Diabetes and Metabolism Journal

Korean Diabetes Association (대한당뇨병학회)
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Protective Effects of Glucagon Like Peptide-1 on HIT-T15 $\beta$ Cell Apoptosis via ER Stress Induced by 2-deoxy-D-glucose

2-deoxy-D-glucose에 의해 유도된 소포체 스트레스에서 글루카곤양 펩티드-1의 HIT-T15 베타세포 사멸 보호효과

Kim, Ju-Young;Lee, Seong-Kyu;Baik, Haing-Woon;Lee, Ki-Ho;Kim, Hyun-Jin;Park, Kang-Seo;Kim, Byung-Joon
김주영;이성규;백행운;이기호;김현진;박강서;김병준

  • Published : 2008.12.01
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Abstract

Background: The characteristic feature of pancreatic β cells is highly developed endoplasmic reticulum (ER) due to a heavy engagement in insulin secretion. The ER serves several important function, including post-translational modification, folding, and assembly of newly synthesized secretory proteins, and its proper function is essential to cell survival. Various stress conditions can interfere with ER function. Pancreatic β cells may be particularly vulnerable to ER stress that causes to impair insulin biosynthesis and β cell survival through apoptosis. Glucagon like peptide-1 (GLP-1) is a new drug for treatment of type 2 diabetes and has effects on stimulation of insulin secretion and β cell preservation. Also, it may have an antiapoptotic effect on β cells, but detailed mechanisms are not proven. Therefore, we investigated the protective mechanism of GLP-1 in β cells through ER stress response induced by 2-deoxy-D-glucose (2DG). Methods: For induction of the ER stress, HIT-T15 cells (hamster β cell line) were treated with 2DG (10 mM). Apoptosis was evaluated with MTT assay, hoechst 33342 staining and Annexin/PI flow cytometry. Expression of ER stress-related molecules was determined by real-time PCR or western blot. For blocking ER stress, we pretreated HIT-T15 cells with exendin-4 (Ex-4; GLP-1 receptor agonist) for 1 hour before stress induction. Results: After induction with ER stress (2DG), β cells were lost by apoptosis. We found that Ex-4 had a protective effect through ER stress related molecules (GRP78, GRP94, XBP-1, eIF2α, CHOP) modulation. Also, Ex-4 recovered the expression of insulin2 mRNA in β cells. Conclusion: These results suggest that GLP-1 may protect β cells apoptosis through ER stress modulation. (KOREAN DIABETES J 32:477-487, 2008)

연구배경: 췌장 베타세포는 인슐린 분비 작용으로 인해 소포체가 잘 발달되어 있다는 것이 중요한 특징 중의 하나 이다. 소포체는 번역 후 수정, 폴딩, 새롭게 합성된 분비 단 백질의 재배열 등 중요한 기능을 담당하고, 세포의 생존에 있어서도 중요한 역할을 담당한다. 소포체는 다양한 생리, 병리학적 자극에 의한 스트레스를 받게 되는데 이를 소포체 스트레스라 하고, 이러한 소포체 스트레스에 베타세포는 취 약하며, 소포체 스트레스에 의해 베타세포는 인슐린의 합성 및 생존에 문제가 발생하여 세포 자연사를 일으킨다. 글루 카곤양 펩티드-1 (GLP-1)은 제2형 당뇨병의 새로운 치료제 로 인슐린 분비와 베타세포 증식의 촉진한다고 알려져 있다. 또한 이는 베타세포의 사멸에 있어 보호효과를 가지지만, 이에 대한 자세한 기전은 밝혀지지 않았다. 따라서 2-deoxy-D-glucose에 발생된 소포체 스트레스를 통한 베타 세포 사멸에 있어서 GLP-1의 보호효과와 기전을 연구하고 자 한다. 방법: 인슐린 분비 베타세포로 햄스터에서 유래된 HIT-T15 세포주를 사용하였다. 소포체 스트레스를 유도하 기 위해서 HIT-T15 세포에 2-deoxy-D-glucose (2DG, 10 mM)를 처리하였다. 베타세포의 자연사는 MTT 분석, hoechst 33342 염색과 annexinV/PI 이중염색을 통한 FACS 분석을 통해 시행하였고, 소포체 스트레스 관련 유전자 및 단백질은 real-time PCR과 western blot으로 분석하였다. GLP-1 수용체 자극제인 Ex-4를 이용하여 세포 자연사에 대한 보호효과를 증명하기 위해 스트레스 유도 1시간 전에 전처리하였다. 결과: 2DG (10 mM)에 의한 소포체 스트레스를 유도했 을 때, 시간이 경과함에 따라 베타세포의 세포사멸이 증가 된다. 이 때, Ex-4 (25 nM)를 처리하게 되면 GRP78, GRP94, eIF2α, XBP-1과 CHOP 등을 조절함으로써 베타세 포의 세포사멸에 대한 보호효과를 가지고, insulin2 mRNA 의 발현 증가로 인해 인슐린 합성능을 증가시킨다. 결론: GLP-1은 소포체 스트레스 반응 조절을 통하여 베 타세포의 세포사멸에 대한 보호효과를 가지며 인슐린 mRNA 발현도 향상시켰다. 이는 GLP-1이 당뇨병 발생을 예방하고 치료하는데 중요한 역할을 할 수 있음을 제시한다.

Keywords

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