2014-08-012024-05-17https://scholars.lib.ntu.edu.tw/handle/123456789/695165摘要：研究目的： 胰臟所分泌的 insulin 與 glucagon 皆和血糖的代謝與恆定有關，目前糖尿病的研究大多著重於探討 insulin 的角色，然而近幾年糖尿病新藥的研究結果指出，glucagon 也扮演重要的地位，但確切機轉與應用仍有待釐清。因此，本研究的第一部分將探討 glucagon 在糖尿病致病機轉上所扮演的角色。首先是觀察性研究：分析影響血中 glucagon 濃度的因子，包括血糖、胰島素濃度、胰島素敏感性、腰圍、BMI、內臟與皮下脂肪量、以及血脂肪之間的關係 (specific aim 1A)，接著是半介入性研究：隨著時間經過，有些人的血糖或者體型會改變、胰島素濃度、胰島素敏感度也會改變。我們將分析最初血中 glucagon 的濃度是否可用來預測將來新發生糖尿病或代謝症候群(specific aim 1B)。此外，我們將設計 glucagon sensitivity indices，來代表 glucagon 的敏感度並研究這些 index是否可預測糖尿病的發生(specific aim 1C)。 本研究第二部份將尋找可調控胰島細胞分泌的因子，其中主要是著重於 amine oxidase[簡稱 AO，包含 monoamine oxidase(簡稱 MAO)與 semicarbazide-sensitive amine oxidase(簡稱 SSAO)]對於α細胞與β細胞的影響。首先，我們將在細胞模式中，分析胰島細胞內 AO 的表現量與胰島功能的關係，接著加入不同種類的 AO 抑制劑看其對於 insulin 或 glucagon 分泌的影響(specific aim 2)。 研究設計與方法： Specific aim 1A 先利用橫斷式研究法(cross-sectional study)，用 ELISA 法測定血中 glucagon 濃度，並以電腦斷層檢查測定皮下脂肪與內臟脂肪的量，再分析血中 glucagon 濃度與臨床表徵、代謝參數之間的關係。Specific aim 1B 將以世代研究法(cohort study)，分析血中 glucagon 濃度是否可以預測之後新發生的糖尿病。Specific aim 1C 將藉由口服葡萄糖耐受試驗前後血糖與血中 glucagon 濃度的散佈圖，以電腦運算求得數學模式，用來分別代表不同族群的肝臟細胞對於 glucagon的敏感度與 α 細胞對於高血糖的反應，並探討這數學模式是否可以預測將來新發生糖尿病的機率。 Specific aim 2 將針對 MIN6 細胞(為 β細胞株)和 αTC1 clone 9 細胞(為 α 細胞株)，萃取出蛋白質與 mRNA，以分析細胞中 MAO-A、MAO-B 與 SSAO 的表現量是否與 insulin 或 glucagon 的分泌量有關。接著在上述的細胞株中，加入各種 AO的受質(substrate)或酵素活性抑制劑(inhibitor)後，測定培養皿中 H2O2 的濃度以確認AO 活性，並測定 insulin 或 glucagon 的濃度變化，以了解 AO 是否可以調控 insulin或 glucagon 的分泌。<br> Abstract: Objective: Pancreatic bi-hormones insulin and glucagon are associated with glucose metabolism and homeostasis. Over the past few decades, the classical view in diabetes research has mainly focused on the pathogenesis of insulin deficiency, whereas glucagon has long been dismissed as a minor contributor to metabolic disease. Therefore, the first part of this project is to investigate the role of glucagon in the development of diabetes. We will study the relationship between glucagon levels and metabolic parameters (i.e., blood sugar, waist circumference, body mass index, area of subcutaneous and visceral fat, insulin levels and insulin sensitivity) in human subjects (specific aim 1A). Besides, we will explore if glucagon levels can predict incident diabetes and metabolic syndrome in the future (specific aim 1B). Mathematical models of the glucose: glucagon interactions will be used to predict the homeostatic concentrations which arise from varying degree of glucagon sensitivity. In addition, we will investigate if these glucagon sensitivity indices can predict future development of diabetes (specific aim 1C). The second part of this project is to search for potential regulators of islet hormone secretion. We will mainly focus on the function of amine oxidase (AO), including monoamine oxidase (MAO) and semicarbazide-sensitive amine oxidase (SSAO), in pancreatic α- and β-cell. In cell models, we will analyze the relationship between the expression of AO and islet hormone secretion. Besides, we will study if the changes of insulin and glucagon secretion are in response to different kinds of AO substrates and inhibitors. Research design and methods: In specific aim 1A, subjects without the past history of diabetes will be recruited in a cross-sectional study. We will measure plasma glucagon concentration by ELISA kit and compute the area of subcutaneous and visceral fat. We will study the correlations between plasma glucagon levels and clinical characteristics and metabolic parameters. In specific aim 1B, we will investigate if plasma glucagon levels can predict the future development of diabetes and metabolic syndrome in a cohort study. In specific aim 1C, computer-solved models of glucose: glucagon interactions will be used to plot an array of plasma glucose and glucagon concentrations before and after oral glucose tolerance test that will be expected for varying degree of hepatic glucagon sensitivity and glucose sensing of α cell. We will examine if these indices can predict incident diabetes. In specific aim 2, we will use MIN6 cells (β-cell line) and αTC1 clone 9 cells (α-cell line). Protein and RNA will be extracted. We will analyze the correlation between the expression of AO (i.e., MAO-A, MAO-B, SSAO) in islet cells and the secretion of insulin and glucagon. In addition, the substrates and inhibitors of AO will be added in cell culture. We will measure insulin, glucagon and H2O2 concentration in culture medium to evaluate if AO activity can regulate α- and β-cell function.