Expression of the sodium iodide symporter (NIS) in reproductive and neural tissues of teleost fish.

Iodine, an essential component of thyroid hormones, can only be obtained through the diet. The sodium iodide symporter (NIS) transports iodide across mammalian intestinal and thyroid epithelia to deliver iodide for thyroid hormone production. Using reverse transcription-polymerase chain reaction (RT-PCR) we confirmed that mRNA for a homolog of mammalian NIS is expressed in comparable locations, both sub-pharyngeal thyroid tissue and intestine, in multiple teleost fish species, supporting a conserved mechanism for intestinal-thyroid iodine transport across vertebrates. To determine when in embryogenesis NIS expression is initiated we utilized in situ hybridization (ISH) during development of zebrafish (Danio rerio) embryos. This revealed expression of nis as early as 2 days post fertilization (dpf) along the dorsal surface of the yolk sac, suggesting a function to import iodine from yolk. To evaluate the potential for maternal deposition of iodine in yolk, RT-PCR and further in situ staining of ovarian tissue in gravid female zebrafish confirmed NIS mRNA presence in the ooplasm and granulosa layer of early stage follicles. This further suggests that maternally-deposited NIS mRNA may be available for early embryogenesis. Unexpectedly, ISH in embryos revealed robust nis expression in the central nervous system throughout days 2-5 days post fertilization, with adult whole brain ISH localizing expression in the hypothalamus, cerebellum, and optic tectum. RT-PCR on whole brain tissue from five species of adult fish representing three taxonomic orders likewise revealed robust CNS expression. These unexpected locations of nis expression suggest novel, as yet undescribed reproductive and neural functions of NIS in teleost species.

Glucose delays the insulin-induced increase in thyroid hormone-mediated signaling in adipose of prolong-fasted elephant seal pups.

Prolonged food deprivation in mammals typically reduces glucose, insulin, and thyroid hormone (TH) concentrations, as well as tissue deiodinase (DI) content and activity, which, collectively, suppress metabolism. However, in elephant seal pups, prolonged fasting does not suppress TH levels; it is associated with upregulation of adipose TH-mediated cellular mechanisms and adipose-specific insulin resistance. The functional relevance of this apparent paradox and the effects of glucose and insulin on TH-mediated signaling in an insulin-resistant tissue are not well defined. To address our hypothesis that insulin increases adipose TH signaling in pups during extended fasting, we assessed the changes in TH-associated genes in response to an insulin infusion in early- and late-fasted pups. In late fasting, insulin increased DI1, DI2, and THrß-1 mRNA expression by 566%, 44%, and 267% at 60 min postinfusion, respectively, with levels decreasing by 120 min. Additionally, we performed a glucose challenge in late-fasted pups to differentiate between insulin- and glucose-mediated effects on TH signaling. In contrast to the insulin-induced effects, glucose infusion did not increase the expressions of DI1, DI2, and THrß-1 until 120 min, suggesting that glucose delays the onset of the insulin-induced effects. The data also suggest that fasting duration increases the sensitivity of adipose TH-mediated mechanisms to insulin, some of which may be mediated by increased glucose. These responses appear to be unique among mammals and to have evolved in elephant seals to facilitate their adaptation to tolerate an extreme physiological condition.

Chemotaxis of Escherichia coli to norepinephrine (NE) requires conversion of NE to 3,4-dihydroxymandelic acid.

Norepinephrine (NE), the primary neurotransmitter of the sympathetic nervous system, has been reported to be a chemoattractant for enterohemorrhagic Escherichia coli (EHEC). Here we show that nonpathogenic E. coli K-12 grown in the presence of 2 µM NE is also attracted to NE. Growth with NE induces transcription of genes encoding the tyramine oxidase, TynA, and the aromatic aldehyde dehydrogenase, FeaB, whose respective activities can, in principle, convert NE to 3,4-dihydroxymandelic acid (DHMA). Our results indicate that the apparent attractant response to NE is in fact chemotaxis to DHMA, which was found to be a strong attractant for E. coli. Only strains of E. coli K-12 that produce TynA and FeaB exhibited an attractant response to NE. We demonstrate that DHMA is sensed by the serine chemoreceptor Tsr and that the chemotaxis response requires an intact serine-binding site. The threshold concentration for detection is ≤5 nM DHMA, and the response is inhibited at DHMA concentrations above 50 µM. Cells producing a heterodimeric Tsr receptor containing only one functional serine-binding site still respond like the wild type to low concentrations of DHMA, but their response persists at higher concentrations. We propose that chemotaxis to DHMA generated from NE by bacteria that have already colonized the intestinal epithelium may recruit E. coli and other enteric bacteria that possess a Tsr-like receptor to preferred sites of infection.

Prolonged food deprivation increases mRNA expression of deiodinase 1 and 2, and thyroid hormone receptor ß-1 in a fasting-adapted mammal.

Food deprivation in mammals is typically associated with reduced thyroid hormone (TH) concentrations and deiodinase content and activity to suppress metabolism. However, in prolonged-fasted, metabolically active elephant seal pups, TH levels are maintained, if not elevated. The functional relevance of this apparent paradox is unknown and demonstrates variability in the regulation of TH levels, metabolism and function in food-deprived mammals. To address our hypothesis that cellular TH-mediated activity is upregulated with fasting duration, we quantified the mRNA expression and protein content of adipose and muscle deiodinase type I (DI1) and type II (DI2), and TH receptor beta-1 (THrß-1) after 1, 3 and 7 weeks of fasting in northern elephant seal pups (N=5-7 per week). Fasting did not decrease the concentrations of plasma thyroid stimulating hormone, total triiodothyronine (tT3), free T3, total thyroxine (tT4) or free T4, suggesting that the hypothalamic-pituitary-thyroid axis is not suppressed, but rather maintained during fasting. Mean mRNA expression of adipose DI1 and DI2 increased threefold and fourfold, respectively, and 20- and 30-fold, respectively, in muscle. With the exception of adipose DI1, protein expression of adipose DI2 and muscle DI1 and DI2 increased twofold to fourfold. Fasting also increased adipose (fivefold) and muscle (fourfold) THrß-1 mRNA expression, suggesting that the mechanisms mediating cellular TH activity are upregulated with prolonged fasting. The data demonstrate a unique, atypical mechanism of TH activity and regulation in mammals adapted to prolonged food deprivation in which the potential responsiveness of peripheral tissues and cellular TH activity are increased, which may contribute to their lipid-based metabolism.

Effects of dietary arginine on endocrine growth factors of channel catfish, Ictalurus punctatus.

Thyroid (TH) and growth (GH) hormones, and insulin-like growth factor I (IGF-I) are anabolic regulators in fish and responsive to nutrient intake. A study was conducted to determine if previously reported growth effects of dietary arginine (ARG) in channel catfish were related to the activation of endocrine axes. In a first experiment, catfish were fed incremental levels of ARG (0.5 - 4% of diet) for 6 weeks and sampled at 2-week intervals. In a second experiment, fasted (48h) fish were fed a single ration of ARG (0.5 or 4% of diet) and sampled at various intervals (0 to 72h postprandial, PP). Experiment 1 did not reveal any influence of ARG on circulating TH, GH, or IGF-I despite the significantly increased growth of fish fed ARG-enriched diets. In experiment 2, feeding the 4% ARG diet significantly increased the amplitude of pulsatile plasma GH levels and also significantly increased IGF-I mRNA in liver and muscle, (at 2h PP) and plasma IGF-I levels (at 6h PP). Although relatively infrequent sampling failed to reveal alterations in TH or GH levels in response to ARG-induced growth activation, PP high frequency sampling unveiled high amplitude pulsatile GH secretions and may be important in activating IGF production in target tissues. Additionally, expressed and secreted IGF-I exhibited discernible patterns which closely correlate with ARG-induced growth effects in catfish.

Molecular cloning and regulation of mRNA expression of the thyrotropin ß and glycoprotein hormone α subunits in red drum, Sciaenops ocellatus.

Full-length cDNAs for thyrotropin ß (TSHß) and glycoprotein hormone α (GSUα) subunits were cloned and sequenced from the red drum (Sciaenops ocellatus). The cDNAs for TSHß (877 bp) and GSUα (661 bp) yielded predicted coding regions of 126 and 94 amino acid proteins, respectively. Both sequences contain all invariant cysteine and putative glycosylated asparagines characteristic of each as deduced by comparison with other GSUα and TSHß sequences from representative vertebrate species. Multiple protein sequence alignments show that each subunit shares highest identity (79% for the TSHß and 86% for the GSUα) with perciform fish. Furthermore, in a single joint phylogenetic analysis, each subunit segregates most closely with corresponding GSUα and TSHß subunit sequences from closely related fish. Tissue-specific expression assays using RT-PCR showed expression of the TSHß subunit limited to the pituitary. GSUα mRNA was predominantly expressed in the pituitary but was also detected in the testis and ovary of adult animals. Northern hybridization revealed the presence of a single transcript for both TSHß and GSUα, each close in size to mRNA transcripts from other species. Dot blot assays from total RNA isolated from S. ocellatus pituitaries showed that in vivo T3 administration significantly diminished mRNA expression of both the TSHß and GSUα subunits and that goitrogen treatment caused a significant induction of TSHß mRNA only. Both TSHß and GSUα mRNA expression in the pituitary varied significantly in vivo over a 24-h period. Maximal expression for both TSHß and GSUα occurred during the early scotophase in relation to a peak in T4 blood levels previously documented. These results suggest the production of TSH in this species which may serve to drive daily cycles of thyroid activity. Readily quantifiable, variable, and thyroid hormone-responsive pituitary TSH expression, coupled with previously described dynamic daily cycles of circulating T4 and extensive background on the growth, nutrition, and laboratory culture of red drum, suggests that this species will serve as a useful model for experimental studies of the physiological regulation of TSH production.

Thyrotropin in teleost fish.

Thyrotropin (TSH), a pituitary glycoprotein hormone that stimulates the thyroid gland, has been cloned and sequenced from over a dozen teleost fish species. Although TSH is established as a primary driver of systemic thyroid status in mammals, its importance in the regulation of fish thyroid function is still uncertain. We review recent studies indicating that TSH structure is highly conserved across species representing six teleost families. These studies have found TSH messenger RNA consistently expressed in teleost pituitary tissue, although ectopic expression, particularly in gonads, has also been observed. They have also provided evidence for negative feedback inhibition of TSH expression by thyroid hormones, as well as stimulation by hypothalamic peptides. Descriptive studies have found increased TSHbeta expression associated with life history events thought to be promoted by thyroid hormones. These results, coupled with the discovery of a G-protein coupled TSH receptor in several teleost species, supports an active and conserved role for TSH in the regulation of teleost thyroid function. The relative importance of central pathways in regulating thyroid hormone provision to targets and the identity of a proposed thyrotropin-inhibiting factor in teleost fish are still unanswered questions whose resolution will be facilitated by development of methods to measure circulating TSH and its secretion from the pituitary gland.

Biomarkers of exposure and effects of environmental contaminants on swallows nesting along the Rio Grande, Texas, USA.

We collected adult cave swallows (Petrochelidon fulva) and cliff swallows (P. pyrrhonota) during the breeding seasons in 1999 and 2000 from eight locations along the Rio Grande from Brownsville to El Paso (unless otherwise specified, all locations are Texas, USA) and an out-of-basin reference location. Body mass, spleen mass, hepatosomatic index (HSI), gonadosomatic index (GSI), thyroxine (T4) in plasma, DNA damage measured as the half-peak coefficient of variation of DNA content (HPCV) in blood cells, as well as acetylcholinesterase and butyrylcholinesterase in brain were compared with concentrations of organochlorines, metals, and metalloids in carcasses to determine potential effects of contaminants on swallows during the breeding season. Concentrations of 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p'-DDE) were significantly greater in swallows from El Paso than in those from most locations, except for Pharr and Llano Grande. All swallows from these three locations had p,p'-DDE concentrations of 3 microg/g wet weight or greater. Swallows from El Paso either had or shared the highest concentrations of p,p'-DDE, polychlorinated biphenyls, and 13 inorganic elements. Swallows from El Paso exhibited greater spleen mass and HPCV values as well as lower T4 values compared with those from other locations. Thyroxine was a potential biomarker of contaminant exposure in swallows of the Rio Grande, because it was negatively correlated with p,p'-DDE and Se. Spleen mass was positively correlated with selenium and HSI and negatively correlated with body mass, GSI, Mn, and Ni. Overall, the present study suggests that insectivorous birds living in areas of high agricultural and industrial activity along the Rio Grande bioaccumulate environmental contaminants. These contaminants, particularly p,p'-DDE, may be among multiple factors that impact endocrine and hematopoietic function in Rio Grande swallows.

Central regulation of thyroidal status in a teleost fish: nutrient stimulation of T4 secretion and negative feedback of T3.

Several experiments were conducted to investigate the dynamics of central regulation of thyroid function in the red drum, Sciaenops ocellatus, by manipulating a well-characterized circadian rhythm of T(4) secretion. In the first experiment, red drum were reared under either a long (16L:8D) or short (8L:16D) photoperiod and fed at the same time relative to dawn. The same feeding time under different photoperiods maintained the same phase relationship between T(4) cycles under each photoperiod. This suggests that the circadian clock that determines when the hypothalamus-pituitary-thyroid (HPT) axis is activated is comprised of a feeding-entrained oscillator and a light-entrained oscillator that interact to determine the phase of the T(4) rhythm. Additionally, the amplitude of the main T(4) peak of the cycle was inversely related to the frequency of feeding, while the duration of the main T(4) peak was directly related to feeding frequency under a long photoperiod. Feeding time appears to modify the diurnal profile of circulating T(4) by stimulating post-prandial T(4) secretion that subsequently results in negative feedback on the HPT axis to regulate thyroidal status. In following experiments, red drum immersed in T(3), in lieu of a meal at a specific time that would diminish the main T(4) peak, exhibited a dose-dependent decline in amplitude of the T(4) cycle. This demonstrates that T(3) can exert negative feedback on the HPT axis of red drum to maintain appropriate thyroid hormone concentrations. These data are consistent with a dynamic and physiologically important central component of the regulation of thyroid function in fish.