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Negative feedback regulation of thyrotropin subunits and pituitary deiodinases in red drum, Sciaenops ocellatus
General and Comparative Endocrinology 240 (2017) 19–26
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Research paper
Negative feedback regulation of thyrotropin subunits and pituitary deiodinases in red drum, Sciaenops ocellatus R.A. Jones ⇑, W.B. Cohn, A.A. Wilkes, D.S. MacKenzie Department of Biology, Texas A&M University, 3258 TAMUS, College Station, TX 77843-3258, USA
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Article history: Received 20 March 2016 Revised 30 August 2016 Accepted 1 September 2016 Available online 3 September 2016 Keywords: Thyrotropin Deiodinase Fish Thyroid hormone Negative feedback
a b s t r a c t Thyroxine (T4) undergoes dynamic daily cycles in the perciform fish the red drum, Sciaenops ocellatus, that are inversely timed to cycles of thyrotropin (TSH) subunit mRNA expression in the pituitary gland. We have proposed that these daily cycles are regulated by negative feedback of circulating T4 on expression of pituitary thyroid hormone deiodinase type 3 (Dio3), such that elevated circulating T4 results in diminished pituitary thyroid hormone catabolism and consequent increased negative feedback on expression of TSH subunits during the day. To determine whether thyroid hormones function to modulate expression of pituitary deiodinase enzymes we developed an immersion technique to administer physiological doses of T3 and T4 in vivo. Immersion in T4 or T3 significantly inhibited the mRNA expression of the TSH a and b subunits from 4 to 66 h of immersion. Pituitary Dio3 expression was significantly diminished by T3 and T4 at 22 h. These results indicate that both T4 and T3 are capable of negative feedback regulation of TSH subunit expression in red drum at physiological concentrations and on a time scale consistent with the T4 daily cycle. Furthermore, thyroid hormones negatively regulate Dio3 expression in the pituitary in a manner suggesting that negative thyroxine feedback on Dio3 promotes the release of TSH subunits from TH inhibition and may be an important mechanism for generating daily thyroid hormone cycles. These results highlight a potentially important role for D3 in mediating thyroid hormone feedback on TSH expression, not previously described in other species. Published by Elsevier Inc.
1. Introduction Thyroid hormone negative feedback to regulate the synthesis and secretion of thyrotropin (TSH) is an established mechanism for maintenance of thyroid homeostasis in mammals (Gereben et al., 2008). Studies in several mammalian species have indicated that deiodination of thyroxine (T4) to 3,5,30 -triiodothyronine (T3) by Type 2 iodothyronine deiodinase (D2, Dio2) in the pituitary gland is an essential component of this feedback mechanism (Fonseca et al., 2013; Gereben et al., 2008). In contrast, the relative importance of TSH as a central regulator of thyroid homeostasis has been questioned in nonmammalian species (Eales and Brown, 1993). Studies which have examined regulation of TSH expression in teleost fish have supported a role for thyroid Abbreviations: TSH, thyrotropin; TH, thyroid hormones; GSUa, glycoprotein hormone subunit a; D2, type 2 iodothyronine deiodinase; D3, type 3 iodothyronine deiodinase; T4, thyroxine 3,5,30 50 -tetraiodothyronine; T3, 3,5,30 -triiodothyronine. ⇑ Corresponding author. E-mail addresses: [email protected] (R.A. Jones), [email protected] (W.B. Cohn), [email protected] (A.A. Wilkes), [email protected] (D.S. MacKenzie). http://dx.doi.org/10.1016/j.ygcen.2016.09.003 0016-6480/Published by Elsevier Inc.
hormone in negative feedback (MacKenzie et al., 2009) while leaving the question of the importance of deiodination unresolved. Fish feedback studies have generally utilized prolonged hormone (primarily T3) or goitrogenic drug administration and have provided evidence both for a mammalian-like feedback requiring deiodination of T4 or direct feedback of T3 (Campinho et al., 2015; Cohn et al., 2010; Leiner and MacKenzie, 2003; Lema et al., 2009; MacKenzie et al., 2009), which often circulates in fish at higher concentration than in mammals. Eales (2006) has argued that because of pharmacological doses and indirect effects of circulating thyroid hormones through actions on peripheral deiodinases, circulating T3 may not function as a primary regulator of TSH negative feedback. The issue of differential sensitivity of TSH to T4 and T3 feedback in vivo has not subsequently been addressed and is an important consideration in establishing the role of central mechanisms in regulating fish thyroid function. Evidence in fish of a robust daily rhythm of circulating T4, hypothesized to be driven by a central oscillator working through TSH, provides further evidence that TSH may indeed be dynamically regulated by thyroid hormone negative feedback (Leiner and MacKenzie, 2001, 2003). In the perciform teleost, the red drum
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(Sciaenops ocellatus), a robust daily rhythm of circulating T4 is inversely timed to the pituitary expression of mRNAs for both TSH subunits, TSH beta (TSHb) and glycoprotein subunit alpha (GSUa), suggesting that negative feedback of T4 acting upon TSH drives this cycle (Jones et al., 2013). In this same study Dio2 was expressed in the pituitary at the peak of the T4 cycle, but Dio2 expression did not predictably and consistently vary with TSH subunit expression. Interestingly, however, type 3 iodothyronine deiodinase (D3, Dio3) mRNA expression was robustly cyclic with a waveform inversely related to circulating T4, suggesting that TH negative feedback in this species may be mediated in part through regulation of intra-pituitary deactivation of THs (Jones et al., 2013). Whereas pituitary D3 expression has been identified in some mammalian species (Fliers et al., 2006) it has not previously been identified as an important player in the TSH negative feedback mechanism. We have proposed that if expression of D3 in red drum is regulated by circulating thyroid hormones it could serve as a mechanism to rapidly modulate thyroid hormone feedback on TSH through T3 and T4 degradation (Jones et al., 2013). The objective of the current study was to determine if physiological elevations of circulating T3 and T4 do indeed regulate the expression of deiodinases in the pituitary consistent with a role in negative feedback on TSH expression. In particular, because of our previous observation of an inverse temporal relationship between circulating T4 and pituitary Dio3 expression (Jones et al., 2013), we wish to determine whether physiological elevations of circulating T4 are capable of down-regulating Dio3 expression, supporting an important role for D3 in the negative feedback process. Leiner and MacKenzie (2003) employed a T3 immersion technique in red drum that, by elevating T3 over a brief period of hours, provided evidence for negative feedback of physiological doses of T3 by significantly depressing the daily peak in circulating T4 in red drum. We utilized a similar experimental design to noninvasively administer not only T3 but also T4 to red drum to address the question of the relative importance of these hormones in regulating pituitary deiodinase expression. By developing a non-invasive technique for administering thyroid hormones that results in rapid, reversible, physiological increases in blood hormone levels we have been able to characterize differential negative feedback capabilities of T3 vs. T4 on pituitary mRNA expression of TSHb, GSUa, Dio2, and Dio3 during constant TH immersion. Our results support an important role for pituitary D3 in the central regulation of a feedback oscillator system that drives daily circulating thyroxine cycles. 2. Materials and methods 2.1. Animals The Coastal Conservation Association Marine Development Center in Corpus Christi and the Texas Parks and Wildlife Department’s hatchery at Sea Center Texas in Lake Jackson provided red drum fingerlings weighing approximately 0.5 g. Fingerlings were held in the Department of Biology’s BioAquatics Facility at Texas A&M University until they reached a weight of at least 20 g before being used for experiments. Sex in these juvenile fish cannot be readily determined. Animals were housed in a 4000 L recirculating system at 4 ppt salinity (SuperSalt, Fritz Industries, Mesquite, Texas), 26 °C, 12L:12D photoperiod. Fish were fed Aquamax (PMI Nutrition, Brentwood, MO) to apparent satiation once daily. TH immersion experiments were performed in 80 L tanks connected to the 4000 L recirculating system. Fish were euthanized in MS222 (Finquel, Argent Laboratories, Redmond, Washington) prior to obtaining blood and pituitary samples. All procedures were approved by the Texas A&M University Institutional Animal Care and Use Committee.
2.2. TH immersion experiments The thyroid hormone immersion technique of Leiner and MacKenzie (2003) was modified to develop our T3 and T4 immersion protocol. A series of pilot experiments was undertaken to establish ambient water doses of T3 and T4 that resulted in increased circulating thyroid hormone levels in immersed fish within physiological ranges. Thyroid hormones (3,5,30 -triiodothyro nine,T3, and 3,5,30 ,50 -tetraiodothyronine, both obtained from Sigma Chemical Co., St. Louis) were dissolved in 0.1 N NaOH to make stock solutions. Target concentrations of 100 ng/ml T3 (3 ml of a 2 mg/ml T3 stock solution in 60 L tank water) and 200 ng/ml T4 (3 ml of a 4 mg/ml T4 stock solution in 60 L tank water) were found to result in circulating thyroid hormone levels between 15 and 25 ng/ml, equivalent to levels observed during peak thyroid hormone cycles in captive red drum (Leiner et al., 2000). To avoid overcrowding of fish in static tanks at the initiation of long duration immersion experiments, three separate immersion experiments were performed. Five to seven fish per tank were immersed in Experiments 1 (mean weight ± s.e. 22.5 ± 0.8 g), 2 (24.3 ± 1.0 g), and 3 (29.9 ± 1.9 g), sampled at 4.5 h, 22 and 40 h, or 66 h, respectively. The 4.5 h time point was chosen as within the expected time for physiological negative feedback in relation to daily cycles of THs in red drum. The 22, 40, and 66 h time points were chosen as near to 24 h increments as room lighting and personnel schedules would allow and to replicate the time frames for feedback used in studies for other teleost species (MacKenzie et al., 2009). No tank was sampled more than once so that the N for all sampling times and treatments (control, T3, or T4), comprised 5– 7 animals. All three experiments were initiated at 3 h after lights on to control for daily cycles in circulating thyroid hormones. Each immersion tank contained a conditioned corner filter allowing fish to be fed during immersion. Data from these three experiments were combined and graphed together to facilitate visual comparison of changes in hormone levels and gene expression over a 66 h period of immersion. On the day before an immersion experiment fish were moved to flow-through 80 L tanks, all connected to the same recirculating system as their stock tank. At the beginning of the experiment flow to these tanks was turned off, their volume adjusted to 60 L, and thyroid hormone added. Hormone immersion duration ranged from 4.5 to 66 h before flow was returned to the tanks in the three experiments. At the conclusion of each experiment as much water as possible (approximately 90%) was removed from TH treated tanks, discarded from the recirculating system, and replaced with hormone-free system water. Remaining water draining from these tanks back into the recirculating system was passed through activated carbon to remove any residual immersion hormone from the system. 2.3. Blood and tissue analysis Blood was collected from the caudal vasculature at the end of immersion and plasma was separated and frozen at 80 °C. Plasma samples were analyzed for thyroid hormones using Coat-A-Count Total T4 or T3 kits (Siemens, Los Angeles, CA) following the manufacturer’s protocol as described by Cohn et al. (2010). Following terminal anesthesia, tissues for RNA extraction were rapidly removed by dissection and transferred to ZR RNA Microprep kit reagent (Zymoresearch, Irvine, CA). All samples were frozen at 80 °C until RNA extraction according to the manufacturers’ protocols. 2.4. qPCR qPCR primers, probes and techniques have been previously described (Jones et al., 2013). PCR efficiency was above 90% for
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all assays and R2 values were greater than 0.9 for all dilution standard curves. Reference gene (18S) mRNA levels were not detectably different among treatments.
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The 2TDDC method was used to calculate qPCR results (Livak and Schmittgen, 2001). Relative values correspond to the mRNA expression of the gene-of-interest/18S Ribosomal subunit. The average control relative value corresponds to the mean of all relative values from control animals within the same experiment independently determined for each gene-of-interest. All mRNA expression graphs were displayed as percent of the average control relative value to eliminate any bias toward one experimental time point over another. Error bars represent standard error of the mean. SPSS software was used for Kruskal-Wallis ANOVA to compare treatments at each time point followed by Mann-Whitney tests at each sampling time point to compare T4 and T3 groups to control to determine significance using Bonferroni correction. A P value of less than 0.05 was considered significant.
or T4 over time-matched controls between 4.5 and 40 h, but only T3 was elevated at the end of the 66 h immersion (Fig. 1). T4 immersion transiently elevated blood T3, but by 40 h this had reversed (Fig. 1A). T3 immersion significantly depressed blood T4 at every point other than the initial 4.5 h sampling (Fig. 1B). The concentration of THs in tank water steadily decreased over time during the static immersion to final concentrations at 66 h of 40 ng/ml T4 and 0 ng/ml T3. Immersion in T3 or T4 significantly inhibited the expression of both pituitary TSHb (Fig. 2A) and GSUa (Fig 2B) compared to time-matched controls at three out of four time points. In contrast, pituitary Dio2 expression was different from control at only the 22 h time point but once the Bonferroni correction was applied this difference was no longer significant (Fig. 3A). T3 and T4 immersion significantly diminished the expression of pituitary Dio3 with a maximal T4 inhibition compared to control at 22 h (Fig. 3B). At 22 and 40 h, T3 immersion significantly inhibited the hepatic expression of Dio2 and stimulated the hepatic expression of Dio3 compared to time-matched controls. In contrast, T4 immersion did not alter the expression of Dio2 or Dio3 in the liver (Fig. 4).
3. Results
4. Discussion
An immersion dose of T4 double that of T3 was required to elevate circulating T4 above control values. Blood from fish immersed in these T3 or T4 concentrations showed significant elevations of T3
In this study we noninvasively administered thyroid hormones to red drum to assess hepatic and pituitary mRNA responses within a time frame reflecting endogenous thyroid hormone cycles. A
2.5. Data analysis
Fig. 1. (A) T3 and (B) T4 response in red drum to up to 66 h of thyroid hormone immersion. Blood samples were taken from fish in constant static immersion with either T3 (100 ng/ml), T4 (200 ng/ml), or NaOH vehicle. Experiment 1 (E1), experiment 2 (E2), and experiment 3 (E3) are graphed together on a single axis. Significant differences from same time control are denoted by asterisks.
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Fig. 2. Red drum pituitary (A) TSHb and (B) GSUa mRNA expression resulting from thyroid hormone immersion in either T3 (100 ng/ml), T4 (200 ng/ml), or NaOH vehicle. Experiment 1 (E1), experiment 2 (E2), and experiment 3 (E3) are graphed together on a single axis. Significant differences from same time control are denoted by asterisks.
variety of techniques have been utilized to administer thyroid hormones to fish but most either employ pharmacological elevations or involve handling stress and as such complicate interpretation of results (Eales, 2006). Hormone feeding has been used extensively in rainbow trout, but this method fails to account for differential dosing due to variable food consumption among animals (Eales, 2006). Additionally, feeding experiments have traditionally focused on T3 feeding because T4 is not absorbed across the gut of fish (Eales, 2006; Larsen et al., 1997; Lema et al., 2009; PradetBalade et al., 1999). Immersion on the other hand is non-invasive and insures that all fish receive equivalent hormone exposure (Eales, 2006). Immersion has been used successfully to administer physiological doses of thyroid hormones in salmonids (Bres et al., 2006; Eales, 2006). Leiner and MacKenzie (2003) developed an immersion technique for administering T3 which resulted in a reduction in circulating T4, presumably through negative feedback at the pituitary, in red drum. This immersion protocol did not include T4 as a treatment however. The present study confirms that circulating T3 does indeed inhibit pituitary TSH subunit expression at physiological concentrations. Whereas T3 has been found to inhibit TSH expression in a number of teleost studies (MacKenzie et al., 2009), it is necessary to demonstrate a similar action of T4 to support our hypothesis that daily cycles of circulating T4 in red drum are driven by negative T4 feedback on TSH.
In teleost fish, in vitro inhibition of TSH expression by T3 or T4 has been observed, signifying that feedback of both hormones at the pituitary is physiologically possible (Chowdhury et al., 2004; Schmitz et al., 1998). However, the in vivo physiological role of inhibitory thyroid hormone feedback on TSH expression in fish has not been consistently demonstrated (Eales, 2006). In vivo experiments in fish have demonstrated T4 or T3 suppression of TSH expression, but usually employing pharmacological doses or prolonged time courses of administration of 2 weeks or more (MacKenzie et al., 2009). In red drum, we found that the daily rhythm of circulating T4 is mirrored by cyclic expression of TSH subunits (Jones et al., 2013), suggesting that this TSH cycle is regulated by negative T4 feedback at the pituitary. The red drum TSH expression cycle was 6–12 h out of phase with circulating T4 further suggesting that this negative feedback was occurring over a time scale of hours. The thyroid hormone administration results in the present study provide additional support for this negative feedback model. Within 4 h of the initiation of immersion T4 had significantly inhibited the expression of both TSH subunits, indicating that T4 within the range of circulating levels encountered during the daily cycle can feed back rapidly enough to inhibit TSH production and thus drive the TSH cycle in red drum. This inhibition was observed during both T3 and T4 immersion and was sustained for up to 66 h, providing convincing evidence for a
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Fig. 3. Red drum pituitary (A) Dio2 and (B) Dio3 mRNA expression resulting from thyroid hormone immersion in either T3 (100 ng/ml), T4 (200 ng/ml), or NaOH vehicle. Experiment 1 (E1), experiment 2 (E2), and experiment 3 (E3) are graphed together on a single axis. Significant differences from same time control are denoted by asterisks.
powerful negative feedback action of both thyroid hormones at physiological concentrations. Pilot studies for the immersion experiments indicated that red drum take up T3 more readily from water than T4, even in the face of progressively declining water thyroid hormone content. Consequently, the dose of T4 used to achieve comparable levels of circulating T4 in T4-immersed fish was twice as high as the T3 dose, which was equivalent to the ambient concentration employed for physiological supplementation in salmonids (Bres et al., 2006). Currently, the mechanism through which ambient thyroid hormone enters fish circulation is unknown. However, the monocarboxylate transporter MCT8, known to be a thyroid hormone transporter in mammals (Friesema et al., 2003), is expressed in the fish gill (Arjona et al., 2011; Muzzio et al., 2014) and in zebrafish preferentially transports THs in a temperature-dependent fashion, with T3 transport maximal at 26 °C vs. T4 at 37 °C. In mammals, the MCT8 transporter preferentially transports T3, while the Oatp1c1 transporter preferentially transports T4 (Little, 2016; Visser et al., 2011), and the low expression of Oatp1c1 in the fish gill (Muzzio et al., 2014) may explain the differential uptake of T4 and T3. If MCT8 and Oatp1c1 in red drum are expressed in the gills and exhibit similar thyroid hormone transporting properties at our water temperature of 25–29 °C, a preference of T3 over T4 uptake would be expected, as was observed.
Experiments 1, 2, and 3 addressed the time course of responses to T3 and T4 immersion. Circulating T3 initially increased within 4 h of T3 immersion, but its decline resulted in residual levels still significantly above control by 66 h. As previously observed (Leiner and MacKenzie, 2003), T4 was significantly diminished in T3 immersed fish, consistent with a negative feedback action of T3 at the pituitary thyrotroph. Samples from T3 immersed fish taken up to 66 h all showed a reduction in T4 suggesting that T3 has a sensitive and sustained negative feedback effect on TSH. In contrast, circulating T4 significantly also increased within 4 h of initiation of T4 immersion but eventually declined to control levels by 66 h. Interestingly, T4 immersion resulted in a transient (at 4 h only) increase in circulating T3. This increase in circulating T3 in response to brief T4 treatment has not been previously reported in fish, but it is consistent with the initial elevated T4 entering peripheral targets and being converted to T3 by D2. Indeed, the bulk of circulating T3 in fish is thought to be generated by T4 deiodination (Eales and Brown, 1993), particularly at the liver (Orozco and Valverde-R, 2005). That this transient increase in circulating T3 is not sustained suggests a hormone-induced adjustment in peripheral deiodination over time. Hepatic regulation of a blood T3 set-point has been proposed to be an important component of the peripheral regulation of teleost fish thyroid function (Eales and Brown, 1993). Generally, both T3 and T4 challenge rapidly
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Fig. 4. Red drum hepatic (A) Dio2 and (B) Dio3 mRNA expression resulting from thyroid hormone immersion in either T3 (100 ng/ml), T4 (200 ng/ml), or NaOH vehicle. Experiment 1 (E1), experiment 2 (E2), and experiment 3 (E3) are graphed together on a single axis. Significant differences from same time control are denoted by asterisks.
suppress outer ring deiodination and induce inner ring deiodination in multiple fish tissues (Eales, 2006; Garcı´a-G et al., 2004; Orozco et al., 2012), although the simultaneous regulation of inner and outer ring deiodination has only been examined in a few species. Bres et al. (2006) observed in rainbow trout that hepatic Dio2 expression was down-regulated whereas Dio3 expression was upregulated by T3 but neither Dio2 nor Dio3 expression were influenced by T4. We also observed a similar differential hepatic response of Dio2 and Dio3 to T3 vs. T4 immersion, serving as confirmation that our qPCR assays can detect expected physiological changes for these hepatic deiodinase gene products. As Dio3 expression in the liver of fish has been found to be activated by T3, the generation of this T3 should progressively decline over time as hepatic expression of D3 increases, as we observed. This delayed activation of D3 could also contribute to the steady decrease of T3 in T4 treated red drum, where by 40 h circulating T3 had significantly declined below control levels. Whereas D2 and D3 are known to be regulated by thyroid hormones in fish liver, the TH regulation of deiodinase expression in the fish pituitary has, until now, not been studied. In mammals, T3 for negative feedback on TSH expression is generated by D2 deiodination of T4 (Bianco et al., 2002; Nikrodhanond et al., 2006). In the red drum we found that similarly to liver, both T3 and T4 down-regulated the expression of pituitary Dio2 at 22 h.
However, in mice (Christoffolete et al., 2006), this reduction in enzyme expression may not necessarily result in levels of intrapituitary T3 production insufficient for feedback. Conversely, mouse pituitary Dio3 is up-regulated in the face of high circulating THs, likely preventing excessive inhibition of TSH secretion by degrading T3 (Barca-Mayo et al., 2011). Interestingly, in the red drum pituitary we found the opposite result, where elevated TH significantly inhibited the expression of Dio3. Observations in fish that T3 differentially regulates Dio3 in the brain, liver, and gill (Johnson and Lema, 2011; Orozco and Valverde-R, 2005) and in mice that Dio2 is down-regulated by T3 in the brain (Burmeister et al., 1997) while up-regulated by T3 in brown adipose tissue (Mena et al., 2010) suggests that THs regulate deiodinase expression in a tissue-specific manner. Although no TH response element has been discovered in the human Dio3 gene promoter, T3 applied to GH3 cells cotransfected with a human Dio3 gene promoter construct and the thyroid hormone receptor (TRa but not TRb) stimulated luciferase activity from this Dio3 promoter construct, leading Barca-Mayo et al. (2011) to suggest that differential expression of the TRs may mediate tissue-specific stimulatory vs. inhibitory regulation of Dio3 in response to TH. Alternatively, T3 has been suggested to negatively regulate TSH expression in the pituitary through direct activation of co-repressors (Chiamolera and Wondisford, 2009; Shupnik, 2000). A similar mechanism of
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Fig. 5. Model for a feedback oscillator generating daily cycles of circulating T4 in red drum: 1) At the conclusion of the scotophase, minimal circulating T4 and elevated pituitary D3 activity result in diminished pituitary T3 content and consequently minimal negative feedback on TSH subunit and D3 expression. 2) As a result, TSH secretion increases within 6–12 h, stimulating progressively increasing circulating T4. 3) As T4 rises during the photophase D2 supplies T3 available for negative feedback on the expression of D3, preventing destruction of the TH feedback signal. Thus, as circulating T4 rises, negative feedback of T3 formed through pituitary D2 results in a decreased expression of TSH subunits. 4) This inhibition in TSH subunit expression would be followed 6–12 h later by decreased TSH secretion and consequent decreasing circulating T4 during the scotophase. The cycle would then begin again as the expression of D3 increases due to diminished inhibition during the scotophase. The resulting increased production of reverse T3 and T2 at the expense of T3 in thyrotrophs also diminishes inhibition of TSH subunit expression. This diminished feedback reactivates increased expression of TSH subunits, followed once again by increasing TSH secretion driving increased circulating T4 throughout the next photophase.
co-activator or co-repressor direct regulation could be involved in TH mediated expression of deiodinases allowing for inhibition of expression in tissues such as the pituitary and stimulation in tissues such as the liver. In red drum cyclic expression of TSH subunits was closely synchronized with pituitary Dio3 expression, suggesting negative feedback mediated through TH catabolism by D3 may play a role in TSH regulation (Jones et al., 2013). Our finding that rapid, physiological alterations in circulating thyroid hormone levels can elicit rapid responses in pituitary thyroid-related transcript expression, in particular thyroid hormone-induced depression of pituitary Dio3 and TSH subunit expression, further suggests that D3 plays an important role in negative feedback of T4, and that the daily cycle of circulating T4 in red drum is the product of a peripheralpituitary interaction that establishes a self-sustaining endocrine oscillator. We propose a model for this oscillator in Fig. 5. Intriguingly, this oscillator as proposed could function without stimulatory input from a hypothalamic thyrotropin-releasing factor, consistent with the observation that hypothalamic regulation of TSH secretion in teleost fish may be predominantly under inhibitory control, as contrasted with the stimulatory control observed in tetrapod vertebrates (MacKenzie et al., 2009). Although our studies have provided a compelling argument for inhibition of pituitary TSH secretion by circulating thyroid hormones, confirmation of this model requires extension of these studies beyond gene expression to include true measures of TSH secretion and deiodinase enzyme activity over throughout the 24 h cycle. An important extension of these studies would be to examine the relationship among mRNA expression of TSH subunits, TSH secretion, and subsequent T4 release, which cannot
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presently be achieved due to the lack of techniques for measuring circulating TSH in fish. This linkage between TSH mRNA expression and protein secretion has received little attention in any species (although evidence does suggest that LH expression might be linked to secretion (Kandel-Kfir et al., 2002)), but is important to establish if one wishes to utilize subunit mRNA expression as an index of pituitary thyrotroph secretory activity. Similarly, our studies assumed that deiodinase expression reflects subsequent deiodinase activity in the pituitary, but this has rarely been examined in fish. However, in both tilapia (Van der Geyten et al., 2001) and trout (Bres et al., 2006), induced alterations in hepatic deiodinase expression were paralleled by changes in deiodinase enzyme activity, suggesting that much of the regulation of deiodinase function may be pretranslational in these species. Additionally, in rats, experimentally produced decreases in cerebral cortex Dio2 mRNA expression were linked with decreased Dio2 enzyme activity lending further support that deiodinase mRNA expression is a useful index of deiodinase activity (Burmeister et al., 1997). In conclusion, we have shown that both THs can be absorbed from tank water by fish to yield physiological elevations of circulating hormones, eliciting rapid changes in thyroid-related gene expression. Most obvious among these genes are the two subunits for TSH, providing compelling evidence that both thyroid hormones can exert negative feedback at the pituitary, and supporting the hypothesis that the previously described T4 cycle in red drum is at least partially driven by negative feedback. We have also shown that THs negatively regulate Dio3 expression in the pituitary in a manner suggesting that negative T4 feedback on Dio3 promotes the release of TSH subunits from TH inhibition and may be an important mechanism for generating daily thyroid hormone cycles. These results highlight a potentially important role for D3 in mediating thyroid hormone feedback on TSH expression, not previously described in fish or mammals. Unlike mammals, T3 often circulates in the blood of fish at concentrations equivalent to or greater than that of T4. In red drum, and possibly other teleost fish which maintain this high T3 set point, an active inner-ring deiodinating system could serve as an adaptation to protect the T4-driven TSH negative feedback system from interference by circulating T3. Acknowledgments We thank Dr. Scott Jaques of the Texas Veterinary Medical Diagnostic Laboratory for providing facilities and reagents for performing RIAs and Prof. J.G. Eales for helpful comments on the manuscript. We thank Dr. Delbert Gatlin III, Brian Ray, Dr. Thomas Miller, and Rhonda Patterson of Texas A&M University for assistance in obtaining and maintaining fish. We wish to acknowledge undergraduate researchers Cristina Copus, Elizabeth Drone, Quyhn Lam, Jarrett Aldinger, Bryan Berletch, McKensie Daugherty, Michael Hinojosa, Minal Parikh, Thomas McKean, Nery Guerrero, Anthony Martillotti, Ryan Clark, and Laura Ron for assistance with fish maintenance, sample collection, and analysis. Funding for this project was provided in part by the Department of Biology and the Graduate Interdisciplinary Degree Program in Marine Biology, both at Texas A&M University. References Arjona, F.J., de Vrieze, E., Visser, T.J., Flik, G., Klaren, P.H.M., 2011. Identification and functional characterization of zebrafish solute carrier Slc16a2 (Mct8) as a thyroid hormone membrane transporter. Endocrinology 152, 5065–5073. Barca-Mayo, O., Liao, X.-H., Alonso, M., Di Cosmo, C., Hernandez, A., Refetoff, S., Weiss, R.E., 2011. Thyroid hormone receptor a and regulation of type 3 deiodinase. Mol. Endocrinol. 25, 575–583. Bianco, A.C., Salvatore, D., Gereben, B., Berry, M.J., Larsen, P.R., 2002. Biochemistry, cellular and molecular biology, and physiological roles of the iodothyronine selenodeiodinases. Endocr. Rev. 23, 38–89.
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Bres, O., Plohman, J.C., Eales, J.G., 2006. A cDNA for a putative type III deiodinase in the trout (Oncorhynchus mykiss): influence of holding conditions and thyroid hormone treatment on its hepatic expression. Gen. Comp. Endocrinol. 145, 92– 100. Burmeister, L.A., Pachucki, J., St. Germain, D.L., 1997. Thyroid hormones inhibit type 2 iodothyronine deiodinase in the rat cerebral cortex by both pre- and posttranslational mechanisms. Endocrinology 138, 5231–5237. Campinho, M.A., Silva, N., Roman-Padilla, J., Ponce, M., Manchado, M., Power, D.M., 2015. Flatfish metamorphosis: a hypothalamic independent process? Mol. Cell. Endocrinol. 404, 16–25. Chiamolera, M.I., Wondisford, F.E., 2009. Thyrotropin-releasing hormone and the thyroid hormone feedback mechanism. Endocrinology 150, 1091–1096. Chowdhury, I., Chien, J.T., Chatterjee, A., Yu, J.Y.L., 2004. In vitro effects of mammalian leptin, neuropeptide-Y, b-endorphin and galanin on transcript levels of thyrotropin b and common a subunit mRNAs in the pituitary of bighead carp (Aristichthys nobilis). Comp. Biochem. Physiol. Part B: Biochem. Mol. Biol. 139, 87–98. Christoffolete, M.A., Ribeiro, R., Singru, P., Fekete, C., da Silva, W.S., Gordon, D.F., Huang, S.A., Crescenzi, A., Harney, J.W., Ridgway, E.C., Larsen, P.R., Lechan, R.M., Bianco, A.C., 2006. Atypical expression of type 2 iodothyronine deiodinase in thyrotrophs explains the thyroxine-mediated pituitary thyrotropin feedback mechanism. Endocrinology 147, 1735–1743. Cohn, W., Jones, R., Valverde, R., Leiner, K., MacKenzie, D., 2010. Molecular cloning and regulation of mRNA expression of the thyrotropin b and glycoprotein hormone a subunits in red drum, Sciaenops ocellatus. Fish Physiol. Biochem. 36, 1277–1290. Eales, J.G., 2006. Modes of Action and Physiological Effects of Thyroid Hormones in Fish, Fish Endocrinology. Science Publishers, Enfield, NH, pp. 767–808. Eales, J.G., Brown, S.B., 1993. Measurement and regulation of thyroidal status in teleost fish. Rev. Fish Biol. Fish. 3, 299–347. Fliers, E., Unmehopa, U.A., Alkemade, A., 2006. Functional neuroanatomy of thyroid hormone feedback in the human hypothalamus and pituitary gland. Mol. Cell. Endocrinol. 251, 1–8. Fonseca, T.L., Correa-Medina, M., Campos, M.P.O., Wittmann, G., Werneck-deCastro, J.P., Arrojo e Drigo, R., Mora-Garzon, M., Ueta, C.B., Caicedo, A., Fekete, C., Gereben, B., Lechan, R.M., Bianco, A.C., 2013. Coordination of hypothalamic and pituitary T3 production regulates TSH expression. J. Clin. Invest. 123, 1492– 1500. Friesema, E.C.H., Ganguly, S., Abdalla, A., Fox, J.E.M., Halestrap, A.P., Visser, T.J., 2003. Identification of monocarboxylate transporter 8 as a specific thyroid hormone transporter. J. Biol. Chem. 278, 40128–40135. García-G, C., Jeziorski, M.C., Valverde, R.C., Orozco, A., 2004. Effects of iodothyronines on the hepatic outer-ring deiodinating pathway in killifish. Gen. Comp. Endocrinol. 135, 201–209. Gereben, B., Zavacki, A.M., Ribich, S., Kim, B.W., Huang, S.A., Simonides, W.S., Zeöld, A., Bianco, A.C., 2008. Cellular and molecular basis of deiodinase-regulated thyroid hormone signaling. Endocr. Rev. 29, 898–938. Johnson, K.M., Lema, S.C., 2011. Tissue-specific thyroid hormone regulation of gene transcripts encoding iodothyronine deiodinases and thyroid hormone receptors in striped parrotfish (Scarus iseri). Gen. Comp. Endocrinol. 172, 505–517. Jones, R.A., Cohn, W.B., Miller, T.C., Jaques, J.T., MacKenzie, D.S., 2013. Cyclic mRNA expression of thyrotropin subunits and deiodinases in red drum, Sciaenops ocellatus. Gen. Comp. Endocrinol. 194, 248–256. Kandel-Kfir, M., Gur, G., Melamed, P., Zilberstein, Y., Cohen, Y., Zmora, N., Kobayashi, M., Elizur, A., Yaron, Z., 2002. Gonadotropin response to GnRH during sexual
ontogeny in the common carp, Cyprinus carpio. Comp. Biochem. Physiol. BBiochem. Mol. Biol. 132, 17–26. Larsen, D.A., Dickey, J.T., Dickhoff, W.W., 1997. Quantification of salmon a- and thyrotropin (TSH) b-subunit messenger RNA by an RNase protection assay: regulation by thyroid hormones. Gen. Comp. Endocrinol. 107, 98–108. Leiner, K.A., MacKenzie, D.S., 2001. The effects of photoperiod on growth rate and circulating thyroid hormone levels in the red drum, Sciaenops ocellatus: evidence for a free-running circadian rhythm of T4 secretion. Comp. Biochem. Physiol. A: Mol. Integr. Physiol. 130A, 141–149. Leiner, K.A., MacKenzie, D.S., 2003. Central regulation of thyroidal status in a teleost fish: nutrient stimulation of T4 secretion and negative feedback of T3. J. Exp. Zool. 298A, 32–43. Leiner, K.A., Han, G.S., MacKenzie, D.S., 2000. The effects of photoperiod and feeding on the diurnal rhythm of circulating thyroid hormones in the red drum, Sciaenops ocellatus. Gen. Comp. Endocrinol. 120, 88–98. Lema, S.C., Dickey, J.T., Schultz, I.R., Swanson, P., 2009. Thyroid hormone regulation of mRNAs encoding thyrotropin b-subunit, glycoprotein a-subunit, and thyroid hormone receptors a and b in brain, pituitary gland, liver, and gonads of an adult teleost, Pimephales promelas. J. Endocrinol. 202, 43–54. Little, A.G., 2016. A review of the peripheral levels of regulation by thyroid hormone. J. Comp. Physiol. B., 1–12 Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2DDCT method. Methods 25, 402–408. MacKenzie, D.S., Jones, R.A., Miller, T.C., 2009. Thyrotropin in teleost fish. Gen. Comp. Endocrinol. 161, 83–89. Mena, R.M.D., Scanlan, T.S., Obregon, M.-J., 2010. The T3 receptor b1 isoform regulates UCP1 and D2 deiodinase in rat brown adipocytes. Endocrinology 151, 5074–5083. Muzzio, A.M., Noyes, P.D., Stapleton, H.M., Lema, S.C., 2014. Tissue distribution and thyroid hormone effects on mRNA abundance for membrane transporters Mct8, Mct10, and organic anion-transporting polypeptides (Oatps) in a teleost fish. Comp. Biochem. Physiol. A 167, 77–89. Nikrodhanond, A.A., Ortiga-Carvalho, T.M., Shibusawa, N., Hashimoto, K., Liao, X.H., Refetoff, S., Yamada, M., Mori, M., Wondisford, F.E., 2006. Dominant role of thyrotropin-releasing hormone in the hypothalamic-pituitary-thyroid axis. J. Biol. Chem. 281, 5000–5007. Orozco, A., Valverde-R, C., 2005. Thyroid hormone deiodination in fish. Thyroid 15, 799–813. Orozco, A., Valverde-R, C., Olvera, A., García-G, C., 2012. Iodothyronine deiodinases: a functional and evolutionary perspective. J. Endocrinol. 215, 207–219. Pradet-Balade, B., Burel, C., Dufour, S., Boujard, T., Kaushik, S.J., Quérat, B., Boeuf, G., 1999. Thyroid hormones down-regulate thyrotropin b mRNA level in vivo in the turbot (Psetta maxima). Fish Physiol. Biochem. 20, 193–199. Schmitz, M., Balade, B., Lebelle, N., Dufour, S., Querat, B., 1998. In vitro study of the TSH subunit mRNA regulation in primary culture of pituitary cells of the European eel, Anguilla anguilla. Ann. N.Y. Acad. Sci. 839, 466–468. Shupnik, M.A., 2000. Thyroid hormone suppression of pituitary hormone gene expression. Rev. Endocr. Metab. Disord. 1, 35–42. Van der Geyten, S., Toguyeni, A., Baroiller, J.-F., Fauconneau, B., Fostier, A., Sanders, J. P., Visser, T.J., Kühn, E.R., Darras, V.M., 2001. Hypothyroidism induces type I iodothyronine deiodinase expression in tilapia liver. Gen. Comp. Endocrinol. 124, 333–342. Visser, W.E., Friesema, E.C.H., Visser, T.J., 2011. Minireview: thyroid hormone transporters: the knowns and the unknowns. Mol. Endocrinol. 25, 1–14.
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Research paper
Negative feedback regulation of thyrotropin subunits and pituitary deiodinases in red drum, Sciaenops ocellatus R.A. Jones ⇑, W.B. Cohn, A.A. Wilkes, D.S. MacKenzie Department of Biology, Texas A&M University, 3258 TAMUS, College Station, TX 77843-3258, USA
a r t i c l e
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Article history: Received 20 March 2016 Revised 30 August 2016 Accepted 1 September 2016 Available online 3 September 2016 Keywords: Thyrotropin Deiodinase Fish Thyroid hormone Negative feedback
a b s t r a c t Thyroxine (T4) undergoes dynamic daily cycles in the perciform fish the red drum, Sciaenops ocellatus, that are inversely timed to cycles of thyrotropin (TSH) subunit mRNA expression in the pituitary gland. We have proposed that these daily cycles are regulated by negative feedback of circulating T4 on expression of pituitary thyroid hormone deiodinase type 3 (Dio3), such that elevated circulating T4 results in diminished pituitary thyroid hormone catabolism and consequent increased negative feedback on expression of TSH subunits during the day. To determine whether thyroid hormones function to modulate expression of pituitary deiodinase enzymes we developed an immersion technique to administer physiological doses of T3 and T4 in vivo. Immersion in T4 or T3 significantly inhibited the mRNA expression of the TSH a and b subunits from 4 to 66 h of immersion. Pituitary Dio3 expression was significantly diminished by T3 and T4 at 22 h. These results indicate that both T4 and T3 are capable of negative feedback regulation of TSH subunit expression in red drum at physiological concentrations and on a time scale consistent with the T4 daily cycle. Furthermore, thyroid hormones negatively regulate Dio3 expression in the pituitary in a manner suggesting that negative thyroxine feedback on Dio3 promotes the release of TSH subunits from TH inhibition and may be an important mechanism for generating daily thyroid hormone cycles. These results highlight a potentially important role for D3 in mediating thyroid hormone feedback on TSH expression, not previously described in other species. Published by Elsevier Inc.
1. Introduction Thyroid hormone negative feedback to regulate the synthesis and secretion of thyrotropin (TSH) is an established mechanism for maintenance of thyroid homeostasis in mammals (Gereben et al., 2008). Studies in several mammalian species have indicated that deiodination of thyroxine (T4) to 3,5,30 -triiodothyronine (T3) by Type 2 iodothyronine deiodinase (D2, Dio2) in the pituitary gland is an essential component of this feedback mechanism (Fonseca et al., 2013; Gereben et al., 2008). In contrast, the relative importance of TSH as a central regulator of thyroid homeostasis has been questioned in nonmammalian species (Eales and Brown, 1993). Studies which have examined regulation of TSH expression in teleost fish have supported a role for thyroid Abbreviations: TSH, thyrotropin; TH, thyroid hormones; GSUa, glycoprotein hormone subunit a; D2, type 2 iodothyronine deiodinase; D3, type 3 iodothyronine deiodinase; T4, thyroxine 3,5,30 50 -tetraiodothyronine; T3, 3,5,30 -triiodothyronine. ⇑ Corresponding author. E-mail addresses: [email protected] (R.A. Jones), [email protected] (W.B. Cohn), [email protected] (A.A. Wilkes), [email protected] (D.S. MacKenzie). http://dx.doi.org/10.1016/j.ygcen.2016.09.003 0016-6480/Published by Elsevier Inc.
hormone in negative feedback (MacKenzie et al., 2009) while leaving the question of the importance of deiodination unresolved. Fish feedback studies have generally utilized prolonged hormone (primarily T3) or goitrogenic drug administration and have provided evidence both for a mammalian-like feedback requiring deiodination of T4 or direct feedback of T3 (Campinho et al., 2015; Cohn et al., 2010; Leiner and MacKenzie, 2003; Lema et al., 2009; MacKenzie et al., 2009), which often circulates in fish at higher concentration than in mammals. Eales (2006) has argued that because of pharmacological doses and indirect effects of circulating thyroid hormones through actions on peripheral deiodinases, circulating T3 may not function as a primary regulator of TSH negative feedback. The issue of differential sensitivity of TSH to T4 and T3 feedback in vivo has not subsequently been addressed and is an important consideration in establishing the role of central mechanisms in regulating fish thyroid function. Evidence in fish of a robust daily rhythm of circulating T4, hypothesized to be driven by a central oscillator working through TSH, provides further evidence that TSH may indeed be dynamically regulated by thyroid hormone negative feedback (Leiner and MacKenzie, 2001, 2003). In the perciform teleost, the red drum
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(Sciaenops ocellatus), a robust daily rhythm of circulating T4 is inversely timed to the pituitary expression of mRNAs for both TSH subunits, TSH beta (TSHb) and glycoprotein subunit alpha (GSUa), suggesting that negative feedback of T4 acting upon TSH drives this cycle (Jones et al., 2013). In this same study Dio2 was expressed in the pituitary at the peak of the T4 cycle, but Dio2 expression did not predictably and consistently vary with TSH subunit expression. Interestingly, however, type 3 iodothyronine deiodinase (D3, Dio3) mRNA expression was robustly cyclic with a waveform inversely related to circulating T4, suggesting that TH negative feedback in this species may be mediated in part through regulation of intra-pituitary deactivation of THs (Jones et al., 2013). Whereas pituitary D3 expression has been identified in some mammalian species (Fliers et al., 2006) it has not previously been identified as an important player in the TSH negative feedback mechanism. We have proposed that if expression of D3 in red drum is regulated by circulating thyroid hormones it could serve as a mechanism to rapidly modulate thyroid hormone feedback on TSH through T3 and T4 degradation (Jones et al., 2013). The objective of the current study was to determine if physiological elevations of circulating T3 and T4 do indeed regulate the expression of deiodinases in the pituitary consistent with a role in negative feedback on TSH expression. In particular, because of our previous observation of an inverse temporal relationship between circulating T4 and pituitary Dio3 expression (Jones et al., 2013), we wish to determine whether physiological elevations of circulating T4 are capable of down-regulating Dio3 expression, supporting an important role for D3 in the negative feedback process. Leiner and MacKenzie (2003) employed a T3 immersion technique in red drum that, by elevating T3 over a brief period of hours, provided evidence for negative feedback of physiological doses of T3 by significantly depressing the daily peak in circulating T4 in red drum. We utilized a similar experimental design to noninvasively administer not only T3 but also T4 to red drum to address the question of the relative importance of these hormones in regulating pituitary deiodinase expression. By developing a non-invasive technique for administering thyroid hormones that results in rapid, reversible, physiological increases in blood hormone levels we have been able to characterize differential negative feedback capabilities of T3 vs. T4 on pituitary mRNA expression of TSHb, GSUa, Dio2, and Dio3 during constant TH immersion. Our results support an important role for pituitary D3 in the central regulation of a feedback oscillator system that drives daily circulating thyroxine cycles. 2. Materials and methods 2.1. Animals The Coastal Conservation Association Marine Development Center in Corpus Christi and the Texas Parks and Wildlife Department’s hatchery at Sea Center Texas in Lake Jackson provided red drum fingerlings weighing approximately 0.5 g. Fingerlings were held in the Department of Biology’s BioAquatics Facility at Texas A&M University until they reached a weight of at least 20 g before being used for experiments. Sex in these juvenile fish cannot be readily determined. Animals were housed in a 4000 L recirculating system at 4 ppt salinity (SuperSalt, Fritz Industries, Mesquite, Texas), 26 °C, 12L:12D photoperiod. Fish were fed Aquamax (PMI Nutrition, Brentwood, MO) to apparent satiation once daily. TH immersion experiments were performed in 80 L tanks connected to the 4000 L recirculating system. Fish were euthanized in MS222 (Finquel, Argent Laboratories, Redmond, Washington) prior to obtaining blood and pituitary samples. All procedures were approved by the Texas A&M University Institutional Animal Care and Use Committee.
2.2. TH immersion experiments The thyroid hormone immersion technique of Leiner and MacKenzie (2003) was modified to develop our T3 and T4 immersion protocol. A series of pilot experiments was undertaken to establish ambient water doses of T3 and T4 that resulted in increased circulating thyroid hormone levels in immersed fish within physiological ranges. Thyroid hormones (3,5,30 -triiodothyro nine,T3, and 3,5,30 ,50 -tetraiodothyronine, both obtained from Sigma Chemical Co., St. Louis) were dissolved in 0.1 N NaOH to make stock solutions. Target concentrations of 100 ng/ml T3 (3 ml of a 2 mg/ml T3 stock solution in 60 L tank water) and 200 ng/ml T4 (3 ml of a 4 mg/ml T4 stock solution in 60 L tank water) were found to result in circulating thyroid hormone levels between 15 and 25 ng/ml, equivalent to levels observed during peak thyroid hormone cycles in captive red drum (Leiner et al., 2000). To avoid overcrowding of fish in static tanks at the initiation of long duration immersion experiments, three separate immersion experiments were performed. Five to seven fish per tank were immersed in Experiments 1 (mean weight ± s.e. 22.5 ± 0.8 g), 2 (24.3 ± 1.0 g), and 3 (29.9 ± 1.9 g), sampled at 4.5 h, 22 and 40 h, or 66 h, respectively. The 4.5 h time point was chosen as within the expected time for physiological negative feedback in relation to daily cycles of THs in red drum. The 22, 40, and 66 h time points were chosen as near to 24 h increments as room lighting and personnel schedules would allow and to replicate the time frames for feedback used in studies for other teleost species (MacKenzie et al., 2009). No tank was sampled more than once so that the N for all sampling times and treatments (control, T3, or T4), comprised 5– 7 animals. All three experiments were initiated at 3 h after lights on to control for daily cycles in circulating thyroid hormones. Each immersion tank contained a conditioned corner filter allowing fish to be fed during immersion. Data from these three experiments were combined and graphed together to facilitate visual comparison of changes in hormone levels and gene expression over a 66 h period of immersion. On the day before an immersion experiment fish were moved to flow-through 80 L tanks, all connected to the same recirculating system as their stock tank. At the beginning of the experiment flow to these tanks was turned off, their volume adjusted to 60 L, and thyroid hormone added. Hormone immersion duration ranged from 4.5 to 66 h before flow was returned to the tanks in the three experiments. At the conclusion of each experiment as much water as possible (approximately 90%) was removed from TH treated tanks, discarded from the recirculating system, and replaced with hormone-free system water. Remaining water draining from these tanks back into the recirculating system was passed through activated carbon to remove any residual immersion hormone from the system. 2.3. Blood and tissue analysis Blood was collected from the caudal vasculature at the end of immersion and plasma was separated and frozen at 80 °C. Plasma samples were analyzed for thyroid hormones using Coat-A-Count Total T4 or T3 kits (Siemens, Los Angeles, CA) following the manufacturer’s protocol as described by Cohn et al. (2010). Following terminal anesthesia, tissues for RNA extraction were rapidly removed by dissection and transferred to ZR RNA Microprep kit reagent (Zymoresearch, Irvine, CA). All samples were frozen at 80 °C until RNA extraction according to the manufacturers’ protocols. 2.4. qPCR qPCR primers, probes and techniques have been previously described (Jones et al., 2013). PCR efficiency was above 90% for
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all assays and R2 values were greater than 0.9 for all dilution standard curves. Reference gene (18S) mRNA levels were not detectably different among treatments.
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The 2TDDC method was used to calculate qPCR results (Livak and Schmittgen, 2001). Relative values correspond to the mRNA expression of the gene-of-interest/18S Ribosomal subunit. The average control relative value corresponds to the mean of all relative values from control animals within the same experiment independently determined for each gene-of-interest. All mRNA expression graphs were displayed as percent of the average control relative value to eliminate any bias toward one experimental time point over another. Error bars represent standard error of the mean. SPSS software was used for Kruskal-Wallis ANOVA to compare treatments at each time point followed by Mann-Whitney tests at each sampling time point to compare T4 and T3 groups to control to determine significance using Bonferroni correction. A P value of less than 0.05 was considered significant.
or T4 over time-matched controls between 4.5 and 40 h, but only T3 was elevated at the end of the 66 h immersion (Fig. 1). T4 immersion transiently elevated blood T3, but by 40 h this had reversed (Fig. 1A). T3 immersion significantly depressed blood T4 at every point other than the initial 4.5 h sampling (Fig. 1B). The concentration of THs in tank water steadily decreased over time during the static immersion to final concentrations at 66 h of 40 ng/ml T4 and 0 ng/ml T3. Immersion in T3 or T4 significantly inhibited the expression of both pituitary TSHb (Fig. 2A) and GSUa (Fig 2B) compared to time-matched controls at three out of four time points. In contrast, pituitary Dio2 expression was different from control at only the 22 h time point but once the Bonferroni correction was applied this difference was no longer significant (Fig. 3A). T3 and T4 immersion significantly diminished the expression of pituitary Dio3 with a maximal T4 inhibition compared to control at 22 h (Fig. 3B). At 22 and 40 h, T3 immersion significantly inhibited the hepatic expression of Dio2 and stimulated the hepatic expression of Dio3 compared to time-matched controls. In contrast, T4 immersion did not alter the expression of Dio2 or Dio3 in the liver (Fig. 4).
3. Results
4. Discussion
An immersion dose of T4 double that of T3 was required to elevate circulating T4 above control values. Blood from fish immersed in these T3 or T4 concentrations showed significant elevations of T3
In this study we noninvasively administered thyroid hormones to red drum to assess hepatic and pituitary mRNA responses within a time frame reflecting endogenous thyroid hormone cycles. A
2.5. Data analysis
Fig. 1. (A) T3 and (B) T4 response in red drum to up to 66 h of thyroid hormone immersion. Blood samples were taken from fish in constant static immersion with either T3 (100 ng/ml), T4 (200 ng/ml), or NaOH vehicle. Experiment 1 (E1), experiment 2 (E2), and experiment 3 (E3) are graphed together on a single axis. Significant differences from same time control are denoted by asterisks.
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Fig. 2. Red drum pituitary (A) TSHb and (B) GSUa mRNA expression resulting from thyroid hormone immersion in either T3 (100 ng/ml), T4 (200 ng/ml), or NaOH vehicle. Experiment 1 (E1), experiment 2 (E2), and experiment 3 (E3) are graphed together on a single axis. Significant differences from same time control are denoted by asterisks.
variety of techniques have been utilized to administer thyroid hormones to fish but most either employ pharmacological elevations or involve handling stress and as such complicate interpretation of results (Eales, 2006). Hormone feeding has been used extensively in rainbow trout, but this method fails to account for differential dosing due to variable food consumption among animals (Eales, 2006). Additionally, feeding experiments have traditionally focused on T3 feeding because T4 is not absorbed across the gut of fish (Eales, 2006; Larsen et al., 1997; Lema et al., 2009; PradetBalade et al., 1999). Immersion on the other hand is non-invasive and insures that all fish receive equivalent hormone exposure (Eales, 2006). Immersion has been used successfully to administer physiological doses of thyroid hormones in salmonids (Bres et al., 2006; Eales, 2006). Leiner and MacKenzie (2003) developed an immersion technique for administering T3 which resulted in a reduction in circulating T4, presumably through negative feedback at the pituitary, in red drum. This immersion protocol did not include T4 as a treatment however. The present study confirms that circulating T3 does indeed inhibit pituitary TSH subunit expression at physiological concentrations. Whereas T3 has been found to inhibit TSH expression in a number of teleost studies (MacKenzie et al., 2009), it is necessary to demonstrate a similar action of T4 to support our hypothesis that daily cycles of circulating T4 in red drum are driven by negative T4 feedback on TSH.
In teleost fish, in vitro inhibition of TSH expression by T3 or T4 has been observed, signifying that feedback of both hormones at the pituitary is physiologically possible (Chowdhury et al., 2004; Schmitz et al., 1998). However, the in vivo physiological role of inhibitory thyroid hormone feedback on TSH expression in fish has not been consistently demonstrated (Eales, 2006). In vivo experiments in fish have demonstrated T4 or T3 suppression of TSH expression, but usually employing pharmacological doses or prolonged time courses of administration of 2 weeks or more (MacKenzie et al., 2009). In red drum, we found that the daily rhythm of circulating T4 is mirrored by cyclic expression of TSH subunits (Jones et al., 2013), suggesting that this TSH cycle is regulated by negative T4 feedback at the pituitary. The red drum TSH expression cycle was 6–12 h out of phase with circulating T4 further suggesting that this negative feedback was occurring over a time scale of hours. The thyroid hormone administration results in the present study provide additional support for this negative feedback model. Within 4 h of the initiation of immersion T4 had significantly inhibited the expression of both TSH subunits, indicating that T4 within the range of circulating levels encountered during the daily cycle can feed back rapidly enough to inhibit TSH production and thus drive the TSH cycle in red drum. This inhibition was observed during both T3 and T4 immersion and was sustained for up to 66 h, providing convincing evidence for a
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Fig. 3. Red drum pituitary (A) Dio2 and (B) Dio3 mRNA expression resulting from thyroid hormone immersion in either T3 (100 ng/ml), T4 (200 ng/ml), or NaOH vehicle. Experiment 1 (E1), experiment 2 (E2), and experiment 3 (E3) are graphed together on a single axis. Significant differences from same time control are denoted by asterisks.
powerful negative feedback action of both thyroid hormones at physiological concentrations. Pilot studies for the immersion experiments indicated that red drum take up T3 more readily from water than T4, even in the face of progressively declining water thyroid hormone content. Consequently, the dose of T4 used to achieve comparable levels of circulating T4 in T4-immersed fish was twice as high as the T3 dose, which was equivalent to the ambient concentration employed for physiological supplementation in salmonids (Bres et al., 2006). Currently, the mechanism through which ambient thyroid hormone enters fish circulation is unknown. However, the monocarboxylate transporter MCT8, known to be a thyroid hormone transporter in mammals (Friesema et al., 2003), is expressed in the fish gill (Arjona et al., 2011; Muzzio et al., 2014) and in zebrafish preferentially transports THs in a temperature-dependent fashion, with T3 transport maximal at 26 °C vs. T4 at 37 °C. In mammals, the MCT8 transporter preferentially transports T3, while the Oatp1c1 transporter preferentially transports T4 (Little, 2016; Visser et al., 2011), and the low expression of Oatp1c1 in the fish gill (Muzzio et al., 2014) may explain the differential uptake of T4 and T3. If MCT8 and Oatp1c1 in red drum are expressed in the gills and exhibit similar thyroid hormone transporting properties at our water temperature of 25–29 °C, a preference of T3 over T4 uptake would be expected, as was observed.
Experiments 1, 2, and 3 addressed the time course of responses to T3 and T4 immersion. Circulating T3 initially increased within 4 h of T3 immersion, but its decline resulted in residual levels still significantly above control by 66 h. As previously observed (Leiner and MacKenzie, 2003), T4 was significantly diminished in T3 immersed fish, consistent with a negative feedback action of T3 at the pituitary thyrotroph. Samples from T3 immersed fish taken up to 66 h all showed a reduction in T4 suggesting that T3 has a sensitive and sustained negative feedback effect on TSH. In contrast, circulating T4 significantly also increased within 4 h of initiation of T4 immersion but eventually declined to control levels by 66 h. Interestingly, T4 immersion resulted in a transient (at 4 h only) increase in circulating T3. This increase in circulating T3 in response to brief T4 treatment has not been previously reported in fish, but it is consistent with the initial elevated T4 entering peripheral targets and being converted to T3 by D2. Indeed, the bulk of circulating T3 in fish is thought to be generated by T4 deiodination (Eales and Brown, 1993), particularly at the liver (Orozco and Valverde-R, 2005). That this transient increase in circulating T3 is not sustained suggests a hormone-induced adjustment in peripheral deiodination over time. Hepatic regulation of a blood T3 set-point has been proposed to be an important component of the peripheral regulation of teleost fish thyroid function (Eales and Brown, 1993). Generally, both T3 and T4 challenge rapidly
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Fig. 4. Red drum hepatic (A) Dio2 and (B) Dio3 mRNA expression resulting from thyroid hormone immersion in either T3 (100 ng/ml), T4 (200 ng/ml), or NaOH vehicle. Experiment 1 (E1), experiment 2 (E2), and experiment 3 (E3) are graphed together on a single axis. Significant differences from same time control are denoted by asterisks.
suppress outer ring deiodination and induce inner ring deiodination in multiple fish tissues (Eales, 2006; Garcı´a-G et al., 2004; Orozco et al., 2012), although the simultaneous regulation of inner and outer ring deiodination has only been examined in a few species. Bres et al. (2006) observed in rainbow trout that hepatic Dio2 expression was down-regulated whereas Dio3 expression was upregulated by T3 but neither Dio2 nor Dio3 expression were influenced by T4. We also observed a similar differential hepatic response of Dio2 and Dio3 to T3 vs. T4 immersion, serving as confirmation that our qPCR assays can detect expected physiological changes for these hepatic deiodinase gene products. As Dio3 expression in the liver of fish has been found to be activated by T3, the generation of this T3 should progressively decline over time as hepatic expression of D3 increases, as we observed. This delayed activation of D3 could also contribute to the steady decrease of T3 in T4 treated red drum, where by 40 h circulating T3 had significantly declined below control levels. Whereas D2 and D3 are known to be regulated by thyroid hormones in fish liver, the TH regulation of deiodinase expression in the fish pituitary has, until now, not been studied. In mammals, T3 for negative feedback on TSH expression is generated by D2 deiodination of T4 (Bianco et al., 2002; Nikrodhanond et al., 2006). In the red drum we found that similarly to liver, both T3 and T4 down-regulated the expression of pituitary Dio2 at 22 h.
However, in mice (Christoffolete et al., 2006), this reduction in enzyme expression may not necessarily result in levels of intrapituitary T3 production insufficient for feedback. Conversely, mouse pituitary Dio3 is up-regulated in the face of high circulating THs, likely preventing excessive inhibition of TSH secretion by degrading T3 (Barca-Mayo et al., 2011). Interestingly, in the red drum pituitary we found the opposite result, where elevated TH significantly inhibited the expression of Dio3. Observations in fish that T3 differentially regulates Dio3 in the brain, liver, and gill (Johnson and Lema, 2011; Orozco and Valverde-R, 2005) and in mice that Dio2 is down-regulated by T3 in the brain (Burmeister et al., 1997) while up-regulated by T3 in brown adipose tissue (Mena et al., 2010) suggests that THs regulate deiodinase expression in a tissue-specific manner. Although no TH response element has been discovered in the human Dio3 gene promoter, T3 applied to GH3 cells cotransfected with a human Dio3 gene promoter construct and the thyroid hormone receptor (TRa but not TRb) stimulated luciferase activity from this Dio3 promoter construct, leading Barca-Mayo et al. (2011) to suggest that differential expression of the TRs may mediate tissue-specific stimulatory vs. inhibitory regulation of Dio3 in response to TH. Alternatively, T3 has been suggested to negatively regulate TSH expression in the pituitary through direct activation of co-repressors (Chiamolera and Wondisford, 2009; Shupnik, 2000). A similar mechanism of
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Fig. 5. Model for a feedback oscillator generating daily cycles of circulating T4 in red drum: 1) At the conclusion of the scotophase, minimal circulating T4 and elevated pituitary D3 activity result in diminished pituitary T3 content and consequently minimal negative feedback on TSH subunit and D3 expression. 2) As a result, TSH secretion increases within 6–12 h, stimulating progressively increasing circulating T4. 3) As T4 rises during the photophase D2 supplies T3 available for negative feedback on the expression of D3, preventing destruction of the TH feedback signal. Thus, as circulating T4 rises, negative feedback of T3 formed through pituitary D2 results in a decreased expression of TSH subunits. 4) This inhibition in TSH subunit expression would be followed 6–12 h later by decreased TSH secretion and consequent decreasing circulating T4 during the scotophase. The cycle would then begin again as the expression of D3 increases due to diminished inhibition during the scotophase. The resulting increased production of reverse T3 and T2 at the expense of T3 in thyrotrophs also diminishes inhibition of TSH subunit expression. This diminished feedback reactivates increased expression of TSH subunits, followed once again by increasing TSH secretion driving increased circulating T4 throughout the next photophase.
co-activator or co-repressor direct regulation could be involved in TH mediated expression of deiodinases allowing for inhibition of expression in tissues such as the pituitary and stimulation in tissues such as the liver. In red drum cyclic expression of TSH subunits was closely synchronized with pituitary Dio3 expression, suggesting negative feedback mediated through TH catabolism by D3 may play a role in TSH regulation (Jones et al., 2013). Our finding that rapid, physiological alterations in circulating thyroid hormone levels can elicit rapid responses in pituitary thyroid-related transcript expression, in particular thyroid hormone-induced depression of pituitary Dio3 and TSH subunit expression, further suggests that D3 plays an important role in negative feedback of T4, and that the daily cycle of circulating T4 in red drum is the product of a peripheralpituitary interaction that establishes a self-sustaining endocrine oscillator. We propose a model for this oscillator in Fig. 5. Intriguingly, this oscillator as proposed could function without stimulatory input from a hypothalamic thyrotropin-releasing factor, consistent with the observation that hypothalamic regulation of TSH secretion in teleost fish may be predominantly under inhibitory control, as contrasted with the stimulatory control observed in tetrapod vertebrates (MacKenzie et al., 2009). Although our studies have provided a compelling argument for inhibition of pituitary TSH secretion by circulating thyroid hormones, confirmation of this model requires extension of these studies beyond gene expression to include true measures of TSH secretion and deiodinase enzyme activity over throughout the 24 h cycle. An important extension of these studies would be to examine the relationship among mRNA expression of TSH subunits, TSH secretion, and subsequent T4 release, which cannot
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presently be achieved due to the lack of techniques for measuring circulating TSH in fish. This linkage between TSH mRNA expression and protein secretion has received little attention in any species (although evidence does suggest that LH expression might be linked to secretion (Kandel-Kfir et al., 2002)), but is important to establish if one wishes to utilize subunit mRNA expression as an index of pituitary thyrotroph secretory activity. Similarly, our studies assumed that deiodinase expression reflects subsequent deiodinase activity in the pituitary, but this has rarely been examined in fish. However, in both tilapia (Van der Geyten et al., 2001) and trout (Bres et al., 2006), induced alterations in hepatic deiodinase expression were paralleled by changes in deiodinase enzyme activity, suggesting that much of the regulation of deiodinase function may be pretranslational in these species. Additionally, in rats, experimentally produced decreases in cerebral cortex Dio2 mRNA expression were linked with decreased Dio2 enzyme activity lending further support that deiodinase mRNA expression is a useful index of deiodinase activity (Burmeister et al., 1997). In conclusion, we have shown that both THs can be absorbed from tank water by fish to yield physiological elevations of circulating hormones, eliciting rapid changes in thyroid-related gene expression. Most obvious among these genes are the two subunits for TSH, providing compelling evidence that both thyroid hormones can exert negative feedback at the pituitary, and supporting the hypothesis that the previously described T4 cycle in red drum is at least partially driven by negative feedback. We have also shown that THs negatively regulate Dio3 expression in the pituitary in a manner suggesting that negative T4 feedback on Dio3 promotes the release of TSH subunits from TH inhibition and may be an important mechanism for generating daily thyroid hormone cycles. These results highlight a potentially important role for D3 in mediating thyroid hormone feedback on TSH expression, not previously described in fish or mammals. Unlike mammals, T3 often circulates in the blood of fish at concentrations equivalent to or greater than that of T4. In red drum, and possibly other teleost fish which maintain this high T3 set point, an active inner-ring deiodinating system could serve as an adaptation to protect the T4-driven TSH negative feedback system from interference by circulating T3. Acknowledgments We thank Dr. Scott Jaques of the Texas Veterinary Medical Diagnostic Laboratory for providing facilities and reagents for performing RIAs and Prof. J.G. Eales for helpful comments on the manuscript. We thank Dr. Delbert Gatlin III, Brian Ray, Dr. Thomas Miller, and Rhonda Patterson of Texas A&M University for assistance in obtaining and maintaining fish. We wish to acknowledge undergraduate researchers Cristina Copus, Elizabeth Drone, Quyhn Lam, Jarrett Aldinger, Bryan Berletch, McKensie Daugherty, Michael Hinojosa, Minal Parikh, Thomas McKean, Nery Guerrero, Anthony Martillotti, Ryan Clark, and Laura Ron for assistance with fish maintenance, sample collection, and analysis. Funding for this project was provided in part by the Department of Biology and the Graduate Interdisciplinary Degree Program in Marine Biology, both at Texas A&M University. References Arjona, F.J., de Vrieze, E., Visser, T.J., Flik, G., Klaren, P.H.M., 2011. Identification and functional characterization of zebrafish solute carrier Slc16a2 (Mct8) as a thyroid hormone membrane transporter. Endocrinology 152, 5065–5073. Barca-Mayo, O., Liao, X.-H., Alonso, M., Di Cosmo, C., Hernandez, A., Refetoff, S., Weiss, R.E., 2011. Thyroid hormone receptor a and regulation of type 3 deiodinase. Mol. Endocrinol. 25, 575–583. Bianco, A.C., Salvatore, D., Gereben, B., Berry, M.J., Larsen, P.R., 2002. Biochemistry, cellular and molecular biology, and physiological roles of the iodothyronine selenodeiodinases. Endocr. Rev. 23, 38–89.
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