We have shown that TH regulates thermal acclimation of metabolism in an ectothermic vertebrate. Our principal novel findings were that 1) the actions of TH are temperature-specific, and 2) TH regulates thermal acclimation in ectotherms. We also showed that 3) T2 has a functional role in this thermal response, which, to our knowledge has not been shown in any other system. Thus, to our knowledge, this is the first time that an environmental factor as pervasive as temperature has been shown to determine not just the magnitude of a hormone-mediated response, but also the direction.
The traditional model for hormonal regulation is based on homeostatic control , by which the bioavailability of a hormone is adjusted to regulate its action. Opposing responses are typically mediated by antagonistic hormone pairs , but single hormones can also drive different responses depending upon the physiological context . In the current study, we identified a novel signaling response, by which TH elicits a positive or negative response depending on the actual temperature and thermal history of the animal. TH has long been known to act in a tissue-specific manner [38, 39], and it is possible that the same mechanisms that underlie its tissue specificity also underlie its temperature specificity. Phenotypic differences between tissues are primarily driven by differential patterns in gene expression defined during ontogeny [40, 41], but gene-expression patterns, and therefore tissue phenotypes, are plastic, and can be adjusted in response to environmental factors such as temperature [1–3]. Thus, the thermal-acclimation response may change the tissue phenotype temporally to alter sensitivity to TH in a way that may parallel how different tissue types respond to TH.
The temperature specificity of TH action is evident at multiple levels of physiological organization, and mediates performance functions that determine fitness. We have shown that TH regulates energy metabolism and locomotor performance in response to chronic exposure to cold. This is the first time, to our knowledge, that a central regulator of thermal acclimation has been identified in an ectotherm, and provides a model that could explain vertebrate radiation during thermal-niche expansion. TH appears to have evolved as an environmental signaling molecule prior to vertebrate evolution [26–30, 42, 43]. In many invertebrates, TH suppresses larval structures, and promotes the growth and development of the juvenile rudiment . Although many of these animals require exogenous THs ingested from food, others can synthesize THs or TH-like compounds endogenously [25, 32, 44–46]. It is interesting to note that in echinoderms, endogenously synthesized TH has been suggested to be a derived trait . Growth and developmental rates are intrinsically linked to energy metabolism, and it is therefore likely that TH has always regulated these processes, at least in part by regulating metabolism. It is unknown whether the temperature specificity of TH is conserved in invertebrates, but it is conceivable that TH pathways evolved their sensitivity to temperature because both play such major roles in development [47–49]. If TH regulated metabolism to promote development at thermally challenging temperatures, then selection could favor this additional role. With an endogenous store of TH in the form of the thyroid gland, vertebrates could regulate these responses autonomously, and exploit novel thermal environments while maintaining important performance parameters such as locomotor capacity.
The properties that underlie the role of TH in thermal acclimation, temperature sensitivity, and metabolic control may have predisposed this hormone for a regulatory role in the evolution of endothermy. Of the response variables related to cold acclimation that we measured, most were highly sensitive to TH. The genes that were upregulated by TH during cold acclimation in zebrafish are homologous to those that control mammalian thermogenesis. In mammals, TH modulates the transcriptional regulation of metabolism by controlling expression levels of PGC1α , which plays a master role in coordinating the cross-genome expression of transcription factors involved in mitochondrial biogenesis and proteins that drive oxidative metabolism, including COX and F0F1-ATPase . Our findings show that this same pathway underlies cold acclimation in an ectotherm. The most parsimonious explanation is that these pathways were conserved in early vertebrate ancestors; however, without similar analyses of other ectotherms, we cannot preclude the possibility that they were independently derived in both fish and mammals. PGC1α has also been shown to adjust skeletal muscle phenotype in ways that affect locomotion . As is the case in mammals, PGC1α appears to be conserved as a target of TH in zebrafish, and probably also in other ectotherms. With the same pathways underlying both processes, the evolution of thermogenesis in mammals and birds [53–57] may have already been pre-programmed as a component of the cold-acclimation response in ectotherms.
T3 is often considered to be the only TH capable of genomic action because of its unique affinity for thyroid receptors . However, T2 has recently been shown to stimulate metabolism in mammals and fish . Our work supports the notion that T2 is also an important transcriptional regulator [58, 59]. In many cases, T2 was just as effective as, if not more effective than, T3 at regulating the transcription of metabolic genes. Although T2 has poor affinity for thyroid receptors, it could exert its transcriptional control through cell surface receptors that are also known to respond to TH [5, 18, 60], or through reversible epigenetic modifications to histone complexes . Importantly, the current study shows that T2 modulates performance parameters in the whole animal, which means that it is of ecological relevance and probably also of medical relevance. TH is associated with many modern lifestyle-induced conditions, and zebrafish have emerged as an important model for human endocrine diseases including obesity, diabetes, and metabolic syndromes [35, 36]. The fact that T2 regulates metabolism and locomotor performance in a manner that is potentially very different from that of T3 means that the mechanistic basis of TH action is far broader than realized to date. A corollary is that thyroid-related diseases may be more complex, but also that there may be novel avenues for treatments that specifically target T2.
Several studies have measured changes in T3 and T4 plasma levels during thermal response in fish, but the combined results are ambiguous. In the current study we found a high level of variation in T3 levels in warm-acclimated euthyroid fish. There was also much variation in the TH levels of the fish receiving T2 and T3 supplement treatments, although all individuals measured had supplementation levels greater than or equal to their euthyroid counterparts. Overall, the muscle-specific concentrations of T3 and T2 in our study decreased drastically with cold acclimation, when their effects were most pronounced. However, TH concentrations alone do not indicate the bioavailability and/or bioactivity of the hormone within the target tissue. Instead, the action of TH is modulated by downstream regulators, which include plasma distributor proteins, TH transporters, deiodinase enzymes, intracellular reservoir proteins, target proteins (thyroid receptors, cell surface receptors, and specific enzymes), and transcriptional regulators [16–21, 62]. The actions of TH may be more sensitive to changes in these downstream regulatory elements than to absolute free TH levels.
In many cases, the warm-acclimated zebrafish were far less sensitive to TH than cold-acclimated fish. This was especially evident in measures of locomotor performance. Decreased responsiveness to TH at warmer temperatures suggests that chronic increases in temperature, such as those brought about by global warming, will reduce the capacity of animals to adjust to environmental variation. Chronic warming could conceivably alter tissue phenotypes in ways that dampen, or reverse, TH-mediated response. This would interfere with TH as an environmental signaling molecule, and would compromise its crucial role in ectotherm growth and development. Importantly, many aquatic pollutants found worldwide, such as dioxins, bisphenol A, and phthalates, are thyroid-disrupting chemicals (TDCs) and bioaccumulate higher up the food chain . Our findings indicate that the toxic effects of these chemicals may be temperature-specific, which is of crucial importance to the ecological influence of these pollutants. Together, climate change and rising levels of global pollution may amplify these risks. Warming temperatures are likely to result in higher concentrations, longer durations, and increased distributions of TDCs throughout the water column .