魚類離子與滲透壓調節之碳水化合物能量供應

Abstract

本論文的研究，在於釐清魚類鰓與表皮的上皮細胞內，肝醣代謝及接續代謝產物運輸的分子細胞機制，並探討其中對於離子與滲透壓調節的作用。 一個肝糖磷酸化酵素新異構型(tGPGG)在吳郭魚鰓上的表皮細胞被發現，進一步偵測其會表現在離子細胞周邊的富含肝醣細胞(GR cells)中。經由海水適應的吳郭魚鰓上，tGPGG的mRNA、蛋白質、總活性表現，以及肝醣含量與GR cells的表現密度，均會較淡水處理組高。經由離體培養的吳郭魚鰓上發現：鰓上鈉鉀幫浦(NKA)的活性會被GP的抑制劑-caffine所抑制，然而，添加D-glucose可以幫助其活性恢復。本部份研究指出在GR cells表現的tGPGG會受到高滲透壓環境的刺激而活化，分解肝醣提供能量給接鄰的離子細胞進行離子與滲透壓調節。 再一步確認GP、肝醣生合成酵素(GS)、與肝醣均會表現在GR cells後，發現鰓上與肝臟的肝醣含量均會在吳郭魚轉移至海水後顯著的降低，但在降低的時間點上，是鰓早於肝臟。而鰓與腦部的NKA活性會在轉移海水瞬間的1~6小時內提昇，此時鰓與腦部的GP蛋白質表現也會同時上升，而GS的蛋白表現則會降低。然而，肝臟的GP與GS蛋白表現則會在海水轉移後較晚的6~12小時出現變化。總而言之，肝醣儲存細胞在緊急狀況下，可以在短時間內提供即時的能源給鄰近的細胞進行離子調節功能。在數小時後，肝臟的肝醣代謝提供後續的能源供給。 接續的實驗進一步在斑馬魚定義出18型葡萄糖運輸通道(GLUTs)。經由foxi3a/3b的蛋白表現抑制檢測、同位雜合法、與免疫細胞染色等實驗，發現三型的GLUTs (zglut1a, -6, and -13a)與一型的單羧基運輸蛋白(zmct4)會表現在斑馬魚表皮的離子細胞或GR cells上。而且，zGLUT13a被指出是負責冨含氫幫浦細胞 (HR cells)的葡萄糖吸收，並且與HR cells 的Na+吸收有關。而zGLUT6 是負責GR cells葡萄糖的運輸，與離子調節並無直接關聯。這些結果顯示，zGLUT13a與zGLUT6會分別專一地的表現在HR cells與GR cells上，他們擁有對葡萄糖不同的親和力以因應不同型表皮細胞的不同生理需求。

The purposes of the present study were to elucidate the molecular and cellular mechanisms of glycogen metabolism, the subsequent metabolites’ transport in fish gill/skin epithelial cells, and their roles in the iono- and osmoregulatory functions. A novel gill glycogen phosphorylase (GP) isoform (tGPGG) was identified in tilapia (Oreochromis mossambicus) gill epithelia, and was colocalized in glycogen-rich cells (GR cells) which surround ionocytes. The tGPGG mRNA and protein levels and total activity, the glycogen content, and the density of GR cells in fish acclimated to seawater (SW) were significantly higher than those in the freshwater (FW) controls. Na+-K+-ATPase (NKA) activities in cultured gills were inhibited by caffeine (a GP inhibitor), and the addition of D-glucose rescued the inhibited NKA activity. In summary, tGPGG expression in GR cells is stimulated by environmental hyperosmotic challenges, and this may catalyze initial glycogen degradation to provide adjacent ionocytes with energy to carry out their iono- and osmoregulatory functions. GP, glycogen synthase (GS), and glycogen were all immunocytochemically colocalized in GR cells. Glycogen contents in the gills and liver were significantly depleted after transfer to SW, but the depletion occurred earlier in gills than in the liver. Gill and brain NKA activities rapidly increased immediately after transfer to SW. Gill and brain GP protein expressions were subsequently upregulated 1~6 h post-transfer, while GS protein levels were simultaneously downregulated. Similar changes in liver GP and GS protein expressions were also observed, but they occurred later at 6~12 h post-transfer. In conclusion, glycogen storage cells were initially stimulated to provide prompt energy so that adjacent cells could initiate their ion regulation mechanisms, and then several hours later, the liver began to degrade its glycogen stores for an additional energy supply. In subsequent experiments, 18 members of glucose transporters (GLUTs, SLC2A) were cloned from zebrafish (Danio rerio). Based on the experiments of foxi3a/3b knockdown and triple in situ hybridization/immunocytochemistry, 3 GLUT isoforms, zglut1a, -6, and -13a, and a monocarboxylate transporter, zmct4, were specifically localized in zebrafish skin/gill ionocytes and GR cells. Furthermore, zGLUT13a was demonstrated to absorb glucose to provide energy so that H+-ATPase-rich (HR) cells could take up Na+, while zGLUT6 was responsible for transporting glucose into GR cells, but was not directly related to ion regulation. Taken together, zGLUT13a and -6, specific transporters in HR cells and GR cells, absorb glucose into the respective cells with different affinities, but appear to fulfill different physiological demands in different types of epithelial cells.