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標題: 低溫逆境對淡水與海水馴養虱目魚(Chanos chanos)鰓能量代謝變化之影響
Hypothermal effects on energy metabolism in gills of seawater- and fresh water-acclimated milkfish, Chanos chanos
作者: 呂偉杰
Wei-Jie Lu
關鍵字: 葡萄糖轉運蛋白;能量代謝;glucose transporter;energy metabolism
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虱目魚 (Chanos chanos) 是台灣重要的經濟魚種,具廣鹽性,可馴養在淡水及海水魚塭,但虱目魚不耐低溫的特性經常在冬季時造成漁民重大的損失。已知虱目魚馴養在海水中對於低溫的耐受性較優於馴養於淡水的魚隻。鰓是魚體進行離子調節的重要器官,並具有耗能的特性。因此,在魚體面臨溫度及鹽度逆境時,必須有足夠的能量透過有氧代謝與無氧代謝途徑供應給鰓以維持其生理功能。本研究藉由鰓的能量代謝相關產物,探討虱目魚面臨環境中不同的鹽度 (海水、淡水)及溫度 (28 oC、18 oC) 時,其鰓的能量供應變化。葡萄糖為脊椎動物 (包括魚類) 的重要能量來源, 經由葡萄糖轉運蛋白 (glucose transporter; GLUT) 將葡萄糖運輸進出細胞。其中,葡萄糖轉運蛋白1 (GLUT1) 為一種廣泛分布於身體各組織的蛋白質,其功能是將葡萄糖運送至目標細胞供其運用。因此,本研究首先分析GLUT1作為不同鹽度環境中虱目魚面臨低溫逆境時,其鰓的有氧代謝指標。免疫轉漬結果顯示,低溫逆境下的虱目魚鰓GLUT1蛋白表現量在淡水組顯著上升,海水組亦有上升趨勢但無顯著差異;而在常溫和低溫下,海水組蛋白表現量皆高於淡水組。以免疫螢光染色則觀察到GLUT1不論在淡水或海水虱目魚鰓,皆表現在代表離子細胞 (ionocytes)的NKA 免疫反應細胞 (NKA immunoreactive cells)旁邊的細胞上。其次,在有氧代謝方面,經由分析葡萄糖與肝醣含量,以及糖解作用的速率決定酵素,己糖激酶 (hexokinase),之活性變化,探討GLUT1的蛋白表現量是否會影響鰓的能量來源。結果顯示,葡萄糖含量在海水低溫組的鰓顯著高於常溫組,而淡水組別無顯著差異。鰓肝醣的含量,在海水常溫組顯著高於低溫組,而淡水組無顯著差異。己糖激酶活性在常溫或低溫下,海水組皆顯著高於淡水組; 而在海水中,常溫組又顯著高於低溫組; 在淡水中常溫與低溫組的活性則無顯著差異。另一方面,本研究在無氧代謝方面則以乳酸與廣泛分佈型的乳酸運輸蛋白Monocarboxylate transporter 1(MCT1)作為指標。海水組或淡水組虱目魚鰓的乳酸含量皆是在常溫馴養的個體顯著高於低溫馴養的魚隻; 而mct1基因表現,海水組在常溫馴養的個體顯著高於低溫馴養的魚隻。在淡水組雖然表現平均值在低溫中降低,但是統計上無顯著差異。綜上所述,GLUT1蛋白表現確實會影響到葡萄糖在虱目魚鰓的含量,且虱目魚在常溫下,鰓行無氧代謝的狀況較低溫下更為旺盛。海水虱目魚在面臨低溫逆境時,鰓會使用暫存的肝醣作為能量利用的來源,可能是海水虱目魚較淡水虱目魚更能耐受低溫逆境的原因之一。

The euryhaline milkfish (Chanos chanos) is one of the economically important aquaculture species in Taiwan. However, the cold-intolerant characteristic of milkfish usually caused huge economic loss of the fishermen in winter. The seawater-acclimated milkfish has better cold tolerance than the fresh water-acclimated individuals. The gill is an energy-consuming organ mainly for ionoregulation. When fish faced changes in environmental temperatures or salinities, sufficient energy was required through regulating aerobic and anaerobic metabolic pathways to maintain physiological functions in gills. This study compared the mechanisms of energy metabolism, including aerobic and anaerobic pathways, in gills between seawater (SW) and freshwater (FW) milkfish under hypothermal stress (18oC vs. 28oC) to reveal the changes in energy supply. The glucose is an essential energy resource for vertebrates including fish and is transported through facilitative glucose transporters (GLUTs) in cells. Among the GLUTs, the glucose transporter 1 (GLUT1) is widely distributed in various tissues, responsible for transporting glucose into target cells to be utilized. Hence, relative protein abundance and localization of GLUT1 was first studied as the indicator of aerobic metabolism. Upon hypothermal challenge, branchial GLUT1 protein expression of FW milkfish was up-regulated. At normal or hypothermal temperature, relative protein abundance of GLUT1 was higher in gills of SW milkfish than FW ones. In addition, localization of GLUT1 was found in gill epithelial cells adjacent to NKA-immunoreactive (MR) cells (ionocytes). Subsequently, the other indicators of the aerobic metabolism pathway including glucose contents, glycogen contents, and the activities of hexokinase (HK), the rate-limiting enzyme for glycolysis that catalyzed glucose, were compared. Glucose contents were significantly lower in the SW/18oC group than the SW/28oC group, while there was no significant change between the FW/18oC and FW/28oC group. Glycogen contents were not changed in the FW/18oC group. Glycogen contents were down-regulated in the SW/18oC group. In the FW group, HK activities were not changed under hypothermal stress. However, in SW, HK activities were down-regulated in the hypothermal group. On the other hand, lactate and monocarboxylate transporter 1 (MCT1), responsible for lactate uptake in cells, were assayed as the anaerobic metabolism indicators. Under hypothermal environments, lactate contents and mct1 expression were both down-regulated in SW and FW milkfish. Taken together, GLUT1 protein expression was found to influence glucose contents in the SW groups. Anaerobic metabolism was down-regulated in both hypothermal SW and FW milkfish. Under hypothermal stress, SW-acclimated milkfish could use glycogen as an energy resource that might make SW milkfish more tolerant to hypothermal stress.
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