당뇨병제 31 권제 2 호, 2007 고하며, 고농도포도당에의한산화스트레스의증가가원인으로알려져있다 6). Polyol 경로, methylglyoxal 형성, 비효소단백질당화 (nonenzymatic protein glycosylation), protein kinase

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1 당뇨병제 31 권제 2 호, 2007 원저 2-Deoxy-D-ribose 가췌장베타세포의손상을유발하는기전에관한연구 제주대학교의과대학내과학교실, 조직학교실 1, 경희대학교의과대학내분비대사내과 2 고관표, 우정택 2, 이대호, 오승준 2, 김성운 2, 김진우 2, 김영설 2, 박덕배 1 Mechanism of 2-Deoxy-D-ribose-induced Damage in Pancreatic β-cells Gwanpyo Koh, Jeong-taek Woo 2, Dae-Ho Lee, Seungjoon Oh 2, Sung-Woon Kim 2, Jin-Woo Kim 2, Young-Seol Kim 2, Deok-Bae Park 1 Department of Internal Medicine, Cheju National University College of Medicine; Department of Medicine 1, Cheju National University College of Medicine; and Department of Endocrinology & Metabolism 2, Kyung Hee University College of Medicine Abstract Background: Mechanism for glucose toxicity is known to be an increased oxidative stress produced by multiple pathways. In our previous report, 2-deoxy-d-ribose (drib) promoted apoptosis by increasing oxidative stress in a pancreatic β-cell line. We performed this study to investigate the mechanism of drib-induced damage of β-cells. Methods: HIT-T15 cells were cultured in RPMI-1640 medium with 40 mm drib for 24 hours after pretreatment with various concentrations of a metal chelator (DTPA) and inhibitors of protein glycation (aminoguanidine and pyridoxamine). Cell viability was determined by MTT assay. Apoptosis was analyzed by flow cytometry with annexin V/PI double staining. Results: DTPA, which inhibits the monosaccharide autoxidation, partially reversed drib-induced cytotoxicity in a dose-dependent manner (P < 0.01). The cytotoxicity was also suppressed dose-dependently by aminoguanidine (AG) and pyridoxamine (PM) (P < 0.05 and P < 0.01, repectively). Flow cytometric analysis showed that pretreatment of DTPA and AG also reversed the drib-triggered apoptosis in a dose-dependent manner. We assessed the additional protective effects of inhibitors of protein glycation from drib-induced cytotoxiciy in the presence of a metal chelator. The additions of AG (P < 0.05) and PM (P < 0.01) significantly reduced the cytotoxicity compared with DTPA alone group. Conclusion: This results suggest that drib produce cytotoxicity and apoptosis through the mechanisms of advanced glycation endproducts (AGEs) formation including the monsaccharide autoxidation and protein glycation in pancreatic β-cell. Thus, drib could be a surrogate for glucose in the study of glucose toxicity and chronic diabetic complications. (J Kor Diabetes Assoc 31:105~112, 2007) Key Words: Autoxidation, Glucose toxicity, Oxidative stress, Protein glycation, β-cell apoptosis, 2-deoxy-D-ribose 서론제2 형당뇨병의병인은말초조직의인슐린저항성과췌장의베타세포부전으로설명된다. 베타세포부전은인슐린분비이상뿐아니라세포자멸사 (apoptosis) 로인한베타세포용 적 (β-cell mass) 의감소도포함한다 1). 베타세포부전의정확한기전은알려져있지않으나만성적인고혈당, 혈중유리지방산의증가, 아밀로이드폴리펩티드그리고유전적요인등이가설로제기되고있다 2-5). 만성적인고혈당에의한비가역적인베타세포부전을당독성 (glucose toxicity) 이라 접수일자 : 2006 년 10 월 26 일, 통과일자 : 2007 년 1 월 15 일, 책임저자 : 고관표, 제주대학교의과대학내과학교실 * 본연구는 2005 년대한당뇨병학회제 11 회세르비에연구비에의해이루어졌음. 105

2 당뇨병제 31 권제 2 호, 2007 고하며, 고농도포도당에의한산화스트레스의증가가원인으로알려져있다 6). Polyol 경로, methylglyoxal 형성, 비효소단백질당화 (nonenzymatic protein glycosylation), protein kinase C 활성화, hexosamine 경로, 포도당자가산화 (glucose autoxidation) 그리고산화적인산화 (oxidative phosphorylation) 등이당독성의기전으로받아들여지고있다 7,8). 이중비효소단백질당화, 포도당자가산화그리고 methylglyoxal은서로밀접하게연결되어있으며결국최종당화산물들 (advanced glycation endproducts, AGEs) 을형성하게된다. 비효소단백질당화는 Maillard 반응이라고도하며포도당이저절로단백질의아미노기와반응하여 Schiff 염기를형성하고이어서재배열 (rearrangement) 되어비가역적인 Amadori 화합물이만들어지고추가적인농축 (condensation), 탈수 (dehydration) 그리고산화분절 (oxidative fragmentation) 반응을거쳐최종당화산물이형성된다 9,10). Aminoguanidine 과 pyridoxamine 은구체적인기전은다르지만단백질당화반응을억제하여최종당화산물형성을억제시키는것으로알려져있다 11). 포도당자가산화는전이금속 (transition metal) 을촉매로하여포도당이 enediol 을거쳐 α -ketoaldehyde 로변환되는반응을말한다. 이반응은금속킬레이트제 (metal chelator) 인 diethylenetriaminepentaacetic acid (DTPA) 에의해억제되며, 활성화산소종 (reactive oxygen species, ROS) 이중간산물로만들어진다. 자가산화의종산물인 α-ketoaldehyde는단백질과반응하여최종당화산물을형성하며이를 'autoxidative glycosylation' 이라고한다 12,13). Methylglyoxal 은반응성이매우강한 dicarbonyl 로서해당작용의중간산물인 dihydroxyacetone phosphate와 glyceraldehyde 3-phosphate로부터만들어지며최종당화산물의전구체로작용한다 14). 알데하이드기를가지면서카르복실산으로산화되는당을환원당 (reducing sugar) 이라고하며, 단백질당화능력은 glucose < mannose < galactose < xylose < fructose < arabinose < ribose < 2-deoxy-D-ribose (drib) 의순으로알려져있다 10,15). 이중포도당은환원능이가장낮기때문에실험실에서당독성을관찰하기위해서는오랜시간이필요하다. Olson 등 16) 은베타세포주를고농도포도당에약 20주간노출시켰을때비로서인슐린유전자발현이비가역적으로감소된다고보고한바있다. 특히고농도포도당에의한베타세포의자멸사는실험실에서관찰하기가매우어렵다. 따라서포도당대신환원능이아주강한 drib로베타세포를처리하여당독성연구를진행하였다. 저자는최근 drib가베타세포주인 HIT-T15 세포에서 24 시간만에산화스트레스를증가시켜세포자멸사를유발한다고보고하였으나그기전에대한연구는수행하지않았다 17). 따라서본연구는 drib가당독성의기전을통해산화스트레 스를유발한다는사실을밝힘으로써췌장베타세포에서 drib가포도당대신당독성연구에쓰일수있는지알아보기위해시행하였다. 대상및방법 1. 시약 2-Deoxy-D-ribose (drib), aminoguanidine (AG), diethylenetriaminepentaacetic acid (DTPA), pyridoxamine (PM), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) 와 dimethyl sulfoxide (DMSO) 는 Sigma Chemical Co. (St. Louis, MO, USA), RPMI-1640, Dubelco's phosphate-buffered saline (DPBS), trypsin, penicillin과 streptomycin은 GIBCO Invitrogen Co. (Grand Island, NY, USA), 그리고 fetal bovine serum (FBS) 은 HyClone Co. (Logan, UT, USA) 에서구입하였다. 모든배양접시는 BD Falcon (Franklin Lakes, NJ, USA) 제품을사용하였다. 2. 세포배양인슐린분비세포주인 HIT-T15 세포 (24-40 passage) 를 10% FBS, penicillin 100 IU/mL, 그리고 streptomycin 100 μg/ml이들어있는 RPMI-1640 배지로 37, 5% CO 2 환경에서배양하였다. 세포밀도 (confluence) 가약 70% 에이르면 DPBS으로세척하고 0.05% trypsin 용액을처리하여계대배양하였다. 계대배양하고 2일후에 0.5% FBS가들어있는 RPMI-1640 배지로교체하여하루동안방치하였다. 그리고 40 mm 농도의 drib와 0.5% FBS가들어있는 RPMI-1640 배지로다시교체하여 24시간동안배양하였으며, DETAPAC, AG와 PM은 drib 자극 30분전에추가하였다. 3. 세포생존율측정세포생존율은잘알려진 MTT 분석으로측정하였다 18). 24-well 배양접시에 well 당 개의 HIT-T15 세포를분주하여배양하였다. 배지 1 ml 당 MTT 용액 (5 mg/ml) 100 L을추가하여 4시간경과후배양액을버리고각 well에 DMSO 200 μl를가하여세포내 formazan crystal을용해시켰다. 흡광도는 ELISA plate reader (Bio-Rad Laboratories, Hercules, CA, USA) 로 540 nm 파장에서측정하였다. 세포생존율은매실험때마다 drib를처리하지않은대조군의흡광도를 100% 로하였으며각실험군은이에대한비율로표시하였다. 4. 세포자멸사의측정 Annexin V와 propidium iodide (PI) 로이중염색하고유동세포분석법 (flow cytometry) 을이용하여생존세포 (annexin 106

고관표외 7 명 : 2-Deoxy-D-ribose 가췌장베타세포의손상을유발하는기전에관한연구 V-음성 /PI-음성), 초기세포자멸사세포 (annexin V-양성 /PI- 음성 ), 후기세포자멸사또는괴사세포 (annexin V-양성 /PI-양성) 로구분하였으며, 총세포자멸사는 annexin V-양성 /PI-음성또는양성세포의비율로표시하였다 19).

3 고관표외 7 명 : 2-Deoxy-D-ribose 가췌장베타세포의손상을유발하는기전에관한연구 V-음성 /PI-음성), 초기세포자멸사세포 (annexin V-양성 /PI- 음성 ), 후기세포자멸사또는괴사세포 (annexin V-양성 /PI-양성) 로구분하였으며, 총세포자멸사는 annexin V-양성 /PI-음성또는양성세포의비율로표시하였다 19). 6-well 배양접시에 well 당 개의 HIT-T15 세포를분주하여배양하였다. Vibrant Apoptosis Assay Kit #2 (Molecular Probes, Eugene, OR, USA) 를사용하였으며첨부된설명서에따라이중염색을시행하였다. 요약하면 6-well 배양접시로부터세포를수확하여원심분리한후세포 pellets을 DPBS에현탁시킨다. 다시원심분리하여형성된 pellets을 FITC가결합된 annexin V와 PI 염색용액에재현탁시켜실온에 15분간방치하였다. 염색된세포들은 FACScan (BectonDickinson, San Jose, CA, USA) 으로분석하고 CellQuestPro 소프트웨어 (BectonDickinson) 로각사분획의세포들을계산하였다. 각시료당 10,000개의세포를사용하였다. 4. 통계분석군간비교는 SPSS 11.0 for Windows (Chicago, IL, USA) 프로그램을이용하여비모수검정인 Mann-Whitney test로분석하였으며, P 값이 0.05 미만일경우통계적유의성이있는것으로판정하였다. 모든결과는평균 ± 표준편차로기술하였다. 결과 1. 금속킬레이트제와단백질당화억제제가 drib에의한세포독성에미치는영향세포독성이 drib의자가산화에의한반응인지알아보기위하여금속킬레이트제인 DTPA를전처치하였으며, 단백질당화억제제로잘알려진 aminoguanidine과 pyridoxamine 을사용하여 Maillard 반응과의관계를알아보았다. DTPA, aminoguanidine과 pyridoxamine을 30분전에처치하고 40 mm drib와 0.5% FBS가들어있는 RPMI-1640 배지로 HIT-T15 세포를 24시간동안배양한후 MTT 분석을시행하였다. 40 mm drib로만자극하였을때 33% (P < 0.01) 까지감소했던세포생존율은 DTPA, aminoguanidine과 pyridoxamine을전처치함으로써농도의존적으로의미있게회복되었다. DTPA는 0.01, 0.1 그리고 0.3 mm 농도에서세포생존율이각각 48 (P < 0.01), 66 (P < 0.01) 그리고 71% (P < 0.01) 로 drib만투여한군에비해유의하게증가되었다. Aminoguanidine은 0.5, 1 그리고 5 mm 농도로전처치하였을때세포생존율이 38 (P < 0.05), 39 (P < 0.05) 그리고 56% (P < 0.01) 로유의하게증가되었으나 DTPA에비해효과가작았다. Pyridoxamine은 0.5, 1 그리고 3 mm 농도에서 46 (P < 0.01), 53 (P < 0.01) 그리고 72% (P < 0.01) 로회복되어 DTPA와비슷한효과를보였다 (Fig. 1). Fig. 1. Protective effects of DTPA, aminoguanidine (AG) and pyridoxamine (PM) on drib-triggered cytotoxicity. HIT-T15 cells were preincubated with DTPA, AG and PM for 30 min at the indicated concentrations and then cultured with 40 mm drib for 24 h. Cell viability was determined by MTT assay. Data are expressed as the mean ± SD of the percentage of viable cells relative to the untreated control. This experiment was performed twice, in quadruplicate. ** P < 0.01 vs. control. P < 0.05 vs. 40 mm drib alone. P < 0.01 vs. 40 mm drib alone. 107

4 당뇨병제 31 권제 2 호, 2007 Fig. 2. Effect of DTPA on drib-induced apoptosis of HIT-T15 cells. Cells were preincubated with 0.01, 0.1 and 0.3 mm DTPA for 30 min, then cultured with 40 mm drib for 24 h. Cells were stained with Annexin V-FITC (horizontal axis)/pi (longitudinal axis) and analyzed by flow cytometry. The graph is representative of two independent experiments. (A) Control; (B) 40 mm drib; (C) 40 mm drib mm DTPA; (D) 40 mm drib mm DTPA; (E) 40 mm drib mm DTPA. Fig. 3. Effect of AG on drib-induced apoptosis of HIT-T15 cells. Cells were preincubated with 0.5, 1 and 5 mm AGfor 30 min, then cultured with 40 mm drib for 24 h. Cells were stained with Annexin V-FITC (horizontal axis)/pi (longitudinal axis) and analyzed by flow cytometry. The graph is representative of two independent experiments. (A) Control; (B) 40 mm drib; (C) 40 mm drib mm AG; (D) 40 mm drib + 1 mm AG; (E) 40 mm drib + 5 mm AG. 108

고관표외 7 명 : 2-Deoxy-D-ribose 가췌장베타세포의손상을유발하는기전에관한연구 2. 금속킬레이트제와단백질당화억제제가 drib에의한세포자멸사에미치는영향 DTPA, aminoguanidine과 pyridoxamine이단순한세포독성이아닌세포자멸사를억제하는지알아보기위해 annexin V와 PI로이중염색하고유동세포분석법을시행하였다.

5 고관표외 7 명 : 2-Deoxy-D-ribose 가췌장베타세포의손상을유발하는기전에관한연구 2. 금속킬레이트제와단백질당화억제제가 drib에의한세포자멸사에미치는영향 DTPA, aminoguanidine과 pyridoxamine이단순한세포독성이아닌세포자멸사를억제하는지알아보기위해 annexin V와 PI로이중염색하고유동세포분석법을시행하였다. 생존세포비율은대조군이 68.2% 였고 40 mm drib 자극에의해 12.8% 로감소되었으나, 0.01, 0.1 그리고 0.3 mm DTPA를전처치하였을때는각각 44.7, 53.1 그리고 61.2% 로농도에비례하여회복되었다. 총세포자멸사 ( 초기와후기세포자멸사 ) 비율은대조군 29.4% 에서 drib 자극으로 85.5% 까지증가하였으며, DTPA 전처치로인해각각 53.6, 44.9 그리고 37.1% 로농도의존적으로감소되었다 (Fig. 2). 0.5, 1 그리고 5 mm aminoguanidine을전처치하였을때생존세포비율이대조군 63.3% 그리고 drib군 21.4% 에서각각 30.2, 38.4 그리고 40.4% 로증가하였고, 총세포자멸사비율또한대조군 34.8% 와 drib군 76.6% 에서 DTPA를전처치했을때각각 68.4, 60.3 그리고 57.8% 로농도의존적인감소를보였다 (Fig. 3). 3. 금속킬레이트제를전처치하였을때단백질당화억제제가 drib에의한세포독성을억제하는효과 Aminoguanidine과 pyridoxamine을비롯한많은최종당화산물억제제들은금속킬레이트효과를가지고있는것으 로보고된바있다 20). 이를배제하기위해금속킬레이트제인 DTPA를전처치하고나서 aminoguanidine과 pyridoxamine 의세포독성억제효과를관찰하였다. drib에의한세포독성과세포자멸사억제효과가가장큰 0.3 mm 농도의 DTPA 를 30분전에처치하고여러농도의 aminoguanidine과 pyridoxamine과 40 mm drib를자극한후 MTT 분석을시행하였다. drib 자극에의해 11% 까지감소되었던세포생존율은 0.3 mm DTPA 전처치로인해 41% (P < 0.01) 로유의하게회복되었다. 1과 5 mm aminoguanidine를추가하였을때세포생존율이각각 51 (P < 0.05), 52% (P < 0.05) 로 0.3 mm DTPA만전처치했을때보다의미있게증가하였다. Pyridoxamine은 0.5, 1 그리고 3 mm 농도에서각각 72 (P < 0.01), 76 (P < 0.01) 그리고 74 (P < 0.01)% 로더좋은효과를보였다 (Fig. 4). 고찰본연구는췌장베타세포주인 HIT-T15 세포에서 drib에의한세포독성과세포자멸사가금속킬레이트제인 DTPA와단백질당화억제제인 aminoguanidine과 pyridoxamine에의해부분적이지만의미있게억제되는결과를보여주었다. 이전연구에서저자는 HIT-T15 세포를 drib로자극하였을때세포자멸사와산화스트레스가증가되며, N-acetyl-L-cysteine Fig. 4. Additive effects of AG and PM on DTPA-mediated protection from of drib-induced cytotoxicity. HIT-T15 cells were preincubated with DTPA, AG and PM for 30 min at the indicated concentrations and then cultured with 40 mm drib for 24 h. Cell viability was determined by MTT assay. Data are expressed as the mean ± SD of the percentage of viable cells relative to the untreated control. This experiment was performed twice, in quadruplicate. ** P < 0.01 vs. control. P < 0.01 vs. 40 mm drib alone. P < 0.05 vs. 40 mm drib mm DTPA. P < 0.01 vs. 40 mm drib mm DTPA. 109

6 당뇨병제 31 권제 2 호, 2007 (NAC) 으로전처치했을때거의대조군수준으로회복되는결과를보고하였다 17). 당시산화스트레스측정은세포내활성화산소종과반응하는 dichlorodihydrofluorescein diacetate (H 2DCF-DA) 염색을이용하였고전처치물질인 NAC는 H 2O 2 를제거하는 glutathione의전구체이므로 drib 는세포내활성화산소종을증가시켜세포자멸사를일으킨다고결론지었다. 따라서본연구의결과는 drib가베타세포에서단당류자가산화와단백질당화기전을통해산화스트레스를증가시켜세포자멸사를유발한다는사실을시사한다. 1987년까지최종당화산물은 Maillard 반응을통해서만만들어지는것으로알려졌다. 그러나 Wolff 등 12,13) 은포도당이자가산화되면서활성산소종과 α-ketoaldehyde 가만들어지고산소자유라디칼에의해변형된단백질에 α-ketoaldehyde 가결합하여 ketoimmine 부가물 (adduct) 이형성되며, 이부가물은 Amadori 부가물보다더반응성이높아추가 Maillard 반응을거쳐최종당화산물이만들어진다는 autoxidative glycosylation' 의개념을처음으로주장하였다. 이후 Wells -Knecht 등 21) 에의해포도당의 autoxidative glycosylation 반응중만들어지는 α-ketoaldehyde 가 glyoxal로밝혀졌다. 이자가산화반응은금속을촉매로하는비효소반응으로금속킬레이트제인 DTPA에의해억제되며포도당보다는환원능이높은다른단당류에의해잘발생한다 22). 그러나금속을촉매로하는산화반응은단당류자가산화에만국한된것이아니며, 최종당화산물형성기전중 glycoxidation' 반응에서도관찰된다. Glycoxidation 이란단백질당화 (glycation) 에이어산화 (oxidation) 반응이발생하는것으로 23), Maillard 반응중생성된 Amadori 화합물이금속을촉매로하는자가산화반응을거쳐 carboxymethyllysine (CML) 또는 pentosidine 등의산화성최종당화산물을형성하게된다 24,25). 따라서본연구에서관찰된 DTPA의세포손상억제효과는 drib의자가산화반응뿐만아니라 Amadori 중간체의자가산화, 즉 glycoxidation 이억제되어나타났을수있다. 그러나 autoxidative glycosylation 와 glycoxidation은 in vitro와 in vivo에서주위여건에따라동시에발생할수있으며부산물도거의유사하기때문에두반응을감별하는것은매우어렵다 25,26). Aminoguanidine은약 100년전에 nitroguanidine을환원시켜개발된물질로 hydrazine과 guanidium의반응부위를가지고있는친핵성 (nucleophilic) 물질이다. 이물질은현재까지최종당화산물억제제로가장많이쓰이고있으며, 비효소단백질당화의특정경로억제제가아니라여러단계에작용한다. Hydrazine 부위는단백질과반응하기전포도당의 carbonyl 기와결합하여 Schiff 염기형성을방해하며, 또한 Amadori 화합물의 carbonyl 기와반응하여당화반응의진행을억제한다. Guanidium 부위는최종당화산물의강력한전구체인 methylglyoxal, 3-deoxyglucosone, glyoxal 등의 dicarbonyl 화합물들을차단 (trapping) 하며 aminoguanidine 의가장중요한작용으로알려져있다 27,28). Aminoguanidine은많은동물연구에서당뇨병성신증, 망막병증그리고신경병증을예방한다고잘알려져있을뿐만아니라 27) HIT-T15 세포와 Zucker 당뇨병비만쥐에서당독성을억제하여인슐린분비능을회복시킨다고보고된바있다 29). Pyridoxamine 은비타민 B6의일종으로새로운최종당화산물억제제로각광받고있으며, aminoguanidine처럼비효소단백질당화과정중에형성되는 dicarbonyl 화합물들을제거하는효과가있다 30). 또한 aminoguanidine 에서는볼수없는 Amadori 이후경로 (post-amadori pathway) 를억제하는작용이있다 11). Khalifah 등 31) 은반응도가높은고농도의 ribose를단백질과반응시킨후유리 ribose와 Schiff 염기는투석으로제거하고 Amadori 화합물만분리하여 Amadori 이후경로에대해서특이적으로연구할수있는기법을개발하였다. 이연구에서 pyridoxamine은 Amadori 화합물이최종당화산물로이행되는것을효과적으로억제하였으나 aminoguanidine은효과가없었다 32). 동물연구에서도 pyridoxamine은조직의최종당화산물침착을감소시키고당뇨병성신증을의미있게지연시키는것으로밝혀졌다 33). 그러나 dicarbonyl 화합물을제거하는능력은약하므로 aminoguanidine 보다부작용이적을것으로기대되며, 현재당뇨병성신증에대한제3 상임상연구가진행중이다 30). Price 등 20) 은많은최종당화산물억제제들이금속킬레이트작용을가지고있으므로연구결과를해석할때금속킬레이트효과가 carbonyl 또는 dicarbonyl 차단효과로오인될수있다고하였다. 그들은 aminoguanidine과 pyridoxamine 이금속을촉매로하는 ascorbate의자가산화를농도의존적으로억제하였고반응의 50% 를억제할수있는농도 (IC 50) 는각각 2.5와 1 mm라고보고하였으며, 이는본연구에사용된농도와크게다르지않았다. 따라서본연구에서는 aminoguanidine과 pyridoxamine의금속킬레이트효과를배제하기위해 DTPA를전처치한후최종당화산물억제제들의효과를관찰하였다. drib에의한세포손상을최대로억제했던 0.3 mm DTPA을전처치했음에도최종당화산물억제제들은추가보호작용을나타내었다 (Fig. 4). 이는 drib 가금속을촉매로하는자가산화반응뿐만아니라최종당화산물형성과관련된다른기전을통해세포손상을초래한다는사실을시사한다. 또한 pyridoxamine 이 aminoguanidine 보다 drib에의한세포독성을억제하는효과가크기때문에 (Fig. 1, 4) drib는 Maillard 반응중 Amadori 이후경로를거칠가능성이있다. 본연구의결과를종합해보면 drib는 HIT-T15 세포에서자가산화또는 glycoxidation 기전과 Amadori 이후경로등의최종당화산물형성기전을통하여세포독성과자멸사를증가시키는것으로보이며, 이전연구결과를감안할때산 110

7 고관표외 7 명 : 2-Deoxy-D-ribose 가췌장베타세포의손상을유발하는기전에관한연구 화스트레스매개반응으로생각된다. 그러나세부적인경로는알아내지못했으며그이유는다음과같다. 첫째대분분의단백질당화연구는화학반응을통해이루어졌으나저자의연구는세포실험으로중간반응산물의측정이어렵다. 둘째단백질당화는비효소화학반응으로다른효소, 수용체나유전자연구와는달리특이적매개체로사용할수있는물질이존재하지않는다 11). 결론적으로 drib는췌장베타세포에서최종당화산물형성과관련된기전을통해세포손상을유발하는것으로생각된다. 따라서장시간이소요되는포도당을대신하여베타세포에서당독성연구를간편하게수행할수있으며, 더나아가다른장기나조직의당뇨병으로인한만성합병증연구에도응용될수있을것으로생각된다. 요약연구배경 : Polyol 경로, methylglyoxal 형성, 비효소단백질당화, protein kinase C 활성화, hexosamine, 포도당자가산화그리고산화적인산화등의경로를통한산화스트레스의발생은췌장베타세포에서당독성의원인으로받아들여지고있다. 저자는최근 2-deoxy-D-ribose (drib) 가베타세포주에서산화스트레스와세포자멸사를유발한다고보고하였으며, 그구체적인기전을규명하기위해본연구를시행하였다. 방법 : HIT-T15 세포를 RPMI-1640 배지로배양하였다. 자가산화반응을억제하는금속킬레이트제인 DTPA 그리고단백질당화억제제로알려진 aminoguanidine (AG) 과 pyridoxamine (PM) 을 30분전에자극하고 40 mm drib를추가하여 24시간동안배양하였다. 세포생존율은 MTT 방법을이용하였고세포자멸사는 annexin V와 PI로이중염색한후유동세포분석법으로측정하였다. 결과 : 40 mm drib (33%; P < 0.01) 의해감소됐던세포생존율이 0.01, 0.1 그리고 0.3 mm DTPA를전처치했을때농도의존적으로의미있게회복되었으며 ( 각각 48, 66 그리고 71%; P < 0.01), 0.5, 1 그리고 1 mm AG ( 각각 38, 39 그리고 56%; P < 0.05) 과 0.5, 1 그리고 3 mm PM ( 각각 46, 53 그리고 72%; P < 0.01) 을전치치했을때도농도에비례하여유의하게증가되었다. 유동세포분석에서도 DTPA와 AG는농도의존적으로생존세포비율을증가시키고 drib에의한세포자멸사를억제하였다. AG와 PM의금속킬레이트효과를배제하기위하여 0.3 mm DTPA를전처치한실험에서도 AG (P < 0.05) 와 PM (P < 0.01) 은추가보호작용을나타내었다. 결론 : drib는단당류자가산화와비효소단백질당화를비롯한최종당화산물형성과관련된기전을통하여베타세 포의손상을초래하는것으로보이며당독성이나만성당뇨병합병증연구에쓰일수있을것으로생각된다. 참고문헌 1. Weir GC, Laybutt DR, Kaneto H, Bonner-Weir S, Sharma A: Beta-cell adaptation and decompensation during the progression of diabetes. Diabetes 50 Suppl 1:S154-9, Rossetti L, Giaccari A, DeFronzo RA: Glucose toxicity. Diabetes Care 13:610-30, McGarry JD, Dobbins RL: Fatty acids, lipotoxicity and insulin secretion. Diabetologia 42:128-38, Lorenzo A, Razzaboni B, Weir GC, Yankner BA: Pancreatic islet cell toxicity of amylin associated with type-2 diabetes mellitus. Nature 368:756-60, Polonsky KS, Sturis J, Bell GI: Seminars in Medicine of the Beth Israel Hospital, Boston. Non-insulindependent diabetes mellitus-a genetically programmed failure of the beta cell to compensate for insulin resistance. N Engl J Med 334:777-83, Robertson RP, Harmon J, Tran PO, Tanaka Y, Takahashi H: Glucose toxicity in beta-cells: type 2 diabetes, good radicals gone bad, and the glutathione connection. Diabetes 52:581-7, Brownlee M: Biochemistry and molecular cell biology of diabetic complications. Nature 414:813-20, Robertson RP: Chronic oxidative stress as a central mechanism for glucose toxicity in pancreatic islet beta cells in diabetes. J Biol Chem 279: , Hodge JE: Dehydrated foods: Chemistry of browning reactions in model systems. Agric. Food. Chem. 1:928-43, Monnier VM: Nonenzymatic glycosylation, the Maillard reaction and the aging process. J Gerontol 45:B105-11, Khalifah RG, Baynes JW, Hudson BG: Amadorins: novel post-amadori inhibitors of advanced glycation reactions. Biochem Biophys Res Commun 257:251-8, Hunt JV, Dean RT, Wolff SP: Hydroxyl radical production and autoxidative glycosylation. Glucose autoxidation as the cause of protein damage in the experimental glycation model of diabetes mellitus and ageing. Biochem J 256:205-12, Wolff SP, Dean RT: Glucose autoxidation and protein 111

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Pharmacotherapeutics Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee Univ

Pharmacotherapeutics Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee Univ Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee University College of Medicine & Hospital E mail : ycell2@yahoo.co.kr Abstract