摘要:研究目的:近年來糖尿病的治療方式不斷進步,但成效仍不盡理想,糖尿病仍位居主要死因之一。因此,預防糖尿病的發生,就成為促進民眾健康的另一個重要方式。然而,我們先前的研究結果指出,在未來會發生糖尿病的人中,有50%在追蹤前並不被認為是高危險群,換句話說,還有許多糖尿病的危險因子尚未被發現。藉由上次國科會計畫的研究經費補助,我們以nested case-controlstudy 的研究設計,用global profiling 的方式進行代謝體研究,發現了15 種血中的代謝產物與未來是否會發生糖尿病有關。這15 種代謝產物中,有3 個代謝產物跟嘌呤代謝有關,包括hypoxanthine、2-deoxyguanosine 5-monophosphate 與AMP。因此,本研究將透過三部份的研究,深入探討嘌呤代謝在糖尿病所扮演的角色。第一部份將以世代研究的設計,透過targeted metabolomics 的方法測定所有嘌呤代謝相關代謝物,以尋找可預測人類糖尿病發生的代謝物、計算預測力並發展出臨床可用的risk score 或是algorithm。第二部份將利用細胞模式,篩選可以調節胰島素或升糖素分泌的嘌呤代謝相關代謝物,特別是salvage pathway 中重要的代謝物,包括hypoxanthine。此外,由於allopurinol 也會增加hypoxanthine 的濃度,因此也將測試allopurinol 是否可調節胰島細胞的功能。第三部份將進行動物實驗,探討hypoxanthine 的攝取,以及allopurinol 的使用,是否會改變老鼠的insulin secretion 與insulin sensitivity,並用代謝體方法了解嘌呤代謝相關產物在各組織的濃度。研究設計與方法:第一部份將利用一個2048 人、平均追蹤時間3.5 年的世代進行。此世代民眾在追蹤前、以及每兩年的追蹤過程中,都接受75g OGTT 與糖化血色素測定,以得知是否有糖尿病。本計畫將針對其中400 位追蹤時間最長的民眾,將追蹤前的血清檢體以targeted metabolomics 的方式,在液相層析串聯質譜儀上測定所有嘌呤代謝相關代謝物,共35 種,其中也包括hypoxanthine。測定完成後,將以Kaplan-Meier 方法、log-rank test 以及multivariate Cox proportional hazard models 進行分析,以得知可以預測糖尿病發生的嘌呤代謝相關代謝物。針對顯著的代謝物,將以同位素內標定量法,在液相層析串聯質譜儀上進行絕對定量,以得知該代謝物的濃度與參考值範圍。接著將以concordance statistics與ROC curve 來檢驗代謝物的預測力,並發展出臨床可用的risk score 或是algorithm。第二部份將在MIN 6 細胞株與αTC1 clone 9 細胞株,測試salvage pathway 的多種嘌呤代謝物(包括hypoxanthine)、以及allopurinol,在不同濃度與作用時間下,會如何影響這些細胞株在低糖(3.3mM)與高糖(16.7mM)環境下,分泌胰島素或升糖素到culture media 的功能。之後將由male C57B6 mice 的胰臟,分離出isolated islets,測試篩選出的嘌呤代謝物、hypoxanthine 與allopurinol,在不同的濃度與作用時間下,如何影響isolated islets 在低糖(3.3mM)與高糖(16.7mM)環境下,分泌胰島素或升糖素到culture media 的功能,並以targeted metabolomics 方式測定cell lysate 的purine metabolism-relatedmetabolome,以得知嘌呤代謝受到什麼樣的影響。第三部份將動物分為5 組,每組15 隻,包括control 組、purine-free diet control 組、hypoxanthine 0.25mg/ml 組、hypoxanthine 0.5 mg/ml 組與allopurinol 組。在離乳之後4 週,開始接受high-fat, high-sucrosediet,hypoxanthine 與allopurinol 組在飲用水中加入hypoxanthine 或allopurinol。之後每兩週測量體重與空腹血糖,於第10-12 週與第38-40 週時,測量空腹時血中的purine metabolism-related metabolome、insulin tolerance test、intraperitoneal glucose tolerance test 與oral glucose tolerance test。動物於40 週時犧牲,一部份動物測量肝臟、肌肉、脂肪組織與胰臟中purine metabolism-related metabolome,另一部份動物於犧牲前30 分鐘進行insulin tolerance test,之後以western blot 測量肝臟、肌肉與脂肪組織中insulinsignaling cascade 中的重要蛋白,包括p-IR、p-IRS1 與p-Akt。
Abstract: Background and objectives: Diabetes is a major cause of death and results in various complications,despite there is a great improvement in the treatment of diabetes in recent decades. Therefore, prevention fordiabetes is another important way to improve public health. However, current definition of high-risk groupfor diabetes is not good enough and will miss as many as 50% of subjects who develops diabetes in thefuture, based on the findings from our previous report. In our preliminary results, we used a nestedcase-control design to identify 15 metabolites by global profiling metabolomics approach, which can predictthe development of diabetes in the future. Among which, 3 metabolites are involved in purine-metabolismpathways, including hypoxanthine, 2-deoxyguanosine 5-monophosphate, and AMP.In this project, we will investigate the role of purine metabolism in diabetes. In specific aim 1, we willapply cohort study design and measure all purine metabolism-related metabolites by targeted metabolomicsin the serum of study subjects, in order to find metabolites which can predict incident diabetes, and todevelop risk scores or algorithms which can be used clinically. In specific aim 2, we will use cell models toscreen purine metabolism-related metabolites which can regulate insulin or glucagon secretion, especiallymetabolites involved in salvage pathway. Since allopurinol can increase the concentration of hypoxanthine,we will also test if allopurinol can regulate islet function. In specific aim 3, we will test if oral intake ofhypoxanthine or allopurinol can improve insulin secretion and insulin sensitivity in animal models. We willalso measure concentrations of purine metabolism-related metabolites in different organs, in order tounderstand the effect of above treatment on purine metabolism in specific organ.Study Design and Methods: A cohort of 2048 subjects, followed for an average of 3.5 years, will be usedfor specific aim 1. In this cohort, diabetes was diagnosed by 75g OGTT and hemoglobin A1c every 2 years.We will measure 35 purine metabolism-related metabolites, including hypoxanthine, in baseline serum of400 subjects who have longest follow-up duration in this cohort, using liquid chromatography-massspectrometry by targeted metabolomics approach. Data will be analyzed by Kaplan-Meier survival analyses,tested by log-rank tests, and modeled by multivariate Cox proportional hazard models, in order to findmetabolites which significantly predict incident diabetes. These metabolites will be quantified by liquidchromatography-mass spectrometry using isotope-labeled internal standard. Reference range of thesemetabolites will be calculated. Prediction ability will be evaluated by concordance statistics and ROC curve.Based on the data, we will also develop risk scores or algorithms which can be used clinically.In specific aim 2, we will use MIN 6 cell lines and αTC1 clone 9 cell lines to screen several purinemetabolism-related metabolites, including hypoxanthine, and allopurinol, for their effects on insulin andglucagon secretion. Time and dose effects will be tested in low glucose (3.3mM) and high glucose (16.7mM)conditions. Islets from male C57B6 mice will be isolated. Metabolites which can regulate insulin secretionin MIN 6 cell lines and glucagon secretion in αTC1 clone 9 cell lines will be used in experiments in isolatedmice islets. Time and dose effects will be tested in low glucose (3.3mM) and high glucose (16.7mM)conditions. Purine metabolism-related metabolome will be measured in cell lysates using targetedmetabolomics approach, in order to know the effect on purine metabolism.In specific aim 3, C57B6 mice will be divided into 5 groups, with 15 mice in each group, includingcontrol group, purine-free diet control group, hypoxanthine 0.25 mg/ml group, hypoxanthine 0.5 mg/mlgroup, and allopurinol group. High-fat, high-sucrose diet will start 4 weeks after weaning. Hypoxanthineand allopurinol will be added in drinking water. Body weight and fasting plasma glucose will be measuredevery 2 weeks. At week 10-12 and week 38-40, fasting serum purine-metabolism-related metabolome,insulin tolerance test, intraperitoneal glucose tolerance test, and oral glucose tolerance test will be done.Mice will be sacrificed at week 40. Tissue concentration of purine metabolism-related metabolome will bemeasured in liver, muscle, adipose tissue, and pancreas in some mice. Insulin tolerance test will be done 30minutes before sacrifice in some mice. Proteins involved in insulin signaling cascade, including p-IR,p-IRS1, and p-Akt, will be measured by western blot in liver, muscle, and adipose tissue.