Browsing by Author "Liu, Sian-Tai"

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    Brain functioning under acute hypothermic stress supported by dynamic monocarboxylate utilization and transport in ectothermic fish
    (2014-08-08) Tseng, Yung-Che; Liu, Sian-Tai; Hu, Marian Y; Chen, Ruo-Dong; Lee, Jay-Ron; Hwang, Pung-Pung
    Abstract Background The vertebrate brain is a highly energy consuming organ that requires continuous energy provision. Energy metabolism of ectothermic organisms is directly affected by environmental temperature changes and has been demonstrated to affect brain energy balance in fish. Fish were hypothesized to metabolize lactate as an additional energy substrate during acute exposure to energy demanding environmental abiotic fluctuations to support brain functionality. However, to date the pathways of lactate mobilization and transport in the fish brain are not well understood, and may represent a critical physiological feature in ectotherms during acclimation to low temperature. Results We found depressed routine metabolic rates in zebrafish during acute exposure to hypothermic (18°C) conditions accompanied by decreased lactate concentrations in brain tissues. No changes in brain glucose content were observed. Acute cold stress increased protein concentrations of lactate dehydrogenase 1 (LDH1) and citrate synthase (CS) in brain by 1.8- and- 2.5-fold, paralleled by an increased pyruvate to acetyl-CoA transformation. To test the involvement of monocarboxylate transporters (MCTs) under acute cold stress in zebrafish, we cloned and sequenced seven MCT1-4 homologues in zebrafish. All drMCT1-4 are expressed in brain tissues and in response to cold stress drmct2a and drmct4a transcripts were up-regulated 5- and 3-fold, respectively. On the contrary, mRNA levels of drmct1a, -1b and -4b in zebrafish brain responded with a down regulation in response to cold stress. By expressing drMCTs in Xenopus oocytes we could provide functional evidence that hypothermic stress leads to a 2-fold increase in lactate transport in drMCT4b expressing oocytes. Lactate transport of other paralogues expressed in oocytes was unaffected, or even decreased during cold stress. Conclusion The present work provides evidence that lactate utilization and transport pathways represent an important energy homeostatic feature to maintain vital functions of brain cells during acute cold stress in ectotherms.
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    青鱂魚海水型離子細胞之排氨、排鹽以及酸鹼平衡機制研究
    (2015) 劉咸台; Liu, Sian-Tai
    海水硬骨魚皮膚或鰓上的離子細胞主要負責排氨、排酸以及排鹽等功能。過去研究認為酸促進氨排放的機制對於淡水魚排氨相當重要。然而，酸促進氨排放的機制對於海水魚排氨的重要性至今仍未清楚。除此之外，離子細胞的排酸機制在海水魚的研究中也尚未釐清。本研究的目的是利用廣鹽性物種青鱂魚為模式，用來釐清海水魚離子細胞的排氨、排鹽以及酸鹼平衡機制。 在第一章利用選擇性離子掃秒電極的電生理技術來偵測仔魚體表的氫離子濃度梯度，發現海水青鱂魚卵黃囊體表的區域有酸性層的存在。卵黃囊體表的離子細胞其排氫的能力比角質細胞佳。在Tricine buffer和EIPA藥物處理後發現仔魚體表的排酸和排銨皆明顯的下降。藉由原位雜交反應以及化學免疫染色的方式標定出Na+/H+ exchanger 2 (NHE2) mRNA和NHE3蛋白皆表現在同一型海水離子細胞上。經定量即時聚合酶鏈鎖反應分析中發現青鱂魚鰓上的NHE3、Rhesus B glycoprotein (Rhbg)、Rhcg1和Rhcg2的mRNA在海水馴養後表現量下降，NHE2則是表現量上升。然而，在高銨海水馴養後青鱂魚鰓上的NHE3、Rhbg、Rhcg1和Rhcg2的mRNA表現量皆上升。這些發現證實海水型離子細胞同時具有排酸以及排氨的能力，藉由離子細胞的NHE和Rh蛋白來參與酸促進氨排放的排氨機制。 在第二章將利用電生理技術測量海水青鱂魚仔魚體表離子細胞的氫和氯離子排放量。在NHE、Carbonic anhydrase (CA)、anion exchanger (AE)蛋白的抑制劑處理後發現離子細胞的排氫和排氯皆明顯下降。在短期CO2馴養後會同時刺激離子細胞酸和氯的排放量。透過原位雜交反應和化學免疫染色的方式定位CA2和AE1皆表現在同一型海水離子細胞上。青鱂魚鰓上的Na+/K+/2Cl- cotransporter (NKCC1a)、CA2和AE1 的mRNA在海水馴養後皆上升。另外，在酸化的海水馴養後鰓上的AE1a和AE1b 的mRNA皆上升；而NKCC1a則是在鹼化的海水馴養後上升。這些結果解釋海水魚離子細胞的排酸機制並重新定義過去所建立的排氯機制。 在第三章我們將近一步的探討NHE2蛋白在海水型離子細胞的酸鹼平衡和排鹽功能。在抑制NHE2蛋白質表現後離子細胞的排酸和排氯皆明顯的下降，說明NHE2參與了海水型離子細胞的酸鹼平衡和排鹽功能。綜合這些結果，我們解釋海水型離子細胞的排氨、排鹽以及酸鹼平衡機制並且發現這些機制彼此間的是有關聯性的。