魚類在適應鹽度過程中能量代謝之細胞分子機制

Abstract

當廣鹽性魚類面對環境鹽度改變時，魚類鰓上皮細胞許多位於富含粒線體細胞 (mitochondria-rich cell, MR cell)的離子通道就必須及時運作以維持魚體內的滲透壓恆定，由於大多數的離子通道都是需要耗能的主動運輸，故鰓是個需要大量能量的器官。而魚類鰓上皮細胞中究竟是以何種物質作為能量的來源，而其又是如何在細胞間運輸的機制至今尚未清楚。相對於哺乳動物中，中樞神經系統需要充足的能量並以肝醣作為細胞內主要能量儲存形式，下游的能量運輸分子則是以乳酸為主而並非是葡萄糖。而星狀細胞(astrocyte)會儲存大量的乳酸再藉由單羧基運輸蛋白(monocarboxylate transpoter, MCT)提供給神經細胞作為能量來源。其中，乳酸脫氫酶(lactate dehydrogenase, LDH)對於乳酸的生成扮演一重要的角色。 本實驗利用莫三比克吳郭魚進行相關研究。首先利用同功異構酶澱粉凝膠電泳觀察乳酸脫氫酶不同的同功異構酶在吳郭魚鰓上皮細胞的表現情形。結果觀察到在鰓上皮細胞以乳酸脫氫酶1(LDH1)的表現為主。進一步利用LDH1、LDH5、glycogen synthase、glycogen、Na+-K+ ATPase α次單元抗體進行免疫細胞螢光染色發現到LDH1以及LDH5分別在 MR cells和富含肝醣細胞(glycogen-rich cell, GR cell)都有分布。之後利用西方墨點法分析吳郭魚在適應不同環境鹽度時LDH1、LDH5以及檸檬酸合成酶(citrate synthase, CS)蛋白質的表現量，發現到LDH1和 CS的蛋白質表現量在轉移海水後1st h都有顯著的增加，而LDH5的蛋白質表現量則是在轉移海水後的1-3rd h 之中都有顯著的增加。藉由上述結果，我們認為在魚類適應不同鹽度過程中，其鰓上皮細胞能量代謝的機制如下：當魚類轉移到海水後1st h時，其鰓上的MR cells會進入檸檬酸循環(TCA cycle)以產生大量的能量給MR cells。而在轉移到海水3rd h時，MR cells則是進行無氧呼吸為主以繼續產生大量的能量給予離子通道進行滲透壓調節。

Upon a salinity challenge, euryhaline teleosts immediately regulate the functions of many ion transporters and enzymes in the gill mitochondria-rich (MR) cells for maintaining their internal homeostasis. Large amount of energy is necessary for the operations of these transporters and enzymes, however the mechanism of energy metabolism in fish gills is still unclear. On the other hand, in mammalian central nervous system lactates, instead of glucose, are the main energy substrate transported from astrocyte to neuron via monocarboxylate transporter (MCT). Lactate dehydrogenase 1 (LDH1) is the key enzyme to convert the lactate to pyruvate, and LDH5 is responsible for the reverse reaction. In the present study, we used tilapia (Oreochromis mossambicus) as a model animal to investigate the roles of LDHs in the energy metabolism of fish gills during acclimation to seawater (SW). Results of starch gel electrophoresis and western blot demonstrated that LDH1 is the major form and LDH5 the minor form expressed in tilapia gill epithelial cells. Immunocytochemical experiments indicated that both LDH1 and LDH5 were expressed in MR cells (Na+-K+ ATPase as a marker) and glycogen-rich cells. The protein expression level of LDH1 and citrate synthase increased immediately at the 1st h after acclimation to SW, while that of LDH 5 significantly enhanced during the 1st-3rd h. Taking all together, we proposed a energy metabolism model in gill epithelial cells during SW acclimation: The tricarboxylic acid cycle (TCA cycle) in MR cells is stimulated to produce enormous amount of energy for maintaining homeostasis and ion regulation at the 1st h after transfer to SW. Subsequently at the 3rd h, the LDH5 in MR cells is stimulated to enhance anaerobic respiration to produce emergent energy.