2016-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/648128摘要：研究目的：Primary amine oxidase (PAO) 又稱為vascular adhesion protein-1 (非vascular cellular adhesionmolecule-1，也不是VCAM-1)或是semicarbazide-sensitive amine oxidase，可代謝bioactive amines，並產生H2O2、aldehydes 與NH3。以往的研究指出，amines 可經由脂肪細胞上的PAO 代謝，促進glucosetransporter (GLUT)的表現，進而增加脂肪細胞醣類的攝取(glucose uptake)。PAO 除了transmembraneform 之外，也存在有soluble form，最近的文獻指出，soluble PAO 可促進肝臟組織的glucose uptake。我們初步的研究結果發現，人類的胎盤有表現PAO，我們認為胎盤上的PAO 應該會調節胎盤上的醣類運輸，進而影響胎兒的生長，而胎盤分泌的soluble PAO 應該會影響孕婦體內的醣類平衡 (glucosehomeostasis)。因此，本計畫將探討孕婦血中amines 與PAO 濃度、胎盤上PAO 表現量，與胎盤GLUT1, 3, 4, 12 的表現、insulin secretion index、insulin resistance index 以及胎兒出生體重的關係，並比較妊娠糖尿病與健康孕婦，母血amine 濃度、PAO 濃度與胎盤PAO 以及胎盤GLUT 1, 3, 4, 12 的表現是否不同 (specific aim A)； 並於細胞模式中，探討胎盤上的PAO 對於glucose transporters 的表現以及醣類運輸的影響 (specific aim B)，以及soluble PAO 對於胰島β 細胞、肝臟細胞、骨骼肌細胞與脂肪細胞glucose uptake 的影響 (specific aim D)，並研究調節PAO 的調節因子 (specific aim C)。研究設計與方法：本計畫預計進行三年，包括specific aim A-D，分述如下：Specific aim A 將收錄一個孕婦的世代(cohort)，以targeted metabolomics 的方式測定其血中amines濃度，並定量血中PAO 的濃度，以及胎盤中PAO 與glucose transporter 1, 3, 4, 12 的表現量。接著以Pearson's correlation coefficients 以及multiple linear regression models，分析血中母血amine 濃度、母血PAO 濃度、胎盤PAO 的表現、胎盤GLUT 1, 3, 4, 12 的表現、insulin secretion index HOMA2%B、insulinresistance index HOMA2%IR 與出生體重兩兩的關係，並調整相關干擾因子。妊娠糖尿病孕婦與健康的孕婦，血中amines 濃度、血中PAO 濃度、胎盤PAO 與胎盤GLUT 1, 3, 4, 12 表現的不同，將以Student'st test 與multiple linear regression models 比較，同時調整相關干擾因子。Specific aim B 將利用3A-sub E cell line from placenta (ATCC CRL-1584)以及primary humantrophoblasts，於細胞模式下探討PAO 的substrate 可否透過PAO activity 來調節glucose uptake 與GLUT1, 3, 4, 12 的表現，而此作用是否透過H2O2 以及Akt pathway。Specific aim C 將比較生產前後母血中PAO 濃度的變化，並於前述細胞模式中，探討estrogen、progesterone、prolactin、human placenta lactogen、insulin 與metalloprotease inhibitors 是否可以調節PAO與soluble PAO 的表現。Specific aim D 將利用pancreatic β cell line INS1，探討soluble PAO 對於insulin secretion、以及β cellproliferation 的影響，並探討此作用是否透過H2O2；也將利用骨骼肌細胞株L6 cells，以及脂肪細胞株3T3-L1 adipocytes，研究soluble PAO 對於glucose uptake 的作用，並探討此作用是否透過H2O2 以及Akt pathway；最後將利用肝臟細胞株HepG2 cells，探討soluble PAO 對於gluconeogenesis 的影響，並研究此作用是否透過H2O2 以及Akt pathway。<br> Abstract: Background and objectives: Primary amine oxidase (PAO), also called vascular adhesion protein-1 (notvascular cellular adhesion molecule-1, nor ICAM-1) or semicarbazide-sensitive amine oxidase, can catalyzethe breakdown of bioactive amines, to produce H2O2、aldehydes, and NH3. In adipocytes, amines can inducethe translocation of glucose transporters (GLUT) and enhance glucose uptake into adipocytes, through theactivity of adipose PAO. Besides, PAO exists a soluble form, which has been shown to enhance glucoseuptake in liver tissues in a recent report. In our preliminary results, we found that human placenta expressedPAO. We hypothesize that PAO in placenta can regulate glucose transport across placenta and affect thegrowth of the fetus. Besides, we think that soluble PAO from placenta can affect glucose homeostasis inpregnant women. Therefore, this project will investigate the relationships between maternal bloodconcentrations of amines, maternal blood PAO concentrations, placental PAO expressions, placentalexpressions of GLUT 1, 3, 4, 12, insulin secretion index, insulin resistance index, and birth weight. We willalso compare the differences in maternal blood concentrations of amines, maternal blood PAO concentrations,placental PAO expressions, placental expressions of GLUT 1, 3, 4, 12 between women with and withoutgestational diabetes mellitus. (specific aim A) In cell models, we will study the effect of placental PAO on theexpression of GLUTs and glucose uptake (specific aim B), and the effect of soluble PAO on the function andproliferation of pancreatic β cells, on gluconeogesis in hepatic cells, and on glucose uptake in skeletal musclecells and adipocytes (specific aim D). We will also investigate the regulators of both cellular PAO andsoluble PAO (specific aim C).Study Design and Methods: In this 3-year project, we planned to do 4 specific aims A-D:Specific aim A is to recruit a cohort of pregnant women. We will measure blood amines by targetedmetabolomics approach. Their blood PAO concentrations, placental PAO expressions, and placentalexpressions of GLUT 1, 3, 4, 12 will be measured. The relationship between maternal blood concentrationsof amines, maternal blood PAO concentrations, placental PAO expressions, placental expressions of GLUT 1,3, 4, 12, insulin secretion index, insulin resistance index, and birth weight will be analyzed by Pearson'scorrelation coefficients. Confounders will be adjusted in multiple linear regression models. We will useStudent's t test and multiple linear regression models to assess the differences in maternal bloodconcentrations of amines, maternal blood PAO concentrations, placental PAO expressions, placentalexpressions of GLUT 1, 3, 4, 12 between women with and without gestational diabetes mellitus.Specific aim B uses 3A-sub E cell line from placenta (ATCC CRL-1584) and primary humantrophoblasts to investigate if substrates of PAO can regulate the expression of GLUT 1, 3, 4, 12 and glucoseuptake through the activity of PAO, generation of H2O2, and Akt pathway.Specific aim C is to compare maternal blood PAO concentrations before and after delivery. In cellmodels, we will study if estrogen, progesterone, prolactin, human placenta lactogen, insulin, andmetalloprotease inhibitors can regulate the expression of cellular and soluble PAO.Specific aim D is to study the effect of soluble PAO on insulin secretion and cell proliferation usingpancreatic β cell line INS1, and if the effect acts through the generation of H2O2. We will also investigate theeffect of soluble PAO on glucose uptake in skeletal muscle cell line L6 and 3T3-L1 adipocytes, and if theeffects act through the generation of H2O2 and Akt pathway. In hepatic cell line HepG2, we will study ifsoluble PAO can regulate gluconeogenesis, and if the effect acts through the generation of H2O2 and Aktpathway.研究目的：Primary amine oxidase (PAO) 又稱為vascular adhesion protein-1 (非vascular cellular adhesion molecule-1，也不是VCAM-1)或是semicarbazide-sensitive amine oxidase，可代謝bioactive amines，並 產生H2O2、aldehydes 與NH3。以往的研究指出，amines 可經由脂肪細胞上的PAO 代謝，促進glucose transporter (GLUT)的表現，進而增加脂肪細胞醣類的攝取(glucose uptake)。PAO 除了transmembrane form 之外，也存在有soluble form，最近的文獻指出，soluble PAO 可促進肝臟組織的glucose uptake。 我們初步的研究結果發現，人類的胎盤有表現PAO，我們認為胎盤上的PAO 應該會調節胎盤上的醣 類運輸，進而影響胎兒的生長，而胎盤分泌的soluble PAO 應該會影響孕婦體內的醣類平衡 (glucose homeostasis)。因此，本計畫將探討孕婦血中amines 與PAO 濃度、胎盤上PAO 表現量，與胎盤GLUT 1, 3, 4, 12 的表現、insulin secretion index、insulin resistance index 以及胎兒出生體重的關係，並比較妊 娠糖尿病與健康孕婦，母血amine 濃度、PAO 濃度與胎盤PAO 以及胎盤GLUT 1, 3, 4, 12 的表現是否 不同 (specific aim A)； 並於細胞模式中，探討胎盤上的PAO 對於glucose transporters 的表現以及醣類 運輸的影響 (specific aim B)，以及soluble PAO 對於胰島β 細胞、肝臟細胞、骨骼肌細胞與脂肪細胞 glucose uptake 的影響 (specific aim D)，並研究調節PAO 的調節因子 (specific aim C)。 研究設計與方法： 本計畫預計進行三年，包括specific aim A-D，分述如下： Specific aim A 將收錄一個孕婦的世代(cohort)，以targeted metabolomics 的方式測定其血中amines 濃度，並定量血中PAO 的濃度，以及胎盤中PAO 與glucose transporter 1, 3, 4, 12 的表現量。接著以 Pearson's correlation coefficients 以及multiple linear regression models，分析血中母血amine 濃度、母血 PAO 濃度、胎盤PAO 的表現、胎盤GLUT 1, 3, 4, 12 的表現、insulin secretion index HOMA2%B、insulin resistance index HOMA2%IR 與出生體重兩兩的關係，並調整相關干擾因子。妊娠糖尿病孕婦與健康的 孕婦，血中amines 濃度、血中PAO 濃度、胎盤PAO 與胎盤GLUT 1, 3, 4, 12 表現的不同，將以Student's t test 與multiple linear regression models 比較，同時調整相關干擾因子。 Specific aim B 將利用3A-sub E cell line from placenta (ATCC CRL-1584)以及primary human trophoblasts，於細胞模式下探討PAO 的substrate 可否透過PAO activity 來調節glucose uptake 與GLUT 1, 3, 4, 12 的表現，而此作用是否透過H2O2 以及Akt pathway。 Specific aim C 將比較生產前後母血中PAO 濃度的變化，並於前述細胞模式中，探討estrogen、 progesterone、prolactin、human placenta lactogen、insulin 與metalloprotease inhibitors 是否可以調節PAO 與soluble PAO 的表現。 Specific aim D 將利用pancreatic β cell line INS1，探討soluble PAO 對於insulin secretion、以及β cell proliferation 的影響，並探討此作用是否透過H2O2；也將利用骨骼肌細胞株L6 cells，以及脂肪細胞株 3T3-L1 adipocytes，研究soluble PAO 對於glucose uptake 的作用，並探討此作用是否透過H2O2 以及 Akt pathway；最後將利用肝臟細胞株HepG2 cells，探討soluble PAO 對於gluconeogenesis 的影響，並 研究此作用是否透過H2O2 以及Akt pathway。Background and objectives: Primary amine oxidase (PAO), also called vascular adhesion protein-1 (not vascular cellular adhesion molecule-1, nor ICAM-1) or semicarbazide-sensitive amine oxidase, can catalyze the breakdown of bioactive amines, to produce H2O2、aldehydes, and NH3. In adipocytes, amines can induce the translocation of glucose transporters (GLUT) and enhance glucose uptake into adipocytes, through the activity of adipose PAO. Besides, PAO exists a soluble form, which has been shown to enhance glucose uptake in liver tissues in a recent report. In our preliminary results, we found that human placenta expressed PAO. We hypothesize that PAO in placenta can regulate glucose transport across placenta and affect the growth of the fetus. Besides, we think that soluble PAO from placenta can affect glucose homeostasis in pregnant women. Therefore, this project will investigate the relationships between maternal blood concentrations of amines, maternal blood PAO concentrations, placental PAO expressions, placental expressions of GLUT 1, 3, 4, 12, insulin secretion index, insulin resistance index, and birth weight. We will also compare the differences in maternal blood concentrations of amines, maternal blood PAO concentrations, placental PAO expressions, placental expressions of GLUT 1, 3, 4, 12 between women with and without gestational diabetes mellitus. (specific aim A) In cell models, we will study the effect of placental PAO on the expression of GLUTs and glucose uptake (specific aim B), and the effect of soluble PAO on the function and proliferation of pancreatic β cells, on gluconeogesis in hepatic cells, and on glucose uptake in skeletal muscle cells and adipocytes (specific aim D). We will also investigate the regulators of both cellular PAO and soluble PAO (specific aim C). Study Design and Methods: In this 3-year project, we planned to do 4 specific aims A-D: Specific aim A is to recruit a cohort of pregnant women. We will measure blood amines by targeted metabolomics approach. Their blood PAO concentrations, placental PAO expressions, and placental expressions of GLUT 1, 3, 4, 12 will be measured. The relationship between maternal blood concentrations of amines, maternal blood PAO concentrations, placental PAO expressions, placental expressions of GLUT 1, 3, 4, 12, insulin secretion index, insulin resistance index, and birth weight will be analyzed by Pearson's correlation coefficients. Confounders will be adjusted in multiple linear regression models. We will use Student's t test and multiple linear regression models to assess the differences in maternal blood concentrations of amines, maternal blood PAO concentrations, placental PAO expressions, placental expressions of GLUT 1, 3, 4, 12 between women with and without gestational diabetes mellitus. Specific aim B uses 3A-sub E cell line from placenta (ATCC CRL-1584) and primary human trophoblasts to investigate if substrates of PAO can regulate the expression of GLUT 1, 3, 4, 12 and glucose uptake through the activity of PAO, generation of H2O2, and Akt pathway. Specific aim C is to compare maternal blood PAO concentrations before and after delivery. In cell models, we will study if estrogen, progesterone, prolactin, human placenta lactogen, insulin, and metalloprotease inhibitors can regulate the expression of cellular and soluble PAO. Specific aim D is to study the effect of soluble PAO on insulin secretion and cell proliferation using pancreatic β cell line INS1, and if the effect acts through the generation of H2O2. We will also investigate the effect of soluble PAO on glucose uptake in skeletal muscle cell line L6 and 3T3-L1 adipocytes, and if the effects act through the generation of H2O2 and Akt pathway. In hepatic cell line HepG2, we will study if soluble PAO can regulate gluconeogenesis, and if the effect acts through the generation of H2O2 and Akt pathway.初級胺氧化脢妊娠糖尿病醣類代謝primary amine oxidasegestational diabetes mellitusglucose homeostasis