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  • 學位論文

莫三比克吳郭魚適應不同環境鹽度時之肝糖代謝機制

Glycogen metabolism in tilapia (Oreochromis mossambicus) during acclimation to environmental salinity changes

指導教授 : 黃鵬鵬

摘要


肝糖代謝能夠提供動物所需的能量來源。肝糖由許多葡萄糖分子聚合而成,這些小單位以a-1,4-鍵連接成線形結構聚集,直到需要的時候才會被分解,當生物體面臨緊急狀況時,肝醣的分解可以作為急時能量的提供。肝醣代謝過程中,肝糖生合成酶 (Glycogen synthase, GS)及肝糖磷酸化酶 (Glycogen phosphorylase, GP)為最重要互為拮抗之酵素。 廣鹽性魚類在適應不同環境鹽度時,由於體內滲透壓的改變,負責調節體內離子平衡最主要的器官鰓必須活化鰓表皮上富含粒線體細胞 (MR cells)的離子通道及運輸蛋白,以維持滲透壓的恆定。在此調節過程中,魚體消耗體內大量能量,因此魚類面臨環境鹽度改變時,應有相關能量代謝之機制供應能量需求。 我們已成功的從吳郭魚鰓細胞中選殖出GS,並將其氨基酸序列進行演化樹分析。以專一性的GS、GP、Na+-K+-ATPase和肝糖抗體,進行免疫螢光化學染色分析,發現GS、GP與肝糖並非分布於Na+-K+-ATPase分布的富含粒線體細胞 (MR cells)上,而分布於週邊未定義的細胞中。利用qRT-PCR分析長期適應不同環境鹽度的吳郭魚鰓組織,發現GS在鰓細胞中之核酸表現量與環境言度的關係並不顯著。藉由western blot分析短期點適應不同環境鹽度吳郭魚的腮組織,發現GS無論是在鰓、腦、或是肝臟中,其蛋白表現量隨鹽度增加而顯著下降。同時,GP蛋白表現量在各組織中海水適應組顯著高於淡水控制組。基於上述之結果,我們認為吳郭魚在適應不同環境鹽度時,肝糖代謝為其主要之能量來源,以提供吳郭魚進行滲透壓離子調節。

並列摘要


Glycogen, a high-molecular-weight polysaccharide, is a major energy supply for routine and emergency needs. Functions of glycogenesis and glycogenolysis are highly regulated by the relative activities of glycogen synthase (GS; EC 2.4.1.11) and glycogen phosphorylase (GP; EC 2.4.1.1). Gill is the most important extrarenal organ responsible for ion/osmoregulation in teleosts. Furthermore, mitochondrial-rich (MR) cells, which abundantly located in branchial epithelium, have been identified as the major sites for active transport of ions and acid/base regulation. Acclimation to seawater (SW) in euryhaline teleosts is achieved by activation of ion secretion pathway in the gill. The activation of these ion transport processes demands timely and sufficiently extra energy. However, nothing was known about the cellular and molecular basis of the energy metabolism for the ion/osmoregulation in teleosts during salinity challenges. The purpose of the present study was to examine the role of GS and GP in the energy metabolism for the ion/osmoregulation in teleosts. We have successfully cloned and sequenced the full-length cDNA of GS form tilapia gill epithelial cells. The results of deduced amino acid sequence alignment and phylogenetic tree analysis showed that the tilapia GS (tGS) cloned from tilapia gill is a homologue of the mammalian GS1 (muscle form). The results of immunohistochemical experiments demonstrated that GS, GP, and glycogen were co-localized in the un-identified cells of gill. These cells were just adjacent to the Na+-K+-ATPase-expressing mitochondrial-rich cells (MR cells), the major ionocytes in fish gill. The gene expression levels of GS in gill measured by quantitative real-time PCR and semi-quantitative RT-PCR showed no significant change in tilapia after a long-term acclimation from fresh water (FW) to 35-ppt seawater (SW). However, the Western blotting analysis indicated that the GS protein expression in gill decreased immediately after transferring to 25-ppt SW. This finding was opposite to the protein expression profile of GP, which increased right after transfer to SW and eventually recovered. Similar phenomena of up-regulation of GP and down-regulation of GS protein expressions were also found in liver and brain right after SW transfer. However, the differential expressions of GP and GS in brain and liver occurred at 6 and 12 hours post-transfer, respectively. According to these results, we proposed that GS and GP are involved in energy re-storing and degradation to maintain the internal glucose homeostasis in gill and brain of fish during acclimation to environmental salinity. Gill and brain initialize the metabolism of the local glycogen right after salinity challenge, and subsequently liver follows to responsible for the energy supply for longer acclimation to salinity change.

參考文獻


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