2016-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/657692摘要：糖尿病治療的達標率仍不盡理想，已有幾種G protein coupled receptors (GPRs)被研究與 糖尿病有關，並已開發出一些小分子藥物以治療糖尿病。之前我們曾經選殖nocturnin convential knockout (NOC KO)老鼠，並發現此小鼠有血糖不耐症且葡萄糖刺激膜島素分泌的能 力也降低。透過膜島細胞mRNA微陣列分析發現G protein coupled receptor 18 (GPR18)的表 現量在NOC KO小鼠顯著下降。我們也發現GPR18基因下調的胰島貝他細胞(MIN6)，其葡 萄糖刺激膜島素分泌的能力顯著下降。而GPR18已被鑑定為是一個新的abnormal cannabidiol receptor，目前已知N-arachidonoyl glycine是GPR18的内源性配體。已有研究發現NAGly會 刺激胰島素分泌，但此作用是否與GPR18有關目前並不清楚。所以在本研究計晝，我們將 進一步探討GPR18在葡萄糖恆定的角色及其分子機轉。本研究計晝的特定目的與相關實驗設計及研究方法：1.GPR18在掠櫚酸醋（palmitate)抑制胰島素分泌的角色：建立GPR18過度表現 (over-expression)的小鼠胰島細胞瘤細胞株(MIN6)，並比較不同基因型的MIN6細胞，其葡 棕櫚酸酯抑制胰島素分泌的能力。2.GPR18於糖毒性與脂質毒性之下在貝他細胞存活與凋亡的角色：比較上述不同基因型的 MIN6細胞經methylglyoxal或4-hydroxynonenal處理後的細胞存活能力、凋亡、活性氧物 種(ROS)、内質網壓力、與粒線體功能。3.分別建立GPR18剔除鼠與胰島貝他細胞專一性的GPR18剔除鼠以探討GPR18在葡萄 糖恆定、胰島貝他細胞質量與功能並探究其分子機轉：比較不同基因型小鼠餵食常規粉末 飼料與高脂肪高果糖飼料後的代謝表現型、胰島貝他細胞體積、胰臟組織胰島素含量、胰 島細胞分泌胰島素的功能、基因、微小RNA、與蛋白質表現的差異。<br> Abstract: The targets for treatment of DM remain unsatisfactory and that will result in development of chronic diabetic complications. Several G protein coupled receptors (GPRs) have been shown to be related with diabetes and develop small molecules to treat diabetes.Previously, we generated a nocturnin knock-out (NOCKO) mice and fount it was glucose intolerant with decreased glucose-stimulated insulin secretion (GSIS). Through mRNA microarray study of isolated islets, the expression of G protein coupled receptor 18 (GPR18) was significantly lower in NOCKO mice. We also demonstrated knock-down of GPR 18 in MIN6 cells decreased GSIS significantly. GPR18 has been identified as a novel abnormal cannabidiol receptor (Abn CBD receptor) with anti-inflammatory effects and sympathoinhibitory effect in a NO dependent pathway, and N-arachidonoyl glycine (NAGly)has been also identified as its endogenous ligand. It also has been reported that NAGly stimulates insulin• • • 2+ . secretion through stimulation of the voltage-dependent Ca channels (VDCC), but whether GPR18 isinvolved in this effect is still unclear. Therefore, in this project we will further study the role of GPR18 onglucose homeostasis and its molecular mechanism.Secific aims and pertinent design and methods for achieving these goals:1.The role of GPR18 on palmitate-suppressed insulin secretion: Generate GPR18 overexpressed mouse insulinoma cells (MIN6), and check palmitate suppressed insulin secretion2.The role of GPR18 on cell survival and apoptosis in beta cells under glucotoxicity and lipotoxicity: Compare cell viability, apoptosis, reactive oxygen species (ROS), ER stress, mitochondria copy number, mitochondria membrane potential, intracellular ATP concentration and oxygen consumption rate (OCR) on methylglyoxal and 4-hydroxynonenal treatment among wild type, GPR18 over-expressed, GPR18 knock-down and scramble transfected MIN6 cells3.Generate GPR18 conventional knockout (GPRI8KO) and beta cell specific GPR18 knock out (GPR183KO) mice to explore the role of GPR18 on glucose homeostasis, beta cell mass & function, and elucidate the molecular mechanisms: Compare metabolic phenotypes, beta cell mass, insulin content, GSIS, gene expression profile (mRNA microarray), miRNA profile (miRNA next generation sequencing), and protein profile (LC-mass/mass) among WT, GPR18KO and GPR18^KO mice under regular chow diet or high fat high sucrose diet (HFHSD).G protein coupled receptor 18葡萄糖恒定跋島貝他細胞功能騰島貝他細胞存活與 凋亡G protein coupled receptor 18glucose homeostasisislet beta cell functionislet beta cell survival and apoptosis