Abstract
The thyroid gland in fish mainly secretes the thyroid prohormone 3,5,3′,5′-tetraiodothyronine (T4), and extrathyroidal outer ring deiodination (ORD) of the prohormone to 3,5,3′-triiodothyronine (T3) is pivotal in thyroid hormone economy. Despite its importance in thyroid hormone metabolism, factors that regulate ORD are still largely unresolved in fish. In addition, the osmoregulatory role of T3 is still a controversial issue in teleosts. In this study, we investigated the regulation of the ORD pathway by cortisol and T3 in different organs (liver, kidney, and gills) of Solea senegalensis and the involvement of T3 in the control of branchial and renal Na+, K+-ATPase activity, a prime determinant of the hydromineral balance in teleosts. Animals were treated with i.p. slow-release coconut oil implants containing cortisol or T3. Hepatic and renal ORD activities were up-regulated in cortisol-injected animals. T3-treated fish showed a prominent decrease in plasma-free T4 levels, whereas ORD activities did not change significantly. Branchial and renal Na+, K+-ATPase activities were virtually unaffected by T3, but were transiently up-regulated by cortisol. We conclude that cortisol regulates local T3 bioavailability in S. senegalensis via ORD in an organ-specific manner. Unlike T3, cortisol appears to be directly implicated in the up-regulation of branchial and renal Na+, K+-ATPase activities.
Introduction
Thyroid hormone regulates many important functions such as growth, salinity preference, oxygen consumption, nutrient metabolism, and metamorphosis in fish (reviewed by Eales (2006) and Blanton & Specker (2007)). Under normal conditions, the piscine thyroid gland mainly secretes the prohormone 3,5,3′,5′-tetraiodothyronine (T4). T4 must be converted, by removal of an iodine atom, to yield the biologically active hormone 3,5,3′-triiodothyronine (T3; Eales & Brown 1993). This reaction is catalyzed by iodothyronine deiodinases, a family of selenoenzymes, of which three types exist. Deiodinases type 1 (D1) and type 2 (D2) catalyze the outer ring deiodination (ORD, or 5′-deiodination) pathway that converts the prohormone T4 to T3. The ORD pathway is thus of paramount importance for thyroid bioactivity in fish. Liver, gills, and kidney in particular have a high ORD capacity, and can function as an extrathyroidal source of bioactive T3 (Arjona et al. 2010).
Thyroid hormones and the steroid hormone cortisol are thought to be implicated in osmoregulation in teleosts. Cortisol is a gluco- and mineralocorticoid in fish (Mommsen et al. 1999, McCormick 2001). In teleosts acclimating to hyperosmotic environments, cortisol regulates Na+, K+-ATPase activities which are prime determinants of osmoregulatory capacity (Mancera & McCormick 2007). On the other hand, the role of thyroid hormones in osmoregulation is generally much less well investigated (Klaren et al. 2007a).
Several studies have revealed effects of glucocorticoids on thyroid hormone metabolism. Cortisol stimulated the conversion, by ORD, of T4 to T3 in Salvelinus fontinalis liver in vitro (Vijayan et al. 1988), and enhanced liver D1 and D2 activities in Fundulus heteroclitus (Orozco et al. 1998). Negative results have also been reported, i.e. for the hepatic conversion of T4 to T3 in Oncorhynchus mykiss (Brown et al. 1991). I.v. administration of the synthetic glucocorticoid dexamethasone decreased liver D1 and D2 activities in Oreochromis niloticus but left ORD unaffected in gills, kidney, and brain (Walpita et al. 2007). It appears that the regulation of ORD by glucocorticoids in fish depends on the species and organ assessed, and a general picture cannot be constructed.
The Senegalese sole (Solea senegalensis) is a euryhaline species that is cultured at a commercial scale in Spain and Portugal (Dinis et al. 1999). Experimental results show that, following acclimation of S. senegalensis to different osmotic conditions, plasma cortisol and free T4 (fT4) concentrations change in a concerted manner, hinting at an interaction between the hypothalamus–pituitary–inter-renal (HPI) and the thyroidal axes (Arjona et al. 2008). Moreover, during the adjustment period that follows the transfer to a different ambient osmolarity, renal and hepatic ORD activities are elevated, and plasma cortisol concentrations are increased (Arjona et al. 2008).
Whether the bidirectional communication between HPI and thyroidal axes in S. senegalensis occurs at a central and/or peripheral level is as yet unknown. We here investigate the hypothesis that cortisol and T3 affect the ORD pathway, a key metabolic route for thyroid hormones, in Senegalese sole. In particular, liver and kidney, two organs with considerable ORD capacity, and gills, the main osmoregulatory organ, were investigated. We have also analyzed Na+, K+-ATPase activities in gills and kidney, and plasma concentrations of glucose and lactate, which can fuel osmoregulatory processes.
Materials and Methods
Fish and animal care
Juvenile Senegalese sole (S. senegalensis) with a body weight of 38±9 g and a total length of 150±19 mm (mean±s.d., n=90) were provided by Planta de Cultivos Marinos (C.A.S.E.M., Universidad de Cádiz, Puerto Real, Cádiz, Spain). Fish were acclimated for 14 days to full strength seawater (SW, with a nominal salinity of 38% and a nominal osmolality of 1037 mOsm/kg) in three 400 l tanks in a flow-through system until the start of experimentation. Each tank, with a bottom surface of 0.81 m2, contained 30 fish at an initial density of 1.4 kg/m2. Fish were kept under a photoperiod of 10 h light:14 h darkness and at a constant water temperature of 18 °C. Fish were fed daily with commercial dry pellets (Dibaq-Diproteg SA, Segovia, Spain) at a ration of 1% of the total body weight, and were fasted 24 h before sampling. This ration did not lead to detectable nitrogenous waste build-up in the tanks. The experimental procedures comply with the Guidelines of the European Union Council (86/609/EU) and of the University of Cádiz (Spain) for the use of animals in research. No mortality was observed during the experiment.
Experimental design
SW-acclimated fish were randomly divided into three groups of 27 animals. Fish were caught by netting, lightly anesthetized with 0.05% (v/v) 2-phenoxyethanol (Sigma Chemical Co., St Louis, MO, USA.), weighed, and injected intra-peritoneally with slow-release coconut oil (Sigma Chemical Co.) implants. The injection volume was 5 μl/g body weight, and coconut oil was warmed to its melting point (24 °C) prior to injection. Experimental groups received cortisol (hydrocortisone 21-hemisuccinate) in a dose of 50 μg/g body weight, or T3 (3,3′,5-triiodo-l-thyronine, ≥95% HPLC quality) in a dose of 2 μg/g body weight. The control group received only the coconut oil implant. Hormone preparations were obtained from Sigma Chemical Co. The use of coconut oil as a vehicle for i.p. hormonal implants has been shown to be an effective and practical method to raise plasma cortisol or thyroid hormone levels in different teleost species (Laiz-Carrión et al. 2003, Morgado et al. 2007). Nine fish did not receive any treatment and served as the pre-injection group. After injection, fish were returned to three designated 400 l tanks in a flow-through system with SW that was refreshed at a rate of 50 l/h. Per experimental group, nine animals were sampled on days 3, 7, and 14 post-implantation.
Sampling procedures
Fish were anesthetized in 0.1% (v/v) 2-phenoxyethanol and weighed. Mixed arterial and venous blood was collected from the caudal peduncle in 1 ml heparinized syringes. Plasma was obtained by centrifugation (3 min at 10 000 g), immediately frozen in liquid nitrogen, and stored at −80 °C until further analysis. After blood collection, fish were killed by spinal transection. From each fish, the first gill arch on the ocular side was excised as well as a small portion of the caudal zone of the kidney. Excess water was removed using absorbent paper, and tissues were frozen in liquid nitrogen and stored at −80 °C until analysis of Na+, K+-ATPase activities. Liver, the remaining kidney, and the rest of gill arches were removed, frozen in liquid nitrogen, and stored at −80 °C until analysis of ORD activities.
Plasma parameters
Six fish of each group were randomly selected for plasma analyses. Plasma osmolality was measured with a cryoscopic osmometer (Gonotec, Berlin, Germany). Plasma Na+, Cl−, K+, glucose, and lactate concentrations were measured using a Stat Profile pHOx plus analyser (Nova Biomedical, Waltham, MA, USA). Plasma cortisol was measured by RIA as described by Metz et al. (2005). Plasma-free T3 (fT3) and fT4 levels were determined by a commercially available solid-phase time-resolved fluoroimmunoassay (Wallac DELFIA from PerkinElmer Life and Analytical Sciences, Turku, Finland). Samples were diluted with charcoal-stripped plasma from SW-acclimated S. senegalensis prior to fT3 determinations: plasma from T3-injected fish was diluted 1:10 (v/v); plasma obtained from the other groups was diluted 1:3 (v/v). The DELFIA method has previously been validated for use with S. senegalensis plasma (Arjona et al. 2010).
Tissue preparations
To determine Na+, K+-ATPase activities, gills were thawed at room temperature. Branchial tissue was obtained by scraping the gill arch with a glass microscope slide and homogenized in 1 ml of ice-cold sucrose buffer (250 mM sucrose, 1 mM EDTA, and 100 mM trishydroxymethylaminomethane-HCl, pH 7.4) in a glass dounce homogenizer equipped with a tightly fitting Teflon pestle. Kidney fragments were homogenized in 0.5 ml sucrose buffer. To determine ORD activities, gills, liver, and the remaining kidney were homogenized in phosphate buffer (100 mM Na-phosphate, 2 mM EDTA, pH 7.0). Homogenates were stored at −80 °C until further analysis. Protein was measured with a commercial Coomassie Brilliant Blue reagent kit (Bio-Rad Laboratories) using BSA as a standard.
Gill and kidney Na+, K+-ATPase activities
The specific Na+- and K+-dependent, ouabain-sensitive ATPase activity was measured in triplicate in gill and kidney homogenates according to the method described by Flik et al. (1983). The method was adapted to 96-well microplate format by scaling down original volumes. Homogenates were diluted with ice-cold sucrose buffer in order to achieve maximally <15% ATP consumption during the incubation period. Triplicate 5 μl aliquots of diluted homogenates were incubated for 15 min at 37 °C. ATP consumption percentages were 11.8±0.4 for gills and 10.3±0.3 for kidney (n=60, mean±s.e.m.). The specific Na+, K+-ATPase activity is expressed as μmol inorganic phosphate per min per mg protein.
ORD activities
ORD activities were assayed following the method described by Klaren et al. (2005). We used reverse T3 (rT3, 3,3′,5′-triiodothyronine) and T4 as the preferred substrates for 5′-deiodinases (Mol et al. 1998). The requirements of the rT3-ORD and T4-ORD reactions for dithiothreitol (DTT) in S. senegalensis have been determined previously (Arjona et al. 2008, 2010). As DTT inhibited ORD, it was excluded from the assay media. ORD activities were assayed in duplicate using 20–70 μg homogenate protein at 37 °C in 200 μl of 100 mM phosphate buffer (pH 7.0) to which were added: 5 μM of rT3 or T4 (Sigma Chemical Co.), 105 c.p.m. [125I]rT3 or [125I] T4 (NEN Life Science Products, Inc., Boston, MA, USA), and 2 mM EDTA. Incubation period was set at 15 min for rT3-ORD and 12 min for T4-ORD. During this period, substrate consumption was <10%: rT3 consumption percentages were 6.7±0.10 for gills, 5.8±0.19 for kidney, and 2.4±0.07 for liver; T4 consumption percentages were 9.1±0.13 for gills, 6.4±0.18 for kidney, and 8.8±0.3 for liver (n=60, mean±s.e.m.). Measurements were corrected for non-enzymatic ORD activity that was determined in the absence of sample. Radiotracer was purified on a 10% (w/v) Sephadex LH-20 mini-column shortly before use, as described by Mol & Visser (1985). The specific ORD rate was expressed as fmoles rT3 or T4 deiodinated per minute per mg protein. Our calculations included a correction factor of 2 to take into account the random radiolabeling of the 3′- and 5′-positions of [125I]rT3 and [125I]T4.
Statistical analysis
Experimental groups (cortisol- or T3-injected animals) were compared with control groups (coconut oil-injected fish) on days 3, 7, and 14 post-implantation using Student's unpaired t-test or Mann–Whitney's non-parametric rank sum U test, where appropriate. Statistical significance was accepted at P<0.05. The same statistical analyses were applied to test the effect of the coconut oil vehicle, but here groups injected with coconut oil alone were compared with the pre-injection group.
Results
No mortality or pathologies or differences in growth rates were observed in any group throughout the experimental period. No significant differences were observed between untreated fish (pre-injection group, 0 days) and those fish implanted with coconut oil alone (controls) for any parameter assessed.
Treatment with cortisol and T3 implants effectively elevated plasma concentrations of cortisol and fT3 respectively (Fig. 1A and B). Specifically, cortisol implants elicited a transient increase in plasma cortisol levels, while T3 treatment resulted in sustained elevated fT3 levels throughout. Treatment with T3 also produced a ca. twofold increase in plasma cortisol levels on day 3 (Fig. 1A). Both cortisol and T3 treatments decreased plasma fT4 levels (Fig. 1C), where the effect of T3 was more pronounced than that of cortisol.
Plasma Na+, Cl−, and K+ concentrations in cortisol-treated animals decreased significantly on day 3 compared with the control group and returned to basal values from day 7 onwards (Table 1). Plasma osmolality did not vary significantly with the different experimental treatments. Cortisol treatment increased plasma glucose levels transiently on day 3 (P<0.001) but did not change plasma lactate values. Treatment with T3 did not produce statistically significant changes in plasma electrolyte, glucose, and lactate concentrations (Table 1).
Plasma osmoregulatory and metabolic parameters in Solea senegalensis specimens injected with coconut oil alone (control) or containing cortisol (50 μg/g body weight) or T3 (2 μg/g body weight). Values are expressed as means±s.e.m. (n=5–6)
Days post-injection | Control (oil) | Cortisol | T3 | |
---|---|---|---|---|
Parameters assessed | ||||
Osmolality (mOsm/kg) | 3 | 346±4 | 335±3 | 340±2 |
7 | 350±5 | 342±4 | 345±3 | |
14 | 351±5 | 341±1 | 342±4 | |
Na+ (mM) | 3 | 154±4 | 137±2† | 147±2 |
7 | 158±4 | 154±2 | 153±1 | |
14 | 158±1 | 157±4 | 157±2 | |
Cl− (mM) | 3 | 150±4 | 139±1* | 141±4 |
7 | 152±5 | 149±3 | 149±4 | |
14 | 152±4 | 150±7 | 150±4 | |
K+ (mM) | 3 | 4.9±0.27 | 3.8±0.14† | 4.3±0.18 |
7 | 3.9±0.27 | 4.4±0.22 | 4.8±0.4 | |
14 | 4.2±0.16 | 4.4±0.4 | 4.5±0.28 | |
Glucose (mM) | 3 | 3.7±0.15 | 5.4±0.12‡ | 3.5±0.17 |
7 | 3.9±0.3 | 4.4±0.14 | 3.5±0.18 | |
14 | 4.0±0.27 | 4.5±0.14 | 3.7±0.14 | |
Lactate (mM) | 3 | 3.1±0.08 | 3.3±0.4 | 3.3±0.20 |
7 | 3.5±0.7 | 3.2±0.20 | 2.9±0.16 | |
14 | 3.2±0.20 | 3.0±0.6 | 3.2±0.27 |
Symbols indicate significant differences when comparing cortisol- or T3-injected groups with control groups (injected with coconut oil alone) on days 3, 7, and 14 post-implantation (*P<0.05; †P<0.01; ‡P<0.001).
Gill and kidney Na+, K+-ATPase activities were stable in the control fish, and reached a maximum on day 3 in the cortisol-treated fish (Fig. 2A and B), which coincided with the peak in plasma cortisol levels (Fig. 1A). Enzyme activities returned to control levels on day 7. Treatment with T3 did not produce significant changes in Na+, K+-ATPase activities, except at the end of the experimental period (day 14) where lowered Na+, K+-ATPase activities were observed in gills and kidney (P<0.05).
The effects of hormone treatments on kidney, liver, and gill rT3-ORD activities were similar to the respective T4-ORD activities in these organs (Figs 3 and 4). In fish treated with cortisol, renal and hepatic ORD activities were enhanced two- to three-fold at day 3 following injection, and decreased to control levels afterwards (Figs 3A and B, 4A and B). In T3-treated fish, we only observed a significantly decreased renal T4-ORD activity (P<0.01) on day 7 (Fig. 4A). A transient and slight decrease in branchial T4-ORD was observed in cortisol-injected fish (Fig. 4C).
Discussion
Our results demonstrate that cortisol stimulates the ORD pathway, an important peripheral source of T3, in S. senegalensis liver and kidney. This observation supports our hypothesis, and indicates that the activity of thyroid hormones can be regulated not only at a central level in the brain, but also peripherally in extrathyroidal tissues. No effect of cortisol was seen on ORD activity in the gills. The constitutive branchial ORD capacity is comparable in magnitude to that in liver and kidney. It could well be that the control of intracellular thyroid hormone concentrations in gills occurs by means of up- or down-regulation of the activity of T3 membrane transporters rather than de novo production of T3. The increased ORD activities in kidney and liver following cortisol treatment in S. senegalensis (Figs 3 and 4) point to these organs as important determinants of thyroid bioactivity during a stress response when plasma cortisol concentrations increase (Wendelaar Bonga 1997, Barton 2002). It also suggests that at least some of cortisol's actions, i.e. increased renal Na+, K+-ATPase activity and liver gluconeogenesis (as deduced from the increased plasma glucose concentrations), are mediated via T3. Treatment with T3 failed to produce significant changes in renal Na+, K+-ATPase activity or plasma glucose levels, which is consistent with the suggestion that T3 is a mediator of cortisol actions, without osmoregulatory or gluconeogenic activity per se.
A consequence of increased ORD activities is an increase in intracellular T3 availability and, hence, an increased transcription of its genomic targets. The notion of intracellular actions of locally produced T3 also helps explain why plasma fT3 concentrations were unaltered by cortisol injection. Indeed, osmotic challenges changed peripheral ORD activities in S. senegalensis and Sparus auratus but did not result in altered plasma fT3 levels (Klaren et al. 2007b, Arjona et al. 2008). In S. fontinalis, circulating T4 and T3 remained unaltered after a cortisol treatment that had increased hepatic T4-ORD activities (Vijayan et al. 1988). A plausible explanation is that traffic of T3 out of cells is regulated by the activity of membrane transporters, preventing T3 from entering plasma.
The approximately eightfold increase in plasma cortisol levels following treatment with implants is within the physiological range, as it compares very well to the stressor-induced 4- to 40-fold increases observed in S. senegalensis and other teleost species (Woodward & Strange 1987, Goss & Wood 1988, van den Burg et al. 2005, Arjona et al. 2008). There are very few reports on the use of slow-release thyroid hormone implants in fishes. Morgado et al. (2007) used coconut oil to treat gilthead seabream (S. auratus) with a dose of 1 μg T3/g body weight, resulting in a 25-fold increased T3 plasma concentration. We found a similar 27-fold increase using a dose of 2 μg T3/g. There are large differences between species in how plasma thyroid hormone levels respond to a stressor. Zebrafish (Danio rerio) can increase its plasma T3 levels ca. 20-fold when challenged, whereas European flounder (Platichthys flesus) is refractory to a similar stimulus (Kuiper et al. 2008). We conclude that the experimentally increased plasma T3 levels reflect an upper limit of a physiological range in S. senegalensis.
Cortisol affects the metabolism of carbohydrates, proteins, and lipids in fish (Mommsen et al. 1999). In our study, cortisol produced a glucocorticoid effect in S. senegalensis, evoking a mild hyperglycemia, probably by stimulation of gluconeogenic routes in the liver (Mommsen et al. 1999). In addition to its glucocorticoid actions, the role of cortisol as a mineralocorticoid is obvious in S. senegalensis, as cortisol-injected animals showed lowered plasma Na+ and Cl− levels and increased Na+, K+-ATPase activities in gills and kidney 3 days after injection. These results agree with the classical osmoregulatory role of cortisol in teleosts (Seidelin et al. 1999, Laiz-Carrión et al. 2003, Sherwani & Parwez 2008).
A drop in plasma fT4 levels can be caused by a decrease in thyroidal T4 production and secretion and/or changes in peripheral metabolism (Blanton & Specker 2007), a picture similar to that in mammals (Visser & Fliers 2007). The decrease in plasma fT4 levels in cortisol-injected S. senegalensis (Fig. 1) coincided with increased ORD activities in liver and kidney and, hence, an increased T4-to-T3 conversion. However, we cannot exclude a feedback mechanism in which cortisol reduces the activity of hypothalamic corticotropin-releasing hormone (CRH), urotensin-I (UI), and thyrotropin-releasing hormone (TRH), factors with both corticotropic and thyrotropic activities in a number of fish species (reviewed by Bernier et al. (2009)). Indeed, in salmonids and eels, CRH has thyrotropic activity in vitro (Larsen et al. 1998, Rousseau et al. 1999), and, in carp, the interaction between thyroid hormones and the HPI axis is illustrated by the up-regulation of hypothalamic crh-binding protein gene expression after T4 treatment (Geven et al. 2006). It could well be that hypothalamic factors of the HPI axis, namely CRH, UI, TRH, are altered after cortisol treatment in S. senegalensis and then, jointly with the hepatic and renal ORD pathway, have affected plasma fT4 concentrations.
Treatment with T3 reduced plasma fT4 levels ca. fourfold. Besides a 47% reduction measured in the kidney 7 days after injection, no major changes in peripheral ORD activities were observed. It is very likely that the reduction in plasma fT4 levels is the result of a reduced activity of the thyroid gland caused by a negative feedback of T3 on pituitary thyrotropes. As in mammals, T3 represses tsh β-subunit gene expression in fish pituitaries in vitro in a classical negative feedback system (Pradet-Balade et al. 1997, Schmitz et al. 1998, Sohn et al. 1999). Moreover, thyroid hormone economy is complicated by the presence of specific thyroid hormone-binding proteins that facilitate vectorial plasma transport (reviewed by Klaren et al. (2007a)). Morgado et al. (2007) have shown that treatment of S. auratus with T3 increases the plasma concentration of the thyroid hormone-binding protein transthyretin ca. threefold. The concentration of free thyroid hormone levels in the plasma is a complex balance of hormonogenesis in the thyroid gland and the concentration of circulating binding proteins.
In S. senegalensis, T4- and rT3-ORD activities were mainly unaltered after T3 treatment (Fig. 4). In some teleost species, deiodinase activity seems to be regulated by its iodothyronine substrate (Orozco et al. 1997, 2003, Sanders et al. 1997, Valverde-R et al. 1997). The ORD assays as performed in this study do not allow the discrimination between the 5′-deiodinases D1 and D2. Using rT3, we have tested an iodothyronine that is the preferred substrate for many vertebrate D1 enzymes. T4 is the preferred substrate for many deiodinases type 2, but, more importantly, is also the physiologically relevant iodothyronine since it is the endogenous prohormone that needs to be activated by ORD. Our results show that peripheral ORD is not responsive to in vivo T3 treatment. Instead, cortisol appears to be a key regulator of peripheral ORD activity and, hence, extrathyroidal T3 production in S. senegalensis.
This work confirms and extends previous results (Arjona et al. 2008), and provides evidence for the involvement of cortisol in the regulation of the ORD pathway in S. senegalensis in an organ-specific manner as well as in physiological processes related to the hydromineral balance.
Declaration of interest
The authors declare that there is no conflict of interest that would prejudice the impartiality of the research reported.
Funding
This work was supported by the Ministerio de Ciencia e Innovación and FEDER (Spain, grant number AGL2007-61211/ACU); the Consejería de Innovación, Ciencia y Empresa (Junta de Andalucía, Spain, grant number PO7-RNM-02843); and by a pre-doctoral grant to Francisco Jesús Arjona within the program Formación de Profesorado Universitario from the Ministerio de Ciencia e Innovación (Spain, grant number AP-2004-6829).
Acknowledgements
The authors are very grateful to Planta de Cultivos Marinos (C.A.S.E.M., Universidad de Cádiz, Puerto Real, Cádiz, Spain) for providing experimental fish.
References
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