소법을이용하여측정한결과, 당뇨병이유발된생쥐에서혈중인슐린 ( 대조군, 1,303.7 ± 687.6 pmol/l; 실험군, 240.3 ± 26.8 pmol/l, P < 0.01) 과렙틴 ( 대조군, 2,116.5 ± 465 pg/ml; 실험군, 86.5 ± 41.5 pg/ml, P < 0.001) 의농도가유의하게감소하였다 (Table 1). 2. 당뇨병실험동물에서시상하부의식욕조절신경펩타이드와 FoxO1 mrna 합성의변화인슐린과렙틴이결핍된당뇨병실험동물에서시상하부의신경펩타이드를확인한결과식사섭취를자극하는신경펩타이드인 NPY의합성은증가하였으며 (P < 0.05, Fig. 1B) 식사섭취를억제하는신경펩타이드인 POMC는감소하였다 (P < 0.01, Fig. 1C). 시상하부 FoxO1 mrna의합성은대조군에비해당뇨병실험군에서유의하게증가하였다 (P < 0.001, Fig. 1A). 3. 식사제한을한실험동물에서식욕조절신경펩타이드와 FoxO1 mrna 합성의변화 48시간동안식사제한을한실험동물에서시상하부 NPY 의합성은증가하였으며 (P < 0.01, Fig. 2B) POMC는감소하였다 (P < 0.01, Fig. 2C). 시상하부 FoxO1 mrna의합 Table 1. Changes of body weight and plasma metabolic parameters Control Diabetes P Change of body weight (g) 0.9 ± 0.1-2.2 ± 0.5 < 0.01 Daily food intake (g/day) 4.2 ± 0.4 7.4 ± 0.9 < 0.001 Plasma glucose (mmol/l) 6.8 ± 0.8 25.5 ± 2.8 < 0.001 Plasma insulin (pmol/l) 1,303.7 ± 687.6 240.3 ± 26.8 < 0.01 Plasma leptin (pmol/l) 2,116.5 ± 465.0 86.5 ± 41.5 < 0.001 Data are means ± S.D. Change of body weight during the experiment (for 10 days). 377
A B C Fig. 1. Results of hypothalamic gene expression in streptozotocin induced diabetic mice. Levels of FoxO1, NPY and POMC gene expression in the hypothalamus of diabetic mice as determined by real time PCR. A, B, C. Expression levels of FoxO1, NPY and POMC mrna were examined by real time PCR. Results are presented as means ± S.D. n = 5 per group. P < 0.05, P < 0.01 and P < 0.001 vs. control mice. A B C D E Fig. 2. Effect of food deprivation on the mrna level of hypothalamic FoxO1 and serum level of insulin and leptin. A, B, C. Expression levels of hypothalamic FoxO1, NPY and POMC mrna in the animals of food deprivation for two days and in the animals of normally fed. D, E. Serum levels of insulin and leptin. Data are shown as means ± S.D. n = 5 per group; P < 0.05, P < 0.01 vs. control mice. 성은대조군에비해식사제한을한실험군에서유의하게증가하였다 (P < 0.01, Fig. 2A). 혈중인슐린 ( 대조군, 669.7 ± 82.9 pg/ml; 실험군, 208.0 ± 94.6 pg/ml, P < 0.01, Fig. 2D) 과렙틴 ( 대조군, 1,365.7 ± 670.7 pg/ml; 실험군, 186.8 ± 155.5 mg/dl, P < 0.05, Fig. 2E) 은식사를제한한실험 군에서의미있게감소하였다. 4. 당뇨병실험동물의뇌실에인슐린또는렙틴의투여후 FoxO1 mrna 합성변화당뇨병이유발된실험동물의뇌실에인슐린또는렙틴을 378
남궁일성외 8 인 : 스트렙토조토신으로유발된당뇨병모델에서과식증에대한시상하부 FoxO1 의역할 A B C Fig. 3. Effect of i.c.v. administration of saline, insulin or leptin on hypothalamic FoxO1 expression in the streptozotocin induced diabetic mice. A, B, C. Expression levels of hypothalamic FoxO1, NPY, POMC mrna in diabetic mice after i.c.v. administration of saline, insulin or leptin. Data are shown as the means ± S.D. n = 5 per group. P < 0.05 and P < 0.01 vs. control mice. P < 0.05 and P < 0.01 vs. diabetic mice. 주입하고시상하부에서 FoxO1의합성변화를관찰한결과시상하부 FoxO1의합성증가가뇌실에인슐린또는렙틴의투여후유의하게감소하였다 (P < 0.05, Fig. 3A). 또한, 실험동물의뇌실에인슐린또는렙틴을주입후에는당뇨병실험동물에서증가되었던시상하부 NPY mrna 합성이감소되었으며 (P < 0.05; P < 0.01, Fig. 3B), 당뇨병실험동물에서감소되었던 POMC mrna 합성은인슐린또는렙틴을뇌실내투여후유의하게증가하였다 (P < 0.05; P < 0.05, Fig. 3C). 고찰생명유지를위하여에너지항상성조절은필수적인요소이다. 시상하부는말초조직에서기원되는다양한호르몬의신호를통합하여신경계및내분비계를통하여식사섭취행동을조절한다 11,12). 최근여러연구에의해에너지항상성조절신호를통합하며조절하는중추신경계의역할및작용기전에대한연구가활발하게진행되고있지만실험적모델을활용하여시상하부에서식욕을조절하는유전자의기능분석에관한연구는부족한실정이다. 따라서본연구에서는인슐린과렙틴이결핍된당뇨병모델에서나타나는과식증과시상하부에서식욕조절기능을수행하는전사조절인자인 FoxO1과의관계를살펴보았다. 당뇨병에서는인슐린의분비능상실에의해세포내에너지결핍이유도되고이를극복하기위해수반되는지질분해활성화가혈중렙틴의수준을낮춘다. 이러한인슐린과렙틴의결핍은당뇨병에서나타나는과식증의직접적인원인이된다 4,13). FoxO1은인슐린신호전달경로에서중요한 역할을수행하는전사조절인자로서세포의증식과분화에관여하며근육에서당대사조절기능을수행할뿐만아니라지방세포분화조절을수행하는등체내대사조절에중요한전사조절인자이다 14-16). 특히최근에는시상하부에서식욕조절호르몬의신호를매개하여식욕조절신경펩타이드인 AgRP와 POMC의유전자발현을직접적으로조절하는중요한식사섭취의조절인자임이규명되었다 8,9). 본연구에서스트렙토조토신으로유발된당뇨병생쥐에서시상하부의 FoxO1 mrna 발현이대조군보다유의하게증가하였으며이는인슐린과렙틴의결핍으로인한식사섭취의증가를 FoxO1이매개할가능성을제시하였다. 또한당뇨병모델과유사하게과식증이나타나고인슐린과렙틴이낮은수준으로유지되는식사제한모델에서도, 시상하부의 FoxO1 mrna 발현이증가하였다. 흥미롭게도 FoxO1 은 NPY와 AGRP의전사를조절하는전사조절인자이므로 FoxO1이혈중인슐린과렙틴의수준을매개하고식욕조절신경펩타이드의전사를조절하여당뇨병성과식증에관여할것으로판단된다. 실제로당뇨병생쥐의뇌실로인슐린또는렙틴을주입한결과, 이두호르몬의결핍으로유도된 FoxO1의합성증가가유의하게억제되는것을확인하였다. 따라서, FoxO1이당뇨병에서동반되는혈중인슐린과렙틴의저하에의한과식증을매개하는전사조절인자임을확인하였다. 최근 Ropelle 등의연구에서는식사에의해비만이유도된쥐에서 FoxO1 antisense oligonucleotide (ASO) 를이용하여시상하부에서 FoxO1의작용을차단하는실험을하였다. 비만이유도된실험동물은인슐린저항성이동반되어있어뇌실내로투여된인슐린에의해 FoxO1의변화는나타 379
나지않았다. 그러나 FoxO1-ASO을뇌실내로투여하여직접 FoxO1의작용을차단하면실험동물의식사섭취와체중이감소함을확인하였다 17). 결국비만에서나타나는과식증이 FoxO1과연관이있음을시사한다. 본연구에서는 Ropelle 등의연구와는달리스트렙토조토신으로유발된당뇨병모델에서의과식증이 FoxO1을매개하여작용하며여기에는인슐린과렙틴의결핍이모두관여함을확인하였다. 인슐린과렙틴이외에도당뇨병에서현저하게상승하며식욕촉진을자극하는호르몬인 ghrelin의기능에따라식사섭취의증가가유도된다는보고가있다 18). 따라서, 추가적인연구를통해 FoxO1이당뇨병모델에서 ghrelin의신호를매개하여식사섭취를변화시키는지확인할필요가있다고본다. 본연구는스트렙토조토신으로유발된당뇨병에서인슐린과렙틴의신호를전달하여식사섭취를조절하는 FoxO1 의기능을구체화하였다. 이는앞으로당뇨병성과식증을포함한대사질환의치료를위한기초적자료를제공할것으로기대된다. 요약연구배경 : 스트렙토조토신으로유발된당뇨병실험동물은혈중인슐린과렙틴의결핍으로인하여과식증이특징적으로발생된다. 전사조절인자인 FoxO1은인슐린신호전달체계를통하여대사및세포의분화를조절한다. 본연구에서는인슐린과렙틴이결핍된당뇨병동물모델에서시상하부의 FoxO1이당뇨병성과식증과연관되어있는지를알아보고자하였다. 방법 : 생쥐 (C57BL/6) 의복강으로스트렙토조토신 (200 mg/kg) 을투여하여당뇨병을유발하였으며대조군은 50 mm의 sodium citrate buffer (ph4.0) 을복강으로투여였다. 생쥐의뇌실로인슐린및렙틴을주입하기위하여정위뇌수술장치를이용하여스테인리스관을삽관하고 3주후생리식염수 (2 μl), 인슐린 (500 nmol), 렙틴 (1 μg/2 μl) 을각각주입하였다. FoxO1과시상하부신경펩타이드의 mrna 발현을확인하기위하여각각의실험군및대조군생쥐의시상하부를적출하여 RNA을분리하고 cdna를합성하여 real time PCR을수행하였다. 결과 : 스트렙토조토신으로유발된당뇨병생쥐에서는대조군과비교하여유의한혈당증가 (25.5 ± 2.8 mmol/l vs. 6.8 ± 0.8 mmol/l) 및식사섭취의증가 (7.4 ± 0.9 g/day vs. 4.2 ± 0.4 g/day) 가나타났다. 또한당뇨병이유발된생쥐는 정상생쥐보다혈중인슐린 (1,303.7 ± 687.6 pmol/l vs. 240.3 ± 26.8 pmol/l) 과렙틴 (2,116.5 ± 465.0 pg/ml vs. 86.5 ± 41.5 pg/ml) 은현저하게감소하였다. 시상하부 FoxO1의 mrna 합성은당뇨병이유발된생쥐 (P < 0.001) 에서대조군과비교하여유의한증가를관찰하였다 (2.6배증가, P < 0.001). 당뇨병이유발된시험군은시상하부의 NPY mrna의발현이유의하게증가 (1.85배증가, P < 0.05) 하였으며 POMC mrna는유의하게감소 (2.1배감소, P < 0.01) 하였다. 생쥐의뇌실로인슐린또는렙틴을투여하였을때증가된시상하부 FoxO1이유의하게감소하였다 ( 각각 1.5배, 1.8배감소, P < 0.05). 또한, 인슐린또는렙틴을뇌실내로투여한결과 NPY mrna는감소되고 ( 인슐린 1.5배감소, P < 0.05, 렙틴 1.6배감소, P < 0.01) POMC mrna는증가되었다 ( 각각 1.6배, 1.9배증가, P < 0.05). 결론 : 본연구에서는인슐린과렙틴의결핍으로과식증이유발된당뇨병모델에서시상하부 FoxO1의합성이증가하였으며뇌실로인슐린또는렙틴을주입하였을때 FoxO1합성의증가가상쇄됨을확인하였다. 이러한결과는당뇨병에서나타나는과식증이시상하부의전사조절인자인 FoxO1 과연관되어있음을나타낸다. 참고문헌 1. Booth DA: Some characteristics of feeding during streptozotocin-induced diabetes in the rat. J Comp Physiol Psychol 80:238-49, 1972 2. Hidaka S, Yoshimatsu H, Kondou S, Oka K, Tsuruta Y, Sakino H, Itateyama E, Noguchi H, Himeno K, Okamoto K, Teshima Y, Okeda T, Sakata T: Hypoleptinemia, but not hypoinsulinemia, induces hyperphagia in streptozotocin-induced diabetic rats. J Neurochem 77:993-1000, 2001 3. Namkoong C, Kim MS, Jang PG, Han SM, Park HS, Koh EH, Lee WJ, Kim JY, Park IS, Park JY, Lee KU: Enhanced hypothalamic AMP-activated protein kinase activity contributes to hyperphagia in diabetic rats. Diabetes 54:63-68, 2005 4. Havel PJ, Uriu-Hare JY, Liu T, Stanhope KL, Stern JS, Keen CL, Ahrén B: Marked and rapid decreases of circulating leptin in streptozotocin diabetic rats: reversal by insulin. Am J Physiol 274:1482-91, 1998 5. Sipols AJ, Baskin DG, Schwartz MW: Effect of 380
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