Anterior pituitary type II thyroxine 5'-deiodinase activity is not affected by lesions of the hypothalamic paraventricular nucleus which profoundly depress pituitary thyrotropin secretion

Bilateral destruction of the hypothalamic paraventricular nuclei (PVN) produced a profound depression of plasma TSH and the median eminence TRH concentration in hypothyroid rats. Anterior pituitary type II iodothyronine 5'-deiodinase (5'-D) activity was consistently lower but not significantly different in sham- and PVN-lesioned rats. Treatment with suboptimal replacement doses of 0.15 and 0.75 micrograms T4/100 g BW.day produced a graded depression of plasma TSH in the PVN (P less than 0.02), but not in the sham (P greater than 0.8) groups. Adenohypophyseal 5'-D was depressed in both sham and PVN groups by the highest T4 dose. Plasma T4 was much lower in PVN than in sham rats given comparable doses of T4 (P less than 0.001), but plasma T3 was not significantly different. This suggests that an increase in peripheral T4 metabolism was produced by PVN lesions. Our data indicate that changes in adenohypophyseal 5'-D activity are not responsible for the decrease in plasma TSH in PVN-lesioned rats and that neither the PVN nor endogenous TRH plays a significant role in the regulation of anterior pituitary 5'-D activity.     

The paraventricular nucleus of the hypothalamus has a major role in thyroid hormone feedback regulation of thyrotropin synthesis and secretion

The role of the hypothalamic paraventricular nucleus (PVN) in thyroid hormone regulation of TSH synthesis during hypothyroidism was studied in adult male rats that were normal (n = 10), had primary hypothyroidism with sham lesions in the hypothalamus (n = 17), and had primary hypothyroidism with PVN lesions (n = 14). Two and 4 weeks after initiation of treatment, plasma levels of thyroid hormones (TSH, corticosterone and PRL) and pituitary content of TSH beta and alpha-subunit mRNA were measured. TRH mRNA levels in the PVN were determined by in situ hybridization histochemistry. At 2 weeks, despite a decrease in plasma free T4 in both hypothyroid groups, plasma TSH levels increased, but to a lesser degree, in the hypothyroid PVN lesioned compared to hypothyroid sham-lesioned group (7.8 +/- 1.3 vs. 20.5 +/- 1.1 ng/dl; P less than 0.05). Similarly, at 4 weeks, the hypothyroid PVN-lesioned group demonstrated a blunted TSH response compared to the hypothyroid sham-lesioned group (6.8 +/- 0.7 vs. 24.0 +/- 1.3 ng/dl; P less than 0.05). Plasma corticosterone and PRL did not significantly differ between sham-lesioned and PVN-lesioned groups. TSH beta mRNA levels markedly increased in hypothyroid sham-lesioned rats compared to those in euthyroid controls at 2 weeks (476 +/- 21% vs. 100 +/- 39%; P less than 0.05) and 4 weeks (1680 +/- 270% vs. 100 +/- 35%; P less than 0.05). In contrast, TSH beta mRNA levels did not increase with hypothyroidism in the PVN-lesioned group compared to those in euthyroid controls at 2 weeks (140 +/- 16%, P = NS) and only partially increased at 4 weeks (507 +/- 135; P less than 0.05). alpha mRNA levels at 4 weeks markedly increased in hypothyroid sham-lesioned rats compared to those in euthyroid controls (1121 +/- 226% vs. 100 +/- 48%; P less than 0.05), but did not increase in the hypothyroid PVN-lesioned rats (61 +/- 15%; P = NS). TRH mRNA in the PVN increased in the hypothyroid sham-lesioned rats compared to those in euthyroid controls (16.6 +/- 1.3 vs. 4.8 +/- 1.2 arbitrary densitometric units; P less than 0.05), and TRH mRNA was not detectable in the PVN of hypothyroid-lesioned rats at 2 weeks. In summary, lesions in rat PVN prevented the full increase in plasma TSH, pituitary TSH beta mRNA, and alpha mRNA levels in response to hypothyroidism. Thus, factors in the PVN are important in thyroid hormone feedback regulation of both TSH synthesis and secretion.     

Effects of rexinoids on thyrotrope function and the hypothalamic-pituitary-thyroid axis

Retinoid X receptor (RXR)-selective retinoids (rexinoids) can cause central hypothyroidism in humans, and this effect has been confirmed in rodent models. In this report, we characterized the effect of rexinoids on the hypothalamic-pituitary-thyroid axis in mice and TSH regulation in a thyrotrope-derived cell line. The synthetic rexinoid (LG 268) suppressed TSH and T4 levels in mice. Hypothalamic TRH mRNA was unaffected, but steady-state pituitary TSHbeta mRNA levels were significantly lowered, suggesting a direct effect of rexinoids on thyrotropes. LG 268 suppressed TSH protein secretion and TSHbeta mRNA in TalphaT1 thyrotropes as early as 8 h after treatment, whereas the retinoic acid receptor-selective retinoid (TTNPB) had no effect. Type 2 iodothyronine deiodinase (D2) mRNA and activity were suppressed by LG 268 in TalphaT1 cells, whereas only D2 mRNA was suppressed in mouse pituitaries. LG 268 suppressed TSHbeta promoter activity by 42% and the -200 to -149 region accounted for a majority of the LG 268-mediated suppression of promoter activity. The RXRgamma isotype is expressed in thyrotropes. In vitro transfection and in vivo transgenic studies indicate that any RXR isotype can mediate TSH suppression by rexinoids, but the RXRgamma isotype is most efficient at mediating this response. RXRgamma-deficient mice lacked pituitary D2 mRNA suppression by LG 268, but D2 activity remained intact. In summary, RXR-selective retinoids (rexinoids) have multiple effects on the hypothalamic-pituitary-thyroid axis. Rexinoids directly suppress TSH secretion, TSHbeta mRNA levels and promoter activity, and D2 mRNA levels but have no direct effect on hypothalamic TRH levels. Rexinoids also stimulate type 1 iodothyronine deiodinase activity in the liver and pituitary.     

Agouti-related protein (AGRP) has a central inhibitory action on the hypothalamic-pituitary-thyroid (HPT) axis; comparisons between the effect of AGRP and neuropeptide Y on energy homeostasis and the HPT axis

Because alpha-MSH has a potent stimulatory action on hypophysiotropic TRH synthesizing neurons in the hypothalamic paraventricular nucleus (PVN), preventing the effects of fasting on the gene expression of the TRH prohormone (proTRH), we hypothesized that agouti-related protein (AGRP), a melanocortin receptor antagonist, may exert a central inhibitory action on these neurons. To test the hypothesis, the effects of intracerebroventricularly administered AGRP on circulating thyroid hormone levels and proTRH mRNA in the hypothalamic paraventricular nucleus (PVN) were compared with the effects of the recently described central inhibitor of the HPT axis, neuropeptide Y (NPY). AGRP administration increased food consumption and weight gain, suppressed circulating levels of thyroid hormones (T(3) and T(4)), and resulted in an inappropriately normal TSH. These alterations were associated with a significant suppression of proTRH mRNA in the PVN, indicating that AGRP infusion resulted in a state of central hypothyroidism. While similar observations were made in the NPY-infused animals, AGRP-treated animals had higher feeding efficiency, higher T(4) levels, and lower type 2 iodothyronine deiodinase levels in brown adipose tissue than NPY-infused animals. These data demonstrate that AGRP and NPY have a similarly potent inhibitory action on the proTRH gene expression of hypophysiotropic neurons, indicating that both AGRP and NPY may play a major role in the inhibition of the HPT axis during fasting.     

Food-restricted and dehydrated-induced anorexic rats present differential TRH expression in anterior and caudal PVN. Role of type 2 deiodinase and pyroglutamyl aminopeptidase II

TRH synthesized in hypothalamic paraventricular nucleus (PVN) regulates thyroid axis function and is also implicated in anorexigenic effects. Under energy deficit, animals present decreased PVN TRH expression and release, low TSH levels, and increased appetite. Dehydration-induced anorexia (DIA) model allows insight into underlying mechanisms of feeding regulation. Animals drinking a 2.5% NaCl solution for 7 d present body weight reduction; despite their negative energy balance, they avoid food and have increased PVN TRH expression and TSH serum levels. These findings support an inhibiting role of PVN TRH in feeding control. We compared TRH expression by in situ hybridization in PVN subdivisions of 7-d dehydrated male rats to those of a pair-fed group (forced food-restricted) with similar metabolic changes than DIA, but motivated to eat, and to controls. We measured peripheral deiodinase activities, and expression and activity of medial basal hypothalamic type 2 deiodinase and pyroglutamyl-aminopeptidase II, to understand their regulating role in PVN TRH changes between food restriction and anorexia. TRH mRNA levels increased in anterior (aPVN) and medial-caudal subdivisions in DIA rats, whereas it decreased in medial PVN in both experimental groups. We confirmed the nonhypophysiotropic nature of aPVN TRHergic cells by injecting ip fluorogold tracer. Findings support a subspecialization of TRHergic hypophysiotrophic cells that responded differently between anorexic and food-restricted animals; also, that aPVN TRH participates in food intake regulation. Increased type 2 deiodinase activity seemed responsible for low medial PVN TRH synthesis, whereas increased medial basal hypothalamic pyroglutamyl-aminopeptidase II activity in DIA rats might counteract their high TRH release.     

Thyrotropin-releasing hormone gene expression in the hypothalamic paraventricular nucleus is dependent upon feedback regulation by both triiodothyronine and thyroxine.

The biosynthesis of TRH in hypophysiotropic neurons of the paraventricular nucleus (PVN) is inversely regulated by feedback effects of circulating levels of thyroid hormones. As the PVN contains little or no deiodinase activity, the enzyme necessary to convert T4 to biologically active T3, we determined whether feedback inhibition of pro-TRH mRNA in thyroid hormone-sensitive neurons of the PVN is mediated exclusively by circulating levels of T3. The concentration of pro-TRH mRNA in the PVN of hypothyroid male rats receiving constant infusions of T3 over 7 days from ip implanted osmotic minipumps was studied by in situ hybridization histochemistry using computerized image analysis. Pro-TRH mRNA could not be suppressed to euthyroid levels by an infusion of T3 that returned plasma T3 levels to normal and required the infusion of higher concentrations of T3 that elevated plasma T3 into the supranormal range. By regression analysis, the mean concentration of plasma T3 required to suppress pro-TRH mRNA to euthyroid levels was estimated to be 110.3 ng/dl, similar to the amount of T3 estimated to be necessary to suppress TSH secretion from the anterior pituitary (108.7 ng/dl). We conclude that both T3 and T4 contribute to feedback inhibition of TRH biosynthesis in hypophysiotropic neurons of the PVN and propose that the effects of T4 on the PVN could be mediated after its monodeiodination at a different locus within the brain.     

Triiodothyronine exerts direct cell-specific regulation of thyrotropin-releasing hormone gene expression in the hypothalamic paraventricular nucleus

Thyroid hormone administered systemically exerts negative feedback control of biosynthesis of the TRH pro-hormone in the hypothalamic paraventricular nucleus (PVN), the origin of neurons that regulate anterior pituitary TSH secretion, but not in any other group of TRH-synthesizing neurons in the brain. To determine whether this response is mediated by direct effects on PVN neurons, we studied the effect of unilateral stereotaxic implants of L-T3 into the anterior hypothalamus on the concentration of pro-TRH mRNA and pro-TRH in the PVN of hypothyroid rats. Because hypothalamic-pituitary-thyroid function is also regulated by central catecholamines, we also determined the effect of unilateral ablation of ascending catecholaminergic fibers to one side of the PVN by stereotaxic injection of 6-hydroxydopamine or transection of ascending catecholaminergic pathways. T3-implanted hypothyroid animals showed a marked reduction in pro-TRH mRNA and immunoreactive pro-TRH in medial parvocellular neurons of the PVN on the same side as the implant, but not in contralateral PVN neurons or TRH-synthesizing neurons in other hypothalamic regions. In contrast, hypothyroid animals implanted with pellets of hormonally inactive 3,5-diiodo-L-thyronine showed intense symmetric hybridization and immunoreaction product in both wings of the PVN. Despite marked unilateral reduction in the catecholamine innervation to the PVN, no reduction in pro-TRH mRNA or immunoreactive pro-TRH was observed in the PVN on the affected side compared to that on the unaffected side. These studies demonstrate that negative feedback regulation of thyroid hormone occurs directly on TRH neurons and is restricted only to those in the PVN tuberoinfundibular system.     

Expression of type 2 iodothyronine deiodinase in human osteoblast is stimulated by thyrotropin

Thyroid hormones play important roles in bone growth, development, and turnover. To exert its biological activity, T(4) needs to be converted to T(3) by iodothyronine deiodinase. In human thyroid gland as well as rat brown adipose tissue, type 2 iodothyronine deiodinase (D2) expression is regulated by a TSH receptor-cAMP-mediated mechanism. TSH receptor knockout mice demonstrated the direct effects of TSH on bone via TSH receptors found on osteoblast and osteoclast precursors. In the present study we investigated the possible expression and function of iodothyronine deiodinase and TSH receptors in human osteoblast-like osteosarcoma (SaOS-2) cells and normal human osteoblast (NHOst) cells. Iodothyronine deiodinase activity was detected in SaOS-2 cells and NHOst cells, and all of the characteristics of deiodinating activity were compatible with those of D2. Northern analysis demonstrated D2 mRNA expression in SaOS-2 cells and NHOst cells. D2 mRNA levels as well as D2 activities were rapidly increased by dibutyryl cAMP or forskolin in SaOS-2 cells and NHOst cells. TSH receptor mRNA was demonstrated in SaOS-2 cells and NHOst cells, and D2 mRNA and D2 activity were stimulated by TSH in both cells. In addition, all T(3) receptor isoforms were detected by RT-PCR in SaOS-2 cells and NHOst cells. The present results indicate the expression of functional TSH receptors and D2 in human osteoblasts and suggest previously unrecognized roles of TSH receptors and local T(3) production by D2 in the pathophysiology of human osteoblasts.     

Hypothalamic thyrotropin-releasing hormone regulates pituitary thyrotropin beta- and alpha-subunit mRNA levels in the rat.

It has been controversial whether thyrotropin-releasing hormone (TRH) may be involved in the regulation of biosynthesis of anterior pituitary thyroid-stimulating hormone (TSH), whereas TRH is well known to control the secretion of TSH from anterior pituitaries. We therefore studied the effect of the destruction of paraventricular nuclei (PVN), containing TRH neuronal cell bodies, on anterior pituitary TSH beta- and alpha-subunit mRNA levels the thyroidectomized rat. Median eminence TRH contents and serum TSH levels significantly decreased even 1 day after PVN destruction, whereas anterior pituitary TSH beta- and alpha-subunit mRNA levels did not change. In contrast, these mRNA levels significantly decreased by 3 days after PVN destruction. Growth hormone mRNA levels were not affected during 7 days after PVN destruction. Although the involvement of other factors related to the PVN is not totally excluded, these results raise the strong possibility that hypothalamic TRH regulates anterior pituitary TSH mRNA levels.     

Type 1 iodothyronine deiodinase is a sensitive marker of peripheral thyroid status in the mouse

Mice with one thyroid hormone receptor (TR) alpha-1 allele encoding a dominant negative mutant receptor (TR alpha1(PV/+)) have persistently elevated serum T3 levels (1.9-fold above normal). They also have markedly increased hepatic type 1 iodothyronine deiodinase (D1) mRNA and enzyme activity (4- to 5-fold), whereas other hepatic T3-responsive genes, such as Spot14 and mitochondrial alpha-glycerol phosphate dehydrogenase (alpha-GPD), are only 0.7-fold and 1.7-fold that of wild-type littermates (TR alpha1+/+). To determine the cause of the disproportionate elevation of D1, TR alpha1+/+ and TR alpha1(PV/+) mice were rendered hypothyroid and then treated with T3. Hypothyroidism decreased hepatic D1, Spot14, and alpha-GPD mRNA to similar levels in TR alpha1+/+ and TR alpha1(PV/+) mice, whereas T3 administration caused an approximately 175-fold elevation of D1 mRNA but only a 3- to 6-fold increases in Spot14 and alpha-GPD mRNAs. Interestingly, the hypothyroidism-induced increase in cerebrocortical type 2 iodothyronine deiodinase activity was 3 times greater in the TR alpha1(PV/+) mice, and these mice had no T3-dependent induction of type 3 iodothyronine deiodinase. Thus, the marked responsiveness of hepatic D1 to T3 relative to other genes, such as Spot14 and alpha-GPD, explains the relatively large effect of the modest increase in serum T3 in the TR alpha1(PV/+) mice, and TR alpha plays a key role in T3-dependent positive and negative regulation of the deiodinases in the cerebral cortex.     

Exogenous triiodothyronine lowers thyrotropin-releasing hormone concentrations in the specific hypothalamic nucleus (paraventricular) involved in thyrotropin regulation and also in posterior nucleus

Recent evidence indicates that thyroid hormones can regulate thyrotropin secretion in vivo in part by inhibiting thyrotropin-releasing hormone (TRH) secretion itself. Therefore, to explore whether triiodothyronine (T3) interacts with the specific hypothalamic area involved in thyrotropin (TSH) secretory regulation, the paraventricular nucleus (PVN), Palkovitz micropunches from eight nuclear regions were obtained from 1,000-microns frozen coronal brain slices for immunoassay determinations of TRH. Rats were treated either with parenteral L-T3 for 6 days to induce experimental thyrotoxicosis, or 0.15 M saline. The induction of thyrotoxicosis was confirmed by demonstrating that mean plasma TSH concentrations fell from 108 to less than 10 microU/ml (p less than 0.01). TRH concentrations in the PVN were reduced concomitantly after L-T3 from 1.9 to 1.1 ng/mg protein (p less than 0.05). No reductions in TRH concentrations during T3 treatment occurred in other nuclear groupings except in the posterior hypothalamic nucleus. Total TRH content in the median eminence declined also in T3-treated animals from 1.77 to 1.29 ng, representing a 32% reduction (p less than 0.01). No significant change was seen in the median eminence content of the TRH structurally related dipeptide, cyclo(His-Pro). The data herein indicate that experimental thyrotoxicosis in the rat is associated with a selective reduction in TRH concentrations in the PVN, documenting T3 effects upon hypothalamic TRH metabolism per se.     

Hypothalamic secretion of thyrotrophin releasing hormone is decreased in male Japanese quail exposed to long daily photoperiods

Adult male Japanese quail held under short daily photoperiods (8 h light: 16 h darkness; 8L: 16D) had significantly higher plasma concentrations of thyroid-stimulating hormone (TSH), tri-iodothyronine (T3) and thyroxine (T4) than did those kept under long days (16L:8D). When given a single s.c. injection of 50 microgram thyrotrophin releasing hormone (TRH) the birds held under both the 8L: 16D and 16L: 8D photoperiods showed rapid increases in their blood concentrations of TSH, T4 and T3, the amplitude of the TSH response of the birds exposed to 16L: 8D being particularly marked. These results suggest that, in the male quail, long daily photoperiods produce a hypothyroid state as a result of diminished TRH secretion. The synthetic and secretory capacities of the thyroid gland and pituitary thyrotrophs are apparently unimpaired by long days.     

Effects of amiodarone and desethylamiodarone on pituitary deiodinase activity and thyrotropin secretion in the rat

The effect of acute administration of amiodarone, its major metabolite desethylamiodarone and iodine in an amount equal to that contained in amiodarone on serum thyroid hormone and thyrotropin (TSH) concentrations and hepatic and pituitary 5' deiodination of thyroxine (T4) in the euthyroid and hypothyroid rat was evaluated. Amiodarone, desethylamiodarone and iodine all caused a decrease in serum T4 and triiodothyronine (T3) concentrations in euthyroid rats, while serum TSH concentrations and pituitary and hepatic 5' deiodinase activities were decreased only in the amiodarone and desethylamiodarone-treated animals. Serum TSH was increased in the iodine treated rats. Amiodarone, but not iodine, decreased serum T3 and TSH concentrations and pituitary and hepatic 5' deiodinase activities in hypothyroid rats. Inhibition of hepatic 5' deiodinase activity was also observed by the addition of amiodarone in vitro in the absence of dithiothreitol (DTT) but not in the presence of DTT. The decrease in the serum T4 concentration observed with amiodarone and desethylamiodarone administration is probably secondary to the inhibitory effect of iodine released from the drugs on thyroidal T4 synthesis and secretion. Iodine inhibition of thyroidal T3 synthesis and secretion, decreased T4 substrate for a peripheral generation of T3 and inhibition of T4 to T3 conversion all contribute to the decrease in serum T3 observed. The decrease in the serum TSH concentration, despite low serum T4 and T3 concentrations and inhibition of pituitary 5' deiodinase, suggest that amiodarone may function as a thyroid hormone agonist in the pituitary.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional neuroanatomy of thyroid hormone feedback in the human hypothalamus and pituitary gland

A major change in thyroid setpoint regulation occurs in various clinical conditions such as critical illness and psychiatric disorders. As a first step towards identifying determinants of these setpoint changes, we have studied the distribution and expression of thyroid hormone receptor (TR) isoforms, type 2 and type 3 deiodinase (D2 and D3), and the thyroid hormone transporter monocarboxylate transporter 8 (MCT8) in the human hypothalamus and anterior pituitary. Although the post-mortem specimens used for these studies originated from patients who had died from many different pathologies, the anatomical distribution of these proteins was similar in all patients. D2 enzyme activity was detectable in the infundibular nucleus/median eminence (IFN/ME) region coinciding with local D2 immunoreactivity in glial cells. Additional D2 immunostaining was present in tanycytes lining the third ventricle. Thyrotropin-releasing hormone (TRH) containing neurons in the paraventricular nucleus (PVN) expressed MCT8, TRs as well as D3. These findings suggest that the prohormone thyroxine (T4) is taken up in hypothalamic glial cells that convert T4 into the biologically active triiodothyronine (T3) via the enzyme D2, and that T3 is subsequently transported to TRH producing neurons in the PVN. In these neurons, T3 may either bind to TRs or be metabolized into inactive iodothyronines by D3. By inference, local changes in thyroid hormone metabolism resulting from altered hypothalamic deiodinase or MCT8 expression may underlie the decrease in TRH mRNA reported earlier in the PVN of patients with critical illness and depression. In the anterior pituitary, D2 and MCT8 immunoreactivity occurred exclusively in folliculostellate (FS) cells. Both TR and D3 immunoreactivity was observed in gonadotropes and to a lesser extent in thyrotropes and other hormone producing cell types. Based upon these neuroanatomical findings, we propose a novel model for central thyroid hormone feedback in humans, with a pivotal role for hypothalamic glial cells and pituitary FS cells in processing and activation of T4. Production and action of T3 appear to occur in separate cell types of the human hypothalamus and anterior pituitary.     

Hypothyroidism and deficiency of the nocturnal thyrotropin surge in children with hypothalamic-pituitary disorders

The circadian pattern of serum TSH in normal children, aged 5-18 yr, is characterized by a nocturnal surge and is presumably related in some way to a biological clock within the central nervous system. To look for patients deficient in the nocturnal TSH surge, we studied 52 children with hypothalamic-pituitary disorders. Thirteen of the children were hypothyroid, as judged by subnormal serum free T4 (FT4). The hypothyroid patients had a mean nocturnal TSH surge of 22% (range, -30% to +114%), significantly less than that of normal controls (mean, 124%; 95% confidence limits, 47-300%; n = 96; P less than 0.01). Only 1 of the hypothyroid children had a value for the nocturnal TSH surge (114%) that was within the normal range. Nineteen of the 52 patients with hypothalamic-pituitary disorders had subnormal nocturnal TSH surges; their mean iodothyronine values were significantly less than those of the 33 patients with normal surges [total T4, 73 +/- 4 (mean +/- SE) vs. 109 +/- 3 nmol/L (P less than 0.01); FT4, 13 +/- 1.0 vs. 19 +/- 0.5 pmol/L (P less than 0.01)]. These data demonstrate a clear association of a deficient nocturnal TSH surge and low iodothyronine concentration in children with hypothalamic-pituitary disorders. We performed both TRH tests and nocturnal TSH surge tests in 11 of the children with central hypothyroidism; TRH was abnormal in only 2, while the nocturnal surge test was abnormal in 10 of the 11. We suggest that the nocturnal surge of TSH is important for maintenance of thyroid function and conclude that the nocturnal TSH surge is a much more sensitive test than the TSH response to TRH for the diagnosis of central hypothyroidism.     

Differential effects of refeeding on melanocortin-responsive neurons in the hypothalamic paraventricular nucleus

To explore the effect of refeeding on recovery of TRH gene expression in the hypothalamic paraventricular nucleus (PVN) and its correlation with the feeding-related neuropeptides in the arcuate nucleus (ARC), c-fos immunoreactivity (IR) in the PVN and ARC 2 h after refeeding and hypothalamic TRH, neuropeptide Y (NPY) and agouti-related protein (AGRP) mRNA levels 4, 12, and 24 h after refeeding were studied in Sprague-Dawley rats subjected to prolonged fasting. Despite rapid reactivation of proopiomelanocortin neurons by refeeding as demonstrated by c-fos IR in ARC alpha-MSH-IR neurons and ventral parvocellular subdivision PVN neurons, c-fos IR was present in only 9.7 +/- 1.1% hypophysiotropic TRH neurons. Serum TSH levels remained suppressed 4 and 12 h after the start of refeeding, returning to fed levels after 24 h. Fasting reduced TRH mRNA compared with fed animals, and similar to TSH, remained suppressed at 4 and 12 h after refeeding, returning toward normal at 24 h. AGRP and NPY gene expression in the ARC were markedly elevated in fasting rats, AGRP mRNA returning to baseline levels 12 h after refeeding and NPY mRNA remaining persistently elevated even at 24 h. These data raise the possibility that refeeding-induced activation of melanocortin signaling exerts differential actions on its target neurons in the PVN, an early action directed at neurons that may be involved in satiety, and a later action on hypophysiotropic TRH neurons involved in energy expenditure, potentially mediated by sustained elevations in AGRP and NPY. This response may be an important homeostatic mechanism to allow replenishment of depleted energy stores associated with fasting.     

Impact of Oatp1c1 deficiency on thyroid hormone metabolism and action in the mouse brain

Organic anion-transporting polypeptide 1c1 (Oatp1c1) (also known as Slco1c1 and Oatp14) belongs to the family of Oatp and has been shown to facilitate the transport of T(4). In the rodent brain, Oatp1c1 is highly enriched in capillary endothelial cells and choroid plexus structures where it may mediate the entry of T(4) into the central nervous system. Here, we describe the generation and first analysis of Oatp1c1-deficient mice. Oatp1c1 knockout (KO) mice were born with the expected frequency, were not growth retarded, and developed without any overt neurological abnormalities. Serum T(3) and T(4) concentrations as well as renal and hepatic deiodinase type 1 expression levels were indistinguishable between Oatp1c1 KO mice and control animals. Hypothalamic TRH and pituitary TSH mRNA levels were not affected, but brain T(4) and T(3) content was decreased in Oatp1c1-deficient animals. Moreover, increased type 2 and decreased type 3 deiodinase activities indicate a mild hypothyroid situation in the brain of Oatp1c1 KO mice. Consequently, mRNA expression levels of gene products positively regulated by T(3) in the brain were down-regulated. This central nervous system-specific hypothyroidism is presumably caused by an impaired passage of T(4) across the blood-brain barrier and indicates a unique function of Oatp1c1 in facilitating T(4) transport despite the presence of other thyroid hormone transporters such as Mct8.     

Altered thyrotropin (TSH) carbohydrate structures in hypothalamic hypothyroidism created by paraventricular nuclear lesions are corrected by in vivo TSH-releasing hormone administration

TSH is a glycoprotein hormone composed of two subunits with attached carbohydrate chains that have varying structural characteristics. To determine the role of TRH in vivo in regulating structural characteristics of TSH carbohydrate chains, adult rats received paraventricular nuclear (PVN) lesions (n = 6) or sham lesions (n = 6). The PVN contain large amounts of TRH, and rats with lesions in these hypothalamic nuclei have been shown to have decreased plasma thyroid hormone levels. At 10 days after surgery, sc osmotic pumps infusing saline or 1 mg/kg/day TRH were placed. At 14 days after surgery, pituitaries were removed and incubated with [3H]glucosamine for 24 h. Glycopeptides prepared from secreted TSH were sequentially eluted from Concanavalin-A chromatography columns selecting unbound, weakly bound, and strongly bound forms. Plasma free T4 was lower in the PVN lesioned rats treated with saline than sham lesioned rats treated with saline (1.6 +/- 0.4 vs. 5.2 +/- 0.1 ng/dl, P less than 0.001). In vivo TRH administration in the PVN lesioned group normalized plasma free T4 but had no effect on free T4 in the sham group. Secreted TSH glycopeptides in the PVN lesioned rats treated with saline as compared to sham lesioned rats treated with saline had fewer unbound forms reflecting multiantennary structures (43 +/- 4 vs. 57 +/- 1%; P less than 0.05) and more weakly bound forms reflecting biantennary structures (50 +/- 4 vs. 35 +/- 2%; P less than 0.05). TRH administration in vivo normalized the Concanavalin-A binding pattern of secreted TSH glycopeptides in the PVN lesioned group but had no significant effect in the sham lesioned group. TSH alpha-subunit demonstrated both multi- and biantennary forms but TSH-beta subunit showed a predominance of multiantennary forms in both the PVN and sham lesioned groups treated with saline. In vivo changes in TRH levels altered TSH carbohydrate characteristics as described above for both subunits. In summary, hypothalamic hypothyroidism altered TSH carbohydrate structures, and in vivo TRH administration normalized these structures in parallel with the correction of serum free T4. In addition to reported quantitative changes in TSH in response to TRH, these qualitative changes may have an important effect on TSH action.     

Thyrotropin-releasing hormone regulates the diurnal variation of pyroglutamyl aminopeptidase II activity in the male rat adenohypophysis

OBJECTIVE: Thyrotropin-releasing hormone (TRH) is inactivated in the extracellular compartment by pyroglutamyl aminopeptidase II (PPII), a narrow specificity ectopeptidase present in the brain and in the lactotrophs of the adenohypophysis. TRH and various hypothalamic/paracrine agents regulate the activity of PPII on the surface of adenohypophyseal cells in primary culture. The activity of the hypothalamic-pituitary-thyroid axis presents circadian variations including an increase of serum thyrotropin levels in the early hours of the day. The purpose of this study was to determine whether adenohypophyseal PPII activity fluctuates during the daytime in the male rat and the role of TRH in these regulatory events in vivo. RESULTS: Adenohypophyseal PPII specific activity and mRNA levels presented diurnal variations. A decrease in specific activity occurred with a minimum between 0930 and 1130 h, associated with increased serum thyrotropin levels. PPII mRNA levels were lowest at 0800 h. Intraperitoneal injection at 0800 or 1000 h of [3-Me-His(2)]-TRH, a potent agonist of the TRH receptor, reduced PPII specific activity at 30 min post-injection which was followed by a return to basal levels at 2 h. A second phase of decrease occurred between 4 and 8 h post-injection. Intravenous injection of a TRH-immune serum induced, at 2 h post-injection, an increase in adenohypophyseal PPII specific activity, which lasted up to 6 h. CONCLUSIONS: Adenohypophyseal PPII activity and mRNA levels fluctuate during the day; TRH down-regulates PPII activity in vivo, contributing to some of these variations. These new findings, and previous data, suggest that adenohypophyseal PPII activity varies in distinct physiological events, in response to endocrine and hypothalamic/paracrine factors, potentially modulating responses to TRH.