臺灣博碩士論文加值系統

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。

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.

Table of Contents
Acknowledgement
Table of contents
中文摘要........................................................................................................................1
Abstract………………………………………………………………….……………2
Introduction………………………………………………………………………..…4
Glycogen metabolism process…………………………………………………………4
Regulation of glycogen synthesis……………………………………………………...4
Glycogenolysis mechanisms…………………………………………………………..5
Osmoregulation mechanisms in euryhaline teleosts…………………………………..6
Energy requirement for teleost’s osmoregulation……………………………………..7
Aim of the study……………………………………………………………………….9
Materials and Methods……………………………………………………………..10
Experimental animals………………………………………………………………...10
Acclimation experiments…………………………………………………………… 10
Isolation of epithelial cells from tilapia gill………………………………………….10
Preparation of total RNA……………………………………………………………..11
Purification of mRNA………………………………………………………………..11
Amplification of cDNA fragments by RT-PCR………………………………………12
Cloning of tilapia GS cDNA from gill……………………………………………….12
Full-length cDNA sequence………………………………………………………….13
GS expression in different tissues……………………………………………………13
Quantitative RT-PCR…………………………………………………………………14
Protein extraction from the epithelial cells of tilapia gill…………………………….14
Western blotting……………………………………………………………………...14
Computation of the protein amount………………………………………………….15
Immunocytochemistry……………………………………………………………….16
Statistical analysis……………………………………………………………………17
Results……………………………………………………………………………….18
Tilapia glycogen synthase (tGS) cloning and Sequencing………………………..18
a. Full-length GS cDNA from tilapia gill……………………………………………18
b. Phylogenetic analysis of tGS amino acid sequence………………………………18
tGS gene expression in different tissues…………………………………………..19
Long-term acclimation……………………………………………………………..19
a. Effect of environmental salinity on tGS mRNA expression in tilapia gill………...19
b. Localization of GS, Na+-K+-ATPase, and glycogen in tilapia gill………………...19
Short-term acclimation……………………………………………………………..20
a. Time course changes of GS and GP protein amount in tilapia gill after transferred to 25-ppt SW……………………………………………………………………..20
b. Localization of GS and GP in tilapia gill………………………………………….20
c. Time course changes of GS and GP protein amount in tilapia liver after transferred to 25-ppt SW………………………………………………………………………21
d. Localization of GS and glycogen in tilapia liver…………………………………..21
e. Time course changes of GS and GP protein amount in tilapia brain after transferred to 25-ppt SW………………………………………………………………………22
f. Localization of GS and glycogen in tilapia brain………………………………….22
Discussion……………………………………………………………………………23
Summary…………………………………………………………………………….29
References…………………………………………………………………………...30
Figures…………………………………………………………………………...…..39

Alonso MD, Lomako J, Lomako WM and Whelan WJ. (1995). Catalytic activities of glycogenin additional to autocatalytic self-glucosylation. J Biol Chem 270, 15315-15319.
Azpiazu I, Manchester J, Skurat AV, Roach PJ and Lawrence JC, Jr. (2000). Control of glycogen synthesis is shared between glucose transport and glycogen synthase in skeletal muscle fibers. Am J Physiol Endocrinol Metab 278, E234-243.
Bacca H, Huvet A, Fabioux C, Daniel JY, Delaporte M, Pouvreau S, Van Wormhoudt A and Moal J. (2005). Molecular cloning and seasonal expression of oyster glycogen phosphorylase and glycogen synthase genes. Comp Biochem Physiol B Biochem Mol Biol 140, 635-646.
Báez M, Preller A and Ureta T. (2003). Frog oocyte glycogen synthase: enzyme regulation under in vitro and in vivo conditions. Arch Biochem Biophys 413, 9-16.
Bollen M, Keppens S and Stalmans W. (1998). Specific features of glycogen metabolism in the liver. Biochem J 336, 19–31.
Brown AM, Sickmann HM, Fosgerau K, Lund TM, Schousboe A, Waagepetersen HS and Ransom BR. (2005). Astrocyte glycogen metabolism is required for neural activity during aglycemia or intense stimulation in mouse white matter. J Neurosci Res 79, 74-80.
Buschiazzo A, Ugalde JE, Guerin ME, Shepard W, Ugalde RA and Alzari PM. (2004). Crystal structure of glycogen synthase: homologous enzymes catalyze glycogen synthesis and degradation. Embo J 23, 3196-3205.
Cameron JN, Kormanik GA. (1982). The acid-base responses of gills and kidneys to infused acid and base loads in the channel catfish, Ictalurus punctatus. J Exp Biol 99,143-160.
Chen YY, Lu FI, Hwang PP. (2003). Comparisons of calcium regulation in fish larvae. J Exp Zoology A Comp Exp Biol 295(2), 127-135.
Cohen P. (1982). The role of protein phosphorylation in neural and hormonal control of cellular activity. Nature 296, 613-620.
Evans DH, Piermarini PM and Choe KP. (2005). The multifunctional fish gill: dominant site of gas exchange, osmoregulation, acid-base regulation, and excretion of nitrogenous waste. Physiol Rev 85, 97-177.
Feng SH, Leu JH, Yang CH, Fang MJ, Huang CJ and Hwang PP. (2002). Gene expression of Na+-K+-ATPase alpha 1 and alpha 3 subunits in gills of the teleost Oreochromis mossambicus, adapted to different environmental salinities. Mar Biotechnol (NY) 4, 379-391.
Fernandez-Novell JM, Arino J, Vilaro S and Guinovart JJ. (1992). Glucose induces the translocation and the aggregation of glycogen synthase in rat hepatocytes. Biochem J 281, 443-448.
Fernandez-Novell JM, Bellido D, Vilaro S and Guinovart JJ. (1997). Glucose induces the translocation of glycogen synthase to the cell cortex in rat hepatocytes. Biochem J 321, 227-231.
Fisher JS, Nolte LA, Kawanaka K, Han DH, Jones TE and Holloszy JO. (2002). Glucose transport rate and glycogen synthase activity both limit skeletal muscle glycogen accumulation. Am J Physiol Endocrinol Metab 282, E1214-1221.
Franch J, Aslesen R and Jensen J. (1999). Regulation of glycogen synthesis in rat skeletal muscle after glycogen-depleting contractile activity: effects of adrenaline on glycogen synthesis and activation of glycogen synthase and glycogen phosphorylase. Biochem J 344, 231-235.
Frolow J and Milligan CL. (2004). Hormonal regulation of glycogen metabolism in white muscle slices from rainbow trout (Oncorhynchus mykiss Walbaum). Am J Physiol Regul Integr Comp Physiol 287, R1344-1353.
Garcia-Rocha M, Roca A, De La Iglesia N, Baba O, Fernandez-Novell JM, Ferrer JC and Guinovart JJ. (2001). Intracellular distribution of glycogen synthase and glycogen in primary cultured rat hepatocytes. Biochem J 357, 17-24.
Gibbs SR and deRoos RM. (1991). Plasma levels of glucose, ketone bodies, lactate, and alanine in the vascular supply to and from the brain of the adult American bullfrog (Rana catesbeiana). J Exp Zool 258, 14-23.
Gomis RR, Cid E, Garcia-Rocha M, Ferrer JC and Guinovart JJ. (2002). Liver glycogen synthase but not the muscle isoform differentiates between glucose 6-phosphate produced by glucokinase or hexokinase. J Biol Chem 277, 23246-23252.
Goss GG, Perry SF, Fryer JN and Laurent P. (1998). Gill morphology and acid-base regulation in freshwater fishes. Comp Biochem Physiol A Mol Integr Physiol 119, 107-115.
Halse R, Fryer LG, McCormack JG, Carling D and Yeaman SJ. (2003). Regulation of glycogen synthase by glucose and glycogen: a possible role for AMP-activated protein kinase. Diabetes 52, 9-15.
Hardy TA and Roach PJ. (1993). Control of yeast glycogen synthase-2 by COOH-terminal phosphorylation. J Biol Chem 268, 23799-23805.
Higgins DG and Sharp PM. (1988). CLUSTAL: a package for performing multiple sequence alignment on a microcomputer. Gene 73, 237-244.
Higgins DG, Thompson JD and Gibson TJ. (1996). Using CLUSTAL for multiple sequence alignments. Methods Enzymol 266, 383-402.
Hirose S, Kaneko T, Naito N and Takei Y. (2003). Molecular biology of major components of chloride cells. Comp Biochem Physiol B Biochem Mol Biol 136, 593-620.
Howlett RA, Parolin ML, Dyck DJ, Hultman E, Jones NL, Heigenhauser G.J and Spriet LL. (1998). Regulation of skeletal muscle glycogen phosphorylase and PDH at varying exercise power outputs. Am J Physiol 275, R418-425.
Huang D, Wilson WA and Roach PJ. (1997). Glucose-6-P control of glycogen synthase phosphorylation in yeast. J Biol Chem 272, 22495-22501.
Hwang PP. (1987). Tolerance and ultrastructural response of branchial chloride cells on salinity changes in euryhaline teleost, Oreochromis mossambicus. Marine Biol 94, 643-649.
Hwang PP, Sun CM and Wu SM. (1989). Changes of plasma osmolality, chloride concentration, and gill Na,K-ATPase activity in tilapia, Oreochromis mossambicus, during seawater acclimation. Mar Biol 100, 295-299.
Hwang YH. (2001). Differential stimulation of enzyme activity and protein level of Na+,K+-ATPase in the organs of Tilapia (Oreochromis mossambicus) upon acute seawater challenge. Master thesis. Institute of Fisheries Science, National Taiwan University.
Jensen MK, Madsen SS and Kristiansen K. (1998). Osmoregulation and salinity effects on the expression and activity of Na+,K+-ATPase in the gills of European sea bass, Dicentrarchus labrax (L.). J Exp Zool 282, 290-300.
Johnson LN. (1992). Glycogen phosphorylase: control by phosphorylation and allosteric effectors. FASEB J 6, 2274-2282
Johnston M. (1999). Feasting, fasting and fermenting glucose sensing in yeast and other cells. Trends Genet 15, 29-33.
Lee TH, Feng SH, Lin CH, Hwang YH, Huang CL and Hwang PP. (2003). Ambient salinity modulates the expression of sodium pumps in branchial mitochondria-rich cells of Mozambique tilapia, Oreochromis mossambicus. Zoolog Sci 20, 29-36.
Lee TH, Tsai JC, Fang MJ, Yu MJ and Hwang PP. (1998). Isoform expression of Na+-K+-ATPase α subunit in gills of the teleost, Oreochromis mossambicus. Am J Physiol 275, R926-932.
Lin CH, Huang CL, Yang CH, Lee TH and Hwang PP. (2004). Time-course changes in the expression of Na, K-ATPase and the morphometry of mitochondrion-rich cells in gills of euryhaline tilapia (Oreochromis mossambicus) during freshwater acclimation. J Exp Zoolog A Comp Exp Biol 301, 85-96.
Lin LY and Hwang PP. (2004). Mitochondria-rich cell activity in the yolk-sac membrane of tilapia (Oreochromis mossambicus) larvae acclimatized to different ambient chloride levels. J Exp Biol 207, 1335-1344.
Mancera JM and McCormick SD. (2000). Rapid activation of gill Na(+),K(+)-ATPase in the euryhaline teleost Fundulus heteroclitus. J Exp Zool 287, 263-274.
Mancera JM and McCormick SD. (1998). Evidence for growth hormone/insulin-like growth factor I axis regulation of seawater acclimation in the euryhaline teleost Fundulus heteroclitus. Gen Comp Endocrinol 111, 103-112.
Marshall WS, Emberley TR, Singer TD, Bryson SE and McCormick SD. (1999). Time course of salinity adaptation in a strongly euryhaline estuarine teleost, Fundulus heteroclitus: a multivariable approach. J Exp Biol 202, 1535-1544.
Marshall WS. (2002). Na(+), Cl(-), Ca(2+) and Zn(2+) transport by fish gills: retrospective review and prospective synthesis. J Exp Zool 293, 264-283.
McCormick SD, Hasegawa S and Hirano T. (1992). Calcium uptake in the skin of a freshwater teleost. Proc Natl Acad Sci USA 89, 3635-3638.
McCormick SD. (1995). Hormonal control of gill Na+,K+-ATPase and chloride cell function. Fish Physiology 14, 285-315.
Milligan CL. (2003). A regulatory role for cortisol in muscle glycogen metabolism in rainbow trout Oncorhynchus mykiss Walbaum. J Exp Biol 206, 3167-3173.
Moon TW, Busby ER, Cooper GA and Mommsen TP. (1999). Fish hepatocyte glycogen phosphorylase: a sensitive indicator for hormonal modulation. Fish Physiol Biochem 21, 15 24.
Morgan JD and Iwama GK. (1991). Effects of salinity on growth, metabolism, and ion regulation in juvenile rainbow trout (Oncorhynchus mykiss) and fall Chinook salmon (Oncorhynchus tshawytscha). Can J Fish Aquat Sci 48, 2083-2094.
Newgard CB, Hwang PK and Fletterick RJ. (1989). The family of glycogen phosphorylases: structure and function. Crit Rev Biochem Mol Biol. 24, 69-99.
Oliveira GT, Rossi IC, Kucharski LC and Da Silva RS. (2004). Hepatopancreas gluconeogenesis and glycogen content during fasting in crabs previously maintained on a high-protein or carbohydrate-rich diet. Comp Biochem Physiol A Mol Integr Physiol 137, 383-390.
Ozcan S, Dover J and Johnston M. (1998). Glucose sensing and signaling by two glucose receptors in the yeast Saccharomyces cerevisiae. Embo J 17, 2566-2573.
Pan TC. (2003). Calcium uptake mechanism in Zebrafish embryos. Master thesis. Institute of Fisheries Science, National Taiwan University.
Pederson BA, Chen H, Schroeder JM, Shou W, DePaoli-Roach AA and Roach PJ. (2004). Abnormal cardiac development in the absence of heart glycogen. Mol Cell Biol 24, 7179-7187.
Pederson BA, Cheng C, Wilson WA and Roach PJ. (2000). Regulation of glycogen synthase. Identification of residues involved in regulation by the allosteric ligand glucose-6-P and by phosphorylation. J Biol Chem 275, 27753-27761.
Perry SF. (1997). The chloride cell: structure and function in the gills of freshwater fishes. Annu Rev Physiol 59, 3253-3247.
Pfeiffer-Guglielmi B, Fleckenstein B, Jung G and H amprecht B. (2003). Immunocytochemical localization of glycogen phosphorylase isozymes in rat nervous tissues by using isozyme-specific antibodies. J Neurochem 85, 73-81.
Reshkin SJ and Ahearn GA. (1987). Intestinal glucose transport and salinity adaptation in a euryhaline teleost. Am J Physiol 252, R567-578.
Roach PJ. (1990). Control of glycogen synthase by hierarchal protein phosphorylation. FASEB J 4, 2961-2968.
Sangiao-Alvarellos S, Laiz-Carrion R, Guzman JM, Martin del Rio MP, Miguez JM, Mancera JM and Soengas JL. (2003). Acclimation of S aurata to various salinities alters energy metabolism of osmoregulatory and nonosmoregulatory organs. Am J Physiol Regul Integr Comp Physiol 285, R897-907.
Sardella BA, Cooper J, Gonzalez RJ and Brauner CJ. (2004). The effect of temperature on juvenile Mozambique tilapia hybrids (Oreochromis mossambicus x O. urolepis hornorum) exposed to full-strength and hypersaline seawater. Comp Biochem Physiol A Mol Integr Physiol 137, 621-629.
Sardella BA, Matey V, Cooper J, Gonzalez RJ and Brauner CJ. (2004). Physiological, biochemical and morphological indicators of osmoregulatory stress in ''California'' Mozambique tilapia (Oreochromis mossambicus x O. urolepis hornorum) exposed to hypersaline water. J Exp Biol 207, 1399-1413.
Seidelin M, Madsen SS, Blenstrup H and Tipsmark CK. (2000). Time-course changes in the expression of Na+, K+-ATPase in gills and pyloric caeca of brown trout (Salmo trutta) during acclimation to seawater. Physiol Biochem Zool 73, 446-453.
Seoane J, Gomez-Foix AM, O''Doherty RM, Gomez-Ara C, Newgard CB and Guinovart JJ. (1996). Glucose 6-phosphate produced by glucokinase, but not hexokinase I, promotes the activation of hepatic glycogen synthase. J Biol Chem 271, 23756-23760.
Skurat AV and Roach PJ. (1995). Phosphorylation of sites 3a and 3b (Ser640 and Ser644) in the control of rabbit muscle glycogen synthase. J Biol Chem 270, 12491-12497.
Skurat AV, Dietrich AD and Roach PJ. (2000). Glycogen synthase sensitivity to insulin and glucose-6-phosphate is mediated by both NH2- and COOH-terminal phosphorylation sites. Diabetes 49, 1096-1100.
Smythe C and Cohen P. (1991). The discovery of glycogenin and the priming mechanism for glycogen biogenesis. Eur J Biochem 200, 625-631.
Soengas JL and Aldegunde M. (2002). Energy metabolism of fish brain. Comp Biochem Physiol B Biochem Mol Biol 131, 271-296.
Tagaya M, Nakano K and Fukui T. (1985). A new affinity labeling reagent for the active site of glycogen synthase. Uridine diphosphopyridoxal. J Biol Chem 260, 6670-6676.
Tipsmark CK, Madsen SS and Borski RJ. (2004). Effect of salinity on expression of branchial ion transporters in striped bass (Morone saxatilis). J Exp Zoolog A Comp Exp Biol 301, 979-991.
Tseng YC. (2004). Role of glycogen phosphorylase in energy supplying for osmoregulation in gills of tilapia (Oreochromis mossambicus). Master thesis. Institute of Fisheries Science, National Taiwan University.
Vijayan M, Morgan J, Sakamoto T, Grau E and Iwama G.. (1996). Food-deprivation affects seawater acclimation in tilapia: hormonal and metabolic changes. J Exp Biol 199, 2467-2475.
Weng CF, Chiang CC, Gong HY, Chen MH, Lin CJ, Huang WT, Cheng CY, Hwang PP and Wu JL. (2002). Acute changes in gill Na+-K+-ATPase and creatine kinase in response to salinity changes in the euryhaline teleost, tilapia (Oreochromis mossambicus). Physiol Biochem Zool 75, 29-36.
Wilson JM and Laurent P. (2002). Fish gill morphology: inside out. J Exp Zool 293, 192-213.
Wright PA, Perry SF and Moon TW. (1989). Regulation of hepatic gluconeogenesis and glycogenolysis by catecholamines in rainbow trout during environmental hypoxia. J Exp Biol 147, 169-188.
Wullimann MF, Rupp B and Reichert H. (1996). Neuroanatomy of the zebrafish brain. Birkhäuser Verlag, Basel, Switzerland.
Zadunaisky JA. (1996). Chloride cells and osmoregulation. Kidney Int 49, 1563-1567