The effects of non-specific potassium channel blocker tetraethylammonium on insulin secretion in perfused rat pancreas

第2型糖尿病通常源自於營養代謝的異常，且伴隨著胰島素阻抗及胰島素分泌不良的情形。Sulfonylurea類藥物則是現今廣泛用於治療非胰島素依賴型糖尿病患者的口服藥物，其作用主為關閉ATP-sensitive K+（KATP）通道，造成細胞膜電位的去極化，從而開啟電位控制的鈣離子通道，細胞外鈣離子流入細胞內，使細胞內鈣離子濃度上升，因而導致胰島素之釋放。本實驗以活體灌流大白鼠胰臟的模式來探討glibenclamide與tetraethylammonium（TEA）合併或單獨使用，對大白鼠胰臟胰島素分泌之影響。結果發現glibenclamide與TEA皆可直接刺激胰臟分泌胰島素，同時也皆能增強葡萄糖刺激胰島素分泌的作用，但兩者胰島素的釋放趨勢並不相同。實驗濃度範圍之glibenclamide（15—100 nM）或TEA（1—100 mM）之下，其刺激作用與其劑量呈正比。Diazoxide是KATP通道的活化劑，能夠有效阻斷葡萄糖所刺激β細胞的胰島素釋放，結果發現diazoxid能夠抑制glibenclamide及TEA所刺激的胰島素釋放。研究指出TEA能夠阻斷voltage-dependent K+（KV）通道，延緩再極化及延長動作電位的持續時間，並增強葡萄糖所刺激的胰島素釋放。比較單獨使用glibenclamide，與glibenclamide合併TEA灌流的反應，結果顯示TEA在0—30 mM的濃度範圍能夠增強glibenclamide（15 nM）所引起的胰島素釋放，且呈現劑量依賴反應關係。故由實驗得知TEA可以直接刺激胰島素之分泌，並對glibenclamide刺激胰島素之分泌有增強的效果，這些作用可能是經由抑制KATP通道與KV通道所達成。因此我們推測KV通道對於調節β細胞電位活性，與胰島素之釋放扮演著一定的作用。Type 2 diabetes mellitus was an abnormal nutritional metabolic symptom associated with insulin resistance and impaired insulin secretion. Sulfonylureas were the most common therapeutic agents used orally in patients with non-insulin-dependent diabetes mellitus. Sulfonylureas enhanced β-cell insulin secretion by blocking ATP-sensitive K+ (KATP) channels, depolarizing membrane, and stimulating Ca2+ influx through voltage-gated Ca2+ channels. By using pancreatic perfusion technique, glibenclamide and tetraethylammonium (TEA) were perfused together or alone into rat pancreas. We found that glibenclamide and TEA were able to stimulate insulin secretion and enhanced 10 mM glucose-induced insulin secretion, but their secretion patterns were different. In addition, both glibenclamide (15-100 nM) and TEA (1-100 mM) stimulated insulin secretion in a dose-dependent manner. The KATP channel activator diazoxide blocked the effect of glucose on β-cell insulin release. In addition, the application of diazoxide into perfusate also inhibited glibenclamide- and TEA-induced insulin secretion. It was recognized that TEA blocked voltage-dependent K+ (KV) channels, prevented the repolarization, prolonged β-cell action potentials and enhanced insulin secretion in a glucose-dependent manner. By comparing the stimulatory response of glibenclamide with or without TEA, TEA (1-30 mM) potentiated the glibenclamide (15 nM)-induced insulin secretion in a dose-dependent manner. These results suggested that TEA had a direct effect on insulin secretion and potentiated glibenclamide-stimulated insulin secretion that was mediated through the inhibition of KATP and KV channels. In addition, KV channels were active participants in the regulation of β-cell electrical activity and insulin secretion.中文摘要 ............................................. i 英文摘要 ............................................. ii 目錄 ................................................. iv 圖次 ..................................................vi 第一章 緒言 ..........................................01 第二章 文獻探討 ......................................03 第一節 糖尿病簡介 ................................03 第二節 胰島素訊息傳遞與作用 ......................06 第三節 胰臟β細胞之胰島素分泌 .....................09 第四節 Voltage-dependent K+（KV）通道與胰臟β細胞 .18 第五節 口服治療第2型糖尿病之藥物 .................22 第六節 Sulfonylureas .............................26 第三章 材料與方法 ....................................32 實驗動物 ..........................................32 灌流系統 ..........................................32 灌流方法 ..........................................33 實驗一：不同劑量之glibenclamide對大白鼠胰臟胰島素分 泌之影響 ..................................34 實驗二：Glibenclamide對葡萄糖刺激大白鼠胰臟胰島素分 泌之影響 ..................................35 實驗三：不同劑量之tetraethylammonium（TEA）對大白鼠 胰臟胰島素分泌之影響 ......................36 實驗四：TEA對葡萄糖刺激大白鼠胰臟胰島素分泌之影響 .36 實驗五：KATP通道活化劑diazoxide對葡萄糖刺激胰島素分 泌作用之影響 ..............................37 實驗六：KATP通道活化劑diazoxide對glibenclamide刺激 胰島素分泌作用之影響 ......................38 實驗七：KATP通道活化劑diazoxide對TEA刺激胰島素分泌 作用之影響 ................................39 實驗八：TEA與glibenclamide合併使用對胰島素分泌之影 響 ........................................39 胰島素之測定 ......................................40 資料分析與統計 ....................................41 第四章 結果 ..........................................42 實驗一：不同劑量之glibenclamide對大白鼠胰臟胰島素分 泌之影響 ..................................42 實驗二：Glibenclamide對葡萄糖刺激大白鼠胰臟胰島素分 泌之影響 ..................................44 實驗三：不同劑量之tetraethylammonium（TEA）對大白鼠 胰臟胰島素分泌之影響 ............ .........47 實驗四：TEA對葡萄糖刺激大白鼠胰臟胰島素分泌之影響 .49 實驗五：KATP通道活化劑diazoxide對葡萄糖刺激胰島素分 泌作用之影響 ..............................51 實驗六：KATP通道活化劑diazoxide對glibenclamide刺激 胰島素分泌作用之影響 ......................54 實驗七：KATP通道活化劑diazoxide對TEA刺激胰島素分泌 作用之影響 ................................57 實驗八：TEA與glibenclamide合併使用對胰島素分泌之影 響 ........................................60 第五章 討論 ..........................................63 第六章 結論 ..........................................71 參考文獻 ..............................................7