| Abstract: | 標題：厭食劑在腦內之分子機制：NPY 受器、GRE-DNA 結合位、及STAT3 轉錄因子 之角色 本文所指厭食劑是指安非他命(amphetamine, AMPH)及其類似物。安非他命的厭食作 用，是透過腦內catecholamine 的釋放，去調控腦內Y型神經胜肽(neuropeptide Y, NPY) 基 因及原始類鴉片黑色素皮質素(proopiomelanocortin, POMC)基因所致。NPY 是腦內含量 豐富可興奮食慾的神經傳遞物，而POMC 是腦內厭食性神經傳遞物。安非他命若每天 注射一次，持續數天時，在第一天及第二天可引發最顯著的厭食效應(本文稱為AMPH anorexia)，但在隨後數天，則逐漸產生厭食耐受性(本文稱為AMPH tolerance)。最近， 筆者研究團隊發現：AMPH anorexia 與「抑制NPY 基因表現及興奮POMC 基因表現」 有關，而AMPH tolerance 則與「NPY 及POMC 基因表現的逐漸回復正常」有關。筆者 由蛋白激酶(PKA、PKC)及轉錄因子(AP-1、CREB、NF-κB)等訊息傳遞實驗發現，NPY 及POMC 基因二者呈逆向交互作用的方式，去調控安非他命厭食作用；此外，抗游離 基酶(SOD、nNOS)也參與調控安非他命厭食作用。本計畫將探討其他因子參與之可能 性。計畫名稱定為：厭食劑在腦內之分子機制：NPY 受器、GRE-DNA 結合位、及STAT3 轉錄因子之角色。擬分三年探討，第一年子計畫名稱為：「下視丘中NPY 受器在類安非 他命厭食劑作用之角色」，第二年名稱為：「糖皮質素反應元素(GRE)-DNA 結合位在類 安非他命厭食劑作用之角色」,第三年名稱為：「STAT3 轉錄因子在類安非他命厭食劑作 用之角色」。第一年預期完成三項工作：(1) 安非他命注射對Y1R、Y5R、POMC、α-MSH、 MC4R 及NPY 基因表現的影響，(2) 以雙重免疫顆粒染色法觀察Y1R(或Y5R)及NPY 免疫顆粒之表現，(3) 腦內Y1R(或Y5R)基因剃除(knock down)對NPY、POMC、α-MSH、 MC4R 及厭食行為的影響。第二年預期完成四項工作：(1) 安非他命注射對POMC、 MC4R、GR、CREB、PKA 及NPY 基因表現的影響，(2) 以EMSA (electromobility shift assay)方法分析下視丘GR 及GRE-DNA binding activity，(3) GR 基因剃除(knock down) 或RU486 對NPY、POMC 及厭食行為的影響、(4) 去腎上腺及糖皮質素回復的影響。 第三年預期完成三項工作：(1) 安非他命注射對JAK、STAT3、NPY、POMC、MC4R 及SOD 基因表現的影響，(2) )以「雙重免疫顆粒染色法」觀察STAT3、MC4R 及NPY 免疫顆粒之表現，(3) 腦內STAT3 訊息基因剃除(knock down)對NPY- and POMC-ergic pathway 、及厭食行為的影響。以上安非他命之研究完成後， 將繼續其類似物 (phenylpropanolamine)的研究。
Title: Molecular mechanisms of anorectic drugs in the brain: roles of NPY receptor, GRE-DNA binding site, and STAT3 transcription factor The mechanism for the anorectic effect of amphetamine (AMPH), an anorectic drug, is due to the central release of catecholamine which controls the expression of neuropeptide Y (NPY) and proopiomelanocortin (POMC) genes. NPY is an anorexigenic neurotransmitter, while POMC is an orexigenic neurotransmitter. Daily treatment with AMPH for several days induced a marked anorexia (AMPH anorexia) on Day 1 and Day 2 and a gradual tolerance to this anorexia (AMPH tolerance) on the following days. Recently, we found that the response of AMPH anorexia was relevant to the inhibition of NPY gene and the activation of POMC gene. However, the response of AMPH tolerance was relevant to the restorations of both NPY and POMC genes toward normal. Moreover, based on the changes of expression levels of PKA,PKC, CREB, AP-1 and NF-kB during AMPH treatment, we found that NPY and POMC were expressed reciprocally to control both AMPH anorexia and AMPH tolerance. Besides, we also found that some anti-free radicals, such as SOD and nNOS, were involved in AMPH’s action. Contents of the present study can be divided into three Parts that will be completed within 3 years. The protocol title for Part 1 is to investigate the role of NPY receptor subtype in the mediation of AMPH-induced anorexia, for that in Part 2 is to investigate the role of glucocorticoid response element (GRE)-DNA binding site in the regulation of AMPH-induced anorexia, and for that in Part 3 is to investigate the role of STAT3 transcription factor in the regulation of AMPH-induced anorexia. There are three or four experiments that need to be carried out for each separate Part of investigations. In Part 1, we will examine: (1) the effects of AMPH treatment on NPY receptor 1 (Y1R), NPY receptor 5 (Y5R), NPY, POMC, α-MSH and melanocortin receptor 4 (MC4R) gene expression, (2) the effect of AMPH treatment on Y1R (orY5R) and NPY double immunoreactivity, and (3) the effect of ICV injection of Y1R (or Y5R) antisens on AMPH-induced anorexia and the expression of NPY、POMC、α-MSH、MC4R. In Part 2, we will examine: (1) the effect of AMPH treatment on POMC、MC4R、glucocorticoid receptor (GR)、CREB、PKA and NPY gene expression, (2) the effect of GR and GRE-DNA binding activity using the technique of EMSA, and (3) the effect of GR knock down or GR inhibition (RU486) on AMPH anorexia as well as NPY and POMC gene expression. In Part 3, we will examine: (1) the effect of AMPH treatment on JAK、STAT3、NPY、POMC、MC4R and SOD gene expression, (2) the effect of AMPH treatment on STAT3, MC4R, and NPY immunoreactivities, and (3) the effect of ICV injection of STAT3 antisense on the changes of NPY- and POMC-ergic pathway as well as AMPH anorexia. In addition to AMPH, we will explore similar effects of other AMPH-like anorectic drugs, such as phenylproipanolamine. |
