系統識別號 U0026-0611201714081500
論文名稱(中文) 探討三磷酸腺苷敏感型鉀離子通道的運輸對於脂肪酸誘導胰島貝他細胞凋亡中之角色
論文名稱(英文) Study on ATP sensitive potassium (KATP) channel trafficking in free fatty acid induced apoptosis of pancreatic β; -cells in vivo and in vitro
校院名稱 成功大學
系所名稱(中) 生理學研究所
系所名稱(英) Department of Physiology
學年度 106
學期 1
出版年 106
研究生(中文) 阮瀞萱
研究生(英文) Jing-Syuan Ruan
電子信箱 dory81423@hotmail.com.tw
學號 S36034091
學位類別 碩士
語文別 英文
論文頁數 40頁
口試委員 指導教授-陳珮君
中文關鍵字 三磷酸線苷敏感型鉀離子通道  脂毒性 
英文關鍵字 KATP channel  lipotoxicity 
中文摘要 代謝性症候群中以第二型糖尿病為最普遍,當過多脂肪酸累積在胰臟的
貝他細胞中會造成脂毒性的產生,譬如,促使貝他細胞進行自我凋亡導致功能逐漸喪失,因而引起胰島素釋放失去調控‧貝他細胞中胰島素的分泌主要與細胞膜上的三磷酸線苷敏感型鉀離子通道有關,此通道由四個Kir6.2 次單元SUR1 次單元所組成‧通道的開關主要受細胞內核苷酸的濃度的影響,故可以因應內分泌訊號及細胞代謝的狀況來分泌胰島素‧此研究主要目的為,探討過多的脂肪酸對於三磷酸線苷敏感型鉀離子通道運輸至膜上的影響‧棕梠酸是最廣泛存在於飲食及血液中的飽和脂肪酸‧我們發現長期由皮下給予棕梠酸的老鼠有著第二型糖尿病的疾病表徵‧從其胰臟切片可看到細胞內的胰島素及SUR1次單位的表現量上升,接下來我們利用INS-1 細胞株,給予不同濃度的棕梠酸並維持在不同的時間點來找尋適合的藥物劑量及給予時間‧由結果可以看到,在0.4 毫莫爾濃度進行72 小時刺激之下,增加的了細胞自我凋亡,但仍有百分之二十五的細胞仍然存活‧我們也發現到細胞表面上的SUR1 次單位表現量明顯下降‧過去研究指出利用藥理學的方法,給予其抑制劑glibenclamide 可以校正三磷酸線苷敏感型鉀離子通道運輸至膜上的的缺失‧而在我們的結果當中,glibenclamide 確實增加了細胞表面SUR1 次單位的表現量上升且下降貝他細胞的死亡‧而除了glibenclamide,當同時給予其活化劑diazoxide,發現可以抑制胰島素分泌來改善胰島素過多的情形‧而同時給予兩個藥物可以看到最顯著病徵改善,包括內質網壓力的減緩及粒線體膜電位的回覆‧更重要的是glibenclamide 改善了老鼠的葡萄糖耐受性,胰島素分泌及下降三酸甘油酯的濃度‧總結以上,三磷酸線苷敏感型鉀離子通道運輸至膜上的的缺失不只影響了胰島素的分泌更影響了貝他細胞的存活‧而透過給予glibenclamide 來校正此運輸的缺失後,進而使內質網壓力下降來增加細胞的存活率及改善葡萄糖平衡‧
英文摘要 Type 2 diabetes is the most prevalent metabolic syndrome worldwide and elevated systemic levels of fatty acids are now considered significant contributors towards the progression of this syndrome. Lipotoxicity is associated with the high level of fatty acid accumulation observed in the pancreatic β-cells. The overload of free fatty acid contributes to pancreatic β-cells apoptosis and
dysfunction. Insulin secretion is controlled by KATP channel which is an octameric complex of four pore-forming inwardly rectifying potassium channel Kir6.2 subunits and four sulfonylurea receptor 1 (SUR1) regulatory subunits. In addition to gating regulation by intraceullar nucleotides, KATP conductance is also determined by channel abundance in the plasma membrane. This study was aimed to investigate effects of lipotoxicity on trafficking of KATP channel in pancreatic β cell using chronic palmitic acid mice and cell models. Palmitic acid (Palm) is the most abundant free fatty acids (FFA) present in the diet and in serum. Chronic Palm-injected mice displayed type II diabetic characteristics. The pancreatic sections of these mice showed upregulated intracellular insulin granules
and protein expression of SUR1. Then, we tested time and dose effects of palmitic acid on the cell viability using rat insulinoma (INS-1) cells. Our results indicated that 25 % of cells after 72 hours of 0.4 mM palmitic acid stayed viable. We also found a significant decrease in the surface expression of SUR1. In addition, this treatment induced pancreatic β cells apoptosis by increasing cleaved caspase 3 proteins. Researches have shown that the pharmacological chaperone, glibenclamide, corrects trafficking defect of KATP channels. Our results showed glibenclamide increased surface SUR1 expression and decreased pancreatic β cell death. Upon increasing surface KATP channels in the plasma membrane, diazoxide, the KATP channel opener, can reduce insulin secretion.
Co-treatment with glibenclamide and diazoxide in Palm-treated cells showed the most promising rescued effect, the lowest ER stress and better mitochondrial membrane potential. Importantly,glibenclamide also improved glucose tolerance, triglyceride concentration and increased insulin release in Palm-injected mice. In conclusion, impaired trafficking of KATP channels not only affects insulin secretion but also pancreatic β cell death. The underlying mechanisms in which chronic palmitic acid-impaired KATP channel trafficking is associated ER stress, hence,glibenclamide as the pharmacological chaperone can rescue cells and improve glucose homeostasis.
論文目次 口試合格證明------------------- I
Abstract --------------------- II
中文摘要 ---------------------- III
誌謝--------------------------- IV
Contents ---------------------- VI
Figure Contents --------------- VII
Introduction ------------------- 1
Roles of free fatty acids (FFAs) in glucose homeostasis-1
Regulation of ATP sensitive potassium channel (KATP) --4
KATP channel and cell survival ---------------------- 7
Research rationales and hypothesis ----------------- 9
Specific aims -------------------------------------- 9
Materials and methods ------------------------------ 10
Preparation of Palm-BSA complex Solution ----------- 10
Animals and experimental Protocol ------------------ 11
Tissue samples harvesting -------------------------- 11
Cell culture and palmitic acid treatment ----------- 11
Oil red O staining --------------------------------- 12
Cell viability assay ------------------------------- 12
Detection of mitochondrial depolarization ---------- 12
Immunocytochemistry Examination -------------------- 13
Glucose tolerance test (GTT) ----------------------- 13
Immunohistochemical (IHC) Examination -------------- 13
Glucose stimulated insulin secretion assay --------- 14
Western blot --------------------------------------- 14
Surface biotinylation ------------------------------ 15
Microscope examination ----------------------------- 15
Image analysis ------------------------------------- 15
Statistical analysis ------------------------------- 16
Results -------------------------------------------- 16
Discussion ----------------------------------------- 21
Conclusion ----------------------------------------- 24
Figures -------------------------------------------- 25
References ----------------------------------------- 35
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