Yung Che Tseng

Some insights into energy metabolism for osmoregulation in fish

A sufficient and timely energy supply is a prerequisite for the operation of iono- and osmoregulatory mechanisms in fish. Measurements of whole-fish or isolated-gill (or other organs) oxygen consumption have demonstrated regulation of the energy supply during acclimation to different osmotic environments, and such regulation is dependent on species, the situation of acclimation or acclimatization, and life habits. Carbohydrate metabolism appears to play a major role in the energy supply for iono- and osmoregulation, and the liver is the major source supplying carbohydrate metabolites to osmoregulatory organs. Compared with carbohydrates, the roles of lipids and proteins remain largely unclear. Energy metabolite translocation was recently found to occur between fish gill ionocytes and neighboring glycogen-rich (GR) cells, indicating the physiological significance of a local energy supply for gill ion regulatory mechanisms. Spatial and temporal relationships between the liver and other osmoregulatory and non-osmoregulatory organs in partitioning the energy supply for ion regulatory mechanisms during salinity challenges were also proposed. A novel glucose transporter was found to specifically be expressed and function in gill ionocytes, providing the first cue for investigating energy translocation among gill cells. Advanced molecular physiological approaches can be used to examine energy metabolism relevant to a particular cell type (e.g., gill ionocytes), and functional genomics may also provide another powerful approach to explore new metabolic pathways related to fish ion regulation.

Role of SLC12A10.2, a Na-Cl cotransporter-like protein, in a Cl uptake mechanism in zebrafish (Danio rerio)

The thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC), a member of the SLC12 family, is mainly expressed in the apical membrane of the mammalian distal convoluted tubule (DCT) cells, is responsible for cotransporting Na(+) and Cl(-) from the lumen into DCT cells and plays a major role in the mammalian renal NaCl reabsorption. The NCC has also been reported in fish, but the functional role in fish ion regulation is yet unclear. The present study used zebrafish as an in vivo model to test the hypothesis of whether the NCC plays a role in Na(+) and/or Cl(-) uptake mechanisms. Four NCCs were cloned, and only one of them, zebrafish (z) slc12a10.2 was found to predominately and specifically be expressed in gills. Double in situ hybridization/immunocytochemistry in zebrafish skin/gills demonstrated that the specific expression of zslc12a10.2 mRNA in a novel group of ionocytes differed from those of the previously-reported H(+)-ATPase-rich (HR) cells and Na(+)-K(+)-ATPase-rich (NaR) cells. Gill mRNA expression of zslc12a10.2 was induced by a low-Cl environment that stimulated fish Cl(-) influx, while a low-Na environment suppressed this expression. Incubation with metolazone, a specific inhibitor of the NCC, impaired both Na(+) and Cl(-) influx in 5-day postfertilization (dpf) zebrafish embryos. Translational knockdown of zslc12a10.2 with a specific morpholino caused significant decreases in both Cl(-) influx and Cl(-) content of 5-dpf zebrafish embryos, suggesting that the operation of zNCC-like 2 results in a net uptake of Cl(-) in zebrafish. On the contrary, zslc12a10.2 morphants showed increased Na(+) influx and content that resulted from upregulation of mRNA expressions of Na(+)-H(+) exchanger 3b and carbonic anhydrase 15a in HR cells. These results for the first time provide in vivo molecular physiological evidence for the possible role of the NCC in the Cl(-) uptake mechanism in zebrafish skin/gills.

Effects of stanniocalcin 1 on calcium uptake in zebrafish (Danio rerio) embryo

Stanniocalcin (STC) formerly called hypocalcin or teleocalcin, is a 50-kDa disulfide-linked homodimeric glycoprotein that was originally identified in fish and secreted from the corpuscles of Stannius (CS). One of the main functions of STC-1 is Ca(2+) uptake inhibition; however, the mechanisms remain unknown. In the present study, we provide molecular evidence to elucidate how zebrafish STC-1 regulates Ca(2+) uptake in zebrafish embryos. In a wide variety of tissues including the kidney, brain, gill, muscle, and skin, zstc-1 was expressed. Incubating zebrafish embryos in low-Ca(2+) (0.02 mM) freshwater stimulated whole body Ca(2+) influx and zebrafish epithelial Ca(2+) channel (zECaC) mRNA expression, while downregulated zstc-1 expression. A morpholino microinjection approach was used to knockdown the zSTC-1 protein, and the results showed that the Ca(2+) content, Ca(2+) influx, and zECaC mRNA expression all increased in morphants. These data suggest that zSTC-1 negatively regulates ECaC gene expression to reduce Ca(2+) uptake in zebrafish embryos.

Elevated seawater pco2 differentially affects branchial acid-base transporters over the course of development in the cephalopod Sepia officinalis

The specific transporters involved in maintenance of blood pH homeostasis in cephalopod molluscs have not been identified to date. Using in situ hybridization and immunohistochemical methods, we demonstrate that Na(+)/K(+)-ATPase (soNKA), a V-type H(+)-ATPase (soV-HA), and Na(+)/HCO(3)(-) cotransporter (soNBC) are colocalized in NKA-rich cells in the gills of Sepia officinalis. mRNA expression patterns of these transporters and selected metabolic genes were examined in response to moderately elevated seawater Pco(2) (0.16 and 0.35 kPa) over a time course of 6 wk in different ontogenetic stages. The applied CO(2) concentrations are relevant for ocean acidification scenarios projected for the coming decades. We determined strong expression changes in late-stage embryos and hatchlings, with one to three log2-fold reductions in soNKA, soNBCe, socCAII, and COX. In contrast, no hypercapnia-induced changes in mRNA expression were observed in juveniles during both short- and long-term exposure. However, a transiently increased ion regulatory demand was evident during the initial acclimation reaction to elevated seawater Pco(2). Gill Na(+)/K(+)-ATPase activity and protein concentration were increased by ~15% during short (2-11 days) but not long-term (42-days) exposure. Our findings support the hypothesis that the energy budget of adult cephalopods is not significantly compromised during long-term exposure to moderate environmental hypercapnia. However, the downregulation of ion regulatory and metabolic genes in late-stage embryos, taken together with a significant reduction in somatic growth, indicates that cephalopod early life stages are challenged by elevated seawater Pco(2).

Exploring Uncoupling Proteins and Antioxidant Mechanisms under Acute Cold Exposure in Brains of Fish

Exposure to fluctuating temperatures accelerates the mitochondrial respiration and increases the formation of mitochondrial reactive oxygen species (ROS) in ectothermic vertebrates including fish. To date, little is known on potential oxidative damage and on protective antioxidative defense mechanisms in the brain of fish under cold shock. In this study, the concentration of cellular protein carbonyls in brain was significantly increased by 38% within 1 h after cold exposure (from 28°C to 18°C) of zebrafish (Danio rerio). In addition, the specific activity of superoxide dismutase (SOD) and the mRNA level of catalase (CAT) were increased after cold exposure by about 60% (6 h) and by 60%–90% (1 and 24 h), respectively, while the specific glutathione content as well as the ratio of glutathione disulfide to glutathione remained constant and at a very low level. In addition, cold exposure increased the protein level of hypoxia-inducible factor (HIF) by about 50% and the mRNA level of the glucose transporter zglut3 in brain by 50%–100%. To test for an involvement of uncoupling proteins (UCPs) in the cold adaptation of zebrafish, five UCP members were annotated and identified (zucp1-5). With the exception of zucp1, the mRNA levels of the other four zucps were significantly increased after cold exposure. In addition, the mRNA levels of four of the fish homologs (zppar) of the peroxisome proliferator-activated receptor (PPAR) were increased after cold exposure. These data suggest that PPARs and UCPs are involved in the alterations observed in zebrafish brain after exposure to 18°C. The observed stimulation of the PPAR-UCP axis may help to prevent oxidative damage and to maintain metabolic balance and cellular homeostasis in the brains of ectothermic zebrafish upon cold exposure.

Regulation of glycogen metabolism in gills and liver of the euryhaline tilapia (Oreochromis mossambicus) during acclimation to seawater.

SUMMARY Glucose, which plays a central role in providing energy for metabolism, is primarily stored as glycogen. The synthesis and degradation of glycogen are mainly initialized by glycogen synthase (GS) and glycogen phosphorylase (GP), respectively. The present study aimed to examine the glycogen metabolism in fish liver and gills during acute exposure to seawater. In tilapia (Oreochromis mossambicus) gill, GP, GS and glycogen were immunocytochemically colocalized in a specific group of glycogen-rich (GR) cells, which are adjacent to the gills main ionocytes, mitochondrion-rich (MR) cells. Na+/K+-ATPase activity in the gills, protein expression and/or activity of GP and GS and the glycogen content of the gills and liver were examined in tilapia after their acute transfer from freshwater (FW) to 25‰ seawater (SW). Gill Na+/K+-ATPase activity rapidly increased immediately after SW transfer. Glycogen content in both the gills and liver were significantly depleted after SW transfer, but the depletion occurred earlier in gills than in the liver. Gill GP activity and protein expression were upregulated 1–3 h post-transfer and eventually recovered to the normal level as determined in the control group. At the same time, GS protein expression was downregulated. Similar changes in liver GP and GS protein expression were also observed but they occurred later at 6–12 h post-transfer. In conclusion, GR cells are initially stimulated to provide prompt energy for neighboring MR cells that trigger ion-secretion mechanisms. Several hours later, the liver begins to degrade its glycogen stores for the subsequent energy supply.

A new model for fish ion regulation: identification of ionocytes in freshwater- and seawater-acclimated medaka (Oryzias latipes)

The ion regulation mechanisms of fishes have been recently studied in zebrafish (Danio rerio), a stenohaline species. However, recent advances using this organism are not necessarily applicable to euryhaline fishes. The euryhaline species medaka (Oryzias latipes), which, like zebrafish, is genetically well categorized and amenable to molecular manipulation, was proposed as an alternative model for studying osmoregulation during acclimation to different salinities. To establish its suitability as an alternative, the present study was conducted to (1) identify different types of ionocytes in the embryonic skin and (2) analyze gene expressions of the transporters during seawater acclimation. Double/triple in situ hybridization and/or immunocytochemistry revealed that freshwater (FW) medaka contain three types of ionocyte: (1) Na+/H+ exchanger 3 (NHE3) cells with apical NHE3 and basolateral Na+-K+-2Cl− cotransporter (NKCC), Na+-K+-ATPase (NKA) and anion exchanger (AE); (2) Na+-Cl− cotransporter (NCC) cells with apical NCC and basolateral H+-ATPase; and (3) epithelial Ca2+ channel (ECaC) cells [presumed accessory (AC) cells] with apical ECaC. On the other hand, seawater (SW) medaka has a single predominant ionocyte type, which possesses apical cystic fibrosis transmembrane conductance regulator (CFTR) and NHE3 and basolateral NKCC and NKA and is accompanied by smaller AC cells that express lower levels of basolateral NKA. Reciprocal gene expressions of decreased NHE3, AE, NCC and ECaC and increased CFTR and NKCC in medaka gills during SW were revealed by quantative PCR analysis.

Ghrelin affects carbohydrate-glycogen metabolism via insulin inhibition and glucagon stimulation in the zebrafish (Danio rerio) brain

Carbohydrate-glycogen metabolism (CGM) is critical for emergency energy supplies in the central nervous system (CNS). Ghrelin (GHRL) in pancreas is known to significantly regulate a dominant player in CGM, insulin (INS). However, its regulatory effect on extrapancreatic INS synthesis is yet unknown. In this study, we used adult zebrafish to elucidate the expression and role of zebrafish GHRL (zGHRL) in genes primarily involved in the brains CGM. Results showed that zebrafish brain expressed zghrl and its receptor, growth hormone secretagogue-receptor (GHS-R: zghs-r1a and zghs-r2a), according to RT-PCR and in situ hybridization. Protein localization coupled with mRNA spatial expression further verified zGHRLs presence in the brain. For the in vivo study, significant increases in zghs-r1a and zghs-r2a synthesis were observed after injection of synthetic peptide goldfish GHRL-12 (gGHRL) using brain templates analyzed by quantitative real-time PCR (qPCR). Ligand-receptor synthesis of INS (zinsa; zins-r1 and zins-r2) significantly decreased, while glucagon (GCG) (zgcgb1 and zgcgb2; zgcg-r1 and zgcg-r2) exhibited a significant transient increase. In CGM, subsequent processes indicate urgent glucose-sensing response that will balance glycogen degradation and energy storage. Taken together, these findings suggest that GHRL regulates INS synthesis by mediating its action on GHS-R in the CNS and partly involved in CGM.

CO2-driven seawater acidification differentially affects development and molecular plasticity along life history of fish (Oryzias latipes)

Fish early life stages have been shown to react sensitive to simulated ocean acidification. In particular, acid-base disturbances elicited by altered seawater carbonate chemistry have been shown to induce pathologies in larval fish. However, the mechanisms underlying these disturbances are largely unknown. We used gene expression profiling of genes involved in acid-base regulation and metabolism to investigate the effects of seawater hypercapnia on developing Japanese ricefish (medaka; Oryzias latipes). Our results demonstrate that embryos respond with delayed development during the time window of 2-5 dpf when exposed to a seawater pCO(2) of 0.12 and 0.42 kPa. This developmental delay is associated with strong down-regulation of genes from major metabolic pathways including glycolysis (G6PDH), Krebs cycle (CS) and the electron transport chain (CytC). In a second step we identified acid-base relevant genes in different ontogenetic stages (embryos, hatchlings and adults) and tissues (gill and intestine) that are up regulated in response to hypercapnia, including NHE3, NBCa, NBCb, AE1a, AE1b, ATP1a1a.1, ATP1a1b, ATP1b1a, Rhag, Rhbg and Rhcg. Interestingly, NHE3 and Rhcg expressions were increased in response to environmental hypercapnia in all ontogenetic stages and tissues tested, indicating the central role of these proteins in acid-base regulation. Furthermore, the increased expression of genes from amino acid metabolism pathways (ALT1, ALT2, AST1a, AST1b, AST2 and GLUD) suggests that energetic demands of hatchlings are fueled by the breakdown of amino acids. The present study provides a first detailed gene expression analysis throughout the ontogeny of a euryhaline teleost in response to seawater hypercapnia, indicating highest sensitivity in early embryonic stages, when functional ion regulatory epithelia are not yet developed.

Involvement of calcitonin and its receptor in the control of calcium-regulating genes and calcium homeostasis in zebrafish (Danio rerio)

Calcitonin (CT) is one of the hormones involved in vertebrate calcium regulation. It has been proposed to act as a hypocalcemic factor, but the regulatory pathways remain to be clarified. We investigated the CT/calcitonin gene–related peptide (CGRP) family in zebrafish and its potential involvement in calcium homeostasis. We identified the presence of four receptors: CTR, CRLR1, CRLR2, and CRLR3. From the phylogenetic analysis, together with the effect observed after CT and CGRP overexpression, we concluded that CTR appears to be a CT receptor and CRLR1 a CGRP receptor. The distribution of these two receptors shows a major presence in the central nervous system and in tissues involved in ionoregulation. Zebrafish embryos kept in high‐Ca2+‐concentration medium showed upregulation of CT and CTR expression and downregulation of the epithelial calcium channel (ECaC). Embryos injected with CT morpholino (CALC MO) incubated in high‐Ca2+ medium, showed downregulation of CTR together with upregulation on ECaC mRNA expression. In contrast, overexpression of CT cRNA induced the downregulation of ECaC mRNA synthesis, concomitant with the downregulation in the calcium content after 30 hours postfertilization. At 4 days postfertilization, CT cRNA injection induced upregulation of hypercalcemic factors, with subsequent increase in the calcium content. These results suggest that CT acts as a hypocalcemic factor in calcium regulation, probably through inhibition of ECaC synthesis.